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Polyploidy and Genome Evolution
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2012
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Polyploidy and Genome Evolution
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Pamela S. Soltis Douglas E. Soltis
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Polyploidy and Genome
Evolution
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Editors
Pamela S. Soltis
Florida Museum of Natural History
University of Florida
Gainesville, FL
USA
Douglas E. Soltis
Florida Museum of Natural History
University of Florida
Gainesville, FL
USA
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ISBN 978-3-642-31442-1
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ISBN 978-3-642-31441-4
DOI 10.1007/978-3-642-31442-1
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Preface
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Polyploidy (whole-genome duplication; WGD) is common in plants and has long
been considered as both an important speciation mechanism and a crucial component of plant genome structure. Analyses of chromosome numbers and
hypothesized breaks between diploid and polyploid base numbers have suggested
anywhere from 30 to 80 % of all angiosperms are polyploid. While recent
polyploids may be easily detected through comparison of chromosome numbers,
various processes of diploidization or fractionation may substantially alter chromosome numbers and structure, ultimately masking the evolutionary history of
duplication events. In contrast, other footprints of ancient WGD may remain in the
genome, even when chromosome numbers no longer carry the signature of past
WGDs. Genome sequences and other sources of genomic data tell us that, in fact,
all angiosperms, as well as all seed plants, have undergone one or more rounds of
polyploidy. Furthermore, ancient WGD characterizes all vertebrates, with subsequent, more recent polyploidization in fishes and amphibians. Ancient WGD is
also evident in the genomes of yeast and other fungi. While more common in
plants than other major lineages of life, polyploidy is now recognized as a fundamental process in all crown eukaryotes. Polyploidy plays a major role in shaping
genome structure and organization and in establishing patterns and mechanisms of
gene regulation. In fact, it is now impossible to construct models of genome
evolution that do not account for genomic content and genetic interactions contributed by WGD.
It has been over 30 years since the publication of a comprehensive treatment of
polyploidy [Polyploidy: Biological Relevance, W. H. Lewis (ed.), 1980]. The
intervening years have witnessed a technological revolution with a transition from
the early days of recombinant DNA to nearly routine genome sequencing of nonmodel organisms and from limited biological computing to high-performance
computing networks for the biological sciences. These transformations in methodology and computation permit fresh perspectives on polyploidy and the ability
to ask old questions with new tools.
Over the past decade, it has been a dream of ours to publish a book that
synthesizes the rapid progress in understanding the role of polyploidy in genome
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Gainesville, April 2012
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evolution, and this book is now a reality. In the current volume, we have compiled
the expertise of scientists studying polyploid genome evolution from multiple
perspectives in phylogenetically diverse organisms. Topics range from the conceptual and theoretical underpinnings of polyploidy (chapters by McGrath and
Lynch, Birchler) to processes at work in polyploid genomes (Zielinski and
Mittelsten Scheid, Finigan et al., Evans et al.), to patterns of ancient polyploidy
and its detection (Burleigh, Paterson et al.), to a series of case studies that both
document attributes of genome evolution in focal species and address general
properties of polyploid genomes, from ancient polyploids [maize (Schnable and
Freeling), legumes (Doyle), vertebrates (Cañestro), fishes (Braasch and Postlethwait), yeast (Hudson and Conant)] to classic model polyploids [cotton (Wendel
et al.), tobacco (Kovarik et al.), wheat (Feldman et al.)] to very recent ones
[Spartina (Ainouche), Senecio (Hegarty et al.), and Tragopogon (Soltis et al.)].
The emerging paradigm from these studies is that polyploidy—through alterations
in genome structure and gene regulation, some of which occur shortly after
polyploid formation—generates genetic and phenotypic novelty that manifests
itself at the chromosomal, physiological, and organismal levels, with long-term
ecological and evolutionary consequences.
We thank our many colleagues, students, and postdocs for lively and challenging discussions on polyploidy and its many evolutionary consequences. We
further acknowledge the support of the U.S. National Science Foundation (Grants
9624643, 0346437, 0614421, 0919254, and 0922003).
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Contents
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1
Evolutionary Significance of Whole-Genome Duplication . . . . . . .
C. L. McGrath and M. Lynch
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Genetic Consequences of Polyploidy in Plants . . . . . . . . . . . . . . .
James A. Birchler
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Meiosis in Polyploid Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Marie-Luise Zielinski and Ortrun Mittelsten Scheid
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Origins of Novel Phenotypic Variation in Polyploids . . . . . . . . . .
Patrick Finigan, Milos Tanurdzic and Robert A. Martienssen
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Identifying the Phylogenetic Context of Whole-Genome
Duplications in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
J. Gordon Burleigh
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Genomic Plasticity in Polyploid Wheat . . . . . . . . . . . . . . . . . . . .
Moshe Feldman, Avraham Levy, Boulos Chalhoub
and Khalil Kashkush
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Maize (Zea mays) as a Model for Studying the Impact
of Gene and Regulatory Sequence Loss Following
Whole-Genome Duplication . . . . . . . . . . . . . . . . . . . . . . . . . . . .
James C. Schnable and Michael Freeling
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Ancient and Recent Polyploidy in Monocots . . . . . . . . . . . . . . . .
Andrew H. Paterson, Xiyin Wang, Jingping Li and Haibao Tang
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Polyploidy in Legumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Jeff J. Doyle
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Polyploid Evolution in Spartina: Dealing with Highly
Redundant Hybrid Genomes. . . . . . . . . . . . . . . . . . . . . . . . . . . .
M. Ainouche, H. Chelaifa, J. Ferreira, S. Bellot, A. Ainouche
and A. Salmon
Allopolyploid Speciation in Action: the Origins and Evolution
of Senecio cambrensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Matthew J. Hegarty, Richard J. Abbott and Simon J. Hiscock
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The Early Stages of Polyploidy: Rapid and Repeated
Evolution in Tragopogon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Douglas E. Soltis, Richard J. A. Buggs, W. Brad Barbazuk,
Srikar Chamala, Michael Chester, Joseph P. Gallagher,
Patrick S. Schnable and Pamela S. Soltis
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Evolutionary Implications of Genome and Karyotype
Restructuring in Nicotiana tabacum L. . . . . . . . . . . . . . . . . . . . .
Ales Kovarik, Simon Renny-Byfield, Marie-Angèle Grandbastien
and Andrew Leitch
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Yeast as a Window into Changes in Genome Complexity
Due to Polyploidization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corey M. Hudson and Gavin C. Conant
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Two Rounds of Whole-Genome Duplication: Evidence
and Impact on the Evolution of Vertebrate Innovations . . . . . . . .
Cristian Cañestro
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Jeans, Genes, and Genomes: Cotton as a Model
for Studying Polyploidy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Jonathan F. Wendel, Lex E. Flagel and Keith L. Adams
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Contents
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Polyploidy in Fish and the Teleost Genome Duplication . . . . . . . .
Ingo Braasch and John H. Postlethwait
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Polyploidization and Sex Chromosome Evolution
in Amphibians . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ben J. Evans, R. Alexander Pyron and John J. Wiens
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Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Book Title
Polyploidy and Genome Evolution
Series Title
Chapter Title
Evolutionary Significance of Whole-Genome Duplication
Copyright Year
2012
Copyright HolderName
Springer-Verlag Berlin Heidelberg
Corresponding Author
Family Name
Lynch
Particle
Given Name
M.
Suffix
Author
Division
Department of Biology
Organization
Indiana University
Address
Bloomington, IN, USA
Email
milynch@indiana.edu
Family Name
McGrath
Particle
Given Name
C. L.
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Division
Department of Biology
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Indiana University
Address
Bloomington, IN, USA
Email
Abstract
Whole-genome duplication (WGD) appears to be a widespread phenomenon, occurring in diverse taxa
including many of the model organisms used in molecular, cellular, and developmental biology. It is therefore
essential to understand the potential evolutionary consequences for individual duplicated genes, as well as
for the lineage as a whole. For example, duplicate genes may undergo pseudogenization or may be maintained
due to neofunctionalization, subfunctionalization, or selection for increased dosage or dosage balance.
Duplicates created via WGD are maintained at higher rates than single-gene duplicates, perhaps due to dosagebalance constraints. Duplicate-gene maintenance may lead to heterodimerization of an existing homodimer
or to the divergence of an entire duplicated network or pathway. Allopolyploids and autopolyploids are likely
to undergo different evolutionary pressures due to increased divergence between allopolyploid paralogs and
an increased prevalence of multivalent formation at meiosis in autopolyploids. Perhaps most importantly,
duplicate-gene loss following a WGD may significantly increase the rate of reproductive isolation between
geographically isolated subpopulations and may therefore temporarily increase the speciation rate within
polyploid lineages.
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1.1 Introduction
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Abstract Whole-genome duplication (WGD) appears to be a widespread
phenomenon, occurring in diverse taxa including many of the model organisms
used in molecular, cellular, and developmental biology. It is therefore essential to
understand the potential evolutionary consequences for individual duplicated
genes, as well as for the lineage as a whole. For example, duplicate genes may
undergo pseudogenization or may be maintained due to neofunctionalization,
subfunctionalization, or selection for increased dosage or dosage balance. Duplicates created via WGD are maintained at higher rates than single-gene duplicates,
perhaps due to dosage-balance constraints. Duplicate-gene maintenance may lead
to heterodimerization of an existing homodimer or to the divergence of an entire
duplicated network or pathway. Allopolyploids and autopolyploids are likely to
undergo different evolutionary pressures due to increased divergence between
allopolyploid paralogs and an increased prevalence of multivalent formation at
meiosis in autopolyploids. Perhaps most importantly, duplicate-gene loss following a WGD may significantly increase the rate of reproductive isolation between
geographically isolated subpopulations and may therefore temporarily increase the
speciation rate within polyploid lineages.
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One of the major findings of the new field of evolutionary genomics is that
duplication events involving individual genes or multigene segments arise at rates
comparable to the rate of mutation at single-nucleotide sites (Lynch and Conery
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e-mail: milynch@indiana.edu
P. S. Soltis and D. E. Soltis (eds.), Polyploidy and Genome Evolution,
DOI: 10.1007/978-3-642-31442-1_1, Springer-Verlag Berlin Heidelberg 2012
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2000, 2003a, b), or possibly at even higher rates (Lipinski et al. 2011). Such
observations lend credibility to Ohno’s (1970) early speculation that gene duplication is a major resource for the origin of evolutionary novelties. Moreover, it is
now clear that whole-genome duplication (WGD) events have occurred in a wide
diversity of phylogenetic lineages, including most of the model systems relied
upon in molecular, cellular, and developmental biology. For example, budding
yeast is a descendant of an ancient genome duplication (Wolfe and Shields 1997;
see Chap. 15, this volume), as is the frog Xenopus laevis (Morin et al. 2006; see
Chap. 18, this volume) and the zebrafish (Postlethwait et al. 2000; see Chap. 17,
this volume). Many ray-finned fish lineages have experienced additional rounds of
WGD (Meyer and Van de Peer 2005; see Chap. 17, this volume), and Ohno’s
(1970) suggestion that two WGD events preceded the radiation of the vertebrate
lineage has become increasingly credible (Panopoulou and Poustka 2005; Hughes
and Liberles 2008; Putnam et al. 2008; see Chap. 16, this volume). Finally, three
WGD events are recorded within the genome of Arabidopsis thaliana (Simillion
et al. 2002), and nearly all other land-plant genomes appear to harbor a legacy of at
least one polyploidization event (Doyle et al. 2008), with a proposed WGD in the
ancestor of all seed plants and another in the ancestor of all angiosperms (Jiao
et al. 2011). Thus, it is clear that understanding the mechanisms of origin and
preservation of duplicate genes promises to reveal not only the ways in which
genes acquire new functions and organisms respond to natural selection, but also
the roots of organismal diversity across the tree of life.
Because genome duplication adds thousands of duplicate genes to the genome,
understanding the evolutionary forces that act on individual duplicate genes is
critical to our understanding of polyploidization. Processes such as neofunctionalization and subfunctionalization have the potential to influence all gene duplicates, whether created through polyploidization or smaller scale duplication
events. It has become increasingly clear, however, that duplicates that arise via
polyploidization are subject to unique evolutionary forces, such as increased
retention due to dosage-balance constraints. Further, there may be processes that
are exclusive to gene duplicates that arise via specific types of polyploidization,
such as changes in duplicate-gene expression due to the genomic merger that
occurs with allopolyploidization. The relative contributions of these evolutionary
forces that give rise to the maintenance and evolution of duplicate genes that arise
via WGD, or to the evolution of the genome or species as a whole, are currently
unknown. However, discriminating between these forces and their effects is likely
to be the subject of much research over the next several years.
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1.2 Fates of Duplicate Genes
The fate of the vast majority of duplicate genes arising by segmental duplication is
nonfunctionalization of one member of the pair (Lynch and Conery 2000, 2003a, b),
and this is expected to occur within a few million years in the absence of any
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intrinsic advantage of a duplicate copy (Watterson 1983; Lynch et al. 2001). Despite
this, most genomes that have been studied contain a large number of duplicate
genes, some of which are clearly quite ancient (Lynch and Conery 2000). Based on
this observation, several mechanisms have been proposed for the permanent preservation of duplicate genes (Hughes 1994; Force et al. 1999; Lynch et al. 2001;
Taylor and Raes 2004; Lynch 2007; Innan and Kondrashov 2010): (1) neofunctionalization, whereby one copy acquires a novel, beneficial function at the expense
of an essential ancestral function; (2) subfunctionalization, whereby complementary
mutations lead to a partitioning of independently mutable subfunctions in the
ancestral gene; (3) selection for increased gene product; and (4) the masking of
nonfunctional alleles.
When a duplicate is maintained by selection for increased gene product, it
experiences purifying selection (and may also undergo repeated gene conversion)
in order to maintain its ancestral function; this process is likely responsible for the
multiple copies of ribosomal RNA genes present in many genomes (e.g., Pinhal
et al. 2011). Neofunctionalization, on the other hand, is thought to involve positive
selection for the mutation(s) responsible for the new function, generally arising at
the expense of an essential original function, thereby preserving both copies. There
are many examples of neofunctionalization giving rise to novel gene functions in a
variety of organisms, including Arabidopsis (Erdmann et al. 2010), fish (Ngai et al.
1993), vertebrates (Layeghifard et al. 2009), and yeast (Byrne and Wolfe 2007;
Tirosh and Barkai 2007). Because one duplicate is undergoing positive selection
for a new function while the other is under purifying selection to maintain the
ancestral function, asymmetric evolutionary rates between duplicates are often
thought to be a hallmark of neofunctionalization (Johnson and Thomas 2007; Han
et al. 2009), though purely stochastic mechanisms can also give rise to apparent
rate asymmetry (Lynch and Katju 2004).
Subfunctionalization may involve positive selection acting on both duplicates if
the partitioning of the ancestral functions leads to relaxation of pleiotropic
constraints, enabling each ancestral function to be fine-tuned and improved
through mutation independently in each copy (Piatigorsky and Wistow 1991;
Hughes 1994; Des Marais and Rausher 2008). Alternatively, subfunctionalization
may be a completely neutral process if each duplicate copy simply acquires a
degenerative mutation that renders it unable to perform one of the ancestral
functions (Force et al. 1999). At this point, both copies are needed in order to
provide the organism with all of the functionality of the original, single-copy gene,
and so both will be maintained in the genome by selection. Although identifying
definitive cases of subfunctionalization requires determining that the ancestral
gene carried multiple functions that have been partitioned in the daughter duplicates, there are nonetheless several compelling examples (e.g., Force et al. 1999;
Altschmied et al. 2002; Yu et al. 2003; Adams and Liu 2007; MacNeil et al. 2008;
Semon and Wolfe 2008; Buggs et al. 2010; Deng et al. 2010; Hickman and Rusche
2010; Colon et al. 2011; Froyd and Rusche 2011).
In addition to these cases of qualitative subfunctionalization, where duplicates
eventually come to be expressed in different tissues or at different times or carry out
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different functions relative to each other, quantitative subfunctionalization, in which
reduction-of-expression (Force et al. 1999) or activity-reducing mutations (Stoltzfus
1999; Scannell and Wolfe 2008) affect both duplicates, is also possible. In quantitative subfunctionalization, both duplicates acquire partial loss-of-function
mutations that affect the same function, again rendering both copies essential for the
proper dosage or activity of the gene products. In this case, both copies are preserved
while retaining the ancestral gene function. Although few studies demonstrating
quantitative subfunctionalization exist, Qian et al. (2010) estimated that this process
has been responsible for the maintenance of a large proportion of duplicates in yeast
and mammals, whereas Woolfe and Elgar (2007) postulated that sequence evolution
in cis-regulatory elements may have caused quantitative subfunctionalization
among Fugu duplicates.
A related consequence of gene duplication is that it can allow for the differentiation of multimeric subunits, such as the evolution of heterodimers from
homodimers. Consider a gene whose protein product forms a homodimer. After
duplication of this gene, protein subunits produced by the two duplicates (denoted
A and B) may randomly associate to make mixtures of dimers in the ratio 1 AA: 2
AB: 1 BB. If the duplicate genes are identical initially, the AA, AB, and BB dimers
will be identical as well. However, subsequent differentiation of the duplicate
genes causes the three types of dimer to become distinct. This differentiation could
be neutral, or it could be selective. If, for example, there were pleiotropic
constraints on the form or function of the pre-duplication homodimer, duplication
could allow for escape from these constraints in the AB heterodimer, as each
subunit (A and B) can now evolve independently. This can be viewed as a special
type of subfunctionalization of duplicates. Winter et al. (2002) showed that a classB floral protein heterodimer had evolved from an ancestral homodimer via this
mechanism during the gymnosperm/angiosperm transition. In gymnosperms,
GGM2-like genes form homodimers, while the duplicated homologs in eudicots,
DEF-like genes and GLO-like genes, form heterodimers. Monocots also have
duplicated DEF-like genes and GLO-like genes, but, interestingly, it appears the
GLO-like proteins of monocots can both homodimerize and heterodimerize with
DEF-like proteins, perhaps representing the transition between the homo- and
heterodimerized states (Winter et al. 2002; Kanno et al. 2003; Soltis et al. 2006).
A similar process appears to have occurred several times in the evolution of the
DUF606 family of transmembrane proteins in bacteria (Lolkema et al. 2008). In
bacteria with a single DUF606 gene, the DUF606 proteins are able to insert into the
membrane in both orientations, and functional homodimers are formed by two
subunits in opposite (antiparallel) orientations. Other species of bacteria, however,
have duplicated DUF606 genes located tandemly in an operon. In all of these latter
cases, the two protein subunits each have a fixed but opposite orientation in the
membrane, and they heterodimerize to form the necessary antiparallel two-domain
complex. A phylogenetic analysis of the DUF606 gene family reveals that this
process of duplication followed by heterodimerization likely occurred five different
times in the history of this gene family lineage (Lolkema et al. 2008). Other
proposed examples of this mechanism include SMC proteins (Surcel et al. 2008),
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1.3 Fates of Duplicate Genes Arising via WGD
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In addition to the general preservational processes just mentioned, paralogs
resulting from WGD events are subject to unique mechanisms of duplicate-gene
maintenance and evolution (Force et al. 1999; Lynch and Conery 2000; Yang et al.
2003; Davis and Petrov 2005; Veitia et al. 2008). Well-studied polyploid species
commonly exhibit 25–75 % retention of paralogous gene pairs from the most
recent WGD event (reviewed in Lynch 2007; Otto 2007), budding yeast being an
exception with only *8 % duplicate-gene preservation (Wolfe and Shields 1997).
These are surprisingly high preservational levels, when, as discussed above, the
fate of the vast majority of duplicate genes arising by segmental duplication is
nonfunctionalization of one duplicate (Lynch and Conery 2000, 2003a, b).
Although it is possible that many polyploid species have not yet reached equilibrium and are still in an ongoing phase of duplicate-gene loss, it has become
increasingly clear that there are likely to be additional forces acting to preserve
duplicate genes arising via WGD.
A simple explanation for the large number of preserved duplicates within
polyploids is that, unlike single-gene duplicates, WGD duplicates exhibit complete
conservation of surrounding regulatory sequences, chromosomal environments, etc.
Although this likely contributes somewhat to the pattern of higher duplicate
retention in polyploids, it does not explain the observation that different types of
genes seem to be preserved following WGD compared to smaller scale duplications.
This fact can be better explained by selection for dosage balance among proteins.
Due to stoichiometric relationships with other interacting genes (e.g., multi-subunit
complexes and numerous pathways involved in metabolism and transcriptional
regulation), the functions of a subset of protein-coding loci can be highly influenced
by dosage imbalances (Veitia 2002; Papp et al. 2003; Birchler et al. 2005; Veitia
et al. 2008). In such cases, duplication of just a single member of a gene interaction
may be detrimental and actively selected against. In contrast, following a WGD
event, most stoichiometric relationships are initially intact, and therefore
subsequent losses of interacting paralogs will be inhibited by selection for proper
dosage relationships. Thus, for dosage-dependent genes, the dosage-balance
hypothesis predicts an under-representation among duplicates created by singlegene duplications, but an over-representation among those created by WGD (Yang
et al. 2003; Davis and Petrov 2005; Veitia et al. 2008). For example, Davis and
Petrov (2005) showed that the pool of preserved duplicates from the WGD event in
S. cerevisiae is enriched for ribosomal genes (which form a large complex) and
regulatory genes encoding transferases, kinases, and transcription factors, while
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adenylyl cyclases (Sinha et al. 2005), and mitochondrial peptidases (Brown et al.
2007), all gene families that contain duplicates that form heterodimers in eukaryotes
(or eukaryotic mitochondria) with single-copy homologs that form homodimers in
prokaryotes.
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those involved in ion transport are under-represented. Likewise, the Paramecium
tetraurelia genome exhibits elevated retention of duplicate genes involved in known
complexes (Aury et al. 2006) and in metabolic pathways (Gout et al. 2009). As in
yeast, ribosomal genes, transferases, and kinases are over-represented among
surviving paralogs, while ion-transport genes are underrepresented. In Paramecium,
there also appears to be an additional effect whereby highly expressed genes are
over-retained in duplicate following the most recent polyploidization event (Gout
et al. 2010). That certain types of genes are maintained preferentially following a
WGD has achieved fairly convincing empirical support from other studies as well
(Papp et al. 2003; Yang et al. 2003; Barker et al. 2008; Liang et al. 2008; Qian and
Zhang 2008; Edger and Pires 2009), including studies in Arabidopsis (Blanc and
Wolfe 2004; Maere et al. 2005; Thomas et al. 2006), vertebrates (Makino and
McLysaght 2010), and across divergent species (Paterson et al. 2006). Selection to
maintain dosage balance following WGD has also been hypothesized to be the
driving force behind the original selective advantage of the WGD in the Saccharomyces cerevisiae lineage (Conant and Wolfe 2007). In this scenario, the maintenance of glycolytic genes and the loss of non-glycolytic genes following WGD
might have increased the relative dosage of glycolytic genes, thereby increasing flux
through the glycolysis pathway and providing polyploid yeast with a growth
advantage over non-polyploids due to increased glucose fermentation ability.
Duplicate genes that arise via WGD are further unique in that entire (or partial)
duplicated pathways or networks of interacting proteins can diverge in concert. For
example, Evlampiev and Isambert (2007) modeled the evolution of protein–protein
interaction networks following WGD and concluded that such networks grow under
exponential, rather than time-linear, dynamics following WGD. Interestingly, they
also found that these exponential dynamics relied on asymmetric divergence
between duplicates.
Another intriguing possibility is that following WGD, a whole ancestral
network may become neofunctionalized or subfunctionalized following polyploidization, with one set of paralogs carrying out one task or reaction and a parallel
set of paralogs carrying out a related, but largely independent, task. Obviously,
such innovations require the establishment of multiple mutations and the avoidance of pathway crosstalk. Although the essential population genetic theory
remains to be worked out, several examples of such paralog coevolution appear to
have followed the WGD in yeast: parallel paralogous networks have been identified where the expression of each gene is highly correlated with the other genes
within its network but poorly correlated with its paralog (Blanc and Wolfe 2004;
Conant and Wolfe 2006). In this way, polyploidy provides a unique mechanism for
the evolution of gene networks with new (or subdivided) functions.
A final consideration in duplicate-gene evolution is whether the forces that act
to preserve duplicates change over evolutionary time. For example, it seems
possible that following WGD, a large proportion of genes could be initially
maintained due to dosage-balance constraints. Subsequently, however, over longer
periods of evolutionary time, some duplicates might accumulate mutations that
could lead to neofunctionalization or subfunctionalization. Because these genes
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1.4 Autopolyploidy Versus Allopolyploidy
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are dosage sensitive (hence their initial preservation due to selection for dosage
balance), it is likely that such neo- or subfunctionalizing mutations would need to
be preceded or rapidly followed by mutations affecting the dosage of one or both
copies. For a more detailed example, imagine proteins A and B that must interact in
a 1:1 ratio for proper functioning. Both genes become duplicated during a WGD,
giving rise to duplicates A1 and A2 and B1 and B2. Initially, all four genes are
preserved by selection for dosage balance, as loss of any one gene interrupts the
1:1 interaction ratio. Over evolutionary time, however, slightly deleterious
mutations in the A1 promoter that decrease its expression level become fixed due
to drift. To compensate, mutations in the A2 promoter that increase its expression
level are fixed, which helps to restore the 1:1 A/B ratio. At this point, A1 is
contributing fewer products to the overall A protein pool. A subsequent mutation
that changes the function of A1, allowing it to take on a new role completely, is
now more easily accommodated, as A2 is better able to compensate and take on the
full load of the ancestral A activity. Note that, instead of A1 and A2 dosage
evolving in concert, as above, A1 and B1 dosage could also evolve in concert to
maintain the proper 1:1 A/B ratio, allowing both A1 and B1 to take on new
functions.
While still just a verbal theory, this scenario has two advantages in terms of
allowing for neofunctionalization (or subfunctionalization) of WGD duplicates.
First, there is a longer time frame in which neo- or subfunctionalizing mutations
can arise, as duplicates are maintained for longer time-scales without becoming
nonfunctionalized. This is important because neofunctionalization requires the
accumulation of beneficial mutation(s), which are thought to be rare. Second, this
process would allow for neo- or subfunctionalization of dosage-sensitive duplicates, both of which might otherwise be constrained to maintain their ancestral
function indefinitely following WGD.
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Whether polyploidization occurs by autopolyploidy or allopolyploidy can have a
significant impact on the expression and evolution of duplicate genes. Autopolyploids arise when there is an increase in ploidy within a single species (often
within a single individual), while allopolyploids are created by hybridization
between two different species, each of which contributes a full complement of
chromosomes to the hybrid, thus doubling the genome (reviewed in Coyne and Orr
2004). Many plant and frog polyploids are the result of allopolyploidization
(Adams 2007; Evans 2008), while the yeast WGD appears to have been an
autopolyploidization event (Scannell et al. 2007), although in practice it is difficult
to ascertain the ancestral state once paralog divergence has become high.
It has long been assumed that autopolyploids would initially form multivalents
at meiosis, with all four homologous chromosomes pairing randomly, while
allopolyploids would be more likely to form bivalents, with homologous
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chromosomes from each diploid ancestor pairing independently. This would mean
that duplicate copies in autopolyploids would not represent true paralogs as the
term is usually understood, but would instead represent a doubling of the number
of homologs (i.e., four homologs instead of two). The presence of multivalents is
significant biologically, as multivalent pairing can lead to intergenomic recombination via segregation, crossing-over, and double reduction. Certain duplicates
from one diploid parent could be lost completely via this process, leaving only
duplicates from the other diploid parent. This would not represent gene silencing
as it is typically understood then, but would rather be a byproduct of multivalent
formation and segregation. Evidence from plants indicates that multivalent pairing
is indeed more prevalent among autopolyploids, though the difference between the
two forms of polyploidy is perhaps less than originally expected: a survey of plant
polyploids indicated that the mean percent occurrence of multivalents is 28.8 % in
autopolyploids and 8.0 % in allopolyploids (Ramsey and Schemske 2002).
Although multivalent formation occurs at a lower rate in allopolyploids, it may be
more biologically significant than multivalent formation in autopolyploids, as
intergenomic recombination is likely to have a greater effect when genomes are
more divergent. Over time, divergence between duplicated chromosomes would
lead to increased bivalent formation.
Because allopolyploids are the result of a genomic merger between two species,
duplicate genes in allopolyploids are already differentiated to some extent
immediately after polyploidization, while duplicates in autopolyploids are likely to
be more similar in sequence and may even be identical. Allopolyploids often
exhibit immediate changes in gene expression due to the genetic differentiation
present between homeologs. This can lead to changes in methylation (Salmon
et al. 2005; Gaeta et al. 2007), changes in heterochromatin formation and transposable element suppression (Josefsson et al. 2006), biased expression of
homeologs (Adams et al. 2003; Bottley et al. 2006; Tate et al. 2006; Udall et al.
2006; Rapp et al. 2009), and non-additive expression effects between homeologs
(Hegarty et al. 2006; Wang et al. 2006; Rapp et al. 2009). These initial expression
differences between homeologs can, in turn, impact the long-term evolution of
duplicates, as selection pressures may be expected to act differently on genes that
are differentially expressed. For example, Anderson and Evans (2009) showed that
in octoploid and dodecaploid Xenopus species, paralogs of RAG1b were more
likely to become pseudogenized than paralogs of RAG1a (the homeolog of
RAG1b from an earlier allopolyploidy event), and they inferred that this was due
to differences in ancestral expression between RAG1a and RAG1b.
Many of these effects seen in allopolyploids are believed to be due to the
hybridization between two divergent genomes, rather than genome doubling per se.
Flagel et al. (2008) estimated that of the genes with biased expression between
homeologs in the allopolyploid Gossypium hirsutum, 24 % exhibit a bias due to the
genomic merger (i.e., the bias existed immediately when the allopolyploidization
occurred, at time zero), while the bias in the remaining 76 % is due to long-term
evolutionary forces such as neofunctionalization and subfunctionalization. The
relationship between the magnitude of these alterations in gene expression and the
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1.5 Polyploidization and Speciation
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Perhaps the most pivotal role that polyploidization plays in evolution is in the
creation of new species. The polyploidization event itself can lead to instantaneous
reproductive isolation and speciation, as the cross between a new tetraploid (4n)
and its diploid progenitor (2n) yields triploid (3n) offspring, which are often sterile
due to problems with chromosome pairing/segregation during meiosis and the
production of aneuploid gametes (reviewed in Coyne and Orr 2004). It is for this
reason that models predict that species capable of self-fertilization are more likely
to give rise to a successful polyploid lineage (Rodriguez 1996; Baack 2005;
Rausch and Morgan 2005).
Perhaps more importantly, however, once a polyploid lineage is established,
subsequent silencing of duplicate genes can lead to further reproductive isolation
among subpopulations of the polyploids themselves and, therefore, give rise to
additional daughter species (Oka 1988; Werth and Windham 1991; Lynch and
Conery 2000; Lynch and Force 2000). In this model, we assume a pair of fully
functional and redundant duplicate genes, A and B, in an ancestral population, such
that each member of the initial population has the genotype AABB (Fig. 1.1). If two
subpopulations become geographically isolated and one duplicate becomes
nonfunctionalized in each subpopulation, there is a 50 % probability that a different
duplicate copy will be lost in each of the two groups. This reciprocal gene loss (or
divergent resolution) would result in the genotypes aaBB and AAbb for the two
subpopulations, where a and b denote null alleles. Hybridization between the two
groups would then lead to offspring with the genotype AaBb. Gametes produced by
these F1 individuals would have a 1/4 probability of carrying an ab genotype and
would therefore be inviable if a functional copy of the A/B gene were essential for
gamete survival or function. Even if this were not the case, 1/16 of the F2 individuals
would have the aabb genotype and, if a functional copy were essential for zygote
viability or sterility, would be inviable or sterile, whereas another 1/4 would have
three null alleles and might experience reduced viability or sterility. Up to 50–65 %
of the genes encoding transcription factors, membrane receptors, and members of
macromolecular protein complexes are estimated to be haploinsufficient (JimenezSanchez et al. 2001; Veitia 2002), suggesting that only one functional allele of such
genes is indeed likely to be deleterious.
An appealing aspect of the divergent resolution model is that it is a natural
consequence of degenerative mutations, requiring no adaptive evolution at the
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genetic divergence between the two parental genomes is not well understood,
however, as demonstrated by Brassica allopolyploids (Pires and Gaeta 2011). While
the parental species that gave rise to the allopolyploid Brassica napus are more
similar to each other than those that gave rise to B. juncea, resynthesized B. napus
polyploids exhibit more genomic rearrangements, changes in gene expression, and
epigenetic alterations than do resynthesized B. juncea polyploids.
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Fig. 1.1 Divergent
resolution of duplicate genes
can lead to hybrid sterility/
inviability and reproductive
isolation. Red bar represents
a gene that becomes
duplicated. See text for
details
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Population Isolation and
Divergent Resolution
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Hybridization
F1 progeny
1/4 F1 gametes
(no functional copy)
1/16 F2 progeny
(no functional copy)
OR
1/4 F2 progeny
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molecular level for speciation to occur. Moreover, in addition to genes that are
reciprocally silenced, duplicate pairs that undergo neofunctionalization or
subfunctionalization may also contribute to hybrid sterility/inviability in a similar
fashion—for example if a different duplicate becomes neofunctionalized and loses
its ancestral function in each subpopulation, or if the two duplicates become
subfunctionalized in complementary ways in the two subpopulations (Lynch and
Force 2000).
The process of reciprocal gene loss has been shown to be responsible for male
sterility between hybrids of Drosophila melanogaster and D. simulans (Masly
et al. 2006). A gene essential for male fertility, JYAlpha, is located on the fourth
chromosome in D. melanogaster and on the third chromosome in D. simulans.
This translocation presumably occurred via duplication of the JYAlpha gene and
subsequent silencing of one copy. The difference in chromosomal location of the
gene in the two species causes a proportion of hybrids to completely lack JYAlpha,
leading to their sterility.
Two similar cases were recently identified in rice. The first involves reproductive isolation between two subspecies of Oryza sativa. The ancestral O. sativa
genome appears to have had a pair of duplicates termed DOPPELGANGER1
(DPL1) and DOPPELGANGER2 (DPL2) (Mizuta et al. 2010). The subspecies
japonica and indica have experienced independent losses of one copy each: DPL1
has become a pseudogene in indica, while DPL2 has been nonfunctionalized in
japonica. Hybrid pollen lacking a functional copy of either DPL1 or DPL2 is
nonfunctional and does not germinate, contributing to the partial reproductive
isolation present between the subspecies. This validates an earlier hypothesis by
Oka (1988) that F1 sterility between japonica and indica was caused by ‘‘duplicate
gametophytic sterility genes’’, japonica being homozygous for one nonfunctional
copy and indica being homozygous for another nonfunctional copy. In the second
rice example, reciprocal loss of one of the duplicated nuclear genes encoding
mitochondrial ribosomal protein L27 in O. sativa and O. glumaepatula again
causes a proportion of the pollen produced by F1 hybrids to be sterile (Yamagata
et al. 2010).
The final example of reproductive isolation through reciprocal gene loss comes
from A. thaliana, where the histidinol-phosphate amino-transferase gene appears
in different chromosomal locations (as in the Drosophila example, presumably via
duplication and subsequent silencing of one copy) in the Columbia and Cape
Verde Island accessions (Bikard et al. 2009). F2 offspring homozygous for both
null alleles completely lack the gene’s product, HPA, which results in arrested
embryo development and seed abortion. In addition, in at least one intermediate
heterozygote, a quantitative phenotype termed ‘‘weak root’’ was observed,
suggesting that the presence of three null alleles is somewhat deleterious in this
cross. As these four examples constitute *1/3 of the dozen or so successful
searches for the genes underlying the speciation process (most in Drosophila
species; Presgraves 2010), there now seems little question that the passive nonfunctionalization of duplicate genes is a major mechanism of speciation.
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1.6 Unsolved Problems
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The maintenance of duplicate genes via selection for increased gene product,
neofunctionalization, and subfunctionalization has been hypothesized for nearly
40 years (Ohno 1970). Recent genetic and genomic data have now identified
compelling examples of these processes and have further contributed to our
understanding of the prevalence of whole-genome duplications and the dosagebalance theory of duplicate maintenance. However, a number of unresolved
questions related to WGDs and duplicate maintenance merit further scrutiny.
The first avenue for future study involves a more comprehensive understanding
of the relative importance of the forces behind duplicate-gene maintenance,
including maintenance for increased dosage, dosage-balance constraints,
neofunctionalization, and subfunctionalization. All of these mechanisms have been
demonstrated to be responsible for duplicate maintenance in certain cases, but it
remains unclear which, if any, is responsible for maintaining the majority of
duplicate genes or how such contributions vary among phylogenetic lineages. Most
likely, there will be no single driving force for duplicate maintenance but the relative
strength of these forces will differ among taxonomic groups or among functional
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These examples demonstrate that the divergent resolution of even one duplicated gene can lead to detectable reproductive isolation. However, genetic
incompatibility between two populations can be magnified substantially when
reciprocal gene loss occurs at hundreds or thousands of duplicated loci simultaneously, as is the case in polyploid lineages. The probability that an F2 offspring
obtained by outcrossing will be double null for at least one of n pairs of divergently resolved loci is 1-(15/16)n, which takes on values of 0.063, 0.276, 0.476,
and 0.998 for n = 1, 5, 10, and 100, respectively. Moreover, in species that
undergo autogamy or selfing, such as Paramecium, this probability can be as high
as 1-(3/4)n, giving probabilities of 0.250, 0.763, 0.944, and & 1 for n = 1, 5, 10,
and 100. Speciation events will continue to occur as long as duplicates are still
being resolved between subpopulations, leading to nested rounds of speciation,
and, because a large number of duplicates are thought to be silenced quickly
following WGD (Scannell et al. 2006), a cluster of speciation events might occur
within a brief period of time. The net result is the expected generation of a species
radiation following a WGD event.
It has been suggested that this nested speciation process might be responsible
for the radiations of the polyploid yeast species (Scannell et al. 2006), teleost fishes
(Semon and Wolfe 2007; see Chap. 15, this volume), angiosperms (Soltis et al.
2009; though see Mayrose et al. 2011), and the Paramecium aurelia species
complex (Aury et al. 2006). This mechanism may also be responsible for
reproductive isolation between mutagenized lines of an experimentally derived
allotetraploid created by hybridizing two species of Saccharomyces (Maclean and
Greig 2010).
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classes of genes. For example, subfunctionalization of duplicates may be more
likely within species that have evolved a modular (and therefore independently
mutable) regulatory structure. Such modular systems are predicted to arise more
easily within species with smaller population sizes (Force et al. 2005), demonstrating how species-level features of an organism may influence the evolutionary
forces acting upon duplicate genes. More comprehensive studies of large numbers
of duplicates from a variety of organisms are required to address what other features
might influence the relative strengths of mechanisms of duplicate maintenance.
Such studies must not only detail what genes remain duplicated vs. single-copy, but
must also detail whether existing duplicates have the same function as each other
(to assess rates of neofunctionalization) or share functions with the pre-duplicated
ancestor (to assess rates of subfunctionalization) (Fig. 1.2).
There are few data on the rate of duplicate-gene loss over time following a
WGD, though data from polyploid yeast species suggest that the rate changes over
time (Scannell et al. 2006). Data from additional taxa would aid in determining
whether this is a general pattern among all WGDs (Fig. 1.3). A related unresolved
question is whether the evolutionary forces controlling duplicate maintenance
change over time following a WGD, e.g., whether a dosage-sensitive gene may
initially be preserved due to selection for dosage balance, but then evolve a new
function concurrent with its release from such dosage constraints. An analysis of
this question could be made by comparing the fates of duplicate genes in multiple
lineages descended from a single WGD event. Such an analysis might identify
duplicates that had been maintained due to dosage constraints in the majority of
daughter lineages but that had become neofunctionalized in one lineage, perhaps
suggesting a secondary mechanism of retention.
The unsolved question that promises to be the hardest to answer is why certain
lineages or taxonomic groups appear to contain more WGD events than others.
This is not the same as asking why certain groups contain more polyploid species,
as this may be a simple reflection of the fact that WGD may promote subsequent
reproductive isolation and speciation. The pattern remains, however, that some
phylogenetic groups seem to contain more independent WGD events than others in
their evolutionary pasts. For example, a recent analysis estimated that among
ferns, 31 % of speciation events involve polyploidization, while the value for
angiosperms is only 15 % (Wood et al. 2009). Similarly, in the history of the
Xenopus lineage, there are many more instances of WGD events than compared to,
say, mammals. Several factors could contribute to such patterns, such as the ability
to hybridize and form allopolyploids, or the ability to self-fertilize (at least transiently) or undergo asexual reproduction, which helps a polyploid lineage become
abundant in a surrounding world of diploids. It is not even understood whether
mechanistic reasons (at meiosis, say) or differences in developmental programs
would facilitate or hinder creation of a viable polyploid in certain lineages, or
whether discrepancies in ecological persistence of polyploid species alone are able
to explain the patterns that we see. Perhaps the best way to approach such a
question is to study closely related lineages where one exhibits several WGD
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Expression Level
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Fig. 1.2 Distinguishing
between the evolutionary
forces that maintain duplicate
genes. Panel A shows the
expression level of a gene
(purple) across different
conditions or tissues before
duplication. The bottom six
panels (B–G) show patterns
that might be seen for the two
copies (red and blue) once the
gene has been duplicated and
what evolutionary processes
these patterns would indicate.
Note that panel D might
indicate maintenance for
increased dosage in the case
of a single-gene duplicate or
maintenance for dosage
balance in the case of a
duplicate arising via WGD
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Nonfunctionalization
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Dosage/dosage balance
F
Qualitative
Subfunctionalization
C
Nonfunctionalization
E
Neofunctionalization
G
Quantitative
Subfunctionalization
events and the other does not, though teasing apart the mechanistic and ecological
differences is certain to remain a challenge for decades to come.
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1 Evolutionary Significance of Whole-Genome Duplication
(a)
D
C
B
Outgroup
A
D
% of duplicates lost
(b)
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Species 4
B
C
D
Time
Whole-genome duplications are widespread across the tree of life and appear in
the evolutionary history of a large number of model organisms. Processes such as
neo- and subfunctionalization affect retention of individual gene duplicates, and
dosage-balance constraints promote the retention of large sets of genes following
polyploidization. Allopolyploidization, through hybridization and subsequent
changes or biases in homeolog expression, has the ability to instantaneously create
a population of individuals that are ecologically and epigenetically unique from
either parent lineage, providing a new lineage upon which natural selection can
act. Both allo- and autopolyploidization provide a unique opportunity for the
differentiation of new gene networks and pathways through concerted evolution of
duplicated, interacting proteins. Most importantly, WGD can lead to reproductive
isolation through divergent resolution of duplicated genes, thus creating new
species and species groups. Further understanding of the relative importance and
the temporal properties of the forces acting on polyploid species and the duplicate
genes within their genomes promises to enhance our knowledge of the origins of
species as well as genetic, protein network, and organismal complexity.
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Species 3
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Species 1
Species 2
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Fig. 1.3 Determining the
rate of duplicate-gene loss
over time. a An example of a
tree for four species that share
a whole-genome duplication
(WGD). Duplicate-gene
presence/absence information
for each of the four species
could be used to infer the
number of duplicate genes
lost on each of the branches
labeled A, B, C, and D. b The
data on gene retention/loss
gathered in A could be used to
plot the percentage of
duplicate genes lost per unit
time (or divergence).
Depending on where points
A, B, C, or D land on the
graph, the data may indicate
that the rate of gene loss
remains constant (top dashed
line) or changes (bottom
dotted line) over time
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Metadata of the chapter that will be visualized in
SpringerLink
Book Title
Polyploidy and Genome Evolution
Series Title
Chapter Title
Genetic Consequences of Polyploidy in Plants
Copyright Year
2012
Copyright HolderName
Springer-Verlag Berlin Heidelberg
Corresponding Author
Family Name
Birchler
Particle
Given Name
James A.
Suffix
Abstract
Division
Division of Biological Sciences
Organization
University of Missouri
Address
117 Tucker Hall, 65211, Missouri, Colombia
Email
birchlerj@missouri.edu
Most eukaryotes have a history of whole-genome multiplication events followed by a progressive return to
a more diploid state. The initial state of polyploidization, in which more than two copies of the genome are
present, is considered here and the various types of genetic consequences that occur depending on the nature
of the polyploid formed. The degree of association of chromosomes in meiosis is determined by the relative
homology and will affect the segregation of the chromosome which determines the genetic properties. If all
the chromosomes are quite similar and form associations of like type, this situation is referred to as
autopolyploidy. If the different sets of multiple chromosomes are sufficiently dissimilar to each other, then
the homologs will pair in meiosis with themselves and segregate independently of the different but related
chromosome pair. This situation is referred to as allopolyploidy. Gene expression in ploidal series typically
follows a per cell level correlated more or less with the number of sets of chromosomes present. Variation of
individual chromosomes, or aneuploidy, produces a greater number of modulations of gene expression in
parallel to classical studies noting that aneuploids have greater impact on the phenotype than changes in the
copy number of the whole genome. The genetic properties of odd-number ploidies, such as triploids, are also
described as well as higher ploidal levels such as hexaploidy and octoploidy.
1
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Abstract Most eukaryotes have a history of whole-genome multiplication events
followed by a progressive return to a more diploid state. The initial state of
polyploidization, in which more than two copies of the genome are present, is
considered here and the various types of genetic consequences that occur
depending on the nature of the polyploid formed. The degree of association of
chromosomes in meiosis is determined by the relative homology and will affect the
segregation of the chromosome which determines the genetic properties. If all the
chromosomes are quite similar and form associations of like type, this situation is
referred to as autopolyploidy. If the different sets of multiple chromosomes are
sufficiently dissimilar to each other, then the homologs will pair in meiosis with
themselves and segregate independently of the different but related chromosome
pair. This situation is referred to as allopolyploidy. Gene expression in ploidal
series typically follows a per cell level correlated more or less with the number of
sets of chromosomes present. Variation of individual chromosomes, or aneuploidy,
produces a greater number of modulations of gene expression in parallel to classical studies noting that aneuploids have greater impact on the phenotype than
changes in the copy number of the whole genome. The genetic properties of oddnumber ploidies, such as triploids, are also described as well as higher ploidal
levels such as hexaploidy and octoploidy.
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Genetic Consequences of Polyploidy
in Plants
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J. A. Birchler (&)
Division of Biological Sciences, University of Missouri, 117 Tucker Hall,
65211 Missouri, Colombia
e-mail: birchlerj@missouri.edu
P. S. Soltis and D. E. Soltis (eds.), Polyploidy and Genome Evolution,
DOI: 10.1007/978-3-642-31442-1_2, Springer-Verlag Berlin Heidelberg 2012
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2.1 Introduction
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2.2 Allopolyploids
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Most eukaryotes have a history of polyploidization followed by fractionation back
to a near diploid level (Wolfe and Shields 1997; Simillion et al. 2002; Bowers
et al. 2003; Blanc and Wolfe 2004; Chapman et al. 2006; Maere et al. 2005;
Blomme et al. 2006; Freeling and Thomas 2006; Barker et al. 2008). Thus, at the
least, polyploidy in essence is a matter of degree, and it has played an important
role in the composition of the gene repertoire of many species. Typically, it is
defined as the presence of more copies of the whole genome than the normal two
that constitute a diploid (Stebbins 1947). However, from the standpoint of gene
content, the determination of whether a species is a polyploid is somewhat arbitrary and dependent on the time before the present when the copy number of the
genome was increased. Nevertheless, for evolutionarily ‘‘recent’’ events, certain
principles can apply which will be summarized in this chapter.
In the polyploidy literature, the basic chromosome number is designated by
x and consists of the complete set of chromosomes, or a genome. The number of
chromosomes in the gametophyte generation and hence the gametes is referred to
as the gametic chromosome number or n. In diploids, x = n, but at higher levels of
polyploidy, this is not the case.
Polyploidy is typically divided into at least two categories that are determined
by the type of chromosome pairing in meiosis I and the distribution of chromosomes during this process. Indeed, the type of chromosome pairing that occurs in
meiosis affects the genetic properties of the species so such classifications have
value. If the increase in genome copy number results from the combination of
chromosome sets from divergent species, the different types of chromosomes will
usually not pair with each other in prophase of meiosis I. In the case of tetraploids,
if both divergent genomes are doubled by whatever means, those sets of chromosomes that are similar or identical will preferentially pair with each other to the
exclusion of the other genome. This type of pairing is referred to as ‘‘disomic’’ in
analogy with the situation in a diploid. A species with this type of scenario is
referred to as an allopolyploid because the contributing genomes are different from
each other.
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Genetic ratios in an allotetraploid depend on the constitution of each genome
(Clausen and Goodspeed 1925; Clausen 1941; Clausen and Cameron 1944). The
different sets of related chromosomes are referred to as homoeologues. If both the
homoeologues possess the homoeologous gene copies that are expressed similarly,
then both would need to be mutant in order to express a recessive phenotype.
Under these circumstances, duplicate gene ratios would typically be observed. In
other words, recessive phenotypes would be found in 1/16 (1/4 9 1/4) of the F2
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2.3 Autopolyploids
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If on the other hand the increase in genome copy number in a polyploid results
from the same species such that the chromosomes are all quite similar, the pairing
in prophase of meiosis I forms conglomerates that switch pairing partners along
the length of the chromosome (Fig. 2.1). This type of pairing is referred to as
‘‘quadrivalent’’ pairing because all four chromosomes present can be involved
with each other. However, 3:1 and 2:2 associations are also observed. The segregation in this case will depend on the position of the locus in question in the
chromosome and relative to the respective centromere (Blakeslee et al. 1923;
Haldane 1930; Bartlett and Haldane 1934; Mather 1935, 1936; Randolph 1935;
Little 1945, 1958; Doyle 1973). Those genes near the centromere will be distributed to the diploid gametes based on the usual case that pairs of centromeres
will separate from each other at meiosis I and that the two sets from each chromosome of the complement will do so at random. A homozygous dominant
autotetraploid (AAAA) is referred to as a quadruplex and the homozygous recessive
(aaaa) as a nulliplex. There are three types of heterozygotes: AAAa (triplex), AAaa
(duplex), and Aaaa (simplex). If one designates a hybrid autotetraploid as AA0 aa0 ,
then there are six types of possible gametes that will be formed: AA0 , Aa, Aa0 , aA,
a0 A0 , aa0 . The frequency of diploid homozygous gametes under these circumstances is 1/6 (0.167). A self-pollination will produce 2.77 % of the progeny that
are homozygous for the recessive allele (Fig. 2.2). However, as the distance of a
gene from the centromere increases, recombination between the locus and the
centromere will randomize the distribution of the different alleles into the diploid
gametes to the point that the frequency of homozygous diploid gametes will be
(4/8 9 3/7 = 0.21) as a maximum. In this case, a self-pollination will produce
4.41 % of the progeny that are homozygous.
Recombination between the monitored locus and the centromere can also
produce homozygous spores from a triplex heterozygote (AAAa) to produce aa
gametes (Catcheside 1956). This process is called double reduction. Again, this
result is affected by the position of the locus under consideration from the centromere with greater double reduction increasing with distance. Another factor
affecting segregation in autotetraploids is aneuploidy, i.e., altered copy number of
individual chromosomes. This circumstance would change the pairing and segregation properties of individual chromosomes. Autotetraploids can also generate
spontaneous diploid progeny via parthenogenesis (Randolph and Fischer 1939).
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from a self of an F1 between parental types that are dominant and recessive.
However, if one of the gene copies is missing or expressed in other tissues from
one of the homoeologous chromosomes, then genetic ratios typical of a diploid
will be found because only one genome will have different alleles in an F1, and
they will segregate to produce a 3:1 ratio because the single genome will behave as
a diploid.
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Fig. 2.1 Comparison of meiotic anaphase I in matched diploid and tetraploid maize plants.
a Gray value image of anaphase I of diploid inbred line B73. Note that membrers of each pair of
homologs separate from each other. Modified from (Birchler 2011). b Gray value image of
anaphase I of a tetraploid derivative of inbred line B73. Note the multivalent associations of
chromosomes. Photos by Zhi Gao and Fangpu Han
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Fig. 2.2 Genotypes in a self-pollination of an autotetraploid heterozygote AAaa. In an
autotetraploid, the gametes are diploid. With two chromosomes carrying the dominant A allele
and two carrying the recessive a allele, the distribution of gametes from the heterozygote is
shown across the top and along the side. The combinations of these gametes to produce the
tetraploid progeny are shown in the grid. Only one out of 36 are homozygous for A, and one out
of 36 are homozygous for the recessive a. Other combinations of A and a are shown. These
conditions hold for genes closely linked to centromeres as described in the text
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2.4 Segmental Allopolyploids
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2.5 Heterosis and Ploidy
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Because of the chromosome pairing considerations noted above, allopolyploids
will have a diversity of gene products ‘‘fixed’’ in their genomic structure. This
circumstance will basically maintain the essence of hybrid vigor even though
technically an otherwise high degree of homozygosity might be present. Thus,
allopolyploids typically exhibit robust biomass and excellent fertility compared to
the diploid progenitor species. Nevertheless, crosses between different isolates of
allopolyploids can show even greater heterotic effects when each genome is heterozygous as well (Gustafson 1946). Autopolyploids, which can have up to four
different alleles at one locus, also exhibit hybrid vigor and with increasing
diversity of alleles present, a phenomenon known as progressive heterosis, there is
increasing biomass and fertility (Busbice and Wilsie 1966; Levings et al. 1967;
Mok and Peloquin 1975; Groose et al. 1989). However, autotetraploids can be
subject to inbreeding depression in which the potential exists for all copies of a
chromosome to become homozygous (Busbice and Wilsie 1966; Sockness and
Dudley 1989a, b). Polyploids that are entirely homozygous exhibit extreme
depression and reduction of stature and fertility (Busbice and Wilsie 1966; Riddle
et al. 2006; Abel and Becker 2007; Stupar et al. 2007; Redei 1964; d’Erfurth et al.
2009; Yao et al. 2011). This situation is unlikely under natural circumstances.
A discussion of the vigor of polyploids needs to consider the intersection with
heterosis or hybrid vigor. In recent years, it has been possible to produce ploidy
series for completely or highly homozygous materials (Riddle et al. 2006; Abel
and Becker 2007; Stupar et al. 2007; d’Erfurth et al. 2009; Yao et al. 2011). The
general rule that emerges from these studies is that with increasing ploidy and the
maintenance of homozygosity, there is usually a decline in stature and fertility.
The cell and pollen size increases with ploidy and the plants typically take on a
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A third classification based upon empirical chromosome associations is segmental
allopolyploid (Stebbins 1947). In this case, some chromosomes exhibit bivalent
pairing and others show quadrivalent pairing. The basis of such behavior was not
clear until recently. Xiong and colleagues (Xiong et al. 2011) found that in
resynthesized Brassica napus derived from the diploid progenitors, B. oleracea and
B. rapa, different lineages could form compensating nullisomic-tetrasomic
configurations for different chromosomes. In this case, the tetraploid will have some
chromosomes that are basically identical and other members of the set that will be
divergent. Similar results were reported for naturally occurring tetraploid
Tragopogon miscellus (Chester et al. 2012). Such a species will be a composite of
allo- and autotetraploid chromosomes for different members of the karyotype and
would be expected to exhibit the pairing characterized by a segmental allopolyploid.
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2.6 Aneuploidy Relative to Ploidy
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In contrast to a ploidy series, changes in dosage of individual chromosomes (or
substantial parts of chromosomes) have a more dramatic effect on the phenotype
(Blakeslee et al. 1920; Blakeslee 1934). Typically, the removal of a chromosome
or chromosomal segment has the strongest effects and is lethal in some cases
(Kush and Rick 1968; Vizir and Mulligan 1999). All of the monosomics for each
of the ten chromosomes in maize have been recovered and studied (Weber 1983),
but this is not the case in other species in which this issue has been examined such
as tomato (Kush and Rick 1968) and Arabidopsis (Vizir and Mulligan 1999). The
addition of a chromosome to produce a trisomic usually also has a detrimental
effect on plant vigor but the usual circumstance is that the impact is much less than
monosomics (Lee et al. 1996). Indeed, full sets of trisomics have been produced
for many plant species (Singh 1993). Tetrasomics for whole chromosome arms,
otherwise called secondary trisomics, have been produced in Datura by recovery
of extra chromosomes that are duplicated for one or the other arm of the progenitor
chromosome (Blakeslee 1934). These secondary trisomics usually have more
intensified phenotypic effects and are more intensified when present in haploids
(Satina et al. 1937a, b).
Extra or missing chromosomes in higher ploidies have less severe phenotypic
effects. A comprehensive set of aneuploids was generated in hexaploid wheat
(Sears 1944; Sears 1953, 1954). Monosomics and trisomics are regularly produced, and because of the high ploidy state, nullisomics, which are missing both
copies of a chromosome, can be produced (Sears 1953, 1954). Nullisomics have a
more severe effect than the corresponding monosomic. Tetrasomics can be produced and have a more severe effect than the respective trisomic. Compensating
nullisomics for one homoeologue and tetrasomics for another return to a more
normal phenotype than exhibited by the nullisomic or tetrasomic alone (Sears
1953, 1954). Newly synthesized B. napus (Xiong et al. 2011) and natural neopolyploids of T. miscellus (Chester et al. 2012) will exhibit aneuploidy that
resolves into compensating 4:0 or 3:1 contributions from different progenitor
genomes illustrating that the compensating balanced condition is favored in
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‘‘stocky’’ appearance but with extreme ploidies, the plants are depauperate
(Blakeslee 1941; Randolph 1942; Rhoades and Dempsey 1966; d’Erfurth et al. 2009;
Yao et al. 2011). In contrast, hybrids with increasing ploidy tend to exhibit greater
biomass and in the species in which it has been examined closely, there is an increase
in heterosis with increasing diversity of alleles, i.e., progressive heterosis (Busbice
and Wilsie 1966; Mok and Peloquin 1975; Levings et al. 1967; Groose et al. 1989;
Bingham et al. 1994; Riddle and Birchler 2008). The common view that polyploids
exhibit more robust stature derives from experience with allopolyploids or with
heterotic autopolyploids, which are the situations most commonly encountered.
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2.7 Gene Expression Studies
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laboratory or natural selection. Together, these results further illustrate that the
greater the deviation from the standard set of chromosomes, the more severe the
impact on the phenotype.
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2.8 Genomic Balance
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Studies on gene expression in ploidy and aneuploid series parallel the phenotypic
results. When individual genes are sampled in a ploidy series, the expression level
is more or less proportional to the ploidal level, although there are examples of
genes whose expression deviates from this trend both positively and negatively
(Birchler and Newton 1981; Guo et al. 1996). Genome-wide studies of gene
expression in ploidy series demonstrate a similar pattern (Wang et al. 2004;
Albertin et al. 2005; Stupar et al. 2007; Riddle et al. 2010; Yu et al. 2010). In
contrast, sampling of individual genes or protein patterns in aneuploids reveals a
greater set of changes from the diploid level of expression (Birchler 1979; Birchler
and Newton 1981; Guo and Birchler 1994). A dosage series for a particular
chromosomal region would alter the amount of expression of a portion of the total
gene products encoded across the genome. The effects could be positive or negative correlations with the change in dosage. The more common effect especially
with trisomics was a negative correlation between the dosage and the target gene
expression (Birchler 1979; Birchler and Newton 1981; Guo and Birchler 1994).
Thus, the gene expression patterns show changes in a ploidy series but aneuploid
series exhibit greater effects in parallel with the phenotypic relationships.
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This gene expression relationship led to the suggestion that the stoichiometry of
regulatory genes affected the outcome of gene expression (Birchler and Newton
1981) and ultimately the phenotype (Guo and Birchler 1994). Studies to identify
single genes that would mimic the aneuploid effects using a partial loss of function
mutation in the white eye color gene in Drosophila produced single-gene mutations that would modulate the target’s expression either positively or negatively
(Rabinow et al. 1991; Birchler et al. 2001). The molecular identification of many
of these genes revealed them to be transcription factors, chromatin modifiers, and
components of signal transduction (Birchler et al. 2001).
Interestingly, these same classes of genes are typical of those that exhibit
preferential retention following a polyploidization event (Blanc and Wolfe, 2004;
Freeling and Thomas, 2006) and underrepresentation in segmental duplications
(Maere et al. 2005; Freeling et al. 2008). Thus, this evidence suggests that if these
classes of genes are out of register with each other, there is a negative fitness
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2.9 Triploids
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consequence. Thus, the phenotypic, gene expression and evolutionary studies form
a coherent picture that these types of genes form a balance. When individual
components exhibit a dosage effect, this will ultimately produce a fitness consequence due to the impact of the altered gene expression on the phenotype (Birchler
et al. 2001; Veitia 2002; Veitia 2004; Birchler et al. 2005, 2007; Veitia et al. 2008;
Birchler and Veitia 2007, 2010).
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2.10 Higher Ploidal Levels
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Ploidal levels above the tetraploid level most commonly involve hexaploids and
octoploids although much higher levels have been documented. Chromosome
pairing in allohexaploids has been studied in detail, for example in wheat, which is
ordinarily disomic in nature (Kihara 1919; Lilienfeld 1951; Dvorak et al. 1988).
The Ph system insures pairing of homologs and against pairing of homoeologues
but homoeologues can pair in mutant plants (Yousafzai et al. 2010; see Chap. 7,
this volume). The wheat genome is composed of three different slightly diverged
genomes tracing back through the joining of an allotetraploid composed of two
genomes with the third. The Ph system maintains disomic pairing and hence
excellent fertility. Octoploids, using sugar cane as an example, have variable
chromosome numbers due to the minimal detrimental effects of aneuploidy at this
level (Piperidis et al. 2010). In contrast, triticale, which is an octoploid consisting
of hexaploid wheat with the addition of a rye genome, exhibits faithful chromosomes numbers.
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Triploids are a polyploid level between diploid and tetraploid. They arise from
crosses between diploid and tetraploids of the same or related species or from
unreduced gametes from one diploid parent. In meiosis, the chromosomes associate in trivalents, which consists of pairing of any two chromosomes at any one
point (McClintock 1929; Punyasingh 1947; Upcott 1935). The distribution of
chromosomes is nearly random, resulting in spores that range from 1x to 2x. As a
consequence, the gametophytes are mostly highly aneuploid and in some cases
abort (Satina and Blakeslee 1937a, b). Fertilization involving gametes of different
chromosome numbers in the endosperm will often cause endosperm abortion
(Satina et al. 1938; Punyasingh 1947; Brink and Cooper 1947; Cooper 1951). The
gametes that are successful tend to be those at or near the 1x or 2x level. Because
of the variability of the chromosome numbers in gametes from triploid individuals,
this ploidal level is not stable.
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2.11 Concluding Remarks
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Acknowledgments Research supported by National Science Foundation grant DBI 0733857.
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References
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Abel S, Becker HC (2007) The effect of autopolyploidy on biomass production in homozygous
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Albertin W, Brabant P, Catrice O, Eber F, Jenczewski E, Chèvre AM, Thiellement H (2005)
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Barker MS, Kane NC, Matvienko M, Kozik A, Michelmore RW, Knapp SJ, Rieseberg LH (2008)
Multiple paleopolyploidizations during the evolution of the compositae reveal parallel
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Bartlett MS, Haldane JBS (1934) The theory of inbreeding in Autotetraploids. J Genet 29:175–180
Bingham ET, Groose RW, Woodfield DR, Kidwell KK (1994) Complementary gene interactions
in alfalfa are greater in autotetraploids than diploids. Crop Sci 34:823–829
Birchler JA (1979) A study of enzyme activities in a dosage series of the long arm of
chromosome one in maize. Genetics 92:1211–1229
Birchler JA (2011) Epigenetic aspects of centromere function in plants. Curr Opin Plant Biol
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Birchler JA, Newton KJ (1981) Modulation of protein levels in chromosomal dosage series of
maize: the biochemical basis of aneuploid syndromes. Genetics 99:247–266
Birchler JA, Bhadra U, Pal Bhadra M, Auger DL (2001) Dosage dependent gene regulation in
multicellular eukaryotes: implications for dosage compensation, aneuploid syndromes and
quantitative traits. Dev Biol 234:275–288
Birchler JA, Riddle NC, Auger DL, Veitia RA (2005) Dosage balance in gene regulation:
biological implications. Trends Genet 21:219–226
Birchler JA, Veitia RA (2007) The gene balance hypothesis: from classical genetics to modern
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The genetics of polyploids depends essentially on the pairing properties of the
multiple chromosomes in meiosis. If the multiple copies of a genome are sufficiently dissimilar from each other, they tend to pair among themselves and
maintain the genetic variation within each genome. If the multiple copies of a
genome are similar to each other, then all copies are free to pair and recombine
among themselves. In this circumstance, the genetic behavior of a particular gene
is dependent on its position on the chromosome and the fidelity of the pairing of
homologs. Aneuploidy, i.e., the variation of a single chromosome or chromosomal
segment, can have more severe consequences than varying the whole genome.
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change of aneuploidy becomes less. The phenotypic effects, gene expression
patterns and the evolutionary results of differential gene retention following
whole-genome duplications versus segmental duplication suggest the importance
of genomic balance.
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Blakeslee AF, Belling J, Farnham ME (1923) Inheritance in tetraploid Daturas. Bot Gaz
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Blanc G, Wolfe KH (2004) Functional divergence of duplicated genes formed by polyploidy
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Chapman BA, Bowers JE, Feltus FA, Paterson AH (2006) Buffering of crucial functions by
paleologous duplicated genes may contribute cyclicality to angiosperm genome duplication.
Proc Natl Acad Sci USA 103:2730–2735
Chester M, Gallagher JP, Symonds VV, Cruz da Silva AV, Mavrodiev EV, Leitch AR, Soltis PS,
Soltis DE (2012) Extensive chromosomal variation in a recently formed natural allopolyploid,
Tragopogon miscellus (Asterasceae). Proc Natl Acad Sci USA (in press)
Clausen RE (1941) Polyploidy in Nicotiana. Am Nat 75:291–306
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Book Title
Polyploidy and Genome Evolution
Series Title
Chapter Title
Meiosis in Polyploid Plants
Copyright Year
2012
Copyright HolderName
Springer-Verlag Berlin Heidelberg
Corresponding Author
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Mittelsten Scheid
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Given Name
Ortrun
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Division
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Gregor Mendel Institute of Molecular Plant Biology
Address
Vienna, Austria
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ortrun.mittelsten_scheid@gmi.oeaw.ac.at
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Zielinski
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Marie-Luise
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Gregor Mendel Institute of Molecular Plant Biology
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Vienna, Austria
Email
Abstract
Meiosis is an obligate process during sexual reproduction, which involves the combination of parental
genomes and the coordinated segregation of the recombined chromosomes to the gametes. Polyploidy has
direct and fundamental consequences on meiosis, which are gradually and individually different between the
extreme cases of auto- and allopolyploids. Multiple chromosome complements have a major impact,
especially on chromosome pairing during pachytene and on the segregation of genotypes and phenotypes in
progeny. At the same time, irregularities during meiosis are a major source of naturally occurring
polyploidization events by the formation of unreduced gametes. Although individuals originating from
nonhaploid gametes may suffer from reduced vigor and fecundity, their gametogenesis can produce many
more chromosomal combinations than regular diploids, and thereby expose more diversity to natural selection.
A more relaxed control of pairing and segregation in polyploids, possibly also with increased recombination
rates, might be an important contribution to evolution and adaptation potential, especially under drastic or
frequent changes in environmental conditions.
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Meiosis in Polyploid Plants
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Marie-Luise Zielinski and Ortrun Mittelsten Scheid
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Abstract Meiosis is an obligate process during sexual reproduction, which involves
the combination of parental genomes and the coordinated segregation of the
recombined chromosomes to the gametes. Polyploidy has direct and fundamental
consequences on meiosis, which are gradually and individually different between the
extreme cases of auto- and allopolyploids. Multiple chromosome complements have
a major impact, especially on chromosome pairing during pachytene and on the
segregation of genotypes and phenotypes in progeny. At the same time, irregularities
during meiosis are a major source of naturally occurring polyploidization events by
the formation of unreduced gametes. Although individuals originating from
nonhaploid gametes may suffer from reduced vigor and fecundity, their gametogenesis can produce many more chromosomal combinations than regular diploids,
and thereby expose more diversity to natural selection. A more relaxed control of
pairing and segregation in polyploids, possibly also with increased recombination
rates, might be an important contribution to evolution and adaptation potential,
especially under drastic or frequent changes in environmental conditions.
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3.1 Introduction
Polyploidization can affect the size of the nucleus and the cell, stimulate genetic
rearrangements, or modify gene expression patterns, but these changes very likely
operate by the same mechanistic principles of nuclear organization, recombination,
or gene regulation, as in diploid cells. In contrast, reducing the number of
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M.-L. Zielinski O. Mittelsten Scheid (&)
Gregor Mendel Institute of Molecular Plant Biology, Vienna, Austria
e-mail: ortrun.mittelsten_scheid@gmi.oeaw.ac.at
P. S. Soltis and D. E. Soltis (eds.), Polyploidy and Genome Evolution,
DOI: 10.1007/978-3-642-31442-1_3, Springer-Verlag Berlin Heidelberg 2012
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chromosomes during the formation of gametes requires a more elementary
modification in polyploids. At the same time, irregular gamete formation is a
major trigger for polyploidization. Both processes converge in meiosis. This is a
sequence of usually two special, subsequent cell divisions in all sexually reproducing eukaryotic organisms in which the number of chromosomes is reduced to
half, before two gametes of different parental origin fuse to form a zygote to
produce the next generation.
Like mitosis, the cell division of somatic cells, meiosis is also preceded by the
replication of the nuclear DNA into duplicate sister chromatids which are attached at
the centromeres. While in mitosis the replicated chromosomes are arranged individually on the metaphase plate, and chromatids become separated in the subsequent
anaphase, each replicated chromosome in meiotic cells is first aligned with its
respective homologous partner. This pairing is followed by programed induction of
DNA double-strand breaks (DSB), from which at least one per chromosome becomes
converted into a recombination event between non-sister chromatids (crossover, CO).
Later, these COs become cytologically visible as chiasmata. Recombination within
chromosome pairs results in new and unique combinations of paternal and maternal
genetic information per chromosome, while it is also prerequisite for subsequent
organized chromosome segregation. Paired chromosomes become physically
connected by the synaptonemal complex (SC). The process of pairing, synapsis, and
recombination takes most of the time during prophase I, which is subdivided into
leptotene, zygotene, pachytene, diplotene, and diakinesis according to chromosome
condensation and configuration. In the subsequent metaphase I, the recombined
meiotic chromosome pairs are arranged on the equatorial plane, sister kinetochores of
each pair attach to opposite spindles and are pulled apart in anaphase I. Correct and
synchronous pairing is a prerequisite for equal distribution of the chromosomes to the
two daughter cells. After telophase I and prophase II, sister chromatids become
separated during meta-, ana-, and telo-phase II, resulting in a classical meiosis with
four nuclei. Faultless meiosis is an essential factor for fertility and thereby decisive for
evolutionary success. Therefore, it is not surprising that it is a tightly controlled
process and intensely studied in many organisms.
The majority of meiosis research is performed with diploid organisms, and
there are excellent reviews available that describe morphological, genetic, and
mechanistic aspects of meiosis in many different systems (Dawe 1998; Bhatt et al.
2001; Armstrong et al. 2003; Mezard et al. 2007; Mercier and Grelon 2008;
Harrison et al. 2010; Pawlowski 2010). However, characteristic differences in all
stages of meiosis in many different polyploids have been observed repeatedly
(reviewed in Ramsey and Schemske 2002). Therefore, we will focus on those
aspects that are known or expected to be different between meiosis in diploids and
polyploids. Although mechanistically intertwined, we will separate three aspects,
(1) chromosome pairing, (2) recombination and crossover, and (3) segregation.
Whenever relevant, we will distinguish between autopolyploids, with multiples of
similar chromosomes, and allopolyploids, with chromosomes of different origin,
and therefore higher divergence.
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3.2 Recognition, Pairing, and Synapsis
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Early in meiosis, homologous chromosomes have to find and recognize each other.
Interphase chromosomes are thought to occupy nonrandom territories (Schubert
and Shaw 2011), and nonrandom spatial organization of homologous chromosomes has been recognized (reviewed in Avivi and Feldman 1980). In many
eukaryotes, telomeres and centromeres can cluster at opposite poles of the nucleus
(Rabl 1885; Cowan et al. 2001). This Rabl-configuration (Fig. 3.1a) is found in
wheat, rye, oats, and barley, but not in maize or Arabidopsis (reviewed in Schubert
and Shaw 2011). It is assumed to be related to large genome size and/or
chromosome length (Dong and Jiang 1998). Rabl-configuration may support gene
expression control and the onset of meiosis (Cowan et al. 2001).
After the premeiotic DNA replication, the two sister chromatids are held together
by sister chromatid cohesion and gradually become condensed. During leptotene, their
chromatin is folded into loops attached to a protein fiber core, the axial element (AE)
(Harper et al. 2004). During transition to zygotene, the so-called telomere bouquet is
formed, in which the chromosome ends attach to the inner nuclear envelope and cluster
(Fig. 3.1b). This association is likely to be actively regulated (Scherthan 2007).
Recognition between the homologous chromosomes appears to occur between subtelomeric regions (Corredor et al. 2007). The telomere bouquet, found in almost all
studied organisms except Drosophila and C. elegans (Harper et al. 2004), is not
absolutely required for the following steps but is proposed to make progression of
meiosis more efficient. Both Rabl-configuration and telomere bouquet bundle chromosomes and thereby help reduce the spatial distance between them. After telomere
bouquet formation in yeast, the centromeric regions are distributed throughout the
nucleus and oscillate to enhance finding homologous sequences (Bass et al. 1997;
Scherthan 2007). Chromosome movements are extremely dynamic at this stage
(Sheehan and Pawlowski 2009), which increases physical encounters and makes
meeting and recognition of the homologs more likely (Pawlowski and Cande 2005).
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Since polyploidy in animal germ lines is restricted to relatively few groups, and
the research literature about meiotic mechanisms is scarce, most of the evidence
summarized here stems from investigations in fungi and plants. There, polyploidy is
by far most common among angiosperms, but genome research provides growing
evidence for ancient polyploidization events in other groups (Sundstrom et al. 2008;
Jiao et al. 2011). Independent of how and when polyploids originate, mastering
meiosis is an important checkpoint for survival and evolutionary success.
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3.2.1 Homology Recognition in Polyploids
It seems plausible to expect that higher chromosome numbers, and larger nuclei in
general, but especially more potential homologous partner chromosomes in
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(a)
Fig. 3.1 Chromosome arrangement (red circles centromeres; orange triangles telomeres).
a Rabl-configuration in interphase, with centromeres clustered at one pole of the nucleus and
telomeres oriented toward the other. b Bouquet formation during meiosis at the onset of zygotene,
with telomeres clustered at the inner nuclear envelope
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3.2.2 Pairing in Autopolyploids
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There is one important difference that distinguishes the pachytene stage in
autopolyploids: the possibility of pairing between more than two chromosomes,
resulting in the formation of multivalents. This requires a certain degree of
homology between pairing partners, and this is reflected in different frequency of
multivalent occurrence depending on the type of polyploidy, species, individual
chromosomes, and chromosome segments (Sybenga 1996). While allopolyploids
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autopolyploids, could delay progression through meiosis, since the early stages of
recognition and pairing can be more complex. Indeed, meiosis in autotetraploid
Saccharomyces cerevisiae strains is delayed in comparison to its diploid counterpart (Trelles-Sticken et al. 2003). While higher C-values (genomic DNA content) in
different plant species also increase the duration of meiosis when compared at the
same ploidal level, the comparison of plants with different ploidy within the same
species revealed a surprising, negative correlation: higher polyploidy seems to
shorten the time needed for completing meiosis (Bennett 1977). These discrepancies might be created by defining the duration only by cytologically visible events.
Analysis with higher resolution techniques and consideration of molecular events
(Carlton et al. 2006) at the onset of meiosis might provide more accurate information on meiosis kinetics in polyploids. In spite of individual differences, it is
remarkable that a wide range of genome and nuclear size variation does not modify
the length of the recognition process (Moore and Shaw 2009). It appears that
homology recognition is not the limiting step, due to supportive elements like Rablconfiguration and telomere bouquet , specialized chromosome pairing sites as found
in D. melanogaster and C. elegans (McKee 1996; McKim 2007), and intensive
chromosome movements that are effective in diploids and polyploids.
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with genomes composed of genetically divergent parents often form only bivalents
aligned over the whole length of the chromosome, autopolyploids may have
multivalents. These are generated by simultaneous alignment of different partners
and at different chromosome ends. The probability depends on the degree of ploidy
and homology (random-end pairing model by John and Henderson 1962) and is
controlled by genetic factors (see below), but it is independent of chromosome
length (Morrison and Rajhathy 1960). Although multivalent formation is more
often associated with autopolyploidy, many exceptions delimit the universality of
this correlation: newly formed autopolyploids often have a lower rate of multivalents than expected, and allopolyploids may form multivalents to some extent
(Ramsey and Schemske 2002). A few of these cases might be due to tri- or
tetrasomy compensated by lack of other chromosomes, resulting in apparent
euploidy (Mestiri et al. 2010).
Progressive pairing starting simultaneously from opposite ends can result in
multivalents with pairing partner switches (PPS), so that one chromosome can be
aligned with two or more others in different segments (Fig. 3.2a). The distribution
of PPS is variable but probably not totally random. More than one switch per
chromosome (Fig. 3.2b) indicates the existence of additional, autonomous pairing
sites (APS) along the chromosomes, each with a uniform and low probability of
generating a PPS (Jones and Vincent 1994). The number of switches indicates the
minimal number of pairing sites (Loidl 1995). These are likely not only determined genetically, since a higher number of PPS in autotriploid Crepis capillaris,
compared to autotetraploid (Jones 1994), indicates other factors than just APS
distribution, but probably some interference between pairing initiation sites.
Pairing with one partner at one pairing site preferentially promotes continuation of
pairing in a zipper-like manner, due to steric constraints. However, this preference
is valid only once synapsis has been initiated.
According to the random-end pairing model (John and Henderson 1962),
alignment of two chromosome ends enhances the two remaining to pair, although
the opposite ends of the chromosome still can pair randomly, resulting in 2/3
multivalents and 1/3 bivalents in tetraploids (only chromosome ends are considered under this model). Exceptions with fewer than expected multivalents (Weiss
and Maluszynska 2000; Santos et al. 2003; Carvalho et al. 2010) or bivalent
formation due to selective pairing (Simioni and do Valle 2011, and references
within) reflect genetic and/or epigenetic heterozygosity between parental chromosome sets, due to ongoing diploidization. This can be different between individual plants or individual chromosomes (Santos et al. 2003) and extremely
divergent in complex polyploids, as deduced from genetic maps of sugarcane
(Jannoo et al. 2004). Higher than expected ratios of multivalents can also occur, as
in newly generated autotetraploids of Arabidopsis thaliana, indicating multiple
active pairing initiation sites even in small chromosomes (Santos et al. 2003).
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3.2.3 Pairing in Allopolyploids
Although newly formed hybrids combining different genomes and different chromosome numbers and shapes can exhibit erroneous pairing or lack of alignment
(Ozkan and Feldman 2009), established allopolyploids show usually diploid-like
pairing and formation of bivalents. Nevertheless, multivalents or segmental multivalent pairing can be found in allopolyploids, depending on external or genetic factors. In many polyploids, a genetic control system has evolved that regulates the
pairing behavior during meiosis (Watanabe 1981; Gupta and Fedak 1985; MartinezPerez et al. 2001; Comai et al. 2003; Martinez-Perez et al. 2003; Jenczewski and Alix
2004). Probably the best studied example was discovered in the hexaploid bread
wheat (Triticum aestivum, composed of genomes A, B, and D). In the presence of the
Ph1 locus, pairing and recombination occur preferentially between homologous
chromosomes (Riley and Chapman 1958); lack of Ph1 leads to a substantial increase
of homoeologous pairing. The Ph1 locus from wheat can also affect pairing after
introgression into rye (Lukaszewski and Kopecky 2010). The absence of Ph1 in
diploids suggests its emergence through polyploidization (Griffiths et al. 2006). The
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Fig. 3.2 Pairing partner switches during multivalent formation in polyploids. Alignment and
partial synapsis between four chromosomes with one (a) or two (b) pairing partner switches
(PPS) on the long arm
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As soon as meiotic chromosomes align, they become joined by a stable proteinaceous
structure, the SC. Its formation starts mainly at telomeres (Stack and Anderson 2002)
and is intimately connected with DSBs and recombination (see next paragraph). The
SC consists of three components: the two parallel AEs, the connecting central element, and periodically occurring recombination nodules (RNs) (Lohmiller et al.
2008). The RNs are multiprotein complexes thought to be involved in synapsis and
recombination and can be distinguished by their structure and appearance as early or
late RNs (Stack and Anderson 2002; Anderson and Stack 2005).
Synapsis is expected to follow once pairing is established (Loidl 1995). However,
unequal chromosome numbers can make a difference in polyploids: in autotriploid,
but not in autotetraploid, yeast, trivalent SC formation was observed (Loidl 1995).
Such fixed multivalents are prone to nondisjunction at metaphase I. Multivalents with
equal numbers seem to resolve more often into regular bivalents, although SC
connections between several AEs can occur and become fixed by crossovers. Without
crossover at the region of the PPS, the central region is twisted and bivalents are
formed (von Wettstein et al. 1984). When, and how, are interesting questions,
especially for meiosis in autopolyploids.
In summary, recognition, pairing, and synapsis of homologous chromosomes in
diploids and polyploids are different in several aspects and determined by various
parameters. The high rate of multivalent formation in autopolyploids depends on
the type and history of the polyploidization, the number of pairing initiation sites,
and odd or even chromosome multiplication. Allopolyploids rather generally form
bivalents, but segment-, chromosome-, or genome-specific multivalent formation,
and genetic and environmental influence, can also result in meiotic progression
different from that in diploids.
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Ph1 locus is a complex rearrangement between subtelomeric heterochromatin
translocated within a cluster of cdk2-like genes localized on one of the chromosomes
of the B genome (Griffiths et al. 2006).
Ph1 controls meiosis at several levels. While telomere pairing is not affected,
Ph1 prevents pairing between centromeres of nonhomologous chromosomes, keeps
centromeres at the nuclear periphery, synchronizes chromatin condensation, and
controls the expression of synapsis and recombination components (Naranjo and
Corredor 2004; Moore and Shaw 2009; Knight et al. 2010; Yousafzai et al. 2010).
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3.3 Recombination and Crossover
Meiotic recombination in plants is initiated by multiple DSBs. Some of the breaks
are repaired by homologous recombination using the non-sister chromatid as a
template, resulting in reciprocal strand exchange (CO events). As the number of
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initial DSBs exceeds the number of COs, the majority of DSBs are processed
otherwise, either by separating double Holliday junction into non-crossover (NCO)
products or by the synthesis-dependent strand annealing (SDSA) pathway. There
are excellent recent reviews that describe details of DSB processing and components of meiotic recombination (Hamant et al. 2006; Mercier and Grelon 2008;
Sanchez-Moran et al. 2008; De Muyt et al. 2009; Edlinger and Schlogelhofer
2011; Osman et al. 2011), and there is no reason to assume that the principal
mechanisms are different in polyploids. However, there is growing evidence that
the frequency with which DSBs give rise to COs can be regionally or generally
modified by polyploidy. Therefore, we will describe some factors that influence
crossover frequencies.
Due to the role of recombination in the regulation of chromosome pairing and
segregation, there appears to be a minimum of one CO per chromosome in many
species (Youds and Boulton 2011). Beyond this, and in spite of variation between
different species, there seems to be no correlation between the length of chromosomes and the number of COs (Brubaker et al. 1999; Mezard 2006). The genomes
are rather composed of ‘hot spots’ and ‘cold spots’, with high and low probabilities
for meiotic recombination (Drouaud et al. 2006; Mezard 2006; Kim et al. 2007).
Even small local sequence divergence, like a transgene insertion, can modify
meiotic recombination locally (Sun et al. 2008). Variation in recombination or CO
frequency among Arabidopsis accessions (Barth et al. 2001; Sanchez-Moran et al.
2002) also indicates genetic components that regulate the meiotic recombination
frequency in trans. Further, temperature and age of flowers within the plant can
modify CO events (Francis et al. 2007). Another aspect to consider is CO interference, the inhibition of additional recombination events by COs in their proximity
or even along the whole chromosome (Holliday 1977; van Veen and Hawley 2003;
Baudat and de Massy 2007; Youds and Boulton 2011).
Differences in the number of CO events in polyploids have been described in
several systems. The A and D genome components of allotetraploid Gossypium
(cotton) are very different in size but marker pairs have, nevertheless, comparable
genetic distances if compared between diploid or allopolyploid mapping populations, respectively. Surprisingly, the comparison for the same markers between
diploids and allotetraploids indicated a higher recombination rate in the latter
(Brubaker et al. 1999; Desai et al. 2006). F1 hybrids between the diploid parents
Brassica oleracea and Brassica rapa, compared with F1 plants derived from the
same hybrid with doubled chromosome numbers after colchicine treatment,
produced more progeny with intergenome recombination (Szadkowski et al.
2010, 2011). A comparison of the CO number among diploid, allotriploid, and
allotetraploid hybrids from crosses between Brassica oleracea and Brassica rapa
revealed the highest number for allotriploids, intermediate values for allotetraploids, and the lowest numbers in diploids (Nicolas et al. 2008; Leflon et al. 2010).
This indicates stimulation of recombination by hybridity and/or polyploidy,
however, in a nonlinear correlation with the number of homologous chromosomes. This might be coupled with the occurrence of univalents, in addition to
bivalents, in the triploids, whereas the tetraploids exclusively form bivalents
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3.4 Chromosome and Allele Segregation
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3.4.1 Chromosome Segregation
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Following pairing and recombination in prophase I, chromosomes are arranged at
the equatorial plane during metaphase I. The following anaphase I is distinct from
that in mitosis since both centromeres of sister chromatids attach to spindles of the
same pole and retain their cohesion when the chromosome pairs get dragged apart,
moving to opposite poles. This ‘reductional’ division, therefore, halves the
chromosome number. It is followed by metaphase II with the formation of two new
equatorial planes, and anaphase II, in which the sister chromatids now become
separated and distributed to the resulting four postmeiotic nuclei. A tightly
controlled order of maintenance and stepwise release of cohesion specifies all stages
of meiosis (Sakuno and Watanabe 2009), but there is no evidence that an increased
chromosome number in polyploids affects this control.
In contrast, due to the effects of polyploidy on pairing and/or recombination,
there is a much higher probability of unequal segregation of chromosomes into the
postmeiotic nuclei. Unequal distribution of the chromosomes can occur in many
different ways (Pagliarini 2000). It can decrease the regular nuclear DNA content, if
unresolved multivalents, unpaired or laggard chromosomes, or parts of chromosomes are not included or translocated to other chromosomes (Madlung et al. 2005;
Charles et al. 2010; Gaeta and Pires 2010; Wang et al. 2010). Extreme cases of
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(Leflon et al. 2010), indicating a control mechanism to prevent multivalents. A
good candidate is the PrBn locus (Jenczewski et al. 2003; Nicolas et al. 2009;
Cifuentes et al. 2010). Exploring an assay system for meiotic recombination
based on fluorescent proteins expressed in seeds (Melamed-Bessudo et al. 2005),
a comparison among diploids, isogenic autotetraploids, and allotetraploids generated by interspecies hybridization revealed an unexpected increase of recombination between the markers for both types of polyploids (Pecinka et al. 2011).
The presence of mainly multivalent formation in one, and bivalent formation in
the other, argue against a correlation with the pairing behavior.
In summary, the frequency of recombination between any two markers is
determined by interplay of physical distance, local cis- and trans-acting genetic
control elements, the degree of overall genetic divergence, and sex-specific differences (Vizir and Korol 1990; De Vicente and Tanksley 1991; Drouaud et al.
2007; Nelson et al. 2005; Pecinka et al. 2011), regardless of ploidy. The few briefly
described examples of increased recombination frequency in polyploid plants
suggest that pairing behavior, potential modification of crossover interference by
PPS, distinctive segregation patterns (see below), or other still unknown factors
add to the complexity. However, it is tempting to speculate that high(er) recombination rates, at least in part, contributed to the prevalence of polyploidy among
angiosperms and crop plants bred within the last 12,000 years.
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systematic chromosome elimination were observed in the allopolyploid Paspalum
subciliatum (Adamowski et al. 1998) and in pentaploid Brachiaria decumbens
(Ricci et al. 2010), due to kinetic asynchrony of the two different genomes after
diakinesis. Alternatively, the nuclear DNA may be increased if restitution of nuclei
occurs prior to division or around incompletely separated chromosomes (Ramsey
2007; Brownfield and Kohler 2011). This can occur either after the first or the
second meiotic division, differing in the degree of heterozygosity transmitted to the
progeny (Peloquin et al. 2008).
While loss of chromosomal material is often deleterious for the resulting cell and
leads to cell death or reduced viability and fertilization, a gain, and optimally a
balanced multiplication of all chromosomes, is much less detrimental. Accordingly,
unreduced gametes occur in numerous species and are a major source of polyploidization events (Leitch and Leitch 2008; Koehler et al. 2010). The frequency of
unreduced gametes varies significantly between hybrids (e.g., Ortiz 1997; Lim et al.
2004), between different ploidal levels (e.g., Burton and Husband 2001; Ramsey
2007), and also depends on genetic factors (reviewed in Brownfield and Kohler
2011), external conditions, and stress factors (reviewed in Ramsey and Schemske
1998). The frequent occurrence of unreduced gametes has been explained by the
lack of, or less effective, ‘pachytene checkpoint’ in plants (Li et al. 2009), but may
be an inherent element of circumventing meiotic problems in interspecies hybrids
by polyploidization.
The occurrence of diploid or other unreduced post-meiotic cells also has
consequences for the subsequent phase of the life cycle. Haploid gametophytes
from diploid parents depend on functionality of each single genomic copy that
encodes factors for proper gametophyte viability and fertilization potential.
Gametophytes from polyploid species contain more than one copy, providing
backups for defective alleles and the potential for heterozygosity even during the
two and three postmeiotic cell divisions forming the male and female gametophytes, respectively. Since at least pollen selection is efficient at several levels
(Ottaviano et al. 1990), avoiding haploidy might provide reduced selection
pressure and increased variability of transmitted genetic information.
Not directly related to meiosis but with consequences for gamete formation and
fertility is the phenomenon of cytomixis, the fragmentation of chromatin and
transfer of fragments among cells through cytoplasmic channels. Observed in
many species, and especially in pollen mother cells (e.g., Mursalimov and Deineko
2011), one published comparison between diploid and tetraploid varieties of
Withania somnifera indicated that the resulting reduction of fertility is much less
pronounced in the tetraploids, along with a reduced extent of intercellular
connections (Singhal and Kumar 2008). However, a correlation with polyploidy in
other species awaits investigation.
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The genetic segregation of traits in polyploids can be quite different from that in
diploids. Heterozygosity and multiple alleles per gamete increase the combinatorial
possibilities in the progeny population substantially and make the segregation patterns
deviate from the Mendelian ratios for diploids. This makes QTL analysis or any
mapping process complex, as in the extreme case of highly polyploid and partially
aneuploid sugarcane (Andru et al. 2011). Naturally, analysis of inheritance requires
consideration of pairing behavior and the resulting chromosome segregation. Variation among hybrids, individuals, chromosomes, and chromosome segments does not
allow establishing general rules. Therefore, we will depict the extremes: autotetraploids with quadrivalent formation, unbiased chromosome segregation, and tetrasomic
inheritance on one hand, and allopolyploids with strict bivalent formation and disomic
inheritance on the other. A Punnett square for segregation of one trait in the progeny of
heterozygous autotetraploid parents with two copies of two different alleles has 36
fields (Fig. 3.3b), rather than four in the case of a heterozygous diploid (Fig. 3.3a).
Deleterious recessive alleles become apparent as affected phenotypes in only 1:35,
rather than 1:3 in diploids. Each individual tetraploid can have one, two, three, or four
identical alleles (termed simplex, duplex, triplex, or quadruplex) at each locus, and the
expected segregation of the different genotypes upon crossing with a partner of the
same genotype is 1:8:18:8:1 (Fig. 3.3b). In the case of allopolyploids with strict
bivalent formation between the chromosomes of common origin, each gamete carries
one of each chromosome type so that the progeny are uniform (Fig. 3.3c). If the
chromosomes carry different alleles, autotetraploids can produce 19 different genotypes at one locus (Fig. 3.4a), while disomic inheritance in allotetraploids is limited to
9 combinations (Fig. 3.4b). While exclusive tetrasomic or disomic inheritance in
polyploids does occur, intermediate forms and changes in both directions over time
are frequently observed in nature and need to be considered in genetic and population
studies (Stift et al. 2008).
A genetic peculiarity of polyploid meiosis that does not occur in diploid
organisms is the chance of double reduction (Darlington 1929a; Butruille and
Boiteux 2000). This term describes the possibility that regions from two sister
chromatids become combined in the same gamete. Double reduction results from
recombination events between the locus under observation and the centromere. If
the two chromosomes that have recombined move to the same pole in anaphase I,
there is a high probability that the distal end of the recombined chromatid will be
included in the same nucleus as the distal end of the non-recombined chromatid,
after anaphase II (locus B in 2 gametes in Fig. 3.5). The frequency of double
reduction for a genetic locus depends on its distance from the centromere and the
frequency of multivalent formation.
Since segregation of traits in polyploids is so complex, but of high importance
for population geneticists and plant breeders, there were many attempts to
approach the problem via mathematical modeling. Ground-breaking theoretical
work (Mather 1936; Fisher 1947) has more recently stimulated several
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Fig. 3.3 Comparison of
segregation for an individual
locus with two alleles upon
bivalent or quadrivalent
formation. Two different
alleles (red, blue) in diploid
parents are transmitted with
equal probability, resulting in
three genotypes, forming
twice as many heterozygotes
as each homozygote (a). Two
different alleles with two
copies each in tetraploids
with random pairing between
each of the four
chromosomes result in five
genotypes, with only one
homozygote among 36
individuals (b). The same
situation in tetraploids with
strict bivalent formation
between the more similar
chromosome pairs renders
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(c)
computational methods based on maximum-likelihood and other statistical
approaches to estimate double reduction frequency and recombination and to
support mapping of quantitative trait loci in polyploids with multivalent formation
(Doerge and Craig 2000; Ridout et al. 2001; Wu et al. 2001a, b; Luo et al. 2004,
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Fig. 3.4 Comparison of
segregation for an individual
locus with four alleles upon
bivalent or quadrivalent
formation. Four different
alleles (red, orange, dark
blue, light blue) in tetraploids
with random pairing between
each of the four
chromosomes result in
nineteen genotypes (a). The
same situation in tetraploids
with strict bivalent formation
between the more similar
chromosome pairs renders
only nine combinations (b)
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2006; Li et al. 2010). However, different modeling approaches do not always
support the same conclusions (Ma et al. 2002; Cao et al. 2004), and it is likely that
we need to extend and refine experimental data collection as well as modeling
approaches to provide satisfying tools. Already difficult for strict autopolyploids
with mostly polysomic inheritance, the situation is even more complex for
polyploids with intermediate types of inheritance, as mentioned above. These can
be caused either by segment- or chromosome-specific homology differences, or by
stochastic pairing differences. Not even the situation in tetraploid yeast strains,
where tetrad analysis provides excellent resolution of segregation analysis, is easy
to interpret (Albertin et al. 2009; Stift et al. 2010). Allelic segregation in
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Fig. 3.5 Double reduction. Limited to polyploids, there is a certain probability that distal regions
(around locus B) from two sister chromatids are included in the same gamete. This exceptional
configuration occurs if two recombined chromosomes move to the same pole in anaphase I,
which is excluded in diploids
allopolyploid plants, as described, depends very much on the ratio of multivalent
formation at the genetic locus under investigation.
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As for other developmental and physiological processes, progress and efficiency of
meiosis can be modified by environmental influences. Temperature has been most
extensively studied, with a range of different responses. Higher temperatures
reduced regular meiosis in diploid Allium ursinum (Loidl 1989) and Rosa (Pecrix
et al. 2011) and increased the number of unreduced pollen (reviewed in Ramsey
and Schemske 1998). This could enhance the formation of polyploids under heat
stress and thereby support the described adaptive role of polyploidization under
adverse conditions. However, data for polyploids are rare, and partially divergent.
In allopolyploid Brassica hybrids, more unreduced pollen was observed at lower
temperatures (Mason et al. 2011), and meiosis in wheat was not affected within a
physiological range of temperatures (Bayliss and Riley 1972).
A more uniform effect across species (yeast, worms, and plants) appears to be a
stimulation of meiotic recombination rates at higher temperatures (Rose and
Baillie 1979; Borner et al. 2004; Francis et al. 2007). It is likely that adaptation to
habitats with more or less drastic environmental challenges has selected a
matching range of meiotic responses. Further, the more recently established
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polyploid hybrids generated by breeders might respond quite differently than
polyploids originating from spontaneous events long ago.
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Ancient polyploidization events and the prevalence of polyploids among living
plants leave no doubt that the ecological and metabolic advantages of multiple
chromosome complements preponderate over possible disadvantages (Soltis and
Soltis 1999; Ramsey and Schemske 2002; Comai 2005; Otto 2007; Parisod et al.
2010; Jiao et al. 2011). As described, successful meiosis and gamete formation are
mastered in different ways. Nevertheless, the survival and success of each new
hybridization or polyploidization event depends on multiple factors during
meiosis. Besides the requirements to adapt chromosome pairing and segregation,
asynchrony, incompatibility of protein complex subunits, dosage effects, or
differences in gene regulation can present meiotic barriers.
One solution in plants to avoid such a decrease in reproduction is to avoid
meiosis by apomixis. Apomixis, the asexual production of seeds, leads to offspring
that is genetically identical to the mother plant and originate via different modifications of the double fertilization pathway (Gustaffson 1946; Asker and Jerling
1992). Gametophytic apomixis (Nogler 1984) is divided into apospory, where the
unreduced embryo sac is formed by a cell of the nucellus, and diplospory, where
the unreduced embryo sac is formed by bypassing meiosis I during megasporogenesis. Both result in an embryo sac that contains an unreduced egg cell, which
develops into an embryo independent of fertilization. Although apomicts are found
in diploids and polyploids, they are more common among polyploids (Asker and
Jerling 1992). Apomixis is thought to have evolved several times from sexual
ancestors in over 400 plant species (Nogler 1984). It is clearly associated with
changes in gene expression, kinetics, or epigenetic regulation (Carman 1997;
Sharbel et al. 2010). Whether polyploidization is a driving force toward apomixis,
or a consequence of apomictic propagation, is a matter of debate. Apomixis
harbors the potential to avoid sexual sterility caused by multivalent formation in
polyploids (Horandl et al. 2011). On the other hand, and as described, polyploidy
promotes the formation of unreduced gametes (Ramsey and Schemske 1998) and
might thereby foster the establishment of higher ploidal levels. Polyploidization of
apomicts could also protect them against extinction, by buffering against the
irreversible accumulation of deleterious mutations in the absence of recombination
as depicted by the metaphor of ‘‘Muller’s ratchet’’ (Darlington 1929b). Nevertheless, it is likely that these conditions are relevant in varying and individual
combinations, with additional components such as highly efficient DNA repair
system in asexually reproducing species (Schoen and Martens 1998) or occasional
or conditional sexual reproduction (D’Souza et al. 2004).
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Adamowski ED, Pagliarini MS, Batista LAR (1998) Chromosome elimination in paspalum
subciliatum (Notata group). Sex Plant Reprod 11(5):272–276
Agashe B, Prasad CK, Siddiqi I (2002) Identification and analysis of DYAD: a gene required for
meiotic chromosome organisation and female meiotic progression in Arabidopsis. Development 129(16):3935–3943
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While natural selection in sexually reproducing species rapidly eradicates failures
in meiosis, these are of interest for plant breeders. If not the seed, but the
surrounding fruit is the wanted product, for example in Citrus species or melons,
customers prefer products with small, reduced, or no seeds. Besides exploiting
developmental mutations, one effective and popular way to achieve such fruits is
the generation of triploids (Sanchez-Moran et al. 2002) by crossing tetraploids
with diploids. Triploid embryos are prone to abort subsequently (Kamiri et al.
2011). Since fruit development is often regulated by phytohormones originating
from the developing seeds (Dorcey et al. 2009), this strategy depends on their
seed-independent substitution (Pandolfini 2009). However, once generated, triploid plants are often seedless, due to a high number of univalents in meiosis, a
resulting low number of balanced gametes, and poor pollination and fertilization
rates. This principle is applied in banana (Heslop-Harrison and Schwarzacher
2007) and watermelon (Beaulieu and Lea 2006), or is combined with selection for
developmental mutants as for squash (Menezes et al. 2005).
Once a beneficial combination of genetic traits by crossing is achieved, breeders
want to fix this for further progeny (Wijnker and de Jong 2008; Chan 2010).
Programed switches between crossing and apomixis would, therefore, be desirable
(Spillane et al. 2004), especially for polyploids with their complex segregation
patterns. Recent advances in provoking apomixis have been made by strategies to
disturb meiosis. A mutation in the Arabidopsis gene SWI/DYAD (Mercier et al. 2001;
Agashe et al. 2002) leads to the formation of unreduced egg cells, albeit at low
frequencies. This is due to failure in female meiosis and maintains complete maternal
heterozygosity in the triploid progeny (Ravi et al. 2008). Combining three mutations
affecting different steps of meiosis (osd1/Atspo11-1/Atrec8) turned meiosis into
mitosis and resulted in unreduced male and female gametes (d’Erfurth et al. 2009).
Consequently, ploidy in subsequent generations was doubled, accompanied by
reduced fertility. Nevertheless, the achievement of fertilization-independent seed
development is a step toward introducing apomixis into crop plants, and of special
interest for polyploid plants.
The work in the OMS lab was supported by grants from the Austrian Science
Fund FWF P18986 and I489.
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Santos JL, Alfaro D, Sanchez-Moran E, Armstrong SJ, Franklin FC, Jones GH (2003) Partial
diploidization of meiosis in autotetraploid Arabidopsis thaliana. Genetics 165(3):1533–1540
Scherthan H (2007) Telomere attachment and clustering during meiosis. Cell Mol Life Sci 64(2):
117–124
Schoen I, Martens K (1998) DNA repair in ancient asexuals: a new solution to an old problem? J
Nat Hist 32:943–948
Schubert I, Shaw P (2011) Organization and dynamics of plant interphase chromosomes. Trends
Plant Sci 16(5):273–281
Sharbel TF, Voigt ML, Corral JM, Galla G, Kumlehn J, Klukas C, Schreiber F, Vogel H, Rotter B
(2010) Apomictic and sexual ovules of Boechera display heterochronic global gene
expression patterns. Plant Cell 22(3):655–671
Sheehan MJ, Pawlowski WP (2009) Live imaging of rapid chromosome movements in meiotic
prophase I in maize. Proc Natl Acad Sci USA 106(49):20989–20994
Simioni C, do Valle CB (2011) Meiotic analysis in induced tetraploids of Brachiaria decumbens
Stapf. Crop Breed Appl Biotech 11(1):43–49
Singhal VK, Kumar P (2008) Cytomixis during microsporogenesis in the diploid and tetraploid
cytotypes of Withania somnifera (L.) Dunal, 1852 (Solanaceae). Comp Cytogenet 2(1):85–92
Soltis DE, Soltis PS (1999) Polyploidy: recurrent formation and genome evolution. Trends Ecol
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Spillane C, Curtis MD, Grossniklaus U (2004) Apomixis technology development-virgin births in
farmers fields? Nat Biotechnol 22(6):687–691
Stack SM, Anderson LK (2002) Crossing over as assessed by late recombination nodules is
related to the pattern of synapsis and the distribution of early recombination nodules in maize.
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Stift M, Reeve R, van Tienderen PH (2010) Inheritance in tetraploid yeast revisited: segregation
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Stift Marc, Berenos C, Kuperus P, van Tienderen PH (2008) Segregation models for disomic,
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Szadkowski E, Eber F, Huteau V, Lode M, Huneau C, Belcram H, Coriton O, ManzanaresDauleux MJ, Delourme R, King GJ et al (2010) The first meiosis of resynthesized Brassica
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Arabidopsis thaliana. Hereditas 133(3):255–261
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Wu SS, Wu RL, Ma CX, Zeng ZB, Yang MC, Casella G (2001b) A multivalent pairing model of
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3 Meiosis in Polyploid Plants
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Book Title
Polyploidy and Genome Evolution
Series Title
Chapter Title
Origins of Novel Phenotypic Variation in Polyploids
Copyright Year
2012
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Springer-Verlag Berlin Heidelberg
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Family Name
Martienssen
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Robert A.
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Cold Spring Harbor Laboratory
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One Bungtown Road, 11724, Cold Spring Harbor, NY, USA
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martiens@cshl.edu
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Finigan
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Patrick
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Cold Spring Harbor Laboratory
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One Bungtown Road, 11724, Cold Spring Harbor, NY, USA
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Tanurdzic
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Milos
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Cold Spring Harbor Laboratory
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Abstract
Polyploid species represent a special type of organism in nature, one that can survive and compete with three
or more full sets of homologous chromosomes. While less common in the animal and fungal kingdoms,
polyploid species are highly prevalent in the plant kingdom. Indeed, most agricultural crops are polyploids,
typically because polyploidy confers greater robustness and therefore higher yields. Among many examples
of novel phenotypic variation exhibited by polyploids are the production of larger fruits, reduced tillering,
delays in the reproductive transition, and even the creation of visually stunning flower pigmentation patterns
coveted by gardeners. The source of this novel variation in polyploids is still largely unclear. However,
multiple cellular mechanisms have been proposed, with some supporting evidence, to explain novel variation.
We review some of these mechanisms here.
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Abstract Polyploid species represent a special type of organism in nature, one
that can survive and compete with three or more full sets of homologous
chromosomes. While less common in the animal and fungal kingdoms, polyploid
species are highly prevalent in the plant kingdom. Indeed, most agricultural crops
are polyploids, typically because polyploidy confers greater robustness and
therefore higher yields. Among many examples of novel phenotypic variation
exhibited by polyploids are the production of larger fruits, reduced tillering, delays
in the reproductive transition, and even the creation of visually stunning flower
pigmentation patterns coveted by gardeners. The source of this novel variation in
polyploids is still largely unclear. However, multiple cellular mechanisms have
been proposed, with some supporting evidence, to explain novel variation. We
review some of these mechanisms here.
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Origins of Novel Phenotypic Variation
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4.1 Prevalence and Significance of Polyploids
Polyploid organisms have three or more complete sets of homologous chromosomes (Winge 1917 #1369; Ramsey 2002 #1334). Polyploidy, or whole-genome
duplication (WGD), can arise through multiple ways—most often from unreduced
gametes following meiosis (Ahloowalia 1961 #1252; Harlan 1975 #1285;
Jørgensen 1928 #1290; Newton 1929 #1318; Skalinska 1946 #1344; Ramsey 1998
#1333; Bretagnolle 1995 #1258). In mammals, WGD events are considered to be
rare and typically lethal, presumably due to deleterious effects associated with
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Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring Harbor, NY 11724, USA
e-mail: martiens@cshl.edu
P. S. Soltis and D. E. Soltis (eds.), Polyploidy and Genome Evolution,
DOI: 10.1007/978-3-642-31442-1_4, Springer-Verlag Berlin Heidelberg 2012
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dosage (Bertrand 2010 #1255; Van de Peer 2009 #1361) see also Chap. 18, this
volume). However, genomic sequencing has revealed evidence for a few widespread ancient WGD events in the animal kingdom (Maere 2005 #1308; Dehal
2005 #1270; Kellis 2004 #1293; Ohno 1970 #1322; Meyer 2005 #1314, 1999
#1313) (see also Chaps. 16 and 17, this volume). Extant vertebrate genomes in
particular are believed to be the result of two or three separate WGD events during
evolution (Maere 2005 #1308; Dehal 2005 #1270; Kellis 2004 #1293; Ohno 1970
#1322; Meyer 2005 #1314, 1999 #1313) (Chap. 16, this volume). In plants,
polyploidy is generally tolerated, and most plant species are recent or ancient
polyploids (Lexer 2003 #1302; Soltis et al. 2009 #1346; Jiao 2011 #1289).
Polyploidy is thought to play a major role in speciation (Clausen 1945 #1267;
Grant 1981 #1282; Lumaret 1988 #1306), as polyploids are often reproductively
isolated from their progenitors—crosses between diploids and tetraploids generally yield triploid progeny with unbalanced meiosis, leading to polyploid infertility
(Darlington 1963 #1269; Grant 1981 #1282).
On a simplified level, Polyploid organisms can be either autopolyploids or
allopolyploids (Kihara 1926 #1296). Following one definition, an autopolyploid is
a Polyploid organism in which all of the chromosome sets are derived from the
same species, whereas an allopolyploid organism has chromosome sets derived
from different species (Kihara 1926 #1296). Allopolyploids are therefore permanent hybrids, but with a complete chromosome set from each parental species
(Kihara 1926 #1296). The complete parental chromosome sets in allopolyploids
allow for proper pairing of homologous chromosomes during meiosis (disomic
segregation), rather than missegregation of trivalents and quadrivalents as may be
found in autopolyploids (multisomic segregation) (Stebbins 1971 #1355; Ramsey
2002 #1334). Thus, allopolyploids have often been considered more stable than
autopolyploids, and ‘‘fix’’ hybrid genotypes in successive generations, along with
any beneficial or detrimental phenotypes that result (Winge 1932 #1370). However, it has been recognized that autopolyploids may have functional (either
disomic or multisomic) chromosome pairing and be highly fertile (Soltis et al.
2007 #1349; Soltis and Rieseberg 1986 #1347).
Many important agricultural crops are polyploids (Eigsti 1957 #1275). Polyploid crops typically demonstrate increased growth, including flower and fruit size,
and novel variation compared to their diploid counterparts that can make them
better suited as agricultural products (Stebbins 1971 #1355; Ramsey 2002 #1334;
Grant 1981 #1282). The modern-day bread wheat, Triticum aestivum, is an allohexaploid that is the combination of three different diploid species (Dubcovsky
2007 #1272) (see also Chap. 7, this volume). T. aestivum has largely replaced its
diploid progenitors and now accounts for about 95 % of the entire wheat crop
produced around the world (Dubcovsky 2007 #1272). In addition to wheat, many
staple crops (including corn, cotton, coffee, oat, canola, rye, apple, banana,
watermelon, potato, sugar cane, and soybean) are also recent or ancient polyploids
(Gaut 1997 #1281; Stebbins 1971 #1355; Ohno 1970 #1322; Shoemaker 1996
#1341; Lagercrantz 1996 #1298; Eigsti 1957 #1275). The reduced seed set of
certain polyploids (odd ploidal levels) can make them more desirable for
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4.2 Origin of Novel Variation in Neopolyploids
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The prevalence of polyploids in the plant kingdom despite the rarity of their
formation (Ramsey 1998 #1333) indicates that polyploids may have a fitness
advantage compared to their progenitors (Stebbins 1950 #1354). The explanation
for this fitness advantage stems from the observation that many polyploids display
novel phenotypic variation compared to their progenitors (Randolph 1941 #1335;
Levin 1983 #1300, 2002 #1301; Lumaret 1988 #1306; Ramsey 2002 #1334;
Müntzing 1936 #1316). This novel phenotypic variation is believed to enable
polyploids to exploit different environmental niches better than their progenitors;
this variation bestows a fitness advantage on the polyploids (Clausen 1945 #1267;
Stebbins 1950 #1354). One caveat of this hypothesis is that studies documenting
the geographic distribution of polyploids and their diploid progenitors used extant
diploid relatives instead of the exact diploid progenitors (usually unknown)
(Stebbins 1971 #1355; Ramsey 2002 #1334). Thus, it is possible the real progenitors had the same geographic range distribution as the current polyploids. To
address this problem, many current studies of polyploids use synthetic polyploids
that can be compared to their exact diploid progenitors (Stebbins 1971 #1355;
Ramsey 2002 #1334). Future studies should help to clarify if range expansion
relative to their progenitors is a general characteristic of polyploids.
The emergence of novel phenotypes in polyploids, especially neoallopolyploids
(defined as early-generation polyploids), compared to their progenitors has been of
great interest because of its application in plant breeding and relevance to biodiversity and evolution (Eigsti 1957 #1275; Stebbins 1971 #1355; Ramsey 2002
#1334; Grant 1981 #1282). Genetic mechanisms encompassing gene dosage/allelic
combinations, novel gene interactions, genomic alterations, and epigenomic
reorganization have all been proposed to explain the origin of novel phenotypes of
polyploids (Fig. 4.1). The relevance of these different mechanisms for causing
novel variation is still largely unclear, with important distinctions between autopolyploids and allopolyploids.
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consumption, like seedless watermelons and bananas. Moreover, allopolyploids
can overcome hybrid incompatibility between different species, allowing production of new varieties and introgression of favorable traits into important crops
(Eigsti 1957 #1275). Allohexaploid Nicotiana plants were used as a bridge to
transfer the gene responsible for tobacco mosaic virus resistance from wild to
commercial varieties of Nicotiana species (Eigsti 1957 #1275). This was only
possible through a Polyploid intermediate, as the interspecies Nicotiana hybrids
were sterile (Eigsti 1957 #1275) (see also Chap. 11, this volume).
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4.2.1 Gene Dosage and Allelic Combinations May Result in Novel
Variation in Neopolyploids
Genome-wide increases in DNA content have been proposed to contribute to novel
phenotypes in neopolyploids (Randolph 1941 #1335). The ‘‘gigas effect’’ is a welldocumented phenotype associated with polyploid formation, where certain organs
are larger in the polyploids than their progenitors—but not necessarily the entire
plant (Randolph 1941 #1335; Stebbins 1971 #1355). The gigas effect is believed to
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Fig. 4.1 Molecular mechanisms that may contribute to novel phenotypic variation. Four distinct
molecular mechanisms have been suggested to contribute to changes in gene expression and
function that may result in novel phenotypic variation in neoallopolyploids compared to their
progenitors—adapted from (Osborn 2003 #1324). The figure diagrams each mechanism by
showing the two progenitor states in comparison with their resultant polyploid offspring.
a Changes in genic or allelic dosage are diagramed by the inheritance of novel chromosome sets.
b Genomic alterations are shown by the inheritance of recombined chromosomes. c Novel gene
regulatory interactions are diagrammed by unique protein–protein interactions. d Epigenomic
reorganization is shown as the inheritance of large-scale changes in chromatin compaction. The
progenitors and their polyploid offspring are diagrammed by ellipses, with the progenitors
represented by the ellipses furthest from the central ‘‘novel phenotypic variation’’ ellipse. The
polyploids are represented by the ellipses closest to the central ellipse.
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be a consequence of increasing the genome size, leading to the increase in cell size
and reduction of cell divisions that are commonly associated with neopolyploids
compared to their progenitors (Noggle 1946 #1321; Stebbins 1971 #1355).
Additionally, some evidence suggests that neopolyploids have a protracted
reproductive growth phase, with a delay in the onset of flowering and longer
reproductive growth (Stebbins 1971 #1355). While these changes in morphology
and growth may be the result of simply increasing the amount of DNA in neopolyploids, they may also stem from increasing the relative level of gene and
protein expression.
Gene and protein expression may be sensitive to gene dosage effects, whereby
increases in ploidy result in changes in expression that are not proportional to the
increase in gene dosage. Gene and protein expression studies in Zea, Solanum,
Helianthus, and Arabidopsis autoploid series have suggested that the vast majority
of changes in expression are proportional to gene dosage (Yu 2010 #1376; Pignatta
2010 #1327; Wang 2006a, b #1366; Stupar 2007 #1357; Guo 1996 #1283; Yao
2011 #1375; Riddle 2010 #1337; Church 2009 #1266). Pignatta et al. (2010)
created independent neo-autotetraploid lines of Arabidopsis thaliana (Columbia)
from the same homogeneous diploid progenitor. The researchers performed genome-wide gene expression studies to identify reproducible changes in gene
expression in the autotetraploids compared to the diploid progenitor. Only a few
genes, out of 26,107, were found that potentially displayed changes in gene
expression that were not proportional to the ploidal levels. Of note, resolution or
sampling limitations may have missed some dosage-sensitive genes (Pignatta 2010
#1327). A recent study with Columbia and Landsberg accessions of Arabidopsis
ploidal series found similar results to those above, with slightly higher numbers of
genes whose expression was disproportionately affected by changes in ploidy (Yu
2010 #1376). A greater role for gene dosage was identified in potato and maize
studies, as increasing the ploidal levels resulted in roughly 10 % of genes with
disproportionate effects on expression (Stupar 2007 #1357; Guo 1996 #1283; Yao
2011 #1375; Riddle 2010 #1337; Church 2009 #1266). Taken together, these
studies suggest that most gene expression is proportional to ploidal level. None of
these studies identified dosage-sensitive genes whose increase in expression correlated with any ploidy-dependent phenotype. One unanswered question is: what
effect does increasing the overall level of gene expression, without changing the
relative abundance of gene products, have on biological pathways?
In theory, increased gene dosage and mixtures in polyploids could result in
unique allelic combinations that would expand phenotypic diversity (Bingham
1979 #1256; Grant 1981 #1282; Stebbins 1971 #1355). Instead of possessing only
two alleles for a given gene as in a diploid, a tetraploid could possess four alleles
that may allow additional variability for a phenotypic trait. This mechanism of
allelic interaction is a natural extension of phenotypic variation in diploids
attributed to combinations of different alleles that affect quantitative traits (Guo
1994 #1377; Birchler 2001 #1257; Osborn 2003 #1324). However, it remains
unclear what allelic combinations contribute to novel variation in polyploids
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Hybrid gene interactions may contribute to phenotypic variation in allopolyploids
(Osborn 2003 #1324; Ramsey 2002 #1334). Similar to interspecies hybrids, allopolyploids have divergent genetic contributions from their progenitors. This
heterogeneity could result in perturbations in regulatory networks that could dramatically alter their outcomes (Ramsey 2002 #1334; Stebbins 1947 #1353;
Stebbins 1950 #1354; Osborn 2003 #1324). In synthetic Arabidopsis allopolyploids, altered regulatory interactions between the two progenitor-derived chromosome sets are thought to be responsible for delaying flowering time (Wang
2006a, b #1365). The A. arenosa FRIGIDA (FRI) gene is a transcription factor that
trans-activates the A. thaliana (Landsberg) Flowering Locus C (FLC) gene (Wang
2006a, b #1365). FLC is a major negative regulator of the reproductive transition in
Arabidopsis species, and its over-expression is predicted to delay flowering in the
Arabidopsis neoallopolyploids (Shindo 2005 #1340; Simpson 2002 #1342). While
FRI and FLC genes are present in both the progenitors, A. thaliana (Landsberg) FRI
and A. arenosa FLC alleles have reduced expression or function (Wang 2006a, b
#1365). Therefore, it is possible that the flowering delay might be explained by the
novel genic interaction between A. arenosa FRI and A. thaliana FLC in the neoallopolyploids (Wang 2006a, b #1365).
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4.2.2 Hybrid Regulatory Interactions Can Cause Novel
Phenotypic Variation
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(Osborn 2003 #1324), and indeed allelic variation in neopolyploids may be limited
due to genetic ‘‘bottlenecking’’ in their formation.
4.2.3 Chromosome Missegregation Can Lead to Novel Variation
in Neopolyploids
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Perturbations in chromosome segregation have been proposed to be a major source
of novel phenotypic variation in polyploids (Stebbins 1971 #1355; Müntzing 1937
#1317; Ramsey 2002 #1334; Song 1993 #1352, 1995 #1351; Soltis and Soltis 1999
#1348). During meiotic prophase, the increased chromosome sets in polyploids
will often lead to multivalent chromosome pairing that can result in slower cell
division, unequal chromosome partitioning to daughter cells, and even homeologous recombination (Storchova 2006 #1356; Stebbins 1971 #1355; Ramsey 2002
#1334). For example, autotetraploids have four homologous chromosomes instead
of two, like their diploid counterparts. These four homologous chromosomes may
form univalents, trivalents, or quadrivalents during prophase that will cause lagging or unequal chromosome partitioning during anaphase and result in aneuploid
gametes (Ramsey 2002 #1334). The prevalence of multivalent formation in
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polyploids is widespread; a survey of the available literature by Ramsey et al.
(2002) found that the mean multivalent frequency for auto- and allopolyploids was
estimated to be 28.8 and 8.0 %, respectively. The lower percentage of allopolyploids with multivalent pairing versus autopolyploids, is most likely the result of
sequence divergence between homeologous chromosomes in allopolyploids that
favors disomic pairing.
Multivalent pairing is expected to result in a much higher degree of aneuploidy
than disomic pairing and ultimately reduce the fertility of polyploids (Ramsey
2002 #1334; Stebbins 1971 #1355). Ramsey et al. (2002) compared the occurrence
of aneuploidy in gametic and sporophytic cells in different Polyploid species. They
found that the mean frequency of aneuploid pollen was approximately 40 %, while
the mean frequency of aneuploid progeny was an estimated 29 %. Importantly,
there were no studies on the frequency of aneuploid ovules, but if one were to
assume a similar ratio of aneuploidy as found in pollen, it would imply that 64 %
of all zygotes would be aneuploids. These differences in the expected and observed
percentages of aneuploid progeny suggest that some aneuploid gametes or progeny
do not survive and that chromosome segregation defects correlate with the reduced
fertility of polyploid. In contrast to the very different multivalent frequencies
observed at meiosis, the frequency of aneuploid gametes and progeny was similar
in auto- and allopolyploids. If aneuploidy requires multivalent pairing, it is unclear
how allopolyploids could have a similar level of aneuploidy as autopolyploids.
Either there is a much higher level of multivalent pairing in allopolyploids than
reported, or other factors contribute to the unexpected level of aneuploidy in
allopolyploids (Ramsey 2002 #1334; Chester 2012 #1265).
In addition to aneuploidy, multivalent pairing of homeologous chromosomes
can also result in genetic recombination that can severely disrupt genome organization and contribute to phenotypic variation (Stebbins 1971 #1355; Ramsey
2002 #1334; Song 1993 #1352, 1995 #1351). In allopolyploids, unequal recombination between chromosomes can result from synteny between homeologous
chromosomes. Changes in gene dosage, gene expression, the epigenetic landscape,
and even gene conversion are all possible outcomes of homeologous recombination in allopolyploids. Homeologous recombination has been observed in many
different resynthesized allopolyploid species (Wendel 2000 #1368; Osborn 2003
#1324; Doyle 2008 #1271; Hegarty 2008 #1287; Leitch 2008 #1299; Soltis and
Soltis 2009 #1346; Stebbins 1971 #1355; Grant 1981 #1282; Ramsey 2002 #1334;
Chester 2012 #1265; Pires 2004 #1328; Buggs 2009 #1259), suggesting it may be
a major mechanism of genome alteration and evolution. In synthetic Arabidopsis
allopolyploids derived from A. thaliana and A. arenosa progenitors, the nucleolus
organizer region (NOR) and 5S rDNA genetic regions have been shown to be lost
or recombined between the chromosomes derived from A. thaliana and A. arenosa
by the F3 generation (Pontes 2004 #1329). Rearrangements of the NOR and 5S
rDNA regions were also demonstrated in the naturally occurring Arabidopsis
allopolyploid, A. suecica. Genetic rearrangements in resynthesized Brassica napus
allopolyploids have been linked to phenotypic variation (Gaeta 2007 #1280; Pires
2004 #1328; Xiong 2011 #1372; Song 1995 #1351). In one striking example,
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4.2.4 Epigenetic Remodeling Can Result in Novel Variation
in Neopolyploids
Epigenetic remodeling of polyploid genomes has also been suggested to play a role
in the origin of novel phenotypes (Osborn 2003 #1324). Epigenetic changes
involving DNA methylation, histone post-translational modifications (PTMs),
histone replacement, and sRNA-mediated silencing have all been demonstrated to
affect gene expression levels in diploids (Wolffe 1999 #1371; Calarco 2011
#1378). Moreover, the dynamic nature of these epigenetic modifications in combination with the instability of neopolyploid genomes would suggest that there is
the potential for rampant epigenetic remodeling in polyploids that could affect
gene expression (Osborn 2003 #1324; Wolffe 1999 #1371; Ramsey 2002 #1334;
Matzke 1999 #1310).
Concurrently, there have been many studies documenting novel changes in
DNA methylation (Li 2010 #1303; Chen 2008 #1264; Lukens 2006 #1305; Xu
2009 #1373; Wang 2009 #1364, 2004 #1367; Liu 2003 #1304; Madlung 2002
#1307; Kenan-Eichler 2011 #1294; Parisod 2009 #1325; Yaakov 2011 #1374) as
well as changes in sRNA profiles (Kenan-Eichler 2011 #1294; Ha 2009 #1284;
Preuss 2008 #1331) within allopolyploid genomes compared to their progenitors,
but there has been limited data demonstrating links of these epigenetic changes to
phenotypic consequences. One classic example of epigenetic reprogramming in
allopolyploids is the occurrence of nucleolar dominance among interspecies
hybrids, including allopolyploids (McStay 2006 #1311; Preuss 2007 #1330).
Nucleolar dominance occurs when rRNA genes from one parent, or progenitor
species, are preferentially silenced in a hybrid; this silencing is neither the result of
random inactivation, nor correlated with imprinting and sexual parent of origin
(McStay 2006 #1311; Preuss 2007 #1330). In Arabidopsis allopolyploids, small
interfering RNAs (sRNA), DNA methylation, and histone deacetylation result in
the silencing of A. thaliana-derived rRNA genes in A. suecica (Preuss 2007 #1330,
2008 #1331; Earley 2006 #1274). While no clear phenotype is associated with
nucleolar dominance, it is believed to be important for genome stability and
prevention of premature aging, which has been linked to perturbations in rDNA
genic regions (Finigan 2008 #1277).
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variation for flowering time between two different B. napus allopolyploid lines was
found to correlate with a genomic rearrangement disrupting the expression of FLC
(Pires 2004 #1328). These allopolyploid lines also displayed phenotypic variation
in flowering time that exceeded the range of their progenitors (Gaeta 2007 #1280;
Pires 2004 #1328). Together, these results provide powerful evidence for the role
of genomic rearrangements in neopolyploids driving the creation of novel phenotypic variation that is not present in their diploid progenitors.
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For histone PTMs, a few studies in Arabidopsis neoallopolyploids demonstrate
changes in histone modification patterns that correlate with changes in gene
expression (Wang 2006a, b #1365; Ni et al. 2009 #1320). Interestingly, these
changes in gene expression patterns were linked to phenotypic changes in flowering time (Wang 2006a, b #1365) and chlorophyll and sugar content (Ni et al.
2009 #1320). However, these perturbations are most likely the downstream consequences of novel regulatory interactions (Ni et al. 2009 #1320; Wang 2006a, b
#1365). Additionally, a tentative link between seed death and perturbations in
histone H3 lysine 27 methylation (H3K27me) has been identified in Arabidopsis
neoallopolyploids (Josefsson 2006 #1291; Walia 2009 #1363). Josefsson et al.
(2006) reported that the expression of PHERES1, MEDEA, and other imprinted
genes were deregulated in developing Arabidopsis neoallopolyploid seeds. In
addition to the imprinted genes, Athila retrotransposons from the pollen parent
were also deregulated in Arabidopsis allotriploid seed, which are inviable. This
deregulation of imprinted genes and Athila elements was suggested to result from
a lack of silencing of their pollen parent copies by the POLYCOMB REPRESSIVE
COMPLEX (PRC), which is responsible for H3K27me in Arabidopsis plants
(Josefsson 2006 #1291). Subsequent work suggested that the PRC complex was
not functioning in these neoallopolyploids because of a 2–10-fold down-regulation
in the expression of FERTILIZATION-INDEPENDENT SEED 2 (FIS2), one of the
subunits of the PRC (Walia 2009 #1363). Why the PRC complex, and FIS2, are
misregulated in the developing neoallopolyploids is still an unanswered question,
but major disruptions of at least H3K27 methylation patterns in these neoallopolyploids would be predicted by this model (Josefsson 2006 #1291; Walia 2009
#1363). Future studies to analyze H3K27 methylation patterns in the neoallopolyploids will be important to validate this hypothesis. This misregulation of the
PRC complex is only present in the neoallopolyploids but absent in its autopolyploid progenitors and depends on both the ploidy level and genetic diversity of
the progenitors (Josefsson 2006 #1291; Walia 2009 #1363).
So far, the limited evidence for epigenetic remodeling has been primarily
identified in allo-, and not autopolyploids. While allopolyploids inherit epigenetic
and genomic divergence from their progenitors, one might still expect to see
epigenomic remodeling in autopolyploids due to their dynamic nature. One role
for epigenetic mechanisms in autopolyploids is in the origin of unreduced gametes
from which they arise. In the ovules of most sexual flowering plants, female
gametogenesis is initiated from a single surviving gametic cell, the functional
megaspore, formed after meiosis of the somatically derived megaspore mother cell
(MMC). The Arabidopsis small RNA binding protein ARGONAUTE 9 (AGO9)
controls female gamete formation by restricting the specification of gametophyte
precursors to the MMC (Olmedo-Monfil 2010 #1323; Durán-Figueroa 2010
#1273). Mutations in AGO9 lead to the differentiation of diploid (unreduced)
gametic cells from the surrounding ovule that are able to initiate gametogenesis.
Mutations in the maize AGO9 homolog also lead to unreduced gametes, through a
related but distinct mechanism (Singh 2011 #1343). AGO9 preferentially interacts
with 24-nucleotide sRNAs derived from transposable elements (TEs), and its
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4.2.5 Nonadditive Gene Expression and Novel Phenotypic
Variation
Mechanisms encompassing gene dosage/allelic combination, novel gene interactions, genomic alterations, and epigenomic reorganization must all converge on
gene expression and function to induce novel phenotypic variation. While little is
known about perturbations in gene function in neopolyploids, changes in gene
expression (Wang 2006a, b #1366, #1365; Akhunova 2010 #1253; Pumphrey 2009
#1332; Chaudhary 2009 #1263; Chagué 2010 #1261; Flagel 2008 #1278, 2010
#1279; Hegarty 2006 #1286) and protein levels (Albertin 2006 #1254; Ng 2011
#1319) have been demonstrated in allopolyploids compared to their progenitors.
Transcript profiling experiments in synthetic Arabidopsis allopolyploids were
the first demonstrations of genome-wide nonadditive gene expression in allopolyploids (Wang 2006a, b #1366). Nonadditive gene expression occurs when the
expression level of genes in hybrids differs from the average expression level of
the orthologous genes in the parental species (i.e. midparent level). In Arabidopsis
neoallopolyploids, an estimated 5–35 % of all genes were nonadditively expressed—depending on the statistical method employed (Wang 2006a, b #1366).
Subsequent research in other neoallopolyploid species has suggested that nonadditive gene expression is a general trend found associated with recent allopolyploidization; however, the amount of nonadditive gene expression in specific
allopolyploids is highly variable (Hegarty 2006 #1286; Chaudhary 2009 #1263;
Flagel 2008 #1278, 2010 #1279; Akhunova 2010 #1253; Pumphrey 2009 #1332;
Chagué 2010 #1261). For example, in bread wheat allohexaploids nonadditive
gene expression has been reported as anywhere from 7 to 40 % (Pumphrey 2009
#1332; Chagué 2010 #1261; Kashkush 2002 #1292; Akhunova 2010 #1253).
The discovery of nonadditive gene expression in neoallopolyploids implies that
the progenitor-derived chromosome sets must be interacting to result in novel gene
expression patterns. How these chromosome sets interact is just beginning to be
unraveled; but, by definition, these novel interactions must stem directly from
changes in cis- and trans-regulatory divergence between the progenitor-derived
genomes. Cis- and/or trans-regulatory differences could result in changes in the
expression of homeologous genes (homeoalleles) from each progenitor-derived
chromosome set. Cis-regulatory divergence can act directly on single genes or on
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activity is necessary to silence TEs in female gametes and their accessory cells
(Olmedo-Monfil 2010 #1323; Durán-Figueroa 2010 #1273). That AGO9-dependent sRNA silencing is also crucial to specify ploidy in the gametes indicates that
epigenetic reprogramming may link transposon silencing to germ cell fate (Slotkin
2009 #1345). This may reflect the coevolution of transposon activity and sexual
reproduction, as well as the increased tolerance of polyploids to mutations caused
by TE insertions (Martienssen 2010 #1309).
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localized chromatin domains, such as promoters or enhancers, resulting in
asymmetric accumulation of homeologous transcripts in allopolyploids. Transregulatory divergence between the parental genomes will affect homeologous
genes equally and result in equal accumulation of homeologous transcripts. Thus,
both cis- and trans-regulatory differences could result in nonadditive homeologous
gene expression, but only cis-regulatory differences could lead to biased homeoallele-specific gene expression. The relative contribution of cis- and trans-regulatory divergence to nonadditive gene expression is still unclear, but some
evidence has arisen recently implicating a major role of cis-regulatory differences
for this phenomenon.
Transcriptome profiling studies in Gossypium (Flagel 2010 #1279; Doyle 2008
#1271), Arabidopsis (Wang 2006a, b #1366), and maize (Schnable 2011 #1339),
and small-scale analyses in Tragopogon (Buggs 2011 #1260; Tate 2006 #1359)
polyploids have suggested that there is a common theme of ‘‘genome dominance’’
in homeologous gene expression in allopolyploids (Rapp 2009 #1336). In the
genome dominance model, one of the progenitor transcription profiles outcompetes the other to shift the overall expression in the allopolyploids toward a progenitor-specific expression profile. In Gossypium allopolyploids, five different
allopolyploids have been suggested to have a 54–60 % transcriptional bias in favor
of the D genome versus the A genome (Flagel 2010 #1279). A similar phenomenon has been reported in Arabidopsis allopolyploids, where a 55 % bias toward
A. arenosa over A. thaliana homeoallele expression has been reported for natural
allopolyploids (Chang 2010 #1262) and suggested for synthetic allopolyploids
(Wang 2006a, b #1366). In contrast, the evidence for genome dominance has been
inconclusive in Triticum (Akhunova 2010 #1253; Pumphrey 2009 #1332; Chagué
2010 #1261). However, this phenomenon is still un-resolved, because the majority
of the evidence for genome dominance are based on comparing the expression
profiles between the progenitors and their allopolyploid offspring, without distinguishing between homeoalleles in the allopolyploids (Flagel 2008 #1278, 2010
#1279; Wang 2006a, b #1366). Further, in cases where genome-wide profiles of
homeologous gene expression have suggested biased expression of one genome
over the other (Akhunova 2010 #1253; Chang 2010 #1262; Schnable 2011 #1339),
the exact progenitors of these established allopolyploids were extinct and true
comparisons could not be made. Future studies should address these discrepancies
and help to unravel the phenotypic consequences of nonadditive gene expression.
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4.3 Technical Considerations When Comparing Gene
Expression Studies in Polyploids
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Many of the controversies surrounding genome dominance and the variability of
nonadditive gene expression in neoallopolyploids may stem from the differences in
the methodologies employed and the organisms studied. One important distinction
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(a)
Species α‘ (4x = 2n)
Polyploidization
Species α‘ β‘ (4x = 2n)
Hybridization
Polyploidization
(b)
Species α (2x = 2n)
‘
Species α‘ β (2x = 1n)
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is whether polyploidization preceded hybridization or vice versa (Hegarty 2008
#1287). If polyploidization precedes hybridization then the resulting neoallopolyploid will have duplicate chromosomes that can accurately pair at meiosis
(Fig. 4.2). Synthetic Arabidopsis allopolyploids are an example, and natural
Arabidopsis allopolyploids are believed to have arisen this way (Hegarty 2008
#1287; Josefsson 2006 #1291). On the other hand, if polyploidization follows
hybridization, then the resulting hybrid will first transit through an unbalanced
chromosome state, most likely severely infertile, before restoration with a polyploidization event to create a neoallopolyploid (Fig. 4.2) (Hegarty 2006 #1286,
2008 #1287). Gossypium, Triticum, Tragopogon, Spartina, and Senecio allopolyploids are examples of the latter type (Hegarty 2006 #1286). Transcriptome
profiling studies in Senecio (Hegarty 2006 #1286) and Triticum (Feldman 2005
#1276) hybrids before and after polyploidization suggested that the homologous
gene expression levels were more perturbed (vs. the midparent) in the sterile
hybrids before chromosome doubling. These results may suggest that gene
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Fig. 4.2 The origin of allopolyploids. Two types of allopolyploids are distinguished by the
relative order of polyploidization and hybridization. a Type I allopolyploids arise when
polyploidization precedes hybridization, typically via unreduced gametes. Type I neoallopolyploids will have duplicate chromosomes that properly pair in meiosis. b Type II allopolyploids are
created when hybridization precedes polyploidization. Type II allopolyploids transit through a
hybrid state, which is usually sterile, as chromosomes do not pair during meiosis. A subsequent
polyploidization event restores 2n chromosome number in allopolyploids
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4.4 Conclusions
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expression profiles in the resulting allopolyploid may be influenced by the creation
of a hybrid intermediate.
Another important distinction is the nature of the progenitor species used to
compare with allopolyploid expression profiles. Many allopolyploids do not have
extant progenitors, and relatives must be used instead (Chang 2010 #1262; Rapp
2009 #1336; Flagel 2010 #1279). These relatives may not be exact genetic matches (Chang 2010 #1262; Rapp 2009 #1336; Flagel 2010 #1279) and may contain
epigenetic differences as well, as is the case for different accessions of A. thaliana
(Vaughn 2007 #1362; Kliebenstein 2006 #1297). A recent study illustrates this
point. An estimated 6,790 homologous genes were differentially expressed
between A. suecica and synthetic Arabidopsis allopolyploids (Chang 2010 #1262),
and comparative genome sequencing revealed that 938 homeologous genes were
missing from A. suecica compared to the synthetic allopolyploid (Chang 2010
#1262). These genomic differences are comparable to those between different
Arabidopsis thaliana accessions (Vaughn 2007 #1362; Kliebenstein 2006 #1297)
and may represent differences between the A. suecica progenitors and their extant
relatives.
Additionally, some studies employ progenitor species that have been severely
genetically manipulated (Akhunova 2010 #1253; Chang 2010 #1262; Kerber 1964
#1295; Wang 2006a, b #1366). Previous analysis of fifth-generation Arabidopsis
allopolyploids have relied on a tetraploidized A. thaliana (Landsberg accession) line
produced spontaneously from root explant regenerants that underwent a callus phase
and cell culture treatment (Chang 2010 #1262; Valvekens 1988 #1360; Wang 2006a,
b #1366; Comai 2000 #1268). Plant lines regenerated from callus/cell cultures
typically display heritable changes in genetic and epigenetic regulation—namely,
somaclonal variation (Phillips 1994 #1326; Meins 2003 #1312; Mohan Jain 2001
#1315; Tanurdzic 2008 #1379). These changes could have a profound effect on gene
expression and epigenetic regulation in neoallopolyploids derived from such progenitors. Classical approaches based on colchicine treatment have also been applied
to the generation of tetraploid A. thaliana lines (Henry 2005 #1288; Santos 2003
#1338). Studies in Gossypium (Rapp 2009 #1336; Chaudhary 2009 #1263), Triticum
(Akhunova 2010 #1253; Pumphrey 2009 #1332; Chagué 2010 #1261), and Tragopogon (Tate et al. 2009) have also relied on colchicine treatments to polyploidize
sterile triploid hybrids after fertilization. Importantly, there was no difference in
genetic or DNA methylation changes between spontaneous or colchicine produced
Brassica polyploids (Gaeta 2007 #1280).
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The prevalence of polyploidy in nature suggests it has been positively selected for
during evolution. Indeed, the higher yields of polyploids among agricultural crops
and their usefulness to bridge species barriers have led to their widespread use and
creation for the agricultural industry (Eigsti 1957 #1275). Understanding the
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Ahloowalia B, Garber F (1961) The genus Collinsia. XIII. Cytogenetic studies of interspecific
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Akhunova A, Matniyazov R, Liang H, Akhunov E (2010) Homoeolog-specific transcriptional
bias in allopolyploid wheat. BMC Genomics 11(1):505
Albertin W, Balliau T, Brabant P, Chevre A-M, Eber F, Malosse C, Thiellement H (2006)
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Bingham E (1979) Maximizing heterozygosity in autopolyploids. Basic Life Sci 13:471–489
Birchler JA, Bhadra U, Bhadra MP, Auger DL (2001) Dosage-dependent Gene regulation in
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Bretagnolle F, Thompson J (1995) Tansley review no. 78. Gametes with the somatic chromosome
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Buggs RJA, Doust AN, Tate JA, Koh J, Soltis K, Feltus FA, Paterson AH, Soltis PS, Soltis DE
(2009) Gene loss and silencing in Tragopogon miscellus (Asteraceae): comparison of natural
and synthetic Allotetraploids. Heredity 103 (1):73–81. doi: http://www.nature.com/hdy/
journal/v103/n1/suppinfo/hdy200924s1.html
Buggs Richard JA, Zhang L, Miles N, Tate Jennifer A, Gao L, Wei W, Schnable Patrick S,
Barbazuk WB, Soltis Pamela S, Soltis Douglas E (2011) Transcriptomic shock generates
evolutionary novelty in a newly formed. Natural allopolyploid plant. Curr Biol
21(7):551–556. doi: 10.1016/j.cub.2011.02.016
Calarco JP, Martienssen RA (2011) Genome reprogramming and small interfering RNA in the
Arabidopsis germline. Curr Opin Genet Dev 21(2):134–139. doi: 10.1016/j.gde.2011.01.014
Chagué V, Just J, Mestiri I, Balzergue S, Tanguy A-M, Huneau C, Huteau V, Belcram H, Coriton
O, Jahier J, Chalhoub B (2010) Genome-wide gene expression changes in genetically stable
synthetic and natural wheat allohexaploids. New Phytol 187(4):1181–1194. doi: 10.1111/
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emergent properties of neopolyploids, such as novel phenotypic variation and
reproductive barriers, is vitally important to further advance the usefulness of
polyploids for crop breeding. While we are beginning to understand the molecular
mechanisms that contribute to novel variation in polyploids, there is still a lack of
specific links between genes, proteins, and phenotypes. We also do not understand
the interplay between these different mechanisms and the important drivers of
phenotypic diversity. What role do genomic alterations or allelic diversity play in
the origin of this novel phenotypic variation? Many homeologous genes are
nonadditively expressed in neoallopolyploids, but what is the source of this differential expression? If nonadditive gene expression is a result of genome dominance, than how is such a mechanism established? The answers to these questions
are likely to emerge in the coming years.
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Chang P, Dilkes B, McMahon M, Comai L, Nuzhdin S (2010) Homoeolog-specific retention and
use in allotetraploid Arabidopsis suecica depends on parent of origin and network partners.
Genome Biol 11(12):R125
Chaudhary B, Flagel L, Stupar R, Udall J, Verma N, Springer N, Wendel J (2009) Reciprocal
silencing, transcriptional bias and functional divergence of homeologs in polyploid cotton
(Gossypium). Genetics 182:503–517
Chen M, Ha M, Lackey E, Wang J, Chen Z (2008) RNAi of met1 reduces DNA methylation and
induces genome-specific changes in gene expression and centromeric small RNA accumulation in Arabidopsis Allopolyploids. Genetics 178:1845–1858
Chester M, Gallagher JP, Symonds VV, Cruz da Silva AV, Mavrodiev EV, Leitch AR, Soltis PS,
Soltis DE (2012) Extensive chromosomal variation in a recently formed natural allopolyploid
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SpringerLink
Book Title
Polyploidy and Genome Evolution
Series Title
Chapter Title
Identifying the Phylogenetic Context of Whole-Genome Duplications in Plants
Copyright Year
2012
Copyright HolderName
Springer-Verlag Berlin Heidelberg
Corresponding Author
Family Name
Burleigh
Particle
Given Name
J. Gordon
Suffix
Abstract
Division
Department of Biology
Organization
University of Florida
Address
32611, Gainesville, FL, USA
Email
gburleigh@ufl.edu
Although evolutionary biologists have long recognized the transformative evolutionary potential of wholegenome duplications (WGDs) in plants, identifying the precise phylogenetic location of WGDs presents many
challenges. This chapter reviews some new approaches to map WGDs on a phylogeny, the first step for
understanding the large-scale evolutionary and ecological consequences of WGDs in plants. Specifically, it
examines approaches for using chromosome and gene copy number data, gene trees, and other genomic
insights to identify the evolutionary location of WGDs. The abundance of genomic sequence data and
advances in phylogenetic methods present unprecedented opportunities to place WGDs within the plant tree
of life. Still, there exist few direct tests to identify and place WGDs, and analyses of complex data are often
susceptible to error.
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A central challenge in evolutionary biology is to determine the genetic mechanisms
that generate species diversity as well as new traits, functions, and adaptations. Plant
evolutionary biologists have long recognized the transformative evolutionary potential of polyploidy or whole-genome duplication (WGD) (e.g., Stebbins 1950; Grant
1963; Levin 2002; Soltis and Soltis 2000; Soltis et al. 2009). However, to study the
evolutionary consequences of whole-genome duplications (WGDs) and link WGDs to
phenotypic changes or diversification, the WGDs must first be placed in a phylogenetic context. Unfortunately, the rapid gene loss and genome rearrangements that
frequently follow WGDs erase evidence of WGD (e.g., Wolfe 2001; Doyle et al.
2008). This process of diploidization brings about a paradox of the study of polyploidy; despite its apparent pervasiveness throughout the evolutionary history of land
plants and its evolutionary importance, clear, unambiguous evidence of ancient
WGDs can be remarkably difficult to locate within a phylogeny.
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Abstract Although evolutionary biologists have long recognized the transformative evolutionary potential of whole-genome duplications (WGDs) in plants,
identifying the precise phylogenetic location of WGDs presents many challenges.
This chapter reviews some new approaches to map WGDs on a phylogeny, the first
step for understanding the large-scale evolutionary and ecological consequences of
WGDs in plants. Specifically, it examines approaches for using chromosome and
gene copy number data, gene trees, and other genomic insights to identify the
evolutionary location of WGDs. The abundance of genomic sequence data and
advances in phylogenetic methods present unprecedented opportunities to place
WGDs within the plant tree of life. Still, there exist few direct tests to identify and
place WGDs, and analyses of complex data are often susceptible to error.
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Identifying the Phylogenetic Context
of Whole-Genome Duplications in Plants
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e-mail: gburleigh@ufl.edu
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DOI: 10.1007/978-3-642-31442-1_5, Springer-Verlag Berlin Heidelberg 2012
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5.1 Chromosome Evolution
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Many early surveys of polyploidy in plants used chromosome counts to estimate
the percentage of polyploid species (e.g., Grant 1963, 1982; Stebbins 1938, 1950,
1971; Goldblatt 1980). This work laid the foundation for understanding the role of
WGDs in plant evolution. However, lacking a phylogenetic context, it is difficult
to estimate the frequency of WGD events, let alone the evolutionary placement of
WGDs simply from the percentage of polyploids (although see Levin and Wilson
1976; Otto and Whitton 2002). For example, if a plant family has 100 species, 50
of which are recent polyploids, this could be the result of as few as one WGD or as
many as 50 WGDs (assuming at most one WGD per lineage). Understanding the
relationships among species is necessary to infer the history of WGD. Furthermore, chromosome number alone is not necessarily indicative of polyploidy. Zea
mays, with a haploid chromosome number of 10, has had multiple WGDs in the
last *20 million years (Gaut and Doebley 1997; Gaut 2001), and Arabidopsis
thaliana, with a haploid chromosome number of five, has experienced 3–5 WGDs
since the origin of seed plants (Vision et al. 2000; Blanc et al. 2003; Bowers et al.
2003; Jiao et al. 2011). Yet few chromosome number surveys would not have
considered either polyploid.
Mapping chromosome numbers on a phylogenetic hypothesis can help reveal
the frequency and evolutionary placement of WGD events. Informal phylogenetic
observations were first used to deduce ancestral chromosome numbers. For
example, numerous studies surmised a low base chromosome number for angiosperms based on the chromosome counts of the ‘‘basal’’ angiosperm lineages (e.g.,
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WGD in plants has been and studied for more than 100 years (e.g., Digby 1912;
Winge 1917). Although sometimes cryptic, evidence of WGDs may be gleaned
from such disparate sources as chromosome counts (e.g., Stebbins 1971), guard
cell size (Masterson 1994), age distributions of gene copies (e.g., Blanc and Wolfe
2004), or large segmental duplications within genomes (e.g., Vision et al. 2000).
Still, it is difficult to infer the phylogenetic location of the WGDs with any precision from these observations alone. To place WGDs in an evolutionary context
requires a robust, and ideally well–sampled, phylogenetic hypothesis, large-scale
comparative data indicating WGDs, and models and methods to map these data
onto a phylogeny. Only recently have advances in phylogenetics, comparative
methods, and genome sequencing made this possible on a large scale. These
advances have enabled the first studies addressing some of the basic evolutionary
questions about polyploidy, such as the frequency of polyploid speciation (Wood
et al. 2009) or the effect of WGDs on diversification (Vamosi and Dickinson 2006;
Soltis et al. 2009; Mayrose et al. 2011), in a rigorous phylogenetic framework.
This chapter reviews some new approaches to map WGDs on a phylogeny, the first
step for understanding the large-scale evolutionary and ecological consequences of
WGDs in plants.
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Ehrendorfer et al. 1968; Stebbins 1971; Walker 1972; Raven 1975). These
observations implied a history of WGD near the root of angiosperms, although
they could not map this precisely; indeed, the relationships among these lineages
were unknown. With the growth of phylogenetic methods and data, more formal
maximum parsimony approaches were used to reconstruct ancestral chromosome
number on the inner nodes of phylogenetic trees (e.g., Stace et al. 1997; Schultheis
2001; Mishima et al. 2002; Guggisberg et al. 2006; Hipp et al. 2007). To do this,
chromosome number can be treated as a discrete variable, and the ancestral states
can be reconstructed using linear or squared change parsimony. If the ancestral
states are far higher than the base chromosome number, then that ancestral node
may represent a polyploid. It is possible to construct elaborate chromosome
number transition matrices for parsimony analyses, for example, allowing chromosome doubling as well as single chromosome changes, or to weight different
changes, like down-weighting chromosome losses, but these analyses are rarely
performed. In any case, parsimony reconstructions often have difficulty accounting
for multiple transitions on a single branch or quantifying uncertainty in ancestral
state reconstructions.
More recently, probabilistic models of chromosome number evolution have
been developed (Meyers and Levin 2006; Mayrose et al. 2010). In a simple formulation, the chromosome models allow transitions that add a chromosome,
remove a chromosome, or double the number of chromosomes (Mayrose et al.
2010). Thus, although the transition matrix among chromosome states (chromosome numbers) may be extremely large, the evolutionary process can be modeled
with only a few parameters. The performance of these models has not been
characterized in detail; however, they appear to infer more ancient WGDs than
parsimony methods (Wood et al. 2009) and may also provide quite different
ancestral state reconstructions of chromosome number (Cusimano et al. 2012). In
the future, these models may link chromosome evolution to diversification rates or
phenotypes related to the frequency of WGDs to obtain even more accurate
estimates of WGDs in a phylogeny.
Studies of plant chromosome numbers have provided a wealth of insight into
polyploidy in plants and have contributed substantially to canonical views of plant
speciation and evolution. Yet chromosome number is a sort of summary statistic
for WGD, a simple observation that is meant to represent a complex, large-scale
genomic change, and chromosome number alone may not be sufficient to detect
WGDs. A small chromosome number, as in Arabidopsis thaliana, does not necessarily imply the absence of historical WGDs, and high chromosome numbers are
not necessarily evidence of WGDs. Without additional cytological or genetic data,
it is impossible to distinguish between a WGD and increasing dysploidy, a change
in the chromosome number that is not associated with a change in the amount of
genetic material, based solely on chromosome counts.
Also, as with any phenotype, there are limitations and biases associated with
ancestral state reconstruction (e.g., Schluter et al. 1997; Ané 2008). Often
reconstruction is most difficult for characters with high rates of evolution or high
degrees of homoplasy. Chromosome numbers may be unstable following a WGD
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5.2 Gene Copy Models
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Form availability of large-scale genomic data from an increasing number of plant
species, estimates of copy numbers for gene families are increasingly available for
many plant species. Since WGD should change not only the chromosome numbers
but also the copy numbers for all gene families, gene family copy number should
provide more data to estimate a WGD than simply a single chromosome number.
Like chromosome number evolution, ancestral gene copy numbers can be reconstructed using parsimony methods (Snel et al. 2002; Kunin and Ouzounis 2003;
Mirkin et al. 2003; Csürös 2010; Ames et al. 2012; Librado et al. 2012). The
parsimony models can be implemented in numerous ways, including weighting
gains and losses differently. It is not necessarily easy to find evidence of WGDs
based on the number of gene gains or losses on a branch in the species tree, but we
might expect WGDs will result in far more gains, and subsequently losses, per unit
time than are found on other branches. Hahn et al. (2005) developed a stochastic
birth and death model that assumes a homogeneous process of duplication and loss
throughout the species tree. The ML implementation of this model in CAFÉ
(De Bie et al. 2006), as well as a similar Bayesian approach (Liu et al. 2011),
estimates gene family gain and loss rates across the tree and can identify
anomalous gene families and branches on the tree. These branches may reflect the
effects of WGDs. More complex models that account for heterogeneity in the rates
of duplications and losses across lineages, and in some cases also allowing gains of
genes or gene families by lateral transfer, also have been proposed (Iwasaki and
Takagi 2007; Csurös 2010; Ames et al. 2012; Librado et al. 2012).
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(see Lim et al. 2008; Chester et al. 2012) and can decrease quickly following a
WGD. Thus, modeling approaches likely will have difficulty for identifying
ancient WGDs. In fact, it appears that the frequency of chromosome loss and
diploidization was not always appreciated in studies that only examined chromosome numbers, and even with large-scale genomic data, the mechanisms for
rapid chromosome loss are not clear (Doyle et al. 2008). This lack of appreciation
for the lability of chromosome numbers may have contributed to the failure to
detect, or even surmise, the extent of ancient WGDs, and also may have
encouraged the idea that WGDs were evolutionary dead ends (e.g., Stebbins 1950).
Despite the limitations of chromosome numbers alone, data are available for
many thousands of plant species, for example on the online Index to Plant
Chromosome Numbers (IPCN) database (http://www.tropicos.org/Project/IPCN).
Thus, until large-scale genomic sequence data sets become available for thousands
of phylogenetically diverse taxa, chromosome number may provide the best
opportunity to identify putative WGD events, especially recent events, with
phylogenetic precision and to examine the macroevolutionary consequences of
WGD throughout the history of all plants.
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In spite of much recent work on developing models of gene family copy number,
all of the gene copy models assume that gene duplications or gene gains are
independent. Thus, a WGD in a plant might be viewed as 20,000 gene duplications
rather than a single duplication event. Consequently, while an increased duplication
rate on a branch or increased loss rates on subsequent branches may suggest a
WGD, there is no definitive test of WGD, and it may be difficult to distinguish
WGD from simply an elevated duplication rate or a large-scale duplication. One
approach may be to create a model that could estimate a rate of doubling for all gene
family numbers. This transition matrix could be applied to different branches to test
for either a rate of WGD [0 or different rates between clades or branches.
Although gene copy number provides more detailed assessment of genomic
content than chromosome numbers, inferring the histories of gene copy number
and chromosome number have similar limitations. For example, gene copies
appear to be rapidly silenced and lost immediately following a WGD (e.g., Tate
et al. 2006; Buggs et al. 2009, 2012; see Chap. 14, this volume), which may
quickly obscure the evidence for WGDs. However, simply obtaining accurate
estimates of gene copy number for extant taxa may be a challenge. Without
complete genome sequencing, it can be difficult to distinguish a gene loss from
simply a failure to sample a gene. In fact, the lack of complete sequencing across a
broad range of plant species may explain the lack of studies of gene copy number
evolution in plants. Even with complete genome sequences, estimates of gene copy
number depend on the vagaries of the extremely complex genome annotation and
gene family circumscription problems. Furthermore, there are high levels of
intraspecific variation in gene copy number in some plants (e.g., Springer et al.
2009; Debolt 2010; Zheng et al. 2011). It is possible to account for uncertainty in
the gene copy numbers or incomplete sampling in a likelihood model, although
such approaches have not been implemented.
Still, gene copy number does not always provide direct evidence for the location
of gene or WGDs. For example, take the case in Fig. 5.1, in which an outgroup has a
single gene copy, and two sister taxa each have two gene copies. The parsimonious
explanation for these data would be that a gene duplication preceded the most
recent common ancestor of the sister taxa, although it is possible that there were
independent duplications in each sister lineage. In this case, the gene topologies can
provide much more insight into the history of duplication than simply looking at
copy number and can easily distinguish between the two duplication scenarios in
Fig. 5.1. The additional information from evolutionary history of the genes can
further help identify the placement of historical duplications and WGDs.
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5.3 Gene Tree Reconciliation
The general problem of gene tree reconciliation is based on the observation that
population-level processes, such as coalescence (lineage sorting), as well as
evolutionary events, such as gene duplications and loss, recombination,
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Fig. 5.1 Gene copy number data and corresponding gene trees. The gene copy number data
mapped on a species tree implies at least one gene duplication, but do not specify the location of the
duplication(s). Gene tree 1 implies a single duplication preceding the most recent common ancestor
of A and B. Gene tree 2 implies independent duplications in the lineages leading to A and B
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5.3.1 Parsimony Approaches
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hybridization, or lateral gene transfer can result in gene topologies that differ from
the phylogeny of the species in which the genes evolve (e.g., Maddison 1997). The
general challenge of gene tree reconciliation is to find the evolutionary scenario
that best explains the gene tree topologies. For the case of WGD, we might ask if a
collection of gene trees is consistent with a WGD event, or at least a large number
of duplications, at a particular point in the species phylogeny. While this approach
seeks to directly map the location of gene duplications, and thus provide more
direct evidence of WGD than simply examining changes in chromosome or gene
copy numbers, in practice it often is complicated by the number of different
scenarios that can cause gene tree incongruence and the inherent difficulty of
accurately inferring a gene tree from only a gene sequence alignment (for more
detailed, general reviews see Eulenstein et al. 2010; Doyon et al. 2011b).
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Much of the initial work in gene tree reconciliation was based on optimizing a parsimony criterion; that is, finding a mapping of a gene tree topology onto the species tree
that implies the fewest evolutionary events. The gene duplication model was first
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introduced by Goodman et al. (1979; also see Page 1994 and Guigó et al. 1996) to find
the minimum number of gene duplications needed to explain the incongruence
between a gene tree and species tree. To do this, a gene tree can be embedded into a
species tree through least common ancestor mapping (lca-mapping), which maps
every node in the gene tree (tips and internal nodes) to the most recent node in the
species tree that could have contained the gene node (Fig. 5.2). A duplication occurred
if a parent and child node in the gene tree share the same lca-mapping in the species
tree. The lca-mapping identifies the most recent possible location of the gene duplication in the species tree, but this often is not the only possible location of the gene
duplication. In many cases, the duplication could have preceded the lca-mapping,
although the earlier placement of the duplication may imply additional gene losses.
However, as in the gene copy number analyses, gene losses are often difficult to
distinguish from incomplete sampling.
This simple gene reconciliation can provide a direct estimate of the phylogenetic location of gene duplication events with a precision that is impossible with
chromosome or gene copy number data. Also, it does not even require the presence
of duplicated, paralogs genes; incongruence between a single-copy gene tree
topology and the species phylogeny may be evidence of a hidden history of gene
duplication and loss. On the other hand, gene duplications and losses are not the
only explanation of gene tree incongruence. For example, incomplete lineage
sorting or reticulation also can confound gene tree topologies. In this case, the
gene duplication model may mistakenly imply a large number of duplications
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Fig. 5.2 An example of
lca-mapping of gene
duplications. The parent and
child nodes in the gene tree
(h and t) map to the same
node (X) in the species tree.
This implies a single
duplication occurring prior to
or at node X in the species
tree
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preceding rapid cladogenesis in the species tree, where we might expect high
levels of incomplete lineage sorting.
Perhaps the most difficult problem underlying the gene reconciliation approaches
is that, in many cases, the incongruence between a gene tree and the species phylogeny may simply be due to error. The gene tree model will interpret any topological
error as evidence of duplications. Consequently, this approach often implies far more
duplications rather than biologically plausible (e.g., Rasmussen and Kellis 2011).
The errors in the gene tree tend to place erroneously large numbers of duplications
near the root of the species tree, which may falsely suggest large-scale duplication
events at the origins of major clades (Hahn 2007; Burleigh et al. 2010). In the
parsimony context, several strategies have been proposed to ameliorate the problems
of gene tree error. First, poorly supported clades in the gene tree may be collapsed.
Several algorithmic approaches have extended the gene duplication model to deal
with reconciling nonbinary trees (Berglund-Sonnhammer et al. 2006; Chang and
Eulenstein 2006; Durand et al. 2006). Also, several approaches have been developed
to allow minor modifications of the gene tree topology if they reduce the number of
implied duplications (e.g., Chen et al. 2000; Chaudhury et al. 2011, 2012; Gorecki
and Eulenstein 2012). For example, Chaudhury et al. (2012) introduced an algorithm
that, given a gene tree and a species tree, finds a gene topology in a subtree pruning
and regrafting (SPR) neighborhood of the original gene tree that minimizes the
number of implied duplications. These local rearrangements can massively reduce
the number of estimated gene duplication events.
In spite of the many issues related to gene tree reconciliation, simple and
informal gene tree reconciliations have been effective at helping to identify the
phylogenetic location of WGDs in plants. These approaches are usually limited to
small gene trees with paralogs, that is, gene trees in which at least one duplication
must have occurred. In a simple three-taxon approach, a gene tree is constructed
with a pair of paralogs genes from a test taxon, and the best homologs from a sister
taxon and from an outgroup taxon (e.g., Bowers et al. 2003). If paralogs from the
test taxon form a clade, they diverged after the common ancestor with the sister
taxon; if they do not, they diverged before the most recent common ancestor. This
three-taxon phylogenetic approach provides only a limited phylogenetic context
for the duplications, but it has been used to determine the timing of WGDs in
Arabidopsis relative to its divergence from pines, rice, and other eudicots (Bowers
et al. 2003) and rice relative to its divergence from pines, Arabidopsis, and other
monocots (Vandepoele et al. 2003; Chapman et al. 2004). More recently, Jiao et al.
(2011) counted the gene trees that were consistent with different scenarios of
WGD to infer WGDs at the root of angiosperms and seed plants.
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5.3.2 Parsimony Methods to Identify WGDs
The gene duplication problem described above treats each duplication independently. Although it may identify places in the species tree with high numbers of
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gene duplications, it does not attempt to find large-scale duplication events.
Several proposed approaches attempt to identify the minimum number of gene
duplication events, where an event may include duplications of many or all genes,
rather than simply the number of duplications. One indirect approach is to examine
all the possible locations of each gene duplication and find a mapping that minimizes the number of locations (nodes) on the species tree where gene duplications
occur (Guigó et al. 1996; Page and Cotton 2002; Burleigh et al. 2009; Luo et al.
2009). This approach does not directly infer WGDs; but ideally, it can identify
places in the species tree that are possible locations of clusters of many duplications. With a limited number of gene trees, this approach appears to be effective at
identifying some locations of ancient WGDs in plants (Burleigh et al. 2009).
Unfortunately, with a large number of gene trees, it is likely that all possible
duplication mappings will require duplication events at every node in the species
tree. In this case, every possible mapping of gene duplications will be equally
optimal, and this approach will be uninformative.
Another approach seeks to find a gene duplication mapping that implies the
fewest gene duplication episodes (Guigó et al. 1996; Page and Cotton 2002;
Bansal and Eulenstein 2008; Luo et al. 2009). Given a single gene tree and species
tree, any set of gene duplications, from the same or different gene trees, that occur
on the same node in a species tree can be explained by a single gene duplication
episode (or event) as long as none of the gene duplications in the set have an
ancestor–descendant relationship with each other (Fig. 5.3). This approach appears
to help identify WGDs, which should be very large episodes, but the largest
episode is simply the largest episode found on any single gene tree (Page and
Cotton 2002; Burleigh et al. 2010). In practice, the mapping that minimizes the
number of episodes is determined by only the largest gene trees. Furthermore,
randomizing the leaf labels (taxon names) on the gene trees can result in gene tree
mappings that imply fewer episodes (Burleigh et al. 2010). Thus, although the
notion of finding gene tree mappings that are consistent with large-scale duplications is desirable, it is not clear that this problem has been properly formulated.
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If the gene trees are accurate, the parsimony criterion for mapping gene duplications appears to perform well when the rates of duplication and loss are low
(Åkerborg et al. 2009; Doyon et al. 2009). However, these approaches do not
consider branch lengths in the gene or species trees, and they have a limited ability
to allow multiple duplications and losses on a single branch. Perhaps more
important, it is difficult to incorporate the parsimony criterion into a rigorous
statistical framework to examine evolutionary hypotheses associated with gene
duplication. Numerous likelihood-based models of gene duplication and loss for
reconciling gene trees and species trees have been proposed (e.g., Arvestad et al.
2003, 2004, 2009; Åkerborg et al. 2009; Doyon et al. 2009, 2011a; Rasmussen and
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Kellis 2011; Gorecki et al. 2011). Most modeling approaches are based on using
birth–death processes to model duplications and losses of genes as they evolve
within a species tree (e.g., Arvestad et al. 2003, 2004, 2009; Åkerborg et al. 2009;
Rasmussen and Kellis 2011). Also, Doyon et al. (2009, 2011a) introduced a reconciliation algorithm that calculates the likelihood of possible reconciliations
based on a constant duplication and loss rate model, and this appears to produce
similar reconciliations as a parsimony approach. Gorecki et al. (2011) used a
Poisson model to identify the most likely distribution of reconciliations across
branches in the tree based on the species branch lengths and the gene tree
topologies.
Most of the likelihood model-based approaches require as input a species tree
with branch lengths. The method of Gorecki et al. (2011) also requires gene tree
topologies. Thus, it may be susceptible to the same problems with gene tree error
as parsimony approaches. Several approaches, however, take a gene sequence
alignment as input and use a Markov chain Monte Carlo (MCMC) approach to
simultaneously obtain the posterior distributions of gene tree topologies and gene
duplication and loss mappings (e.g., Arvestad et al. 2004, 2009; Åkerborg et al.
2009; Rasmussen and Kellis 2011). This approach provides an elegant, although
computationally difficult, way to incorporate gene tree uncertainty into the gene
tree reconciliation. Rasmussen and Kellis (2011) demonstrated that this approach
can produce more accurate gene trees and consequently greatly reduce the number
of implied duplication events compared to a parsimony approach.
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Fig. 5.3 Examples of gene duplication episodes. Duplications in the gene trees are noted with a
* followed by their location in the species tree. Both gene trees have three duplications, one at the
root node and two at species node C. In gene tree 1, the two duplications at species node C can be
explained by a single duplication episode (since there is not a parent–child relationship between
the duplication nodes). However, in gene tree 2, the two duplication episodes must have occurred
at species node C since one duplication preceded the other
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5.4 Other Genomic Data
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From availability of large-scale genomic data, evidence of cryptic ancient WGDs
often comes from either identifying large, syntenic (duplicated) blocks within a
single genome, or by looking at the age distribution of duplicated genes within a
chromosome (see Van de Peer 2004). Since these approaches use only data from
a single species and are focused more on identifying ancient WGDs than placing
the WGDs in an evolutionary context, I will not cover them in detail. The presence
of duplicated chromosomal segments may provide direct, unambiguous evidence
of WGDs that may be difficult to obtain from simply gene copy numbers or gene
trees (Vision et al. 2000). However, in practice, rapid gene losses and rearrangements after polyploidy can make it extremely difficult to detect such duplications, and different methods of detecting duplicated blocks and using different
criteria for defining a syntenic block can greatly affect interpretations of the history
of large-scale duplications (see Durand and Hoberman 2006). Although simply
examining the genome of a single species cannot reveal the phylogenetic context
of a WGD, the dates of the ancient divergences can be estimated based on the
molecular divergence of paralogs. It may be possible to map the evolution of large
duplicated segments on a tree, but in plants, this may require extending the taxonomic sampling of species with adequate genomic mapping data. Perhaps the
greater contribution of these duplicated regions is that they can define sets of
paralogs that originated from WGDs, and this information can be used to validate
mappings of duplications from gene copy number or gene reconciliation analyses.
The rapidly increasing abundance of large-scale transcriptome data sets for
plants provides an opportunity to define WGDs based on the age distribution of
duplicated genes (see Cui et al. 2006). The methods first can define pairs of most
recent gene duplicates using methods such as an all-by-all BLAST. If gene
duplication and loss occur at a constant rate, the frequency of duplicated genes in a
genome will decrease exponentially with time. In contrast, a large-scale duplication
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The computational complexity of these likelihood-based approaches raises concerns that they may have difficulty exploiting the magnitude of new genomic data.
Yet, in many ways, they still greatly simplify the complexity of genome evolution.
Rasmussen and Kellis (2012) have described the first models of both duplication and
coalescence, and other modeling approaches estimate the effects of hybridization and
coalescence, but not duplication and loss (e.g., Meng and Kubatko 2009; Gerard et al.
2011). These represent important steps in simultaneously accounting for the many
processes that affect gene tree topologies. Still, all of the modeling approaches for
duplications and losses assume that the genes and all gene duplications are independent. Thus, they may detect branches in the species tree with high rates of
duplication or loss, but they do not directly assess the likelihood of WGDs. The
development of such models that allow for simultaneous duplications across many
genes can allow for rigorous statistical tests of the placement of WGD events.
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5.5 Conclusions
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event, like a WGD, should result in an overrepresentation of duplicated gene pairs
at the time corresponding to the large-scale duplication event. In practice, in a plot
of the age distribution, usually represented by synonymous substitution distance, of
duplicated genes, peaks in the age distribution curves or evidence of multiple
distributions, may indicate WGDs. Again, it is difficult to precisely place a WGD
just from the pairwise divergence of duplicated sequences, but with data available
from many taxa, comparisons of these age plots from related species can be
informative. In some cases, analyses of the age distribution plots have failed to
detect known WGDs (e.g., Blanc and Wolfe 2004; Paterson et al. 2004). However,
unlike gene tree reconciliation methods, they will not be misled by incomplete
lineage sorting or gene tree error.
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Acknowledgments This chapter was developed and written in parts with support from the Gene
Tree Reconciliation Working Group at NIMBioS through NSF award EF-0832858, with additional support from the University of Tennessee. Discussions with members of the working group
including Cecile Ané, Oliver Eulenstein, Pawel Gorecki, and Brian O’Meara were helpful for this
manuscript.
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New genomic sequence data and advances in phylogenetic methods presents
unprecedented opportunities to place WGDs within the plant tree of life. Still, there is
much work to do. Although numerous data sources and methods may provide evidence of WGDs, there exist few statistical tests of WGD. A rigorous statistical
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Wood TE, Takebayashi N, Barker MS, Mayrose I, Greenspoon PB, Rieseberg LH (2009) The
frequency of polyploidy speciation in vascular plants. Proc Natl Acad Sci USA
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Zheng L-Y, Guo X-S, He B, Sun L-J, Peng Y, Dong S-S, Liu T-F, Jiang S, Ramachandran S, Liu C-M,
Jing H-C (2011) Genome-wide patterns of genetic variation in sweet and grain sorghum (Sorghum
bicolor). Genome Biol 12:R114
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Metadata of the chapter that will be visualized in
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Book Title
Polyploidy and Genome Evolution
Series Title
Chapter Title
Ancient and Recent Polyploidy in Monocots
Copyright Year
2012
Copyright HolderName
Springer-Verlag Berlin Heidelberg
Corresponding Author
Family Name
Paterson
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Given Name
Andrew H.
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Author
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Plant Genome Mapping Laboratory
Organization
University of Georgia
Address
Athens, GA, USA
Email
paterson@plantbio.uga.edu
Family Name
Wang
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Given Name
Xiyin
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Author
Division
Plant Genome Mapping Laboratory
Organization
University of Georgia
Address
Athens, GA, USA
Division
Center for Genomics and Computational Biology
Organization
Hebei United University
Address
Tangshan, People’s Republic of China
Email
wang.xiyin@gmail.com
Family Name
Li
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Given Name
Jingping
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Plant Genome Mapping Laboratory
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University of Georgia
Address
Athens, GA, USA
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jingpingli@gmail.com
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Tang
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Haibao
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University of Georgia
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Abstract
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J. Craig Venter Institute
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Rockville, MD, USA
Email
tanghaibao@gmail.com
At least two whole-genome duplications (WGD) have profoundly influenced the evolution of most, if not all,
grass (Poaceae) genomes, with the most recent of these predating the divergence of these lineages by 20
million or more years. Taxa within each major lineage of Poaceae (e.g., Panicoideae, Ehrhartoideae, Pooideae)
have independently experienced additional polyploidizations that have been of central importance to the
evolution and productivity of some of our most important crop plants [for example, sugarcane (Saccharum
spp.), and durum and bread wheat (Triticum spp.)]. Following polyploidy, adaptation to the duplicated state
is evident at the levels of transmission genetics, chromosome structure, and gene repertoire. While most
duplicated chromosomal regions re-establish largely independent evolution within a few million years, 70million-year-old duplicated chromosome segments in one unusual region of the rice genome and its orthologs
in other grasses have continued to exhibit concerted evolution more recently than the divergence of rice
subspecies japonica and indica an estimated 400,000 years ago.
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Abstract At least two whole-genome duplications (WGD) have profoundly influenced the evolution of most, if not all, grass (Poaceae) genomes, with the most recent
of these predating the divergence of these lineages by 20 million or more years. Taxa
within each major lineage of Poaceae (e.g., Panicoideae, Ehrhartoideae, Pooideae)
have independently experienced additional polyploidizations that have been of central
importance to the evolution and productivity of some of our most important crop
plants [for example, sugarcane (Saccharum spp.), and durum and bread wheat
(Triticum spp.)]. Following polyploidy, adaptation to the duplicated state is evident at
the levels of transmission genetics, chromosome structure, and gene repertoire. While
most duplicated chromosomal regions re-establish largely independent evolution
within a few million years, 70-million-year-old duplicated chromosome segments in
one unusual region of the rice genome and its orthologs in other grasses have continued to exhibit concerted evolution more recently than the divergence of rice subspecies japonica and indica an estimated 400,000 years ago.
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Ancient and Recent Polyploidy
in Monocots
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A. H. Paterson (&) X. Wang J. Li H. Tang
Plant Genome Mapping Laboratory, University of Georgia, Athens, GA, USA
e-mail: paterson@plantbio.uga.edu
X. Wang
e-mail: wang.xiyin@gmail.com
J. Li
e-mail: jingpingli@gmail.com
H. Tang
e-mail: tanghaibao@gmail.com
X. Wang
Center for Genomics and Computational Biology, Hebei United University, Tangshan,
People’s Republic of China
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H. Tang
J. Craig Venter Institute, Rockville, MD, USA
P. S. Soltis and D. E. Soltis (eds.), Polyploidy and Genome Evolution,
DOI: 10.1007/978-3-642-31442-1_6, Springer-Verlag Berlin Heidelberg 2012
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Monocotyledons, also known as monocots, are one of the major clades of
angiosperms. Most recent phylogenetic analyses (based largely on plastid
sequence data) reveal a successive series of basal angiosperm lineages, with
monocots often sister to Ceratophyllum plus eudicots (e.g., Soltis et al. 2011).
According to the IUCN there are 59,300 species of monocots (http://
cmsdata.iucn.org/). The most species-rich family in this clade (and indeed, one
of the largest of all angiosperms) are the orchids (family Orchidaceae), with more
than 20,000 species (Raven et al. 2005). Among the best-studied monocots are the
grasses, family Poaceae (Gramineae), which provide much of the world’s food and
plant biomass and include economically important grains, such as rice (Oryza),
wheat (Triticum), maize (Zea), barley (Hordeum), and sorghum (Sorghum), turf
and forage/pasture grasses, sugar cane, and the bamboos. Other economically
important monocot families are the palms (Arecaceae), bananas (Musaceae),
gingers (Zingiberaceae), and the onion family Alliaceae.
As of this writing, the vast majority of monocot genetic and genomic information, including all whole-genome-scale DNA sequences of sufficient contiguity
for synteny analysis, are for members of Poaceae, necessarily constraining the
focus of this chapter. However, this constraint is expected to be relieved in the
very near future, with genome sequences in progress for several non-grass
monocots (e.g. Phoenix dactylifera, Musa acuminata, Ananas comosus, Asparagus
officinalis, Phalaenopsis equestris, Zostera marina), adding important new
dimensions to understanding of monocot gene and genome evolution. A draft of
the date palm (P. dactylifera) genome is publicly available, but is presently of too
low contiguity for robust synteny analysis.
High-quality sequences for representatives of all three major grass clades have
been published, including rice (International Rice Genome Sequencing Project
2005; Yu et al. 2005) (Ehrhartoideae), sorghum (Paterson et al. 2009b) and maize
(Schnable et al. 2009) (Panicoideae), and Brachypodium (The International
Brachypodium Initiative 2010) (Pooideae) (Fig. 6.1). Draft genome sequence of
barley (Hordeum) and wheat (Triticum) group 1 chromosomes were recently made
available (Mayer et al. 2011; Wicker et al. 2011).
Initial analyses of the available genome sequences available for Poaceae have
shown at least two whole-genome duplications (WGD) influencing most, if not all,
grass genomes, with the most recent of these predating the divergence of these
lineages by an estimated 20 million or more years. Taxa within each lineage have
independently experienced additional polyploidizations and readaptation to the
duplicated state (for example, sugarcane and durum and bread wheat), with the
model genomes constituting a good starting point for accelerating progress in the
study and improvement of many additional taxa. Although chloridoid and arundinoid grasses are explored only at the EST level to date, these data show that at
least the Chloridoideae experienced an evolutionary history similar to those of the
other major grass clades, including both of the two WDGs shared by other
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6.1 Monocot Comparative Genomics
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Fig. 6.1 Phylogenetic relationships among major lineages of monocots and estimated positions
of early paleopolyploidy events (modified from Paterson et al. 2009a). Notice that some lack
sufficient contiguity (Phoenix) or data (Ananas, Zostera) to infer genome duplication, and
accordingly the position of sigma (r) (Tang et al. 2010) cannot be precisely determined at this
time; this uncertainty is indicated by arrows
Poaceae. Chloridoids also have an additional lineage-specific genome duplication
(Kim et al. 2009).
By developing and using comparative genomics tools, research starting from
the sequenced genomes may transitively shed light on monocots that are still
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6.2 Rho (q), the Most Recent Pan-Poaceae Polyploidy
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The notion that even relatively small and structurally simple grass genomes may
be paleopolyploid is of long-standing interest. Indeed, secondary associations of
grass chromosomes have been known for 80 years (Lawrence 1931), and genetic
mapping nearly 2 decades ago suggested duplication of scattered chromosome
segments in both rice (Kishimoto et al. 1994; Nagamura et al. 1995) and sorghum
(Chittenden et al. 1994).
Paleopolyploidy in the grasses was demonstrated beyond a reasonable doubt with
the first availability of the whole-genome sequence of rice, Oryza sativa ssp.
japonica (Goff et al. 2002). A brief controversy about whether the scope of duplication was confined to specific chromosomes (Vandepoele et al. 2003) or the whole
genome (Paterson et al. 2003, 2004) was soon reconciled, by analyzing the genome
sequence of another rice cultivar, O. sativa ssp. indica (Yu et al. 2005), with the
controversy attributed to differences in the approaches used to infer the duplicated
blocks (Wang et al. 2005). This polyploidy event (q) was dated to *70 million years
ago (mya) based on putatively neutral DNA substitution rates between duplicated
genes, and suggested to be shared by all main lineages of grasses.
The genome sequences of sorghum (Sorghum bicolor) and Brachypodium (The
International Brachypodium Initiative 2010) confirmed that these taxa share the
paleoduplication first discerned in rice, and that neither has been affected by
additional polyploidization after the evolutionary split of their respective lineages.
Moreover, Brachypodium and rice have preserved a very high level of gene collinearity (Paterson et al. 2009b; The International Brachypodium Initiative 2010),
making it possible to take them as a single genetic system to perform transitive
genetics research across different grasses (Freeling 2001). Only a small fraction of
genes shows differential gene losses after the split of rice (1.8 %) and sorghum
(3.1 %). These findings suggest that, after the 70-mya polyploidization event, the
genome of the last common ancestor of grasses had already experienced most gene
loss and reached a relatively stable state prior to the divergence of the extant major
grass lineages about 50 mya (Paterson 2008; The International Brachypodium
Initiative 2010). Some prior reports of deviations from collinearity in the grasses
may be accounted for by these low rates of differential gene loss on homoeologous
chromosomes.
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lacking de novo genomic data (Van de Peer 2004; Wang et al. 2006; Lohithaswa
et al. 2007; Tang et al. 2008b). The sequences of additional grasses and non-grass
monocots such as Elaeis (Arecaceae), Musa (Musaceae), and Zostera (Zosteraceae) will clarify the functional innovation of their gene sets, further elucidating
the structural and functional evolution of this ecologically and economically
important plant family.
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Rho accounted for much, but not all, of the paleo-duplication that could be discerned
in modern cereal genomes (Paterson et al. 2004). Shortly after the discovery of rho,
an early report (Zhang et al. 2005), later supported by two independent studies
(Jaillon et al. 2007; Salse et al. 2008) hinted at the presence of additional, earlier
monocot duplications.
Detailed elucidation of sigma (r), an additional duplication event preceding rho
in the grass lineage, utilized a ‘bottom–up’ approach, first collapsing 15,640 rice
genes and 15,636 sorghum genes into 13,308 rho-nodes that computationally
reverse post-rho gene loss, increasing the sensitivity of subsequent analysis.
These nodes (genes) were compared among themselves, revealing collinear
patterns of correspondence that involve all nine major synteny blocks resulting
from the rho duplication. Some collinear patterns between pairs of rho-blocks are
one-to-one, while others are higher order, suggesting that multiple events may
have been identified. The eight largest synteny blocks that retained collinearity
following sigma contain a total of 4,168 sigma-nodes, covering 5,747 rice genes
and 5,738 sorghum genes (*20 % of the rice and sorghum transcriptomes).
Further study of these sigma-duplicated regions highlights an important
constraint in monocot genomics, albeit one that soon will be relieved. There is
little remaining intragenomic correspondence between sigma-derived rice
segments, although relationships between some of these duplicated segments can
still be identified through transitive comparisons of cereal genomes to outgroups.
At present, however, the only available outgroups are eudicots such as grape
(Vitis). Similarities between monocot and eudicot genomes resulting from
common ancestry have been obscured by many paleo-polyploidy events and
numerous genome rearrangements (Liu et al. 2001; Jaillon et al. 2007). The
availability of non-cereal monocot outgroup genomes would significantly increase
power to study these segments, particularly outgroups with genomes that have not
experienced lineage-specific duplication and associated fractionation of ancestral
gene orders.
The lack of an ideal (non-cereal non-reduplicated monocot) outgroup
notwithstanding, better understanding of monocot paleopolyploidy has improved
our ability to compare monocot and eudicot genomes, a long-standing goal
(Paterson et al. 1996) that has been complicated by paleopolyploidy events. Toward
this goal, we applied a hierarchical clustering approach, first dividing the chromosomes into small bins and then comparing all pairs of rice and grape bins.
Duplicated segments retained in grape following the eudicot ‘gamma’ hexaploidy
event (3), and homologous segments retained in rice following at least two rounds of
duplication (rho and sigma), contain 38 ‘‘putative ancestral regions’’ (PAR) that
collectively explain 19.1 % of all observed homolog pairs and 31.0 % of reciprocal
best hits between grape and rice genes, *10-fold more than would be explicable by
chance. The PARs interleave multiple grape and rice genomic regions collectively
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covering *70 % of each genome. By consolidating much of the redundancy in each
genome, the PARs create syntenic blocks with much less ambiguity and in most
cases show association between one gamma block and one sigma block. We did not
find any PAR that simultaneously mapped to two different gamma or sigma blocks
(Tang et al. 2010). Some ‘‘ghost duplications’’ (Van de Peer 2004) in rice that we
failed to identify through intragenomic comparisons (due to reciprocal gene losses
in largely complementary fashion) are much clearer in cross-species comparisons
(PARs).
Compared to the WGD events in grape where 94.5 % of the genome appears
duplicated (Jaillon et al. 2007), the rice WGDs are more complicated and
degenerate. The 38 grape–rice PARs are a qualitative advance toward a global
view of monocot-eudicot synteny. Collinearity appears to be disrupted around the
peri-centromeric regions in 10 of the 12 rice chromosomes, suggesting dynamic
reorganization of heterochromatic portions of the rice genome that may result from
massive transpositions or gene losses (Bowers et al. 2005).
Our unique approach to synteny analysis provides new insight into the number(s)
of WGD events experienced by modern cereal genomes. In many lineages, the
existence and the numbers of WGD events have been contentious. In 22 of the 38
PARs, grape–rice collinearity is clear, with 12 PARs being threefold redundant in
grape, consistent with hexaploidy (Jaillon et al. 2007). The level of redundancy in
rice is less clear, ranging from as little as twofold (1 PAR) to sevenfold (3 PARs) and
eightfold (5 PARs). In line with the intragenomic evidence from our bottom–up
analysis, these high redundancies suggest that the rice lineage experienced more
than two, perhaps three, rounds of ancient WGD that have collectively been lumped
into the single event that we are presently referring to as ‘sigma’ (r). Once again, the
availability of non-cereal and non-reduplicated monocot outgroup genomes would
significantly improve our understanding of monocot evolutionary history.
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Polyploidy in monocots is, of course, not limited to ancient events, and indeed
more recent events have been integral to the evolution and productivity of many
major crops. A classical textbook example of a more recent polyploidization is the
evolution of wheat (see also Chap. 7, this volume), with formation of a tetraploid
and subsequently a hexaploid leading to independent crops, durum wheat used for
pasta (Triticum turgidum) and bread wheat (T. aestivum), respectively (Feldman
and Levy 2005). Indeed, the synthesis by humans of octoploid triticale (9 Triticosecale a hybrid between Triticum and Secale) in the past century illustrates that
there may be further gains to be made by mimicking the natural tendency of some
plant lineages to form polyploids.
Polyploidy has been particularly closely associated with productivity in
autopolyploids such as many forage and biomass grasses. One of the most extreme
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cases of a successful autoploid is sugar cane (Saccharum spp.), in which cultivated
forms are typically octoploid—dodecaploid (i.e., with 8–12 chromosome sets, and
sometimes variations in between!). These are interspecific hybrids (and therefore
not strict autopolyploids, but a combination of auto- and allopolyploidy) composed
of about 85–90 % chromatin from Saccharum officinarum and 10–15 % from
S. spontaneum, a wild relative. Studies using DNA markers to quantify ‘dosages’ of
homologous genomic regions in sugar cane show clearly nonlinear consequences of
allele dosage, with one or two copies of favorable alleles at homologous loci usually
having favorable effects, but additional copies yielding diminishing or even reduced
returns (Ming et al. 2001, 2002a, b). Comparative genomic studies, in particular
with sorghum, show that the Saccharum lineage experienced at least one WGD
since its divergence from the Sorghum lineage something less than 10 mya (Ming
et al. 1998; Jannoo et al. 2007).
Ploidy in Saccharum is particularly interesting, in that it has been further
complicated by a high frequency of chromosome non-reduction, yielding
2n ? n progeny in S. officinarum (female) 9 S. spontaneum (male) crosses (Bremer
1923). In efforts to broaden genetic variability in ‘noble canes’ that were the foundation of sugar cane production until the early part of the twentieth century, F1
progeny of interspecific crosses between S. officinarum and S. spontaneum were
found to be distinctively more robust than either parent. When S. officinarum clones
were used as the female parent, progeny tended to be larger stalked, higher in sucrose
levels, and generally more vigorous than when S. spontaneum clones were used as the
female parent. The pollination of noble cane with S. spontaneum followed by
repeated backcrosses to the noble canes has come to be called ‘nobilization’, with
selected hybrid progenies referred to as ‘nobilized’ canes (Bremer 1961). A key
event in the evolution of modern sugarcane cultivars was the production of the
nobilized cultivar, ‘POJ2878’, of Proefstation Oost, Java, in 1921 (Jeswiet 1929).
Further study of the clade that includes Saccharum may prove intriguing. This
clade also includes Miscanthus, which has biomass yields that are similarly high as
Saccharum, but is better adapted to temperate climates and therefore has stimulated
much interest for bioenergy production in the USA, Europe, and China (Heaton et al.
2008). Miscanthus species have a basic set of n = x = 19 chromosomes, versus the
x = 10 that is characteristic of many Saccharinae including Saccharum. One
attractive hypothesis to explain the transition from 10 to 19 chromosomes is that
Miscanthus, like Saccharum (Ming et al. 1998), may have experienced a polyploidization event in the 8–9 my since its divergence from sorghum—but unlike
Saccharum, which is largely autopolyploid, Miscanthus homologs have diverged
sufficiently that they no longer normally pair with one another—that is, there is now
preferential pairing of chromosomes. Genome evolution may have included a
chromosomal fusion to get from n = 20 to 19. If Miscanthus and Saccharum shared
a genome doubling event, a possibility that we are continuing to investigate, it would
be an intriguing and perhaps unprecedented case in which one lineage (Miscanthus)
adapted to the duplicated state by re-establishing disomy, while a sister lineage
continued to have the option of polysomy.
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A polyploidy event may result in genomic instability, consequently incurring a
process of diploidization characterized by widespread DNA rearrangements often
accompanied by large-scale gene losses (Paterson et al. 2004; Van de Peer 2004;
Wang et al. 2006; Xiong et al. 2011). These DNA rearrangements may result in
chromosome number variations. Grasses range from n = 2 to 18 in their basic
chromosome sets (Soderstrom et al. 1987; Hilu 2004). In the sequenced genomes,
rice, sorghum, and Brachypodium have n = 12, 10, and 5 chromosomes, respectively. Although maize (Zea mays) experienced a WGD since divergence from
sorghum, modern-day maize retains the same chromosome number (n = 10) as
sorghum. Some lineages of Sorghum have experienced chromosome condensations
even in the absence of polyploidization (Spangler et al. 1999; Spangler 2003).
Comparison of grass genomes has shed light on chromosome number evolution and
ancestral grass karyotypes (Salse et al. 2009; Murat et al. 2010). An ancestral
karyotype of n = 5 chromosomes was inferred (Salse et al. 2009; Murat et al. 2010)
before the pan-grass polyploidization, with n = 2x = 10 chromosomes after the
duplication, then two fissions to result in n = 2x = 12 chromosomes in the common
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One important challenge facing a newly formed autopolyploid may be that the
presence of sets of four homologous chromosomes may tend to hinder purging of
deleterious alleles. Transition to diploid inheritance would, in principle, allow
more rapid allele frequency changes and reduced genetic load.
Centromeric divergence may have been a mechanism by which paleopolyploid
grasses restored diploid inheritance (Bowers et al. 2005). A high concentration of
rice genes duplicated by ancient polyploidy falls near Ks 0.85, while rice gene pairs
with Ks 0.2–0.6 tend to be located in peri/centromeric regions. This suggests that
shortly after polyploidization, a substantial restructuring of centromeric regions
began that lasted until about 16 mya [based on the synonymous substitution rate
used (Lynch and Conery 2000)]. About 18 % of the rice genome shows highly
significant concentrations of matches (p \ 1 9 10-5) in the Ks range of 0.2–0.6.
The restructuring of non-syntenic regions largely involves migration of DNA
between pericentromeric regions of different chromosomes. The concentrations of
relatively recent single–locus duplications near the centromeres may, but does not
necessarily, reflect a higher duplication rate in these regions. Alternatively, recent
duplications may be preserved more frequently in pericentromeric regions, i.e.,
there may be more rapid loss of single-gene duplications in euchromatic regions
(Bowers et al. 2005).
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Among the genes duplicated in the polyploidy events shared by the grass lineage,
the vast majority of pairs have lost at least one duplicated copy. The finding that
only a small fraction of genes show differential gene losses after the split of rice
(1.8 %) and sorghum (3.1 %) demonstrates empirically that, after the 70-mya
polyploidization, the genome of the last universal common ancestor of grasses had
already experienced most gene loss and reached a relatively stable state prior to the
divergence of the major grass lineages about 50 mya (Paterson 2008; The International Brachypodium Initiative 2010).
Gene losses have often occurred in a complementary and segmental manner,
that is, with non-random patterns of retention/loss on corresponding duplicated
DNA segments, in a process known as fractionation (Thomas et al. 2006). Genes
may be removed by a short-DNA deletion mechanism (Woodhouse et al. 2010),
and in a pair of duplicates, gene loss may be universally biased to preserve the
gene that is responsible for the majority of expression (Schnable et al. 2011). For
more than 90 % of the preserved duplicated genes, the two copies have the same
transcriptional orientations (Wang et al. 2005), and the exceptions may be a result
of local DNA inversions or differential gains/losses of new tandemly duplicated
genes in the paleo-duplicated regions.
In grasses, and indeed across angiosperms, we find three ‘fates’ of individual gene
pairs following duplication (see also Chap. 1, this volume). Most gene functional
groups show post-duplication gene preservation/loss rates that are indistinguishable
from the genome-wide average. Such ‘neutral’ loss of duplicated genes presumably
involves inactivating mutations opposed by very weak selection (Haldane 1933), as
the fate of the vast majority of duplicated genes. Population genetic models suggest
that loss of duplicated genes may happen over a few million years (Lynch and
Conery 2000). Genes in some specific functional categories duplicate and reduplicate
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ancestor of major cereals. However, the authors noted that an ancestral karyotype of
n = 6–7 was also possible. They inferred that chromosome number variation/
reduction from the common ancestor may be attributable to non-random centric
double-strand break repair events. It was suggested that centromeric/telomeric
illegitimate recombination between non-homologous chromosomes led to nested
chromosome fusions and synteny break points, and concluded that these break
points were meiotic recombination hotspots that corresponded to high sequence
turnover loci through repeat invasion. These rules seem to explain most chromosome number changes in the grass genomes sequenced so far, especially the previously observed nested chromosome fusions in Brachypodium (The International
Brachypodium Initiative 2010). However, many details related to dynamics of
centromeres and telomeres during the rearrangements remain unclear, and the wide
range in possible ancestral karyotypes (from 12 to 24) suggests that further revision
of thinking on this subject is likely.
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6.6 The Unique Case of Rice Chromosomes 11 and 12
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Rice chromosomes 11 and 12 (R11 and R12) are a striking exception among
chromosomes affected by the 70-mya polyploidization (Wang et al. 2011). R11
and R12 share a *3-Mb duplicated DNA segment at the termini of their short
arms, the formation of which had been dated based on synonymous substitutions to
*5–7 mya (The Rice Chromosomes 11 and 12 Sequencing Consortia 2005; Wang
et al. 2005; Yu et al. 2005). Remarkably, in the second grass genome sequence,
sorghum, the corresponding region(s) of its orthologous chromosomes (S5 and S8,
respectively) also contained such an apparently recent duplication despite having
diverged from rice about 50 mya (Paterson et al. 2009b). Physical and genetic
maps also suggest shared terminal segments of the corresponding chromosomes in
wheat (4, 5), foxtail millet (VII, VIII), and pearl millet (linkage groups 1, 4)
(Devos et al. 2000; Singh et al. 2007). It would be exceedingly unlikely for
segmental duplications to happen independently at such closely corresponding
locations in reproductively isolated lineages. A much more parsimonious
hypothesis is that the R11/12 and S5/8 regions each resulted from the pan-grass
duplication 70 mya but have an unusual evolutionary history (Paterson et al.
2009b).
Detailed analysis of R11 and R12 suggested that illegitimate recombination has
continued for millions of years after the 70-mya divergence of these homoeologs,
indeed remaining ongoing in the past 400,000 years since divergence of subspecies japonica and indica (Wang et al. 2007). Gradual and step-by-step restrictions
on recombination, starting around the time of the 70-mya polyploidization, have
resulted in ‘strata’ along the chromosome pair that differ in the degree of DNA
sequence similarity between homoeologous genes (Wang et al. 2011). Sequence
similarity between homoeologs in the strata reflects the time(s) of recombination
suppression rather than the times of their origination. Indeed, the most terminal
stratum in rice (RSA) appears \0.5 my old, while two more internal strata (RSB,
RSC) appear to have been restricted in their ability to recombine 9.4 and 39.1 mya,
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(Blanc and Wolfe 2004; Seoighe and Gehring 2004; Maere et al. 2005; Chapman et al.
2006; Paterson et al. 2006; Tang et al. 2008a), and in many instances can be related to
the ‘‘gene balance’’ hypothesis, that stoichiometry among members of pathways and
networks is important to biological function (Birchler et al. 2005; Birchler and Veitia
2007; Veitia et al. 2008). Other specific genes and gene functional groups show more
extensive loss of duplicate copies than the genome-wide average, and this loss has
often been convergent following independent duplications separated by hundreds of
millions of years during the evolution of grasses, Arabidopsis, yeast, and Tetraodon
(pufferfish) (Paterson et al. 2006). Much greater knowledge of gene functions,
particularly regarding those less-explored genes that are recurrently restored to the
singleton state, may provide new insights into the ‘adaptation’ of a newly formed
polyploid to the duplicated state.
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Fig. 6.2 Gene repertoire and organization in a terminal segment of homoeologous rice and
sorghum chromosomes experiencing concerted evolution. This region includes 4 rice-sorghum
quartets (Q), 3 triplets [T: losses on S5 (2), S8], 13 pairs of taxon-specific genes [P: 5 sorghum, 8
rice]; 8 taxon-specific singletons [S: 6 sorghum, 2 rice], and one lineage-specific duplication
(X: two terminal genes on S5 and S8 share common ancestor with single rice genes).
Classification letters for a gene family appear in only one member (members connected by lines).
Reprinted from Wang et al. (2011) with permission. Copyright is owned by the American Society
of Plant Biology
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respectively. In sorghum, even the most terminal (of only two) strata dates to
13.4 mya, reflecting the parallel but independent evolution of this unusual chromosome segment in divergent lineages (Wang et al. 2011). The corresponding
regions in maize and Brachypodium also show prominent homoeologous recombination. However, widespread chromosomal rearrangement, especially in maize
after its lineage-specific polyploidization, makes the stratification patterns more
difficult to compare than in rice and sorghum.
Both intriguing and perplexing is that the distal chromosomal region with the
greatest DNA similarity between surviving duplicated genes also has the highest
concentration of lineage-specific gene pairs found anywhere in these genomes, and a
significantly elevated gene evolutionary rate (Wang et al. 2011). Of 33 and 23 riceand sorghum-specific gene pairs on these chromosomes, a respective 100 and 90 %
of them are in the young strata. Both members of a remarkable 50 % of the 16
duplicated RSA gene pairs are absent from sorghum, and 15 (38 %) of 39 SSA pairs
are absent from rice (Fig. 6.2). Gene losses on either one of a pair of homoeologs
experiencing concerted evolution may be commuted to the other, perhaps
explaining the more than tenfold higher rate of gene loss in the RSA and SSA
regions than the genome-wide averages of 1.8 % in rice and 3.1 % in sorghum since
their divergence about 50 mya.
The singular evolutionary history of this pair of grass chromosomes needs
further exploration. Elevated gene loss rates and elevated evolutionary rates of the
preserved genes in young strata may facilitate speciation in that the loss of
alternative copies of duplicated genes leads to reproductive isolation (Werth and
Windham 1991; Lynch and Force 2000; Scannell et al. 2006; see also Chap. 1, this
volume). The recently proposed inter-relationship between reproductive isolation
and autoimmune responses (Bomblies et al. 2007; Yin et al. 2008) draws attention
to the finding that orthologs R11 and S5 each contain *25 % of the NBS-LRR
resistance genes (Zhou et al. 2004; Paterson et al. 2009b). Second, a high level of
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D, Hilu KW, Latvis M, Wurdack K, Xi Z, Davis C, Donoghue M, Soltis PS (2011) Inferring
angiosperm phylogeny: a 17-gene analysis. Am J Bot 98:704–730
Spangler R (2003) Taxonomy of Sarga, Sorghum, and Vacoparis (Poaceae: Andropogoneae).
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Spangler R, Zaitchik B, Russo E, Kellogg E (1999) Andropogoneae evolution and generic limits
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Tang H, Wang X, Bowers JE, Ming R, Alam M, Paterson AH (2008) Unraveling ancient
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Tang H, Bowers JE, Wang X, Ming R, Alam M, Paterson AH (2008) Synteny and colinearity in
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Tang HB, Bowers JE, Wang XY, Paterson AH (2010) Angiosperm genome comparisons reveal
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Van de Peer Y (2004) Computational approaches to unveiling ancient genome duplications. Nat
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Vandepoele K, Simillion C, Van de Peer Y (2003) Evidence that rice and other cereals are ancient
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Book Title
Polyploidy and Genome Evolution
Series Title
Chapter Title
Genomic Plasticity in Polyploid Wheat
Copyright Year
2012
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Springer-Verlag Berlin Heidelberg
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Kashkush
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Department of Life Sciences
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Ben-Gurion University
Address
84105, Beer-Sheva, Israel
Email
kashkush@bgu.ac.il
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Feldman
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Moshe
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Plant Sciences Department
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The Weizmann Institute of Science
Address
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Levy
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Avraham
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Abstract
The importance of hybridization and polyploidization in wheat speciation has been recognized for close to a
century (Sakamura 1918; Kihara 1919, 1924, 1954; Percival 1921; Sax 1927). Following these pioneering
works, it quickly became apparent that polyploid wheats are not the sum of their constituent genomes. This
is not unexpected because the nascent hybrids/polyploids are equipped with a complex set of regulatory
elements and of copy number variation that originate from two or more divergent genomes and that generate
novel types of interactions and dosage effects. Moreover, they have to adjust at the cytological level, at the
level of gene expression, and at the protein level. They also have to maintain genome stability through the
regulation of meiotic pairing and recombination, the orchestration of cell division, and the silencing of
transposons. The recent studies described here provide an impressive account with regard to the extent and
the rapid time course at which a new genetic variant was established upon hybridization and polyploidization.
We describe here the current knowledge on the changes that occurred in the wheat genome upon
allopolyploidization, starting from the early evolutionary and cytological studies to the recent genomic
analyses.
Book ISBN: 978-3-642-31441-4
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Chapter 7
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Genomic Plasticity in Polyploid Wheat
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Moshe Feldman, Avraham Levy, Boulos Chalhoub
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Abstract
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The importance of hybridization and polyploidization in wheat speciation has been
recognized for close to a century (Sakamura 1918; Kihara 1919, 1924, 1954;
Percival 1921; Sax 1927). Following these pioneering works, it quickly became
apparent that polyploid wheats are not the sum of their constituent genomes. This
is not unexpected because the nascent hybrids/polyploids are equipped with a
complex set of regulatory elements and of copy number variation that originate
from two or more divergent genomes and that generate novel types of interactions
and dosage effects. Moreover, they have to adjust at the cytological level, at the
level of gene expression, and at the protein level. They also have to maintain
genome stability through the regulation of meiotic pairing and recombination, the
orchestration of cell division, and the silencing of transposons. The recent studies
described here provide an impressive account with regard to the extent and the
rapid time course at which a new genetic variant was established upon hybridization and polyploidization. We describe here the current knowledge on the
changes that occurred in the wheat genome upon allopolyploidization, starting
from the early evolutionary and cytological studies to the recent genomic analyses.
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Book ID: 272454_1_En
Date: 16-8-2012
M. Feldman A. Levy
Plant Sciences Department, The Weizmann Institute of Science,
76100, Rehovot, Israel
B. Chalhoub
UMR INRA 1165—CNRS 8114—UEVE,
Organization and Evolution of Plant Genomes (OEPG),
Unité de Recherche En Génomique Végétale (URGV),
2 Rue Gaston Crémieux, 91057, Evry Cedex, France
UN
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Chapter No.: 7
K. Kashkush (&)
Department of Life Sciences, Ben-Gurion University, 84105,
Beer-Sheva, Israel
e-mail: kashkush@bgu.ac.il
P. S. Soltis and D. E. Soltis (eds.), Polyploidy and Genome Evolution,
DOI: 10.1007/978-3-642-31442-1_7, Springer-Verlag Berlin Heidelberg 2012
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Diploids
(2n = 2x = 14)
Tetraploids
(2n = 4x = 28)
Hexaploids
(2n = 6x = 42)
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Spelt (hulled)
wheats
Naked (free threshing)
wheats
The discovery of wild emmer wheat, the progenitor of most domesticated wheats
(Aaronsohn and Schweinfurth 1906; Aaronsohn 1910), made it possible for Schulz
(1913) to assemble the first natural classification of the wheats. He divided the
genus Triticum into three major groups: einkorn, emmer, and dinkel. This classification was supported by the pioneering cytological study of Sakamura (1918),
who was the first to determine the correct chromosome number of the wheats.
Sakamura discovered that Schulz’s three groups of wheats differ in their chromosome number: the einkorns are diploids (2n = 14), the emmers are tetraploids
(2n = 28), and the dinkels are hexaploids (2n = 42) (Table 7.1). It then became
obvious that the species of Triticum represent a polyploid series with diploid,
tetraploid, and hexaploid species.
Since then, the species of the wheat group (Triticum and its closely related
genus Aegilops) have been subjected to extensive taxonomic, cytogenetic, genetic,
biochemical, molecular, and evolutionary study by numerous scientists (see
reviews of Kihara 1954; Mac Key 1966; Morris and Sears 1967; Kimber and Sears
1987; Feldman et al. 1995; Feldman 2001; Gupta et al. 2005; Dvorak 2009). Due
to these extensive studies the allopolyploid species of the wheat group became a
classic example of evolution through allopolyploidy. The cytogenetic studies of
Kihara (1919, 1924, 1954), Percival (1921), Sax (1927), and others on chromosome pairing in hybrids among species of different ploidy levels showed that all
the polyploid species of the group form an allopolyploid series based on x = 7.
Each allopolyploid species was identified as a product of hybridization followed
by chromosome doubling (Fig. 7.1). Von Tschermak and Bleier (1926) were the
first to identify a spontaneous chromosome doubling in the cross of wild emmer
(T. turgidum ssp. dicoccoides) with Aegilops geniculata, thus demonstrating the
possibility of species formation via allopolyploidy in the wheat group. Subsequent
studies showed that the frequency of unreduced gametes in intergeneric hybrids of
wheat could be in some hybrids as high as 50 % (Kihara and Lilienfeld 1949).
Therefore, one might assume that there is a high potential for the frequent and
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Domesticated wheats
Einkorn (one-grained T. aegilopoides T. monococcum None
wheat)
—[
Emmer (two-grained T. dicoccoides T. dicoccum — T. durum
wheat)
—[
[
T. turgidum
T. polonicum
Dinkel
None
T. spelta — [ T. compactum
T. vulgare
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Wild
progenitors
7.1 The Wheat Group: Natural and Synthetic Polyploids
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Ploidy level
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Table 7.1 Superimposition of Sakamura’s (1918) finding of the right chromosome number on
Schulz’s (1913) natural classification of the wheats
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recurrent formation of interspecific or intergeneric hybrids and allopolyploids in
the wheat group.
The discovery by Blakeslee (1937) that colchicine can induce chromosome
doubling opened new possibilities for the study of wheat evolution through allopolyploidization. It also provided an easy method to synthesize different wheat
allopolyploids, some of which have similar genomes to natural allopolyploids and
others having new genomic combinations (see examples in Ozkan et al. 2001).
Synthetic allopolyploids, either induced or occurring spontaneously, offer excellent tools to mimic the evolutionary speciation events that occurred in nature and
to test in a controlled manner; the new features of the hybrid/polyploid genome
compared to those of its parents.
Since the discovery that the polyploid species of wheat comprise an allopolyploid series, attempts have been made to identify the diploid donors of the different
genomes to the allopolyploids of the wheat group. Most of these attempts used the
cytogenetic approach of genome analysis, developed by Kihara (1919, 1924) and
based on the concept of genome stability, assuming that the genomes of the
allopolyploid species remain similar to those of their diploid parents. However, the
accumulating cytogenetic and molecular evidence has indicated that this is not the
case; while one genome remains relatively unchanged, the second genome(s) of
the allopolyploids have changed considerably from those of their parental diploids.
These genomes were termed modified genomes by Kihara (1954) and other wheat
cytogeneticists. Every allopolyploid species of Aegilops and Triticum contains an
unchanged genome side-by-side with a modified one whose diploid origin has
been intricate and difficult to trace (Zohary and Feldman 1962).
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Fig. 7.1 Evolutionary history of allotetraploid and allohexaploid wheat. Diploid wheats
(2n = 2x = 14) from the Triticum-Aegilops group diverged *4 million years ago (MYA) from
a common progenitor (Huang et al. 2002). Interspecific hybridization between the diploid
T. urartu (genome AA) as male and the donor of the B genome (an unknown species similar to
Ae. speltoides) as female, followed by chromosome doubling, gave rise, *0.5 MYA, to wild
allotetraploid wheat, Triticum turgidum ssp. dicoccoides (2n = 4x = 28, genome BBAA), an
allotetraploid considered as the direct progenitor of durum wheat. Domestication of allotetraploid
wheat took place *10,500 YA and was rapidly followed (*9,500 YA) by a second round of
intergeneric hybridization and chromosome doubling between domesticated allotetraploid wheat
and the donor of the D genome, Ae. tauschii (2n = 2x = 14, genome DD), giving rise to bread
wheat, an allohexaploid with 2n = 6x = 42 chromosomes (genome BBAADD). Adapted from
Levy and Feldman (2004)
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7.2 Cytological Diploidization
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Because of the close relationship of the progenitors and the similarity of the two
different genomes of allotetraploid wheat and of the three different genomes of
allohexaploid wheat (Morris and Sears 1967), the successful establishment of the
polyploid species in nature required the acquisition during the allopolyploidization
process of molecular and genetic systems that would prevent intergenomic pairing
and recombination. By restricting pairing to fully homologous chromosomes
(intragenomic pairing), the cytological-diploidizing systems ensure exclusive
bivalent pairing at meiosis and, consequently, regular segregation of genetic
material, complete fertility, and genetic stability. Cytological diploidization has
been brought about in allopolyploid wheat by two independent systems that
complement each other: One system is based on genetic control of pairing, and the
second is based on physical divergence of chromosomes.
Historically, the first mechanism of cytological diploidization that received
much attention was a genetic system involved in sustaining the exclusive bivalent
pairing in allopolyploids. It consisted of the activity of the genetic loci Ph1 on
chromosome arm 5BL and Ph2 on chromosome arm 3DS (Sears 1976). These loci
suppress pairing of homoeologous chromosomes while allowing homologs to pair
regularly. The mechanism controlling the Ph1 mode of action is still unclear.
Mapping data suggests the involvement of a complex locus that affects cell cycle
progression through the regulation of Cyclin-dependent kinases (Griffiths et al.
2006); however, direct evidence through complementation studies is still missing.
It was suggested by several authors that Ph-like genes exist in diploid species of
the wheat group (Okamoto and Inomata 1974; Avivi 1976; Waines 1976; Maan
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Nevertheless, genome analysis studies revealed that allotetraploid wheat
(genome BBAA) originated from hybridization events involving two diploid
progenitors classified in the genera Aegilops and Triticum. Genome B, which is a
modified genome, was derived from Ae. speltoides and underwent changes on the
polyploid level or, more likely, from a closely related species to Ae. speltoides
which is extinct or extant (Feldman et al. 1995). Genome A, which has been
modified relatively little, was derived from T. urartu (Chapman et al. 1976;
Dvorak 1976). Allohexaploid wheat (genome BBAADD) originated from
hybridization between allotetraploid wheat and Ae. tauschii, the donor of the
D genome (Kihara 1944; McFadden and Sears 1944, 1946). The evolution of the
wheats is presented in Fig. 7.1 (for details, see Feldman et al. 1995; Feldman
2001). Modern classification for the Triticum group (Van Slageren 1994;
Table 7.2) recognizes two diploid species, T. monococcum L. and T. urartu Tum.
ex Gand., two tetraploid species, T. turgidum L. and T. timopheevii (Zhuk.) Zhuk.,
and two hexaploid species, T. aestivum L. and T. zhukovskyi Men. & Er. The
economically important wheats are T. aestivum (bread wheat, comprising 95 % of
the global wheat production) and T. turgidum (macaroni wheat).
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AmAm
AA
Tetraploids (2n = 4x = 28) GGAA
Triticum urartu
Triticum timopheevii
ssp. armeniacum
ssp. timopheevii
Triticum turgidum
ssp. dicoccoides
ssp. dicoccon
ssp paleocochicum
ssp. parvicoccumb
ssp. durum
ssp. turgidum
ssp. polonicum
ssp. turanicum
ssp. carthlicum
Triticum zhukovskyi
D
BBAA
Triticum monococcum
ssp. aegilopoides
ssp. monococcum
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Hexaploids (2n = 6x = 42) GGAA
AmAm
BBAADD Triticum aestivum
ssp. spelta
ssp. macha
ssp. aestivum
ssp. compactum
ssp. sphaerococcum
a
Wild einkorn
Domesticated einkorn or
small spelt
None (wild form)
F
Diploids (2n = 2x = 14)
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Table 7.2 The nomenclature of the commercially cultivated wheats and their immediate wild
relatives (after van Slageren, 1994)a
Ploidy level
Genome
Species and subspecies Common name
Wild timopheevii
Domesticated timopheevii
Wild emmer
Domesticated emmer
Georgian wheat
None
Macaroni or hard wheat
Rivet, cone or pollard wheat
Polish wheat
Khorassan wheat
Persian wheat
None
Dinkel or large spelt
None
Common or bread wheat
Club wheat
Indian dwarf or short wheat
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1977) and they became more effective at the polyploid level as a result of
duplication. This dosage-dependent effect might have been selected to improve the
fertility of the allopolyploid. This genetic system superimposes itself on, takes
advantage of, and thereby reinforces the system of physical homoeologous differentiation already in existence and described below. The genetic system is very
effective in suppressing homoeologous pairing in interspecific and intergeneric F1
hybrids. However, its suppressive effect on homoeologous pairing in allopolyploid
wheat might not be essential since in plants deficient for Ph1 there is relatively
very little such pairing (Sears 1976). Interestingly, and in accord with the above,
gene(s) like Ph were not found in all the allopolyploid species of the closely
related genus Aegilops. In spite of this, these species, relying solely on the
structural homoeologous differentiation, exhibit exclusive bivalent pairing of fully
homologous chromosomes, i.e., exclusive intragenomic pairing. It might be that
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Taxa derived from a single spontaneous or induced mutation and are not commercially cultivated, such as diploid T. sinskajae Filat and Kurk., tetraploid T. militinae Zhuk. and Migush.,
and hexaploid T. vavilovii (Tum.) Jakubz., are not included
b
Extinct, described by Kislev 1980
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Occur during or immediately after allopolyploidization
F
Genetic and epigenetic changes
Species-specific
Lead to cytological diploidization
Occur during the life of the
allopolyploid species
Mostly genetic changes
Population- or biotype-specific
Promote genetic diversity,
flexibility, and adaptability
Improve harmonic functioning of the divergent genomes
Stabilize the nascent allopolyploid and facilitate its
establishment as a new species in nature
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Table 7.3 Types and characteristics of genomic changes in allopolyploid wheat
Revolutionary changes (triggered by allopolyploidization) Evolutionary changes (Facilitated
by allopolyploidy)
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7.3 Genomic Structural Changes
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Studies with synthetic polyploids as well as genome sequencing data indicate that
a broad range of DNA rearrangements occurred during, or soon after, hybridization and polyploidization. What triggers these changes is a fascinating and still
open question, but what is clear is that these changes are extensive, including DNA
loss, transposon activation, gene duplication, and pseudogenization, and are relatively rapid. Feldman and Levy (2005) have distinguished between the revolutionary changes that occur rapidly and evolutionary changes that take place
throughout the evolution of the allopolyploid (Tables 7.3 and 7.4). Note that
evolutionary changes might also occur in an accelerated manner, thanks to the
buffering of mutations in the polyploid background (Mac Key 1954, 1958; Sears
1972; Thompson et al. 2006), leading to rapid neo- or subfunctionalization of
genes and to a process of diploidization and of divergence from the diploid progenitor genomes.
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stringent selection for fertility under domestication has favored the development of
two systems to ensure suppression of multivalent formation and to promote
bivalent pairing. Moreover, the cytological diploidization of the allopolyploid
wheats that leads to disomic inheritance prevents independent segregation of genes
from the different genomes. This mode of inheritance leads to permanent maintenance of favorable intergenomic genetic interactions. It enables fixation of heterotic interaction between genomes and sustained division of tasks (genome
asymmetry) between genomes. A series of DNA rearrangements in the allopolyploid further contributes to the physical divergence between the homoeologous
chromosomes and to the strengthening of the disomic genetic system. These
changes are described below.
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Structural
• Elimination of low-copy DNA sequences
Chromatin remodeling
Chromatin modifications
Heterochromatinization
DNA methylation
Small RNAs activation or
repression
Methylation (leading to
silencing)
Demethylation (leading to
gene activation)
Release transposons from
silencing
Silencing transposons
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• Elimination, reduction, or amplification of highcopy sequences
• Inter-genomic invasion of DNA sequences
• Elimination of rRNA and 5S RNA genes
Functional • Gene loss
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Table 7.4 Revolutionary changes induced by allopolyploidization in wheat
Level
Genetic
Epigenetic
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Allopolyploidization causes immediate nonrandom elimination of specific noncoding, lowcopy, and high-copy DNA sequences. These sequences are present in
all the diploid species of Aegilops and Triticum but occur in only one pair of
chromosomes (chromosome-specific sequences) or in several chromosome pairs of
one genome (genome-specific sequences) at the polyploid level (Fig. 7.2)
(Feldman et al. 1997; Liu et al. 1998a; Liu et al. 1998b; Ozkan et al. 2001; Shaked
et al. 2001; Han et al. 2003; Salina et al. 2004; Han et al. 2005). The extent of
DNA elimination was estimated by the determination of the nuclear DNA amount
in natural allopolyploids and in their diploid progenitors as well as in newly
synthesized allopolyploids and in their parental plants (Ozkan et al. 2003; Eilam
et al. 2008; Eilam et al. 2010). Natural wheat allopolyploids contain 2–10 % less
DNA than the sum of their diploid parents, and synthetic allopolyploids exhibit a
similar loss, indicating that DNA elimination occurs soon after allopolyploidization (Eilam et al. 2008; Eilam et al. 2010). Also, from the very little variation in
DNA amount that exists at the intraspecific level, it was concluded that the
reduction of DNA content occurred immediately after the formation of the
polyploids, and that after this there was almost no change in DNA amount during
the life of the allopolyploid species. In triticale (an allopolyploid between wheat
and rye, Secale cereale), Boyko et al. (1984, 1988) found that there was a great
reduction in DNA content in the course of triticale formation with about 9 % for
octoploid and 28–30 % for hexaploid triticale. The different genomes were not
affected equally in triticale; wheat genomic sequences were relatively conserved,
whereas rye genomic sequences were predominantly involved in a very high
level of variation and elimination (Ma et al. 2004; Ma and Gustafson 2005, 2006).
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• Rewiring of gene expression through novel inter- •
genomic interactions
• New dosage response (positive, negative, dosage •
compensation)
• Gene suppression or activation
•
• Transcriptional activation of transposons (that may
affect nearby genes in cis)
• New transpositions of transposons
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Also in hexaploid wheat the genomes were not affected equally; genome D
underwent considerable reduction in DNA amount whereas the wheat A and B
genomes did not shrink in size (Eilam et al. 2008; Eilam et al. 2010). DNA
elimination seems to be nonrandom at the intrachromosomal level as well: Liu
et al. (1997) found in allohexaploid wheat that the chromosome-specific sequences
of chromosome arm 5BL are not distributed at random along this chromosome arm
but cluster in terminal (subtelomeric), subterminal, and interstitial regions of this
arm, making these regions extremely chromosome-(homologous-)specific. Hence,
it was tempting to suggest that these homologous-specific regions play a critical
role in homology search and initiation of meiotic pairing (the classical pairing
initiation sites) (Feldman et al. 1997). In some studies, DNA elimination was not
observed in the first generations of synthetic allopolyploids (Mestiri et al. 2010;
Zhao et al., 2011). It is possible that the timing, as well as the extent of elimination
depends on the type of genomic combinations involved or on the type of sequences
analyzed.
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Fig. 7.2 Schematic representation of the wheat karyotype. The wheat karyotype is arranged into
genomes A, B, and D and into seven homoeologous groups. Examples of the different types of
sequences are drawn on top of the chromosomes, namely: triplicated sequences (group-specific
sequences), chromosome-specific sequences (CSSs) that are present in only one chromosome
pair, genome-specific sequences (GSSs) that can be on more than one chromosome pair but only
in one of the genomes, and dispersed repeats that are present on both homoeologous and
nonhomoeologous chromosomes. Adapted from Levy and Feldman (2004)
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Table 7.5 Evolutionary changes facilitated by allopolyploidy in wheat
Level
Type
• Chromosomal repatterning (intra- and inter-genomic translocations)
• Introgression of chromosomal segments from alien genomes and production of
recombinant genomes
Functional • Nonfunctionalization (deletion or pseudogenization)
• Subfunctionalization
• Neofunctionalization
• Copy number changes
• New allelic variation
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Several structural changes occur in the allopolyploid wheat genomes, which
generate new variants that could not take place in the diploid parental genomes and
that occur almost exclusively in an allopolyploid background (Table 7.5). This
includes events, such as intergenomic horizontal transfer of chromosomal segments, repetitive sequences, transposons, or genes among the constituent genomes.
These events may occur sporadically throughout the history of the allopolyploid
species. Intergenomic translocations that are population- or biotype-specific are
widespread in allohexaploid wheat (Maestra and Naranjo 1999). Invasion of the A
genome by sequences from the B genome—most probably transposons—was
detected in wild allotetraploid wheat using GISH (Belyayev et al. 2000). The
possibility of intergenomic transfer adds to the allopolyploid genomes’ plasticity
and enables the creation of new genetic combinations that are beyond the addition
of two genomes.
Moreover, in contrast to diploids, which are genetically isolated from each
other and have undergone divergent evolution, allopolyploids in the wheat group
exhibit convergent evolution, because they contain genetic material from two or
more different diploid genomes and can exchange genes with each other via
hybridization and introgression, resulting in the production of new genomic
combinations. Examples of such introgression between allotetraploid Aegilops
species that share one genome and differ in the other genome(s) were provided by
Zohary and Feldman (1962) and Feldman (1965a, b, c). Such hybridizations are
eased by the shared genome, which acts as a buffer and ensures some fertility in
the resulting hybrids. In such hybrids, chromosomes of two dissimilar genomes,
brought together from different parents, may pair and exchange genetic material
and recombine (Feldman 1965a). Additional evidence for the existence of introgressed genomes in allopolyploid Aegilops was obtained from C-banding analysis
(Badaeva et al. 2004). Introgression of a DNA sequence from allopolyploid wheat
to the allotetraploid Aegilops species, Ae. peregrina, was described by Weissmann
et al. (2005).
In addition to evolutionary changes that are almost unique to an allopolyploid
background, it might be that other types of mutations that can cause structural or
functional changes on an evolutionary scale (e.g., point mutations, satellite
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7.4 Functional Changes
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instability, transposition, etc.) can occur in an accelerated manner in the polyploid
background. The presence of duplication or triplication of the genetic material in
wheat allopolyploids might have relaxed constraints on gene structure and function. Thus, the accumulation of genetic variation through mutations or hybridization might be tolerated more readily in allopolyploid than in diploid species.
While there is no direct evidence for this assertion, there is indirect support from
experimental data showing a higher resistance of allohexaploid wheat to irradiation compared to the diploid progenitors (Mac Key 1954, 1958; Sears 1972). Such
increase in resistance to mutation with increased ploidy level was shown to be
correlated with increased evolvability and fitness in yeast (Thompson et al. 2006).
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7.4.1 Functional Diploidization
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Allopolyploidy affects gene or protein function through a variety of mechanisms
(Table 7.4). It has been widely suggested that following polyploidization,
individual genes follow one of many possible evolutionary fates including
nonfunctionalization (deletion or pseudogenization), neofunctionalization (evolution of novel functions among alleles or homoeoalleles), generating new phenotypes, or subfunctionalization (evolution of partitioned ancestral functions among
alleles or homoeoalleles) (Lynch and Force 2000; Prince and Pickett 2002;
Chaudhary et al. 2009). Functional changes are often regulated by genetic and
epigenetic interactions among homoeoalleles and might provide the plasticity that
is required to improve the fitness and adaptation of the newly formed allopolyploid
and to increase its competitive efficiency with its parental species as well as other
plant species, leading to its successful establishment in nature. Moreover, as
discussed above, polyploidization seems to facilitate gene evolvability.
241
Increased gene dosage may lead to redundancy or in some cases may have a
deleterious effect, for example, due to the formation of an unbalanced system
(Veitia et al. 2008). Regulating gene action in a duplicated genome might be
achieved through dosage compensation (nonlinear gene dosage response). A
regulatory process that can bring redundant or unbalanced gene systems in
polyploids toward a diploid-like mode of expression is functional diploidization
(Ohno 1970). Functional diploidization is the process whereby existing genes in
multiple doses can be eliminated, become inactive via mutations (base substitution, insertion, or deletion leading to pseudogenization) or diverted to new
functions.
Examples of functional diploidization in polyploid wheat involve mainly genes
that code for structural or storage proteins, e.g., histones, subunits of tubulins,
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subunits of glutenins and gliadins, and ribosomal RNA (and possibly also tRNA).
In such genes, expression of all homoeoalleles might be redundant and even
deleterious, due to overproduction and inefficiency. Also, activity of all homoeoalleles may produce intermediate phenotypes in several traits that decrease the
viability of the plants. In this case, traits controlled by genes from only one
genome may have a higher adaptive value. It is, therefore, expected that such gene
loci would have been targets for genetic diploidization. In hexaploid wheat, intergenomic suppression, as seen by the disappearance of a storage protein subunit,
was observed immediately upon formation of a wheat allohexaploid (genome
BBAADD; see Galili and Feldman 1984; Galili et al. 1986). This is a common way
to instantaneously reduce the negative effect of overproduction and inefficiency of
genes that exist in super-optimal dose. Fascinatingly, suppression was reversible:
the storage protein reappeared upon extraction of the tetraploid BBAA genomes
and disappeared when the D genome was added. Similarly, attempts to transfer a
leaf-rust resistance gene from tetraploid to hexaploid wheat failed because of a
suppressor gene that was mapped to the D genome (Kerber and Green 1980).
Intergenomic suppression of disease resistance genes is a common phenomenon as
was noticed in natural and in several newly formed allopolyploids (Anikster, Y.,
Manisterski, J., and Feldman, M., unpublished data). Comparable results were
obtained by Dhaliwal and co-workers (Aghaee-Sarbarzeh et al. 2001) who found
that in Triticum durum-Aegilops amphiploids dominant leaf rust and stripe rust
resistance genes from the Aegilops parents were suppressed by genes on the AB
genomes of the wheat parent. Another well-studied example of intergenomic
suppression is the silencing of rye ribosomal RNA genes in the presence of the
wheat genome. Cytosine methylation is involved in this silencing, as suggested by
reactivation of the rye ribosomal RNA genes upon treatment with 5-aza-cytidine
and analyzed by the use of methylation sensitive/insensitive isoschizomers
(Houchins et al. 1997).
Recent analysis of the sequences of wheat group 1 chromosomes shows significant deviations from synteny with many of the nonsyntenic genes representing
pseudogenes (Wicker et al. 2011). Part of this pseudogenization might have
occurred after polyploidization as suggested from the analysis of the Q-factor. A
recent study shows that a combination of mutations in Q genes contributed to the
domestic spike phenotype, namely nonfragile, soft glumes, and free threshing
(Zhang et al. 2011). The mutation with the most significant phenotypic effect is an
amino acid substitution in the protein coded by the 5A locus, while other mutations, such as pseudogenization of the locus on 5B or subfunctionalization of the
locus on 5D, also contributed to the domestication phenotype, but to a lesser extent
(Zhang et al. 2011). Remarkably, these mutations occurred after polyploidization.
The Hardness (Ha) gene constitutes another example of genetic diploidization,
through gene deletion, in polyploid wheat (Chantret et al. 2005).
Genetic diploidization might also be achieved through epigenetic control.
Epigenetic silencing can be brought about via cytosine methylation of DNA
sequences (Kashkush et al. 2002) or chromatin modifications or remodeling as
well as the activity of small RNA molecules (Kenan-Eichler et al. 2011). Gene
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silencing can also be achieved via novel regulatory interactions such as intergenomic suppression (Galili and Feldman 1984). Shaked et al. (2001) and Kashkush
et al. (2002) reported the occurrence of alterations in cytosine methylation in 13 %
of the loci affecting both repetitive DNA sequences and low-copy DNA in
approximately equal proportions. Changes in microRNAs, such as miR168 which
targets the Argonaute1 gene, were shown to occur in newly synthesized wheat
(Kenan-Eichler et al. 2011).
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and Compensation of Duplicated Gene Expression
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7.4.3 New Interactions Between Maintained Genes/Proteins
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Functional diversification of duplicated genes (subfunctionalization), i.e., differential or partitioning of expression of homoeoalleles in different tissues and/or in
different developmental stages, is also a form of genetic diploidization (Adams
et al. 2003). Subfunctionalization, an important aspect of allopolyploidy, has been
studied relatively little in allopolyploid wheat. Koebner and co-workers (Bottley
et al. 2006) found that differential expression of homoeoalleles in different plant
tissues is common in hexaploid wheat. The activity of silenced genes could be
restored in aneuploid lines, suggesting that no mutation was involved but rather
new cis–trans interactions or reversible epigenetic alterations were responsible.
The data of Bottley et al. (2006) suggest that for leaf transcripts, there is a modest
bias toward silencing of the D genome copies, but this pattern does not extend to
root transcripts. Mochida et al. (2006) also presented evidence for differential
expression of homoeoalleles in wheat and suggested that inactivation of homoeoalleles is a nonrandom effect. The molecular basis for these cases of instant
subfunctionalization remains to be determined. Similarly, subfunctionalization of
all three homoeologs of the Q-locus in hexaploid wheat was recently described
(Zhang et al. 2011).
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In allohexaploid wheat many gene loci exist in triplicate dose, and the extra gene
dosage per se may produce improved or even novel traits. Homoeoalleles may
differ from one another by allelic variation, and in this case, activity of all the
duplicated genes may produce desirable intergenomic interactions and heterotic
effects. These are mainly genes that code for functional proteins (enzymes). This
occurrence of enzyme diversity (isozymes) increases the biochemical potential of
the allopolyploids. In fact, in wheat allopolyploids the expression of most homoeoalleles coding for functional enzymes is retained (Mitra and Bhatia 1971;
Hart 1983a, b, 1987). Also, intergenomic gene interactions may be, in some cases,
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expressed in novel traits that do not exist in their parental diploids. Some of these
traits may have great adaptive value. Intergenomic gene interactions have direct
relevance also to wheat cultivation. For example, the baking quality of allohexaploid wheat (bread wheat) is due to the unique properties of its gluten—a product
derived from the combined contribution of the three genomes of hexaploid wheat,
and thus exists only at the hexaploid level. In addition, the combination of a large
number of spikelets per spike derived from T. urartu (the donor of the A genome),
with several fertile florets per spikelet originating from the donors of the B genome, facilitated the high fertility of durum and bread wheat. Likewise, merging of
Ae. tauschii (the donor of the D genome) displaying the cold hardiness phenotype
with the prolific nature of allotetraploid wheat (the donor of the A and B genomes)
enabled the expansion of wheat cultivation into colder regions. Enzyme multiplicity, derived from the activity of all homoeoalleles, increases the ability of the
allopolyploid to adapt to a wider range of environments. This might account for
the very wide distribution of wheat under cultivation—much wider than that of any
other cultivated plant.
Genome-wide reprograming in gene expression may occur in a polyploid as a
result of new interactions among regulatory factors of the parents. For example,
novel interactions between the trans factor from one species and the cis or trans
factors of the other parental species, as was shown in yeast in a cross between wide
hybrids (Tirosh et al. 2009), may account for the observed cases of gene repression
or for cases of activation via overexpression.
The consequences of allopolyploidy on gene expression have been widely
studied at the genome-wide level in several natural and synthetic allopolyploids of
Triticum. The majority of studies relied on the comparison of the expression level
in the allopolyploid to those of its parents and/or to the average of its parents,
expressed as the mid-parental value (MPV). In hexaploid wheat, Pumphrey et al.
(2009) found that approximately 16 % of the 825 analyzed genes displayed
nonadditive expression in the first generation of synthetic hexaploid wheat.
Chagué et al. (2010) analyzed 55,052 transcripts in two lines of synthetic allohexaploid wheat and found that 7 % of the genes had nonadditive expression,
while Akhunova et al. (2010) found in synthetic allohexaploid wheat that about
19 % of the studied genes showed nonadditive expression. Similar studies (He
et al. 2003) showed that the expression of a significant fraction of genes (7.7 %)
was altered in the synthetic allohexaploid T. turgidum-Ae. tauschii, and that Ae.
tauschii genes were affected much more frequently than those of T. turgidum.
Strikingly, these different studies show that deviation of gene expression in allopolyploids from their parents or average of parents appears to be a common
feature, although evaluated in various allopolyploids and using different techniques and approaches. Interestingly, silencing of the same genes was also found
in natural hexaploid wheat, i.e., in the variety Chinese Spring (He et al. 2003).
Chagué et al. (2010) suggested, based on similar gene expression patterns
observed between natural and synthetic wheat allohexaploids, that regulation of
gene expression is established immediately after allohexaploidization and maintained over generations. It is of interest to note that several genes that are silent in
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7.5 Genome asymmetry
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The rapid processes of cytological and genetic diploidization allow for the
development and occurrence of two contrasting and highly important genetic
phenomena in allopolyploid wheat that contribute to the evolutionary success of
these polyploids: (1) build up and maintenance of enduring intergenomic favorable
genetic combinations, and (2) genome asymmetry in the control of a variety of
morphological, physiological, and molecular traits, i.e., complete or principal
control of certain traits by only one of the constituent genomes. However, while
the first phenomenon was taken for granted by plant geneticists, genomic asymmetry in interspecific hybrids and allopolyploids was mainly known in ribosomal
RNA genes (reviewed in Pikaard 2000) and only recently has also been documented in other traits (Peng et al. 2003a, b; Fahima et al. 2006; Feldman and Levy
2009; Flagel et al. 2009; Rapp et al. 2009; Flagel and Wendel 2010). The phenomena of diploidization and of dominance in gene expression lead to genome
asymmetry, which is manifested in a clear-cut division of tasks among the constituent genomes of allopolyploid wheat (Levy and Feldman 2004; Feldman and
Levy 2009; Feldman et al., in press). Genome A controls morphological traits
while genome B in allotetraploid wheat and genomes B and D in allohexaploid
wheat control the reaction to biotic and abiotic factors (Tables 7.6 and 7.7). Intergenomic pairing would lead to both disruption of the linkage of the homoeoalleles that contribute to positive intergenomic interactions and segregation of
genes that participate in the control of certain traits by a single genome. Intergenomic recombination may, therefore, result in many intermediate phenotypes
that may affect, in a negative manner, the functionality, adaptability, and stability
of the allopolyploids.
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the parental species became active in the newly formed allohexaploid (He et al.
2003). Similarly, cDNA-AFLP gels also revealed several cDNAs that were
expressed only in the allopolyploids and not in the diploid progenitors (Shaked
et al. 2001; Kashkush et al. 2002).
The proportion of genes for which expression in the polyploids is different from
the average of the parents is underestimated in these studies, as most of the
technologies used do not allow separate analysis of the expression level of each of
the gene copies (homoeoalleles) and their respective contribution to overall gene
expression (Pumphrey et al. 2009; Chagué et al. 2010; Akhunova et al. 2010). It is
not possible to detect, for example, a situation where repression of one homoeoallele is compensated by the activation of the other using microarray technologies. The progress towards sequencing of wheat genomes and the promise of
next-generation technologies should allow in the future the resolution of expression at the level of individual homoeologues.
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Glumes with keels
The shape of the edge of the glumes (beaked
glumes)
Hairs at the base of every spikelet
Plant habitus
Growth habit
Autogamous behavior
Many domestication genes
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In the preceding sections, we have discussed structural changes and changes in
gene expression resulting from genetic or epigenetic phenomena. Transposable
elements (TEs) are discussed here separately due to their specific mode of action,
their abundance (up to 90 % of the wheat genomes; Sabot et al. 2005), and their
impact on both structure and expression of the genome.
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7.6.1 Transcriptional Activation of Retrotransposons in Synthetic
Wheat Polyploids
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Higher polymorphism of HMW glutenin
genes
Larger amount of repetitive sequences
Activity on nucleolar organizers
Larger number of rRNA genes
7.6 Response of Transposable Elements
to Allopolyploidization
It is now clear that some eukaryote retrotransposon promoters retain activity under
normal conditions and initiate either read-in transcripts of the transposon itself or
read-out transcripts into flanking host sequences (Vicient et al. 2001; Kashkush
et al. 2002; Nigumann et al. 2002; Kashkush et al. 2003; Kashkush and Khasdan
2007). Following allopolyploidization events in wheat, the steady-state level of
expression of LTR retrotransposons was massively elevated (Kashkush et al. 2002,
2003, unpublished data), similarly to what was observed in synthetic Arabidopsis
allopolyploid hybrids (Madlung et al. 2005). In addition, the transcriptional
activity of a LTR element termed Wis2-1A (Lucas et al. 1992) leads to the production of read-out transcripts toward flanking host DNA sequences, a process that
occurred in a genome-wide manner (Kashkush et al. 2003). In many cases, these
read-out transcripts were associated with the expression of adjacent genes,
depending on their orientation: knocking down or knocking out the gene product if
the read-out transcript was in the antisense orientation relative to the orientation of
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Regulation of ecological adaptation
Double the number of disease resistance
genes
Contains more stress–related genes?
Higher polymorphism of molecular markers
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Inflorescence morphology
Free caryopsis
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Table 7.6 Genome asymmetry in the control of various traits in the wild allotetraploid wheat,
T. turgidum subsp. dicoccoides (genome BBAA)
Traits under control of Genome A
Traits under control of Genome B
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Table 7.7 Genome asymmetry in the control of agronomic traits in domesticated durum (genome BBAA) and bread wheat (genome BBAADD)a
Traits
Traits under control of
br B1 on 3BS
tg2 on 2BS
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Rht B1 on 4BS; Rh4
on 2Bl;
Ga1, Ga3 on 4BS
W1 on 2BS
W1I on 2BS; W3I on
1BL
ms1 on 4BS
Ph1 on 5BL
Ne1 on 5BL; Ne2 on
2BS
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Hybrid chlorosis
Ch1 on 2A
Aluminum tolerance
Boron tolerance
Low cadmium uptake
Iron deficiency
Herbicide response
Difenzoquat insensitivity
Chlortoluron insensitivity Imi3 on 6AL
Imidazolinone resistance
Response to photoperiod
Response to vernalization Vrn-A1 on
5AL
Response to salinity
Frost resistance
Number of resistance
genes to diseases and
pests
a
Fr1 on 5AL
45
tg1 on 2DS
Rht D1 on 4DS; Rht8 on
2DL;
Rht5 on 3BS; Rht9 on
7BS; Rht13 on
7BS
Gpc B1 on 6BS
Pro1 on 5DL; Pro2 on 5Ds
Ha on 5DS
Pin D1 on 5DS
TE
Grain protein content
Grain hardness
Puroindolines and grain
softness protein
Gibberellic acid response
Waxiness
Epistatic inhibitors of
waxiness
Male sterility
Ms3 on 5AS;
ms5 on 3A
Pairing homoeologous
Hybrid necrosis
Genome D
F
Genome B
D
Genome A
Inflorescence morphology
Elongated glumes
Eg P1 on 7AL
Branched spikes
Bh on 2AS
Nonbrittle rachis
br A1 on 3AS
br A2 on 2A
Nontenacious glume (lax
glume)
Reduce plant height
Rht7 on 2A;
Rht12 on
5AL;
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Bo1 on 7BL
Cdu1 on 5BL
Fe2 on 7BS
Dfg 1 on 2BL
Su1 on 6BS
Imi2 on 6BL
Ppd-B1 on 2BS
Vrn-B1 on 5BL;
Vrn-B3 on 7BS
88
Ga2 on 4DS
W2 on 2DS (?)
W2I on 2DS
Ms2 on 4DS; Ms4 on 4DS
Ph2 on 3DS
Ch2 on 3DL
Alt2 on 4DL
Fe1 on 7DL
Imi1 on 6DL
Ppd-D1 on 2DS
Vrn-D1 on 5DL; Vern-D4
on 5DL;Vern-D5 on
5DL
Kna1 on 4DL
Fr2 on 5DL
51
Data from the 2008 Wheat Gene Catalogue (http://wheat.pw.usda.gov/GG2/index.shtml)
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7.6.2 Massive Methylation of TE-Adjacent DNA Sequences
Following Allopolyploidization
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Alterations in the genomic methylation patterns following allopolyploidization
have been examined in several polyploid systems, including Arabidopsis (Madlung
et al. 2002; Belzile et al. 2009), Spartina (Salmon et al. 2005; Parisod et al. 2009),
Brassica (Lukens et al. 2006; Wang et al. 2009), and wheat (Shaked et al. 2001).
The methylation alterations are either hyper- or hypomethylation, depending on the
sequence analyzed, and are reproducible. Recent studies in wheat have investigated
in detail the methylation of CCGG sites flanking several TE families (Kraitshtein
et al. 2010; Yaakov and Kashkush 2011a, b; Zhao et al. 2011). In one study
(Kraitshtein et al. 2010), transposon methylation display (TMD) analysis was
applied (see Kashkush and Khasdan 2007) to analyze a terminal-repeat retrotransposon in miniature (TRIM), termed Veju, in Triticum turgidum ssp. durum
(genome AABB) and Aegilops tauschii (genome DD), and the first four generations
of the derived allohexaploid. It was estimated that over 50 % of the CCGG sites
flanking Veju elements showed altered TMD patterns in the first four generations of
the newly formed allohexaploid. Hypomethylation of Veju-flanking CCGG sites
was predominant in the first generation of the newly formed allohexaploid, while
hypermethylation was predominant in subsequent generations. This might indicate
reduced Veju transcriptional activity after the third generation of the synthetic
allohexaploid. A similar pattern of hypomethylation of Veju elements was also
observed in the first three generations of a synthetic allotetraploid that was derived
from a cross between Ae. sharonensis (genome SlSl) and T. monococcum ssp.
aegilopoides (genome AmAm) (Yaakov and Kashkush 2011b). However, unlike in
the synthetic allohexaploid, Veju elements remained hypomethylated up to the
fourth generation of the synthetic allotetraploid. In support of these studies, Zhao
et al. (2011) observed massive methylation changes around Veju in three different
combinations of newly formed wheat allohexaploids.
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the gene transcript (such as the iojap-like gene) or overexpressing the gene if the
read-out transcript was in the sense orientation (such as the puroindoline-b gene).
The mechanisms by which transcriptional activation of TEs influences the
expression of neighboring genes are poorly understood. In some cases, the correlation between the reduction of the sense expression of the gene and the production of the antisense strand that initiated from the adjacent transposon promoter
(Kashkush et al. 2003; Puig et al. 2004) might indicate that post-transcriptional
gene silencing is a major mechanism for inactivating adjacent genes. Recent
studies on tracking methylation changes around a LTR retrotransposon in the first
four generations of a newly formed wheat allopolyploid (Kraitshtein et al. 2010)
may indicate that this read-out activity is restricted to the first generations of the
nascent polyploid species.
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The prevalence of TEs and their inherent sequence similarity make them a prime
target for illegitimate and nonhomologous recombination. TEs have been shown to
undergo rearrangements following allopolyploidization in Spartina (Parisod et al.
2009), tobacco (Petit et al. 2010), and Triticale (Bento et al. 2008). Recent data for
synthetic allohexaploid wheat indicate that rearrangements of retrotransposoncontaining sequences occur rapidly and reproducibly (repeated in independently
newly formed allopolyploid lines) in the first generations following polyploidization. In addition, a change in the methylation status (usually hypomethylation) in
the first generation was followed by deletion of retrotransposon-containing
sequences in subsequent generations (Kraitshtein et al. 2010). These data suggest a
correlation between methylation and post-allopolyploidization rearrangements that
occur via a mechanism that has yet to be identified. One possible explanation is
that hypomethylation confers an open chromatin structure to the TE sequences,
which exposes these demethylated elements to be targeted for deletion by the host.
There is evidence that small RNAs corresponding to Veju elements might play a
pivotal role in Veju methylation in the newly formed wheat allohexaploid (KenanEichler et al. 2011).
Despite the altered methylation status and transcriptional activation of TEs
following allopolyploidization, there are very few reports on the transpositional
activity of transposons. Madlung et al. (2005) showed both methylation alterations
and limited transpositional activation of a Sunfish transposon in polyploid
Arabidopsis. Petit et al. (2010) reported an increase in the copy number of a Tnt1
retrotransposon in allotetraploid tobacco. No transposition bursts were reported in
Spartina (Parisod et al. 2009) or in wheat (Kashkush et al. 2003; Kraitshtein et al.
2010; Yaakov and Kashkush 2011a). These reports suggest that the transpositional
activity of TEs following allopolyploidization might be restricted to specific TE
families (Parisod et al. 2010). Recently, it was shown that the immense loss of
Veju sequences in the first generation of the synthetic allohexaploid is probably
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The methylation patterns of three TEs [Balduin (belonging to the CACTA
superfamily), Apollo (belonging to the MuDR/Foldback superfamily), and Thalos
(a stowaway-like MITE belonging to the Tc1/mariner superfamily)] have also been
analyzed in allopolyploid wheat. The CCGG sites flanking the three elements
underwent massive hypermethylation in the first four generations of the synthetic
allohexaploid (Yaakov and Kashkush 2011a, b), while they underwent massive
hypomethylation in the first four generations of the synthetic allotetraploid
(Yaakov and Kashkush 2011b). The massive hypermethylation of these elements
in the synthetic allohexaploid might be connected to the lack of transpositional
activity (Yaakov and Kashkush 2011a). It is important to mention that transcriptional activation of LTR retrotransposons does not correlate with transpositional
activity (Kashkush et al. 2003).
495
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7.7 Concluding Remarks
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The studies reviewed above indicate that, in the wheat group, hybridization and
chromosome doubling induce a burst of genomic alterations, some of which could
not occur at the diploid level. Some of these changes might improve the ability of
the newly formed allopolyploids to survive in nature and to compete with their
parental species, corresponding thus to phenotypic and adaptive novelty. Other
changes are probably deleterious. TEs seem to play an important role in the
various responses to hybridization and polyploidization due to their abundance and
also due to their tendency to be dysregulated as a result of genomic shocks. The
balance between the beneficial and deleterious changes associated with allopolyploidization is probably what determines the fate of the nascent species.
The formation of an allopolyploid species is accomplished rapidly via the
combined processes of hybridization and genome doubling, but its establishment
in nature as a successful species probably depends on a high degree of plasticity
that enables it to overcome potential incompatibilities and to gain new traits. The
studies reported here suggest that wheat can achieve genomic plasticity through
the induction of a series of cardinal nonadditive genomic changes. Some changes,
genetic and epigenetic, are rapid and nonMendelian, occurring during or immediately after the formation of the F1 hybrid or the allopolyploid (revolutionary
changes). Other changes occur sporadically over a long time period during the life
of the allopolyploid species (evolutionary changes). From a population point of
view, the chance of a new individual, such as a nascent hybrid/allopolyploid, to
establish itself as a new species is almost nil, unless it has some increased fitness
over its parents. This fitness advantage must be manifested within a few generations of formation or the new species will rapidly be extinct. The revolutionary
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followed by retrotransposition in subsequent generations, a process that causes
new insertions to accumulate in allohexaploids (Kraitshtein et al. 2010). The same
study also suggests that these new insertions are targeted for methylation. Methylation of the new Veju elements protects the genome from deleterious transposon
insertions. Investigating the scale of eliminated DNA sequences, including TE
sequences, by identifying the deletion breakpoints will allow a better understanding of the mechanism(s) involved and of the nature of the connection between
methylation and rearrangements.
In summary, different classes of wheat TEs appear to respond differently to the
allopolyploidization event. Table 7.8 summarizes the type of response of 12 different class I and class II elements. It can be seen clearly that epigenetic response,
mainly methylation changes, is a common factor for all studied TEs, while genetic
response that includes rearrangements and/or transpositions is restricted to specific
TE families. Transcriptional data for most studied TEs are still lacking (Table 7.8).
However, there is a good basis for suggesting a connection among methylation
changes in TEs with alteration in their expression patterns.
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Table 7.8 Summary of the genetic and epigenetic responses of several TE families to allopolyploidization in wheat
Reference
Family
TE
Genetic and epigenetic alterations in synthetic
allopolyploids (compared to parental lines)a
Methylation Transcription Rearrangementb Transpositionc
4
4
4
Copia
Angela
BARE1
Wis2-1
A
4
4
NA
NA
NA
9
NA
4
9
NA
NA
9
9
MuDR/
Foldback
Apollo
4
NA
4
NA
a
b
c
578
579
580
581
582
583
584
585
4
9
9
9
9
9
9
4
4
4
4
?
4
?
?
EC
NA
NA
NA
NA
Kraitshtein
et al.
(2010)
KenanEichler
et al.
(2011)
Zhao
et al.
(2011)
Unpublished
Zhao et al.
(2011)
Kashkush
et al.
(2003)
Unpublished
Yaakov and
Kashkush
(2011a)
Yaakov and
Kashkush
(2011a)
Yaakov and
Kashkush
(2011a)
Unpublished
Unpublished
Unpublished
Unpublished
4 altered, 9 no change, ? not validated, NA data not available
Include deletion and/or insertion
Typical TE transposition
changes described here may contribute to the establishment of the new species.
Instantaneous elimination of sequences from one genome in the newly formed
allopolyploids increases the divergence of the homoeologous chromosomes, and
thus leads to exclusive intragenomic pairing that improves fertility. Mechanisms
such as loss of deleterious genes (e.g. genetic incompatibilities) or positive dosage
effects or new intergenomic heterotic interactions may all rapidly increase the
fitness of the nascent species. The evolutionary changes, on the other hand, contribute to the build-up of genetic variability and thus increase adaptability, fitness,
competitiveness, and colonizing ability. It is clear that most hybridization events in
UN
577
4
4
CO
RR
Stowaway- Thalos
like
MITE
Eos
Minos
Oleus
Fortuna
9
D
Sabrina 4
Balduin 4
9
9
TE
Gypsy
CACTA
?
F
Veju
PR
OO
TRIM
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618
619
620
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623
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625
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7 Genomic Plasticity in Polyploid Wheat
Metadata of the chapter that will be visualized in
SpringerLink
Book Title
Polyploidy and Genome Evolution
Series Title
Chapter Title
Maize (Zea Mays) as a Model for Studying the Impact of Gene and Regulatory Sequence Loss Following
Whole-Genome Duplication
Copyright Year
2012
Copyright HolderName
Springer-Verlag Berlin Heidelberg
Corresponding Author
Family Name
Freeling
Particle
Given Name
Michael
Suffix
Author
Division
Department of Plant and Microbial Biology
Organization
University of California
Address
Berkeley, USA
Email
freeling@uclink.berkeley.edu
Family Name
Schnable
Particle
Given Name
James C.
Suffix
Division
Department of Plant and Microbial Biology
Organization
University of California
Address
Berkeley, USA
Email
Abstract
Modern maize (2n = 20) is functionally diploid, and its chromosomes pair normally, forming 10 bivalents
during meiosis. Sufficient genomic rearrangement has occurred that no two maize chromosomes are
homologous across their entire lengths. Yet comparisons of genetic maps, duplicate gene sequences, and later
genome assemblies revealed maize is descended from a polyploid ancestor which lived 5–12 million years
ago. In the time since that polyploid ancestor lived 8,000–9,000 genes conserved at syntenic positions in other
grass species have been reduced to single copy in maize while 4,000–5,000 genes are still retained as
homologous gene pairs. The consequences of this polyploidy are continuing to resolve in modern maize
accessions. With a wide range of data sets generated by an active research community, maize is an unparalleled
model for the in silico study of the changes in genome structure, gene content, and gene regulation that a
successful polyploidy brings about in a plant lineage.
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Abstract Modern maize (2n = 20) is functionally diploid, and its chromosomes
pair normally, forming 10 bivalents during meiosis. Sufficient genomic rearrangement has occurred that no two maize chromosomes are homologous across
their entire lengths. Yet comparisons of genetic maps, duplicate gene sequences,
and later genome assemblies revealed maize is descended from a polyploid
ancestor which lived 5–12 million years ago. In the time since that polyploid
ancestor lived 8,000–9,000 genes conserved at syntenic positions in other grass
species have been reduced to single copy in maize while 4,000–5,000 genes are
still retained as homologous gene pairs. The consequences of this polyploidy are
continuing to resolve in modern maize accessions. With a wide range of data sets
generated by an active research community, maize is an unparalleled model for the
in silico study of the changes in genome structure, gene content, and gene regulation that a successful polyploidy brings about in a plant lineage.
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Maize (Zea Mays) as a Model for Studying
the Impact of Gene and Regulatory
Sequence Loss Following Whole-Genome
Duplication
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8.1 Background on the Maize Polyploidy
Suspicion of a polyploid origin for maize first came from the prevalence of
duplicate mutants identified at unlinked locations throughout the genome; these
have been observed since the early days of maize genetics (Rhoades 1951). Over
time, the evidence that the maize lineage was descended from an ancient polyploid
grew to include the arrangement of duplicate genes and markers in similar orders
on multiple maize chromosomes reported in a number of genetic maps developed
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J. C. Schnable M. Freeling (&)
Department of Plant and Microbial Biology, University of California, Berkeley, USA
e-mail: freeling@uclink.berkeley.edu
P. S. Soltis and D. E. Soltis (eds.), Polyploidy and Genome Evolution,
DOI: 10.1007/978-3-642-31442-1_8, Springer-Verlag Berlin Heidelberg 2012
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8.2 Timing of the Maize Polyploidy
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Unlike other important crops species, such as bread wheat and potato, the maize
polyploidy was not directly associated with domestication, but occurred millions
of years earlier. Analysis of divergence between duplicate genes on opposite maize
subgenomes—known as homeologs—has placed the split of the two progenitor
genomes found within modern maize at *12 million years before present (Swigoňová et al. 2004). The date of divergence between the two subgenomes of maize
does not necessarily reflect how long ago the actual event of polyploidization
occurred, as fertile allopolyploids can form between related species that have been
evolving independently for millions of years. However, based on a single case of
gene conversion it can be concluded that the two subgenomes of maize have
shared a single nucleus for at least 5 million years (Swigoňová et al. 2004).
Recent evidence has allowed the phylogenetic placement of the maize tetraploidy to be narrowed to a discrete interval within the diversification of the Andropogoneae—the tribe of grasses within which maize is placed (Mathews et al.
2002). Phylogenetic trees of the homologous genes zfl1 and zfl2 show that these
whole genome duplicates had already diverged in the common ancestor of the
genus Zea (maize and teosinte) and the sister genus Tripsacum (Bomblies and
Doebley 2005). Comparison of genetic maps in maize and sorghum prior to the
publication of complete genome sequences for these species demonstrated that
sorghum—and by extension other relatives within the ‘‘core’’ Andropogoneae—
did not share the maize polyploidy (Wei et al. 2007). Following the publication of
complete genome assembles for both maize (Schnable et al. 2009) and sorghum
(Paterson et al. 2009), a study of thousands of maize homologous gene pairs found
that both maize subgenomes appear equally diverged from sorghum (Woodhouse
et al. 2010). Given these constraints, the phylogenetic placement of the maize
tetraploidy can be inferred (Fig. 8.1).
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during the 1980s (Goodman et al. 1980; Wendel et al. 1986; Helentjaris et al.
1988). With the availability of abundant sequence data for cloned duplicate genes,
it became possible to definitively classify maize as an ancient polyploid (Gaut and
Doebley 1997).
In the absence of diploid species showing higher genetic similarity to one maize
subgenome or the other, it will be impossible to conclusively prove maize is not an
ancient autopolyploid. The most closely related genus to Zea, Tripsacum, is descended from the same polyploid ancestor (Bomblies and Doebley 2005). The most
closely related species with a sequenced genome is Sorghum bicolor, which shows
an equal divergence from both subgenomes of maize (Swigoňová et al. 2004;
Woodhouse et al. 2010). Almost no molecular data are available for the most
closely related, and apparently diploid, genera to maize, Elionurus (13 sequences
in GenBank) and Coelorachis (14 sequences in GenBank). The question of autoversus allo-polyploidy may never be conclusively answered for maize.
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8 Maize (Zea Mays) as a Model for Studying the Impact of Gene
Fig. 8.1 A highly pruned tree of grass species. Branches are scaled by modal synonymous
substitutions per site among all syntenic orthologs as measured by SynMap and QuotaAlign
(Lyons et al. 2008b; Tang et al. 2011). The position of the branch between Zea and Tripsacum is
only approximated, as the genome of Tripsacum has not yet been sequenced
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While all plant genomes sequenced to date have experienced at least one ancient
polyploidization (Paterson et al. 2010), in the vast majority of cases, duplicate
regions have been too heavily rearranged to reconstruct which regions can be
grouped together as originating from the same parental genome. Such was not the
case with maize. The polyploid ancestor of maize possessed 20 chromosomes, 2
equivalent to each of the 10 chromosomes of sorghum. Researchers could assign
segments of the ten chromosomes of modern maize back to each of these ancestral
chromosomes even before the completion of either the sorghum or maize genomes
(Wei et al. 2007). With the publication of the completed maize and sorghum
genomes, the same reconstruction can now be carried out using web-based tools by
anyone with a fondness for puzzles (Fig. 8.2).
A cursory examination of Fig. 8.2 will reveal for any position in the sorghum
genome there are two syntenic orthologous regions in maize, while for any
position in the maize genome there is only one syntenic orthologous region in
sorghum. In no case are the two regions orthologous to the same region in sorghum
are present on the same maize chromosome. In 15 of the 20 inferred ancestral
chromosomes, all segments are contained within a single chromosome of modern
maize. In the remaining five cases, orthologous regions belonging to a single
inferred ancestral maize chromosome are split between two—or in one case
three—modern maize chromosomes. Fusion of chromosomes has often resulted
from the insertion of one chromosome in between the arms of another—often
linked with inversions of whole chromosome arms—as previously observed in the
reduction of the Brachypodium distachyon genome to five chromosomes from the
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8.3 Changes in Genome Arrangement Following Polyploidy
in Maize
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Fig. 8.2 A dotplot generated using SynMap (Lyons et al. 2008b) to compare the arrangement of
orthologous regions of the sorghum (x-axis) and maize (y-axis) genome. Each dot represents a
pair of homologous genes in maize and sorghum within a syntenic region, and each dot is color
coded by synonymous substitutions per site between those two genes. In general, purple lines
represent syntenic orthologous regions between the maize and sorghum genomes whilegreen
diagonals represent homologous syntenic regions from a more ancient whole-genome duplication
shared by all grass species. An interactive version of this graphic can be regenerated using the
following link: http://genomevolution.org/r/3vpl
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8.4 Ancient Gene Loss in Maize
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inferred ancestral number of 12 (The International Brachypodium Initiative 2010).
Researchers can search for differences across whole pairs of reconstructed maize
ancestral chromosomes, and, by using differential gene loss as a mark to distinguish chromosome copies, even compare entire subgenomes (Schnable et al.
2011).
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Being able to compare pairs of complete ancestral chromosomes enabled a thorough investigation of an odd observation reported for Arabidopsis paleopolyploidy: the loss of genes was biased between homologous regions of the genome
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8.5 Ongoing Gene Loss in Maize
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(‘‘biased fractionation’’), with one copy of each region retaining more total genes
than the other (Thomas et al. 2006). Depending on the definition of a functional
gene, between 4,000 and 5,000 homologous gene pairs are retained within the
maize genome (Schnable et al. 2009; Woodhouse et al. 2010). Another
8,000–9,000 genes conserved at syntenic location in both rice and sorghum have
fractionated back to a single copy in maize (Schnable et al. 2011). The same
fractionation bias was observed across each of the ten pairs of ancestral maize
chromosomes, with one chromosome copy retaining 70–90 % of the ancestral
grass gene content and the other chromosome copy retaining only 40–60 %. By
grouping high gene loss ancestral chromosomes together into one parental subgenome and low gene loss ancestral chromosomes into the other parental subgenome and using the wealth of maize RNA-seq data being produced by other
research projects, it was shown that genes on the subgenome with low gene loss,
referred to as maize1, tend to be expressed at higher levels than their duplicates on
the subgenome with high gene loss, or maize2 (Schnable et al. 2011). This
inequality of expression between parental subgenomes has previously been
reported in allopolyploid cotton—among other species—where it was termed
‘‘genomic dominance’’ (Flagel and Wendel 2010).
The correlation between bias in gene expression levels and bias in gene loss
rates suggests a simple explanation for the bias in gene loss rates observed in all
ancient polyploid species studied to date (Sankoff et al. 2010). The deletion of the
gene copy that contributed the majority of total gene pair expression should be
more likely to have a functional impact than the deletion of the gene copy which
contributes the minority of total gene pair expression. Therefore, even if the base
rate of gene deletion is equal on both subgenomes, null alleles of more highly
expressed gene copies would be more likely to be purged from the population by
purifying selection. Over time, deletion of redundant gene copies would tend to
cluster on the less expressed subgenome. In support of this hypothesis, it was noted
that known maize morphological mutants are significantly more likely to result
from the disruption of genes on the high-expression subgenome than expected
given the overall distribution of expressed genes between the subgenomes (Schnable and Freeling 2011).
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In addition to ancient fixed patterns of gene loss, fractionation continues within
maize today, with genes present in some maize inbreds lost in other accessions.
Concurrent with the publication of the maize genome, the first report emerged
describing a high incidence of presence–absence variation (PAV) between different
maize inbreds. A comparison of two of the most-studied maize lines, B73 and
Mo17 (the former being the line used to generate the reference genome), determined that thousands of sequences were present in the former but entirely absent
from the latter, including at least 180 single-copy genes (Springer et al. 2009).
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The loss of genes is the result of the deletion of sequence from the genome. Given
the extensive observation of chromosomal mispairing, rearrangements, and partial
or complete aneuploidy in synthetic and recent natural polyploids, it might be
expected that much duplicate gene loss is the result of large deletions which
remove whole chromosomal segments from one subgenome. The fact that only a
portion of the genomes of ancient polyploid species such as rice—65.7 % of the
genes covered by duplicated blocks (Yu et al. 2005)—and Arabidopsis—89 % of
genes covered (Bowers et al. 2003)—would seem consistent with this expectation.
However, the pattern of gene loss in maize is not consistent with large deletions
following whole-genome duplication. A search for regions of the sorghum genome
represented by only one syntenic orthologous region within maize revealed only
one putative deletion of [30 genes (Schnable et al. 2012). A comparison of the
patterns of gene loss observed in duplicate regions of the maize genome to simulations assuming different lengths of sequence deletion found that the pattern
observed is consistent with [85 % of deletions removing only a single gene, or a
portion there of, with the remainder of deletions removing two, or occasionally
three, adjacent genes (Woodhouse et al. 2010). The present genome of maize is
littered with the partially deleted fragments of homologous genes (Fig. 8.3a).
The prevalence of short direct repeats flanking deletions within these fragmentary genes indicates the loss of genes in polyploids is a result of intrastrand
non-homologous recombination: short direct repeats found by chance throughout
the genome pair with each other, splicing out any intervening sequence, and
leaving a single copy of the repeat sequence (Woodhouse et al. 2010).
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A follow-up study which examined 33 inbreds and accessions of wild teosinte
identified 3,410 high-confidence genes in the genome of B73 which had been lost in
one or more of these lines—a total that represents more than 10 % of all highconfidence genes present in the maize genome (Swanson-Wagner et al. 2010).
While most PAV in maize was initially observed using comparative genomic
hybridization to microarrays, the same variation in gene content was observed in a
recent study that resequenced six inbreds using Illumina short read technology
(Lai et al. 2010).
While many genes which exhibit PAVs between B73 and other maize inbreds
possess no syntenic orthologs in other grass species and may have recently inserted
into their current locations, 4–6 % of maize genes with syntenic orthologs in both
sorghum and rice—indicating these genes have been functionally conserved for at
least 50 million years—are also involved in PAV between diverse maize lines
(Schnable et al. 2011). Among genes where both homologous are still retained in
B73, genes on the ‘high-gene-loss, low-expression’ subgenome (maize2) are more
likely to be involved in PAV (Swanson-Wagner et al. 2010; Schnable et al. 2011).
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Fig. 8.3 a GEvo panel (Lyons et al. 2008a) illustrating an example of a gene conserved in rice,
sorghum, and one of two maize subgenomes (maize1), while on the second maize subgenome
(maize 2) only the 5’ and 3’ ends of the gene remain conserved. The colored boxes mark regions
of sequence similarity, as determined by blastn, between the orthologous genomic regions in this
figure. An interactive version of the same panel can be regenerated at the following link: http://
genomevolution.org/r/3vbu. b A second GEvo panel comparing a single sorghum gene to its two
coorthologs in maize. Purple rectangles represent functionally constrained noncoding sequences
identified by the comparison of this gene to its syntenic ortholog in rice. The red box highlights a
cluster of conserved noncoding sequences conserved upstream of one maize gene but lost from
the promoter of the other. c A comparison of the relative expression of these two maize genes in a
wide range of tissues using RNA-seq data produced by a number of maize research groups (Wang
et al. 2009; Jia et al. 2009; Li et al. 2010; Davidson et al. 2011; Waters et al. 2011)
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8.7 Future Prospects
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The mechanism responsible for the unequal expression of duplicate genes from
different parents in polyploid species remains unknown. With an extensive collection
of well-characterized mutants, including knockouts of a variety of epigenetic
mechanisms, maize is an excellent system for the investigation of this inexplicable
behavior. If the whole-genome duplication in maize was indeed the result of allopolyploidy, the identification of a wild species more closely related to one maize
subgenome than the other remains an exciting possibility. The wealth of RNA-seq
data being generated in maize for unrelated purposes creates an opportunity to
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Fractionation and sequence deletion are not confined to only the protein-coding
regions of genes. Sequence deletion, presumably by the same mechanism of intrastrand non-homologous recombination, can also remove conserved noncoding regulatory sequences from one of two duplicate copies of a maize gene (Fig. 8.3b).
These genes often show different patterns of tissue-specific expression in existing
maize RNA-seq data sets, allowing researchers to develop testable hypotheses about
the function of specific regulatory sequences (Fig. 8.3c) (Freeling et al. 2012).
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Acknowledgments We thank Vincent Li, a high school intern in the Freeling lab from Project
SEED, for identifying the gene fragment shown in Fig. 8.3 and Addie M. Thompson for critical
reading of an early version of this text. Funding provided by NSF Plant Genome Research
Program grant 0701871 to MF and a Chang-Lin Tien Graduate Fellowship to JCS.
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Bomblies K, Doebley JF (2005) Molecular evolution of FLORICAULA/LEAFY orthologs in the
Andropogoneae (Poaceae). Mol Biol Evol 22:1082–1094
Bowers JE et al (2003) Unravelling angiosperm genome evolution by phylogenetic analysis of
chromosomal duplication events. Nature 422:433–438
Davidson RM et al (2011) Utility of RNA sequencing for analysis of maize reproductive
transcriptomes. Plant Genome 4:191–203
Flagel LE, Wendel JF (2010) Evolutionary rate variation, genomic dominance and duplicate gene
expression evolution during allotetraploid cotton speciation. New Phytol 186:184–193
Freeling M et al (2012) Fractionation mutagenesis and similar consequences of mechanisms
removing dispensable or less-expressed DNA in plants. Curr Opin Plant Biol, Advance Online
Publication
Gaut BS, Doebley JF (1997) DNA sequence evidence for the segmental allotetraploid origin of
maize. Proc Natl Acad Sci USA 94:6809–6814
Goodman MM et al (1980) Linkage relationships of 19 enzyme Loci in maize. Genetics 96:697–710
Helentjaris T et al (1988) Identification of the genomic locations of duplicate nucleotide sequences
in maize by analysis of restriction fragment length polymorphisms. Genetics 118:353–363
Jia Y et al (2009) Loss of RNA–dependent RNA polymerase 2 (RDR2) function causes
widespread and unexpected changes in the expression of transposons, genes, and 24-nt small
RNAs. PLoS Genet 5:e1000737
Lai J et al (2010) Genome-wide patterns of genetic variation among elite maize inbred lines. Nat
Genet 42:1027–1030
Li P et al (2010) The developmental dynamics of the maize leaf transcriptome. Nat Genet
42:1060–1067
Lyons E et al (2008a) Finding and comparing syntenic regions among Arabidopsis and the
outgroups papaya, poplar, and grape: CoGe with Rosids. Plant Physiol 148:1772–1781
Lyons E et al (2008b) The value of non-model genomes and an example using SynMap within
CoGe to dissect the hexaploidy that predates the Rosids. Trop Plant Biol 1:181–190
Mathews S et al (2002) Phylogeny of Andropogoneae inferred from phytochrome B, GBSSI, and
ndhF. Int J Plant Sci 163:441–450
Paterson AH et al (2010) Insights from the comparison of plant genome sequences. Annu Rev
Plant Biol 61:349–372
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investigate how the expression patterns of homologous gene pairs in maize have
diverged in silico. Soon, it may also be possible to study how the functions of
duplicate genes have diverged in the 5–12 million years since the maize polyploidy in
silico, as high-resolution genome-wide associate studies begin to identify the loci
responsible for variation in a wide range of maize phenotypes. In addition to a higher
likelihood of being lost entirely, has the reduced importance of less expressed genes
on the non-dominant subgenome also given these genes greater freedom for innovation, even if their new role comes at the express of their ancestral function? We
predict that higher levels of neofunctionalization will be observed on the nondominant subgenome, but this hypothesis remains untested.
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Paterson AH et al (2009) The sorghum bicolor genome and the diversification of grasses. Nature
457:551–556
Rhoades MM (1951) Duplicate genes in maize. Am Nat 85:105–110
Sankoff D et al (2010) The collapse of gene complement following whole genome duplication.
BMC Genomics 11:313
Schnable JC et al (2011) Differentiation of the maize subgenomes by genome dominance and
both ancient and ongoing gene loss. Proc Natl Acad Sci USA 108:4069–4074
Schnable JC et al (2012) Genome-wide analysis of syntenic gene deletion in the grasses. Genome
Biol Evol 4:265–277
Schnable JC, Freeling M (2011) Genes identified by visible mutant phenotypes show increased
bias toward one of two subgenomes of maize. PLoS ONE 6:e17855
Schnable PS et al (2009) The B73 maize genome: complexity, diversity, and dynamics. Science
326:1112–1115
Springer NM et al (2009) Maize inbreds exhibit high levels of copy number variation (CNV) and
presence/absence variation (PAV) in genome content. PLoS Genet 5:e1000734
Swanson-Wagner RA et al (2010) Pervasive gene content variation and copy number variation in
maize and its undomesticated progenitor. Genome Res 20:1689–1699
Swigoňová Z et al (2004) Close split of sorghum and maize genome progenitors. Genome Res
14:1916–1923
Tang H et al (2011) Screening synteny blocks in pairwise genome comparisons through integer
programming. BMC Bioinf 12:102
The International Brachypodium Initiative (2010) Genome sequencing and analysis of the model
grass Brachypodium distachyon. Nature 463:763–768
Thomas BC et al (2006) Following tetraploidy in an Arabidopsis ancestor, genes were removed
preferentially from one homologue leaving clusters enriched in dose-sensitive genes. Genome
Res 16:934–946
Wang X et al (2009) Genome-wide and organ-specific landscapes of epigenetic modifications and
their relationships to mRNA and small RNA transcriptomes in maize. Plant Cell 21:1053–1069
Waters AJ et al (2011) Parent-of-origin effects on gene expression and DNA methylation in the
maize endosperm. Plant Cell 23:4221–4233
Wei F et al (2007) Physical and genetic structure of the maize genome reflects its complex
evolutionary history. PLoS Genet 3:e123
Wendel JF et al (1986) Duplicated chromosome segments in maize (Zea mays L.): further
evidence from hexokinase isozymes. Theoret Appl Genet 72:178–185
Woodhouse MR et al (2010) Following tetraploidy in maize, a short deletion mechanism
removed genes preferentially from one of the two homeologs. PLoS Biol 8:e1000409
Yu J et al (2005) The Genomes of Oryza sativa: a history of duplications. PLoS Biol 3:e38
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Metadata of the chapter that will be visualized in
SpringerLink
Book Title
Polyploidy and Genome Evolution
Series Title
Chapter Title
Polyploidy in Legumes
Copyright Year
2012
Copyright HolderName
Springer-Verlag Berlin Heidelberg
Corresponding Author
Family Name
Doyle
Particle
Given Name
Jeff J.
Suffix
Abstract
Division
Department of Plant Biology
Organization
Cornell University
Address
14850, Ithaca, NY, USA
Email
jjd5@cornell.edu
Legumes are the third largest family of flowering plants, with over 700 genera and more than 19,000 species.
Genomic evidence has shown that a whole-genome duplication (WGD) occurred shortly after the origin of
the family, in an ancestor that gave rise to the papilionoids, the clade that comprises 65 % of the genera and
71 % of the species, including nearly all of the economically important crop legumes. This polyploidy event
may have been associated with the origin of nitrogen-fixing symbiosis (nodulation) in the papilionoids.
Nodulation most likely evolved independently in other legumes outside the papilionoids, hence there appears
to be no requirement for polyploidy in the evolution of this important symbiosis. More recent polyploidy, as
inferred from chromosome counts, occurs in approximately a quarter of all legume genera for which data are
available. In most cases, polyploidy is confined to individual genera, species within genera, or cytotypes
within species. An exception is the core clade of the genistoid legumes, a major papilionoid group that includes
lupines (Lupinus). This group is probably fundamentally polyploid and also has a propensity for further
polyploidy and aneuploidy in many of its genera. The frequency of polyploidy varies considerably among
clades of the family, being most common (outside the genistoids) in the largely temperate, herbaceous
Hologalegina (including pea and clover), and low in woody tropical groups such as the caesalpinioids.
Book ID: 272454_1_En
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Chapter 9
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Polyploidy in Legumes
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Jeff J. Doyle
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Abstract Legumes are the third largest family of flowering plants, with over 700
genera and more than 19,000 species. Genomic evidence has shown that a wholegenome duplication (WGD) occurred shortly after the origin of the family, in an
ancestor that gave rise to the papilionoids, the clade that comprises 65 % of the
genera and 71 % of the species, including nearly all of the economically important
crop legumes. This polyploidy event may have been associated with the origin of
nitrogen-fixing symbiosis (nodulation) in the papilionoids. Nodulation most likely
evolved independently in other legumes outside the papilionoids, hence there
appears to be no requirement for polyploidy in the evolution of this important
symbiosis. More recent polyploidy, as inferred from chromosome counts, occurs in
approximately a quarter of all legume genera for which data are available. In most
cases, polyploidy is confined to individual genera, species within genera, or cytotypes within species. An exception is the core clade of the genistoid legumes, a
major papilionoid group that includes lupines (Lupinus). This group is probably
fundamentally polyploid and also has a propensity for further polyploidy and
aneuploidy in many of its genera. The frequency of polyploidy varies considerably
among clades of the family, being most common (outside the genistoids) in the
largely temperate, herbaceous Hologalegina (including pea and clover), and low in
woody tropical groups such as the caesalpinioids.
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Department of Plant Biology, Cornell University, Ithaca, NY 14850, USA
e-mail: jjd5@cornell.edu
P. S. Soltis and D. E. Soltis (eds.), Polyploidy and Genome Evolution,
DOI: 10.1007/978-3-642-31442-1_9, Springer-Verlag Berlin Heidelberg 2012
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9.1 Introduction
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9.2 A Brief Overview of Legume Phylogeny
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Along with Polygalaceae, Surianaceae, and Quillajaceae, Leguminosae form the
order Fabales, one of eight-orders in the Fabidae clade of rosid eudicots (Wang
et al. 2009). Bello et al. (2009) suggested that the Fabales are the product of a rapid
radiation, with legumes probably sister to Surianaceae plus Quillajaceae.
The Leguminosae has been the focus of considerable phylogenetic study, culminating in solid, chloroplast based, working hypotheses of generic relationships
(Fig. 9.1), notably those of Wojciechowski et al. (2004) and Bruneau et al. (2008).
The older classification of the family into three subfamilies, Caesalpinioideae,
Mimosoideae, and Papilionoideae (sometimes treated as separate families), is not
supported by molecular phylogenetic studies, in that although the Mimosoideae
(mimosas, acacias) and Papilionoideae (pea, bean, soybean, etc.) are monophyletic, the former is embedded in one clade of a paraphyletic caesalpinioid grade.
Relationships at the base of the family are uncertain and differ among the studies
of Wojciechowski et al. (2004), which focused most heavily on Papilionoideae,
and Bruneau et al. (2008), which emphasized caesalpinioids and included few
papilionoids. However, both studies identified caesalpinioids as the earliestdiverging lineages, including such taxa as the tribe Cercideae, which includes
Cercis (the redbud or Judas tree) and the large genus, Bauhinia (orchid tree).
Relationships at the bases of the two monophyletic subfamilies are also uncertain.
Fossil evidence places the origin of the family in the Paleocene, around
60 million years ago (MYA; see Lavin et al. 2005 for discussion; see also Bell
et al. 2010). Divergence times of all major groups within the family have been
estimated from fossil-calibrated molecular data (Lavin et al. 2005; Bruneau et al.
2008) and suggest rapid diversification of many clades, such that within
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The legumes (Leguminosae or, less preferably, Fabaceae, according to Lewis et al.
2005) are the third largest family of flowering plants, and are tremendously diverse
ecologically, morphologically, chemically, and cytologically (Doyle and Luckow
2003; Lewis et al. 2005). Not surprisingly, the family is also cytologically diverse.
As in other families, polyploidy is implicated as a major force at all levels of
legume evolution, from the early stages of radiation in the family to the origin and
recent diversification of modern genera, such as Glycine (soybean and allies) and
species within genera, such as the Medicago sativa complex (alfalfa and allies).
After summarizing progress in understanding the phylogeny of the family, this
review will discuss the role of paleopolyploidy during the early stages of the
radiation of the entire family and the possible connections with nodulation. The
occurrence of polyploidy in each of the major clades of the family will then be
reviewed.
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x = 14
} Early-diverging
papilionoid lineages
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polyploidy
(to x= 28)
Genistoids x = 9
Dalbergioids x = 10
Baphioids x = 11
Hypocalyptus
Mirbelioids x = 9
H-Loteae x = 7, 8
H-Robinieae x = 10, 11
H-IRLC x = 8
Indigofereae x = 7, 8
Millettioids x = 10, 11
F
Cercideae x = 7 or 14
Detarieae x = 12
Duparquetia
Dialiinae x = 14
MCC clade
(incl. Mimosoideae) x = 14
(incl. Phaseoleae)
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Fig. 9.1 Phylogeny of legumes. Caesalpinioids are shown in green, papilionoids in black.
Relationships among caesalpinoid lineages are shown as unresolved due to conflict among
published studies. Papilionoid taxa marked ‘‘H’’ are members of the Hologalegina. Divergence
dates for the origins of major clades are from Lavin et al. (2005). Base chromosome numbers are
given for groups with published counts. Minimum (solid arrow) and maximum (dashed arrow)
dates for the papilionoid polyploidy event are shown
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9.3 Polyploidy and the Early Diversification of Legumes
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To what degree has polyploidy shaped the radiation of legumes? Given the
growing understanding that polyploidy can drive phenotypic diversification (e.g.,
Freeling et al. 2006) and has played a role in the preservation of lineages during
periods of extinction (Fawcett et al. 2009), it might be expected that polyploidy
would be an important feature of evolution in a family that is ‘‘successful’’ as
judged by its sheer size and ecological dominance in some tropical biomes (e.g.,
rain forests, woody savannas, and dry forests), and in which a significant adaptive
novelty—the symbiotic association with nitrogen-fixing soil bacteria, termed
nodulation—has arisen (e.g., Doyle 2011).
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10 million years of divergence from a common ancestor, all of the major lineages
in the family had evolved, including the two monophyletic subfamilies and all of
the major clades within the Papilionoideae.
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Until the advent of genomic data, chromosome number was the prime source of
information available for inferring the existence of polyploidy. Goldblatt’s (1981)
review of the distribution of chromosome numbers in Leguminosae, published in
Advances in Legume Systematics, Part 2, remains the most comprehensive treatment of chromosomal variation in the family, and includes hypotheses concerning
the base numbers and ploidy levels of its constituent subfamilies and tribes. The
information from which his summary was drawn was included in the descriptions
of genera in Advances in Legume Systematics, Part 1 (Polhill and Raven 1981),
known to researchers in the family as the legume ‘‘bible.’’ The taxonomic treatments provided by this key resource were recently updated in Legumes of the
World (Lewis et al. 2005), taking into account the rapid progress in legume
phylogenetics. The phylogenetic studies that have revolutionized our understanding of relationships within the family also provide a new phylogenetic context for understanding chromosome number evolution that was not available
previously, but unfortunately the otherwise excellent Legumes of the World does
not include any cytological information.
The key contribution of objective phylogenetic data to our understanding of
cytological evolution in the family is the confirmation of caesalpinioid legumes as
a grade rather than as a natural subfamily. Chromosome numbers for the major
clades that comprise the caesalpinioid grade are relatively constant, principally
2n = 24–28 (Fig. 9.1). Standing out from these higher chromosome numbers are
Chamaecrista and Cercis. The large, mostly, pantropical genus, Chamaecrista, is
cytologically complex, with 2n = 14, 16, and 28. Goldblatt (1981) considered its
lower numbers to be the products of aneuploid reduction, and this hypothesis has
been recently supported (Torres et al. 2011, see below). Phylogenetic studies
(Wojciechowski et al. 2004; Bruneau et al. 2008) now nest Chamaecrista and
other Cassieae s.s. within the Mimosoideae-Caesalpinioideae-Cassieae (MCC)
clade (2n = 28), supporting this hypothesis.
Cercis is a small genus (10 species) with disjunct worldwide distribution and
2n = 14. It is a member of the Cercideae, all other members of which are
2n = 28, including the large pantropical genus, Bauhinia s.l. (ca. 250 species).
Phylogenetic studies show that Cercis is sister to the remaining genera (Bruneau
et al. 2008), which may be consistent with Goldblatt’s (1981) conclusion that it is
diploid and the remainder of the tribe is fundamentally polyploid. This is of some
importance given the relatively early divergence of Cercideae in some phylogenies. In the rbcL phylogeny of Kajita et al. (2001) the tribe was sister to the
remainder of the family, though with relatively weak support, and this topology
also appears in the phylogenetic summary of Lewis et al. (2005). The chloroplast
matK tree of Wojciechowski et al. (2004), which emphasized Papilionoideae,
placed Cercideae, and Detarieae (mainly 2n = 24) together as the first-diverging
legume lineage. In these topologies, it is possible that, as Goldblatt (1981) suggested, the legumes are fundamentally x = n = 7, with subsequent independent
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chromosomal increase both within Cercideae and in the ancestor of all remaining
legumes.
In contrast, the concatenated chloroplast matK/trnK ? trnL-F tree of Bruneau
et al. (2008) placed Detarieae as the first branch in the legume phylogeny, sister to
a trichotomy composed of Cercideae, Duparquetia, and the remainder of the
family. In this phylogeny, then, the base number for the family would be
x = n = 12, with 2n = 14 in Cercis representing a reduction. Interestingly, the
genome size of Cercis canadensis is comparable to measurements from the several
species of Bauhinia in the Kew C-value database (http://data.kew.org/cvalues/;
Leitch and Doyle, unpublished data), supporting this reduction hypothesis.
Even a high base number for early diverging lineages, as suggested by the
Bruneau et al. (2008) topology, would not definitively suggest polyploidy at the
base of the family, given what is known of chromosome numbers from other
Fabales. No information is available for Quillaja in the Index of Plant Chromosome Numbers (IPCN; http://www.tropicos.org/Project/IPCN), but Surianaceae is
represented by a single species of Stylobasium, with a number of 2n = 30, suggesting that the common ancestor of legumes and Surianaceae could have had a
high chromosome number.
Patterns of chromosomal evolution among major groups of legumes are complex even outside of the earliest branching. The bulk of the family belongs to two
sister clades: the MCC clade and the Papilionoideae. The two tribes that comprise
the MCC clade along with Mimosoideae (Caesalpinieae and Cassieae s.s.) are both
diploid based on x = 14. Given the presence of taxa with 2n = 28 in the grade at
the base of Papilionoideae, it is likely that the common ancestor of that group and
the MCC was diploid based on x = 14 as well. The majority of papilionoids,
however, have lower base chromosome numbers, ranging from x = 7–11,
depending on the tribe. These presumably represent reductions in chromosome
number, as discussed below; they certainly give no evidence for polyploidy.
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9.3.2 Genetic and Genomic Evidence for Polyploidy in the Early
Evolution of Legumes
Genomic studies, starting with linkage maps and continuing through studies of
expressed sequence tags (ESTs) and genome sequencing, have revolutionized
understanding of polyploidy in seed plants. Although it was long known that
diploidization can erase chromosomal evidence of polyploidy over time, it is now
clear that plant genomes comprise nested sets of WGD. The common ancestor of
all seed plants underwent a polyploid duplication, with a later WGD in the
ancestor of all angiosperms (Jiao et al. 2011) and numerous lineage-specific
duplications in various groups of flowering plants (Soltis et al. 2009).
It has been known for some time that cryptic polyploidy occurs in legume
genomes. For example, Shoemaker et al. (1996) used linkage map information to
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hypothesize that the soybean genome shows evidence of a more ancient duplication than the one that is responsible for its high chromosome number relative to
allied phaseoloid genera (millettioid clade, Fig. 9.1). In 2004, two different groups
mined the extensive EST collections of soybean and the diploid model legume,
Medicago truncatula (2n = 14; a member of the Hologalegina IRLC clade), to
search for the genomic signature of ancient polyploidy events (Blanc and Wolfe
2004; Schleueter et al. 2004). This signature is produced when all genes in the
genome are duplicated by autopolyploidy or when homoeologous loci are brought
together by allopolyploidy. It is observed by plotting the frequency distribution of
pairwise Ks (synonymous substitutions per synonymous site—a stand-in for time)
values for hundreds to thousands of paralogous gene pairs. Simple gene duplication is an ongoing phenomenon in all eukaryotes, but most duplicates are purged
from the genome rapidly, producing a characteristic distribution with many recent
duplicates with low Ks values and relatively few older pairs with high Ks (Lynch
and Conery 2003). Polyploid duplications appear as additional components
(‘‘peaks’’) against this background; the mode of such a Ks peak is taken as an
estimate for the age of the polyploid, though it is generally an overestimate of that
age (Doyle and Egan 2010).
Both Blanc and Wolfe (2004) and Schleueter et al. (2004) identified two Ks
peaks in soybean, as expected (Fig. 9.2); both reported similar Ks modes for these
peaks but because they used different substitution rates for plant nuclear genes, this
led to different estimates of the age of polyploidy (or homoeologue divergence).
The Schleueter et al. (2004) estimates are more in keeping with divergence dates
for papilionoid legume taxa (Lavin et al. 2005) and are preferred for that reason
(Shoemaker et al. 2006); in addition, the rate used by Schleueter et al. (2004) is
much closer to the rate recently estimated for Arabidopsis (Ossowski et al. 2010).
Of great interest was the finding, by both groups, of two Ks peaks in the M.
truncatula EST collection (Fig. 9.2). The younger of the two peaks is recent
enough that if due to polyploidy, it would most likely have left chromosomal
evidence, and has yet to be explained (Young et al. 2011). The older Medicago
peak, on the other hand, was estimated by Schleueter et al. (2004) to be around
54.6 MYA, very close to the 54 MY age estimated by Lavin et al. (2005) for the
divergence of the soybean (millettioid) and Medicago (Hologalegina) lineages,
and also similar to the age estimated for the older soybean peak (41.6 MYA). This
raised the possibility that the two species shared an ancient WGD.
This hypothesis was tested by Pfeil et al. (2005) using a phylogenomic
approach with 39 gene pairs chosen from among those used by Schleueter et al.
(2004) to identify the Ks peak in soybean. Topologies of gene trees overwhelmingly favored the hypothesis that their common ancestor was polyploid. Comparisons of linkage relationships between Medicago and the other legume model
species, Lotus japonicus (in the Hologalegina Loteae clade, Fig. 9.1), provided
further support for this hypothesis, and also showed that the duplication was not
found in poplar. Using the Lavin et al. (2005) date for the divergence of the
millettioid (Glycine) and Hologalegina (Lotus, Medicago) clades, the WGD event
had taken place by around 54 MYA. Thus, the common ancestor of the two major
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Fig. 9.2 Evidence for polyploidy in the genomes of: a Glycine max and b Medicago truncatula.
The graphs plot the number of pairs of paralogous sequences (‘‘density’’ or ‘‘percent of pairs’’)
versus binned Ks (‘‘synonymous distances’’) classes. Pairs with very low divergence (produced by
ongoing recent duplications) were not plotted. Curves were fit to the binned divergence data and
are interpreted as groups of genes duplicated simultaneously in large-scale genomic events such
as polyploidy; modes of peaks provide a maximum age for allopolyploid events (Doyle and Egan
2010). Divergence time was estimated from synonymous distances using standard clock methods;
note the different estimated ages (modes of curves) for the older event in the two species. Data are
from Schleueter et al. (2004), who used expressed sequence tags (ESTs). Figure courtesy of
Jessica Schlueter (UNC-Charlotte)
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9.3.3 Polyploidy and Nodulation in Legumes
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sister clades that comprise nearly 9,000 species––around 45 % of all legumes and
63 % of papilionoids––was polyploid.
Subsequently, Bertioli et al. (2009) studied Arachis (peanut) and showed that its
genome also shows evidence of the [54 MYA event, indicating that the large
dalbergioid clade is also fundamentally polyploid. Unpublished information
reported by McClean (personal communication) at the 2009 International Conference of Legume Genetics and Genomics showed that Lupinus, and hence the
genistoid clade, also shares this WGD. Thus, all of the lineages of the main
radiation of the papilionoids share a polyploid ancestor.
The possibility that all legumes share this polyploidy event remained tenable
until transcriptomic data from the caesalpinioid genus, Chamaecrista, became
available (Singer et al. 2009). Cannon et al. (2010) produced and analyzed over
1,200 gene phylogenies from these data, which overwhelmingly supported the
conclusion that the Chamaecrista genome shows no evidence of any polyploidy
event subsequent to the prerosid triplication, and notably lacks the WGD found in
core papilionoids (all but the early diverging lineages in Fig. 9.1). Chamaecrista
belongs to the MCC clade, which is sister to the papilionoid clade. Therefore, the
absence of the WGD in Chamaecrista indicates that the common ancestor of the
MCC clade and papilionoids was not polyploid, placing the WGD within papilionoids (Fig. 9.1). Whether the WGD took place in the papilionoid common
ancestor is still unknown, because the lineages that comprise the paraphyletic
grade lacking the putative molecular synapomorphy for the major papilionoid
radiation (chloroplast genome 50 kb inversion) remain to be sampled.
Thus, it is possible that this core papilionoid WGD facilitated the radiation of
the most species-rich lineage of legumes, comprising 69 % of the species (13,390/
19,327) and 59 % (438/741) of the genera of the third largest family of flowering
plants. This is the group that is more uniformly characterized by the eponymous
legume fruit, by the bilaterally symmetric papilionoid flower, and by the ability to
nodulate. The early diverging grade of papilionoids does contain some genera with
papilionoid flowers and legume fruits, but many lineages in this part of the tree are
characterized by unusual, nonpapilionaceous corollas and drupaceous or samaroid
fruits (Pennington et al. 2000); this grade also contains nearly all of the papilionoid
genera that do not nodulate (Doyle 2011).
204
The correspondence between nodulation and polyploidy in the family is interesting. Core papilionoids nearly all nodulate, but this is also true of Mimosoideae,
and Chamaecrista is among a handful of caesalpinioids known to be able to form a
nodulation symbiosis (Sprent 2009). It remains unclear whether there was a single
origin of nodulation in the common ancestor of the papilionoid and MCC clades,
followed by many losses of nodulation, or whether there were multiple origins of
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Whatever the original basic chromosome number of the family, the earliest radiation from the common ancestor does not seem to have involved polyploidy,
despite Goldblatt’s (1981, p. 457) conclusion that the ‘‘… initial phase of polyploidy is probably very ancient and may have taken place in the late Cretaceous,
when major groups of Leguminosae began differentiating and were probably
evolving rapidly into new habitats.’’ Evidence against Goldblatt’s view of polyploidy in the ancestor of the entire family is the absence of any trace of polyploidy
in gene families of Chamaecrista other than the prerosid whole-genome triplication
(WGT; Jaillon et al. 2007). This indicates that the ancestor of the MCC and older
ancestors back to the prerosid WGT did not experience polyploid duplications. The
uniformity of base chromosome numbers in the major radiations of the family—
Cercideae, detarioids, MCC, and probably papilionoids (see above) suggest that
relatively high numbers (2n = 24–28) are plesiomorphic in the family.
The most parsimonious hypothesis for papilionoids is that the earliest papilionoid ancestor was also 2n = 28. Shortly after the divergence of this ancestor
from the MCC ancestor, the papilionoids radiated rapidly, and polyploidy occurred
nearly simultaneously, no later than the divergence of the first major lineage to
diverge in the core clade (genistoids, e.g., Lupinus). This WGD did not leave
evidence in higher chromosome numbers; to the contrary, polyploidy is associated
with chromosome number reduction in core papilionoids (Fig. 9.1). Goldblatt
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nodulation in the MCC clade and an independent origin in the ancestor of the core
papilionoids (Doyle 2011).
The demonstration that Chamaecrista not only lacks the core papilionoid
WGD, but also does not show any genomic evidence of other polyploidy events,
indicates that polyploidy is not a prerequisite for nodulation in legumes as a whole
(Cannon et al. 2010). Thus, in a model of a single origin of nodulation in the
family, polyploidy would have played no role (Fig. 9.3). At the other extreme,
polyploidy could not have been involved in an origin of nodulation unique to
Chamaecrista, nor would it have been essential for the origin of nodulation in a
model where the symbiosis evolved in a common ancestor of Chamaecrista and
other members of the MCC clade (e.g., Mimosoideae).
This is not to say, of course, that polyploidy was not important in the origin or
evolution of nodulation in core papilionoids or in other nodulating taxa whose
genomes have yet to be explored, such as mimosoids. In the case of papilionoids, it
is possible that nodulation and the WGD will be found to coincide, either in the
ancestor of the main radiation of core papilionoids or at the first papilionoid
ancestor, but better phylogenetic resolution is required before this can be tested
(Pennington et al. 2001). In either case, refinement of the nodulation symbiosis in
taxa such as Medicago may well have been facilitated by the availability of homoeologues produced in the core papilionoid WGD (Young et al. 2011).
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Cercideae
Detarieae
Amherstieae
Dialiinae
Umtiza clade
Batesia
Chamaecrista
Melanoxylon
Cassia
Senna
Caesalpinia clade
Moldenhawera
Erythrophleum
Calpocalyx
Pentaclethra
Parkia
core Mimosoideae
Campsiandra
Dimorphandra
Burkea
Dinizia
Arapatiella
Jacqueshuberia
Sclerolobium
Tachigali
Peltophorum clade
Swartzia clade
Non-nod. papil.
Mimosoideae-Caesalpinieae-Cassieae (MCC) clade
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million years
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Fig. 9.3 Relationship between polyploidy and nodulation in Leguminosae. Genera known to
nodulate are shown in bold face; Pentaclethra includes both nodulating species and species that
apparently cannot nodulate. Possible origins of nodulation are indicated with symbols and colors.
A single origin of nodulation for the entire family could have occurred in the common ancestor of
the papilionoids and the MCC clade (indicated by ‘‘N’’); this would have required many
independent losses in the course of legume evolution. Independent origins within the MCC clade
and Papilionoideae would require fewer losses. In the MCC clade, a single origin could have
occurred in the common ancestor of all genera known to nodulate (triangle), or once in each
major lineage of nodulating taxa (circles), or additional times within some clades (squares).
Similarly, a single origin could be hypothesized for papilionoids (green circle), or twice
(squares). The placement of the papilionoid polyploidy event (red ‘‘P’’) is indicated as in
Fig. 9.1. Polyploidy is associated with nodulation only in the papilionoids and might not be
directly associated there, given the uncertainty about the placement of both the polyploidy event
and the origin(s) of nodulation. Figure adapted from Doyle (2011)
(1981, p. 457) notes that ‘‘the cytological history of legumes seems to involve
some descending aneuploidy in every major evolutionary line but is most pronounced in Papilionoideae, in which most predominantly herbaceous tribes or
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9.4 Polyploidy in Tribes and Genera of Legumes
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Genomic and phylogenomic information currently is limited to the few genera
discussed above. More information can be expected with the advent of new
sequencing technologies, notably (at this writing) Illumina and 454. Twenty-five
legume genera, representing all three subfamilies, are included in the 1,000 Transcriptomes project (1 kp, see http://www.onekp.com/angiosperms.html), but results
from these species have not yet been analyzed (Steven Cannon, personal communication). Chromosome numbers provide a better guide at this level, but clearly
should be interpreted with the caveat that high numbers are likely to indicate
polyploidy, but low numbers cannot be assumed to be fundamentally diploid, given
the potential for cryptic polyploidy, a phenomenon discussed further below.
Three sources of data were used for the following survey of polyploidy in
legume clades: Goldblatt (1981); chromosome numbers provided for each genus
listed in the tribal treatments in Advances in Legume Systematics, Part 1 (Polhill
and Raven 1981); and the on-line Index of Plant Chromosome Numbers (IPCN;
http://www.tropicos.org/projectwebportal.aspx?pagename=Home&projectid=9).
IPCN was searched for all genera in Lewis et al. (2005) for which no data were
available in Goldblatt (1981) or Polhill and Raven (1981), and for genera with
polyploid counts, to identify infrageneric patterns of polyploidy, particularly when
published phylogenies were available. For genera with evidence of polyploidy,
BIOSIS was searched using the genus name in conjunction with either \polyploid*[and/or\phylogeny[. For some larger genera, IPCN was also consulted to
search for polyploid counts published since 1981. There remain many gaps in our
knowledge of legume chromosome numbers. Overall, only around 54 % of legume
genera have counts reported (Table 9.1), and the percentage is far lower for
tropical woody groups such as the caesalpinioid tribe Detarieae (30 %). This is
perhaps not too surprising, given the size of the family, the large number of small
genera, and the tropical distributions of many groups. It is also no doubt a commentary on how counting chromosomes has fallen from favor in this age of highthroughput science.
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genera have achieved relatively low base numbers.’’ The details of this process
await elucidation of phylogenetic relationships of the early diverging papilionoid
lineages, and sampling of these taxa for the presence of polyploidy. What is clear
is that, at some point early in the history of Papilionoideae, chromosome numbers
fell from as high as n = 28 to n = 7–11. This process could have begun in, or
prior to, the common ancestor of the core papilionoids (Fig. 9.1). In any case, the
initial reduction was rapid, taking place within 10 MY after the polyploid event,
and perhaps within only 5 MY of the WGD. Detailed studies of synteny in the
various core papilionoid lineages should help elucidate whether initial reorganization occurred in a common ancestor or was completely independent in the longdiverged major clades of the core papilionoids.
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171 (2251)
82 (3271)
40 (415)
83 (2354)
53 (1514)
7 (58)
32 (763)
34 (414)
62
39
15
59
32
6
22
23
4
10
2
24
11
1
5
9
6
26
13
41
34
17
23
39
54 (4351)
7 (768)
40
2
15
1
38
50
56 (1104)
22
1
5
112 (2064)
731
71
393
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17
24
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F
Percent genera
with polyploidy
(%)
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Mimosoideae
Swartzieae ? Sophoreae
Genistoids
Dalbergioids
Baphioids
Mirbelioids
Hologalegina:
Robinioids ? Loteae
Hologalegina: IRLC
Millettioids:
indigofereae
Millettioids: core
Millettioids
Millettioids: phaseoloids
Total
based on chromosome numbers
Genera with
Number of
chromosome
genera with
counts
polyploidy
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Table 9.1 Polyploidy in legume clades,
Clade
Number of
genera
(species)a
Source: Lewis et al. (2005)
Summation of the following monophyletic groups: Cercideae, 12 genera total, 2 of 6 genera
with chromosome counts have reports of polyploidy, 33 % (12, 2/6, 33 %); Dialiinae (17, 0/7,
0 %); Detarieae (82, 2/25, 8 %); MCC clade minus mimosoids (59, 0/24, 0 %)
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Goldblatt (1981) made a distinction among genera that are exclusively polyploid relative to genera in the same tribe, and genera that include species with both
‘‘diploid’’ and ‘‘polyploid’’ chromosome numbers. In light of our current understanding of polyploidy, this is an artificial distinction, but exclusively polyploid
genera are perhaps worth noting, because they represent lineages where polyploids
may have replaced their diploid progenitors entirely. Such lineages would constitute evidence of polyploid ‘‘success’’ (e.g., Mayrose et al. 2011) in measures of
diversity a million years from now.
The results of this survey show that polyploidy, as inferred solely from chromosome numbers, occurs in nearly a quarter of all legume genera, but varies
widely in frequency among different lineages (Table 9.1). As Goldblatt (1981)
noted, polyploidy is rare in woody, tropical groups such as caesalpinioids and early
diverging papilionoid lineages.
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9.4.1.1 Clades of the Caesalpinioid Grade, Excluding the MCC Clade
Goldblatt (1981) stated that polyploidy (assuming a basic diploid number of
2n = 28) is uncommon in caesalpinioid groups. As noted above, unpublished
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Chromosome numbers of the caesalpinioid members of the MCC clade range from
2n = 20–28, with the exception of Chamaecrista, in which numbers of 2n = 14,
16, and 28 occur. Goldblatt (1981) raised the possibility that low chromosome
numbers in this genus are not ancestral, but instead represent aneuploid reduction
from ancestral x = 14 species. This makes good sense given the prevalence of
2n = 28 counts throughout much of the MCC clade. A recent molecular phylogenetic analysis of Chamaecrista (Torres et al. 2011) supported the hypothesis that
the 2n = 14 species of sect. Xerocalyx form a monophyletic group nested within
species having higher chromosome numbers. Chamaecrista is a genus of considerable interest as a potential model for nonpapilionoid legumes (Singer et al.
2009), making it an attractive system for exploring chromosome evolution in the
caesalpinioids.
Mimosoideae comprise a monophyletic group embedded in the MCC clade, and
the subfamily is dominated by taxa with n = 13. Goldblatt (1981) listed only three
genera with base numbers higher than n = 14: Schleinitzia (2n = 52, 54), Leucaena (2n = 52, 56, 104, 112), and Dichrostachys (2n = 50, 56). These genera are
all members of tribe Mimoseae and are relatively closely related within that tribe
(Lewis et al. 2005), with the former two being members of the same clade and
Dichrostachys being part of a sister clade (Luckow et al. 2003). However,
Schleinitzia and Leucaena are not sisters within their clade (Hughes et al. 2003;
Luckow et al. 2005), suggesting that polyploidy has originated independently in
each case.
Leucaena itself has been fertile ground for systematic investigation. Boff and
Schifino-Wittmann (2003) concluded that its species are segmental paleopolyploids. A series of studies has built a strong foundation for understanding the
complex history of hybridization and polyploidy in the genus, and the impact of
these phenomena on characters such as nrDNA ITS pseudogene evolution, and
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genome size data do not support the hypothesis that Bauhinia (commonly
2n = 28) is tetraploid relative to Cercis (2n = 14). However, this is negative
evidence, and given the prevalence of genome downsizing (Leitch and Bennett
2004) in polyploids, it remains possible that Bauhinia (along with Adenolobus and
Griffonia) is polyploid. More recent polyploidy occurs in the group, with Tylosema
having 2n = 52; Tylosema is nested within Bauhinia s. l. (Sinou et al. 2009). The
large Detarieae s.l. clade is overwhelmingly 2n = 24. The two detarioid genera
having higher chromosome numbers (Hardwickia, 2n = 34; Colophospermum,
2n = 36) are strongly supported as sisters within the Prioria clade of Bruneau
et al. (2008); they could represent independent aneuploid reduction from a polyploid ancestor. Goldblatt (1981) mentioned polyploidy in Anthonotha (2n = 24,
28, 72), but only 2n = 24 and 28 are listed by Cowan and Polhill (1981), and no
counts are listed in IPCN. All known counts from the Dialiinae clade are 2n = 24
or 28.
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their role in domestication (Hughes et al. 2002, 2007; Govindarajulu et al. 2011a,
b). Govindarajulu et al. (2011b) concluded that ‘‘… a comprehensive picture of the
complex evolutionary dynamics of polyploidy in Leucaena is emerging. This
includes paleotetraploidization, diploidization of the last common ancestor to
Leucaena, allopatric divergence among diploids, and recent allopolyploid origins
for tetraploid species likely associated with human translocation of seed.’’
Acacia (sens. lat.) is reported to have 2n = 26, 52, 76, and 104, though the
majority of its species are diploid (Gallagher et al. 2011). Polyploidy has not been
a focus of recent phylogenetic analyses (e.g., Brown et al. 2010; Murphy et al.
2010). Similarly, phylogenetic studies of Prosopis (2n = 28, 52, 56) or Prosopidastrum (2n = 28, 56) do not discuss polyploidy (e.g., Bessega et al. 2006;
Catalano et al. 2008). No phylogenetic studies appear to have addressed polyploidy in Neptunia (2n = 28, 36, 54, 56, 72, 78), though Pandit et al. (2006) note
that N. plena, an invasive species in Singapore, is a polyploid (2n = 72). No
studies appear to exist on polyploidy and phylogeny of Inga (2n = 26, 52; the
latter reported by Hanson 1995), Albizia (2n = 26, c. 78), or Calliandra (2n = 16,
22, 32, 44).
Mimosa (2n = 24, 26, 28, 40, 52) is a genus of around 500 species; a second
genus whose species vary in ploidy, Schrankia (2n = 16, 22, 24, 26, 52), is deeply
nested within Mimosa (Simon et al. 2011). Dahmer et al. (2011) concluded that the
phylogenetic pattern ‘‘… suggests that duplication of chromosome numbers
evolved several times in the genus and that polyploidy is not restricted to any
particular clade within Mimosa. On the contrary, it seems that polyploids arose
independently from ancestors with lower ploidy levels and are present in divergent
lineages in the genus.’’ Seijo and Fernandez (2001) reported chromosome numbers
from the southern extreme of the range and discovered polyploidy within M.
balansae. Chromosomal and morphological studies by Morales et al. (2010)
clarified relationships in the M. debilis-M. nuda complex, demonstrating that
hybridization and polyploidy are responsible for taxonomic complexity in the
group.
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Relatively few papilionoid genera appear to be exclusively polyploid based on
chromosome number. As noted above, the early diverging papilionoid lineages
have relatively high numbers, like the caesalpinioid and mimosoid groups. Ateleia
(Swartzieae) is 2n = 40, presumably representing a second polyploidy event
followed by aneuploid reduction. Goldblatt listed Dipteryx (Dipterygeae) as being
a polyploid genus but with a questionable count of 2n = 32; this number was not
reported in the treatment of the tribe by Polhill (1981a), nor is a count for the
genus listed in IPCN.
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The genistoid clade, which is weakly supported as sister to the remaining core
papilionoids (Fig. 9.1), is one of the most complex groups with respect to polyploidy, and detailed discussion is beyond the scope of this review. The frequency
of polyploidy in the genistoid clade is the highest for any well-sampled group of
legumes (Table 9.1). Cusma-Velari and Feoli-Chiapella (2009) discussed cytology
in ‘‘so-called ‘primitive’ genera of Genisteae’’ in light of molecular phylogenetic
data. Genistoids clearly have a base number of x = 9 (Goldblatt 1981), with
2n = 18 being common in most of its tribes.
Sophora s.l. has been divided into several segregates that vary in chromosome
number. Sophora s.s. is part of the genistoid clade and is x = 9. Boatwright and
van Wyk (2011) reported on the relationships of several of these based on nrDNA
ITS sequences. They focused on the placement of the South African species, S.
ihambanensis, which is polyploid (2n = 36); in their tree it is sister to S. tomentosa, a diploid, but they do not discuss origins of the polyploid. A count of
2n = 18 is common in Sophora s.l., and several additional species are polyploids
with 2n = 36 (S. alopecuroides, S. pachycarpa, and S. songarica); S. leachiana is
listed by ICPN as having 2n = 36 and 2n = 54 cytotypes. The small segregate,
Calia, has 2n = 18 and may be sister to the entire genistoid clade. The remainder
of Sophora s.l. comprises 2n = 28 species transferred to Styphnolobium in the
early diverging papilionoid grade.
In Thermopsideae, Thermopsis has both diploid and polyploid species
(2n = 18, 36). In Podalyrieae, two genera are exclusively polyploid: Virgilia
(2n = 54) and Cyclopia (2n = 36); they are not supported as sisters in Boatwright
et al. (2008). Crotalarieae are sister to Genisteae and include the exclusively
polyploid Buchenroedera (2n = 28; Van Wyk and Schutte 1988), as well as
polyploids within Crotalaria (2n = 14, 16, 32) and Lotononis (2n = 18, 28, 36).
Genisteae are by far the most cytologically complex group in the entire Leguminosae. Even genera with low numbers may be polyploid, such as Anarthrophyllum (2n = 24; Goldblatt 1981) and Dichilus (2n = 28). These genera, along
with the polyploid Polhillia (2n = 32) and complex Melolobium (2n = 18, 32),
were once placed in Crotalarieae. It is in the core Genisteae that polyploidy and
aneuploidy have run rampant. The group includes Argyrolobium (2n = 24, 26, 30,
32, 48), Adenocarpus (2n = 26, 46, 48, 52, 54), Laburnum (2n = 48, 50), Cytisophyllum (2n = 50, 52), Petteria (2n = 52), Argyrocytisus (2n = 50), Chamaecytisus (2n = 48, 96), Cytisus (2n = 22, 24, 46, 48, 92, 96), Calicotome
(2n = 24, 48, 50), Erinacea (2n = 52), Spartium (2n = 48, 52, 54, 56), Retama
(2n = 48), Genista (2n = 18, 22, 24, 26, 28, 30, 32, 36, 40, 42, 44, 46, 48, 50, 52,
56, 72, 80, 96), Echinospartium (2n = 44, 52), Stauracanthus (2n = 28, 48, ca.
128), Ulex (2n = 32, 64, 80, 96), and Lupinus (2n = 24, 30, 32, 34, 36, 38, 40, 42,
48, 50, 52, 96).
Bisby (1981) considered the plethora of chromosome numbers attributed to
individual genera to be partly a real phenomenon, but also due to difficulties in
obtaining reliable counts given the small size and high numbers of chromosomes,
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combined with the taxonomic complexity of the groups. Some of the taxonomic
complexity is being resolved by molecular phylogenetic studies focused on Cytisus
and Genista (Cubas et al. 2002; Pardo et al. 2004), but these studies do not discuss
polyploidy per se. For Lupinus, three studies have documented the rapid radiation
of the genus in its New World center of diversity (Hughes and Eastwood 2006;
Drummond 2008; Drummond et al. 2012), and another phylogenetic study focused
on the Old World species (Ainouche et al. 2004). Drummond (2008) noted that,
‘‘While a complex history of aneuploidy (2n = 32, 34, 36, 38, 40, 42, 50, 52) in
the Old World and eastern New World … implies that allopolyploidy may have
provided an additional mechanism for reproductive isolation and evolutionary
divergence, chromosomal numbers in the western New World species (2n = 48
with occasional autopolyploids of 2n = 96) are relatively stable.’’ It is this western
group that has radiated explosively, presumably driven by ecology and not due to
polyploidy per se (Drummond 2008). Conterato and Schifino-Wittmann (2006)
described chromosome numbers and meiotic behavior in diploid and polyploid
American lupines, and noted consistencies with phylogenetic relationships in the
genus.
The placement of polyploid former Crotalarieae in the same clade with core
Genisteae may lend support to the idea that polyploidy arose early in the entire
clade, as suggested by Goldblatt (1981), Lavin et al. (2005) dated the common
ancestor of Crotalarieae and Genisteae at around 41 MYA. On the other hand,
Goldblatt also noted (1981, p. 452) that ‘‘basic numbers for these genera (Genista,
Ulex, Cytisus) are however in the diploid range and a basic number of x = 12 for
the group as a whole and for several genera has been suggested …’’. Genomic data
for members of Genisteae should eventually allow the determination of the number
and relative timing of polyploid events in the group.
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The dalbergioid s.l. clade is split into two major subclades, Amorpheae and a
second clade comprising Adesmieae, Aeschynomeneae, and many members of the
polyphyletic Dalbergieae (Lewis et al. 2005). The entire dalbergioid clade is
dominated by 2n = 20 species. Within Amorpheae, polyploidy occurs in genera
from each of the major subclades described by McMahon (2005). In the daleoid
clade, Dalea has 2n = 14, 16, 28, and 42. Spellenberg (1981) hypothesized that
tetraploids and hexaploids of D. formosa (2n = 28, 42) were autopolyploids
derived from the diploid (2n = 14) cytotype. In the amorphoid clade, Amorpha
includes both diploids and polyploids (2n = 20, 40), all native to the New World.
The widespread A. fruticosa is exclusively polyploid and morphologically complex (Wilbur 1975). It has become an invasive weed in Europe (e.g., Hulina 2010),
illustrating a common feature of polyploidy (e.g., Pandit et al. 2011; te Beest et al.
2011). Its relationships to other members of the genus appear to be complex,
sharing chloroplast haplotypes with different sympatric diploids across its range
(Straub and Doyle, unpublished data). Studies of the A. georgiana complex
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9.4.2.3 Baphioids
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identified mixed populations of diploids and polyploids in what had been assumed
to be an exclusively diploid species; these included an apparent allopolyploid
between A. georgiana and A. herbacea (Straub and Doyle 2009). Straub
(unpublished data) has identified additional polyploid species in the genus and
hypothesized their origins.
The core dalbergioid clade is split into the small Adesmia clade (six genera) and
the much larger clade comprising the Dalbergia and Pterocarpus sister clades
(Lewis et al. 2005). Polyploidy occurs in all three clades. In the Adesmia clade,
Adesmia includes both diploids and polyploids (2n = 20, 40), and Amicia is
exclusively polyploid (2n = 38).
In the Dalbergia clade, Smithia (2n = 38) is exclusively polyploid and is
considered closely related to Kotschya (Lewis et al. 2005), a genus that includes
species with chromosome numbers indicative of polyploidy and aneuploidy
(2n = 28, 30, 36, 40). These two genera are grouped with Aeschynomene species
(Lavin et al. 2001), a genus that also includes diploids and polyploids (2n = 18,
20, 40). Another dalbergioid-clade genus, Ormocarpum (2n = 24, 26), was not
listed in Goldblatt’s discussion of polyploid genera, but could potentially be a
cryptic polyploid with aneuploid reduction. Information on chromosome numbers
of other members of the Ormocarpum group (Thulin and Lavin 2001) would be
useful in addressing this issue.
The Pterocarpus clade also includes several genera with both diploids
(including presumed aneuploids) and polyploids scattered among its subclades:
Platymiscium (2n = 16, 18, 20, 32), Pterocarpus (2n = 22, 24, 44), Geoffroea
(2n = 20, 60), and Arachis (2n = 20, 40). Arachis includes the tetraploid peanut
or groundnut (A. hypogaea), as well as three other tetraploid species, one of which
(A. glabrata) is a tropical forage crop. Peanut is hypothesized to be an allopolyploid derived from A- and B-genome species (e.g., Burow et al. 2009). Seijo et al.
(2007) provide a useful summary of hypotheses concerning the origin (or origins)
of peanut; controversy exists concerning such issues as the exact progenitor species of both homoeologous genomes, mode and number of origins, and whether
there was subsequent introgression from wild species into the cultigen. They
identified likely diploid progenitors of A. hypogaea using GISH and studied
meiotic behavior of two other tetraploids (Ortiz et al. 2011) and a spontaneous
autotriploid of A. pintoi (Lavia et al. 2011).
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This small group appears to be interesting from the standpoint of polyploidy.
Chromosome numbers are known from six of its seven genera. Of these, four are
listed as 2n = 22, one (Dalhousiea) is 2n = 44, and Baphia was reported in
Polhill and Raven (1981) to have both numbers, though no reports for any of the
47 species of the genus exists in IPCN.
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The Hologalegina clade includes robinioids and the Inverted Repeat Loss Clade
(IRLC; named for the absence of a prominent feature of the chloroplast genome).
Goldblatt (1981) concluded that ‘‘Species polyploidy is overwhelmingly concentrated in temperate to cool Eurasia’’ so it is not surprising that this largest clade of
legumes, which includes many temperate genera, has a higher frequency of
polyploidy than the family average, nearly 40 % in both of its major subclades
(Table 9.1).
The robinioids comprise two clades: one with Sesbania plus Loteae (including
Coronilleae), the other being Robinieae (s.s.). Diploid chromosome numbers vary
considerably within Robinieae s.s., and Goldblatt (1981) suggested several possible base numbers, the most likely being x = 10 or 11. There is one apparently
exclusively polyploid genus, Poissonia, which Goldblatt (1989) counted as
(2n = ca. 32). Although only numbers of 2n = 10 and 11 were given for Robinia
in Polhill and Sousa (1981), more recent counts of 2n = 30 for R. hispida suggest
polyploidy within this small genus.
Even with the limited sampling of Loteae in Wojciechowski et al. (2004), it is
clear that numerous problems exist with the genera as circumscribed, notably that
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Polyploidy in the mirbeliod clade (23 %) is close to the average for the whole
family (Table 9.1). Isotropis (2n = 16, 18, 32) is sister to the large ‘‘Pultenaea s.l.
group’’ in Orthia et al. (2005), which includes Oxylobium (2n = 16). Chandler et al.
(2001) sank both Brachysema (2n = 16, 32) and the monotypic Jansonia
(2n = 32) in Gastrolobium, previously a genus with only 2n = 16 species.
Chandler et al. (2001) placed the single sampled species of Jansonia sister to
Brachysema celsianum (not listed in IPCN), in a clade that also included B.
praemorsum; that species is a diploid at 2n = 16, as is at least one species in the
sister clade, Nemcia coriacea. Thus, it is likely that polyploidy has arisen more
than once just within Gastrolobium s.l., and another time in Isotropis. Among
several ‘‘strongly paraphyletic’’ Pultenaea s.l. genera, nearly all of which are
2n = 16, are two genera with polyploidy: Pultenaea s.s. (2n = 8, 12, 14, 16, 18,
27, 32) and Chorizema (2n = 16, 32). Sorting out how the various chromosome
numbers in Pultenaea s.s. are related will be of considerable interest but will
require more complete phylogenies than appear to be available at present. Of the
10 Chorizema species (out of 27 in the genus) listed in IPCN, polyploidy is only
reported from C. aciculare, which has both diploid and tetraploid cytotypes.
Smaller genera in Pultenaea s.l. with known polyploidy are Eutaxia (2n = 16,
32) and Dillwynia (2n = 14, 21, 28), which are in the same weakly supported
clade in Orthia et al. (2005). One of the two Dillwynia species (D. phylicoides)
included in the Orthia et al. (2005) tree has both diploid and tetraploid cytotypes
listed in IPCN.
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Anthyllis (2n = 10, 12, 14, 16, 28) and Ornithopus (2n = 14) are nested within
Lotus (2n = 10, 12, 14, 24, 28). Other genera with known polyploidy are Coronilla (2n = 12, 20, 24); Hippocrepis (2n = 14, 28); Dorycnium (2n = 14, 28;
both cytotypes in D. axilliflorum), and Scorpiurus (S. muricatus has 2n = 14, 16,
28; other species are 2n = 14 or 28). Degtjareva et al. (2006) provided phylogenetic hypotheses for Lotus, with sampling of other genera, but did not discuss
polyploidy. Rosello and Castro (2008) discussed polyploidy in the flora of the
Balearic Isles, among which are species of Anthyllis and Coronilla.
The genus Lotus includes the genomic model legume, L. japonicus (Sato et al.
2008), which is part of the L. corniculatus (birdsfoot trefoil) polyploid complex.
Grant and Small (1996) summarized many studies of this complex and concluded
that it was a fertile topic for further study, particularly to identify the diploid
progenitors of L. corniculatus itself, which they considered to be an allopolyploid.
Gauthier et al. (1998a, b) discussed evolutionary patterns in the L. corniculatus/L.
alpinus polyploid complex in the Alps of Europe; they described morphological
and genetic consequences of autopolyploidy in L. alpinus and suggested introgression at the tetraploid level between it and L. corniculatus.
The majority of genera and species in the IRLC clade belong to two sister
clades in Wojciechowski et al. (2004): one includes the Astragalean clade
(Astragalus and allies) and Hedysareae, and the second includes the Vicioid clade.
The remainder of the IRLC phylogeny, moving successively further from these
clades, consists of a clade with Wisteria (2n = 16) and one species of Callerya,
followed by a clade with Glycyrrhiza (2n = 16) and a second species of Callerya.
The Astragalean clade has extensive polyploidy. Perhaps most striking is the
clade that includes the New Zealand endemic tribe Carmichaelieae plus the
Australian Swainsona and an additional New Zealand genus, Montigera, all of
which are polyploid (Wagstaff et al. 1999). The only exclusively polyploid genera
that Goldblatt (1981) listed for the IRLC clade belong to this clade: Swainsona
(2n = 32); Clianthus (2n = 32); Carmichaelia (2n = 32, ca. 96), Chordospartium
(2n = 32); and Corallospartium (2n = 32), with the latter two subsumed in
Carmichaelia in Lewis et al. (2005). Wagstaff et al. (1999) concluded that the New
Zealand radiation was recent, involved an already polyploid colonizer, and may
have been associated with orogeny and glaciation.
Elsewhere in the Astragalean clade are two large genera with extensive polyploidy and aneuploidy, Oxytropis (300–400 species: 2n = 16, 32, 48, 64, 96) and
Astragalus (ca. 2500 species: 2n = 16, 22, 24, 26, 28, 32, 44, 48, 64). In
Astragalus, polyploidy appears to be more common among Old World than among
New World species. Wojciechowski (2005) summarized phylogenetic results for
this huge genus, showing that aneuploid species form a clade. According to Gohil
and Ashraf (2008), polyploidy occurs in around only 17 % of Astragalus species.
However, Astragalus is one of the largest genera of plants with as many as 2500
species (Lewis et al. 2005), so if this percentage is correct, then there are over 400
polyploid species in the genus. There does not seem to be a comprehensive
phylogeny that discusses origins of polyploidy in Oxytropis. However, a series of
papers describe autopolyploidy, including multiple autopolyploid origins, in
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Oxytropis chankaensis (e.g., Artyukova et al. 2011). Jorgensen et al. (2003) suggested ‘‘a scenario of multiple formations of polyploids, possibly including
hybridization among diverged Alaskan Oxytropis populations.’’
Within the Hedysareae, phylogenetic studies of Caragana (2n = 16, 24, 32, 48)
suggest that polyploidy is confined to a single group of species, and that triploids,
tetraploids, and hexaploids may all be autopolyploid in origin (Zhang et al. 2009).
Neither Hedysarum (2n = 14, 16, 48) nor Onobrychis (2n = 14, 16 28, 32)
appears to be monophyletic on the basis of nrDNA ITS phylogenies (Ahangarian
et al. 2007). Hejazi et al. (2010) discussed karyotypic evolution in diploid and
polyploid species but did not provide a phylogenetic context or identify origins of
polyploids. Based on IPCN listings, most polyploidy reported for Hedysarum in
IPCN appears to involve multiple cytotypes within a single species (e.g., H.
arcticum and H. hedysaroides, both 2n = 14, 28; H. dasycarpum and H. mackenziei, both 2n = 16, 32; H. gmelinii, 2n = 16, 28, 56), but some species are
exclusively polyploid (e.g., H. inundatum, 2n = 28). Similarly, in Onobrychis
there is variation within species (e.g., O. aequidentata, 2n = 14, 16, 28; O. arenaria, O. bobrovii, 2n = 14, 28; O. crista-galli, 2n = 16, 32), with other species
being exclusively polyploid (e.g., O. biebersteinii, O. cyri, O. dielsii, all 2n = 28).
There appear to be no phylogenies or evolutionary studies of polyploidy in Alhagi
(2n = 16, 28; the latter number is not listed in IPCN).
The majority of the vicioid clade forms two sister clades, one with Fabeae
(Vicieae) plus Trifolium, and a second comprising the remaining Trifolieae genera;
polyploidy occurs in both clades. Successive sisters to this clade (Cicer, Galega,
and Parochetus) are all 2n = 16.
Vicia includes both diploids and tetraploids (2n = 10, 12, 14, 24, 28), but
polyploidy was considered rare in the genus by Kupicha (1981). Indeed, Vicia is
best known for its extensive non-polyploid variation in genome size (Chooi 1971;
Neumann et al. 2006), which shows only weak correlation with ploidy: diploid
(2n = 14) V. peregrina has a genome size of 9.48 pg/1C, double that of tetraploid
(2n = 24) V. tenuifolia (4.73 pg/1C). Endo et al. (2008) did not discuss either
issue in their phylogenetic study of New Wold Vicia. Travnicek et al. (2010)
studied the history of polyploidy in V. cracca, determining the ploidy of over
6,500 individuals at more than 250 localities in Europe and mapping the distributions of diploids, triploids, and tetraploids; they noted the rarity of triploids,
suggesting strong reproductive barriers between diploids and tetraploids.
Polyploidy is also noted to be rare in Lathyrus (2n = 14, 28, 42; Kupicha
1981). Gutierrez et al. (1994) hypothesized autopolyploid origins of L. pratensis
and L. palustris from conspecific diploids, but an allopolyploid origin of L.
venosus from two diploid species (L. ochroleucus and L. palustris). Only 2n = 14
counts are listed for the closely related Pisum in IPCN, for which Kupicha (1981)
listed polyploidy as ‘‘rare.’’
Turini et al. (2010) reconstructed nrDNA ITS and chloroplast phylogenies for
69 of the 86 species of Ononis (2n = 16, 20, 30, 32, 60, 64) and identified several
well-supported clades. They concluded that, ‘‘Unfortunately, only limited information is available … on chromosome numbers to test support for these groups’’.
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However, chromosome counts are available for nearly half of the species in their
phylogeny in IPCN, and some conclusions can be drawn. For example, the clade
that is strongly supported as sister to the remainder of the genus in their nrDNA
ITS tree includes only polyploids (O. tridentata and O. fruticosa are both 2n = 30;
O. rotundifolia is 2n = 32), suggesting that the genus as a whole could be polyploid. Only three species have low, potentially non-polyploid numbers in IPCN.
These occur in different clades, and in two cases species with low numbers have
higher numbers as well (O. variegata, 2n = 16, 30; O. ornithopodioides, 2n = 20,
32), raising the possibility that they are independent reductions from typical
polyploid numbers. The exception, O. adenotricha, is only reported as 2n = 16; its
position varies between the nrDNA ITS and trnL-F trees of Turini et al. (2010),
being sister to the tridentata clade in the trnL-F tree; however, this entire group is
not resolved as sister to the remainder of the genus in that tree. Elsewhere in the
genus, O. spinosa has multiple cytotypes (2n = 30, 32, 60), whereas O. pendula is
only known at 2n = 64. Kloda et al. (2008) studied patterns of genetic diversity in
several diploid and polyploid species in England and concluded that gene flow was
occurring within ploidy levels, but not between diploids and tetraploids.
Medicago includes the genomic model legume, M. truncatula (Young et al.
2011). Steele et al. (2010) provided a phylogeny for Medicago (2n = 14, 16, 32,
48), including multispecies sampling of its sister clade, which comprises the
interdigitated species of the two paraphyletic genera Melilotus (2n = 16, 24, 32;
though tetraploids are not reported in IPCN) and Trigonella (2n = 16, 28, 32, 44).
Aneuploid change from 2n = 16 to 2n = 14 has occurred several times in
Medicago (Steele et al. 2010). Polyploidy is concentrated in a clade that comprises
most species of sect. Medicago, along with M. arborea (sect. Dendrotelis); an
additional polyploid species, M. scutellata, occurs in the clade sister to this sect.
Medicago clade. Rosato et al. (2008) used fluorescence in situ hybridization
(FISH) to study relationships between polyploids and diploids in sect. Dendrotelis.
Some Medicago species are exclusively polyploid whereas others possess
multiple cytotypes. The M. sativa complex, which includes cultivated autotetraploid alfalfa (M. sativa ssp. sativa) as well as other diploid (2n = 16) and autopolyploid (2n = 32) species and their hybrids, has been the focus of several recent
studies (Sakiroglu et al. 2010; Havananda et al. 2010, 2011, and unpublished data).
Two major autopolyploid pairs in the complex are: (1) M. s. caerulea and M. s.
sativa, both with blue flowers and coiled pods, distinguishable by the larger size of
the tetraploid (M. s. sativa) for several characters; and (2) M. s. falcata, a yellowflowered taxon with falcate pods whose diploid and polyploid cytotypes are
indistinguishable morphologically. Interestingly, although M. s. sativa and M. s.
caerulea are undifferentiated for chloroplast haplotypes, the two cytotypes of M. s.
falcata possess nearly mutually exclusive sets of haplotypes, with haplotypes in
the tetraploid most likely derived by introgression from M. prostrata, a species
from outside the complex (Havananda et al. 2011). Jenczewski et al. (1999)
reported gene flow between wild and cultivated M. sativa populations; however,
based on chloroplast data, there does not appear to be significant gene flow
between blue- and yellow-flowered taxa in the complex either at the diploid or
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9.4.2.6 Indigofereae
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Schrire et al. (2009) provided a detailed phylogeny for this tribe, a monophyletic
group that is sister to the millettioid clade. Schrire et al. (2009) did not comment
on chromosomal variation or polyploidy, but numerous records are readily
available in IPCN, and mapping these onto the phylogeny provides some insights
into cytological evolution of the group.
The tribe is dominated by the very large genus Indigofera (ca. 700 species),
which Goldblatt (1981) and Polhill (1981b) listed as having 2n = 14, 16, 32, 48.
The higher numbers thus would be interpreted as representing tetraploids and
hexaploids. However, Frahm-Leliveld (1966), summarizing the cytotaxonomy of
the tribe, cited two x = 6 species, I. macrocalyx (2n = 12) and I. emarginella
(2n = 24), and concluded that ‘‘… the 48-chromosome Himalayan and EastAsiatic shrubby Indigoferas may not be hexaploids with base number x = 8, but
octoploids in an x = 6 range.’’ None of the species is listed in IPCN, but Reddy
and Revathi (1993) reported 2n = 12 for I. anil, confirming the presence of x = 6
in the genus.
The Schrire et al. (2009) phylogeny does not support the Frahm–Leliveld
(1966) hypothesis. One of the two 2n = 12 species, I. macrocalyx, is placed in the
large Palaeotropical clade of Schrire et al. (2009) and is sister to a group of species
that includes I. pulchra (2n = 16). All three sampled species with 2n = 48 are in
the Palaeotropical clade, but are placed nowhere near I. macrocalyx. In the Pantropical clade, I. rhynchocarpa (2n = 16) is sister to the clade that includes I.
emarginella, which is on a long branch sister to several other species; the only
other species counted from this subclade is also 2n = 16. Thus, there is no evidence that 2n = 48 species are derived from x = 12 species.
Tetraploids based on x = 8 are scattered throughout the phylogeny (Schrire
et al. 2009), supporting the observation of Frahm-Leliveld (1966) that 2n = 32 is
common in the genus. In the Palaeotropical clade, I. atriceps (2n = 32) is in a
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tetraploid levels, despite the existence of morphologically intermediate hybrid
subspecies (Havananda et al. 2011, and unpublished data). Much is known about
the genetics of polyploidy in alfalfa, where unreduced gametes have received
considerable study as a breeding tool (e.g., Bingham 1972; Veronesi et al. 1986;
Tondini et al. 1993; Calderini and Mariani 1997).
Ellison et al. (2006) constructed a phylogeny of Trifolium (2n = 10, 14, 16, 28,
32) that included 218 of its ca. 255 species, as well as species from 11 genera of
the vicioid clade. They showed that the genus is monophyletic; incongruence
within the genus between nuclear and chloroplast markers suggests considerable
hybridization. They also hypothesized a minimum of 19 shifts to aneuploidy and
22 instances of polyploidy from a base number of 2n = 16. They identified the
progenitors of two important species, both shown to be allopolyploids: the
widespread weed, T. dubium, and the most commonly cultivated clover species, T.
repens (Ellison et al. 2006).
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9.4.2.7 Millettioids
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With the recognition that Wisteria and Callerya are part of the IRLC, and that
Cyclolobium and Poecilanthe belong in the Brongniartieae, chromosome numbers
in the Millettieae (Tephrosieae in Polhill and Raven (1981)) are mostly 2n = 20 or
22, with 2n = 24 in Xeroderris, though many genera have no reported counts in
IPCN. Interestingly, Xeroderris is placed as sister to the remainder of the entire
millettioid clade in Wojciechowski et al. (2004), suggesting that 2n = 20 or 22
may be synapomorphic for the remainder of the millettioid clade (including
phaseoloids; see below). Millettieae comprises the bulk of one of the two major
millettioid clades (core millettioids), along with Abrus (Abreae), and much of the
subtribe Diocleinae of tribe Phaseoleae. Both diploid and tetraploid cytotypes
(2n = 22, 44) have been reported in three species of the ca. 40 IPCN records for
the large (ca. 350 spp.) genus Tephrosia (e.g., Srivastav and Raina 1986). The low
frequency of polyploidy in the core millettioid clade (5 %) is nearly identical to
the frequency in caesalpinioids, both of which are largely woody, tropical groups.
The other large clade (phaseoloids) contains most of the tribe Phaseoleae as
well as the tribes Desmodieae and Psoraleeae and is dominated by 2n = 20 or 22
counts. Polyploidy is more frequent in the phaseoloid clade (17 %), but still less
than half as common as in Hologalegina (Table 9.1). Within the phaseoloid clade,
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subclade that also includes diploids. In another subclade, I. mysorensis includes
both 2n = 16 and 32 cytotypes; other two members of its subclade are diploid.
Indigofera microcalyx, in yet another subclade, is 2n = 32; no other members of
its subclade has been counted, but the only counts from its sister clade are diploid.
In the pantropical clade, a small subclade in Schrire et al. (2009) includes I.
koreana (2n = 32), I. grandiflora (2n = 32, 48), I. decora (2n = 48 in Choi and
Kim (1997) but not listed in IPCN), as well as I. venulosa (no count available) and
I. kirilowii (2n = 16). Topologies differ between Schrire et al. (2009) and Choi
and Kim (1997), who focused on this group of mostly Korean endemics. Choi and
Kim (1997) listed I. grandiflora as 2n = 16, and given this count their topology
could suggest independent derivation of polyploidy in I. koreana (from I. grandiflora) and I. decora (from I. venulosa if it is diploid). An alternative explanation
is a single derivation of polyploidy within this clade. Elsewhere in the pantropical
clade, I. heterantha (2n = 48) is sister to I. hebepetala (2n = 16); the clade sister
to these two species includes I. amblyantha (2n = 48) and I. cassioides (2n = 16).
In a different subclade, I. suffruticosa is reported to have both 2n = 16 and 32
cytotypes; the closest reported species to it is diploid. In the Tethyan clade, I.
sessiliflora (2n = 32) is the only member of its subclade with a count in IPCN.
Indigofera hochstetteri has both 2n = 16 and 32 counts; its sister species, I.
arabica, is diploid. Indigofera angulosa is 2n = 32; no other species in its clade
has counts in IPCN.
Thus, there appear to be no large clades composed exclusively of polyploids in
Indigofera. Instead, as in other large legume genera, polyploidy is sporadic.
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chromosome numbers suggest that several genera are polyploid. The best known
of these is Glycine, with around 30 species whose lowest chromosome numbers are
2n = 38 and 40, in contrast to most of its phylogenetic neighbors (e.g., Doyle et al.
2003; Stefanovic et al. 2009) which are typical millettioids with 2n = 20 or 22. As
noted above, genomic data confirm the presence of two cycles of polyploidy in G.
max (soybean) since the origin of the legumes. The more recent of these has
resulted in homoeologous gene pairs that diverged around 10 MYA (Shoemaker
et al. 2006; Egan and Doyle 2010), setting a maximum date for the polyploidy
event, with the minimum date set by the earliest divergence of the various Glycine
species around 5 MYA (Innes et al. 2008; Doyle and Egan 2010). Phylogenetic
evidence is consistent either with autopolyploidy or with allopolyploidy from
extinct species more closely related to one another than to any extant genera
outside of Glycine (Straub et al. 2006). The presence of two classes of centromeric
heterochromatin repeats suggests that Glycine could be an allopolyploid, with the
two repeat types each derived from one of the diploid progenitor species (Gill et al.
2009). Such a hypothesis is difficult to test due to the extensive rearrangement of
homoeologous segments in the soybean genome (Schmutz et al. 2010) and also
requires complex patterns of concerted evolution among repeats on different
chromosomes.
At least three other phaseoloid genera are likely to be exclusively polyploid
based on chromosome number alone (Lackey 1981). In Erythrina (coral bean), all
sampled species are 2n = 42. The single count in IPCN for the small genus
Cologania is 2n = 44, and the monotypic Teyleria is also 2n = 44 (Kumar and
Hymowitz 1989). Goldblatt (1981) considered Calopogonium, with counts of
2n = 36 and ca. 37 in C. mucunoides, to be a polyploid, presumably with aneuploid reduction from a base of x = 10; however, Gill and Husaini (1986) reported
a count of 2n = 24, which could suggest a more recent derivation of polyploidy
within the genus. Similarly, counts of 2n = 28—considered polyploid in T. mollis
by Kumari and Bir (1990)—predominate in Teramnus species, though T. labialis is
variously listed as 2n = 20, 22, and 28. Strongylodon is also 2n = 28.
Polyploidy appears to be rare within Phaseoleae genera. Even relatively large
and well-studied genera such as Rhynchosia (ca. 230 species), Phaseolus (60–65
species), and Vigna (ca. 100 species) were reported in Polhill and Raven (1981) as
being exclusively diploid, though Sen and Bhattacharya (1988) later reported a
count of 2n = 44 in V. glabrescens. Polyploidy has also been reported within
species of Amphicarpaea and Neonotonia by Kumar and Hymowitz (1989; both
2n = 22, 44). Apios americana includes both diploid and triploid cytotypes
(2n = 22, 33); Joly and Bruneau (2004) reported multiple origins of autotriploidy
and high heterozygosity in this species. Glycine not only is a relatively recent
polyploid at the generic level (see above) but also includes several recently formed
allopolyploid species whose genomic relationships to extant diploids have been
worked out using molecular phylogenies (reviewed by Doyle et al. 2004), and
which are the focus of physiological and transcriptomic studies (Coate and Doyle
2010; Ilut et al. (in press)).
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9.5 Searching for Cryptic Polyploidy in the Phaseoloid
Legumes
Clearly, all papilionoids are fundamentally polyploid, even those with low chromosome numbers. The tempo and mechanism(s) of chromosomal diploidization
are unknown (Doyle et al. 2008; Soltis et al. 2010), and without that information it
is difficult to estimate the prevalence of cryptic polyploidy. As noted above,
consideration of the divergence times for major lineages suggests that the rate of
chromosomal diploidization is rapid—likely 10 MY or less.
On the other hand, ‘‘polyploid’’ chromosome numbers have persisted for at
least 5–10 MY in Glycine (Fig. 9.2). A cryptic polyploid papilionoid legume is a
taxon that has experienced an additional polyploidy event subsequent to the ca. 50
MYA papilionoid WGD but has a low chromosome number typical of its clade.
Thus, in the phaseoloid clade, cryptic polyploids would have chromosome numbers of 2n = 20 or 22. Polyploids on the way to diploidization would have
chromosome numbers between these numbers and 2n = 40-44. As noted above,
Calopogonium and Teramnus are candidates for this class; one perennial Glycine
species with 2n = 38 is likely at the first stages of this process.
We know from the paralog Ks profile of Glycine (Schleueter et al. 2004) that no
additional polyploidy events took place between the two WGD episodes detectable
in its genome. Therefore, we can infer that all of the ancestors of Glycine experienced only the papilionoid WGD; this includes the ancestors that form the backbone of the phaseoloid clade (Fig. 9.4), as well as the common ancestor of
phaseoloids and Indigofereae, and also its common ancestor with the IRLC clade.
Given these conditions, candidates for cryptic polyploidy include lineages that are
connected to the phaseoloid backbone by branches longer than 5–10 MY. This
includes several major groups, such as subtribes Phaseolinae and Cajaninae
(Fig. 9.4). Initial sampling of one species of each lineage would provide information on another set of ancestors, and subsequent searches can then be focused on
lineages connected to these ancestors by suitably long branches. This has now been
done for Cajanus cajan (pigeonpea), which Varshney et al. (2011) have shown has
no history of recent polyploidy. We are sampling other phaseoloids using 454
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Polyploidy occurs within at least one genus of Desmodieae, Lespedeza, which
was listed in Polhill and Raven (1981) as 2n = 18, 20, 22, 36. However, IPCN
gives higher numbers, for example L. bicolor with both 2n = 22 and 42 cytotypes,
as well as L. daurica and L. potaninii, both exclusively 2n = 42. Triploidy occurs
in Campylotropis polyantha var. leiocarpa (2n = 22, 33) and possibly in the genus
Pseudarthria, listed as 2n = 22, 26, 34. Only diploid counts (2n = 20, 22) were
reported by Ohashi et al. (1981) from the large (ca. 275 spp.) genus, Desmodium.
However, additional counts are found in IPCN, both at the diploid (2n = 24, 26)
and tetraploid levels, the latter in D. styracifolium (2n = 42) and D. incanum
(2n = 22, 44).
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Bituminaria
Cullen
Glycine 2n = 38, 40
Teramnus 2n = 28
Amphicarpaea 2n = 22, 44
Pueraria lobata
Neonotonia 2n = 22, 44
Pachyrhizus
Pueraria phaseoloides
Pseudovigna
Dumasia
Cologania 2n = 44
Macroptilium
Vigna
Macrotyloma
Psophocarpus
Erythrina 2n = 42
Cajanus
Bolusafra
Hardenbergia
Kennedia
Lespedeza
Campylotropis
Desmodium pauciflorum
Desmodium barbatum
Mucuna
Shuteria
Apios 2n = 22, 33
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Fig. 9.4 Phylogeny and polyploidy in the phaseoloid clade. Topology and dates of the
chronogram are taken from Stefanovic et al. (2009). Chromosome numbers of the species used in
that study are color coded as follows: Green, 2n = 20; blue, 2n = 22; black, 2n = 18; red,
known or possible polyploid numbers, with the numbers shown following the taxon name.
Species known to have multiple cytotypes are indicated by multiple colors corresponding to
chromosome numbers listed above. The two numbers shown for Glycine are from different
species, only one of which (G. max, 2n = 40) was used in the Stefanovic et al. (2009) study. The
range of dates for the polyploidy event in Glycine is indicated. Yellow dots indicate ancestral
nodes that lacked any polyploidy event subsequent to the papilionoid WGD
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transcriptome sequencing, including taxa with both low and high chromosome
numbers; for the latter we wish to estimate maximum ages of polyploidy. Thus far
we have not found examples of cryptic polyploidy, but have determined that
polyploidy in Erythrina probably took place on roughly the same time scale as in
Glycine (\10 MYA; Egan and Doyle, unpublished data).
898
Among the most persistent questions concerning polyploidy in plants are how
successful the phenomenon is as an evolutionary mechanism. Is polyploidy a ticket
to innovation, adaptation, invasiveness, survival in the face of global catastrophes,
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Acknowledgments I thank many colleagues and lab members for discussions of polyploidy, and
Jane Doyle for her support and encouragement. I also thank Jane Doyle, Sue Sherman-Broyles,
Iben Sorensen, and Toby Pennington for critical reading of the manuscript, and Melissa Luckow
for help with mimosoid systematics. I am grateful for many years of funding from the National
Science Foundation for work on polyploidy, most recently grants DEB-0948800, IOS-0939423,
IOS-0822258, and IOS-0744306. I thank Doug Soltis for helpful suggestions in review of the
manuscript.
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or is it an evolutionary dead end … or both? Based on rates of polyploid formation
and extinction in the phylogenetic record, Mayrose et al. (2011) conclude that
‘‘polyploidy is most often an evolutionary dead end, but the possibility remains
that the expanded genomic potential of those polyploids that do persist drives longterm evolutionary success.’’
Legumes may illustrate both of these points. The most diverse and species-rich
clade of this third largest family of flowering plants, the core papilionoids, is
ancestrally polyploid. Clearly, the ancestor of this group of around 450 genera and
13,000 species, like the ancestor of seed plants and the ancestor of flowering plants
(Jiao et al. 2011), was most emphatically not a ‘‘dead end.’’ It remains to be
determined whether there is a perfect correlation between the papilionoid polyploidy event and the origin of nodulation in core papilionoids, and it will take
much more work to demonstrate that the two are causally related (Doyle 2011;
Young et al. 2011). It is also clear that nodulation is not sufficient to explain the
explosive radiation of papilionoid legumes, because other nodulating groups both
in legumes and elsewhere in the rosids have not proliferated to the same extent as
papilionoids (Doyle 2011).
Despite the obvious success of the core papilionoid lineage, the pattern of
evolution within the core papilionoids suggests that polyploidy has not been a
major feature in establishing new lineages, similar to the conclusion of Mayrose
et al. (2011) for angiosperms generally. It is not that polyploidy is rare within the
family—indeed, around a quarter of all legumes for which chromosome data are
available have one or more species that are polyploid (Table 9.1). However, much
of the polyploidy in the family occurs as single polyploid genera embedded within
diploids, as scattered species within genera, or as multiple cytotypes within species. Two significant exceptions are the Genisteae, which may be entirely polyploid and within which nearly all genera show a propensity for polyploidy and
aneuploidy, and the lineage that includes the IRLC tribe Carmichaelieae. The
largest papilionoid genera, including Astragalus, are not fundamentally polyploid.
‘‘Success’’ is a very ambiguous term and can be measured in many ways.
Species with short evolutionary histories that have not been involved in subsequent
speciation, yet have invaded extensive new territories and had major impact on the
environment, certainly could be considered ‘‘successful.’’ Many plant polyploids,
including genera and species of legumes, fit this description. So does Homo
sapiens.
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Metadata of the chapter that will be visualized in
SpringerLink
Book Title
Polyploidy and Genome Evolution
Series Title
Chapter Title
Jeans, Genes, and Genomes: Cotton as a Model for Studying Polyploidy
Copyright Year
2012
Copyright HolderName
Springer-Verlag Berlin Heidelberg
Corresponding Author
Family Name
Wendel
Particle
Given Name
Jonathan F.
Suffix
Author
Division
Department of Ecology, Evolution, and Organismal Biology
Organization
Iowa State University
Address
50011, Ames, IA, USA
Email
jfw@iastate.edu
Family Name
Flagel
Particle
Given Name
Lex E.
Suffix
Author
Division
Department of Biology
Organization
Duke University
Address
90338, 27708, Durham, NC, USA
Email
lex.flagel@duke.edu
Family Name
Adams
Particle
Given Name
Keith L.
Suffix
Abstract
Division
Department of Botany, and UBC Botanical Garden and Centre for Plant
Research
Organization
University of British Columbia
Address
6268 University Blvd, V6T 1Z4, Vancouver, BC, Canada
Email
keitha@mail.ubc.ca
We present an overview of the cotton genus (Gossypium) as a model for the study of polyploidy. A synopsis
of the origin and evolution of polyploid cotton is provided, offering an organismal framework and
phylogenetic perspective that is critical for understanding modes and mechanisms of gene and genome
evolution. Sequence data from thousands of genes implicate a mid-Pleistocene (1–2 mya) origin of polyploid
cotton, following trans-oceanic dispersal of an Old World, A-genome diploid to the New World and
subsequent hybridization with an indigenous D-genome diploid. This chance biological reunion, occurring
after 5–10 million years of diploid evolution in isolation, has led to an array of molecular genetic interactions
in the newly formed allopolyploid lineage, including nonreciprocal homoeologous recombination and perhaps
other forms of interlocus concerted evolution, differential rates of genomic evolution, intergenomic spread
of transposable elements, and myriad forms of alterations in duplicate expression relative to that experienced
in the ancestral diploids. The latter include developmental, organ-, tissue-, and cell-specific biases in
homoeologous gene expression, which can be sensitive to various forms of environmental perturbation and
stress. The allopolyploid Gossypium transcriptome is exceptionally dynamic, with homoeolog expression
ratios being subject to change even during development of the single-celled cotton fiber. Expression evolution
is temporally partitioned into changes accompanying genome merger (hybridization) at the diploid level,
polyploidization, and longer term evolution at the allopolyploid level. Evidence indicates that allopolyploidy
facilitated colonization of a new ecological niche for the genus and led to an enhanced capacity for developing
agronomically superior cotton varieties. The myriad mechanisms that underlie genomic and regulatory
evolution are suggested to have contributed to both ecological success and agronomic potential.
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Abstract We present an overview of the cotton genus (Gossypium) as a model for
the study of polyploidy. A synopsis of the origin and evolution of polyploid cotton
is provided, offering an organismal framework and phylogenetic perspective that is
critical for understanding modes and mechanisms of gene and genome evolution.
Sequence data from thousands of genes implicate a mid-Pleistocene (1–2 mya)
origin of polyploid cotton, following trans-oceanic dispersal of an Old World,
A-genome diploid to the New World and subsequent hybridization with an indigenous D-genome diploid. This chance biological reunion, occurring after 5–10
million years of diploid evolution in isolation, has led to an array of molecular
genetic interactions in the newly formed allopolyploid lineage, including nonreciprocal homoeologous recombination and perhaps other forms of interlocus
concerted evolution, differential rates of genomic evolution, intergenomic spread
of transposable elements, and myriad forms of alterations in duplicate expression
relative to that experienced in the ancestral diploids. The latter include developmental, organ-, tissue-, and cell-specific biases in homoeologous gene expression,
which can be sensitive to various forms of environmental perturbation and stress.
The allopolyploid Gossypium transcriptome is exceptionally dynamic, with
homoeolog expression ratios being subject to change even during development of the
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Jeans, Genes, and Genomes: Cotton
as a Model for Studying Polyploidy
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Book ID: 272454_1_En
Date: 16-8-2012
J. F. Wendel (&)
Department of Ecology, Evolution, and Organismal Biology,
Iowa State University, Ames, IA 50011, USA
e-mail: jfw@iastate.edu
L. E. Flagel
Department of Biology, Duke University, 90338 Durham, NC 27708, USA
e-mail: lex.flagel@duke.edu
UN
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K. L. Adams
Department of Botany, and UBC Botanical Garden and Centre for Plant Research,
University of British Columbia, 6268 University Blvd, Vancouver, BC V6T 1Z4, Canada
e-mail: keitha@mail.ubc.ca
P. S. Soltis and D. E. Soltis (eds.), Polyploidy and Genome Evolution,
DOI: 10.1007/978-3-642-31442-1_10, Springer-Verlag Berlin Heidelberg 2012
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single-celled cotton fiber. Expression evolution is temporally partitioned into
changes accompanying genome merger (hybridization) at the diploid level, polyploidization, and longer term evolution at the allopolyploid level. Evidence indicates
that allopolyploidy facilitated colonization of a new ecological niche for the genus
and led to an enhanced capacity for developing agronomically superior cotton
varieties. The myriad mechanisms that underlie genomic and regulatory evolution
are suggested to have contributed to both ecological success and agronomic
potential.
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Because of its economic importance, the cotton genus (Gossypium L.) has long
attracted the attention of agricultural scientists, taxonomists, and biologists in
multiple disciplines. Accordingly, a great deal is understood about the origin and
diversification of the genus, its basic plant biology, and its properties as a crop
plant (Paterson 2009; Stewart et al. 2010; Wendel et al. 2009). One of the most
salient features of the genus is that its history is so encompassing in scope,
involving a global (mostly austral) phylogenetic diversification and a repeated
history of trans-oceanic dispersal. Superimposed on this natural diversification has
been a many-thousand-year history of human manipulation, tracing to ancient
human cultures on several continents who independently domesticated four species, two from the Americas, G. hirsutum and G. barbadense, and two from
Africa-Asia, G. arboreum and G. herbaceum. In each of these four cases,
aboriginal peoples discovered that the unique properties of cotton ‘‘fibers’’, which
are unicellular epidermal seed trichomes, made them useful for ropes, textiles, and
other applications. Each of these crop species has its own history of domestication,
diversification, and current utilization (Brubaker et al. 1999a; Hutchinson 1951,
1954; Hutchinson et al. 1947; Percy and Wendel 1990; Wendel et al. 2009).
This rich history of scientific study has made Gossypium one of the best systems
for studies of polyploidy in plants. As testified by the many papers in this volume,
plant evolution has been characterized by repeated rounds of whole-genome doubling. Evolutionary footprints of paleopolyploidy in ‘‘diploid’’ (n = 13) cotton
have long been evidenced using both classic and more modern techniques,
including chromosome banding, comparative genome mapping, and analysis of
synonymous substitution rate curves (Muravenko et al. 1998; Brubaker et al. 1999b;
Reinisch et al. 1994; Paterson 2009; Lin and Paterson 2009; Lin et al. 2011). These
more ancient events, which must have profoundly impacted morphological, ecological, and physiological diversification, predate a more recent allopolyploidy
event that traces to the mid-Pleistocene (Wendel 1989; Wendel et al. 2009; Wendel
and Cronn 2003). The most experimentally tractable polyploid event is this most
recent one, resulting from the merger that led to the evolution of the five extant
species of New World allopolyploid cotton (2n = 4x = 52), including the two
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10.1 Introduction
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10.1.1 Origin and Diversification of the Diploid Cottons
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The cotton genus belongs to a small tribe, the Gossypieae, that includes only eight
genera and *120 species (Fryxell 1968, 1979). Four of these genera are either
monotypic or contain only several species with restricted geographic distributions,
Lebronnecia (Marquesas Islands), Cephalohibiscus (New Guinea, Solomon Islands),
Gossypioides (east Africa, Madagascar), and Kokia (Hawaii). The tribe also includes
four moderately sized genera with broader ranges: Hampea, with 21 neotropical
species; Cienfuegosia, with 25 species from the neotropics and parts of Africa;
Thespesia, with 17 tropical species; and last but not least, Gossypium, the largest and
most widely distributed genus in the tribe with more than 50 species (Fryxell 1992).
Gossypium species collectively have achieved a nearly worldwide distribution,
with several primary centers of diversity in the arid or seasonally arid tropics and
subtropics. Species-rich regions include Australia, especially the Kimberley region
in NW Australia, the Horn of Africa and southern Arabian Peninsula, and the
western part of central and southern Mexico. Recognition of these groups of
species reflects decades of accumulated understanding that emerged from basic
plant exploration and taxonomic analysis (Fryxell 1979, 1992; Hutchinson et al.
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cultivated species G. hirsutum (Upland cotton) and G. barbadense (Pima cotton,
Sea Island cotton). Gossypium hirsutum presently is responsible for over 90 % of
the cotton crop internationally, having spread from its original home in Mesoamerica to over 50 countries in both eastern and western hemispheres. Accumulating evidence indicates that polyploidy per se has played a critical role in enabling
the development of modern, agronomically elite varieties. Thus, it is of interest to
explore what cotton has taught us about the evolutionary consequences of genome
doubling, and in turn, what the process of genome doubling might reveal about
phenotypic diversification and the goals of agronomic improvement.
We begin by reviewing our current understanding of the diversity of the genus
and the origin of the Gossypium clade, which includes one of the classic examples
of polyploidy. This phylogenetic and temporal perspective provides the organismal
framework that serves as the foundation for all analyses of the consequences of
polyploid evolution, including those focused on genomic, epigenomic, and phenotypic levels. We then provide a synopsis of the myriad genomic consequences
that were set in motion by the evolutionary processes of genome merger, chromosome doubling, and subsequent evolutionary diversification which collectively
gave rise to modern allopolyploid cottons, drawing attention to the relationships
among evolutionary processes and temporal scale of divergence. Thus, we distinguish phenomena and processes that might characterize the earliest stages of
polyploid formation from those that are responsible for longer term genomic and
phenotypic changes. Finally, we summarize evidence that polyploidy enables the
evolution of transgressive or novel phenotypes in cotton, as exemplified by the
differences between modern cultivated diploid versus allopolyploid cotton.
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1947; Saunders 1961; Watt 1907; Wendel et al. 2009). The genus is extraordinarily
diverse; species morphologies range from fire-adapted, herbaceous perennials in
NW Australia to trees in SW Mexico that escape the dry season by dropping their
leaves. Corolla colors span a rainbow of blue to purple (G. triphyllum), mauves
and pinks (‘‘Sturt’s Desert Rose’’, G. sturtianum, is the official floral emblem of
the Northern Territory, Australia), whites and pale yellows (NW Australia,
Mexico, Africa-Arabia), and even a deep sulfur-yellow (G. tomentosum from
Hawaii). Seed coverings range from nearly glabrous (e.g., G. klotzschianum and
G. davidsonii), to short stiff, dense, brown hairs that aid in wind-dispersal
(G. australe, G. nelsonii), to the long, fine white fibers that characterize highly
improved forms of the four cultivated species (Fig. 10.1). There are even seeds
that produce fat bodies to facilitate ant-dispersal (Seelanan et al. 1999). At the
other end of the ant coevolution spectrum is G. tomentosum from the Hawaiian
Islands, which lost the foliar and extra-floral nectaries that are common in other
Gossypium species, presumably in response to the absence of native ants.
The evolution of this morphological and geographic diversity was accompanied
by a parallel diversification at the chromosomal level (Lin and Paterson 2009).
Although all diploid species share the same chromosome number (n = 13), there
is more than 3-fold variation in DNA content per genome (Hendrix and Stewart
2005). Chromosome morphology is similar among closely related species, as
reflected in the ability of related species to form hybrids that display normal
meiotic pairing and sometimes high F1 fertility. In contrast, crosses among more
distant relatives may be difficult to achieve, and those that are successful are
characterized by meiotic abnormalities. The collective observations of pairing
behavior, chromosome size, and relative fertility in interspecific hybrids led to the
designation of single-letter genome symbols (Beasley 1941) for related clusters of
species. Presently, eight diploid genome groups (A through G, plus K) are recognized (Endrizzi et al. 1985; Stewart 1995).
A genealogical framework for the genus is provided from analyses of multiple
molecular phylogenetic investigations (reviewed in Wendel and Cronn 2003).
A key phylogenetic conclusion has been the demonstration that the group of species
recognized as belonging to Gossypium do, in fact, constitute a single natural
lineage, despite their exceptionally broad geographic distribution and extraordinary
morphological and cytogenetic diversity. A second important result is that the
closest relative of Gossypium is the sister clade that includes the AfricanMadagascan genus Gossypioides and the Hawaiian endemic genus Kokia; these
latter genera may thus be used as phylogenetic outgroups for studying evolutionary
patterns and processes within Gossypium. A third phylogenetic conclusion is that
each of the classically recognized genome groups comprises a monophyletic group.
This information is summarized in a depiction of our present understanding of
relationships (Fig. 10.2), which shows four major lineages of diploid species
corresponding to three continents: Australia (C-, G-, K-genomes), the Americas
(D-genome), and Africa/Arabia (two lineages: one comprising the A-, B-, and
F-genomes, and a second containing the E-genome species). Embedded in this
result is the observation that the earliest divergence in the genus separated the New
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World D-genome lineage from the ancestor of all Old World taxa, and thus, that
New World and Old World diploids are phylogenetic sister groups. Following this
split in the genus, cottons comprising the Old World lineage divided into three
groups, namely, the Australian cottons (C-, G-, and K-genome species), the
African-Arabian E-genome species, and the African A-, B-, and F-genome cottons.
The African F-genome clade, which consists of the sole species G. longicalyx, is
diagnosed as sister to the A-genome species, an important realization in that this
relationship identifies the wild forms most closely related to the clade (the
A-genome) that ‘‘invented’’ long, or spinable, fiber since these two clades (A- and
F-) shared a common ancestor.
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Fig. 10.1 Representative seed and trichome diversity in Gossypium. Seed and trichome size and
morphology are exceedingly variable in the genus. Most wild species have relatively small seeds
(\5 mm in any dimension) with equally short fibers. Long (spinnable) fiber evolved only once, in
the ancestor of modern A-genome cottons, which subsequently donated this capacity to modern
tetraploid species, including the commercially important G. hirsutum and G. barbadense, at the
time of allopolyploid formation. Key to species: Cult. AD1 = G. hirsutum TM1; Wild AD1 = G.
hirsutum Tx2094 from the Yucatan Peninsula; AD3 = G. tomentosum WT936 from Hawaii;
C1 = G. sturtianum C1-4 from Australia; Cult. A2 = G. arboreum AKA8401; Wild A1 = G.
herbaceum subsp. africanum from Botswana; D5 = G. raimondii from Peru; D3 = G. davidsonii
D3d-32 from Baja California; F1 = G. longicalyx F1-3 from Tanzania; B1 = G. anomalum B1-1
from Africa
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A temporal framework for the origin of Gossypium and its diversification is
provided by sequence divergence data, which may serve as a proxy for time
(Senchina et al. 2003). These analyses indicate that Gossypium diverged from its
closest relatives during the Miocene, perhaps 10–15 mya, subsequently spreading
around the world via trans-oceanic dispersal to acquire its modern geographic
range. These early estimates of divergence, and hence times, have recently been
supported by an enormous amount of new sequence data derived from a global
assembly of ESTs (Flagel et al. 2012). Consideration of the phylogeny of Fig. 10.2
in a temporal context and in light of plate tectonic history leads to the inference
that the history of Gossypium has entailed multiple episodes of trans-oceanic
dispersal. These include at least one dispersal between Australia and Africa,
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Fig. 10.2 Evolutionary history of Gossypium, as inferred from multiple molecular phylogenetic
data sets. The closest relative of Gossypium is a lineage containing the African-Madagascan
genus Gossypioides and the Hawaiian endemic genus Kokia. Following its likely origin 5–10
mya, Gossypium split into three major diploid lineages: the New World clade (D-genome); the
African-Asian clade (A-, B-, E-, and F-genomes); and the Australian clade (C-, G-, and
K-genomes). This global radiation involved several trans-oceanic dispersal events and was
accompanied by morphological, ecological, and chromosomal differentiation (2C genome sizes
shown in white ellipses). Allopolyploid cottons formed following trans-oceanic dispersal of an
A-genome diploid to the Americas, where the immigrant underwent hybridization, as female,
with a native D-genome diploid similar to modern G. raimondii. Polyploid cotton probably
originated during the Pleistocene (1–2 mya), with the five modern species comprising the
descendants of an early and rapid colonization of the New World tropics and subtropics
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10.1.2 Origin and Diversification of the Polyploid Cottons
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A rich body of cytogenetic and experimental evidence has demonstrated that
the tetraploid species, which are entirely New World in their distribution, are
allopolyploids containing two genomes, an A-genome from Africa or Asia, and a
D-genome similar to those found in the American diploids (Endrizzi et al. 1985;
Wendel et al. 2009; Wendel and Cronn 2003). The hemisphere-scale allopatry of
these two diploid genome groups led to more than 50 years of mystery surrounding
the timing of formation and parentage of the New World allopolyploids. With
respect to the question of ‘‘when’’, gene sequence data convincingly demonstrate
that allopolyploid Gossypium originated prior to the evolution of modern humans
but relatively recently in geological terms, perhaps 1–2 mya, or in the midPleistocene (Senchina et al. 2003; Wendel 1989; Flagel et al. 2012). With respect to
the second part of the question, that of polyploid parentage, it is now clear that both
extant A-genome species (G. arboreum, G. herbaceum) are equally divergent from
the A-genome of allopolyploid cottons and that the closest living relative of the
progenitor D-genome donor is G. raimondii (Endrizzi et al. 1985; Wendel et al.
2009; Wendel and Cronn 2003). Studies using nuclear (biparentally inherited)
genes led to the same conclusion. Additionally, all allopolyploids contain an
A-genome cytoplasm, as evidenced from analysis of both mitochondrial and plastid
genomes (Galau and Wilkins 1989; Small and Wendel 1999; Wendel 1989).
Finally, the studies mentioned above, and additional, extensive DNA sequence data
from ongoing studies (Grover et al. unpublished), support a single origin for
allopolyploid cotton.
Given a Pleistocene origin for allopolyploid cotton species, one may infer that
their morphological diversification and spread must have been relatively rapid
following polyploidization. At present, five allopolyploid species are widely recognized, although a sixth species (G. ekmanianum) was recently proposed
(Krapovickas and Seijo 2008). Gossypium darwinii is native to the Galapagos
Islands, where it may form large populations in some areas (Percy and Wendel
1990). Gossypium tomentosum, from the Hawaiian Islands, has a more diffuse
population structure, occurring mostly as scattered individuals and small populations on several islands (DeJoode and Wendel 1992). A third allopolyploid,
G. mustelinum, is restricted to a small region of northeast Brazil (Wendel et al.
1994). In addition to these three truly wild species, there are two cultivated species
(G. barbadense and G. hirsutum), each of which has a large indigenous range,
collectively encompassing a wealth of morphological forms that span the wildto-domesticated continuum (Brubaker and Wendel 1993, 1994, 2001; Fryxell 1979;
Hutchinson 1951; Percy and Wendel 1990). Gossypium hirsutum is widely
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another to the Americas leading to the evolution of the D-genome diploids, and a
second, much later colonization of the New World by the A-genome ancestor of
the AD-genome allopolyploids (see below).
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10.2 Evolution Following Genome Duplication
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10.2.1 Chromosomal Stabilization and Structural Stasis
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Classical cytogenetic evidence indicates that chromosomes of the A- and D-genomes
of allopolyploid Gossypium are less able to pair with one another than are chromosomes of the living descendants of their diploid progenitors (reviewed in Endrizzi
et al. 1985). For example, allopolyploid-derived haploids form an average of less
than one bivalent per cell at meiotic metaphase, whereas chromosomes in hybrids
between extant A- and D- genome diploids average 5.8 and 7.8 bivalents (reviewed in
Endrizzi et al. 1985). These and similar observations indicate that natural selection
has favored the evolution of mechanisms that promote exclusive bivalent formation
in the allopolyploid. Neither the pace at which such mechanisms operate nor their
nature are understood, but it seems rational to postulate that selection would be most
intense in the first generations following allopolyploid formation, where the fitness
cost of unbalanced gametes would be the greatest.
One hypothesis for this apparent lack of bivalent formation is that genome
stabilization following polyploidization involved genomic reorganization of the
two resident genomes such that they no longer are capable of homoeologous
pairing. To evaluate the extent of structural change, genetic maps were generated
and compared among interspecific F2 progenies for diploid (both A- and D-) and
allopolyploid cottons (Brubaker et al. 1999b; Rong et al. 2004). Comparisons of
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distributed in Central and northern South America, the Caribbean, and even reaches
distant islands in the Pacific (eg., Solomon Islands, Marquesas). Gossypium
hirsutum is thought to have a more northerly distribution than G. barbadense, with
wild populations occurring as far north as Tampa Bay, Florida (27̊38’N) (Stewart,
personal observation). Gossypium barbadense has a more southerly indigenous
range, centered in the northern third of South America but with a large region of
range overlap with G. hirsutum in the Caribbean.
Consideration of the distribution of the allopolyploid species suggests that
polyploidy led to the invasion of a new ecological niche. Fryxell (1965, 1979)
noted that in contrast to the majority of diploid species, allopolyploid species
typically occur in coastal habitats, at least those forms that arguably are truly wild.
Two species, both island endemics (G. darwinii and G. tomentosum), are restricted
to near coastlines, and for two others (G. barbadense and G. hirsutum), wild forms
occur in littoral habitats ringing the Gulf of Mexico, northwest South America, and
distant Pacific Islands. Fryxell speculated that following initial formation, adaptation of the newly evolved allopolyploid to littoral habitats enabled it to exploit
the fluctuating sea levels that characterized the Pleistocene. This ecological
innovation is envisioned to have facilitated initial establishment of the new
polyploid lineage and also may have provided a means for the rapid dispersal of
the salt-water-tolerant seeds.
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10.2.2 Genome Sizes, Transposable Element Mobilization,
and Genomic Downsizing
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gene order and synteny among the A- and D-genome maps, as well as those
for both genomes of the allopolyploid (A vs. AT and D vs. DT), demonstrate
that relatively few structural rearrangements have arisen in the 1–2 my since
allopolyploid formation; conservation of collinear linkage groups is the rule rather
than the exception. Thus, allopolyploidy in Gossypium has not been accompanied
by extensive chromosomal rearrangement. This implies that structural rearrangement has not been a significant aspect of the process of polyploid genome
stabilization in cotton. Additional support for this idea emerges from experiments
involving synthetic allopolyploids; for example, Liu et al. (2001) used AFLP
analysis to demonstrate almost exclusive fragment additivity for 22,000 genomic
loci in nine sets of newly synthesized allotetraploid and allohexaploid Gossypium.
Thus, and in contrast to some other plant models described in this volume (e.g.,
wheat, Tragopogon), the polyploid Gossypium genome appears to be relatively
quiescent, at least with respect to the phenomenon of rapid genome change.
A corollary is that the evolutionary enforcement of homologous pairing discussed
above originated through means other than structural rearrangements.
As shown in Fig. 10.2, genome sizes vary widely among diploid cotton species,
from *900 Mb in the D-genome diploids to *2,600 Mb in the Australian diploids
(Hendrix and Stewart 2005), reflecting primarily the differential and punctuated
proliferation of various families of copia and gypsy transposable elements (TEs), as
well as lineage-specific differences in the rate of deletions (Hawkins et al. 2008,
2006, 2009). The two progenitor genomes of allopolyploid cotton differ 2-fold in
size, and moreover, they differ in their complement of resident TEs. Thus, allopolyploidization entailed the merger of two different complements of TEs, creating
the potential for activation of TEs due to the generalized disruption of epigenetic
suppression of TE activity following the merger of two diverged regulatory systems, a process commonly referred to as ‘‘genomic shock’’. To evaluate the possibility that polyploidization in Gossypium was accompanied by a transpositional
burst, as in some other species (Kashkush et al. 2002; Shan et al. 2005; Ungerer
et al. 2006), Hu et al. (2010) used phylogenetic and quantitative methods to identify
changes in TE populations. These data showed that the major LTR retrotransposon
classes in the AD genome phylogenetically clustered with either their A- or Dgenome antecedent elements in a genome-specific fashion, with no evidence of an
impressive, recent, TE burst. Thus, hybridization and polyploidy do not appear to
have stimulated a massive TE proliferation in Gossypium.
Notwithstanding the relative TE quiescence indicated by these studies, evidence
using FISH (Hanson et al. 1999, 1998) implicates at least a modest level of TE
activity in allopolyploid cotton. These data show that a family of copia-like
retrotransposable elements ‘‘horizontally’’ transferred across genomes following
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10.2.3 Genic Evolution in Diploid Versus Allopolyploid Cotton
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allopolyploid formation. This result highlights the phenomenon of TE spread
across previously separated genomes following polyploid formation, raising the
possibility that this process has played a role in diversification and adaptation via
novel TE insertions.
An attractive feature of the Gossypium model system is that species representing both progenitor genomes remain extant, and that they vary so dramatically
(nearly 2-fold) in genome size yet retain collinearity with their orthologs in the
allopolyploid genome. Although the Gossypium genome has yet to be sequenced,
it is likely that a high-quality D-genome sequence (from G. raimondii) will be
published by the time this volume is published, with perhaps the A-genome
sequence not far behind. It is an exciting prospect to contemplate the availability
of both diploid sequences as well as one or more from allopolyploid Gossypium.
These data will offer a veritable gold mine for generating insight into the pace,
patterns, and dynamics of genome evolution that accompany diploid divergence,
allopolyploid formation, and subsequent evolution at the polyploid level.
A glimpse of this promise is provided by current work focused on comparative
sequencing of bacterial artificial chromosomes (BACs) from Gossypium in polyploid and diploid species. Two comparative BAC sequencing studies have been
published, a region surrounding the CesA1 gene (Grover et al. 2004) from both
homoeologous genomes of G. hirsutum, and a region surrounding the AdhA gene
(Grover et al. 2007) from G. hirsutum and diploids representing models of the two
progenitor genomes, i.e., G. arboreum and G. raimondii. Data generated to date
indicate that small deletions are more prevalent in the polyploid genomes (AT and
DT) than in either diploid genome, illustrative of the general phenomenon of
genomic downsizing in polyploid genomes (Bennett and Leitch 2005), while
providing a glimpse into an underlying mechanism (illegitimate recombination,
which is biased in the allopolyploid toward deletion). Extensions of this work into
20 regions and in more genomes, including a phylogenetic outgroup are underway
(Grover, Wendel, and Paterson, unpubl.), offering an opportunity to learn more
about features of genome evolution that distinguish polyploid cotton from its
diploid progenitors.
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The most immediate and important genomic consequence of allopolyploid formation in Gossypium was simultaneous duplication of all nuclear genes. From a
phylogenetic perspective, the various fates of gene duplication may partially be
modeled as shown in Fig. 10.3. The null hypothesis for sequence evolution in
allopolyploids derives from the organismal history; if both duplicated genes evolve
independently following allopolyploid formation, then each homoeolog should be
phylogenetically sister to its ortholog from its progenitor diploid, rather than to the
other homoeolog. Similarly, if rates of sequence evolution are maintained between
the diploid and allopolyploid level, branch lengths for the two A-genome sequences
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Recombination
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Novel expression
Intergenomic
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Fig. 10.3 A model of various possibilities for duplicate gene evolution after allopolyploidy in
Gossypium. The null expectation (center) derives from the organismal history (left): if
homoeologs evolve independently following allopolyploid formation, then each should be
phylogenetically sister to its ortholog from the donor diploid, rather than to the other homoeolog.
Similarly, if rates of sequence evolution are similar at the diploid and allopolyploid level, branch
lengths for the two A-genome sequences (one from the diploid, ‘‘A’’, and the other from the
allopolyploid, ‘‘AT’’) should be similar, as they should for the two D-genome sequences (‘‘D’’
and ‘‘DT’’). The utility of this null hypothesis lies in its falsification; if homoeologous sequences
interact via concerted evolutionary forces or nonreciprocal homoeologous recombination, for
example, a different tree may be recovered (‘‘Recombination’’, top center), or if there is strong
directional selection or pseudogenization, rate inequalities may become evident (‘‘Unequal
rates’’, bottom center). Additional possibilities include loss of one of the homeologs (‘‘Gene
loss’’, top right), replicative transfer of sequences from one genome to the other (‘‘Intergenomic
transfer’’, bottom right), and evolutionary divergence in duplicate gene expression domains or
amounts (‘‘Novel expression’’, right middle). This latter category, novel expression, encompasses
multiple phenomena, including developmentally or environmentally regulated biases in
homoeolog expression ratios, organ- or tissue- or cell-specific homoeolog silencing, novel
expression domains, and transgressive (higher or lower than either progenitor diploid) expression
amounts
(one from the diploid, the other from the allopolyploid) should be similar, as they
should for the two D-genome sequences. The utility of the null hypothesis lies in its
falsification; if homoeologous sequences interact, for example, a different tree may
be recovered, or if there is strong directional selection or pseudogenization, rate
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10.2.3.1 Molecular Evolution of Homoeologs and Orthologs
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Early work (Cronn et al. 1999; Senchina et al. 2003) demonstrated that duplicated
genes typically are both retained even in natural (1–2 mya -ld) allopolyploids, and
that they evolve essentially as modeled in the central panel of Fig. 10.3, i.e.,
equally, additively, and at equivalent rates. A more recent analysis (Flagel et al.
2012), based on a global assembly of 5 million Sanger and 454 ESTs supplemented by *150 million 82-bp Illumina reads, has provided a vastly expanded
and detailed view of genic evolution in diploid and allopolyploid cotton, perhaps
the most extensive yet for any plant genus. These data, representing analysis of
*10,000 genes in each comparison, show that rates of synonymous substitution
(Ks) between A- and D-genome orthologs (0.036) are nearly identical to that
experienced by their homoeologous descendants in the allopolyploid genome
(0.037), with non-synonymous substitution rates being identical in the two contrasts (0.009). These results demonstrate unequivocally that allopolyploidy has not,
in general, been accompanied by an enhanced rate of nucleotide substitution in
coding regions, as might be expected from an assumption of rapid decay of
‘‘redundant’’ duplicated copies.
An additional perspective provided by Flagel et al. (2012) is that the data also
provide an extraordinarily accurate depiction of the ancestry of polyploid cotton.
By using thousands of genic alignments, the relative distances of the A- and
D-genome diploids to their counterparts in the allopolyploid (AT, DT) can be
calculated and effectively translated into relative divergence times because of the
demonstration of rate homogeneity discussed above. The results show that the A
genome diploid has a mean Ks of 0.009 from the AT-genome, whereas the
comparable figure for the D genome comparison is 0.015. From this we infer that
modern A-genome diploids are a better model (by about 50 %) of the actual
genome donor of allopolyploid cotton than are extant D-genome diploids,
consistent with previous suggestions based on diverse data sources (Wendel et al.
2009; Wendel and Cronn 2003; Senchina et al. 2003). These new data provide
deep insight into the cotton model system, furthering its value for comparative
analyses.
A general expectation of molecular evolution in allopolyploids is that mutations
occur randomly among homoeologs, and hence that evolutionary rates will be
equivalent for homoeologs (Fig. 10.3). A corollary expectation is that duplicated
gene copies will accumulate diversity, within and among populations, at equivalent rates. Small et al. (1999) tested this hypothesis of rate equivalence among
homoeologs using the gene AdhA, for which both homoeologs were sequenced for
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inequalities may become evident. Additional possibilities include silencing or loss
of one of the duplicated copies, ‘‘horizontal transfer’’ of sequences from one
genome to the other, and novel expression, the latter encompassing a variety of
phenomena discussed under Duplicate gene expression (Sect. 10.4).
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10.2.3.2 Interaction Among Homoeologs at the DNA Level
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22 accessions (44 alleles per genome) of G. hirsutum and for five accessions (10
alleles per genome) of G. barbadense. In both allopolyploids, estimates of
nucleotide diversity were higher for AdhA from the D-genome than from the Agenome. In a follow-up study (Small and Wendel 2002) using a second ADH gene
(AdhC), this conclusion was even more strongly supported; here 24 different
alleles were detected for the D-genome homoeolog versus only 7 for the
A-genome homoeolog, with a similar increase in allelic diversity for the
D-homoeolog in G. barbadense. These observations indicate that at least for some
genes, homoeologs may accumulate synonymous substitutions at vastly different
rates. At present, the responsible forces and underlying molecular mechanisms are
obscure, but a logical suggestion is that they are causally connected to the nearly 2fold difference in genome size between the co-resident genomes in the allopolyploid nucleus (the AT genome is approximately twice the size of the DT genome).
At present, little information exists for Gossypium that enables a thorough analysis of the relative rates of pseudogenization between diploids and allopolyploids, or
between the two homoeologous genomes of allopolyploid cotton. The ongoing
extensions of the comparative BAC sequencing approach taken by Grover and
colleagues (Grover et al. 2004, 2007) are promising in this respect, as are the ongoing
genome sequencing projects. Similarly, relatively little information exists on rates of
gene loss in cotton, though evidence to date based on comparative BAC sequencing
(Grover et al. 2004, 2007), comparative mapping analyses (Rong et al. 2004;
Brubaker et al. 1999b), EST collections (Flagel et al. 2012), and AFLP studies (Liu
et al. 2001) suggests that rates of gene loss are neither high nor particularly biased
with respect to genomic origin. A recent study using Southern hybridization,
however, detected three losses (of 27 genes studied) of D-homoeologs from
allopolyploid cotton and no losses of the A-homoeolog, suggesting a possible bias
(Rong et al. 2010). Again, key data likely will be generated soon that will permit these
speculations to be evaluated.
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One of the first indications that duplicated genes could behave in an evolutionarily
dependent fashion was the study of Wendel et al. (1995), who demonstrated
interaction among the 18S–26S ribosomal genes that exist at multiple loci in the
A- and D-genomes. Instead of evolving independently, as expected if homoeologous repeats did not interact, repeats at the different arrays in allopolyploid cotton
have been ‘‘homogenized’’ to the same sequence (either ‘‘A-like’’ or ‘‘D-like’’) by
one or more processes of concerted evolution (reviewed by Elder and Turner
1995). In four of the five allopolyploid species, interlocus homogenization has
created exclusively D-genome like rDNAs, whereas in G. mustelinum nearly all
rDNA repeats have been homogenized to an A-like form. This example showed
that since polyploid formation 1–2 mya, some 3,800 repeats, each approximately
10 kb in length, were ‘‘overwritten’’ with the alternative form originating from the
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other parental genome, probably through unequal crossing over or gene conversion, and that this phenomenon operated bidirectionally, in different directions in
different allopolyploid lineages. Interlocus concerted evolution of rDNA repeats
has since been documented in many other plant polyploids, including Nicotiana
and Tragopogon (see this Chaps. 10 and 14, this volume). From a mechanistic
perspective, it seems likely that the homogenization has resulted from unequal
crossing over within and among arrays and nonreciprocal homoeologous recombination events, with homoeolog exchanges being facilitated by the sub-telomeric
location of rDNAs in many plant lineages, including Gossypium (Wendel 2000).
This demonstration that some repeated sequences could interact across genomes
in the allopolyploid nucleus led to additional investigations of the scope of the
phenomenon. In an analogous study, Cronn et al. (1996) showed that in contrast to
18S–26S arrays, 5S rDNA genes are not homogenized by concerted evolutionary
forces in the allopolyploid. Similarly, and as noted above, early studies demonstrated apparent independent evolution of homoeologs (Cronn et al. 1999;
Senchina et al. 2003), consistent with the null hypothesis of Fig. 10.3. Thus, until
recently it was thought that most nuclear genes duplicated by allopolyploidy largely evolve independently of one another in the polyploid nucleus, a proposition
that seemed entirely reasonable given the absence of evident cytogenetic interactions among homoeologs.
This presumption of homoeolog independence turns out to be incorrect; however, and for a surprisingly large percentage of duplicated genes. Using ESTs
generated from both A- and D-genome diploids and AD-genome allopolyploids,
Salmon et al. (2010) sought evidence of small ‘‘gene conversion’’ or nonreciprocal
exchanges among homoeologs, based on the presence in allopolyploid cotton of
genes that have genome-diagnostic (A or D) SNPs (single nucleotide polymorphisms) that occurred in patterns suggestive of genic interactions (e.g., a single
expressed sequence containing diagnostic SNPs in the following order, AADDAA,
implicating conversion of the middle section of an AT homoeolog by the DT
homoeolog). These bioinformatic inferences were validated by de novo PCR and
sequencing. Results were convincing and compelling, demonstrating that about
2 % of contigs in G. hirsutum have experienced nonreciprocal homoeologous
exchanges since the origin of polyploid cotton 1–2 mya. Moreover, when a
sampling of these homoeologous interactions was studied throughout the polyploid
clade using a phylogenetic approach, nonreciprocal homoeologous exchanges
were shown to have occurred throughout polyploid divergence and speciation, as
opposed to being concentrated at the root of the polyploid tree. Among six
homoeologous interactions, five occurred in only one species, with the sixth event
being shared. This result refutes a logical a priori prediction, namely that interactions among homoeologs should be most frequent early in allopolyploid evolution (and hence shared among species), prior to reinforcement of apparently
strict bivalent pairing. Finally, some genomic regions showed multiple patterns of
homoeologous recombination among species, suggesting that some regions or
genes may be ‘‘hot-spots’’ for nonreciprocal homoeologous exchanges.
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In addition to evolutionary changes in gene and genome structure, a key component of polyploid evolution concerns the consequences of genome doubling on
gene expression. The biological reunion in the allopolyploid nucleus of two regulatory systems that had evolved in isolation for 5–10 my (A- and D-genome), in
only one of the two parental cytoplasms (A-genome), might be expected to lead to
violations of the equal and additive expression modeled in Fig. 10.3. In principle,
both genomes in an allotetraploid could contribute equally to the transcriptome, for
any pair of homoeologs or overall, or alternatively, there may be preferential
transcription of one genome due to intergenomic interactions that could bias the
transcription machinery. Many aspects of this problem are of interest, including
the scope and scale of preferential homoeolog expression, its tissue and organ
specificity, the level of genomic bias, the mechanistic underpinnings that result in
regulatory responses, the temporal scale at which alterations in homoeolog expression evolve, and ultimately, its physiological and evolutionary relevance.
Steps toward answering these questions for Gossypium have been taken using
many different experimental approaches over the last decade. We summarize these
results below, focusing on lessons regarding (1) developmental and environmental
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This demonstration of non-independent evolution of homoeologs has recently
been confirmed and extended to tens of thousands of genes using an expanded EST
set (Flagel et al. 2012). This analysis resulted in an even higher percentage (5 %)
of contigs in the assembly that show evidence of homoeolog contact in at least one
of the two polyploid species studied (G. hirsutum and G. barbadense), as well as
confirm the suggestion of hot-spots for homoeologous recombination. This high
frequency of homoeologous contact is an astonishing result given the absence of
prior cytogenetic or other observations that would have suggested this possibility,
and the apparently complete bivalent formation at meiosis. Flagel et al. (2012)
also detected 50 % more homoeologous exchanges in G. hirsutum than in
G. barbadense, suggesting that following polyploid formation, rates of nonreciprocal homoeologous recombination may diverge, even among closely related
species that share nearly all evident ecological and life-history characteristics.
These results are fascinating and lead to a number of questions. For example, what
is the genomic distribution of genes subject to nonreciprocal recombination events,
and does this information suggest a causal mechanism? Are nonreciprocal
recombination events random with respect to outcome (A vs D), or alternatively, is
the nuclear genome of allopolyploid cotton slowly becoming more ‘‘A-like’’ or
D-like’’? Do any of the detected ‘‘gene conversion’’ events have physiological
consequences and hence possible adaptive significance? Insight into these and
related questions in cotton and in other allopolyploids is likely to emerge in the next
several years.
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The first indication that polyploidy in Gossypium is accompanied by extensive
organ-specific changes in duplicate gene expression emerged nearly a decade ago in
a study of 40 homoeologous gene pairs in different organs of G. hirsutum using
SSCP-cDNA (Adams et al. 2003). Almost one-third of the genes revealed bias
toward one homoeolog or the other, or only expression of one homoeolog, in at least
one organ. Transcript levels for the two members of each gene pair varied by gene
and, unexpectedly, by organ type. Especially noteworthy were genes that showed
organ-specific, reciprocal silencing of alternative homoeologs; that is, one member
of a duplicated gene pair displayed minimal to no transcription in some organs,
whereas a reciprocal pattern was exhibited by the alternative homoeolog in other
organ(s). In particular, floral organs showed dramatic expression patterns in this
regard, with major differences among petals, stamens, and stigmas/styles (Adams
et al. 2003, 2004). Organ-specific expression of homoeologs was assayed more
extensively by Chaudhary et al. (2009), who employed a novel high-resolution
mass-spectrometry technology (Sequenom) to investigate relative expression levels
of each homoeolog for 63 gene pairs in 24 tissues. Results from over 2,000 assays
demonstrated that 40 % of homoeologs are transcriptionally biased in at least one
stage of cotton development, that genome merger per se has a large effect on
relative expression of homeologs (see section on temporal partitioning, below), and
that the majority of alterations are caused by cis-regulatory divergence between
diploid progenitors. The study also revealed 15 cases of probable regulatory
neofunctionalization among 8 tissues, perhaps the first such demonstration in
allopolyploid plants.
These surprising indications of partitioning of duplicate gene expression during
development have since been found in many other allopolyploid plants (see also
Chap. 14, this volume, and, e.g., Buggs et al. 2010; Bottley and Koebner 2008;
Bottley et al. 2006; Wang et al. 2006), opening the experimental floodgates on the
study of duplicate gene expression. Because differences in duplicate gene
expression were found between different organ types, the question arose as to
patterns of duplicate gene expression during development of any single organ. Liu
and Adams (2007) examined the expression ratio of homoeologous AdhA genes in
eight developmental stages of hypocotyls, cotyledons, and roots, and 11 developmental stages of ovary walls and ovules. They showed that expression ratios of
the two homoeologs changed considerably when comparing some stages of organ
development, indicating that differential expression of homoeologous genes is
developmentally regulated and that determining the extent of homoeologous gene
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Gene Expression
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effects on gene expression; (2) global analyses of genomic interactions; and (3) the
temporal scale at which expression evolution arises.
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expression changes requires analysis of multiple developmental stages and organs.
An important extension of this line of analysis was to ask whether biased
expression of homoeologs can be cell type-specific. In this respect, cotton offers an
excellent opportunity because the single-celled fibers are easily accessible. To
address this question, Hovav et al. (2008a, b) used homoeolog-specific microarrays
to assay homoeologous gene expression during fiber development. In these
remarkable studies, discussed further below, a full range of duplicate gene
expression was observed among the approximately 1,400 genes, from balanced
expression of homoeologs, to varying homoeolog ratios that shifted during
development. Notably, four genes showed complete reciprocal silencing of alternative homoeologs during fiber development. Collectively, the foregoing studies
show that variation in homoeologous gene expression during development is the
rule rather than the exception, and that it occurs even during development of a
single cell type.
An additional twist on duplicate gene expression has been the demonstration that
homoeolog expression patterns are not simply developmentally fixed, but instead
are responsive to environmental conditions. Liu and Adams (2007) examined
expression of AdhA homoeologs in response to abiotic stress conditions, with a
focus on cold and water submersion (flooding simulation), at different developmental stages. They found that some stress treatments significantly altered AT to DT
expression ratios, including a case of reciprocal silencing of homoeologs in
response to abiotic stress, an unprecedented finding. In a more recent study, Dong
and Adams (2011) extended their analysis to 30 gene pairs using three organs
(leaves, roots, and cotyledons) and five abiotic stress treatments (heat, cold,
drought, high salt, and water submersion). Over 70 % of the genes showed stressinduced changes in the relative expression levels of the duplicates under one or
more stress treatments, and 12 sets of homoeologs showed opposite changes in
expression levels in response to different abiotic stress treatments. These results
indicate that abiotic stress conditions can have considerable effects on duplicate
gene expression in a polyploid, with the effects varying by gene, stress, and organ
type. Together with the profound developmental partitioning noted above, differential expression in response to environmental stresses or cues may all be factors
that contribute to the preservation of duplicated genes in polyploids.
544
To provide a more global perspective on homologous gene expression in
allopolyploids, Udall et al. (2006) developed homoeolog-specific microarrays that
utilized genome-diagnostic SNPs from ESTs generated from the two genomes of
allopolyploid cotton and the diploids G. arboreum (A-genome) and G. raimondii
(D-genome). Using leaf RNA, they found that 199 of 461 gene pairs (43 %)
deviated from equal expression, thereby providing an initial quantitative
perspective on the scale of biased homoeolog expression. This microarray
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approach was refined and extended to a larger set of gene pairs (n = 1383) by
Flagel et al. (2008), who reported that 70 % of the genes in petals have biased
homoeolog expression ratios, and that more of these genes are D-genome (39.5 %)
than A-genome (30.5 %) biased. In addition, they found that the D-genome copies
of 69 genes and the A-genome copies of 46 genes were silenced, collectively
representing about 8 % of all genes. Similarly, Hovav et al. (2008a, b) used the
same SNP-specific microarray technology on RNAs extracted from trichomes
harvested from three developmental time points in wild and modern accessions of
two independently domesticated cotton species, G. hirsutum and G. barbadense.
Among these species 25–37 % of genes were significantly biased toward one of
the two genomes at each developmental stage, but these biases were not random
with respect to genome-of-origin; instead, duplicate gene expression was biased
toward the D-genome at all three time points studied, accounting for 63–76 % of
the biased genes. Finally, and to place homoeolog expression evolution in a
phylogenetic context, Flagel and Wendel (2010) extended their analysis of
duplicate gene expression in petals to all five extant allopolyploid species. Several
aspects of this study are notable, including the demonstration that all five species
display an overall preference for D-genome expression (D-genome bias accounting for 54–60 % of genes with biased homoeolog expression), that the percentage
of duplicate genes that are biased varies widely among species (from 48 to 88 %),
and that the overall magnitude of bias (as opposed to simply whether or not a gene
exhibits bias) similarly varies widely among species.
The foregoing synopsis shows that gene expression is massively altered in
polyploid cotton relative to its diploid progenitors. Unequal expression of one of
the two homoeologs likely is the rule rather than the exception, when integrated
across organs and tissues, with gene silencing representing only the endpoint in a
continuum of biased homoeolog expression. A global bias in gene expression from
one of two co-resident genomes (the D-genome; see, however, Yang et al. 2006) is
evident in allopolyploid cotton. At present, little evidence connects these observations to physiology or metabolism, and so connections to function and
ecological relevance remain obscure (see, however, section on fiber below).
Recent data (Hu et al. 2011) have extended the concept of bias to the proteomic
level, where allopolyploid cotton seeds have been shown to preferentially
accumulate proteins from the D-genome diploid parent, suggesting the possibility
of a functional connection. A promising direction for future research is targeted,
functional studies of specific gene families, protein complexes, and metabolic
pathways and networks, aiming to place homoeolog expression into a context that
permits insight into the biological significance of expression bias.
An emerging phenomenon in the study of polyploid gene expression on a large
scale is the concept of genomic dominance in expression. This phenomenon,
originally discovered and elaborated in cotton (Flagel and Wendel 2010; Rapp
et al. 2009), and just recently detected in Coffea allopolyploids (Bardil et al. 2011),
is defined as the state where total expression of a homoeolog pair mimics the
expression level of one of two diploid parents of an allopolyploid. That is, if parent
A is upregulated relative to parent B, the allopolyploid would exhibit the
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10.3.3 Temporal Dynamics of Duplicate Gene Expression
Evolution
One of the early (Adams et al. 2003) and subsequently further characterized
findings about duplicate gene expression in cotton is that expression evolution has
a temporal dimension; phenomena that typify genome merger and doubling need
not be the same, qualitatively or quantitatively, as those that characterize natural
allopolyploids that are 1–2 million years distant from polyploidization (Fig. 10.4).
In some cases, such as organ-specific silencing of particular homoeologs, it has
been shown that this can arise immediately upon allopolyploid formation (Adams
et al. 2003, 2004; Chaudhary et al. 2009), leading to the notion (Adams et al. 2003;
Rapp and Wendel 2005) that homoeolog silencing may be significant in duplicate
gene retention and that it may actually represent a form of instantaneous
subfunctionalization. These studies of individual genes have demonstrated that
some of the changes detected in synthetic allopolyploids are mirrored in natural
allopolyploids, whereas others are distinct between the synthetic and natural
allopolyploids (see examples in Adams et al. 2004; Chaudhary et al. 2009).
One possibility raised by these observations is that in some cases there is an
immediate epigenetic response to genome merger and doubling that becomes
either epigenetically stable or genetically fixed, for 1–2 million years.
With respect to the phenomena of genomic bias, the evolution of novel quantitative (transgressive) expression states, and genomic dominance, the studies of
Flagel et al. (2008, 2010) have been particularly illuminating with respect to this
temporal dimension to expression evolution. For example, whereas only about
one-third of genes exhibit biased homoeolog expression in petals of AxD-genome,
diploid hybrids, half or more do in petals of natural allopolyploids. Moreover, many
of the genes that display bias in the initial F1 retain or even magnify the degree of
bias after 1–2 million years. Also, whereas allopolyploid formation appears to
result in relatively few genes that exhibit quantitatively transgressive expression
levels, relative to their diploid parents, this number rises sharply in natural allopolyploids, suggesting novel opportunities for exploring gene expression space.
Finally, a comparison of genomic dominance in all five natural allopolyploids
(Flagel and Wendel 2010) to that observed in synthetic allopolyploids (Rapp et al.
2009) demonstrated that the magnitude of genomic dominance remains high after
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expression state of parent A; in the case where parent A is downregulated relative
to parent B, so is the allopolyploid. In this example, the A parent is genomically
dominant with respect to gene expression. Using two different genomic combinations in synthetic allopolyploid Gossypium, Rapp et al. (2009) demonstrated that
there exists a quantitative bias in genomic dominance toward one of the two
parents. In the case of the synthetic AD allopolyploid, more than 10,000 genes
display this dominant, quantitative expression phenotype. At present, the functional significance of genomic dominance is unknown.
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G. hirsutum
AD
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G. barbadense
G. hirsutum
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G. tomentosum
G. darwinii
G. barbadense
G. mustelinum
“Gene conversion”
Gene loss
Genomic dominance
Concerted evolution
Genetic sub- and neofunctionalization
Intergenomic transfer
Biased homoeolog expression
Transgressive expression
Homoeologous expression change
Biased genomic dominance
Epigenetic subfunctionalization
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1–2 my of evolution, but that the bias in its direction dissipates (shifting from
predominantly D-genome dominance in the synthetics to similar levels of A- and
D-genome dominance in the natural allopolyploids). Thus, it appears that natural
allopolyploids have adjusted to more equally utilize the transcriptomes of the two
co-resident genomes, notwithstanding the residual D-genome homoeolog bias
described above. An intriguing possibility is that this D-genome homoeolog bias is
connected causally to the biased D-genome expression dominance that arises following genome merger; to the extent that it is, it leads to the suggestion that
homoeolog bias in other allopolyploids may be predicted by the initial conditions
established by genomic merger in the distant past, and which may be experimentally mirrored in many systems through the use of synthetic hybrids and
allopolyploids.
The partitioning of gene expression evolution into its temporal components
leads to the suggestion that these different components may entail different or at
least complementary mechanisms. The first, involving rapid or instantaneous gene
expression alteration as a consequence of genome merger and doubling, reflects
the myriad novel interactions accompanying a biological reunion of two differentiated genomes into a common nucleus. The precise nature of these interactions
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Fig. 10.4 A temporal depiction of phenomena that characterize polyploid evolution in
Gossypium. Shown are the formation and diversification of the allopolyploid clade, several
accompanying phenomena (dispersal, chromosomal stabilization), and the parallel domestication
of G. hirsutum and G. barbadense. Images to the right represent wild and domesticated states,
with close-ups of single seeds and the transformations in morphology accompanying domestication and crop improvement. Genetic and epigenetic phenomena associated with polyploidy in
cotton are shown below, with a timeline (bottom), illustrating the temporal context for each
phenomenon and whether there are increases or decreases (where known) in magnitude over time
(blue to purple gradation indicates increase, the reverse indicates decrease)
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10.4 Polyploidy and Ecological Novelty
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The foregoing discussion of the genomic and genetic attributes of allopolyploid
cotton demonstrates that polyploid formation has led to a diverse array of genetic
and genomic responses, including non-Mendelian transmission and various forms
of non-additive gene expression. The question naturally arises as to whether
allopolyploidy has stimulated novel adaptation or physiological capability.
A voluminous literature in plants documents the frequency of polyploids in various
habitats, their morphological and physiological attributes, and their ecological
success relative to diploids (Grant 1981; Stebbins 1947, 1950; Soltis and Soltis
2000). One generalization that has emerged is that polyploidy often is associated
with broader ecological amplitude and novel evolutionary opportunity, often
suggested to be mediated by the increased ‘‘buffering’’ capacity afforded by
duplicated genes and the enhanced vigor resulting from the ‘‘fixed heterozygosity’’
of their duplicated genomes. We might now rephrase or expand these suggested
mechanistic explanations to encompass a network perspective, and the vastly
increased combinatorial possibilities for regulation and evolution enabled by a
suddenly duplicated complement of genes and merged regulatory systems.
With respect to Gossypium, allopolyploidy led to the apparent invasion of a new
ecological niche. In considering the Pleistocene origin of allopolyploid cotton,
Fryxell (1965, 1979) noted that in contrast to the majority of diploid species, which
occur mostly inland in various arid to seasonally arid environments, allopolyploid
species typically occur in coastal habitats, at least those forms that arguably are
truly wild (see also Brubaker and Wendel 1994). Thus, among the five allopolyploid species, two are completely restricted to near coastlines, in that they
are island endemics (G. darwinii and G. tomentosum), and for two others
(G. barbadense and G. hirsutum), wild forms occur disparately in littoral habitats
ringing the Gulf of Mexico, northwest South America, and distant Pacific Islands.
The capacity for oceanic dispersal in Gossypium (Fryxell 1965, 1979; Stephens
1958, 1966) was associated at the allopolyploid level with specialization for
establishment in coastal communities. Fryxell (1965, 1979) forwarded the tantalizing suggestion that following initial formation, adaptation of the newly evolved
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is not known, but probably includes disruptions in gene dosage balance, stoichiometric changes resulting from differences in competition for transcription factors,
differences in microRNA expression, and a host of novel cis and trans interactions
(Birchler et al. 2005; Birchler and Veitia 2010; Chen 2007; Osborn et al. 2003;
Veitia 2005) (see also Chaps. 1, 2, and 4, this volume). Superimposed on these
rapid evolutionary responses to polyploidy are those that arise more slowly during
the stabilization of the new polyploid genome and during evolution and speciation
over much longer periods of time. Thus, the presence of duplicated genomes
would seem to provide evolutionary opportunity and consequences immediately
on polyploid formation and for millions of years thereafter.
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10.5 Polyploidy and Fiber
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Finally, we would be remiss if we failed to consider the consequences of polyploidy on the development of agronomically advanced cultivars of allopolyploid
cotton. Although four separate species of Gossypium were independently
domesticated for their seed hairs, the characteristic that attracted the attention of
the earliest domesticators, the seed ‘‘lint’’ itself, evolved only once in the progenitor of the A-genome diploids (Hovav et al. 2008c; Wendel et al. 2009).
Applequist et al. (2001) generated growth curves for trichomes from cultivated and
wild diploid and allopolyploid species and demonstrated that the evolution of an
extended primary wall elongation occurred in the ancestor of wild A-genome
cotton prior to domestication and in Africa. Follow-up comparative expression
profiling experiments (Hovav et al. 2008a, b; Rapp et al. 2010) further identify
some of the metabolic pathways that were modified to enable this evolution in
fiber properties. These results led to the fascinating implication that domestication
of the New World allopolyploid cottons (which contain an A-genome, in addition
to a D-genome) that presently dominate cotton agriculture worldwide was first
precipitated by developmental and physiological transformations that occurred
hundreds of thousands of years ago in a different hemisphere.
Because fibers from all D-genome diploids are short and non-spinnable, it is
particularly interesting that fiber from the cultivated (New World) allopolyploids is
agronomically superior to that of the cultivated A-genome diploids; in this sense,
the cultivated allotetraploid fiber morphology is ‘‘non-additive’’, or perhaps
‘‘heterotic’’. A number of studies have noted this point (Jiang et al. 1998; Paterson
2005; Wright et al. 1998), suggesting that allopolyploidization provided novel
opportunities for agronomic improvement. A recent meta-analysis (Rong et al.
2007) of a large number of QTL studies in allopolyploid cotton leads to a general
picture consistent with this interpretation; more loci affecting fiber yield and
quality traits are found in the DT (n = 221) than the AT (n = 184) genome,
possibly explaining the superiority of the lint of the allopolyploids relative to the
A-genome diploids. Support for this speculation that ‘‘recruitment’’ of D-genome
genes has been important in enabling the development of advanced allotetraploid
cultivars is also emerging from comparative expression profiling studies (Hovav
et al. 2008a, b; Rapp et al. 2010), which reveal in exquisite detail the thousands of
gene expression differences that distinguish wild from domesticated cotton fiber
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allopolyploid to littoral habitats enabled it to exploit the fluctuating sea levels
that characterized the Pleistocene. This ecological innovation not only is
envisioned to have permitted the initial establishment of the nascent polyploid
lineage, but is also suggested to have provided a means for the rapid dispersal of
the salt-water-tolerant seeds. By this means, perhaps, the mobile shorelines of the
Pleistocene facilitated exploitation of a new ecological niche, and hence colonization of the New World tropics.
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Acknowledgments Research in the Wendel lab has largely been funded by the NSF Plant
Genome Program, with additional support from other NSF programs, the USDA NRI, and Cotton
Incorporated. Research in the Adams lab has been supported by the Natural Science and Engineering Research Council of Canada and by the USDA NRI program. We gratefully acknowledge
all of these sources of support.
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Adams KL, Cronn R, Percifield R, Wendel JF (2003) Genes duplicated by polyploidy show
unequal contributions to the transcriptome and organ-specific reciprocal silencing. Proc Nat
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Adams KL, Percifield R, Wendel JF (2004) Organ-specific silencing of duplicated genes in a
newly synthesized cotton allotetraploid. Genetics 168:2217–2226
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development, as well as a bias toward preferential expression of D-genome
homoeologs. Finally, similar implications emanate from genetic mapping experiments, where it has been shown that for 535 genes implicated in cotton fiber
development, more transcription factors were from DT than AT genome, whereas
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interpreted to suggest that the D-genome ancestor provided key transcription
factors that regulate the expression of fiber genes donated by the ancestral
A-genome parent. Taken together, these studies may provide actual genetic evidence for a speculation forwarded 75 years ago by Harland (1936), who stated If
as a consequence of polyploidy a large number of genes become duplicated, and
the characters governed by such genes are of importance to the species, one of the
members may mutate, leaving the character unimpaired, with the further possibility that the mutation may be of benefit to the species. An exciting prospect is that
in the near future we will develop a deeper understanding of the nature of these
genes, the molecular genetic meaning of Harland’s invocation of the word
‘‘mutation’’, and their effects on the developmental networks that underlie altered
morphology and agronomic improvement.
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Book Title
Polyploidy and Genome Evolution
Series Title
Chapter Title
Evolutionary Implications of Genome and Karyotype Restructuring in Nicotiana tabacum L
Copyright Year
2012
Copyright HolderName
Springer-Verlag Berlin Heidelberg
Corresponding Author
Family Name
Leitch
Particle
Given Name
Andrew
Suffix
Author
Division
School of Biological and Chemical Sciences
Organization
Queen Mary University of London
Address
London, UK
Email
a.r.leitch@qmul.ac.uk
Family Name
Kovarik
Particle
Given Name
Ales
Suffix
Division
Organization
Institute of Biophysics
Address
Brno, Czech Republic
Email
Author
Family Name
Renny-Byfield
Particle
Given Name
Simon
Suffix
Division
School of Biological and Chemical Sciences
Organization
Queen Mary University of London
Address
London, UK
Email
Author
Family Name
Grandbastien
Particle
Given Name
Marie-Angèle
Suffix
Division
Organization
Institute Jean-Pierre Bourgin
Address
Versailles, France
Email
Abstract
Nicotiana tabacum is an allopolyploid that formed within the last 200,000 years from relatives of the extant
diploids N. sylvestris and N. tomentosiformis, the donors of the S- and T-genomes, respectively. Here we
review progress in our understanding of the divergence of N. tabacum subsequent to its formation, by
comparing the N. tabacum genome with those of its diploid progenitors. We also review the data from
synthetic N. tabacum, where there is evidence for much genetic change in early generations, including various
chromosomal translocations, allopolyploid-induced retroelement mobility and loss, and reductions in the copy
numbers of some tandem repeats. These observations are similar to patterns found in natural N. tabacum,
suggesting that rapid genetic divergence is induced by allopolyploidy. The T-genome of N. tabacum shows
the greatest number of genetic changes and appears to be less stable than the S-genome. We describe possible
mechanisms that may have stimulated these genetic changes and propose that these can lead to enhanced
fertility, more regular chromosome pairing, and the evolution of disomic inheritance.
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Ales Kovarik, Simon Renny-Byfield, Marie-Angèle Grandbastien
and Andrew Leitch
Abstract Nicotiana tabacum is an allopolyploid that formed within the last
200,000 years from relatives of the extant diploids N. sylvestris and N. tomentosiformis, the donors of the S- and T-genomes, respectively. Here we review progress in our understanding of the divergence of N. tabacum subsequent to its
formation, by comparing the N. tabacum genome with those of its diploid progenitors. We also review the data from synthetic N. tabacum, where there is
evidence for much genetic change in early generations, including various chromosomal translocations, allopolyploid-induced retroelement mobility and loss, and
reductions in the copy numbers of some tandem repeats. These observations are
similar to patterns found in natural N. tabacum, suggesting that rapid genetic
divergence is induced by allopolyploidy. The T-genome of N. tabacum shows the
greatest number of genetic changes and appears to be less stable than the
S-genome. We describe possible mechanisms that may have stimulated these
genetic changes and propose that these can lead to enhanced fertility, more regular
chromosome pairing, and the evolution of disomic inheritance.
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Evolutionary Implications of Genome
and Karyotype Restructuring in Nicotiana
tabacum L.
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A. Kovarik
Institute of Biophysics, Brno, Czech Republic
S. Renny-Byfield A. Leitch (&)
School of Biological and Chemical Sciences, Queen Mary University of London,
London, UK
e-mail: a.r.leitch@qmul.ac.uk
UN
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M.-A. Grandbastien
Institute Jean-Pierre Bourgin, Versailles, France
P. S. Soltis and D. E. Soltis (eds.), Polyploidy and Genome Evolution,
DOI: 10.1007/978-3-642-31442-1_11, Springer-Verlag Berlin Heidelberg 2012
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The genus Nicotiana is the sixth largest genus in the family Solanaceae and
includes 76 species found naturally in the Americas, Australia and surrounding
islands, and with a single species in Namibia (Goodspeed 1954; Knapp et al. 2004).
However, several species, including N. tabacum (tobacco), now occur more widely.
The genus has received much recent attention because of evidence for recurrent
polyploidy, resulting in nearly half the species in the genus being chromosomally
polyploid. It is likely there have been six independent polyploidy events within the
genus: at about 10 million years ago (mya) (section Suaveolentes, c. 24 species), 5
mya (section Repandae, 4 species), 1–2 mya (section Polydicleae, 2 species), and
within the last 200,000 years (N. rustica, N. arentsii, and N. tabacum) (Lim et al.
2004a). There is also reticulation at the diploid level, with at least three species
likely to be of homoploid hybrid origin (N. glauca, N. linearis, and N. spegazzinii)
(Goodspeed 1954; Kelly et al. 2010).
N. tabacum is an autotetraploid (2n = 4x = 48) derived from diploid species that
most closely resemble N. sylvestris (2n = 2x = 24), the maternal S-genome donor,
and N. tomentosiformis (2n = 2x = 24, section Tomentosae), the paternal T-genome
donor. Nicotiana sylvestris is the only species in section Sylvestres, and detailed
genetic analysis of multiple accessions indicates that there is little genetic diversity in
this species (4.2 % polymorphisms in AFLPs—Amplified Fragment Length
Polymorphisms, Petit et al. 2007). However, there is considerably more genetic
diversity among accessions of N. tomentosiformis (31.7 % AFLP polymorphism),
which form two distinct groups, one of which most closely resembles the T-genome of
N. tabacum (Murad et al. 2002; Petit et al. 2007). These data indicate that N. tabacum
formed subsequent to the divergence of the two groups of N. tomentosiformis.
Molecular clock estimates of plastid and internally transcribed spacer sequences (ITS) of nuclear ribosomal DNA (rDNA) suggest that N. tabacum formed less
than 200,000 years ago. These estimates were calibrated using likely maximum
ages of some endemic species occurring on islands of known geological ages
(Clarkson et al. 2005; Leitch et al. 2008). However, given that only feral populations of N. tabacum have been found and no truly ‘‘wild’’ population exists
(Knapp personal communication), it is quite possible that N. tabacum was born
subsequent to the origin of human agriculture, within the last 10,000 years.
The purpose of this paper is to review current understanding of the genetic
consequences of polyploidy in N. tabacum. We show that genetic change can occur
rapidly within a few generations, perhaps as a consequence of the ‘‘genomic shock’’
of allopolyploidy (McClintock 1984), a process that generates variants from which
selection favors those with enhanced fertility. This unstable phase is presumably
transient; however, there remains evidence for genome dynamism over time scales
of thousands of years, including the replacement of multiple copies of rDNA
sequences, the turnover of retroelements, and the loss of sequences targeted at the
T-genome. We discuss how such changes may promote fertility and lead to the
fixation of rearranged karyotypes during polyploid species establishment.
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Kenton et al. (1993) were the first to use genomic in situ hybridization (GISH) to
study the chromosomes of N. tabacum. The fluorescent probes derived from total
genomic DNA of N. sylvestris and N. tomentosiformis hybridized to a separate
subset of 24 chromosomes, corresponding to the chromosomes of the S- and
T-genomes, respectively. However, the efficacy of the T-genome labeling was
inferior (Lim et al. 2000b), likely because of a loss of T-genome sequences
(Renny-Byfield et al. 2011). GISH also revealed in all N. tabacum accessions 4–9
intergenomic translocations (Kenton et al. 1993; Lim et al. 2004a; Moscone et al.
1996). Similar translocations (up to 3) were also observed in some synthetic
N. tabacum lines that were only a few generations old (Skalicka et al. 2005).
Perhaps these translocations have arisen as a consequence of multivalent formation
(see Chap. 7), or they may represent hotspots of recombination. While some
translocations are fixed (i.e. occur in all varieties), others are specific for particular
accessions. There are more translocations of S-genome origin chromatin to
T-genome chromosomes (T/s chromosome) than the reverse (S/t chromosome).
Kenton et al. (1993) hypothesized that this could be caused by selection against
S/t chromosomes. Alternatively, some of the ‘‘translocations’’ may actually be
S-genome subtelomeric satellite sequences that now occur on T-genome chromosomes, perhaps arising in their new location via recombination-based homogenization processes (Koukalova et al. 2010). Next-generation sequencing (NGS)
may enable us to distinguish among these alternative hypotheses.
As with most other plant species (Heslop-Harrison and Schwarzacher 2011), a
large fraction of Nicotiana genomes is composed of various types of retroelements. Genome sampling using NGS revealed that the major component of the
genome of N. tabacum and its diploid relatives comprises long terminal repeat
(LTR)-retrotransposons, with at least 17.1–22.5 % of the genome being Ty3/gypsy
elements and 2.2–3.4 % being Ty1/copia elements. DNA transposons comprise
around 1.7 % of the genome of these species (Renny-Byfield et al. 2011).
In addition, there are several unrelated satellite repeat families that have been
characterized (the distributions of some are shown Fig. 11.1, Lim et al. 2000b). These
repeats include: (1) The HRS60 family, which is the best-characterized tandem repeat
family in Nicotiana and includes an interstitial repeat in N. tomentosiformis and
N. tabacum (called GRS, Gazdova et al. 1995) and predominantly subtelomeric repeats
in N. sylvestris and N. tabacum (called HRS60 and NSYL2, Koukalova et al. 2010;
Koukalova et al. 1989); (2) NTS9 in N. sylvestris and N. tabacum (Jakowitsch et al.
1998); (3) NTRS in N. tomentosiformis and N. tabacum (Fig. 11.1, Matyasek et al.
1997), and (4) NicCL3, a long 2.2 kb tandem repeat comprising *2 % of the
N. tomentosiformis genome and in lower abundance in N. tabacum (Fig. 11.2,
Renny-Byfield et al. 2011 and Renny-Byfield et al. 2012). There are also a number of
satellite repeat families of known origin; these are: (5) A1/A2 satellite repeats derived
from the intergenic spacer (IGS) of rDNA, which have transposed and amplified to
multiple locations across the genome (Lim et al. 2004b); (6) tandem repeats of
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geminivirus-related DNA (GRD), occurring in two distinct families in some accessions of N. tomentosiformis and inherited to N. tabacum (Murad et al. 2004; Murad
et al. 2002), the first endogenous viruses discovered in plants (Bejarano et al. 1996); (7)
sequences of pararetroviral origin (Matzke et al. 2004), including distinct variants
found in N. tomentosiformis (NtoEPRV) and N. sylvestris (NsEPRV), both of which
have been inherited in N. tabacum.
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11.3 Retroelement Response to Allopolyploid ‘‘Genomic
Shock’’
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Fig. 11.1 Ideograms showing the distribution of tandem repeat sequences in N. tabacum and its
diploid progenitors, N. sylvestris and N. tomentosiformis. Taken with permission from Murad
et al. (2002). See text for a description of the repeats shown
Nicotiana hybrids were among the first in which chromosomal changes following
hybridization were demonstrated. Gerstel and Burns (1967) reported that
N. otophora 9N. tabacum hybrids have genetic instabilities of two kinds. Firstly, the
heterochromatin from N. otophora undergoes breakage causing chromatin loss; this
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Fig. 11.2 Reduction in copy number of T-genome repeats in N. tabacum from expectation given
their abundance in N. tomentosiformis. a Fluorescence in situ hybridization (FISH) showing the
distribution of dispersed A1/A2 repeats (green fluorescence). The 18-5.8-26S rDNA locus on
chromosome 3 (N. tomentosiformis) and T3 (N. tabacum) is labeled with yellow fluorescence due
to the overlap of red and green rDNA fluorescence. Note that there is much reduced A1/A2 signal
in N. tabacum (for further information see Lim et al. 2004b). b FISH showing the distribution of
NicCL3 in both species (green fluorescence). Note the reduced abundance of this repeat in
N. tabacum. The 18-5.8-26S rDNA locus on chromosome 3 (N. tomentosiformis) and T3
(N. tabacum) is labeled with red fluorescence. For further information see Renny-Byfield et al.
(2012). c, d. NGS analysis of repeat clusters in N. tabacum and its diploid progenitors. The graphs
show the genome proportions (arcsine transformed) of repeats uniquely inherited in N. tabacum
from c N. tomentosiformis and d N. sylvestris. If N. tabacum had faithfully inherited the repeats as
found in the diploids, then all repeats would fall on the blue lines. However, in N. tomentosiformis
c most repeats at higher genome proportions are underrepresented in N. tabacum. This is not the
case for N. sylvestris d, where repeats are more or less in their expected abundance. This trend is
reflected in the abundance of repeats that are biparentally inherited, i.e., the more abundant they
are in N. tomentosiformis, the more likely they will be underrepresented in N. tabacum. For
further information see Renny-Byfield et al. (2011)
can be observed cytologically and also phenotypically by the leaf variegation it
causes. Secondly, in some cells heterochromatic blocks from N. otophora proliferate
enormously to many times their normal length, forming ‘‘megachromosomes’’. This
pioneering work revealed that interspecific hybridization can induce chromosomal
rearrangements and rapid sequence losses and gains.
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11.4 Loss of DNA: Targeting the T-Genome
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Melayah et al. (2001) were the first to show, using N. tabacum, that environmental stresses can induce retrotransposition. There is also evidence that allopolyploidy can stimulate transposition in N. tabacum and synthetic mimics of
N. tabacum made from the diploid progenitors (Petit et al. 2007, 2010). These
authors analyzed four populations of Ty1/copia LTR-retrotransposons (Tnt1-ol16,
Tnt1-ol13, Tnt2, and Tto1) using sequence-specific amplification polymorphism
(SSAP). They showed that the parental diploid species share essentially similar
classes of retroelements. In N. tabacum there is evidence of retrotransposon
diversification subsequent to allopolyploidy, with sequence losses concomitant
with gains. Losses of retroelements were more frequent to the T-genome, while
novel insertions of some populations, such as Tnt2, were shown to preferentially
locate to the S-genome. However, each retrotransposon population seems to
behave differently, with some populations undergoing rapid turnover, while others
display relative stasis. There was little or no diversification of retrotransposons
among individual accessions of natural N. tabacum, indicating that retroelement
diversification occurred before the accessions analyzed had diverged, perhaps
early in the species’evolution and arising as a consequence of allopolyploidy and
induced ‘‘genomic shock’’ (McClintock 1984).
To test the hypothesis that allopolyploidy can induce retroelement diversification,
Petit et al. (2010) compared insertion patterns of the Tnt1 family in the synthetic
N. tabacum Th37 (Burk 1973) with its diploid progenitors. In some Th37 individuals
in the fourth synthetic generation (S4), there was evidence of new Tnt1 insertions.
Newly, transposed copies were amplified from elements located on the N. sylvestrisderived genome and were highly similar to the Tnt1A tobacco copies amplified in
response to microbial factors (Grandbastien et al. 2005). Furthermore, a high proportion of parental SSAP bands was lost in Th37, particularly those from the
N. tomentosiformis-derived genome, again as observed in natural N. tabacum.
Together, these data indicate that retrotransposon amplification and molecular
restructuring in, or around, insertion sites occur rapidly in response to allopolyploidy,
and that similar sequences have responded in the same way to allopolyploidy in
natural and synthetic N. tabacum.
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As with many other allopolyploid species (Leitch and Bennett 2004), N. tabacum has
a lower genome size than is expected from the sum of parental genomes (Leitch et al.
2008, N. tabacum genome size, 1C = 5110 Mpb; N. sylvestris, 1C = 2636 Mbp;
N. tomentosiformis, 1C = 2683 Mbp (http://data.kew.org/cvalues/). Recently,
(Renny-Byfield et al. 2011) used NGS analysis of N. tabacum and its two diploid
progenitors and showed that repeats derived from N. tomentosiformis was underrepresented in N. tabacum, a trend that was not observed for repeats from N. sylvestris
(Fig. 11.2). These observations lead to the conclusion that the T-genome of
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11.5 Ribosomal DNA Homogenization is Rapid and Ongoing
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In most eukaryotes, 5S and 18–5.8–26S rDNA units occur in tandem arrays at one
or several loci. Each large rDNA unit contains the 18S, 5.8S, and 26S ribosomal
RNA (rRNA) genes, ITS, and IGS sequences. Whilst these sequences are vital for
cell functioning, they can also be highly recombinogenic and labile sequences
influencing genome stability (Kobayashi 2011). The genes themselves are highly
conserved; however, their spacers diverge at suitable rates for resolving species
relationships within most genera, including Nicotiana (Chase et al. 2003). Early
studies revealed that the restriction fragment length polymorphism (RFLP) patterns
of the N. tabacum IGS is not additive of the diploid progenitors (Kovarik et al.
1996), but that N. tabacum has evolved its own distinct rRNA gene family(ies).
Sequence analysis revealed that the tobacco-specific units arose by reorganization
of N. tomentosiformis-inherited units followed by their subsequent amplification
(Volkov et al. 1999). The sequence changes mainly involved amplification and
reduction of subrepeats upstream and downstream of the transcription start site
within the IGS. However, the newly evolved units still occur at the four rDNA loci
that N. tabacum inherited from its parents (see Fig. 11.1). Thus, it is likely that
the parental units of S-genome origin were overwritten by the newly amplified
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N. tabacum has undergone extensive sequence losses, and that genetic changes have
occurred more rapidly to the paternally derived T-genome than the S-genome.
N. tabacum harbors two families of endogenous pararetrovirus DNA, one from
each progenitor (Jakowitsch et al. 1999). These are NsEPRV from N. sylvestris,
which in N. tabacum is at the expected abundance, and NtoEPRV from
N. tomentosiformis, which occurs in N. tabacum with reduced copy number.
Likewise, the A1/A2 repeats (Lim et al. 2004b) and NicCL3 (Renny-Byfield et al.
2012), which are found in multiple locations around the genome in N. tomentosiformis (Fig. 11.2), are also underrepresented in N. tabacum. Similarly, NGS data
show that all families of Ty3/gypsy elements have undergone sequence loss in
N. tabacum (Renny-Byfield et al. 2011), and SSAP analysis of Tnt retroelements
(Ty1/copia) reveals more deletion events influencing loci from the T-genome than
the S-genome of N. tabacum (Petit et al. 2007). There is evidence of rapid and
targetted loss of repeats from the T-genome even from the earliest generations,
since in synthetic N. tabacum lines, many individuals already have reduced copy
numbers of NtoEPRV, NTRS repeats (Skalicka et al. 2005), and NicCL3
(Renny-Byfield et al. 2012), whilst most losses of Tnt1 retroelements were from
the T-genome (Petit et al. 2010). All these data suggest rapid, targetted changes
early in allopolyploid evolution. The targeting of repeat losses from the T-genome
may support the nuclear-cytoplasmic interaction hypothesis, whereby the paternally derived genome functions in a maternal cytoplasm (Gill 1991; Leitch et al.
2006), which in allopolyploids may lead to incompatibilities that preferentially
destabilize the paternally derived genome.
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N.tabacum-specific units (Lim et al. 2000a), although a few units of S genomeorigin remain intact, perhaps because they are methylated and inactive (Kovarik
et al. 2008). In contrast, the 5S rDNA follows a different evolutionary pattern in
N. tabacum, with no evidence for intergenomic homogenization, although shifts in
copy numbers of parental gene families have been observed (Fulnecek et al. 2002).
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11.6 The Fate of Duplicated Genes
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Although there has been substantial restructuring of the repetitive fraction of the
N. tabacum genome since its formation, most genic sequences analyzed have
remained in duplicate copies, e.g., genes for drug resistance (Schenke et al. 2003),
putrescine N-methytransferases (Riechers and Timko 1999), a family of small
GTP-binding proteins (Takumi et al. 2002), lignin forming peroxidase (Matassi
et al. 1991), nitrite reductase (Kronenberger et al. 1993), nitrate reductase
(Vaucheret et al. 1989), glutamine synthetase (Clarkson et al. 2010), phytochrome
A (Intrieri et al. 2008), the developmental gene LEAFY (McCarthy 2010), and
families of DNA methyltransferases (Fulnecek et al. 2009). The only current
exception is a family of N. tabacum glucan endo-1,3-beta-glucosidase genes that
appeared to be recombinants of both ancestral sequences (Sperisen et al. 1991).
Locus additivity does not always seem to be a rule even in recently formed
synthetic allopolyploids. For example, frequent deletions of homoeologs were
found among 70 protein-coding loci in Tragopogon miscellus that formed within
the last 80 years (Buggs et al. 2009, 2012; see also Chap. 14, this volume). These
changes may not be random, as Buggs et al. (2012) showed that clusters of genes
are repeatedly lost or retained and the likelihood of retention reflects gene
ontology categories or their predicted levels of dosage sensitivity. Similarly,
synthetic lines of Brassica napus seem to eliminate much of the parental DNA,
although it is currently unknown whether coding or noncoding sequences are
preferentially targeted (Song et al. 1995; Szadkowski et al. 2011). The occurrence
of both homoeologs in N. tabacum may reflect limited sampling, or high levels of
retention. Additivity is observed in synthetic lines of Gossypium (Liu et al. 2002;
see also Chap. 10, this volume) and in synthetic wheat allopolyploids, although in
the latter case there remains some controversy (Mestiri et al. 2010; Feldman and
Levy 2009; see also Chap. 7, this volume). Additivity has also been demonstrated
at the expression level. Alleles of phytochrome A, lignin forming peroxidases,
nitrite reductase, and DNA methyltransferases are expressed from both parental
homeologs. There are also reports of epigenetic silencing of one subset of the
parental alleles, including rRNA genes that have escaped homogenization
(Kovarik et al. 2008) and the CYP82E4 locus (Chakrabarti et al. 2007) involved in
the alkaloid biosynthesis pathway.
Duplicate copies arising through polyploidy can be retained through a number
of mechanisms (Doyle et al. 2008): (1) one copy can evolve a new function
(neofunctionalization) that can become fixed through a selective advantage
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11.7 Genome Revolution and Allopolyploid Establishment
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Until the early 1990s, it was commonly thought that chromosomal changes
establish isolation barriers among populations because of reduced fertility in
heterozygotes. Rearranged karyotypes may, then, become fixed through inbreeding
or meiotic drive, particularly in small populations (King 1993). However, it is
difficult to distinguish between karyotype changes that drive speciation events and
those arising by genetic drift after speciation. Certainly, many rearrangements are
deleterious and can cause embryo lethality or hybrid breakdown prior to reproduction (Rieseberg 1997). Only neutral or advantageous recombinants will pass
the bottleneck of selection during the early phase of speciation. Furthermore,
evolutionary geneticists pointed out that genetic mutations at the DNA level are
more common than karyotype change and are therefore likely to be more
important in speciation processes (Butlin 1993). However, recent evidence from a
number of sources, e.g., Anopheles gambiae (Turner and Hahn 2010) and
Helianthus homoploid hybrids (Strasburg et al. 2009), indicates that chromosomal
inversions can, indeed, have a role in establishing barriers between populations
and drive speciation. Strasburg et al. (2009) suggested that inversions may become
foci for the accumulation of adaptive genes, particularly at the junctions between
collinear and inverted regions, where gene flow is likely to be most impeded.
Likewise, computer modeling has shown that when recombination is eliminated at
an inverted region, and in the presence of strong selection and minimal or no gene
flow, then species isolation can be driven by such a rearrangement (Feder and
Nosil 2009).
In the context of early polyploid evolution, karyotype rearrangement may drive
enhanced fertility and potentially be fundamental to allopolyploid species establishment. Certainly, high levels of chromosomal change have been observed in
young polyploids, e.g., karyotype variability (dysploidy and intergenomic translocations) within and among populations of Tragopogon polyploids that formed
within the last 80 years (Chester et al. 2010, 2012; Lim et al. 2008), and largescale chromosomal deletions occurring in individual plants of early generation
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(Lynch et al. 2001); (2) duplicate copies can diverge through complementary loss
of function at a particular point in the development or in particular tissues (subfunctionalization), a process that can occur through the action of drift alone; and
(3) selection can occur against gene loss because that loss would compromise
appropriate levels of gene product in relation to another gene(s) (gene balance
hypothesis, cf. Birchler and Veitia 2010). In N. tabacum, a mechanism termed
‘‘nonfunctionalization’’ has been proposed which considers a combination of
degenerative mutation and epigenetic silencing (Chakrabarti et al. 2007). Here,
‘‘non-functionalization’’ has altered alkaloid metabolism through reduced conversion of nicotine to nornicotine. Perhaps this mutation has been important to the
‘‘success’’ of N. tabacum as a recreational drug.
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• Reduced chiasma frequency. Selection may favor the formation of fewer and/or
focussed chiasma at recombination hotspots. This would result in quadrivalents
formed in prophase I falling into two bivalents in metaphase I and then segregating normally (Fig. 11.3a). Computer modeling has shown that reduced
chiasma frequency results in the evolution of disomic inheritance (Le Comber
et al. 2010), although we are unaware of empirical evidence to support this
assertion.
• Structural and epigenetic divergence of homeologues. Allopolyploidy can
trigger extensive karyotype and molecular restructuring in early generations
(Gaeta et al. 2007; Renny-Byfield et al. 2011). This may result in genetic
divergence of the parental genomes, e.g., through the loss or rearrangement of
sequences shared by homeologues and recognized by the homolog recognition
machinery in meiosis (Fig. 11.3b). Such sequences are unlikely to be those that
are highly repeated across the genome since they are not chromosome specific.
Nevertheless, genetic changes may introduce regions among homeologues that
do not recombine, forming ‘‘islands of divergence’’, as in the establishment of
homoploid hybrid species (Strasburg et al. 2009). Without recombination, the
local regions around these islands will continue to diverge, leading to regular
homolog pairing, and bivalent formation. Similarly, allopolyploidy can trigger
epigenetic changes across the genome (Parisod et al. 2009). Epigenetic changes,
which are thought to alter patterns of ectopic recombination (Colot et al. 1995),
may also influence patterns of meiotic recombination and initiate islands of
divergence. In addition, newly formed epi-alleles may establish tissue or temporal patterns of gene expression (subfunctionalization, Adams and Wendel
2005). Subfunctionalization is likely to favor the evolution of regular disomic
inheritance and to select against multivalent formation. This is because multivalents result in the inappropriate segregation of homeoalleles and the
generation of individuals with reduced fitness, because they have aberrant
patterns of gene expression (Le Comber et al. 2010).
• Intergenomic translocations. In the context of a diploid, translocations among
heterologues can promote quadrivalent formation, leading to chromosome loss,
and reduced fertility. Similarly, in the context of polyploids, translocations and
aneuploidy are associated with low fertility, as observed in synthetic B. napus
lines (Xiong et al. 2011). However, it is possible that a more complex dynamic
can occur (Fig. 11.3c). If homeologues already form multivalents, then it is
conceivable that the gain of genetic material from a heterologous chromosome
may promote bivalent formation, disomic inheritance, and increased fertility.
Alternatively, complex rearrangements can result through cascades of induced
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synthetic allopolyploids of B. napus (Gaeta et al. 2007). Typically, a major hurdle
that a newly formed polyploid must overcome is reduced fertility, often arising
through multivalent formation, where chromosomes can segregate aberrantly
leading to aneuploidy and reduction in fertility. We can envisage several allopolyploid-induced processes that may act to reduce multivalent formation and be
favored by selection, these are:
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(a) Reduced chiasma frequency
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formation -enhancing fertility
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(b) Structural divergence of homologues
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(c) Intergenomic translocations
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Rapid homeologue
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enhancing fertility
Promotes
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Homeologous translocations
promote multivalent
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translocations. Meiotic
abnormalities stimulate
unequal reciprocal
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end-to-end chromosome
fusions, resulting in dysploidy
.
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pairing problems resulting from recurrent multivalent formation (unequal reciprocal translocations, inversions, end-to-end chromosome fusions), potentially
generating novel chromosomes with segments from multiple ancestral chromosomes. Such a process may be associated with a reduction in chromosome
number to a diploid-like level, as reported in the divergence of Arabidopsis
karyotypes (Mandakova et al. 2010).
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Fig. 11. 3 Fast molecular and chromosome evolution may enhance bivalent formation, driving
disomic inheritance, and fertility in young polyploids. Homologs are shown in the same color
(pink or pale green). Homeologues diverge over time, this is reflected in their colors becoming
more distinct (red and green). A translocation from a heterologue is shown in blue. Crossovers
among chromosomes are shown with crosses
From Nicotiana allopolyploids, we have evidence for several of the proposed
modes of chromosome evolution. First, in synthetic N. tabacum, we have observed
plants that were homozygous for an intergenomic translocation that is similar to
translocations observed in natural N. tabacum (Lim et al. 2004a). Perhaps this
translocation is selected because it enhances fertility, and that in the synthetic
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N. tabacum evolution is repeating itself. Second, the allotetraploids of sections
Polydicliae and Repandae, which formed c. 1 mya and 5 mya, respectively, have
chromosome numbers that are additive of the progenitor diploids (2n = 4x = 48).
However, the sequence organization along their chromosomes has diverged considerably, particularly in the latter. Indeed, Lim et al. (2007) suggested that in
some species of section Repandae there has been near complete genome turnover.
Perhaps these changes contributed to the establishment of disomic inheritance.
Finally, in section Suaveolentes, several species show evidence of chromosome
number reduction from expectation (chromosome numbers range from
2n = 4x = 32 to 46, depending on species, Goodspeed 1954; Knapp et al. 2004).
We anticipate that this reduction is associated with karyotype restructuring, as in
the polyploids of Arabidopsis (Mandakova et al. 2010).
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11.8 Advantages of the Nicotiana System and Future
Perspectives
369
Acknowledgments We thank NERC and the Czech Science Foundation for support.
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Book Title
Polyploidy and Genome Evolution
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Polyploid Evolution in Spartina: Dealing with Highly Redundant Hybrid Genomes
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Abstract
Polyploidy and recurrent interspecific hybridization represent major features of Spartina evolution, resulting
in several superimposed divergent genomes that coexist in the currently living species. This chapter
summarizes what we presently know about Spartina history, emphasizing the recent hybridization and
polyploidization events that have important ecological and evolutionary consequences. Particular attention
is devoted to the recent formation of the allododecaploid invasive Spartina anglica, a salt-marsh “ecosystem
engineer” that resulted from hybridization between the hexaploid S. alterniflora (introduced from North
America) and tetraploid S. maritima (a European native) and subsequent genome duplication of the F1 hybrid
S. x townsendii during the nineteenth century in Western Europe. Allopolyploidy was not accompanied by
substantial restructuring of the parental genomes, as observed in some other allopolyploid systems. The major
evolutionary events affect the regulatory systems controlling gene expression (including epigenetic
regulation), which appear to have been profoundly altered by the merger of different genomes.
Methodological challenges in exploring non-model, highly redundant genomes resulting from superimposed
events of polyploidization (such as those encountered in Spartina) and the contribution of the new massive
parallel sequencing technologies are discussed.
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M. Ainouche, H. Chelaifa, J. Ferreira, S. Bellot, A. Ainouche
and A. Salmon
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Abstract Polyploidy and recurrent interspecific hybridization represent major
features of Spartina evolution, resulting in several superimposed divergent genomes that coexist in the currently living species. This chapter summarizes what we
presently know about Spartina history, emphasizing the recent hybridization and
polyploidization events that have important ecological and evolutionary consequences. Particular attention is devoted to the recent formation of the allododecaploid invasive Spartina anglica, a salt-marsh ‘‘ecosystem engineer’’ that resulted
from hybridization between the hexaploid S. alterniflora (introduced from North
America) and tetraploid S. maritima (a European native) and subsequent genome
duplication of the F1 hybrid S. x townsendii during the nineteenth century in
Western Europe. Allopolyploidy was not accompanied by substantial restructuring
of the parental genomes, as observed in some other allopolyploid systems. The
major evolutionary events affect the regulatory systems controlling gene expression (including epigenetic regulation), which appear to have been profoundly
altered by the merger of different genomes. Methodological challenges in
exploring non-model, highly redundant genomes resulting from superimposed
events of polyploidization (such as those encountered in Spartina) and the contribution of the new massive parallel sequencing technologies are discussed.
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Polyploid Evolution in Spartina: Dealing
with Highly Redundant Hybrid Genomes
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M. Ainouche (&) H. Chelaifa J. Ferreira S. Bellot A. Ainouche A. Salmon
University of Rennes 1, UMR CNRS 6553 Ecobio, Bât. 14A, Campus Scientifique de
Beaulieu, 35042, Rennes Cedex, France
e-mail: malika.ainouche@univ-rennes1.fr
P. S. Soltis and D. E. Soltis (eds.), Polyploidy and Genome Evolution,
DOI: 10.1007/978-3-642-31442-1_12, Springer-Verlag Berlin Heidelberg 2012
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Polyploidy and recurrent interspecific hybridization represent major features of
Spartina evolution, resulting in several superimposed divergent genomes that
coexist in the currently living species. Particularly fascinating is the rapid range
expansion of the recently formed allododecaploid species S. anglica Hubbard that
formed in Western Europe during the end of the nineteenth century. The ecological
impact and genetic determinants of the spectacular propagation of this invasive
species that is now (deliberately or accidentally) introduced on several continents
generated an abundant literature (reviewed in Gray et al. 1990, Ainouche et al.
2004a, Triplet and Gallicé 2008, Ainouche et al. 2009) and much interest, as
illustrated by the successive International Invasive Spartina Conferences or Forums held in 1990 (Seattle WA, USA), 1997 (Olympia, WA, USA), 2004 (San
Francisco, CA, USA), and 2011 (Oakland, CA, USA). In the field of evolutionary
biology, Spartina has long represented a textbook example of recent allopolyploid
speciation in which the historical context of species formation is particularly well
documented (Huskin 1930; Stebbins 1950).
In this chapter, we summarize the current state of knowledge about Spartina
evolutionary history, including recent insights from evolutionary genetics and
genomic approaches. We also examine how genome evolution following natural
interspecific hybridization and polyploidization has contributed to diversification
and adaptation. Methodological challenges in exploring highly redundant genomes
resulting from superimposed events of polyploidization will be discussed.
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12.1 Introduction
12.2 Recurrent Reticulate Evolution and Polyploidy
in Spartina
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The grass genus Spartina (‘‘cordgrasses’’) belongs to the Chloridoideae subfamily
(Fig. 12.1), one of the most poorly understood lineages of the Poaceae. Divergence
between Spartina and various grass model species is currently estimated as 35–40
MYA with Sorghum—maize–sugar cane (subfamily Panicoideae) and 50 MYA
with rice (subfamily Erhartoideae) (Christin et al. 2008, but see Prasad et al. 2011).
Phylogenetic relationships among genera of Chloridoideae are not fully resolved
and still are under debate (Hilu and Alice 2001, GPWG 2001). In recent molecular
phylogenies, Spartina appears closely related to Sporobolus, Calamovilfa and
Zoysia (Columbus et al. 2007, Fortune et al. 2007). Species from these genera
share a C4-type photosynthetic system that evolved in the Chloridoideae 25–32
MYA (Christin et al. 2008). C4 photosynthesis is generally considered an adaptation conferring higher productivity under warm temperatures. However, species
of Spartina exhibit geographic distributions that cover a range of climatic conditions, from temperate to tropical–subtropical regions; the species exhibit salt
and/or drought tolerance on coastal or inland marshes or sand dunes.
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(c)
Panicoideae
Cynodon dactylon
S. argentinensis
Hybrids (9x)
6x
S. foliosa
CHLORIDOIDEAE
Hybrids (6x)
4x
Cynodonteae
(b)
Zoy sieae
Pooideae
S. pectinata
6x , 8x
S. cynusoroides
Hybrids
S. densiflora (7x)
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Aristidoideae
Bambusoideae
S. anglica
(12x)
S. alterniflora
Danthonioideae
Ehrhartoideae
S. x townsendii
(6x)
S. maritima
Arundinoideae
+ Micrairoideae
S. x neyrautii
(6x)
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S. x caespitosa
SPARTINA
S. bakeri
Calamovilfa
Sporobolus
Zoysia
S patens
Eragrostideae
S. arundinacea
Hybrids
Fig. 12.1 Phylogeny of Spartina (see references in the text) a position within the grass family
b position within the Chloridoideae subfamily c phylogenetic relationships among Spartina
species, recurrent hybridization and polyploidy
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Marchant (1968a)
Marchant (1968a)
Marchant (1968a)
Marchant (1968a)
Marchant. (1968a)
Marchant (1968a)
Kim et al. (2010)
Ayres et al. (2008), Fortune et al. . (2008)
Marchant. (1968b)
Ayres et al. (2008)
Marchant (1968b)
Marchant (1968b)
Marchant (1977)
Marchant. (1968b)
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densiflora Brongn.
maritima (Curtis) Fern.
foliosa Trin.
alterniflora Loisel.
x townsendii H & J Groves
x neyrautii Foucaud
anglica C.E. Hubbard
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patens (Aiton) Muhl.
cynusoroides (L.) Roth
backeri Merr.
gracilis Trin.
arundinacea (Thouars) Carmich.
pectinata Link
Spartina is composed of about 15 perennial species that have mostly diversified
in the New World (Mobberley 1956). Accidental or deliberate introduction of
species outside their native range over the past 150 years has accelerated
diversification by facilitating hybridization with native species, introgression or
speciation. The basic (haploid) chromosome number in this lineage is considered
to be x = 10 (Marchant 1968a), and all Spartina species recorded to date are
polyploid, ranging from tetraploids to dodecaploids (Table 12.1).
Molecular phylogenies based on nuclear and chloroplast DNA sequences
indicate that Spartina comprises two main lineages that include tetraploid and
hexaploid species, respectively (Baumel et al. 2002a). The tetraploid lineage is
composed of species native to the New World; these species colonize coastal or
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Table 12.1 Chromosome numbers reported for Spartina. Additional chromosome numbers
resulting from interspecific hybridization and/or backcrosses are presented in the text
Taxa
2n
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inland salt marshes from either North America (S. patens, S. bakeri, S. gracilis,
S. cynusoroides, S. pectinata) or South America (S. ciliata, S. arundinacea).
S. argentinensis (syn. S. spartinae), which has a disjunct distribution in Central
America and South America, is sister to the hexaploid lineage (Fig. 12.1c). This
hexaploid clade is composed of S. maritima, S. alterniflora, and S. foliosa, all
colonizing low-marsh zones. S. maritima, native to the Atlantic coasts of Western
Europe and Africa was, until the nineteenth century, the only Old World native
species, if we exclude recent taxa of hybrid origin. S. foliosa, a species limited to
the Pacific coast of North America (California and Mexico), is a weakly supported
sister species to S. alterniflora (Baumel et al. 2002a; Ainouche et al. 2004b) and is
distributed along the east coast of North and South America from Canada to
Argentina. This species has now one of the largest geographic distributions in the
genus, as it has been introduced to several continents (Europe, Asia).
The nature of polyploidy (auto- vs. allopolyploidy) and the origin of the
hexaploid clade are not fully understood. Up to three different duplicated (homoeologous) genes were distinguished in hexaploids for the low-copy nuclear
gene Waxy, with substitution rates ranging from 2.18 to 4.79 % among homoeologs (Fortune et al. 2007). The presence of three different homoeologous copies of
Waxy could support a hybrid (allopolyploid) origin of this lineage (Fortune et al.
2007). These hexaploid species exhibit regular bivalent pairing (Marchant 1968b).
Although allopolyploidy is the currently favored hypothesis for the origin of the
hexaploid clade, and is supported by the propensity for interspecific hybridization
in this genus, an autopolyploid origin cannot be ruled out. It has been shown that
genetic diploidization via disomic inheritance may occur rapidly following autopolyploid formation (Le Comber et al. 2010), leading to a progressive divergence
of genes duplicated by polyploidy, and to a gene topology that becomes similar to
what would be expected for allopolyploids (Staub et al. 2003).
Dating the origin of a polyploid species from (biparentally inherited) nuclear gene
divergence data is difficult in that (selective and neutral) population-level evolutionary processes and differential evolution of genes duplicated by polyploidy may
give different estimates of the date of origin at different loci. Moreover, in allopolyploids, the divergence of homoeologs actually reflects the divergence between the
parental species, thus leading to an overestimated age of the polyploid (Doyle and
Negan 2009). We recently attempted to estimate the divergence times between
different Spartina lineages from maternally inherited chloroplast sequences. Using
reconstruction of the Spartina chloroplast genome from 454 Roche GS-FLX (Titanium) pyrosequencing (Bellot S. and Ainouche M., unpublished), we were able to
analyze three coding (matK, rbcL, ndhF) and eight non-coding (intergenic spacers or
introns) chloroplast sequences. Using a Bayesian phylogenetic analysis calibrated
with known divergence times in the grass family (Wolfe et al. 1989; Gaut 2002;
Prasad et al. 2005; Chalupska et al. 2008; Christin et al. 2008), we estimated the
divergence between the tetraploid and hexaploid Spartina maternal genomes at 5
MYA, and the divergence time between the two hexaploid S. alterniflora and S.
maritima chloroplast genomes at approximately 3 MYA.
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Several additional recent hybridization events between Spartina species have
led to the formation of homoploid hybrids or new allopolyploids. These events not
only have involved parental species of the same ploidal level within the tetraploid
or the hexaploid clades, but also involved crosses between tetraploid and hexaploid species (Fig. 12.1c). In the tetraploid lineage, early morphological analyses
suggested that S. x caespitosa was a hybrid between S. patens (Eastern North
America) and S. pectinata, which has a wide distribution across much of North
America (Mobberley 1956). Moreover, a recent survey of S. pectinata populations
across North America has revealed hexaploid and octoploid cytotypes that are
morphologically indistinguishable from the tetraploids (Kim et al. 2010). The
origin of these octoploids is unknown, and the current hypothesis that the
hexaploids derive from backcrosses between octoploids and tetraploids has to be
verified. Genetic and genomic resources are currently being developed for this
species, which is considered a good candidate for bioenergy due to its high biomass production (Gedye et al. 2010).
The South American species S. densiflora illustrates well the genetic exchanges
occurring between the tetraploid and hexaploid lineages. The history of this species was only recently elucidated: this vigorous, high marsh species originated
from southeastern coast of South America, but has been introduced to Chile,
California and Spain (Bortolus 2006). Genetic analyses and molecular phylogenies
(Baumel et al. 2002a; Ayres et al. 2008; Fortune et al. 2008) have revealed that this
species is heptaploid (2n = 70), with a hybrid origin from a tetraploid maternal
species closely related to S. arundinacea (with which it shares high chloroplast
sequence similarity), which occurs in Southern Hemisphere islands (Amsterdam,
St Paul and Tristan de Cunha islands), and a hexaploid paternal species related to
S. alterniflora that also occurs in Argentina. Interestingly, S. densiflora appears to
have hybridized with each species from the hexaploid clade in both its native area
and in more recently colonized regions where it came in contact with native
species: (1) At the mouth of the Rio de la Plata in Argentina and Uruguay,
morphologically intermediate plants named Spartina longispica were early recognized as hybrids between S. densiflora and S. alterniflora (Saint-Yves 1932;
Mobberley 1956). (2) S. densiflora was accidentally introduced to California,
probably from Chile, as suggested by geographic, historical, and molecular data
(Bortolus 2006; Fortune et al. 2008). In the San Francisco Bay, Ayres et al. (2008)
have reported hybrids between S. densiflora and the native S. foliosa (2n = 62).
Different chromosome numbers (2n = 66, 94–96) suggest that the F1 hybrids
deriving from a heptaploid and a hexaploid parent might also have backcrossed
with the parental species. (3) On the Atlantic coast of the Iberian Peninsula,
Castillo et al. (2010) have recently detected hybrids between introduced highmarsh S. densiflora and the native low-marsh hexaploid S. maritima (2n = 60). As
in California, different chromosome numbers (2n = 64–66, 2n = ca. 94) were
recorded for the hybrids, with either S. densiflora or S. maritima as maternal
genome donors. No genome doubling (allopolyploid speciation) is recorded to date
in these perennial (apparently sterile) hybrids, as occurred for S. anglica.
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Within the hexaploid clade, the outcomes of interspecific hybridization are
consistent with the phylogenetic relationships and genetic divergence among
species (Baumel et al. 2002a; Ainouche et al. 2004b): production of fertile invasive
hybrids between closely related taxa on the one hand, and formation of sterile
hybrids (followed by genome doubling) between more divergent species on the
other (Fig. 12.1c). These hybridizations resulted from recent introductions (nineteenth and twentieth centuries) of S. alterniflora from the eastern coast of the
Americas to the Pacific coast of North America and to the Atlantic coast of
Western Europe.
In California, plants of S. alterniflora spread rapidly (Daehler and Strong 1997;
Civille et al. 2005). Hybridization with its sister species S. foliosa (native to
California) has been shown to occur in both directions (Ayres et al. 1999). The
greater male fitness of S. alterniflora and recurrent backcrosses has resulted in
hybrid swarms that progressively replace original S. foliosa plants (Antilla et al.
2000; Ayres et al. 2007). Adding an additional layer of complexity, some of these
plants might also be involved in the formation of new hybrid genotypes with the
introduced S. densiflora (Ayres et al. 2008).
In Europe, S. alterniflora was accidentally introduced by ship ballast in southern
England and western France, where it hybridized with S. maritima. In England,
hybridization recorded in 1870 gave rise to S. x townsendii, a perennial, sterile
hybrid (Groves and Groves 1880). Another sterile hybrid between S. alterniflora
and S. maritima was discovered in 1892 in southwestern France in the Bidassoa
Estuary (Foucaud 1897) and named Spartina x neyrautii (Jovet 1941). Because of
their different morphology, some authors suggested that S. x neyrautii and S. x
townsendii might result from reciprocal crosses; however, molecular data revealed
that both hybrids share the same chloroplast genome of S. alterniflora, identifying it
as the maternal parent of both hybrids (Baumel et al. 2003).
After 1890 in England, fertile plants were recorded that appeared to have
resulted from chromosome doubling in S. x townsendii (Marchant 1963), thus
leading to the formation of a new allododecaploid species named S. anglica
(Hubbard 1968), with chromosome numbers 2n = 120, 122, or 124 (Marchant
et al. 1968b) suggesting aneuploidy. The vigorous plants (Fig. 12.2) have rapidly
colonized Western European salt marshes (Raybould et al. 1991a; Thompson
1991; Genegou and Levasseur 1993). Robust shoots, rhizomes and root systems
enable this new species to accumulate large volumes of tidal sediments. For this
reason, S. anglica was deliberately introduced in several parts of the world
(northern Europe, Australia, New Zealand, China, North America) for land reclamation and marsh restoration purposes. S. anglica has rapidly expanded in its
introduced range and now has a worldwide distribution (Ainouche et al. 2009).
The rapid spread of the introduced populations has led to various attempts to
control or eradicate the species; in fact, it is now listed among the 100 ‘‘world’s
worst’’ invaders (IUCN 2000). A recent survey of the ploidal levels in the original
population (Southampton area, UK) where S. anglica and S. x townsendii still
coexist, has revealed that the perennial, sterile F1 hybrids represent more than
90 % of the population (Renny-Byfield et al. 2010). Using genomic in situ
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Fig. 12.2 Spartina anglica in the Baie des Veys (Cotentin), first colonized site (1906) in France
(Corbière 1926)
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hybridization (GISH), this study also confirmed the existence of nonaploid plants
(2n = ca. 90), most likely resulting from backcrosses between S. anglica and its
maternal parent S. alterniflora.
The recurrent and continuing hybridization and genome duplication in Spartina
(Fig. 12.1) make it a particularly useful model with which to explore the consequences of these processes at various evolutionary time scales. Recent events
allow ecological, phenotypic, and genetic comparisons between the newly formed
hybrids or polyploids and their parents that are still extant in natural populations, a
situation that is only met in a few biological systems (see also Chaps. 13 and 14,
this volume).
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12.3 Ecological and Adaptive Consequences of Hybridization
and Polyploidy in Spartina
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Spartina species play an important ecological role in the sedimentary dynamics of
salt marshes, where the plants are considered to be ‘‘ecosystem engineers’’(Crooks
2002). The ecological range of S. anglica along the shore is larger than either of its
parents. Spartina anglica tolerates several hours of immersion at high tides and
thus is able to occupy a vacant niche as a pioneer species in the low-tide zone. This
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species may accrete large volumes of tidal sediments, making the habitat more
terrestrial, and allowing colonization by other salt marsh plant species, which
modifies the physical structure of intertidal coastal zones.
Recently formed Spartina hybrids and allopolyploids display hybrid vigor and
rapid expansion in their native range and invasive abilities when introduced, with
important implications for ecosystem management (Lambrinos 2008). S. alterniflora x foliosa hybrids have rapidly invaded the San Francisco Bay (Ayres et al.
2007). Several generations of introgressive hybridization make it difficult to differentiate invasive ‘‘cryptic hybrids’’ from ‘‘pure’’ native S. foliosa plants, and this
complicates eradication plans (The Invasive Spartina Project, www.spartina.org).
The heptaploid South American S. densiflora has also rapidly colonized Californian marshes where it increased fivefold in distribution during the last 25 years
following its introduction (Ayres et al. 2004). In Spain, the hybrid S. densiflora
x maritima exhibits greater ecological amplitude than either parental species:
hybrids are able to survive both in lower elevations in the intertidal zone where
S. maritima naturally grows and also in high marshes where S. densiflora invades
(Castillo et al. 2010).
Hybridization and polyploidy in Spartina are accompanied by various
biological changes that have influenced important adaptive traits such as breeding
system, physiology, and morphology. The Californian S. densiflora x foliosa
hybrids, derived from self-incompatible, outcrossing parents, have evolved selffertility that has contributed substantially to their rapid spread (Sloop et al. 2009).
This breakdown in self-incompatibility, also observed following allopolyploidy in
Brassica and Arabidopsis, is most likely triggered by epigenetic mechanisms
(Nasrallah et al. 2007). Several transgressive traits in height and biomass, vegetative growth rates, intertidal amplitude, and salinity tolerance are also reported in
the Spartina hybrids from California (Ayres et al. 2007).
Hybridization between S. alterniflora and S. maritima had very different morphological consequences in the two independent events that occurred in England
(S. x townsendii) and France (S. x neyrautii), even though these hybridization
events involved crosses in the same direction (S. alterniflora being the maternal
genome donor) and similar parental genotypes (Baumel et al. 2003; Yannic et al.
2004; Salmon et al. 2005). S. x neyrautii has shorter spikelets and is distinctly
more slender than S. x townsendii, which has longer fleshy leaves, resembling
more closely the maternal parent S. alterniflora, whereas S. x townsendii has
intermediate morphological features between S. maritima and S. alterniflora
(Mobberley 1956). The phenotypic differences between these two F1 hybrids of
similar genetic origin are puzzling and most likely represent differential effects of the
‘‘genomic shock’’ resulting from the merger of divergent genomes. S. x townsendii is
almost indistinguishable from its allopolyploid derivative S. anglica; moreover, the
latter species exhibits larger phenotypic plasticity (Thompson et al. 1991).
Physiological and anatomic adaptations are important components of Spartina
ecology and distribution (Maricle et al. 2006, 2009). As observed in many
polyploids (Otto 2007), stomatal cell size increases with ploidy level in Spartina
(Marchant 1967; Kim et al. 2010), which may affect photosynthetic rates (Warner
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and Edwards 1993). The larger ecological amplitude of the allopolyploid
S. anglica compared to its parents has to be related to increased tolerance to highly
reducing and sulfidic sediment conditions. This increased tolerance may explain
the ability of S. anglica to colonize low-marsh zones (Maricle et al. 2006).
Survival of S. anglica in anoxic sediments likely is facilitated by its particular
ability to develop aerenchyma systems that supply the submerged plants with
atmospheric oxygen and efficiently transport oxygen to the roots (Maricle and Lee
2002). S. anglica displays enhanced mechanisms to transport O2 and exhibits five
times greater H2S removal than its progenitor species S. alterniflora (the other
parental species, S. maritima, was not investigated; Lee 2003).
An interesting function seems to have accompanied the formation of the
hexaploid lineage of Spartina: the ability to produce dimethylsulfoniopropionate
(DMSP), an osmoprotectant and anti-stress molecule (Larher et al. 1977). DMSP is
environmentally important as the main biogenic precursor of atmospheric dimethyl
sulfide (DMS), which has roles in the biogeochemical sulfur cycle, in cloud formation and in acid precipitation (Kocsis et al. 1998). DMSP is commonly produced by many marine algae, but this capacity is rare in angiosperms, where it has
been found only in three genera, one in the Asteraceae (Wollastonia) and two in
the Poaceae (Saccharum and Spartina) (Otte et al. 2004). Asteraceae and Poaceae
have independently developed different metabolic pathways to achieve this synthesis (Kocsis et al. 1998; Kocsis and Hanson 2000).
High DMSP concentrations are found in leaves of the hexaploids S. alterniflora,
S. foliosa and S. maritima (Otte et al. 2004), providing these plants with a characteristic ‘‘unpleasant sulphurous odor’’ noticed by early taxonomists (Mobberley
1956). In contrast, no DMSP is detected in the tetraploid species analyzed to date.
Although the evolutionary steps giving rise to this physiological novel capacity are
yet to be elucidated, the comparative data generated to date indicate that it may have
been enabled by the transition to the hexaploid condition. If so, this would represent
an example of a presumably important ecological adaptation arising from
polyploidy.
Physiological (and more generally phenotypic) evolution following hybridization and polyploidy is a direct outcome of the genomic consequences of genome
merger and duplication. Although we are still far from obtaining an exhaustive
knowledge of the complex gene and regulatory networks involved in most phenotypic traits of adaptive importance, significant progress has been made in recent
years regarding genetic and genomic processes accompanying polyploid evolution.
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It is now well established that hybrid and allopolyploid genomes are not simply
additive with respect to their parental genomes and that myriad novel interactions
at both the structural and functional levels may lead to rapid evolution and
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evolutionary novelties. It also may be that these phenomena play a critical role in
the evolutionary success of newly formed species in both short-term and long-term
evolutionary time (e.g., Wendel 2000; Chen 2007; Doyle et al. 2008; Van de Peer
et al. 2009).
The genetic context of the hybrid or allopolyploid species formation is an
important parameter to consider for subsequent evolution. Recurrent hybridization
events in natural populations provide new lineages with an expanded genetic base,
as observed in the introgressant populations resulting from multiple hybridization
and recurrent backcrosses involving different S. alterniflora and S. foliosa genotypes in California (Ayres et al. 2007). In contrast, low genetic diversity is
encountered in the parental populations of S. maritima and S. alterniflora that
hybridized in southern England and southern France (Baumel et al. 2003; Yannic
et al. 2004; Ainouche et al. Ainouche et al. 2004a). As a result, the F1 hybrids S. x
townsendii and S. x neyrautii share very similar parental genotypes and exhibit
similar, additive genetic composition. A strong genetic bottleneck seems to have
affected the new allopolyploid S. anglica as a result of a unique origin from S. x
townsendii: a very low interindividual genetic diversity is encountered within and
among populations in both its native and introduced ranges (Guénégou 1988;
Raybould et al. 1991b; Baumel et al. 2001; Ainouche et al. 2004a), although some
genetic variants may be encountered (Ayres et al. 2001).
Contrasting with most other young or experimentally resynthesized allopolyploid systems that exhibit rapid genome structural evolution (Ozkan et al. 2001;
Skalika et al. 2005; Gaeta et al. 2007; Lim et al. 2008; Tate et al. 2009; Szadowski
et al. 2010; Buggs et al. 2012; Chester et al. 2012), the new allododecaploid
S. anglica exhibits relative genome stability (Baumel et al. 2002b; Ainouche et al.
2004a). Most of the early evolutionary changes following allopolyploid speciation
in Spartina seem to affect the regulation of gene expression, including epigenetic
and transcriptome changes (Fig. 12.3). DNA methylation alterations revealed by
Methylation Sensitive AFLP (MSAP) appear triggered by hybridization in both
S. x townsendii and S. x neyrautii (Salmon et al. 2005). Genome duplication does
not entail significant additional changes, as S. anglica has inherited most of the
changes observed in S. x towsendii but exhibits few specific methylation
alterations. Parisod et al. (2009) have shown that an important fraction of these
methylation changes affect regions flanking transposable elements, which agrees
with the general view of methylation having evolved to control transposable
elements in eukaryotic genomes (Slotkin and Martienssen 2007) and with the fact
that no burst of transposition was detected following allopolyploid speciation
(Baumel et al. 2002b; Parisod et al. 2010). The hexaploid parental species
S. maritima and S. alterniflora have a genome size of 2C = 3.8 pg and 4.3 pg,
respectively (Fortune et al. 2008), which suggest that the basic haploid genome
ranges between 600 and 700 MB in these species. A preliminary investigation
from 454 pyrosequencing of genomic DNA in S. maritima revealed that about
27 % of the analyzed sequences were recognized as repetitive, with Gypsy-like
elements being mostly represented.
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Transcriptome evolution was first investigated in Spartina using oligo-microarrays. As no Expressed Sequence Tag (EST) database was then available for
Spartina, heterologous hybridization was performed using the related model
species Oryza sativa (Chelaifa et al. 2010a, b). The hexaploid species S. maritima
and S. alterniflora that exhibit high exon sequence identity (94.0–99.7 %) at
homologous loci displayed 1,247 differentially expressed genes in leaves from
plants grown in the same controlled conditions (Chelaifa et al. 2010a). Most of
these genes were found to be up-regulated in S. alterniflora. Similar levels of nonadditive parental patterns of gene expression were observed in both of the hybrids
S. x townsendii and S. x neyrautii (6.1 and 6.4 % of the analyzed genes, respectively, Fig. 12.3), including parental (mostly maternal) gene expression dominance
and transgressively expressed genes. However, the dominance of maternal
expression appeared more pronounced in S. x townsendii than in S. x neyrautii.
About 8.7 % of the analyzed genes were found differentially expressed between
these two F1 hybrids (Fig. 12.3), and interestingly, most transgressively expressed
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Fig. 12.3 Genome evolution following recent hybridization and allopolyploidy in Spartina.
Percentages in red represent transcriptome changes evaluated using microarrays as described in
Chelaifa et al. (2010a, b). Effects of hybridization were estimated by comparing expression
profiles in the two independently formed natural F1 hybrids to a theoretical mid-parent value
representing additive parental expression. Effects of genome duplication were evaluated by
comparing S. anglica to S. x townsendii. Percentages in black represent DNA methylation
alterations evaluated using methylation sensitive AFLP, following the procedure employed by
Salmon et al. (2005) and Parisod et al. (2009)
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genes were different, with genes up-regulated in S. x townsendii being related to
transport, detoxification and stress, and genes up-regulated in S. x neyrautii being
related to cellular growth and development. The two independent hybridization
events involving the same parental species appear to have generated differential
consequences in terms of gene expression. The functions of these differentially
expressed genes are consistent with the phenotypic differences previously mentioned between the two hybrids. Genome duplication in S. anglica entailed additional transcriptome changes (Fig. 12.3), consisting of the attenuation of the
maternal dominance observed in the F1 hybrid and an increased number of
transgressively overexpressed genes (Chelaifa et al. 2010b). Thus, both hybridization and genome duplication appear to have important, though different, effects
on the Spartina transcriptome, occurring shortly after genome merger and polyploidization. For the first time, these decoupled effects were analyzed during the
allopolyploid speciation process, by comparing the actual (naturally formed) F1
hybrid to its immediately derived allopolyploid that formed and survived in natural
conditions. Interestingly, our findings seem to parallel the conclusions emerging
from similar comparisons involving natural, more or less recent allopolyploids
and/or synthetic F1 hybrids (Hegarty et al. 2006; Flagel et al. 2008; Flagel and
Wendel 2010; Buggs et al. 2011).
The microarray analyses allowed the first large-scale investigation of the
Spartina transcriptome. However, it should be kept in mind that the expression
changes reported in these studies are most likely underestimated: only global
expression of the genes that hybridized on the rice microarrays were examined,
and homoeologous gene expression could not be distinguished. Similar levels of
expression might be attained via biased parental expression, which represents an
important component of the functional plasticity of polyploid genomes (Adams
et al. 2003; Chaudhary et al. 2009; Flagel et al. 2009; Buggs et al. 2011).
Distinction of homoeologous gene expression has been studied in several allopolyploid models where diploid representatives of the parental species are identified
(e.g. cotton: Udall et al. 2006; Flagel et al. 2008; Arabidopis: Chang et al. 2010;
Tragopogon: Buggs et al. 2010; Buggs et al. 2011). In Spartina, this task is particularly challenging as the parents of the young allopolyploid S. anglica are hexaploids
(expected to have retained up to three more-or-less divergent duplicated homoeologs
per locus) and because no diploid species is known in the genus (Fig. 12.4a).
Next-Generation Sequencing (NGS) technologies offer unique avenues to distinguish homoeologous copies in highly redundant genomes from natural, nonmodel species that have experienced successive polyploidization events. This
procedure is being developed for Spartina as follows (Fig. 12.4b): (1) construction
of a Spartina reference transcriptome for the hexaploid parental species using 454
Roche GS-FLX pyrosequencing; (2) Single Nucleotide Polymorphism (SNP)
detection among reads per annotated contig; (3) haplotype assembly to discriminate homoeoalleles; and (4) Illumina RNA-Sequencing (RNA-Seq) to explore
variation of homoeolog expression in the parental species.
Using the procedure outlined above, 38,000 contigs representing *17,000
unigenes were annotated for S. maritima and S. alterniflora from leaf and root
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cDNA libraries, which represent the first reference transcriptome for the hexaploid
Spartina species (Ferreira et al. submitted ). An example of SNP detection among
reads is presented in Fig. 12.4b for an aligned portion of two homologous contigs
annotated in S. maritima and S. alterniflora as HECT-domain-containing protein
(Oryza sativa annotation LOC_Os12g24080.1|13112.m02448|cDNA). Reads were
assembled using a 95 % identity threshold, to avoid potential comparisons
involving paralogs. Six polymorphic sites are detected in this region, including
four polymorphic sites shared between S. maritima and S. alterniflora and two
species-specific polymorphic sites. The shared polymorphisms allow distinction of
two divergent haplotypes (1 and 2, Fig. 12.4b) present in both hexaploids, and one
(in S. maritima) or two (in S. alterniflora) additional minor variants. Screening of a
larger number of polymorphic sites and loci will provide information about the
number and divergence of (homoeo) alleles encountered in the hexaploids and
shed light on the evolutionary history of this lineage. A variable number of
retained copies per homologous locus may be expected. For instance, Fortune et al.
(2007) analyzed the low-copy nuclear gene Waxy that is present in two paralogs
(A and B) in Spartina. Only one B copy was encountered in S. maritima, whereas
three distinct B copies were found in S. alterniflora. These two species have
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Fig. 12.4 a Expected nuclear homoeologous gene copies per locus in the hexaploid parents, F1
hybrid and allododecaploid Spartina species b Procedure for sequence heterogeneity detection at
homologous nuclear coding loci; example from two homologous contigs in the hexaploid S.
maritima (contig length = 4,294, number of read = 127) and S. alterniflora (contig
length = 3,961, number of reads = 85) assembled from 454 Roche pyrosequencing of cDNA
libraries. Arrows of similar color (green or pink) represent divergent alleles (1 and 2) present in
the two hexaploids and detected from shared polymorphic sites between the two species, in the
central zone of the compared region. Other haplotypes represent slight variants of these two
copies
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Acknowledgments This work benefited from the financial support of the Centre National de la
Recherche Scientifique (CNRS), University of Rennes 1, the French National Research Agency
(ANR), Region Bretagne, Genoscope, and from the Biogenouest (Transcriptomics and Environmental Genomics) platform facilities. P. Wincker, J. Poulain, Corinne Da Silva, O. Lima, S.
Coudouel, D. Naquin, A. Deillhy are thanked for their contribution to the 454 pyrosequencing
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apparently lost the A copy that is still retained in the hexaploid S. foliosa (Fortune
et al. 2007). The large-scale detection of homoeologs from massive parallel
sequencing will provide a genome-wide view of the retention-loss process at
various evolutionary time scales (in the hexaploid parents and the nascent allododecaploid). Transcript loss might result from either homoeolog silencing as
observed in the various cases of subfunctionalization reported in allopolyploids
(reviewed in Osborn et al. 2003; Chen 2007; Doyle et al. 2008), physical loss of
the duplicated copies that may occur more or less rapidly following polyploid
speciation (e.g. Gaeta et al. 2007; Tate et al. 2009; Koh et al. 2010; Buggs et al.
2012) or from homoeologous recombination (Cifuentes et al. 2010; Salmon et al.
2010; Gaeta et al. 2010). High-throughput sequencing of genomic DNA and targeted sequencing (e.g. Grover et al. 2012) offer new possibilities to differentiate
the effects of these alternatives. Finally, distinction among Spartina homoeologs
will allow more accurate analysis of homoeologous expression in the F1 hybrids
and the allododecaploid using RNA-Seq data for various organs from plants collected in different ecological conditions.
In conclusion, the well-established framework now available for Spartina offers
important opportunities to elucidate the phenotypic, ecological, and genomic consequences of recurrent hybridization and polyploidy. Based on the dramatic increase
in knowledge that has accumulated for various polyploid systems in recent years
(Ainouche and Jenczewski 2010), it has become clear that hybridization and polyploidy generate a range of possible responses that vary among genera. Recent allopolyploidy in S. anglica was not accompanied by rapid restructuring of the parental
genomes as has occurred in other polyploid systems. The major evolutionary events
in S. anglica appear to affect the regulation of gene expression (including epigenetic
regulation); these appear profoundly altered by the merger of different genomes. The
rapid advances of NGS technologies will allow more exhaustive exploration of
highly redundant genomes (which until now suffered from severe technical limitations) such as those of Spartina. These will provide a better understanding of the
genetic and epigenetic mechanisms underlying expression plasticity, and their effect
on adaptive and ecologically relevant functions.
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Adams KL, Cronn R, Percifield R, Wendel JF (2003) Genes duplicated by polyploidy show
unequal contributions to the transcriptome and organ-specific reciprocal silencing. Proc Natl
Acad Sc USA 100(8):4649–4654
Ainouche ML, Jenczewski E (2010) Focus on polyploidy. New Phytol 186:1–4
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Ainouche ML, Baumel A, Salmon A (2004a) Spartina anglica Schreb. A natural model system
for analysing early evolutionary changes that affect allopolyploid genomes. Biol J Linn Soc
82:475–484
Ainouche ML, Baumel A, Salmon A, Yannic G (2004b) Hybridization, polyploidy and speciation
in Spartina Schreb (Poaceae). New Phytol 161:165–172
Ainouche ML, Fortune PM, Salmon A, Parisod C, Grandbastien M-A, Fukunaga K, Ricou M,
Misset M-T (2009) Hybridization, polyploidy and invasion: lessons from Spartina (Poaceae).
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Antilla CK, King RA, Ferris C, Ayres DR, Strong DR (2000) Reciprocal hybrid formation of
Spartina in San Francisco Bay. Mol Ecol 9:765–770
Ayres DR, Strong DR (2001) Origin and genetic diversity of Spartina anglica (Poaceae) using
nuclear DNA markers. Am J Bot 88:1863–1867
Ayres DR, Garcia-Rossi D, Davis HG, Strong DR (1999) Extent and degree of hybridization
between exotic (Spartina alterniflora) and native (S. foliosa) cordgrass (Poaceae) in
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Ayres DR, Smith DL, Zaremba K, Klohr S, Strong DR (2004) Spread of exotic cordgrass and
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Ayres DA, Zaremba K, Sloop CM, Strong DR (2007) Sexual reproduction of cordgrass hybrids
(Spartina foliosa 9 alterniflora) invading tidal marshes in San Francisco Bay. Divers Distrib
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Ayres DR, Grotkopp E, Zaremba C, Sloop CM, Bloom MJ, Bailey JP, Anttila CK, Strong DR
(2008) Hybridization between invasive Spartina densiflora (Poaceae) and native S. foliosa in
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Baumel A, Ainouche ML, Levasseur JE (2001) Molecular investigations in populations of
Spartina anglica C.E. Hubbard (Poaceae) invading coastal Brittany (France). Mol Ecol
10:1689–1701
Baumel A, Ainouche ML, Bayer RJ, Ainouche AK, Misset M-T (2002a) Molecular phylogeny of
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22:303–314
Baumel A, Ainouche ML, Kalendar R, Schulman AH (2002b) Retrotransposons and genomic
stability in populations of the young allopolyploid species Spartina anglica C.E. Hubbard
(Poaceae). Mol Biol Evol 19:1218–1227
Baumel A, Ainouche ML, Misset MT, Gourret JP, Bayer RJ (2003) Genetic evidence for
hybridization between the native Spartina maritima and the introduced Spartina alterniflora
(Poaceae) in South-West France: Spartina x neyrautii re-examined. Plant Syst Evol 237:87–97
Bortolus A (2006) The austral cordgrass Spartina densiflora Brong.: its taxonomy, biogeography
and natural history. J Biogeogr 33:158–168
Buggs RJA, Chamala S, Wu W, Gao L, May GD, Schnable PS, et al. (2010) Characterization of
duplicate gene evolution in the recent natural allopolyploid Tragopogon miscellus by next
generation sequencing and Sequenom iPLEX MassARRAY genotyping. Mol Ecol 19:132–146
Buggs RJA, Zhang L, Miles N, Gao L, Wu W, Schnable P, Barbazuk WB, Soltis PS, Soltis DE
(2011) Transcriptomic shock generates evolutionary novelty in a newly formed, natural
allopolyploid plant. Curr Biol 21:551–556
Buggs RJA, Chamala S, Wu W, Tate JA, Schnable PS, Soltis DS, Soltis PS, Barbazuk WB (2012)
Rapid, repeated, and clustered loss of duplicated genes in allopolyploid Tragopogon
populations of independent origin. Curr Biol 22:248–252
Castillo JM, Ayres DR, Leira-Doce1 P, Bailey J, Blum M, Strong DR, Luque T and Figueroa E
(2010) The production of hybrids with high ecological amplitude between exotic Spartina
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Evolutionary genetics of genome merger and doubling in plants. Annu Rev Genet 42:443–461
Ferreira de Carvalho J, Poulain J, Da Silva C, Wincker P, Michon-Coudouel S, Dheilly A, Naquin
D, Boutte J, Salmon A, Ainouche M. Transcriptome de novo assembly and comparative
analysis of the hexaploid salt marsh species Spartina maritima and Spartina alterniflora
(Poaceae) using high-throughput 454 Roche pyrosequencing. Heredity, submitted
Flagel LE, Wendel JF (2010) Evolutionary rate variation, genomic dominance and duplicate gene
expression evolution during allotetraploid cotton speciation. New Phytol 186:184–193
Flagel L, Udall J, Nettleton D, Wendel JF (2008) Duplicate gene expression in allopolyploid
Gossypium reveals two temporally distinct phases of expression evolution. BMC Biol 6:16
Flagel LE, Chen L, Chaudhary B, Wendel JF (2009) Coordinated and fine-scale control of
homoeologous gene expression in allotetraploid cotton. J Hered 100:487–490
Fortuné PM, Schierenbeck K, Ainouche A, Jacquemin J, Wendel JF, Ainouche ML (2007)
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Book Title
Polyploidy and Genome Evolution
Series Title
Chapter Title
Allopolyploid Speciation in Action: the Origins and Evolution of Senecio cambrensis
Copyright Year
2012
Copyright HolderName
Springer-Verlag Berlin Heidelberg
Corresponding Author
Family Name
Hegarty
Particle
Given Name
Matthew J.
Suffix
Author
Division
Institute of Biological, Environmental and Rural Sciences
Organization
Aberystwyth University
Address
Penglais Campus, SY23 3DA, Aberystwyth, Ceredigion, UK
Email
ayh@aber.ac.uk
Family Name
Abbott
Particle
Given Name
Richard J.
Suffix
Author
Division
School of Biology
Organization
University of St. Andrews
Address
Harold Mitchell Building, KY16 9TH, St. Andrews, Fife, UK
Email
rja@st-andrews.ac.uk
Family Name
Hiscock
Particle
Given Name
Simon J.
Suffix
Abstract
Division
School of Biological Sciences
Organization
University of Bristol
Address
Woodland Road, BS8 1UG, Bristol, UK
Email
simon.hiscock@bristol.ac.uk
Senecio cambrensis is one of a few allopolyploid plant species known to have originated in the recent past
and, therefore, provides excellent material for analysing allopolyploid speciation. This allohexaploid species
originated in the UK within the last 100 years following hybridization between diploid S. squalidus and
tetraploid S. vulgaris. In this chapter, we first describe the events leading up to hybridization between these
two species, focusing mainly on the origin and spread of S. squalidus in the UK. We then consider alternative
pathways by which S. cambrensis might have originated and conclude that current evidence suggests an origin
via formation of the triploid hybrid (S. x baxteri) followed by chromosome doubling. We next review our
investigations into levels of genetic diversity and also changes to gene expression and the possible causes of
this (epigenetic effects) during the origin of S. cambrensis. High levels of genetic diversity, assessed by
surveys of allozyme and AFLP variation, have been recorded in S. cambrensis, and it is likely that
intergenomic recombination was an important generator of this diversity. Our studies of ‘resynthesized’ S.
cambrensis have shown that the initial genome merger (hybridization) producing S. x baxteri generates
genome-wide, non-additive alterations to parental patterns of gene expression and DNA methylation, with
genome duplication resulting in a secondary burst of both transcriptional and epigenetic modification. In
synthetic allohexaploid lines of S. cambrensis phenotypic changes become apparent from the second to fifth
generations, possibly as a consequence of recombination or epigenetic effects; these include changes in ray
flower form and emergence of self-incompatible individuals. We conclude by considering the future of S.
cambrensis from the standpoint of it being a model species for further study of allopolyploid speciation, and
second its long-term success in the wild. Ongoing work to produce a draft reference genome for S.
squalidus will underpin future research in S. cambrensis, enabling a more thorough survey of changes to DNA
methylation, small RNA activity and promoter binding in the hybrids, as well as comparison with the related
allotetraploid S. eboracensis to determine the effects of genome dosage. The future of the species in the wild
is currently uncertain. The population in Edinburgh that represented a separate origin of the species in the
wild during the 1970s is now extinct, and there has been a marked decline in the number of populations and
individuals of the species in its heartland, North Wales, since the 1980s. An analysis of how its ecology
compares with those of its parents is lacking. However, it appears to share the same habitats in the wild with
its parents, which might have contributed to its decline. Although S. cambrensis may become extinct in the
wild in the near future, the potential will remain for it to originate again in the UK providing that conditions
prevail for its parents to hybridize.
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Abstract Senecio cambrensis is one of a few allopolyploid plant species known to
have originated in the recent past and, therefore, provides excellent material for
analysing allopolyploid speciation. This allohexaploid species originated in the
UK within the last 100 years following hybridization between diploid S. squalidus
and tetraploid S. vulgaris. In this chapter, we first describe the events leading up to
hybridization between these two species, focusing mainly on the origin and spread
of S. squalidus in the UK. We then consider alternative pathways by which S.
cambrensis might have originated and conclude that current evidence suggests an
origin via formation of the triploid hybrid (S. x baxteri) followed by chromosome
doubling. We next review our investigations into levels of genetic diversity and
also changes to gene expression and the possible causes of this (epigenetic effects)
during the origin of S. cambrensis. High levels of genetic diversity, assessed by
surveys of allozyme and AFLP variation, have been recorded in S. cambrensis, and
it is likely that intergenomic recombination was an important generator of this
diversity. Our studies of ‘resynthesized’ S. cambrensis have shown that the initial
genome merger (hybridization) producing S. x baxteri generates genome-wide,
non-additive alterations to parental patterns of gene expression and DNA
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Allopolyploid Speciation in Action:
the Origins and Evolution of Senecio
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Date: 16-8-2012
M. J. Hegarty (&)
Institute of Biological, Environmental and Rural Sciences, Aberystwyth University,
Penglais Campus, Aberystwyth, Ceredigion SY23 3DA, UK
e-mail: ayh@aber.ac.uk
R. J. Abbott
School of Biology, University of St. Andrews, Harold Mitchell Building,
St. Andrews, Fife KY16 9TH, UK
e-mail: rja@st-andrews.ac.uk
UN
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Chapter No.: 13
S. J. Hiscock
School of Biological Sciences, University of Bristol,
Woodland Road, Bristol BS8 1UG, UK
e-mail: simon.hiscock@bristol.ac.uk
P. S. Soltis and D. E. Soltis (eds.), Polyploidy and Genome Evolution,
DOI: 10.1007/978-3-642-31442-1_13, Springer-Verlag Berlin Heidelberg 2012
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methylation, with genome duplication resulting in a secondary burst of both
transcriptional and epigenetic modification. In synthetic allohexaploid lines of S.
cambrensis phenotypic changes become apparent from the second to fifth generations, possibly as a consequence of recombination or epigenetic effects; these
include changes in ray flower form and emergence of self-incompatible individuals. We conclude by considering the future of S. cambrensis from the standpoint
of it being a model species for further study of allopolyploid speciation, and
second its long-term success in the wild. Ongoing work to produce a draft reference genome for S. squalidus will underpin future research in S. cambrensis,
enabling a more thorough survey of changes to DNA methylation, small RNA
activity and promoter binding in the hybrids, as well as comparison with the
related allotetraploid S. eboracensis to determine the effects of genome dosage.
The future of the species in the wild is currently uncertain. The population in
Edinburgh that represented a separate origin of the species in the wild during the
1970s is now extinct, and there has been a marked decline in the number of
populations and individuals of the species in its heartland, North Wales, since the
1980s. An analysis of how its ecology compares with those of its parents is
lacking. However, it appears to share the same habitats in the wild with its parents,
which might have contributed to its decline. Although S. cambrensis may become
extinct in the wild in the near future, the potential will remain for it to originate
again in the UK providing that conditions prevail for its parents to hybridize.
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13.1.1 General Introduction
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Polyploidization (genome duplication) is an important evolutionary process in plants
(Grant 1981) that appears to have accompanied major transitions in land plant
evolution, including the evolution of the seed habit and the evolution of angiosperms
(Jiao et al. 2011). All angiosperms are thought to have a polyploid ancestry (Jiao et al.
2011) but most of these are paleopolyploids that now function essentially as diploids.
Nevertheless, there are also numerous examples of recently formed polyploids
(Adams and Wendel 2005; Wood et al. 2009). Most recent polyploids have formed in
association with interspecific hybridization (Grant 1981; Leitch and Bennett 1997;
Soltis and Soltis 1999; Otto and Whitton 2000; Leitch and Leitch 2008; Hegarty and
Hiscock 2008)—allopolyploidy, which is now recognised as perhaps the most
important mechanism of abrupt speciation in plants (Grant 1981; Leitch and Leitch
2008; Hegarty and Hiscock 2008). Allopolyploidy confers rapid fertility to hybrids
because duplicated parental chromosomes pair ‘normally’ during meiosis; it also
confers reproductive isolation of the hybrids from their parental species because of
aberrant or failed chromosome pairing (Soltis and Soltis 1999; Rieseberg et al. 2003;
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13.1.2 The Genus Senecio and the Origins of UK Allopolyploids
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The genus Senecio (Asteraceae), which includes ragworts and groundsels, is one of
the largest and most morphologically diverse genera of flowering plants. With
between 1000 and 3000 species, the genus has a worldwide distribution with species
described from almost every land mass on Earth (Vincent 1996). Recent revisions,
following molecular phylogenetic analysis, of the genus have assigned many former
Senecio species to other genera leaving a conservative 1200 species of Senecio sensu
stricto (Pelser et al. 2007). Within the genus there are numerous examples of
hybridization and polyploidy, and many species have been proposed to have an
allopolyploid origin (Abbott and Lowe 1996; Coleman et al. 2001; Kadereit et al.
2006; Pelser et al. 2012). In the UK, three new polyploid taxa have arisen within the
last 100 years as a consequence of hybridization between native tetraploid Senecio
vulgaris (common groundsel) and the introduced invasive diploid species S.
squalidus. The origins of these new polyploid taxa (see below) provide one of the best
examples of ‘evolution in action’ (Abbott and Lowe 1996, Hegarty and Hiscock
2008). The introduction and rapid spread of alien S. squalidus across the UK was the
main catalyst for this burst of hybrid speciation, which was further facilitated by the
obligate outcrossing mating system of S. squalidus increasing the frequency of
interspecies pollinations (Brennan and Hiscock 2010).
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Rieseberg and Willis 2007). Numerous examples of allopolyploid speciation have
been described in the literature, including at least six new plant species that have
arisen in the last century: Tragopogon mirus and T. miscellus (Novak et al. 1991,
Chap. 14, this volume); Spartina anglica (Gray et al. 1991, Chap. 12, this volume),
Senecio cambrensis and S. eboracensis (Ashton and Abbott 1992, Abbott and Lowe
1996, 2004) and Cardamine schulzii (Urbanska et al. 1997). In this chapter, we
review the origins of the Senecio allopolyploid species S. cambrensis (Welsh
groundsel). We show how resynthesised forms of this allopolyploid can reveal
important insights into the genetic and genomic consequences of allopolyploidization and how parental traits, both morphological and physiological, recombine in
neopolyploids.
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13.1.3 Oxford Ragwort in the British Isles: Introduction
and Spread
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Senecio squalidus (2n = 2x = 20), commonly known as Oxford ragwort, is itself
a recently evolved (homoploid) hybrid species (James and Abbott 2005; Abbott
et al. 2010; Brennan et al. 2012) that originated on Mount Etna, Sicily, as a result
of hybridization between S. aethnensis (2n = 2x = 20, a Mount Etna endemic of
higher altitudes) and S. chrysanthemifolius (2n = 2x = 20, a native Sicilian
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species of lower altitudes). At mid-altitudes on the volcano the distribution of
these species overlaps, leading to the formation of a stable hybrid zone (Brennan
et al. 2009). Material from this hybrid zone was introduced to the Oxford Botanic
Garden in the early 1700s, but records of the collection locality or localities,
methods of cultivation, and numbers of plants cultivated in the garden have been
lost. Plants subsequently escaped from the Botanic Garden and colonised the
masonry of the old college walls from the end of the eighteenth century (Harris
2002). During the industrial revolution of the nineteenth and early twentieth
centuries S. squalidus moved rapidly out of Oxford by colonising the clinker beds
of the expanding UK railway, of which Oxford was a key hub. The chronology of
this famous plant invasion is meticulously documented in a set of papers by Kent
(reviewed in Abbott et al. 2009), which record that it began to spread northwards
in the late nineteenth century, reaching different parts of northern England during
the middle of the twentieth century, before becoming established in the Central
Belt of Scotland by the mid-1950s. Today, S. squalidus continues to spread north
in Scotland and across Northern Ireland.
The rapidity of this invasion is intriguing because S. squalidus is self-incompatible (Abbott and Forbes 1993; Hiscock 2000a, b, and obligate outcrossers are generally thought not to make good colonizers or invasives (Baker 1967). Most invasive
species tend to have uniparental reproduction, either sexual (selfing) or asexual
(apomixis or vegetative reproduction), although there are exceptions, most notably
other species of Asteraceae, such as yellow star thistle, Centaurea solstitialis (Sun
and Ritland 1998). According to Baker’s ‘rule’ (Baker 1967), successful colonising
and invasive plants are usually self-fertile (self-compatible [SC]) (Stebbins 1957;
Baker 1967). Studies of the mating system of S. squalidus, however, have shown that
individuals exhibit strong self-incompatibility (SI) across its entire British range,
and, as in other species of Asteraceae, this SI is regulated sporophytically by a single
polymorphic S locus (Hiscock 2000a, b; Brennan et al. 2002, 2005, 2006). The
finding of strong SI in S. squalidus is intriguing, particularly in the light of the
extreme population bottleneck that its ancestors must have experienced during its
introduction and early colonisation. Following a population bottleneck, allelic
diversity at the S locus will be lowered and opportunities for mating (between
individuals carrying different S alleles) correspondingly reduced (Hiscock 2000b;
Brennan et al. 2002). An extensive survey of SI in S. squalidus across the UK showed
that a combination of substantial between-population sharing of the seven S alleles
contained in the entire UK population and low levels of selfing (‘pseudo-self-compatibility’) were the most likely cause of S. squalidus’ reproductive success as a
colonizer (Brennan et al. 2005, 2006).
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13.1.4 The Origins of Senecio cambrensis
During its spread across the UK, S. squalidus (2n = 2x = 20) hybridized with the
self-compatible native groundsel, S. vulgaris (2n = 4x = 40), resulting in the recent
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origin of three hybrid taxa. These are the allohexaploid S. cambrensis
(2n = 6x = 60), the recombinant tetraploid S. eboracensis (2n = 4x = 40), and the
stabilized introgressant radiate form of S. vulgaris, S. vulgaris var. hibernicus
(2n = 4x = 40). Interestingly, all three new hybrid taxa are self-compatible, suggesting that this SC mating system, inherited from S. vulgaris, is ‘dominant’ over the
SI mating system present in S. squalidus. Detailed descriptions of these new taxa and
what is known about their origins are presented elsewhere (see Abbott et al. 1992;
Lowe and Abbott 2003; Abbott and Lowe, 2004; Kim et al. 2008).
Here, we briefly summarise the information available on the origin of S. cambrensis (Fig. 13.1) focussing particularly on the possible pathways of its origin.
Knowing the pathway of origin of a polyploid taxon is helpful in regard to
understanding the species’ potential to generate genetic diversity during its initial
stages of development, and also for accurate production of synthetic forms of the
polyploid used to study possible genetic and epigenetic changes that occurred in
the taxon immediately following its origin in the wild (Hegarty et al. 2006, 2008,
2011; Lukens et al. 2006; Buggs et al. 2009).
Senecio cambrensis was described by Rosser (1955) from material provided by
H.E. Green, who first observed the plant growing at Ffrith and Ceffn-y-bedd, North
Wales, UK, in 1948. The plant was described as an annual or short-lived perennial
herb that was hexaploid with flower heads (capitula) containing ray florets having
short ligules (*4.8 mm in length). Rosser (in Crisp 1972) later determined a herbarium specimen, collected in Denbigh, North Wales in 1925 and originally named
as S. squalidus x S. vulgaris, to be S. cambrensis. However, in the absence of a
chromosome count there remains some doubt as to whether this specimen is
S. cambrensis or, alternatively, a fertile hybrid of S. squalidus and S. vulgaris
(see below). Fertile, hexaploid plants with similar morphology to the wild form of
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Fig. 13.1 The neoallohexaploid Senecio cambrensis (centre) flanked by its parents, tetraploid S.
vulgaris (left) and diploid S. squalidus (right). Here, S. cambrensis has flower heads (capitula) of
intermediate type to its parents; however, its ray florets can vary in length, and occasionally nonradiate forms (lacking ray florets) are also found in the wild
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S. cambrensis can be produced by treating synthetic triploid hybrids between S.
squalidus and S. vulgaris with colchicine (Harland 1955, Weir and Ingram 1980,
Hegarty et al. 2005). On this basis, Rosser (1955) concluded that S. cambrensis was a
new species that originated by hybridization between native S. vulgaris and introduced S. squalidus followed by chromosome doubling. The species is likely to have
originated shortly before it was first recorded in North Wales and after S. squalidus
had spread to the region in the early part of the twentieth century (Kent 1963).
In 1982, S. cambrensis was found growing in Edinburgh, UK (Abbott et al.
1983), and subsequent molecular analysis involving surveys of allozyme and
chloroplast DNA variation showed that it had originated independently in Edinburgh rather than being dispersed there from North Wales (Ashton and Abbott
1992; Harris and Ingram 1992). Herbarium records indicate that the Edinburgh
lineage may have existed since at least 1974; however, it is now thought to be
extinct as the species has not been recorded in the Edinburgh area or nearby since
1993 (Abbott and Forbes 2002).
Because S. cambrensis is readily synthesised by treating the triploid hybrid
between S. squalidus and S. vulgaris with colchicine, it has been assumed that
chromosome doubling of the triploid hybrid was the likely pathway of origin of the
allopolyploid species in the wild (Rosser 1955). In theory, however, the species could
have originated along several possible pathways (Table 13.1) with the first step
involving formation of a triploid, tetraploid, pentaploid or hexaploid hybrid. Of these
alternatives, the formation of a triploid hybrid is more likely in that it results from
fusion of normal haploid gametes produced by each parent. In contrast, formation of
higher ploidy hybrids relies on the production of unreduced gametes, which will be
generated at a much lower frequency in each parent species.
There are many records of the triploid hybrid (Senecio x baxteri Druce)
occurring in the wild (Crisp 1972; Benoit et al. 1975; Marshall and Abbott 1980).
It is easily recognised because of its intermediate morphology and its almost
complete seed sterility. Progeny tests of S. vulgaris plants have shown that this
hybrid is generated regularly but at very low frequencies in the wild where S.
vulgaris and S. squalidus co-occur (Marshall and Abbott 1980). In contrast, the
tetraploid hybrid has never been reported unequivocally in the wild, although
Crisp (1972) described a plant likely to have been such a hybrid based on morphology and an analysis of its offspring. Although no chromosome count was
made of the plant, all of its offspring were approximately tetraploid and segregated
for a range of morphological, reproductive and disease resistance traits. Crisp
(1972) suggested that such offspring could become stabilized in the wild to form
distinct taxa, and it is feasible that the tetraploid S. eboracensis originated in this
way (Lowe and Abbott 2000). Whether such a hybrid might have contributed to
the origin of S. cambrensis as detailed in Table 13.1 remains unknown, but seems
less likely than an origin involving the triploid hybrid, given the apparent rarity of
the tetraploid hybrid in the wild. Similarly, origins involving the formation of
either a pentaploid or hexaploid hybrid are less parsimonious than one involving
the triploid hybrid, although cannot be ruled out entirely. Further support for the
hypothesis that formation of a triploid hybrid was the first step in the origin of
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S. cambrensis comes from reports by Vosa (in Crisp 1972) and Ingram (1978) that
rare allohexaploid offspring were produced spontaneously by natural selfing of the
synthetic triploid hybrids they made. However, Weir and Ingram (1980) also
reported the production of an allohexaploid plant directly from a cross between S.
vulgaris and S. squalidus. This could have been formed by fusion of unreduced
gametes or alternatively by chromosome doubling of a triploid hybrid early in its
development. We shall never know exactly how the different lineages of S.
cambrensis originated in the UK, but given that the triploid hybrid is regularly
encountered in the wild and is capable of producing allohexaploid offspring
spontaneously, an origin involving doubling of the chromosome number of a
triploid hybrid seems the most likely route of origin.
Because S. cambrensis is self-fertile, one newly formed individual of the species would have been able to reproduce sexually and successfully following the
species’ origin, i.e. without need of a mate. Moreover, if the species had originated
through chromosome doubling of a triploid hybrid, it would be expected initially
to be homozygous at all loci within its parental genomes, but to exhibit frequent
fixed heterozygosity at duplicated loci among parental genomes. Somewhat
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Table 13.1 Some possible pathways of origin for Senecio cambrensis in the wild
(1) Via Triploid hybrid (2n=30)
• Step 1 - Formation of triploid hybrid (2n=30) through fusion of haploid gametes
produced by each parent species.
• Step 2 - Chromosome doubling of triploid hybrid by: (i) fusion of ‘unreduced’ triploid gametes
(n=30); or (ii) doubling of chromosome number of a somatic cell ancestral to a floret or flower
head producing ‘reduced’ triploid gametes (n=30).
(2) Via Tetraploid hybrid (2n=40)
• Step 1 - Formation of tetraploid hybrid (2n=40) by: (i) fusion of a haploid gamete (n=20)
of S. vulgaris and a diploid (unreduced) gamete (n=20) of S. squalidus; or (ii) fusion
of an ‘unreduced’ triploid gamete of triploid hybrid (n=30) and haploid gamete of
S. squalidus (n=10).
• Step 2 - Production of hexaploid hybrid by: (i) fusion of an ‘unreduced’ tetraploid gamete
(n=40) generated by tetraploid hybrid and a ‘reduced’ diploid gamete (n=20) of same hybrid
or of S. vulgaris or an unreduced gamete of S. squalidus; or (ii) fusion of ‘balanced’ triploid
gametes (n=30) produced by same hybrid.
(3) Via Pentaploid hybrid (2n=50)
• Step 1 - Formation of pentaploid hybrid by: (i) fusion of a haploid gamete of S. squalidus
(n=10) and an unreduced gamete of S. vulgaris (n=40); or (ii) fusion of unreduced gamete
of triploid hybrid (n=30) with haploid gamete of S. vulgaris (n=20) or unreduced gamete
of S. squalidus (n=20).
• Step 2 - Production of hexaploid hybrid by: (i) fusion of gametes with same or different
‘balanced’ chromosome numbers (i.e. n=10, n=20, n=30, n=40, n=50) generated by
pentaploid hybrid such that the zygote produced is hexaploid (2n=60); (ii) fusion of tetraploid
‘balanced’ gamete (2n=40) produced by pentaploid hybrid with reduced gamete (n=20) of
S. vulgaris or unreduced gamete of S. squalidus. (iii) fusion of diploid ‘balanced’ gamete
(n=20) produced by pentaploid hybrid with unreduced gamete (n=40) of S. vulgaris.
4) Direct formation of Hexaploid hybrid (2n=60)
• Step 1 - Formation of hexaploid hybrid by fusion of unreduced gametes from both
S. squalidus (n=20) and S. vulgaris (n=40).
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surprisingly, however, the species has been shown through surveys of allozyme
variation (Ashton and Abbott 1992) and particularly AFLP variation (Abbott et al.
2007) to contain high levels of genetic diversity, indicating that it rapidly generated this diversity following its origin. Abbott et al. (2007) considered the ways in
which such genetic diversity was produced and concluded that intergenomic
recombination would most likely have been an important mechanism, although
other mechanisms such as aneuploidy, gene conversion, activation of transposons
and retroelements, other forms of mutation and gene flow from parental species
could not be ruled out. The occurrence of radiate and non-radiate forms of S.
cambrensis as well as variation in the ligule length of ray florets have been
attributed to intergenomic recombination (Ingram and Noltie 1984), while the
observation of multivalent formation occurring at low frequency in meiotic cells of
the species (Ingram and Noltie, 1989) provides a mechanism for such recombination to occur. Although not reported by Ingram and Noltie (1989), Crisp (1972)
observed up to eight chromosomes with subterminal centromeres in the somatic
complement of S. cambrensis plants (based on root tip squashes). He pointed out
that as neither parent species possessed such chromosomes they were probably the
products of chromosome rearrangements following meiotic abnormalities. In
addition to generating genetic diversity, intergenomic recombination resulting in
chromosome rearrangements could lead to the formation of reproductive barriers
between divergent lineages of S. cambrensis. Clearly, further work on the frequency of intergenomic recombination and its possible effects in S. cambrensis
would be worthwhile.
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13.2 Consequences of Hybridization and Polyploidy
in Natural and Resynthesised Senecio cambrensis
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13.2.1 Transcriptome Shock
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In common with many other allopolyploid species, the merger of two divergent
genomes during the formation of Senecio cambrensis has had a dramatic impact at
the level of gene expression. As part of our investigation of the allopolyploid
origins of S. cambrensis, we conducted gene expression analysis using a custom
cDNA microarray platform (Hegarty et al. 2005) to survey the transcript levels of
floral genes in both the intermediate triploid hybrid S. x baxteri and wild S.
cambrensis, relative to their progenitor species. The experimental design of this
comparison is shown in Fig. 13.2. This experiment revealed an initial large change
in floral gene expression in S. x baxteri, with approximately 475 cDNA clones
showing up- or down-regulation relative to its parental taxa or, also importantly,
relative to natural S. cambrensis, from which it differs primarily by a change in
ploidal level (Hegarty et al. 2005). Thus, the greatest changes in gene expression
relative to the parents appeared to be associated with the hybridization step to form
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S. x baxteri. This initial burst of altered gene expression we termed ‘‘transcriptome
shock’’, after the phenomenon of ‘‘genome shock’’ described by McClintock in her
seminal work on transposable elements in plant hybrids (McClintock 1984). The
‘transcriptome shock’ effect in S. x baxteri was confirmed in our later analysis of
resynthesised S. cambrensis, which further showed that the polyploidization event
(here induced by colchicine) had an immediate calming (ameliorating) effect on
altered patterns of gene expression detected in S. x baxteri (Hegarty et al. 2006).
Importantly, this altered pattern of gene expression, apparent in first-generation
allopolyploids, was preserved in four successive generations of the synthetic allopolyploids and in wild S. cambrensis (Hegarty et al. 2006). Previous research in
resynthesised wheat (Feldman and Levy 2005) identified separate effects of
hybridization and polyploidization on the genome and transcriptome, but our
findings in S. cambrensis represented one of the first indications that these changes
in gene expression were genome-wide. Interestingly, the putative functional
classes of genes affected by hybridization and allopolyploidization were remarkably similar, with no functional class of genes being overly affected by hybridization or allopolyploidization (Fig. 13.3). However, perhaps not surprisingly,
when compared with functional classes of genes not affected by either process,
there was a greater representation of genes potentially involved in flower/inflorescence and pollen developments, which may reflect the transitions in floral
phenotypes observed after hybridization and allopolyploidization.
We later reassessed the data (Hegarty et al. 2008) in light of a new approach
used by Wang et al. (2006a) in their studies of allotetraploid Arabidopsis suecica.
In this study, they focused on the identification of genes whose expression in
hybrids differed from the additive expression midpoint of the two different parental
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Fig. 13.2 Experimental design employed in microarray comparisons of gene expression between
the allopolyploid S. cambrensis and its progenitor taxa, S. vulgaris and S. squalidus, and their
sterile triploid F1 hybrid, S. x baxteri. Experimental details can be found in Hegarty et al. (2005),
but, briefly, mature flower bud tissue was harvested from a mixed population of approximately 30
plants and pooled prior to RNA extraction to create an ‘average’ for each taxon. Labelled cDNA
for each taxon was hybridized to a custom floral cDNA microarray. Two taxa were differentially
labelled and compared per array hybridization (with 10 replicate hybridizations performed per
comparison) using dye swaps to account for any bias in labelling efficiency. Each taxon was
compared with the other three, for a total of 30 array hybridizations per taxon. Raw expression
data for each taxon were extracted from these 30 replicates and imported separately into the
GENESPRING microarray analysis software (Silicon Genetics) to enable comparison between all
four taxa. Figure reproduced from Hegarty et al. (2008)
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gene copies. A similar approach had also been used to analyse gene expression
change in maize hybrids (Stupar et al. 2007). Such an approach provides a consistent and unified methodology for identifying genes affected by hybridization
and/or polyploidization in different model study systems. We therefore reanalysed
the microarray data from our original study to identify specific genes and classes of
genes affected by hybridization and polyploidization. Using methods similar to
Stupar et al. (2007), we tested whether changes in gene expression observed in
synthetic S. x baxteri and wild allohexaploid S. cambrensis were additive or nonadditive (Hegarty et al. 2008). By averaging the parental expression values for
each feature on the array showing differential expression in the hybrids, a parental
midpoint expression value (MPV) was obtained. The derived midpoint values were
then used to calculate a ratio of hybrid and parental expression values compared to
the MPV for each array feature. A ratio of -0.33 indicates additive gene
expression, whilst ratios below -1 or above 1 represent expression in the hybrid
outside the range of either parent. Statistical analysis of differentially expressed
genes from our previous microarray experiment (Hegarty et al. 2005) showed that,
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Fig. 13.3 Functional classes of genes affected by allopolyploidization and hybridization. Basic
gene ontologies for cDNA clones displaying a conserved expression changes in both wild and
synthetic Senecio cambrensis relative to S. x baxteri (genes affected by allopolyploidy, 540
clones), b expression changes relative to the parental taxa S. squalidus and S. vulgaris in both
hybrid taxa (genes affected by hybridization, 99 clones) and c genes showing no expression
difference between the parental and hybrid taxa (unaffected by hybridization or polyploidy, 289
clones). With the exception of a higher proportion of floral/pollen-related genes in (a, b)
compared with (c), there are no substantial differences between the classes of affected genes
(adapted with permission from Hegarty et al. 2006)
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for both hybrids, the median ratio was significantly different from -0.33, allowing
us to reject the null hypothesis of largely additive gene expression changes.
Instead, for both hybrids, the majority of the data were skewed towards one of the
parents; in the case of S. x baxteri, expression was skewed towards that of the
lower expressing parent, whereas in S. cambrensis it was skewed towards that of
the higher expressing parent. Further analysis of the data showed that for both
hybrids S. vulgaris was the lower expressing parent in 70 % of cases. Expression
outside the parental range was observed in a substantial proportion of cases in both
hybrids: 7.42 % in S. x baxteri and 3.03 % S. cambrensis (Hegarty et al. 2008).
Having identified a pool of cDNA clones displaying non-additive changes to
gene expression in both hybrid taxa, we then tested these clones for evidence of
expression beyond the parental ranges, i.e. transgressive gene expression. In S. x
baxteri, 80.4 % of non-additively expressed clones differed from the MPV by
[1.5-fold, with 42.2 % of clones in S. cambrensis showing the same effect. Within
both of these groups, the majority of cases involved upregulation compared with
the MPV (66.9 and 70.4 % in S. x baxteri and S. cambrensis, respectively). Aside
from the genes for which no functional class could be ascribed (49.2 %), the major
functional groups affected in S. x baxteri were genes involved in development
(6.6 %), nucleotide binding (6.1 %), mitochondrial activity (4.76 %) and cell wall
function (3.97 %). Within the development category, a high proportion of clones
(32 %) were found to encode tubulins, profilins or senescence-associated proteins.
Of the clones involved in nucleotide binding, 34 % were transcription factors. In S.
cambrensis, the majority of clones (58 %) could not be assigned to a functional
category. Of the remainder, the largest categories were defense (11.1 %) and cell
wall-related genes (6.17 %).
Our reanalysis of the microarray data therefore revealed a relatively high
proportion of non-additive gene expression change in the hybrids relative to their
parental expression levels (Hegarty et al. 2008). In addition, the degree of nonadditive gene expression was lower in allohexaploid S. cambrensis compared with
its triploid intermediate S. x baxteri. This finding was consistent with our previous
observation that the ‘‘transcriptome shock’’ resulting from allopolyploidization is
largely due to hybridization, with polyploidization resulting in a distinct secondary
shift (amelioration) in gene expression (Hegarty et al. 2006). The fairly diverse
nature of the genes affected was consistent with other findings in Arabidopsis
(Wang et al. 2006b), cotton (Adams et al. 2004) and maize (Stupar et al. 2007) that
non-additive changes to gene expression are genome-wide.
Interestingly, similar functional classes of genes were affected by hybridization
in Senecio, Arabidopsis and maize (Fig. 13.4), suggesting that certain gene networks may be particularly susceptible to perturbation by hybridization; the functional categories of nucleotide binding, defense and mitochondria being good
examples. In terms of the classes of genes affected in Senecio, it is also noteworthy
that one of the major affected groups in S. x baxteri was nucleotide binding. In
addition to a number of (primarily down-regulated) transcription factors that have
shown similar alterations in expression pattern in the polyploid Arabidopsis suecica (Wang et al. 2006b), this category also contained clones encoding cytidine
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deaminase (CDA) and 8-oxoG-DNA glycosylase (OGG1). OGG1 has been
implicated in DNA base excision repair (García-Ortiz et al. 2001), while CDA has
been suspected to be involved in RNA editing, although it now appears that
pentatricopeptide repeat proteins that contain CDA-like domains are the more
likely candidates (Salone et al. 2007). These genes were of interest, given that we
also observed a relatively high number of clones encoding proteins involved in
either DNA modification or cell division. In addition to cytidine deaminase and
OGG1 (both of which were upregulated compared with the parental midpoint in S.
x baxteri), there was also up-regulation of adenosylhomocysteinase and adenosyl
kinase, the genes involved in S-adenosylmethionine (SAM) dependent methylation
(Moffatt et al. 2002; Mull et al. 2006). The expression of SAM synthetase was also
increased relative to both parents. SAM-dependent methylation is used in gene
silencing and also in pectin methylation (Pereira et al. 2006), and, indeed, we
observed an increase in the expression of pectin methylesterase as well as another
SAM-dependent enzyme, caffeic acid 3-O-methyltransferase (Hegarty et al. 2008).
It is clear from these analyses that hybridization and polyploidy have separate,
distinct effects on gene expression in Senecio. These changes in gene expression
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Fig. 13.4 Comparison of non-additive expression changes resulting from hybridization in maize
and hybridization/polyploidy in Senecio and Arabidopsis. The formation of the hybrids is shown
in each case, together with the level of non-additive gene expression in the hybrids expressed as a
percentage of the features on the microarray platform used. Finally, the top five functional gene
classes affected (ignoring unknowns) for each hybrid are displayed for comparison. Red indicates
a functional gene class affected in all four hybrid systems, blue indicates a functional class
affected in at least one of the Senecio hybrids and one of the other two hybrid taxa, and green
indicates a functional class affected in both Senecio hybrids but not in either Arabidopsis or
maize. For Arabidopsis suecica, gene function data were taken from Wang et al. (2006b), and for
maize from an extrapolation of the supplementary data given in Stupar et al. (2007). Sb, S. x
baxteri; Sc, S. cambrensis. Reproduced from Hegarty et al. (2008)
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13.2.2 Epigenetic Modification
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While we were unable to demonstrate complete silencing of any clones in our
microarray analyses, it is important to note that the cDNA-based microarrays used
could not distinguish between different parental homeologues. Therefore, in the
cases where a hybrid showed down-regulation of a gene relative to its parental
taxa, it may have been due to either homeologue loss or silencing as a consequence
of DNA methylation. Both phenomena were observed using cleaved amplified
polymorphic sequence (CAPS) analysis in natural and resynthesised allotetraploid
Tragopogon miscellus (Buggs et al. 2009) and suggested that homeologue loss was
not an immediate consequence of hybridization or polyploidy but occurs slightly
later due to recombinational events. Silencing was also not observed to occur
immediately in Tragopogon synthetics, but observations in natural populations
suggest that silencing of one homoeologue is more prevalent than sequence loss.
Studies in other allopolyploid systems such as wheat, Arabidopsis and Spartina
have demonstrated that methylation status in F1 hybrids and first-generation
allopolyploids displays similar changes to those observed for gene expression.
To determine whether methylation in Senecio x baxteri and S. cambrensis was
affected in a similar manner, we undertook a methylation-sensitive AFLP (MSAP)
analysis of the parental taxa, three triploid lines and their S0–S1 allohexaploid
derivatives (Hegarty et al. 2011). The MSAP technique (Xiong et al. 1999)
involves digestion of genomic DNA with a standard, rare-cutter enzyme (i.e.,
EcoRI) and one of a pair of isoschizomeric enzymes that share a restriction site but
are either sensitive or insensitive to cytosine methylation. In this case, we
employed HpaII, which is sensitive to methylation of either cytosine in its recognition site (CCGG) and MspI, which is sensitive only to methylation of the
external cytosine. Comparing the two AFLP profiles produced from each isoschizomer enables identification of the methylation status of a given locus, as
described in Table 13.2. Five selective primer combinations were used to screen
33 plants as detailed in Table 13.3.
We successfully amplified MSAP products from all 33 individuals and obtained
a total of 408 reliable MSAP loci, which were then surveyed to determine levels of
non-additivity in the hybrids. Of the 408 loci, 264 (64.7 %) showed polymorphisms between the parental taxa S. squalidus and S. vulgaris. In the remaining
144 loci that were monomorphic in the parents, 75.7 % were monomorphic across
all hybrid samples tested. Surveying the polymorphic loci, it was found that the
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affect a wide spectrum of transcripts from a number of functional groups and are
non-additive in nature. A good proportion of these changes are also transgressive—outside the range of either parent species—representing a possible source of
heterotic success in interspecific hybrids. To investigate possible mechanisms for
these changes in gene expression, we focused our attention on epigenetic modification in Senecio hybrids.
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Table 13.2 HpaII% MspI banding patterns and locus methylation state (reproduced from Hegarty et al, 2011)
HpaII
MspI
Methylation status
+
CCGG (unmethylated)
+
CmCGG (methylation of internal cytosine)
m
CCGG or mCmCGG (methylation of external cytosine)
hm
+
CCGG (hemimethylation of external cytosine)
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determine whether the internal cytosine is also methylated
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triploid hybrids each displayed similar overall methylation patterns (Table 13.4),
with a strong bias in favour of the S. vulgaris (maternal) methylation state for each
locus (an average of 57.4 % of loci between the three triploid lines). In addition,
the triploids also displayed approximately equivalent levels of non-additive
methylation (13.4 % on average). To determine whether methylation patterns were
maintained following genome duplication, the methylation status of the S0 allohexaploids was compared to the triploid lines from which they were derived. In the
vast majority of cases, the synthetic allohexaploids retained the methylation state
observed in the triploid. On average, 78.2 % of these cases involved additive
methylation, whereas 9.7 % of loci involved retention of a non-additive methylation profile. An average of 10.1 % of loci displayed a shift relative to the triploid
in the specific parental methylation state favoured (4.5 % shift to S. squalidus,
3.8 % shift to S. vulgaris, 1.8 % shift to both). Finally, an average of 2 % of loci
showed novel non-additive methylation not observed in the triploids.
The analysis was then extended to the S1 allohexaploids, comparing them to
both their triploid ancestors and the preceding S0 generation. As with the S0
allohexaploids, overall methylation state was highly similar among the three lines.
Again, the most common result was retention of methylation status compared to
both the triploid and the S0 allohexaploid, with an average of 70.3 % of loci
showing retention of additive methylation patterns and 7.3 % of non-additive
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Table 13.3 Oligonucleotides for methylation-sensitive AFLP and number of loci (reproduced
from Hegarty et al, 2011)
Oligo
Sequence (5’-3’
Primer Combination
# Loci
EcoRI adaptor 1
CTCGTAGACTGCGTACC
Eco+AGC/Hpa+CTG
118
EcoRI adaptor 2
AATTGGTACGCAGTC
Eco+AAC/Hpa+CTT
88
HpaII adaptor 1
GATCATGAGTCCTGCT
Eco+AGC/Hpa+CTT
80
HpaII adaptor 2
CGAGCAGGACTCATGA
Eco+AAC/Hpa+AAG
Failed
Eco+A
GACTGCGTACCAATTCA
Eco+ACG/Hpa+AAG
69
Hpa
ATCATGAGTCCTGCTCGG
Eco+AAC/Hpa+CTT
53
Eco+AAC (NED)
GACTGCGTACCAATTCAAC
Eco+ACG (FAM)
GACTGCGTACCAATTCACG
Hpa+CTG
ATCATGAGTCCTGCTCGGCTG
Hpa+CTT
ATCATGAGTCCTGCTCGGCTT
Hpa+AAG
ATCATGAGTCCTGCTCGGAAG
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Table 13.4 Summarised methylation status of hybrid lines (reproduced from Hegarty et al.
2011)
Methylation
Percentage of Loci (triploids)
State
Line 1
Line 2
Line 3
Additive (SS)
24.58%
24.58%
22.26%
Additive (SV)
56.15%
57.14%
58.80%
Additive (monomorphic)
4.65%
6.98%
4.65%
Nonadditive
14.62%
11.30%
14.29%
Percentage of Loci (S0 allohexaploids)
Same as triploid (additive)
76.17%
78.86%
79.53%
Same as triploid (nonadditive)
9.73%
9.73%
9.73%
Differs from triploid (additive SS)
5.03%
4.36%
4.03%
Differs from triploid (additive SV)
5.37%
3.69%
2.35%
Differs from triploid (monomorphic)
2.01%
0.67%
2.68%
Novel nonadditive methylation
1.68%
2.68%
1.68%
Percentage of Loci (S1 allohexaploids)
Same as triploid+S0 (additive)
72.06%
72.38%
66.35%
Same as triploid+S0 (nonadditive)
7.62%
7.94%
6.35%
7.62%
5.71%
3.49%
Same as S0 not triploid (additive)
Same as S0 not triploid (nonadditive)
0.32%
1.27%
0.63%
Same as triploid not S0 (additive)
3.81%
3.17%
1.90%
0.95%
0.00%
2.54%
Same as triploid not S0 (nonadditive)
Differs from triploid+S0 (additive SS)
2.54%
4.76%
7.62%
Differs from triploid+S0 (additive SV)
3.49%
2.22%
7.62%
Novel nonadditive methylation
1.59%
2.54%
3.49%
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methylation. A further 5.6 % of loci showed retention of additive methylation in
the two allohexaploid generations, whereas the triploid had displayed non-additivity. Methylation status was not always consistent between the two allohexaploid
generations; however, in 4.1 % of cases, loci displayed a shift in methylation state
to that seen in the triploid but not in the S0 allohexaploid. Furthermore, the S1
generation showed a return to additivity in 9.4 % of cases, where both the triploid
and the S0 allohexaploids had been non-additive. As with the S0 generation,
though, there was a small (2.5 % average) degree of novel non-additivity.
The results were in accordance with our previous studies of gene expression
(Hegarty et al. 2006, 2008): we found that, while cytosine methylation in both
hybrid taxa was largely additive between the two parental patterns, a significant
degree of non-additivity also exists. Overall methylation status was well conserved
between different hybrid lines; while individual loci displayed differences, the
global percentages of different methylation states were highly similar between
lines (Hegarty et al. 2011). In all three triploid lines, approximately 13.4 % of loci
showed non-additive methylation, although the precise type of methylation was
not identical between lines in all cases. Levels of non-additive methylation
observed in other allopolyploid systems are variable: 8.3 % in Arabidopsis
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Note: SS = S. squalidus, SV = S. vulgaris. ‘‘Monomorphic’’ refers to loci with a common
methlyation state in the parents, rather than across hybrid lines
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(Madlung et al. 2002), 9 % in Brassica (Gaeta et al. 2007), 13–20 % in wheat
(Dong et al. 2005; Pumphrey et al. 2009) and as high as 30 % in Spartina (Salmon
et al. 2005). It has been speculated that the higher genome copy number in wheat
and Spartina might explain their greater levels of methylation, although Doyle
et al. (2008) point out that both species are monocots, which tend to possess a
higher GC content (and thus greater potential for methylation) than eudicots. The
fact that conserved methylation changes between the Senecio hybrids are more on
a par with the levels seen in Arabidopsis and Brassica suggests that this latter
hypothesis may be correct, as S. cambrensis exhibits the same ploidy as wheat. We
should note, however, that the wheat genome is significantly larger than that of
Senecio (2C genome sizes of 33.96 Gbp wheat; 5.05 Gbp S. cambrensis) and is
known to contain a significant amount of repetitive DNA including large numbers
of retroelements. It is therefore probable that alterations to methylation are more
necessary to prevent widespread activation of these genetic regions in wheat and
similar polyploid monocots. Indeed, studies of methylation in Spartina (Parisod
et al. 2010) showed that such changes frequently occur in the vicinity of transposable elements and, perhaps as a result, no transposition burst was detected in
the Spartina hybrids analysed. Methylation change therefore appears to play a
frequent role in genome mergers, but there are exceptions: despite significant
differential gene expression in the allotetraploid Gossypium hirsutum, almost no
differences in methylation could be observed between the hybrid and its parental
taxa (Liu et al. 2001) nor do the parental genomes undergo any significant rearrangement. In this situation, it appears that subfunctionalization of the two genomes is the primary cause of phenotypic variation (Adams et al. 2004; Liu and
Adams 2007), although a recent study by Chaudhary et al. (2009) demonstrated
that neofunctionalization and divergence in parental cis-regulatory sequences also
play a significant role. Exactly what factors determine the response of the parental
genomes to hybridization are largely unknown, although the degree of parental
divergence is speculated to play a large role (Chapman and Burke 2007; Buggs
et al. 2008; Paun et al. 2009).
In further accordance with our expression analyses, we observed that nonadditive methylation in S. x baxteri triploids was maintained, on average, in only
73.6 and 55.6 % of cases in the S0 and S1 allohexaploids, respectively (Hegarty
et al. 2011). In approximately 73 % of cases observed in our microarray studies,
the resynthesised allohexaploid lines (S0–S4) displayed either an immediate or
gradual shift towards an expression pattern similar to that of wild S. cambrensis
(Hegarty et al. 2006). It seems likely, therefore, that our previous observation that
non-additivity results from hybridization but can be partially reduced by genome
duplication, also holds true when applied to DNA methylation. This again matches
observations from MSAP analysis of Spartina allopolyploids (Salmon et al. 2005),
which showed that the allopolyploid S. anglica retained 71.4 % of the non-additive
methylation patterns observed in the F1 hybrid. These findings were again confirmed when assessing methylation changes associated with transposable elements
(Parisod et al. 2010), with the additional observation that many of the changes seen
in the F1 hybrid involved loss of parental markers (usually in the maternal
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genome), indicating that such changes involved structural rearrangements to the
parental genomes. A similar process may be at play in S. x baxteri, because MSAP
markers also detect structural changes: indeed, wild populations of S. cambrensis
show evidence of intergenomic recombination (Abbott et al. 2007, and above),
again favouring the S. vulgaris genome as with our triploid lines here. By contrast,
most of the differences observed in allopolyploid Spartina involved alterations to
methylation status, rather than structural changes. Similar findings have also been
identified in Brassica, where most of the methylation changes identified in the S0
allotetraploid were maintained in S5 lines, but with a number of revertants and
novel changes present as well (Gaeta et al. 2007).
A key finding from our analysis was that the global patterns of DNA methylation change observed in our experiments strongly mirror the global changes in
gene expression observed in our earlier microarray analyses, indicating a possible
underlying causation. Whilst further investigation of specific loci showing methylation differences is required to make a definitive case, the similarities between
changes in gene expression and DNA methylation are nevertheless striking. For
example, we noted that a number of loci displayed novel non-additivity in both the
S0 and S1 allohexaploids (2.01 % on average in the S0 lines, 2.54 % in the S1),
again a point of consistency between the methylation study and our earlier
microarray expression analysis (Hegarty et al. 2008). The overall level of nonadditive methylation may therefore not actually decrease as a consequence of
genome duplication, but instead the level of conserved methylation may be lessened. A proportion of loci also displayed unstable methylation patterns across
generations in the hexaploids, with an average of 16.08 % of loci showing differences between the S0 and S1 lines (including the aforementioned novel nonadditive methylation). Of these, the majority consisted of cases where the S1
allohexaploids revert to additivity or favoured a different parental methylation
state to the S0 line. This reversion to an additive profile was also observed between
the triploids and the S0 plants and agreed with observations from the microarray
data that wild S. cambrensis often showed an opposing expression pattern to S. x
baxteri.
However, approximately one-quarter of loci also displayed a shift relative to the
S0 allohexaploids to favour the methylation state seen in the triploid. These
findings suggest that the methylation state of some loci may vary as a consequence
of segregation. This may explain the novel changes observed by Gaeta et al. (2007)
in their S5 allopolyploids of Brassica napus. Similarly, an analysis of natural
populations of the allopolyploid Tragopogon miscellus (Buggs et al. 2009), where
hybridization occurred at least 40 generations ago, identified a random loss of one
parental homoeologue at a rate of 3.2 % across 10 loci in 57 natural hybrids from
five populations. In addition, a further 6.8 % of loci showed evidence of gene
silencing in one parental copy. The loci lost/silenced were not consistent across
populations or individuals, although within populations, there was some conservation in the loci affected. Whilst Buggs et al. (2009) did not note any homoeologue loss/silencing in resynthesised S0 hybrids, the variability in silencing after
such an extended period of time since hybrid formation suggests that
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13.2.3 Phenotypic Change
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Senecio x baxteri F1 hybrids generated by crossing S. vulgaris, as female, with S.
squalidus were all self-sterile in contrast to previous studies which reported some
self-fertility (Crisp 1972; Ingram 1978). Treating shoots of S. x baxteri plants with
colchicine produced ‘chimeric’ plants with allohexaploid branches that produced
flower heads that were fully self-fertile. Seed from these flower heads was then
used to found the synthetic S. cambrensis lines (S0–S5) used in the transcriptomic
and epigenetic analyses described above. The first wholly allohexaploid plants
generated from this seed (S0 lines), and their progeny (S1 lines), showed similar
ray flower structure and self-compatibility (Hiscock and Hegarty, unpublished).
However, in subsequent lines, from the S2 onwards, variation in ray flower form
was detected between individuals within and between the nine independent lineages of synthetic S. cambrensis. Some individuals were observed with no ray
flowers, some had short partially tubular ray flowers, while in others ray flowers
were observed of different lengths and number (Fig. 13.5.) Observations on the
progeny of these different individuals showed that these various forms of ray
flower are heritable (Hiscock, unpublished). Comparable variation in S. cambrensis ray flower form was previously attributed to intergenomic recombination
(Ingram and Noltie 1984), but here we suggest another possibility for such abrupt
changes to ray flower phenotype—epigenetic effects associated with the
epigenetic instability observed in early-generation synthetic S. cambrensis
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independently formed hybrids can still display significant levels of epigenetic
variation. This study, as well as that of Gaeta et al. (2007), was based on a survey
of a limited number of loci using cDNA-AFLP or CAPS assay. A later study by
Buggs et al. (2011) used the Sequenom MassARRAY allelotyping technology to
survey a much larger set of loci in Tragopogon miscellus and confirmed that
natural hybrids displayed altered patterns of tissue-specific gene expression, whilst
resynthesised hybrids demonstrated relaxed control of tissue-specificity. This latter
finding suggests a possible mechanism for the ‘transcriptome shock’ effect we
observed in Senecio x baxteri and S. cambrensis, resulting from a loss of tissuespecific expression patterns seen in the parent taxa. Further work will be needed to
confirm if this is the case in Senecio. The Sequenom assay used by Buggs et al.
(2011) shows the benefits of new molecular tools for studies of allopolyploid
systems: with the advent of new technologies for global analysis of DNA methylation such as MSAP and next-generation sequencing (Salmon and Ainouche
2010), it would also be interesting to analyse our later-generation allohexaploid
derivatives at a global scale to investigate the longer term changes in methylation
as hybrid genomes undergo recombination and adaptation. Such studies may
therefore provide further insights into which epigenetic changes are mandated by
hybridity, and which may vary between populations and serve as a source of
novelty upon which selection may act.
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(Hegarty et al. 2011). Genes shown to be involved in ray flower development in
Senecio (RAY1 and RAY2) are orthologues of CYCLOIDEA (CYC) (Kim et al.
2008), which has been shown to occur as a stable mutant (hypermethylated) epiallele that in Linaria (toadflax, Plantaginaceae) manifests itself in a change in
flower symmetry from bilateral to radial symmetry (Cubas et al. 1999). We
therefore hypothesise that the observed changes in ray flower form may in part be
associated with epigenetic modifications to RAY1 and/or RAY2. Testing this
hypothesis will be a focus for future work.
Another unexpected observation in the synthetic S. cambrensis lines was the
appearance of self-sterile individuals in otherwise SC lines, again from the S2
generation onwards. Subsequent analyses using controlled self- and crosspollinations confirmed that these self-sterile individuals possessed functional
sporophytic self-incompatibility (Brennan and Hiscock 2010). This is the first time
that sporophytic SI has been shown to be inherited and expressed in an allopolyploid and raises intriguing questions about the mechanism regulating this
important trait. All S0 and S1 lines of synthetic S. cambrensis were highly selffertile (SC) indicating that the SI system, present in parental S. squalidus, was
repressed in these allopolyploids, only to be reactivated/derepressed later. The
emergence of SI individuals may be a consequence of recombination or might also
be associated with epigenetic changes observed in the early-generation synthetic
allopolyploids. Most observations of wild S. cambrensis have reported it to be SC
(Abbott and Lowe 2004), but the finding of SI in synthetic S. cambrensis prompted
a re-examination of the mating system of wild S. cambrensis. An analysis of
selfing rates in 41 wild S. cambrensis individuals from Edinburgh and North Wales
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Fig. 13.5 Variation in ray flower morphology observed in individuals of synthetic lines of
allohexaploid S. cambrensis from the S2 to S5 generation
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identified five SI individuals (Brennan and Hiscock 2010) implying, albeit from a
relatively small sample size, that SI may be present in wild S. cambrensis at a
frequency of *12 %. This important finding means that S. cambrensis should now
be considered as possessing a mixed mating system that has the potential to evolve
towards either outcrossing or selfing.
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13.3 Future Prospects
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We are currently engaged in the generation of a draft reference sequence of the Senecio
squalidus ‘‘gene-space’’ (low-copy, non-methylated regions of the genome). Nextgeneration sequencing (NGS) platforms enable a variety of potential experiments to
examine the consequences of polyploidy and hybridization in Senecio further. For
example, we intend to identify promoter regions using chromatin-immunoprecipitation sequencing (ChIPseq) to enrich for DNA fragments bound by enzymes involved in
transcription. We can then determine whether hybrids and polyploids display alterations in promoter sequence/binding that may explain the altered patterns of expression
observed in our microarray experiments. Further, once a reference sequence is available, we can consider bisulphite sequencing to identify genomic regions which show
differential methylation in hybrids relative to their progenitors. One key target for
bisulphite sequencing will be the RAY1 and RAY2 genes (Kim et al. 2009) which we
suspect may show differential methylation associated with the observed variation in
ray flower morphology that appears in synthetic S. cambrensis lines. Identification of
small interfering RNAs (siRNAs) and their targets will enable analysis of changes to
epigenetic regulation of gene expression in hybrids. At the structural level, resequencing of hybrid ‘‘gene-space’’ and comparative sequence analysis may allow us to
detect genomic rearrangements and sequence loss, plus the activity of transposable
elements. The increasing capability of genotyping-by-sequencing (GBS) approaches
such as restriction-associated DNA (RAD) sequencing (Baird et al. 2008) may also
prove useful in detecting structural rearrangements in hybrid genomes. Finally,
comparative sequencing of Senecio cambrensis and the two other hybrid derivatives of
S. vulgaris and S. squalidus, i.e. S. vulgaris var. hibernicus and S. eboracensis, may
enable identification of dosage effects, since the hybrids share parental genomes but
differ in the specific combinations thereof.
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13.3.2 S. cambrensis in the Wild
Senecio cambrensis now exists in the wild only in North Wales, UK, following
extinction of the Edinburgh population in 1993 (Abbott and Forbes 2002).
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Although the species was recorded at numerous sites and in relatively high
numbers in North Wales in the early 1980s (Ingram and Noltie 1995; Fig. 13.6), it
has undergone a significant decline in this region since then. In the most recent
census of the species undertaken in 2004, it was found at only nine sites in North
Wales with a total of 349 individuals recorded across all sites (Abbott et al. 2007;
Fig. 13.6). It has been speculated that this decline in numbers has been caused by a
reduction of available sites for colonisation by the species (e.g., waste ground) and
also to increased use of herbicide to control weed populations (Abbott et al. 2007).
Its disappearance from the thin layer of soil that collects along roads between the
road edge and verge, where it was found often in the past, is most likely due to
increased use of herbicides on plants growing along road sides. However, it has
also been noted that plants of the species are frequently heavily infected with the
rust Puccinia lagenophorae. Although this rust also infects both parent species, it
is possible that its effects on S. cambrensis are more dramatic in terms of production of offspring for colonising new sites, given that numbers of S. squalidus
and S. vulgaris that reproduce each year in North Wales and elsewhere in the UK
are vast relative to those of S. cambrensis. Although no detailed analysis has been
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Fig. 13.6 Decline in the number of a sites and b flowering individuals across sites, recorded for
S. cambrensis in North Wales in different years from 1982 to 2004. Records are from Abbott et al.
(2007) and were made in June each year except in 1987 when they were collected in May
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Acknowledgments Our research on Senecio cambrensis and its parents has been supported by
grants from the NERC, BBSRC and Leverhulme Trust to whom we are grateful. We would also
like to acknowledge our long-term collaboration with Keith Edwards and the considerable efforts
of former research students, technicians and postdocs who contributed to the research over the
past 25 years. These include Alexandra Allen, Paul Ashton, Garry Barker, Tom Batstone, Adrian
Brennan, Mark Chapman, David Forbes, Amanda Gillies, Helen Ireland, Juliet James, Joanna
Jones, Andrew Lowe, David Tabah, and Ian Wilson.
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undertaken on whether S. cambrensis is ecologically divergent from its two parents, it tends to grow in sympatry with one or both parent species in the wild. Thus,
there is likely competition between the three species for occupation of available
open sites, and this might place S. cambrensis at a disadvantage if the seed it
produces is less numerous relative to that of S. vulgaris and/or S. squalidus
occurring in the same area. Whatever the cause of its marked decline in numbers in
North Wales over the last 25 years or so, it is clear that S. cambrensis has reached
the stage where its presence in the wild is endangered and that a conservation plan
is required to prevent it from possibly becoming extinct in the near future.
Given the decline in numbers of S. cambrensis in recent years it is important to
gain a better understanding of the nature of its mating system and mating
dynamics, especially in the light of our findings of SI in wild populations (Brennan
and Hiscock 2010). If there has been a recent shift in mating system from predominantly SC towards SI, it is possible that mating potential has become compromised due to obligate outcrossing. Given the possibility of a single hybrid
origin for S. cambrensis in Wales, it is likely that wild populations possess very
few S alleles. Whilst a small number of shared S alleles are not necessarily a
problem when populations are large, it becomes a problem when populations are in
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uncontrollable spiral of extinction. A reappraisal of the mating system of S.
cambrensis is thus urgently needed.
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Polyploidy and Genome Evolution
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The Early Stages of Polyploidy: Rapid and Repeated Evolution in Tragopogon
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Abstract
Elucidating the causes and consequences of polyploidy (whole-genome duplication; WGD) is arguably central
to understanding the evolution of most eukaryotic lineages. However, much of what we know about these
processes is derived from the study of crops and synthetic polyploids. Tragopogon provides the unique
opportunity to investigate the genetic and genomic changes that occur across an evolutionary series from
F1 hybrids, synthetic allopolyploids, independently formed natural populations of T. mirus and T.
miscellus that are 60–80 years post-formation, to older Eurasian polyploids that are dated by molecular clocks
at several million years old, and finally to a putative ancient polyploidization thought to have occurred prior
to or early in the history of the Asteraceae (40–43 mya). Tragopogon joins other well-studied natural polyploid
systems (e.g., Glycine, Nicotiana, Gossypium, Spartina, Senecio), but presents a range of research possibilities
that is not available in any other system. We have shown in T. mirus and T. miscellus that upon
allopolyploidization, massive gene loss occurs in patterns that are repeated across populations of independent
origin and with a bias against genes derived from T. dubius, the diploid parent shared by both new
allotetraploids. We have also shown significant changes in gene expression (transcriptomic shock) in the early
generations of allopolyploidy in these species. Massive and repeated patterns of chromosomal variation
(intergenomic translocations and aneuploidy) have been revealed by fluorescence in situ hybridization.
Aneuploidy results in substitutions between homeologous chromosomes, through reciprocal monosomytrisomy (1:3 copies) or nullisomy-tetrasomy (0:4 copies). We propose that substantial chromosomal instability
results in karyotype restructuring, a likely common process following WGD and a driver of allopolyploid
speciation, which has largely unexplored implications for gene losses, gains, and expression patterns. But
gene loss and expression changes as well as karyotypic changes are ongoing in T. mirus and T. miscellus, in
that no population is fixed for any of these events; thus, we have literally caught evolution in the act.
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Douglas E. Soltis, Richard J. A. Buggs, W. Brad Barbazuk, Srikar
Chamala, Michael Chester, Joseph P. Gallagher, Patrick S. Schnable
and Pamela S. Soltis
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Abstract Elucidating the causes and consequences of polyploidy (whole-genome
duplication; WGD) is arguably central to understanding the evolution of most
eukaryotic lineages. However, much of what we know about these processes is derived
from the study of crops and synthetic polyploids. Tragopogon provides the unique
opportunity to investigate the genetic and genomic changes that occur across an
evolutionary series from F1 hybrids, synthetic allopolyploids, independently formed
natural populations of T. mirus and T. miscellus that are 60–80 years post-formation, to
older Eurasian polyploids that are dated by molecular clocks at several million years
old, and finally to a putative ancient polyploidization thought to have occurred prior to
or early in the history of the Asteraceae (40–43 mya). Tragopogon joins other wellstudied natural polyploid systems (e.g., Glycine, Nicotiana, Gossypium, Spartina,
Senecio), but presents a range of research possibilities that is not available in any other
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and Repeated Evolution in Tragopogon
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D. E. Soltis (&) R. J. A. Buggs W. B. Barbazuk S. Chamala
M. Chester J. P. Gallagher
Department of Biology, University of Florida, Gainesville,
FL 32611, USA
e-mail: dsoltis@botany.ufl.edu
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Present Address:
J. P. Gallagher
Department of Ecology, Evolution, and Organismal Biology,
Iowa State University, Ames, IA 50011, USA
R. J. A. Buggs
School of Biological and Chemical Sciences, Queen Mary University of London,
London, E1 4NS, UK
P. S. Schnable
Center for Plant Genomics, Iowa State University, Ames, IA 50011, USA
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Florida Museum of Natural History, University of Florida, Gainesville,
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P. S. Soltis and D. E. Soltis (eds.), Polyploidy and Genome Evolution,
DOI: 10.1007/978-3-642-31442-1_14, Springer-Verlag Berlin Heidelberg 2012
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system. We have shown in T. mirus and T. miscellus that upon allopolyploidization,
massive gene loss occurs in patterns that are repeated across populations of independent origin and with a bias against genes derived from T. dubius, the diploid parent
shared by both new allotetraploids. We have also shown significant changes in gene
expression (transcriptomic shock) in the early generations of allopolyploidy in these
species. Massive and repeated patterns of chromosomal variation (intergenomic
translocations and aneuploidy) have been revealed by fluorescence in situ hybridization. Aneuploidy results in substitutions between homeologous chromosomes, through
reciprocal monosomy-trisomy (1:3 copies) or nullisomy-tetrasomy (0:4 copies).
We propose that substantial chromosomal instability results in karyotype restructuring,
a likely common process following WGD and a driver of allopolyploid speciation,
which has largely unexplored implications for gene losses, gains, and expression
patterns. But gene loss and expression changes as well as karyotypic changes are
ongoing in T. mirus and T. miscellus, in that no population is fixed for any of these
events; thus, we have literally caught evolution in the act.
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14.1 Introduction
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14.1.1 General Introduction
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Polyploidy, or whole-genome duplication (WGD), is currently recognized as a major
evolutionary force in eukaryotes (e.g., Mable 2003; Gregory and Mable 2005; Mable
et al. 2011). Polyploidy generally results in instant speciation, increasing biodiversity
and providing new genetic material for evolution (e.g., Levin 1983, 2002). Some
evidence suggests that two polyploid events occurred in ancestors of vertebrates (Ohno
1970; Panapoulou and Poustka 2005; see Chap. 16 of this volume), with subsequent
polyploidy in amphibians and fish (Mable et al. 2011; see Chaps. 18 and 17, respectively, this volume). The genomes of yeast and other Saccharomyces also appear to be
anciently duplicated (Wolfe and Shields 1997; Kellis et al. 2004; Dujon et al. 2004; see
Chap. 15 of this volume). Researchers have long recognized that polyploidy is an
inseparable part of angiosperm biology; the polyploidy process has, in fact, been
studied in plants for a little over a century. Early reviews of polyploidy in plants include
now classic papers by Müntzing (1936), Darlington (1937), Clausen et al. (1945), Löve
and Löve (1949), and Stebbins (1950, 1971). However, these seminal works did not
anticipate the huge role for polyploidy in evolution that genomic studies now suggest.
During the past decade there has been a tremendous resurgence of interest in
polyploidy, stimulated in large part by the development of increasingly powerful
genetic and genomic tools. The result has been numerous new insights into the
genomic and genetic consequences of polyploidy (other chapters of this volume).
Recent discoveries have dramatically reshaped traditional views and concomitantly revealed that polyploidy is a highly dynamic and ubiquitous process. For
example, studies of many duplicated genes across genomes suggest that all
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Tragopogon now provides a well-known, textbook example of recent allopolyploid speciation; two new allotetraploid species originated within the last 80 years
(Soltis et al. 2004, 2009b) (Fig. 14.1). Tragopogon mirus and T. miscellus
(Fig. 14.1) formed repeatedly following the introduction of three diploids from
Europe into the Palouse region of North America in the early 1900s (Ownbey
1950; Soltis et al. 2004); the tetraploids have not formed in Europe. The parentage
of T. mirus (T. dubius and T. porrifolius) and T. miscellus (T. dubius and
T. pratensis) is well documented (Figs. 14.1 and 14.2) and confirmed with multiple
markers and approaches (Soltis et al. 1995, 2004, 2009b).
Ownbey (1950) described the few populations of the newly formed allotetraploids (each consisting of fewer than 100 individuals) as ‘‘small and precarious’’, but noted that they had ‘‘attained a degree of success’’ and they appeared to
be ‘‘competing successfully’’ with their diploid parents. He also stated that it
would be ‘‘important to follow the ecological development of the newly formed
polyploids’’ through time. Significantly, both tetraploids have been highly successful since their formation. Novak et al. (1991) conducted a survey to determine
the distributions of the two polyploids 40 years after Ownbey’s discovery. One or
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angiosperms have undergone at least one round of genome doubling. Significantly,
genomic and phylogenetic analyses also associate polyploidy with major diversifications (e.g., within Poales, Solanaceae, Fabaceae; Soltis et al. 2009a), and the
origin of angiosperms and seed plants (Jiao et al. 2011).
As a result of a diverse array of studies, we have learned a great deal about the
interactions that occur among the diploid genomes forced together via allopolyploidy. Because of its apparent prevalence, elucidating the causes and consequences of polyploidy is arguably central to understanding the origin and
diversification of most major lineages of eukaryotes. Significantly, however, most
of what we know about the genetic and genomic consequences of polyploidy is
derived from the study of synthetic polyploids, crops (e.g., cotton, wheat, Brassica,
Nicotiana), and genetic models (Arabidopsis) (see refs above, Chen et al. 2004;
Soltis and Soltis 2009). To understand better how polyploidization impacts genome evolution and gene function in natural populations, we must extend from a
few crops, genetic models, and synthetics to naturally occurring polyploids.
Three systems are known that permit insights into the early stages of polyploidy
in nature: Spartina anglica (Ainouche et al. 2004, 2009; Salmon et al. 2005; see
Chap. 12 of this volume), Senecio cambrensis (Ashton and Abbott 1992; Abbott
and Lowe 2004; Hegarty et al. 2005, 2006; see Chap. 13, this volume), and
Tragopogon (T. mirus, T. miscellus, Soltis et al. , 2009b). Research has progressed
on all, and they are complementary. In addition, a2004s stressed here, Tragopogon
also affords unique opportunities to investigate polyploidy over a continuum of
ages, as well as the consequences of recent and frequently repeated polyploidy.
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14.1.3 Tragopogon as a Unique Evolutionary Model
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For many reasons Tragopogon is a novel system that affords the opportunity to
examine the early stages of polyploidization in nature. The natural populations are
approximately 80 years (40 generations in these biennials) old—this time frame
and the fact that they have experienced natural selection provide a window into
polyploidization that cannot be matched via the study of synthetic polyploids (e.g.,
crops and genetic models). Furthermore, molecular studies suggest that the diploid
parents diverged *2.5–5 MYA and that there may be as many as 21 lineages of
separate origin of T. miscellus and 11 of T. mirus just in the Palouse (Soltis et al.
1995, 2004, 2009b; Symonds et al. 2010); the polyploids have also formed in
Arizona, Oregon, Wyoming, and Montana (Soltis et al. 2012; Ownbey unpublished
data). Recent studies employing microsatellite markers (Symonds et al. 2010)
reveal multiple origins on a small geographic scale (see Sect. 14.1.4). Given that
multiple polyploidizations are common in plants (Soltis and Soltis 1993, 1999,
2000, 2009), Tragopogon represents in microcosm what occurs in other polyploids
over much larger geographic areas and longer time frames. These repeated origins
in a small geographic area and a narrow time frame also provide the unique
opportunity to ask if evolution repeats itself across these many lineages.
Adding to the utility of the Tragopogon system as an evolutionary model is the recent
production of multiple synthetic lines of both T. mirus and T. miscellus (Tate et al.
2009a), providing the added opportunity of examining both species from polyploidization onward. Morphologically, the synthetics resemble the natural polyploids with
short- and long-liguled forms of T. miscellus resulting when T. pratensis and T. porrifolius are reciprocally crossed (Tate et al. 2009a). In nature, all formations of T. mirus
have T. porrifolius as the maternal parent and T. dubius as the paternal parent, but we
have synthesized T. mirus reciprocally. We also produced allotetraploids between
T. porrifolius and T. pratensis, which are not known from nature (Fig. 14.1). All of these
synthetic lines are now in the fourth generation and offer the unique opportunity for
comparative study of repeated formations of both natural and synthetic polyploids.
Further adding to the allure of Tragopogon are Old World polyploids that are
much older than the recently formed New World polyploids. Tragopogon comprises *150 species, 12 of which are Eurasian polyploids (Mavrodiev et al.
2008a). Of several Eurasian polyploids for which we clarified parentage
(Mavrodiev et al. 2008a, b, c), T. castellanus (2n = 24) from Spain (Blanca and
Díaz de la Guardia 1996) has emerged as a promising new model. Estimating the
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both polyploids were found in most towns of the Palouse with populations ranging
from small (fewer than 100 individuals) to many thousands of individuals.
Tragopogon miscellus is now one of the most common weeds in and around
Spokane, WA, as well as in Moscow, ID, and Spangle, WA. Populations of
T. mirus and T. miscellus often form dense stands and are, in fact, displacing their
parents, particularly T. pratensis and T. porrifolius.
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date of origin of a polyploid is difficult, but given well-known caveats, we used
DNA sequence data to estimate the age of T. castellanus as 0.8–2.8 million years
(Mavrodiev et al. 2008a, b, c). This agrees with other evidence in that T. castellanus occurs in well-known Pleistocene glacial refugia that harbor other paleoendemics (e.g., Petit et al. 2003). Thus, in Tragopogon we have the opportunity to
extend our analyses to older Eurasian polyploids, providing a continuum of ages
from F1 hybrids and raw synthetics, to 80-year-old natural polyploids, to natural
polyploids that are perhaps several million years old.
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Fig. 14.1 Summary of parentage of tetraploid Tragopogon species comparing natural with
synthetic allopolyploids. The diploid parents (with 2n = 12) are at the corners of the triangle;
polyploids (2n = 24) are in between the corners. Synthetic polyploids are on the outside of the
triangle (connected by white lines); those polyploids forming naturally are to the inside of the
triangle. Polyploids are placed closer to the maternal parent. In nature, T. miscellus has formed
reciprocally, and T. mirus has formed only with T. porrifolius as the maternal parent. However,
we have made reciprocal synthetic lines of both and have also made reciprocal polyploids of
T. pratensis 9 T. porrifolius; this polyploid has not formed in nature. Note that populations of
T. miscellus of reciprocal origin differ in morphology. Those with T. pratensis as the maternal
parent have short ligules, and those with T. dubius as the maternal parent have long ligules
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14.2 Does Evolution Repeat Itself?
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The Tragopogon system is comparable in some ways to an island biogeography
scenario. The diploids and the derivative polyploids only occur in small towns that
are scattered across the Palouse area of eastern Washington and adjacent Idaho and
not in the intervening areas, which are large tracts of agricultural land. This begged
the question—how did the polyploids spread so quickly to many towns? Certainly,
seed dispersal is one possibility given the wind-dispersed nature of the achenes.
Molecular data indicate instead that repeated formation has played the major role
in range expansion. This was suspected by Ownbey and McCollum (1953), who
surmised that T. miscellus had formed reciprocally with the long-liguled form
(found only in Pullman, WA) having T. dubius as the maternal parent and all other
populations having T. pratensis as the maternal parent; this reciprocal parentage
results in distinctive morphologies (Fig. 14.1) and was later confirmed by
molecular methods (Soltis and Soltis 1989).
A suite of molecular markers including allozymes, AFLPs, sequence data, and
microsatellites has now revealed that most populations of the allotetraploids are of
distinct origin (Soltis et al. 2004; Symonds et al. 2010). Furthermore, microsatellites
have documented multiple origins on a fine geographic scale, revealing in several
cases that distinct populations in the same town separated by only 1–2 km are of
separate origin (Symonds et al. 2010). Interestingly, microsatellite data reveal that of
the many genotypes of T. dubius currently in nature, only three general types appear
to have contributed to the repeated formations of both polyploids, and there are no
exact matches to present-day T. dubius genotypes. Hence, the genotypes detected in
the two polyploid species appear to represent a snapshot of the historical population
structure in the diploid progenitors, rather than modern diploid genotypes.
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Evolutionary biologists have long wondered if evolution would repeat itself, given
the chance. Gould (1994) suggested that, on a broad evolutionary scale, if we
could replay the evolutionary tape of life on Earth, it would play differently—
‘‘history involves too much chaos,’’ and too many chance events are involved for
the evolutionary process to be repetitive. He stated that ‘‘chains of historical events
are so intricate, so imbued with random and chaotic elements, so unrepeatable in
encompassing such a multitude of unique objects, that standard models of simple
prediction and replication do not apply.’’ In contrast, other researchers have argued
that ‘‘within certain limits the outcome of evolutionary processes might be rather
predictable’’ (Morris 1998; see also Stern et al. 2009). However, is this true on a
finer scale? Are certain aspects of the polyploidy process actually ‘‘hard-wired’’?
Preservation of duplicated gene copies following genome duplication appears far
from random, with specific functional categories preferentially retained (Blanc and
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Wolfe 2004; Freeling 2009) and reduplicated in subsequent polyploidizations
(Paterson et al. 2006). Independent WGDs in the ancestors of Arabidopsis, Oryza
(rice), Saccharomyces (yeast), and Tetraodon (pufferfish) have been followed by
convergent fates of many gene families (Paterson et al. 2006). Collectively, these
observations indicate that at deep timescales there may exist certain ‘‘principles’’
that govern the fates of gene and genome duplications. But is this true in the early
stages following polyploid formation?
In the sections that follow we review data from different sets of molecular
markers as well as chromosomal data across polyploid populations of separate
origin and ask if aspects of polyploid evolution are indeed hard-wired or if
stochastic processes prevail.
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Fig. 14.2 Maps showing the location of Tragopogon allotetraploid populations that are found
within the Palouse region of the Pacific Northwest of the U.S.A. Populations of T. miscellus
(squares) and T. mirus (circles) located in towns are indicated in the relevant counties of
Washington and Idaho. A contiguous urbanized area containing multiple T. miscellus populations
is indicated (shaded)
14.2.1 rDNA Loci/Concerted and Repeated Evolution
Concerted evolution, which results in the homogenization of gene sequences to
one type, is a common feature of ribosomal RNA genes (e.g., Zimmer et al. 1980).
In both T. mirus and T. miscellus, concerted evolution is ongoing, but incomplete
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14.2.2 Homeolog Loss and Gene Silencing
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Although Tragopogon affords unique opportunities for evolutionary study, it is not
a genetic model organism; hence, until recently, genetic resources have not been
available (but see Sect. 14.2.2.2), which has slowed research progress. As a result,
genetic and genomic changes in the newly formed tetraploids were initially
examined one gene at a time for upto 29 loci (Tate et al. 2006, 2009b; Buggs et al.
2009, 2010b; Koh et al. 2010). Initially, we used AFLP-cDNA display to screen
plants of T. miscellus, T. mirus, and parental diploids to look for promising candidate genes—that is, fragments that did not show additivity in the allopolyploids
as would be expected (Tate et al. 2006; Koh et al. 2010). Additional genes were
surveyed because they were orthologous to genes that were singletons in other
Asteraceae species (Buggs et al. 2009; Koh et al. 2010); the fate of such genes
seemed of particular interest in new polyploids.
The results of these one-gene-at-a-time surveys are presented in detail elsewhere (Tate et al. 2006, 2009b; Buggs et al. 2009, 2010b; Koh et al. 2010).
Significantly, most of the changes observed in populations of both young polyploids are homeolog loss events, which far outnumber gene-silencing events in all
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(Kovarik et al. 2005). In contrast to 80-year-old natural polyploids, F1 hybrids
have equal contributions of the diploid parents, as do raw (S0) synthetic polyploids, as well as the earliest natural populations of T. mirus and T. miscellus (based
on DNA from herbarium specimens). But in all modern-day natural populations
except one, each representing a distinct origin, the rDNA type of T. dubius is
consistently in very low abundance, with either the T. pratensis rDNA type (in
T. miscellus) or T. porrifolius rDNA type (in T. mirus) in much greater abundance.
In only one population of T. mirus are the parental contributions balanced (Malinska et al. 2011). Thus, concerted evolution has consistently occurred in these
new polyploid lines of separate origin, and it has repeatedly operated ‘‘against’’
T. dubius, homogenizing those copies in the direction of the other parent. Surprisingly, despite being the least abundant in terms of rDNA gene copy number,
T. dubius is by far the most abundant transcript in natural polyploid populations
(Matyasek et al. 2007).
This bias against the rDNA cistron in natural polyploid populations is already
apparent in the S1 generation of synthetic polyploids (Malinska et al. 2010, 2011). For
example, in four lines of synthetic T. miscellus, only three individuals (4 %) had
balanced parental gene ratios while 65 individuals (92 %) inherited more T. pratensisorigin units than would be expected under additivity. In seven lines of synthetic
T. mirus, 32 individuals (29 %) exhibited balanced rDNA genotypes, 69 individuals
(63 %) showed more 35S rDNA of T. porrifolius origin than expected, and only 9
plants (8 %) had more T. dubius-origin rDNA (Malinska et al. 2010, 2011).
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Fig. 14.3 Considerable variation from plant to plant within and among populations in the
amount of homeolog loss detected in natural populations of T. miscellus. Population and plant
designations are given on the x-axis; proportion of loci on the y-axis. Colors designate genotype
as indicated in the figure. D = T. dubius allele; P = T. pratensis allele
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populations examined of both polyploids. Furthermore, most of the homeolog
losses in both polyploids were from T. dubius, the diploid parent that is shared by
both T. mirus and T. miscellus.
It is also noteworthy that the same suite of genes consistently shows additivity
of the parental gene copies (no loss or silencing) in polyploid populations of
separate origin, whereas some of the genes analyzed consistently show some
evidence of loss across at least some of the populations surveyed. Thus, these early
analyses of Tragopogon polyploids are also in agreement with the hypothesis that
there may be some underlying ‘‘principles’’ to polyploidization at the genetic or
biochemical level.
But stochasticity is operating as well in these young polyploids. Although
homeolog loss is present in the polyploid populations, the process is ongoing, and
appears to be stochastic within individual populations. In no population examined
has silencing or loss been complete (i.e., observed in all individuals of a population). The amount of gene loss within populations varies (Fig. 14.3). Furthermore, these losses and gene silencing events were not detected in F1 hybrids or
early-generation synthetic lines (S1). Hence, loss of homeologs and gene silencing
are not immediate consequences of hybridization or polyploidization in Tragopogon, but appear to occur several generations after polyploid formation and for
certain genes.
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Fig. 14.4 Chromosome
diagram for the alloetraploid
T. miscellus (representative of
other allotetraploids)
illustrating terminology used
throughout this chapter—
chromosome, homeolog, and
allele
14.2.2.2 Tragopogon Goes Genomic
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Using Sequenom genotyping, we examined patterns of homeolog presence/
absence in 70 sets of homeologs in 59 plants from five independently formed
populations of allotetraploid T. miscellus (Buggs et al. 2012). Extensive gene loss
occurred in the B40 generations since polyploidization in T. miscellus. An average
of *20 % of the 70 loci investigated in each plant of T. miscellus was missing one
or both alleles of one homeolog (excluding assays where neither homeolog was
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Although important insights were obtained in Tragopogon, as well as other nonmodel systems, using the methods described above, these approaches have
shortcomings. These analyses were based on examination of specific homeolog
pairs using cleaved amplified polymorphic sequence analysis (CAPS). This
approach uses restriction enzymes and hence has limitations, as discussed by
Gaeta and Pires (2010). In addition, such surveys are slow and labor-intensive
(reviewed in Soltis et al. 2009b; Buggs et al. 2009). Because of these limitations,
we have sought ways to quickly and inexpensively build a framework for
addressing genome-scale questions in Tragopogon.
Newly developed genomic resources quickly facilitated the use of Tragopogon as
a model for the study of recent and repeated polyploidization. Using a combination of
454 and Illumina sequencing of genomic and cDNA, we identified SNPs (singlenucleotide polymorphisms) between the parental species T. dubius and T. pratensis
for analysis of the allotetraploids (Buggs et al. 2010a, b). Thousands of SNPs
distinguish the three parental diploids. Following SNP discovery, we designed
primers for Sequenom analysis (see Buggs et al. 2010a). Briefly, Sequenom iPLEx
genotyping uses mass spectrometry to carry out high-throughput and highly accurate
genotyping with multiple SNPs multiplexed in one reaction (Jurinke et al. 2005). The
assays permitted detection of gene loss and changes in allele number (‘allele’ is used
here to designate alleles at both homologous and homeologous loci; Fig. 14.4) within
polyploids versus changes in gene expression (see Sect. 14.2.3 below).
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detected). Approximately one-third of these cases were single-allele absences.
There was an overall bias toward loss of paternally derived homeologs. Assuming
that immediately after polyploidization each individual had two alleles from
T. dubius and two from T. pratensis at each pair of homeologous loci, on average
at least 7.7 % of the original 280 allele copies have been lost in an individual
plant’s lineage since polyploidization occurred. In only one case did we find that
absence of a homeolog was fixed in a population. The fact that few gene losses
were fixed in populations, and many individuals were missing single alleles,
indicates that allele loss is ongoing in these populations (Buggs et al. 2012).
These genomic data confirmed and extended our previous results and provided
more insight into the processes involved. Certain loci were repeatedly missing in
independently formed populations; the loci studied could be grouped into 12
clusters that followed recurrent patterns of presence/absence in populations with
unique origins. Therefore, evolution is repeated in separate lineages of independent origin.
Homeolog loss is also repeated at deeper phylogenetic scales. We compared
patterns of gene loss found in T. miscellus with patterns found in 12 other species
of Asteraceae that are considered ancient polyploids (Barker et al. 2008). Eighteen
genes in our study had GO categories that tended to be lost in Asteraceae (Barker
et al. 2008), and these had median homeolog absence of 7.0 % in T. miscellus. A
comparison of these 18 genes versus all other genes (i.e., those that tend to be
retained in Asteraceae, plus those with no preferential loss or retention: 45 genes,
with a median absence of 4.0 %) showed significantly higher homeolog absence in
the 18 genes. Thus, gene loss in T. miscellus, a very young Asteraceae polyploid,
appears to repeat patterns occurring in older Asteraceae polyploids.
One hypothesis for the repeated patterns of duplicate gene retention is the
gene balance hypothesis (Freeling 2009; Freeling and Thomas 2006; Birchler
et al. 2005; Papp et al. 2003). This hypothesis maintains that genes coding for
products that are highly connected (i.e., within protein complexes or biochemical
pathways) are dosage-sensitive in that they must be present in the nucleus in the
same number of copies as the genes for products with which they interact. Thus,
‘‘connected’’ genes are hypothesized to be retained together as duplicate copies
so as to preserve stoichiometry rather than reverting to singleton status one by
one over time. In contrast, genes whose products are less connected are dosageinsensitive and are expected to revert gradually to singleton status. Our analyses
so far suggest that dosage sensitivity may in fact be playing a role in Tragopogon
(Buggs et al. 2012).
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14.2.3 Tissue-Specific Silencing
Divergence of duplicate gene expression patterns among tissues has been suggested as a precursor of future evolution (Ohno 1970). Expression of a gene
duplicate in a tissue where the progenitor copy was not expressed may indicate
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neofunctionalization (Ohno 1970; Duarte et al. 2006; see Chap. 1 of this volume),
while division of ancestral patterns of tissue-specific expression among duplicates
suggests subfunctionalization (Lynch and Conery 2000; Rodin and Riggs 2003;
Duarte et al. 2006) and silencing of a gene duplicate in all tissues points to
nonfunctionalization (Duarte et al. 2006). Both neofunctionalization and subfunctionalization will lead to long-term retention of duplicated genes, whereas
silencing/nonfunctionalization will generally lead to loss of a duplicate. Tissuespecific expression of duplicated genes has been studied in older gene duplicates in
model organisms and crops (Adams et al. 2003; Duarte et al. 2006; Ganko et al.
2007; Semon and Wolfe 2008; Chaudhary et al. 2009), but in these species the
ancestral patterns of gene expression and the ages of duplicates are not known
precisely. Cases of tissue-specific expression patterns of very young gene duplicates are restricted to a few synthetic polyploids (Adams et al. 2003, 2004; Wang
et al. 2004; Chaudhary et al. 2009).
We examined the expression of 13 homeolog pairs in seven tissues of 10 plants
of T. mirus from two natural populations of independent origin (Buggs et al.
2010b). Of the 910 assays in T. mirus, 63 % showed expression of both homeologs, 7 % showed no expression of either homeolog, 20 % showed non-expression
of one homeolog across all tissues of a plant, and 8 % showed non-expression of a
homeolog in a particular tissue within a plant. We found two cases of reciprocal
tissue-specific expression between homeologs, potentially indicative of subfunctionalization. This study therefore showed that tissue-specific silencing, and even
apparent subfunctionalization, can arise rapidly in the early generations of natural
allopolyploidy. Similar results were found for 18 homeolog pairs using the same
approaches in T. miscellus populations (Buggs et al. 2011b).
In T. miscellus we also examined tissue-specific gene expression using the
previously described Sequenom assays (Buggs et al. 2011b). Tissue-specific
expression of 144 homeolog pairs in two natural populations was compared with
patterns of allelic expression in both in vitro ‘‘hybrids’’ and hand-crossed F1
hybrids between the parental diploids T. dubius and T. pratensis, and with patterns
of homeolog expression in synthetic (S1) allotetraploids. Tissue-specific homeolog
expression was frequent in natural allopolyploids, but F1 hybrids and S1 allopolyploids showed less tissue-specific homeolog expression than the natural allopolyploids and the in vitro ‘‘hybrids’’ of diploid parents. These results suggest that
‘‘transcriptomic shock’’ upon hybridization (McClintock 1984) includes the activation of allele/homeolog expression in all tissues, causing a loss of tissue-specific
expression patterns seen in the diploid parents. Such activation has seldom been
considered in terms of the tissue-specific activation of protein-coding genes.
Activation of homeologs has also been found in cotton F1 hybrids and allopolyploids (Chaudhary et al. 2009), who termed it ‘‘transcriptional neofunctionalization’’. We showed this to be widespread in Tragopogon. This may fit a scenario
in which activity of small interfering RNA molecules, which influence gene
expression, is temporarily lost in F1 hybrids and early allopolyploids, but restored
subsequently.
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Despite enormous progress in our understanding of many aspects of polyploidy,
little attention has been paid to chromosomal constitution, structure, and organization. The New World Tragopogon allotetraploids illustrate the valuable role of
cytology in examining hybridization and polyploidy in plants. Our recent studies
have revealed high levels of chromosomal variation in *80-year-old natural
populations, as well as the newly synthesized allotetraploids T. mirus and T. miscellus. The earliest studies of T. mirus and T. miscellus identified mitotic complements of 2n = 24 and typically 12 bivalents at meiosis (Ownbey 1950;
Ownbey and McCollum 1954). This supported the expectation that allotetraploids
were chromosomally additive of the diploid progenitors, which are both 2n = 12.
Lim et al. (2008) used genomic and fluorescence in situ hybridization (GISH/
FISH) in a preliminary survey of several T. mirus and T. miscellus plants and
found a few plants of each species that were not chromosomally additive of the
diploid parents. Several natural T. mirus and T. miscellus plants were found to be
aneuploid, with intergenomic translocations. Although most of the aneuploid
individuals examined were 2n = 24, not all chromosomes were present in two
copies as expected. GISH conducted on synthetic T. mirus pollen mother cells at
the diplotene stage of meiosis showed allosyndetic pairing within multivalents.
Meiotic instability, in the form of anaphase bridges and lagging chromosomes, was
also observed by Tate et al. (2009a) in the first synthetic polyploid generation (S1)
in T. mirus and T. miscellus lines. Thus, gametes with aneuploid and/or rearranged
chromosome complements can be potentially generated as early as the first meiosis
following genome doubling, which has been shown to be the case for synthetic
allotetraploid B. napus (Szadkowski et al. 2010).
The preliminary work of Lim et al. (2008) prompted a detailed examination
using GISH and FISH of the chromosomal variation generated in six T. miscellus
populations of independent origin (Chester et al. 2012). In all six populations, both
aneuploidy and translocations were common (Fig. 14.5). Only 3 of the 58 plants
exhibited the expected additivity of the diploid parental karyotypes (with neither
aneuploidy nor translocations). Although approximately 70 % of polyploid plants
were aneuploid for one or more chromosomes, variation in copy number appears
to be constrained. Most plants were 2n = 24, and the total copy number for each
homeologous group of chromosomes was typically four as a result of aneuploidy
being reciprocal between homeologous chromosomes (Fig. 14.5). Thus, most
deviations from disomy were in the form of monosomy-trisomy or nullisomytetrasomy, between homeologous chromosomes. This pattern of extensive aneuploidy while maintaining the overall copy number closely resembles cytological
changes in synthetic neoallotetraploid Brassica napus (Xiong et al. 2011). Gene
dosage has been implicated as a major factor constraining chromosomal changes
such that imbalances, which arise, require compensation by chromosomes (compensatory aneuploidy) or homeologous segments (compensatory translocations).
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In T. miscellus, genome rearrangements via intergenomic translocations were
common; 76 % of the plants showed evidence of at least one translocation. The two
largest chromosomes (groups A and B) showed the highest incidence of translocations.
Across all T. miscellus plants at least six different translocation breakpoint positions
were observed along the A group chromosomes. In one T. miscellus population in
Spokane, WA, an A-chromosome intergenomic translocation appeared close to fixation, being homozygous in 8 of the 10 individuals analyzed. Individuals from six
populations also appear to have undergone reciprocal translocations on the B-group
chromosomes, but they mostly occur at a similar position, near the end of the long arm.
The observed chromosomal changes in the recently formed Tragopogon allotetraploids provide two possible explanations for the loss of DNA that has been
observed using molecular SNP-based assays. (1) If a translocation was in a nonreciprocal state and homozygous, this could lead to the loss of DNA from one of
the parental diploids in the translocated region. (2) Nullisomy would lead to the
complete loss of DNA from one of the parental chromosomes. This may have
contributed to the clustering of patterns of gene loss and retention found using
Sequenom assays (see above). However, the diverse patterns of homeolog losses
that have been detected are not readily explainable by only these large-scale
chromosomal changes detectable with GISH.
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Fig. 14.5 Mitotic GISH karyotypes of three aneuploid T. miscellus individuals. Chromosomes
derived from each parental genome are shown, i.e., the T. dubius genome (D-subgenome) and the
T. pratensis genome (P-subgenome). Chromosomes deviating from disomy are as follows: top,
monosomy: trisomy for E chromosomes; middle, nullisomy: tetrasomy for E chromosomes;
bottom, four cases of monosomy: trisomy for chromosomes B, C, D, and E. All three individuals
are 2n = 24 and have four copies of each homeologous group (A–F). Scale bar: 5 lm
14.3 Comparing Tragopogon to Other Well-Studied Systems
Genome evolution in other well-studied polyploid systems (e.g., Gossypium,
Triticum, Nicotiana, Arabidopsis, Brassica, Senecio, Spartina) exhibits important
similarities and differences. Tragopogon is noteworthy in that initial studies show
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that while true expression changes play a major role, homeolog losses are also very
prominent in these young polyploids (Tate et al. 2006, 2009b; Koh et al. 2010;
Buggs et al. 2009, 2010a, b, 2011b, 2012). Of the initial 23 genes analyzed in
T. miscellus, 15 showed homeolog loss in one or more plants from nature, and 8
showed true expression changes (results for T. mirus are comparable); these patterns were confirmed via genomic analyses of many more genes. In contrast, in
synthetic wheat (Triticum) and synthetic Arabidopsis thaliana and A. suecica
polyploids, as well as in tetraploid cotton (Gossypium), expression changes
dominate (Adams et al. 2003, 2004; Hovav et al. 2008a, b; Udall et al. 2006; Flagel
et al. 2008; Madlung et al. 2004; Kashkush et al. 2002; Wang et al. 2006).
Tragopogon may be most similar to the allotetraploid B. napus, in which most of
the apparent gene silencing events observed in later generations were due to gene
losses, most likely resulting from genomic rearrangements (Song et al. 1995; Gaeta
et al. 2007). Across 50 lines of B. napus, genetic changes are equally distributed
between the parental diploid genomes (Gaeta et al. 2007). The system is dynamic—
some lines become more ‘‘oleracea like’’ and others more ‘‘rapa like’’ in terms of
losses and corresponding expression differences. In Arabidopsis suecica allopolyploids, silencing of homeologs from one parent (A. thaliana) was observed more
frequently than the silencing of homeologs from the other parent, Arabidopsis
arenosa (Wang et al. 2006). In synthetic polyploids in Triticale, the contribution of
the rye genome (Secale cereale) is preferentially silenced (Ma et al. 2004; Ma et al.
2006). In cotton, tissue-specific subfunctionalization occurs for some loci (Adams
et al. 2003, 2004; Adams and Wendel 2004), but overall gene expression is biased
toward one parent (Udall et al. 2006; Flagel et al. 2008; Rapp et al. 2009; Flagel and
Wendel 2010). In both T. miscellus and T. mirus, homeologs of one diploid genome
(T. dubius) are more often lost or not expressed; that is, T. dubius is often the ‘‘loser
genome’’ based on the set of genes surveyed to date.
Tragopogon also exhibits cytogenetic similarities to Brassica. Both T. mirus
and T. miscellus exhibit numerous translocations as well as extensive aneuploidy
while maintaining the overall copy number expected in an allotetraploid (reciprocal monosomy: trisomy and nullisomy: tetrasomy); this closely resembles
cytological changes in synthetic neoallotetraploid B. napus (Xiong et al. 2011).
It is also now possible to compare results for Tragopogon with several of the
other recently formed natural polyploids. Great research progress has now been
made on four of the five polyploids known to have formed in the past two centuries. In addition to T. mirus and T. miscellus, numerous insights into recent
polyploidy have been obtained for Senecio cambrensis (also in the Asteraceae) and
Spartina anglica (Poaceae). Both these model systems are reviewed elsewhere in
this volume (see Chaps. 13 and 12, respectively). Interestingly, all of these recent
polyploids have formed following introductions of one or both progenitors into a
completely new geographic area. Some of these new polyploids have been successful in nature, particularly Spartina anglica, which is now distributed worldwide with major ecological impact (Ainouche et al. 2004, 2009; see Chap. 12 of
this volume). The Tragopogon polyploids are now major weeds in the Palouse
region of northwestern USA. In contrast, following initial range expansion,
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Senecio cambrensis now seems to be disappearing from parts of its original range
(Abbott et al. 2007; see Chap. 13 of this volume).
Senecio cambrensis formed from parents of different ploidal levels (one parent
is itself a polyploid), whereas in Spartina anglica both parents are polyploids but
of the same ploidal level. In Senecio cambrensis there is an additional level of
complexity in that the diploid parent is itself of homoploid hybrid origin. In
contrast, both parents of the recent Tragopogon polyploids are diploid. The
multiple layers of recent hybridization and polyploidization in Senecio and
Spartina could influence the genetic and expression changes detected in the recent
polyploids in those genera.
Molecular studies have revealed striking genetic and genomic changes in all of
these recently formed polyploids. Transcriptomic shock has now been shown
following hybridization in both Senecio (Hegarty et al. 2005, 2006) and Tragopogon (Buggs et al. 2011b), and substantial expression changes have also been
reported in F1 hybrids and polyploids in Spartina (Cheilafa et al. 2010a, b) Thus,
in recent polyploids in all three genera, hybridization seems to result in a major
burst of altered gene expression.
Changes in gene expression have been shown to be important in all of the
systems, but have been best characterized in Spartina (Ainouche et al. 2009, Chap.
12 of this volume) and Senecio (Hegarty et al. 2008, Chap. 13 of this volume).
Methylation alterations have been detected in both Spartina (Salmon et al. 2005)
and Senecio (Hegarty et al. 2011), with epigenetic changes now considered to play
a major role in these new polyploids (Parisod et al. 2009; Ainouche et al. 2009;
Hegarty et al. 2011). However, epigenetic changes have not yet been analyzed in
Tragopogon. Dramatic changes in gene expression have been documented in
Spartina anglica, Senecio cambrensis, and the recently formed Tragopogon
polyploids. However, homeolog loss seems to have played a more prominent role
in young Tragopogon polyploids than in these other recent polyploids.
In both the recently formed Tragopogon polyploids and in S. cambrensis, genomic studies reveal that one parental genome predominates over the other in terms of
global patterns of expression and gene retention. In the allohexaploid S. cambrensis,
gene expression is more similar to that of the tetraploid parent (S. vulgaris) than to the
diploid parent, S. squalidus (Hegarty et al. 2006). In the young Tragopogon polyploids, homeologs of the diploid parent T. dubius are more often lost or not expressed.
That is, following polyploidy there are clear winner and loser parental genomes, and
these patterns are established quickly and repeatedly.
Chromosomal changes have been investigated in detail in both Tragopogon and
Spartina using FISH/GISH; recent polyploids in both genera exhibit substantial
change, but different types of variation are evident. In Spartina, nonaploid plants
(2n = ca. 90) were detected (rather than the expected 2n = 120–124), most likely
resulting from backcrosses between S. anglica and its maternal parent S. alterniflora
(Renny-Byfield et al. 2010). In Tragopogon, frequent translocations were detected as
well as frequent reciprocal monosomy: trisomy and nullisomy: tetrasomy—these
have not been previously reported from nature (Chester et al. 2012). Chromosomal
pairing abnormalities (multivalents) have been detected in both Tragopogon
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A diverse array of experimental approaches has helped us to understand the
evolution of young, natural Tragopogon polyploids at the genomic and transcriptomic levels. These have demonstrated: (1) the allopolyploids have multiple
origins; (2) substantial gene loss occurs within the first 40 generations in nature,
and is still ongoing; (3) patterns of gene loss are repeated among independent
origins of the allopolyploids; (4) transcriptomic shock occurs upon allopolyploidization, involving a reduction in tissue-specific expression; (5) major chromosomal changes have occurred and are ongoing in the natural allopolyploids.
The dynamic evolutionary processes that appear to be underway in Tragopogon
polyploids may be representative of those that have occurred in other groups, and
after past polyploidization events. Care is needed when extrapolating from a single
system, because patterns of change may be influenced by the genetic background
of the polyploid, such as the level of genetic differentiation between the two
parental species of an allopolyploid (reviewed in
Buggs et al. 2011a). Studies of polyploids in many plant families, of different
ages and of different parental combinations, are needed to provide a comprehensive understanding of the evolution of allopolyploids.
Tragopogon has great potential for further development as a model system for
polyploidy. Older Eurasian polyploids will allow placement of another time point
in the evolutionary series. Research is also underway on natural hybrids between
T. pratensis and T. porrifolius (T. 9 mirabilis) that occur in the United Kingdom.
Further development of genetic resources—sequencing of the T. dubius genome
and production of a genetic map—will allow new questions to be addressed.
In recent years, progress in understanding the genetic consequences of polyploidy has outstripped progress in understanding the ecological background in
which evolution occurs (but see Ramsey 2011). As a genus that occurs almost
exclusively in the wild, Tragopogon affords a system in which relevant ecological
studies can be carried out, comparing the fitness of polyploids and diploids, and the
different chromosomal variants of the polyploids. Such studies will contribute to a
comprehensive view of polyploid evolution.
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allopolyploids as well as in synthetic polyploids (Ownbey and McCollum 1953; Tate
et al. 2009a). FISH/GISH has been problematic in Senecio, but traditional cytogenetic analysis suggests possible structural chromosomal changes, as well as some
meiotic pairing irregularities (Crisp 1972). In addition to generating genetic diversity, intergenomic recombination resulting in chromosome rearrangements could
lead to the formation of reproductive barriers between lineages of separate or
independent origin, which is under investigation in Tragopogon.
532
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Date: 16-8-2012
Acknowledgments Funding for this research was provided by the University of Florida and
NSF grants MCB-0346437, DEB-0614421, DEB-0919254, DEB-0922003, and DEB-0919348.
R.J.A.B. has been supported since March 2010 by NERC Fellowship NE/G01504X/1.
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Book Title
Polyploidy and Genome Evolution
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Yeast as a Window into Changes in Genome Complexity Due to Polyploidization
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conantg@missouri.edu
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Hudson
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Given Name
Corey M.
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University of Missouri
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Abstract
Due to the long history of genetic analyses in yeasts and their experimental tractability, the yeast genome
duplication provides important perspectives on the genome and population-level processes that follow wholegenome duplication (WGD). We discuss the history of the discovery of the Saccharomyces cerevisiae WGD,
with special emphasis on the role of comparative genomics in its analysis. We then explore models of the
WGD shaped population and species divergence, both at a gene level (e.g., Dobzhansky-Muller
incompatibility) and from the perspective of recent work on secondary allopolyploidy in Saccharomyces
pastorianus. Finally, we explore the selective forces that act on the WGD-produced paralogs and shape their
patterns of loss and retention. In addition to discussing the dosage balance hypothesis as it applies to the yeast
WGD, we explore the role of the WGD in shaping several complex metabolic and regulatory phenotypes.
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Abstract Due to the long history of genetic analyses in yeasts and their experimental tractability, the yeast genome duplication provides important perspectives
on the genome and population-level processes that follow whole-genome duplication (WGD). We discuss the history of the discovery of the Saccharomyces
cerevisiae WGD, with special emphasis on the role of comparative genomics in its
analysis. We then explore models of population and species divergence, both at a
gene level (e.g., Dobzhansky-Muller incompatibility) and from the perspective of
recent work on secondary allopolyploidy in Saccharomyces pastorianus. Finally,
we explore the selective forces that act on the WGD-produced paralogs and shape
their patterns of loss and retention. In addition to discussing the dosage balance
hypothesis as it applies to the yeast WGD, we explore the role of the WGD in
shaping several complex metabolic and regulatory phenotypes.
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15.1 Introduction
Researchers have found remnants of ancient whole-genome duplications (WGDs)
preserved in the genomes of many and diverse eukaryotes. This book, in fact, is a
testament to that diversity, illustrating the sheer number of independent events in
plants as well as the evolutionarily basal events in vertebrates and other, more
recent WGDs in teleost fishes and frogs. Although we have still not fully validated
C. M. Hudson G. C. Conant
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15.2 Evidence for WGD in Yeast
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Although early analyses of genes potentially created by the vertebrate 2R events
used phylogenetic approaches (Hughes 1999; Furlong and Holland 2002), most
current studies of WGD rely on one or both of two methods: (1) finding numerous
blocks of paralogous genes in multiple chromosomes with similar gene orders and
(2) clustering homologs into groups by measuring the rate of synonymous substitutions (Ks or dS). This second method assumes that the gene pairs created by
WGD cluster about some mean Ks value (Lynch and Conery 2000). The simultaneous application of both methods has been used to group multiple WGD events
within species (e.g., Arabidopsis thaliana and Tetraodon nigroviridis; Jaillon et al.
2004; Van de Peer et al. 2009). However, the yeast genomes present an interesting
challenge in this respect because the synonymous substitutions between yeast
paralogs produced by WGD (hereafter ohnologs; Wolfe 2000) are often saturated
(Byrne and Wolfe 2007). In other words, identical synonymous positions between
two ohnologs occur almost as often due to repeated convergent substitutions as due
to common ancestry, a fact pointed out by Smith (1987), who attempted to date
histone gene duplicates in yeast. While the genomic structure of the core histone
genes suggested that they were all duplicated simultaneously, these genes show
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Susumo Ohno’s claim for the primacy of polyploidization in the generation of new
adaptations (Ohno 1970), it is clear that WGD events have had a massive influence
on the content and structure of the genomes of their possessors. The next step in
exploring Ohno’s hypothesis is to link genome evolution to known changes in
function. This goal, however, remains challenging, primarily because our
knowledge of how genotype links to phenotype remains woefully incomplete
(Pigliucci 2010). However, one group of organisms in which we can at least begin
to make such associations is in the polyploid yeasts. Our knowledge of the
functional genomics of yeast is drawn primarily from Saccharomyces cerevisiae,
which has a well-annotated genome, decades of biochemical, genetic, and cell
biology research, a relatively small genome, and a life cycle that lends itself to
scalable laboratory analyses. Taken together, these facts have allowed yeast
researchers not only to understand the structure of the genome following WGD but
also to experimentally evaluate hypotheses regarding the evolution of particular
complex phenotypes. As we will stress throughout this chapter, one of the themes
that emerges from all of these analyses is the degree to which the outcome of a
WGD depends as much on the interactions between genes as on the role of any
particular locus. Of equal importance evolutionarily, we now also have data from
other polyploid yeast species, which are valuable both as a point of comparison to
S. cerevisiae and for their own sakes. This wealth of data affords us insight into the
mechanisms that drive the preferential loss and preservation of gene duplicates
after polyploidy, lead to the functional divergence of genes, and are behind the
evolutionary origins of complex phenotypes.
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15.2.1 Synteny-Based Evidence for WGD in Saccharomyces
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considerable variation in the numbers of synonymous substitutions separating
them. This observation led Smith (1987) to hypothesize that S. cerevisiae underwent a WGD ancient enough that the duplicates surviving from it had saturated.
However, it was not until the genome sequence of S. cerevisiae became available
that this speculation could be confirmed (see below). Another similar but subtler
problem in using Ks as a means of dating duplicate genes is the issue of gene
conversion. Gene conversion was presumed to be quite common in yeast (Petes
and Hill 1988), even prompting some authors (Gao and Innan 2004) to suggest that
estimates of duplication rates based on duplicate divergences were inapplicable
due to the homogenization of duplicate loci by conversion. Fortunately, although
gene conversion is very common among yeast ribosomal proteins, it does not
appear to be a general characteristic of the genome (Evangelisti and Conant 2010).
Nonetheless, these various issues collectively meant that comparisons of paralogous sequence divergence were deemed unhelpful as a means to detect WGD in
yeast.
Given that paralogous sequence comparisons were generally unhelpful in finding
WGD relics, another tactic was to consider gene order. In fact, even before the S.
cerevisiae genome sequence was completed in 1996, it was clear to many
researchers that it contained numerous, long, homologous clusters of ordered
genes (Goffeau et al. 1996). Melnick and Sherman (1993) found ordered homologous gene clusters in chromosomes V and X covering 7.5 kb. Lalo et al. (1993)
similarly found ordered homologous gene clusters in chromosomes XIV and III
covering 15 kb. When the genome was sequenced, researchers found 18 ordered
homologous genes in chromosomes IV and II that covered 120 and 170 kb,
respectively (Goffeau et al. 1996). Just how to interpret these redundant regions
remained a challenge at that time (Goffeau et al. 1996; Oliver 1996), and, in spite
of Smith’s prior hypothesis (Smith 1987), few, if any, of the contemporaneous
explanations included an ancient WGD.
However, opinions changed the next year when Wolfe and Shields (1997)
presented a thorough, genome sequence-based, analysis that gave strong evidence
for WGD in S. cerevisiae. To find syntenic regions, they conducted a BLASTP
search of amino acid sequences throughout the yeast genome and made a dot plot
of the results. They then created gene blocks from these data, where each block
was required to have at least three homologous pairs with intergenic distances B
50 kb and conservation of gene order and orientation. This analysis yielded 55
duplicated regions containing a total of 376 pairs of ohnologs. The large number of
duplicated regions led Wolfe and Shields to posit two explanations: (1) successive
independent gene duplications, and (2) a single duplication of the entire genome,
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15.2.2 Comparative Genomics and Proof of WGD
in S. cerevisiae
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A number of researchers disputed the claims of Wolfe and Shields (1997), arguing
that, because the syntenic regions identified made up only a small part of the
genome, independent duplications better explained the genomic structure of S.
cerevisiae (Coissac et al. 1997; Mewes et al. 1997; Hughes et al. 2000; Llorente
et al. 2000a; Llorente et al. 2000b; Friedman and Hughes 2001; Piskur 2001;
Koszul et al. 2004). However, this independent duplication hypothesis became
untenable following the genome sequencing of other yeasts that proved to lack
these syntenic paralog blocks. These sequences were described by three independent groups. The comparison of S. cerevisiae with Kluyveromyces waltii
(Kellis et al. 2004) and the comparison of S. cerevisiae with Ashbya gossypii
(Dietrich et al. 2004) involved different genomes, but effectively made the same
argument: that the 2:1 mapping of blocks of paralogs from S. cerevisiae to
homologous single-copy genes in K. waltii/A.gossypii could best be explained by
WGD. This explanation was particularly striking because the doubly conserved
synteny blocks cover 90 % of the genome in K. waltii (Kellis et al. 2004) and 96 %
of that in A. gossypii (Dietrich et al. 2004). Furthermore, both studies found a large
number of 2:1 pairing of centromeres in the species-respective chromosomes.
There were 16:8 such pairings between S. cerevisiae and K. waltii and 14:7
between S. cerevisiae and A. gossypii with a subsequent break at the expected
centromere position in S. cerevisiae chromosomes X and XII that are syntenic with
regions in A. gossypii chromosomes I and III. Finally, and perhaps most strikingly,
both groups also showed that the single-copy orthologs of genes from A. gossypii
or K. waltii in the genome of S. cerevisiae are interleaved between two paralogous
chromosomes in S. cerevisiae that nonetheless retain the relative gene order of the
single chromosome in the non-WGD yeast (see Fig. 15.1). Such a pattern is only
explicable under the hypothesis of a WGD event followed by massive gene losses.
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followed by massive gene loss. There were two lines of evidence discounting the
first possibility. First, 90 % (50/55) of the gene regions shared the same orientation
with respect to the centromeres of the duplicated regions when we would expect
independent duplications to be instead randomly distributed about the centromeres. Second, there were no examples of triplicated regions in the S. cerevisiae
genome. If the duplications involved several distinct events separated in time, such
a pattern would be highly unlikely, because it would require that later duplication
events never overlapped with prior ones. Given these arguments, Wolfe and
Shields (1997) argued for a single ancient WGD, which they dated to be hundreds
of millions of years old (note that attempts to conclusively date this event have
been difficult, due to a lack of fossils and the previously mentioned saturation of
substitutions; see Taylor and Berbee 2006; Rolland and Dujon 2011).
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Fig. 15.1 Yeast gene order browser (YGOB) screenshots with a window size of six. Each box
represents a gene; each color, a chromosome. The gene in focus, the A. gossypii gene ABR086W,
is highlighted by an orange border. Each vertical column (‘‘pillar’’) represents a single gene prior
to the WGD (hence, all genes in a column are homologs, and the paired upper and lower genes,
when present, are paralogs). The ancestral order of these genes (pink boxes) just prior to the
WGD has also been exhaustively inferred (Gordon et al. 2011). Connectors join nearby genes: a
solid bar for adjacent genes, two bars for loci less than five genes apart, and one bar for loci \20
genes apart. The connectors are extended in gray over intervening space. The end of a
chromosome or contig is denoted by a brace. Arrows denote transcriptional orientation. The
browser also includes a control panel that allows users to select the window size and the gene to
focus on. This panel also has buttons for running BLAST searches against YGOB’s database,
outputting YGOB data in tabular format, obtaining pairwise Ka and Ks values among genes, and
computing multiple sequence alignments and phylogenetic trees of individual pillars. Species
names for each track are labeled at right (Byrne and Wolfe 2005)
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15.2.3 Yeast Gene Order Browser
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The argument of Dujon et al. (2004) is subtly different. They sequenced and analyzed four other genomes. One genome that of Candida glabrata, shares the genome
duplication with S. cerevisiae. This was determined by comparing syntenic blocks in S.
cerevisiae and C. glabrata with the other three sequenced genomes, Kluveromyces
lactis, Debaryomyces hansenii, and Yarrowia lipolytica. Dujon et al. (2004) found 20
distinct blocks of paralogs shared by both S. cerevisiae and C. glabrata. These blocks
allowed them to map the WGD onto a phylogeny, rather than do a simple pairwise
comparison. Mapping this WGD phylogenetically creates distinct hypotheses as to
where in the tree we expect to find polyploid yeasts (c.f., Fig. 15.1), predictions that
have been confirmed with each of the subsequently sequenced genomes of known
phylogenetic position (Wapinski et al. 2007; Scannell et al. 2011).
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15.2.4 Additional Non-Saccharomyces-Specific WGDs
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One of the major benefits of studying the yeast WGD is that the relatively slow rates of
gene order change in yeast genomes and the compactness of their genomes means that
an exhaustive enumeration of all WGD-produced ohnologs is possible. Just such a
project was carried out, with the results presented as the web-based Yeast Gene Order
Browser (YGOB) (Byrne and Wolfe 2005), which illustrates a number of non- and
post-WGD yeasts in a graphical framework (Fig. 15.2). This work has been followed
by a reconstruction of the set of genes and their relative orders that existed just prior to
the WGD (Gordon et al. 2009) and by a likelihood-based model of post-WGD
duplicate loss that attempts to quantify the orthology inferences made by YGOB
(Conant and Wolfe 2008a). On the basis of these three projects, the post-WGD evolutionary history of virtually every locus in the S. cerevisiae genome can be traced
(Fig. 15.1 is thus illustrative of the predominant pattern seen across the genome).
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In addition to the ancient WGD that characterizes the Saccharomyces clade
(Fig. 15.2), several cases of allopolyploidy have been discovered in yeasts. Some
of these occur in species within the Saccharomyces sensu stricto clade (Scannell
et al. 2011), while others are independent.
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15.2.4.1 Secondary Allopolyploidy in Saccharomyces pastorianus
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Several cases of allopolyploidy are known from within S. sensu stricto (Dequin
and Casaregola 2011). One of the most well studied is that of the lager yeast,
S. pastorianus (syn. S. carlsbergensis). It has long been known that the polyploid
S. pastorianus and other members of the complex of related lager yeasts are
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allotetraploids of diploid S. cerevisiae and some other unknown diploid species
(Martini and Kurtzman 1985; Kielland-Brandt et al. 1995). However, aside from
the general difficulties facing anyone interested in identifying the origins of hybrid
genomes, the debate surrounding the origin of the second parental diploid species
was further complicated by a difficulty in delimiting species within these groups
(Rainieri et al. 2006). The tetraploid S. pastorianus belongs to a group of yeast
species which, until recently, was represented as a phylogenetically unresolved
species complex including S. pastorianus, S. monacensis (S. pastorianus strain
CBS 1503), S. bayanus, and S. bayanus var. uvarum (Casaregola et al. 2001;
Rainieri et al. 2006). This taxonomic confusion has recently been partially
resolved through the sequencing of the genomes of both S. pastorianus and one of
its presumed parental diploid species, S. eubayanus (Nakao et al. 2009). The
genome history that has emerged is a complicated story of allopolyploidy followed
by genomic transformation forming the related species S. bayanus (Libkind et al.
2011). As summarized by Libkind et al. (2011), S. cerevisiae hybridized
with S. eubayanus (a species recently recovered in Patagonia) with subsequent
genome doubling producing the allotetraploid progenitor of modern S. pastorianus. Following domestication, smaller regions of the S. pastorianus genome were
then apparently transferred into the genome of the diploid parent S. eubayanus
(which is nonetheless a descendant of the ancient polyploidy). This hybrid form of
S. eubayanus, with contributions from S. pastorianus, then proceeded to interbreed
with diploid S. uvarum to produce the modern, diploid, S. bayanus (Fig. 15.3).
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Fig. 15.2 Consensus view of the evolutionary relationships between the yeast taxa discussed.
Black branches indicate relationships described by both Kurtzman and Robnett (2003) and
Fitzpatrick et al. (2006). Red branches indicate conflicts between the two phylogenies, in which
case Fitzpatrick et al. (2006) is presented. Curved grey branches illustrate the allopolyploidy
events between two species (S. cerevisiae and S. bayanus, Z. rouxii, and Z. pseudorouxii). Taxa in
blue are reported in text (e.g., S. pastorianus and Z. rouxii ATCC 42981). Stars mark wholegenome duplications. Note that genus names are an imperfect guide to the relationships
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natural environments
brewing environment
early
S. pastorianus
1 50:50 hybrid of
S. eubayanus and
S.cerevisiae
modern
S. pastorianus
3 S. pastorianus
cells lyse and
release large
DNA fragments
4 transformation
of S. eubayanus
by S. pastorianus
2 SUL1 inactivation,
loss-of-heterozygosity
on the right arm of chromosome VII adds an extra copy
of ScerIMA1, aneuploidy and
chromosome rearrangements
S. bayanus
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S. eubayanus
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domestication
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Fig. 15.3 Genome evolution in S. pastorianus. A model of the formation of S. pastorianus and the
hybrid strains of S. bayanus. First, wild S. eubayanus and ale-type S. cerevisiae hybridized to form
an allotetraploid that became the ancestor of the modern (doubly paleopolyploid) S. pastorianus.
Second, domestication imposed strong selective pressure for strains with the most desirable brewing
properties. Third, in the brewing vats with high densities of S. pastorianus, cell lysis releases large
DNA fragments that occasionally transform, fourth, contaminating wild strains of S. eubayanus
(which possesses only the ancient WGD shared with S. cerevisiae) because of the lack of pure
culture techniques. Fifth, multiple hybridization events between S. eubayanus and wild strains of
S. uvarum gave rise to CBS 380T and NBRC 1948. This model does not exclude prior or parallel
involvement of S. uvarum in brewing or contamination. Reprinted from Libkind et al. (2011)
15.2.4.2 Allopolyploidy in Zygosaccharomyces rouxii
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The spoilage agent and industrial yeast Zygosaccharomyces rouxii strain ATCC
42981 was identified as another allopolyploid by James et al. (2005) and Gordon
and Wolfe (2008). This hybridization/polyploidy event is significant for two reasons. First, unlike all of the previous examples, it occurs outside of S. sensu stricto.
Second, Gordon and Wolfe (2008) determined that most of the paralogs produced
by WGD are still present, presumably due to the recentness of the event. Thus,
while other yeast genome duplications are ancient and show considerable gene loss
and rearrangement (Wolfe and Shields 1997), the Z. rouxii genome retains most of
the ‘‘new’’ genes produced by its WGD. Since the survival time of ohnologs has
been modeled to follow a power law, most of the duplicates are expected to be lost
very rapidly (Maere et al. 2005), suggesting that Z. rouxii represents an example of
the early features of genome evolution following WGD.
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An important potential outcome of polyploidy is in altering patterns of speciation.
This change can happen in at least two ways. First, the WGD can relax selective
constraints resulting in an adaptive radiation by means of ecological speciation.
Another, more neutral mechanism, is a special case of the Dobzhansky-Muller
(DM) process of speciation, in which species lose reciprocal paralogs following
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some period of isolation (Lynch and Force 2000; Werth and Windham 1991; see
also Chap. 1, this volume). WGD potentially increases the probability of this
simply by increasing the number of paired genes in a genome. The fertility of
hybrids is 0.75n, where n is the number of reciprocal losses of essential genes
among populations (Werth and Windham 1991). Clearly, for any significant
number of reciprocal losses (such as occur after WGD), the number of viable,
fertile offspring of a crossing of two such populations is negligible. Both phylogenetic and experimental studies of the DM process after WGD have been carried
out in yeast. Scannell et al. (2006) showed that the number of reciprocal gene
losses in several species of yeast sharing the S. cerevisiae WGD was sufficient to
induce such inviability. This observation suggests that a DM mechanism was
partly responsible for the multiple speciation events among the Saccharomyces
species (e.g., S. cerevisiae, S. bayanus, and C. glabrata) following WGD.
An advantage of studying the DM process in yeast is the ability to experimentally create and cross artificial polyploids. This possibility has been highlighted in experimental studies of reproductive isolation. Polyploid yeasts have
been allowed to evolve in different selective environments (Dettman et al. 2007)
and in neutral environments subject to random mutagenesis (Maclean and Greig
2011). These two experiments have shown that moderate reproductive isolation,
coupled with reciprocal gene loss, results in a clear loss of fitness when independently derived polyploids are crossed. Similarly, Lee et al. (2008) showed that
hybrids of S. cerevisiae and S. bayanus were less fit than their parental phenotypes,
due primarily to incompatibility between their nuclear and mitochondrial genomes. Chou et al. (2010) extended this analysis, providing another pair of mitochondrial and nuclear genes and posited nuclear-mitochondrial incompatibility as
a common mechanism in species formation. In another twist, Anderson et al.
(2010) demonstrated the existence of alleles with depressed hybrid fitness in lowglucose environments, which argues for a model in which neutral changes in
paired genes are followed by strong selection, a sequence of events that promotes
rapid reproductive isolation. Kao et al. (2010), however, argue against the existence of a small number of so-called speciation genes, instead claiming that
genome scans provide no evidence of any single paired dominant or recessive
genic incompatibilities. They instead argue that following WGD, many changes in
loci of little effect resulted in lowered fitness due, in part, to the rewiring of
transcriptional and metabolic networks.
Another debate that has emerged in this field is whether these changes are due
primarily to the decrease in the fertility of hybrids (Xu and He 2011) or a decrease
in their viability (Greig 2008). This question ultimately amounts to a debate about
what stage in the yeast life cycle the genetic incompatibilities occur—sporulation
or clonal growth, and whether the decrease in fitness is the result of competition
for resources or offspring. The discontinuity between these ideas likely represents
an opportunity to explain speciation as a process across different genomic and
temporal scales, and we would speculate that the process of DM incompatibility
induces selection for the evolution of some form of prezygotic barrier.
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Duplicate retention and evolutionary models. In addition to such population
processes as speciation, WGD also altered many other aspects of the S. cerevisiae
lifestyle. For instance, several pairs of ohnologs have been shown to have
undergone various types of functional divergence, allowing the study of some of
the proposed mechanisms of duplicate divergence after duplication (Conant and
Wolfe 2008b). In an elegant series of experiments, van Hoof (2005) showed that
two ohnologs, ORC1 and SIR3, have distinct and non-overlapping functions (in
DNA replication and gene silencing, respectively). Strikingly, however, the mutual
ortholog of these genes from the non-WGD yeast S. kluyveri is able to complement
both functions, constituting a clear example of subfunctionalization. An apparently
similar case, involving the S. cerevisiae ohnolog pair GAL1 and GAL3, which
presently functions, respectively, as an enzyme and as a transcriptional regulator,
was complicated by the discovery of an adaptive conflict between the shared
regulator and enzymatic function of their ortholog in the non-WGD K. lactis.
Thus, although the K. lactis GAL1 gene does indeed serve the functions of both
GAL1 and GAL3 in S. cerevisiae, it does so in a suboptimal way, being unable to
tune its expression to both roles simultaneously (Hittinger and Carroll 2007). This
conflict illustrates an important point about subfunctionalization, namely that the
original neutral model of subfunctionalization proposed by Force and coauthors
(Force et al. 1999) is not the only possible mechanism for such functional partitioning (Des Marais and Rausher 2008). Other examples of divergence among
ohnologs where the mechanism of that divergence is less clear include ribosomal
proteins (Ni and Snyder 2001; Komili et al. 2007; Kim et al. 2009), glucose
sensors (Özcan et al. 1998), and glycolysis enzymes (Boles et al. 1997).
The dosage balance hypothesis (DBH). In addition to facilitating the above
work, the wealth of functional data from S. cerevisiae also provides an excellent
opportunity to test hypotheses explaining the differences in gene retention patterns
after WGD and small-scale duplications (hereafter SSD). Chief among these is
probably the DBH (Papp et al. 2003; Freeling and Thomas 2006; Birchler and
Veitia 2007; Freeling 2009), which states that, in eukaryotes, there is selection
operating to disfavor duplications of central network genes due to the imbalance in
network stoichiometry that results. This situation is reversed for WGD because in
that case, the loss of a second copy of a gene introduces imbalances relative to the
remaining duplicated genes. In keeping with the DBH, several classes of genes are
over-retained after several evolutionarily ancient WGD events, including that in
yeast. They include ribosomal proteins, protein kinases and transcription factors
(Seoighe and Wolfe 1999; Blanc and Wolfe 2004; Maere et al. 2005; Aury et al.
2006; Conant and Wolfe 2008a). Similarly, genes that tend to have been fixed by
WGD are less likely to have undergone SSD in other yeast species (Wapinski et al.
2007). However, duplicates produced by WGD have more protein interactions
(Guan et al. 2007; Hakes et al. 2007), more phosphorylation sites (Amoutzias et al.
2010), and tend to be highly expressed (Seoighe and Wolfe 1999) than those from
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SSD. Although genes retained in duplicate after WGD are rarely essential on an
individual basis (Guan et al. 2007), this dispensability appears to be due to
functional compensation by the other ohnolog (DeLuna et al. 2008). Thus, it
appears that while ohnologs are less likely to be essential than their SSD counterparts today, their ancestral genes were actually at least as essential as current
single-copy genes (DeLuna et al. 2008).
System-level changes produced by WGD. Of course, one of the unique features of
polyploidy relative to SSD is the possibility of coordinated changes among multiple
sets of ohnologs. At the simplest level, we have previously illustrated examples of
what appears to be network subfunctionalization where a number of ohnologs collectively divided two expression domains among themselves (Conant and Wolfe
2006). A more complex and interesting example is the role of the WGD (Piškur et al.
2006) in shaping S. cerevisiae’s propensity for aerobic glucose fermentation
(the Crabtree effect; Geladé et al. 2003; Johnston and Kim 2005), a novel and
somewhat paradoxical phenotype. There is a general association between the presence of the WGD and the Crabtree effect across yeast species (Merico et al. 2007).
As a result, we and others have argued that dosage effects among the glycolysis
enzymes post-WGD helped to increase flux through glycolysis (Blank et al. 2005;
Kuepfer et al. 2005; Conant and Wolfe 2007; Merico et al. 2007; van Hoek and
Hogeweg 2009). Such increased flux likely could only be accommodated through
fermentation pathways, given the complex spatial organization of the competing
respiratory pathway (Conant and Wolfe 2007). Supporting this hypothesis is an
elegant computational analysis by van Hoek and Hogeweg (2009) showing that
future WGD events in modern S. cerevisiae could also be expected to provide a
selective advantage in glucose-rich environments through the preferential retention
of duplicated glycolysis enzymes. Note that the apparently ‘‘wasteful’’ fermentation
can actually be selectively advantageous in the context of rich but ephemeral
resource patches (Pfeiffer et al. 2001; Pfeiffer and Schuster 2005), a phenomenon that
has been experimentally confirmed in yeast (MacLean and Gudelj 2006). Such a
change in the yeast lifestyle likely led to other, later changes in the genome.
One suggestive example concerns the decoupling of cytosolic and mitochondrial
ribosomal protein expression post-WGD (Ihmels et al. 2005). Prior to WGD, bakers’
yeast was likely similar to other yeasts in having a strong association in the
expression of the two types of ribosomal proteins. After WGD, however, cisregulatory element evolution diverged in the two groups of genes (Ihmels et al.
2005), allowing S. cerevisiae to express only cytosolic proteins at high levels during
fermentation, an important refinement in a fermentative lifestyle.
Connecting the DBH to large-scale evolutionary changes following WGD,
Conant (2010) and Fusco et al. (2010) found transcriptional regulatory motifs to be
over-retained in ohnologs. Modeling network evolution after WGD, these authors
find the network enriched for transcription factors and particular network motifs.
Duplicated transcription factors still show some relics of the WGD, being more
likely to share targets than are random transcription factors, but on the whole show
considerable divergence post-WGD (Conant 2010). Given this rapid regulatory
evolution, it may not be easy to ascertain the role of WGD in the evolution of
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the modern S. cerevisiae regulatory network. Nonetheless, the retention of many
transcription factors that have acquired distinct sets of target genes may imply that
the WGD served to ‘‘relax’’ the regulatory complexity of this organism, which
may have implications for its future ability to adapt (as seen for the GAL1/GAL3
example).
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Acknowledgments We would like to thank Michaël Bekaert, Patrick Edger, and Chris Pires for
helpful discussions. This work was supported by the National Library of Medicine Biomedical and
Health Informatics Training Fellowship [LM007089-19] (CMH) and the Reproductive Biology
Group of the Food for the twenty-first century program at the University of Missouri (GCC).
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The S. cerevisiae WGD has been implicated in a number of evolutionarily complex
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Book Title
Polyploidy and Genome Evolution
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Chapter Title
Two Rounds of Whole-Genome Duplication: Evidence and Impact on the Evolution of Vertebrate Innovations
Copyright Year
2012
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Abstract
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Departament de Genètica, Facultat de Biologia
Organization
Universitat de Barcelona
Address
Av. Diagonal, 643, edifici Prevosti, 2a planta, 08028, Barcelona, Spain
Email
canestro@ub.edu
The origin and evolution of the vertebrates have been linked to the study of genome duplications since Susumo
Ohno ventured the 2R-hypothesis, suggesting that the successful diversification of complex vertebrates was
facilitated by polyploidization in the stem vertebrate ancestor due to two rounds of whole-genome duplication
(2R-WGD). This chapter first reviews evidence supporting Ohno’s 2R-hypothesis and gathers information
about the timing and mechanisms underlying the 2R-WGD. Second, this chapter describes the impact of the
2R-WGD on the evolution of the vertebrate genome structure, gene number, and the evolutionary dynamics
of the functional fate of vertebrate ohnologs (paralogous genes that originated by WGD) in comparison with
non-vertebrate chordate gene homologs. Finally, this review discusses the functional consequences of the
2R-WGD on the origin and evolution of vertebrate innovations compared with urochordates and
cephalochordates, paying special attention to the origin of neural crest cells, placodes, and the big complex
brain, key features that probably facilitated the transition from ancestral filter-feeding non-vertebrate
chordates to voracious vertebrate predators. Currently available data, however, seem to suggest that these
putative key features were present to at least some extent in stem Olfactores; hence, the impact of the 2RWGD may not be related to the immediate origin of vertebrate innovations, but to the subsequent
diversification of a wide variety of complex structures that facilitated the successful radiation of vertebrates.
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Abstract The origin and evolution of the vertebrates have been linked to the study of
genome duplications since Susumo Ohno ventured the 2R-hypothesis, suggesting that
the successful diversification of complex vertebrates was facilitated by polyploidization in the stem vertebrate ancestor due to two rounds of whole-genome duplication
(2R-WGD). This chapter first reviews evidence supporting Ohno’s 2R-hypothesis and
gathers information about the timing and mechanisms underlying the 2R-WGD.
Second, this chapter describes the impact of the 2R-WGD on the evolution of the
vertebrate genome structure, gene number, and the evolutionary dynamics of the
functional fate of vertebrate ohnologs (paralogous genes that originated by WGD) in
comparison with non-vertebrate chordate gene homologs. Finally, this review
discusses the functional consequences of the 2R-WGD on the origin and evolution of
vertebrate innovations compared with urochordates and cephalochordates, paying
special attention to the origin of neural crest cells, placodes, and the big complex
brain, key features that probably facilitated the transition from ancestral filter-feeding
non-vertebrate chordates to voracious vertebrate predators. Currently available data,
however, seem to suggest that these putative key features were present to at least some
extent in stem Olfactores; hence, the impact of the 2R-WGD may not be related to the
immediate origin of vertebrate innovations, but to the subsequent diversification of a
wide variety of complex structures that facilitated the successful radiation of
vertebrates.
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Two Rounds of Whole-Genome
Duplication: Evidence and Impact
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Innovations
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C. Cañestro (&)
Departament de Genètica, Facultat de Biologia, Universitat de Barcelona,
Av. Diagonal, 643, edifici Prevosti, 2a planta, 08028 Barcelona, Spain
e-mail: canestro@ub.edu
P. S. Soltis and D. E. Soltis (eds.), Polyploidy and Genome Evolution,
DOI: 10.1007/978-3-642-31442-1_16, Springer-Verlag Berlin Heidelberg 2012
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The study of the origin and evolution of vertebrates, the subphylum to which we
belong, has stood at the crossroad between genome evolution and molecular
developmental biology since the late 1960s, when Susumo Ohno published his
famous work on Evolution by Gene Duplication and proposed his hypothesis about
the pivotal role of genome duplication in the origin of vertebrates and their
diversification (Ohno et al. 1968; Ohno 1970). Vertebrates comprise all animals
that have a backbone and include mammals, birds, reptiles, amphibians, fishes, and
agnathans—the jawless lampreys and hagfishes. Vertebrates together with
urochordates (tunicates) form the Olfactores, which together with cephalochordates (amphioxus or lancelets) constitute the chordates (Fig. 16.1). All chordates
share a common basic body plan at least during the larval stage of their life cycle,
consisting of a notochord running through a post-anal tail, with a dorsal hollow
nerve cord, longitudinal blocks of muscle along the notochord, and ciliated
pharyngeal gill slits (Brusca and Brusca 2002). Recent phylogenomic analyses
have dethroned cephalochordates from the long-assumed position as sister group
of the vertebrates; this position is now occupied by urochordates, which include
ascidians, larvaceans and thaliaceans (Wada et al. 2006; Oda et al. 2002; Bourlat
et al. 2006; Delsuc et al. 2006; Putnam et al. 2008) (Fig. 16.1).
Ohno’s 2R-hypothesis was based on comparative analyses of genome sizes and
isozyme complexity among chordate taxa. Ohno found that basally divergent
chordate subphyla had smaller genomes and less isozyme complexity than
vertebrate lineages. This observation led him to suggest that the combination of
tandem gene duplication and in particular an octoploidization event involving two
rounds of whole-genome duplication were key to the invertebrate–vertebrate
transition, and for the subsequent successful vertebrate diversification (Ohno et al.
1968).
Ohno was one of the pioneers in conceiving the evolutionary significance of
whole-genome duplication (see also Chap. 1, this volume). Ohno emphasized the
importance of gene duplication as probably the main source of raw genetic material
for the evolution of new gene functions (reviewed in Taylor and Raes 2004). In
Ohno’s classical model, one of the duplicated genes retains the original function
whereas its duplicate either disappears by accumulation of detrimental mutations
(called pseudogenization or nonfunctionalization) or it is preserved after gaining
advantageous mutations that confer positively selected novel functions (neofunctionalization) (Ohno 1970; Nowak et al. 1997; Force et al. 1999). The duplication,
degeneration, complementation hypothesis (or DDC model) suggests a third
possibility for duplicate gene preservation: subfunctionalization, the complementary partitioning of ancestral structural and/or regulatory subfunctions between two
duplicate genes, so that the sum of their functions provides at least that of the
original pre-duplication gene (Force et al. 1999). The DDC model predicts that
subfunctionalized genes will have lower pleiotropy than the original pre-duplicated
gene and lower evolutionary constraints, and thereby will be more permissive to the
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CHORDATES
OLFACTORES
UROCHORDATES
teleost fish
sharks - rays
amphibians
birds
mammals
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lamprey
VERTEBRATES
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amphioxus
ascidianslarvaceans
tetrapods
cartilaginous
vertebrates
agnathans
(jawless vert.)
R1
R2
R1+R2 gnathostomes
(jawed vert.)
R1+R2
3rd. Scenario:
R1+R2 after the agnathan-gnathostome split
?
2nd. Scenario: (Panvertebrate Quadruplication PV4)
R1+R2 prior to agnathan-gnathostome split
1st. Scenario:
R1 prior to agnathan-gnathostome split
R2 in stem jawed vert. prior to cartilagenous-bony vert. split
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Vertebrate Innovations:
Big Brain
Neural Crest Cells
Placodes
bony vertebrates
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accumulation of mutations that might confer novel functions. The acquisition of
new functions is favored if the duplication affects the entire genome at once, as
opposed to multiple individual gene duplications, because when entire gene
networks are duplicated, gene stoichiometry is maintained, and therefore deleterious gene dosage effects can be counteracted (Birchler and Veitia 2007, 2010; Van de
Peer et al. 2009; Makino and McLysaght 2010; see also Chap. 2, this volume).
Genes that originated by gene duplication are called paralogs. Genes that have been
duplicated via genome duplication, however, are a special type of paralogs referred
as ohnologs, a term suggested by Wolfe (2000) in honor of Ohno’s contribution.
This term is useful because of the special properties that ohnologs possess at their
birth compared to duplications that arise by other local mechanisms such as unequal
crossing-over, tandem gene duplication, or retrotransposition.
The sudden creation and evolution of ohnologs by the 2R-WGD that occurred
in the stem vertebrate lineage has been suggested as one of the potential key events
underlying the increase of morphological complexity, facilitating the acquisition
of genetic and developmental innovations of vertebrates (Shimeld and Holland
2000; Aburomia et al. 2003). Genome duplication doubles the number of genes,
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Fig. 16.1 Evolutionary tree of chordates representing the three possible scenarios for the timing
of the 2R-WGD and the origins for vertebrate innovations. Evidence suggests that the 2R-WGD
might have had a significant impact on the diversification (black star) of vertebrate innovations,
including structures derived from neural crest cells and placodes, as well as the development of a
big complex brain in the stem vertebrate. But whether the 2R-WGD was crucial for the
evolutionary origin of these structures remains unclear, and that the hypothesis that the origin of
these innovations dated back to at least stem olfactores (gray star) cannot be dismissed. (Lamprey
picture courtesy of Juan Pascual-Anaya)
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many of which have the chance of evolving ‘novel’ functions that might provide
new selectable advantages promoting species diversification (Lynch et al. 2001;
Van de Peer et al. 2009). Some vertebrate species that have undergone recent
polyploidization, such as the frog Xenopus laevis that experienced tetraploidization *40 million years ago (Hellsten et al. 2007), show a higher adaptability to a
variety of different environments, such as drought, salt, cold, and disease resistance, than closely related diploid species, such as Silurana tropicalis (for further
details on frog polyploidization see also Chap. 18, this volume). Interestingly, only
a limited number of retained ohnologs present evidence of neofunctionalization or
subfunctionalization in X. laevis, suggesting that additional selective mechanisms
might act on preserving gene duplicates that could promote species diversification
(Chain and Evans 2006; Semon and Wolfe 2008).
In addition to the evolutionary significance of novel fates of duplicate genes on
species biology, another mechanism that might contribute to species diversification
after genome duplication is reciprocal ohnolog loss between different populations, a
concept known as ‘divergent resolution’ that can lead to reproductive isolation
(Lynch and Force 2000). Reciprocal ohnolog loss is likely to occur in the period of
relaxed selection that duplicate genes experience while they are functionally
redundant (Werth and Windham 1991; Lynch and Conery 2000; Lynch and Force
2000; Scannell et al. 2006; Taylor et al. 2001; Semon and Wolfe 2007). The
divergent resolution of gene redundancies, such that one population loses one
ohnolog copy while the second population loses the other ohnolog copy, leads to
chromosomal restructuring such that gametes produced by hybrid individuals can be
completely lacking in functional genes for a duplicate pair. In addition to the
isolation due to reciprocal gene losses, this model can be further expanded to
isolation due to independent processes of gene duplicate subfunctionalization
between two populations, in which hybrids will lack one or more subfunctions
(Force et al. 1999; Lynch and Force 2000). Hence, large-scale reciprocal ohnolog
loss and independent subfunctionalization of ohnologs can be the cause of reproductive isolation of two populations after polyploidization, favoring genetic
divergence of these newly incipient future species. This hypothesis is supported by
the analyses of both fish and angiosperm lineages that have undergone polyploidization and include more species diversity (e.g. salmonids, catostomids, eudicots,
grasses) than their sister groups that did not go through polyploidization and include
a lower number of species (Nelson 1994; Ferris et al. 1979; Soltis et al. 2009).
Recent integrated approaches of comparative genomics and gene expression analyses in teleosts, however, provide limited evidence supporting the significance of
differential ohnolog loss in reproductive isolation and diversification (Kassahn et al.
2009) (see also Chap. 17, this volume).
Many studies have tackled the central question of whether or not the 2R-WGD
had a significant impact on the origin of vertebrate innovations and their subsequent
diversification. This chapter first reviews evidence supporting the 2R-hypothesis
and information regarding the timing and potential mechanisms underlying the
2R-WGD in vertebrates. Second, this chapter examines the impact that the
2R-WGD may have had on the evolution of vertebrate genome structure, number of
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16.2 Supporting Evidence for the 2R-Hypothesis
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If two rounds of genome duplication (2R-WGD) have occurred, we would expect
the presence of many paralogs (ohnologs) in conserved, syntenic genomic regions,
which are known as paralogons (Coulier et al. 2000) (or ohnologons (Gout et al.
2009)). Paralogons, therefore, consist of series of linked (but frequently functionally and phylogenetically unrelated) genes on one chromosome region, many
of which have linked paralogs on at least one other chromosome region. The
discovery of paralogy groups made of four paralogons in the genome of human and
mouse was interpreted as remnants of the two events of tetraploidization that
occurred early during vertebrate evolution and therefore provided the earliest
strong evidence supporting Ohno’s 2R-hypothesis (Lundin 1979, 1993; Pebusque
et al. 1998). Possibly one of the best and first examples of a paralogy group
supporting the 2R-hypothesis is the case of the four Hox-bearing regions on human
chromosomes Hsa2, Hsa7, Hsa12, and Hsa17 (Fig. 16.2) (Kappen et al. 1989;
Bailey et al. 1997; Larhammar et al. 2002; Lundin et al. 2003). In contrast, only a
single Hox cluster is present in the cephalochordate amphioxus (Garcia-Fernàndez
and Holland 1994). This 4:1 ratio is consistent with Ohno’s hypothesis of two
tetraploidization events after the cephalochordate–vertebrate split (Holland et al.
1994; Sidow 1996; Garcia-Fernandez 2005). In addition to the Hox paralogy
group, several other similar examples have been identified (e.g. MHC (Katsanis
et al. 1996), Tbx (Ruvinsky and Silver 1997), G-protein-coupled receptors
(Fredriksson et al. 2003), ParaHox clusters (Ferrier et al. 2005), linked receptor
tyrosine kinases (Siegel et al. 2007), endothelin ligands and receptors (Braasch
et al. 2009), Fox cluster (Wotton and Shimeld 2006), and the EGF ligand paralogons (Laisney et al. 2010)).
Several databases of conserved syntenic chromosomal regions between different
species are available, and these provide additional evidence of two rounds of
genome duplication. Popovici et al. (2001), for instance, identified 14 paralogons
containing more than 1600 genes assembled in a human genome paralogy map
(http://u119.marseille.inserm.fr/Db/paralogy.html). The ParaDB (http://abi.mars
eille.inserm.fr/paradb) predicted that the human genome far exceeds 1000 paralogons that contain more than three pairs of duplicated genes (Leveugle et al. 2003).
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genes, and the fate of retained ohnologs in comparison with non-vertebrate chordate
paralogs. Finally, this chapter discusses how the 2R-WGD might have affected the
origin and evolution of vertebrate innovations, with special emphasis on the vertebrate big complex brain, and structures derived from neural crest cells and placodes. Recent data, however, suggest that neural crest cells and placodes could
already have been present in stem chordates. Hence, the impact of the 2R-WGD
cannot be related to the origin of neural crest cells and placodes, but it could be
related to their subsequent diversification and development of a wide variety of
complex structures.
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Genomic Synteny Conservation
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(b)
Hsa2
R2
HOX-D cluster
THSD7B
UPP2
ITGB6FIGN
COBLL1
GRB14
HOXD1
OSBPL6
HOXD9 PLEKHA3
HNRNPA3
CDCA7HOXD10 DFNB59
FKBP7
PDE1A
SP9SP3 CHN1
FRZB
NEUROD1
ATF2 EVX2
SCRN3
STK17B
IGFBP2 VIL1
ERBB4
ABCA12 IGFBP5 TNS1
BZW1
HECW2 FAM126BICA1L
HOXB1
HOXB3
HOXB5
HOX-B cluster HOXB6
ADAP2
CCT6B
MYO1D
ERBB2RAPGEFL1
CDK12GRB7 IGFBP4
NEUROD2
FKBP10
NT5C3L
HOXB7
ARL4D
NFE2L1 HOXB8
TMEM106A
CRHR1ITGB3SP2 HOXB9
FAM117A
OSBPL7
ETV4SOSTHDAC5
SNX11
SCRN2
MPP2
CALCOCO2
MMD
PRKAR1A
AC113554.1
AMZ2
TTYH2
NACA2
GNA13WIPI1 SDK2
FAM20A
ITGB4
DNAH17
USP36
CYTH1
FOXK2
FSCN2 RAC3
ACTG1 RFNG
MAFG
Hsa7
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2R-WGD: R1
TTYH3 FOXK1
SP4 DFNA5
AMZ1
CREB5PLEKHA8
FSCN1 TMEM106BTSPAN13 DNAH11 NFE2L3
CDK13 STK17APPIAIGFBP1ABCA13
EGFR
ARL4ABZW2
LFNG
TAX1BP1
RAC1GLCCI1
FAM20C MAFK SDK1 WIPI2USP42
SCIN
ITGB8 MPP6SNX10
EVX1 CHN2 AQP1
POU6F2
HECW1
ADCY1 UPP1
CCT6A
PRKAR1B
GNA12 ACTB
ETV1
SCRN1
CRHR2
PDE1C
DPY19L1
INHBA
FAM126A
HOXA1
NACADTNS3FIGNL1
DGKB
NT5C3
SFRP4
SOSTDC1 RAPGEF5
HNRNPA2B1 HOXA3 FKBP14NEUROD6
COBL
AMPH GLI3 MYO1G
MMD2 CYTH3ICA1
ADAP1
IGFBP3 GRB10
HOXA4
SP8 OSBPL3
THSD7A HDAC9 CDCA7L
HOXA5
HOXA6
HOXA7
HOXA9
HOXA10
HOXA11
HOXC11
HOXC9
HOXC8
GLI1
HOXC6
AQP5
ERBB3 INHBE TSPAN31
AQP2
SP1 HOXC5 PDE1B DGKA
INHBC
NFE2
MIP
AVIL
HDAC7ADCY6
BIN2
CALCOCO1
NACA
DPY19L2
NEUROD4
TENC1
POU6F1
ITGB7 HNRNPA1
RAPGEF3
SP7 ATF7
IGFBP6
Hsa12
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HOX-A cluster
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HOX-C cluster
Fig. 16.2 Conserved synteny in the vertebrate genome generated by the 2R-WGD. a Simplified
representation of a genomic region that has been amplified by the 2R-WGD (R1 and R2) showing
conserved genes (colored boxes) in four syntenic regions (a–d), which have suffered genomic
rearrangements and gene loss (white boxes) and different degrees of conservation (green and red
lines label ohnologs preserved in two or more than two regions, respectively). b Representation of
the four human paralogons containing Hox A-D clusters in chromosomes Hsa2, Hsa7, Hsa12, and
Hsa17, displaying high amounts of conserved synteny between ohnologs in two (green lines) or
at least three (red lines) different paralogons. This representation has been generated using a 100gene sliding window in the Synteny Database (Catchen et al. 2009)
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The Genomicus database v60.01 (http://www.dyogen.ens.fr/genomicus) predicted
that 18,228 ancestral vertebrate genes were grouped in 2,642 conserved ancestral
synteny blocks with a median N50 size of 5 genes (Muffato et al. 2010). The Synteny
Database (http://teleost.cs.uoregon.edu/synteny_db) predicted 231 paralogy clusters with more than 5 genes, and 102 paralogy clusters with more than 10 genes, in a
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more rigorous count using a 100-gene sliding window and taking amphioxus genes
as the outgroup for paralogy assignment in human (Catchen et al. 2009, 2011).
Until recently, however, available evidence did not permit us to discard the
possibility that these groups of paralogous genes originated by multiple independent block duplications, rather than two duplications of the entire genome
(Skrabanek and Wolfe 1998; Wolfe 2001; Larhammar et al. 2002). An initial
hypothesis, based on extensive phylogenetic analysis and dating of the duplications that produced hundreds of vertebrate gene families, proposed a ‘big-bang
mode’ of sudden large-scale gene origin resulting from two waves of gene
duplications, rather than the alternative hypothesis of a constant generation by
small-scale duplications (Gu et al. 2002). Wave-I was suggested to consist of
tandem or segmental duplications that occurred after the mammalian radiation, and
wave-II was interpreted as a rapid increase of paralogs in the early stage of
vertebrate evolution after their split from non-vertebrate chordates, consistent with
one round of whole-genome duplication (Gu et al. 2002). The first analyses of the
human genome draft led to the conclusion that the most parsimonious explanation
of the current structure of the human paralogons was a ‘big-bang’ expansion event
by a paleopolyploidy that included the whole genome or substantial sections of it.
However, no specific evidence was found for two rounds of polyploidy as opposed
to one (Venter et al. 2001; McLysaght et al. 2002; Panopoulou et al. 2003).
Recently, however, several analyses have provided definitive support for the
2R-hypothesis (reviewed in Kasahara 2007). Dehal and Boore (2005) developed
an elegant, compelling approach to test the 2R-hypothesis by plotting the genomic
map position of only those genes that were duplicated prior to the fish–tetrapod
split, which rendered a clear global physical pattern of four-way paralogon organization covering most of the human genome. Dehal and Boore’s work therefore
provided unmistakable evidence of two distinct rounds of genome duplication
during early vertebrate evolution.
Furthermore, the recent sequencing of the whole genome of the chordate
amphioxus Branchiostoma floridae (sister to all other chordates) provided even
more indisputable evidence supporting the 2R-hypothesis (Putnam et al. 2008).
Despite the fact that small-region comparison between human, chicken, teleost
fish, and amphioxus genomes revealed low gene-order conservation at the local
level (microsynteny), striking extensive gene linkage conservation was observed
when entire chromosomes were considered (macrosynteny). Syntenic analysis
reconstructed 17 chordate linkage groups (CLG) that might represent the protochromosomes of the last common chordate ancestor (Putnam et al. 2008).
Exhaustive evaluation of the 17 CLGs revealed that most of the human genome
(112 segments spanning 2.68 Gb, which is the equivalent of 95 % of the
euchromatic genome) was affected by large-scale duplication events that occurred
on the stem vertebrate lineage before the teleost/tetrapod split. Analysis of the
distribution of the human segments among the 17 CLGs showed that nearly all
ancient chordate chromosomes were quadruplicated (Putnam et al. 2008)
(Fig. 16.3). This result robustly demonstrated the occurrence of two rounds of
genome duplication, corroborating previous lines of evidence based on analysis of
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16.2.1 Timing of the Vertebrate 2R-WGD
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Analyses of the completely sequenced genome of the cephalochordate amphioxus
(Putnam et al. 2008; Holland et al. 2008) and the genomes from various urochordates
(Dehal et al. 2002; Small et al. 2007; Denoeud et al. 2010) validated Ohno’s
hypothesized lower-bound timing for the 2R-WGD as after the split between vertebrates and non-vertebrate chordates. Regarding the upper-bound timing, extensive
analysis of gene duplicates (Robinson-Rechavi et al. 2004) and the identification of
the four clusters in the genome of the elephant shark suggested that the 2R-WGD took
place before the cartilaginous/bony vertebrate split (Venkatesh et al. 2007; Ravi et al.
2009).
Within this time window, the most prevalent hypothesis suggests a scenario in
which the first round (R1) occurred before the split between gnathostome and
jawless vertebrates, and the second (R2) occurred in the stem jawed vertebrates
after their divergence from jawless vertebrates (Fig. 16.1). However, a second
scenario proposes that both rounds (R1 ? R2) of genome duplication took place
before the split between gnathostome and jawless vertebrates (pan-vertebrate
quadruplication (PV4) hypothesis (Kuraku et al. 2009)) (Fig. 16.1). Comparative
analysis of 55 gene families revealed a common expansion in both jawless and
jawed vertebrates, which has been interpreted as evidence supporting this second
scenario (Kuraku et al. 2009). Available information from sea lampreys and
hagfish does not permit us to discern between these two hypothetical scenarios,
because these organisms also appear to have suffered lineage-specific duplications
and reciprocal gene losses compared to vertebrates, which together obscure the
assessments of orthology/paralogy (reviewed in Kuraku 2008, 2010). For instance,
multiple Hox gene surveys in different species of sea lampreys and hagfish
suggested that extensive independent duplications of Hox genes might have
occurred during the evolution of jawless vertebrates (Pendleton et al. 1993;
Sharman and Holland 1998; Takio et al. 2004; Force et al. 2002; Irvine et al. 2002;
Fried et al. 2003; Stadler et al. 2004; Kuraku et al. 2009). Some of the jawless Hox
clusters might have disintegrated, casting doubt as to the usefulness of Hox genes
as reliable markers to trace duplications during genome evolution in stem vertebrates (Kuraku 2011). Finally, recent phylogenetic analysis of the degenerated
ParaHox cluster in hagfish has opened the possibility of a third scenario, in which
both rounds (R1 ? R2) occurred in stem jawed vertebrates after their divergence
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specific regions of interest, such as the Hox-bearing regions (Garcia-Fernàndez and
Holland 1994; Larhammar et al. 2002) and the major histocompatibility regions
(Vienne et al. 2003; Danchin and Pontarotti 2004). Spring (1997) proposed the
term ‘‘tetralogs’’ to refer groups of quadruplicated vertebrate genes at four
different chromosomal locations formed by the 2R-WGD corresponding to a single
invertebrate gene, with all four more similar to each other than to members of the
other tetralogy group.
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Fig. 16.3 Quadruplicated conserved syntenic pattern between the amphioxus and the human
genome as a result of the 2R-WGD. Dot-plots display the distribution throughout human
chromosomes (y-axes) of human orthologs (blue dots) of amphioxus genes (black dots) located in
two arbitrarily selected genomic regions of approximately 1 Mb (a) and 10 Mb (b) (x-axes). The
dot-plots reveal four major human chromosomes (yellow shadow) of conserved synteny as the
product of the two rounds of whole-genome duplication. In panel (a), the four paralogons
coincide with the Hox-cluster bearing chromosomes 2, 7, 12, and 17, whereas in panel B the four
paralogons coincide with the endothelin receptors and ParaHox-cluster bearing chromosomes
2/5, 4, 13, and X. The dot-plots were generated as described in Canestro et al. (2009) using the
Synteny Database (Catchen et al. 2009)
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16.2.2 Mechanisms Underlying the Vertebrate 2R-WGD
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A question that still remains is how did the stem vertebrate genome become
octoploid by two rounds of tetraploidization. There are two main mechanisms of
tetraploidization observed in many species of plants and animals (Van de Peer et al.
2009). The first mechanism is allotetraploidy, which occurs when two related but
not identical genomes are combined by hybridization of closely related species and
associated (often subsequent) genome duplication. In the case of allotetraploidy, the
pairs of distinct ‘homologous’ chromosomes that are sufficiently different due to
their separated origin are called homeologs. The second mechanism is autotetraploidy, which occurs when the genomes are not sufficiently diverged into
homeologous sets; autotetraploidy therefore ranges from the combination of
genomes of two conspecific individuals (perhaps from different populations) to the
combination of identical genomes from a single individual. The genetic attributes
of allo- and autotetraploids differ and may have substantial effects at individual,
population, and species levels (see also Chap. 2, this volume). Both allotetraploidy
and autotetraploidy could be generated by several processes such as: (i) an
abnormal non-disjunction of sister chromatids at meiosis; (ii) the uncoupling of
mitotic DNA replication and cell division during early development of the germ
line (this process, for instance, occurs normally during the endoreplication of
megakaryocytic bone-marrow precursors of blood platelets, or during the development of the oikoblastic epithelia that secrete the house in basal urochordate
larvaceans); (iii) potential cell fusion during early embryo development or in germline precursors in syncytial gametogenesis (cell fusion is observed naturally, for
instance, in skeletal muscle cells and placenta) (reviewed in Storchova and Pellman
2004; Shemer and Podbilewicz 2000).
In the case of allotetraploids, each pair of homologous chromosomes should
segregate normally during meiosis, and genetic interchange between homeologous
chromosomes is rare. If two consecutive events of allotetraploidization occurred in
stem vertebrates, we would predict that in the ideal situation in the absence of gene
losses, a phylogenetic tree of homeologs will render a symmetrical (A,B) (C,D)
topology (Furlong and Holland 2002).
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from jawless vertebrates (Furlong et al. 2007) (Fig. 16.1). The validation of this
third scenario could have a significant impact on our understanding of vertebrate
evolution, because it would imply that the 2R-WGD would have not been
important for the origin of vertebrate innovations (i.e. big brain, neural crest cells,
and placodes, which clearly exist in jawless vertebrates (Kuratani and Ota 2008;
Kuratani 2009)). According to this third scenario, however, the 2R-WGD would
have been important for the radiation of gnathostomes into cartilaginous fish, bony
fishes, and tetrapods. A solid answer about the timing of the 2R-WGD may have to
wait until larger-scale comparisons of the whole-genome organization of hagfish
and lampreys are available.
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16.3 Consequences of the 2R-WGD on the Evolution
of Vertebrate Genome Structure
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It has been suggested that polyplodization events, at least in plants, can trigger
genomic stress associated with major genomic rearrangements, in many cases
mediated by a burst of mobilization of transposable elements (Matzke and Matzke
1998; Comai 2000). Transposable elements can be substrates for unequal and
illegitimate recombination and can be responsible for a variety of genome
reorganizations associated with the transposition, including chromosomal insertions, deletions, inversions, translocations, and duplications. Lineage-specific
genome rearrangements mediated by transposable elements might facilitate rapid
evolution, reproductive isolation of different populations, and consequently species
diversification (Parisod et al. 2010).
Contrary to possible genome reorganization after polyploidization, as noted
above for plants, in stem vertebrates, recent work based on proximate gene pair
methods and measurement of syntenic clustering conservation found that the 2RWGD in vertebrates were not followed by an increase of genome rearrangement
(Hufton et al. 2008). Unexpectedly, this work measured massive genome rearrangements prior to the 2R-WGD, which has been interpreted as a pre-existing
‘disposition’ toward genomic structural change (Hufton et al. 2008). Interestingly, in contrast to the archetypal condition that has been described in the
organization of particular genomic regions (e.g. Hox-cluster region (GarciaFernàndez and Holland 1994)), the amphioxus genome structure is not exceptionally well conserved, evolving its own particular type of repetitive elements
(e.g. ‘mirage’ minisatellites (Cañestro et al. 2002b; Ebner et al. 2010)), undergoing extensive local tandem gene duplications (see section below), and experiencing a moderate rate of synteny loss similar to that of sea urchin or sea
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In autopolyploids, however, meiotic pairing might occur between any of the four
identical chromosomes at meiosis I, facilitating genetic interchanges freely among
the four alleles, and leading to ‘tetrasomic inheritance’. Eventually the alleles, and
chromosomes, might diverge, starting a process of diploidization that reestablishes
diploidy. Randomly one of the chromosomes will diverge first and no longer form
homologous structures, while the other three will keep pairing until another further
divergence. Hence, if two consecutive events of autotetraploidy occurred in quick
succession (pseudo-octoploidy) in stem vertebrates, we would predict that in the
absence of gene losses, gene family phylogenetic trees will likely render
asymmetrical (((A,B),C),D) topologies (Furlong and Holland 2002). Because many
vertebrate gene families do render asymmetrical tree topologies (Friedman and
Hughes 2001; Hughes 1999; Hughes and Friedman 2003), two quick consecutive
events of autotetraploidy have been considered a likely mechanism for the 2R-WGD
in stem vertebrates (Furlong and Holland 2002; Lynch and Wagner 2009).
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anemone (Hufton et al. 2008). Therefore, the amphioxus genome structure
cannot be considered a fossil genome representing the pre-duplication condition,
at least in terms of genome structure (Garcia-Fernàndez et al. 2001; Hufton et al.
2008), although it is far less divergent from the vertebrate genome structure than
is any known urochordate genome (Dehal et al. 2002; Denoeud et al. 2010;
Louis et al. 2012).
There have been several attempts to infer the karyotype and genome structure
from common chordate ancestors and to reconstruct the evolutionary history
leading to present chromosome structures. The first comparisons of conserved
syntenic associations in different vertebrate karyotypes, using an in silico chromosome painting approach, allowed reconstructions of the ancestral vertebrate
genome containing 10–13 ancestral proto-chromosomes (Kohn et al. 2006;
Nakatani et al. 2007). Recently, the sequencing of the amphioxus genome has
allowed researchers to reconstruct the ancestral chordate genome as consisting of
17 conserved syntenic blocks, which might represent the ancient chordate protochromosomes (Putnam et al. 2008).
After the 2R-WGD, under the naive assumption of absence of loss or fusions of
chromosomes, we would expect 68 (17 9 4) proto-vertebrate segments, but
parsimonious reconstruction of chromosome history revealed that numerous
chromosomal fusions and translocations have occurred. These reconstructions
predict at least 20 fusions that led to 37–49 chromosomes in the bony vertebrate
ancestor, which became 12 chromosomes in the stem teleost ancestor due to many
additional fusions, and 33–45 chromosomes in the stem tetrapod ancestor due to at
least 4 fusions shared between human and chicken genomes (Putnam et al. 2008;
Naruse et al. 2004; Nakatani et al. 2007). An excellent example of chromosomal
rearrangement after the 2R-WGD has been recently provided by a phylogenetic
analysis of members of the four Hox paralogons that resulted in a (B(A(C,D)))
topology. These results suggest that two chromosomal rearrangements between
protochromosomes 11 and 4, and 7 and 5 occurred after the clusters duplicated but
before the diversification of extant vertebrates 450 million years ago (Lynch and
Wagner 2009). These chromosomal rearrangements resolve conflicting data
regarding the order of linked genes and support the hypothesis that the 2R-WGD
occurred by two consecutive events of autotetraploidy, and thereby the ancestral
vertebrate might have been ‘‘pseudo-octoploid’’. Interestingly, the asymmetrical
(B(A(C,D))) topology of the vertebrate Hox cluster (Lynch and Wagner 2009)
contrasts with the symmetrical (A,B) (C,D) topology inferred from the cartilaginous elephant shark using the amphioxus Hox cluster as the outgroup (Ravi et al.
2009). Further extensive analyses including HoxA-D clusters from a broader
representation of cartilaginous and bony vertebrates will be required to resolve
these conflicting topologies, which could suggest that the Hox-cluster rearrangement took place after the cartilaginous/bony vertebrate split and not immediately
subsequent to the 2R-WGD.
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16.4.1 Function of Gene Duplicates After 2R-WGD
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After polyploidization, a period of transilience may follow in which genes might
enjoy extra ‘degrees of freedom’ to mutate without selective penalty (reviewed in
Soltis and Soltis 1999; Otto 2007). Understanding the processes by which genome
duplication might influence the fate of duplicated genes is crucial to evaluate how
the 2R-WGD might have impacted the evolution of vertebrate innovations.
Neofunctionalization and subfunctionalization are the two main processes driving
the functional fate of newly generated ohnologs after the 2R-WGD and have been
extensively discussed in the literature (Hughes 1994; Force et al. 1999; Lynch and
Conery 2000; Durand 2003; Postlethwait et al. 2004; Hoekstra and Coyne 2007;
Conant and Wolfe 2008; Semon and Wolfe 2007, 2008; Jimenez-Delgado et al.
2009). A prominent example of neofunctionalization related to the 2R-WGD
occurred during the expansion of the vertebrate retinoic acid receptor (RAR)
family, which acquired new functions in both their expression domains and in their
structural protein activities (Escriva et al. 2006). There are also examples,
however, in which neofunctionalization and subfunctionalization are related to
both 2R-WGD and local tandem duplications (e.g. the expansion of the vertebrate
globin superfamily, which promotes the vertebrate innovation related to oxygen
transport and storage (Hoffmann et al. 2011)), or merely related to local tandem
duplications, and not the 2R-WGD, such as the expansion of the vertebrate
Alcohol Dehydrogenase (Adh) family, which promotes the acquisition of new
enzymes for the synthesis of retinoic acid (Cañestro et al. 2000, 2002a, 2003b).
Therefore, not all vertebrate innovations can be exclusively attributed to the
2R-WGD, and the global weight of the impact of the 2R-WGD on the evolution of
vertebrate gene functions remains unknown.
391
In many cases, neofunctionalization and subfunctionalization can be due to
alterations in cis-regulatory elements that might lead to adaptative changes in
duplicated genes (Force et al. 1999). Many of the cis-regulatory elements appear to
be embedded in distinct repeat families, especially in transposable elements (TE)
(Thornburg et al. 2006; Polak and Domany 2006; Bourque et al. 2008). Analysis of
the distribution of 10,000 TEs in the human genome, for instance, revealed that
most TEs are concentrated under strong purifying selection near regulatory and
developmental genes (Lowe et al. 2007). Most of the described examples of TE
mobilization and rewiring of gene regulatory networks have been associated with
relatively recent events of TE mobilizations. For instance, TE-mediated rewiring
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for neofunctionalization after gene duplication has been recently described for the
sex-determining gene dmrt1bY in medaka fish, in which a novel regulatory
element driving a negative feedback on dmrt1bY has been acquired due to the
insertion of an Izanagi transposon (Herpin et al. 2010), or for the origin of a novel
gene regulatory network dedicated to pregnancy in placental mammals, which was
due to a transposition of the MER20 TE (Lynch et al. 2011).
A massive expansion of TEs appears, therefore, as a powerful mechanism that
could boost a vast redeployment of cis-regulatory elements into new gene
regulatory networks (Feschotte 2008), promoting large-scale events of neofunctionalization and subfunctionalization (van de Lagemaat et al. 2003; Bennetzen
2005; Bejerano et al. 2006). Polyploidization can trigger the mobilization of
transposable elements (Matzke and Matzke 1998; Parisod et al. 2010), because
recently duplicated genomes contain many redundant genes and substantial
repetitive DNA, which serve as buffer against TE insertional mutagenesis (Matzke
et al. 2000). According to this expectation, bursts of TE mobilization have been
described after polyploidization in different organisms (Matzke and Matzke 1998;
Comai 2000; SanMiguel et al. 1996, 1998).
A question that remains unclear is whether there was or was not a massive
TE mobilization after the 2R-WGD that could have favored a significant
redeployment of cis-regulatory elements into new gene regulatory networks in the
stem vertebrate lineage. Recent comparison of the diversity and content of TEs
between vertebrates and amphioxus has provided some insights that might help to
answer this question (Canestro and Albalat 2012). The dynamics of the TE content
within a genome follows a competition model in which the expansion of a
particular TE might cause the reduction of other types of TEs, consequently
reducing the TE diversity, until a new equilibrium that preserves the functionality
of the genome is reached (Abrusán and Krambeck 2006). According to this model,
if a massive expansion of TEs occurred after the 2R-WGD, we expect that the
diversity of TEs shared among vertebrates should be smaller than in cephalochordates. Consistent with this prediction, a recent comparative study reveals that
the shared TE diversity of vertebrates (14 superfamilies in lampreys, 28 in
ray-finned fishes, 20 in amphibians, 14 in reptiles, 10 in birds, and 15 in mammals)
is lower than the TE diversity in amphioxus (33 superfamilies), which makes
plausible the hypothesis that a TE burst could have occurred after the 2R-WGD in
the stem vertebrate lineage (Canestro and Albalat 2012). Further comparative
genomic analysis between different vertebrates and cephalochordates will be
required to test this hypothetical burst of TEs, and especially to evaluate its
putative impact on the evolution of gene functions after the 2R-WGD.
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While several studies focus on the functional fate of retained gene duplicates, less
attention has been paid to how losses of paralogs or ohnologs might impact the
evolution of the functions of other genes (reviewed in Cañestro et al. 2007). Loss
of one copy of two fully redundant gene duplicates should not usually have
significant impact, but loss of one of the paralogs after functional divergence likely
has evolutionary consequences. Recent analyses of gene losses by comparative
genomics have led to the unexpected finding that significant components of the
developmental toolkit might be lost without major changes to the body plan
(Cañestro and Postlethwait 2007; Holland 2007), which suggests the presence of
compensatory mechanisms or the acquisition of innovations that have preserved
unaltered the ancestral condition (Cañestro et al. 2007). Tracing the evolution of
gene families throughout ancestral proto-chromosomes using blocs of conserved
synteny has become a powerful tool to clarify uncertain phylogenies, to detect
ohnologs gone missing (OGM) (Postlethwait 2007; Catchen et al. 2009, 2011), to
provide robust assessments of orthology and paralogy between different species,
and to discern evolutionary innovations from losses of ancestral features in sister
lineages (Canestro et al. 2009).
There are cases in which different ohnologs in different species acquire the same
expression pattern, which has been called function shuffling (McClintock et al.
2001) and synfunctionalization (Gitelman 2007), and in some cases the convergence
of expression patterns between paralogs can be related to OGM (Postlethwait 2007;
Canestro et al. 2009). The evolution of the vertebrate retinaldehyde dehydrogenease
Aldh1a family provides a paradigmatic example of how uncovering the evolution of
gene family members through the 2R-WGD has been fundamental to illuminating
how gene functions evolve among newly generated paralogs after genome duplications in the face of loss of ohnologs (Canestro et al. 2009). For instance, analysis
of conserved synteny revealed that the presence of Aldh1a1 in tetrapods and its
absence in teleost fish was not due to a tetrapod innovation, but to an OGM in the
teleost stem lineage, which was accompanied by a re-acquisition of ancestral
functions by surviving paralogs (Canestro et al. 2009). Medaka provides a more
radical example in which aldh1a2, the only survivor of the aldh1a family in this
species, recapitulates the expression pattern of all other aldh1a paralogs that have
been lost in medaka. This result is in agreement with a model of functional evolution
in which surviving genes re-acquire ancestral gene family roles in the face of loss of
ohnologs. Other examples that illustrate the importance of identifying OGMs
ohnologs are shown in the endothelin and agouti systems, in which the exclusive
presence of endothelin 4 (edn4) and the agouti-signaling protein 2 genes (asip2a/
b) in teleost fish was not due to a fish innovation related to the teleost-specific wholegenome duplication, but instead to a loss of ohnologs that originated in the 2R-WGD
in the tetrapod lineage (Braasch et al. 2009; Braasch and Postlethwait 2011). To
understand acquisition of functions of vertebrate ohnologs that were generated by
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on Surviving Ohnologs
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16.5 Consequences of the 2R-WGD on Vertebrate Gene
Number and Functional Evolution
How many of the genes that were part of the original fourfold increase in genes
generated by 2R-WGD in the stem vertebrate have actually survived nonfunctionalization? And importantly, how significant have the functional consequences of
those retained genes been for promoting the origin and evolution of vertebrate
innovations? Estimates on gene retention in other organisms that have experienced a
WGD have reported *13 % retention over *100 million years (MY) in yeast
(Wolfe and Shields 1997), *72 % in maize over *11 MY (Ahn and Tanksley 1993;
Gaut and Doebley 1997), and *77 % in Xenopus over *40 MY (Hellsten et al.
2007). In vertebrates, a *33 % retention of divergent functional genes after the
2R-WGD over *500 MY was inferred initially based on theoretical models applied
to 270 gene families of the human genome (Nadeau and Sankoff 1997). More recent
and broader analyses based in the complete catalog of human ohnologs estimated a
rate of retention between 20 and 30 % (Putnam et al. 2008; Huminiecki and Heldin
2010; Makino and McLysaght 2010).
But how can we assess the impact of the 2R-WGD on the origin of vertebrate
complex features? A naive approach to estimating this impact could be to perform a
comparison of the total number of retained paralogs and their distribution among
functional categories in vertebrates and non-vertebrate chordates that did not undergo
any WGD since their split from our last common chordate ancestor. Comparison of
the gene catalog of the three chordate subphyla (i.e. cephalochordates, urochordates,
and vertebrates) has allowed us to identify a lower bound of 8,437 gene families with
members that descend from a single gene in the last common chordate ancestor
(Putnam et al. 2008). Through subsequent genome or local duplication, these families
account for 13,610 amphioxus genes, 13,401 human genes, and 7,216 ascidian genes,
the latter being a significantly lower number due to the extensive gene losses that have
occurred in urochordate lineages (Dehal et al. 2002; Cañestro et al. 2003a; Edvardsen
et al. 2005; Denoeud et al. 2010). Although it is frequently true that the multiple
ohnologs of a vertebrate gene family are represented by a single gene in amphioxus,
the total number of paralogs derived from a single-copy gene in the last common
ancestor is surprisingly similar between amphioxus (13,610) and human (13,401)
(Putnam et al. 2008). Therefore the mere total numbers of retained genes after the
2R-WGD duplicates might not be the key to explain the gain of complexity during the
evolution of the vertebrate lineage in comparison with amphioxus.
In vertebrates, analysis of the functional categories of the gene families that
have expanded after the 2R-WGD revealed that cell signalers and transcriptional
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the 2R-WGD, both the impact of the retention of neo- or subfunctionalized
ohnologs, as well as the impact of OGM, on the functions of other survivor gene
family members should be studied.
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regulators of developmental pathways are generally retained as multiple ohnologs
(Roux and Robinson-Rechavi 2008; Putnam et al. 2008; Hufton et al. 2008;
Huminiecki and Heldin 2010). Genes associated with basic cellular functions (i.e.
translation, replication, splicing, and recombination, with the important exception
of cell cycle), however, have been less successfully retained after the 2R-WGD
(Huminiecki and Heldin 2010) (although see Gout et al. (2009) for different results
in other organisms that have also undergone WGD). Analysis of the human
genome reveals that dosage-balance constraints act on the retention of ohnologs,
resulting in an enrichment of dosage-balanced genes, an observation predicted
following WGD (Birchler and Veitia 2007, 2010) and also reported for other
vertebrates, plants, and yeast (e.g., Paterson et al. 2006). Interestingly, many of
these retained ohnologs in humans are refractory to copy number variation, have
rarely experienced subsequent small-scale duplication, and are frequently associated with diseases related to dosage-imbalance such as down syndrome (Makino
and McLysaght 2010). Analysis of retained genes that have originated in vertebrates by local duplications revealed a strong underrepresentation of genes related
to cell communication, cell cycle, and embryo development (Huminiecki and
Heldin 2010).
In amphioxus, although a thorough analysis of amphioxus-specific gene family
expansions has not been performed, Table 16.1 shows an extensive list of
amphioxus-specific duplicated genes reported in the literature (this list is probably
not complete, and may be biased toward the research with which I am most
familiar). This list shows numerous retained duplicates from a broad array of
functional categories, including metabolic enzymes, members of transduction and
signaling cascades, members of the immunity system, as well as pivotal transcription factors of developmental pathways. Awaiting a more exhaustive analysis,
including different amphioxus species to infer the ancestral cephalochordate
condition, the list in Table 16.1 shows no obvious bias toward any particular
functional category, although it is noticeable that duplicated developmental transcription factors do not account for more than two paralogs (with the exception of
the eight hairy amphioxus paralogs (Minguillon et al. 2003)).
Remarkably, the main difference between the newly acquired paralogs in
amphioxus and ohnologs in vertebrates is the mechanism of duplication. While
approximately 25 % of the ancestral chordate gene families have two or more
ancient vertebrate ohnologs generated by the 2R-WGD, there is strong evidence that
most amphioxus paralogs originated by local tandem duplications rather than largescale chromosomal duplications (Table 16.1). Therefore, considering the functional
bias of retention of genes duplicated by WGD or local duplication, it is reasonable to
speculate that the key influence of the 2R-WGD promoting the successful diversification of vertebrate features resides in the fact that whole networks were
duplicated, in contrast to local duplications such as those that occurred in amphioxus, an organism that seems to have maintained morphological and genetic stasis
during the last 200 million years (Garcia-Fernàndez and Holland 1994; Cañestro
et al. 2002a; Somorjai et al. 2008; Canestro and Albalat 2012; Paps et al. 2012).
Duplication of whole gene networks is dosage-balanced and increases the
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Table 16.1 List of paralogs originated independently in the amphioxus lineage
Functional category
Gene
Number of
References
amphioxus paralogs
28
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C1q-like
LRR-containing
gene models
Hairy
Brachyury
bHLH
Emx
Hox13–14
Evx
Mnx
Uncx
Pou3
Lhx2/9
Iro
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Cañestro et al. (2006)
Albalat (2009)
Albalat et al. (2011)
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Transduction and
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Aldh1a
Cyp26
Hsd11b1
Rdh cluster
Bdh1
Rdh11/12
Hsd17b8
ApoD
Cyp2
Cyp11/24/27
Hsd3b
Fabp
Nos
Opsins
GPR54
GnRH
CRHR
Somatostatine
receptors
LGR7/8
PTHR
Secretin receptors
Estrogen receptors
NOK
ACK
TIE
MARTK
EXTK
HNK-Ras
Calmoduline-like
Andreakis et al. (2011)
Holland et al. (2008)
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Metabolic proteins
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Bridgham et al. (2008)
D’Aniello et al. (2008)
Bertrand et al. (2009)
Karabinos and
Bhattacharya (2000)
Holland et al. (2008)
Minguillon et al. (2003)
Holland et al. (1995)
Araki et al. (1996)
Minguillon et al. (2002)
Ferrier et al. (2000)
Ferrier et al. (2001)
Holland et al. (2008)
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evolvability to generate novel functions, which in the case of the vertebrate 2RWGD could have led to an increase in complexity of the signaling and developmental regulatory networks that facilitated the acquisition of innovations.
In addition to the evolutionary role of coding genes in the acquisition of innovations, microRNAs (miRNAs) also play crucial roles during development and have
been postulated as important players for the evolution of organismal complexity (Lee
et al. 2007; Sempere et al. 2006). Analysis of miRNAs in chordate species showed that
the 2R-WGD has increased the diversity of the inventory of miRNAs in vertebrates,
which correlated with the increase of complex patterns of tissue specificity of
miRNAs (Heimberg et al. 2008; Campo-Paysaa et al. 2011). However, the finding of
41 vertebrate-specific miRNA families, absent in non-vertebrate chordates, suggests
that their origin must have occurred in stem vertebrates after their separation from
urochordates and is not explained by the 2R-WGD (Heimberg et al. 2008). The
appearance of these 41 vertebrate-specific miRNA families has been proposed as a
potential key evolutionary force lying behind the dramatic increase of vertebrate
complexity (Heimberg et al. 2008). Future exhaustive analysis of the expression
patterns of the members of these 41 vertebrate-specific families, and an understanding
of their roles, will allow a reevaluation of the importance that this innovation could
have had on the origin of vertebrate features.
16.6 Consequences of the 2R-WGD on the Innovation
of Vertebrate Features
A major question not resolved yet is the precise impact of the 2R-WGD on the
innovation of particular vertebrate features. Three vertebrate features are perhaps
the most prominent innovations: derivatives from neural crest cells, sensory organs
concentrated in the head derived from ectodermal placodes, and a big complex
brain. When taken together, these features probably allowed the transition from
ancestral, peaceful, filter-feeding, non-vertebrate chordates to active, voracious,
vertebrate predators (Northcutt and Gans 1983; Gans and Northcutt 1983);
reviewed in Yu et al. 2008; Holland 2009) (Fig. 16.1).
Vertebrate neural crest cells are a transient population of developmental cells that
delaminate at the border of the neural plate through an epithelial–mesenchymal
transition, migrate, and differentiate at their final destination into a variety of
structures such as sensory neurons, glial cells, peripheral nervous system, pigment
cells, smooth muscle cells, connective tissue, cranio-facial cartilage, skeletal bones,
and teeth (Weston 1970). Vertebrate crest development depends on four crucial sets
of genes that form what is called the neural crest gene regulatory network
(NC-GRN): (1) patterning signal genes establish the expression of (2) neural plate
border specifier genes, which activate (3) crest specifier genes, which turn on (4)
neural crest effector genes that provide differentiated products (Meulemans and
Bronner-Fraser 2004, 2005; Ota and Kuratani 2007; Sauka-Spengler et al. 2007).
Analysis of the amphioxus genome has revealed the presence of cephalochordate
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orthologs from all of these four sets of genes, including (1): Fgf, Wnt, Bmp, Notch
Dlx, AP2, SoxB, Zic, and islet; (2): Pax3/7, Msx, Dlx5, and Zic; (3): Snail, SoxE,
AP2, Twist, Id, FoxD, and Myc; and (4): Rho, cRet, Erbb3, Mitf, tyrosinase, and
tyrosinase-related genes, with the remarkable exception of the tyrosine kinase c-Kit
essential for migration and survival of crest cells, and the gene for myelin protein
P0, consistent with the notion that the glial myelin sheath is a vertebrate innovation
(Meulemans and Bronner-Fraser 2007; Holland et al. 2008; Holland and Short 2008;
Nikitina et al. 2009).
The fact that most of the specifier genes are present as single copy in amphioxus,
but multiple paralogs in vertebrates, presumably due to the 2R-WGD, has led to the
hypothesis that neofunctionalization and subfunctionalization of paralogs may have
facilitated the co-option of ancestral genes into the NC-GRN (Sauka-Spengler et al.
2007; Meulemans and Bronner-Fraser 2007; Holland et al. 2008; Holland and Short
2008). Gene ontology (GO) analysis estimates that 91 % of the neural crest genes in
vertebrates have been co-opted from genes already present in basal metazoans,
while the remaining 9 % of the neural crest genes are vertebrate innovations
(Martinez-Morales et al. 2007), including the assembly of new signaling pathways
like the endothelin system (Braasch et al. 2009). The evolution of the vertebrate
NC-GRN, therefore, appears as the result of a combination of ancestral gene cooption, newly evolving genes, and amplification of these components by the 2RWGDs (Braasch et al. 2009).
Interestingly, however, the recent description in urochordates of neural crest-like
cells that express typical vertebrate crest marker orthologs, migrate, and differentiate into pigments challenges the idea that neural crest cells are a vertebrate
innovation (Jeffery et al. 2004, 2008; Jeffery 2007). Thus, it cannot be discounted
that some types of neural crest cells might have been present in the last common
ancestor of olfactores (urochordates ? vertebrates), followed by losses during the
significant morphological and genetic simplification suffered by urochordate
lineages (Seo et al. 2004; Cañestro et al. 2005; Cañestro and Postlethwait 2007;
Holland 2007). Therefore, it seems plausible to consider that the 2R-WGD might
have not been crucial for the origin of the neural crest cells, but the 2R-WGD might
have been important for increasing the evolvability of the NC-GRN and the
diversification of derivative structures.
Similar conclusions have been reached through studies of the gene regulatory
network underlying placode and brain development. Analysis of placode-marking
genes (e.g. Eye, Pitx, Six, and Pax) in ascidian and larvacean urochordates suggested
that the last common olfactore ancestor already presented multiple placode derivatives, such as olfactory and adenohypophyseal. Additional and independent
proliferation and loss of a variety of placodes probably occurred in both urochordate
and vertebrate lineages (Bassham and Postlethwait 2005; Mazet et al. 2005), in
some cases recruiting paralogs that had been independently duplicated in both
urochordates and vertebrates (Bassham et al. 2008).
Despite the fact that non-vertebrate chordates have a simple brain lacking a
midbrain and a midbrain–hindbrain organizer (MHB), most brain-making gene
orthologs are present in non-vertebrate chordates, suggesting that vertebrate brain
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features were built on a foundation already present in the ancestral chordate
probably facilitated by the new ohnologs created by the vertebrate 2R-WGD
(reviewed in Holland 2009). Recent analysis of developmental genes in the
ascidian brain revealed that the expression of Fgf8 can reorganize the expression
of other brain genes and transform hindbrain structures into an expanded
mesencephelon, recapitulating the organizing activity of the vertebrate MHB and
therefore suggesting that the MHB was already present at least in the last common
ancestor of olfactores (Imai et al. 2009). Analysis of urochordate genomes
revealed that important genes (i.e. Gbx) for the positioning of the MHB have been
lost in stem urochordates (Cañestro et al. 2005), as has the retinoic acid dependent
anterior–posterior axial patterning of the central nervous system (Cañestro et al.
2006), making plausible the hypothesis that the absence of midbrain in urochordates is not due to a vertebrate innovation of a midbrain, but a simplification in
urochordates of an ancestral tripartite brain structure (Cañestro and Postlethwait
2007; Cañestro et al. 2007). Evolutionary analysis of the origin of the complex
Nova-regulated splice variants of the vertebrate brain genes revealed that many of
these variants were already present in the last common olfactore ancestor (Irimia
et al. 2011). It is possible that the 2R-WGD promoted the increase of the
complexity of Nova-dependent splice variants in the vertebrate brain, although a
simplification of this system during urochordate evolution cannot be discarded.
In conclusion, it is likely that the origin of vertebrate features such as neural
crest cells, placodes, and a complex tripartite brain are not related to the 2R-WGD,
but that these features were already present to some extent in stem non-vertebrate
chordates (Fig. 16.1) (reviewed in Donoghue et al. 2008). However, it is likely that
the subsequent evolution of these three features has been strongly influenced by
the new ohnologs that originated after the 2R-WGD, due to processes of
neofunctionalization, subfunction partitioning and subsequent refinement,
recruitment of cis-regulatory elements driven by genome rearrangement and
transposable element activity, inventions of novel miRNA families, and evolution
of novel splice variants, which overall increased the complexity of duplicated
developmental gene regulatory networks after the 2R-WGD. Future integrative
analysis of comparative genomics, functional evo-devo, and examinations of gene
regulatory networks in a wide variety of non-vertebrate chordates as well as
basally divergent jawless vertebrates will help to narrow down the precise timing
of the 2R-WGD and evaluate its actual impact on the origin and evolution of
vertebrate features.
Probably the new ‘2R, or not 2R’ question (Hughes and Friedman 2003) is now
to ascertain whether the origins of vertebrate innovations were, or were not, the
consequence of the 2R-WGD, and to understand the mechanisms by which the
2R-WGD increased the evolvability of developmental gene regulatory networks
that facilitated the diversification of complex vertebrate features.
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Acknowledgments This work has been supported by grant BFU2010-14875 from the Ministerio
de Ciencia e Innovación (Spain). I would like to thank Julian Catchen for his generous support on
the Synteny Database, and Ingo Braasch, John H. Postlethwait, Ricard Albalat, and Adriana
Rodriguez for their valuable comments on the chapter, and cheerful and endless discussions on
‘‘2R, or not 2R, that is the question … on vertebrate innovations’’.
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Metadata of the chapter that will be visualized in
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Book Title
Polyploidy and Genome Evolution
Series Title
Chapter Title
Polyploidy in Fish and the Teleost Genome Duplication
Copyright Year
2012
Copyright HolderName
Springer-Verlag Berlin Heidelberg
Corresponding Author
Family Name
Postlethwait
Particle
Given Name
John H.
Suffix
Author
Division
Institute of Neuroscience
Organization
University of Oregon
Address
97403, Eugene, OR, USA
Email
jpostle@uoneuro.uoregon.edu
Family Name
Braasch
Particle
Given Name
Ingo
Suffix
Abstract
Division
Institute of Neuroscience
Organization
University of Oregon
Address
97403, Eugene, OR, USA
Email
ibraasch@uoneuro.uoregon.edu
Multiple rounds of whole-genome duplications (WGDs) punctuated the evolution of rayfin fish, a speciesrich group comprising about half of all vertebrates. Rayfin fish, along with lobefin vertebrates including
humans, derive from early vertebrate ancestors that evolved through two rounds of polyploidization (the first
and second rounds of vertebrate genome duplication, VGD1 and VGD2) at the dawn of the vertebrate lineage.
Furthermore, teleost fish underwent an additional round of polyploidization in their stem lineage, the teleost
genome duplication (TGD). Additional WGD events occurred independently in numerous species and higher
level taxa of teleosts and other rayfin fish, for example in salmonids, carp, and sturgeon, so that some fish
lineages experienced at least four rounds of WGD since the origin of vertebrates. This chapter provides an
overview of these polyploidization events in the fish lineage and focuses on the impact these genome
duplications (GD) had on genome evolution in selected fish taxa. We then review evidence for the TGD and
discuss its consequences for the evolution of gene content, order, and functions in the teleost lineage. We
argue that, although evidence remains sparse, the TGD may have had a profound influence on the evolutionary
success and the biodiversity of teleosts. Importantly, an in-depth understanding of the causes and
consequences of the TGD and other teleost GD events will help to inform us about the evolution of our own
paleopolyploid genome.
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Chapter 17
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Ingo Braasch and John H. Postlethwait
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Abstract Multiple rounds of whole-genome duplications (WGDs) punctuated the
evolution of rayfin fish, a species-rich group comprising about half of all vertebrates. Rayfin fish, along with lobefin vertebrates including humans, derive from
early vertebrate ancestors that evolved through two rounds of polyploidization (the
first and second rounds of vertebrate genome duplication, VGD1 and VGD2) at the
dawn of the vertebrate lineage. Furthermore, teleost fish underwent an additional
round of polyploidization in their stem lineage, the teleost genome duplication
(TGD). Additional WGD events occurred independently in numerous species and
higher level taxa of teleosts and other rayfin fish, for example in salmonids, carp,
and sturgeon, so that some fish lineages experienced at least four rounds of WGD
since the origin of vertebrates. This chapter provides an overview of these polyploidization events in the fish lineage and focuses on the impact these genome
duplications (GD) had on genome evolution in selected fish taxa. We then review
evidence for the TGD and discuss its consequences for the evolution of gene
content, order, and functions in the teleost lineage. We argue that, although evidence remains sparse, the TGD may have had a profound influence on the evolutionary success and the biodiversity of teleosts. Importantly, an in-depth
understanding of the causes and consequences of the TGD and other teleost GD
events will help to inform us about the evolution of our own paleopolyploid
genome.
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Polyploidy in Fish and the Teleost
Genome Duplication
2
I. Braasch J. H. Postlethwait (&)
Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA
e-mail: jpostle@uoneuro.uoregon.edu
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I. Braasch
e-mail: ibraasch@uoneuro.uoregon.edu
P. S. Soltis and D. E. Soltis (eds.), Polyploidy and Genome Evolution,
DOI: 10.1007/978-3-642-31442-1_17, Springer-Verlag Berlin Heidelberg 2012
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The problems of this world are only truly solved in two ways:
by extinction or duplication.
Susan Sontag
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In common usage, ‘‘fish’’ applies to any aquatic vertebrate that possesses gills and
fins (if any appendages) (Nelson 2006). Following this phenotypic definition,
‘‘fish’’ include jawless fish such as lampreys and hagfish, cartilaginous fish, such as
sharks and rays, lobefin fish including coelacanth and lungfish, and rayfin fish such
as teleosts. ‘‘Fish’’, however, are a paraphyletic assemblage because it excludes
tetrapod vertebrates, which share a more recent common ancestor with lungfish
than lungfish share with teleost fish. Until we tend to call tetrapods (including
ourselves) lobefin fish and accept that all living vertebrates are fish, the term ‘‘fish’’
should be used with caution (see Fig. 17.1).
This chapter focuses on the monophyletic group of rayfin fish, or Actinopterygii, and, in more detail, their largest subgroup, teleost fish. The jawless,
cartilaginous, and lobefin fish, however, are important for understanding evolution
of the vertebrate genome by whole-genome duplications (WGDs) at the dawn of
the vertebrate lineage and are thus featured in Chap. 12 (this volume).
Among actinopterygian fish, the teleosts are by far the largest subgroup in terms
of extant species numbers. Nelson (2006) lists 26,891 living rayfin fish species
(453 families), of which only 51 species (5 families) are not teleosts. The teleosts,
in contrast, comprise 26,840 living species (448 families), an impressive 99.8 % of
all rayfins. Even more impressive, adding all living vertebrates to the calculation
gives around 50 % of all vertebrates being teleost fish.
Draft genome assemblies of ten rayfin species (all of them clupeocephalan
teleosts) are currently publicly available (Fig. 17.1): zebrafish (Danio rerio), threespined stickleback (Gasterosteus aculeatus), medaka (Oryzias latipes) (Kasahara
et al. 2007), fugu (Takifugu rubripes) (Aparicio et al. 2002), spotted green pufferfish (Tetraodon nigroviridis) (Jaillon et al. 2004), Atlantic cod (Gadus morhua)
(Star et al. 2011), and four species of East African cichlids, including tilapia
(Oreochromis niloticus). More species such as platyfish (Xiphophorus maculatus),
Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), and the nonteleost spotted gar (Lepisosteus oculatus; see below) are soon to be added to this
list (Fig. 17.1), and numerous additional rayfins will be sequenced as part of the
GENOME 10K Project (Haussler et al. 2009).
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17.1 A Brief Introduction to ‘‘Fish’’
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In their great numbers and degree of anatomical diversity, the
modern ray-finned fishes may be considered the most
successful of all vertebrates.
Robert L. Carroll
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Spotted green pufferfish
Tetraodon nigroviridis
Medaka
Oryzias latipes
Platyfish
Xiphophorus maculatus
Atlantic cod
Gadus morhua
SaGD
Salmonids
Zebrafish
Danio rerio
TGD
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rayfin fish
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East African cichlids
clupeocephala
teleosts
Three-spined stickleback
Gasterosteus aculeatus
percomorpha
Japanese pufferfish
Takifugu rubripes
Carps + Goldfish
CGD
D
Goldeye
Hiodon alosoides
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Bowfin
Amia calva
Spotted gar
Lepisosteus oculatus
AGDs
VGD1
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Bichirs
Tetrapods
Lungfish
lobefin fish
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Coelacanth
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Jawless fish
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0 MYA
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Fig. 17.1 A phylogeny of all kinds of ‘‘fish’’. Tree topology and timing of divergence events
follows data from Setiamarga et al. (2009) and www.timetree.org. The figure shows the timing of
genome duplication (GD) events (VGD1/VGD2 the vertebrate GDs, TGD the teleost GD, AGDs
acipenserid GDs, SaGD the salmonid GD, CGD the carp GD). Whether the second vertebrate
genome duplication (VGD2) occurred before or after the divergence of jawless fish remains
controversial. MYA million years ago
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The species richness of rayfin and teleost fish poses the question of the secret of
their evolutionary success. A recurring pattern of genome evolution in rayfin fish is
their propensity to polyploidization. In line with this idea, a positive correlation of
genome size and species richness has been reported among actinopterygians as
well as for specific teleost clades (Mank and Avise 2006a).
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17.2 Duplications in the Crown: Polyploidy in Rayfin Fish
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17.2.1 Twigs and Leaves: Polyploidization Events in Fish
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In comparison to plants, polyploidizations are rare in the animal kingdom (Muller
1925), yet the reasons for this genomic discrepancy between fauna and flora
remain controversial (Mable 2004). In the vertebrate lineage, polyploids are most
often found among amphibians and rayfin fish (Mable et al. 2011; Otto 2007;
Comai 2005). Factors that may favor the proclivity of rayfin fish to generate
surviving polyploid lineages compared to other groups of vertebrates (e.g., high
production rate of unreduced gametes, their propensity for hybridization, genomic
flexibility, etc.) have been extensively discussed recently elsewhere, yet a clear
explanation remains elusive (Mable et al. 2011). Given that polyploid events also
appear to have occurred in some jawless, cartilaginous, and lobefin fish (see
Leggatt and Iwama 2003 and references therein), it may be worth considering that
a paucity of genome duplication (GD) is an amniote vertebrate-specific phenomenon. Several works provide comprehensive lists of known cases of polyploidy in
fish (Otto and Whitton 2000; Leggatt and Iwama 2003; Le Comber and Smith
2004; Mable et al. 2011).
Here, we give an overview of the few general trends that can be inferred from
the phylogenetic distribution of polyploidization events and will then discuss
several specific cases in more detail.
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Despite large variations in genome size, rayfin fish have surprisingly conserved
karyotypes, with the majority of diploid genomes having 48 or 50 chromosomes
(Ohno et al. 1968; Mank and Avise 2006b); thus, chromosome counts have been
used as indicators of polyploidizations in fish. A study based on chromosome
counts of 615 rayfin species concluded that at least 7–20 polyploidization events
occurred in the evolution of extant rayfins (Mank and Avise 2006b); this estimate
is likely an underestimate because less than 3 % of all living rayfin species were
included in this study.
Polyploidization events in fish are phylogenetically restricted, i.e., are unevenly
distributed across the actinopterygian tree. Some groups, especially those occupying early diverging branches in rayfin phylogeny, and among teleosts particularly the Ostariophysi (including carps and suckers and many others), seem to be
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particularly prone to polyploidization in their lineage, while other groups,
including more derived lineages such as the percomorphs (perch and relatives, like
pufferfish, stickleback, cichlids and many more), have few or no GDs after the
teleost genome duplication (TGD) (Leggatt and Iwama 2003; Mable et al. 2011).
Polyploidizations have occurred in many individual fish genera, species, and
populations (i.e., the ‘‘leaves’’ of the rayfin tree), as for example in multiple species
of barbs (genus Barbus, family Cyprinidae) (Chenuil et al. 1999), in the cyprinid
Squalius alburnoides species complex (Alves et al. 2001), in the pond loach
(Misgurnus anguillicaudatus) (Li et al. 2011) and other loach species (Cobitidae,
order Cypriniformes) (Ferris and Whitt 1977a), and in the stinging catfish
(Heteropneustes fossilis, order Siluriformes) (Pandian and Koteeswaran 1999). On
the other hand, piscine GD events have also occurred in the last common ancestor
of higher level taxa (some of the tree’s ‘‘twigs’’). All members are polyploid in the
three families Salmonidae (salmonids) (Allendorf and Thorgaard 1984), Callichthyidae (armored catfish) (Oliveira et al. 1992), and Catostomidae (suckers) (Ferris
1984; Uyeno and Smith 1972). Common carp and goldfish (subfamily Cyprininae)
also arise from a shared tetraploid origin (Ohno et al. 1967; David et al. 2003;
Schultz 1980; Larhammar and Risinger 1994). Among rayfins diverging before
teleosts, the Acipenseridae (sturgeons) are famous for their multiple ploidy levels,
up to at least hexadecaploid (Birstein et al. 1997; Ludwig et al. 2001). In addition,
artificial polyploids, particularly triploids, are routinely produced for multiple
commercially important fish species to induce sterility and thereby improve somatic
growth rate (Piferrer et al. 2009).
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17.2.2 Piscine Polyploidy and the Evolution of Genome Function:
Salmon, Carp, and Calandinos
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17.2.2.1 Salmonids
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Traditional studies of polyploidy in fish generally involve the analysis of karyotypes and allozymes. Only recently, with the advent of genome sequencing
techniques, we have begun to understand some of the evolutionary footprints left
by polyploidization events in fish genomes.
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Salmonids (salmon, trout, whitefish, grayling) are among the fish of highest economic importance (Davidson et al. 2010). Salmonids have been suspected to be
polyploids since the 1940s (Svärdson 1945; Kupka 1948), but it was several
decades until it was generally accepted that all living salmonids are of autotetraploid origin due to a GD event that occurred in their stem lineage 25–100 million
years ago (Ohno et al. 1968; Allendorf and Thorgaard 1984). This WGD is
unlikely to have occurred by segmental duplications because all segments of each
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chromosome appear to be in duplicate (although of course not every gene in each
segment is still present in duplicate); if segmental duplications had been responsible, then some chromosome segments would be present in three or five copies.
Salmonids still show multivalent chromosomes during meiosis as well as tetrasomic inheritance of some loci (Allendorf and Thorgaard 1984; Phillips and Rab
2001) and are thus considered pseudotetraploids, i.e., the process of diploidization
has not yet concluded in these fish (Danzmann et al. 2008).
The autotetraplodization in salmonids is sometimes referred to as ‘‘4R’’,
because it is the fourth round of WGD in this lineage since the rise of the vertebrate lineage (see later this chapter and Chap. 16, this volume). This numerating
terminology, however, imposes difficulties to distinguish independent GD events
in various lineages of rayfins as discussed above. Therefore, we suggest using the
term salmonid genome duplication (SaGD). Recent progress in salmonid genomics
(Davidson et al. 2010; Miller et al. 2011) has provided early insights into the
evolutionary dynamics of the salmonid genome after the SaGD.
In line with tetraploidization in a salmonid ancestor, the diploid genomes of
most salmonids contain 96–104 chromosome arms. The Atlantic salmon is a
special case among salmonids, because its more derived karyotype has been
secondarily reduced to 72–74 chromosome arms by chromosome fusions (Phillips
and Rab 2001; Phillips et al. 2009). Conserved synteny analysis comparing genetic
linkage maps of Atlantic salmon and rainbow trout (Danzmann et al. 2008; Lien
et al. 2011) and analyzing the genomic distribution of conserved non-coding
elements (CNEs) (Moghadam et al. 2009) show first that gene order in salmonids
generally reflects the inferred ancestral rayfin karyotypes (Nakatani et al. 2007;
Kasahara et al. 2007), and second that two salmonid homeologous chromosomes
share conserved synteny with one teleost outgroup chromosome (e.g., medaka,
zebrafish, and stickleback), as would be expected from an additional round of GD.
The duplication of hox gene clusters, generally a good indicator for GDs in
animals (see Chap. 16, this volume), has been studied in detail in salmonids.
Salmonid genomes contain at least 13 hox clusters, nearly twice as many as most
diploid teleost genomes (Moghadam et al. 2005a, b; Mungpakdee et al. 2008a).
Given that gene loss (nonfunctionalization, or pseudogenization) is the most frequent fate of gene duplicates (Lynch and Conery 2000), it is surprising that the
salmonid hox cluster repertoire, as judged by collapsing duplicated clusters, shows
no loss of hox paralogy groups between the ancestral teleost and today’s Atlantic
salmon genome, which has retained more hox paralogy groups than any other
studied teleost (Mungpakdee et al. 2008a). After the SaGD, several hox paralogs
evolved asymmetrically, as manifested by pseudogenization, elevated rates of
molecular evolution, and/or divergence of non-coding regions in one of the two
hox gene duplicates. This asymmetry is also apparent over entire hox clusters
(Mungpakdee et al. 2008a). Overall, however, expression domains of duplicated
Atlantic salmon hox genes are conserved with their unduplicated orthologs in other
teleosts, although salmon hox paralogs show evidence for quantitative subfunctionalization (Mungpakdee et al. 2008b), a predicted type of functional evolution
of duplicate genes (Force et al. 1999).
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17.2.2.2 Carp and Goldfish: Facilitated Domestication by Genome
Duplication?
An allotetraploidization occurred in a shared ancestor of common carp (Cyprinus
carpus; 2n = 100) and goldfish (Cyprinus carpio; 2n = 100). The carp genome
duplication (CaGD) occurred between 11 and 21 million years ago (Risinger and
Larhammar 1993; Ohno et al. 1967; Larhammar and Risinger 1994; David et al.
2003; Yuan et al. 2010). Evidence for the hybrid (i.e., allotetraploid) origin of
these polyploids comes from the absence of quadrivalents (Ohno et al. 1967) and
the disomic inheritance of genetic markers in view of the short time period since
the GD event (David et al. 2003). Independent chromosomal rearrangements took
place in goldfish and carp after the allotetraploidization (Ohno et al. 1967). Around
52–60 % of genomic loci of the common carp are still duplicated (David et al.
2003; Ferris and Whitt 1977b), but duplicate retention seems to be considerably
lower in goldfish (Woods and Buth 1984). As in salmonids, several duplicates of
hox cluster genes have become pseudogenes after the CaGD (Luo et al. 2007;
Yuan et al. 2010).
Like salmonids, the common carp and its relatives are important economic
species in aquaculture as food sources and as ornamental species. Common carp
and goldfish are the oldest domesticated fish species, bred in central Europe for
*2,000 years and in China for *1,000 years, respectively (reviewed in Balon
2004). During domestication of carp and goldfish, significant morphological
change has been fixed in populations compared to their wild ancestors in a short
period of time, as exemplified by the multiple, independent loss of scales in the
carp (Balon 2004), the color morphs of koi carp (David et al. 2004), and the
selection for monstrosities such as telescope eyes, lion-head, or fin loss in ornamental goldfish (Komiyama et al. 2009).
The molecular basis of the strong response to artificial selection in carp and
goldfish has rarely been explored. Based on the multigenic inheritance of coloration
in ornamental koi carp, it has been suggested that the propensity to generate the
many color morphs may be related to the tetraploidization of carps (David et al.
2004). Is it possible that allopolyploidization provided carp and goldfish with a high
degree of genomic flexibility similar to domestic polyploid plants (see Chaps. 7 and
10, this volume)—made them particularly responsive to morphological selection
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Overall, around 50 % of SaGD-duplicated loci have been retained in salmonids
(Allendorf and Thorgaard 1984). Although purifying selection appears to be the
prevalent mode of salmonid gene duplicate evolution, asymmetric divergence of
salmonid gene duplicates was also observed in an analysis of EST sequences when
compared to the diploid Northern pike (Esox lucius) as outgroup (Koop et al. 2008;
Leong et al. 2010). The asymmetric pattern of molecular evolution among paralogs, observed particularly often for DNA binding proteins, is thought to be caused
by relaxed functional constraints on one of the two paralogs after the SaGD (Leong
et al. 2010; Mungpakdee et al. 2008a) as for GDs in general.
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17.2.2.3 Gene Expression Regulation in Triploid Calandino
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and domestication? A recent study of the common carp suggests that the duplication
of the fgfr1a gene during the course of the CaGD may have permitted the breeding
of ‘‘mirror’’ forms that lack almost all scales (Rohner et al. 2009). Importantly, two
different domesticated forms of mirror carp have been bred by selection of two
different loss-of-function mutations in the same paralog, fgfr1a1 (Rohner et al.
2009).
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Although polyploidization events in salmonids and carp are fairly recent events on
the evolutionary timescale, they are nevertheless too old to study changes in gene
expression pattern following soon after polyploidization and hybridization in fish
and the interplay of these two factors. The relationship of polyploidization and
hybridization, however, has been analyzed in naturally occurring populations of an
Iberian cyprinid, the calandino (S. alburnoides).
The allopolyploid S. alburnoides species complex consists of di-, tri-, and
tetraploid fish of different genomic compositions derived from interspecific
hybridizations of a paternal ancestor (the A genome) and different, geographically
separated, maternal genome contributors, the southern S. pyraneicus (P genome)
or the northern S. caroliterii (C genome) (Alves et al. 2001; Pala et al. 2010).
Expression studies of seven genes in different adult tissues revealed that dosage
compensation occurs in polyploids, so that overall gene expression is reduced to
the diploid level (Pala et al. 2008, 2010). In S. alburnoides, gene dosage compensation is accomplished by a complex pattern of gene copy silencing in a genespecific and tissue-specific manner: for example, in southern triploids of the PAA
genome composition, some genes are expressed only from the A genome, others
from P and A genomes; some tissues express only A genes, while others express
both P and A genes; some genes are expressed from A in one tissue, but from P and
A in another tissue (Pala et al. 2008, 2010).
The gene expression patterns also depend on genomic composition: while the
A genome allele is dominant in southern individuals, C and A alleles are
co-dominant in northern polyploids (CAA, CCA, CCAA, or C-A-) (Pala et al. 2010).
Although the exact molecular mechanism leading to dosage compensation in the
calandino remains elusive at this point, these studies illustrate the potential these
animals provide for the study of gene regulation in diverging polyploid populations.
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Although recent polyploidization events appear to be comparatively common in
rayfin fish compared to other vertebrate clades, the most important polyploidization event for genome evolution in fish took place on the branch leading to the
teleosts, the largest clade of rayfin fish. Initial arguments for and against the
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17.3.1 Evidence for the Teleost Genome Duplication
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occurrence of such a third round of vertebrate WGD (alias 3R, fish-specific genome duplication, FSGD, or teleost genome duplication, TGD) have been overcome
by sequencing the genomes of several teleost species.
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17.3.1.2 Teleost Hox Gene Clusters
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Initially, the branch leading to the teleosts did not raise suspicion of polyploidization because the ancestral vertebrate karyotype, like that of teleosts, was initially
inferred to be 48 chromosomes (Ohno et al. 1968).
A general observation since the rise of allozyme data, however, was that many
enzyme loci with a single locus in tetrapods appeared to have multiple copies in
fish (see Morizot 1990 and references therein). With the advent of the zebrafish
and other teleosts as model organisms for developmental and genetic studies in the
1990s and the accompanying cloning of teleost DNA sequences, this impression of
‘‘more genes in fish’’ (Wittbrodt et al. 1998) was substantially reinforced.
Three alternative explanations were initially considered to explain these
observations: (1) higher frequencies of individual or local or tandem gene duplication in teleosts than in tetrapods; (2) higher retention of gene duplicates from the
ancestral vertebrate genome duplications (VGD) in teleosts than in tetrapods; or
(3) a WGD in the rayfin fish lineage after the divergence from tetrapods (Morizot
1990; Postlethwait et al. 1998; Wittbrodt et al. 1998; Meyer 1998; RobinsonRechavi et al. 2001).
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Sequencing the hox gene clusters, which had already suggested the earlier VGDs
(see Chap. 16, this volume), settled the argument toward the teleost-specific GD
hypothesis: since the cloning of the first teleost hox genes in the late 1980s (Eiken
et al. 1987; Njolstad et al. 1988), it became apparent that hox genes were no
exception to the generalization of ‘‘more genes in fish’’ and additional paralogs of
hox genes were found in different teleosts (e.g., Misof and Wagner 1996; Aparicio
et al. 1997; Prince et al. 1998). The genomes of tetrapods and other non-teleost
vertebrates generally have four hox gene clusters (Graham et al. 1989). The discovery that hox genes are actually organized in seven gene clusters in the zebrafish, that two copies of the hoxA cluster were present in fugu, and that each
duplicated hox cluster in fugu was orthologous to a single duplicated copy in
zebrafish provided the first significant support that a GD had occurred in the
lineage leading to zebrafish and that the event was shared by fugu (Amores et al.
1998; Aparicio et al. 1997). Importantly, the hox clusters and genes closely linked
to the hox clusters were found to be distributed over eight different chromosomes
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in zebrafish, as would be expected if a WGD increased the hox cluster number
from four to eight, followed by the loss of one of the seven hox complements
(Amores et al. 1998). Seven or eight hox gene clusters have since been identified in
several other teleosts (see e.g. Prohaska and Stadler 2004; Hoegg and Meyer 2005;
Hoegg et al. 2007, and references therein).
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Although the genomic location of hox gene clusters and linked gene families could be
best explained by a WGD in the lineage leading to teleosts (Meyer and Schartl 1999),
several alternative possibilities were discussed, including: (1) that the seven hox
clusters in zebrafish were due to polyploidization specific to the zebrafish lineage
(Stellwag 1999); (2) that hox cluster-containing chromosomal segments had been
amplified in teleosts by local duplications (Elgar et al. 1999); and/or (3) that teleosts
had an unusually high gene duplication rate (Robinson-Rechavi et al. 2001).
Phylogenetic analyses of numerous duplicated zebrafish genes, however,
including those not related or linked to the hox genes and those distributed all over
the genome, provided evidence for their origin after the divergence of zebrafish
and tetrapod lineages (Taylor et al. 2001; Van de Peer et al. 2001). Additional
work on specific gene families was also in line with this hypothesis [e.g. mitf
(Altschmied et al. 2002), midkines (Winkler et al. 2003), sox9 (Cresko et al. 2003),
egfr (Gomez et al. 2004), pomc (de Souza et al. 2005), and receptor tyrosine
kinases (Braasch et al. 2006)].
At the same time, putting more and more genes on the genetic maps of zebrafish
(Gates et al. 1999; Postlethwait et al. 2000; Woods et al. 2000, 2005) and medaka
(Naruse et al. 2004) provided additional evidence for a teleost WGD beyond just
the duplication of hox cluster-bearing chromosomes.
Importantly, the inclusion of gene sequences from the Japanese pufferfish
(T. rubripes) revealed that these fish-specific gene duplications within multigene
families usually date back to a point during rayfin fish evolution before the
divergence of zebrafish and pufferfish (Amores et al. 1998; Taylor et al. 2003).
Therefore, it became apparent that the GD shared by these teleost fish most likely
occurred somewhere along the branch to the teleost fish; but did it occur after the
origin of teleosts? Did it include non-teleost rayfin fish? Or did it include all
teleosts and only teleosts?
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The sequence of the fugu (T. rubripes) genome was the second vertebrate and the
first teleost fish draft genome assembly (Aparicio et al. 2002). The authors were
cautious about making conclusions from the Takifugu genome assembly with
respect to the TGD hypothesis, stating that the distribution of gene duplicates in
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the Takifugu genome indicated segmental or large-scale, but not tandem, duplications (Aparicio et al. 2002).
Two follow-up large-scale analyses, however, came to the conclusion that the
distribution and age of gene duplicates in the Takifugu genome were indeed
consistent with the TGD hypothesis (Vandepoele et al. 2004; Christoffels et al.
2004). Both studies found that the majority of gene duplicates in Takifugu were
located in paralogons, i.e., chromosomal blocks that share paralogous syntenies
within the genome. Also, molecular clock-based analyses of gene families containing fish-specific gene duplicates helped to date the duplication event and hinted
at their occurrence around the origin of the teleost lineage.
In contrast to the genome assembly of Takifugu, the genome assembly of the
spotted green pufferfish (T. nigroviridis) was anchored onto chromosomes, which
made possible a more detailed analysis of conserved synteny blocks within a
teleost genome (Jaillon et al. 2004). The publication of the Tetraodon genome
assembly put the TGD beyond doubt, by showing that internal paralogy and
chromosomal blocks of doubled conserved synteny extended genome-wide within
this pufferfish genome. Doubled conserved synteny refers to a human chromosomal region sharing conserved synteny with two pufferfish paralogons (see Sect.
17.3.2.2). These patterns of conserved synteny also led to reconstructions of
ancestral karyotypes (Jaillon et al. 2004).
With the publication of the medaka genome assembly, patterns of conserved
synteny within and between the genomes of medaka, pufferfish, and zebrafish,
using human as an outgroup, allowed more refined reconstruction of ancestral, preWGD karyotypes (see Sect. 17.3.2.5) (Kasahara et al. 2007).
Formal publications of the zebrafish and stickleback genome assemblies are
pending, but analyses of these two species (as exemplified in Sect. 17.3.2) overall
confirm observations made from the genomes of pufferfish and medaka: the
occurrence of a WGD in the lineage leading to the teleosts is the most parsimonious explanation of all available genome data.
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Genome analyses of zebrafish, pufferfish, medaka, and stickleback pointed to a GD
event somewhere in the rayfin fish lineage prior to the divergence of these teleosts,
but when exactly did the event occur? Is it, for example, a trait shared only by
clupeocephalan teleosts, to which the aforementioned sequenced species belong
(Fig. 17.1)? Or did it happen in an ancestor of all teleosts? Or even earlier, before
the rise of the teleost lineage and as such represents a trait shared with earlier
diverging rayfin branches, such as those leading to bowfin, gar, sturgeon, or bichir?
Of course, the phylogenetic timing of the TGD is essential for understanding any
possible causal relationships between the TGD and the teleost radiation. This
question is further complicated by difficulties in establishing historical relationships of various rayfin fish lineages diverging basal to the teleosts as well as of the
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17.3.2 The TGD and Gen(om)e Evolution in Teleosts
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basal relationships within teleosts themselves based on morphological and
molecular data.
The sequencing of four hox clusters from the bichir (Polypterus senegalus), a
representative of the most basally branching extant lineage of rayfin fish, the
polypteriforms, indicated that the GD did not occur in an ancestor of all rayfins
(Chiu et al. 2004; Raincrow et al. 2011). Subsequently, several studies cloned and
sequenced homeobox genes and several other nuclear markers from different
species representing major phylogenetic lineages of rayfins (Hoegg et al. 2004;
Crow et al. 2006; Mulley et al. 2006; Hurley et al. 2007). These studies did not find
any evidence for the presence of paralogs derived from the TGD in the genomes of
extant nonteleosts, i.e., Lepisosteiformes (gars), Amiiformes (bowfin), and Acipenseriformes (sturgeons and paddlefish, which have, however, experienced their
own lineage-specific polyploidizations; see above). In contrast, paralogs were
found in all teleost lineages analyzed, including representatives of the early
branching teleost lineages, i.e., Osteoglossomorpha (bonytongues and mooneyes),
Elopomorpha (tarpons and eels), and Clupeomorpha (herrings and shads) (see
Fig. 17.1). Finally, Amores et al. (2011) recently presented the first genome-wide
synteny comparison between a non-teleost and teleost rayfins through the generation of a high-density genetic map for the spotted gar (L. oculatus). A clear
pattern of double conserved synteny became apparent between the gar and teleost
genomes showing that the gar lineage diverged from the teleost lineage before the
GD shared among teleosts (Amores et al. 2011).
To summarize, the GD initially detected in zebrafish, medaka, and pufferfish
was an event shared by all teleosts but not by non-teleost rayfin fish, and it
occurred (depending on the use of different molecular clocks) around 226–350
million years ago (Vandepoele et al. 2004; Christoffels et al. 2004; Hoegg et al.
2004; Hurley et al. 2007) (Fig. 17.1). It thus seems better to call this tetraploidization event the TGD (The often-used term Fish-Specific Genome Duplication, or
FSGD, is ambiguous due to the many GD events in multiple fish lineages, such as
salmonids, sturgeons, and cyprinids, and the paraphyletic term ‘‘fish’’.).
Reviewing morphological characters defining the teleost lineage, de Pinna
(1996) articulated: Intriguingly, molecular data have yet to provide consistent
support for teleostean monophyly. Fifteen years later, apparently it is the TGD that
provides the best synapomorphy, a molecular character supporting the monophyly
of teleosts and probably the best we will ever obtain from extant molecules.
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17.3.2.1 The TGD and Conserved Syntenies
A pair of genes that are syntenic are on the same chromosome in one species.
Conserved syntenies are situations in which a pair of genes that are syntenic in one
species have orthologs that are syntenic in another species. People often use the
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term ‘syntenic’ erroneously to mean ‘conserved synteny’. Conserved syntenies give
strong evidence for the TGD. The Synteny Database (http://teleost.cs.uoregon.edu/
synteny_db/) uses BLAST scores to identify groups of paralogous genes in a
genome rather than a single pair of genes showing the ‘‘best hit’’; the algorithm then
anchors paralogy groups to an ortholog in another genome, and finally plots
orthologs and paralogs along chromosomes (Catchen et al. 2009, 2011). Results
show that segments of human chromosomes generally have orthologs and coorthologs in paralogons on two zebrafish chromosomes. For example, the horizontal
axis of Fig. 17.2 displays genes ordered along human chromosome 16 (Hsa16) and
on the vertical axis shows the zebrafish orthologs and co-orthologs of each Hsa16
gene plotted on the appropriate linkage groups directly above the human gene. The
short arm of Hsa16 (Hsa16p) has orthologs and co-orthologs mainly on zebrafish
chromosomes Dre3 and Dre12, including duplicated hemoglobin loci, and the long
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Fig. 17.2 Conserved syntenies are as predicted by GD. a Genes on human chromosome 16 are
aligned along the horizontal axis and their zebrafish orthologs and co-orthologs are marked
directly above each human gene on the zebrafish chromosome on which it resides. Results reveal
duplicate ohnologons on Dre3 and Dre12 for Hsa16p and Dre7 and Dre18 for Hsa16q. A gray
circle marks the location of the centromere on Hsa16. b Zebrafish ohnologons for Hsa17 are
mainly on Dre3 and Dre12. c Human orthologs of Dre12 genes occupy single portions of human
chromosomes, mainly Hsa10 and Hsa17. d Human orthologs of Dre12 are widely scattered on
mostly Hsa16 and Hsa17. Individual genes are located on each plot
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arm (Hsa16q) has orthologs and co-orthologs on Dre7 and Dre18, including
co-orthologs of CDH1 and many other genes. Other zebrafish chromosomes also
have strings of genes that appear on the plot, including Dre24 and Dre25, but most
of these genes are more distantly related paralogs resulting from the VGDs.
The content of other human chromosomes appears to have involved more
rearrangements than Hsa16 since the divergence of the human and zebrafish lineages, either in the rayfin or the lobefin lineage or both. For example, in zebrafish,
the genetic content now on Hsa17 occupies duplicates of at least seven distinct
large chromosome segments, mostly on Dre3 and Dre12 (Fig. 17.2b), which
indicates several major translocations with respect to human, some of which
occurred before the TGD because they are shared by both copies, and some after
the TGD, because they differ between the two paralogons (or ohnologons, paralogons arising from GD).
Comparisons in the other direction, zebrafish to human, generally display a
one-to-one relationship with a substantial number of translocations and inversions
(Fig. 17.2c). For example, about half of the human orthologs of Dre12 genes
are located on Hsa17, and about half on Hsa10, each with several rearrangements;
minor numbers are located on four other zebrafish chromosomes. Other
chromosomes, like Dre3, appear to have experienced an even greater number of
chromosome rearrangements (Fig. 17.2d).
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17.3.2.3 An Example: The Hox Clusters
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The most frequent fate of a pair of ohnologs after WGD is nonfunctionalization
(Lynch and Conery 2000); hence, ohnologons often experience reciprocal gene
loss, like the regions containing two zebrafish co-orthologs of SLC16A 5 on Dre3
and Dre12 (Figs. 17.2b and 17.3). Of the string of four genes on Hsa17 including
human SLC16A 5 , different sets are missing from each duplicated zebrafish
segment, so that the content of both zebrafish ohnologons must be summed to get
the orthologous human gene content in this region (Fig. 17.3). In the extreme, all
WGD duplicates will have resolved to singletons, and so conserved syntenies will
be maintained but with no co-orthologs to anchor the segments in a doubled
conserved synteny.
457
Circle plots can display conserved paralogs on a genome-wide scale. For example,
the Synteny Database shows that paralogs of genes within 10 Mb of the four
human HOX clusters preferentially occupy regions near each of the other clusters
(Fig. 17.4a) (For nomenclature, see https://wiki.zfin.org/display/general/ZFIN+
Zebrafish+Nomenclature+Guidelines.). Note stronger links between the pairs
HOXA/HOXB and HOXC/HOXD, which may reflect the order of their origin, with
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Fig. 17.3 Doubled
conserved synteny. The
region at about 73 Mb on
Hsa17 including SLC16A5
shares syntenies with small
regions of Dre3 and Dre12.
Zebrafish has two coorthologs of SLC16A5
(boxed), but some of its
neighbors were reciprocally
lost from one or the other
ohnologon. In addition, an
inversion on Dre12 reversed
gene orders and caused other
genes to disrupt the ancestral
group. This pattern of
doubled conserved synteny
with disrupting inversions is
quite common
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17.3.2.4 Symmetry of Gene Loss Between Members of Ohnologons
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Immediately after GD, both ohnologons have identical gene compositions. As
nonfunctionalization events occur, ohnologons begin to diverge from each other.
Gene loss events raise the question: Do both members of a pair of ohnologons tend
to lose genes at the same rate? Or do genes tend to disappear more frequently from
one duplicated chromosome segment than the other? According to one hypothesis,
gene losses would be purely stochastic. According to an alternative hypothesis,
expression of a number of genes in the same neighborhood might be regulated by a
central element, for example, an element that helped to regulate local chromosome
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VGD1 giving rise to HOXA/B and HOXC/D clusters (Amores et al. 1998) and
VGD2 providing all four clusters. A circle plot of orthologs and paralogs of genes
surrounding zebrafish hox clusters (Fig. 17.4b) shows that zebrafish has eight hox
cluster-containing ohnologons even though only seven of them contain proteincoding hox genes (Amores et al. 1998). While zebrafish has two copies of the
hoxa, hoxb, and hoxc clusters, it has a single hoxd cluster that contains proteincoding genes. The hoxdb cluster, while lacking protein-coding genes, retains a
microRNA-10 paralog, which is embedded in Hox clusters from flies to fish to
humans (Woltering and Durston 2006), as well as paralogs of many surrounding
genes easily detected in the plot (Fig. 17.4b). Likewise, stickleback lacks a second
hoxc cluster, but the location where it ‘should’ go is apparent from the circle plot
(Fig. 17.4c).
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(a)
I. Braasch and J. H. Postlethwait
HOXC
(b)
HOXB
(c)
hoxaa
hoxda
hoxab
hoxca
hoxca
(hoxcb)
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hoxab
hoxaa
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Fig. 17.4 Hox cluster ohnologons. a Paralogs within 10 Mb of human HOX clusters mapped on
the human genome. b Paralogs within 5 Mb of zebrafish hox clusters mapped on the zebrafish
genome. Although only a microRNA gene remains in the hoxdb cluster, the paralogy of this
segment and its relationship to hoxda is evident. c Paralogs within 3 Mb of stickleback hox
clusters mapped on the stickleback genome
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structure, and loss of that element would then lead to rapid loss of the several
genes that element controls and hence asymmetric gene loss. Analysis of a few
individual chromosome regions suggests that asymmetries often occur in teleosts
(Canestro et al. 2009; Braasch et al. 2006; Siegel et al. 2007), but this question has
not been explored sufficiently on a genome-wide scale.
To study this question, we used the Synteny Database to plot the zebrafish and
stickleback chromosomes on which orthologs, co-orthologs, and paralogs of each
human gene lie for all human chromosomes. Figure 17.5 displays results for two
human chromosomes (Hsa7 and Hsa18) for both zebrafish and stickleback. Figure 17.5a shows genes on Hsa7 as gray dots along the bottom with the zebrafish
orthologs and paralogs directly above; note that gene order in these plots reflects
gene arrangements in the human genome, not the fish genome.
Results revealed several principles (Fig. 17.5a–d). (1) Many regions of
ohnology are clear, as marked by rectangles. For example, the region from 18 to
38 Mb on Hsa7 has two clear ohnologons in both zebrafish and stickleback, with
orthologous chromosome segments marked by color (Fig. 17.5a, b). (2) In contrast, many regions have ambiguous ohnologons, such as the region from the left
telomere of Hsa7 to about 18 Mb for both zebrafish and stickleback. (3) Some
ohnolog pairs are clear in one species but ambiguous in the other; for example,
from 38 to 64 Mb of Hsa7, where three or four zebrafish chromosomes have
strings of ‘hits’ compared to two clear regions in stickleback. Regions like these
(also note for stickleback region 101–142 Mb the ‘ghost’ paralogons on groupXII
and groupXVIII) are often due to paralogs arising in the VGD1 and VGD2 events
rather than the TGD. (4) Most chromosome translocations occurred before the
divergence of the zebrafish and stickleback lineages. Evidence supporting this
conclusion is that, with respect to the human chromosome, orthologous blocks
tend to have the same termini in both fish species. And (5), a minority of translocations occurred in one or the other fish lineage after divergence; for example,
the region from 77 to 88 Mb and the region from 101 to 142 Mb are syntenic (on
the same chromosome) in human (Hsa7) and in both ohnologons in stickleback
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10Mb
20Mb
30Mb
40Mb
50Mb
60Mb
70Mb
Hsa18 Orthologs
Fig. 17.5 Ohnologons in zebrafish (a, c) and stickleback (b, d) for Hsa7 (a, b) and Hsa18 (c, d).
These plots show doubled conserved synteny as well as the pattern of chromosome translocations
in various lineages
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(groupIV and groupXX)—thus representing the ancestral condition—but for
zebrafish, these two regions are syntenic for only one of the copies (Dre4) but not
the other (Dre18 and Dre25). These results demonstrate a translocation in the
zebrafish lineage after it diverged from the stickleback lineage (Fig. 17.5a, b).
Note that the effects of inversions in the fish lineage are invisible in these plots
because gene orders in the fish genomes are displayed according to their order in
the human genome.
Having identified ohnologons, we can now pose the question of gene loss
asymmetries. Over the human genome, we identified 49 unambiguous ohnologon
pairs in zebrafish or stickleback or both, and counted the number of fish orthologs
and paralogs of human genes occupying each segment. For zebrafish, 7 of 39
(17.9 %), and for stickleback, 13 of 44 (29.5 %) ohnologon pairs had significantly
different number of genes [v2 test, 1 df, p \ 0.01; 10/39 (25.6 %), which increased
to 18/44 (40.9 %) if p \ 0.05]. We conclude: (1) that most ohnologons appear to
lose genes approximately randomly between the two duplicated copies, but (2) that
about 20–40 % of ohnologons in these fish species have resolved TGD ohnologs
asymmetrically between the two duplicated chromosome segments, with one
member of the pair retaining significantly more genes than the other. (3) Of 13
segments with significance at the 0.01 level in one or the other fish, 6 are significant at 0.01 or 0.05 level in both species, which diverged deeply in teleost
phylogeny (Fig. 17.1), and for all of those pairs, chromosome segments retaining
the most genes in zebrafish are orthologs of those retaining the most in stickleback.
This result is consistent with the idea that the basis for most asymmetric gene
losses occurred before the divergence of zebrafish and stickleback lineages.
A question that remains is the mechanism that led to asymmetric resolution of
gene duplicates after the TGD. One hypothesis is that, if a long-range regulatory
function that controls the expression of many genes in a neighborhood disappears
from one ohnologon, then nonfunctionalization of the genes that the regulatory
function controls can follow without penalty given the maintenance of that longrange function in the sister ohnologon. That hypothesis is yet to be seriously
investigated. An example of resolution asymmetries is shown in Fig. 17.6 for
Hsa15 in zebrafish and stickleback, along with chromosome diagrams comparing
genes in their order along a segment of Hsa15 and their fish orthologs and coorthologs in zebrafish (52 ortholog pairs in one ohnologon vs. 4 ortholog pairs in
the other) and stickleback (54 ortholog pairs vs. 16 ortholog pairs) (Fig. 17.6c, d).
A related question is whether ohnologs residing in ohnologons that suffer
asymmetric rates of gene loss also experience asymmetric rates of gene evolution.
Asymmetric rates of molecular evolution have been found for a substantial number
of TGD paralogs (Van de Peer et al. 2001; Steinke et al. 2006; Brunet et al. 2006),
sometimes correlating with asymmetric loss of genes from ohnologons (Braasch
et al. 2006; Siegel et al. 2007). Available evidence from other WGDs, however,
suggests that gene evolutionary rate asymmetry does not strongly depend on the
conservation of syntenies (Bu et al. 2011).
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groupXII
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groupXX
Hsa15
0Mb
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(c)
PAK6(1of2)
sb:cb730
srp14
zgc:136872
FMN1
GJD2 MEIS2 SRP14
C15orf57
C15orf24 ACTC1
PLCB2
AQR
C15orf52
ATPBD4
EXD1
EHD4 LRRC57
RPAP1 PLA2G4F
CDAN1
VPS39
TTBK2
ZFP106 UBR1
spint1b
cx35
zgc:86709
meis2.1
SCG5
ENSGACG00000007697
ENSGACG00000007661
ENSGACG00000005610
GJD2(2of2)
CASC5
GacgroupXVIII
54 pairs
im:7147183
80Mb
GREM1
SCG5CHRM5
ARHGAP11A RYR3
Hsa15
16 pairs
GacgroupXV
FMN1
AC012652.1
EIF2AK4
CASC5
CHAC1
MAPKBP1
FAM98B
RPUSD2 SPINT1
TYRO3
SPRED1
BUB1B DISP2
ZFYVE19 NUSAP1
JMJD7
C15orf41
THBS1 PAK6
IVD GCHFR DLL4
RTF1 MGA
GJD2
MEIS2
FSIP1 PLCB2
DNAJC17
C15orf57
AVEN
ACTC1 RASGRP1
GPR176
C15orf24
AQR
SRP14
C15orf29
ATPBD4
MAPKBP1(2of2)
ENSGACG00000003916
TYRO3
LRRC57 JMJD7
100Mb
NDUFAF1
RPAP1
CHAC1(1of2)
DLL4(2of2)
LOC100334937
vps39
LOC100331590
si:ch211-283f6.8
zgc:63611
zgc:154068
zgc:110758
meis2.2
MEIS2(2of2)
fam98b
spred1
zgc:154061
CAPN3(2of2)
VPS39
CDAN1
TMEM87A
TTBK2
ZFP106
UBR1
LRRC57
TMEM87A
ZFP106
capn3
LOC100331516
ubr1
ENSGACG00000009824
FMN1(2of2)
CHRM5(2of2)
THBS1(1of2)
RYR3(2of2)
STARD9
HAUS2
SNAP23
CAPN3
GANC
TMEM62
CAPN3(1of2)
VPS39
GANC
ENSGACG00000005683
ENSDARG00000091548
LOC565314
zgc:77419
PAK6(1of2)
FMN1(1of2) GCHFR
BUB1B
FSIP1 EIF2AK4
SRP14
RASGRP1 CHRM5(1of2) DNAJC17 ZFYVE19
THBS1(2of2)
SPINT1(1of2)
RYR3(1of2)
PLCB2
GREM1
ENSGACG00000006109
C15orf57
DISP2
UBR1
RPUSD2 IVD
RPAP1
FAM98B
SPRED1
C15orf41
GJD2(1of2) MEIS2
ENSGACG00000010421
AQR
ATPBD4
TMEM62
SNAP23
ENSGACG00000011127
CDAN1
TTBK2(1of2)
RTF1
NUSAP1
ENSGACG00000012183
NDUFAF1
EC
HAUS2
C15orf24
C15orf29
GPR176
AVEN
zgc:123218
zgc:56072
LOC565088 ivd
LOC564712
RPAP1
si:dkey-170l10.1
TE
4 pairs
Dre20
NP_001185682.1
disp2 itpka
MAPKBP1
jmjd7
mga
tyro3
JMJD7
STARD9
RPUSD2
MAPKBP1
HAUS2
FAM98B PAK6 BAHD1
MGA
SNAP23
SPRED1 BUB1B
IVD
SPINT1 TYRO3
CAPN3
C15orf41 THBS1
DISP2 ZFYVE19
ITPKA
GANC TMEM62
CHRM5
GREM1
ARHGAP11A
Hsa15
LOC565386
ENSDARG00000056535
lrrc57
ENSDARG00000079066
actc1b
PLCB2
chrm5a
spint1a
LOC100005728 zgc:92683
arhgap11a
LOC796966
52 pairs
D
ENSDARG00000088538
THBS1(2of2)
zgc:195050
Dre17
(d)
60Mb
Hsa15 Orthologs
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17.3.2.5 Reconstruction of the Ancestral Karyotype and Teleost Chromosome
Rearrangements
As we discussed earlier, the ancestral haploid teleost (post-TGD) genome was
inferred to contain 24 chromosomes based on karyotype data (Ohno et al. 1968;
Mank and Avise 2006b). Using several teleost genetic maps as well as the genome
assemblies of Tetraodon and medaka, several attempts have been made to
reconstruct the ancestral pre-TGD protokaryotype (Naruse et al. 2004; Jaillon et al.
2004; Woods et al. 2005; Kohn et al. 2006; Kasahara et al. 2007; Nakatani et al.
2007). All studies largely agree that the teleost karyotype is derived from 11 to 12
pre-TGD protochromosomes, which is in agreement with the inferred number of
24 post-TGD chromosomes (Mank and Avise 2006b). There are, however, some
differences in the assignment of chromosomal blocks from different extant teleosts
to the protochromosomes (Woods et al. 2000; Kasahara et al. 2007). The reconstruction of the ancestral teleost karyotype has so far relied on using tetrapod
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Fig. 17.6 Asymmetrically resolved ohnologons for Hsa15 in zebrafish (a) and stickleback (b).
Chromosome contents displayed with proper gene orders for zebrafish duplicates (c, 52 vs. 4
ortholog pairs) and stickleback (d, 54 vs. 16 pairs)
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genomes as outgroups, but the genome sequence of gar or bowfin, rayfin fish that
can serve as outgroup to the TGD, will help to provide a more detailed picture of
the ancestral teleost genome.
Nevertheless, the current state of karyotype reconstruction is helpful to compare
and cross-refer different types of data on duplicated genes in teleosts. As an
example, for zebrafish and stickleback we downloaded all pairs of predicted
duplicated genes and their genomic locations from the EnsemblCompara GeneTree set (Vilella et al. 2009) and parsed them for the ‘Clupeocephala’ duplication
node, which indicates a duplication on the branch leading to teleosts, after
divergence from tetrapods, but before the divergence of teleosts. We then plotted
the location of these pairs as Oxford grids for zebrafish and stickleback genomes
(Fig. 17.7) and overlaid them with the 12 ancestral pre-TGD protochromosomes
a–m inferred from the analysis of the medaka genome (Kasahara et al. 2007). This
analysis clearly shows that paralogs are nonrandomly distributed over the genome;
for example, of the 228 genes in the analysis that reside on zebrafish chromosome
Dre3 and have TGD duplicates, the vast majority, 105 genes, are on Dre12 and a
sizable minority on Dre1, and of the 167 genes on stickleback groupXIV that are
duplicated and assigned to chromosomes, 152 are on groupXIII. These results
show that the vast majority of ‘Clupeocephala-duplicated’ genes obtained from the
tree-only method used by Ensembl supports a TGD origin of the paralogs and is in
line with conserved synteny data and ancestral karyotype reconstructions.
Teleost genomes are rearranged with respect to tetrapod genomes, which may
be due to chromosome rearrangements that were facilitated by the TGD, for
example through illegitimate recombination between homeologous (paralogous)
chromosomes. This hypothesis, however, is controversial (Comai 2005; Semon
and Wolfe 2007a; Hufton et al. 2008), because chromosome restructuring may
have likewise occurred before the TGD, on the long branch separating teleosts and
tetrapods after the divergence of rayfin fish from lobefin fish.
Using the genetic map of the spotted gar, which contains nearly 1,000 coding
markers, Amores et al. (2011) recently showed unexpectedly high conservation of
synteny between human and gar when compared to zebrafish and stickleback. This
suggests that chromosome rearrangements and the loss of ancestral syntenies
accelerated after the TGD, but before the divergence of stickleback from zebrafish,
and supports the hypothesis that WGD can facilitate syntenic rearrangements.
The zebrafish genome appears to be more rearranged than the genome of
percomorph teleosts (stickleback, medaka, pufferfish). Chromosomal blocks of the
inferred ancestral pre-TGD karyotype (Kasahara et al. 2007; Nakatani et al. 2007)
are generally distributed over many more chromosomes in zebrafish than in percomorphs. For example, as shown in Fig. 17.7, chromosomal blocks of the
ancestral pre-TGD chromosome m are found on four stickleback chromosomes
(GacI, GacIII, GacVIII, GacXXI), but on six chromosomes in zebrafish (Dre2,
Dre6, Dre8, Dre11, Dre22, Dre24); pieces of ancestral chromosome j are found on
two chromosomes in stickleback (GacII, GacXIX), but on three in zebrafish (Dre7,
Dre18, Dre25), and so forth. A genome sequence of another representative of the
ostariophysians, which, after percomorphs, is the next most species-rich group of
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7
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12
0
7
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0
22
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0
0
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0
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0
0
2
60
1
0
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8
0
1
13
1
0
1
2
0
0
0
41
0
0
18
0
0
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0
0
17
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0
14
54
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0
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1
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0
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teleosts and includes zebrafish and carps (Nelson 2006), will be necessary to
clarify whether this enhanced rearrangement rate is specific to the zebrafish genome or a more general phenomenon on this major branch of teleost diversity.
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Fig. 17.7 Oxford grid of duplicated genes in zebrafish (a) and stickleback (b). Chromosomal
location of paralogs determined by EnsemblCompara GeneTrees (Vilella et al. 2009) to be
generated at the ‘Clupeocephala’ node. Each box in the grid shows the number of duplicated
genes shared between the indicated chromosomes. The distribution of duplicated genes is
nonrandom as expected from the TGD. Most clusters of duplicated genes can be traced back to
the ancestral protochromosome (Kasahara et al. 2007), from which they are derived (see color
code)
17.3.2.6 Global Patterns of Gene Retention and Loss After the TGD
The TGD obviously had a major impact on the evolution of genome structure in
teleosts. We still do not know, however, how many paralogs from the TGD have
escaped nonfunctionalization and have been retained in duplicate in extant
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teleosts. Genome-wide estimates range between 12 and 24 % depending on the
approach and dataset (Postlethwait et al. 2000; Jaillon et al. 2004; Woods et al.
2005; Brunet et al. 2006; Kassahn et al. 2009) and do not differ significantly
among sequenced teleost genomes (Kassahn et al. 2009). Between *1,800 and
*2,200 TGD-derived pairs of gene duplicates can be identified in the five
sequenced teleost genomes (I. Braasch, ‘‘unpublished data’’). Loss of paralogs
after the TGD appears to be time-dependent, with most losses occurring soon after
the GD event (Braasch et al. 2009a; Sato et al. 2009). A similar trend can be
observed when comparing paralog loss after different GD events in teleosts, from
the CaGD *11–21 million years ago (mya) with a paralog retention rate of
*60 % (David et al. 2003), over the sucker genome duplication (SuGD) and
SaGD, which occurred *50 mya and 25–100 mya, respectively, and which both
have estimated paralog retention rates of *50 % (Ferris 1984; Allendorf and
Thorgaard 1984), to the TGD that happened *226–350 mya and that has an
estimated paralog retention of *12–24 % (see above).
Retained paralogs from the TGD are enriched for transcription factors, developmental genes, and cell communication proteins (Brunet et al. 2006; Kassahn
et al. 2009), similar to results for the earlier rounds of WGD in vertebrates (see
Putnam et al. 2008; Huminiecki and Heldin 2010; Chap. 16, this volume). Some
gene classes appear to have a high rate of TGD paralog retention, such as genes
encoding components of pigmentation pathways (Braasch et al. 2007, 2009a) and
of signaling pathways involved in long-term potentiation of synaptic transmission,
and olfactory and taste transduction (Sato et al. 2009). However, a more detailed
analysis of gene family evolution after the TGD awaits the genome assembly of a
rayfin outgroup to the TGD, the spotted gar (see below).
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After a polyploidization event, ohnolog pairs are initially identical, both in regulatory elements and in protein-coding areas, so they are fully redundant. Ohnologs,
however, soon begin to accumulate mutations that become fixed in populations
and distinguish the a copy from the b copy; these mutations can occur in both
regulatory and protein coding regions.
Some mutations can cause one copy of a duplicated pair of genes to become
nonfunctional, either because they inactivate the protein by premature stop
mutation or destructive amino acid substitution, or because they eliminate gene
expression. Nonfunctionalization is the most frequent fate of gene duplicates
(Lynch and Conery 2000). For genes that are not dosage-sensitive, nonfunctionalization of one copy is likely to carry little selective penalty (and may provide in
some cases an advantage), reflecting the fact that most mutations are recessive in a
diploid.
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17.3.3.1 Subfunctionalization
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The classic example that led to the idea of subfunctionalization—duplicate gene
preservation by the reciprocal loss of ancestral gene subfunctions—is the zebrafish
pair of engrailed-1 co-orthologs (Force et al. 1999). The mouse En1 gene is
expressed in both the pectoral appendage bud and in a specific set of hindbrain
interneurons (Joyner and Martin 1987; Davis et al. 1991; Gardner and Barald
1992). In contrast, the zebrafish co-orthologs eng1a and eng1b partitioned these
two expression domains between them, with eng1a expressed in the pectoral
appendage bud and, reciprocally, eng1b expressed in the hindbrain interneurons, a
case of spatial subfunctionalization (Force et al. 1999). The reciprocal loss of
ancestral regulatory domains like this, which can occur purely by neutral evolution, makes both copies essential, with eng1a essential to provide Engrailed
function to the pectoral fin bud and eng1b necessary to deliver Engrailed protein to
interneurons.
Many genes evolved like eng1 genes to show spatial subfunctionalization, but
some genes evolved by temporal subfunctionalization, duplicate gene expression
in the same tissue but at multiple developmental stages. For example, Sox1 is
expressed in tetrapods at several developmental stages in the lens, but after the
TGD, sox1a came to be expressed several hours earlier in the lens than is
sox1b (Okuda et al. 2006). Subfunctionalization is pervasive: a global analysis of
TGD ohnologs showed that nearly all that were examined had diverged in their
patterns of spatial and/or temporal expression during embryogenesis and that such
regulatory changes were more frequent than changes at the level of protein
function (Kassahn et al. 2009). Tetrapod enhancers, which represent a type of
regulatory subfunction, and CNEs, many of which may be enhancers, evolved
divergent sequences in teleosts by altering a few specific bases that underlie
divergent functions, as for medaka and fugu ohnologs derived from Hoxa2
(Tumpel et al. 2006) or zebrafish and stickleback Fgf8 co-orthologs (Canestro
et al. 2007); this result would be expected to occur by subfunctionalization. Cases
of preserved ohnologs that have no documented differences in embryonic
expression domains may display differences at developmental stages or tissues that
have not yet been examined.
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Besides nonfunctionalization of one member of a duplicate pair, reciprocal
changes to both members of the pair cause both ohnologs to become essential.
Reciprocal changes can be of two main sorts, either reciprocal loss of ancestral
gene subfunctions that occur purely by neutral processes (subfunctionalization), or
by mutation in one or both copies that result in both ohnologs being positively
selected for new functions (neofunctionalization) (Force et al. 1999). Mutations
that lead to the partitioning of subfunctions between ohnologs do not preclude the
later origin of novel subfunctions [called subneofunctionalization (He and Zhang
2005; Rastogi and Liberles 2005)] and vice versa. Here, we note a few examples
for each of several fates of TGD ohnologs.
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Besides spatial or temporal differences in expression domains, some ohnologs
may achieve quantitative subfunctionalization, the accumulation of activityreducing mutations in both ohnologs, so that neither gene by itself can provide
sufficient product to achieve normal function (Force et al. 1999; Stoltzfus 1999).
Mutations that partially reduce gene function should be relatively common, and if
a population fixes a partial loss-of-function allele at one copy, then it is unlikely to
fix a null allele in the gene’s ohnolog. Although quantitative subfunctionalization
may be common after WGD in yeast (Scannell and Wolfe 2008), few examples
exist from the TGD, probably because of the lack of appropriate analyses. We do
know that quantitative subfunctionalization occurred in many of the hox gene
duplicates after the SaGD (Mungpakdee et al. 2008b). The finding that most pairs
of CNEs in teleost ohnologs experience substantial degeneration in element length
might also reflect cases of quantitative subfunctionalization (Woolfe and Elgar
2007). The evolutionary significance of quantitative subfunctionalization may be
that it preserves both duplicates long enough to increase the opportunity for later
mutations that might secondarily increase gene activity, as appears to have happened for the medaka copy of aldh1a2, a gene originating in VGD2 (Canestro
et al. 2009).
Protein structure evolution: Individual tetrapod genes can encode proteins with
multiple functions. In some cases, a single protein sequence performs several
functions; in other cases, a single gene encodes different protein sequences, often
with different functions, due to alternative splicing or start sites.
Amino acid substitutions can cause proteins encoded by a pair of ohnologs to
diverge in sequence in functionally important ways. For example, the chemokine
Cxcl12 guides gastrulation and the migration of primordial germ cells (PGCs) in
several vertebrates (Nair and Schilling 2008; Richardson and Lehmann 2010).
Zebrafish has co-orthologs of Cxcl12 that appear to have arisen in the TGD
(Doitsidou et al. 2002). Cxcl12 functions by binding Cxcr4 receptor, which is also
duplicated in zebrafish. Cxcl12a, but not Cxcl12b, guides PGCs to their target, the
gonad, and a single amino acid substitution causes the relative affinity of Cxcl12
ligands to switch from one of the duplicated Cxcr4 receptors to the other, thereby
controlling whether the Cxcl12:Cxcr4 ligand:receptor system controls PGC
migration or gastrulation (Boldajipour et al. 2011). These experiments are a particularly deeply studied case study supporting the concept of protein
subfunctionalization.
Alternative splicing can generate different proteins with variant functions. For
example, the human STAT1 gene, which mediates interferon signaling, makes two
different proteins by alternative splicing: STAT1-alpha, which contains a TAZ2binding domain, and STAT1-beta, which lacks that domain and may act as a
negative regulator of STAT1-alpha (Bromberg et al. 1996). The TGD provided
teleosts with duplicate stat1 genes, and, due to reciprocal mutations that affect
transcript splicing, the zebrafish stat1a gene came to encode a protein that contains
the TAZ2-binding domain like the human STAT1-alpha isoform, while
stat1b evolved to encode a protein that lacks this domain like STAT1-beta (Song
et al. 2011). Thus, due to subfunctionalization of splicing signals, two genes in
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17.3.3.2 Neofunctionalization
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In the race between nonfunctionalization, subfunctionalization, and neofunctionalization, nonfunctionalization generally rides the fastest horse. Subfunctionalization
appears to place second, and neofunctionalization—the preservation of ohnologs by
the origin of new functions (Ohno 1970)—shows in third. As with subfunctionalization, neofunctionalization can occur at the level of either gene regulation or protein
structure. Preservation by neofunctionalization differs from subfunctionalization in
two main ways: first, it involves positive selection rather than neutral events, and
second, it involves the origin of gene subfunctions that did not exist in the ancestral
gene rather than the partitioning of ancestral subfunctions. Genome-wide studies
suggest that the acquisition of novel protein domains might have occurred in a quarter
of duplicate pairs after the TGD and happened more frequently in duplicates than in
single-copy genes (Kassahn et al. 2009). Although several specific cases of
neofunctionalization after the TGD have been suggested (Yao and Ge 2010; Sha et al.
2008; Howarth et al. 2008), conclusions are often difficult due to assumptions about the
ancestral state, which is usually based on tetrapods, species that are far removed from
the teleost lineage.
To distinguish neofunctionalization from subfunctionalization, one must
accurately identify the ancestral state. Inferences about the ancestral state can be
difficult with WGD but are often easier with tandem duplications, which have led
to neofunctionalization of vitellogenin and aquaporin genes in teleost egg hydration (Finn and Kristoffersen 2007; Zapater et al. 2011; Cerda 2009), the evolution
of antifreeze proteins by Antarctic teleosts (Deng et al. 2010), and the preservation
of fatty acid-binding protein genes (Karanth et al. 2009).
Several examples of neofunctionalization after the TGD have been suggested.
(1) Phosphoglucose isomerase (PGI) ohnologs from the TGD, after the partitioning
of tissue-specific regulatory elements for expression, diverged so that the copy
expressed in organs of the body cavity became more negative in charge while the
muscle isoform became more positively charged, although the functional significance of these changes is not yet well understood (Sato and Nishida 2007). (2)
After the TGD, cyprinid fish, like zebrafish, retained only one copy of the
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zebrafish encode two protein variants that are encoded by a single gene in human.
Another example is the synapsin gene in fugu (Yu et al. 2003).
Alternative start sites can also result in alternative exon usage. The human
MITF gene controls pigment production both in melanocytes and in the pigmented
retina, with different transcription start sites employed by the two different
cell types (Udono et al. 2000). Zebrafish and other teleosts have two MITF
co-orthologs, with mitfa expressed in melanophores and mitfb in the pigmented
retina; correspondingly, evolution of the two ohnologs resulted in mitfa using one
transcription start site and mitfb using the other (Lister et al. 2001; Altschmied
et al. 2002). Again, two different tissue-specific proteins encoded by one gene in
mammals are formed from sister ohnologs in teleosts due to subfunctionalization.
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androgen receptor gene (ar), but percomorph fish, like pufferfish and medaka,
retained both ohnologs, one of which (arb) experienced substantial sequence
alterations that are consistent with neofunctionalization; further, these changes
occurred after the divergence of basal teleost lineages, including eels, but before
the radiation of percomorphs (Douard et al. 2008). (3) Some TGD duplicates show
elevated evolutionary rates, like csf1rb and its neighbor pdgfrbb in cichlids, which
could be due either to neofunctionalization or to relaxed selection; it has been
suggested that such behavior could be a causal factor in the evolution of the
spectacular coloration patterns of some teleost fish (Braasch et al. 2006). To learn
whether neofunctionalization is at play in these examples and many others, the
duplicates must be compared to each other by function in addition to sequence and
compared to the structure and function of the ancestral genes in a rayfin fish that
diverged before the TGD.
In two cases of neofunctionalization—a hox gene and a sodium channel gene—
TGD duplicates have been investigated functionally in a phylogenetic context that
leaves little doubt about the functions of the preduplication gene. Zebrafish has coorthologs of Hoxa13 derived from the TGD (Amores et al. 1998), and, as determined
by a broad phylogenetic study, these ohnologs began to diverge asymmetrically long
after their initial preservation subsequent to the TGD (Crow et al. 2009). Ohnolog
knockdown experiments in zebrafish, coupled with rescue experiments, showed that
hoxa13a, the faster evolving ohnolog, contains many derived a clade-specific amino
acid replacements that are necessary for development of the yolk sac extension (the
hind yolk), a derived feature in zebrafish and its relatives (Crow et al. 2009).
A great example of neofunctionalization after the TGD occurred in electric fish.
The distantly related African elephantfish (Mormyridae; Osteoglossiformes) and
South American knifefish (Gymnotideae; Gymnotiformes) independently evolved a
muscle-derived electric organ, which facilitates electrical communication and
utilizes the voltage-gated sodium channel subunit Scn4a. Teleosts have two
scn4a genes from the TGD, both expressed in skeletal muscle in most teleosts; in
two lineages of electric fish, however, scn4aa independently lost its expression in
skeletal muscle and gained expression in the electric organ (Arnegard et al. 2010).
Phylogenetic analyses showed that this neofunctionalization event occurred more
than 100 million years after the TGD with a signal of positive selection (Arnegard
et al. 2010). Meanwhile, scn4ab maintained the ancestral expression in skeletal
muscle. These results for Scn4a and Hoxa13 both show that, long after the preservation of ohnologs by subfunctionalization, one copy can still provide the genetic
variation necessary to lead to the innovation of new morphologies and functions.
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17.3.3.3 Spotted Gar as a Rayfin Outgroup for Functional Studies of TGD
Paralogs
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As pointed out above, knowledge of the functions of the preduplication gene is
essential for inferring subfunctionalization versus neofunctionalization. Investigators often infer the properties of the pre-TGD gene from the phenotype of well-
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studied tetrapods, especially mouse. The tetrapod lineage, however, diverged from
the teleost lineage about 450 million years ago and involved the evolution of
substantial differences in morphologies from ancestral bony fish. In addition, rayfin
fish followed an independent lineage for about 200 million years before the TGD;
thus, what appears to involve neofunctionalization when comparing teleost
co-orthologs to tetrapod genes (Fig. 17.8a) could instead be due to the tetrapod
lineage having lost a subfunction originally present in the single-copy gene in the
last common ancestor of teleosts and tetrapods (Fig. 17.8b). A third alternative is
the reciprocal loss of ancestral VGD ohnologs, which could also mimic neofunctionalization, pointing out the importance of careful distinction between
orthologs and ohnologs (Fig. 17.8c). Finally, the novel function thought to have
arisen after the TGD might have actually originated in ancestral rayfin fish,
pointing out the importance of identifying a fish representing this node
(Fig. 17.8d). In addition, distinguishing among the possibilities shown in
Fig. 17.8b–d requires examination of an outgroup diverging basal to the diagrammed tree, such as a cartilaginous fish.
Recently, the spotted gar L. oculatus has been identified as an unduplicated
rayfin outgroup to the TGD (see Sect. 17.3.1.5 and Amores et al. 2011). The
spotted gar occupies a lineage of ‘ancient fish’ that includes not only lepisosteids
(gars), but also bowfin (Amia calva) and possibly acipenseriforms (sturgeons, e.g.
Acipenser transmontanus, and paddlefish, e.g. Polyodon spathula) (Inoue et al.
2003; Kikugawa et al. 2004; and Fig. 17.1). Among these ‘ancient fish’, spotted
gar is the species that has the smallest genome and can most readily be fertilized in
vitro, providing embryos that are amenable to in situ hybridization studies and that
grow to adults in laboratory aquaria with little care. Furthermore, no lineagespecific polyplodization has occurred in gar, in contrast to acipenseriforms. Thus,
the spotted gar makes an ideal outgroup for the investigation of the mechanisms of
evolution of gene function after GD.
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Polyploidy in Fish and the Teleost Genome Duplication
17.3.4 Mind the Gap: The TGD and the Teleost Radiation—Is
There a Connection?
The preceding paragraphs demonstrate that rayfin species experienced many WGD
events and that teleosts are the most species-rich group of vertebrates. Importantly,
many lineages experiencing WGD, such as barbs, armored catfish, and salmonids,
appear to be particularly species-rich (Le Comber and Smith 2004). These considerations raise the question of whether WGD facilitates lineage diversification in
fish. Ever since its discovery, the TGD has been suggested to have had a major
impact on the radiation and biodiversity of the teleost lineage as a whole (Amores
et al. 1998; Wittbrodt et al. 1998; Meyer and Schartl 1999; Taylor et al. 2001,
2003; Postlethwait et al. 2004; Volff 2005; Meyer and Van de Peer 2005;
Froschauer et al. 2006; Ravi and Venkatesh 2008; Volff et al. 2011; Christoffels
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(a) Neofunctionalization and
(b) Subfunction loss in tetrapods;
subfunctionalization after TGD
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et al. 2004; Van de Peer et al. 2009), but likewise, this has been questioned by
others based on the fossil record and speciation rate analyses (Donoghue and
Purnell 2005; Hurley et al. 2007; Santini et al. 2009).
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Fig. 17.8 Functional analysis of a pre-TGD diverging rayfin fish is important to distinguish
neofunctionalization from subfunctionalization. a Preservation of one ohnolog by neofunctionalization (the function symbolized by a star) and preservation of the other by subfunctionalization. b Preservation of teleost ohnologs by subfunctionalization; the ‘star’ function was
ancestral, not newly originated after the TGD, but was lost in the tetrapod lineage, as
demonstrated by the pre-TGD-diverging rayfin outgroup. c The ‘star’ function that appears to be
newly originated after the TGD was ancestral in a VGD ohnolog that was lost in the tetrapod
lineage, while the rayfin lineage reciprocally lost the other ohnolog. d. A novel function not
present in the ancestral gene could have originated in stem rayfins and then have been lost in one
TGD ohnolog. Distinguishing among b, c and d requires an additional outgroup, such as a
cartilaginous fish
17.3.4.1 Testing Speciation Models in the Fish World
Genetic principles argue that a WGD event that is followed by population isolation, lineage-specific reciprocal nonfunctionalization events, and later hybridization would tend to promote speciation (Lynch and Force 2000; Werth and
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ancestral population AA
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ohnolog divergence
Aa Aa ; Ab Ab
lineage divergence
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Fig. 17.9 How WGD could
facilitate lineage
diversification. ‘A’ indicates
the original gene. ‘Aa’ and
‘Ab’ indicate functional but
diverged ohnologs. ‘A-’
indicates a nonfunctionalized allele. A ‘-’
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Windham 1991). As Fig. 17.9 shows, the fertility of hybrids would be expected to
be somewhat reduced if even a single duplicated gene had reciprocal nonfunctionalization, but if reciprocal nonfunctionalization (or reciprocal gene loss)
occurred at least once on several or many chromosomes, which is likely, then
hybrids would have greatly reduced fertility, thus strengthening lineage
divergence.
Regarding the TGD, while one study estimated *7 % reciprocal gene loss rate
between zebrafish and Tetraodon, which would amount to *1,700 genes (Semon
and Wolfe 2007b), more recent work including more teleost species found little
support for reciprocal gene loss coinciding with speciation events in teleosts
(Kassahn et al. 2009). The latter is more in line with our observation that differences in asymmetric gene loss from paralogons are rare between zebrafish and
stickleback (see Sect. 17.3.2.4).
Similar to reciprocal nonfunctionalization, speciation after WGD may also be
promoted by reciprocal subfunctionalization that results in genomic incompatibilities in post-WGD hybrids (Lynch and Force 2000; Werth and Windham 1991).
Differential functional evolution of TGD paralogs in divergent teleost lineages is
being found in more and more cases (see below), yet associations with genetic
incompatibilities and speciation in teleosts remain to be shown.
Finally, it has also been suggested that the TGD may have caused reduced
probability of extinction in teleosts (Crow and Wagner Crow and Wagner 2006).
The extinction rate of teleost lineages, however, is apparently higher than in nonteleost rayfin lineages (Santini et al. 2009).
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Besides their species richness, teleosts are also extremely diverse in terms of
morphology, physiology, behavior, ecology, and biogeography. If the TGD laid
the groundwork for divergence in such traits, one would expect to find differential
function of TGD paralogs in various teleost lineages. Testing this proposition
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involves experimentally challenging studies in different fish species, and only a
handful of examples are currently available. For teleost sox9 co-orthologs, several
ancient expression domains are differentially retained in zebrafish, stickleback, and
medaka, indicating lineage-specific subfunctionalization (Cresko et al. 2003;
Kluver et al. 2005; Yokoi et al. 2009); similar lineage-specific partitioning of
expression domains have been reported for fgf8 (Jovelin et al. 2007) and tyrp1
(Braasch et al. 2009b) co-orthologs.
A better way to compare gene functions across teleosts is to compare mutant
phenotypes. Unfortunately, few genes have yet been mutated in teleosts other than
zebrafish and medaka, and still fewer have been mutated in both species. Most
teleosts have two TGD ohnologs encoding FGF receptor-1; in zebrafish and carp,
both cyprinids, mutation of fgfr1a causes a scaleless phenotype (Rohner et al.
2009). In contrast, mutation of fgfr1a in medaka, a perciform fish, deletes the trunk
and tail (the headfish mutant) (Yokoi et al. 2007; Shimada et al. 2008). This
finding shows that subfunction partitioning in fgfr1 ohnologs occurred after the
divergence of medaka and zebrafish lineages. Such divergence in subfunction
partitioning after the TGD may provide the genomic basis for some morphological
divergence in teleosts.
And what about the TGD and morphological complexity? The identification of
the seven hox clusters in teleosts, generally not considered to be more complex
than other vertebrate groups, called into question the idea that morphological
complexity along the body axis and the number of hox clusters and genes may be
directly linked (Amores et al. 1998; Prince 2002). The high retention rate of TGD
paralogs for developmentally important genes (Brunet et al. 2006; Kassahn et al.
2009), however, argues for an involvement of at least some TGD paralogs in
teleost morphological evolution. Also, some aspects of teleost physiology and
morphology are unique among vertebrates. To these belong, for example, the
complexity and diversity of teleost body pigmentation and color patterning
(Braasch et al. 2008); some functional modules of pigment cell development and
differentiation have been retained in large part in duplicate in teleosts after the
TGD (Braasch et al. 2009a).
Another question that has so far been rarely addressed but will need to be
investigated in more detail is the possible involvement of TGD duplicates in
morphological and physiological novelties. For example, is functional divergence
of TGD paralogs causing some of the synapomorphies of the teleost lineage
(de Pinna 1996), such as the truly symmetric (homocercal) tail fin? Or are
functional shifts in TGD paralogs involved in lineage-specific key innovations?
We met one intriguing case earlier, the parallel, independent gain of electric
organs used for electrical communication in knifefish and elephantfish by
neofunctionalization of the scn4aa paralog (Arnegard et al. 2010). A more
comprehensive picture, however, can only be gathered by studying TGD paralogs
in more than just a few teleost representatives and adding an appropriate rayfin
outgroup to the analyses.
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Based on the fossil record, a major temporal delay of about 150 million years
separated the TGD event and the major radiation of the teleost lineage in the
percomorphs (Donoghue and Purnell 2005; Hurley et al. 2007). Recent diversification rate shift analyses lead to the recognition that three incidents of accelerated
speciation rates occurred within rayfin fish: on the branches leading (1) to the
teleosts, (2) to the ostariophysians (zebrafish and relatives), and (3) to the percomorphs (perch-related fish, including medaka, stickleback, and pufferfish),
respectively (Santini et al. 2009; Alfaro et al. 2009). Diversification rate shifts
shortly after the TGD accounted for about 10 % of species diversity in teleosts
(Santini et al. 2009). As noted above, however, TGD duplicates can alter their
functions hundreds of millions of years after the GD itself. Whether individual fish
taxa with additional WGD events (salmonids, barbs, etc.) are actually more species
rich than diploid taxa has not been critically tested.
For plants, it has recently been shown that recent polyploids had lower rates of
speciation and higher rates of extinction than diploid control lineages (Mayrose
et al. 2011). In contrast to recent polyploids, the number of ancient polyploidization events that provided today’s flora is higher than expected if diversification
rates of diploids and polyploids were equal (Mayrose et al. 2011). Furthermore,
ancient WGDs appear to be associated with several major radiations in the
angiosperms (Soltis et al. 2009). In contrast, the ratio of species death rate
(extinction) to species birth rate is 1.5 times higher in teleosts than in non-teleost
rayfin fish (Santini et al. 2009). Thus, crucial questions are, when and under which
circumstances do we expect a polyploidization event to deploy its full potential for
lineage diversification and radiation? Given the deep influence of the TGD on
genome evolution in teleosts illustrated in this chapter and given the described
potential for ongoing non-, sub-, and neofunctionalization long after the TGD
event, its major impact may not be apparent immediately after the GD event.
Obviously, the TGD event was not an evolutionary dead-end, but quite the
contrary should be considered a genomic exaptation that generated countless
‘‘spandrels’’ (Gould and Lewontin 1979) in the teleost genome upon which
selection could act long after the actual polyploidization event, when ‘‘opportunity
meets preparation’’.
964
As we have seen—although recognized since early studies of vertebrate genomes—the apparent propensity for polyploidization in the rayfin fish lineage, and
among them, of cyprinid teleosts in particular, still requires an explanation. Since
the first genomic evidence was published in 1998, the TGD hypothesis has
overcome initial criticism, and the TGD is so far the best-studied GD in ‘‘fish’’. We
are confident that many questions regarding the evolution of genome structure,
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Fig. 17.10 The cover of Susumo Ohno’s book (1970) underlines the importance of fish for
understanding our own past
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gene families, and regulatory circuitry after the TGD can be solved by adding the
sequences of basal rayfins (such as the spotted gar) and of basal teleosts to the
genomic pond. The even harder question about the significance of the TGD for the
teleost radiation will require extensive work on gene function evolution at all
levels of teleost diversity, including numerous non-model fish species. The advent
of next-generation sequencing techniques and the application of developmental
biology studies to an increasing arsenal of fish species are promising for the
acceleration of important research opportunities in this direction. Yet other
questions, for example whether the TGD was an auto- or an allopolyploidization
event, may never be solved due to the millions of years that elapsed since the
event.
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Acknowledgments We would like to thank Cristian Canestro for extensive discussions as well
as Irene Pala for discussions of gene expression in the calandino. This work was supported by a
grant from the Volkswagen Foundation Germany (IB) and National Institutes of Health grant R01
RR020833 (JHP).
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the evolutionary processes that have shaped the vertebrate genome after VGD1
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Book Title
Polyploidy and Genome Evolution
Series Title
Chapter Title
Polyploidization and Sex Chromosome Evolution in Amphibians
Copyright Year
2012
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Springer-Verlag Berlin Heidelberg
Corresponding Author
Family Name
Evans
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Ben J.
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Department of Biology
Organization
McMaster University
Address
Life Sciences Building Room 328, 1280 Main Street West, L8S 4K1,
Hamilton, ON, Canada
Email
evansb@mcmaster.ca
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Alexander Pyron
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Department of Biological Sciences
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The George Washington University
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2023 G St. NW, 20052, Washington, DC, USA
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Wiens
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John J.
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Department of Ecology and Evolution
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Stony Brook University
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Abstract
Genome duplication, including polyploid speciation and spontaneous polyploidy in diploid species, occurs
more frequently in amphibians than mammals. One possible explanation is that some amphibians, unlike
almost all mammals, have young sex chromosomes that carry a similar suite of genes (apart from the genetic
trigger for sex determination). These species potentially can experience genome duplication without
disrupting dosage stoichiometry between interacting proteins encoded by genes on the sex chromosomes and
autosomal chromosomes. To explore this possibility, we performed a permutation aimed at testing whether
amphibian species that experienced polyploid speciation or spontaneous polyploidy have younger sex
chromosomes than other amphibians. While the most conservative permutation was not significant, the frog
genera Xenopus and Leiopelma provide anecdotal support for a negative correlation between the age of sex
chromosomes and a species’ propensity to undergo genome duplication. This study also points to more
frequent turnover of sex chromosomes than previously proposed, and suggests a lack of statistical support
for male versus female heterogamy in the most recent common ancestors of frogs, salamanders, and
amphibians in general. Future advances in genomics undoubtedly will further illuminate the relationship
between amphibian sex chromosome degeneration and genome duplication.
Book ISBN: 978-3-642-31441-4
Page: 385/410
Chapter 18
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Ben J. Evans, R. Alexander Pyron and John J. Wiens
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Abstract Genome duplication, including polyploid speciation and spontaneous
polyploidy in diploid species, occurs more frequently in amphibians than mammals.
One possible explanation is that some amphibians, unlike almost all mammals, have
young sex chromosomes that carry a similar suite of genes (apart from the genetic
trigger for sex determination). These species potentially can experience genome
duplication without disrupting dosage stoichiometry between interacting proteins
encoded by genes on the sex chromosomes and autosomal chromosomes. To explore
this possibility, we performed a permutation aimed at testing whether amphibian
species that experienced polyploid speciation or spontaneous polyploidy have
younger sex chromosomes than other amphibians. While the most conservative
permutation was not significant, the frog genera Xenopus and Leiopelma provide
anecdotal support for a negative correlation between the age of sex chromosomes
and a species’ propensity to undergo genome duplication. This study also points to
more frequent turnover of sex chromosomes than previously proposed, and suggests
a lack of statistical support for male versus female heterogamy in the most recent
common ancestors of frogs, salamanders, and amphibians in general. Future
advances in genomics undoubtedly will further illuminate the relationship between
amphibian sex chromosome degeneration and genome duplication.
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Polyploidization and Sex Chromosome
Evolution in Amphibians
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Book ID: 272454_1_En
Date: 16-8-2012
B. J. Evans (&)
Department of Biology, McMaster University, Life Sciences Building Room 328,
1280 Main Street West, Hamilton, ON L8S 4K1, Canada
e-mail: evansb@mcmaster.ca
R. Alexander Pyron
Department of Biological Sciences, The George Washington University,
2023 G St. NW, Washington, DC 20052, USA
UN
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Layout: T1 Standard SC
Chapter No.: 18
J. J. Wiens
Department of Ecology and Evolution, Stony Brook University,
Stony Brook, NY 11794-5245, USA
P. S. Soltis and D. E. Soltis (eds.), Polyploidy and Genome Evolution,
DOI: 10.1007/978-3-642-31442-1_18, Springer-Verlag Berlin Heidelberg 2012
385
Layout: T1 Standard SC
Chapter No.: 18
Book ID: 272454_1_En
Date: 16-8-2012
Book ISBN: 978-3-642-31441-4
Page: 386/410
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Why polyploidization is more common in plants than in animals is a central
question in biology (Mable 2004; Muller 1925; Orr 1990), and multiple explanations have been put forward (reviewed in Gregory and Mable 2005; Mable 2004;
Orr 1990; Otto and Whitton 2000). One possibility is that the propensity for a
species to undergo polyploidization is related to the extent of sex chromosome
degeneration. Sex chromosome degeneration is the evolution of differences in gene
content that goes beyond the fundamental difference in the presence or absence of
a genetic trigger for sex determination. Degenerate sex chromosomes could
present problems during ‘‘diploidization’’ of a polyploid genome. Diploidization
refers to the phenomenon by which a polyploid species transitions to a mode of
chromosomal inheritance that is similar to a diploid species. A key feature of
diploidization is the switch from polysomic inheritance, where multivalents form
during cell division, to disomic inheritance, where only bivalents form (Wolfe
2001). This phenomenon is probably achieved via divergence between duplicated
pairs of homologous chromosomes. Diploidization therefore could be instantaneous when polyploidization occurs via allopolyploidization (genome duplication
associated with hybridization among diverged species) because duplicated
homologous chromosome pairs already diverged from one another in the ancestral
species. When a polyploid genome with duplicated sex chromosomes becomes
diploidized, one pair of sex chromosomes presumably begins to segregate autosomally. With a degenerate Y-chromosome, for instance, the nascent autosomal
pair that was previously a pair of sex chromosomes would initially have three
possible genotypes: AXAX, AX0, and 00 where AX refers to an autosomal allele
derived from an ancestral X-chromosome and 0 refers to a missing allele that was
lost on the ancestral Y-chromosome. If the 00 genotype is deleterious or lethal,
there would be reproductive incompatibilities in the early stages of diploidization
until the degenerate chromosome is lost. This fitness cost could be mitigated if a
functional paralogous allele were still present on the sex chromosomes, as would
be expected in an autopolyploid (formed from genome duplication within a species). In allopolyploids, however, sex chromosomes could degenerate in unique
ways in each ancestral species, giving rise to diverged gene content, so homozygous null genotypes would be a bigger problem. In both types of polyploids,
dosage balance requirements—natural selection favoring a specific relative
expression level of interacting genes (Papp et al. 2003; Qian and Zhang 2008)—
could impose a fitness cost on a homozygous or heterozygous null autosomal
genotype in a polyploid.
Polyploidization could also present challenges to species with degenerate sex
chromosomes that have also evolved mechanisms for dosage compensation (Orr
1990). Dosage compensation equilibrates expression levels of genes that have one
allele in one sex and two alleles in the other sex (for example, X-linked genes have
one allele in XY males but two alleles in XX females). In this way the stoichiometry
of expression of X-linked and autosomal genes is constant, or ‘‘balanced’’, in males
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18.1.1 Sex Chromosome Evolution
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and females. Orr (1990) argued that a polyploid lineage would initially be established via backcrossing a new polyploid individual to diploid individuals, and that
this would disrupt this balance.
The evolution of differences in gene content is a combined consequence of the
migration of genes, especially genes with sex-specific function, to one or the other
sex chromosome, and also the loss of genes from the region of suppressed recombination on the sex-specific sex chromosome (for example, the Y-chromosome).
The disparity in gene content between the sex chromosomes is thought to increase
over time as a consequence of natural selection (Bergero and Charlesworth 2009;
Charlesworth et al. 2005). Substantial disparity in gene content between the sex
chromosomes could be coupled with selective pressure favoring the evolution of
mechanisms of dosage compensation. For this reason, the proposal that degenerate
sex chromosomes deter polyploidization (including species that lack dosage compensation) is not independent of Orr’s (1990) proposal that dosage compensation
deters polyploidization. In either case, if sex chromosome degeneration acts as a
barrier to polyploidization, this would predict that polyploid species or species with
polymorphism in ploidy levels would have relatively ‘‘young’’, minimally degenerate sex chromosomes as compared with other species.
In this chapter we briefly review polyploidization in frogs and salamanders and
general features of sex chromosome evolution. Using previously published
information, we then use a maximum likelihood approach to analyze the evolution
of new sex-determining mechanisms in frogs and salamanders in a phylogenetic
context, where new mechanisms are inferred either from a change in which
chromosomes are the sex chromosomes, the evolution of a new trigger for sex
determination, or from observed polymorphism in sex-determining mechanisms.
Following this, we explore whether polyploidization occurs more frequently soon
after a new sex-determining mechanism evolves using a permutation test that
accommodates uncertainty in ancestral reconstruction. We conclude that novel
sex-determining mechanisms have evolved in amphibians even more frequently
than previously proposed, and that amphibians with young sex chromosomes may
be more likely to experience genome duplication, resulting either in polyploid
speciation or in spontaneous polyploidy of individuals of an otherwise diploid
species. A major caveat to the latter result is that information on the age of sexdetermining mechanisms of most polyploid amphibians is lacking.
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Sex chromosomes originate from autosomes (Ohno 1967) but differ in carrying
genetic information that (a) differs between the sexes and (b) triggers or represses
sex-specific gonadal differentiation. The ‘‘heterogametic’’ sex produces two types
of gametes, each type with a different sex chromosome, and the ‘‘homogametic’’
sex produces only one type of gamete with respect to the sex chromosomes. The sex
chromosomes of species with male heterogamy are called ‘‘X’’ and ‘‘Y’’ (females
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have two X chromosomes and males have an X and a Y), and the sex chromosomes
of species with female heterogamy are called ‘‘Z’’ and ‘‘W’’ (males have two Z
chromosomes, and females have a Z and a W). In species with genetic sex determination, gonadal differentiation—also known as primary sex determination—is
achieved either using a sex chromosome-specific genetic trigger or by gene dosage,
where the homogametic sex carries two doses of an activator of that sex, or a
repressor of the heterogametic sex.
The age of sex chromosomes influences important aspects of their evolution
and divergence, including divergence in gene content, and the origin of dosage
compensation. For example, the sex chromosomes of therian (placental and
marsupial) mammals are extremely old ([180 million years; Graves 2008), and
the Y chromosome is much smaller than the X and carries fewer genes. This
disparity in size and gene content arose due to Y chromosome ‘‘degeneration’’ as a
consequence of suppressed recombination with the X chromosome (Charlesworth
and Charlesworth 2000). Suppressed recombination ensures that male progeny
inherit an intact copy of the genetic trigger for testis formation, which in therians is
the SRY gene. But this also permits deleterious mutations to accumulate in
Y-linked genes (Muller 1964), leading to loss of function and deletion. ‘‘Muller’s
ratchet’’, the stochastic loss of the least deleterious allele in a population
(Felsenstein 1974), leads to a decline in fitness. This decline occurs more quickly
in regions of the genome that do not recombine. Hill-Robertson effects, background selection, and hitchhiking of deleterious alleles also contribute to fitness
declines of non-recombining portions of the genome (reviewed in Charlesworth
and Charlesworth 2000). Degeneration of the therian Y-chromosome occurred in a
stepwise fashion as the region of suppressed recombination expanded in large
increments (Skaletsky et al. 2003). In therians the disparity in gene content
increased over time after the origin of SRY and associated suppression of
recombination between the X and Y chromosomes. Most angiosperm plants that
have separate sexes (dioecy), in contrast, have comparatively young sex chromosomes that are not substantially differentiated, although exceptions exist
(Bergero and Charlesworth 2011; Charlesworth 2002). Polyploid species are
prevalent in angiosperms (Otto and Whitton 2000) but absent in therians (Svartman et al. 2005), and these observations thus provide anecdotal support for the
contention that the extent of sex chromosome degeneration is negatively correlated
with the incidence of polyploid speciation. Amphibians offer an interesting focal
group with which to further evaluate this hypothesis because some features of
amphibian genome evolution resemble plants more than other animal groups such
as therian mammals. In particular, unlike therian mammals, sex chromosomes in
many amphibians are relatively young, chromosome degeneration is modest or
absent, and polyploidization is fairly common.
Species that determine sex exclusively using environmental triggers do not
have genomic differences between the sexes and therefore have no sex chromosomes. In amphibians, sex determination is genetic so all species are expected to
have sex chromosomes. In addition, temperature has been reported to influence
offspring sex ratios of various species of the salamander genera Pleurodeles and
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Hynobius and the frog genera Bufo, Rana, and laboratory-generated polyploids of
the genus Xenopus (Hayes 1998; Kobel 1996; Schmid and Steinlein 2001). Sex
chromosomes are cytologically distinct in some amphibian species (Schmid et al.
2010). Differences in gene content between the sex chromosomes, which is suggested by cytologically distinct sex chromosomes, led to the independent evolution
of dosage compensation mechanisms in placental mammals, birds, and other
groups such as Drosophila and Caenorhabditis (Arnold et al. 2008; Straub and
Becker 2007). However, in amphibians evidence of dosage compensation has not
been found (Hayes 1998; Schmid et al. 1986; Schmid and Steinlein 2001). One
possible reason for this is that we do not yet know the identity of any amphibian
genes that are hemizygous in the heterogametic sex, so a rigorous test for dosage
compensation in amphibians is not yet possible. These genes would be restricted to
the portion of the X or Z chromosome that does not recombine with the Y or W
chromosome, respectively.
A recent study of European tree frogs identified one way that amphibians
circumvent sex chromosome degeneration (Stöck et al. 2011). In three species, no
recombination occurred between the sex chromosomes in males generated from
intraspecific crosses, yet no intraspecific sex chromosome divergence was observed
(Stöck et al. 2011). This suggests that Muller’s ratchet is periodically reset in these
species by infrequent recombination between the sex chromosomes. Sex chromosome degeneration can also be circumvented by genomic translocation of the sexdetermining locus to another chromosomal pair, or by re-assignment of the sexdetermining function to a gene located elsewhere in the genome. Both of these
phenomena result in a change in which chromosomes are the sex chromosomes
(hereafter ‘‘sex chromosome turnover’’). Ancient examples of sex chromosome
turnover are evinced in amniotes by homology between the sex chromosomes of
platypuses and those of birds but not those of therian mammals (Graves 2008). In
amphibians, sex chromosome turnover is common and is suggested by variation
among and within species in male versus female heterogamy (Ezaz et al. 2006; Hillis
and Green 1990). Using maximum parsimony, Hillis and Green (1990) analyzed
variation in male and female heterogamy in amphibians in a phylogenetic context
and concluded that sex chromosome turnover occurred at least seven times.
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18.1.2 How Many Frog and Salamander Species
are Polyploid?
Comprehensive reviews of polyploidization in amphibians are available in Bogart
(1980), Kawamura (1984), Duellman and Trueb (1994), Beçak and Beçak (1998),
Otto and Whitton (2000), Gregory and Mable (2005), Schmid et al. (2010), and
Mable et al. (2011). The two most recent of these reviews have up-to-date lists of
known polyploid species and associated citations that document polyploidy.
Schmid et al. (2010) also summarize male and female heterogamy in frogs and
salamanders, including information on species with cytologically detectable sex
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chromosome divergence (their Table 8, pp 160–161). In their Supplementary
Information, Mable et al. (2011) provide data on confirmed diploid species that are
closely related to the polyploids. A key difference between these two reviews is
that Mable et al. (2011) include only bisexually reproducing polyploids whereas
Schmid et al. (2010) also include unisexual polyploids. We have attempted to
compile this information as inclusively as possible in Table 18.1, including some
minor corrections and a few additional species and associated citations. Thus, not
all polyploids listed in this table are bisexual, and some are diploid species in
which polyploid individuals occur spontaneously or by induction due to laboratory
manipulation.
Fifty polyploid frog species have been described, including seven triploids, 30
tetraploids, 11 octoploids, and two dodecaploids derived from 15 families and 20
genera (Table 18.1). Three tetraploids and two dodecaploids have been reported
from the genus Xenopus but not yet formally described as species (Evans 2007,
2008; Evans et al. 2004a, 2005a; Tymowska 1991). Stable triploids are known from
three frog genera (Bufo, Eupsophus, and Rana), tetraploids from 16 (Aphantophryne, Astylosternus, Bufo, Chiasmocleis, Dicroglossus, Eleuthrodactylus, Hyla,
Neobatrachus, Odontophrynus, Phyllomedusa, Pleurodema, Pyxicephalus,
Scaphiophryne Silurana, Tomopterna, and Xenopus), octoploids from three
(Ceratophrys, Pleurodema, and Xenopus), and dodecaploids only from Xenopus.
Spontaneous or experimentally induced polyploidy has been reported in at least five
frog species. Six polyploid species of salamander, including four triploids and two
tetraploids, are known from only two genera (Ambystoma and Siren) from two
families (Table 18.1). Spontaneous or experimentally induced triploidy or tetraploidy has been reported in eight salamander species.
The origin of polyploidy necessarily is preceded by the existence of one or more
diploid ancestors. Interestingly, a number of polyploid frog species are inferred to
have originated from ancestral diploid species that do not have known extant diploid
descendants. In Xenopus and Silurana, for example, three currently unknown
diploid species contributed their genomes to extant tetraploid species (reviewed in
Evans 2008). There are also three currently unknown tetraploid species that contributed their genomes to extant octoploid and dodecaploid Xenopus species
(reviewed in Evans 2008). The tetraploid Hyla versicolor is thought to be derived
from multiple independent allopolyploidization events between three diploid species, two of which are currently unknown, and probably extinct given that the region
in which they occur (temperate North America) is well studied (Holloway et al.
2006). In Ceratophrys, there are no known tetraploid species even though three
species in this genus are octoploid (Table 18.1). Similarly, the tetraploid species
Bufo pewzowi is thought to be derived from the diploid B. turanensis and another
unidentified diploid (Stöck et al. 2009), and various tetraploid species of Neobatrachus are derived from diploid ancestors whose diploid descendants are currently
unknown (Mable and Roberts 1997). It is tempting to speculate from these
observations that polyploidization contributed to the long-term survival of these
lineages, given that the diploid ancestors of extant polyploids seem to have gone
extinct in many cases. However, we lack information on how frequently
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Table 18.1 A list of known polyploid amphibians compiled primarily from Schmid et al. (2010)
, Mable et al. (2011) and citations therein
Family and species
Ploidy
Family and species
Ploidy
(Frogs)
(Frogs continued)
Tetraploid
Tetraploid
Tetraploid
Tetraploid
F
Myobatrachidae
Neobatrachus aquilonius
Neobatrachus centralis
Arthroleptidae
Neobatrachus sudelli
Astylosternus diadematus Tetraploid Neobatrachus kunapalari
Microhylidae
Scaphiophryne gottlebei
Bufonidaea
Bufo asmaerae /
Tetraploid Aphantophryne(Cophixalus)
Amietophrynus
pansa
asmaerae
Bufo baturae
Triploid
Chiasmocleis leucosticta
Bufo kerinyagae
Tetraploid Odontophrynidae
Bufo oblongus and
Tetraploid Odontophrynus americanus
subspecies (synonym
of ‘‘B. danatensis’’)
Bufo pewzowi and
Tetraploid Pipidae
subspecies (also a
synonym of
‘‘B. danatensis’’)
Bufo poweri
Triploid
Silurana epitropicalis
Triploid
‘‘Silurana new tetraploid 1’’
Bufo pseudoraddeia
(‘‘Silurana sp. Nov VII’’,
‘‘Silurana paratropicalis’’)b,c
Bufo viridis
Triploid
‘‘Silurana new tetraploid 2’’b
a
Triploid
Xenopus borealis
Bufo zugmayeri
Xenopus clivii
Ceratophryidae
Xenopus fraseri
Tetraploid Xenopus gilli
Craugastoridae
Eleutherodactylus
(Haddadus) binotatus
Ceratophrys dorsata /
Octoploid Xenopus laevisd
Ceratophrys aurita
Ceratophrys joazeirensis Octoploid Xenopus muelleri
Ceratophrys ornata
Octoploid Xenopus pygmaeus
Xenopus largeni (‘‘Xenopus
sp. Nov. III’’)e
Triploid
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Alsodidae
Eupsophus vertebralis
Tetraploid
Tetraploid
Tetraploid
Tetraploid
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Tetraploid
Tetraploid
Tetraploid
Tetraploid
Tetraploid
Tetraploid
Tetraploid
Tetraploid
Tetraploid
Tetraploid
Tetraploid
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Family and species
(Frogs continued)
Dicroglossus
(Hoplobotrachus)
occipitalis
Tetraploid
‘‘Xenopus new tetraploid 1’’ (‘‘Xenopus
sp. Nov. VI’’,’’ Xenopus muelleri
west’’)e,f
Xenopus amieti
Tetraploid
Tetraploid
Xenopus andrei
Xenopus boumbaensis
Xenopus itombwensisg
Xenopus lenduensish
Xenopus vestitus
Xenopus wittei
‘‘Xenopus sp. nov. X’’e,i
Hylidae
Ploidy
Octoploid
Octoploid
Octoploid
Octoploid
Octoploid
Octoploid
Octoploid
Octoploid
Dodecaploid
Dodecaploid
Dodecaploid
Dodecaploid
Tetraploid
Tetraploid
Ranidae
Rana esculenta
Rana japonica*k
Triploid
Triploid
Triploid /
tetraploid
Rana nigromaculata**
Triploid /
Triploid
Triploid
Triploid
Rana pipiens*l
Rana rugosa*m
Triploid
Triploid
Triploid
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Leiopelmatidae
Leiopelma
hochstetteri**
Family and species
(Salamanders)
Ambystomatidae
Ambystoma
jeffersonianum
Ambystoma
mexicanum**
tetraploid
Ambystoma nothagenesn
Ambystoma platineumn
Ambystoma tremblayin
Plethodontidae
Eurycea bislineata**
Xenopus longipes
Xenopus ruwenzoriensis
Tetraploid ‘‘X. cf. boumbaensis’’j
Octoploid ‘‘Xenopus sp. Nov. VIII’’e,i
Tetraploid Pyxicephalidae
Pyxicephalus (Tomopterna) delalandii
Tomopterna tandyi
Triploid
D
Leptodactylidae
Pleurodema bibroni
Pleurodema cordobae
Pleurodema kriegi
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Hyla versicolor
Phyllomedusa
tetraploidea
Tetraploid
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Dicroglossidae
Ploidy
F
Ploidy
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Table 18.1 (continued)
Family and species
(Frogs)
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Table 18.1 (continued)
Family and species
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Ploidy
Family and species
(Frogs continued)
393
Ploidy
Salamandridae
F
Triploid
Triploid
Triploid
Triploid
PR
OO
Pleurodeles waltl*
Ichthyosaura alpestris*
Cynops pyrrhogaster**o
Notophtalamus
viridescens**p
Lissotriton vulgaris**q
Sirenidaer
Siren intermedia
Siren lacertina
Triploid
Tetraploid
Tetraploid
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Additional citations are provided for examples not included in these references, and for unnamed
species
* Experimentally induced
** Spontaneously observed, in some cases also experimentally induced
a
Following taxonomy of Stöck et al. (2009). Further work is needed to confirm stable triploid
ploidy of B. pseudoraddei and B. zugmayeri (Stöck et al. 2009)
b
Evans et al. (2004b)
c
‘‘Silurana paratropicalis’’ is a nomen nudem; see Blackburn DC and Beier M (2011)
d
Here we consider as X. laevis all diverged populations within this clade as identified by Evans
et al. (2004). This includes Xenopus sp. Nov. IX (a.k.a. X. congo 3) from Tymowska (1991), X.
petersi, and X. victorianus
e
Tymowska (1991)
f
Kobel et al. (1996)
g
Table 13 of Schmid et al. (2010) incorrectly lists Xenopus itombwensis as a dodecaploid
h
Evans et al. (2011)
i
Species status requires further investigation
j
Evans (2007)
k
Kawamura and Tokunaga (1952)
l
Briggs (1947)
m
Kashiwagi (1993)
n
These names refer to individuals that are not species in the sense of being reproductively
isolated lineages that persist through time. They are unisexual progeny resulting from hybridization of other species
o
Fankhauser et al. (1942)
p
Fankhauser (1941); Fankhauser and Watson (1942)
q
Litvinchuk et al. (1998)
r
Morescalchi and Olmo (1974) found Pseudobranchus striatus to be polyploid but this result
was not supported by the analysis of Moler and Kezer (1993)
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polyploidization occurs and how frequently diploids outcompete polyploids, so it is
difficult to test this. It is also plausible, for example, that variation in ploidy level is a
neutral phenomenon influenced by stochastic survival and extinction of polyploids
and diploids, or by variation among lineages, including polyploids (Mayrose et al.
2011), in their ability to speciate by polyploidization.
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About one third of the described polyploid frog species belong to the genus
Xenopus. At least six independent instances of genome duplication gave rise to the
ploidy levels seen among extant species in this group, including multiple episodes
that generated the highest ploidy level of any vertebrate—dodecaploidy (reviewed
in Evans 2008). Tetraploid Xenopus evolved at least once, octoploid Xenopus
evolved independently at least three times, and dodecaploid Xenopus evolved
independently at least two times (and possibly more depending on the species
status of Xenopus cf. boumbaensis and of Xenopus sp. nov. VIII; Table 1, Evans
2007, 2008; Evans et al. 2008a, 2011, 2005a; Tymowska 1991). Tetraploidy also
occurred independently in Silurana (Evans 2007; Evans et al. 2005a).
With respect to genome duplication, something is clearly special about Xenopus—
but what? One possible clue emerges from the recent discovery of the first known
genetic trigger of sex determination in amphibians by Yoshimoto et al. (2008). These
researchers identified a female-specific gene called DMW in the tetraploid species
Xenopus laevis. DMW is a W-chromosome linked gene that evolved via gene duplication from another important regulator of sexual differentiation called DMRT1
(Yoshimoto et al. 2008). DMW may function by blocking DMRT1 induction of testis
differentiation (Yoshimoto et al. 2010, 2008). Potentially relevant to the high incidence
of polyploidization in Xenopus is the discovery that DMW originated extremely
recently in amphibian evolution—after divergence of Silurana and Xenopus, but
before diversification of most or all extant species of Xenopus (Bewick et al. 2011). Not
surprisingly, the sex chromosomes of Xenopus are not cytologically distinct (Tymowska 1991). Gene contents of the W and Z chromosomes of Xenopus are therefore
probably very similar, and Xenopus species presumably lack mechanisms of dosage
compensation operating over most sex-linked genes because both sexes have two
alleles at most loci on the sex chromosomes. The preponderance of polyploids in
Xenopus is therefore consistent with the proposal that polyploidization is more likely to
occur in lineages with young, minimally degenerate sex chromosomes.
Another possible link between sex chromosome evolution and polyploidization is provided by Leiopelma hochstetteri. This species has intraspecific variation in the presence of a recently evolved univalent W chromosome that
governs sex determination in females (Green 1988). Leiopelma hochstetteri is
diploid but also has spontaneous triploidy (that is, polyploidy without speciation;
Green et al. 1984). It is not clear whether novel mechanisms for sex determination are more likely to evolve and persist in species that have nondegenerate
sex chromosomes, but this seems plausible under the same reasoning discussed
above with respect to the propensity for lineages to experience polyploidization.
More specifically, if a new pair of sex chromosomes appears in a population then
the old ones would segregate as a newly established autosomal pair. For this
reason, ancestral sex chromosomes with similar gene content would lack or have
few null alleles when they segregate autosomally. While the observation of
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18.1.3 Examples of polyploidy in species with demonstrably
young sex chromosomes
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18.2 Evolution of Sex Determination Systems in Amphibians
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Changes in the heterogametic sex, evolution of new triggers for sex determination,
and polymorphism in sex chromosomes mark the origin of novel features in
genetic pathways for sex determination. In order to quantify how many times this
has happened in amphibians, we began with the large amphibian phylogeny
reported by Pyron and Wiens (2011). We trimmed from this tree all species except
those for which we had information on either heterogamy or polyploidy, or both,
and retained the original maximum likelihood branch lengths among the retained
species. For illustrative purposes, we also retained diploid species (confirmed or
presumed) from the phylogeny of Pyron and Wiens (2011) that are closely related
to polyploid species. In many cases diploidy has been confirmed in these species or
other closely related species (see Supplementary Information of Mable et al.
2011). We had heterogamy information for Physalaemus (Engystomops) petersi,
but this species was not included in the phylogeny. Therefore, we used a closely
related species (Physalaemus cuvieri) that is included in the phylogeny to represent Physalaemus petersi. Physalaemus petersi was the only species from this
genus that was analyzed, so this substitution should be uncontroversial (note that
placing some Physalaemus in Engystomops makes no difference as Engystomops
and Phsalaemus are sister taxa). To better illustrate the phylogenetic distribution
of polyploid species in Fig. 18.1, we also substituted Chiasmocleis hudsoni, which
was present in the phylogeny of Pyron and Wiens (2011), with the tetraploid
species C. leucosticta, which was not present in the phylogeny of Pyron and Wiens
(2011). However, C. leucosticta was not included in any of the analyses described
below because we lack data on heterogamy for this species.
A total of 143 species (97 frogs, 45 salamanders, and one caecilian as an
outgroup) were included. We then converted this tree to a chronogram (a timecalibrated phylogeny) using the penalized likelihood approach (Sanderson 2002),
implemented in r8s version 1.71 (Sanderson 2003). We used the calibration points
detailed in Wiens (2011) but some had to be excluded given the more limited
taxon sampling used here, and some were added or modified given the differences
in taxon sampling (e.g., we added two calibration points within the genus Hyla,
given our more extensive sampling of species in that genus relative to Wiens
(2011). We used the following 17 calibration points. The first 16 were treated as
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spontaneous triploidy suggests a tolerance of polyploidy, L. hochstetteri is not a
polyploid species, so a direct link between the age of the sex chromosomes and
polyploid speciation (as opposed to the toleration of polyploidy) is not established by this species.
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Necturus punctatus
Necturus alabamensis
Necturus maculosus
Necturus beyeri
Necturus lewisi
Aneides ferreus
Hydromantes flavus
Hydromantes supramontis
Hydromantes imperialis
Hydromantes italicus
Hydromantes ambrosii
Eurycea junaluska
Eurycea bislineata
Nototriton abscondens
Nototriton picadoi
Nototriton richardi
Oedipina parvipes
Oedipina pseudouniformis
Oedipina cyclocauda
Oedipina poelzi
Cryptotriton veraepacis
Dendrotriton rabbi
Chiropterotriton dimidiatus
Thorius dubitus
Pleurodeles waltl
Pleurodeles poireti
Triturus marmoratus
Triturus pygmaeus
Triturus dobrogicus
Triturus karelinii
Triturus cristatus
Triturus carnifex
Notophthalmus perstriatus
Notophthalmus viridescens
Ambystoma laterale
Ambystoma mexicanum
Ambystoma dumerilii
Ambystoma tigrinum
Pseudobranchus striatus
Pseudobranchus axanthus
Siren intermedia
Siren lacertina
Hynobius quelpaertensis
Hynobius tokyoensis
Hynobius hidamontanus
Leiopelma hamiltoni
Leiopelma pakeka
Leiopelma archeyi
Leiopelma hochstetteri
Bombina orientalis
Discoglossus pictus
Xenopus gilli
Xenopus laevis
Silurana epitropicalis
Silurana tropicalis
Hymenochirus boettgeri
Pelodytes punctatus
Rana clamitans
Rana catesbeiana
Rana pipiens
Rana sphenocephala
Rana blairi
Rana berlandieri
Rana tagoi
Rana japonica
Rana temporaria
Rana tsushimensis
Rana rugosa
Rana esculenta
Rana lessonae
Rana ridibunda
Rana plancyi
Rana nigromaculata
Buergeria buergeri
Hoplobatrachus crassus
Hoplobatrachus occipitalis
Tomopterna cryptotis
Tomopterna tandyi
Tomopterna delalandii
Pyxicephalus adspersus
Pyxicephalus edulis
Aphantophryne pansa
Cophixalus humicola
Scaphiophryne spinosa
Scaphiophryne gottlebei
Chiasmocleis leucosticta
Chiasmocleis shudikarensis
Astylosternus batesi
Astylosternus diadematus
Strabomantis biporcatus
Pristimantis pulvinatus
Pristimantis shrevei
Pristimantis euphronides
Eleutherodactylus johnstonei
Eleutherodactylus glamyrus
Eleutherodactylus cavernicola
Eleutherodactylus turquinensis
Eleutherodactylus cuneatus
Eleutherodactylus casparii
Eleutherodactylus emiliae
Eleutherodactylus albipes
Physalaemus petersi
Pleurodema thaul
Pleurodema bibroni
Pseudopaludicola falcipes
Vitreorana antisthenesi
Bufo boulengeri
Bufo viridis
Bufo balearicus
Bufo oblongus
Bufo pewzowi
Bufo bufo
Bufo regularis
Bufo poweri
Eupsophus calcaratus
Eupsophus roseus
Eupsophus insularis
Eupsophus migueli
Eupsophus vertebralis
Proceratophrys boiei
Odontophrynus cultripes
Odontophrynus americanus
Ceratophrys cranwelli
Ceratophrys ornata
Phyllomedusa bahiana
Phyllomedusa burmeisteri
Phyllomedusa tetraploidea
Phyllomedusa distincta
Hyla femoralis
Hyla chrysoscelis
Hyla versicolor
Hyla arborea
Hyla intermedia
Hyla sarda
Hyla meridionalis
Pseudis tocantins
Gastrotheca riobambae
Gastrotheca pseustes
Gastrotheca walkeri
Crinia signifera
Neobatrachus pictus
Neobatrachus sudelli
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Fig. 18.1 Heterogamy and polyploidy in salamanders and frogs. The phylogeny is from Pyron
and Wiens (2011) with branch lengths proportional to time based on a relaxed molecular clock
and calibration points described in the text. A black scale bar indicates 40 million years of
evolution. Red and blue rectangles on tips indicate male or female heterogamy respectively;
green, orange, and yellow rectangles indicate de novo sex determining systems; missing data on
heterogamy have no rectangles in this column. In the right column, black rectangles indicate
polyploid species; other species are either confirmed or assumed diploid. Some polyploid species
listed in Table 18.1 are not included in this figure due to a lack of phylogenetic information. Pie
charts on nodes indicate ancestral reconstructions of heterogametic state
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(1) Most recent common ancestor (MRCA) of extant salamanders, at least 150.8
Mya (Millions of years ago), based on the fossil Iridotriton hechti of the
Kimmeridgian/Early Tithonian (Late Jurassic), which is considered to be a
crown-group caudate (Evans et al. 2005b).
(2) MRCA of Salamandroidea (all salamanders exclusive of cryptobranchids,
hynobiids, and sirenids), at least 125.0 Mya (early Barremian, Cretaceous),
based on Galverpeton and Valdotriton (Evans and Milner 1996).
(3) MRCA of plethodontids and proteids, at least 65.5 Mya. The oldest known
amphiumid fossil (Proamphiuma cretacea) is late Maastrichtian or early
Paleocene, and thus from approximately 65.5 Mya (Gardner 2003). The split
between Plethodontidae and Amphiumidae must be at least this old. We do
not have amphiumids included here, but the clade of plethodontids and
proteids must be at least this old given the well-supported clade consisting of
proteids, rhyacotritonids, amphiumids, and plethodontids; see Pyron, Wiens
(2011) and earlier studies.
(4) MRCA of Aneides and Hydromantes, at least 19 Mya. Given the presence of
an Aneides vertebra in the Arikareean period (Tihen and Wake 1981), the
MRCA of the clade containing modern Aneides must be at least 19 Myo
(Millions of years old).
(5) MRCA of Triturus and Notophthalmus at least 33.9 Mya, based on fossils of
Triturus from the Eocene of Europe (33.9–55.8 Mya; Milner 2000)
(6) MRCA of Ambystomatidae and Salamandridae, at least 56.8 Mya, based on a
fossil dicamptodontid (Paleocene; Tiffanian; 60.2–56.8 Mya; Naylor and Fox
1993), and given that the ambystomatidae is the sister group to the Dicamptodontidae (so the sister group to Ambystomatidae ? Dicamptodontidae
must be at least this old).
(7) MRCA of frogs and salamanders, at least 245 Mya, based on a fossil anuran
(Triadobatrachus) from the Early Triassic (251–245 Mya) of Madagascar
(Carroll 1988; Rage and Rocek 1989)
(8) MRCA of pipoids and all other frogs, at least 145.5 Mya, given Rhadinosteus
parvus, ostensibly a rhinophrynid and clearly a pipoid, from the Late Jurassic
(Tithonian, 145.5–150.8 Mya; Rocek 2000).
(9) MRCA of Hymenochirus and Xenopus, at least 83.5 Mya, given the pipid Pachybatrachus taqueti from the Upper Cretaceous (Coniacian-Santonian, 83.5–89.3
Mya), which is thought to be closely related to Hymenochirus (Rocek 2000).
(10) MRCA of Myobatrachidae (represented here by the limnodynastine Neobatrachus and the myobatrachine Crinia) at least 54.6 Mya, given fossils
assigned to the limnodynastine genus Lechriodus (Evans et al., 2008b;
Sanchiz, 1998).
(11) MRCA of Bufonidae ? Leptodactylidae ? Centrolenidae, at least 55.8 Mya,
given putative fossil Bufo from the late Paleocene (55.8–58.7 Mya; Baéz 2000).
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constraints on the minimum age of each clade, and the final calibration point was a
fixed age for the root of the tree.
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(12) MRCA of Ranidae (sensu Wiens et al. 2009) at least 33.9 Mya, given fossil
Rana from the Late Eocene (37.2–33.9 Mya; Rocek and Rage 2000).
(13) Crown group of Terrarana (the clade including the families Brachycephalidae,
Ceuthomantidae, Craugastoridae, Eleutherodactylidae, and Strabomantidae,
or more simply, the clade including Eleutherodactylus and related genera) at
least 35 Mya, based on an Eleutherodactylus fossil in amber from the La Toca
formation (Dominican Republic) estimated to be *35 Myo (Poinar and
Cannatella 1987).
(14) Stem group of Ceratophryidae (the clade including the genera Ceratophrys,
Chacophrys, and Lepidobatrachus) at least 65.5 Mya based on the late Cretaceous fossil genera Beelzebufo and Baurubatrachus (Evans et al. 2008b). Evans
et al. (2008b) considered the Madagascan taxon Beelzebufo to be a ceratophryine. This taxon is of Maastrichtian (Late Cretaceous) age (65.5–70.6
Mya). The South American genus Baurubatrachus is also considered to be a
ceratophryine (Evans et al. 2008b; Rocek 2000). Although the exact relationships of these taxa are somewhat uncertain, the presence of seemingly ceratophryine fossils in South America suggests that the stem group age of
Ceratophryidae is at least 65.5 Mya. The relationships of ceratophryids are
uncertain, but in this molecular analysis, they appear as the sister group to a
clade including Odontophrynidae and Alsodidae (Eupsophus).
(15) Crown-group age of North American and European Hyla clade, at least 16
Myo; given fossil Hyla similar to extant H. arborea and H. meridionalis in
the Lower Miocene of Austria (*16 Myo; Sanchiz 1998). We assume that
these Hyla are closely related to Hyla presently extant in Europe. However,
we cannot assume that these fossils are younger than the crown-group age
of the extant European species. We assume instead that the crown group of
the clade of Hyla is at least 16 Myo based on these European fossils.
(16) MRCA of H. avivoca-H. chrysocelis-H. versicolor clade; H. miocenica is
thought to be closely related to H. chrysocelis and H. versicolor and occurs
in the Barstovian of the Middle Miocene (14–16 Myo; Holman 2003). In
our phylogeny, H. avivoca, H. chrysocelis, and H. versicolor form a clade.
We assume that the stem group age of these three species is at least 14 Myo.
(17) We fixed the root age of the tree using the estimated age from Wiens (2011)
for the MRCA of lissamphibians (frogs, salamanders, caecilians) of 368.3
Mya, using penalized likelihood. Although the use of a fixed calibration
point (rather than a minimum constraint) may seem controversial, it should
be noted that at least one node must be given a fixed age. Furthermore, our
focus here is not on re-estimating these ages, but providing relative
assessments of clade ages (see below).
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Construction of a chronogram using r8s requires a cross-validation step that
identifies a best-fitting value for the ‘‘smoothing parameter’’, which specifies the cost
of differing rates of evolution between neighboring branches (Sanderson 2002).
Cross-validation considered smoothing parameter values from 100 –105.5 in exponential increments of 0.5. These cross-validation analyses failed until a species with a
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zero-length branch (Bufo pewzoi) was removed. After removing this species, the
cross-validation analyses showed that a value of 101 gave the lowest Chi-squared
error. We then generated a chronogram for the 142 remaining species using this
smoothing parameter. We then performed a second analysis using this same
smoothing parameter but including all 143 species. This second analysis gave
identical divergence-date estimates throughout the tree as the first analysis with 142
species. The resulting chronogram (Fig. 18.1) shows species for which we have
information on phylogenetic relationships and either heterogamy and/or ploidy.
Using the R package ‘‘ape’’ (Paradis et al. 2004), which is a software package for
phylogenetic analysis, we tested the fit of alternative models for the evolution of new
sex determination systems for a total of 90 species (55 frogs and 35 salamanders) for
which we had data on heterogamy or sex chromosome polymorphism. This was done
to select an appropriate model for ancestral reconstruction and for use in a permutation test described below. We coded all species as having either male heterogamy
(0), female heterogamy (1), or, for each of three species (Xenopus laevis, Leiopelma
hochstetteri, and Rana rugosa), a unique ‘‘de novo’’ sex-determining mechanism (2,
3, and 4), in which categories we include species with polymorphisms in mechanisms
for sex determination. Xenopus laevis was assigned a unique heterogametic state in
order to account for the finding that this species evolved its W-linked sex-determining gene after the split from S. tropicalis (Bewick et al. 2011), which also has
female heterogamy. Leiopelma hochstetteri was assigned a unique heterogametic
state in order to account for the finding that this species recently evolved a derived
W0 sex-determining system that is unique to this lineage (Green et al. 1993; Sharbel
et al. 1998). Rana rugosa was assigned a unique heterogamy state in order to
accommodate evidence for recent and possibly repeated instances of sex chromosome turnover (Ogata et al. 2008). The three de novo states were coded as separate
character states in order to ensure that known instances of novel sex determination
mechanisms were included in the analysis even though they did not necessarily
involve a change in heterogamy. We note that the newly evolved sex chromosomes
of L. hochstetteri and R. rugosa are polymorphisms, and it is not clear whether these
new polymorphisms will eventually fix in each species, and thus actually constitute a
sex chromosome turnover. However, in both of these examples, at least one of the
polymorphic systems for sex determination is species-specific and therefore new.
Using the ‘‘ace’’ function of the ‘‘ape’’ package, we then evaluated the
following models for evolution of the five heterogamy states:
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(1) All rates equal (one rate for all possible transitions between states, one parameter).
(2) One reversible rate between XY and ZW, and one reversible rate to and from
any of the de novo states (two rates in total, two parameters). By reversible, we
mean, for example, that the rate of change from XY to ZW is equal to the rate
of change from ZW to XY.
(3) One rate for XY to ZW, another rate for ZW to XY, and one reversible rate to
and from any of the de novo states (three rates in total, three parameters).
(4) Rates between each of the five heterogamy states are reversible and unique
(ten rates in total, ten parameters).
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(5) All rates unique (twenty rates in total, twenty parameters).
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The likelihoods of each of these models were compared using the Akaike Information Criterion (Akaike 1974) calculated as 2k–2ln(L) where k is the number of
parameters in the model and L is the maximum value of the likelihood function of the
model. A P value was generated with a hierarchical likelihood ratio test (hLRT) with
degrees of freedom equal to the difference in free parameters of the models under the
assumption of a Chi-squared distribution. For the hLRT we evaluated whether
adding complexity to the models resulted in a significant increase in model fit. The
likelihoods of Models 1, 2, 3, 4 and 5 were -70.74572 (AIC = 143.5), -53.82688
(AIC = 111.7), -53.81533 (AIC = 113.6), -52.31287 (AIC = 124.6), and
-49.97701 (AIC = 140.0) respectively. Model 2 thus was favored by the Akaike
Information Criterion. According to the hLRT, Model 2 was also preferred over
Model 1 (P\0.0001), but Model 3 was not preferred over Model 2 (P = 0.879).
Model 4 was not preferred over Model 2 (P = 0.932) or Model 3 (P = 0.885), and
Model 5 was not preferred over Model 2 (P = 0.982), or Model 3 (P = 0.999), or
Model 4 (P = 0.912). These results suggests that the transition rate from ZW to XY
is not significantly higher than the transition rate from XY to ZW. Model 2 was
therefore used to reconstruct ancestral heterogamy states and also used for simulations in our permutation test described below. The model used in the ancestral
reconstructions differs slightly from the model used in the permutation test in that the
rate of reversal from the de novo heterogamy states to other heterogamy states was set
to zero for the ancestral reconstructions. This was not possible with the permutation
test, which requires a reversible model. We present results from this slightly different
version for the ancestral reconstructions for illustrative purposes because with this
model there is zero likelihood for all of the de novo states in all of the ancestral
reconstructions. Other inferences, such as the likelihood of male and female heterogamy in the most recent common ancestor of frogs, of salamanders, and of frogs and
salamanders discussed below, are identical with both of these models.
The ancestral state reconstructions estimated from the analysis with 90 species
but plotted on the chronogram with 143 species (Fig. 18.1) suggest that there is not
strong statistical support to distinguish whether the ancestral hetrogamy state was
female or male heterogamy (that is, ZW females and ZZ males) in frogs or
salamanders. The marginal likelihood of female heterogamy (ZW) for the most
recent common ancestor of frogs is 0.544, for the most recent common ancestor of
salamanders is 0.499, and for the most recent common ancestor of frogs and
salamanders is 0.513. Thus, the support for ZW versus XY heterogamy as the
ancestral state in each group is effectively equivocal.
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In our analysis, we consider the sex-determining system to have changed every
time that a change in heterogamy occurred from XY to ZW, from ZW to XY, or
from ZW or XY to one of the three de novo sex-determining systems. To quantify
the number of times that the system for sex determination changed in amphibians,
we used the stochastic character mapping approach proposed by Nielsen (2002) as
implemented by the R package ‘‘phytools’’ (Revell 2011). This approach simulates
character evolution on a phylogeny, conditioning on the observed character states
of the terminals. In this way, one can estimate the number of character-state
transitions that occurred and also evaluate where in the phylogeny changes are
likely to have occurred. The stochastic mapping simulations were performed using
a reversible version of Model 2 described above.
As discussed earlier, recent discoveries implicate sex chromosome turnover in
facilitating polyploid speciation or the tolerance of polyploidization, at least in
Xenopus and Leiopelma. These observations raise the question: is this a general
phenomenon in amphibians? Since a change in heterogamy necessarily involves a
change in the sex-determining system, we predicted that the time since a change in
heterogamy (XY to ZW or ZW to XY) would be lower in species that are polyploid or
that tolerate polyploidy than expected by chance, if the same number of polyploid
species (or polyploid-tolerant species) were to evolve randomly on the phylogeny.
We note that this hypothesis does not involve correlation between polyploidy and a
particular heterogamy state, so standard approaches to test for phylogenetic correlation among traits cannot be used to test this prediction. Instead, we developed a
novel permutation test that accommodates uncertainty in when and in which lineages
sex chromosome turnover occurred during amphibian evolution.
From the set of 90 species for which heterogamy information was available, we
identified five phylogenetically independent instances of stable or spontaneous
polyploidy. We emphasize that this is an underestimate of the number of independent polyplodization events, and we were able to use only five instances of
polyploidization because we lack heterogamy and/or phylogenetic information for
the other examples listed in Table 18.1. Diploidy has either been confirmed for the
other species for which heterogamy information was available (Mable et al. 2011)
or was assumed. The five examples of independent polyploidization or tolerance of
polyploidization are:
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(1) Siren intermedia, a tetraploid, which may have descended from a tetraploid
ancestor that also gave rise to S. lacertina (Morescalchi and Olmo 1974). We
note that the polyploid status of the family Sirenidae has not been confirmed
by additional studies (Mable et al. 2011).
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For this permutation test, we used as a test statistic the mean time since the origin
of the current heterogamy state for the five polyploid lineages. This mean was
calculated from 1,000 simulations that are conditioned on the observed heterogamy
states, using the stochastic character mapping approach described by Nielsen (2002)
and implemented by the R package ‘‘phytools’’ (Revell 2011). Each stochastic
mapping simulation provides one possible evolutionary scenario that is consistent
with the data. It was necessary to perform many (1,000) stochastic mapping
simulations for the observed data in order to accommodate uncertainty in these
evolutionary scenarios (that is, to accommodate uncertainty in the ancestral
reconstruction of the evolution of heterogamy).
If polyploidy tends to occur soon after a change in heterogamy, then the
observed test statistic should be smaller than the distribution of statistics calculated
after repeatedly randomly selecting five species across the tree to be polyploid, and
performing 1,000 stochastic character mapping simulations for each of the randomizations. The observed test statistic was therefore compared to a distribution of
statistics generated from 100 randomizations where, in each randomization, five
species are selected to be polyploid, with 1,000 stochastic character mapping
simulations that were conditioned on the observed heterogamy states performed
for each of the randomizations. The difference between the test statistic and the
randomizations therefore is that the mean path length (that is, for each polyploid,
the path length between the most recent change in heterogamy and the branch tip)
was calculated respectively either from real polyploid species (for the test statistic), or from five species selected at random from the 90 species for which we have
heterogamy data (for each of the randomizations).
This test is conservative in the sense that it does not consider subsequent, phylogenetically independent instances of polyploidization that occurred in Xenopus (three
additional independent instances of octoploidization and at least two additional independent instances of dodecaploidization). More specifically, because Xenopus has a
relatively young sex-determining system, a test statistic generated by counting each of the
independent polyploidization events in Xenopus is even lower than the test statistic that
counts polyploidization of Xenopus only once (see below).
One limitation of this analysis is that the frequency of transitions in amphibian
sex-determining systems is undoubtedly underestimated. It is possible, for example,
that changes in heterogamy occurred in other species that were excluded in the
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(2) Ambystoma mexicanum, a species with spontaneous triploidy (Humphrey 1963)
that is closely related to the unisexual triploids A. jeffersonianum, A. platineum,
and A. tremblayi (see notes on species status of unisexuals in Table 18.1).
(3) Xenopus laevis, a tetraploid species (Tymowska 1991). We excluded X. gilli
from this analysis even though we have heterogamy data for this species
because it shares a polyploid ancestor with X. laevis.
(4) Rana esculenta, a naturally occurring diploid/triploid hybridogenic species
formed from hybridization of R. lessonae and R. ridibunda (Uzzell et al. 1975).
(5) Leiopelma hochstetteri, a diploid species with spontaneous triploidy (Green
et al. 1984).
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Stochastic mapping of heterogamy state, including the independent evolution of
three de novo sex chromosomes provides an average estimated number of times
that the sex chromosomes turned over of 32 (95% confidence interval: 25–41)
based on 1,000 simulations that were conditioned on the observed heterogamy
states. This is much higher than the maximum parsimony inference of only seven
changes by Hillis and Green (1990). In the example simulation depicted in
Fig. 18.2a, for instance, there are 28 changes in heterogamy.
These simulations also suggest that more changes occurred from male heterogamy to female heterogamy than the reverse, even though our model comparison
suggested that the rate of change in each direction was not significantly different.
Out of 1,000 stochastic mapping simulations, the mean number of changes from
male to female heterogamy (XY to ZW) was 22 (95 % confidence interval: 13–28)
and the mean number of changes from female to male heterogamy (ZW to XY)
was 7 (95 % confidence interval: 2–14). The example simulation depicted in
Fig. 18.2a is typical of the other simulations in the sense that there are 19 changes
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analysis because differences between the sex chromosomes were not cytologically
detectable. It is also possible that some species experienced a change in the sexdetermining system that did not involve a change in heterogamy. Gastrotheca
pseustes, for instance, is known to have polymorphism in the morphology of the Y
chromosome (Schmid et al. 1990), but this species was coded as XY because these
size variants may involve homologous chromosomes and no change in the sexdetermining system. Intraspecific polymorphism in sex-determining mechanisms
has also been observed in Rana narina, Eleutherodactylus maussi, Rana japonica
and R. narina (Eggert 2005), but phylogenetic information was lacking from these
species in the phylogeny of Pyron and Wiens (2011). Furthermore, adding more taxa
might influence the inferred timing of the transitions to heterogamy, even if the
phylogeny remains the same (e.g., added taxa could subdivide long branches and
help clarify where on a given branch the heterogamy transition occurred).
Another limitation of this analysis is that we use the total time that the polyploid species have been in the observed heterogamy states for our test statistic,
rather than the difference between these times and the age of each polyploid
species. The latter difference would be a better metric for this test because it
focuses on events prior to polyploidization. However, it is difficult to estimate the
age of each polyploid (or of a randomly selected species in the permutation)
because in some cases the diploid ancestor of the polyploid is unknown or extinct
(see above), or other cases because we lack phylogenetic information from the
sister taxon. We note that results are contingent on the phylogeny and evolutionary
model, and that this analysis does not accommodate uncertainty in divergence
times and phylogenetic relationships.
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from male to female heterogamy but only 6 changes from female to male
heterogamy.
The observed average path length to a change in the sex-determining system for
the five polyploids averaged over 1,000 stochastic mapping simulations, was
89.0 million years. This is not to suggest that no sex chromosome degeneration
occurred within this period. In fact, if sex chromosome degeneration in amphibians
occurred at a similar rate as it did in therian mammals (4.6 genes per million years;
Graves 2004), the ancestors of these polyploids probably did have somewhat
degenerate sex chromosomes. Rather, this result suggests that the amount of sex
chromosome degeneration that typically occurs within this period of time was not
of sufficient magnitude to prevent polyploidization or the tolerance of spontaneous
polyploidization. The permutation test indicates that this test statistic is not significantly lower than the distribution of statistics calculated when polyploids
evolved five times on random branches in this phylogeny (P = 0.059; average
path length in permutations was 141.2 million years and the standard deviation
was 32.7 million years), although the P value is close to 0.05. If we include six
independent polyploidizations in Xenopus (one tetraploidization, three
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Fig. 18.2 Simulations that stochastically map evolution of heterogamy form the basis of the
permutation test. Shown here is (a) an example of a stochastic mapping simulation for the
evolution of the observed heterogamy states and (b) highlighted simulated paths to each of the
five observed polyploid species. Species with missing heterogamy data from Fig. 18.1 have been
removed for these analyses and species names are omitted for clarity. In (b) simulated ages of the
observed heterogamy states for Ambystoma mexicanum, Siren intermedia, Leiopelma hochstetteri, Xenopus laevis, and Rana esculentia are labeled 1, 2, 3, 4, and 5 respectively. The test
statistic is the average age of the observed heterogamy state for these five polyploid species,
averaged over 1,000 stochastic mapping simulations. This test statistic is compared to analogous
calculations from 100 permutations where five species were randomly selected to be polyploid
and 1,000 stochastic mapping simulations were performed for each randomization
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octoploidizations, and at least two dodecaploidizations; reviewed in Evans 2008)
in the observed test statistic, in addition to the four other examples of polyploidization itemized above, the average observed path length to a change in the sexdetermining system for the ten independent polyploid lineages is 68.1 million
years. This test statistic is significantly smaller than statistics calculated from 100
permutations where one of the five randomly selected polyploids is also assumed
to undergo six independent polyploidizations (P = 0.020; average path length in
permutations was 169.8 million years and the standard deviation was 55.3 million
years). Although there are at least six independent polyploidizations in Xenopus,
this test suffers from pseudoreplication in that these polyploid lineages may share
the same system for sex determination (i.e., DMW). Additional data on whether
other polyploid species of amphibians have male or female heterogamy would
clearly help illuminate the question of whether species with young sex chromosomes are more tolerant of polyploidization. It is surprising how little is known
about heterogamy of polyploid amphibians given that karyotypes of essentially all
of these species were inspected in order to identify polyploidy in the first place.
One possible reason for this dearth of information on heterogamy of polyploid
species is that many of these species may lack morphologically distinct sex
chromosomes. This proposal, if accurate, would be consistent with the contention
that polyploidization is better tolerated by species with minimally degenerate sex
chromosomes.
Additional insights into the influence of sex chromosome evolution and polyploidization may be gained from studies of laboratory-generated polyploid
Xenopus. Laboratory allopolyploidization in Xenopus duplicates autosomal chromosomes but generates female polyploid individuals with 3 Z chromosomes and 1
W chromosome and male polyploid individuals with 4 Z chromosomes (Kobel and
Du Pasquier 1986). Thus, the W chromosome of one of the ancestral diploids is not
inherited by Xenopus allopolyploids, and therefore never gets a chance to segregate as an autosome. In this way, the mechanism of Xenopus allopolyploidization
in nature may circumvent autosomal segregation of a W chromosome (whether
degenerate or not) (reviewed in Evans 2008), and this could account for the
unusually high incidence of polyploidization in this genus.
Analyses presented here show that changes in the system for sex chromosome
turnover were much more common in amphibians than previously proposed [*32
versus 7 as proposed by Hillis and Green (1990)], and that there is not strong
support for female versus male heterogamy in the ancestor of salamanders, frogs,
or the most recent common ancestor of salamanders and frogs. These changes need
not involve the evolution of completely novel systems for sex determination, and
some of these inferred changes may be reversals to an ancestral system. We also
found that changes from male to female heterogamy occurred more frequently
than changes from female to male heterogamy, although the transition rates
between each state were not significantly different. This result also contradicts the
conclusions of Hillis and Green (1990), who stated that there was a bias in
evolution from female heterogamy to male heterogamy. One reason for these
differences is that our analysis included new data on heterogamy (reviewed in
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Schmid et al. 2010), more species, and a more comprehensive phylogeny
(Pyron and Wiens 2011). Another reason for our higher estimate in the number of
changes is that we considered three de novo changes in sex determination as a new
heterogamy state, thereby forcing a sex chromosome turnover in these lineages
(but this is only 3 out of *32 changes). Differences in the resolution, relationships,
and branch lengths of the phylogenies used in each study are also likely to have
played a role in these differing conclusions. Finally, and importantly, the different
analytical approaches may have influenced the results (that is, the use of maximum
parsimony by Hillis and Green (1990) and maximum likelihood here).
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Acknowledgements We are particularly grateful to Barbara Mable who provided a comprehensive critical assessment of an earlier version of this chapter. We also thank Liam Revell for
advice and assistance with the R package ‘‘phytools’’ and Jim Bogart and Ben Bolker for
comments.
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Y chromosome is a mosaic of discrete sequence classes. Nature 432:823–837
Stöck M, Horn A, Grossen C et al (2011) Ever-young sex chromosomes in European tree frogs.
PLoS Biol 9:e1001062
Stöck M, Ustinova J, Lamatsch DK et al (2009) A vertebrate reproductive system involving three
ploidy levels: Hybrid origin of triploids in a contact zone of diploid and tetraploid Paleartic
green toads (Bufo viridis subgroup). Evolution 64:944–959
Straub T, Becker PB (2007) Dosage compensation: the beginning and end of a generalization. Nat
Rev Genet 8:47–57
Svartman M, Stone G, Stanyon R (2005) Molecular cytogenetics discards polyploidy in
mammals. Genomics 85:425–430
Tihen JA, Wake DB (1981) Vertebrae of plethodontid salamanders from the Lower Miocene of
Montana. J Herpetology 15:35–40
Tymowska J (1991) Polyploidy and cytogenetic variation in frogs of the genus Xenopus.
In: Green DS, Sessions SK (eds) Amphibian cytogenetics and evolution. Academic Press, San
Diego, pp 259–297
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Uzzell T, Berger L, Günther R (1975) Diploid and triploid progeny from a diploid female of Rana
esculenta (Amphibia Salientia). Proc Acad Nat Sci Philadelphia 127:81–91
Wiens JJ (2011) Re-evolution of lost mandibular teeth in frogs after more than 200 million years,
and re-evaluating Dollo’s Law. Evolution 65:1283–1296
Wiens JJ, Sukumaran J, Pyron RA, Brown RM (2009) Evolutionary and biogeographic origins of
high tropical diversity in Old World frogs (Ranidae). Evolution 63:1217–1231
Wolfe KH (2001) Yesterdays’s polyploids and the mystery of diploidization. Nat Rev Genet
2:333–341
Yoshimoto S, Ikeda K, Izutsu Y et al (2010) Opposite roles of DMRT1 and its W-linked paralog,
DM-W, in sexual dimorphism of Xenopus laevis: implications of a ZZ/ZW-type sexdetermining system. Development 137:2519–2526
Yoshimoto S, Okada E, Umemoto H et al (2008) A W-linked DM-domain gene, DM-W, participates
in primary ovary development in Xenopus laevis. Proc Nat Acad Sci 105:2469–2474
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Index
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1
2
19
20
21
22
23
24
25
26
27
28
29
B
Background
selection, 388
Bichir, 352
Bivalent formation/bivalent, 218, 219
Bivalent(s), 37–41, 43–45
Bottom-up approach
(to genome alignment), 97
Bowfin, 351, 360, 367
Brachypodium, 96
Brassica napus, 283, 285
30
31
32
33
34
35
Cephalochordate, 309, 310, 313, 316, 325
Chiasma, 218, 219
Chromosome, 272, 280, 283, 284, 287
non-reduction, 99
number, 78–80
Cod, 343
Comparative expression
profiling, 202
Compensating
aneuploidy, 283
Concerted evolution, 103, 193
Conserved synteny, 346, 351–355, 360
Cotton, 183, 273, 282, 285
Crossover (co), 34, 39–41
Cytomixis, 42
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
D
Detecting whole genome duplication, 77–82,
84, 85, 87, 88
Diakinesis, 34, 42
Diploidization, 386
Diplotene, 34
Disomic, 43
Disomic inheritance, 218–220
Divergent resolution/reciprocal gene loss after
polyploidization, 9, 10, 12
DNA replication, 34, 35
Dosage
balance, 293, 302
compensation, 386, 387
sensitivity, 281
Dosage/dosage balance and
duplicate genes, 2, 5, 14
Double reduction, 43, 44, 46
Double strand breaks (DSB), 34, 39, 40
Duplicate gene expression, 192, 195
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
D
A
Allopolyploidy, 7, 8, 9, 22, 183, 184, 245–247,
250, 252, 254, 255, 257, 259–263,
272, 282
Amphibians, 385, 387–389, 394, 395, 401,
404–406
Amplification fragment length polymorphism
(AFLP), 245, 252, 257, 262
Anaphase, 34, 41, 43, 46
Aneuploidy, 26, 218, 272, 283, 285
Apomixis, 47, 48
Arabidopsis suecica, 285
Arabidopsis thaliana, 285
Autonomous pairing sites (APS), 37
Autopolyploids, 7, 8, 23, 98
Axial element (AE), 35
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C
Calandinos, 345
Carp, 341, 345, 347, 348
Catfish, 345, 367
Centromere, 34, 35, 39, 41, 43
Centromeric divergence, 100
P. S. Soltis and D. E. Soltis (eds.), Polyploidy and Genome Evolution,
DOI: 10.1007/978-3-642-31442-1, Springer-Verlag Berlin Heidelberg 2012
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Index
E
Ecological and adaptive consequences of
hybridization and polyploidy in
spartina, 231
Epigenetic, 216–218, 245, 246, 249,
257, 262–264
Expression, 272, 280–282, 285–287
Expression stoichiometry, 386
79
80
81
82
83
84
85
86
87
88
89
90
F
Fertility, 210, 217–219
Fiber, 202
Fish, 341–344, 349, 350, 352
Fluorescence in situ hybridization (fish), 213,
283, 286, 287
Fractionation
Biased, 141
Mechanism, 143
Fugu, 342, 349, 350, 363, 365
Functional
coherence, 104
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
G
Gamete(s), gametogenesis, 33, 34, 42, 43,
46–48
Gametophyte, 42, 47
Gar, 342, 351, 352, 360, 366, 367
Gene
balance hypothesis, 281
conversion, 194
copy number, 80, 81
duplication and loss, 85–87
Chordate, 309, 310, 313, 315, 322, 324,
325, 327, 328
Vertebrate, 309, 310, 313, 315, 316,
319, 321, 327, 328
dosage, 283
expression, 27, 245, 252, 256, 257, 261,
262, 264
function evolution, 310, 321, 322
loss, 101, 191
Gene tree
parsimony, 82, 83–85
reconciliation, 81–87
Genome
dominance, 141
doubling, 127
duplication, 293, 300, 341, 343, 344, 349,
351, 353
duplication and evolution, 309, 310, 313,
316, 323
evolution, 112, 117, 127
evolution following hybridization and
allopolyploid speciation in spartina,
233
groups, 187
rearrangement, 139, 140
sequence(s), 94
size, 189
Genomic balance, 27
Genomic dominance, 141, 198
Genomic downsizing, 189
Genomic in situ hybridization (GISH), 211,
283, 284, 286, 287
Genomic plasticity, 109, 127
Ghost duplication, 98
Goldfish, 345, 347
Gossypium, 188, 271, 284
barbadense, 188
hirsutum, 187
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
H
Heterodimerization of duplicate genes, 4
Heterogametic;Heterogamy, 387–389, 400,
403, 405
Heterosis, 25
Hexaploids, 28
Hierarchical clustering approach
(to genome alignment), 97
Hill–Robertson effect, 388
Hitchhiking, 388
Homeolog, 195, 278–282, 284–286
Homoelogous (nonhomologous
chromosomes), 38, 39
Homoeolog expression, 197–200
Homoeologous gene expression, 197
Homoeologous pairing, 188
Homoeologue, 257, 261
Homogametic;Homogamy, 387
Homogenization/intergenomic homogenization, 211, 217, 218
Homologous (chromosomes)
homology, 34–40, 45
Hox, 313, 314, 316, 318, 320, 346, 347, 349,
350, 352, 354, 355, 363, 364, 366, 370
Hybridization, 38, 40–43, 46, 47, 245–247,
250, 252–257, 260, 261, 264
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
I
Illegitimate recombination, 190
Introduction, 226, 227, 230, 232
Inversion(s)/chromosomal inversion(s), 217,
219
ITS
165
166
167
168
169
170
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413
K
Karyotype, 272, 283, 284
evolution, 100
restructuring, 210, 217, 218, 220
175
176
L
Leptotene, 34, 35
177
178
179
180
181
182
183
184
185
186
187
188
189
190
M
Medaka, 342, 350–352, 359, 360, 363, 366,
370, 371
Meiosis, 33
Metaphase, 33, 34
Methylation-sensitive amplification
polymorphism (msap), 257, 260–262
Microarray, 197, 252–257, 260, 261, 264
Monosomy, 272, 283–286
Muller’s ratchet, 388
Multivalent(s), 36–39, 41, 43, 44, 46, 47
formation, 7, 8
formation after
polyploidization, 211, 218, 219
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
N
Neofunctionalization, 363, 365–367,
370, 371
of duplicate genes, 3, 11, 14
of pathways, 6
Next-generation sequencing (NGS), 211,
213–215
Nobilization, 99
Non-crossover, 40
Nonfunctionalization, 346, 354, 355, 358,
361–363, 365, 369
Nonfunctionalization
of duplicate genes, 2
Nonreciprocal
exchange, 194
homoeologous recombination, 191
Nuclear-cytoplasmic interaction
hypothesis, 215
Null allele, 394
Nullisomy, 272, 283, 284, 286
211
212
213
214
215
216
P
Pachytene, 33, 34, 36, 42
Pairing (of chromosomes), 33
Pairing-partner switch(es) (PPS), 37–39, 41
Paleopolyploidy, 95
Ph1 locus, 38, 39
Placode, 309, 313, 318, 327–329
Platyfish, 342
Poaceae (gramineae), 94
Polyploidization, 341, 344, 345, 348,
350, 362, 371
Polyploidy, 129
Polysomic, 45
Presence absence variation, 141, 142
Prophase, 34, 41
Psuedogenization, 191, 193
Pufferfish, 345, 350–352, 366
Punnett square, 43
Putative ancestral region (PAR), 97
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
Q
Quadrivalent(s), 43–45, 218
236
237
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O
Octoploids, 28
Ohnolog, 309, 311, 312, 314, 321,
323–325, 329, 354, 356, 358,
362–367, 370
Ohnologon, 354–356, 358
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R
Rabl-configuration, 35, 36
Random-end pairing model, 37
Rates
of gene duplication, 1, 2
of speciation after polyploidization, 13
Ray flower/ray floret, 262, 263, 264
rDNA, 277, 278
Recent polyploidy, 273, 274, 285, 286
Recombination, 33, 34, 38–41, 43, 44, 46, 47
Recombination nodules (RNS), 39
Recurrent reticulate evolution and polyploidy
in spartina, 226
Repeated polyploidy, 273, 274, 276
Retention rates of duplicate genes, 2, 5, 15
Retroelements/retrotransposons, 210, 211,
212, 214, 215
Rho, 96
Ribosomal DNA (rDNA), 210, 211, 213,
215, 216
Rice, 96
Rice chromosomes 11 and 12, 102
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
S
Saccharomyces cerevisiae, 294
Salmon, 342, 345, 346
Satellite repeat(s), 211
Secondary association(s), 96
260
261
262
263
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Index
F
T
Tandem repeat(s), 211, 212
Teleost, 341, 349–352, 359, 360, 366, 369
Telomere, 35, 36, 39
Telomere bouquet (formation), 35, 36
Telophase, 34
Tetraodon, 351, 359, 369
Tetrasomic (inheritance), 43
Tetrasomy, 272, 283–286
Tilapia, 342
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307
308
309
310
311
312
313
314
Tissue-specific silencing, 281, 282
Tobacco, 210, 214, 215
Tragopogon, 271, 273–288
castellanus, 274, 275
mirus, 271–275, 277–279, 282, 283
miscellus, 271–274, 276–285
x mirabilis, 287
Transcriptome shock, 252, 253, 255, 262
Transcriptomic shock, 272, 282, 286, 287
Transgressive, 255
Translocation(s)/intergenomic
translocation(s), 211, 217, 218, 219
Transposable elements, 123, 189
Transposon, 322
Transposon/transposable
element, 252, 253, 260
Trichomes, 182, 198, 202
Tripartite brain, 329
Triploids, 28
Trisomy, 272, 283–286
Triticum, 110, 111, 112, 115, 119, 121, 125,
284, 285
Trout, 342, 346
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Segmental allopolyploids, 25
Segregation, 33, 34, 40, 41, 43–45, 47, 48
Self-incompatibility, 248, 263
Senecio
cambrensis, 245–266, 273, 285, 286
eboracensis, 246, 247, 249, 250, 264
squalidus, 245–251, 253, 254, 257, 258,
262, 263, 266
vulgaris, 248–251, 253–255, 257, 258, 261,
264–266
x baxteri, 245, 250, 252–257, 260–262
Sequenom, 280, 282, 284
Sex chromosome, 385–389, 394, 399, 401,
404, 405
degeneration, 386, 387, 404
turnover, 389, 405
Sigma, 97
Silencing, 192, 196–199
Sister chromatid, 34, 35, 39, 41, 43, 46
Spartina anglica, 273, 285, 286
Speciation, 245–247
Speciation after polyploidization, 9, 11–13
Squalius alburnoides, 345, 348
Stickleback, 342, 345, 351, 355, 356, 358–360,
370, 371
Subfunctionalization, 282, 285, 363–366,
369, 370
Subfunctionalization of duplicate genes, 3, 4,
11, 13, 15
Synapsis, 34, 35, 37–39
Synaptonemal complex (SC), 34, 39
Synonymous substitution, 182, 192
Synteny
blocks, 97
conservation, 314
Synthetic nicotiana tabacum, 211, 214–216,
219, 220
Synthetic polyploids, 271, 273–275, 278,
282, 285, 287
D
S (cont.)
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
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318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
U
Unequal crossing over, 194
Unreduced gametes, 33, 42, 46–48
Urochordate evolution, 329
338
339
340
341
V
Vertebrate, 341, 342, 344, 348–350, 362, 364,
367, 370, 372
Vertebrate Origin and Innovations
Neural Crest, 309, 313, 327, 328
342
343
344
345
346
W
Wheat, 109–123, 125–129, 273, 285
Whole-genome duplication, 94, 271, 272, 277
347
348
349
X
Xenopus, 391, 393, 394, 399, 401, 402, 404
350
351
Z
Zebrafish, 353, 355, 364–366
Zygotene, 34–36
352
353
354
AQ2
356
F
Chapter No.:
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365
Query Refs.
Details Required
370
368
367
369
371
AQ1
Please provide appropriate page number for the index
term ‘ITS’.
376
373
374
375
AQ2
Please provide index terms with appropriate page
numbers for chapters 4 and 9.
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