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Evolution of Asparagus L. (Asparagaceae): Outof-South-Africa and multiple origins of sexual
dimorphism
Article in Molecular Phylogenetics and Evolution · June 2015
DOI: 10.1016/j.ympev.2015.06.002 · Source: PubMed
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Molecular Phylogenetics and Evolution 92 (2015) 25–44
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
Evolution of Asparagus L. (Asparagaceae): Out-of-South-Africa
and multiple origins of sexual dimorphism q
Maria F. Norup a, Gitte Petersen a, Sandie Burrows b, Yanis Bouchenak-Khelladi c, Jim Leebens-Mack d,
J. Chris Pires e, H. Peter Linder c, Ole Seberg a,⇑
a
Natural History Museum of Denmark, Sølvgade 83, Opg. S, K-1307 Copenhagen K, Denmark
Buffelskloof Nature Reserve Herbarium, P.O. Box 710, Lydenburg 1120, South Africa
Institute of Systematic Botany, University of Zurich, Zollikerstrasse 107, 8008 Zurich, Switzerland
d
Department of Plant Biology, University of Georgia, Athens, GA 30602, USA
e
Division of Biological Sciences, 371 B Life Sciences Center, 1201 Rollins Road, University of Missouri-Columbia, Columbia, MO 65211-7310, USA
b
c
a r t i c l e
i n f o
Article history:
Received 26 May 2014
Revised 3 June 2015
Accepted 4 June 2015
Available online 14 June 2015
Keywords:
cpDNA
Sexual dimorphism
Infrageneric structure
Phylogeny
PHYC
Species complexes
a b s t r a c t
In the most comprehensive study to date we explored the phylogeny and evolution of the genus
Asparagus, with emphasis on the southern African species. We included 211 accessions, representing
77 (92%) of the southern African, 6 (17%) of the tropical African, 10 (56%) of the strictly European and
6 (9%) of the Eurasian species. We analyzed DNA sequences from three plastid regions (trnH-psbA,
trnD-T, ndhF) and from the nuclear region phytochrome C (PHYC) with parsimony and maximum likelihood methods, and recovered a monophyletic Asparagus. The phylogeny conflicts with all previous
infra-generic classifications. It has many strongly supported clades, corroborated by morphological characters, which may provide a basis for a revised taxonomy. Additionally, the phylogeny indicates that
many of the current species delimitations are problematic. Using biogeographic analyses that account
for phylogenetic uncertainty (S-DIVA) and take into account relative branch lengths (Lagrange) we confirm the origin of Asparagus in southern Africa, and find no evidence that the dispersal of Asparagus follow
the Rand flora pattern. We find that all truly dioecious species of Asparagus share a common origin, but
that sexual dimorphism has arisen independently several times.
Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction
Although the few commercially important species of the monocot genus Asparagus L. (Asparagaceae) (edible, e.g. A. officinalis, A.
albus; ornamental or medicinal, e.g. A. asparagoides, A. falcatus, A.
setaceus, A. scandens) are well known, the remaining species are
poorly studied. The exact number of species in the genus is uncertain with estimates between 120 and 300+ spp. (Kubitzki and
Rudall, 1998; Mabberley, 2008; World Checklist of Selected Plant
Families, 2009). Many of these are listed as threatened or vulnerable, but the status of even more species is not sufficiently known
(Raimondo et al., 2009; Red List of Threatened Species of Japan,
2007; Sánchez Gómez et al., 2009; Santana et al., 2004). A few species are even a general threat to biodiversity, as they have become
invasive weeds in e.g. Australia, New Zealand and Lord Howe
Island (Auld and Hutton, 2004).
q
This paper was edited by the Associate Editor Timothy Evans.
⇑ Corresponding author.
E-mail address: Oles@snm.ku.dk (O. Seberg).
http://dx.doi.org/10.1016/j.ympev.2015.06.002
1055-7903/Ó 2015 Elsevier Inc. All rights reserved.
Asparagaceae subfamily Asparagoideae as currently delimited
contains the two genera, Asparagus and Hemiphylacus S. Watson
(Chase et al., 2009; Fay et al., 2000; APG III, 2009; Pires et al.,
2006; Seberg et al., 2012), with the large and morphologically variable Asparagus encompassing the vast majority of species.
Asparagus is strictly Old World with a diversification hotspot in
South Africa (Fig. 1), and occurs in regions with semiarid to arid
and Mediterranean-type climates. Hemiphylacus, with only five
known species, is found exclusively in Mexico (Raimondo et al.,
2009). All species of Hemiphylacus are herbaceous, with characters
reminiscent of e.g. Chlorogalum (Lindl.) Kunth, Hastingsia S.
Watson, Schoenolirion Durand, Hesperocallis A. Gray and
Leucocrinum Nutt. ex. A. Gray (Hernandez, 1995). Asparagus, however, contains both herbaceous and sub-shrubby members, as well
as scramblers/climbers, and is generally recognized by the presence of reduced, scale-like leaves, subtending the usually
needle-like, fascicled cladodes. The plants often bear spines and
the flowers are small (2–12 mm diameter), white or whitish, and
the fruit is a berry or rarely a nutlet. The African species of
26
M.F. Norup et al. / Molecular Phylogenetics and Evolution 92 (2015) 25–44
Fig. 1. Distribution map of the 209 currently accepted Asparagus species (Govaerts et al., 2009). Borders represent TDWG regions (Biodiversity Information Standards; www.
tdwg.org), and regions are coloured (see colour bars on left side of figure) according to the number of species in the area. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
Asparagus generally have hermaphroditic flowers, whereas the
majority of Eurasian species are dioecious.
There are two competing hypotheses on the evolutionary pathway to dioecy: either dioecy evolved via monoecy through fixation
of the male/female ratio (Renner and Ricklefs, 1995), or it first
included the coexistence of male-sterile plants (females) in a hermaphroditic population (gynodioecy) followed by female sterility
in the remaining hermaphrodites (Darwin, 1877; Charlesworth
and Charlesworth, 1978). A recent study on dioecy in the monocots
suggests that dioecy in this group evolves more often from hermaphroditism than from monoecy (Weiblen et al., 2000). Despite
lack of direct observations, it has been suggested that the pathway
would possibly include a gynodioecious phase, which is more plausible than a direct transition (involving an unlikely simultaneous
suppression of both male and female development) or androgynoecy (extremely rare), but should be focus of further studies
(Weiblen et al., 2000).
The infrageneric classification in Asparagus has been contentious since Willdenow (1808) described the genus
Myrsiphyllum, that included all known bisexual Asparagus species
with flattened cladodes and axillary flowers. Since then, authors
have either divided the genus into two or more genera (Dahlgren
et al., 1985; Huber, 1969; Kunth, 1850; Obermeyer, 1983, 1984;
Willdenow, 1808), or recognized a single genus (Baker, 1875;
Malcomber and Demissew, 1993; Roemer and Schultes, 1829),
based on different opinions on the importance of various
morphological characters (e.g. spines, flowers, cladodes, or inflorescence structure). Dividing the genus into subgenera was
another approach, i.e. Baker (1875) based his divisions on lowering
the rank of the genera described by Kunth (1850) (Asparagus (L.)
Kunth, Asparagopsis Kunth and Myrsiphyllum), thus resulting in
three subgenera (Eu)asparagus Baker (= Asparagus; the dioecious
species), Asparagopsis (Kunth) Baker (hermaphroditic species with
linear cladodes) and Myrsiphyllum (Willd.) Baker (hermaphroditic
species with flattened, leaf-like cladodes).
Jessop (1966) challenged Baker’s infrageneric classification in
the first complete revision of a major part of the species in which
he recognized 40 southern African species in one genus
(Asparagus). He rejected the split between Asparagopsis and
Asparagus (sensu Kunth (1850) and Baker (1875)), at least as applicable to the South African species, as none of the species which
Kunth (1850) placed in subgenus Asparagus were in fact dioecious.
Jessop instead subdivided Asparagus into eight sections, based on
what he regarded as natural groupings, and with Myrsiphyllum
retained as one of the sections. Obermeyer (1983, 1984) advocated
a split into three genera sensu Kunth: Asparagus, Protasparagus
Oberm. and Myrsiphyllum. Thus, Asparagus again comprising the
dioecious (Eurasian) species; Myrsiphyllum, the hermaphroditic,
southern African species with flattened, leaf-like cladodes, no
spines, and usually connivent filaments and tepals; and
Protasparagus containing the remaining hermaphroditic species
in southern Africa. This circumscription was retained in the
M.F. Norup et al. / Molecular Phylogenetics and Evolution 92 (2015) 25–44
treatment for the Flora of southern Africa (Obermeyer and
Immelman, 1992), which remains the most recent taxonomic
treatment of the genus in the region.
Malcomber and Demissew (1993) disputed the validity of the
subdivision at a generic level, with only flower sexuality differing
between Asparagus and Protasparagus. Instead they proposed a unified genus Asparagus with only two subgenera, Asparagus and
Myrsiphyllum (sensu Obermeyer, but including only the species
with connivent filaments). Subgenus Asparagus thus contained
species with unisexual or hermaphrodite flowers with free and
spreading filaments, and linear to filiform cladodes. However, taking into account the species not included in the treatment by
Malcomber and Demissew (1993), the morphological characters
used to distinguish subgenus Myrsiphyllum become less clear-cut,
and consequently the subdivision was rejected by Fellingham
and Meyer (1995), at least for the southern African species. As a
result the last publication on the subject recognized an undivided
genus, Asparagus, without an infrageneric classification.
Considering the difficulties in delimiting distinct recognizable
groupings based on morphological characters alone, reconstructing
the phylogeny of the genus based on molecular data is the next
logical step. So far, only a few attempts have been made, and most
have a very limited number of species. Thus, both Lee et al. (1997)
and Stajner et al. (2002) included 10 species in their studies based
on Restriction Fragment Length Polymorphism (RFLP), with only
three species overlapping between the studies. Fukuda et al.
(2005) included 24 species in a study using the chloroplast
(cpDNA) loci petD and rpoA. Wiegand (2006) used the ribosomal
ITS and included 53 species. However, our study, using a considerably denser taxon sampling, indicates that ITS is highly problematic for phylogenetic purposes in Asparagus. Despite the
widespread use of ITS in phylogenetic reconstructions,
non-uniform concerted evolution leads to the existence of several
incompletely homogenized rRNA copies, and results in an array
of orthologs and paralogs, which confound the phylogenetic signal
(reviewed e.g. in Álvarez and Wendel, 2003; Feliner and Rosselló,
2007) and render ITS of limited use. The most recent phylogenetic
study of Asparagus (Kubota et al., 2012) used 5 non-coding regions
of cpDNA but only included 23 species. Both Fukuda et al. (2005)
and Kubota et al. (2012) indicate that the species previously placed
in subgenus Myrsiphyllum do not form a monophyletic group,
but find the species included in subgenus Asparagus to be
monophyletic.
The historical biogeography of Asparagus has received little
attention. However, the pattern of an Old World distribution with
a massing of species in southern Africa is not unusual, and is also
found, inter alia, in Aloe L., Gladiolus L., Scrophulariaceae and
Thesium L. Fukuda et al. (2005) and Kubota et al. (2012) suggest
that Asparagus initially radiated and subsequently dispersed out
of southern Africa, a pattern also proposed for Thesium (Moore
et al., 2010), Scrophulariaceae (Oxelman et al., 2005),
Crassulaceae (Mort et al., 2001) and Amaryllidoideae (Rønsted
et al., 2012).
Although the delimitation of most species in Asparagus appears
to be largely unproblematic, there are several difficult species complexes. Consequently the two most recent revisions, by Jessop
(1966) and Obermeyer and Immelman (1992), recognized very different numbers of species. The differences appear to be based on
different interpretations of character variation, with Jessop interpreting many characters as ecologically plastic, while Obermeyer
and Immelman used all variation to define taxa. While molecular
data might not be suitable to determine species limits, they may
still be helpful in circumscribing species.
Using a much broader taxon sampling, the present paper
focuses on the 81 species occurring in southern Africa (Burrows
and Burrows, 2008; Fellingham and Meyer, 1995; National Red
27
List of South African plants, 2009). The species in this region exhibit substantial ecological variation, with both shade and sun tolerant species, shrubs and climbers, as well as the occasional,
introduced European species (A. officinalis) that dies back each winter. Furthermore, there is a great diversity of growth forms and
rooting systems, as well as an interesting variation in leaf (cladode)
morphology (Jessop, 1966; Obermeyer and Immelman, 1992; personal observations).
With data sampled from both cpDNA (trnH-psbA, trnD-trnT and
30 ndhF) and nuclear DNA (phytochrome C (PHYC)) we (1) reconstruct the phylogeny of Asparagus with focus on southern African
taxa, (2) make an assessment of the infrageneric delimitation and
species circumscription in some larger species complexes in
Asparagus, (3) explore the evolution of dioecy and (4) test the
‘‘out of Africa’’ hypothesis of Fukuda et al. (2005) and Kubota
et al. (2012).
2. Materials and methods
2.1. Taxon sampling
A total of 211 accessions of Asparagus were sampled and used
for DNA sequencing (Table 1), representing 77 southern African
species (92% of all currently accepted in the region Obermeyer
and Immelman, 1992), six (of 36) other African species, ten (of
18) European species (including the Canary Islands) and six (of
67) species from temperate and tropical Asia. Species with known
species delimitation problems were represented by multiple samples in order to investigate their delimitation. Table 1 also include
the author abbreviations. Several new and as yet undescribed species were also included in the analysis; these are given provisional
names in quotation marks. Outgroup species consisted of a number
of near-relatives of Asparagus, all selected on the basis of previous
molecular phylogenetic studies of the order Asparagales (Fay et al.,
2000; Pires et al., 2006; Seberg et al., 2012). Due to sampling issues
it was not always possible to obtain sequences for the same species
for all regions. Following the procedure of Campbell and Lapointe
(2009), we combined the sequences building composite taxa to
represent some of the outgroup genera. This is readily discernible
from Table 1.
2.2. DNA extraction, amplification and sequencing
Material was collected in the field and immediately dried in
silica. Total genomic DNA was extracted from the silica-dried
samples using the DNeasy Plant Mini Kit (Qiagen Nordic,
Copenhagen, Denmark).
The primers used for amplification and sequencing are presented in Table 2. For PCR amplification of the cpDNA regions, reactions (per 50 lL) included the following: 5 lL buffer, 10 lL GATC,
1 lL BSA, 0.25 lL taq polymerase, 4 lL Mg2+, 25.75 lL H2O,
1.5 lL of each of the two primers and 1 lL DNA. For PHYC we
included 1 lL DMSO and only 24.75 lL H2O. The products were
purified using the QIAquick PCR purification kit (Qiagen) according
to the manufacturer’s instructions. PCR reactions were run on
a Bio-Rad C1000 Thermal Cycler (Bio-Rad Laboratories,
Copenhagen, Denmark) according to the manufacturer’s protocol.
Thermocycler conditions employed for trnH-psbA amplification
were an initial 3 min at 94 °C, followed by 32 cycles of 94 °C for
1 min, 53 °C for 1 min, 72 °C for 1 min, and a final extension at
72 °C for 7 min. Amplification of 30 ndhF was conducted using conditions similar to the above, except for an annealing temperature of
47 °C. Amplification of trnD-trnT was similar, except we ran 34
cycles and the final extension at 72 °C was only 5 min.
Thermocycler conditions for PHYC were 5 min at 94 °C followed
28
M.F. Norup et al. / Molecular Phylogenetics and Evolution 92 (2015) 25–44
Table 1
Specimen and voucher information for the taxa included in this study. Herbarium acronyms are in accordance with Index Herbariorum (http://sciweb.nybg.org/science2/
IndexHerbariorum.asp). Voucher information for accessions in GenBank is not indicated. Note: All LPA numbers refer to numbers in Banco ADN at Jardin Canario ‘‘Viera y Clavijo’’.
Species
Agavoideae
Agave sp.
Agave parviflora Torr.
Agave striata Zucc.
Lomandroideae
Arthropodium cirrhartum (G.Forst.) R.Br.
Cordyline stricta (Sims) Endl.
Lomandra hastilis (R.Br.) Ewart
Lomandra sp.
Thysanotus spiniger Brittan
Thysanotus sp.
Voucher
M.W. Chase s. n.
Herbarium acronym
K
GenBank accession no.
trnH-psbA
trnD-trnT
30 ndhF
PHYC
–
–
AM884850
–
AF508399
–
JX574227
–
–
–
JX574446
–
–
–
–
–
JX574655
–
–
–
–
AY191184
JX574027
–
AY225024
–
AY225026
–
EU850207
–
–
JX574447
–
JX574448
–
EU850212
JX574449
–
–
–
–
–
JX574656
–
JX574657
–
–
JX574658
–
–
AF508404
–
AY191186
JX574028
–
EU850060
EU850041
JX574029
AY225018
JX574230
–
JX574231
–
–
JX574659
–
JX574233
–
–
–
–
M.W. Chase 651
Frederiksen et al. C206
M.W. Chase 2215
K
C
K
P.J. Rudall
K
M.W. Chase 496
K
J.C. Pires s. n.
MU
M.W. Chase 2053
P. Goldblatt 9463
G. Petersen C421
M.W. Chase 492
K
Unknown
C
K
H. Meusel 379
Unknown
Brodiaeoideae
Brodiaea coronaria (Salisb.) Jeps.
J.C. Pires 96-044
Unknown
–
–
AF508356
JX574229
Asparagoideae
Hemiphylacus latifolius S. Watson
Hemiphylacus alatostylus L.
Asparagus acocksii Jessop
Asparagus acutifolius L.
Asparagus aethiopicus L.
Asparagus aethiopicus L.
Asparagus aethiopicus L.
Asparagus africanus Lam.
Asparagus africanus Lam.
Asparagus aggregatus (Oberm.) Fellingham & N.L. Mey.
Asparagus albus L.
Asparagus altissimus Munby
Asparagus angusticladus (Jessop) J.-P. Lebrun & Stork
Asparagus aphyllus L. ssp. orientalis (Baker) P.H. Davis
Asparagus arborescens Willd. ex Schult. & Schult.f.
Asparagus arborescens Willd. ex Schult. & Schult.f.
Asparagus aridicola Sebsebe
Asparagus asparagoides (L.) W. Wight
Asparagus asparagoides (L.) W. Wight
Asparagus asparagoides (L.) W. Wight
Asparagus asparagoides (L.) W. Wight
Asparagus asparagoides (L.) W. Wight?
Asparagus aspergillus Jessop
Asparagus biflorus (Oberm.) Fellingham & N.L. Mey.
Asparagus buchananii Baker
Asparagus buchananii Baker
Asparagus burchellii Baker
Asparagus burchellii Baker
Asparagus capensis var. capensis L.
Asparagus capensis var. capensis L.
Asparagus capensis var. capensis L.
Asparagus capensis var. capensis L.
Asparagus capensis var. capensis L.
Asparagus capensis var. capensis L.
Asparagus capensis var. litoralis Suess. & Karling
Asparagus capensis var. litoralis Suess. & Karling
Asparagus cf. aethiopicus L.
Asparagus cf. alopecurus (Oberm.) Malcomber & Sebsebe
Asparagus cf. alopecurus (Oberm.) Malcomber & Sebsebe
Asparagus cf. bechuanicus Baker
Asparagus cf. bechuanicus Baker
Asparagus cf. concinnus (Baker) Kies
Asparagus cf. cooperi Baker
Asparagus cf. cooperi Baker
M.W. Chase 668
K.H. Hertweck
S.S. Burrows 9379
A. Marrero s. n.
M.V. Norup 17
M.V. Norup 111
M.V. Norup 103
M.V. Norup 84
S. Burrows 9445
M.V. Norup 141
A. Marrero s.n.
A. Marrero s.n.
S. Burrows 7771
G.&J. Petersen 06-11
J. Caujape et al. s.n.
Acevedo & Siverio s.n.
S. Demissew s.n.
M.V. Norup 55
M.V. Norup 81
M.V. Norup 83
S. Burrows 7762
S. Burrows 8184
M.V. Norup 148
Edwards & Craig s.n.
S. Burrows 9455
M.V. Norup 140
S. Burrows 8209
M.V. Norup 73
M.V. Norup 26
S. Burrows 9422
M.V. Norup 25
M.V. Norup 89
M.V. Norup 52
S. Burrows 9404
M.V. Norup 48
M.V. Norup 49
M.V. Norup 7
M.V. Norup 54
M.V. Norup 60
S. Burrows 9342
M.V. Norup 136
M.V. Norup 110
S. Burrows 9715
S. Burrows 9675
K
Unknown
BNRH
LPA 1037
C
C
C
C
BNRH
C
LPA 1040
LPA 1060
BNRH
C
LPA 1735
LPA 1714
ETH
C
C
C
BNRH
BNRH
C
No Voucher
BNRH
C
BNRH
C
C
BNRH
C
C
C
BNRH
C
C
C
C
C
BNRH
C
C
BNRH
BNRH
JX574445
–
JX574290
JX574271
JX574294
JX574311
JX574312
JX574336
JX574354
JX574327
JX574276
JX574267
JX574326
JX574256
JX574272
JX574273
JX574285
JX574236
JX574237
JX574238
JX574239
JX574240
JX574277
JX574283
JX574321
JX574322
JX574397
JX574398
JX574387
JX574389
JX574390
JX574391
JX574395
JX574396
JX574393
JX574394
JX574298
JX574241
JX574242
JX574344
JX574350
JX574367
JX574337
JX574338
JX574654
–
–
JX574486
JX574507
JX574524
JX574525
JX574548
JX574565
JX574539
JX574491
JX574482
–
JX574471
JX574487
JX574488
JX574500
JX574451
JX574452
JX574453
JX574454
JX574455
JX574492
JX574498
JX574534
JX574535
JX574608
JX574609
JX574598
JX574600
JX574601
JX574602
JX574606
JX574607
JX574604
JX574605
JX574511
JX574456
JX574457
JX574555
JX574561
JX574578
JX574549
–
AY225020
–
JX573880
JX573863
JX573884
JX573900
JX573901
JX573921
JX573939
JX573912
JX573867
JX573859
JX573911
JX573849
–
JX573864
JX573875
JX573829
JX573830
JX573831
JX573832
JX573833
JX573868
–
JX573907
JX573908
JX573980
JX573981
JX573970
JX573972
JX573973
JX573974
JX573978
JX573979
JX573976
JX573977
JX573888
JX573834
JX573835
JX573929
JX573935
JX573952
JX573922
JX573923
–
JX574660
JX574079
JX574062
JX574083
JX574100
JX574101
JX574124
JX574141
JX574115
JX574066
JX574059
JX574114
JX574051
–
JX574063
JX574074
JX574031
JX574032
JX574033
JX574034
JX574035
JX574067
JX574072
–
JX574110
JX574181
JX574182
JX574171
JX574173
JX574174
JX574175
JX574179
JX574180
JX574177
JX574178
JX574087
JX574036
JX574037
JX574132
JX574138
–
JX574125
JX574126
Nolinoideae
Convallaria keiskei Miq.
Convallaria majalis L.
Dracaena reflexa Lam.
Dracaena aubryana Brongn. ex Morren
Eriospermum parvifolium Jacq.
Eriospermum cernuum Baker
Maianthemum bifolium (L.) F.W. Schmidt
Polygonatum hookeri Baker
Polygonatum cirrhifolium (Wall.) Royle
Ruscus hypophyllum L.
Ruscus aculeatus L.
JX574228
–
JX574232
JX574234
29
M.F. Norup et al. / Molecular Phylogenetics and Evolution 92 (2015) 25–44
Table 1 (continued)
Species
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Asparagus
Voucher
cf. cooperi Baker
cf. cooperi Baker
cf. cooperi Baker
cf. cooperi Baker
cf. cooperi Baker
cf. cooperi Baker
cf. cooperi Baker
cf. cooperi Baker
cf. cooperi Baker
cf. cooperi Baker
cf. cooperi Baker
cooperi Baker
cooperi Baker
cooperi Baker ’forma’
cooperi Baker sensu stricto
cooperi Baker sensu sticto
cooperi Baker sensu sticto
cooperi Baker sensu stricto
cf. juniperoides Engl.
cf. laricinus Burch.
cf. suaveolens Burch.
cf. suaveolens Burch.
cf. suaveolens Burch.
cf. suaveolens Burch.
cf. suaveolens Burch.
cf. suaveolens Burch.
cf. suaveolens Burch.
cf. suaveolens Burch.
cf. suaveolens Burch.
cf. suaveolens Burch.
cf. suaveolens Burch.
cf. suaveolens Burch.
cf. suaveolens Burch.
clareae (Oberm.) Fellingham & N.L. Mey.
cochinchinensis (Lour.) Merr.
coddii (Oberm.) Fellingham & N.L. Mey.
concinnus (Baker) Kies
confertus K. Krause
cf. bechuanicus
crassicladus Jessop
crassicladus Jessop
declinatus L.
declinatus L.
declinatus L.
densiflorus (Kunth) Jessop
densiflorus (Kunth) Jessop
densiflorus (Kunth) Jessop
densiflorus ‘‘Licuati’’
denudatus (Kunth) Baker
devenishii (Oberm.) Fellingham & N.L. Mey.
divaricatus (Oberm.) Fellingham & N.L. Mey.
edulis (Oberm.) J.-P. Lebrun & Stork
elephantinus S.M.S. Burrows
exsertus (Oberm.) Fellingham & N.L. Mey.
exsertus (Oberm.) Fellingham & N.L. Mey.
exuvialis Burch.
exuvialis Burch.
falcatus L.
falcatus L.
fasciculatus Thunb.
filicinus Buch.-Ham. ex D.Don
filicladus (Oberm.) Fellingham & N.L. Mey.
flagellaris (Kunth) Baker
flagellaris (Kunth) Baker
flavicaulis (Oberm.) Fellingham & N.L. Mey.
fouriei (Oberm.) Fellingham & N.L. Mey.
fractiflexus (Oberm.) Fellingham & N.L. Mey.
graniticus (Oberm.) Fellingham & N.L. Mey.
hirsutus S.M.S. Burrows
humilis Engl.
inderiensis Blume ex Ledeb.
sp. indet. 2 (‘‘mueda’’)
intricatus (Oberm.) Fellingham & N.L. Mey.
kiusianus Makino
S. Burrows 9151
S. Burrows 9526
S. Burrows 9569
M.V. Norup 139
M.V. Norup 144
M.V. Norup 151
S. Burrows 9446
M.V. Norup 135
S. Burrows 9527
M.V. Norup 119
M.V. Norup 122
M.V. Norup 27
S. Burrows 9501
S. Burrows 7790b
S. Burrows 7792
S. Burrows 9529
S. Burrows 9553
M.V. Norup 127
M.V. Norup 56
M.V. Norup 137
M.V. Norup 88
S. Burrows 9420
M.V. Norup 112
S. Burrows 9429
M.V. Norup 11
M.V. Norup 12
M.V. Norup 65
M.V. Norup 19
M.V. Norup 97
M.V. Norup 18
M.V. Norup 23
S. Burrows 9418
M.V. Norup 104
M.V. Norup 40
S. Burrows 7922
S. Burrows 9572
S. Burrows 9353
S. Burrows 9539
S. Burrows 8370
M.V. Norup 114
M.V. Norup 61
S. Burrows 8465
S. Burrows 9413
M.V. Norup 113b
S. Burrows 7823
M.V. Norup 80
S. Burrows 8155
S. Burrows 8392
M.V. Norup 134
M.V. Norup 41
S. Burrows 7757
S. Burrows 8781
M.V. Norup 85
M.V. Norup 79
M.V. Norup 143
M.V. Norup 22
S. Burrows 8153
M.V. Norup 133
M.V. Norup 67
M. Suzuki et al. s.n.
S. Burrows 9451
S. Burrows 9708
S. Demissew s.n.
S. Burrows 9003
S. Burrows 8784
S. Burrows 9511
M.V. Norup 50
S. Burrows 5352
S. Burrows 9755
Sagalev s. n.
S. Burrows 9786
M.V. Norup 29
Watanabe 93
Herbarium acronym
BNRH
BNRH
BNRH
C
C
C
BNRH
C
BNRH
C
C
C
BNRH
BNRH
BNRH
C
BNRH
C
C
C
C
BNRH
C
BNRH
C
C
C
C
C
C
C
BNRH
C
C
No voucher
BNRH
BNRH
BNRH
BNRH
BNRH
C
C
BNRH
BNRH
C
BNRH
C
BNRH
BNRH
C
C
BNRH
BNRH
C
C
C
C
BNRH
C
C
TUS
BNRH
BNRH
ETH
BNRH
BNRH
BNRH
C
BNRH
BNRH
Unknown
BNRH
C
Unknown
GenBank accession no.
trnH-psbA
trnD-trnT
30 ndhF
PHYC
JX574339
JX574345
JX574346
JX574347
JX574348
JX574349
JX574355
JX574356
JX574357
JX574358
JX574359
JX574342
JX574343
JX574341
JX574360
JX574433
JX574434
JX574435
JX574243
JX574370
JX574401
JX574402
JX574412
JX574413
JX574421
JX574413
JX574414
JX574426
JX574427
JX574429
JX574430
JX574431
JX574432
JX574299
JX574259
JX574325
JX574368
JX574300
JX574351
JX574291
JX574292
JX574246
JX574247
JX574248
JX574308
JX574309
JX574310
JX574441
JX574269
JX574340
JX574380
JX574352
JX574287
JX574386
JX574388
JX574318
JX574319
JX574288
JX574289
JX574249
JX574257
JX574293
JX574252
JX574253
JX574414
JX574281
JX574268
JX574314
JX574323
JX574275
JX574263
JX574328
JX574378
JX574260
JX574550
JX574556
JX574557
JX574558
JX574559
JX574560
JX574566
JX574567
JX574568
JX574569
JX574570
JX574553
JX574554
JX574552
JX574571
JX574642
JX574643
JX574644
JX574458
JX574581
JX574612
JX574613
JX574621
JX574622
JX574630
JX574631
JX574632
JX574635
JX574636
JX574638
JX574639
JX574640
JX574641
JX574512
JX574474
JX574538
JX574579
JX574513
JX574562
JX574504
JX574505
JX574461
JX574462
JX574463
JX574521
JX574522
JX574523
JX574650
JX574484
JX574551
JX574591
JX574563
JX574502
JX574597
JX574599
JX574531
JX574532
–
JX574503
JX574464
JX574472
JX574506
JX574467
JX574468
JX574623
JX574496
JX574483
JX574527
JX574536
JX574490
JX574478
JX574540
JX574589
JX574475
JX573924
JX573930
JX573931
JX573932
JX573933
JX573934
JX573940
JX573941
JX573942
JX573943
JX573944
JX573927
JX573928
JX573926
JX573945
JX574015
JX574016
JX574017
JX573836
JX573955
JX573984
JX573985
JX573995
JX573996
JX574004
JX574005
JX574006
JX574009
JX574010
JX574011
JX574012
JX574013
JX574014
JX573889
JX573852
JX573910
JX573953
JX573890
JX573936
JX573881
JX573882
JX573839
JX573840
JX573841
JX573897
JX573898
JX573899
JX574023
JX573861
JX573925
JX573964
JX573937
JX573877
JX573969
JX573971
JX573905
JX573906
JX573878
JX573879
JX573842
JX573850
JX573883
JX573845
JX573846
JX573997
JX573872
JX573860
JX573902
–
JX573866
JX573855
JX573913
JX573962
JX573853
JX574127
JX574133
JX574134
JX574135
JX574136
JX574137
JX574142
JX574143
JX574144
JX574145
JX574146
JX574130
JX574131
JX574129
JX574147
JX574216
JX574217
JX574218
JX574038
JX574156
JX574185
JX574186
JX574196
JX574197
JX574204
JX574205
JX574206
JX574209
JX574210
JX574212
JX574213
JX574214
JX574215
JX574088
JX574053
JX574113
JX574154
JX574089
JX574139
JX574080
JX574081
JX574041
JX574042
JX574043
JX574097
JX574098
JX574099
JX574223
JX574061
JX574128
JX574166
JX574140
JX574076
JX574170
JX574172
JX574107
JX574108
JX574077
JX574078
JX574044
–
JX574082
JX574047
JX574048
JX574198
JX574070
JX574060
JX574103
JX574111
JX574065
–
JX574116
JX574164
JX574054
(continued on next page)
30
M.F. Norup et al. / Molecular Phylogenetics and Evolution 92 (2015) 25–44
Table 1 (continued)
Species
Asparagus krebsianus (Kunth) Jessop
Asparagus krebsianus (Kunth) Jessop
Asparagus krebsianus or confertus
Asparagus laricinus Burch.
Asparagus lignosus Burm.f.
Asparagus lignosus Burm.f.
Asparagus lignosus or laricinus
Asparagus lynettae (Oberm.) Fellingham & N.L. Mey.
Asparagus macowanii Baker
Asparagus mariae (Oberm.) Fellingham & N.L. Mey.
Asparagus maritimus (L.) Mill.
Asparagus microraphis (Kunth) Baker
Asparagus microraphis (Kunth) Baker
Asparagus minutiflorus (Kunth) Baker
Asparagus sp. indet.
Asparagus mucronatus Jessop
Asparagus mucronatus Jessop
Asparagus multiflorus Baker
Asparagus myriocladus Baker
Asparagus natalensis (Baker) J.-P. Lebrun & Stork
Asparagus natalensis (Baker) J.-P. Lebrun & Stork
Asparagus nelsii Schinz
Asparagus nelsii Schinz
Asparagus nesiotes ssp. purpuriensis Marrero Rodr. &
A. Ramos
Asparagus pauli-guilelemi Solms
Asparagus officinalis L.
Asparagus oligoclonos Maxim.
Asparagus ovatus T.M. Salter
Asparagus oxyacanthus Baker
Asparagus oxyacanthus Baker
Asparagus petersianus Kunth
Asparagus cf. racemosus Willd.
Asparagus racemosus Willd.
Asparagus radiatus Sebsebe
Asparagus ramosissimus Baker
Asparagus recurvispinus (Oberm.) Fellingham & N.L. Mey.
Asparagus recurvispinus (Oberm.) Fellingham & N.L. Mey.
Asparagus retrofractus L.
Asparagus rigidus Jessop
Asparagus rubicundus P.J. Bergius
Asparagus scandens Thunb.
Asparagus schoberioides Kunth
Asparagus sekukuniensis (Oberm.) Fellingham & N.L. Mey.
Asparagus setaceus (Kunth) Jessop
Asparagus setaceus (Kunth) Jessop
Asparagus sp. D
Asparagus spinescens Steud. ex Schult. & Schult.f.
Asparagus spinescens Steud. ex Schult. & Schult.f.
Asparagus spinescens Steud. ex Schult. & Schult.f.
Asparagus stellatus Baker
Asparagus stipulaceus Lam.
Asparagus stipularis Forssk.
Asparagus striatus (L.f.) Thunb.
Asparagus suaveolens Burch.
Asparagus suaveolens Burch.
Asparagus suaveolens Burch.
Asparagus suaveolens Burch.
Asparagus suaveolens Burch.
Asparagus suaveolens Burch.
Asparagus suaveolens Burch.
Asparagus suaveolens Burch.
Asparagus suaveolens Burch.
Asparagus suaveolens Burch.
Asparagus suaveolens Burch.
Asparagus suaveolens Burch.
Asparagus suaveolens Burch.
Asparagus subulatus Thunb.
Asparagus sylvicola S.M.S. Burrows
Asparagus transvaalensis (Oberm.) Fellingham & N.L. Mey.
Asparagus umbellatus ssp. umbellatus Link
Asparagus virgatus Baker
Asparagus virgatus Baker
Asparagus virgatus Baker
Voucher
Herbarium acronym
GenBank accession no.
trnH-psbA
trnD-trnT
30 ndhF
PHYC
M.V. Norup 108
S. Burrows 9403
M.V. Norup 100
M.V. Norup 28
S. Burrows 8761
M.V. Norup 93
M.V. Norup 86
M.V. Norup 36
S. Burrows 8159
S. Burrows 9423
Fukuda 041402
S. Burrows 9442
M.V. Norup 123
S. Burrows 7885
S. Burrows 7817
M.V. Norup 9
M.V. Norup 69
M.V. Norup 20
S. Burrows 10090
S. Burrows 8081
S. Burrows 9481
M.V. Norup 142
M.V. Norup 35
Scholz & Jaen s.n.
C
BNRH
C
C
BNRH
C
C
C
BNRH
BNRH
C
BNRH
C
BNRH
BNRH
C
C
C
BNRH
BNRH
BNRH
C
C
LPA 2285
JX574295
JX574301
JX574297
JX574369
JX574365
JX574366
JX574364
JX574307
JX574331
JX574424
JX574261
JX574372
JX574373
JX574284
JX574437
JX574332
JX574333
JX574335
JX574306
JX574279
JX574280
JX574315
JX574316
JX574270
JX574508
JX574514
JX574510
JX574580
JX574576
JX574577
JX574575
JX574520
JX574543
JX574633
JX574476
JX574583
JX574584
JX574499
JX574646
JX574544
JX574545
JX574547
JX574519
JX574494
JX574495
JX574528
JX574529
JX574485
JX573885
JX573891
JX573887
JX573954
JX573950
JX573951
JX573949
JX573896
JX573916
JX574007
JX573854
JX573956
JX573957
JX573874
JX574019
JX573917
JX573918
JX573920
–
JX573870
JX573871
JX573903
JX573904
JX573862
JX574084
JX574090
JX574086
JX574155
JX574152
JX574153
JX574151
JX574096
JX574119
JX574207
JX574055
JX574158
JX574159
JX574073
JX574220
JX574120
JX574121
JX574123
JX574095
–
JX574069
JX574104
JX574105
–
S. Burrows 9774
G. Petersen & O. Seberg C471
Deguchi 8090
S. Burrows 9424
S. Burrows 9433
M.V. Norup 115
S. Burrows 8852
S. Burrows 8872
Sebsebe s.n.
S. Burrows 9653
S. Burrows 7759
S. Burrows 9365
M.V. Norup 99
M.V. Norup 66
S. Burrows 7791
M.V. Norup 64
M.V. Norup 68
M. Maki s.n.
M.V. Norup 34
M.V. Norup 101
S. Burrows 9371
S. Burrows 7761
S. Burrows 9430
S. Burrows 9434
S. Burrows 9449
S. Burrows 10073
M.V. Norup 82
A. Marrero s.n.
M.V. Norup 107
S. Burrows 7884
S. Burrows 7744
S. Burrows 8197
S. Burrows 8527
S. Burrows 9346
M.V. Norup 131
M.V. Norup 138
M.V. Norup 145
M.V. Norup 31
M.V. Norup 43
M.V. Norup 46
S. Burrows 9408
M.V. Norup 128
M.V. Norup 105
S. Burrows 7895
S. Burrows 8926
Scholz s.n.
S. Burrows 7760
M.V. Norup 130
M.V. Norup 150
BNRH
C
Unknown
BNRH
BNRH
C
BNRH
BNRH
JX574255
JX574262
JX574264
JX574235
JX574329
JX574330
JX574254
JX574320
JX574317
JX574286
JX574250
JX574383
JX574384
JX574362
JX574376
JX574374
JX574251
JX574265
JX574282
JX574438
JX574439
JX574361
JX574415
JX574416
JX574428
JX574371
JX574392
JX574258
JX574377
JX574399
JX574403
JX574400
JX574404
JX574405
JX574406
JX574407
JX574408
JX574409
JX574410
JX574411
JX574420
JX574425
JX574379
JX574440
JX574324
JX574274
JX574442
JX574443
JX574444
JX574470
JX574477
JX574479
JX574450
JX574541
JX574542
JX574469
JX574533
JX574530
JX574501
JX574465
JX574594
JX574595
JX574573
JX574587
JX574585
JX574466
JX574480
JX574497
JX574647
JX574648
JX574572
JX574624
JX574625
JX574637
JX574582
JX574603
JX574473
JX574588
JX574610
JX574614
JX574611
JX574615
JX574616
JX574617
JX574618
JX574619
JX574620
–
–
JX574629
JX574634
JX574590
JX574669
JX574537
JX574489
JX574651
JX574652
JX574653
JX573849
–
JX573856
JX573828
JX573914
JX573915
JX573847
–
–
JX573876
JX573843
JX573966
JX573967
JX573947
JX573960
JX573958
JX573844
JX573857
JX573873
JX574020
JX574021
JX573946
JX573998
JX573999
–
–
JX573975
JX573851
JX573961
JX573982
JX573986
JX573983
JX573987
JX573988
JX573989
JX573990
JX573991
JX573992
JX573993
JX573994
JX574003
JX574008
JX573963
JX574022
–
JX573865
JX574024
JX574025
JX574026
JX574050
JX574056
–
JX574030
JX574117
JX574118
JX574049
JX574109
JX574106
JX574075
JX574045
JX574167
JX574168
JX574149
JX574162
JX574160
JX574046
JX574057
JX574071
JX574221
JX574222
JX574148
JX574199
JX574200
JX574211
JX574157
JX574176
JX574052
JX574163
JX574183
JX574187
JX574184
JX574188
JX574189
JX574190
JX574191
JX574192
JX574193
JX574194
JX574195
JX574203
JX574208
JX574165
–
JX574112
JX574064
JX574224
JX574225
JX574226
BNRH
BRNH
BNRH
C
C
BNRH
C
C
TUS
C
C
BNRH
BNRH
BNRH
BNRH
BNRH
BNRH
C
LPA 1038
C
BNRH
BNRH
BNRH
BNRH
BNRH
C
C
C
C
C
C
BNRH
C
C
BNRH
BNRH
LPA 1731
BNRH
C
C
(continued on next page)
31
M.F. Norup et al. / Molecular Phylogenetics and Evolution 92 (2015) 25–44
Table 1 (continued)
Species
Voucher
Herbarium acronym
GenBank accession no.
trnH-psbA
trnD-trnT
30 ndhF
PHYC
Asparagus volubilis (L.f.) Thunb.
Asparagus volubilis (L.f.) Thunb.
S. Burrows 9425
M.V. Norup 95
BNRH
C
JX574244
JX574245
JX574459
JX574460
JX573837
JX573838
JX574039
JX574040
Manuscript names
Asparagus ‘‘arenosus’’
Asparagus ‘‘barbertonicus’’
Asparagus ‘‘candelus’’
Asparagus ‘‘ferox’’
Asparagus ‘‘iinoinatus’’
Asparagus ‘‘jessopii’’
Asparagus ‘‘karooicus’’
Asparagus ‘‘longissimus’’
Asparagus ‘‘macrocarpa’’
Asparagus ‘‘muedaensis’’
Asparagus ‘‘odoratus’’
Asparagus ‘‘petiolatus’’
Asparagus plocamoides Webb ex Svent.
Asparagus ‘‘pongolanus’’
Asparagus ‘‘praetermissus’’
Asparagus ‘‘pseudoconfertus’’
Asparagus ‘‘vulpicaudatus’’
Asparagus ‘‘vulpicaudatus’’
Asparagus ‘‘Zululand’’
M.V. Norup 47
S. Burrows 8542
M.V. Norup 30
S. Burrows 9439
S. Burrows 9762
S. Burrows 10304
S. Burrows 9428
S. Burrows 7846
S. Burrows 10060
S. Burrows 9778
S. Burrows 8991
Winston Park ex Hort. Bot.
Acevedo & Siverio s.n.
S. Burrows 9530
S. Burrows 9360
S. Burrows 9363
S. Burrows 9440
S. Burrows 7820
S. Burrows 9537
C
BNRH
C
BNRH
BNRH
BNRH
BNRH
BNRH
BNRH
BNRH
BNRH
No voucher
LPA 1163
BNRH
BNRH
BNRH
BNRH
BNRH
BNRH
JX574363
JX574334
JX574417
JX574418
JX574278
JX574302
JX574385
JX574419
JX574303
JX574353
JX574382
JX574381
JX574266
JX574375
JX574313
JX574296
JX574304
JX574305
JX574436
JX574574
JX574546
JX574626
JX574627
JX574493
JX574515
JX574596
JX574628
JX574516
JX574564
JX574593
JX574592
JX574481
JX574586
JX574526
JX574509
JX574517
JX574518
JX574645
JX573948
JX573919
JX574000
JX574001
JX573869
JX573892
JX573968
JX574002
JX573893
JX573938
JX573965
–
JX573858
JX573959
–
JX573886
JX573894
JX573895
JX574018
JX574150
JX574122
JX574201
JX574202
JX574068
JX574091
JX574169
–
JX574092
–
–
–
JX574059
JX574161
JX574102
JX574085
JX574093
JX574094
JX574219
Table 2
Primer regions used for PCR amplification and sequencing.
Marker
trnH-psbA
30 ndhF
trnD-trnT
PHYC (degenerate)
PHYC (Asparagus specific)
Primer name
GUG
/psbA
trnH
1318F/2110R
GUC
trnD
/trnTGGU
Asp_PhyC_F1/Asp_PhyC_R1
PHYC-F1/PHYC-R1
by 39 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for
1.5 min followed by a final extension at 72 °C for 7 min.
Cycle sequencing was performed using the ABI PRISM BigDye
Terminator Cycle Sequencing Ready Reaction Kit (Applied
Biosystems, Foster City, CA, USA), and the products were purified
using the DyeEX 96 kit (Qiagen) according to the manufacturer’s
instructions. The cycle sequencing reactions were run on a
Bio-Rad DNA-Engine Thermal Cycler (Bio-Rad Laboratories). The
protocol consisted of 30 s at 96 °C followed by 29 cycles of 10 s at
96 °C, 15 s at 50 °C and 4 min at 60 °C. Cleaned cycle-sequencing
products were analyzed using an automatic sequencer 3130xl
Genetic Analyzer (Applied Biosystems). Both strands were analyzed
for each region for all taxa, however, despite repeated attempts,
some accessions failed to amplify for some regions. As all of these
accessions did amplify for at least one region, we judged this sufficient to place them in a phylogenetic context.
2.3. Alignment and phylogenetic analysis
Sequences were edited and assembled using Sequencer 4.9
(Gene Codes, Ann Arbor, Michigan, USA). All sequences were initially aligned using ClustalX ver.2 (Larkin et al., 2007) using default
settings, and followed by manual adjustment in MacClade 4.08
(Maddison and Maddison, 2005). Ambiguous parts of the alignment (e.g., some rather long homopolymer regions) and parsimony
uninformative characters were excluded from the analysis.
The four regions were analyzed separately to identify phylogenetic conflicts prior to performing a combined analysis. In addition,
the data were also partitioned into cpDNA and nrDNA datasets. A
‘‘hard’’ incongruence test was performed by directly comparing
Reference/sequence
Shaw et al. (2005)
Olmstead and Sweere (1994)
Shaw et al. (2005)
Hertweck et al. (2015)
50 CAG TTA ACC CTG CTG ATG TAC C 30 ; 50 ACC TCG CCA CTT TAC AAC CT 30
topologies from the separate analyses (i.e., looking for incongruent
clades with bootstrap percentages (BP) > 85 (Sheahan and Chase,
2000; Wiens, 1998)). Only one clade was found to be incongruent,
and consequently the data partitions were combined.
We searched for the optimal cladogram for each dataset using
parsimony as implemented in PAUP⁄ 4.10b (Swofford, 2002). Due
to the large size of the datasets we used the parsimony ratchet
(Nixon, 1999) as implemented in PAUPRat ver.1 (Sikes and Lewis,
2001). All character changes were treated as unordered and
weighted equally (Fitch, 1971), and indels treated as missing data.
We performed ten parsimony ratchet searches of 200 iterations
each with 15% of the characters perturbed. The trees from these
searches were imported into PAUP⁄ and filtered for the best trees,
and these were then used to create a strict consensus tree (Forest
et al., 2007). Bootstrap values (Felsenstein, 1985) were calculated
in PAUP⁄ using 1000 replicates of simple taxon addition, TBR swapping and retaining a maximum of 10 trees per replicate.
The combined dataset was also analyzed under maximum likelihood (ML). A best fitting model of molecular evolution was
selected using jModeltest 0.1.1 (Guindon and Gascuel, 2003;
Posada, 2008). The GTR + I + G model thus selected was then used
in an ML analysis using GARLI v. 0.96 (Zwickl, 2006). As recommended by Zwickl (2006), multiple runs were performed to test
whether the results were consistent. The log-likelihood values of
each run were retained in order to compare the individual runs,
and the tree corresponding to the best score was chosen as the
ML tree. ML bootstrap values were estimated from 100 bootstrap
replicates in GARLI. We refer to nodes with 50–74% bootstrap support as weakly supported, 75–84% as moderately supported and
85–100% as strongly supported (Chase et al., 2000).
32
M.F. Norup et al. / Molecular Phylogenetics and Evolution 92 (2015) 25–44
2.4. Mapping plant reproductive mode
The reproductive mode of the included Asparagaceae species
were scored from own observations in the field, from personal
comments (Julia Pérez de Paz, Jardín Botánico Canario ‘‘Viera y
Clavijo’’), from observations in C, and from the literature (Baker,
1875; Demissew et al., 2008; Feinbrun-Dothan, 1986; Hernandez,
1995; Jessop, 1966; Kunth, 1850; Malcomber and Demissew,
1993; Obermeyer, 1983, 1984; Obermeyer and Immelman, 1992;
Roemer and Schultes, 1829; Sánchez Gómez et al., 2009; Santana
et al., 2004; Valdés, 1979, 1980). Species were scored for the following character states: (1) bisexual, (2) dioecious, (3) gynodioecious or (4) having an ‘‘unidentified sexual dimorphism’’ (for
species in which dimorphism was noted but not specified). Using
Mesquite version 2.72 (Maddison and Maddison, 2009) character
states were optimized onto one of the most parsimonious trees
from the combined analysis. However, due to the consistency in
topology between the parsimony tree topology and the best scoring ML tree (see Section 3.2), the result will be shown on the ML
tree.
2.5. Biogeographic analysis
In order to reconstruct the historical biogeography for
Asparagus despite phylogenetic uncertainty, we used the program
S-DIVA 1.1b (Yu, 2009). This implements the same methodology
as Bayes-DIVA (Nylander et al., 2008). In S-DIVA the ancestral
reconstructions are averaged over all trees and each alternative
ancestral area at each node is weighted by the probability
(S-DIVA; Yu et al., 2010), thus generating credibility support values
for alternative biogeographical scenarios.
S-DIVA has the same computational constraints as DIVA 1.2
(Ronquist, 1997), and can include only trees with fewer than 128
terminals and maximally 15 areas. The number of terminals on
the MP trees from the combined dataset were therefore trimmed
by (1) reducing monophyletic species to one accession each (this
does not change the distribution pattern), (2) deleting undescribed
taxa without provisional names (the distribution areas of these
species were unknown), (3) deleting terminals that had not been
referred to a particular species (e.g. the sample referred to as ‘‘krebsianus’’ or ‘‘confertus Norup 100’’) (these have the same distribution
pattern, and the deletion would not change the overall pattern in
the clades), (4) reducing monophyletic groups of accessions of
the same species or ‘‘cf. species’’ (also within the polyphyletic species) to one accession (these all have the same distribution pattern,
and the reduction to one accession would not change the overall
pattern in the clades), 5) by retaining only a single accession of
each ‘‘cf. species’’ (see above) and 6) combining sister taxa with
same distribution (e.g. cf. alopecurus/cf. juniperoides). S-DIVA
requires fully resolved phylogenies, and as the trees from the combined analysis included a few non-resolved nodes, e.g. in the heavily sampled suaveolens and cooperi clades (however, see below), the
trees were dichotomized using R, ver. 2.10 (Team, 2009).
The 15 areas (northern Europe, southern Europe, North Africa,
Macaronesia, West Africa, East Africa, South Central Africa,
Madagascar, southern Africa, Siberia, Middle East and western
Asia, Far East, India to the Philippines, Australasia and the
Americas) were simplified from the areas from which
Asparagaceae is recorded by Govaerts et al. (2009) by combining
areas with similar species compositions. Analyses allowing an
unconstrained number of ancestral area combinations resulted in
uninterpretable results (i.e. > eight ancestral areas) for some nodes,
thus we constrained the maximum number of areas in ancestral
distributions to two (see also del Hoyo et al., 2009; Guo and
Wang, 2007; Nylander et al., 2008). As only 18 of the 93 investigated species are found in more than two areas, a constraint of
two areas for ancestral nodes is reasonable. Species ranges were
recorded from Govaerts et al. (2009). These maps are biased
against the widespread species, which also occur north of southern
Africa.
Because S-DIVA does not take branch lengths into account
when inferring ancestral ranges (Clark et al., 2008), we also used
the dispersal-extinction-cladogenesis (DEC) analysis (Ree and
Smith, 2008a,b). It is a continuous-time model for geographic
range evolution in which dispersal events cause range expansion,
local extinction events cause range contraction and the probability
of each event along a phylogenetic internode is proportional to
evolutionary time (Clark et al., 2008). To obtain branch lengths
and ultrametricize the phylogeny, we used BEAST v 1.7.5
(Drummond et al., 2006, 2012; Drummond and Rambaut, 2007).
Because we do not have an easily justifiable primary calibration
point, and because the biogeography reconstruction only need relative branch lengths, we constrained the root node to 1.0 under a
normal distribution. We performed one run of 100,000,000 chains,
sampling every 1000 generations. After removing 30,000,000
burn-in samples, we summarized the results using a Maximum
Clade Credibility (MCC) tree. DEC analysis was performed using
LAGRANGE (Ree and Smith, 2008a) on the MCC tree. We wanted
to test the ‘‘out of Africa’’ hypothesis, and DEC requires relatively
few areas, which are occupied by numerous species, to obtain
robust results. Consequently the 15 areas used in the S-Diva analysis were reduced to five as follows: (A) Northern and southern
Europe, (B) North Africa and Macaronesia, (C) Western, Eastern
and Central Africa including Madagascar, (D) southern Africa and
(E) Siberia, Asia, southern Asia, Australasia and the Americas.
Maximum range sizes and dispersals were unrestricted. Area optimizations were reported and considered significant only if the fraction of the global likelihood at each split exceeded 0.5 (Clark et al.,
2008; Drummond and Rambaut, 2007; Drummond et al., 2006,
2012; Ree and Smith, 2008a,b).
3. Results
3.1. Phylogeny – infrageneric delimitation and species circumscription
The phylogenetic hypotheses (the strict consensus trees from
the sets of bootstrapped trees) for the individual cpDNA data sets
were largely congruent, except for a single strongly supported conflict. In the ndhF data set, one accession of doubtful identity, here
tentatively assigned to A. asparagoides (Burrows 8184) is placed
as sister to another accession of A. asparagoides (Burrows 7762)
with strong bootstrap support (88 BP%), whereas in the trnD-T data
set it is even stronger supported (98 BP) as sister to A. ovatus
(Burrows 9424). Combining the cpDNA datasets resulted in an
increase in the resolution and the number of moderately to
strongly supported nodes (P75 BP), compared to each of the individual analyses (bootstrap consensus shown in Fig. S1, Suppl. Mat.;
Table 3). Compared to the cpDNA sequence data, the PHYC data
(bootstrap consensus shown in Fig. S2, Suppl. Mat.) included less
than half the number of characters, but the percentage of variable
characters is 2.3 times larger, and in the most parsimonious tree
there are 32 (rather than 22) nodes with more than 75 BP
(Table 3). There was no strongly supported conflict between the
bootstrap consensus trees from the combined cpDNA data set
and from the PHYC-dataset.
The matrix of the combined cpDNA and nrDNA included a total
of 3969 characters. Of these, 1080 were variable and 633 (18.1%)
were parsimony informative. The combined MP analysis yielded
1904 shortest trees of length 1828, with a CI = 0.49 and an
RI = 0.81 (Table 3, Figs. S3a–3c, Suppl. Mat.). The resolution and
number of highly supported nodes in the combined tree increased
33
M.F. Norup et al. / Molecular Phylogenetics and Evolution 92 (2015) 25–44
Table 3
Statistics of the included datasets (variable and PI characters calculated after exclusion of ambiguous data).
trnD-trnT
trnH-psbA
ndhF
Combined cpDNA
PHYC
cpDNA + PHYC
Aligned matrix
length
Included
characters
Variable
characters
Parsimony informative
characters (PICs)
CI
RI
Length of
shortest tree
Number of nodes
with P75BP
1314
677
803
2794
1175
3969
1222
585
731
2538
962
3500
231
97
306
634
446
1080
126 (10.3%)
41 (7%)
158 (21.6%)
325 (12.8%)
308 (32%)
633 (18.1%)
0.783
0.838
0.700
0.609
0.458
0.489
0.913
0.916
0.860
0.870
0.786
0.812
302
130
523
768
1080
1828
9
4
14
22
32
52
significantly compared to both the combined cpDNA analysis and
the PHYC analysis. The parsimony tree topology is congruent with
the best scoring ML tree ( ln = 21976.467), with the latter being
better resolved. We therefore base the following discussion on the
ML tree (Figs. 2a–2c, with MP bootstrap support indicated above
the branches).
The combined analysis strongly supports the sister group relationship between Hemiphylacus and Asparagus (100 BP, Fig. 2a)
and Asparagus itself is clearly monophyletic (100 BP), too. The
Setaceus clade is weakly supported as sister to the remaining species of Asparagus (70 BP). Several other major, moderately to
strongly supported clades are resolved: the Africani-Capenses
clade (93 BP), the Asparagus clade (82 BP), and the Myrsiphyllum
clade (82 BP), as well as weakly supported clades: the Racemose
clade (64 BP) (sister to a clade of A. macowanii and A. mucronatus),
and the Lignosus clade (66 BP), sister to A. scandens. The overall
relationships among these major clades within Asparagus are not
strongly supported.
Within the clades, the internal relationships receive varying
support. The internal structure within the Africani-Capenses clade
is generally strongly supported. Thus within the Africani clade (65
BP, Fig. 2c) the Cooperi clade (as defined here the Cooperi clade
excluded three collections (Burrows 9529, Burrows 9553 and
Norup 127) actually found in the Setaceus clade), the
Sympodioidi clade and the Canary Island clade (which includes
the gynodioecious Canary Islands species A. plocamoides and A. acutifolius) are all strongly supported (90, 99 and 95 BP, respectively).
Asparagus multiflorus is resolved in an unsupported weak
sister-group relationship (<50 BP) to the Cooperi clade. Within
the Capenses clade (73 BP; Fig. 2c), the Recurvispinus clade (95
BP) consisting of A. recurvispinus and the undescribed A. ‘‘karooicus’’
is sister to the remaining members of the clade. The relationships
within rest of the Capenses clade are not strongly supported. The
Asparagus clade (Fig. 2a) is composed of a moderately to strongly
supported group (82 BP) including bisexual African species (with
distributions from southern to East Africa) plus a few
Mediterranean species, and a monophyletic group of all the dioecious Eurasian species included in the study (Dioecy clade, 60
BP) with moderate to strongly supported internal relationships.
The Racemose clade (Fig. 2b) consists of the Racemose 1 clade
(including the Falcatus clade; 80 BP), the Racemose 2 clade that
includes A. racemosus and allies (with distributions in
Mozambique, Ethiopia and NE South Africa) as well as the
Mediterranean species A. albus. Sister to the rest of the racemose
species is a strongly supported clade of A. graniticus and A. nelsii
(98 BP).
bootstrap support in only three cases: A. asparagoides, A. africanus
and A. cooperi (however, see above). Paraphyletic species are significantly more likely to have wide distribution ranges, than to be
geographically restricted (Mann–Whitney U = 19.00, Z = 2.649,
p = 0.007). A few collection of A. cooperi occur in the Setacues clade
making A. cooperi polyphyletic.
3.3. Analysis of plant reproductive mode
Sexual dimorphism has evolved several times in Asparagus
(Fig. 3). True dioecy seems to have originated twice in the
Asparagus clade, within both the Dioecy clade and in a clade consisting of A. stipularis and A. aphyllus ssp. orientalis. Gynodioecy has
also evolved twice, once in the Setaceus clade and once in the
Canary Islands clade; the latter includes two additional taxa which
hae been scored as having ‘‘unidentified sexual dimorphism’’.
However, Valdés (1980) considers A. acutifolius from the Canary
Island clade, dioecious, but based on our own observation in
herbarium C and observations done by Julia Pérez de Paz (Jardín
Botánico Canario ‘‘Viera y Clavijo’’), A. acutifolius has here been
scored as gynodioecious. This is supported by Feinbrun-Dothan
(1986) who considers all four Asparagus species in Flora
Palaestina, excluding A. stipularis, but including A. acutifolius,
polygamous.
3.4. Biogeographic analysis
The S-DIVA analysis (Fig. 4) gives a 100% probability that the
most recent common ancestor of Asparagus is southern African
(I), and that the common ancestor of Asparagaceae subfamily
Asparagoideae (sensu APG III) originated in the New World (O),
more specifically in Mexico. There were at least five range expansions from southern into tropical Africa, one expansion into North
Africa and Macaronesia. Eurasia was occupied at least twice from
Africa. The dioecious Asian and European species included in the
study originated from a single common ancestor in southern or
South Central Africa.
The DEC analysis (Fig. 5S, Suppl. Mat.) gives similar results,
albeit with a relative probability of the basal Asparagus nodes being
southern Africa of between 0.35 and 0.5, probably because several
species in the first two diverging clades are more widespread in
Africa. DEC finds at least five range expansions into North Africa
or Macaronesia, 10 range expansions into Eurasia and numerous
expansions into tropical Africa. Many of these expansions are by
widespread species.
4. Discussion
3.2. Species delimitation
4.1. Phylogeny – infrageneric delimitation and species circumscription
Of the almost 100 taxa included in the analyses, 69 were represented by single accessions. Hence, the species delimitation of
these could not be evaluated. Of the 30 taxa with more than one
accession, 21 were grouped in the same clade, and nine were largely paraphyletic. However, their paraphyly received significant
4.1.1. Relationship of Asparagus
As in previous analyses of Asparagales (Fay et al., 2000; Pires
et al., 2006; Seberg et al., 2012) we find the Asparagus and
Hemiphylacus to be closely related and their relationship to be
34
M.F. Norup et al. / Molecular Phylogenetics and Evolution 92 (2015) 25–44
98
91
95
90 91
90 94
86
92 100
100
88
99
100
100
100
100
100
100
79
100
74
82
81
79
81
66
71
60
71
62
61
62
100
98
100
94
75
56
60
69
81
89
82
89
Asparagus
62
88
83
67
88 97 64
60
72
91
70
68
100
100
65
67
64
65
100
100
95
92
93
93
100
100
Myrsiphyllum
clade
Lignosus
clade
Asparagus
clade
Racemose Clade
90
87
Asparagaceae
ovatus Burrows 9424
asparagoides winter rainfall Burrows 8184
asparagoides summer rainfall Burrows 7762
asparagoides Norup 55
alopecurus or juniperoides Norup 54
cf. alopecurus Norup 60
cf. juniperoides Norup 56
asparagoides Norup 81
asparagoides Norup 83
volubilis Burrows 9425
volubilis Norup 95
declinatus Norup 61
declinatus Burrows 8465
declinatus Burrows 9413
ramosissimus Burrows 7759
fasciculatus Norup 67
scandens Norup 68
lignosus or laricinus Norup 86
lignosus Norup 93
lignosus Burrows 8761
cf. concinnus Norup 110
concinnus Burrows 9572
stellatus Burrows 10073
microraphis Burrows 9442
microraphis Norup 123
laricinus Norup 28
cf. laricinus Norup 137
rubicundus Norup 64
flagellaris Burrows 9708
flagellaris Sebsebe s.n.
petersianus Burrows 8852
aphyllus ssp. orientalis
stipularis
pauli-guilelemi Burrows 9774
filicinus
schoberioides
kiusianus
oligoclonos
Dioecy
maritimus
clade
inderiensis
officinalis
cochinchinensis
exuvialis Limpopo Prov. Norup 143
exuvialis W. Cape Norup 22
macowanii Burrows 8159
mucronatus Norup 9
mucronatus Norup 69
Africani-Capenses Clade
97
98
retrofractus Norup 66
”arenosus” Norup 47
90
89
63
88 67
88
54
73 67 52
67
68
64
82
70
67
86
75 52
80 92
85
99
100
99
100
56
89
59 52
100 62
100
arborescens Gran C.
umbellatus ssp. umbellatus
arborescens Tenerife
humilis Burrows 9755
cooperi s. str. Burrows 9529
cooperi s. str. Burrows 9553
cooperi s. str. Norup 127
"Zululand” Burrows 9537
mollis Burrows 7817
”barbertonicus” Burrows 8542
setaceus Norup 101
setaceus Burrows 9371
sylvicola Burrows 7895
densiflorus "Licuati" Burrows 8155
virgatus Burrows 7760
virgatus Norup 130
virgatus Norup 150
Hemiphylacus
Cordyline
Arthropodium
Thysanotus
Lomandra
Eriospermum
Maianthemum
Outgroup
Convallaria
Setaceus
clade
Ruscus
Polygonatum
Dracaena
Agave
Brodiaea
Fig. 2a. The GARLI ML tree with the best log likelihood score. Bootstrap values from the analyses are indicated above (MP) and below (ML) the branches, respectively. Nodes
supported by one of the analyses only have just a single value above or below the branch. Undescribed species with manuscript names are indicated as e.g. ‘‘bamboosicolus’’.
Name and number following species epithets in the Southern African Asparagus is the collection name and number. Clade designations in the figure are discussed in the text.
strongly supported (100 BP; Fig. 2a). Apart from the molecular support, there are certain shared morphological and embryological
characters that support this sister group relationship: Rudall
et al. (1998) found that the shape and histology of the fertilized
ovule in Hemiphylacus resembled that of Asparagus. Furthermore,
both genera possess true rhizomes and all species of
Hemiphylacus bear underground tubers, as do several Asparagus
species (Hernandez, 1995; Obermeyer and Immelman, 1992).
M.F. Norup et al. / Molecular Phylogenetics and Evolution 92 (2015) 25–44
35
Fig. 2b. The Racemose clade from the ML analysis shown in Fig. 2a. Bootstrap values from the analyses are indicated above (MP) and below (ML) the branches, respectively.
Nodes supported by one of the analyses only have just a single value above or below the branch. Undescribed species with manuscript names are indicated as e.g. ‘‘biflorus’’.
Name and number following species epithets in the Southern African Asparagus is the collection name and number. Clade designations in the figure are discussed in the text.
However, most of the characters shared by the two genera are plesiomorphic (Rudall et al., 1998). There are several very noticeable
morphological differences which corroborate the generic distinction between Asparagus and Hemiphylacus. Asparagus species vary
from herbaceous climbers to woody shrubs, with flowers placed
singly, a few together, or in raceme-like inflorescences, the fruit
is a berry and the leaves are reduced to scales (the plants instead
bearing needle-like cladodes).
In contrast, Hemiphylacus species are more reminiscent of species of Asparagaceae subfamily Agavoideae (sensu APG III) in being
erect, with normal leaves arranged in a basal rosette and bearing
1–2 reduced, woody stems each producing a 0.5–1.5 m naked,
racemose or panicle-like inflorescence (Hernandez, 1995).
4.1.2. Infrageneric delimitation
The species of Asparagus are resolved as a strongly supported
monophyletic group (100 BP). A subdivision into three separate
genera (Asparagus, Protasparagus and Myrsiphyllum) sensu
Obermeyer (Obermeyer, 1983,1984; Obermeyer and Immelman,
1992) is clearly not warranted, as Asparagus and Myrsiphyllum then
become nested within Protasparagus (Fig. 2). Either a very large
number of genera should be recognized, breaking up the paraphyletic Protasparagus, or a single genus, Asparagus, has to be
maintained. In these circumstances the current taxonomic trend
is to maintain a single, large genus (Humphreys and Linder,
2009). This facilitates recognition and should lead to more nomenclatural stability.
Due to the high number of currently described Asparagus
species, a division of the genus into series or other subgeneric
divisions, as previously done (Jessop, 1966; Obermeyer and
Immelman, 1992) is merited. We tested the validity of the subgeneric divisions described in the latest treatment of Asparagus
(Obermeyer and Immelman, 1992) by mapping these on a species
level phylogeny (Fig. 2), and our results indicate that the majority
of the previous divisions cannot be readily adopted. Rather than
modify the previous classification by transferring species between
the sections to make them monophyletic, it might be better to use
the main clades in our results to provide the backbone of a new set
of subgeneric groups within Asparagus. It is possible that the southern African bias in our sampling could impact the internal structure of the phylogeny, and thus the subgeneric division based on
this. However, based on morphology all non-sampled species can
be readily assigned to the proposed clades. This is consistent with
the observation that the sampled non-South African species are all
included within strongly supported southern African clades, rather
than forming separate clades.
We suggest that the Asparagus species might all be accommodated in six clades. Most of these molecular defined clades can also
be diagnosed morphologically. The formal reclassification will be
done in a separate publication.
Clade 1: Asparagus clade: This moderately supported clade (82
BP, Fig. 2a) consists of (a) the bisexual, semi-woody African species,
A. exuvialis, A. flagellaris, A. petersianus and A. ‘‘pauli-guilelemi’’, (b)
the black-fruited, semi-woody Mediterranean species, A. aphyllus
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M.F. Norup et al. / Molecular Phylogenetics and Evolution 92 (2015) 25–44
Fig. 2c. The Africani-Capenses clade from the ML analysis shown in Fig. 2a. Bootstrap values from the analyses are indicated above (MP) and below (ML) the branches,
respectively. Nodes supported by one of the analyses only have just a single value above or below the branch. Undescribed species with manuscript names are indicated as
e.g. ‘‘candelus’’. Name and number following species epithets in the Southern African Asparagus is the collection name and number. Clade designations in the figure are
discussed in the text.
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M.F. Norup et al. / Molecular Phylogenetics and Evolution 92 (2015) 25–44
bisexual
dioecious
Myrsiphyllum clade
scandens Norup 68
gynodioecious
Lignosus clade
Asparagus
flagellaris Burrows 9708
flagellaris Sebsebe s.n.
petersianus Burrows 8852
aphyllus ssp. orientalis
stipularis
pauli-guilelemi Burrows 9774
filicinus
schoberioides
kiusianus
oligoclonos
Dioecy
maritimus
clade
inderiensis
officinalis
cochinchinensis
exuvialis Limpopo Prov. Norup 143
exuvialis W. Cape Norup 22
Asparagus
clade
Racemose clade
macowanii Burrows 8159
mucronatus Norup 9
mucronatus Norup 69
Asparagaceae
plocamoides
altissimus
nesiotes ssp. purpuriensis
acutifolius
fractiflexus Burrows 9511
denudatus Burrows 8392
multiflorus Norup 20
Cooperi clade
Sympodioidi clade
Canary Island
clade
Africani
clade
AfricaniCapenses
clade
Capenses clade
retrofractus Norup 66
”arenosus” Norup 47
umbellatus ssp. umbellatus
arborescens Gran C.
arborescens Tenerife
humilis Burrows 9755
cooperi s. str. Burrows 9529
cooperi s. str. Burrows 9553
cooperi s. str. Norup 127
setaceus bushveld form Burrows 9537
mollis Burrows 7817
”Zululand” Burrows 8542
setaceus Norup 101
setaceus TOPO Burrows 9371
sylvicola Burrows 7895
densiflorus "Licuati" Burrows 8155
virgatus Burrows 7760
virgatus Norup 130
virgatus Norup 150
Hemiphylacus
Outgroup
Setaceus clade
Fig. 3. The ML tree from Fig. 2a–c redrawn to show evolution of reproductive mode in Asparagus. Clades in which no shifts have occurred (or are known) have been collapsed.
See the text for further explanation.
ssp. orientalis and A. stipularis (both of which express sexual dimorphisms as they have been described as diclinous (Roemer and
Schultes, 1829) and functionally unisexual (Valdés, 1979)), respectively, and (c) the truly dioecious, mainly herbaceous Eurasian species (Dioecy clade; 60 BP), belonging to subgenus Asparagus. The
majority of the species in this clade are spineless; however, the
tropical A. flagellaris and A. petersianus (possibly also A.
‘‘pauli-guilelemi’’) do possess leaf-derived spines. These three species are morphologically similar scramblers with orange fruits,
possibly part of a species complex (to be reviewed by Burrows
et al., in preparation). The sampling in the distribution range of this
clade is sparse; in total it might include 4–5 species.
Clade 2: Racemose clade: The Racemose clade does not receive
high bootstrap support (64 BP; Fig. 2b), but includes all species
with raceme-like inflorescences, many of them being scramblers
or climbers. It also includes the majority of the Asparagus species
with ‘‘normal’’ spines (hard, woody spines modified from leaves
supporting the side-branches). Obermeyer dispersed the species
from this clade in four series: Globosi p.p., Protasparagus,
Racemosi and Exuviali p.p. The species within the Racemose clade
also differ from the rest of the known Asparagus species in their
rooting structures. Within the clade, two unequally-sized, weakly
supported subclades (Racemose 1 and Racemose 2 clade, supported by 55 and 57 BP respectively; Fig. 2b) and one strongly
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M.F. Norup et al. / Molecular Phylogenetics and Evolution 92 (2015) 25–44
Fig. 4. Phylogeny of the combined analysis showing the result of the biogeographic analysis. The trees used for the S-DIVA analysis were the 1720 MP trees. Node charts are
the results of averaging ancestral reconstructions over all trees, with each alternative ancestral area at a node weighted by its probability. The range for each accession/group
of accessions are given after the accession names and is from Govaerts et al. (2009). Clade designation follows Fig. 2. Areas used in the biogeographic analysis are based on the
TDWG regions used in Govaerts et al. (2009): A = Northern Europe + Western Europe + Eastern Europe; B = South-western Europe + South-eastern Europe; C = Northern
Africa; D = Macaronesia (Canary Islands only); E = West Tropical Africa + West-Central Tropical Africa; F = East Tropical Africa + Northeast Tropical Africa; G = South Tropical
Africa; H = Western Indian Ocean (Madagascar only); I = Southern Africa; J = Siberia + Russian Far East; K = Arabian Peninsula + Middle Asia + Caucasus + Western Asia;
L = China + Mongolia + Eastern Asia; M = Indian Subcontinent + Indo-China + Malesia (Philippines only); N = Australia + New Zealand; O = New World (all New World regions
combined). Grey circles represent area optimizations = ABCDEFGHIJKLM.
M.F. Norup et al. / Molecular Phylogenetics and Evolution 92 (2015) 25–44
supported subclade (Graniticus clade, 98 BP) can be recognized, all
of which have specialized root structures. The Racemose 1 subclade, which has tubers on the lateral roots, is strongly supported
in the PHYC dataset (91 BP; Fig. S2), though not so in the combined
analysis. This is probably due to incongruence between the cpDNA
dataset and the PHYC dataset with respect to the position of A.
‘‘inopinatus’’. The Racemose 2 subclade and the Graniticus clade
lack tubers, but instead have swollen main roots (although in A.
graniticus the main roots are thick and woody). Similar fleshy roots
are also found in the sister group to the Racemose clade (A.
mucronatus and A. macowanii), however, the roots in A. mucronatus
bear tubers (absent in A. macowanii) and the flowers in these species are solitary among the cladode fascicles, not in compound
inflorescences.
Clade 3: The Lignosus clade (66 BP; Fig. 2a) includes six morphologically similar species with erect, woody stems, leaf-derived
spines, orange anthers and usually 10–15 cladodes. Remarkably,
this clade brings together species from four of Obermeyer’s series:
Protasparagus, Myrsiphyllum, Globosi and Africani.
Clade 4: Africani-Capenses clade: The species within this clade
(93 BP; Fig. 2c) do not possess the woody leaf-derived spines that
are often associated with Asparagus. In the Africani clade, the
leaves are either modified into hard, woody scales (Sympodioidi
clade) or forming brown, papery spine-structures (Cooperi clade),
whereas in the Capenses clade the leaves are present as soft, papery scales.
The Africani clade (65 BP) includes three distinctive elements:
(a) the Cooperi clade (90 BP), which includes species from
Obermeyer’s series Retrofracti and Africani and mainly grows in
exposed habitats (i.e. to sun, wind and weather impacts), and often
is thicket-forming, with papery spine-structures (sometimes suppressed) and red berries; (b) the Sympodioidi clade (99 BP), which
corresponds to Obermeyer’s series Sympodioidi (Obermeyer and
Immelman, 1992) and is easily identified by combing divaricating
stems with hard, woody scales and umbellate inflorescences and
(c) the Canary Island clade (95 BP) which includes semi-woody
and woody species from southern Africa (A. denudatus and A. fractiflexus), West and North Africa (A. altissimus) and the Canary
Islands (the gynodioecious species A. nesiotes, A. plocamoides and
A. acutifolius). We have not been able to identify morphological
characters supporting this, by molecular data strongly supported,
clade.
The Capenses clade is a molecularly weakly (73 BP; Fig. 2c), but
morphologically strongly, supported clade, congruent with series
Suaveolens (Obermeyer and Immelman, 1992). The species in the
Suaveolens clade are low, woody, erect shrubs, and are easy to distinguish by their branch-derived spines (branch-tips modified into
spines)
and
compound
cladodes
(deciduous
ultimate,
cladode-bearing branches). There is an unsupported division
between species with a main distribution area in the Western
Cape Province of South Africa (A. capensis and allies), and the widespread A. suaveolens plus allies distributed in the eastern and
northern parts of South Africa. The Capenses clade includes a distinct Recurvispinus clade (95 BP) in which the branch-spines are
recurved when young (compared to straight in the rest of the
Capenses clade), reminiscent of the leaf-derived spines seen in
the rest of the spine-bearing Asparagus. Also, the fruit of A.
recurvispinus and A. ‘‘karooicus’’ is a nutlet, compared to the black
berry found in the rest of the Capenses species.
Clade 5: Myrsiphyllum clade: This moderately supported
monophyletic clade (82 BP) includes almost all species which
Obermeyer placed in Myrsiphyllum, except A. scandens, which is
resolved in an unsupported relationship to the Lignosus clade.
However, the lack of bootstrap support for the position of A. scandens still leaves the possibility that Myrsiphyllum sensu Obermeyer
(1984) might be monophyletic. Flattened cladodes and tubers,
39
which have often been used as key characters of Myrsiphyllum,
are homoplasious, and are also found within the Racemose and
Asparagus clades (although the tubers here are usually found on
lateral roots, compared to on the main roots in the Myrsiphyllum
clade). Obermeyer (1984) used connivent filaments together with
connivent perianth to separate Myrsiphyllum from Protasparagus,
but in A. scandens and A. ramosissimus the tepals are spreading
rather than tube-forming (Malcomber and Demissew, 1993), supporting the exclusion of at least A. scandens from the
Myrsiphyllum clade. Our results do place A. ramosissimus within
the Myrsiphyllum clade together with A. declinatus and A. fasciculatus, as sisters to the strongly supported group of species with
connivent filaments within the Myrsiphyllum clade (99 BP).
We were unable to include some of the species previously
placed in Myrsiphyllum (A. multituberosus, A. kraussianus and A.
undulatus) in our analysis. However, due to their great morphological similarity to A. asparagoides and A. volubilis we anticipate that
they will fall within the Myrsiphyllum clade, most likely within the
group with connivent filaments.
Clade 6: Setaceus clade: This clade (70 BP; Fig. 2a) is mainly
composed of shade-loving, non-spiny, semi-woody climbers and
shrubs and includes species from Obermeyer’s series Penduli and
Globosi. Embedded in the Setaceus clade is a strongly supported
group of non-South African species (90 BP) composed of the East
African A. humilis plus a group (88 BP) of three Macaronesian accessions (A. arborescens ‘‘Tenerife’’, A. arborescens ‘‘Gran Canaria’’ and
A. umbellatus ssp. umbellatus). Additionally it includes a weakly
supported clade of three species assigned to A. cooperi (see above).
A few species fall outside these major clades. These include (1)
A. macowanii and A. mucronatus (strongly supported as sister species by 90 BP), which are most closely related to the Racemose
clade; (2) A. retrofractus which, together with the Setaceus Clade,
is resolved in a weak sistergroup relationship to the remaining
Asparagus species; and (3) Asparagus scandens, sister to the
Lignosus clade, which is morphologically very similar, but its phylogenetic position is still uncertain.
4.1.3. Species circumscription in larger complexes
We tested 33% of all species for their monophyly by including at
least two specimens in the phylogenetic analysis. At least a third
(27) are not monophyletic, but only in three cases do those groups
receive significant support. These results may be expected, as
reviews by Crisp and Chandler (1996) and Funk and Omland
(2003) suggest that paraphyletic species may be quite common
and indeed their surveys produced rather similar percentages of
paraphyletic species (23% of all tested species being paraphyletic).
In some cases, such as the genus Kniphofia, all tested species have
been shown to be paraphyletic (Ramdhani et al., 2009). The potential explanations for the occurrence of paraphyletic species have
attracted much attention (Meimberg et al., 2010; Ramdhani
et al., 2009; Rieseberg and Brouillet, 1994). A simple approach is
to argue that only monophyletic species should be recognized
(Baum and Donoghue, 1995; Mishler and Theriot, 2000), or that
the concepts of mono- or paraphyly do not apply to species
(Davis and Nixon, 1992). However, this cannot negate the observation that often specimens from the same taxonomic species do not
form clades on the optimal phylogeny. Here we group the potential
explanations into three sets: genetic, taxonomic and phylogenetic.
Genetic explanations are most often invoked – these assume
that the species delimitations (taxonomy) are correct, and that
the species are reciprocally monophyletic, but that the molecular
data are misleading. Two mechanisms are commonly proposed:
post-speciation gene flow (e.g., hybridization) and/or lineage sorting (Meimberg et al., 2010; Ramdhani et al., 2009). There is evidence of polyploidy in Asparagus (Jessop, 1966), and although
hybrids can be made between some closely related species in the
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M.F. Norup et al. / Molecular Phylogenetics and Evolution 92 (2015) 25–44
genus, there appear to be sterility barriers among many species
(Falavigna et al., 2008; Ito et al., 2008). Furthermore, there are no
field observations of hybrids in southern Africa. The absence of
robust conflict between the nuclear and plastid partitions in the
phylogenetic analyses is not a good indicator of the absence of
hybridization, since the resolution of these partial phylogenies is
not robust enough to reveal conflict with confidence.
Consequently, we currently have no convincing indications of the
presence of natural gene flow between closely related species.
Lineage sorting is most commonly observed in recently diverged
clades, and is often interpreted as a signal for recent radiation
(Ramdhani et al., 2009). This might apply in some clades in
Asparagus, where the resolution within the species complexes
(e.g. the asparagoides-, the cooperi- or suaveolens-species complexes) is poor.
Taxonomic explanations are rarely invoked; these assume that
the phylogenetic signal is correct, but the species delimitation is
incorrect. For the southern African species of Asparagus two species level taxonomies are available. The earlier taxonomy, of
Jessop (1966), uses a broad species concept, while that of
Obermeyer and Immelman (1992) uses a very narrow species concept. These different taxonomic philosophies are most evident in
the three large species complexes mentioned above (Table 4). For
the delimitation of A. asparagoides, A. cooperi and A. suaveolens,
Jessop (1966) includes several taxa (separated as species by
Obermeyer), which he found to be embedded within these three
widespread species. Jessop argues that the characters used to separate the segregates are ecologically labile, and that this in particular applies to the widespread species, where geographically
separated populations are responding differently to different habitats. However, he also notes that considerable variation may be
found within populations. The segregates are generally separated
based on vegetative characters, such as cladode shape and size,
branching patterns, and the habit in general. If variable within populations, these characters are of limited use in separating species
(Davis, 1997; Du Rietz, 1930). However, we have no quantified
data supporting this pattern. Jessop (1966) also notes that certain
characters change gradually geographically, with transition zones
(e.g. the growth form differences between A. burchellii and A. suaveolens). Such gradual clines may define ecotypes (Turesson, 1922),
but not species. This sort of explanation might well apply to the
Cooperi clade (Fig. 2c), in which the relationships between species
are unclear, and most likely reflect that most of these species are
common and widespread throughout southern Africa, and usually
identified by overlapping character sets. From the variety of small
subclades within the Cooperi clade, it is evident that there is a lot
of morphological variation to account for within the complex. The
Table 4
Summary of the species included in the three largest species complexes.
Clade
Jessop (1966)
Obermeyer and
Immelman (1992)
Range
asparagoides
asparagoides
asparagoides
asparagoides
asparagoides
absent
asparagoides
kraussianus
asparagoides
ovatus
multituberosus
alopecuroides
juniperoides
8
47
9
8
5
5
cooperi
cooperi
cooperi
cooperi
africanus
cooperi
bechuanicus
20
79
17
suaveolens
suaveolens
suaveolens
suaveolens
absent
absent
suaveolens
burchellii
spinescens
mariae
flavicaulis
76
17
3
4
10
many accessions, which we could only refer to as ‘‘cf. cooperi’’ and
the three cooperi accessions found in the Setaceus clade (see
above), makes it evident that species delimitations within this
group are strongly in need of revision. Separating traits, which vary
within populations from characters suitable to delimit species, will
require substantial fieldwork, in order to provide a solid information base on which to base species delimitations.
Phylogenetic explanations are based on the assumption that
both the phylogenetic signal and species delimitations are correct
and that the species are genuinely phylogenetically nested.
Rieseberg and Brouillet (1994) presented convincing arguments
that paraphyletic species are a necessary consequence of speciation, as most speciation is allopatric, by divergence of isolated populations, thus leaving a paraphyletic rest and only following
differentiation of the ‘‘parent species’’ will both species become
monophyletic. Numerous recent population-level investigations
have demonstrated this predicted pattern (e.g. Albach et al.,
2004; Funk and Omland, 2003; Patton and Smith, 1994). This sort
of pattern could well apply to the A. suaveolens-complex, with A.
mariae restricted to limestone near the southern tip of Africa, A.
burchellii and A. spinescens found along the all-year rainfall
south-eastern part of the subcontinent (Schulze, 1997) and A. flavicaulis on rocky outcrops. Following this interpretation A. spinescens and A. burchellii (represented by more than two accessions)
are both nested within A. suaveolens, which is the paraphyletic
‘‘ancestor’’ widespread in the bushveld.
4.2. The evolution of sexual dimorphism in Asparagus
The most plausible evolutionary pathway to dioecy in Asparagus
has been from hermaphroditism via gynodioecy. The presence of
female flowers as well as bisexual flowers has been observed in
several Canary Islands species (e.g., A. umbellatus, A. nesiotes, A. plocamoides and A. acutifolius) (Julia Pérez de Paz, Jardín Botánico
Canario ‘‘Viera y Clavijo’’, personal comments; Baker, 1875;
Kunth, 1850, checked on material in herbarium C). Furthermore,
these species originated within separate Asparagus clades (Fig. 3),
indicating several independent origins of gynodioecy correlated
with the dispersal from Africa to the Canary Islands. Multiple origins of gynodioecy in a genus is documented in dicots (Fritsch,
2003; Navajas-Perez et al., 2005; Weller et al., 1998) although
not commonly described and is so far almost undocumented in
monocots (Connor, 1973). Some sources indicated that A. acutifolius is dioecious (e.g., Sica et al., 2005; Kubota et al., 2012), but
this is apparently not based on observations and cannot be verified
as no vouchers are indicated.
It appears evident that the transitions from hermaphroditism to
sexual dimorphism (dioecy or gynodioecy) are closely related to
the dispersal out of southern Africa. This is consistent with the theory that the evolution of dioecy in plants may be the result of selection for outcrossing and that this mechanism often evolve during
bottlenecks; e.g., inbreeding depression due to long-distance dispersal and colonization of new habitats) (see e.g. Barrett, 2002;
Charlesworth and Charlesworth, 1978; Pannell, 2006; Weiblen
et al., 2000).
It has furthermore been proposed that a link exists between
sexual dimorphism and polyploidization, as seen in e.g. Lycium L.
(Solanaceae), Rubus L. and Fragaria L. (Rosaceae) and Astilbe
Buch.-Ham. (Saxifragaceae), where chromosome doubling is correlated with the evolution of self-compatibility in otherwise
self-incompatible plants, giving rise to polyploid species with gender dimorphism (Barrett, 2002; Miller and Venable, 2000). This
could be a possible pathway for the evolution of sexual dimorphism in Asparagus, as in general Eurasian species are reported to
have larger genomes than southern African species (Kar and Sen,
1985; Kuhl et al., 2005; Moreno et al., 2008), although a few
M.F. Norup et al. / Molecular Phylogenetics and Evolution 92 (2015) 25–44
widespread southern and south central African species are
reported to be tetraploid or hexaploid (e.g. A. falcatus, A. cooperi:
2n = 40; Kar and Sen (1985) and A. densiflorus: 2n = 60; Jessop
(1966)).
4.3. Out-of-Africa – the origin of Asparagus and dispersal to Eurasia
The genus Asparagus is widely distributed in the Old World,
growing in semiarid to arid areas in Africa and Eurasia. However,
due to insufficient analyses or sampling, most recent treatments
of the genus have reserved judgement as to its area of origin (e.g.
Dahlgren et al., 1985; Kubitzki and Rudall, 1998). The S-DIVA
(Fig. 4) and DEC analyses (Fig. S5, Suppl. Mat.) unequivocally infer
a southern African ancestral distribution of Asparagus (area I) and
thus confirm the hypothesis presented by Fukuda et al. (2005).
We do not believe that our sampling imbalance (towards southern
Africa, which is by far the most species rich region) had a significant influence in placing southern Africa as the area of origin, as
all included non-southern African species are found to have common ancestors within southern Africa. These non-southern
African species are shown to be the result of not one, but multiple
independent dispersal events out of southern Africa.
Our results demonstrate that all truly dioecious species of
Asparagus have a single common ancestral area – which the
S-DIVA analysis infers as area L (China, Mongolia and Eastern
Asia) – with later dispersal back to Europe (areas A and B), a lineage from which the widespread A. officinalis and the more local
Mediterranean species A. maritimus evolved. Confidence in this
result is weakened by the poor sampling from the non-African
areas and the low bootstrap support of the Dioecy clade (Fig. 2).
41
The Dioecy clade originated within a group of hermaphroditic
species from southern and South Tropical Africa (areas G and I),
East and Northeastern Tropical Africa (area F), North Africa (area
C), the Canary Islands (area D), the Mediterranean areas (area B;
Southwestern + Southeastern Europe) and the Arabian Peninsula
(area K; Arabian Peninsula + Middle Asia + Caucasus + Western
Asia). Looking at the entire clade including the Dioecy clade, we
conclude that the dispersal of Asparagus seems to have followed
a route (1) from southern Africa through a tropical
East-Northeastern African corridor westwards to North Africa,
Macaronesia and Mediterranean Europe (reflected in the clade
including A. flagellaris, A. petersianus, A. ‘‘pauli-guilelemi’’, A. aphyllus
and A. stipularis) and (2) eastwards to Eurasia (the Dioecy clade)
(see Fig. 5). Part of this dispersal pattern is repeated in the
Setaceus clade (Figs. 2a and 3), where S-DIVA infers the distribution of the most recent common ancestor of the Canary Island species A. arborescens, A. umbellatus ssp. umbellatus plus A. humilis
(East Africa) to have been widespread through South Central
Africa (area G), East and Northeastern Tropical Africa (area F) to
the Canary Islands (area D).
The distribution pattern of Asparagus does not fit the ‘‘Rand
Flora Pattern’’ which is defined as an old xerophylic tropical
African flora (Quezel, 1978), which shows a distribution pattern
also described as the ‘‘arid track’’ (de Winter, 1971; Jürgens,
1997; Verdcourt, 1969). The Rand flora was supposed to have originally diversified in south-western Africa, but also to link the arid
elements of the Canary Islands with those of north-east Africa
(Marrero et al., 1998). Asparagus largely avoids the dry areas and
is centred on the more mesic habitats in southern Africa, from
the fynbos flora to the montane grassland and subtropical
Fig. 5. A map of the Old World showing the main dispersal routes of Asparagus (arrows) derived from the DIVA analysis (Fig. 4), including known radiation centres (stars) (the
major radiation centre still being South Africa).
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M.F. Norup et al. / Molecular Phylogenetics and Evolution 92 (2015) 25–44
savannas. However, Sanmartin et al. (2010) used a broader definition of the Rand flora, which encompasses all open country lineages, and as such would also include Asparagus. Asparagus fits
the model of a southern radiation with a northward dispersal. A
radiation pattern like this may be congruent with, inter alia, Aloe
(Holland, 1978), Crassula L. (Mort et al., 2001), Scrophulariaceae
(Oxelman et al., 2005), Amaryllideae (Meerow et al., 1999;
Motomi et al., 1999; Rønsted et al., 2012), Gladiolus (e.g.
Goldblatt and Manning, 1998) and Lotononis (DC) Eckl. & Zeyh.
(Linder et al., 1992). These clades all have their ancestral areas in
mesic, open habitats in southern Africa (in the heathlands of the
Cape flora, or the grasslands of the central southern African plateau) and achieved global or near-global distributions. Although
they are currently also found in the semi-arid Succulent Karoo, this
biome is of Late Miocene origin (Dupont et al., 2011), younger than
the original radiations of these clades.
5. Conclusion
Asparagus is a complex genus that shows a notable discrepancy
between the molecular phylogeny and the morphologically defined
species in the existing taxonomy, with e.g. several species belonging to larger species complexes, most of which are paraphyletic,
though a few are polyphyletic. Despite the fact that our study is
the most complete and well supported to date, the lack of an
up-to-date taxonomy makes the interpretation of patterns at species level difficult. We provide the phylogenetic basis for a new
infrageneric classification of Asparagus and conclude that species
delimitation needs to be based on both molecular and morphological data. Incongruence of sequence and morphological data indicates problems with species delimitation that need further
investigation, but large-scale morphological and biogeographical
patterns are evident (e.g. dioecy). Our analysis provides no support
for the classical interpretation of the evolution of dioecy through
gynodioecy.
Acknowledgments
The authors thank Akira Kanno and Tatsuya Fukuda for providing DNA material for the Asian species; Juli Caujapé-Castells and
Ruth Jaén Molina at Jardin Botanico Canario ‘‘Viera y Clavijo’’, Las
Palmas, Gran Canaria, for help with samples and information;
Sebsebe Demissew for samples; Terry Trinder-Smith for help in
the Bolus Herbarium; Michael S. Kinney for sharing information
on PHY-C primer optimization and outgroup sequences;
Charlotte Hansen for assistance in the laboratory; Nina Rønsted
and Alexandre Antonelli for assistance and discussions; Justin
Moat for assistance with the map; Reto Nyfeller and Melanie
Ranft for help with GenBank submission. This research was supported by grants to the first author from the EDIT Women
Scientists Grant, the Christian and Ottilia Brorsons Grant, the
Augustinus Foundation, the Oticon Foundation and the foundation
of Svend G. Fiedler and wife.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ympev.2015.06.
002.
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