Timing and Consequences of Recurrent Polyploidy in Meadow-Rues (Thalictrum, Ranunculaceae)

Valerie Soza

Distinct floral pollination syndromes have emerged multiple times during the diversification of flowering plants. For example, in western North America, a hummingbird pollination syndrome has evolved more than 100 times, generally from within insect-pollinated lineages. The hummingbird syndrome is characterized by a suite of floral traits that attracts and facilitates pollen movement by hummingbirds, while at the same time discourages bee visitation. These floral traits generally include large nectar volume, red flower colour, elongated and narrow corolla tubes and reproductive organs that are exerted from the corolla. A handful of studies have examined the genetic architecture of hummingbird pollination syndrome evolution. These studies find that mutations of relatively large effect often explain increased nectar volume and transition to red flower colour. In addition, they suggest that adaptive suites of floral traits may often exhibit a high degree of genetic linkage, which could...

Apomictic plants expand their geographical distributions more to higher elevations compared to their sexual progenitors. It was so far unclear whether this tendency is related to mode of reproduction itself or represents a side effect of polyploidy. Apomixis is advantageous for range expansions as no mating partners and pollinators are needed (Baker's rule). Polyploidy is thought to infer fitness advantages and a higher vigor that would enable plants to adjust better to more extreme climatic conditions. However, little is known about actual performance of plants at higher elevations. We analyzed 81 populations of Ranunculus kuepferi from the whole distribution area in the European Alps to quantify apomictic versus sexual seed formation via flow cytometric seed screening. Seed set and vegetative growth were measured as fitness parameters. All parameters were correlated to geographical distribution, elevation, temperature, and precipitation. Flow cytometric seed screening revealed...

Polyploidy plays a major role in plant evolution. The establishment of new polyploids is often a consequence of a single or few successful polyploidization events occurring within a species' evolutionary trajectory. New polyploid lineages can play different roles in plant diversification and go through several evolutionary stages influenced by biotic and abiotic constraints and characterized by extensive genetic changes. The study of such changes has been crucial for understanding polyploid evolution. Here, we use the multiploidspecies Paspalum intermedium to study population-level genetic and morphological variation and ecological differentiation in polyploids. Using flow cytometry, amplified fragment length polymorphism (AFLP) genetic markers, environmental variables, and morphological data, we assessed variations in ploidy, reproductive modes, and the genetic composition in 35 natural populations of P. intermedium along a latitudinal gradient in South America. Our analyses show that apomictic auto-tetraploids are of multiple independent origin. While overall genetic variation was higher in diploids, both diploids and tetraploids showed significant variation within and among populations. The spatial distribution of genetic variation provides evidence for a primary origin of the contact zone between diploids and tetraploids and further supports the hypothesis of geographic displacement between cytotypes. In addition, a strong link between the ecological differentiation of cytotypes and spatial distribution of genetic variation was observed. Overall, the results indicate that polyploidization in P. intermedium is a recurrent phenomenon associated to a shift in reproductive mode and that multiple polyploid lineages from genetically divergent diploids contributed to the successful establishment of local polyploid populations and dispersal into new environments.

Timing and Consequences of Recurrent Polyploidy in Meadow-Rues (Thalictrum, Ranunculaceae) Valerie L. Soza,*,1 Kendall L. Haworth,1 and Verónica S. Di Stilio*,1 1 Department of Biology, University of Washington *Corresponding authors: E-mail: vsoza@u.washington.edu; distilio@u.washington.edu. Associate editor: Juliette de Meaux Abstract Key words: genome size, dioecy, wind pollination, molecular dating. Introduction Article Historically, polyploidy has been considered rarer in animals than plants (Muller 1925; Orr 1990), but with the advent of next-generation sequencing techniques and more sophisticated genomic analyses, ancient whole-genome duplications (WGDs) have been detected in vertebrates (reviewed in Canestro 2012) and other eukaryotes (reviewed in Jaillon et al. 2009) as well as plants (reviewed in Soltis et al. 2009; Jiao et al. 2011; Fawcett et al. 2013). Moreover, multiple recent episodes of polyploidy have been identified in both vertebrates and invertebrates (reviewed in Mable 2004a; Gregory and Mable 2005; Otto 2007) as well as fungi (Albertin and Marullo 2012), suggesting that polyploidy may be important to the diversification of eukaryotes in general. Unlike animals and fungi (Stebbins 1950), however, polyploidy has a long history of recognized relevance to the evolution of plants (reviewed in Tate et al. 2005; Soltis et al. 2009), and both allo- and autopolyploidy are contributors to plant speciation (Soltis et al. 2007). Discovery of ancient WGDs in the common ancestors of seed plants (" WGD, Jiao et al. 2011), angiosperms ( WGD, Jiao et al. 2011), and core eudicots (Vekemans et al. 2012) has renewed interest in polyploidy and its potential effect on the diversification of major plant groups. WGD in angiosperms can result in novel phenotypes as a result of changes in gene expression, transposable element reactivation, altered gene dosage, and/or changes in regulatory/epigenetic interactions (reviewed in Osborn et al. 2003; Chen and Ni 2006; Finigan et al. 2012; McGrath and Lynch 2012; Fawcett et al. 2013). The large-scale effects on gene expression resulting from polyploidy can produce immediate changes in morphology, breeding system, and ecological interactions (Otto and Whitton 2000; Chen 2007). Furthermore, the effects from molecular mechanisms following polyploidization vary among different polyploid populations, creating plasticity for adaptation to new environments (Jackson and Chen 2010). Finally, polyploidy promotes reproductive isolation between diploid progenitors and resultant polyploids, as well as among polyploid populations, through reciprocal gene loss of duplicate genes (reviewed in Edger and Pires 2009; McGrath and Lynch 2012; Fawcett et al. 2013). Consequently, polyploidization can provide angiosperms with novel phenotypes that may lead to speciation. Polyploidy is defined as the combination of three or more genomes in a single nucleus and is therefore expected to result in an instant increase in genome size (Bennett and Leitch 2005). In fact, the two main factors contributing to genome size expansions in plants are polyploidy and the accumulation of transposable elements (reviewed in Hawkins et al. 2008). As a result, an enormous amount of variation in genome size occurs in angiosperms, partly due to recurrent polyploid events over evolutionary time (reviewed in Wendel et al. 2002). In recently formed polyploids, genome ß The Author 2013. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com 1940 Mol. Biol. Evol. 30(8):1940–1954 doi:10.1093/molbev/mst101 Advance Access publication May 31, 2013 Downloaded from http://mbe.oxfordjournals.org/ at University of Washington on July 3, 2014 The discovery of ancient whole-genome duplications in eukaryotic lineages has renewed the interest in polyploidy and its effects on the diversification of organisms. Polyploidy has large-scale effects on both genotype and phenotype and has been linked to the evolution of genome size, dioecy, and changes in ecological interactions, such as pollinator visitation. Here, we take a molecular systematics approach to examine the evolution of polyploidy in the plant genus Thalictrum (Ranunculaceae) and test its correlation to changes in genome size, sexual system, and pollination mode. Thalictrum is an ideal study system due to its extensive ploidy range and floral diversity. Phylogenetic analyses were used for character reconstructions, correlation tests, and dating estimates. Our results suggest that polyploidization occurred frequently and recently in the evolution of Thalictrum, mostly within the last 10.6–5.8 My, coinciding with the diversification of particular clades. In spite of an overall trend of genomic downsizing accompanying polyploidy in angiosperms and proportional increases observed at finer scales, our genome size estimates for Thalictrum show no correlation with chromosome number. Instead, we observe genomic expansion in diploids and genomic contraction in polyploids with increased age. Additionally, polyploidy is not correlated with dioecy in Thalictrum; therefore, other factors must have influenced the evolution of separate sexes in this group. A novel finding from our study is the association of polyploidy with shifts to wind pollination, in particular, during a time period of global cooling and mountain uplift in the Americas. Timing and Consequences of Recurrent Polyploidy . doi:10.1093/molbev/mst101 (Kaplan and Mulcahy 1971). Moreover, the genus exhibits an enormous range of ploidy, from 2n = 2x = 14 to 2n = 24x = 168 (Löve 1982; Tamura 1995), with very small chromosomes known as the T-type in Ranunculaceae (Langlet 1927). We previously identified the origins of the different sexual systems and pollination modes within the genus (Soza et al. 2012) but had yet to investigate the origins of polyploidy and its correlated evolution with transitions in sexual system or pollination mode. The overall goal of this study was to take a molecular systematics approach to examine the evolution of polyploidy in the genus Thalictrum and determine whether it is correlated to changes in genome size, sexual system or pollination mode. Our specific goals were to 1) investigate the frequency and timing of polyploidization, 2) characterize the relationship between polyploidy and genome size, and 3) determine whether polyploidy is associated with dioecy or pollination mode. Results A Revised Phylogeny We increased taxonomic and DNA sampling within Thalictrum to two to three loci from 69 species (supplementary tables S1 and S2, Supplementary Material online) using likelihood and Bayesian analyses to obtain a better estimate of phylogenetic relationships. As before (Soza et al. 2012), we recovered two strongly supported main clades within the genus (I and II, bootstrap [bs] = 100%, posterior probability [pp] = 1.00, fig. 1), with the difference that Thalictrum ichangense and T. macrocarpum are now sister to the rest of clades I and II, respectively (fig. 1). Within clade II, we identified three new strongly supported subclades: 1 (bs = 84%, pp = 1.00), 2 (pp = 0.99), and 3 (bs = 97%, pp = 1.00); relationships among these subclades were unresolved (fig. 1). In clade II, we recovered two dioecious and one andromonoecious, strongly supported subclades (A–C, bs = 100%, pp = 1.00, fig. 1) as previously shown (Soza et al. 2012). This revised phylogeny had better resolution and support within both clades I and II from previous studies (Soza et al. 2012) and was used in subsequent character reconstructions, correlation tests, and dating analyses. Three species were repositioned in the current phylogeny with respect to our former study (Soza et al. 2012). Previously, T. macrocarpum was identified as sister to the rest of clade I (Soza et al. 2012), but our current analyses show this taxon as sister to the remainder of clade II (fig. 1). The repositioning of this gynomonoecious species results in extant species with unisexual flowers occurring only in clade II. Another species, T. omeiense, was repositioned out of clade I (Soza et al. 2012) to clade II based on our current data (fig. 1); this placement has no bearing on the evolution of sexual systems or pollination modes in the genus. Last, we found T. sparsiflorum occurring in clade 3 rather than in clade C as previously reported (Soza et al. 2012). Insect pollination in this species is therefore maintained from an ancestrally insect-pollinated species, rather than a reversal from wind pollination. We believe these differences are due to sample misidentification in the 1941 Downloaded from http://mbe.oxfordjournals.org/ at University of Washington on July 3, 2014 size is expected to increase in direct proportion with ploidy; however, contrary examples in angiosperms are documented (reviewed in Bennett and Leitch 2005). For example, overall genomic downsizing is observed across angiosperms (reviewed in Hawkins et al. 2008), especially after the formation of polyploids (Leitch and Bennett 2004). This downsizing may occur due to rampant changes in genomes following allopolyploidization, as evidenced by chromosomal rearrangements (translocations), concerted evolution of loci, gene silencing, and epigenetic gene regulation (reviewed in Leitch and Bennett 1997). In contrast to broad trends across angiosperms, finer scale studies of closely related species illustrate that genome size evolution in polyploids is variable, either reflecting proportional increases or downsizing (Ozkan et al. 2003; Pires et al. 2004; Garnatje et al. 2006). Polyploidy has been correlated to variation in sexual system in angiosperms. Gender dimorphism (gynodioecy and dioecy) was shown to follow polyploidization in 12 genera, presumably due to chromosomal rearrangements that facilitate the evolution of sex chromosomes or the breakdown of gametophytic self-incompatibility (GSI) followed by inbreeding depression (Miller and Venable 2000, 2002; Spigler et al. 2010). The breakdown of self-incompatibility as the result of polyploidy, however, is assumed to occur only in species with single-locus GSI and not in species with multilocus complementary GSI or sporophytic self-incompatibility (De Nettancourt 2001). The breakdown of certain self-incompatibility systems following polyploidy should, therefore, result in an increased association between polyploidy and self-compatibility (reviewed in Mable 2004b). Yet large-scale studies across angiosperms have found conflicting evidence for the association of polyploidy with self-compatibility (Mable 2004b; Barringer 2007), highlighting lineage-specific trends. Ever since Charles Darwin, pollination mode has been recognized as an important contributor to the diversification of angiosperms (reviewed in Friedman 2009). In fact, pollinator shifts are known to increase diversification in a variety of angiosperm groups (van der Niet et al. 2006; Whittall and Hodges 2007; Johnson 2010). Few studies, however, have examined whether polyploidy is associated with ecological interactions, such as shifts in pollinators (reviewed in Soltis et al. 2010). On the basis of the geographic distribution of polyploidy and pollination modes, Vamosi et al. (2007) hypothesized an association between wind pollination and polyploidy in angiosperms. Cox and Grubb (1991) also suggested that wind pollination could facilitate the colonization of new habitats by polyploids, as observed in auto- and allopolyploid Atriplex (Stutz and Sanderson 1979). Nevertheless, no studies to date have examined whether polyploidy may be linked to shifts between insect and wind pollination. Thalictrum is an ideal genus to examine the correlated evolution of polyploidy, sexual system, and pollination mode because, unlike most genera, it contains variation in all three features. Specifically, hermaphroditic, dioecious, andromonoecious, and gynomonoecious species are found within the group (Boivin 1944; Guzmán 2005); and both wind and insect pollination are observed within the genus MBE Soza et al. . doi:10.1093/molbev/mst101 1942 Thalictrum amurense W Thalictrum lucidum 1.00 95 1 1.00 99 0.99 58 Thalictrum simplex W 56 Thalictrum foetidum W Thalictrum flavum 0.94 1.00 84 Thalictrum ramosum 96 Thalictrum squarrosum W Thalictrum rochebrunnianum 1.00 Thalictrum uchiyamae 100 0.97 Thalictrum pubescens D,W 55 Thalictrum revolutum D,W 63 Thalictrum dasycarpum D,W 1.00 Thalictrum cooleyi D,W 100 Thalictrum coriaceum D,W 55 A Thalictrum macrostylum D,W Thalictrum delavayi Thalictrum grandiflorum 1.00 Thalictrum squamiferum 86 Thalictrum tenue W 1.00 Thalictrum arsenii c1 A,W 1.00 97 Thalictrum arsenii c2 A,W 84 Thalictrum guatemalense c1 A,W 0.98 Thalictrum henricksonii A,W Thalictrum podocarpum A,W 55 Thalictrum steyermarkii c1 A,W 0.93 Thalictrum hernandezii A,W Thalictrum strigillosum A,W 71 52 1.00 0.96 Thalictrum steyermarkii c2 A,W 58 Thalictrum grandifolium A,W 100 Thalictrum guatemalense c2 A,W Thalictrum tripeltiferum A,W 1.00 Thalictrum fendleri D,W 99 Thalictrum heliophilum D,W Thalictrum dioicum D,W 1.00 Thalictrum pinnatum D,W 1.00 Thalictrum occidentale D,W 100 0.99 1.00 0.95 82 Thalictrum polycarpum D,W 97 Thalictrum venulosum D,W 51 Thalictrum atriplex 1.00 Thalictrum rutifolium 100 Thalictrum decipiens W Thalictrum lecoyeri 0.92 Thalictrum reticulatum 0.98 55 Thalictrum uncatum W 53 1.00 Thalictrum cultratum W Thalictrum isopyroides W 83 Thalictrum omeiense 1.00 Thalictrum aquilegiifolium 1.00 Thalictrum petaloideum 97 0.91 67 Thalictrum actaeifolium W 0.94 Thalictrum minus W Thalictrum baicalense 59 Thalictrum smithii c1 G 1.00 Thalictrum smithii c2 G 100 Thalictrum alpinum W Thalictrum zernyi 1.00 Thalictrum diffusiflorum 84 55 Thalictrum sparsiflorum 1.00 Thalictrum finetii 97 Thalictrum przewalskii 57 Thalictrum leuconotum W Thalictrum virgatum 1.00 Thalictrum reniforme 94 Thalictrum rhynchocarpum A,W Thalictrum macrocarpum G,W 1.00 Thalictrum kiusianum 0.98 Thalictrum punctatum 100 1.00 67 Thalictrum filamentosum 1.00 75 Thalictrum urbainii 1.00 Thalictrum rubescens 100 79 1.00 Thalictrum clavatum 100 Thalictrum thalictroides Thalictrum ichangense Leptopyrum fumarioides Paraquilegia microphylla 1.00 84 1.00 95 B 2 C II 3 1.00 100 1.00 100 1.00 100 50 1.00 95 Aquilegia formosa I 0.2 substitutions/site MBE FIG. 1. Phylogenetic relationships in the genus Thalictrum and outgroups (Ranunculaceae). Bayesian 50% majority-rule consensus tree based on the combined chloroplast trnV-ndhC and nuclear external and internal transcribed spacer regions. Ancestral conditions for sexual system and pollination mode in the genus are hermaphroditic and insect, respectively. Andromonoecious (A), dioecious (D), gynomonoecious (G), and wind-pollinated (W) taxa are indicated on the phylogeny. Two major clades, I and II, indicated within Thalictrum. Three strongly supported clades, 1–3, two dioecious clades, A and C, and one andromonoecious clade, B, within clade II. Bayesian pps 0.90 displayed above branches. Likelihood bs values 50% displayed below branches; c, clonal sequence. Downloaded from http://mbe.oxfordjournals.org/ at University of Washington on July 3, 2014 Timing and Consequences of Recurrent Polyploidy . doi:10.1093/molbev/mst101 former phylogeny. We have re-extracted DNA from original herbarium specimens, reamplified, and resequenced to verify our current results. Chromosome Number and Genome Size Evolution To study the evolution of polyploidy in Thalictrum, we reconstructed haploid chromosome number using a likelihood framework in chromEvol (Mayrose et al. 2009). Approximately 1–82 individuals were represented for each species from published chromosome counts or our own (supplementary table S3, Supplementary Material online). For species lacking published counts or with conflicting published counts, we obtained chromosome counts from one to three cultivated accessions (fig. 2, supplementary tables S3 and S4, Supplementary Material online). On the basis of two coding schemes of chromosome number (polymorphic vs. most common, supplementary table S3, Supplementary Material online), we obtained the same best-fitting model of chromosome evolution from the eight models tested. Both analyses indicated the M2 constant-rate model, where chromosomes are gained and lost individually, as the best model (Akaike Information Criterion [AIC] = 268 or 282.4, respectively). Differences between the analyses were in part due to missing data for T. minus in the polymorphic coding scheme, which reduced the number of genome duplication events inferred in this analysis. More importantly, the polymorphic coding scheme failed to account for genome duplications in terminal lineages that were polymorphic. Therefore, only the results from the most common coding scheme are shown (fig. 3). The ancestral haploid chromosome number at the root of the tree and for the common ancestor of Thalictrum is 7 (probability [P] = 0. 93 and 1.00, respectively; fig. 3), indicating diploidy. Twenty-four genome duplication events were inferred with probabilities > 0.5 (fig. 3). No single chromosome losses or gains (aneuploidy events) were inferred from our analyses. All 24 genome duplications occurred in clade II; no duplications were identified in clade I, where all species are diploids (fig. 3). Of these 24 genome duplications, 14 occurred within clade 1, five within clade 2, and one within clade 3 (fig. 3). Additionally, we observed that both dioecious and andromonoecious clades (A–C) are polyploid (fig. 3) and further tested the significance of this observation. In summary, our results show that 1) diploidy is the ancestral condition in Thalictrum, 2) polyploidy occurred multiple times as a result of doubling or tripling of genomes, 3) aneuploidy did not contribute to the evolution of the genus, and 4) polyploidy occurred only in clade II, especially in clades 1 and 2. To characterize the relationship between polyploidy and genome size evolution within Thalictrum, we gathered relative holoploid genome size data from one to five accessions or replicates for 35 species (supplementary table S4, Supplementary Material online) and denoted this data as 1C values (as defined by Greilhuber et al. 2005). The variation of holoploid genome size within clades I and II, expressed as 1C value (pg) ± standard error, was indicative of the frequency of genome duplications within each clade: 0.37–0.57 and 0.24–3.57 pg, respectively (fig. 3). Of the strongly supported clades within clade II, clade 1 showed the most variation in 1C values, 0.64–3.57 vs. 0.52–1.31 pg in clade 2 (fig. 3). Holoploid genome size variation in Thalictrum corresponds with the number of duplications inferred from our likelihood reconstructions of chromosome number (fig. 3). Therefore, we proceeded to test the correlation between 1C value and ploidy in a subset of 20 species, excluding outliers from clade A (fig. 3), and for which we had estimates of both 1C value and chromosome number from the same source population or from singlepublished chromosome counts (supplementary table S4, Supplementary Material online). Using a phylogenetic generalized least squares regression, the haploid chromosome number and 1C value of sampled Thalictrum species showed no correlation ( = 1.61, 1 = 0.00, 0 = 0.66, r = 0.06, R2 = 0.00; fig. 4), indicating that holoploid genome size does not proportionally decrease or increase with ploidy. Correlations between Polyploidy and Sexual System or Pollination Mode To determine whether polyploidy was associated with sexual system or pollination mode in Thalictrum, we conducted correlation tests in SIMMAP (Bollback 2006). On one hand, polyploidy was not significantly associated with dioecy (P = 0.100) or any other sexual system in the genus (table 1). On the other hand, polyploidy was significantly associated with wind pollination (P = 0.025, table 1), and diploidy was significantly associated with insect pollination (P = 0.025, table 1). In summary, polyploidy is not correlated with dioecy but appears to correlate with shifts to wind pollination in Thalictrum. 1943 Downloaded from http://mbe.oxfordjournals.org/ at University of Washington on July 3, 2014 FIG. 2. Chromosome spreads from PMCs of Thalictrum species. (A) Thalictrum punctatum: 2n = 14. (B) T. delavayi: 2n = 28. (C) T. simplex: 2n = 56. (D) T. dasycarpum: 2n = > 100. Scale bar = 10 mm. MBE 1.00 1.00 7 0.64 0.95 1.00 14 21 1.00 1.00 28 1.00 0.99 A 42 1 70 1.00 1.00 0.97 1.00 84 0.57 105 0.77 Uncertain 0.83 0.87 0.82 B 0.98 1.00 0.99 0.92 1.00 0.66 C 2 1.00 0.99 1.00 0.75 1.00 0.57 1.00 0.89 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 3 1.00 II 1.00 1.00 1.00 1.00 0.51 1.00 1.00 0.98 1.00 I 1.00 0.65 1.00 1.00 1.00 0.93 0.99 T. amurense 14 T. lucidum 14 T. simplex 14, 21, 28, 35 T. foetidum 7, 21 T. flavum 14, 42 T. ramosum T. squarrosum 21 T. rochebrunnianum 14 T. uchiyamae 7 T. pubescens 42, 63, 77 T. revolutum 70 T. dasycarpum 77, 84 T. cooleyi 105 T. coriaceum 35, 70 T. macrostylum 28 T. delavayi 14, 21 T. grandiflorum T. squamiferum T. tenue T. arsenii T. henricksonii T. podocarpum T. steyermarkii T. hernandezii 14 T. strigillosum 14 T. grandifolium T. guatemalense 14 T. tripeltiferum T. fendleri 14, 21, 28, 35 T. heliophilum T. dioicum 7, 14, 21 T. pinnatum T. occidentale 28 T. polycarpum 14 T. venulosum 21 T. atriplex T. rutifolium T. decipiens 14 T. lecoyeri T. reticulatum T. uncatum T. cultratum 7, 21 T. isopyroides 21 T. omeiense T. aquilegiifolium 7, 14 T. petaloideum 7 T. actaeifolium 7 T. minus 21 (7 - 42) T. baicalense 7 T. alpinum 7, 11 T. smithii T. zernyi T. diffusiflorum T. sparsiflorum 7, 21 T. finetii 21 T . p r z e w a l s k i i 7, 35 T. leuconotum T. virgatum T. reniforme 14 T. rhynchocarpum 14 T. macrocarpum 21, 28 T. kiusianum 7 T. punctatum 7 T . f i l a m e n t o s u m 7, 21 T. urbainii T. rubescens T. clavatum 7 T. thalictroides 7 T. ichangense 7 Leptopyrum fumarioides 7 Paraquilegia microphylla 7 0 7 Soza et al. . doi:10.1093/molbev/mst101 1 2 3 4 1C-VALUE (pg) MBE FIG. 3. Likelihood reconstruction of haploid chromosome number evolution (left) and holoploid genome size (right) for Thalictrum and outgroups (Ranunculaceae). Reconstruction based on the Bayesian 50% majority-rule consensus tree, resulting from combined chloroplast trnV-ndhC and nuclear ETS and ITS regions. Likelihood reconstruction is based on the most commonly reported haploid chromosome number for taxa with published or observed chromosome counts. For polymorphic taxa, all reported haploid chromosome numbers are displayed at tips (except for T. minus with 14 different counts, in which case, only range is reported); the most commonly reported haploid chromosome number is in bold. Ancestral states with probabilities >0.5 are displayed in colored branches with corresponding probabilities above branches. For ancestral nodes with multiple states reconstructed with probabilities < 0.5 each, reconstructions are displayed as uncertain. Ancestral states at strongly supported nodes (i.e., Bayesian pps 0.95 or likelihood bs values 70%) indicated in bold. Two major clades in the genus, I and II; three strongly supported clades, 1–3, two dioecious clades, A and C, and one andromonoecious clade, B (within clade II). Outgroups in gray. Mean 1C value in pg and associated standard error shown for 35 Thalictrum species (right). Downloaded from http://mbe.oxfordjournals.org/ at University of Washington on July 3, 2014 1944 1.00 1N CHROMOSOME NUMBER alpha = 1.61 r = -0.06 0.4 0.6 0.8 1.0 1.2 1C-value (pg) Timing and Consequences of Recurrent Polyploidy . doi:10.1093/molbev/mst101 10 15 20 25 Haploid chromosome number FIG. 4. Phylogenetic generalized least squares regression of holoploid genome size (1C value) and haploid chromosome number for 20 species of Thalictrum (Ranunculaceae) and associated alpha and correlation coefficient between these two features. Trait/State Sexual systems Hermaphroditism Dioecy Monoecy Pollination modes Insect Wind Ploidy Diploid Polyploid + 0.045 (0.068) 0.021 (0.100) 0.024 (0.108) 0.045 (0.068) + 0.021 (0.100) + 0.024 (0.108) + 0.041 (0.025)* 0.041 (0.025)* 0.041 (0.025)* + 0.041 (0.025)* NOTE.—Positive association ( + ); negative association ( ). *Statistically significant at P < 0.05. Timing of Polyploidization Events and Major Divergences in Thalictrum To estimate ages for major clades within Thalictrum and for reconstructed polyploidization events, we conducted Bayesian analyses in BEAST (Drummond and Rambaut 2007; Drummond et al. 2012) to obtain 95% highest posterior densities (HPDs) for nodes. We used our combined data set for chloroplast and nuclear ribosomal DNA and three previous age estimates for Thalictrum and outgroups (Anderson et al. 2005; Bastida et al. 2010) in BEAST analyses. The genus itself diverged 27.6–19.5 Ma with a crown age of 22.8–14.0 Ma (fig. 5, supplementary table S5, Supplementary Material online). Clades I and II diverged from each other 22.8–14.0 Ma, with crown ages for clades I and II of 11.7–4.4 Ma and 17.9–10.1 Ma, respectively (fig. 5, supplementary table S5, Supplementary Material online). These age estimates place the divergence of Thalictrum from its sister lineage Leptopyrum + Paraquilegia in the Cenozoic, during the late Oligocene, with its initial diversification in the Miocene. Polyploidization does not appear to have had an impact on the split between clades I and II, yet it occurred frequently and recently within clade II. The oldest genome duplication in clade II occurred between 16.7–9.5 and 11.9–3.9 Ma (fig. 5, supplementary table S5, Supplementary Material online). However, the majority of genome duplications that resulted in more than one extant species occurred more recently, in clades 1 and 2, between 10.8–5.6 and 1.1–0.0 Ma (fig. 5, supplementary table S5, Supplementary Material online). Interestingly, two genome duplications in clade 2 coincide with the origin of andromonoecious and dioecious clades (B–C), between 10.8–5.6 and 5.2–1.9 Ma (fig. 5, supplementary table S5, Supplementary Material online). Multiple genome duplications within clade 1 occurred within the last 7.7–3.3 to 4.3–0.6 My (fig. 5, supplementary table S5, Supplementary Material online). Only one of these events is tied to the origin of dioecy (Clade A), between 2.5–0.6 and 6.2–2.5 Ma (fig. 5, supplementary table S5, Supplementary Material online). In summary, the timing of the majority of polyploidization events within Thalictrum is recent and appears to coincide with the diversification of clades 1 and 2, with clade 1 containing most of the genome duplications. Discussion Our reconstructions of species relationships, ploidy, and age estimates in Thalictrum highlight that polyploidization occurred frequently, at least 24 times, and relatively recently, mostly between 10.8–5.6 and 1.1–0.0 Ma. Most of the genome duplications occurred within two clades (clades 1–2), coincident with the highest diversity in both sexual system and pollination mode. Despite these recent polyploidization events, 1C value and chromosome number are evolving independently in the genus, as no correlation was found between holoploid genome size and ploidy. We found no evidence for polyploidy linked to the evolution of dioecy in Thalictrum. Given that such a link has been connected to the evolution of sex chromosomes or disruption of genetic self-incompatibility in other systems (Miller and Venable 2000, 2002; Spigler et al. 2010), this highlights that the evolution of dioecy in Thalictrum likely involved other pathways. Interestingly, we found a strong and novel association between polyploidy and wind pollination and proceed to discuss possible causes. Relationships and Ages Our new estimates for stem and crown ages of Thalictrum, in the late Oligocene and Miocene, respectively, are consistent with dating of the oldest fossils for the genus from Europe (Szafer 1961; Dorofeev 1963; Mai 1995). Our estimate for the crown age of Thalictrum, 22.8–14.0 Ma (fig. 5, supplementary table S5, Supplementary Material online), is older than a previous estimate of 14.5–2.3 Ma (Bastida et al. 2010), which was based on a reduced sampling of three species. Other divergence dates reported here are consistent with previous estimates and fossils for Thalictrum and outgroups. For example, our estimate for the divergence between Aquilegia and Thalictrum was 28.7–24.5 Ma (fig. 5, supplementary table S5, Supplementary Material online), compared with previous estimates of 24.9 Ma (Anderson et al. 2005) and 28.6–26.6 Ma (Bastida et al. 2010). Additionally, the divergence between Thalictrum and Leptopyrum + Paraquilegia was 27.6–19.5 Ma (fig. 5, supplementary table S5, 1945 Downloaded from http://mbe.oxfordjournals.org/ at University of Washington on July 3, 2014 Table 1. Character Correlation d-Statistic (P value) among Sexual Systems, Pollination Modes, and Ploidy in Thalictrum (Ranunculaceae), Calculated in SIMMAP (Bollback 2006). MBE 3.0 1.3 W 4.1 W P D P P 1 P 2.1 W P A 3.4 W P D 3.6 2 0.4 1.4 0.5 5.2 8.0 P 6.9 T. amurense T. lucidum T. simplex T. foetidum T. flavum T. ramosum T. squarrosum T. rochebrunnianum T. uchiyamae T. pubescens T. revolutum T. dasycarpum T. cooleyi T. coriaceum T. macrostylum T. delavayi T. grandiflorum T. squamiferum T. tenue T. arsenii c1 T. arsenii c2 T. guatemalense c1 T. henricksonii T. podocarpum T. steyermarkii c1 T. hernandezii T. strigillosum T. steyermarkii c2 T. grandifolium T. guatemalense c2 T. tripeltiferum T. fendleri T. heliophilum T. dioicum T. pinnatum T. occidentale T. polycarpum T. venulosum T. atriplex T. rutifolium T. decipiens T. lecoyeri T. reticulatum T. uncatum T. cultratum T. isopyroides T. omeiense T. aquilegiifolium T. petaloideum T. actaeifolium T. minus T. baicalense T. smithii c1 T. smithii c2 T. alpinum T. zernyi T. diffusiflorum T. sparsiflorum T. finetii T. przewalskii T. leuconotum T. virgatum T. reniforme T. rhynchocarpum T. macrocarpum T. kiusianum T. punctatum T. filamentosum T. urbainii T. rubescens T. clavatum T. thalictroides T. ichangense Leptopyrum fum f arioides Paraquilegia microphylla Aquilegia formosa Soza et al. . doi:10.1093/molbev/mst101 1946 1.0 1.5 P II W 3 12.9 13.8 P 7.6 18.4 23.9* 7.5 26.6* 16.6* Oligocene 30.0 Miocene 25.0 20.0 15.0 Pliocene 10.0 5.0 I Pleistocene 0.0 MBE FIG. 5. Chronogram of Thalictrum and outgroups (Ranunculaceae), using the Bayesian 50% majority-rule consensus tree based on the combined chloroplast trnV-ndhC and nuclear ETS and ITS regions. Estimated mean ages and corresponding 95% HPD intervals (blue/gray) are indicated for both stem and crown group nodes for the genus Thalictrum and its two major clades, I and II; for chromosome number duplication events (P = polyploidization) inferred from ML reconstructions that affected more than one taxon; and for origins of andromonoecious (A), dioecious (D), and wind-pollinated (W) clades. Ancestral conditions for ploidy, sexual system, and pollination mode in the genus are diploid, hermaphroditic, and insect, respectively. Estimated ages at strongly supported nodes (i.e., Bayesian pps 0.95 or likelihood bs values 70%) are indicated in bold. Three calibration nodes are indicated with asterisks. Three strongly supported clades, 1–3, within clade II. Outgroups are in gray. Geologic epochs (Walker et al. 2012). Downloaded from http://mbe.oxfordjournals.org/ at University of Washington on July 3, 2014 Timing and Consequences of Recurrent Polyploidy . doi:10.1093/molbev/mst101 Supplementary Material online), compared with a previous estimate of 28.6–13.0 Ma (Bastida et al. 2010). Polyploidy in Thalictrum Genome Size Evolution Although trends of genomic downsizing or increasing accompanying polyploidy have been observed in angiosperms (Ozkan et al. 2003; Leitch and Bennett 2004; Pires et al. 2004; Garnatje et al. 2006), our limited dataset of 1C value estimates for Thalictrum show no correlation with haploid chromosome number (fig. 4). Broader sampling across higher ploidy levels (i.e., > 4x, see outliers in clade A, fig. 3) may show a positive correlation between genome size and ploidy. This uncorrelated evolution could be due to chromosome fission and/or fusion. Although undetected by chromEvol, we do see the potential for chromosomal fission and/or fusion to contribute to genome size variation in Thalictrum as reported in other studies (Chung et al. 2011, 2012; Lipnerová et al. 2013). For example, chromosome fission could explain the difference in chromosome number but similarity in holoploid genome size observed in T. rochebrunnianum and T. uchiyamae (fig. 4). Additionally, chromosome fusion could explain the similarity in chromosome number but difference in holoploid genome size observed in diploids in clade I versus those in clade II (fig. 3). We also observed variation within a given ploidy level, as inferred by chromosome number, that could be related to lineage age. On one hand, within diploids, we see a general increase in 1C value with age. For example, T. kiusianum and T. punctatum diverged from each other 1.1–0.0 Ma and display holoploid genome sizes of 0.4 pg (figs. 3 and 5, supplementary table S4, Supplementary Material online). In contrast, an older diploid, T. ichangense, diverged 11.7–4.4 Ma and displays a larger 1C value of 0.57 pg (figs. 3 and 5, supplementary table S4, Supplementary Material online). These results suggest that mechanisms of genome expansion besides polyploidy are contributing to genome size evolution in Thalictrum. On the other hand, we observed differences in 1C value between recent and older polyploids, indicating a general decrease in 1C value with age within a certain polyploid level. For example, the tetraploid T. rhynchocarpum arose from a genome duplication event as early as 11.9–3.9 Ma and displays a 1C value of 0.25 pg (figs. 3 and 5, supplementary table S4, Supplementary Material online). In contrast, a recent tetraploid, T. lucidum, arose from a genome duplication as early as 1.9–0.3 Ma and displays a larger 1C value of 0.64 pg (figs. 3 and 5, supplementary table S4, Supplementary Material online). These results highlight that genomes of older polyploids may become contracted over longer periods, in comparison to recent polyploids, who have not had as much time to undergo mechanisms of DNA loss (reviewed in Bennett and Leitch 2005). In spite of rampant polyploidy, our study indicates that the genus Thalictrum has relatively small 1C values (0.25–3.7 pg, fig. 3), compared with angiosperm-wide estimates (0.06– 152.23 pg; Greilhuber et al. 2006; Pellicer et al. 2010). This could be due to their small T-type chromosomes (Langlet 1927) that would better tolerate recurrent polyploidy. An angiosperm-wide study observed a negative correlation between genome size (4C value) and percentage of polyploids due to an intolerance for large amounts of DNA (Grif 2000). Therefore, polyploidy would be expected to occur more readily in groups with small genomes and may have become rampant in Thalictrum because of its relatively small holoploid genome size, especially as observed in older, diploid species (i.e., T. ichangense, 0.57 pg, figs. 3 and 5). No Association between Dioecy and Polyploidy Unlike previously suggested (Miller and Venable 2000), we found no significant association between polyploidy and dioecy in Thalictrum (table 1). In spite of limited information on mating systems within Thalictrum (except Westergaard 1958), at least two taxa with diploid populations have been identified as self-compatible (T. alpinum and T. sparsiflorum; Steven and Waller 2004). Plants do appear to set seed readily in cultivation and do not exhibit self-incompatibility, but this remains to be formally tested in the majority of species. Additionally, we have not observed evidence for heteromorphic sex chromosomes during cytological investigations, yet there is evidence for homomorphic sex chromosomes in a few species, based on sex ratios in inter- or intraspecific crosses (Kuhn 1930, 1939; Westergaard 1958; Di Stilio et al. 2005). In conclusion, polyploidy does not appear to be correlated with the evolution of dioecy in Thalictrum; therefore, factors other than GSI and polyploidy must have influenced 1947 Downloaded from http://mbe.oxfordjournals.org/ at University of Washington on July 3, 2014 Our reconstructions of polyploidy suggest that WGDs may have contributed to floral diversity in certain Thalictrum lineages (clades 1–2, fig. 3), as these contain variation in both sexual system and pollination mode: hermaphroditism, andromonoecy and/or dioecy, and insect and wind pollination. Population-level studies in species with multiple cytotypes (i.e., T. coriaceum, T. pubescens, and T. simplex) are needed to further investigate whether recent polyploidization events are contributing to incipient speciation in this genus. Despite the fact that polyploidy has arisen recently and frequently in Thalictrum, we found no obvious evidence of reticulation resulting from allopolyploid hybridization among species. Although most polyploids in the genus had previously been assumed to be of allopolyploid origin (Kuzmanov and Dutschewska 1982), the only hint of reticulation we found was in T. guatemalense and T. steyermarkii, which had divergent clonal sequences (fig. 1). This evidence of reticulation, however, could result from a number of processes in addition to hybridization, such as gene duplication or incomplete lineage sorting. Our inability to detect potential allopolyploids may be due to a prevalence of autopolyploid events or, more likely, to the nature of the DNA regions used in our analyses. Given that chloroplast regions are maternally inherited and nuclear ribosomal regions are known to undergo concerted evolution (Baldwin et al. 1995), they alone are ill suited to detect hybridization events. Future studies using single-copy nuclear regions may provide a better understanding of the origin of polyploid species in this genus. MBE MBE Soza et al. . doi:10.1093/molbev/mst101 the evolution of dioecy, as observed in other genera (Pannell et al. 2008; Volz and Renner 2008). Association between Wind Pollination and Polyploidy Material and Methods Sampling For phylogenetic reconstruction of the genus, we sampled 69 of the 196 species from 13 of the 14 Thalictrum sections (Tamura 1995), to represent the breadth of taxonomic classification and geographic distribution (supplementary table S1, Supplementary Material online). We also sampled three outgroups: Aquilegia formosa, Leptopyrum fumaroides, 1948 Molecular Methods For amplification and sequencing of samples, we extracted genomic DNA from herbarium specimens, field collections, and cultivated accessions using the DNeasy Plant Kit (Qiagen, Valencia, CA), FastDNA Kit (MP Biomedicals, Solon, OH), or following the protocol of Hughey et al. (2001). Voucher specimens are listed in supplementary table S1, Supplementary Material online. We selected three DNA regions, two nuclear and one chloroplast, for phylogenetic analyses of samples. We used the nuclear ribosomal internal transcribed spacer region (ITS: ITS1, ITS2, and 5.8S) from taxa previously amplified in the genus and outgroups (Soza et al. 2012), plus several additional taxa (supplementary table S1, Supplementary Material online). We also used the nuclear ribosomal external transcribed spacer (ETS) region and a more variable chloroplast (cpDNA) region than previously utilized (Soza et al. 2012). To identify the cpDNA region, we conducted preliminary phylogenetic analyses with the five most variable cpDNA regions from Shaw et al. (2007) from a subset of Thalictrum taxa: rpl32-trnL, trnQ-5’rps16, 3’trnV-ndhC, ndhF-rpl32, and trnS-trnG-trnG. We found the 3’trnV-ndhC (trnV-ndhC) intergenic region as the most suitable cpDNA region to use in subsequent analyses of Thalictrum, as it provided the most resolution and support among taxa. For amplification and sequencing of DNA regions, we used published primers (Nickrent et al. 1994; Baldwin and Markos 1998; Goodwillie and Stiller 2001; Wright et al. 2001; Shaw et al. 2007; Soza et al. 2012) and the T. thalictroides transcriptome (Johnson et al. 2012) to design Thalictrum-specific primers for subsequent amplification and sequencing (supplementary table S6, Supplementary Material online). Polymerase chain reaction conditions for ITS and trnV-ndhC followed the protocol of Shaw et al. (2007). Polymerase chain reaction conditions for ETS were 94 C for 2 min, followed by 35 cycles of 94 C for 30 s, 55 C for 30 s, and 72 C for 1 min, with a final extension step at 72 C for 10 min. Amplified DNA was purified using ExoSAP-IT (USB Corporation, Cleveland, OH) and directly sequenced by GENEWIZ (Seattle, WA). We cloned ITS and ETS sequences that could not be directly sequenced, due to allelic differences in insertions and deletions, using the TA Cloning Kit (Invitrogen Corporation, Carlsbad, CA). Three to 24 positive clones per accession, depending on known ploidy or genome size, were screened by colony polymerase chain reaction, purified as outlined earlier, and sequenced by GENEWIZ using the T7 primer (Invitrogen Corporation, Carlsbad, CA). Phylogenetic Analyses Sequences were edited in Sequencher version 4.9 (Gene Codes Corporation, Ann Arbor, MI) and aligned manually for each DNA region using MacClade version 4.08 (Maddison DR and Maddison WP 2005). Alignments are Downloaded from http://mbe.oxfordjournals.org/ at University of Washington on July 3, 2014 We found a strong statistical association between polyploidy and wind pollination in Thalictrum (table 1). This association is intriguing and given the diverse suite of floral traits that contribute to a pollination mode, which specific traits are affected by polyploidy remains to be tested. In Ranunculaceae, a primarily insect-pollinated family, polyploids have been correlated with smaller flowers and multiflowered inflorescences (Vamosi et al. 2007). Given that these features are often characteristic of wind-pollinated flowers, this data offer tantalizing evidence in light of results presented here, that polyploidy may have facilitated the evolution of wind pollination in members of this family. Wind-pollinated Thalictrum clades evolved in concert with polyploidy in the Americas between 10.8–5.6 and 2.5–0.6 Ma. This time period included significant mountain uplift in North and Meso-America and global cooling that culminated in the ice ages (Graham 1999, 2011). The creation of higher elevation habitat and colder, drier environments may have contributed to the diversification of polyploid Thalictrum species, as polyploids have been associated with colonization of recently deglaciated areas (Brochmann et al. 2004; Li et al. 2010) due to their fixed heterozygosity (Brochmann et al. 2004) and with increasing aridity due to higher drought tolerance (Manzaneda et al. 2012). Additionally, insect pollinators are limited at higher elevations or during glaciation periods, thus giving wind pollination a selective advantage in open habitats (reviewed in Gomez and Zamora 1996; Totland and Sottocornola 2001; Duan et al. 2009). Another potential explanation for the link between polyploidy and wind pollination is a prior association between alkaloid biosynthesis and ploidy in Thalictrum (Kuzmanov and Dutschewska 1982; Kuzmanov 1986), whereby polyploids produce more types of isoquinoline alkaloids than diploids. Differential biosynthesis of such compounds could have impacted pollination as these isoquinoline alkaloids are toxic to insects (Miller and Feeny 1983; Philogene et al. 1984), offering a selective advantage to morphologies predisposed to wind pollination. Many genetic and epigenetic changes follow WGDs, and the effect on chemical biosynthesis is just one potential outcome. Having identified relatively recent polyploids in Thalictrum, future research in the group will address the underlying causes of polyploidization and the potential effects of WGDs on transitions from insect to wind pollination. and Paraquilegia microphylla, identified from previous molecular phylogenies of the family (Wang and Chen 2007; Wang et al. 2009). Timing and Consequences of Recurrent Polyploidy . doi:10.1093/molbev/mst101 To assess the reliability of clades in the resulting likelihood trees for each data set, we conducted 1,000 nonparametric bs replicates (Felsenstein 1985) in GARLI. bs replicates were conducted under the above settings but included one search replicate and 10,000 generations as the first part of the termination condition. bs trees were summarized using NCLconverter version 2.1 (Lewis and Holder 2010) and CONSENSE version 3.66 (Felsenstein 2006) via the CIPRES Science Gateway. Cytology Chromosome counts were obtained from one to three cultivated accessions for species lacking published counts or for those with variable published counts. Chromosomes were observed from root tips (T. decipiens, T. hernandezii, and T. strigillosum) or pollen mother cells (PMCs; T. clavatum, T. dasycarpum, T. delavayi, T. filamentosum, T. foetidum, T. guatemalense, T. punctatum, and T. simplex; fig. 2, supplementary tables S3 and S4, Supplementary Material online). Initially, chromosomes were observed using root tips from several Meso-American species, for which we had wildcollected seed. Subsequently, chromosomes were observed from PMCs from freshly collected floral buds from cultivated accessions at the UW Greenhouse, using a modified protocol of Kato (1999) that has worked well in condensing and spreading chromosomes in a variety of plants. Root tips were treated with 0.05% colchicine for 4 h at room temperature, fixed in 3:1 ethanol:acetic acid overnight, hydrolyzed with hydrochloric acid for 15 min, stained with Feulgen for 3 h, and rinsed in sulfurous acid before mounting (Haskins E, personal communication). Floral buds were treated according to the protocol of Matsushita et al. (2012) and Wright et al. (2009), modified with a N2O treatment for 3 h at 160 PSI and enzyme digestion for 3.5 h. PMCs were mounted in VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA), observed and photographed using a Nikon Microphot-FX microscope (Nikon Instruments, Inc., Melville, NY) and a Retiga 1300 monochrome camera (QImaging, Surrey, BC, Canada). Genome Size Estimation One to five accessions or replicates for 35 species cultivated at the UW Greenhouse were analyzed to obtain relative holoploid genome size, expressed as 1C value (as defined by Greilhuber et al. 2005; supplementary table S4, Supplementary Material online). Nuclei were extracted from fresh leaf tissue and combined with chicken erythrocyte nuclei (CEN singlets, BioSure, Grass Valley, CA) before staining with propidium iodide and analyzed with a flow cytometer, as outlined in Davison et al. (2007). CEN, with a 1C value of 1.25 pg (Gregory 2013), were used as an internal calibration standard. Animal standards have been discouraged by some authors for plant studies because 1) they cannot account for the huge range of plant genome sizes, 2) their nuclei structure may be different from plant nuclei, and 3) their precise genome size is unknown (Dolezel and Greilhuber 2010). 1949 Downloaded from http://mbe.oxfordjournals.org/ at University of Washington on July 3, 2014 available through TreeBASE (http://purl.org/phylo/treebase/ phylows/study/TB2:S13801). Molecular features for each DNA region are summarized in supplementary table S2, Supplementary Material online. We reconstructed phylogenies for each molecular data set separately (supplementary figs. S1–S3, Supplementary Material online) and for all regions combined using Bayesian and likelihood analyses. For taxa with various clonal ITS sequences, we constructed a consensus sequence inclusively from each monophyletic group of sequences representing a given taxon or from a monophyletic group of taxa with unresolved relationships that included clones of a given taxon. All other clonal sequences, not forming groups with other clones from the same accession, were included in analyses. For accessions with more than one included ETS or ITS sequence, the corresponding ITS or ETS and cpDNA sequences were duplicated for use in the combined data set. For Bayesian and likelihood analyses, models of evolution for each data set were determined separately by jModelTest version 2.1 (Guindon and Gascuel 2003; Posada 2008). The models selected under the AIC (Akaike 1974) for ETS, ITS, and trnV-ndhC were GTR + , GTR + I + , and TPM1uf + I + , respectively. Bayesian analyses were conducted separately in MrBayes version 3.1.2 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003) via the CIPRES Science Gateway version 3.1 (Miller et al. 2010) for each data set and the combined data set, with data partitioned under the selected models for each DNA region. We used default priors of no prior knowledge for the parameters of these models. Parameters for nucleotide frequencies, substitution rates, and gamma shape were unlinked across data partitions. All partitions were allowed to evolve under different rates, and site-specific rates were allowed to vary under a flat Dirichlet prior across partitions. Bayesian analyses were conducted with three independent Markov chain Monte Carlo (MCMC; Yang and Rannala 1997) analyses of 10 million generations for individual DNA regions and 20 million generations for the combined data set. Metropolis coupling for each analysis was conducted under the default settings. Convergence was determined when the average standard deviation of split frequencies remained less than 0.01. For the trnV-ndhC, ETS, ITS, and combined data set, the first 37%, 60%, 83%, and 28% of trees, respectively, were discarded before convergence. For each analysis, the remaining trees from each run were pooled to construct a 50% majority rule consensus tree or a consensus tree with all compatible groups to obtain pps. Likelihood analyses (maximum likelihood [ML]) were conducted separately in GARLI version 2.0 (Zwickl 2006) via the CIPRES Science Gateway for each data set and the combined data set, with data partitioned under the selected models for each DNA region. All analyses were run under the default settings but included 5 or 10 search replicates, model parameters unlinked across subsets, and different subset rates allowed. MBE MBE Soza et al. . doi:10.1093/molbev/mst101 Character Reconstructions To identify the number of independent origins of polyploidization within Thalictrum, ancestral character states of ploidy were reconstructed using likelihood in chromEvol version 1.3 (Mayrose et al. 2009). This program is advantageous because it allows both polytomies and polymorphic character states. However, the polymorphic assignment of states has not been rigorously tested in the program (Mayrose I, personal communication). Ancestral states were reconstructed under the default settings, using the combined 50% majority-rule Bayesian consensus tree with clonal sequences pruned to one per taxon, with all branch lengths multiplied by one. We also reconstructed ancestral states using the Bayesian consensus tree but with nodes collapsed that had no support in either the ML or Bayesian phylogenetic analyses (supplementary fig. S4, Supplementary Material online) to confirm our results. The character states for ploidy were coded as haploid chromosome number (as required by the program) and were obtained from the literature and/or our cytological data (supplementary table S3, Supplementary Material online). The haploid chromosome number of aneuploid or triploid populations was rounded up to the next whole integer. We attempted two coding assignments for chromosome number: 1) using all published counts for a taxon (polymorphic) or 2) using the most commonly reported count for a taxon (most common). For polymorphic coding, we used the percentage of reports in the literature and/or from personal observations to obtain the probability for each state. Only precise reports were used in obtaining probabilities. One exception was T. minus, which has widely varying counts in the literature (~14 different counts); chromEvol was unable to accommodate this computationally. Therefore, we coded this taxon as missing for the polymorphic analysis. For most common coding, we took the state with the highest probability for each species from the polymorphic coding and assigned this state to each species. Several exceptions were T. coriaceum, T. dasycarpum, and T. pubescens, for which, two equally likely counts are published. Taxa with unknown states were coded as missing. 1950 Character Correlation Tests To evaluate whether a correlation exists between genome size and ploidy within Thalictrum, we used our estimates of holoploid genome size and chromosome number for 20 species grown at the UW Greenhouse, for which we had both estimates from the same source population or from published chromosome counts that did not vary (supplementary table S4, Supplementary Material online). We performed phylogenetic generalized least squares regression on this reduced data set of 20 species, excluding three species considered outliers (T. dasycarpum, T. pubescens, and T. revolutum; clade A, fig. 3), in COMPARE version 4.6b (Martins 2004). We used the fully resolved all-compatible Bayesian consensus tree (as polytomies are not allowed in COMPARE) and pruned taxa in Mesquite version 2.75 (Maddison WP and Maddison DR 2011), for which we did not have both genome size and ploidy estimates. We incorporated our estimates of standard error for genome size data and used estimates of zero for chromosome number and genome size samples of one. The correlation coefficient (r), regression slope ( 1), and intercept ( 0) were determined by the ML estimate of . To evaluate whether a correlation exists between dioecy or pollination mode and polyploidy in Thalictrum, correlation analyses were conducted in SIMMAP version 1.5 (Bollback 2006). SIMMAP is a Bayesian approach for testing correlated evolution, allows for multiple-state characters, incorporates phylogenetic uncertainty over a set of trees, and determines the amount of time along a branch each state occurred. For analyses in SIMMAP, morphological models for each character were configured using the combined Bayesian 50% majority-rule consensus tree and rescaling the tree length to one, using an approach by Schultz and Churchill (1999). For the bias parameter, we used beta distribution priors with 31 categories and empirical priors for two-state and three-state characters, respectively. For all characters, we used gamma distribution priors for the overall evolutionary rate parameter with 90 categories. To determine values of for the beta distribution prior, and and for the gamma distribution prior, we obtained values for each character by running an MCMC analysis under the default settings. Samples from the posterior distribution of these parameters were used to obtain best-fitting distributions and parameter values of and using the R version 2.15.1 (R Development Core Team 2011) script available with SIMMAP. All character states were unordered. To account for phylogenetic uncertainty and computational time, character correlations were conducted in SIMMAP with 100 postburnin trees from each MrBayes run, for a total of 300 trees. We used the character states and priors determined above, with five samples for each tree and from the prior distribution for each character, and five posterior predictive samples simulated to determine P values for the correlation D statistic (Huelsenbeck et al. 2003). The character states used for pollination mode and sexual system were insect or wind, and dioecy, hermaphroditism, Downloaded from http://mbe.oxfordjournals.org/ at University of Washington on July 3, 2014 However, for our purposes, the genome size of CEN falls within the range of Thalictrum genome size estimates. Additionally, our goal was to produce relative holoploid genome size estimates, because absolute estimates are not feasible due to the lack of complete genome coverage in most model taxa due to repetitive regions in the genome (Davison et al. 2007; Dolezel and Greilhuber 2010). Samples were analyzed on a FACScan flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ) with FlowJo software (Tree Star, Ashland, OR) at the UW Department of Immunology Cell Analysis Facility. The 2C median nuclear peak of propidium iodide fluorescence in Thalictrum samples was compared with that of the CEN standard to estimate the 2C nuclear DNA content of Thalictrum using the CEN 2C DNA content (2.5 pg) and equation in Dolezel and Bartos (2005). Timing and Consequences of Recurrent Polyploidy . doi:10.1093/molbev/mst101 or monoecy, respectively, as outlined in Soza et al. (2012; supplementary tables S7 and S8, Supplementary Material online). The character states for ploidy were diploid or polyploid. For polymorphic taxa, the maximum ploidy reported was used (supplementary table S7, Supplementary Material online). Molecular Dating Supplementary Material Supplementary figures S1–S4 and tables S1–S8 are available at Molecular Biology and Evolution online (http://www.mbe. oxfordjournals.org/). Acknowledgments The authors thank Johanne Brunet, Hongzhi Kong, Rebecca Penny, Rick Ree, Rui Zhang, and University of California Botanical Garden at Berkeley for tissue samples; Aaron Liston, David Guzman Otano, and the US National Plant Germplasm System, North Central Regional Plant Introduction Station for seed; Starr Matsushita and Delene Oldenberg for assistance with cytology; Rebecca de Frates, Sarah Choe, Patricia Salles Smith, Xuening Chen, YangBo, and My Linh for assistance with genome size data collection; Cajsa Lisa Anderson and Jesús Bastida for divergence estimates; Michael Karcher, Itay Mayrose, and Cheng Zheng for assistance with data analysis; 1KP project (T. thalictroides material provided by V.S. Di Stilio) for sequencing the T. thalictroides floral transcriptome, and Jim Leebens-Mack for providing the data in searchable format; three anonymous reviewers for valuable comments; and the following herbaria: CS, GH, MO, OSC, ORE, RSA, and TEX. 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