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Letters Are polyploids really evolutionary dead-ends (again)? A critical reappraisal of Mayrose et al. (2011) Introduction Background Throughout the past century, hybridization and polyploidization have variously been viewed as drivers of biodiversity (e.g. Arnold, 1997) or evolutionary noise, unimportant to the main processes of evolution (e.g. Stebbins, 1950; Wagner, 1970). Wagner (1970) argued that while polyploids have always existed, they have never diversified or played a major role in the evolution of plants, and that the study of polyploidy (as well as inbreeding, apomixis, and hybridization) has led researchers to be ‘carried away with side branches and blind alleys that go nowhere’. However, the use of molecular tools revolutionized the study of polyploidy, revealing that a given polyploid species often forms multiple times (reviewed in Soltis & Soltis, 1993, 1999, 2000, 2009). The realization that recurrent polyploidization from genetically differentiated parents is the rule that shattered the earlier perceptions of polyploids as genetically depauperate (Stebbins, 1950; Wagner, 1970). Because of multiple origins, polyploid species can maintain high levels of segregating genetic variation through the incorporation of genetic diversity from multiple populations of their diploid progenitors (e.g. Soltis & Soltis, 1993, 1999, 2000; Tate et al., 2005). Numerous studies have also shown that polyploid genomes are highly dynamic, with enormous potential for generating novel genetic variation (e.g. Gaeta et al., 2007; Doyle et al., 2008; Leitch & Leitch, 2008; Flagel & Wendel, 2009; Hawkins et al., 2009; Chester et al., 2012; Hao et al., 2013; Roulin et al., 2013). Furthermore, genomic studies have also revealed numerous ancient polyploidy events across the angiosperms (e.g. Vision et al., 2000; Bowers et al., 2003; Blanc & Wolfe, 2004; Paterson et al., 2004; Schlueter et al., 2004; Van de Peer & Meyer, 2005; Cannon et al., 2006; Cui et al., 2006; Tuskan et al., 2006; Jaillon et al., 2007; Barker et al., 2008, 2009; Lyons et al., 2008; Ming et al., 2008; Shi et al., 2010; Van de Peer, 2011; Jiao et al., 2012; McKain et al., 2012; Tayale & Parisod, 2013; reviewed in Soltis et al., 2009); all angiosperms have undergone at least one round of polyploidy (e.g. Jiao et al., 2011; Amborella Genome Consortium, 2013).

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Polyploidy is now viewed not as a mere side branch of evolution, but as a major mechanism of evolution and diversification. Evolutionary dead-ends again? Based on analyses of ferns and angiosperms, Mayrose et al. (2011) and Arrigo & Barker (2012) revived the concept of polyploids as ‘blind alleys’. Arrigo & Barker (2012, p. 140) refer to ‘rarely successful polyploids’ and state that ‘despite leaving a substantial legacy in plant genomes, only rare polyploids survive over the long term and most are evolutionary dead-ends’. Mayrose et al. (2011) also refer to polyploids as ‘dead-ends’. However, these authors use ‘evolutionary dead-end’ in a sense that differs from the traditional view of Stebbins (1950) and Wagner (1970). Whereas Stebbins (1950) and Wagner (1970) were concerned that polyploids did not contribute significantly to evolution, Arrigo & Barker (2012) argue that the analyses of Mayrose et al. (2011) indicate that polyploids are more likely to go extinct than are diploids and in this sense are typically ‘dead-ends’. We agree that most new polyploids likely go extinct early, probably at the population level before they are even detected; this has long been espoused (e.g. Levin, 1975; Ramsey & Schemske, 1998; reviewed in Rieseberg & Willis, 2007; Soltis et al., 2010). Ramsey & Schemske (1998) estimated that the rate of autotetraploid formation is high – comparable to the genic mutation rate – but most do not survive. However, these very early, extinctionprone stages of polyploids are much younger than the timeframe considered by Mayrose et al. (2011) and Arrigo & Barker (2012), who concluded that established polyploid species are more likely to go extinct than diploid congeners. Goals We revisit the analyses and conclusions of Mayrose et al. (2011) and Arrigo & Barker (2012) and ask: Is there convincing evidence to support the hypothesis that extinction rates in established polyploids are higher than those of diploids? Perhaps this is true, but we do not feel that these authors have made a compelling case. While we have focused our review and examples on angiosperms, most of our criticisms are equally applicable to the fern dataset included in their studies. We emphasize philosophical, statistical, and analytical arguments against the study of Mayrose et al. (2011), as well as problems with sampling and their data-mining methods. We support our views by drawing on numerous examples from the recent and classic literature on polyploid complexes (most not included in Mayrose et al., 2011). We stress that our goals are to correct errors and stimulate discussion, not attack the authors of Mayrose et al. (2011) and Arrigo & Barker (2012).

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Philosophical arguments Clade age, size, and opportunity for diversification Increased diversification rates are more likely to arise in large clades than in small ones, simply because there are more opportunities for increased speciation or retarded extinction rates. Thus, large, old clades will, on average, yield more clades with increased diversification rates than young, small ones, and this probabilistic statement has relevance for patterns of diversification in polyploids relative to their diploid relatives (including their parents). By definition, a polyploid species is younger than its parents. The parental species are part of a clade that is more numerous and older than the single polyploid derivative – thus, through chance, greater diversification could occur at the diploid than the polyploid level. This relationship between clade age, clade size, and diversification – and its particular relevance to diploid–polyploid comparisons – was not considered by Mayrose et al. (2011). Because a polyploid can never be the same age as its parents, analyses within genera with both diploids and polyploids are biased in favor of greater diversification at the diploid level. A more appropriate comparison would be between related diploid and polyploid clades of the same age. Reticulate evolution Although evolutionary history is represented using bifurcating phylogenetic trees, it is well known that complex sequences of evolutionary events are poorly represented with a branching tree structure (Doolittle, 1999; Huson & Bryant, 2006). Hybridization, hybrid speciation, and allopolyploidy are not described well by bifurcating trees (e.g. Wagner, 1983; Linder & Rieseberg, 2004; McBreen & Lockhart, 2006; Brysting et al., 2007; Soltis & Soltis, 2009; Huson & Scornavacca, 2010). Thus, the evolutionary history of many plant groups is not tree-like, but a network with numerous reticulation events (e.g. Guggisberg et al., 2009; Marcussen et al., 2012; Pellicer et al., 2012; Abbott et al., 2013; Garcia et al., 2014). To reconstruct and visualize complex reticulate evolutionary scenarios, various methods may be employed, including splits graphs, other networks, and analysis of incongruence among gene trees (McBreen & Lockhart, 2006). Comparison of numerous nuclear gene trees is perhaps the best approach to tease apart the complexity of reticulation events (e.g. Linder & Rieseberg, 2004), but to date few studies have used this approach. Mayrose et al. (2011), however, relied entirely on phylogenetic and diversification analyses that have as an underlying assumption that evolution is strictly bifurcating. While we agree that diversification methods have not been developed for nonbifurcating evolutionary histories, we urge caution in interpreting results derived from analyses that violate many of the underlying assumptions of the algorithms used. How polyploids affect phylogeny reconstruction is poorly understood – even at the level of how the topology may be influenced (Soltis et al., 2008; Lott et al., 2009; Marcussen et al., 2012). How those effects cascade onto the accuracy of branch length estimation is unexplored. Finally, how these effects on topology and branch lengths influence diversification analyses is also untested. Again, New Phytologist (2014) www.newphytologist.com

improved methods and understanding of these nonbifurcating evolutionary influences will help address the question. However, we conclude that current methods are poorly suited when it comes to the complex evolution of polyploids.

Statistical and analytical arguments The conclusions of Mayrose et al. (2011) hinge upon accurate estimation of speciation and extinction rates from molecular phylogenies. Here, we focus on the problems with estimating speciation and extinction rates, as well as the statistical interpretation of the results. Estimating extinction rates from molecular phylogenies based solely upon extant taxa in the absence of a fossil record, as in Mayrose et al. (2011), is problematic (Rabosky, 2009, 2010; Quental & Marshall, 2010). Rabosky (2010) showed that when diversification rates vary among lineages, ‘simple estimators based on the birth–death process are unable to recover true extinction rates’ (p. 1816). Because variation in diversification rate is ‘ubiquitous,’ Rabosky (2010) cautioned that extinction rates should not be estimated in the absence of fossils. Similarly, Quental & Marshall (2010) stress that ‘a wide range of processes can give similarly shaped molecular phylogenies’. Mayrose et al. (2011) found relatively little difference in speciation rates between diploids and polyploids; their conclusion that polyploids diversify at a lower rate is based on higher inferred extinction rates in polyploids – if that rate is unreliably estimated, as others have cautioned, their conclusions cannot be supported. Speciation and diversification rates were estimated with BiSSE (Maddison et al., 2007; FitzJohn et al., 2009). However, Davis et al. (2013) examined the power of the BiSSE method and showed that phylogenies with fewer than 300 taxa should be treated with extreme caution. However, only three datasets in Mayrose et al. (2011) have over 100 terminals. Additionally, Davis et al. (2013) caution against using datasets where the frequency of one of the two states is under 10% – 11 of the 63 genera studied have frequencies outside this range (nine with polyploidy frequencies under 10% and two with polyploidy frequencies over 90%, another is reported as 10% polyploid species). Mayrose et al. (2011) cannot be criticized based on conclusions reached in 2013. However, even when BiSSE was published (Maddison et al., 2007), the authors stressed that large phylogenetic trees would be needed, suggesting c. 500 terminals. ‘It remains to be studied how the probability of rejecting a false null hypothesis (power) varies with number of species and with the degree of difference in rates. From our initial exploration, however, we suspect that large phylogenetic trees will generally be needed to have sufficient power’ (p. 708). They also note (p. 706), ‘This shows at least some power to reject the null hypothesis with trees of 500 species when there is a twofold difference in speciation rates’. Mayrose et al. (2011) show the results of the significance tests for each study but do not draw the reader’s attention to the meaning of the information. The last three columns of supplementary material table S2 from Mayrose et al. (2011) show the percent of the MCMC chains where the difference in diversification, speciation, and extinction estimates of diploids minus polyploids is positive (higher in diploids than polyploids). Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist The test of significance is whether zero (no difference in rates) is outside of the 95% credible interval – or outside of the range seen in 95% of the MCMC steps. Thus, under equal rates, the percent is expected to be 50%, while if 95% of the MCMC steps show higher parameter estimates in diploids than polyploids, this would be a significant result (similarly, if only 5% show higher rates, this would be a significant result in favor of polyploids having higher rates). For diversification rate, only six of 63 genera support significantly higher diversification in diploids, and three of the 63 support significantly lower diversification in diploids relative to polyploids – hardly results that support a conclusion of dramatic differences in diversification rates. For speciation, none of the differences support significantly higher speciation in diploids, while 13 of 63 support higher speciation rates in polyploids – in fact, when examined within each MCMC step (as these data do), speciation is higher in polyploids on average, with only an average of 22% of the MCMC steps showing higher speciation in diploids than polyploids vs 50% for equal rates. While the estimated average speciation rates are higher in diploids (columns 1 and 2 of table S2 in the supplementary material of Mayrose et al. (2011)), when association with MCMC steps is accounted for, the trend is the opposite. For the extinction results, we assume that the table is mislabeled (while the file has been corrected once since publication, the latest version was updated September 1, 2011, and remains labeled %(lD > lP), that is, that extinction in diploids is higher than polyploids), and the authors intended the label to be ‘%(lD < lP)’, as this would be consistent with their assertion that extinction is higher in polyploids than diploids. With that assumption, 32 of the 63 genera indicate significantly higher extinction in the polyploids. We again point to the Rabosky (2010) caution regarding estimation of extinction rates, as well as the following section as cautionary examples of how even this result may be questionable. In summary, while on average, speciation rates are higher in diploids, the only significant results are in the opposite direction, where polyploids have significantly higher speciation rates in 20% of the genera studied. Additionally, when the estimates are considered within MCMC steps, accounting for correlation among parameters, diploid species only have a higher rate of speciation 22% of the time – indicating that 78% of the time, polyploids have the same or higher speciation rates. The significant extinction rate differences (and thus diversification) seem compromised by the difficulties in estimating extinction rates. These findings alone call into serious question the validity of the conclusions of Mayrose et al. (2011). While further study and better techniques may well support the results, at this point, we argue that the conclusion that polyploids have lower rates of diversification is unjustified.

Sampling arguments Underestimating the frequency of polyploids Many polyploids are not recognized taxonomically. Many autopolyploids in particular are unnamed, and these may be Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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especially numerous. J. Ramsey and B. C. Husband (Soltis et al., 2007) found that of 2647 species from 346 genera in 62 angiosperm families in the California Flora, 334 species (13%) have multiple cytotypes (3x, 4x, or higher multiples of the base chromosome number for the genus). Because some of these chromosomally polymorphic taxonomic species actually comprise more than two cytotypes, if each cytotype represented a distinct species, the total number of unrecognized species would be 483, or c. 15% of the flora. Although we, and many others, might consider each cytotype to represent a distinct species, others might advocate a more conservative species concept. If only one quarter of these cytotypes represent distinct species, the impact is considerable (121 species, c. 4% of the flora). This tremendous source of biodiversity has not been captured in the phylogenetic analyses from which Mayrose et al. (2011) drew their data. These enormous gaps in knowledge make it difficult to conduct meaningful analyses and reach firm conclusions on the extinction rate of diploid vs polyploid species. An unrepresentative sample Mayrose et al. (2011) rely on the dataset of Wood et al. (2009) and assume that the angiosperm and fern genera analyzed therein are representative of these groups as a whole. Focusing on angiosperms, this assumption is incorrect for several reasons, and its violation has major implications. First, the Wood et al. (2009) sampling is very small.Theysampledonly49ofc.16 000genera(0.31%)and1984of c. 300 000 species (0.66%) of angiosperms (www.theplantlist.org). In addition, phylogenetic analyses conducted to date represent the initial phase of phylogenetic inquiry. Investigators have not yet analyzed many of the really problematic angiosperm genera (of which there are many) in a thorough manner. Several genera with a large proportion of polyploid species have been analyzed phylogenetically, but were not included in Mayrose et al. (2011) (Supporting Information Table S1). Furthermore, because polyploidization violates the model of bifurcating evolution, systematists have often avoided polyploid complexes in phylogenetic analyses and have removed polyploid species from analyses of genera with high proportions of polyploids. As reviewed later, both factors suggest bias in the dataset of Wood et al. (2009). Using the data from Mayrose et al. (2011), for each angiosperm genus sampled, we plotted the % estimated polyploids vs % total sampled species for that genus. The more complete the sampling, the higher the percentage of polyploids (Fig. 1). These analyses support the idea that polyploid species are the ones excluded in the analyses and that the overall sampling approach is not representative and will lead to biased results. Datasets not included in Mayrose et al. We provide a list of genera (Supporting Information Table S1) with a large proportion of polyploids, all of which have been analyzed phylogenetically. Many of these datasets and topologies (e.g. Aegilops, Draba, Elymus, Festuca, Hordeum, Hedera, Gossypium, Nicotiana, Paeonia, Rubus, and Triticum) could have been included in Mayrose et al. (2011). The percentage of polyploids in most of New Phytologist (2014) www.newphytologist.com

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these genera is high (e.g. Draba, 78%; Festuca, 70%; Hedera, 60%; Hordeum, 50%; Nicotiana, 40%; Triticum, 70%). The frequency of polyploidy is only 10% in Gossypium (Grover et al., 2012) and 25% in Paeonia (Sang et al., 1997); in Gossypium, the polyploids form a clade – from a single origin. Other examples of polyploid complexes have only recently been published (e.g. Opuntia, Majure et al., 2012a; Viola, Marcussen et al., 2012; Fragaria, Njuguna et al., 2013) and hence could not have been included in Mayrose et al. (2011). In most of these recent studies, close to half or more than half of the species are polyploid (e.g. Fragaria, 45%; Opuntia, 59%). A high frequency of polyploids does not necessarily equate to diversification at the polyploid level. However, inclusion of a more representative set of taxa having a high frequency of polyploidy (see later) could greatly alter the results of Mayrose et al. (2011), if many of the polyploid species arose via cladogenesis from other polyploid species rather than from separate polyploidization events from diploid parents. Nicotiana (Solanaceae) comprises 75 species (40 diploids, 35 allopolyploids), with the polyploids ranging in age from 200 000 yr to c. 10 million yr (My) old (Clarkson et al., 2004; Leitch et al., 2008; Kelly et al., 2012). The older polyploid clades generally have more species (Leitch et al., 2008); for example, sect. Suaveolentes contains 26 species, all of which are polyploid (Chase et al., 2003; Marks et al., 2011). Opuntia (Cactaceae) is a young clade (5.6  1.9 million yr old; Arakaki et al., 2011) of c. 200 species with a base chromosome number of x = 11 (Pinkava, 2002). Most species of Opuntia are polyploid, ranging from triploids (2n = 33) to nonaploids (2n = 99). Of 150 species with reported chromosome numbers, 59% were polyploid, 12% had both diploid and polyploid counts, and only 29% were diploid (Majure et al., 2012b). Most species of Triticum/Aegilops (Poaceae) are polyploid. Given that the parentage of polyploid species of Triticum includes several species of Aegilops, neither Triticum nor Aegilops is monophyletic (Petersen et al., 2006; Bordbar et al., 2011), and we consider them together here. Aegilops comprises 22–29 species. Using the higher species estimate, 11 are diploid (2n = 14), 13 are New Phytologist (2014) www.newphytologist.com

tetraploid (2n = 28), and five are hexaploid (2n = 42). Triticum comprises 11 species: three diploids (2n = 14), six tetraploids (2n = 28), and two hexaploids (2n = 42), including T. aestivum (common wheat). Viola (Violaceae) contains 500–600 species with numerous hybrid and polyploid complexes. From a putative base number of x = 6 or x = 7, extant chromosome numbers range from 2n = 4 (V. modesta) to at least 20-ploid, 2n = c. 160 (V. arborescens). The high polyploids (with > six sets of chromosomes) are monophyletic (Marcussen et al., 2012) and resulted from allodecaploidization 9–14 million yr ago (Mya), involving diploid and two paleotetraploid ancestors. Two of the high-polyploid lineages remained decaploid; recurrent polyploidization with tetraploids within the last five million yr has resulted in two 14-ploid lineages and one 18-ploid lineage (Marcussen et al., 2012). Polyploid speciation within polyploid clades has been a major contributor to diversification in Viola (Marcussen et al., 2012). ‘Messy’ polyploid genera Many angiosperm genera contain numerous polyploids but lack phylogenetic data. Indeed, classic polyploid complexes remain understudied simply because they are so complex. Thus, the examples analyzed by Mayrose et al. (2011; based on Wood et al., 2009) are not representative of angiosperm diversity, and their conclusions (and Arrigo & Barker, 2012) are premature. Systematists continue to avoid some of the most problematic polyploid complexes (Supporting Information Table S2) in favor of clades for which models of cladogenesis are appropriate and clear results may be obtained. Classic examples of such ‘messy’ genera (Stebbins, 1950, 1971; Grant, 1981) are Claytonia, Crepis, and Crataegus. Claytonia virginica itself comprises over 50 cytotypes in this single recognized species, with numbers ranging from 2n = 12 to c. 191 (Lewis, 1970; reviewed in Doyle, 1983). Salix (Salicaceae) comprises perhaps 400 species, approximately half of which are polyploid (Brunsfeld et al., 1991; Mabberley, 1997). Furthermore, genetic data and the genome sequence of Populus trichocarpa confirmed the prediction based on high chromosome number (Stebbins, 1950, 1971) that Salix and Populus are ancient polyploids (Soltis & Soltis, 1990; Tuskan et al., 2006). Other well-known examples of ‘messy’ groups (due in part to polyploidy) include Castilleja (Tank & Olmstead, 2008), Saxifraga, Micranthes (Webb & Gornall, 1989), Sedum and several other genera of Crassulaceae (Mort et al., 2010), as well as multiple genera of Cactaceae. Some examples are confounded by hybridization and apomixis, but such cases need to be factored into the polyploid equation if we are to ascertain whether polyploids have higher extinction rates than diploids. One can argue that we are preferentially seeking out polyploid complexes to counter Mayrose et al. (2011) and Arrigo & Barker (2012) and that these complexes are also not representative of angiosperms. However, our point is simple – many complex areas of the angiosperm tree of life are highly complicated due to polyploidy. Until more of those complexes are included in comparative studies, any broad assertions of diploid vs polyploid success and extinction are premature. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Data-mining arguments Background Throughout their paper, Mayrose et al. (2011) adhere to the methods that they describe. However, as we show later, in so doing they ultimately misrepresent some of the originally published trees, yielding unsupported conclusions regarding the impact of polyploidy. We review the methods used by Mayrose et al. (2011), highlighting areas of concern. Inconsistent data The bulk of the dataset is based on Wood et al. (2009), which collected data for 143 groups. Chromosome counts were taken from the phylogenetic study, or when not included, obtained from other sources such as the Index to Plant Chromosome Numbers (IPCN) and the Plant DNA C-values Database. Where there were discrepancies, the data reported in the phylogenetic study were favored. Unstated in the methods, but apparent in the supplementary data provided, for species with multiple chromosome numbers, the lowest value was used, generating a bias against polyploids within genera. In many cases, known chromosome counts were omitted with no explanation. Inconsistencies between the dataset of Mayrose et al. (2011) and the original sources were noted. For example, for Physalis (Solanaceae), Mayrose et al. (2011) listed only two polyploids: P. longifolia and P. minima (2n = 48). However, the ICPN lists values for several additional Physalis species, including three with polyploid counts of 2n = 48 (P. angulata (all reports), P. hederaefolia (also 2n = 12, 24), P. peruviana (2n = 48, 72)). In Achillea, A. asiatica, A. biebersteinii, A. crithmifolia, and

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A. holoserica are all listed in the original study as both 2x and 4x (Guo et al., 2004), but all were considered only 2x in Mayrose et al. (2011). Additional inconsistencies occur for Betula, Erodium, and Centaurium. This treatment of chromosome counts uniformly biased results toward having fewer polyploid counts in the trees, falsely creating a tree that is more diploid than reality. Deleted taxa in Mayrose et al. trees For each study, Mayrose et al. (2011) gathered molecular data and re-analyzed the published datasets. For studies with multiple loci, only those taxa included in the combined analysis were included. In some cases, this led to excluding whole clades of polyploids that had only been sequenced for some loci. While Mayrose et al. (2011) were clear about how the datasets were selected, and why some taxa were removed, the effects of their choices are not explored, either in terms of how many studies were affected or in how the exclusions may have biased the results. In some cases, the dataset chosen by Mayrose et al. (2011) resulted in what seems to be an arbitrary use of 35 to 98% of taxa from the original studies instead of the entire published dataset (Table 1). The removal of taxa can have a major impact. Specifically, in Cerastium, 21 taxa were omitted from the 57 species used in Scheen et al. (2004) – because they were not all sequenced for all genes – with major consequences (see later and Brysting et al., 2007); in Graptopetalum and allies, 15 of 43 taxa were removed (Acevedo-Rosas et al., 2004); in Penstemon, 31 of 163 species were omitted (Wolfe et al., 2006). Table 1 lists 21 examples where Mayrose et al. (2011) excluded species from their analyses – one-third of the studies analyzed. Yet there is no discussion of how this may have affected the results, or whether the removal was biased in terms of ploidy.

Table 1 Genera (with original references) from which taxa were omitted in Mayrose et al. (2011)

Focal group

Number of taxa included in Mayrose et al. (2011)

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Original reference

Sium s.l. Antirrhinum + allies Cuphea Lathyrus Gaura/Stenosiphon Phacelia subg. Phacelia Vaccinium sect. Macropelma/Myrtillus/Hemimyrtillus Achillea Pelargonium Houstonia Penstemon Digitalis/Isoplexis Mimulis Cerastium Gunnera Aichryson Graptopetalum + allies Coreopsis Aristolochia s.l. Arisaema Trillium s.l./Paris s.l.

5 17 52 52 18 50 50 59 142 15 132 23 86 36 20 14 28 22 78 75 25

14 32 53 53 21 51 52 63 149 17 163 26 95 57 22 15 43 23 85 81 26

Spalik & Downie (2006) Oyama & Baum (2004) Graham et al. (2006) Kenicer et al. (2005) Hoggard et al. (2004) Gilbert et al. (2005) Powell & Kron (2002) Guo et al. (2004) Bakker et al. (2004) Church & Taylor (2005) Wolfe et al. (2006) Brӓuchler et al. (2004) Beardsley et al. (2004) Scheen et al. (2004) Wanntorp et al. (2002) Fairfield et al. (2004) Acevedo-Rosas et al. (2004) Crawford & Mort (2005) Ohi-Toma et al. (2006) Renner et al. (2004) Farmer & Schilling (2002)

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Artificial diploidization In some cases, the dataset chosen by Mayrose et al. (2011) resulted in the deletion of polyploid taxa, making the trees ‘more diploid’ than they really are. Tiquilia (Boraginaceae), comprising c. 27 species, is an example in which several polyploids were excluded (Moore et al., 2006) (Fig. 2). Mayrose et al. (2011) only used those taxa that were included in the Moore et al. (2006) 5-gene combined analysis. However, six additional polyploid species were included in Moore et al. (2006). Including these polyploids would have increased the sampled polyploids from three out of 18 species (17%) to nine out of 28 (32%) (Fig. 2). We repeated the analyses of Mayrose et al. (2011) using a combined ITS and rps16 dataset from Moore et al. (2006), but including all 28 taxa for which data were available. With most of the polyploids removed, Mayrose et al. (2011) report that diversification is higher in diploid Tiquilias in 38% of the MCMC steps; with the full dataset, this is only the case in 24% of the steps. Notably,

(a) Mayrose et al.

speciation is estimated to be higher in polyploids in both datasets. Extinction was estimated to be higher in polyploids in 82% of the MCMC steps by Mayrose et al. (2011), but in the larger dataset, only 23% of the MCMC steps show this (Fig. 2). This is consistent with Moore et al. (2006), who found that the large polyploid clade in Tiquilia shows high diversification in a short period of time (c. six million yr). Moore et al. (2006) demonstrated that this rapid polyploid diversification is associated with migration into a new geographic area and the evolution of novel morphologies. The deletion of taxa from Cerastium, as well as missing data, by Mayrose et al. (2011) also has a major impact. Cerastium comprises c. 100 species, nearly all of which are polyploid. Only a few species have the lowest (diploid) number of 2n = 18 (C. semidecandrum has 2n = 18, 36, 37; C. lithospermifolium has 2n = 18); many species have 2n = 36. The arctic high-polyploid species (2n = 72 and above) form a clade with relationships best represented as a polytomy. The lack of genetic variation and resolution among the arctic species indicates a recent origin and rapid radiation (Scheen

(b) Including all polyploid species T canescens T greggii T mexicana T turneri T gossypina T tuberculata T durango T purpusii T latior T hispidissima T nuttallii T elongata T grandiflora T atacamensis T ferreyrae T dichotoma T litoralis T conspicua T tacnensis T palmeri T plicata T nesiotica T fusca T galapagoa T darwinii T paronychioides T cuspidata

Tiquilia paronychioides Tiquilia darwinii Tiquilia cuspidata Tiquilia plicata Tiquilia nuttallii Tiquilia conspicua Tiquilia elongata Tiquilia palmeri Tiquilia canescens Tiquilia greggii Tiquilia hispidissima Tiquilia latior Tiquilia purpusii Tiquilia gossypina Tiquilia mexicana Tiquilia tuberculata Tiquilia turneri Tiquilia durango

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Fig. 2 Phylogenies showing inferred ploidy (blue, diploid; red, polyploid) for Tiquilia. (a) Based on the supplemental data in Mayrose et al. (2011). (b) Based on the tree using ITS and rps16 data from the original paper (Moore et al., 2006), including all of the polyploid taxa for which sequence data were given. (c) Posterior probability distributions of the differences in speciation, extinction and diversification rates (rate of diploid minus rate of polyploid) inferred with BiSSE using the coding in (b). New Phytologist (2014) www.newphytologist.com

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et al., 2004). However, Mayrose et al. (2011) left the chromosome count for C. lithospermifolium as unknown when they ran ChromEvol to infer ploidy. Thus, 2n = 36 was considered the lowest number in Cerastium, and taxa with 2n = 36 were scored as diploid, resulting in a percentage of polyploids in the genus of 42% (Fig. 3). However, C. lithospermifolium is listed in Scheen et al. (2004) as 2n = 18. It is unclear why the known value of 2n = 18 was left as unknown in Mayrose et al. (2011). When ploidy is reconstructed with ChromEvol including the count for C. lithospermifolium, the actual percentage of polyploidy in Cerastium is c. 97% (Fig. 3). When BiSSE is run with the corrected data, instead of diversification being higher in diploids in 88% of the MCMC steps as reported by Mayrose et al. (2011), it is only higher in 76% of the MCMC steps, an odd result, given the distribution of ploidy, that clearly demonstrates the limits of BiSSE for these datasets. (a) Mayrose et al.

The results for Cerastium are a powerful demonstration of the lack of significance in the results reported by Mayrose et al. (2011). Superficially, 76% of the steps indicating higher diversification in diploids sounds like a strong conclusion in favor of the Mayrose et al. (2011) position that diversification is higher in diploids. However, we challenge the reader to look at the tree (Fig. 3) and conclude that this topology supports higher diversification of diploids. The results of the BiSSE analysis actually indicate that there is great uncertainty in many of the estimated parameters, and there is no significant difference in diversification rates between diploids and polyploids. This is a conclusion that Mayrose himself made when shown these results (I. Mayrose, pers. comm.). As discussed earlier, the power to find significance is quite low in BiSSE analyses with 36 terminals, as in Cerastium (and most of the other genera studied), and there is no significant difference. (b) Including chromosome count for C. lithospermifolium Cerastium danguyi Cerastium nutans Cerastium pauciflorum Cerastium hemschinicum Cerastium lithospermifolium n = 9 Cerastium falcatum Cerastium kasbek Cerastium multiflorum CERASTIUM PURPURASCENS Cerastium argenteum Cerastium sosnowskyi Cerastium gnaphalodes Cerastium banaticum Cerastium uniflorum Cerastium eriophorum Cerastium latifolium Cerastium pumilum CERASTIUM DIFFUSUM Cerastium semidecandrum CERASTIUM VELUTINUM CERASTIUM TOMENTOSUM CERASTIUM BEERINGIANUM BEERINGIANUM Cerastium biebersteinii CERASTIUM ARVENSE CERASTIUM ALPINUM CERASTIUM NIGRESCENS CERASTIUM REGELII CERASTIUM ARCTICUM CERASTIUM FONTANUM VULGARE Cerastium tianschanicum Cerastium szechuense Cerastium fischerianum Cerastium pusillum CERASTIUM BRACHYPETALUM Cerastium dubium Cerastium cerastoides

Cerastium danguyi Cerastium nutans Cerastium pauciflorum Cerastium hemschinicum Cerastium lithospermifolium n = ? Cerastium falcatum Cerastium kasbek Cerastium multiflorum Cerastium purpurascens Cerastium argenteum Cerastium sosnowskyi Cerastium gnaphalodes Cerastium banaticum Cerastium uniflorum Cerastium eriophorum Cerastium latifolium Cerastium pumilum Cerastium diffusum Cerastium semidecandrum Cerastium velutinum Cerastium tomentosum Cerastium beeringianum beeringianum Cerastium biebersteinii Cerastium arvense Cerastium alpinum Cerastium nigrescens Cerastium regelii Cerastium arcticum Cerastium fontanum vulgare Cerastium tianschanicum Cerastium szechuense Cerastium fischerianum Cerastium pusillum Cerastium brachypetalum Cerastium dubium Cerastium cerastoides

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Fig. 3 Phylogenies showing inferred ploidy (blue, diploid; red, polyploid) for Cerastium. (a) Based on the supplemental data in Mayrose et al. (2011). (b) Based on the same tree, but adding the published chromosome count for C. lithospermifolium of n = 9, as presented in Scheen et al. (2004). (c) Posterior probability distributions of the differences in speciation, extinction and diversification rates (rate of diploid minus rate of polyploid) inferred with BiSSE using the coding in (b). Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Changes in topology Mayrose et al. (2011) re-analyzed the original data of other investigators, which in some cases resulted in topological differences compared to the original publications, most likely due to the elimination of taxa. We noted at least eight instances where this occurred (Centaurium, Cucumis, Erodium, Fuchsia, Geum, Lathyrus, Mimulus, and Senecio). In Centaurium, the relationships among major clades are incorrectly depicted in Mayrose et al. (2011). In Fuchsia the position of the polyploids differs in the tree given in Mayrose et al. (2011) from the original tree of Berry et al. (2004). The differences in topology are likely the result of the reanalyses conducted by Mayrose et al. (2011) – different methods were sometimes used compared to the original papers (e.g. maximum likelihood vs maximum parsimony). Although most of the topological differences between the original phylogenies and those of Mayrose et al. (2011) are minor, the use of different topologies from those used by the original authors improperly assumes that the analyses of Mayrose et al. (2011) are superior to those of the original authors. One small step In several examples from Mayrose et al. (2011), the interpretation of diploid vs polyploid is dramatically altered by including just one more branch on a phylogenetic tree – that is, considering the sister group to the genus analyzed completely alters the results. Although Mayrose et al. (2011) typically restricted their analyses to ‘genera’, some datasets ‘focused on sections, subgenera, or a cluster of closely related genera’ (Mayrose et al., 2011 supporting online material p. 1). The artificial nature of genera as a basis for investigating the evolution of polyploidy is not addressed, nor the reason for exceptions. Mayrose et al. (2011) considered the Macaronesian ‘GAMA’ clade of Greenovia, Aeonium, Monanthes, and Aichryson (Crassulaceae) to represent primarily diploids (with 2n = 36), with just four polyploids (2n = 58; Fig. 4). Here Mayrose et al. (2011) did not limit their analyses to individual genera, but used a clade of four genera. However, Sedum modestum and S. jaccardianum are sister to the GAMA clade, and their inclusion dramatically changes the interpretation (Fig. 4). These two species of Sedum share 2n = 16, indicating that the core of the GAMA clade is polyploid, apparently derived from diploid ancestors with x = 8 (Mort et al., 2001). Including these Sedum species and rerunning ChomEvol indicates that the entire GAMA clade is actually polyploid, has diversified at the polyploid level, and is now undergoing a new round of polyploidization (the four taxa with 2n = 58) (Fig. 4). When BiSSE is run on the dataset, the diversification results go from 96% of MCMC steps being higher in diploids (one of the few significant results in Mayrose et al. (2011)) to only 6% – nearly significant in the opposite direction – with polyploids having higher diversification in 94% of the MCMC steps. Actinidia (Actinidiaceae) is another genus included in Mayrose et al. (2011) whose ploidal level needs to be reconsidered in the context of one more branch of a phylogenetic tree. The sister genera to Actinidia, Saurauia and Clematoclethra, have base numbers of New Phytologist (2014) www.newphytologist.com

x = 13 and 12 (2n = 26, 24), respectively. Actinidia has x = 29 (2n = 58), suggesting a polyploid event and a subsequent aneuploid event shared by all species of the genus (He et al., 2005). Gene duplication data support this polyploid event in the evolutionary history of Actinidia (Shi et al., 2010). Hence, all species of Actinidia are polyploid, but this is not the interpretation of Mayrose et al. (2011). Many species in Actinidia have undergone secondary polyploidy, producing tetraploids (2n = 4x = 116) in 11 species, pentaploids (2n = 5x = 174) in three species, and one (2n = 8x = 232) octoploid (Yan et al., 1997). In addition, many species of Actinidia have multiple ploidal levels, which Mayrose et al. (2011) did not take into account. There are 62 species in Actinidia, 30 with chromosome counts, 19 of which have 2n = 58; all others have higher ploidal levels. Genera differ in age Mayrose et al. (2011) assume that all genera are roughly the same age, which is far from correct. Even the data of Mayrose et al. (2011) indicate a several-fold difference in age among genera included. This problem is confounded by the arbitrary nature of ‘genera’, which are shaped as much (or more) by tradition as biology. Our own analyses using a relaxed molecular clock approach suggest an order of magnitude difference in age among the genera that Mayrose et al. (2011) employed. Older genera have more time to spawn polyploids than very young lineages; hence, including ‘young’ genera while excluding ‘old’ genera will bias the outcome. Some angiosperm genera are very old. Magnolia, not included in Mayrose et al. (2011), has a long fossil history. The oldest Magnolia fossils are c. 65 million yr old (Knobloch & Mai, 1986; Azuma et al., 2001, p. 228); other Magnolia fossils are 47– 50 million yr old (Reid & Chandler, 1933; Manchester, 1994). Other old genera include Cercidiphyllum (65–71 million yr; Magall
on et al., 1999), Altingia (88.5–90.4 million yr; Zhou et al., 2001), and Platanus (c. 110 million yr; Friis et al., 1988; Crane et al., 1993). These ages are older than the estimated ages of many families in Asteridae and Rosidae (see Bell et al., 2010) and large, species-rich families such as Cactaceae (c. 35 million yr; Arakaki et al., 2011), Crassulaceae (36–59 million yr), Solanaceae (29–47 million yr), Asteraceae (31–48 million yr), and Boraginaceae (39–68 million yr) (Bell et al., 2010), families for which generic examples are given in Mayrose et al. (2011). By contrast, the estimated ages of several Mediterranean genera are much younger than the age of Magnolia: Tragopogon 1.7–5.4 million yr (Bell et al., 2012), Dianthus 1.2–7.0 million yr (Valente et al., 2010), Antirrhinum c. 4.1million yr (Vargas et al., 2009), and Cistus 4.25 million yr (Fern
andez-Mazuecos & Vargas, 2010). Hence, there is a 10-fold or more difference in age among angiosperm genera. Old genera such as Magnolia, for which diploids are now extinct, reveal details of the polyploid process that have important implications for assessing the number of ‘polyploids’ in a genus. Masterson (1994), comparing leaf guard cell size in fossil and extant taxa from several angiosperm families to estimate polyploid occurrence through time, concluded that Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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(a) Mayrose et al.

(b) Using Sedum as outgroup to infer ploidy Aichryson laxum Aichryson palmense Aichryson porphyrogennetos Aichryson pachycaulon Aichryson punctatum Aichryson tortuosum Monanthes icterica Aeonium pseudourbicum Aeonium hierrense Aeonium gomerense Aeonium haworthii Aeonium urbicum Aeonium nobile Aeonium decorum Aeonium castello-paivae Aeonium davidbramwellii Aeonium spathulatum Aeonium ciliatum Aeonium glandulosum Aeonium glutinosum Aeonium subplanum Aeonium palmense Aeonium canariense Aeonium virgineum Aeonium tabuliforme Aeonium rubrolineatum Aeonium balsamiferum Aeonium leucoblepharum Aeonium lancerottense Aeonium mascaense Aeonium volkeri Aeonium undulatum Aeonium simsii Aeonium percarneum Aeonium vestitum Aeonium holochrysum Aeonium korneliuslemsii Aeonium gorgoneum Greenovia aurea Greenovia aizoin Aeonium viscatum Greenovia diplocycla Aeonium saundersii Aeonium lindleyi Aeonium goochiae Monanthes subcrassicaulis Monanthes amydros Monanthes brachycaulon Monanthes laxiflora Monanthes adenoscepes Monanthes minima Monanthes anagensis

Aenoium gorgonium Aeonium korneliuoslemsii Aeonium holocryson Aeonium vestitum Aeonium holochrysum Aeonium simsii AEONIUM BALSAMIFERUM Aeonium rubrolineatum Aeonium leucoblepharum AEONIUM UNDULATUM Aeonium mascaense Aeonium lanceroftense Aeonium percameum Aeonium percameum Aeonium glutinosum Aeonium glandulosum Aeonium tabuliforme Aeonium nobile Aeonium davidbramwellii Aeonium ciliatum Aeonium spathulatum Aeonium pseudourbicum Aeonium pseudourbicum AEONIUM HAWORTHII Aeonium urbicum Aeonium decorum Aeonium castello Aeonium ciliatum Aeonium gomerense Aeonium hierrense Aeonium canariense Aeonium lindleyi Aeonium goochiae Aeonium saundersii Greenovia diplocycla Greenovia aizoin Greenovia aureum Aeonium viscatum Greenovia aureum Aeonioum subplanum Aeonioum virgineum Monanthes brachycaulon Monanthes laxiflora Monanthes adenoscepes Monanthes minima MONANTHES ANAGENSIS Monanthes subcrassicaulis Monanthes amydros Aichryson tortuosum Aichryson laxum Aichryson palmense Aichryson punctatum Aichryson porphyrogennetos Aichryson pachycaulon

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Fig. 4 Phylogenies showing inferred ploidy (blue, diploid; red, polyploid) for the GAMA clade. (a) Based on the supplemental data in Mayrose et al. (2011). (b) With ploidy inferred including the immediate sister clade, Sedum, as the outgroup for the ChromEvol analysis. Also indicated by taxon names in ALL CAPS are higher-level polyploids that have undergone an additional round of polyploidization (see Mort et al., 2001). (c) Posterior probability distributions of the differences in speciation, extinction and diversification rates (rate of diploid minus rate of polyploid) inferred with BiSSE using the coding in (b).

now-extinct members of those groups, including species of Magnolia, were diploid. These diploid Magnolias left a legacy of extant ‘polyploids’ with 2n = 38. Genetic data confirm that living species with 2n = 38 are polyploids (Soltis & Soltis, 1990; Cui et al., 2006). Extant species have given rise to additional higherlevel polyploids with 2n = 76. However, if Magnolia had been included in Mayrose et al. (2011), and the phylogeny of Kim et al. (2001) employed, the genus would have been incorrectly considered primarily diploid, giving rise to a few polyploids. A recurring pattern is that, in many old genera, the diploids are now extinct, leaving a legacy of surviving polyploids (e.g. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

Aesculus (n = 20), Cercidiphyllum (n = 19), Trochodendron (n = 19), and Calycanthus (n = 11, 12)). Again, following Mayrose et al. (2011), all of these old lineages would be considered diploid, incorrectly introducing a bias against diversification at the polyploid level. Conclusions We agree with the long-espoused view that most polyploid entities likely go extinct shortly after formation, at the scale of small populations, before becoming established as evolutionarily New Phytologist (2014) www.newphytologist.com

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significant entities or receiving taxonomic recognition. In that sense, polyploids are often ‘dead-ends’. But Mayrose et al. (2011) and Arrigo & Barker (2012) address a timescale different from this initial phase of polyploidization. For multiple reasons reviewed here, we maintain that it is premature to conclude that polyploid species undergo higher extinction rates than diploid species. There are important philosophical, statistical, analytical, sampling, and methodological issues with their approach to the problem. While we fundamentally disagree with their approach – which forces the products of reticulate evolution into bifurcating trees – we note that even if one accepts this framework, their analyses suffer from multiple errors and shortcomings. The main conclusion is based on a faulty statistical comparison, and the correct statistical tests do not generally support their conclusion. The methods employed have little power to find significant differences on datasets of the size used, so it is not surprising that few significant differences were found. We have highlighted a number of data-mining errors and possible biases, the effects of which have not been explored. In most cases, these errors and biases led to trees that are more ‘diploid’ than they really are. Other errors in Mayrose et al. (2011) include incorrect rooting and incorrect or incomplete chromosome numbers. We also argue that the sampling of Mayrose et al. (2011) is not representative of angiosperms as a whole for the simple reason that the most taxonomically and cytologically complex genera have not been investigated. Even perusing the published literature reveals case studies that would argue for, not against, the success of polyploids compared to diploid congeners: Nicotiana, Cerastium, Viola, Gossypium, Triticum, Opuntia, and Draba. To be representative, more of these complexes need to be included in analyses that try to ascertain polyploid vs diploid success and extinction. In addition, the assumption of Mayrose et al. (2011) that all genera are roughly the same age is also highly problematic. On a broader level, a major question remains: how many polyploids are present but not yet recognized due to poor understanding of a group? Many polyploids, in particular autopolyploids or segmental polyploids, are not even named, and these may represent a substantial component of biodiversity. All of these issues make it extremely difficult to conduct meaningful analyses and reach the sweeping conclusion that polyploids undergo higher extinction rates than diploids. Perhaps polyploidy can be viewed as analogous to a baseball slugger like Bonds, Aaron, or Ruth – polyploidy strikes out a lot, but when it connects, the result is frequently a home run. However, in our view, what remains unclear is whether established polyploids actually strike out more than diploids.

Acknowledgements This project is the result of a systematics paper discussion group, and other participants in that group who are not coauthors are acknowledged: Xinshuai Qi, Xiaoxian Liu, and Nicol
as Garc
ıa. Our research on polyploid angiosperms has been supported in part by NSF grants MCB-0346437, DEB-0614421, DEB-0919254, DEB-0922003, and DEB-1146065. The authors thank Itay Mayrose in particular, as well as Sally Otto, Mike Barker, Brian New Phytologist (2014) www.newphytologist.com

O’Meara, Rafa Rubio de Casas, and Rich FitzJohn, for helpful input and discussions. Douglas E. Soltis1,2,3*, Mar
ıa Claudia Segovia-Salcedo1,4, Ingrid Jordon-Thaden1,5, Lucas Majure1, Nicolas M. Miles1, Evgeny V. Mavrodiev2, Wenbin Mei3, Mar
ıa Beatriz Cortez1, Pamela S. Soltis2,3 and Matthew A. Gitzendanner1,3 1

Department of Biology, University of Florida, Gainesville, FL 32611, USA; 2 Florida Museum of Natural History, University of Florida, Gainesville, FL 32611, USA; 3 Genetics Institute, University of Florida, Gainesville, FL 32610, USA; 4 Grupo de Investigacı´on Conservacio´n de Bosques de Polylepis, Departamento de Ciencias de la Vida y de la Agricultura, Universidad de la Fuerzas Armadas - ESPE, Sangolquı´, Ecuador; 5 University and Jepson Herbaria, University of CaliforniaBerkeley, Berkeley, CA 94720, USA (*Author for correspondence: tel +1 352 273 1963; email [email protected])

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Supporting Information Additional supporting information may be found in the online version of this article. Tables S1 Polyploid complexes analyzed phylogenetically, but not included in Mayrose et al. (2011) Table S2 Other polyploid complexes in need of investigation Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Key words: diversification, evolutionary dead end, extinction, genome doubling, polyploidy.

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