The Evolution of Colchicaceae, with a Focus on Chromosome Numbers Author(S): Juliana Chacón, Natalie Cusimano, and Susanne S

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The Evolution of Colchicaceae, with a Focus on Chromosome Numbers Author(s): Juliana Chacón, Natalie Cusimano, and Susanne S. Renner Source: Systematic Botany, 39(2):415-427. 2014. Published By: The American Society of Plant Taxonomists URL: http://www.bioone.org/doi/full/10.1600/036364414X680852

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BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. Systematic Botany (2014), 39(2): pp. 415–427 © Copyright 2014 by the American Society of Plant Taxonomists DOI 10.1600/036364414X680852 Date of publication 04/23/2014 The Evolution of Colchicaceae, with a Focus on Chromosome Numbers

Juliana Chaco´n,1,2 Natalie Cusimano,1 and Susanne S. Renner1

1Department of Biology, University of Munich, 80638 Munich, Germany. 2Author for correspondence: ([email protected])

Communicating Editor: Mark P. Simmons

Abstract—The lily family Colchicaceae consists of geophytic herbs distributed on all continents except the Neotropics. It is particularly diverse in southern Africa, where 80 of the 270 species occur. Colchicaceae exhibit a wide range of ploidy levels, from 2n =14to2n = 216. To understand where and how this cytogenetic diversity arose, we generated multilocus phylogenies of the Colchicaceae and the Colchicum clade that respectively included 85 or 137 species plus relevant outgroups. To infer the kinds of events that could explain the observed numbers in the living species (dysploidy, polyploidization, or demi-duplication, i.e. fusion of gametes of different ploidy), we compared a series of likelihood models on phylograms, penalized likelihood ultrametric trees, and relaxed clock chronograms that con- tained the 58 or 112 species with published chromosome counts. While such models involve simplification and cannot address the processes behind chromosomal rearrangements, they can help frame questions about the direction of change in chromosome numbers in well-sampled groups. The results suggest that dysploidy played a large role in the Colchicaceae, with the exception of Colchicum itself for which we inferred frequent demi-duplication. While it is known that triploids facilitate the fixation of tetraploidy and that plant species often include individuals of odd ploidy level (triploids, pentaploids), we hesitate to accept the phylogenetically inferred scenario without molecular-cytogenetic work and data from experimental hybridizations. Keywords—African Colchicaceae, ancestral chromosome number, maximum likelihood inference, polyploidy.

With some 270 species in 15 genera, the Colchicaceae A striking feature of the Colchicaceae is their high karyo- are the third largest family of the Liliales, after the Liliaceae logical variation (Table 1), with chromosome numbers and Smilacaceae. They occur in Africa, Asia, Australasia, ranging from 2n =14(e.g.Uvularia grandiflora;Therman North America and Europe, but not in South or Central and Denniston 1984) to 2n =216(inColchicum corsicum; America (Vinnersten and Manning 2007). Their closest rela- Persson 2009). Such variation contrasts with the sister tives are the Alstroemeriaceae, which have most of their family, Alstroemeriaceae, in which the chromosome num- species in South America (Chaco´n et al. 2012a). Together, bers vary between 2n =16and2n =20(Chaco´netal. the two form the sister clade to the Petermanniaceae, a 2012b: 44 of the 200 species of Alstroemeriaceae have monospecific family restricted to tropical Australia (Vinnersten been counted). The cytogenetics of Colchicum is espe- and Reeves 2003; Fay et al. 2006). All Colchicaceae contain cially complex, with different species having variable colchicine, an alkaloid traditionally used in the treatment chromosome numbers as well as ploidy levels (from tetra- of gout, and also in cytogenetics for its properties as a to 24-ploid; Persson et al. 2011), perhaps related with the microtubule polymerization inhibitor (Vinnersten and Larsson presence of colchicine (Nordenstam 1998). The effect of 2010). Ecologically, Colchicaceae are long-lived cormose or colchicine on the separation of chromosomes after the rhizomatous geophytes with rather large, animal-pollinated anaphase of mitosis was discovered by B. Pernice (1889) flowers offering nectar at the base of their tepals (Nordenstam and described more fully by Eigsti et al. (1945); it revolu- 1998). African Colchicaceae in the Namaqualand desert often tionized cytogenetics because it permitted experimental have leaves with helical shapes and hairy margins that serve doubling of the entire complement of a cell’s chromosome set. to harvest water from dew and fog, which then drips to the Besides by polyploidy, chromosome numbers can change soil and reaches the root zone where it is ultimately stored through chromosome fission (ascending dysploidy) or chro- in the corms (Vogel and Mu¨ller-Doblies 2011). mosome fusion (descending dysploidy; Schubert and Colchicaceae have been the subject of several molecular- Lysak 2011). Polyploidization can represent an evolu- phylogenetic studies that have clarified relationships and tionary dead end (Mayrose et al. 2011), but there is also circumscriptions of the Australian/African genus Wurmbea, abundant evidence of the adaptive success of polyploid the Mediterranean/Irano-Turanian species of the genus populations and the contribution of polyploidy to the for- Colchicum (the latter extending east to Afganistan and mation of new species (Levin 1983; Abbott et al. 2013; Kyrgyzstan; Persson 2007), and the genus Gloriosa, with Weiss-Schneeweiss et al. 2013 and references therein). By 10 species in Africa, India, and southeastern Asia (Vinnersten contrast, dysploidy is thought to arise accidentally, and and Reeves 2003; Vinnersten and Manning 2007). A we know of no proposed adaptive reason for its pre- redefinition of Colchicum to include all ca. 60 species of ponderance in certain clades. Knowing the distribution Androcymbium (distributed in southern and northern Africa of polyploidy and dysploidy in a particular clade or geo- as well as the Mediterranean) was proposed by Manning graphic region can help set up testable hypotheses about et al. (2007) and Persson (2007), while Del Hoyo and evolutionary pathways, for example about the likelihood Pedrola-Monfort (2008) and Del Hoyo et al. (2009) pre- that hybridization played a large role in the recent past. ferred to keep Androcymbium and Colchicum separate. The Here we investigate chromosome number evolution in most comprehensive analysis is that of Thi et al. (2013), the Colchicaceae using the likelihood approach of Mayrose who used patchy matrices of 3 or 6 combined plastid regions et al. (2010), which models the change rates and relative from 70 species, representing most genera, to infer sub- frequencies of several kinds of past events that could plau- family and tribal relationships. sibly explain the observed haploid chromosome numbers 415 416 SYSTEMATIC BOTANY [Volume 39

Table 1. Chromosome numbers available for the Colchicaceae genera (see details of the species and references in the Table S1)

Genus No. of species No. of species counted Chromosome number n2n Baeometra Salisb. ex Endl. 1 1 22 Burchardia R. Br. 6 5 48 24 Camptorrhiza Hutch. 2 1 22 Colchicum L. ca. 157 97 14, 18, 20, 21, 22, 24, 27, 32, 40, 42–44, 36, 38, 46, 48, 50, 52, 54, 58, 90, 92, 94, 96, 102, 106, 108, ca. 110, ca. 120, 140, 146, 182, ca. 216 Disporum Salisb. ex G. Don 20 11 14, 16, 18, 30, 32 Gloriosa L. 10 7 20, 21, 22, 44, 66, 88 Hexacyrtis Dinter 1 1 22 Iphigenia Kunth 12 6 11 22 Kuntheria Conran & Clifford 1 1 14 Ornithoglossum Salisb. 8 4 24 Sandersonia Hook. 1 1 24 Shelhammera R. Br. 2 2 14, 36 Tripladenia D. Don 1 1 14 Uvularia L. 5 3 7 14 Wurmbea Thunb. ca. 50 3 14, 20, 40 in a group. The approach requires either a phylogram, that with penalized likelihood (below), and a chronogram obtained under a is, a tree in which branch lengths are proportional to num- relaxed clock model (below) to reconstruct the evolution of chromosome numbers. The first data set included 85 species of Colchicaceae (from bers of DNA substitutions, or an ultrametric tree in which all 15 genera) plus nine outgroups (representing the Alstroemeriaceae, branch lengths are proportional to time. Such ultrametric Petermanniaceae, Ripogonaceae, and Philesiaceae), each sequenced for trees can come from strict clock models or relaxed clock five plastid regions (matK, ndhF, rbcL, rps16, and trnL-F), one mito- models, and they can also be calibrated (typically in mil- chondrial gene (matR), and the internal transcribed spacer of nuclear lion years), in which case the tree is called a time-tree or ribosomal DNA (ITS). Species authors, geographic origin, herbarium voucher specimen, and GenBank accession numbers are listed in Chacoń chronogram. The kinds of past events that are modeled are and Renner (2014). The second data set included 187 accessions of duplication of the entire chromosome complement, descend- Colchicum representing 137 species, 96 of them traditionally placed in ing dysploidy, ascending dysploidy, and demi-duplication Colchicum and 41 transferred there from Androcymbium by Manning (the formation of polyploids via the fusion of gametes of et al. (2007) plus two outgroups (Hexacyrtis dickiana and Ornithoglossum vulgare), each sequenced for trnL intron, trnL-trnF intergenic spacer different ploidy, leading to triploidy or pentaploidy). The (IGS), trnY-trnD IGS, trnH-psbA IGS, atpB-rbcL IGS, and rps16 intron. method was tested using simulated and empirical datasets Sequences came from the studies of Del Hoyo et al. (2009), Vinnersten in the original work by Mayrose and colleagues, and has and Reeves (2003), and Persson et al. (2011). The number of Colchicum so far been used in seven studies (Mayrose et al. 2011: across species in the two matrices are thus different because the Colchicaceae 63 plant groups; Ness et al. 2011: Pontederiaceae; Cusimano matrix consisted of plastid, mitochondrial, and nuclear DNA sequences, while the Colchicum matrix consisted only of plastid sequences from etal.2012:Araceae;OcampoandColumbus2012:Portulaca; different genes (see above); we decided not to combine them in order Harpke et al. 2013: Crocus; Metzgar et al. 2013: fern genus to minimize the number of empty cells in the alignment. Instead we ran Cryptogramma; Sousa et al. 2014). Based on these studies, it separate analyses for each matrix. does not appear to be biased towards inferring predomi- Sequences were concatenated and aligned with MAFFT v. 7 (Katoh and Standley 2013) using the L-INS-i algorithm (Katoh et al. 2005), fol- nantly polyploidy, chromosome losses or gains, or demi- lowed by manual adjustment in the program MacClade v. 4.8 (Maddison polyploidy. Mayrose et al. (2010) also demonstrated that and Maddison 2002) based on the similarity criterion of Simmons (2004). the accuracy of the method depends on the number of taxa The resulting alignments were used for phylogenetic tree reconstruction and on branch length (because the approach takes long under maximum likelihood (ML; Felsenstein 1973) using RAxML v. 7.0.4 branches as meaning much time for change). The effects of (Stamatakis 2006) through the CIPRES Science Gateway (Miller et al. 2010). The substitution model used was the GTR + G model, this tree branch lengths indeed can be dramatic, an issue we being the best-fitting model identified by the Akaike Information Cri- take up in the Discussion (Cusimano and Renner, in review). terion (AIC; Akaike 1974) in FindModel (http://hcv.lanl.gov/content/ We here use almost 140 published chromosome counts sequence/findmodel/ findmodel.html). Statistical support for nodes was for Colchicaceae species (52% of their 270 total spe- assessed by 1,000 ML bootstrap replicates (Felsenstein 1985) under the cies), a modified phylogeny of the family from Chacoń same model. The maximum likelihood phylogram from each matrix was trans- and Renner (2014), and a newly compiled phylogeny of formed into an ultrametric tree with the R function “chronopl” of Colchicum with 187 accessions representing 137 species the APE package v. 3.0–6 (Paradis et al. 2004), which implements the to infer the chromosomal history of the family. Our main penalized likelihood (PL) method of Sanderson (2002), including appro- questions were: (i) are there predominant modes of chro- priate cross-validation to find the best smoothing parameter. As an mosome number change in the family’s different clades, alternative to the PL approach, we also used two time-calibrated trees obtained with the Bayesian program BEAST v. 1.7.5 (Drummond et al. and (ii) can changes in chromosome number plausibly be 2012), in which we opted for a relaxed clock model with uncorrelated related to coincidental arrival in a new region or habitat log-normal (UCLN) rate variation, meaning that ancestors and descen- type where a single polyploid or dysploid ancestor might dants are allowed to have rather more different substitution rates than then have radiated? is the case in the PL approach. The substitution model for both matrices was again the GTR + G model, and the tree prior was a Yule process. The length of the Monte Carlo Markov Chain (MCMC) was set to Materials and Methods 90 million generations with parameters sampled every 1000 generations and a burnin of 10%. Following Chacoń et al. (2012a) we applied four Taxon Sampling and Phylogenetic Analyses—We used two data sets calibration points, three of them from fossils and one a secondary cali- and from each inferred a phylogram (below), an ultrametric tree obtained bration from another study. 2014] CHACO´ N ET AL.: COLCHICACEAE CHROMOSOME EVOLUTION 417

The alignments and phylogenetic trees obtained in this study have mum number to 1. In the runs conducted on the Colchicum trees, we been deposited in TreeBASE (study accession No. 14230). fixed the haploid chromosome number at the Colchicum+outgroups Inference of Chromosome-Number Change—The chromosome num- root node to a = 11 with a probability of 1 because this was the bers for 144 species of Colchicaceae and eight outgroup taxa were number inferred for this node with the highest posterior probability obtained from the Index to Plant Chromosome Numbers (http://www in the family-wide analysis using the phylogram. To assess the influ- .tropicos.org/Project/IPCN; October 2012) and other literature (Table S1 ence of outgroup taxa on the estimates, we compared results with or in the supplemental online data; this includes all Colchicaceae and without outgroups (with their corresponding chromosome counts). outgroup species with published chromosome numbers). Chromosome numbers were available for 58 of the 85 species included in the family trees and for 112 of the species included in the Colchicum trees. Only Results accessions with a herbarium voucher were included. When conspecific accessions grouped together and the chromosome number of the Molecular Phylogeny of Colchicaceae—The combined respective species was stable, then only one accession was included. plastid, mitochondrial and nuclear data (6,451 aligned nucleo- When conspecific accessions did not group together, we checked if published counts were associated with the respective accessions. If that tides) yielded a robust phylogeny for the 85 Colchicaceae was the case we included these accessions, otherwise all accessions of species with most clades having >80% bootstrap support that species were excluded. (Fig. 1). The six species of Burchardia,analyzedtogetherfor For maximum likelihood and Bayesian phylogenetic inferences of the first time here, form a clade that is sister to all other ancestral haploid chromosome numbers we relied on ChromEvol v. 1.3 (Mayrose et al. 2010; http://www.tau.ac.il/itaymay/cp/chromEvol/ Colchicaceae. The monotypic genus Kuntheria forms a clade index.html) with an extension provided by I. Mayrose (Tel Aviv Uni- with Schelhammera undulata and Tripladenia cunninghamii. versity; pers. comm., 29 January, 2013) that allows fixing the root node Reconstruction of Ancestral Chromosome Numbers in number. ChromEvol implements eight models of chromosome-number Colchicaceae—Minor differences were found between ana- change, which include the following six parameters: polyploidization lyses that included outgroups and those that did not, (chromosome number duplication) with rate r, demi-duplication (fusion of gametes of different ploidy) with rate m, and dysploidization (ascend- besides the trivial difference that trees with outgroups ing: chromosome gain rate l; descending: chromosome loss rate d)as include more branches and therefore lead to higher num- l d well as two linear rate parameters, 1 and 1, for the dysploidization bers of overall events (Fig. 2 A–C without outgroups vs. l d rates and , allowing them to vary based on the current number of Fig. S1 with outgroups; see supplemental online data). The chromosomes. Four of the models have constant rates, whereas the other four include two linear rate parameters. Both model sets also ancestral chromosome number inferred for the crown have a null model that assumes no duplication events. We first fit all node of the Colchicaceae was a = 7 (Fig. 2A–C) except on models to the data without performing simulations to infer the best-fit the phylogram and chronogram with outgroups (a = 8 and model. For the best-fit model we reran the analysis fixing the parame- a = 6; Fig. S1). The chronograms with or without out- ters to those optimized in the first run and using 10,000 simulations groups also differed in six chromosome gains inferred on the to compute the expected number of changes along each branch as well as the ancestral haploid chromosome numbers at nodes. The null following nodes only when the outgroups were included: hypothesis (no polyploidy) was tested using an AIC test. crown of Camptorrhiza/Iphigenia,crownofGloriosa,crownof Ancestral haploid chromosome numbers, which we refer to as a, Tripladenia/Schelhammera/Kuntheria,crownofUvularia/Disporum, were inferred on the two ML phylograms, the two PL ultrametric trees, crown and stem of Wurmbea; see Fig. S1). As the pattern and the two BEAST chronograms. Species for which no chromosome- number information was available were cut from the trees, resulting of chromosome-number change appears to be quite differ- in 58 species in the Colchicaceae tree (instead of 85) and 112 species ent between the ingroup and outgroup, we focused on (126 accessions) instead of 137 (187 accessions of Colchicum and two out- inferences from trees that included only the ingroup. groups) in the Colchicum tree. Before running ChromEvol the branch Results with Input Trees Differing in Branch Length—The lengths of some trees were adjusted (Table 2) because the root-to-tip model that best fit all three types of trees (phylogram, PL distance was large, which can cause ChromEvol to overestimate the number of transitions. Using artificial data, Mayrose et al. (2010) showed ultrametric tree, BEAST UCLN chronogram) was the con- that reliable reconstructions are obtained with root-to-tip distances rang- stant rates model with the duplication rate equal to the ing from 0.1–0.8. We therefore adjusted branch lengths such that the demi-duplication rate (Table 2). However, dissimilar ances- total tree lengths were between 0.1 and 0.2 (Table 2). We ran addi- tral haploid numbers where inferred on the three types tional analyses with double or half these tree lengths to test if the results would differ substantially; this was not the case. of trees, specially at nodes along the tree backbones, while The maximum haploid number of chromosomes was set to ten more numbers inferred for the crown nodes of most genera and than the highest empirical number (i.e. 108 + 10 = 118), and the mini- on internal nodes near the tips (i.e. near the present) were

Table 2. Results of the analyses carried out in ChromEvol to infer chromosome number changes in the Colchicaceae and the Colchicum clade using a phylogram (ML phyl), an ultrametric tree (PL ultra) or an chronogram from a BEAST analysis (B chrono). The factor with which the branches of the tree have been adjusted for the analysis and the resulting tree length are given. AIC scores; best model: crd = constant rate model with duplication rate equal to the demiduplication rate; crde = constant rate model with duplication rate different to the demi-duplication rate; rate parameters: l = chromosome gain rate, d = chromosome loss rate, r = duplication rate, m = demi-duplication rate; haploid chromosome number a inferred at the root node of the Colchicaceae family and the root node of Colchicum, respectively, under Bayesian optimization with the respective PP, and under maximum likelihood optimization (ML).

Rates Inferred chrom. No. at root a

Resulting Resulting Best Bayes: Bayes: second Tree Factor root-tip length total tree length model AIC ldrmbest a -PP best a -PP ML Colchicaceae ML phyl 1 0.1 0.79 crd 250.3 15.55 14.03 12.05 = r 7–0.68 8–0.25 7 PL ultra 0.5 0.2 2.53 crd 248.1 3.62 0.0 2.21 = r 7–0.98 – 7 B chrono 0.0015 0.13 1.55 crd 235.4 12.7 0.0 5.9 = r 7–0.77 6–0.21 7 Colchicum clade ML phyl 1 0.98 0.76 crde 739.4 45.83 39.45 23.98 48.01 10–0.79 11–0.1 10 PL ultra 1 0.1 4.53 crde 658.6 3.17 11.21 3.39 9.3 11–0.96 – 11 B chrono 3 0.11 2.32 crde 675.2 11.82 13.74 6.4 18.03 10–0.75 11–0.18 10 418 SYSTEMATIC BOTANY [Volume 39

Fig. 1. Maximum likelihood phylogeny for Colchicaceae based on the combined analysis of plastid, mitochondrial, and nuclear markers. The tree is rooted on the sister clade, Alstroemeriaceae, plus species of Petermanniaceae, Ripogonaceae, and Philesiaceae. Bootstrap support for each clade is indicated with the circles according to the values explained in the inset. 2014] CHACO´ N ET AL.: COLCHICACEAE CHROMOSOME EVOLUTION 419

Fig. 2. Chromosome-number reconstructions for the Colchicaceae. Numbers at the tips are the haploid chromosome numbers of species. Pie charts at nodes and tips represent the probabilities of the inferred haploid chromosome numbers; the color-coding of the chromosome numbers is explained in the inset. Numbers inside the pie charts are the chromosome numbers with the highest probability. Numbers above branches represent the expected number of the four possible events, i.e. gains, losses, duplications, and demi-duplications occurring along that branch inferred by simulation with an expectation >0.5. The color-coding of events, the sum of the single events, and the total number of events are explained in the insets. The black arrows indicate the crown group of the African Colchicaceae and the gray arrows the Mediterranean/Irano-Turanian Colchicum clade. A. Reconstruction inferred without outgroups on the UCLN relaxed clock chronogram obtained with BEAST. B. Reconstruction inferred without outgroups on the ultrametric tree obtained with penalized likelihood. C. Reconstruction inferred without outgroups on the phylogram. 420 SYSTEMATIC BOTANY [Volume 39

Fig. 2. Continued. 2014] CHACO´ N ET AL.: COLCHICACEAE CHROMOSOME EVOLUTION 421

Fig. 2. Continued. 422 SYSTEMATIC BOTANY [Volume 39 less affected by tree branch lengths (Fig. 2A–C). In gen- with two subclades (A and B in Fig. 3), one with 17 spe- eral, reconstructions on the UCLN chronogram and the cies (21 accessions) and one with 14 species (22 acces- PL ultrametric tree (Figs. 2A, B) are more similar to each sions), is sister to a clade containing the remaining other than either is to those on the phylogram (Fig. 2C). Colchicum species. While some of the nodes along the As mentioned before, a = 7 (with different probabilities) is backbone lack statistical support (bootstrap < 80%), the dis- inferred for the root, and this number is maintained along tribution of chromosome numbers (next section) matches the early branches of Colchicaceae (Fig. 2). Nevertheless, the topology (Figs. 3, 4A , B). on the phylogram a switch from a =7toa = 11 appears at Reconstruction of Ancestral Chromosome Numbers in the root node of the African Colchicaceae (indicated with Colchicum—For the Colchicum data set (126 accessions of an arrow in Fig. 2C), then changing to a =10 in the crown Colchicum representing 112 species with chromosome counts), node of Colchicum, while on the other two trees the ances- the best-fitting model of chromosome number evolution is tral number a = 7 changed to a = 9 on the root node of the constant rate model with different rates for duplication Colchicum, either through a gradual increase from a = 7–8 and demi-duplication events (Table 2). The reconstructions and 9 (Fig. 2A) or from a =7toa = 9 (Fig. 2B). In the made on the ultrametric tree and the phylogram are shown family-level analyses, ChromEvol assumed a = 9 as the in Fig. 4 (the latter being almost identical to the results ancestral number for the Mediterranean and Irano/Turanian obtained with the chronogram; see Fig. S2). Chromosome species of Colchicum on all trees (Fig. 2A–C; but see next numbers were constant among the species in ten clades section about Colchicum). (labeled A to J in Fig. 3), and in Fig. 4, we collapsed these In all trees, the ancestral haploid number inferred at the clades for easier visualization of the overall tree. The uncol- crown node of Burchardia was a = 24; other early derived lapsed trees are shown in Figs. S2–S5. On the phylogram, genera have numbers based on a =7(Uvularia, Kuntheria, the inferred ancestral number for Colchicum is a =10 Schelhammera,andTripladenia), with the exception of Disporum (PP = 0.79; Fig. 4A) while on the ultrametric tree it is with a = 8 in the phylogram and the chronogram (Figs. 2A, C). a = 11 (PP = 0.96; Fig. 4B). In the phylogram, there is a Other differences between inferences on the UCLN chrono- reduction from a = 10–9 on the branch leading to clade C gram (Fig. 2A), the PL ultrametric tree (Fig. 2B), and the (seeFigs.3,4A)followedbyanincreasetoa =12througha phylogram (Fig. 2C) concern the numbers for the Wurmbea demi-duplication (Fig. S5). On the ultrametric tree (Fig. 4B), crown and stem group, which in the UCLN and the PL ChromEvol instead assumes an increase from a = 11–12 trees are a = 7, while in the phylogram the crown numbers through a chromosome gain for the same clade C. is a = 10, the stem number a =11. To explain the empirical chromosome numbers in five As a consequence of the different most likely ancestral Colchicum clades composed exclusively of species with numbers, the frequencies of events with an expectation >0.5 n = 27 (clades C, D, G, H, J; Fig. 4), the program inferred inferred by simulation also differed among trees (Fig. 2). A duplications and demi-duplications on the phylogram (from summary is provided in Table 3; 26.2 events were inferred a = 9–27, from a = 12–27, and from a = 12–18 and to 27; on the phylogram while ca. 30 events were inferred on Figs. 4A, S4, S5), while on the ultrametric tree it inferred the PL and UCLN ultrametric trees. The best-fit model for only demi-duplications (from a =18–27,froma = 12–27, the Colchicaceae assumed more duplications than demi- and a = 12–18–27; Figs. 4B, S3). The most frequent of the duplications on the phylogram (8 vs. 5.9) while the opposite four possible types of events in the chronogram and the was assumed on the PL tree (7.3 vs. 9.1) and the UCLN tree phylogram is the demi-duplication, while in the ultrametric (6.4 vs. 8.7). Other inferred parameter values also differ (see tree, chromosome losses are the most frequent event, fol- Table 2), especially the number of dysploidy events, with lowed by demi-duplications (Table 3). chromosome gains and losses inferred on the phylogram with similar frequencies (5.8, 6.5; Table 3), but only chromo- Discussion some gains inferred on the PL and UCLN trees (13.5 and 15.6, respectively; Table 3). Chromosome Number Evolution in Colchicaceae—Maximum Molecular Phylogeny of Colchicum—Figure 3 shows a likelihood phylogenies for 85 species of Colchicaceae phylogeny for 187 accessions representing 137 species of (Fig. 1) or 137 species of Colchicum (some represented by Colchicum, rooted on the two outgroup taxa and with several accessions; Fig. 3) were here used to infer events maximum likelihood bootstrap values. A large clade of that could explain the observed range of haploid chro- species previously placed in Androcymbium (see Table S1) mosome numbers in this family (Figs. 2, 4). Genera first completely sampled in the present study are Burchardia, for which we included all its six species, and the monospecific Australian Kuntheria. The latter forms a clade with Table 3. Number of events inferred in ChromEvol by simulation with an expectation >0.5 for the Colchicaceae and the Colchicum data Schelhammera undulata, the type species of an Australian sets, using a phylogram (ML phyl), an ultrametric tree (PL ultra) or a genus that has two other species, and the monospecific chronogram from a BEAST analysis (B chrono). Australian Tripladenia (Fig. 1), all three with a chromosome number of 2n = 14 (Table S1) and an inferred haploid ances- Colchicaceae Colchicum tral number of a = 7 (Fig. 2). The six species of Burchardia Events ML phyl B chrono PL ultra ML phyl B chrono PL ultra form a clade that is sister to all other Colchicaceae (Fig. 1). Gains 5.8 15.6 13.5 20.5 13 4.4 The ancestral haploid chromosome number of the Losses 6.5 0 0 18.9 20.2 37.1 Colchicaceae may have been a = 7, which apparently was Dupl. 8.0 6.4 7.3 14.8 13.4 13.8 maintained in early-diverging non-African groups such as Demi. 5.9 8.7 9.1 24.8 31 31.2 Sum 26.2 30.7 29.9 79 77.6 86.5 the North American Uvularia and the Australian Kuntheria, Schelhammera,andTripladenia.Increasestoa =8anda =24 2014] CHACO´ N ET AL.: COLCHICACEAE CHROMOSOME EVOLUTION 423

Fig. 3. Maximum likelihood phylogeny of Colchicum based on plastid sequences from the studies of Persson et al. (2011), Del Hoyo et al. (2009), and Vinnersten and Reeves (2003). The tree is rooted on Hexacyrtis dickiana and Ornithoglossum vulgare. Bootstrap support for each clade is indicated with the circles according to the values explained in the inset. The gray bars indicate the clades mentioned in the text. The gray arrow shows the placement of Colchicum melanthioides and the Mediterranean/Irano-Turanian clade. 424 SYSTEMATIC BOTANY [Volume 39

Fig. 4. Chromosome-number reconstructions in Colchicum. In these analyses the root node number has been fixed to a = 11. Numbers at the tips are the haploid chromosome numbers of species. The ten clades shown in Fig. 3 were collapsed here for easier visualization and are indicated in front of the corresponding branches with bold letters, and with the number of original tips in parenthesis. The haploid chromosome numbers for those clades are also shown. Pie charts at nodes and tips represent the probabilities of the inferred haploid chromosome numbers; the color-coding of the chromosome numbers is explained in the inset. Numbers inside the pie charts are the chromosome numbers with the highest probability. A. Reconstruction inferred on the phylogram. B. Reconstruction inferred on the penalized likelihood ultrametric tree. C. spec. = Colchicum speciosum. 2014] CHACO´ N ET AL.: COLCHICACEAE CHROMOSOME EVOLUTION 425

Fig. 4. Continued. took place in the Asian Disporum and in the Australian result of a demi-duplication (Fig. 2C). The initial diversifica- Burchardia (Fig. 2). The younger, mainly African taxa (indi- tion of the African clade began during the Eocene, appar- catedwithanarrowinFig.2),wereinferredtohavea =7 ently after a single long-distance dispersal event from or 11, depending on the tree type used for the inference, Australia about 48 Ma (Chaco´n and Renner, 2014) and with a = 7 on the chronogram and the ultrametric tree involved expansion into arid-adapted vegetation. From the (Fig. 2A–B), but a = 11 assumed on the phylogram as the present data, we found no clear signal of the mode of 426 SYSTEMATIC BOTANY [Volume 39 chromosomal change (e.g. polyploidy, dysploidy) related to not understood, and an experimental investigation of the the expansion of the family Colchicaceae in Africa. Perhaps topic concludes that it is advisable to carry out ChromEvol these events are too far in the past to be inferred just from runs on ultrametric trees as well as phylograms and then extant species and their chromosome numbers. to focus on the findings supported by most reconstructions Wurmbea, a genus with 20 species in Africa and 30 in (Cusimano and Renner, in review). Australia (9 from Africa and 7 from Australia included The Mediterranean and northern African species of in the phylogeny), likely is the result of a “return” dis- Colchicum (clade A in Fig. 3) apparently descend from South persal event from Africa eastwards across the Indian Ocean African ancestors that dispersed from the Namib Desert north- (Chaco´n and Renner, 2014). Unfortunately, there are ward sometime during the Pliocene (ca. 3.5 Ma; Del Hoyo only three chromosome numbers, two from South African et al. 2009). These North African species have asymmetrical species (W. variabilis and W. marginata, both 2n = 14) and karyotypes and 2n = 18, while the South African species have one from Australia for W. dioica with 2n = 20 and 40 symmetrical karyotypes and 2n =20or22(Caujape´-Castells (Table S1). The ancestral chromosome number for Wurmbea et al. 2001). Caujape´-Castells et al. (2001) proposed that inferred with ChromEvol is a = 7 (Fig. 2A–B) or a =10 descending dysploidy (from 22 or 20–18) might explain (Fig. 2C). Different from all other Colchicaceae, the these numbers, which is also inferred here (Fig. S5). Australian species of Wurmbea usually have unisexual For many other nodes in Colchicum, ChromEvol inferred flowers in addition to, or instead of, bisexual flowers. demi-duplications, a term introduced by Mayrose et al. Species can be dioecious or gynodioecious and are insect- (2010) to refer to situations where the fusion of gametes of pollinated (Barrett and Case 2006; Case et al. 2008). In the different ploidy (e.g. haploid and diploid or diploid and polyploid W. dioica, which is gynodioecious, individuals triploid) appears to best explain the numbers seen in with bisexual flowers suffer high levels of selfing (Vaughton related species. While the large role of demi-duplications and Ramsey 2003). It would be interesting to test if poly- in Colchicum (Table 3) initially may seem implausible, ploidy is widespread in the Australian clade of Wurmbea triploids are often found in natural populations and are and perhaps associated already with the ancestor arriving expected to play a role in promoting autotetraploid estab- in Australia from Africa lishment (Husband 2004). Recent empirical and theoretical Chromosome Number Evolution in Colchicum—Previous work also stresses complex ploidy-generating processes, less-densely sampled phylogenies already suggested that especially for populations undergoing autopolyploidy (Suda Colchicum and Androcymbium are not mutually monophy- and Herben 2013). So far, hybridization in Colchicum has letic (Vinnersten and Reeves 2003 and Manning et al. 2007: been discussed based on observations of intermediate mor- both with the same 18 species of Androcymbium and nine phologies, sterility in some cultivars, and mathematical species of Colchicum; Del Hoyo and Pedrola-Monfort 2008: addition of haploid chromosome numbers (Persson 1999; 29 species of Androcymbium and five species of Colchicum; Persson et al. 2011), but there are no experimental crosses Del Hoyo et al. 2009: 41 species of Androcymbium and or other studies addressing hybridization. The extent to six species of Colchicum; Persson et al. 2011: 3 species which past allopolyploidy or conversely autopolyploidy of Androcymbium and 96 species of Colchicum; Thi et al. explain the lability of Colchicum chromosome numbers 2013: 11 Androcymbium and 3 Colchicum species). The phy- therefore remains an open question. logeny presented here with 41 species previously placed Acknowledgments. in Androcymbium and 96 of Colchicum shows beyond doubt We thank I. Mayrose, Tel Aviv University, for support with analyses using ChromEvol, and M. P. Simmons that the type species of Androcymbium, A. melanthioides and two anonymous reviewers for constructive suggestions; A. (C. melanthioides), is more closely related to species of Vinnersten, Uppsala Botanical Garden, for samples of Colchicaceae, Colchicum than it is to many species placed in Androcymbium, J. G. Conran, University of Adelaide, for material of Petermanniaceae, supporting Manning et al.’s (2007) sinking of Androcymbium Philesiaceae, Ripogonaceae, and Colchicaceae from Australia; G. Petersen, Natural History Museum of Denmark, and K. Persson, into Colchicum (see the arrows in Fig. 3). Go¨teborg Botanical Garden, for material of Colchicum from Europe; The ancestral haploid chromosome number of Colchicum J. C. Manning, Compton Herbarium, for samples of Colchicaceae from inferred here is either a = 10 (Fig. 4A) or a = 11 (Fig. 4B). South Africa; I. Telford, University of New England, and G. Keighery, Persson et al. (2011), using parsimony-based trait recon- Department of Environment and Conservation Western Australia, for struction with the chromosome numbers coded as seven material of Burchardia;A.Gro¨ger and J. Wainwright-Klein, Munich Botanical Garden, for material of Colchicaceae from Iran and Georgia; states: 0 = 9; 1 = 8; 2 = 7; 3 = 10; 4 = 11; 5 = 12; ? = andM.W.Chase,RoyalBotanicGardens,Kew,forDNAsamplesof unknown (aneuploid?), inferred a Colchicum base number Colchicaceae. We thank M. Silber, University of Munich, for DNA of x = 9. (Note that the ancestral haploid numbers inferred sequences of Burchardia. The first author thanks H. P. Linder, Univer- by ChromEvol for observed numbers at the tip of a tree sity of Zurich, for the opportunity to join a field trip in South Africa, and T. Trinder-Smith for help in the Bolus Herbarium. This project are not the same as the so-called “base number” or “x”, was funded by grants from the Deutsche Forschungsgemeinschaft which is a confusing concept that suffers from contradictory (DFG RE 603/10-2 and DFG RE 603/7-1) and the Extreme Science and definitions; Cusimano et al. 2012). As in the case of our Engineering Discovery Environment (XSEDE), which is supported by family-level trait reconstructions, the tree type used greatly National Science Foundation grant number OCI-1053575. influenced the ancestral numbers inferred for Colchicum, stressing the uncertainty of all such inferences. For instance, Literature Cited the clade formed by C. szovitsii / C. raddeanum / C. kurdicum (clade E in Fig. 3 or branch E in Fig. 4) is inferred to have Abbott, R., D. Albach, S. Ansell, J. W. Arntzen, S. J. E. Baird, N. a = 9 on the phylogram and the chronogram (Figs. 4A, S2), Bierne, J. Boughman, A. Brelsford, C. A. Buerkle, R. Buggs, R. K. Butlin, U. Dieckmann, F. Eroukhmanoff, A. Grill, S. H. Cahan, but a = 10 on the ultrametric tree (Fig. 4B). How exactly J. S. Hermansen, G. Hewitt, A. G. Hudson, C. Jiggins, J. Jones, branch lengths influence chromosome number reconstruction B. Keller, T. Marczewski, J. Mallet, P. Martinez-Rodriguez, M. 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