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

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Systematic Botany (2014), 39(2): pp. 415–427© Copyright 2014 by the American Society of Plant TaxonomistsDOI 10.1600/036364414X680852Date of publication 04/23/2014

The Evolution of Colchicaceae, with a Focus on Chromosome Numbers

Juliana Chacón,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 particularlydiverse in southern Africa, where 80 of the 270 species occur. Colchicaceae exhibit a wide range of ploidy levels, from 2n = 14 to 2n = 216.To understand where and how this cytogenetic diversity arose, we generated multilocus phylogenies of the Colchicaceae and theColchicum clade that respectively included 85 or 137 species plus relevant outgroups. To infer the kinds of events that could explain theobserved numbers in the living species (dysploidy, polyploidization, or demi-duplication, i.e. fusion of gametes of different ploidy), wecompared 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 theprocesses behind chromosomal rearrangements, they can help frame questions about the direction of change in chromosome numbersin well-sampled groups. The results suggest that dysploidy played a large role in the Colchicaceae, with the exception of Colchicum itselffor which we inferred frequent demi-duplication. While it is known that triploids facilitate the fixation of tetraploidy and that plantspecies often include individuals of odd ploidy level (triploids, pentaploids), we hesitate to accept the phylogenetically inferred scenariowithout 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 Colchicaceaeare the third largest family of the Liliales, after the Liliaceaeand Smilacaceae. They occur in Africa, Asia, Australasia,North America and Europe, but not in South or CentralAmerica (Vinnersten and Manning 2007). Their closest rela-tives are the Alstroemeriaceae, which have most of theirspecies in South America (Chacón et al. 2012a). Together,the two form the sister clade to the Petermanniaceae, amonospecific family restricted to tropical Australia (Vinnerstenand Reeves 2003; Fay et al. 2006). All Colchicaceae containcolchicine, an alkaloid traditionally used in the treatmentof gout, and also in cytogenetics for its properties as amicrotubule polymerization inhibitor (Vinnersten and Larsson2010). Ecologically, Colchicaceae are long-lived cormose orrhizomatous geophytes with rather large, animal-pollinatedflowers offering nectar at the base of their tepals (Nordenstam1998). African Colchicaceae in the Namaqualand desert oftenhave leaves with helical shapes and hairy margins that serveto harvest water from dew and fog, which then drips to thesoil and reaches the root zone where it is ultimately storedin the corms (Vogel and Müller-Doblies 2011).

Colchicaceae have been the subject of several molecular-phylogenetic studies that have clarified relationships andcircumscriptions of the Australian/African genus Wurmbea,the Mediterranean/Irano-Turanian species of the genusColchicum (the latter extending east to Afganistan andKyrgyzstan; Persson 2007), and the genus Gloriosa, with10 species in Africa, India, and southeastern Asia (Vinnerstenand Reeves 2003; Vinnersten and Manning 2007). Aredefinition of Colchicum to include all ca. 60 species ofAndrocymbium (distributed in southern and northern Africaas well as the Mediterranean) was proposed by Manninget al. (2007) and Persson (2007), while Del Hoyo andPedrola-Monfort (2008) and Del Hoyo et al. (2009) pre-ferred to keep Androcymbium and Colchicum separate. Themost comprehensive analysis is that of Thi et al. (2013),who used patchy matrices of 3 or 6 combined plastid regionsfrom 70 species, representing most genera, to infer sub-family and tribal relationships.

A striking feature of the Colchicaceae is their high karyo-logical variation (Table 1), with chromosome numbersranging from 2n = 14 (e.g. Uvularia grandiflora; Thermanand Denniston 1984) to 2n = 216 (in Colchicum corsicum;Persson 2009). Such variation contrasts with the sisterfamily, Alstroemeriaceae, in which the chromosome num-bers vary between 2n = 16 and 2n = 20 (Chacón et al.2012b: 44 of the 200 species of Alstroemeriaceae havebeen counted). The cytogenetics of Colchicum is espe-cially complex, with different species having variablechromosome numbers as well as ploidy levels (from tetra-to 24-ploid; Persson et al. 2011), perhaps related with thepresence of colchicine (Nordenstam 1998). The effect ofcolchicine on the separation of chromosomes after theanaphase of mitosis was discovered by B. Pernice (1889)and described more fully by Eigsti et al. (1945); it revolu-tionized cytogenetics because it permitted experimentaldoubling of the entire complement of a cell’s chromosome set.Besides by polyploidy, chromosome numbers can change

through chromosome fission (ascending dysploidy) or chro-mosome fusion (descending dysploidy; Schubert andLysak 2011). Polyploidization can represent an evolu-tionary dead end (Mayrose et al. 2011), but there is alsoabundant evidence of the adaptive success of polyploidpopulations and the contribution of polyploidy to the for-mation of new species (Levin 1983; Abbott et al. 2013;Weiss-Schneeweiss et al. 2013 and references therein). Bycontrast, dysploidy is thought to arise accidentally, andwe know of no proposed adaptive reason for its pre-ponderance in certain clades. Knowing the distributionof polyploidy and dysploidy in a particular clade or geo-graphic region can help set up testable hypotheses aboutevolutionary pathways, for example about the likelihoodthat hybridization played a large role in the recent past.Here we investigate chromosome number evolution in

the Colchicaceae using the likelihood approach of Mayroseet al. (2010), which models the change rates and relativefrequencies of several kinds of past events that could plau-sibly explain the observed haploid chromosome numbers

415

in a group. The approach requires either a phylogram, thatis, a tree in which branch lengths are proportional to num-bers of DNA substitutions, or an ultrametric tree in whichbranch lengths are proportional to time. Such ultrametrictrees can come from strict clock models or relaxed clockmodels, and they can also be calibrated (typically in mil-lion years), in which case the tree is called a time-tree orchronogram. The kinds of past events that are modeled areduplication of the entire chromosome complement, descend-ing dysploidy, ascending dysploidy, and demi-duplication(the formation of polyploids via the fusion of gametes ofdifferent ploidy, leading to triploidy or pentaploidy). Themethod was tested using simulated and empirical datasetsin the original work by Mayrose and colleagues, and hasso far been used in seven studies (Mayrose et al. 2011: across63 plant groups; Ness et al. 2011: Pontederiaceae; Cusimanoet al. 2012: Araceae; Ocampo and Columbus 2012: Portulaca;Harpke et al. 2013: Crocus; Metzgar et al. 2013: fern genusCryptogramma; Sousa et al. 2014). Based on these studies, itdoes not appear to be biased towards inferring predomi-nantly polyploidy, chromosome losses or gains, or demi-polyploidy. Mayrose et al. (2010) also demonstrated thatthe accuracy of the method depends on the number of taxaand on branch length (because the approach takes longbranches as meaning much time for change). The effects oftree branch lengths indeed can be dramatic, an issue wetake up in the Discussion (Cusimano and Renner, in review).We here use almost 140 published chromosome counts

for Colchicaceae species (52% of their 270 total spe-cies), a modified phylogeny of the family from Chacónand Renner (2014), and a newly compiled phylogeny ofColchicum with 187 accessions representing 137 speciesto infer the chromosomal history of the family. Our mainquestions were: (i) are there predominant modes of chro-mosome number change in the family’s different clades,and (ii) can changes in chromosome number plausibly berelated to coincidental arrival in a new region or habitattype where a single polyploid or dysploid ancestor mightthen have radiated?

Materials and Methods

Taxon Sampling and Phylogenetic Analyses—We used two data setsand from each inferred a phylogram (below), an ultrametric tree obtained

with penalized likelihood (below), and a chronogram obtained under arelaxed clock model (below) to reconstruct the evolution of chromosomenumbers. The first data set included 85 species of Colchicaceae (fromall 15 genera) plus nine outgroups (representing the Alstroemeriaceae,Petermanniaceae, Ripogonaceae, and Philesiaceae), each sequenced forfive plastid regions (matK, ndhF, rbcL, rps16, and trnL-F), one mito-chondrial gene (matR), and the internal transcribed spacer of nuclearribosomal DNA (ITS). Species authors, geographic origin, herbariumvoucher specimen, and GenBank accession numbers are listed in Chacónand Renner (2014). The second data set included 187 accessions ofColchicum representing 137 species, 96 of them traditionally placed inColchicum and 41 transferred there from Androcymbium by Manninget al. (2007) plus two outgroups (Hexacyrtis dickiana and Ornithoglossumvulgare), each sequenced for trnL intron, trnL-trnF intergenic spacer(IGS), trnY-trnD IGS, trnH-psbA IGS, atpB-rbcL IGS, and rps16 intron.Sequences came from the studies of Del Hoyo et al. (2009), Vinnerstenand Reeves (2003), and Persson et al. (2011). The number of Colchicumspecies in the two matrices are thus different because the Colchicaceaematrix consisted of plastid, mitochondrial, and nuclear DNA sequences,while the Colchicum matrix consisted only of plastid sequences fromdifferent genes (see above); we decided not to combine them in orderto minimize the number of empty cells in the alignment. Instead we ranseparate analyses for each matrix.

Sequences were concatenated and aligned with MAFFT v. 7 (Katohand Standley 2013) using the L-INS-i algorithm (Katoh et al. 2005), fol-lowed by manual adjustment in the program MacClade v. 4.8 (Maddisonand Maddison 2002) based on the similarity criterion of Simmons (2004).The resulting alignments were used for phylogenetic tree reconstructionunder maximum likelihood (ML; Felsenstein 1973) using RAxML v. 7.0.4(Stamatakis 2006) through the CIPRES Science Gateway (Miller et al.2010). The substitution model used was the GTR + G model, thisbeing the best-fitting model identified by the Akaike Information Cri-terion (AIC; Akaike 1974) in FindModel (http://hcv.lanl.gov/content/sequence/findmodel/ findmodel.html). Statistical support for nodes wasassessed by 1,000 ML bootstrap replicates (Felsenstein 1985) under thesame model.

The maximum likelihood phylogram from each matrix was trans-formed into an ultrametric tree with the R function “chronopl” ofthe APE package v. 3.0–6 (Paradis et al. 2004), which implements thepenalized likelihood (PL) method of Sanderson (2002), including appro-priate cross-validation to find the best smoothing parameter. As analternative to the PL approach, we also used two time-calibrated treesobtained with the Bayesian program BEAST v. 1.7.5 (Drummond et al.2012), in which we opted for a relaxed clock model with uncorrelatedlog-normal (UCLN) rate variation, meaning that ancestors and descen-dants are allowed to have rather more different substitution rates thanis the case in the PL approach. The substitution model for both matriceswas again the GTR + G model, and the tree prior was a Yule process.The length of the Monte Carlo Markov Chain (MCMC) was set to90 million generations with parameters sampled every 1000 generationsand a burnin of 10%. Following Chacón et al. (2012a) we applied fourcalibration points, three of them from fossils and one a secondary cali-bration from another study.

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

n 2nBaeometra Salisb. ex Endl. 1 1 22Burchardia R. Br. 6 5 48 24Camptorrhiza Hutch. 2 1 22Colchicum 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. 216Disporum Salisb. ex G. Don 20 11 14, 16, 18, 30, 32Gloriosa L. 10 7 20, 21, 22, 44, 66, 88Hexacyrtis Dinter 1 1 22Iphigenia Kunth 12 6 11 22Kuntheria Conran & Clifford 1 1 14Ornithoglossum Salisb. 8 4 24Sandersonia Hook. 1 1 24Shelhammera R. Br. 2 2 14, 36Tripladenia D. Don 1 1 14Uvularia L. 5 3 7 14Wurmbea Thunb. ca. 50 3 14, 20, 40

416 SYSTEMATIC BOTANY [Volume 39

The alignments and phylogenetic trees obtained in this study havebeen deposited in TreeBASE (study accession No. 14230).

Inference of Chromosome-Number Change—The chromosome num-bers for 144 species of Colchicaceae and eight outgroup taxa wereobtained from the Index to Plant Chromosome Numbers (http://www.tropicos.org/Project/IPCN; October 2012) and other literature (Table S1in the supplemental online data; this includes all Colchicaceae andoutgroup species with published chromosome numbers). Chromosomenumbers were available for 58 of the 85 species included in the familytrees and for 112 of the species included in the Colchicum trees. Onlyaccessions with a herbarium voucher were included. When conspe-cific accessions grouped together and the chromosome number of therespective species was stable, then only one accession was included.When conspecific accessions did not group together, we checked ifpublished counts were associated with the respective accessions. If thatwas the case we included these accessions, otherwise all accessions ofthat species were excluded.

For maximum likelihood and Bayesian phylogenetic inferences ofancestral haploid chromosome numbers we relied on ChromEvol v. 1.3(Mayrose et al. 2010; http://www.tau.ac.il/�itaymay/cp/chromEvol/index.html) with an extension provided by I. Mayrose (Tel Aviv Uni-versity; pers. comm., 29 January, 2013) that allows fixing the root nodenumber. ChromEvol implements eight models of chromosome-numberchange, which include the following six parameters: polyploidization(chromosome number duplication) with rate r, demi-duplication (fusionof gametes of different ploidy) with rate m, and dysploidization (ascend-ing: chromosome gain rate l; descending: chromosome loss rate d) aswell as two linear rate parameters, l1 and d1, for the dysploidizationrates l and d, allowing them to vary based on the current number ofchromosomes. Four of the models have constant rates, whereas theother four include two linear rate parameters. Both model sets alsohave a null model that assumes no duplication events. We first fit allmodels to the data without performing simulations to infer the best-fitmodel. For the best-fit model we reran the analysis fixing the parame-ters to those optimized in the first run and using 10,000 simulationsto compute the expected number of changes along each branch aswell as the ancestral haploid chromosome numbers at nodes. The nullhypothesis (no polyploidy) was tested using an AIC test.

Ancestral haploid chromosome numbers, which we refer to as a,were inferred on the two ML phylograms, the two PL ultrametric trees,and the two BEAST chronograms. Species for which no chromosome-number information was available were cut from the trees, resultingin 58 species in the Colchicaceae tree (instead of 85) and 112 species(126 accessions) instead of 137 (187 accessions of Colchicum and two out-groups) in the Colchicum tree. Before running ChromEvol the branchlengths of some trees were adjusted (Table 2) because the root-to-tipdistance was large, which can cause ChromEvol to overestimate thenumber of transitions. Using artificial data, Mayrose et al. (2010) showedthat reliable reconstructions are obtained with root-to-tip distances rang-ing from 0.1–0.8. We therefore adjusted branch lengths such that thetotal tree lengths were between 0.1 and 0.2 (Table 2). We ran addi-tional analyses with double or half these tree lengths to test if theresults would differ substantially; this was not the case.

The maximum haploid number of chromosomes was set to ten morethan the highest empirical number (i.e. 108 + 10 = 118), and the mini-

mum number to 1. In the runs conducted on the Colchicum trees, wefixed the haploid chromosome number at the Colchicum+outgroupsroot node to a = 11 with a probability of 1 because this was thenumber inferred for this node with the highest posterior probabilityin the family-wide analysis using the phylogram. To assess the influ-ence of outgroup taxa on the estimates, we compared results with orwithout outgroups (with their corresponding chromosome counts).

Results

Molecular Phylogeny of Colchicaceae—The combinedplastid, mitochondrial and nuclear data (6,451 aligned nucleo-tides) yielded a robust phylogeny for the 85 Colchicaceaespecies with most clades having >80% bootstrap support(Fig. 1). The six species of Burchardia, analyzed together forthe first time here, form a clade that is sister to all otherColchicaceae. The monotypic genus Kuntheria forms a cladewith Schelhammera undulata and Tripladenia cunninghamii.Reconstruction of Ancestral Chromosome Numbers in

Colchicaceae—Minor differences were found between ana-lyses that included outgroups and those that did not,besides the trivial difference that trees with outgroupsinclude more branches and therefore lead to higher num-bers of overall events (Fig. 2 A–C without outgroups vs.Fig. S1 with outgroups; see supplemental online data). Theancestral chromosome number inferred for the crownnode of the Colchicaceae was a = 7 (Fig. 2A–C) except onthe phylogram and chronogram with outgroups (a = 8 anda = 6; Fig. S1). The chronograms with or without out-groups also differed in six chromosome gains inferred on thefollowing nodes only when the outgroups were included:crown of Camptorrhiza/Iphigenia, crown of Gloriosa, crown ofTripladenia/Schelhammera/Kuntheria, crown of Uvularia/Disporum,crown and stem of Wurmbea; see Fig. S1). As the patternof chromosome-number change appears to be quite differ-ent between the ingroup and outgroup, we focused oninferences from trees that included only the ingroup.Results with Input Trees Differing in Branch Length—The

model that best fit all three types of trees (phylogram, PLultrametric tree, BEAST UCLN chronogram) was the con-stant rates model with the duplication rate equal to thedemi-duplication rate (Table 2). However, dissimilar ances-tral haploid numbers where inferred on the three typesof trees, specially at nodes along the tree backbones, whilenumbers inferred for the crown nodes of most genera andon 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 cladeusing a phylogram (ML phyl), an ultrametric tree (PL ultra) or an chronogram from a BEAST analysis (B chrono). The factor with which the branchesof the tree have been adjusted for the analysis and the resulting tree length are given. AIC scores; best model: crd = constant rate model withduplication rate equal to the demiduplication rate; crde = constant rate model with duplication rate different to the demi-duplication rate; rateparameters: l = chromosome gain rate, d = chromosome loss rate, r = duplication rate, m = demi-duplication rate; haploid chromosome numbera inferred at the root node of the Colchicaceae family and the root node of Colchicum, respectively, under Bayesian optimization with the respectivePP, and under maximum likelihood optimization (ML).

Rates Inferred chrom. No. at root a

Tree FactorResulting

root-tip lengthResulting

total tree lengthBestmodel AIC l d r m

Bayes:best a - PP

Bayes: secondbest 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 7PL ultra 0.5 0.2 2.53 crd 248.1 3.62 0.0 2.21 = r 7–0.98 – 7B 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 10PL ultra 1 0.1 4.53 crde 658.6 3.17 11.21 3.39 9.3 11–0.96 – 11B chrono 3 0.11 2.32 crde 675.2 11.82 13.74 6.4 18.03 10–0.75 11–0.18 10

2014] CHACÓN ET AL.: COLCHICACEAE CHROMOSOME EVOLUTION 417

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 foreach clade is indicated with the circles according to the values explained in the inset.


Fig. 2. Chromosome-number reconstructions for the Colchicaceae. Numbers at the tips are the haploid chromosome numbers of species. Pie chartsat nodes and tips represent the probabilities of the inferred haploid chromosome numbers; the color-coding of the chromosome numbers is explainedin the inset. Numbers inside the pie charts are the chromosome numbers with the highest probability. Numbers above branches represent theexpected number of the four possible events, i.e. gains, losses, duplications, and demi-duplications occurring along that branch inferred bysimulation 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 theinsets. The black arrows indicate the crown group of the African Colchicaceae and the gray arrows the Mediterranean/Irano-Turanian Colchicumclade. A. Reconstruction inferred without outgroups on the UCLN relaxed clock chronogram obtained with BEAST. B. Reconstruction inferred withoutoutgroups on the ultrametric tree obtained with penalized likelihood. C. Reconstruction inferred without outgroups on the phylogram.


Fig. 2. Continued.


less affected by tree branch lengths (Fig. 2A–C). In gen-eral, reconstructions on the UCLN chronogram and thePL ultrametric tree (Figs. 2A, B) are more similar to eachother than either is to those on the phylogram (Fig. 2C).As mentioned before, a = 7 (with different probabilities) isinferred for the root, and this number is maintained alongthe early branches of Colchicaceae (Fig. 2). Nevertheless,on the phylogram a switch from a = 7 to a = 11 appears atthe root node of the African Colchicaceae (indicated withan arrow in Fig. 2C), then changing to a =10 in the crownnode of Colchicum, while on the other two trees the ances-tral number a = 7 changed to a = 9 on the root node ofColchicum, either through a gradual increase from a = 7–8and 9 (Fig. 2A) or from a = 7 to a = 9 (Fig. 2B). In thefamily-level analyses, ChromEvol assumed a = 9 as theancestral number for the Mediterranean and Irano/Turanianspecies of Colchicum on all trees (Fig. 2A–C; but see nextsection about Colchicum).In all trees, the ancestral haploid number inferred at the

crown node of Burchardia was a = 24; other early derivedgenera have numbers based on a = 7 (Uvularia, Kuntheria,Schelhammera, and Tripladenia), with the exception of Disporumwith a = 8 in the phylogram and the chronogram (Figs. 2A, C).Other differences between inferences on the UCLN chrono-gram (Fig. 2A), the PL ultrametric tree (Fig. 2B), and thephylogram (Fig. 2C) concern the numbers for the Wurmbeacrown and stem group, which in the UCLN and the PLtrees are a = 7, while in the phylogram the crown numbersis a = 10, the stem number a = 11.As a consequence of the different most likely ancestral

numbers, the frequencies of events with an expectation >0.5inferred by simulation also differed among trees (Fig. 2). Asummary is provided in Table 3; 26.2 events were inferredon the phylogram while ca. 30 events were inferred onthe PL and UCLN ultrametric trees. The best-fit model forthe Colchicaceae assumed more duplications than demi-duplications on the phylogram (8 vs. 5.9) while the oppositewas assumed on the PL tree (7.3 vs. 9.1) and the UCLN tree(6.4 vs. 8.7). Other inferred parameter values also differ (seeTable 2), especially the number of dysploidy events, withchromosome gains and losses inferred on the phylogramwith similar frequencies (5.8, 6.5; Table 3), but only chromo-some gains inferred on the PL and UCLN trees (13.5 and15.6, respectively; Table 3).Molecular Phylogeny of Colchicum—Figure 3 shows a

phylogeny for 187 accessions representing 137 species ofColchicum, rooted on the two outgroup taxa and withmaximum likelihood bootstrap values. A large clade ofspecies previously placed in Androcymbium (see Table S1)

with two subclades (A and B in Fig. 3), one with 17 spe-cies (21 accessions) and one with 14 species (22 acces-sions), is sister to a clade containing the remainingColchicum species. While some of the nodes along thebackbone lack statistical support (bootstrap < 80%), the dis-tribution of chromosome numbers (next section) matchesthe topology (Figs. 3, 4A , B).

Reconstruction of Ancestral Chromosome Numbers inColchicum—For the Colchicum data set (126 accessions ofColchicum representing 112 species with chromosome counts),the best-fitting model of chromosome number evolution isthe constant rate model with different rates for duplicationand demi-duplication events (Table 2). The reconstructionsmade on the ultrametric tree and the phylogram are shownin Fig. 4 (the latter being almost identical to the resultsobtained with the chronogram; see Fig. S2). Chromosomenumbers were constant among the species in ten clades(labeled A to J in Fig. 3), and in Fig. 4, we collapsed theseclades for easier visualization of the overall tree. The uncol-lapsed trees are shown in Figs. S2–S5. On the phylogram,the inferred ancestral number for Colchicum is a = 10(PP = 0.79; Fig. 4A) while on the ultrametric tree it isa = 11 (PP = 0.96; Fig. 4B). In the phylogram, there is areduction from a = 10–9 on the branch leading to clade C(see Figs. 3, 4A) followed by an increase to a = 12 through ademi-duplication (Fig. S5). On the ultrametric tree (Fig. 4B),ChromEvol instead assumes an increase from a = 11–12through a chromosome gain for the same clade C.

To explain the empirical chromosome numbers in fiveColchicum clades composed exclusively of species withn = 27 (clades C, D, G, H, J; Fig. 4), the program inferredduplications and demi-duplications on the phylogram (froma = 9–27, from a = 12–27, and from a = 12–18 and to 27;Figs. 4A, S4, S5), while on the ultrametric tree it inferredonly demi-duplications (from a = 18–27, from a = 12–27,and a = 12–18–27; Figs. 4B, S3). The most frequent of thefour possible types of events in the chronogram and thephylogram is the demi-duplication, while in the ultrametrictree, chromosome losses are the most frequent event, fol-lowed by demi-duplications (Table 3).

Discussion

Chromosome Number Evolution in Colchicaceae—Maximumlikelihood phylogenies for 85 species of Colchicaceae(Fig. 1) or 137 species of Colchicum (some represented byseveral accessions; Fig. 3) were here used to infer eventsthat could explain the observed range of haploid chro-mosome numbers in this family (Figs. 2, 4). Genera firstcompletely sampled in the present study are Burchardia,for which we included all its six species, and the mono-specific Australian Kuntheria. The latter forms a clade withSchelhammera undulata, the type species of an Australiangenus that has two other species, and the monospecificAustralian Tripladenia (Fig. 1), all three with a chromosomenumber of 2n = 14 (Table S1) and an inferred haploid ances-tral number of a = 7 (Fig. 2). The six species of Burchardiaform a clade that is sister to all other Colchicaceae (Fig. 1).

The ancestral haploid chromosome number of theColchicaceae may have been a = 7, which apparently wasmaintained in early-diverging non-African groups such asthe North American Uvularia and the Australian Kuntheria,Schelhammera, and Tripladenia. Increases to a = 8 and a = 24

Table 3. Number of events inferred in ChromEvol by simulationwith an expectation >0.5 for the Colchicaceae and the Colchicum datasets, using a phylogram (ML phyl), an ultrametric tree (PL ultra) or achronogram from a BEAST analysis (B chrono).

Events

Colchicaceae Colchicum

ML phyl B chrono PL ultra ML phyl B chrono PL ultra

Gains 5.8 15.6 13.5 20.5 13 4.4Losses 6.5 0 0 18.9 20.2 37.1Dupl. 8.0 6.4 7.3 14.8 13.4 13.8Demi. 5.9 8.7 9.1 24.8 31 31.2Sum 26.2 30.7 29.9 79 77.6 86.5


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 indicatedwith the circles according to the values explained in the inset. The gray bars indicate the clades mentioned in the text. The gray arrow shows theplacement of Colchicum melanthioides and the Mediterranean/Irano-Turanian clade.


Fig. 4. Chromosome-number reconstructions in Colchicum. In these analyses the root node number has been fixed to a = 11. Numbers at thetips are the haploid chromosome numbers of species. The ten clades shown in Fig. 3 were collapsed here for easier visualization and are indicatedin front of the corresponding branches with bold letters, and with the number of original tips in parenthesis. The haploid chromosome numbers forthose clades are also shown. Pie charts at nodes and tips represent the probabilities of the inferred haploid chromosome numbers; the color-codingof 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.


took place in the Asian Disporum and in the AustralianBurchardia (Fig. 2). The younger, mainly African taxa (indi-cated with an arrow in Fig. 2), were inferred to have a = 7or 11, depending on the tree type used for the inference,with a = 7 on the chronogram and the ultrametric tree(Fig. 2A–B), but a = 11 assumed on the phylogram as the

result of a demi-duplication (Fig. 2C). The initial diversifica-tion of the African clade began during the Eocene, appar-ently after a single long-distance dispersal event fromAustralia about 48 Ma (Chacón and Renner, 2014) andinvolved expansion into arid-adapted vegetation. From thepresent data, we found no clear signal of the mode of

Fig. 4. Continued.


chromosomal change (e.g. polyploidy, dysploidy) related tothe expansion of the family Colchicaceae in Africa. Perhapsthese events are too far in the past to be inferred just fromextant species and their chromosome numbers.Wurmbea, a genus with 20 species in Africa and 30 in

Australia (9 from Africa and 7 from Australia includedin the phylogeny), likely is the result of a “return” dis-persal event from Africa eastwards across the Indian Ocean(Chacón and Renner, 2014). Unfortunately, there areonly three chromosome numbers, two from South Africanspecies (W. variabilis and W. marginata, both 2n = 14) andone from Australia for W. dioica with 2n = 20 and 40(Table S1). The ancestral chromosome number for Wurmbeainferred with ChromEvol is a = 7 (Fig. 2A–B) or a = 10(Fig. 2C). Different from all other Colchicaceae, theAustralian species of Wurmbea usually have unisexualflowers in addition to, or instead of, bisexual flowers.Species can be dioecious or gynodioecious and are insect-pollinated (Barrett and Case 2006; Case et al. 2008). In thepolyploid W. dioica, which is gynodioecious, individualswith bisexual flowers suffer high levels of selfing (Vaughtonand Ramsey 2003). It would be interesting to test if poly-ploidy is widespread in the Australian clade of Wurmbeaand perhaps associated already with the ancestor arrivingin Australia from AfricaChromosome Number Evolution in Colchicum—Previous

less-densely sampled phylogenies already suggested thatColchicum and Androcymbium are not mutually monophy-letic (Vinnersten and Reeves 2003 and Manning et al. 2007:both with the same 18 species of Androcymbium and ninespecies of Colchicum; Del Hoyo and Pedrola-Monfort 2008:29 species of Androcymbium and five species of Colchicum;Del Hoyo et al. 2009: 41 species of Androcymbium andsix species of Colchicum; Persson et al. 2011: 3 speciesof Androcymbium and 96 species of Colchicum; Thi et al.2013: 11 Androcymbium and 3 Colchicum species). The phy-logeny presented here with 41 species previously placedin Androcymbium and 96 of Colchicum shows beyond doubtthat the type species of Androcymbium, A. melanthioides(C. melanthioides), is more closely related to species ofColchicum than it is to many species placed in Androcymbium,supporting Manning et al.’s (2007) sinking of Androcymbiuminto Colchicum (see the arrows in Fig. 3).The ancestral haploid chromosome number of Colchicum

inferred here is either a = 10 (Fig. 4A) or a = 11 (Fig. 4B).Persson et al. (2011), using parsimony-based trait recon-struction with the chromosome numbers coded as sevenstates: 0 = 9; 1 = 8; 2 = 7; 3 = 10; 4 = 11; 5 = 12; ? =unknown (aneuploid?), inferred a Colchicum base numberof x = 9. (Note that the ancestral haploid numbers inferredby ChromEvol for observed numbers at the tip of a treeare not the same as the so-called “base number” or “x”,which is a confusing concept that suffers from contradictorydefinitions; Cusimano et al. 2012). As in the case of ourfamily-level trait reconstructions, the tree type used greatlyinfluenced the ancestral numbers inferred for Colchicum,stressing the uncertainty of all such inferences. For instance,the clade formed by C. szovitsii / C. raddeanum / C. kurdicum(clade E in Fig. 3 or branch E in Fig. 4) is inferred to havea = 9 on the phylogram and the chronogram (Figs. 4A, S2),but a = 10 on the ultrametric tree (Fig. 4B). How exactlybranch lengths influence chromosome number reconstruction(and ancestral trait reconstruction in general) is currently

not understood, and an experimental investigation of thetopic concludes that it is advisable to carry out ChromEvolruns on ultrametric trees as well as phylograms and thento focus on the findings supported by most reconstructions(Cusimano and Renner, in review).

The Mediterranean and northern African species ofColchicum (clade A in Fig. 3) apparently descend from SouthAfrican ancestors that dispersed from the Namib Desert north-ward sometime during the Pliocene (ca. 3.5 Ma; Del Hoyoet al. 2009). These North African species have asymmetricalkaryotypes and 2n = 18, while the South African species havesymmetrical karyotypes and 2n = 20 or 22 (Caujapé-Castellset al. 2001). Caujapé-Castells et al. (2001) proposed thatdescending dysploidy (from 22 or 20–18) might explainthese numbers, which is also inferred here (Fig. S5).

For many other nodes in Colchicum, ChromEvol inferreddemi-duplications, a term introduced by Mayrose et al.(2010) to refer to situations where the fusion of gametes ofdifferent ploidy (e.g. haploid and diploid or diploid andtriploid) appears to best explain the numbers seen inrelated species. While the large role of demi-duplicationsin Colchicum (Table 3) initially may seem implausible,triploids are often found in natural populations and areexpected to play a role in promoting autotetraploid estab-lishment (Husband 2004). Recent empirical and theoreticalwork also stresses complex ploidy-generating processes,especially for populations undergoing autopolyploidy (Sudaand Herben 2013). So far, hybridization in Colchicum hasbeen discussed based on observations of intermediate mor-phologies, sterility in some cultivars, and mathematicaladdition of haploid chromosome numbers (Persson 1999;Persson et al. 2011), but there are no experimental crossesor other studies addressing hybridization. The extent towhich past allopolyploidy or conversely autopolyploidyexplain the lability of Colchicum chromosome numberstherefore remains an open question.

Acknowledgments. We thank I. Mayrose, Tel Aviv University,for support with analyses using ChromEvol, and M. P. Simmonsand two anonymous reviewers for constructive suggestions; A.Vinnersten, Uppsala Botanical Garden, for samples of Colchicaceae,J. G. Conran, University of Adelaide, for material of Petermanniaceae,Philesiaceae, Ripogonaceae, and Colchicaceae from Australia; G.Petersen, Natural History Museum of Denmark, and K. Persson,Göteborg Botanical Garden, for material of Colchicum from Europe;J. C. Manning, Compton Herbarium, for samples of Colchicaceae fromSouth Africa; I. Telford, University of New England, and G. Keighery,Department of Environment and Conservation Western Australia, formaterial of Burchardia; A. Gröger and J. Wainwright-Klein, MunichBotanical Garden, for material of Colchicaceae from Iran and Georgia;and M. W. Chase, Royal Botanic Gardens, Kew, for DNA samples ofColchicaceae. We thank M. Silber, University of Munich, for DNAsequences of Burchardia. The first author thanks H. P. Linder, Univer-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 projectwas funded by grants from the Deutsche Forschungsgemeinschaft(DFG RE 603/10-2 and DFG RE 603/7-1) and the Extreme Science andEngineering Discovery Environment (XSEDE), which is supported byNational Science Foundation grant number OCI-1053575.

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