Molecular Phylogenetics and Evolution 39 (2006) 688–705 www.elsevier.com/locate/ympev
Molecular phylogenetics of the clover genus (Trifolium—Leguminosae)
Nick W. Ellison a, Aaron Liston b,¤, JeVrey J. Steiner c, Warren M. Williams a, Norman L. Taylor d
a Grasslands Research Centre, AgResearch, Private Bag 11008, Palmerston North, New Zealand b Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA c USDA Agricultural Research Service, National Forage Seed Production Research Center, Corvallis, OR 97331, USA d Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY 40546, USA
Received 31 August 2005; revised 16 December 2005; accepted 3 January 2006 Available online 17 February 2006
Abstract
Trifolium, the clover genus, is one of the largest genera of the legume family. We conducted parsimony and Bayesian phylogenetic analyses based on nuclear ribosomal DNA internal transcribed spacer and chloroplast trnL intron sequences obtained from 218 of the ca. 255 species of Trifolium, representatives from 11 genera of the vicioid clade, and an outgroup Lotus. We conWrm the monophyly of Trifo- lium, and propose a new infrageneric classiWcation of the genus based on the phylogenetic results. Incongruence between the nrDNA and cpDNA results suggests Wve to six cases of apparent hybrid speciation, and identiWes the putative progenitors of the allopolyploids T. dubium, a widespread weed, and T. repens, the most commonly cultivated clover species. Character state reconstructions conWrm 2n D 16 as the ancestral chromosome number in Trifolium, and infer a minimum of 19 instances of aneuploidy and 22 of polyploidy in the genus. The ancestral life history is hypothesized to be annual in subgenus Chronosemium and equivocal in subgenus Trifolium. Transitions between the annual and perennial habit are common. Our results are consistent with a Mediterranean origin of the genus, probably in the Early Miocene. A single origin of all North and South American species is hypothesized, while the species of sub-Saharan Africa may originate from three separate dispersal events. © 2006 Elsevier Inc. All rights reserved.
Keywords: Trifolium; Clover; Leguminosae; Fabaceae; Phylogeny; Biogeography; Hybrid speciation; Chromosome evolution; Life history evolution; DNA barcoding
1. Introduction 2.5 cm) are usually arranged in capitate to spicate heads. The four lower petals (wing and keel) are partially connate The Leguminosae (D Fabaceae) is the third largest fam- and their claws are adnate to the staminal tube; the upper ily of Xowering plants (727 genera and ca. 19,325 species; petal (banner) may also be connate to the lower petals, and Lewis et al., 2005) and the clover genus, Trifolium L., is one sometimes to the free stamen (Hossain, 1961; Zohary and of the largest genera in the family, with ca. 255 species (Gil- Heller, 1984). The corolla and calyx are generally persistent lett and Taylor, 2001; Zohary and Heller, 1984). The genus after anthesis, with one or sometimes both functioning in name refers to the distinctive leaves usually composed of fruit dispersal (Zohary, 1972). Fruits are usually only 1–2 three leaXets (trifoliolate). All species are herbaceous peren- seeded, but may contain up to nine seeds. The pods may be nials or annuals, often prostrate and rarely more than regularly dehiscent, or lacking sutures and irregularly 50 cm tall. The small to medium-sized Xowers (ca. 0.3– dehiscent. In the latter case, the pod is often of papery tex- ture and wholly contained within the persistent and often highly modiWed corolla or calyx. * Corresponding author. Fax: +1 541 737 3573. The native distribution of Trifolium (Table 1) encom- E-mail address: [email protected] (A. Liston). passes the temperate and, to a lesser extent, subtropical
1055-7903/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2006.01.004 N.W. Ellison et al. / Molecular Phylogenetics and Evolution 39 (2006) 688–705 689
Table 1 Proposed subgeneric classiWcation of Trifolium and corresponding taxa in Zohary and Heller (1984) New classiWcation Zohary and Heller taxonomy Native distribution Number of Figure species subgenus Chronosemium (Ser.) sect. Chronosemium Mediterranean region 20 Fig. 2 Reichenb. subgenus Trifolium sect. Glycyrrhizum Bertol. T. alpinum and T. polyphyllum European Alps—Caucasus 2 Fig. 3 of sect. Lotoidea Mts. sect. Paramesus Mediterranean region 2 Fig. 3 sect. Paramesus (C. Presl) Berchtold and J. Presl sect. Lupinaster (Fabricius) Ser. T. eximium, T. gordejevii and E Europe—Siberia 3 Fig. 3 T. lupinaster of sect. Lotoidea sect. Trifolium sect. Trifolium Mediterranean region, 73 Fig. 4 S Africa (1) sect. Trichocephalum Koch sect. Trichocephalum Mediterranean region 9 Fig. 4 sect. Vesicastrum Ser. sects. Mistyllus, Vesicaria, and Mediterranean region, 54 Fig. 5 Lotoidea in part sub-Saharan Africa, Madagascar (1) sect. Trifoliastrum S.F. Gray sect. Lotoidea in part Mediterranean region 20 Fig. 6 sect. Involucrarium Hooker sects. Lotoidea and Lotoidea in part N and S America 72 Fig. 7 Nomenclatural priority for new infrageneric taxa follows Hendrych (1988). Species numbers and geographic distributions are based on Zohary and Heller (1984) with the addition of seven species described since 1984 and 11 species treated by them as synonyms or subspeciWc taxa. The Mediterranean region includes the area bordering the Mediterranean Sea, extending to Europe and Southwest Asia. regions of the Northern and Southern Hemispheres. Native only succeed between closely related taxa (Taylor and clovers are absent from southeast Asia and Australia. The Quesenberry, 1996). This limitation has spurred interest in greatest species diversity is found in three geographic the evaluation of the agronomic potential of locally uti- regions: (1) the Mediterranean basin, (2) western North lized and currently uncultivated species (Morris and America, and (3) the highlands of eastern Africa. Trifolium Greene, 2001). species occur in a wide range of habitats, including meadows The comprehensive monograph of Zohary and Heller and prairies, open woodlands, semi-deserts, mountains, and (1984) summarizes the extensive taxonomic history of Tri- alpine peaks. A common feature of these diverse habitats is folium and provides detailed descriptions and illustrations high solar radiation; few clover species tolerate shade. of all recognized species. They classify the genus into eight Chromosome numbers are known for at least 184 spe- sections (Table 1). Although most authors treat Trifolium cies of Trifolium (summarized in Taylor et al., 1979; Zohary as a single genus, several segregates have been proposed and Heller, 1984; see also Goldblatt and Johnson, 2003). (see Section 4.2.), indicating the need for a critical evalua- Over 80% of the examined species are 2n D 16, and x D 8 is tion of the monophyly of the genus. the inferred base number of the genus (Goldblatt, 1981). Trifolium is a member of the large clade of legumes Aneuploidy (2n D 10, 12, or 14) is known from 31 species, lacking one copy of the chloroplast inverted repeat, the 11 of which have both aneuploid and diploid (2n D 16) or IRLC (Lavin et al., 1990; Liston, 1995). Molecular phylo- polyploid counts. Polyploidy is known from 24 species, of genetic studies have identiWed a strongly supported “vici- which six are exclusively tetraploid, two are hexaploid, and oid clade” within the IRLC composed of the tribes one is dodecaploid (12£). Eleven species have both diploid Trifolieae and Fabeae (Table 2), and the genera Cicer, and polyploid counts, while three have multiple polyploid Galega, and Parochetus (Liston and Wheeler, 1994; San- counts at the tetraploid level and above. Nitrogen-Wxing derson and Wojciechowski, 1996; Wojciechowski et al., root nodules have been reported from over 125 species of 2000, 2004). Within the vicioid clade, Fabeae and Trifoli- clover (Sprent, 2001). Among those species that have been eae comprise a monophyletic group. Steele and Wojcie- studied, nodulation is by Rhizobium leguminosarum biovar. chowski (2003) conducted a phylogenetic analysis of the trifolii (Sprent, 2001). Trifolieae and Fabeae based on cpDNA matK. Their anal- Clovers are widely grown as livestock forage and green ysis provided strong support for the monophyly of Trifo- manure crops, and introduced species have become exten- lium. Surprisingly, the genus was resolved (with moderate sively naturalized worldwide. At least 16 species of Trifo- bootstrap support) as sister lineage to the Fabeae, making lium are actively cultivated (Gillett and Taylor, 2001), a Trifolieae paraphyletic. fairly large number for a single genus. Many native spe- No previous molecular phylogenetic analysis of Trifo- cies are also heavily utilized by grazing animals (Cramp- lium has sampled the taxonomic and geographic breadth of ton, 1985). Fertile interspeciWc hybrids are diYcult to the genus. In a study of 59 Old World Trifolium species achieve in Trifolium (Taylor et al., 1980), and generally based on nrDNA ITS sequences and restriction site 690 N.W. Ellison et al. / Molecular Phylogenetics and Evolution 39 (2006) 688–705
Table 2 Zohary and Heller, 1984), their hypotheses have not been ClassiWcation and geographic distribution of the vicioid clade and the out- examined in a phylogenetic context. group genus, Lotus Our phylogenetic analyses are based on data from the Tribe genus Native distribution Species nuclear and chloroplast (cpDNA) genome. The rate and number pattern of ITS sequence mutation are typically appropriate Trifolieae for resolving relationships among species and genera (Bald- Medicago L. Eurasia, E and S Africa 83 win et al., 1995; Hershkovitz et al., 1999). Although thou- Melilotus Mill. Eurasia, E Africa 20 Ononis L. Mediterranean region— 75 sands of copies of the ITS exist in angiosperm genomes, Central Asia, E Africa they are generally homogenized by concerted evolution, Trifolium L. see Table 1 255 and thus can be treated as a single locus (Baldwin et al., Trigonella L. Mediterranean region— 55 1995). However, phylogenetic estimates based on ITS may Central Asia, S Africa, be compromised by paralogy (due to polyploidy or incom- Australia plete concerted evolution), compensatory base changes, Fabeae and problems in alignment due to length variation (Alvarez Lathyrus L. Eurasia, E Africa, 160 N and S America and Wendel, 2003; Bailey et al., 2003). To complement the Lens Mill. Mediterranean region, 4 nrDNA ITS data set, we sequenced the intron-containing Africa chloroplast tRNA (UAA) for leucine (trnL). Like the Pisum L. Mediterranean region 3 nrDNA ITS, cpDNA introns are widely used in the phylo- Vicia L. Eurasia, E Africa, 160 genetic analysis of land plants (Kelchner, 2000). However, N and S America, numerous studies have observed that lineage sorting, intro- Hawaii gressive hybridization, and cytoplasmic sharing among Other vicioids unrelated sympatric species (Belahbib et al., 2001) can con- Cicer L. Mediterranean region— 43 Central Asia, E Africa found phylogenetic analysis based on plastid loci (Cronn Galega L. Eurasia, E Africa 6 et al., 2002; Wendel and Doyle, 1998). Parochetus D. Don Mountains of tropical 2 Combining data from multiple loci is an eVective Asia, E Africa approach for avoiding problems associated with single Outgroup locus estimates of phylogeny (Gatesy and Baker, 2005; Lotus L. Eurasia, N Africa, N 100 Rokas et al., 2003). Because organellar and nrDNA and S America sequences are prone to diVerent types of potential bias, any Species numbers and distribution data from Lewis et al. (2005). resulting “noise” should be randomly distributed and phy- logenetic signal should be reinforced in a combined analysis analysis of PCR-ampliWed cpDNA, Watson et al. (2000) (Wiens, 1998). We also analyzed the nuclear and organellar provided the Wrst molecular phylogenetic evidence that sequences in separate analyses, to detect incongruence that most of the sections recognized by Zohary and Heller could be the result of interspeciWc hybridization (Wendel (1984) are not monophyletic. A novel Wnding was the reso- and Doyle, 1998). Both maximum parsimony and Bayesian lution of a clade deWned by geography comprising the nine approaches to phylogenetic estimation were used. sampled African species. Steele and Wojciechowski (2003) sampled 23 species of Trifolium, including six North Ameri- 2. Materials and methods can species, in their cpDNA matK analysis. They obtained a clade of North American species and found sect. Lotoidea 2.1. Materials sensu Zohary and Heller to be polyphyletic. In a study focused on three subspecies of Trifolium nigrescens, Wil- A total of 218 species of Trifolium, representing ca. liams et al. (2001) demonstrated the utility of nrDNA ITS 86% of the genus, was sampled (Appendix 1). Five Trifo- sequences to resolve closely related taxa of the genus. lium species were represented by two (T. longipes, The goal of this study was to develop a molecular phylo- T. montanum, T. rusbyi) or three (T. nigrescens, T. subter- genetic framework for Trifolium based on comprehensive raneum) subspecies. Nineteen species from 10 other gen- taxonomic sampling and sequences of two loci, one nuclear era in the vicioid clade were also included (Table 2). The and one organellar. In addition to evaluating the classiWca- outgroup was chosen from the genus Lotus, represented tion of the genus, we use the phylogenetic results to obtain by 11 species. Although this genus is relatively distant evidence for hybrid speciation and to examine patterns of from Trifolium (Wojciechowski et al., 2004), previous life history (annual vs. perennial) and chromosome number cpDNA (Hu et al., 2000) and ITS (Hu et al., 2002) analy- change in Trifolium. Zohary and Heller (1984) considered ses have demonstrated that Lotus is an appropriate out- the perennial growth form to be ancestral in clover, a con- group for the clade of legumes that lacks the cpDNA clusion supported by the phylogenetic results of Watson inverted repeat. To facilitate communication, we use our et al. (2000). Although several previous authors have proposed infrageneric classiWcation of Trifolium through- described patterns of chromosome evolution in Trifolium out the results and discussion (Table 1; see Section 4.2, for (Cleveland, 1985; Goldblatt, 1981; Taylor et al., 1979; justiWcation of the classiWcation). N.W. Ellison et al. / Molecular Phylogenetics and Evolution 39 (2006) 688–705 691
Germplasm collections were the primary source of Trifo- at 65 °C for 5–10 min. The ITS region was ampliWed using lium samples, and represent a valuable resource for studies the primers EC-1 and EC-2 (Williams et al., 2001), and the of this genus (Taylor et al., 1979). Most of the accessions trnL (UAA) intron was ampliWed using the primers “c” and are deposited in the USDA-ARS National Plant Germ- “d” (Taberlet et al., 1991). PCR ampliWcation reactions plasm System (Appendix 1), and are readily available from were performed in a volume of 20 L containing 2 L of the this source. Additional seed accessions, including most of prepared DNA, 1£ PCR buVer (75 mM Tris–HCl, pH 8.8, the species of other genera, were obtained from the Margot 20 mM (NH4)2SO4, 0.1% v/v Tween 20), 1.5 mM MgCl2, Forde Forage Germplasm Centre, Palmerston North, New 200 M of each dNTP, 0.4 M of each primer, and 1 U of Zealand, and the N.L. Taylor Clover Germplasm Center, Taq DNA polymerase. AmpliWcation conditions consisted University of Kentucky. These accessions are available of a single cycle of 94 °C for 4 min followed by 35 cycles of upon request. To date, seed-grown plants from 135 acces- 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, followed sions have been deposited as voucher specimens at the by a Wnal extension of 72 °C for 7 min. PCR products were Dame Ella Campbell Herbarium, Massey University, Pal- electrophoresed directly on a low-melting point agarose gel merston North, New Zealand (MPN). Vouchers are also (Cambrex, Rockland, ME) and subsequently puriWed from maintained at the University of Kentucky, Department of the agarose gel as described in Williams et al. (2001). Plant and Soil Sciences Herbarium. Twenty-two samples Sequencing was performed on both strands using the Big- were obtained from herbarium specimens (Appendix 1). Dye Terminator Cycle Sequencing Kit (Applied Biosys- In addition to the accessions included in the present tems, Foster City, CA) in a volume of 10 L containing study, 167 of the 218 sampled Trifolium species were repre- 1 M of primer, 5 L of puriWed PCR product, and 10% sented by 1–34 additional accessions (Ellison and Williams, DMSO. Sequencing reactions were puriWed on mini spin unpublished data). A total of 686 accessions were columns prepared with Sephadex G-50 (Amersham Biosci- sequenced for both the nrDNA ITS and cpDNA trnL ences, Sweden) and electrophoresed on either an AB310, intron, and an additional 196 were sequenced for only ITS. AB3700, or AB3100 automated sequencer (Applied Biosys- These sequences were included in a series of neighbor-join- tems, Foster City, CA). ing analyses. In most cases, multiple accessions of a single species clustered together, and a single accession was cho- 2.2. Data analysis sen for inclusion in our study. When multiple accessions of the same species did not cluster together, plants were grown For each sequence, the sequence data for both strands from seed to conWrm identiWcation, and when possible, were assembled into a contig and manually edited using additional accessions were sequenced. This process revealed AutoAssembler (Applied Biosystems, Foster City, CA). that most discrepancies resulted from labeling errors, mixed Multiple sequence alignments were generated with the Clus- seed collections or misidentiWcations, and these accessions tal implementation within the MegAlign software (DNA- were excluded. Two species endemic to Ethiopia, Trifolium STAR, Madison, WI), with manual optimization. abyssinicum and T. decorum, are represented by two acces- Maximum parsimony analyses were implemented with sions with divergent sequences, since a single accession PAUP* 4.0b10 (SwoVord, 2002). Gaps were treated as miss- could not be determined as “representative” of the species. ing data. Heuristic searches were conducted on the ITS, For initial experiments, total genomic DNA was isolated cpDNA, and combined ITS and cpDNA data. Fifty shortest using the method of Thompson and Henry (1995) as trees were held at each of 1000 random addition sequences described in Williams et al. (2001). Subsequently, the with TBR swapping. The resulting trees were then used as method of Lefort and Douglas (1999), with minor modiW- the starting trees for a second round of TBR swapping, cations, was found to be more reliable. Using this method, retaining a maximum of 50,000 trees. Bootstrap values were DNA was isolated from ca. 10 mg seed, 5 mg fresh leaf, or obtained from 100 resamplings analyzed with 1000 random 2 mg dried leaf. Seeds were Wrst placed between two sheets addition sequence replicates and TBR swapping, holding a of weighing paper, crushed by striking with a small ham- single shortest tree at each replicate. Although we had a pri- mer, and then transferred to a 1.5 mL microcentrifuge tube; ori decided to combine data sets, we evaluated the degree of a piece of fresh or dried leaf was placed directly into a conXict between the sequence partitions using the relatively 1.5 mL microcentrifuge tube. A 400 L volume of extrac- conservative incongruence length diVerence (ILD) test (Far- tion buVer (50 mM Tris–HCl, pH 8.0, 20 mM EDTA, 0.7 M ris et al., 1995; Hipp et al., 2004), and a more liberal crite- NaCl, 0.4 M LiCl, 1% w/v CTAB, 1% w/v PVP 40, 2% w/v rion, a comparison of clades based on bootstrap support SDS) was added to each tube. Leaf samples were ground values (Binder and Hibbett, 2002). To identify potential con- with a plastic pestle, while seed samples required only brief Xict, plots were made of bootstrap values for all clades with vortexing. Samples were digested at 65 °C for 20 min to 1 h, 770% support in the combined or separate analyses. The and then extracted once with 300 L chloroform–isoamyl ILD test was implemented in PAUP* with 100 replicates of alcohol (24:1). DNA was precipitated from the aqueous the partition homogeneity test using the above heuristic phase by the addition of an equal volume of isopropanol, search parameters. ILD tests were also conducted excluding with centrifugation for 15 min. DNA pellets were rinsed taxa with conXicting placement in the separate nrDNA ITS with 750 L of 70% ethanol and resuspended in 50 L H2O and cpDNA trnL analyses. 692 N.W. Ellison et al. / Molecular Phylogenetics and Evolution 39 (2006) 688–705
Bayesian analyses of the separate and combined data shown) is very similar to the Bayesian tree, diVering primar- were conducted with MrBayes 3.0b4 (Huelsenbeck et al., ily in the larger number of polytomies. Bayesian posterior 2002; Ronquist and Huelsenbeck, 2003). The best-Wt mod- probabilities (PP) and parsimony bootstrap (BP) values are els of sequence evolution were chosen using the hierarchical well correlated, with the Bayesian values consistently higher Likelihood Ratio Test (hLRT) and Akaike Information (Figs. 1–7). In the MP analysis, 119 clades have BP support Criterion (AIC), calculated with MrModeltest 2.0 770%, and 78 have 790% support. (Nylander, 2004). A model with six variable substitution Two key relationships, the monophyly of Trifolium and types, gamma-distributed among site rate variation, and an the sister group relationship between Trifolium and estimated proportion of invariant sites (GTR + +I) was Trigonella + Melilotus, have relatively low parsimony BP chosen for nrDNA ITS. A transition/transversion model (62% and <50%) and moderate to low Bayesian PP support (two variable substitution types), gamma-distributed (0.85 and 0.69, respectively). Both analyses resolve an initial among site rate variation, and an estimated proportion of subdivision of Trifolium between subgenus Chronosemium invariant sites (HKY + + I) was chosen for cpDNA trnL. and the rest of the genus (Fig. 1). Subgenus Chronosemium These models were applied to their respective partitions in is split into two well-supported clades, characterized by the separate and combined analyses. In the combined anal- diVerent chromosome numbers (Fig. 2). ysis, three runs of 1,000,000 generations, and one run of Subgenus Trifolium (Fig. 3) has a paraphyletic grade at 5,000,000 generations were conducted. In the separate anal- its base, comprising sect. Paramesus and Wve Eurasian yses, a single run of 5,000,000 generations was conducted. perennials that we place in sect. Glycyrrhizum and sect. In each run, trees were sampled every 100 generations and Lupinaster (Table 1). These Wve species are sometimes burn-in was determined by inspection of the log-likelihoods placed in two segregate genera (see Section 4.2.). There is of sampled trees. Branch length information was recorded strong support for a sister group relationship between sect. and averaged across all retained trees, and majority rule Lupinaster and the remaining Wve sections of the subgenus consensus trees were computed to obtain posterior proba- (Fig. 3). The monophyly of these Wve sections, and their bilities. Clades with >85% bootstrap support and >0.95 inter-relationships, are well supported by the Bayesian posterior probability are considered well supported. analysis (PP 70.95) but most have <85% BP support. Chromosome numbers (missing for 41 species) and plant Sections Trifolium and Trichocephalum are resolved as life history (annual or perennial) were recorded for Trifo- monophyletic sister taxa (Fig. 4). Section Trichocephalum lium following Gillett and Taylor, 2001 (Appendix 1). For contains two well-supported clades, excluding T. israeliti- the other genera, life history information was obtained cum. This species, that was originally described as a variety from the LegumeWeb database (ILDIS, 2002) and chromo- of T. subterraneum, is distinct from that species, but its some numbers were obtained from the TROPICOS data- position in the section is poorly resolved. Section Trifolium base (Goldblatt and Johnson, 2003). Parsimony can be divided into two large clades: one that is well sup- optimization of character states was performed in Mesquite ported (clade A in Fig. 4) and one that has little statistical 1.05 (Maddison and Maddison, 2004). Character states support (clade B in Fig. 4). In contrast, interspeciWc rela- were unordered and all changes were equally probable. tionships are better resolved within clade B than within clade A. 3. Results Section Vesicastrum (Fig. 5) unites all the sampled sub- Saharan species of Trifolium (clades E and H) together with 3.1. Combined analyses four Eurasian species (clade C) previously classiWed in sect. Lotoidea and all species previously classiWed in sects. Sequences of the nrDNA ITS and cpDNA trnL intron Mistyllus (clades D and J) and Vesicaria (clades F and G). were obtained from 257 accessions (Appendix 1). The ITS The latter two taxa are thus polyphyletic. The clovers of alignment was 828 bp, with 452 variable and 377 parsimony sub-Saharan Africa (clades E and H) are also not mono- informative sites. The trnL intron alignment was 783 bp, phyletic, and their relationships are generally unresolved or with 340 variable and 241 parsimony informative sites. All weakly supported. With the exception of three species for- DNA sequences used in this analysis were generated by us merly placed in sect. Mistyllus (clade J) and one in sect. Tri- and are deposited in GenBank (Appendix 1). Sequence folium (not sampled here), all sub-Saharan Trifolium had alignments are available from TreeBase as Accession been previously classiWed in sect. Lotoidea. Two Ethiopian S1387. species (Trifolium abyssinicum, T. decorum) represented by Four Bayesian analyses (three times 1,000,000 and one two accessions are each resolved as polyphyletic. Whether of 5,000,000 generations; burn-ins of 2000 and 5000 trees, this results from misidentiWcations or unrecognized taxa respectively) of the combined sequence data set consistently remains to be determined. The sub-Saharan clovers are not converged on the same narrow range of log-likelihoods and well studied, and this result may reXect the uncertain taxon- tree topologies. The maximum parsimony (MP) analyses omy of this group. resulted in 50,000 trees (the maxtrees limit) of length 2977 Section Trifoliastrum is restricted to ca. 20 Eurasian spe- (consistency index D 0.44, retention index D 0.84). The cies (Fig. 6) formerly classiWed in sect. Lotoidea. This clade topology of the parsimony strict consensus tree (not includes the economically important white clover, N.W. Ellison et al. / Molecular Phylogenetics and Evolution 39 (2006) 688–705 693
Parochetus communis
100 Galega officinalis 1.00 100 1.001.00 Galega orientalis 1.00 Cicer arietinum Pisum sativum 96 1.001.00 99 1.00 Lathyrus laevigatus 1.00 93 Lathyrus sativus 1.00 Vicia sativa 63
0.92 100 Vicia cracca 0.51 1.00 Vicia villosa Trigonella foenum graecum 100 1.00 98 Melilotus albus 1.00 Melilotus officinalis
60 0.69 0.69 1.00 100 Trifolium subg. Chronosemium 1.00 62 0.85 83 Trifolium subg. Trifolium 0.99
Ononis mitissima Medicago lupulina 0.54 100 Medicago ruthenica 1.00 0.1 Medicago sativa 0.53 98 Medicago polymorpha 1.00 Medicago truncatula
Fig. 1. The position of Trifolium among the genera of the vicioid clade. The outgroup Lotus is not shown. The area of the triangles is proportional to the number of species in each Trifolium subgenus. In Figs. 1–7, the combined analysis Bayesian majority rule consensus tree with average branch lengths are represented; scale bars are substitutions per site. Bayesian posterior probabilities are below branches, parsimony bootstrap values are above. Values below 0.50 or 50% are not shown. Symbols placed after species names represent an autapomorphic occurrence of the character state.
T. repens. Many relationships within this clade are resolved All of the eastern North American species of Trifolium in the Bayesian analysis, but have BP values below 50%. form a clade (N) in which the three annual (sometimes Likewise, the sister-group relationship between this clade biennial) species are monophyletic (Fig. 7). Three South and the American clovers (sect. Involucrarium) has <50% American species comprise a well-supported clade (O). The BP, but PP D 0.99 (Fig. 3). remaining species (clades P, Q, T. brandegei, and T. dasy- All of the species in sect. Involucrarium are native to the phyllum) are primarily distributed in the Intermountain western Hemisphere. Although there are several well-sup- Region of western North America. Neither large clade, nor ported clades, relationships among them are not resolved the majority of inter-relationships among these species are (Fig. 7). The Wrst dichotomy has no BP support and a low well supported (Fig. 7). PP (0.60), thus it is equivocal whether this clade has a North (T. breweri) or South American (T. amabile, T. peru- 3.2. Separate analyses vianum) origin. A polytomy of two species (T. brandegei, T. dasyphyllum) and seven clades (K–Q) make this the most The Bayesian and MP analyses of the cpDNA trnL poorly resolved portion of the phylogenetic results. The intron sequences result in poorly resolved topologies (not species of this section were previously classiWed in sect. shown). In the majority rule consensus of 50,000 MP trees Involucrarium (clades K, L, and T. oliganthum) or sect. (897 steps, CI D 0.56, RI D 0.43) only 43 clades have BP Lotoidea. The species of clades K and L are characterized support 770%, and 22 have 790% support. The Bayesian by an involucre of fused bracts below the inXorescence. and MP analyses of the nrDNA ITS sequences result in Clade M includes the western North American annuals well resolved and similar topologies (not shown). For this that lack an involucre, plus the involucrate T. oliganthum. partition, 101 clades have BP support 770%, and 60 have 694 N.W. Ellison et al. / Molecular Phylogenetics and Evolution 39 (2006) 688–705
T. aureum
96 T. spadiceum 1.00
95 T. sintenisii 1.00 T. badium 92 1.00 T. rytidosemium
100 T. boissieri 1.00 T. phitosianum 93 1.00 T. praetermissum
T. billardieri 97 1.00 T. erubescens 87 100 1.00 0.99 T. philistaeum 1.00 cp aneuploid 2n = 14 T. campestre 96 2n = 14, 16 1.00 0.99 T. dubium 0.99 polyploid 2n = 30 T. grandiflorum
annual T. brutium perennial 70 0.81 T. micranthum 98 1.00 0.05 T. patens
Fig. 2. Subgenus Chronosemium. Branch shading corresponds to life history. All species are 2n D 16 or unknown, except as noted. The arrow denotes the inferred origin of the chloroplast in T. campestre and T. dubium.
790% support in the majority rule consensus of 50,000 The comparison of the cpDNA and nrDNA trees identi- MP trees (2056 steps, CI D 0.39, RI D 0.81). Based on the Wes several cases of potential reticulate evolution in Trifo- ILD test, the two partitions are signiWcantly diVerent lium: (P D 0.01). Removal of all non-Trifolium sequences (where several topological discrepancies were observed) and 12 (1) Trifolium campestre and T. dubium have identical species of Trifolium with well supported but conXicting trnL intron and ITS sequences (Fig. 2). The two spe- positions, still results in signiWcantly diVerent partitions cies are morphologically similar but readily distin- (P D 0.01). guishable, and diVer in chromosome numbers: Despite extensive topological incongruence, few of the 2n D 14 and 2n D 30, respectively (H. Ansari, pers. conXicting clades have high BP support in the separate comm.). In the cpDNA topology they form a sister partitions (Fig. 8). Among the 22 highly supported group to T. billardieri, T. erubescens, and T. philista- (790% BP) clades in the cpDNA bootstrap tree, only eum (all 2n D 16), while in the nrDNA and combined seven have 690% BP in the nrDNA analysis. Six of these topology (Fig. 2) they are sister to T. grandiXorum seven clades have 790% BP support in the combined (2n D 16). This is the only example of a conXict where topology. Due to poor resolution in the cpDNA topology, the alternative single gene topologies are both well 45 of the highly supported nrDNA clades have 690% BP supported (cpDNA BP D 87% and PP D 1.0; nrDNA support with cpDNA. These bootstrap comparisons dem- BP D 97% and PP D 1.0). onstrate that despite topological incongruence, the com- (2) Trifolium pannonicum is a polyploid, with a range of bined topology has more nodes with increased support reported chromosome numbers (see Section 4.4.). and retains all but two of the highly supported clades With cpDNA, this species is placed in clade B found in the separate analyses. Furthermore, the poorly (Fig. 4) of sect. Trifolium (BP D 70%), while the resolved cpDNA topology does not reduce resolution in nrDNA results place it in clade A (BP D 100%). The the combined vs. nrDNA analyses. actual ancestry of T. pannonicum may be complex. In N.W. Ellison et al. / Molecular Phylogenetics and Evolution 39 (2006) 688–705 695
100 rense share identical trnL sequences (Fig. 5); T. stolzii sect. Trichocephalum 1.00 is united with T. rueppellianum and T. cryptopodium D 66 (BP 88%); and the positions of T. masaiense and T. 1.00 semipilosum are not resolved. With nrDNA, T. cheran- ganiense, T. masaiense, and T. semipilosum are mono- sect. Trifolium phyletic (Fig. 5, clade E, BPD79%); T. usambarense 0.95 and T. stolzii comprise another (BP D100%); and T. 99 rueppellianum and T. cryptopodium are unresolved. 52 1.00 sect. Vesicastrum The same clades are seen in the nrDNA and combined 0.98 Bayesian analyses with higher posterior probabilities. The chromosome number for T. stolzii is uncounted, T. cryptopodium is reported as both 2nD16 and 55 2nD 48, and the other species are 2nD16. 0.99 (5) One case of weakly supported conXict involves T. 99 sect. Trifoliastrum cyathiferum (2n D 16), a North American species. 1.00 With cpDNA, T. cyathiferum has the same trnL 87 intron sequence as T. buckwestiorum, T. variegatum, 1.00 0.99 and T. polyodon (Fig. 7). In the nrDNA and com- 72 sect. Involucrarium bined analyses, T. cyathiferum is placed in a clade of 1.00 annual species that share the morphological feature of inXated fruiting corollas. Bootstrap support is annual <50% and the Bayesian PP is only 0.70 (nrDNA and polyploid perennial combined analyses) for a clade with T. physanthum, T. gordejevii T. barbigerum, and T. jokerstii. 83 100 0.10 0.99 1.00 T. eximium sect. Lupinaster 3.3. Character evolution 62 0.93 T. lupinaster 2n = 16,32,48 3.3.1. Life history T. alpinum Due to limited sampling of species diversity in other genera 100 sect. Glycyrrhizum of the vicioid clade (Table 2), the ancestral life history for Trifo- 1.00 T. polyphyllum lium cannot be inferred. The annual habit is the ancestral state reconstruction for subgenus Chronosemium (Fig. 2), while the T. glanduliferum ancestral state for subgenus Trifolium is equivocal (Fig. 3). 100 sect. Paramesus 1.00 T. strictum Within this subgenus, the common ancestor of sects. Trifolium and Trichocephalum is inferred to be annual, while the common Fig. 3. The major clades of subgenus Trifolium. The area of the triangles is ancestor of the three other large sections is inferred to be peren- proportional to the number of species in each clade. Branch shading cor- nial (Fig. 3). Within subgenus Chronosemium there is one responds to life history. All species shown here are 2n D 16, except for T. lupinaster; T. gordejevii is uncounted. change to the perennial habit involving two species (Fig. 2), and within sect. Trifolium there are seven changes (Fig. 4). Five of the cpDNA tree, there is weak BP support (65%) but these are autapomorphic, while the others involve clades of a high PP (0.97) for a clade with T. patulum and three and seven species, respectively. Among the sections with a T. squamosum, two species that are not united in the perennial ancestral state, there are at least Wve changes to the nrDNA or combined results. The nrDNA tree places annual habit in sect. Vesicastrum (Fig. 5), three changes in sect. T. pannonicum in a polytomy at the position seen in Trifoliastrum (Fig. 6), and Wve changes in sect. Involucrarium, the combined Bayesian tree (Fig. 4). including three in clade K (Fig. 7). In sect. Vesicastrum, charac- (3) Trifolium repens, a 2nD32 tetraploid, is sister to T. pal- ter state reconstruction within the large African clade H is ham- lescens (2nD 16) in the cpDNA analysis (BPD67%), pered by limited phylogenetic resolution, taxonomic uncertainty, and to T. occidentale (2nD 16) in the nrDNA analysis and the presence of several species that are polymorphic for life (BPD79%). It must be noted that neither relationship history. However, there appears to be several reversals back to is obtained in the combined analysis (Fig. 6). The sep- the perennial habit (Fig. 5). There is also evidence for two rever- arate Bayesian analyses result in high support sals back to the perennial habit within sect. Trifoliastrum (PPD1.0) for the nrDNA placement, but no support (Fig. 6), but no reversals are reconstructed in sect. Involucrarium (PPD0.58) for the cpDNA placement. This is one of (Fig. 7). the few instances where the posterior probability is lower than the bootstrap value. 3.3.2. Chromosome numbers (4) Several cases of conXict involve a group of East Afri- The ancestral chromosome number in Trifolium is diploid can species. Trifolium cheranganiense and T. usamba- 2nD16. Our sampling included 23 of the 24 species with 696 N.W. Ellison et al. / Molecular Phylogenetics and Evolution 39 (2006) 688–705
100 T. batmanicum 99 1.00 T. globosum 97 T. subterraneum ssp subterraneum Sect. Trichocephalum 100 1.00 87 T. subterraneum ssp brachycalycinum 1.001.00 1.00 0.96 T. subterraneum ssp yanninicum 1.00 T. israeliticum 65 0.81 89 T. pauciflorum 100 T. pilulare 0.81 99 1.00 1.00 T. eriosphaerum 1.00 T. meduseum T. leucanthum 81 T. caucasicum 62 T. ochroleucum 1.00 T. canescens 0.98 100 T. dasyurum 73 0.98 1.00 T. purpureum 59 T. palaestinum 0.69 0.98 T. dichroanthum 66 1.00 0.97 1.00 T. angustifolium T. trichocephalum 1.00 85 T. isodon T. squarrosum 87 0.69 1.00 T. miegeanum 1.00 T. obscurum 96 1.001.00 T. clypeatum A 1.00 96 T. constantinopolitanum 1.00 59 100 T. echinatum 1.00 1.00 T. salmoneum 0.94 T. vavilovii 59 0.990.99100 T. plebeium 0.99 62 1.00 T. scutatum T. alexandrinum 0.98 55 T. apertum Sect. 0.95 0.97 T. berytheum T. pannonicum Trifolium 0.95 100 T. haussknechtii 81 T. sylvaticum 1.00 T. trichopterum 1.00 100 T. cherleri T. hirtum 1.00 100 T. scabrum 90 T. dalmaticum 96 1.001.00 1.00 B 1.001.00 0.99 T. lucanicum 0.61 1.00 74 T. bocconei annual 90 T. affine 0.99 1.00 T. arvense 88 T. ligusticum perennial T. noricum 1.00 99 T. incarnatum 98 T. stellatum 100 1.00 1.00 T. striatum aneuploid 1.00 T. latinum T. heldreichianum 0.99 T. gemellum polyploid 1.00 T. phleoides 67 T. velebiticum 0.99 68 T. patulum 97 T. rubens 2n = 10 T. medium 2n = 12 1.00 T. alpestre 0.62 73 T. longidentatum 2n = 14 0.57 0.98 T. pignantii 2n = 16 100 T. squamosum 91 T. barbeyi 1.00 2n = 48 67 T. lappaceum 0.93 1.00 100 T. andricum polyploid series 97 0.93 98 1.00 T. diffusum 1.00 T. pallidum 0.05 1.00 T. pratense
Fig. 4. Sections Trifolium and Trichocephalum. Branch color corresponds to chromosome numbers. The chloroplast results place T. pannonicum in a clade with T. patulum and T. squamosum, these three species are marked with stars. See text for clades A–B. known polyploid counts, including 11 species with both dip- glomeratum), and absent in the remaining sections. Within loid and polyploid records. Ancestral state reconstruction subgenus Chronosemium, there have been three reductions (Figs. 2–7) infers at least 22 independent origins of polyploidy to 2n D 14 (Fig. 2). In sects. Trifolium and Trichocephalum (not counting subsequent chromosome number increases in the situation is more complex (Fig. 4). The common ances- species with multiple ploidy levels). Although all of the large tor of both sections is potentially polymorphic for 2n D 14 clades have at least one inferred origin of polyploidy, the larg- and 2n D 16. There are two clades in sect. Trifolium charac- est number (10) occurs in sect. Involucrarium (Fig. 7). terized by 2n D 10, but it is equally parsimonious for their Aneuploidy, unlike polyploidy, is not evenly distributed ancestors to have been 2n D 14 or 2n D 16. Counts of across the phylogeny. It is very common in sect. Trifolium 2n D 12 are reported from three species (two of which are (Fig. 4, at least 12 times), present in subgenus Chronose- polymorphic for 2n D 12 and 2n D 14) in sect. Trifolium and mium (Fig. 2, three times) and sect. Trichocephalum (Fig. 4, one in sect. Trichocephalum. Each case of 2n D 12 appears to two times), restricted to single species in sect. Vesicastrum have evolved independently from the 2n D 14 condition (Fig. 5, T. resupinatum) and sect. Trifoliastrum (Fig. 6, T. (Fig. 4). Although it is possible that reversals from 2n D 14 N.W. Ellison et al. / Molecular Phylogenetics and Evolution 39 (2006) 688–705 697
C 76 T. hybridum T. ornithopodioides 1.00 85 1.00 100 T. balansae 1.00 T. michelianum T. spumosum 99 88 T. vesiculosum 1.00 T. mutabile 52 D 94 1.00 T. setiferum 1.00 0.98 94 T. aintabense 1.00 T. argutum 74 T. cheranganiense E T. masaiense 1.00 1.00 T. semipilosum 63 F 88 T. physodes 0.99 1.00 T. tumens T. fragiferum G 100 1.00 1.00 T. bullatum 1.00 T. tomentosum 0.67 77 0.97 58 T. clusii 0.99 T. resupinatum 59 T. somalense 2n =14,16, 32 0.89 T. petitianum cp 0.66 T. pseudostriatum T. acaule H 97 0.60 T. africanum 2n = 32 1.00 T. burchellianum 2n = 96 T. schimperi 0.54 97 T. usambarense 1.00 T. stolzii T. cryptopodium 2n = 16, 48 0.71 T. rueppellianum 63 T. purseglovei Africa 1.00 0.97 64 T. lanceolatum T. polystachyum 1.00 0.56 T. quartinianum Africa & Eurasia 0.96 T. steudneri 0.99 80 J aneuploid 0.97 T. lugardii T. spananthum polyploid 0.84 T. mattirolianum 0.99 T. simense 58 annual 0.79 T. decorum B 0.91 T. multinerve T. baccarinii perennial T. abyssinicum B 0.54 T. decorum A 0.57 85 T. abyssinicum A 0.05 0.98 T. tembense 55 T. pichisermollii 0.65 T. bilineatum
Fig. 5. Section Vesicastrum. Branch shading corresponds to life history. All species are 2n D 16 or unknown, except as noted. The arrow denotes the inferred origin of the chloroplast in T. usambarense. See text for clades C–H and J. to 2n D 16 have occurred in both sections (T. aYne, T. haus- Wojciechowski, 2003; Watson et al., 2000). A novel Wnding of sknechtii, T. latinum, and T. pauciXorum) all such cases can Steele and Wojciechowski (2003) was the sister group relation- also be explained by lineage sorting of ancestral chromo- ship between Trifolium and the Fabeae (Table 2). In contrast, some number polymorphisms (Fig. 4). Across the genus, our combined nrDNA ITS and trnL intron analysis resolved eight species are reported to have both aneuploid and dip- Trifolium and Trigonella+Melilotus of Trifolieae as sister loid counts, and two have two diVerent aneuploid counts, groups (Fig. 1). Although this relationship is very poorly sup- suggesting that polymorphic ancestry is possible. ported, it is more consistent with traditional classiWcation (Heyn, 1981). Limited sampling of the other genera of the vici- 4. Discussion oid clade (Table 2) has limited our ability to better resolve the sister group of Trifolium, and the phylogenetic relationships 4.1. Comparison to prior studies among Trifolieae and Fabeae genera. In accordance with our study, Steele and Wojciechowski (2003) resolved Trifolium as Our results show both concordance and conXict with previ- monophyletic, however they obtained greater support. Among ous molecular phylogenetic studies of Trifolium (Steele and the 23 Trifolium species they sampled, many of the same 698 N.W. Ellison et al. / Molecular Phylogenetics and Evolution 39 (2006) 688–705
T. montanum ssp montanum 99 1.00 T. ambiguum 2n = 16, 32, 48 98 0.99 T. montanum ssp humboldtianum
T. retusum
T. suffocatum
T. nigrescens ssp nigrescens
99 1.00 T. isthmocarpum 1.00 T. uniflorum 2n = 32
0.80 T. nigrescens 0.88 ssp meneghinianum T. nigrescens ssp petrisavii
0.64 T. occidentale
nr 81 T. repens 2n = 32 1.00 aneuploid T. pallescens cp polyploid T. glomeratum 2n = 14, 16 annual 97 T. thalii perennial 0.57 1.00 T. parnassii
0.05 T. cernuum
Fig. 6. Section Trifoliastrum. Branch shading corresponds to life history. Arrows denote the inferred ancestry of T. repens. All species are 2n D 16, except as noted. relationships reported here were resolved (including the sister nied by diversiWcation of structures associated with seed dis- group status of subgenus Chronosemium to the remainder of persal. In contrast to those genera, the clover fruits are fairly the genus and monophyly of the American species), but often unmodiWed, but the more variable corolla and calyx are with lower bootstrap support. responsible for the diversity of dispersal mechanisms (Zoh- In contrast to the agreement between our results and ary, 1972). This Xoral diversity has led to classical (Presl, Steele and Wojciechowski (2003), the Watson et al. (2000) 1831) and recent proposals to split Trifolium into a series of study contains several conXicts, including a nested position smaller genera (Hendrych, 1976, 1978; Khokhrjakov, 1998; of subgenus Chronosemium and the polyphyly of sect. Tri- Roskov, 1990; Soják, 1986; see also Small, 1987). Based on folium. Relationships in their separate and combined our molecular phylogenetic results, most of these segregates cpDNA restriction site and nrDNA ITS sequence analyses are either polyphyletic (Amoria C. Presl, Lupinaster Fabr., were generally poorly resolved. Comparisons of published Xerosphaera Soják) or create paraphyletic groups (Bobrovia nrDNA ITS sequences suggest that some of the Watson A.P. Khokhrjakov Dsect. Glycyrrhizum) and Ursia I.T. et al. (2000) accessions (e.g., T. alpinum, T. cherleri, T. sca- Vasil’chenko ( D sect. Lupinaster). The only segregate that brum, and T. subterraneum) were misidentiWed or misla- might be considered useful is elevating subgenus Chronose- beled. As a result, these species were resolved in unexpected mium (commonly known as hop clovers) to the genus Chry- positions and apparently contributed to the discrepancies saspsis Desv. (Hendrych, 1976, 1978). To maintain between our studies. In fact, none of the Watson et al. nomenclatural stability, we do not advocate this change, but (2000) ITS sequences match any of ours, and for this reason propose that the hop clovers should be considered one of two we did not integrate their sequences into this study. subgenera of Trifolium. Following this classiWcation, the remaining species of Trifolium are placed in subgenus Trifo- 4.2. Implications for the classiWcation of Trifolium lium. We further propose recognizing eight of its clades as sections (Table 1 and Fig. 3). The designation of this infra- Like other large genera of Leguminosae (e.g., Astragalus, generic classiWcation is greatly facilitated by the thorough Indigofera, and Mimosa), speciation in Trifolium is accompa- nomenclatural work of Hendrych (1988). The larger clades N.W. Ellison et al. / Molecular Phylogenetics and Evolution 39 (2006) 688–705 699
T. breweri 98 T. amabile T. peruvianum 1.00 T. dasyphyllum 2n = 16, 24 T. brandegei 0.99 T. wigginsii 94 T. wormskioldii 2n = 16, 32 72 88 T. buckwestiorum 1.00 T. variegatum 1.00 1.00 1.00 T. polyodon cp T. fucatum 94 T. cyathiferum K 100 T. physanthum 1.00 0.70 100 T. barbigerum 0.62 1.00 1.00 T. jokerstii 97 T. monanthum 1.00 T. pinetorum 0.60 T. depauperatum T. trichocalyx 0.61 58 98 T. chilense 0.94 1.00 T. triaristatum 58 T. willdenovii 0.99 T. obtusiflorum 100 T. microdon L 94 T. microcephalum 1.00 1.00 T. vernum 100 T. bifidum 1.00 T. oliganthum 73 100 T. ciliolatum M T. palmeri 1.00 73 1.00 52 0.78 T. gracilentum T. amoenum 1.00 1.00 100 T. dichotomum 2n = 32 1.00 T. albopurpureum T. macraei 99 T. reflexum T. bejariense 82 1.00 T. carolinianum 82 N 1.00 70 T. virginicum 0.96 T. calcaricum 1.00 T. stoloniferum 95 T. argentinense O T. riograndense 1.00 0.55 T. polymorphum 2n = 16, 32 polyploid T. leibergii .90 T. andersonii P T. nanum .68 T. parryi 2n = 16, 32 South America 0.82 0.70 T. howellii 0.58 T. macilentum T. thompsonii N. & S. America 74 T. macrocephalum 2n = 32, 48 1.00 T. plumosum 2n = 32 0.68 56 T. eriocephalum annual 0.84 T. douglasii 0.83 T. owyheense 76 T. andinum 84 perennial 1.00 T. gymnocarpon 52 1.00 T. haydenii Q T. beckwithii 2n = 48 0.78 52 T. latifolium 2n = 16, 32 70 T. longipes ssp elmeri 0.61 0.99 79 T. longipes ssp longipes 2n = 16, 32, 48 0.99 86 T. rusbyi 1.00 1.00 T. rusbyi ssp oreganum T. kingii T. bolanderi 0.57 T. lemmonii 0.05
Fig. 7. Section Involucrarium. Branch shading corresponds to life history. The arrow denotes the inferred origin of the chloroplast in T. cyathiferum. All species are 2n D 16 or unknown, except as noted. See text for clades K–Q. could be further divided into subsections, but this is prema- possess gland-tipped stipules, a unique feature in the genus. ture pending more intensive sampling and better phyloge- In sect. Trifolium the pod is included in the calyx tube, netic resolution at this taxonomic level. With few exceptions, which is either narrowed or closed by hairs or tissue devel- the subsections and series recognized by Zohary and Heller opment. Section Trichocephalum is characterized by inXo- (1984) are not monophyletic. rescences with a mixture of fertile and sterile, apetalous Subgenus Chronosemium and some of the proposed sec- Xowers. The remaining Wve sections include species that tions of subgenus Trifolium can be characterized morpho- were classiWed by Zohary and Heller (1984) in sect. Lotoi- logically, following the descriptions of Zohary and Heller dea. They characterized this section by the absence of mor- (1984). Species of subgenus Chronosemium are character- phological traits observed elsewhere in the genus. Thus, no ized by persistent banner petals that function in the dis- putative synapomorphies for these Wve sections are appar- persal of the small one-seeded fruit, usually yellow corollas, ent. The character of inXated bladder-like calyces is and pinnate trifoliolate leaves. The latter two characters are restricted to sect. Vesicastrum; however, these species rare, or absent, in subgenus Trifolium and may be symple- (Fig. 5, clades D, F, G, and J) are polyphyletic. Likewise, siomorphic in Trifolieae. The two species of sect. Paramesus sect. Involucrarium contains numerous species with an invo- 700 N.W. Ellison et al. / Molecular Phylogenetics and Evolution 39 (2006) 688–705
100 Trifolieae + Fabeae at 17.1–30.2 million years ago (mya). They did not sample subgenus Chronosemium, thus their 90 matK chronogram places the origin of subgenus Trifolium at 12.4 mya. Because the genus must have originated between these dates, we hypothesize that Trifolium origi- 80 nated in the Early Miocene, 16–23 mya. Watson et al. (2000) sampled nine African species, and 70 resolved them as monophyletic. With our increased taxo- nomic sampling, 29 sub-Saharan African species comprise
ITS and CP combined 60 the majority of sect. Vesicastrum, but are not monophyletic (Fig. 5). The most parsimonious reconstruction implies two 50 dispersal events (clades E and H) from the Mediterranean 50 60 70 80 90 100 region to sub-Saharan Africa. A third event is needed to ITS account for T. stipulaceum Thunb., not sampled here, a South African species of sect. Trifolium. We also did not 100 include the poorly known T. ankaratrense Bosser, endemic to Madagascar (Bosser, 1959). No Trifolium species cur- 90 rently grow in the Saharan or Arabian deserts. However, it is noteworthy that the sister group (clade G) to the large 80 African clade H includes T. clusii and T. tomentosum, two Mediterranean species that occur further into the northern 70 sections of these deserts than any other Trifolium. Further- more, T. fragiferum has been reported for Ethiopia (Zohary and Heller, 1984). If this species is indeed native to that
ITS and CP combined 60 country (Thulin, 1983), it would suggest that the remaining species of clade G represent a secondary dispersal out of 50 50 60 70 80 90 100 Africa (Fig. 5). All of the North and South American species of Trifo- CP lium comprise a monophyletic group (Fig. 7). No morpho- Fig. 8. Bootstrap percentages for clades with 770% support in the com- logical synapomorphies are known for this clade and it has bined and/or separate analyses. The y-axis gives the values from the com- not been recognized in any previous classiWcation. This bined analysis, the x-axis gives the values for the separate nrDNA ITS (above) and cpDNA trnL intron (below) analyses. Values <50% were not result parallels the discovery of species-rich, morphologi- determined, and are treated as equivalent to 50%. cally diverse “cryptic American clades” in other large tem- perate genera (Noyes and Rieseberg, 1999; Wojciechowski lucre of fused bracts below the inXorescence (Fig. 7, clades et al., 1993). In Aster, the North American species had been K, L, and T. oliganthum), but these do not comprise a clade. classiWed in 14 genera and were assumed to represent at The above generalizations remain unproven pending an least Wve independent colonization events (Noyes and explicit phylogenetic analysis of morphological traits in Rieseberg, 1999). Like American Trifolium, their mono- Trifolium. phyly had not been previously hypothesized. In Astragalus, the North American clade shares aneuploid chromosome 4.3. Biogeography numbers (Spellenberg, 1976), but no morphological syna- pomorphies are known. Their monophyly was not con- In agreement with previous phylogenetic analyses of Tri- Wrmed until molecular phylogenetic studies were conducted folium, two largely geographic clades are recognized involv- (Wojciechowski et al., 1993). Like Trifolium, the Astragalus ing the African (sect. Vesicastrum, Fig. 5) and American clade has both North and South American representatives. (sect. Involucrarium, Fig. 7) species, respectively. The In contrast, Astragalus has non-aneuploid boreal and mon- derived position of the African and American clades argues tane North American species that are derived from Eur- against a North American (Zohary, 1972) or African (Prit- asian lineages, while the montane species of Trifolium are chard, 1962) origin of the genus. Subgenus Chronosemium not closely related to ecologically similar Eurasian species. and the early diverging clades of subgenus Trifolium (sects. Trifolium diVers from both of these genera in the absence of Paramesus and Glycyrrhizum) are currently restricted to the Eurasian representatives in the “American” clade (contra Mediterranean basin (and adjacent regions of Eurasia), Astragalus echinatus Murr. and Erigeron uniXorus L.). consistent with a hypothesized Mediterranean origin of Due to poor resolution at the base of sect. Involucrarium Trifolium (Gillett, 1952; Taylor et al., 1979). Based on a fos- (Fig. 7), a North or South American origin cannot be conW- sil-calibrated penalized likelihood approach and the Bayes- dently resolved. Assuming a North American origin, seven ian analysis of two cpDNA genes (matK and rbcL), Lavin independent dispersals to South America can be inferred et al. (2005) estimated the origin of the crown clade of (Fig. 7), with no clade larger than three species. Unlike the N.W. Ellison et al. / Molecular Phylogenetics and Evolution 39 (2006) 688–705 701 situation in Africa, there is not a strong geographic barrier origin of T. repens, impeding eVorts to identify the progeni- to contemporary dispersal. In fact, T. amabile occurs dis- tors of this species. Ansari et al. (2004) included T. palles- continuously at high elevations (1600–3400 m) from south- cens in their molecular cytogenetic survey of 16 Trifolium ern Arizona to northern Argentina. The Texas endemic T. species for the distribution of a centromeric satellite amphianthum Torr. and Gray (previously considered a syn- sequence (TrR350) isolated from T. repens. The satellite onym of T. polymorphum) apparently belongs to clade O sequence is found in several other species of sect. Trifolia- (M. Vincent, pers. comm.). In both of these cases, the direc- strum including T. pallescens, and thus cannot be used tion of dispersal cannot be inferred. The remaining Wve alone to identify the progenitors of T. repens. intercontinental disjunctions are reconstructed as dispersal Trifolium pannonicum (sect. Trifolium) is a perennial spe- events from North to South America (Fig. 7). All involve cies native to the steppes of central and eastern Europe. A annual species, including three (T. depauperatum, T. micr- broad range of polyploid chromosome numbers (2n D 48, odon, and T. macraei) that are considered conspeciWc on 49, 60, 65, 96, 98, 126, 128, 130, and 180) has been reported both continents. These Wve cases can be interpreted as Cali- (Cleveland, 1985; Gillett and Taylor, 2001; Zohary and fornia–Chile “amphitropical” disjunctions (Raven, 1963). Heller, 1984). Many of these reports are unvouchered and The North American origin hypothesized here has also require conWrmation. Our phylogenetic results are the Wrst been found in other examples of this disjunction (Vargas to suggest that T. pannonicum originated through hybrid- et al., 1998). ization between two distantly related species of sect. Trifo- lium (Fig. 4). Unfortunately, the parental species remain 4.4. Potential cases of reticulate evolution unidentiWed. Trifolium pannonicum shares an identical trnL intron sequence with two species in clade B: T. patulum Integrating chromosome number changes and cpDNA/ (chromosome number unknown) and T. squamosum nrDNA conXict provides evidence for several examples of (2n D 16). However, these two species do not form a clade in hybrid speciation in Trifolium. However, some uncertainty the nrDNA or combined analyses. The isolated position of exists in each case, indicating the need for more intensive T. pannonicum in the nrDNA and combined analyses sug- taxonomic sampling, and incorporation of additional gests that the parental species from clade A may be unsam- sequence data (and chromosome counts) to conWrm the pled or extinct. hypotheses of reticulate evolution presented here. Two other examples of incongruence of cpDNA data Trifolium dubium (subgenus Chronosemium), a native of and ITS data may also result from interspeciWc hybridiza- western Eurasia, is widely introduced in North America, tion. The patterns of cpDNA and nrDNA incongruence and it is particularly common along the west coast of the among several African species are suggestive of reticulate continent (Gillett and Taylor, 2001). The species has long evolution (Fig. 5). The most striking example is Trifolium been considered a 2n D 28 tetraploid, but recent studies usambarense, which shares a trnL intron sequence with the have documented 2n D 30 (H. Ansari, pers. comm.). The distantly related T. cheranganiense. However, only diploid new number is consistent with a hybrid origin between 2n D 16 counts are known from T. usambarense, suggesting 2n D 14 T. campestre and a 2n D 16 common ancestor of T. that it may have acquired the chloroplast of T. cherangan- billardieri, T. erubescens, and T. philistaeum (Fig. 2). The iense via cytoplasmic introgression, and not polyploid spe- only discrepant Wnding is that T. campestre also shares the ciation. InterspeciWc hybridization may also explain the fact cpDNA of this latter clade. Cytoplasmic introgression (also that Trifolium rueppellianum, T. cryptopodium, and T. stol- known as chloroplast capture) is a common phenomenon zii comprise a well-supported clade in the cpDNA analyses, (Rieseberg et al., 1996), and is a likely explanation for this but not in the nrDNA analyses. While T. rueppellianum is a observation. It remains to be determined whether such diploid, both diploid and hexaploid counts are known from introgression involved the T. billardieri clade, or occurred T. cryptopodium. Unfortunately, the chromosome number directly between T. campestre and T. dubium. The latter is of T. stolzii is unknown. Until additional well-documented considered more likely, since these two species have no chromosome numbers are known for this group, no deWni- divergence at the trnL locus. tive conclusions can be drawn. A similar situation exists in Trifolium repens (sect. Trifoliastrum) is an allotetraploid the North American species T. cyathiferum, that is associ- (Williams et al., 1998), however attempts to identify its ated with diVerent clades in the cpDNA and nrDNA analy- parental taxa have been inconclusive (Ansari et al., 2004, ses (Fig. 7). The only reported chromosome count, from a 1999; Badr et al., 2002). Our phylogenetic results implicate single individual, is diploid (Gillett and Mosquin, 1967). As T. occidentale and T. pallescens as its diploid progenitors in the case of the above African species, additional chromo- (Fig. 6). Trifolium occidentale has long been considered a some counts are desirable. All of the above examples of potential ancestor of T. repens (but see Kakes and Char- apparent reticulate evolution involve species that are sym- donnens, 2000). The two species are morphologically simi- patric in at least part of their geographic range. lar and interfertile (Gibson and Beinhart, 1969). In It is remarkable that only 5–6 cases of apparent reticu- contrast, T. pallescens, a montane species distributed in the late evolution were discovered in our analysis of 218 Trifo- Alps and Pyrenees of Europe, has rarely been included in lium species. This Wnding is consistent with the presence of crossing studies and molecular genetic investigations of the strong genetic barriers to interspeciWc hybridization in the 702 N.W. Ellison et al. / Molecular Phylogenetics and Evolution 39 (2006) 688–705 genus (Taylor et al., 1980). It is noteworthy that each of the majority of inferred polyploid events occur in the three three apparent allopolyploid species (T. dubium, T. repens, large sections (Figs. 5–7) where aneuploidy is rare. Poly- and T. pannonicum) has putative parental species from well- ploidy is associated with the perennial habit in all but three separated clades. This could indicate that genetic reinforce- species (Taylor et al., 1979), and two of the annual polyp- ment occurs among closely related species, and that these loids (T. dichotomum and T. resupinatum) occur in sections reproductive isolation mechanisms, that are rarely over- that share a perennial ancestry. Among the 22 inferred come in natural populations, can be surmounted in rela- polyploid events, three are clearly allopolyploid (see Sec- tively distantly related species. ArtiWcial crosses between tion 4.4.). There is lability in the cpDNA and nrDNA posi- distantly related species can produce fertile hybrids (Cleve- tions of T. latifolium (results not shown) that is perhaps land, 1985; Taylor et al., 1980), but generally require the also indicative of allopolyploidy. This species is part of the technique of embryo rescue to succeed. Although natural T. longipes–T. rusbyi species complex (Fig. 7). The cytoge- allopolyploidy is apparently an infrequent phenomenon in ography of this widespread western North American clade Trifolium, two of the resulting species, T. dubium and T. has been well documented (Gillett, 1969), and our results repens, are evolutionary “success stories,” spreading with suggest further investigations with molecular markers and the assistance of humans into appropriate habitats around broader sampling would be very informative. The remain- the globe. ing hypothesized polyploidy events show no evidence of interspeciWc hybridization, and thus could be the result of 4.5. Life history evolution autopolyploidy. However, despite the thoroughness of our taxonomic sampling, it is not exhaustive and unsampled While we can be fairly conWdent that the ancestor of taxa could have contributed to these polyploids. Further- subgenus Chronosemium was an annual (Fig. 2), the ances- more, genetic evidence for autopolyploidy has only been tral state of subgenus Trifolium is uncertain (Fig. 3). Reso- reported for T. uniXorum (Chen and Gibson, 1982). lution of the polytomy between the annual sect. Paramesus Aneuploidy is most common in sects. Trifolium and and the perennial sect. Glycyrrhizum should clarify this. Trichocephalum (Fig. 4). These sections are characterized Within subgenus Trifolium, two sections (Trifolium and by apparent ancestral polymorphism for 2n D 16 and Trichocephalum) share an annual ancestry, while three sec- 2n D 14, followed by lineage sorting in descendent species. tions (Vesicastrum, Trifoliastrum, and Involucrarium) share The alternative explanation, the return to the 2n D 16 con- a perennial ancestry. Across the genus, initial transitions to dition through aneuploid increase, cannot be ruled out. the perennial habit (at least nine) are much less common Molecular cytogenetic investigations of these clades could than transitions to the annual habit (at least 19). However, diVerentiate between the two hypotheses. Taylor et al. in sect. Trifoliastrum (Fig. 5) two reversals to the perennial (1979) noted that reduced chromosome numbers are absent habit are reconstructed, and at least four are inferred in in the African and American species of Trifolium, and rare sect. Vesicastrum (Fig. 6). A hypothesis for this observation in perennial species. Our results extend their Wndings by is that the transition from annual to perennial is under demonstrating that aneuploidy is rare in the three sections more developmental constraint in the ancestrally annual (Vesicastrum, Trifoliastrum, and Involucrarium, Figs. 5–7) sections than in the ancestrally perennial sections. In gen- that share a perennial ancestry and contain all of the Afri- eral, perennial species occur at higher elevation habitats can and American species. The two instances of aneuploidy than the annual species. in these three sections involve annual species (T. glomera- tum and T. resupinatum), providing further evidence for a 4.6. Chromosome evolution correlation between these character states. Our results also suggest that aneuploid species have rarely given rise to Although several authors have reviewed chromosome polyploids. The only exception is T. dubium (Fig. 2), that evolution in Trifolium (Cleveland, 1985; Goldblatt, 1981; apparently combines a 2n D 16 and 2n D 14 genome (Ansari Taylor et al., 1979; Zohary and Heller, 1984), ours is the and Ellison, unpublished data). Trifolium trichocephalum Wrst study to examine these data from a phylogenetic per- (Fig. 4) and T. resupinatum (Fig. 5) are the only species with spective. In agreement with previous authors, our results aneuploid and polyploid counts, however in both species support 2n D 16 as the ancestral chromosome number in the the polyploidy is based on 2n D 16. genus. With the exception of aneuploidy in sects. Trifolium and We sampled 23 of the 24 known polyploids and all 31 Trichocephalum, the majority of chromosome number known aneuploids. Despite the greater number of aneup- changes in Trifolium have been autapomorphic. Most loids, there is little diVerence in the inferred number of instances of aneuploidy and polyploidy are known from aneuploid (19) and polyploid (22) events in the genus. species that are polymorphic for chromosome number, sug- These results reXect the fact that polyploidy is almost gestive of recent evolution of these cytologically diVerenti- always autapomorphic in Trifolium, while aneuploid events ated variants. Two cases were found where are more likely to demarcate clades. Apparently only one morphologically distinct species share identical cpDNA instance of polyploidy has been followed by subsequent and nrDNA sequences, but diVer in chromosome number: speciation (T. africanum and T. burchellianum, Fig. 5). The T. macraei (2n D 16) and T. dichotomum (2n D 32) (Fig. 7) N.W. Ellison et al. / Molecular Phylogenetics and Evolution 39 (2006) 688–705 703 and T. haussknechtii (2n D 16), T. sylvaticum (2n D 14), and (Fig. 8). Most importantly, several cases of potential inter- T. trichopterum (2n D 14) (Fig. 4). These cases can be speciWc hybridization were identiWed by comparing the explained by recent diVerentiation of the polyploid or aneu- position of species in the separate analyses (see Section ploid species. 4.4.). While hybridization for some of these species has been previously suspected, others are novel. In contrast to many 4.7. Maximum parsimony vs. Bayesian analyses other plants (Lihova et al., 2004; Nieto Feliner et al., 2004; Sang et al., 1995; Whittall et al., 2000) nrDNA additivity Bayesian approaches to phylogenetic reconstruction are was rarely observed in Trifolium species. Although this enjoying increasing popularity, despite lingering concerns facilitated analysis of these sequences, it provided limited over the robustness of these methods (Kolaczkowski and opportunity for the identiWcation of hybrids using this sin- Thornton, 2004). It has been repeatedly demonstrated that gle locus. In contrast, artiWcial hybrids do show clear Bayesian posterior probabilities are higher than parsimony nrDNA additivity (N. Ellison, unpublished data). bootstrap (Simmons et al., 2004; Suzuki et al., 2002), and thus it is important to not consider them as equivalent. In 4.9. Potential for DNA barcoding of Trifolium species our interpretations of clade support, we took both the bootstrap percentages and posterior probabilities into The use of DNA sequences to identify organisms has account. However, in the comparison of the separate been proposed as a more eYcient approach than traditional nrDNA and cpDNA analyses, we relied primarily on the taxonomic practices (Blaxter, 2004; Tautz et al., 2003). parsimony bootstrap values since they exhibit more even Kress et al. (2005) have demonstrated the eVectiveness of distribution of support values across the resulting clades. It such “DNA barcoding” in angiosperms using nrDNA and is notable in our combined analyses that several clades non-coding cpDNA sequences. In Trifolium, extensive demarcated by morphological characters or geographic dis- germplasm collections of most wild-collected species exist tributions had little or no bootstrap support but posterior (Morris and Greene, 2001); however rates of misidentiW- probabilities >.85. Prominent examples include the mono- cation may exceed 5–10% (R. Morgan, pers. comm.). Grow- phyly of the genus Trifolium, sect. Trifolium and sect. Invo- ing samples to maturity is time consuming, and many of the lucrarium. These examples of concordance with outside perennial species are diYcult to bring to Xower and fruit data suggest that the greater phylogenetic support and res- outside of nature. A DNA barcoding approach would be a olution obtained with the Bayesian approach better reXects useful supplement to existing identiWcation methods. In our evolutionary history in this genus. All of the topological study, 94% of the sampled Trifolium species had unique diVerences between the MP and Bayesian results are due to combinations of nrDNA ITS and cpDNA trnL intron the greater resolution of the latter. One such example sequences. When indels are considered, only Wve species involves the placement of a suspected interspeciWc hybrid, pairs and one triplet lack any sequence divergence. Thus, T. cyathiferum: the combined Bayesian topology resolves DNA-based identiWcation of Trifolium species is quite fea- this species within a clade of species that share inXated sible. It is important to note that DNA-based identiWcation fruiting corollas (Fig. 7), while the MP topology places it in Trifolium would be much more challenging without the unresolved at the base of the large sect. Involucrarium clade. availability of a comprehensive global monograph (Zohary It is a common occurrence for hybrids to be “pulled” to the and Heller, 1984) and biological information for most of base of a clade (McDade, 1992). This is apparent in other the genus (Gillett and Taylor, 2001). Such a robust taxo- cases (e.g., T. repens, Section 3.2). Thus, at least in this nomic foundation is lacking for the great majority of the instance, the Bayesian approach may be less prone to this world’s species. phenomenon than maximum parsimony. Acknowledgments 4.8. Utility of nrDNA ITS and cpDNA trnL intron in Trifolium The contributions of Helal Ansari and Miguel Dall’Agnol were invaluable for the initiation and the con- The topologies resulting from the separate nrDNA and tinuation of this research. The processing of voucher speci- cpDNA analyses were signiWcantly diVerent by the ILD mens at the Dame Ella Campbell Herbarium, Massey test, even after all genera outside of Trifolium and the 12 University, New Zealand (MPN) by Lesley van Essen and potentially discordant Trifolium species were removed. Fur- Barbara Latch is gratefully acknowledged. We thank the thermore, the nrDNA ITS sequences made a much greater curators of the following herbaria for permitting specimen contribution than the cpDNA trnL intron sequences, due sampling: Botanical Museum Berlin-Dahlem (B), Royal to the poorly resolved cpDNA results. Nevertheless, analy- Botanic Garden Edinburgh (E), The Hebrew University sis of the combined data set revealed phylogenetic informa- (HUJ), Royal Botanic Gardens Kew (K), New York tion that would not be available if the cpDNA sequences Botanical Garden (NY), Oregon State University (OSC), were excluded. Despite the conXicting signal in the two data and Rancho Santa Ana Botanic Garden (RSA). Seeds were sets, the combined analysis was better resolved and boot- provided by Stephanie Greene and Gary Pederson, USDA- strap support increased for the great majority of the clades ARS. Alan Stewart, PGG Seeds, New Zealand, contributed 704 N.W. Ellison et al. / Molecular Phylogenetics and Evolution 39 (2006) 688–705 material and encouragement. Zane Webber, Jessica Ellison, Gatesy, J., Baker, R., 2005. Hidden likelihood support in genomic data: Kyra Hay, Margaret Greig, and Serah Nera assisted with can forty-Wve wrongs make a right? Syst. Biol. 54, 483–492. the growing of plants and preparation of herbarium speci- Gibson, P.B., Beinhart, G., 1969. Hybridization of T. occidentale with two other species of clover. J. Hered. 60, 93–96. mens at AgResearch. Chris Sullivan, OSU Center for Gillett, J.B., 1952. 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