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Phylogeny of subtribe Gentianinae (): Biogeographic inferences despite limitations in temporal calibration points

Article in Taxon · December 2010 DOI: 10.2307/41059867

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Phylogeny of subtribe Gentianinae (Gentianaceae): Biogeographic inferences despite limitations in temporal calibration points

Adrien Favre,1 Yong-Ming Yuan,2 Philippe Küpfer2 & Nadir Alvarez2,3

1 Ecological Genetics Group, Institute of Integrative Biology, ETH Zurich, Universitätsstrasse 16, CHN, 8092 Zurich, Switzerland 2 Laboratory of Evolutionary Botany, Institute of Botany, University of Neuchâtel, Emile-Argand 11, 2009 Neuchâtel, Switzerland 3 Department of Ecology and Evolution, University of Lausanne, Biophore building, UNIL-Sorge, 1015 Lausanne, Switzerland Author for correspondence: Adrien Favre, [email protected]

Abstract The subtribe Gentianinae comprises ca. 425 species, most of them within the well-studied genus Gentiana and mainly distributed over the Eurasian continent. Phylogenetic relationships between Gentiana and its closest relatives, the climbing gentians (Crawfurdia, Tripterospermum) and the new genus Metagentiana, remain unclear. All three genera were recently found to be polyphyletic, possibly because of poor sampling of Tripterospermum and Crawfurdia. Highest diversity of Gentianinae occurs in the western Himalaya, but the absence of uncontroversial fossil evidence limits our understanding of its biogeography. In the present study, we generated ITS and atpB-rbcL sequences for 19 species of Tripterospermum, 9 of Crawfurdia and 11 of Metagentiana, together representing about 60 percent of the species diversity of these genera. Our results show that only Metagentiana is polyphyletic and divided into three monophyletic entities. No unambiguous synapomorphies were associated with the three Metagentiana entities. Different combinations of three approximate calibration points were used to generate three divergence time estimation scenarios. Although dating hypotheses were mostly inconsistent, they concurred in associat- ing radiation of Gentiana to an orogenic phase of the Himalaya between 15 and 10 million years ago. Our study illustrates the conceptual difficulties in addressing the time frame of diversification in a group lacking sufficient fossil number and quality.

Keywords Asia; atpB-rbcL; biogeography; Gentianinae; ITS; molecular dating; molecular phylogeny

Supplementary Material Tables S1–S2 and Figures S1–S3 are available in the free Electronic Supplement to the online version of this article (http://www.ingentaconnect.com/content/iapt/tax).

INTRODUCTION The relationships between Gentiana and its close relatives have been discussed repeatedly and resulted in several contro- The tribe Gentianeae includes some of the most species- versial taxonomic treatments. Wallich (1826) and Blume (1826: rich genera of the family Gentianaceae, for example Genti- 849) described Crawfurdia and Tripterospermum the same year, ana L. (361 species; Yuan & al., 1996) and Gentianella Moench but Marquand (1931, 1937) did not accept these new genera, and (260 species; Hagen & Kadereit, 2001). Within this tribe, about included them in Gentiana as a section. Other authors treated 20 genera are attributed either to the subtribe Gentianinae con- Crawfurdia and Tripterospermum as a single genus (Clarke, sisting of Crawfurdia Wall., Gentiana, Metagentiana T.N. Ho 1885; Ridley, 1923; Ubolcholaket, 1987), but more recent au- & S.W. Liu and Tripterospermum Blume (Ho & al., 2002), or thors have retained them as distinct genera (Ho & Pringle, 1995; to the subtribe Swertiinae, which includes among others Gen- Struwe & al., 2002). Nowadays, Crawfurdia encompasses ca. 20 tianella, Halenia Borkh. and the highly polyphyletic Swertia L. species distributed in the eastern Himalayan areas (southwest (Ho & Liu, 1990; Chassot & al., 2001; Struwe & al., 2002). China, Vietnam, Burma, Bhutan, Sikkim, and northeast India) Several studies have confirmed that both subtribes are mono- (Ho & Pringle, 1995; Struwe & al., 2002). Tripterospermum phyletic (Yuan & Küpfer, 1995; Struwe & al., 2002; Yuan & al., comprises ca. 30 species distributed in East and Southeast Asia 2003) and are well defined by morphological synapomorphies and the neighbouring Himalayan area, with the highest diversity such as the presence of plicae between the corolla lobes and occurring in southwestern China (Murata, 1989). Recently, five intracalycinal membranes between the calyx lobes, both traits new species of climbing gentians (three Tripterospermum and lacking in Swertiinae (Struwe & al., 2002). The phylogenetic two Crawfurdia) were described from Vietnam (Hul, 2002) and relationships within the subtribe Gentianinae, however, remain one from Taiwan (C.H. Chen & al., 2006). Species of Triptero- poorly understood because of a low species coverage in the spermum and Crawfurdia are closely related and easily distin- sampling of Tripterospermum and Crawfurdia. Indeed, the guished from Gentiana and Metagentiana by their twining habit latest studies on this group included only three species of each (Ho & al., 2002). Metagentiana is a new genus encompassing of these genera (S.Y. Chen & al., 2005a,b) and concluded that 14 species, established by Ho & al. (2002) from Gentiana sect. they are polyphyletic. Wider sampling and the identification Stenogyne ex Franch. following morphological and phylogenetic of valid synapomorphies for are therefore required results from previous studies (Smith, 1965; Yuan & al., 1996). to validate this result. The creation of this new genus represents another alternative to

1701 Favre & al.• Phylogeny and biogeography of Gentianinae TAXON 59 (6) • December 2010: 1701–1711

the possible inclusion of Gentiana sect. Stenogyne as a subgenus uncontroversial fossil evidence is lacking in the family Gen- of Tripterospermum (Löve & Löve, 1976) or Gentiana (Halda, tianaceae, we use a comparative approach by combining the 1995). Along with other morphological evidence, Metagentiana available calibration points and test whether the different dating differs from Gentiana and Crawfurdia by having unequal and hypotheses are compatible. apically decurved , and from Tripterospermum by hav- ing small nectaries, which are not disk-like, and corolla plicae with erose to fimbriate margins. Unlike Crawfurdia and Tripter- MATERIAL AND METHODS ospermum, Metagentiana does not show a twining habit and is characterized by twelve vascular bundles in the calyx (Ho & Plant material. — The accession numbers of the 155 al., 2002). In the most recent phylogeny of subtribe Gentianinae, analysed taxa are listed in the Appendix. The origins of the S.Y. Chen & al. (2005b) focused their research on Metagentiana samples collected for this study are given as a table in the sup- and concluded that Crawfurdia, Metagentiana, and Tripteros- plementary material (Table S1). Sequence data of the nuclear permum did not merit treatment as three independent genera ITS region and chloroplast atpB-rbcL spacer of 91 species of because the sampled species of Crawfurdia and Tripterosper- Gentianinae as well as 63 species of Swertiinae were used as mum were embedded within Metagentiana. It was inferred that ingroup. Despite our focus on Gentianinae, we used a large neither Crawfurdia nor Tripterospermum were monophyletic, as sample of Swertiinae to verify the validity of both tribes based one specific sample (i.e., C. tibetica) was nested in the Tripteros- on a more complete sampling of the twining genera of subtribe permum clade (S.Y. Chen & al., 2005b). These results contradict Gentianinae. Moreover, a symmetric topology might serve dat- previous conclusions based on anatomical and chromosomal ing purposes better, particularly when calibration points are data (Ho & al., 2002) and suggest that traits used for taxonomy scarce. Included in the analysis as outgroup, Lisianthius jefensis are not relevant in the evolution of that group. However, with represents the tribe Potalieae, sister group to Gentianeae and only three species each of Crawfurdia and Tripterospermum, the Helieae (Molina & Struwe, 2009). Samples were selected to sampling of species was insufficient with respect to the twining maximize the coverage of representatives in all genera within genera, potentially compromising phylogenetic accuracy and Gentianeae. We obtained new samples in several extensive subsequent biogeographical conclusions. field collection trips in Asia between 2002 and 2005, covering The centre of diversity of the tribe Gentianeae is situated in most of the distribution range of Metagentiana, Crawfurdia the eastern region of the Himalayan range, as for other alpine or and Tripterospermum. Specimens were determined using the subalpine genera such as Rhododendron or Primula (Axelrod & new synopsis of Tripterospermum (Murata, 1989), the Flora al., 1996). Whereas Tripterospermum, Crawfurdia and Meta- of China and literature encompassing new species descriptions gentiana are components of subtropical flora, most Gentiana (Hul, 2002; C.H. Chen & al., 2006). New sequence data were species are obligate subalpine or alpine . Whether the obtained for nine species of Crawfurdia, nineteen species of uplift of this mountain range is the cause of the divergence of Tripterospermum, seven species of Metagentiana, as well as the genus Gentiana or if the Himalayas were secondarily colo- three species of Gentiana. Additional ingroup sequences and nized from the northern temperate regions is difficult to know. those from the outgroup species were retrieved from GenBank. However, the high richness of species in this area suggests that All voucher specimens are deposited in the Herbarium of the species radiation occurred in situ, catalyzed by the geological University of Neuchâtel (NEU), Switzerland, and can be ac- uplift of this area. The Himalayan orogenesis started about 50 cessed upon request to the first author. million years (Ma) ago. Evidence of temperate vegetation in the DNA extraction, amplification and sequencing. — The western Himalayas exists (Franceschi & al., 2008) by the end of CTAB method (Doyle & Doyle, 1987) was used to extract the the Oligocene and the beginning of the Miocene (about 23 Ma total genomic DNA from silica-gel dried leaf material (Chase ago). A well-defined orogenic pulse occurred between 15.2 and & Hills, 1991). The ITS region was amplified with the universal 10.9 Ma ago, followed by an abrupt decrease after 7.5 Ma ago primers ITS4 and ITS5 (White & al., 1990), and primers atpB- (Amano & Taira, 1992). A significant increase in altitude of the rbcL-F and atpB-rbcL-R were used to amplify the atpB-rbcL Tibetan plateau is dated at 10–8 Ma ago (Harrison & al., 1992). intergenic spacer (Hoot & al., 1995) via standard PCR reac- The later phase of this orogenic impulse is associated with the tion in 25 µl volume containing 10× PCR buffer (with 1.5 mM establishment of the monsoon regime (Sun & al., 1998). The MgCl2), 0.5 µl 10 mM dNTPs, 0.5 µl of 10 mM each forward and range reached its current elevation about 3.6 Ma ago (Cui & al., reverse primers, 1 Unit Qiagen Taq DNA polymerase (Qiagen 1996; Shi & al., 1998: 417–466; An & al., 2001). The resulting AG, Basel, Switzerland), between 1 µl and 2 µl of genomic DNA increase in the geographical complexity and climatic changes balanced with 19.8 µl to 18.8 µl H2O, respectively. PCRs were might have favoured speciation and radiation, for instance by performed using a T3 thermocycler (Biometra, Göttingen, Ger- successive vicariance events (He & al., 2001; Peng & al., 2006). many) programmed as follows: for ITS, 3 min at 94°C, followed Here, we test whether the emergence of the Himalayas and by 37 cycles of 45 s at 94°C, 45 s at 54°C, 1 min at 72°C and a its climatic side effects had an impact on the radiation of the final extension at 72°C for 6 min; for atpB-rbcL, 3 min at 94°C, different clades of the Gentianeae at their centre of distribu- followed by 39 cycles of 1 min at 93°C, 1 min at 48°C and 1 min tion. After assessing the generic delimitation and relationships at 72°C, with a final extension at 72°C for 5 min. After checking within Gentianeae, we generated different dating hypotheses the quality and concentration of DNA amplification by running and compared their associated biogeographical scenarios. As 5 µl of each PCR product on a 0.8% agarose gel loaded with a

1702 TAXON 59 (6) • December 2010: 1701–1711 Favre & al.• Phylogeny and biogeography of Gentianinae

100 bp ladder (New England Biolabs, Ipswich, Massachusetts, Chains were run for 30 million generations in two independent U.S.A.), the remaining 20 µl PCR products were run out on an runs, saving a tree every 1000 generations. Convergence of agarose gel again and purified with the QIAquick Gel Extraction the MCMC runs was tested by computing the Potential Scale Kit according to the manufacturer’s protocols (Qiagen) in order Reduction Factor (Gelman & Rubin, 1992) criterion as imple- to ensure high quality of single PCR products. The purified ITS mented in MrBayes, and by determining the Effective Sam- and atpB-rbcL fragments were recovered with 15–30 µl of TE ple Sizes (ESS) using Tracer v.1.4.1 (Rambaut & Drummond, buffer depending on the quantity of the amplified products. 2008). Accordingly, the burn-in period was set to 10 million Cycle sequencing was performed on both ends of the PCR generations until stationarity in the likelihood value was es- products using ABI Prism BigDye Terminator (Applied Biosys- tablished among the runs, and the first 10,000 sample points tems, Foster City, California, U.S.A.) in 5 µl reaction according were discarded. The last 20,000 trees were used to calculate the to the following protocol: 1.5–2.0 µl of purified PCR products half-compatible topology (i.e., majority-rule) and the Bayesian balanced with 0.8–1.3 µl of double-distilled H2O depending posterior probability (BPP) at each node. on the concentration of the PCR products, 0.2 µl of sequencing Tracing characters. — We chose to trace few simple mor- primer and 2 µl of BigDye mix. The same PCR primers were phological characters commonly used to discriminate the dif- used for amplification and sequencing. An additional inter- ferent clades of the Gentianinae (Ho & al., 2002). The charac- nal primer, atpB-rbcL-I with the sequence 5′-TCC TAG ATG ter-coding matrix is available as supplementary material (Table TGA AAA TTC AGC-3′, located half the length of the sequence S2). These key characters were mapped on the half-compatible (the exact position changed from one genus to another) was topology in order to investigate evolution in habit and mor- designed to achieve better sequencing result for the atpB-rbcL phological traits using MacClade v.4.07 (Maddison & Maddi- fragment. The cycle sequencing reactions were carried out in a son, 2003) under the accelerated transformation optimization T3 thermocycler (Biometra) with 29 cycles of 96°C for 5 s fol- criterion (ACCTRAN) and with unordered parsimony. Most lowed by 50°C for 4 min. The cycle sequencing products were morphological traits were scored as binary data, and included further purified following the manufacturer’s protocol (Applied the presence or absence of plicae, recurved stamens, lianescent Biosystems) scaled down to the 5 µl reaction. The purified se- habit, and production of berry. An additional qualitative trait quencing products were resuspended in 12 µl TSR (supplied considered was the life cycle, which was scored as binary data, by Applied Biosystems) and finally analyzed onto an ABI 310 with annual (or biennial) and perennial species. automated sequencer (Applied Biosystems) using a 47 cm cap- Dating hypotheses. — The half-compatible tree was illary and polymer POP-6™ polymer (Applied Biosystems). rendered ultrametric and transferred into a chronogram using Automation-generated base-calls were subsequently checked non-parametric rate smoothing (NPRS; Sanderson, 1997) as and edited manually using the software Sequence Navigator implemented in r8s v.1.70 (Sanderson, 2003). There is no uncon- (Applied Biosystems). troversial fossil evidence in the family Gentianaceae. Here, we Data analysis. — The ITS and atpB-rbcL spacer sequences combine dating estimates of former studies and fossil evidences were further edited in ChromasPro v.1.33 (Technelysium Pty of Gentianaceae-related plants to compare different dating hy- Ltd., Tewantin, Queensland, Australia) and then aligned with potheses. Three calibration points were considered: (1) Pistil- Bioedit v.7.0.5.3 (Hall, 1999) using the Clustal X algorithm lipollenites macgregorii, a putative earliest fossil evidence for (Thompson & al., 1997). The aligned sequences were manu- the Gentianaceae from early Eocene – ca. 50 Ma ago. Although ally adjusted with minor corrections following the similarity the status of this species within Gentianaceae is still debated criterion (Morrison, 2006). Boundaries of the sequences in the (Crepet & Daghlian, 1981; Stockey & Manchester, 1986; Struwe studied material were determined by comparison with previ- & al., 2002), it represents the only available maximum age for ously published sequences of the genera of subtribe Gentiani- the family, which is congruent with molecular dating accord- nae retrieved from GenBank (Yuan & Küpfer, 1995; Yuan & ing to Yuan & al. (2003). (2) Lisianthius fossil pollen from late al., 1996, 2003). Eocene – 37 Ma ago (Graham, 1984). Graham (1984) conceded An incongruence length difference test (using PAUP*, that the taxonomic origin of this fossil pollen might not be exact v.4.0b10; Swofford, 2000) showed that the ITS and atpB-rbcL as it differs from other Lisianthius. However, this calibration partitions were significantly congruent (P = 1.0) and subse- point was also used in another study (Yuan & al., 2005). (3) quently, phylogenetic analyses were performed on a concate- Estimated age of the subtribe Swertiinae using a molecular clock nated supermatrix comprising both partitions, according to the – ca. 15 Ma ago (Hagen & Kadereit, 2002). This age is based total evidence approach (Kluge, 1989). Bayesian analyses (Ny- on a study on Gentianella and despite variable rates have been lander & al., 2004) were performed on the supermatrix (with observed among lineages for ITS (Goertzen & al., 2003; Molina each DNA region represented as a separate partition), using Mr- & Struwe, 2009), it serves here as a proxy for either the split with Bayes v.3.2 (Huelsenbeck & Ronquist, 2001), with substitution Gentianinae or for the crown base of Swertiinae. models as estimated by MrModeltest v.1.0 (Nylander, 2004) Three different dating scenarios were considered to fur- and four estimated alpha categories for the gamma term (Yang, ther reconstruct three independent chronograms. In the first 1994), plus one partition for the gap information coded follow- scenario, two calibration points are considered: 37.2 Ma ago for ing the simple method of Simmons & Ochoterena (2000) as the root, referring to the age of the Lisianthius pollen (i.e., this implemented in FastGap v.1.2 (Borchsenius, 2009) and treated age is the only available proxy roughly corresponding to the as restriction data. Four simultaneous Monte Carlo Markov split between Potalieae and Gentianeae); the second calibration

1703 Favre & al.• Phylogeny and biogeography of Gentianinae TAXON 59 (6) • December 2010: 1701–1711

point was the crown of the tribe Gentianeae at 15 Ma ago. In was polyphyletic and its representatives clustered into three the second scenario, only one calibration point was consid- clades, two of them with BPP = 1.0 (Metagentiana 2 and 3). ered, with the root placed at 37.2 Ma ago. In the third scenario The type specimen of this genus (Metagentiana primulifolia) finally, again two calibration points were considered with the is located in Metagentiana 2. The third clade (Metagentiana 1) root placed at 50 Ma ago (i.e., assumed to be the maximum age showed a lower support with BPP = 0.9. Metagentiana 3 and of the family Gentianaceae) and the crown of the Swertiinae Metagentiana 2 diverged successively and were more basal than placed at 15 Ma ago. These three dating hypotheses were then Tripterospermum and Crawfurdia. In contrast, the divergence of compared using the following important geo-climatic events Metagentiana 1 occurred at a further stage (following Crawfur- associated to the Himalayan uplift as guidelines: the probable dia divergence) therefore rendering Metagentiana polyphyletic. occurrence of alpine habitat (23 Ma ago), a major orogenic Additionally, Gentiana sect. Otophora did not cluster with the impulse (15–10 Ma ago) and the establishment of the monsoon other sections of Gentiana but was the most basal clade of the regime (8 Ma ago). We then compared the ages of four main Tripterospermum-Crawfurdia lineage. However, this position splits or nodes in the topologies across the three scenarios, is not supported yet (BPP = 0.6) and should be confirmed by with the time frame of these three events. Those were the split including more samples and sequencing more regions. between Gentianinae and Swertiinae, the divergence of Gen- Character evolution. — Discriminatory morphological tiana, the radiation of the sections of Gentiana, the divergence characters for the major groups within Gentianinae follow a of Metagentiana and Tripterospermum. rather simple evolutionary pattern (Fig. 2): plicae (P) appeared once at the base of the subtribe Gentianinae, recurved sta- mens appeared once after Gentiana core clade and G. sect. RESULTS Otophora diverged (R) with a reversal (Rr) for Crawfurdia, lianescence appeared again once when Crawfurdia diverged DNA sequences and alignments. — Because our focus from the Tripterospermum lineage (L), with a reversal for a is on Gentianinae, we here report only the information of that few Metagentiana species (Lr). Berry-like fruits are exclusive tribe. All newly obtained sequences have been submitted to the to Tripterospermum (B). Early-diverging taxa demonstrate a GenBank database (see Appendix). For ITS, sequence length perennial strategy whereas annuality evolved five times inde- ranged from 607 to 608 bp for Crawfurdia, 623–626 bp for pendently in Metagentiana and Gentiana (with a reversal back Tripterospermum, 624–625 bp for Metagentiana and 624– to perennial for G. boryi). 625 bp for Gentiana. For atpB-rbcL, sequence length ranged Temporal framework. — A conceptualized topology that from 759 to 789 bp for Crawfurdia, 777–807 bp for Triptero- summarizes the major node ages for each scenario is presented spermum, 649–816 bp for Metagentiana, and 716–732 bp for in Fig. 3. More complete dating results are available in the Gentiana. For both ITS and atpB-rbcL the alignment was supplementary material (Figs. S1–S3). Resulting from the use straightforward, unambiguous and required only minor manual of different calibration points, the three dating hypotheses corrections. The ITS matrix had 664 characters of which 336 differed from each other. Scenario one (S1) differed the most (50.6%) are constant, 118 (17.8%) are variable but parsimony- from the two other scenarios (S2 and S3) with younger ages uninformative and 210 (31.6%) are parsimony-informative. associated to different nodes of the topology. S3 displayed the The atpB-rbcL matrix was composed of 981 characters, of oldest ages and S2 was intermediate, although more similar to which 591 (60.2%) are constant, 202 (20.6%) are variable but S3. The age of the split between Gentianinae and Swertiinae parsimony-uninformative, and 188 (19.2%) are parsimony-in- ranged from 15 (S1) to 39.1 Ma ago (S3), Gentiana diverged formative. Finally 247 binary positions, 132 and 115 in ITS and between 12.4 (S1) and 29.8 Ma ago (S3), the age of the different atpB-rbcL, respectively, corresponded to coded gaps. sections of Gentiana is in average between 10 and 7 Ma for Phylogenetic analysis. — The best substitution models S1 or between 23 and 15 Ma for S3. The divergence between suggested by MrModeltest when considering the hierarchical Tripterospermum and Metagentiana 1 was between 6.2 (S1) and likelihood ratio test were: GTR + G + I for ITS and GTR + G for 16.7 Ma ago (S3) and the base of the crown of Tripterospermum atpB-rbcL. The two independent MCMC runs performed for of ranged from 3.5 (S1) to 9.5 Ma ago (S3). The divergence of the Bayesian inference analysis converged with NPRF values clades Metagentiana 2 and Metagentiana 3 were respectively <1.01 and ESS values >100 for all estimated parameters. The 8.1 and 10.2 Ma ago in S1, 21.1 and 27.7 Ma ago in S3. resulting trees were combined in a 50% majority-rule phyloge- netic tree (Fig. 1) presenting an average log (likelihood) equal to –16,552.1 and a total length of 10.06. The topology clearly DISCUSSION segregated the two subtribes Gentianinae and Swertiinae with a high support value (BPP = 1.0). Within Gentianinae, the gen- This study clarifies the phylogenetic relationships between era Gentiana, Crawfurdia and Tripterospermum were mono- the genera of Gentianinae and identifies remaining uncertainties phyletic and highly supported (BPP = 1.0), if we admit that to be targeted by a future sampling effort. Our results partly C. tibetica was misidentified (see Discussion). Metagentiana contrast with the last phylogeny produced (S.Y. Chen & al.,

Fig. 1. Bayesian majority-rule phylogenetic tree of the tribe Gentianeae (subtribes Swertiinae and Gentianinae) obtained by the Bayesian infer- ence analysis on a dataset combining ITS and atpB-rbcL. Only BPP > 0.70 are shown.

1704 1 Bartonia paniculata TAXON 59 (6) • December 2010: 1701–1711 FavreB &ar tal.•onia Phylogenytexana and biogeography of Gentianinae 1 Bartonia verna 0.78 1 1 Bartonia virginica Halenia elliptica 1 0.99 Halenia weddeliana Swertia dichotoma Swertia tetraptera Veratrilla baillonii Swertia leducii Swertia mileensis Swertia luquanensis 1 Chionogentias muelleriana 1 Chionogentias polysperes Gentiana pleurogynoides Gentianella diemensis Gentiana lilliputiana Gentianella antipoda 0.89 Gentianella bellidifolia Gentianella lineata 0.75 Gentianella saxosa Gentianella cerina Gentianella astonii Gentianella montana Gentianella spenceri 1 Gentianella chathamica Gentianella grisebachii Gentianella myriantha Gentianella magellanica Gentianella narcissoides 1 Gentianella quinquefolia Gentianella umbellata Gentianella bohemica Gentianella bulgarica Gentianella campestris 0.99 Gentianella columnae Gentianella engadinensis Gentianella germanica Gentianella pilosa Gentianella lutescens Gentianella ramosa Gentianella styriaca 0.81 Swertia franchetiana 1 Swertia mussoti 1 Comastoma cyananthiflorum Comastoma pulmonarium 1 Comastoma tenellum 1 Lomatogoniopsis alpina 0.77 Lomatogonium macranthum 1 Lomatogonium rotatum Swertia rosulata Swertia chirayta Swertia pianmaensis 1 fastigiata 1 1 Frasera speciosa 1 Gentianopsis barbata Gentianopsis paludosa Pterigocalyx volubilis 1 Swertia bifolia 0.86 Swertia przewalskii 1 Swertia wolfongiana Swertia erythrosticta Swertia perennis Megacodon stylophorus Swertiinae

1 Crawfurdia angustata Crawfurdia speciosa Gentianineae 1 Crawfurdia campanulacea 1 Crawfurdia maculicaulis Crawfurdia Crawfurdia sessiliflora Crawfurdia poilanei 1 Crawfurdia pricei Crawfurdia delavayi Crawfurdia gracilipes 1 Crawfurdia tibetica 0.98 Tripterospermum cordifolioides 0.86 Tripterospermum championii Tripterospermum luteoviride 0.99 Tripterospermum nigrobaccatum 1 Tripterospermum volubile 0.88 Tripterospermum trinerve Tripterospermum australe 0.94 Tripterospermum nienkui Tripterospermum cordatum Tripterospermum 0.98 Tripterospermum pinbianense 1 0.99 Tripterospermum cordifolium 1 Tripterospermum alutaceofolium 0.73 Tripterospermum taiwanense Tripterospermum luzonense 1 1 Tripterospermum microphyllum Tripterospermum robustum Tripterospermum chinense 1 Tripterospermum hirticalyx Tripterospermum lanceolatum 1 Tripterospermum pallidum 1 0.9 Metagentiana pterocalyx Metagentiana souliei 0.95 0.8 Metagentiana striata Metagentiana 1 Metagentiana australis 1 Metagentiana serra Metagentiana primuliflora 1 Metagentiana gentilis Metagentiana 2 1 Metagentiana rodantha Metagentiana villifera Metagentiana eurycolpa 3 Metagentiana leptoclada Metagentiana Gentiana decorata Gentiana otophoroides Gentiana sect. Otophora 1 Gentiana algida 1 1 Gentiana frigida Gentiana atropurpurea 1 Gentiana vernayi 1 1 Gentiana futtereri Gentiana lawrencei Gentiana oreodoxa 0.98 1 Gentiana veitchiorum Gentiana asclepiadea Gentiana lutea 1 Gentiana verna Gentiana bavarica 1 Gentiana alpina 1 Gentiana clusii 0.88 0.98 Gentiana crassicaulis Gentiana lhassica 1 Gentiana waltonii 0.89 Gentiana cruciata 1 Gentiana dahurica 0.94 Gentiana straminea 0.9 Gentiana siphonantha Gentiana decumbens 0.91 Gentiana macrophylla Gentiana remaining sections Gentiana officinalis 0.8 Gentiana zekuensis Gentiana robusta 1Gentiana tibetica 0.95 Gentiana walujewii Gentiana kaufmanniana Gentiana tianschanica 1 Gentiana olivieri 1 Gentiana decora Gentiana parryi Gentiana triflora Gentiana haynaldii 0.99 Gentiana crassuloides 0.87 Gentiana heleonastes Gentiana pantheica 0.92 Gentiana pseudoaquatica Gentiana boryi Gentiana flexicaulis 1 Gentiana intricata 0.97 1 Gentiana piasezkii 0.87 Gentiana squarrosa Gentiana depressa Gentiana urnula Gentiana phyllocalyx 1705 1 Favre & al.• Phylogeny and biogeography of Gentianinae TAXON 59 (6) • December 2010: 1701–1711

2005a,b), and agree with previous studies (Ho & Pringle, 1995; Monophyly and species distribution of Tripterosper- Struwe & al., 2002). Our topology provides a significant im- mum and Crawfurdia. — Both genera of climbing gentians provement in the understanding of the evolution of key traits are likely to be relevant and well-delimited genera as all spe- and confirms that these synapomorphies are valuable characters cies sequenced with both atpB-rbcL and ITS fell in two highly allowing an easy determination of the different taxa. Moreover, supported monophyletic clades (Fig. 1). However, C. tibetica our dating hypotheses underline the limitations of proper bio- falls within Tripterospermum as the sister taxon of T. cordi- geographical conclusions in the absence of valid fossil records. folioides, another species from the Sichuan province. We did

Fig. 2. Character tracing in the Crawfurdia angustata Rr Crawfurdia speciosa subtribe Gentianinae including Crawfurdia maculicaulis Crawfurdia sessiliflora presence of plicae (P), recurved Crawfurdia poilanei stamens (R), lianescence (L), Crawfurdia pricei 1 Crawfurdia campanulacea berry-like fruits (B) and evo- Crawfurdia delavayi Crawfurdia gracilipes lutionary reversal for recurved Crawfurdia tibetica Tripterospermum cordifolioides stamens (Rr) and lianescence Tripterospermum luteoviride (Lr). The species in bold corre- Tripterospermum nigrobaccatum L Tripterospermum volubile sponding to the black sidebands Tripterospermum trinerve Tripterospermum championii are annual (or biennial) plants Tripterospermum australe whereas those identified with Tripterospermum nienkui 1 Tripterospermum cordatum light grey sidebands are peren- Tripterospermum pinbianense Tripterospermum cordifolium nials. Tripterospermum alutaceofolium 0.99 Tripterospermum taiwanense B Tripterospermum luzonense Tripterospermum microphyllum Tripterospermum robustum Tripterospermum lanceolatum 1 Tripterospermum pallidum Tripterospermum hirticalyx R 1 1 Tripterospermum chinense Metagentiana pterocalyx 0.9 Metagentiana souliei Metagentiana striata 1 Metagentiana australis Lr Metagentiana serra Metagentiana primuliflora 1 Metagentiana gentilis 0.6 Metagentiana rodantha Metagentiana villifera 1 Metagentiana eurycolpa Metagentiana leptoclada Gentiana decorata (only atpB) Gentiana otophoroides P Gentiana algida Gentiana frigida 1 Gentiana atropurpurea Gentiana vernayi 1 0.98 Gentiana futtereri Gentiana lawrencei var farreri Gentiana oreodoxa Gentiana veitchiorum Gentiana asclepiadea Gentiana lutea 1 Gentiana verna Gentiana bavarica Gentiana alpina Gentiana clusii 1 Gentiana crassicaulis Gentiana lhassica Gentiana waltonii Gentiana cruciata Gentiana dahurica Gentiana straminea Gentiana siphonantha 0.94 Gentiana decumbens Gentiana macrophylla Gentiana officinalis Gentiana zekuensis 0.95 Gentiana robusta Gentiana tibetica 0.8 Gentiana walujewii Gentiana kaufmanniana Gentiana tianschanica Gentiana olivieri Gentiana decora 1 Gentiana parryi Gentiana triflora Gentiana haynaldii Gentiana crassuloides Gentiana heleonastes Gentiana pseudoaquatica 1 Gentiana pantheica 0.97 Gentiana boryi Gentiana flexicaulis Gentiana intricata Gentiana piasezkii Gentiana squarrosa Gentiana depressa Gentiana urnula Gentianaphyllocalyx

1706 TAXON 59 (6) • December 2010: 1701–1711 Favre & al.• Phylogeny and biogeography of Gentianinae

not collect and sequence C. tibetica (i.e., the ITS sequence was extends to Southeast Asia (Murata, 1989). The phylogenetic retrieved from GenBank; see Appendix), but given the number position of the Tripterospermum species seems to indicate a of species studied here and the clear phylogenetic signal that trend towards a vicariance pattern despite low statistical sup- discriminates Crawfurdia and Tripterospermum, there is strong port within the genus (Fig. 4). The most basal taxa are located in evidence to consider the previously published C. tibetica as a southeast China and Taiwan, including a whole clade of species mis-determined Tripterospermum cordifolioides. This should endemic to Taiwan (T. alutaceofolium, T. cordifolium, T. luzon- be confirmed with new collections of C. tibetica. Furthermore, ense, T. microphyllum, T. taiwanense). Clades consisting of the monophyly of both genera is supported by clear morpho- other Chinese species diverged later and are more widespread logical characteristics that can easily be used to distinguish one in the phylogeny, although a trend towards a biogeographic genus from the other: both genera are twining, but Tripteros- signature also exists: Tripterospermum australe and T. nienkui permum has apically decurved stamens, a simple trait lacking that cluster together originate from the same adjacent Chinese in Crawfurdia. Additional synapomorphies exist, with Craw- provinces of Guangxi, Guangdong and Hong Kong; species furdia having small independent glands at the base of the gy- occupying the southern flank of the Himalayas (T. luteoviride, nophores, and Tripterospermum bearing berry-like fruits and T. nigrobaccatum, T. volubile) and Southeast Asia (T. trinerve, conspicuously developed nectaries forming a basal disc around T. championii) also cluster together. the gynophore (Ho & al., 2002). Both genera co-occur in the Most of the Tripterospermum species thus seem to be peri-Himalayan region but the distribution of Tripterospermum restricted to particular geographical units (mountain ranges,

Scenario 1 Lisianthius Fig. 3. Three molecular dating Tripterospermum scenarios schematically depict- Metagentiana 1 Crawfurdia ing timing of divergence for the Metagentiana 2 main lineages within Gentia- 37 Metagentiana 3 neae (see Figs. S1–S3 for the G. sect. Otophora detailed topologies and dating G. sect. Cruciata G. sect. Chondrophyl. hypotheses). G. sect. Phyllocalyx G. sect. Isomeria G. sect. Ciminalis 15 G. sect. Frigida G. sect. Microsperma G. sect. Kudoa Swertiinae Scenario 2 Lisianthius Tripterospermum Metagentiana 1 Crawfurdia Metagentiana 2 Metagentiana 3 37 G. sect. Otophora G. sect. Cruciata G. sect. Chondrophyl. G. sect. Phyllocalyx G. sect. Isomeria G. sect. Ciminalis G. sect. Frigida G. sect. Microsperma G. sect. Kudoa Swertiinae Scenario 3 Lisianthius Tripterospermum Metagentiana 1 Crawfurdia Metagentiana 2 Metagentiana 3 50 G. sect. Otophora G. sect. Cruciata G. sect. Chondrophyl. G. sect. Phyllocalyx G. sect. Isomeria G. sect. Ciminalis G. sect. Frigida G. sect. Microsperma G. sect. Kudoa 15 Swertiinae Alpine Habitat Orogenesis Phase Monsoon Regime 23 Ma 15–10 Ma 8 Ma 50 Ma Present

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1 Crawfurdia tibetica SW variation raises doubts regarding the validity of this trait as a 0.98 Tripterospermum cordifolioides China synapomorphy in Metagentiana. Nevertheless, our topology 0.86 Tripterospermum championii Malaysia agrees with the idea that the reduction of vascular bundles is Tripterospermum luteoviride an advanced character (Puri, 1951; Eames, 1961; Davis, 1966; 0.99 Tripterospermum nigrobaccatum South Himalayas Tripterospermum volubile Liang & Zhang, 1986), with the different clades of Metagen- 0.88 Tripterospermum trinerve Indonesia tiana showing intermediate and unstable numbers between Tripterospermum australe Gentiana (15) and Tripterospermum (5). Moreover, we did not 0.94 Tripterospermum nienkui SW China find any consistent morphological trait that is exclusively as- Tripterospermum cordatum 0.98 Tripterospermum pinbianense sociated with each Metagentiana clade, rendering the descrip- 0.99 Tripterospermum cordifolium tion of new genera difficult. Alternatively, we considered the 1 Tripterospermum alutaceofolium inclusion of Metagentiana 1 into Tripterospermum as a possible 0.73 Tripterospermum taiwanense Taiwan taxonomic combination. This approach is supported by the Tripterospermum luzonense 1 Tripterospermum microphyllum lack of anthocyanic pigmentation of the corolla in M. souliei, Tripterospermum robustum M. striata and several species of Tripterospermum (a very rare Tripterospermum chinense characteristic in the whole subtribe). Indeed, flower colour is Tripterospermum hirticalyx SW China an important trait as it is associated with pollinator attraction, Tripterospermum lanceolatum Tripterospermum pallidum reproductive success (Wilkin, 1995; Miller & al., 1999) and reproductive isolation (Hodges & al., 2002). However, not all 1 taxa within these genera show such a peculiarity. An alterna- tive would be to merge Metagentiana, Tripterospermum and Fig. 4. Close-up of phylogenetic relationships within the genus Trip­ Crawfurdia into one single genus that would differ from its terospermum, with sidebands indicating the geographical distribution sister clade, Gentiana, by demonstrating bracteate flowers, a of most clades. filiform style as long as or longer than the ovary and a number of vascular bundles in the calyx tube different from 15. Nev- ertheless, the description of this new genus might currently islands) and species that occur in a same region are often closely be inappropriate for several reasons. First, Crawfurdia and related (with the exception of species in the centre of diversity Tripterospermum show clear morphological and genetic dif- across southwestern China). However, our phylogeny comprises ferentiation (see above); ignoring this differentiation would three examples of taxa with disjunctive distributions (Murata, only relegate the discussion at a lower taxonomic level. Second, 1989): Tripterospermum championii (Sri Lanka, Malaysia, since all the species have not been sequenced and included Vietnam), T. luzonense (Taiwan, Philippines, Indonesia) and in the present study, the taxonomic localization of M. alata, T. cordifolium (Taiwan, China). The latter is probably endemic M. expansa, and M. melvillei remains speculative. Given the to Taiwan as the specimen from the Sichuanese population (il- low number of species in Metagentiana, the inclusion of few lustrated in Murata, 1989) lacks particular specific traits: the additional species might challenge the extant topology. exerted style from the corolla and the recurved heart-shaped Uncertain placement for Gentiana sect. Otophora. — The calyx lobes. It was probably mistaken for a local Sichuanese two species of Gentiana sect. Otophora, a section considered T. cordifolioides of which the creeping habit and heart-shaped for the first time in a phylogenetic analysis of Gentianaceae, leaves are shared with T. cordifolium. Allopatric populations did unexpectedly not cluster with the remaining sections of of T. luzonense and T. championii should also be investigated Gentiana (Fig. 1) and were sister to the other genera. However, since, similarly to the sichuanese population of T. cordifolium, this position was supported with a very low statistical prob- they might be wrongly assigned to one or the other species. ability (i.e., BPP = 0.6; see Fig. 1). Although the sampling of Polyphyly of Metagentiana. — Metagentiana was found the present study is clearly not sufficient to address any conclu- to be polyphyletic, being divided into three well-supported sions regarding this section, it reasonably raises doubts on the clades (Fig. 1). The description of Metagentiana as new ge- phylogenetic position of G. sect. Otophora. Our results address nus was largely based on the number of vascular bundles in the need to increase the taxa sampling within this section as the calyx tube (Ho & al., 2002). This number was reported as well as the number of sequenced regions to confirm or reject 12 and differed from Tripterospermum (5), Crawfurdia (10) the polyphyly of Gentiana. and Gentiana (15). A non-pentamerous number of vascular Inferences in the spatio-temporal framework. — When bundles might be compatible with a flower with unequal re- considering the three scenarios produced with controversial curved stamens, as these Gentianaceae show a tendency to calibration points, different conclusions spanning a large part zygomorphy. However, our observations showed some vari- of the Tertiary can be drawn. Former studies have suggested ation in this trait within Metagentiana : based on a sample of that the Gentianinae cradle occurred in an alpine temperate five to ten collected individuals per species, we found that the habitat in the Old World (Yuan & al., 1996), most likely in number of vascular bundles was variable among species at eastern Asia where today it remains most specious (Ho & Liu, anthesis: Metagentiana primuliflora and M. australis possess 1990; Struwe & al., 2002). Evidence of temperate vegetation 5 vascular bundles; Metagentiana rhodantha and M. souliei in the western Himalayas exist (Franceschi & al., 2008) by have 10; finally, M. striata and M. gentilis have 12 to 14. This the end of Oligocene and the beginning of the Miocene (about

1708 TAXON 59 (6) • December 2010: 1701–1711 Favre & al.• Phylogeny and biogeography of Gentianinae

23 Ma ago): pollen deposition shows the presence of a typical CONCLUSIONS AND PERSPECTIVES temperate broad-leaved forest (with Alnus, Quercus and Betula) as well as a higher altitude conifer forest belt (with Cedrus This study encourages further investigation on the subtribe and Picea), suggesting the appearance of high mountains and Gentianinae. We showed the monophyly of Crawfurdia and potentially local alpine habitat. Scenario 1 would support the Tripterospermum, for which simple morphological characters hypothesis that the split between Gentianinae and Swertiinae, were found as synapomorphies. Metagentiana was found to be if it occurred at the actual centre of biodiversity about 15 Ma polyphyletic and the questionable position of Gentiana sect. ago, was in a temperate to alpine habitat. However, this scenario Otophora was presented. Merging the sister lineages of Genti- is likely to underestimate the split between the two subtribes. ana in one single genus remains an open question that should be (Hagen & Kadereit, 2002). The two other scenarios suggest that re-considered once more species of Metagentiana and G. sect. the split occurred before substantial alpine habitat appeared in Otophora have been analyzed. The different dating hypotheses the Himalayas, i.e., under a more subtropical climate or outside only allowed us to associate the radiation of the genus Gentiana of the region studied here. This should be clarified by further with an orogenic phase of the Himalayan range. Further infer- analysis including the whole geographic range of the Genti- ences in the spatio-temporal framework strongly rely on the in- anaceae family. clusion of new fossil evidence that urgently need to be acquired. From a biogeographic perspective, a part of main interest in the topology is the divergence of Gentiana and its further AKNOWLEDGEMENTS radiation. Because this genus is mostly alpine (in contrast to the other Gentianinae), one could argue that diversification hap- We would like to thank Gaelle Bocksberger for help in the lab, pened once major zones of alpine habitat appeared and might Philippe Chassot for sharing samples, Sven Buerki for help in the be associated with the increasing geographic complexity of the NPRS analyses, Jake Alexander and Paul Page as well as two anony- Himalayan region following major orogenic impulse. Accu- mous reviewers for comments on the manuscript. Access to herbarium rately addressing whether or not Gentiana diverged in the Hima- data and specimens were kindly provided by Sovanmoly Hul in Paris layan region is not possible with the present analysis. However, and Chi Hsiung Chen in Taichung. Many thanks to Dr. Yi Song (Basel), all three scenarios concurred in dating the diversification of the Dr. Jian Ying Xiang (Kunming) and Dr. Chi Hsiung Chen (Taichung) genus Gentiana after the Himalayas reached an elevation suf- for fieldwork support and all the helpful people we met during the ficiently high to encompass alpine habitat (after 23 Ma ago). The Asian collection trips. Field trips were partially supported by the Fond three scenarios broadly place diversification of this genus in the Wuthrich/Mathey-Dupraz in Neuchâtel and the ASSN. time frame of the orogenic impulse that occurred 15–10 Ma ago. This orogenic phase might have promoted habitat diversifica- LITERATURE CITED tion, potentially acting as a driver of speciation and radiation in the genus Gentiana and other temperate genera (Axelrod & Amano, K. & Taira, A. 1992. Two-phase uplift of higher Himalayas al., 1996). Our results correspond to another study providing since 17 Ma. 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Appendix. List of taxa studied: Taxon, GenBank accessions for ITS (eventually ITS 1, ITS 2), atpB-rbcL. An n-dash (–) indicates sequences not available. GENTIANINAE: Crawfurdia angustata C.B. Clarke, GU251013, GU250971; Crawfurdia campanulacea Wall. & Griff. ex C.B. Clarke, GU251014, GU250972; Crawfurdia delavayi Franch., GU251015, GU250973; Crawfurdia gracilipes Harry Sm., GU251016, GU250974; Crawfurdia maculicaulis C.J. Wu, GU251017, GU250975; Crawfurdia poilanei Hul, GU251018, GU250976; Crawfurdia pricei (Marquand) Harry Sm., GU251019, GU250977; Crawfurdia sessiliflora (Mar- quand) Harry Sm., GU251020, GU250978; Crawfurdia speciosa Wall., GU251021, GU250979; Crawfurdia tibetica Franch., AY563383, –; Gentiana algida Pall., GU251024, –; Gentiana asclepiadea L., GU251025, GU250980; Gentiana atropurpurea T.N. Ho, AY858678, –; Gentiana crassicaulis Gilg, AY858676, DQ398601; Gentiana cruciata L., DQ398635, DQ398634, DQ398599; Gentiana dahurica Fisch., DQ398633, DQ398632, DQ398597; Gentiana decora Pollard, EU812468, –; Gentiana decorata Diels, GU250981, –; Gentiana decumbens L., DQ398655, DQ398620; Gentiana depressa D. Don, GU251026, GU250982; Gentiana fut- tereri Diels & Gilg, DQ398658, DQ398623; Gentiana haynaldii Kanitz., AY858671, –; Gentiana kaufmanniana Regel & Schmalh., DQ398649, DQ398613; Gentiana lawrencei Burkill, DQ317492, –; Gentiana lhassica Burkill, DQ398629, DQ398594; Gentiana lilliputiana C.J. Webb, AY160218, –; Gentiana lutea L., DQ358878, –; Gentiana macrophylla Pall., DQ398654, DQ398617; Gentiana officinalis Harry Sm., DQ398639, DQ398604; Gentiana olivieri Griseb., DQ398644, DQ398609; Gentiana oreodoxa Harry Sm., DQ398657, DQ398621; Gentiana otophoroides Harry Sm., GU251027, –; Gentiana parryi Engelm., Z48096, X85370, DQ398624; Gentiana pleurogynoides Griseb., AY136506, –; Gentiana robusta King ex Hook. f., DQ398643, DQ398608; Gentiana siphonantha Maxim. ex Kusnezow, DQ497573, DQ398607; Gentiana straminea Maxim., AF346015, DQ398596; Gentiana tianschanica Rupr., DQ398651, DQ398615; Gentiana tibetica King ex Hook. f., DQ398640, DQ398606; Gentiana triflora Pall., DQ398661, –; Gentiana urnula Harry Sm., Z48071, Z48090, GU250983; Gentiana veitchio- rum Hemsl., AY858677, –; Gentiana verna L., GU251028, GU250984; Gentiana vernayi C. Marquand, AY858670, –; Gentiana waltonii Burkill, DQ398626, DQ398591; Gentiana walujewi Regel & Schmalh., DQ398646, DQ398611; Gentiana zekuensis T.N. Ho & S.W. Liu, DQ398656, –; Metagentiana australis (Craib) T.N. Ho & S.W. Liu, GU251029, GU250986; Metagentiana eurycolpa (C. Marquand) T.N. Ho & S.W. Liu, AY858673, –; Metagentiana gentilis (Franch.) T.N. Ho & S.W. Liu, GU251030, GU250987; Metagentiana leptoclada (Balf. f. & Forrest) T.N. Ho & S.W. Liu, AY858674, –; Metagentiana primuliflora (Franch.) T.N. Ho & S.W. Liu, GU251031, GU250988; Metagentiana pterocalyx (Franch.) T.N. Ho & S.W. Liu, AY562171, –; Metagentiana rhodantha (Franch.) T.N. Ho & S.W. Liu, GU251032, GU250989; Metagentiana serra (Franch.) T.N. Ho & S.W. Liu, GU251033, GU250990; Metagentiana souliei (Franch.) T.N. Ho & S.W. Liu, GU251034, GU250991; Metagentiana striata (Maximowicz) T.N. Ho & S.W. Liu, GU251035, GU250992; Metagentiana villifera (H.W. Li ex T.N. Ho) T.N. Ho & S.W. Liu, AY858672, –; Tripterospermum alutaceofolium (T.S. Liu & C.C. Kuo) J. Murata, GU251038, GU250995; Tripterospermum australe J. Murata, GU251039, GU250996; Tripterospermum championii Gardn., GU251040, GU250997; Tripterospermum chinense (Migo) Harry Sm., GU251041, GU250998; Tripterospermum cordatum (Marquand) Harry Sm., GU251042, GU250999; Tripterospermum cordifolioides J. Murata, GU251053, GU251010; Tripterospermum cordifolium (Yamamoto) Satake, GU251037, GU250994; Tripterospermum hirticalyx C.Y. Wu, GU251043, GU251000; Tripterospermum lanceolatum (Hayata) Hara ex Satake, GU251044, GU251001; Tripterospermum luteoviride (C.B. Clarke) J. Murata, GU251045, GU251002; Tripterospermum luzonense (Vidal) J. Mu- rata, GU251046, GU251003; Tripterospermum microphyllum Harry Sm., GU251047, GU251004; Tripterospermum nienkui (Marquand) C.J. Wu, GU251048, GU251005; Tripterospermum nigrobaccatum Hara, GU251049, GU251006; Tripterospermum pallidum Harry Sm., GU251050, GU251007; Tripterospermum pinbianense C.Y. Wu & C.J. Wu, GU251051, GU251008; Tripterospermum robustum Harry Sm. ex Hul, GU251052, GU251009; Tripterospermum taiwanense (Masamune) Satake, GU251054, GU251011; Tripterospermum trinerve Blume, GU251055, GU251012; Tripterospermum volubile (D. Don) Hara, AY858667, –; SWERTIINAE: Bartonia paniculata (Michx.) B.L. Rob., EU812470, –; Bartonia texana Correll, EU812471, –; Bartonia verna Raf. ex Barton, EU812473, –; Bartonia virginica (L.) Britton, Sterns & Poggenb, EU812474, –; Chionogentias muelleriana L.G. Adams, AY136507, –; Chionogentias polysperes L.G. Adams, AY136505, –; Comastoma cyananthiflorum (Franch.) Holub, AJ294585, AJ294645, GU250970; Comastoma pulmonarium (Turcz.) Toyokuni, AF346008, –; Comastoma tenellum (Rottb.) Toyokuni, AJ580552, –; Frasera fastigiata (Pursh) A. Heller, GU251022, –; Frasera speciosa Dougl. ex Griseb., GU251023, –; Gentianella antipoda (Kirk) T.N. Ho & S.W. Liu, AY136500, –; Gentianella astonii (Petrie) T.N. Ho & S.W. Liu, AY136494, –; Gentianella bellidifolia ( Ho o k . f.) Holub, AY136498, –; Gentianella bohemica Skalicky, AJ580570, –; Gentianella bulgarica (Velen.) Holub, AJ580569, –; Gentianella campestris (L.) Harry Sm., AJ580557, –; Gentianella cerina (Hook. f.) T.N. Ho & S.W. Liu, AY136502, –; Gentianella chathamica (Cheeseman) T.N. Ho & S.W. Liu, AY136495, –; Gen- tianella columnae (Ten.) Holub, AJ580555, –; Gentianella diemensis (Griseb.) J.H. Willis, AY136504, –; Gentianella engadinensis (Wettst.) Holub, AJ580559, –; Gentianella germanica (Willd.) E.F. Warb., AJ580562, –; Gentianella grisebachii (Hook. f.) T.N. Ho, AY136489, –; Gentianella lineata (Kirk) T.N. Ho & S.W. Liu, AY136503, –; Gentianella lutescens (Velen.) Holub, AJ580574; Gentianella magellanica (Gaudich.) Fabris, AY160219, –; Gentianella montana (G. Forst.) Holub, AY136491, –; Gentianella myriantha (Gilg) Fabris, AY136488, –; Gentianella narcissoides (Gilg) T.N. Ho & S.W. Liu, AY136486, –; Gentianella pilosa (Wettst.) Holub, AJ580554, –; Gentianella quinquefolia Small, EU812469, –; Gentianella ramosa (Hegetschw.) Holub, AJ580556, –; Gentianella saxosa (G. Forst.) Holub, AY136499, –; Gentianella spenceri (Kirk) T.N. Ho & S.W. Liu, AY136496, –; Gentianella styriaca Jang & Greimler (unpub.), AJ580577, –; Gentianella umbellata (M. Bieb.) Holub, AJ580553, –; Gentianopsis barbata (Froel.) Ma, AF346007, –; Gentianopsis paludosa (Hook. f.) Ma, Z48106, Z48131, GU250985; Halenia elliptica D. Don, AF346012, –; Lomatogoniopsis alpina T.N. Ho & S.W. Liu, AF247660, –; Lomatogonium macranthum Fernald, AF346011, –; Lo- matogonium rotatum (L.) Fr. ex Fernald, AF346010, –; Megacodon stylophorus (C.B. Clarke) Harry Sm., AY858679, –; Pterigocalyx volubilis Maximowicz, GU251036, GU250993; Swertia bifolia Batalin, DQ317490, –; Swertia chirayta H. Karst., AF255917, –; Swertia dichotoma L., DQ317488, –; Swertia erythrosticta Maxim., AF251122, –; Swertia franchetiana Harry Sm., AF255916, –; Swertia leducii Franch., DQ317486, –; Swertia luquanensis S.W. Liu ex T.N. Ho, DQ317487, –; Swertia mileensis, T.N. Ho & W.L. Shih, AY551184; Swertia mussoti Franch., AF255915, –; Swertia perennis L. AJ580550; Swertia pianmaensis T.N. Ho & S.W. Liu, DQ317485, –; Swertia przewalskii Pissjaukova, AF255913, –; Swertia rosulata (Baker) Klack., AJ489922, –; Swertia tetraptera Maxim., AF346013, –; Swertia wolfongiana, AF255914, –; Veratrilla baillonii Franch., AF251123, GU251012. OUTGROUP: Lisianthius jefensis A. Robyns & T. Elias, EU709782, –.

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