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. 2005 Jul 1;96(3):413–424. doi: 10.1093/aob/mci188

Molecular Systematics and Biogeography of Crawfurdia, Metagentiana and Tripterospermum (Gentianaceae) Based on Nuclear Ribosomal and Plastid DNA Sequences

SHENGYUN CHEN 1,2, TAO XIA 1,2, YUJIN WANG 1,2, JIANQUAN LIU 1, SHILONG CHEN 1,*
PMCID: PMC4246770  PMID: 15994844

Abstract

• Background and Aims The systematic position of the genus Metagentiana and its phylogenetic relationships with Crawfurdia, Gentiana and Tripterospermum have not been explicitly addressed. These four genera belong to one of two subtribes (Gentianinae) of Gentianeae. The aim of this paper is to examine the systematic position of Crawfurdia, Metagentiana and Tripterospermum and to clarify their phylogenetic affinities more clearly using ITS and trnL intron sequences.

• Methods Nucleotide sequences from the internal transcribed spacers (ITS) of nuclear ribosomal DNA and the plastid DNA trnL (UAA) intron were analysed phylogenetically. Ten of fourteen Metagentiana species were sampled, together with 40 species of other genera in the subtribe Gentianinae.

• Key Results The data support several previously published conclusions relating to the separation of Metagentiana from Gentiana and its closer relationships to Crawfurdia and Tripterospermum based on studies of gross morphology, floral anatomy, chromosomes, palynology, embryology and previous molecular data. The molecular clock hypothesis for the tested sequences in subtribe Gentianinae was not supported by the data (P < 0·05), so the clock-independent non-parametric rate smoothing method was used to estimate divergence time. This indicates that the separation of Crawfurdia, Metagentiana and Tripterospermum from Gentiana occurred about 11·4–21·4 Mya (million years ago), and the current species of these three genera diverged at times ranging from 0·4 to 6·2 Mya.

• Conclusions The molecular analyses revealed that Crawfurdia, Metagentiana and Tripterospermum do not merit status as three separate genera, because sampled species of Crawfurdia and Tripterospermum are embedded within Metagentiana. The speciation and rapid radiation of these three genera is likely to have occurred in western China as a result of upthrust of the Himalayas during the late Miocene and the Pleistocene.

Keywords: Asia, biogeography, Crawfurdia, Gentiana, Gentianeae, Gentianaceae, Metagentiana, molecular systematics, ITS, trnL (UAA) intron, Tripterospermum

INTRODUCTION

Crawfurdia was established by Wallich in 1826 (Wallich, 1826). In the same year, Blume (1826) described a new genus Tripterospermum based on T. trinerve from Java. These two genera were re-examined by Marquand (1931, 1937), who did not accept Crawfurdia and Tripterospermum and merged them into Gentiana. However, the two have been retained as separate genera by many botanists (Smith, 1965; Ho and Liu, 1990; Struwe et al., 2002). Metagentiana was separated from Gentiana on the basis of observations related to its gross morphology, floral anatomy, chromosomes, palynology, embryology and molecular data (Yuan et al., 1996; Ho et al., 2002a; Struwe et al., 2002). It was previously treated as Gentiana section Stenogyne, established by Franchet (1884) and revised by Kusnezov (1894). Prior to Ho et al. (2002a), most botanists followed Kusnezov (1894) and considered this group as a section of Gentiana (Pringle, 1978; Ho and Liu, 1990; Struwe et al., 2002). Metagentiana consists of 14 species, two of which are endemic to Thailand and Myanmar, and the rest are concentrated in south-west China. Most species of Metagentiana are herbaceous local endemics growing in alpine scrub, meadows and coniferous forests (Ho and Pringle, 1995; Ho et al., 2002a). The systematic position of Metagentiana and its phylogenetic relationships with Crawfurdia, Gentiana and Tripterospermum have not been explicitly addressed. These four genera belong to one of two subtribes (Gentianinae) of Gentianeae (Struwe et al., 2002). The other, larger subtribe is Swertiinae, which includes Bartonia, Comastoma, Frasera, Gentianella, Gentianopsis, Halenia, Jaeschkea, Latouchea, Lomatogonium, Megacodon, Obolaria, Pterygocalyx, Swertia and Veratrilla (Struwe et al., 2002). Smith (1965) suggested that Gentiana section Stenogyne (Metagentiana) had a closer affinity with Tripterospermum and Crawfurdia than with any other section of Gentiana, and Löve and Löve (1976) recommended that it should be transferred to the genus Tripterospermum, tentatively as a subgenus (Tripterospermum subgenus Stenogyne) based on morphological characters. Halda (1995) treated section Stenogyne as a subgenus of Gentiana. Karyological studies in Gentiana have been reviewed by several authors (Yuan, 1993; Yuan and Küpfer, 1993a, b; Küpfer and Yuan, 1996). Yuan and Küpfer (1993a) reported chromosome numbers and karyotype asymmetry for six species of Metagentiana for the first time and suggested that it had a unique and isolated position in the genus Gentiana because of its higher basic chromosome numbers (x = 17, 21 and 23). Ho et al. (2002b) also reported chromosome numbers for two other species (M. souliei, 2n = 46; M. serra, 2n = 34). Yuan et al. (1996) suggested that section Stenogyne (Metagentiana) should be excluded from Gentiana because inclusion of this section in Gentiana makes the genus paraphyletic based on a molecular phylogeny reconstructed from nucleotide sequences of the internal transcribed spacers (ITS) of nuclear ribosomal DNA; this and the plastid trnL (UAA) intron (trnL) have been shown to be suitable markers for phylogenetic reconstruction within a genus or closely related genera (Baldwin et al., 1995; Wang et al., 1999; Bain and Golden, 2000; Liu et al., 2002). Moreover, previous studies at the generic level of subtribe Gentianinae and the sectional level of Gentiana have shown the phylogenetic utility of ITS and trnL sequences (Yuan and Küpfer, 1995; Gielly and Taberlet, 1996; Yuan et al., 1996; Yuan and Küpfer, 1997). Phylogenetic trees generated by parsimony analysis of these data are principally congruent with morphological observations, and they have improved or clarified some morphological misinterpretations and conflicts (Yuan and Küpfer, 1995; Gielly and Taberlet, 1996; Yuan et al., 1996, 2003; Yuan and Küpfer, 1997). However, only four species representing the three genera Crawfurdia, Metagentiana and Tripterospermum have been studied in previous phylogenetic analyses using ITS sequences (Yuan et al., 1996). Although it has been suggested that the section Stenogyne should be excluded to maintain the monophyly of Gentiana, there were insufficient samples in the cited studies (with regards to Metagentiana) to provide deeper insights into the relationships among these genera. Neither divergence dating nor biogeographic analyses of these groups were performed in any previous studies. In addition, major goals of modern biogeography are to reconstruct the phylogenies of genera and evaluate their origin and evolution against the geological and palaeoclimatic histories of their distribution areas (Avise, 2000).

Applying a molecular phylogenetic approach based on ITS and trnL sequences, the present study is focused on the new genus Metagentiana as well as Crawfurdia and Tripterospermum, allowing their systematic position to be examined and their phylogenetic affinities clarified. An attempt is also made to infer the divergence time of these three genera using a molecular clock hypothesis and to assess putative correlations between the origin of the three genera and geological events.

MATERIALS AND METHODS

Plant species and material

Sequence data were acquired for 22 species of Crawfurdia, Gentiana, Metagentiana and Tripterospermum as indicated by asterisks in Table 1. The voucher specimens were deposited in the Herbarium of the Northwest Plateau Institute of Biology, Xining, Qinghai Province. Sequences for 28 additional species were retrieved from GenBank. The origins of samples, voucher information, GenBank accession numbers and chromosome numbers of species studied are listed in Table 1. Representatives of subtribe Swertiinae, the sister clade of subtribe Gentianinae, Gentianella moorcroftiana, Gentianopsis barbata (Gentianopsis crinita for trnL), Megacodon stylophorus and Swertia bimaculata were used as outgroups according to the results of previous studies (Yuan and Küpfer, 1995; Gielly and Taberlet, 1996; Yuan et al., 1996).

Table 1.

Origin of samples, voucher information, chromosome numbers and GenBank accession numbers of DNA sequences of Crawfurdia, Gentiana, Metagentiana and Tripterospermum

GenBank accesssion no.
Genus/section
 Species
 Locality
 Voucher
 Chromosome number: 2n (n)/x
 ITS or ITS1, ITS2
 trnL (UAA) intron
Crawfurdia Wall. x = 23
C. delavayi Franch. Mt Cangshan, Yunnan, 3620 m Chen03140 46 (23) AY562176* AY563391*
C. speciosa Wall. Cuona, Tibet, 3000 m 751592 46 (23) AY858675* AY858682*
C. tibetica Franch. Mt Gongga, Sichuan, 2300 m Chen03097 46 (23) AY563383* AY563393*
Gentiana (Tourn.) L. x = ?
Calathianae Froelich G. terglouensis Hacquet Hochobir, Austria – 42, 38 or 40 – X77897
G. verna L. St Cergue, Switzerland – 28 (14) – X75704
Chondrophylla Bunge G. aristata Maxim. Maqu, Gansu, China; 3500 m Y92-328 14 (7) Z48100/Z48116 –
G. boryi Boissier Sierra Nevada, Spain; 2300 m Z93-S1 20 or 26 – X77874
G. flexicaulis H. Smith ex Marq. Mt Taibai, Shaanxi, China; 3400 m Y92-264 14 (7) Z71937/Z71938 –
G. heleonastes H. Smith ex Marq. Maqu, Gansu, China, 3650 m G032 12 (6) Z71939/Z71940 –
G. piasezkii Maxim. Mingxian, Gansu, China; 2900 m Y92-272 36 (18) Z71955/Z71956 –
G. squarrosa Ledeb. Xiahe, Gansu, China; 3000 m G046 38 (19) Z71965/Z71966 –
Ciminalis (Adanson) G. alpine Villars Grand Chavalard, Switzerland Y93-09 36 (18) – X77868
Dumorti G. angustifolia Villars Hautes, Franch Kadereit 95/24 36 – X75699
G. clusii Perrier and Songeon Grand Chavalard, Switzerland Y93-13 36 (18) – X77879
G. dinarica G. Beck Abruzzi, Italy Hungerer 36 – X77882
G. ligustica R. de Vilmorin and Chopinet Alpes Maritimes, France Merxmuller 36 – X77886
Cruciata Gaudin G. crassicaulis Duthie ex Burk. Mangkang, Tibet, 3800 m Ge015 26 (13) AY858676* AY858684*
G. cruciata L. Botanic Garden, Geneva, Switzerland – 52 – AF102434
G. macrophylla Pall. Dangchang, Gansu, China Y92-271 26 (13) Z48067/Z48086 –
G. straminea Maxim. Maqu, Gansu, China Y92-313 52 (13) AF346015 –
Dolichocarpa T. N. Ho G. tetrasticha Marq. Dangxiong, Tibet, China; 4500 m Y92-128 24 (12) Z71967/Z71968 –
Frigida Kusnez. G. algida Pall. Rocky Mt, Colorado, USA Y91-S10 24 (12) Z48142/Z48117 AJ490239
G. frigida Haenke Mt Rila, Bulgaria NEU93-17 24 (12) – X77883
Isomeria Kusnez. G. depressa D. Don Zhangmu, Tibet, China Y92-118 24 (12) Z48062/Z48081 –
Gentiana G. montserratii Vivant ex Greuter Castanesa, Spain – – – X77887
Monopodiae T. N. Ho G. callistantha Diels et Gilg Luqu, Gansu, China Y92-298 26 (13) Z48095/Z48078 –
G. futtereri Diels et Gilg Dingqing, Tibet, China, 4160 m Liu1056 – – AY858685*
G. veitchiorum Hemsl. Leiwuqi, Tibet, China, 4280 m Liu1042 24 (12) AY858677*
Microsperma T. N. Ho G. atropurpurea T. N. Ho Dangxiong, Tibet, China, 4620 m Liu1095 – AY858678* AY858686*
G. delavayi Franch. Lijiang, Yunnan, China; 2850 m Y92-229 26 (13) Z48099/Z48080 –
Phyllocalyx T. N. Ho G. phyllocalyx C. B. Clarke Makalu, Nepal Neu97-30 26 (13) AJ318537/ AJ315189
Gentianella Moench. AJ410316
G. moorcroftiana (Wall. Ex Griseb.) – R. McBeath 2093 26 AJ294615/ AJ408007
Airy-Shaw AJ294675
Gentianopsis Ma G. barbata (FrÖel.) Ma Mengyuan, Qinghai, China Liu J.Q.654 26 (13) AF346007 –
G. crinita (Froelich) Ma Mainz Botanic Garden, Germany – 78 – AF102433
Megacodon (Hemsl.) x = 7
H. Smith M. stylophorus (C. B. Clarke) H. Smith Lijiang, Yunnan, China Ge106 x = ? AY858679* AY858687*
Metagentiana T. N. Ho and S. W. Liu x = 17, 21, 23
M. eurycolpa (Marquand) T. N. Ho and S. W. Liu Luquan, Yunnan, 2600 m Zhang0274 – AY858673* –
M. gentilis (Franchet) T. N. Ho and S. W. Liu Kunming, Yunnan, 2220 m Chen03122 42 (21) AY562177* AY563386*
M. leptoclada (I. B. Balfour and Forrest) T. N. Ho and S. W. Liu Dali, Yunnan, 3500 m Liu22798 – AY858674* AY858681*
M. primuliflora (Franchet) T. N. Ho and S. W. Liu Kunming, Yunnan, 2290 m Chen03124 42 (21) AY562178* AY563385*
M. pterocalyx (Franchet) T. N. Ho and S. W. Liu Heqing, Yunnan, 3620 m Chen03158 34 (17) AY562171* AY563384*
M. rhodantha (Franchet) T. N. Ho and S. W. Liu Kunming, Yunnan, 2220 m Chen03123 46 (23) AY562174* AY563390*
M. serra (Franchet) T. N. Ho, S. W. Liu and S. L. Chen Lijiang, Yunnan, 2450 m Chen03141 34 (17) AY562175* AY563387*
M. souliei (Franchet) T. N. Ho, S. W. Liu and S. L. Chen Lijiang, Yunnan, 3320 m Chen03146 46 (23) AY562170* AY563388*
M. striata (Maximowicz) T. N. Ho, S. W. Liu and S. L. Chen Daofu, Sichuan, 3510 m Chen03081 46 (23) AY562173* AY563389*
M. villifera (H. W. Li ex T. N. Ho) T. N. Ho and S. W. Liu Yunlian, Sichuan, 800 m Chuan0018 – AY858672* AY858680*
Swertia L. S. bimaculata (Sieb. Et Zucc.) Hook. f. et Thoms ex C. B. Clarke Makalu, Nepal NEU97Z-22 x = 11 AJ318552/AJ410331 AJ315204
Tripterospermum Blume x = 23 or 10
Tripterospermum T. cordatum (Marq.) H. Smith Kunming, Yunnan, 2210 m Chen03112 46 (23) AY562172* AY563392*
T. filicaule (Hemsl.) H. Smith Mt Gongga, Sichuan, 1990 m Chen03098 – – AY858683*
T. volubile (D. Don) Hara Nielamu, Tibet, 2300 m 1279 – AY858667* –
Platyspermum C. J. Wu T. chinense (Migo) H. Smith Mt Tianmu, Zhejiang, 1506 m Liu160810 – AY858668* –
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*

Indicates sequences that were registered in the present study (?, not affirmative; –, not available).

Chromosomal numbers reported by Yuan and Küpfer (1993a, b), Chen et al. (1997) and Ho et al. (2002a, b).

DNA extraction and PCR amplification

DNA was extracted from silica-gel-dried leaf material (Chase and Hills, 1991) or from leaf tissue taken from herbarium sheets. Total genomic DNA was extracted using the 2× CTAB procedure of Doyle and Doyle (1987) or the CASsuper Plant Genomic DNA Isolation Kit (CASarray, Shanghai, China). The ITS region was amplified with universal primers 1 and 4 (White et al., 1990), and primers ‘c’ and ‘d’ were used to amplify the trnL intron (Taberlet et al., 1991) in 25-μL reactions. PCRs were performed in a Biometra thermal cycler programmed for 4 min at 94 °C, followed by 36 cycles of 94 °C for 50 s, 53 °C (46 °C for trnL) for 50 s and 72 °C for 50 s, with a final extension of 72 °C for 7 min.

PCR purification and sequencing

All successfully amplified DNA fragments were purified using a CASpure PCR Purification Kit following the manufacturer's protocol (CASarray) prior to sequencing. The primers used for sequencing were the same as those used for PCR. The sequencing reactions were carried out in a Biometra thermal cycler (Tpersonal 48) using a DYEnamic Dye Terminator Cycle Sequencing Kit (Amersham) following the recommended protocol, but with the reaction volumes scaled down to 10 μL. The cycle sequencing products were cleaned using Autoseq 96 plates (Amersham) and then analysed with a MegaBACE DNA Analysis System (Amersham Biosciences Corp.). Both strands of DNA were sequenced.

Sequence alignment

The ITS and trnL sequences were aligned using ClustalX (Thompson et al., 1997), with additional minor manual adjustments. Potentially informative and unambiguously assessable indels were scored as binary characters regardless of their length, and added to the sequence data matrix (Simmons and Ochoterena, 2000). The boundaries of the sequences in the studied material were made by comparison with the published sequences of the genera of subtribe Gentianinae retrieved from GenBank (Yuan and Küpfer, 1995; Yuan et al., 1996).

Phylogenetic analysis and molecular clock test

Phylogenetic analyses were performed using PAUP* 4.0b10 (Swofford, 2003). In maximum parsimony (MP) analysis, characters were equally weighted and unordered (Fitch, 1971), with all gaps treated as missing data. Heuristic searches with 100 random additions of sequence replicates, in combination with ACCTRAN character optimization, MULPARS, tree-bisection-reconnection branch-swapping and STEEPEST DESCENT on were utilized to search for possible multiple islands of most-parsimonious trees (Maddison, 1991). The relative support for individual clades was evaluated by bootstrap (BS) analysis (Felsenstein, 1985). BS values were calculated using 1000 replicates of heuristic searches, each with 10 random addition sequence replicates using tree-bisection-reconnection and MULPARS on options.

Support for each branch was assessed using both BS and Bayesian analyses. BPP were estimated as the proportion of trees sampled after burn-in that contained each of the observed bipartitions (Larget and Simon, 1999). Analyses were performed with MrBayes v2.01 (Huelsenbeck and Ronquist, 2001), with GTR + Γ + PINVAR parameters being estimated during the run, and using the default value of four Markov chains. Multiple chains can assist in more easily traversing tree-space and help avoid entrapment in local topological optima. The Monte Carlo Markov chain length was 1 000 000 generations, and the chain was sampled every 100 generations. Log-likelihood values for sampled trees stabilized after approx. 200 000 generations. Therefore, the last 8000 sampled trees were used to estimate BPP, also called Bayesian support values. If >95 % of the sampled trees contained a given clade, it was considered to be significantly supported by the data produced.

A maximum likelihood (ML) analysis was also conducted, using PAUP* 4.0b10. For the ML analyses, among-site rate variations were modelled using a gamma distribution and the shortest trees from the MP analyses as starting points for ML estimation of transitions : transversion (ti/tv) ratios and the alpha parameter of the gamma distribution for among-site rate variation. Then an iterative procedure as described in Swofford et al. (1996), in which the most likely tree from each heuristic search was used to re-estimate the ti/tv ratio and alpha parameter, was followed. This procedure was repeated until essentially no change occurred in the likelihood estimate between iterations. The program Modeltest, Version 3.06 (Posada and Crandall, 1998) was used to find the model of sequence evolution that best fits the data set according to hierarchical likelihood ratio (LR) tests (P = 0·05). The GTR + I + G model (GTR = general time reversible; I = proportion of invariable sites; G = gamma distributed among-site rate variation) and TVM + G model (TVM = transversion model) in the ITS and trnL data, respectively, and parameter settings (gamma shape, base frequencies) were selected through the Hierarchical Likelihood Ratio Tests procedure implemented in Modeltest. The LR test statistic is distributed as χ2 with degrees of freedom equal to the number of free parameters between the two models (Goldman, 1993) when the models of sequence evolution are nested. A molecular clock hypothesis was also tested using the LR test, based on twice the difference between the log-likelihoods for trees generated from clock and clock-free analyses (Muse and Weir, 1992; Baldwin and Sanderson, 1998; Wang et al., 2000), using a χ2 distribution with N – 2 degrees of freedom (where N is the number of terminal taxa in the tree). When the LR test rejected a molecular clock hypothesis, ML trees based on the ITS and trnL intron sequences were subjected to non-parametric rate smoothing (NPRS) (Sanderson, 1997) using the default settings in TreeEdit v.1.0a8 (Rambaut and Charleston, 2000; Richardson et al., 2001) to estimate divergence times.

RESULTS

The lengths of the unaligned ITS and trnL sequences varied from 689 to 692 base pairs (bp) and from 252 to 271 bp, respectively, among species of Crawfurdia, Metagentiana and Tripterospermum. The aligned ITS and trnL sequences were 699 bp and 327 bp long, respectively. Mean pairwise distances ranged from 0·96 % (M. euryolapa vs. M. leptoclada) to 7·72 % (M. pterocalyx vs. M. rhodantha) for ITS and from 0·37 % (M. gentiles vs. M. serra and M. leptoclada vs. M. serra) to 7·91 % (M. pterocalyx vs. M. primuliflora) for trnL within Metagentiana. The highest pairwise distance found between Metagentiana and other genera in subtribe Gentianinae was 17·37 % (G. aristata vs. M. rhodantha) and 15·23 % (G. boryi vs. M. primuliflora) for ITS and trnL, respectively (distance matrix not shown).

When gaps were excluded, the ITS sequences used for analyses contained 193 potentially informative changes. The strict consensus tree was generated from 387 most-parsimonious trees in one island each with 740 steps, a consistency index of 0·57 and a retention index of 0·72 (not shown). The Bayesian majority rule consensus tree pooled from the Bayesian trees is shown in Fig. 1. Many of the nodes along the spine of this tree have strong or moderate BPP and BS support. One of the ML trees is also shown (Fig. 2A). The topology of the ML trees is similar to that of the Bayesian majority rule consensus tree with only minor differences between them. The present analysis strongly supports the placement of Crawfurdia, Metagentiana and Tripterospermum (grey frame), as sister to the Gentiana clade (BPP = 100 %; BS = 94 %), as previously reported by Yuan et al. (1996). In the Crawfurdia, Metagentiana and Tripterospermum clade (Fig. 1), M. eurycolpa and M. leptoclada form a clade (BPP = 64 %; BS = 85 %) sister to the rest. Five species of Metagentiana (M. gentiles, M. primuliflora, M. rhodantha, M. serra and M. villifera) form a second clade (BPP = 93 %; BS = 93 %). Three species of Metagentiana (M. pterocalyx, M. striata and M. souliei) form a third clade placed with species of Crawfurdia and Tripteropermum. In this third clade, Crawfurdia tibetica forms a clade with Tripterospermum cordatum and T. volubile; thus the recognition of Metagentiana, Tripterospermum and Crawfurdia as three distinct genera is not supported.

Fig. 1.

Fig. 1.

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Fifty per cent Bayesian majority rule consensus tree based on ITS sequence data. Numbers above and below the branches indicate the Bayesian posterior probabilities and the MP bootstrap values based on 1000 replicates, respectively. Dashed lines denote branches that collapse in the strict consensus tree from the parsimony analysis. The species of Crawfurdia, Metagentiana and Tripterospermum are indicated by grey shading. The thick black line indicates the four outgroups.

Fig. 2.

Fig. 2.

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(A) The best maximum likelihood tree based on the ITS matrix (likelihood score, –ln 4997·54138). (B) The the best maximum likelihood tree based on the trnL matrix (likelihood score, –ln 1598·70584). The species of Crawfurdia, Metagentiana and Tripterospermum are indicated by grey shading. The thick black line indicates the four outgroups.

The aligned trnL data included 75 potentially informative characters when gaps were excluded. The Bayesian majority rule consensus tree is shown in Fig. 3. Parsimony analysis identified 3321 most parsimonious trees with 212 steps, a consistency index of 0·72 and a retention index of 0·85. The ML analysis resulted in a tree with a likelihood score of –ln 1598·70548 (Fig. 2B). Because the trnL sequence of M. eurycolpa was not obtained, this species was omitted in the phylogenetic analyses of trnL. These analyses also support the placement of Crawfurdia, Metagentiana and Tripterospermum (grey frame) together as sister to the Gentiana clade (BPP = 100 %; BS = 93 %). In agreement with the results based on ITS data, Crawfurdia, Metagentiana and Tripterospermum were polyphyletic.

Fig. 3.

Fig. 3.

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Fifty per cent Bayesian majority rule consensus tree based on trnL sequence data. See Fig. 1 for further details.

The LR test rejected a molecular clock hypothesis based on their models and correlative parameters for both ITS and trnL (P < 0·01). The ML trees based on ITS and trnL were subjected to clock-independent NPRS to obtain homogenized rates. There is no fossil record for the tribe Gentianeae to calibrate the molecular phylogeny of Crawfurdia, Metagentiana and Tripterospermum. The lowest rate of 4·48 × 10−9 substitutions per site per year (s/s/y) and the highest rate of 8·41 × 10−9 s/s/y previously reported for ITS data of Gentianella (Hagen and Kadereit, 2001) were used to estimate divergence times within the clade consisting of Crawfurdia, Metagentiana and Tripterospermum. The inferred divergence times were plotted on the ITS phylogenetic tree obtained with ML (Fig. 4). According to the rates and ITS sequence divergence, the clade formed by Crawfurdia, Metagentiana and Tripterospermum diverged from Gentiana about 11·4–21·4 Mya (million years ago). The divergence times between extant species range from 0·4 to 6·2 Mya.

Fig. 4.

Fig. 4.

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The maximum likelihood tree with estimated ages based on NPRS of ITS (8·41 × 10−9 s/s/y of the evolutionary rates). The dashed line denotes the divergence time of Crawfurdia, Metagentiana and Tripterospermum from Gentiana.

DISCUSSION

Polyphyly of Crawfurdia, Metagentiana and Tripterospermum

Crawfurdia, Metagentiana and Tripterospermum are not monophyletic according to the molecular data presented here. In the phylogenetic tree based on ITS, ten species of Metagentiana fell mainly in two clades: M. pterocalyx, M. striata and M. souliei grouped with the six sampled species of Crawfurdia and Tripteropsermum, whereas M. gentiles, M. primuliflora, M. rhodantha, M. serra and M. villifera grouped together as a strongly supported clade (Figs 1, 2A). With trnL, nine species of Metagentiana formed four clades in the Bayesian majority rule consensus tree (Fig. 3) and three clades in the ML tree (Fig. 2B). Metagentiana villifera formed a clade with M. gentiles, M. leptoclada, M. primuliflora and M. serra in the Bayesian majority rule consensus tree and grouped with M. pterocalyx and the three sampled species of Crawfurdia and Tripterospermum as a clade in the ML tree. These findings indicate that Crawfurdia, Metagentiana and Tripterospermum should not be treated as three distinct genera. However, the present study sampled only six species (out of about 41 defined by Ho and Pringle, 1995) of Crawfurdia and Tripterospermum. Analysis including more species of Crawfurdia and Tripterospermum is necessary to clarify the phylogenetic and taxonomic status of the three genera.

Relationships of Crawfurdia, Metagentiana and Tripterospermum

Phylogenetic trees for Crawfurdia, Metagentiana and Tripterospermum based on the ITS and trnL in this study agree to a certain extent with the phylogenetic hypothesis based on analyses of gross morphology, floral anatomy, chromosomes, palynology, embryology and other molecular data (Smith, 1965; Nilsson, 1967; Löve and Löve, 1976; Yuan et al., 1993a, b, 1996; Chen et al., 1999a, b, 2000; Ho et al., 2000, 2002b). The genus Metagentiana is easily recognized by its solitary, sessile flowers, sessile, broad-ovate to ovate-triangular leaves and large leaf-like bracts, which make this genus more similar to Crawfurdia and Tripterospermum than to Gentiana (Ho et al. 2002a). The stems of Gentiana and Metagentiana are erect and branched, whereas in Crawfurdia and Tripterospermum they are twining. The midveins of the calyx lobes are keeled and winged into the calyx tube in Crawfurdia, Metagentiana and Tripterospermum but are not keeled in Gentiana. The style is filiform and about as long as the ovary in these three genera, but is linear to cylindrical and shorter than the ovary in Gentiana (Ho et al., 2002a). Pollen morphology of subtribe Gentianinae was studied by Nilsson (1967), who suggested that there were close relationships between Crawfurdia, Metagentiana and Tripterospermum. Chromosome numbers have been reported for eight species of Metagentiana (Yuan and Küpfer, 1993a; Ho et al., 2002b). Their basic chromosomal numbers are x = 17, 21 and 23. The karyotypes of Metagentiana are 3A and 3B according to Stebbin's classification (Stebbins, 1971). The higher and apparently secondary basic numbers and asymmetrical karyotypes suggested that Metagentiana had an isolated position in the genus Gentiana (Yuan and Küpfer, 1993a; Table 1). Metagentiana is more similar to Crawfurdia and Tripterospermum than to Gentiana in karyological characters because Crawfurdia and Tripterospermum also have high basic numbers (x = 23) and asymmetrical karyotypes (S. L. Chen, unpubl. res.). In embryological characters, Metagentiana has a unitary original tapetum, uninucleate tapetal cells which do not protrude into the anther locule, one-celled middle layers, a typical parietal placenta, a hypertropous ovule type and ovules often arranged in four columns (Ho et al., 2000). Gentiana has a dual original tapetum, binucleate tapetal cells which elongate and protrude into the anther locule to form ‘trabeculae’ and ‘placentoids’, two-celled middle layers, a superficial parietal placenta, an anatropous ovule and ovules arranged in 10–30 columns (Ho and Liu, 1999). Crawfurdia and Tripterospermum share the same unitary original tapetal and typical parietal placenta with Metagentiana (Chen et al., 2000; Ho et al., 2000). Thus previous evidence indicates that Metagentiana has a closer relationship with Crawfurdia and Tripterospermum than with Gentiana. The molecular data in this study indicate that these three genera form a monophyletic group as sister group to Gentiana and that all three genera are polyphyletic as currently circumscribed.

Biogeographic considerations

Tribe Gentianeae is widely distributed, with the highest diversity occurring in the Old World. Of the two major clades of Gentianeae, subtribe Gentianinae is clearly centred in the Old World (Struwe et al., 2002). All members of Crawfurdia, Metagentiana and Tripterospermum grow in central to east Asia. Within Gentiana, some sections are primarily European, with a few species in north-west Africa, north-east North America and central Asia (Meusel et al., 1978). The widespread sections are generally most diverse in eastern Asia (Ho and Liu, 1990). An exception to this is Gentiana sect. Pneumonanthe which is most diverse in eastern North America. In the context of this distributional pattern and the inferred phylogenetic relationships, it seems most likely that the ancestor of Gentianinae occupied an alpine temperate range in the Old World, and that the New World and southern hemisphere were colonized secondarily (Yuan et al., 1996). The distribution of generic and specific diversity would suggest an eastern Asian origin for Gentianinae (Struwe et al., 2002). Virtually nothing is known about the timescale of this diversification, except that the European Gentiana sect. Ciminalis may have begun to radiate 2 Mya according to Hungerer and Kadereit (1998). However, geological and paleobotanical studies in south-east Asia, especially in south-western China, provide a good framework to develop a scenario regarding the divergence and radiation of Crawfurdia, Metagentiana and Tripterospermum. South-east Asia has a relatively high proportion of Tertiary relicts of vascular plants (Wu, 1980; Tiffney, 1985a, b; Qian et al., 2003). During the early Tertiary, a relatively uniform, warm climate covered the northern Hemisphere (Tiffney, 1985a). During this time, a relatively continuous, homogeneous flora with many tropical and subtropical elements, called ‘the boreotropical flora’ (Wolfe, 1975), spanned most of the current Arctic area (Latham and Ricklefs, 1993). This boreotropical flora was gradually shaped into a mixed mesic forest and became fragmented as cooling climates in the middle and late Tertiary towards the Pleistocene forced the flora southward (Wolfe, 1975; Tiffney, 1985a). During the climate cooling, cold-intolerant taxa at higher latitudes either migrated to lower latitudes or went extinct, giving way to cool-adapted taxa derived from the boreotropical flora or which evolved during the climate cooling (Leopold and MacGinitie, 1972; Wolfe, 1975; Tiffney, 1985a; Xiang and Soltis, 2001). The present data from the ITS analysis suggest that the three genera diverged from Gentiana about 11·4–21·4 Mya. This estimated time correlates well with the climate cooling in the Miocene (5·3–23 Mya). During the Miocene, south-western China was apparently occupied by mesic mixed deciduous hardwood forest with numerous broad-leaved evergreens (Hu and Chaney, 1940). Especially during the late Miocene, the drier climate had spread extensively in the western Himalayan region including south-western China, where evergreen forest was replaced gradually by semi-deciduous and dry deciduous forest with a rapid expansion of grasslands (Quade et al., 1989, 1995). A major reason is that the uplift of several thousand metres of the Himalayas with perhaps 2300–3000 m increase since the middle Miocene had resulted in drastic changes in the regional biota and dry climates in the Himalayan region. Gentiana, Primula and Rhododendron are among many genera that radiated widely in the mountains of China and underwent rapid radiation, probably driven by the Himalayan uplift since the late Miocene (Axelrod et al., 1996). Therefore, it is possible that the ancestors of these three genera grew in south-western China during the Miocene and produced more species and occupied a wider distribution in the subsequent radiation. The current species of these three genera are mainly distributed in western China and grow in coniferous forest, alpine shrub and alpine meadow habitats. The divergence time of the current species of three genera (about from 0·4 and 6·2 Mya) estimated from ITS sequences corresponds well with this hypothesis.

Acknowledgments

The authors thank the decision editor Dr Mike Fay and anonymous reviewers for critical comments, Dr Yuan Yongming for constructive comments on the manuscript, and Professor Ho Tingnong for identifying specimens. This work was supported by the key innovation project of the Chinese Academy of Sciences (KSCX2-SW-106) and the National Science Foundation of China (30170066).

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