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Systematics |
2Nationaal Herbarium Nederland, Universiteit Leiden, P.O. Box 9514, 2300 RA Leiden, The Netherlands; 3Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3DS, United Kingdom; 4Institute for Systematics and Ecology, Experimental Plant Systematics, University of Amsterdam, The Netherlands; 5Institute for Plant Genetics and Crop Plant Research, D06466, Gatersleben, Germany
Received for publication August 29, 2000. Accepted for publication March 15, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: Coelogyne • Coelogyninae • matK • molecular phylogeny • nrDNA ITS • Orchidaceae • plastid DNA RFLPs
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Most species are epiphytes, occurring in tropical lowland and montane rainforests. In open, humid environments, some species may also grow as lithophytes or even as terrestrials (Comber, 1990
). Most species are characterized by medium-sized to large, sweet-scented flowers that are pollinated by bees (van der Pijl and Dodson, 1966
), beetles (O'Byrne, 1994
) or wasps (Carr, 1928
; Dressler, 1981
). The number of recent, artificial hybrids being published indicates the growing commercial interest in this group (Erfkamp and Gruss, 1996
).
Although revisions of several sections of Coelogyne were published in the last decade, a comprehensive treatment of all species is still lacking. This is partly due to the problematic delimitation of groups within the genus. Pfitzer and Kränzlin (1907)
grouped the species of Coelogyne into 14 sections. In contrast, Holttum (1964)
proposed only four and De Vogel (1994)
and Clayton (in press)
recognized 23 subdivisions (Table 1). These large differences in opinion are due not only to the rather large number of species in the genus, but also to the relative lack of morphological characters available to define groups of species. For example, the presence of hairs on the ovary has been used to define sect. Tomentosae (De Vogel, 1992
). However, this character is likely to have evolved convergently in the sections/subgenera Coelogyne, Cyathogyne, Rigidiformes, Veitchiae, and Verrucosae. The naturalness and relationships of the sections and subgenera of Coelogyne have not been previously examined in a phylogenetic context.
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550 species (Pedersen, Wood, and Comber, 1997
). Synapomorphies of the subtribe are sympodial growth, pseudobulbs of one internode, terminal inflorescences, a winged column, and massive caudicles (Dressler, 1981
; De Vogel, 1986
; Butzin, 1992
). Coelogyne Lindl. is defined by a free, never-saccate lip with high lateral lobes over the entire length of the hypochile and papillose, toothed or warty keels (Seidenfaden and Wood, 1992
). The genus is distinguished from others in the subtribe merely by the absence of characters, such as a saccate lip base (present in all other genera of the subtribe) or a lip adnate to the column (present in Neogyna Rchb.f. and Gynoglottis J.J. Sm.; Butzin, 1992
). In addition, many characters intergrade among the genera of the subtribe. For example, a lip with small, inconspicuous lateral lobes characterizes both Chelonistele Pfitzer and Panisea (Lindl.) Steudel (De Vogel, 1986
; Lund, 1987
). A phylogenetic survey of Coelogyne and related genera of Coelogyninae using molecular characters can provide a preliminary phylogenetic classification and serve as an historical framework for evaluating hypotheses of morphological character evolution. The aims of this study were to use phylogenetic analyses of molecular data to (1) address the generic circumscription and sectional and subgeneric relationships within Coelogyne, (2) investigate the relationships of Coelogyne with its allies in subtribe Coelogyninae, and (3) determine whether some previously used morphological key characters are phylogenetically informative. To accomplish these goals, parsimony analyses were conducted on PCR (polymerase chain reaction) amplified restriction fragment length polymorphisms (RFLPs) of 11 regions of the plastid genome and sequence data from both the trnK intron (mostly matK) and the nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) regions.
The PCR-RFLPs were expected to be useful in reconstructing phylogenetic relationships within the genus Coelogyne based on previous RFLP studies in Orchidaceae (Chase and Palmer, 1992
; Yukawa et al., 1993
; Freudenstein and Doyle, 1994
); PCR-RFLPs provide a rapid way of sampling many parts of the genome, which have evolved at different rates and under different constraints (Gielly and Taberlet, 1994
).
The trnK intron has been used for phylogeny reconstruction at a variety of taxonomic levels in angiosperms (Soltis and Soltis, 1998
). In Orchidaceae, it has been used at the generic (Whitten, Williams, and Chase, 2000
) and species levels (Ryan et al., 2000
). The nrDNA ITS regions have been used extensively to infer phylogenetic relationships in Orchidaceae at both tribal (Douzery et al., 1999
), generic (Pridgeon et al., 1997
), and species level (Cox et al., 1997
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), ndhF (Neyland and Urbatsch, 1996
), rbcL (Cameron et al., 1999
), and matK evidence (Chase et al., unpublished data). Voucher specimens are listed in Table 2 and deposited at K or L.
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. Some samples were purified through a cesium chloride/ethidium bromide gradient (1.55 g/mL). Leaf material was taken from one individual per species.
PCR-RFLPs
The RFLPs were detected by digesting three coding (16S, psbA, psbD) and eight noncoding regions (trnT-trnL, trnL, trnL-trnF, trnC-trnD, trnS-psaA, atpB-rbcL, psbA-trnH, petA-psbE) of the plastid genome using 19 restriction enzymes: BamHI, BclI, BglII, BsmI, ClaI, DraI, EcoRI, EcoRV, HindIII, NdeI, NsiI, PstI, PvuII, SacI, ScaI, SspI, XbaI (six base cutters), DdeI (five base cutter), and HinfI (four base cutter). Primers used were from Demesure, Sodzi, and Petit (1995)
, Fofana et al. (1997)
, Sang, Crawford, and Stuessy (1997)
, Savolainen et al. (1995)
, Tsumura et al. (1995)
, and Taberlet, Gielly, and Bouvet (1991)
. The thermal cycling protocol comprised 3 min denaturation at 94°C followed by 35 cycles, each with 45 sec denaturation at 94°C, 45 sec annealing at 50°–57°C, and an extension of 2 min at 72°C, concluding with an extension of 10 min at 72°C. Digested PCR products were separated on 1.5–2% agarose gels and stained with ethidium bromide to detect polymorphisms. The sizes of the fragments were determined with reference to two markers, a HindIII-EcoRI digested lambda bacteriophage DNA marker and a 100-base pair (bp) ladder.
matK and ITS amplifications
The trnK intron (mostly matK) was amplified with the following four primers:—19F (5-CGTTCTGACCATATTGCACTATG-3) and 881R (5-TMTTCATCAGAATAAGAGT-3); 731F (5-TCTGGAGTCTTTCTTGAGCGA-3) and 2R, the last of which is from Johnson and Soltis (1994)
. The thermal cycling protocol comprised 28 cycles, each with 1 min denaturation at 94°C, 30 sec annealing at 48°C, an extension of 1 min at 72°C, concluding with an extension of 7 min at 72°C. All PCR products were sequenced directly after purification with QIAquick purification columns (QIAGEN, Amsterdam, The Netherlands). Four sequencing reactions were performed for each completed sequence, one with each of the four PCR primers, and these generated nearly complete overlapping single strand sequences for the trnK intron fragments.
The ITS1 and ITS2 spacers along with the 5.8S gene were amplified with the primers 17 SE and 26SE from Sun et al. (1994)
. The thermal cycling protocol comprised 26 cycles, each with 10 sec denaturation at 96°C, 5 sec annealing at 50°C, and an extension of 4 min at 60°C. All PCR products were cloned following the protocol of Promega's pGEM-T Easy Vector System (Promega Benelux, Leiden, The Netherlands) and then reamplified from transformed bacterial colonies by touching them with a sterile pipette tip and using that as template. Two sequencing reactions were performed for each completed sequence, one with each of the two PCR primers, and these generated nearly complete overlapping singlestrand sequences for the entire ITS fragments.
All amplified, double-stranded DNA fragments were purified using Wizard PCR minicolumns (Promega Benelux) and sequenced on an ABI 377 automated sequencer (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands), using standard dye-terminator chemistry following the manufacturer's protocols.
Phylogenetic analyses
Variable restriction sites were coded as present or absent. Length variations were not included as characters in the analyses due to difficult interpretation. Raw sequence data were processed with Sequencher version 4.01 (Gene Codes Corporation, 1998
). Sequences were aligned by using the clustal option of MegAlign version 4.03 (DNASTAR, 1999, Madison, Wisconsin, USA) with subsequent adjustment by hand. Characters at positions 143–171 bp were excluded from the ITS sequence data due to ambiguous alignment. Sequences are deposited in GenBank (AF302692 until 302761) and TreeBASE (S558; M838). The matK and ITS alignments and the PCR-RFLPs data set are available from the first two authors upon request (gravendeel@nhn.leidenuniv.nl; m.chase@rbgkew.org.uk).
Maximum parsimony (MP) analysis was performed on the RFLP and sequence data with PAUP* version 4.0b64 (Swofford, 1999
) using heuristic search, ten replicates of random-taxon entry, and tree bisection reconnection (TBR) swapping. Arundina graminifolia, Bletia purpurea, and Thunia alba were specified as outgroups in all analyses. All molecular characters were assessed as independent, unordered, and equally weighted using Fitch parsimony (Fitch, 1971
). Synapomorphic indels in the trnK sequences were coded as present/absent characters at the end of the matrix according to Graham et al. (2000)
. Number of transversions and their consistency index (CI) and retention index (RI) were calculated on one of the most parsimonious trees (MPT) of the combined analysis by using a stepmatrix with zero weights for transitions and the TREE SCORE command (ACCTRAN optimization). From these data, the number of transitions and their CIs and RIs were calculated. To evaluate monophyly, trees were constrained using the enforce topological constraints option in PAUP*. The relative robustness for clades found in each parsimony analysis was assessed by performing 1000 replicates of bootstrapping (Felsenstein, 1985
), using simple stepwise additions, subtree pruning and regrafting (SPR) swapping, MULTREES on, and holding only ten trees per replicate. Bremer support (Bremer, 1994
) was also calculated using the branch-and-bound option to examine trees up to six steps longer than the shortest tree found for each data set. Congruence of the separate data sets was assessed by visual inspection of the individual bootstrap consensus trees. Bootstrap trees were considered incongruent only if they displayed "hard" (i.e., bootstrap percentages >80) incongruences (Wiens, 1998
).
To explore the phylogenetic utility of some traditionally used morphological characters in classifications of Coelogyninae, character state evolution of the shape of the lip base and petals, presence of hairs on the ovary, and flower number per inflorescence was reconstructed using the assumptions of maximum parsimony with the Trace Character facility in MacClade version 3.04 (Maddison and Maddison, 1992
). A complete phylogenetic analysis with morphological characters in Coelogyne and allied genera will be addressed in a separate publication.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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matK sequence analysis
Length ranges of the matK gene and its flanking trnK sequences for Coelogyninae were 1536–1544 bp and 221–245 bp, respectively. Boundaries of the matK gene were determined by comparison to published sequences of Orchidaceae and related monocots. The final alignment has a total length of 1921 sites of which 279 are variable and 117 are potentially phylogenetically informative; there is one autapomorphic indel of eight bp in the matK gene and five synapomorphic indels in the flanking trnK sequences, ranging in size from 4 to 19 bp. The transition/transversion (ts/tr) ratio is 0.85, higher than the ratios found in Orchidaceae so far (Whitten, Williams, and Chase, 2000
), but lower than the ratios found in dicots (Soltis and Soltis, 1998
). Third codon positions contributed the most steps (146 on the combined tree), more than first or second positions, but all three sites displayed equal CI and RI values (Tables 5 and 6). The average number of changes per variable site is 1.3 (Table 4). The MP analysis yielded >10 000 most parsimonious trees (length = 368, CI = 0.80, RI = 0.82; Table 4). The matK bootstrap consensus tree is congruent with the results of the RFLP data, but shows more resolution at the (sub)generic level. Therefore, all plastid data were combined in a single analysis. The bootstrap consensus topology and the corresponding bootstrap percentages and Bremer support of this analysis are indicated in Fig. 1.
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The second major subclade consists of species of Neogyna, Panisea, Pholidota, Coelogyne sect. Bicellae, Brachypterae, Coelogyne, Elatae, Flaccidae, Fuliginosae, Hologyne, Lawrenceanae, Lentiginosae, Longifoliae, Moniliformes, Ptychogyne, and Speciosae (79%). Four strongly supported smaller sets of taxa are present in this second major clade: Neogyna gardneriana plus Pholidota imbricata (85%), C. fimbriata (sect. Fuliginosae) plus C. stricta (sect. Elatae) (100%), C. flexuosa (subgenus Ptychogyne) plus C. miniata (subgenus Hologyne) (86%), and sect. Moniliformes (86%).
nrDNA ITS sequence analysis
Length ranges of nrDNA ITS sequences for Coelogyninae were 204–253, 159–163, and 242–271 bp, respectively. Boundaries of the 5.8S gene are taken from Hershkovitz and Lewis (1996)
. One region of 29 bp in ITS1 was considered unalignable and therefore excluded. The final alignment has a total length of 749 sites (285, 169, and 293 sites for ITS1, 5.8S, and ITS2, respectively). Of the included positions, 485 are variable and 261 are potentially phylogenetically informative, which is in accordance with variation levels in most angiosperms (Baldwin et al., 1995
). The ts/tv ratio is 1.57 in the ITS1 spacer and 1.79 in the ITS2 spacer region (Table 6), which is in accordance with ratios found in Orchidaceae so far (Cox et al., 1997
; Pridgeon et al., 1997
; Whitten, Williams, and Chase, 2000
). The average number of changes per variable site is 2.6 (Table 4). The MP analysis yielded 55 most parsimonious trees (length = 1242, CI = 0.58, RI = 0.53; Table 4). The bootstrap consensus topology and the corresponding bootstrap percentages and Bremer support are indicated in Fig. 2.
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Combined analysis
Bootstrap consensus trees of the three individual data sets revealed no hard incongruences except for the placement of Panisea. To improve resolution, a combined analysis of all three data sets (excluding Panisea) was performed (Kluge, 1989
; Huelsenbeck, Bull, and Cunningham, 1996
). The combined matrix yielded four most parsimonious trees (length =1711; CI = 0.60; RI = 0.58) (Table 4). Bootstrap analyses of the combined data set (excluding Panisea) provided more resolution and higher internal support for relationships (>80%) than did any of the individual data sets (Table 4). One of the four most parsimonious trees with the corresponding bootstrap percentages and Bremer support is shown in Fig. 3.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Mes et al., 1997
; Asmussen and Liston, 1998
). Apparently, the rate of plastid DNA evolution is relatively low in Coelogyninae.
The excess of transversions (Table 6), the excess of substitutions at third-codon positions (Table 5), and the multiple stop-codons present in the alignment, all indicate a loss of function for matK in Coelogyninae. For dicots so far, matK data demonstrate ts/tv ratios >1.0 and a relatively even distribution of substitutions across codon positions (Xiang, Soltis, and Soltis, 1998
). A loss of function is also suspected for Maxillarieae (Whitten, Williams, and Chase, 2000
).
The number of phylogenetically informative characters of the nrDNA ITS sequences is much higher than those of the RFLPs and matK data (261 vs. 20 and 117, respectively; Table 4). However, many clades in the ITS bootstrap consensus are only weakly supported. The much lower CI and RI of the nuclear data (0.58 and 0.53) compared with the matK sequences (0.80 and 0.82) indicates a higher level of homoplasy, which is consistent with the higher rate at which the variable sites in this region evolve compared with the matK sequences (2.6 steps/site vs. 1.3 steps/site, respectively; Table 4).
New generic circumscription of Coelogyne
Many recent studies have indicated that combined molecular data sets utilizing regions with different levels of variation provide resolution at different levels of the cladogram and that phylogenetic resolution and bootstrap percentages are improved by directly combining independent molecular data sets (Chase and Cox, 1998
; Soltis and Soltis, 1998
; Whitten, Williams, and Chase, 2000
). The high level of congruence among the three data sets, representing both coding and noncoding as well as plastid and nuclear regions, and the high bootstrap percentages in the combined analysis support the combined tree as a good hypothesis of phylogenetic relationships of Coelogyne and Coelogyninae.
Coelogyne as currently circumscribed is polyphyletic, with species falling into at least two well-supported clades, a separation of the ancestors of these clades by Pholidota carnea, and a transformation from saccate to nonsaccate lip base (the main defining character of Coelogyne) occurring more than once. Constrained analyses of the combined data set showed that the number of additional steps needed to achieve monophyly of Coelogyne is relatively great (at least 154 steps longer).
There are two possible taxonomic solutions for a new phylogenetic classification of Coelogyne in which only monophyletic groups are recognized. The first would be to include all sampled species of Coelogyninae (excluding Pleione) within a single genus. According to the rules of priority, this genus should be called Coelogyne. However, given the large differences in floral morphology, the creation of many synonyms in widespread horticultural use, and the large number of species Coelogyne would then encompass (530), this option is not satisfactory. A second possibility would be to reduce Coelogyne to one of the three main clades found. The type species of Coelogyne (C. cristata) belongs to clade II (Coelogyne s.s.). The best option for reorganizing Coelogyne seems therefore to take the following actions: (1) Restriction of Coelogyne to the Coelogyne s.s. clade, including Neogyna and Pholidota. These two genera were already considered to be just sections of Coelogyne by Lindley, Griffith, and Reichenbach (Lindley, 1830
; Griffith, 1851
; De Vogel, 1988
). All species of Neogyna and Pholidota have glabrous ovaries and a hypochile with broad, erect lateral lobes. These characters are also present in the other species of the Coelogyne s.s. clade. (2) Removal of the species of Coelogyne sect. Coelogyne (in part), Cyathogyne, Rigidiformes, Tomentosae, Veitchiae and Verrucosae. The main morphological characters distinguishing these species from Coelogyne s.s. are the relatively large number of simultaneously opening flowers with persistent floral bracts, hairy ovaries, and ovate-oblong petals. These characters are also present in Bracisepalum, Chelonistele, Dendrochilum, Entomophobia, Geesinkorchis, and Nabaluia.
Sectional and subgeneric relationships within Coelogyne
From the 18 sections of Coelogyne considered here, just two (with only two sampled species each) form strongly supported monophyletic groups in the combined analysis: sects. Moniliformes and Verrucosae (both 100%). This is consistent with the clear morphological synapomorphies that characterize both sections. All species of sect. Moniliformes have elongate, unifoliate pseudobulbs, a rachis with distinctly swollen, short internodes, and many flowers opening in succession (Carr, 1935
). All species of sect. Verrucosae have rounded to strongly flattened bifoliate pseudobulbs, inflorescences with many, simultaneously opening flowers, and a rachis with a few sterile bracts at the base and scattered minute scale-like hairs, which are also present on the pedicel, ovary, and the abaxial side of the sepals and petals (Sierra, Gravendeel, and De Vogel, 2000
).
Coelogyne sect. Longifoliae is also monophyletic, although support for this clade is weak (76%). The species of sect. Longifoliae all have bifoliate pseudobulbs, long and stiff inflorescences, a rachis with long internodes, and intermediate-sized flowers opening in succession (Clayton, in press
). Coelogyne sect. Flaccidae is monophyletic in all shortest trees, but bootstrap support for this clade is low (<50%). This is in accordance with the few and not unique synapomorphies that define this section. Coelogyne sect. Flaccidae is characterized by a small number of simultaneously opening flowers with deciduous floral bracts and undulating keels on the lip, a combination of characters that also defines sect. Ocellatae (Clayton, in press
).
Coelogyne sect. Tomentosae is not monophyletic, but none of the branches separating its two parts receives even low internal support. Likewise, Coelogyne sects. Coelogyne and sect. Elatae are clearly paraphyletic, which is in accordance with the heterogeneity obvious in pseudobulb shape, inflorescence type, flower size, and morphology of the keels on the lip in both sections. The only character present in all species currently assigned to sect. Cristatae is the color of the flowers: white, with yellow/brown spots. The species currently assigned to sect. Elatae share only the sterile bracts at the base of the rachis and simultaneously opening flowers, a combination of characters present in many other Coelogyninae species.
Coelogyne sect. Lawrenceanae and sect. Speciosae are well separated, which is not in accordance with Seidenfaden (1975)
, who suggested that they should be combined. Molecular data support our view that they should be considered distinct sections because of their clear morphological differences. All species of sect. Lawrenceanae have shiny green, smooth pseudobulbs, hysteranthous inflorescences, and flowers with deeply incised, glabrous keels on the lip. All species of sect. Speciosae are characterized by angular, dull-green pseudobulbs, synanthous or proteranthous inflorescences, and flowers with hairy or warty keels on the lip (Gravendeel and De Vogel, 1999
).
A well-supported subset of species is formed by C. multiflora (subgenus Cyathogyne), C. plicatissima (sect. Rigidiformes), and C. veitchii (sect. Veitchiae). Morphological synapomorphies for this clade are the hairy ovaries, persistent floral bracts, and small flowers. Another clade with high support consists of C. fimbriata (sect. Fuliginosae) and C. stricta (sect. Elatae). Both species have sterile bracts on the scape and intermediate-sized flowers. A third group of taxa supported by high bootstrap percentages is made up of C. eberhardtii (sect. Lawrenceanae) and C. miniata (subgenus Hologyne). Both species have bifoliate pseudobulbs and deciduous floral bracts. However, in other characters, such as plant size, leaf texture, inflorescence type, and keel morphology, they show considerable differences. To investigate whether these three clades warrant the status of new sections, a much larger sampling within Coelogyne is needed.
Naturalness and content of subtribe Coelogyninae
Analysis of the combined RFLP, matK, and ITS data set indicates that Coelogyninae are monophyletic and diverged early into three major clades. Clade I comprises species of Coelogyne sect. Coelogyne, Cyathogyne, Rigidiformes, Tomentosae, Veitchiae and Verrucosae, from which Bracisepalum, Chelonistele, Dendrochilum, Entomophobia, Geesinkorchis, and Nabaluia were split by various authors. Synapomorphies for this group of species are the simultaneously opening flowers (with the exception of Geesinkorchis) and inflorescences with relatively many flowers. Many other characters, such as small flower size, persistent floral bracts, hairy ovaries, ovate-oblong petals and a hypochile with inconspicuous lateral lobes, although present in most taxa of clade I, are not perfectly coincident, probably due to considerable convergent evolution in this group of species. The presence of many different generic names in clade I can be explained by the large number of autapomorphies present, such as the presence of stipes in Geesinkorchis and a transverse callus on the lip of Entomophobia. Bracisepalum selebicum and both Dendrochilum species sampled form a well-supported subset of taxa in clade I. These three species have unifoliate pseudobulbs, pendulous inflorescences with sterile bracts on the base of the rachis, and small flowers with a hypochile with inconspicuous lateral lobes. Another well-supported subclade in clade I comprises Chelonistele sulphurea and Entomophobia kinabaluensis. Both species have erect inflorescences with sterile bracts on the base of the rachis and small flowers with a relatively short column. Generic boundaries within clade I are not clear yet, because most internal nodes of this clade are only poorly supported. More data should be collected to improve resolution. Additional taxon sampling is also needed to find the limits of more monophyletic groups.
Clade II (Coelogyne s.s.) subsequently diverged into species of Neogyna and Pholidota nested among species of Coelogyne sects. Bicellae, Brachypterae, Elatae, Flaccidae, Fuliginosae, Hologyne, Lawrenceanae, Lentiginosae, Longifoliae, Moniliformes, Ptychogyne, and Speciosae. Synapomorphies for this group of species are the glabrous ovaries, linear petals (with the exception of Pholidota), and broad, erect lateral lobes of the hypochile. Many other characters, such as a small flower number, deciduous floral bracts, and large flower size are not present in all taxa of clade II. Neogyna gardneriana and Pholidota imbricata form a strongly supported subset of taxa in clade II. Both species have an epichile with semi-orbicular, widely retuse lateral lobes.
Clade III consists of species of Pleione. The relatively isolated position of Pleione is consistent with the purplish pink color of the flowers, short-lived pseudobulbs, and annually deciduous leaves of many species in this genus, which do not occur in any of the other Coelogyninae (Cribb, Butterfield, and Tang, 1983
).
The position of Panisea differs in the plastid and the ITS trees. In all of the plastid cladograms, Panisea is placed in the Coelogyne s.s. clade (clade II), in some of them as sister species to C. fimbriata and C. stricta. In contrast, Panisea appears as sister species to Geesinkorchis in the majority of the ITS trees (clade I). A combined analysis including Panisea (not shown) results in a nearly complete loss of internal support for clades I and II, an indication that its position is incongruent in the trees from each genome. Therefore, we removed Panisea so that clear patterns could be discerned. Hard incongruence between nuclear and organellar phylogenetic trees is often attributed to introgression of a cytoplasmic genome from one species into the nuclear background of another species (Rieseberg and Brunsfeld, 1992
; Rieseberg, Whitton, and Linder, 1996
; Wendel and Doyle, 1998
). Panisea tricallosa, C. fimbriata, and C. stricta show an overlap in distribution area in northern India and Nepal. We suggest that Panisea tricallosa shares a similar matK sequence with these species as a result of introgression. However, introgression is not the only process that could produce such incongruence. A second cause might be coalescence of alleles antedating species divergence (lineage sorting). There are relatively few examples of plastid DNA polymorphisms that transcend species boundaries, probably because of the generally slow rate of plastid DNA evolution (Wendel and Doyle, 1998
). Therefore, introgression due to hybridization appears to be the most probable explanation for the incongruence caused by Panisea. However, it is difficult to distinguish between introgression and lineage sorting because they both may produce similar phylogenetic patterns (Hardig, Soltis, and Soltis, 2000
).
Phylogenetic utility of traditionally used key characters
The shape of the lip base and petals, presence of hairs on the ovary, and number of flowers per inflorescence have been used for diagnosing genera within Coelogyninae and sections/subgenera within Coelogyne (Pfitzer and Kränzlin, 1907
; De Vogel, 1992
; Pedersen, Wood, and Comber, 1997
; Clayton, in press
). To evaluate their phylogenetic significance, we reconstructed their distribution on one of the four cladograms from the combined analysis (optimization on any of the other three trees does not affect our conclusions).
Lip base shape
A saccate lip base (Fig. 4A) is present in all Coelogyninae sampled except for the species of Coelogyne (Fig. 4B). Figure 5A shows the most parsimonious derivation of a saccate lip base. A saccate lip base is gained at least four times and appears not to be phylogenetically useful at the generic level. The evolutionary lability of this character might be caused by a close association with pollination systems, which can be homoplasious in Orchidaceae (Dressler, 1981
; Chase and Palmer, 1992
; Hapeman and Inoue, 1997
). Moreover, lip bases in Coelogyninae might not be derived from the same structure for all taxa studied. For instance, in Bracisepalum the base of the lip has two sac-like extensions, which might not be homologous with the saccate lip base of the other Coelogyninae. Studies of floral development may give additional insight as to whether different lip-base types are derived by common descent or parallelism.
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Ovary indumentum
Hairy ovaries are present in all species of clade I except for Geesinkorchis, whereas all other Coelogyninae have glabrous ovaries (see also Fig. 5B). The presence of hairs on the ovary is a uniquely derived character state supporting clade I as a separate group in Coelogyninae.
Flower number
A clear gap is present in the number of flowers per inflorescence of the taxa analyzed. Inflorescences with 15 or more flowers are present in all species of clade I, whereas all other Coelogyninae (with the exception of Pholidota) have much lower flower numbers (see also Fig. 5B). Large flower numbers represent a synapomorphy for clade I.
Reorganization of Coelogyne
The traditional generic circumscription of Coelogyne is based mainly on the absence of a saccate lip base, which is present in all other genera of Coelogyninae. Absence of characters often indicates symplesiomorphy and generally makes the group defined by such a character paraphyletic. Sectional delimitations were previously based on such characters, which also intergrade considerably among closely related species. The poly- and paraphyletic nature of Coelogyne and several of its sections according to molecular data clearly shows how convergent floral morphology has confounded traditional taxonomy. Traditionally used classifications of Coelogyne and Coelogyninae are not supported by the molecular data presented here and should be abandoned.
When plotted on the molecular cladograms, some of the traditionally used key characters for generic delimitation in Coelogyninae, such as lip base and petal shape, seem unacceptably homoplasious. In contrast, ovary indumentum and flower number are good diagnostic characters. We propose to redefine the genus Coelogyne by the following two actions: (1) inclusion of Neogyna and Pholidota; these two genera fit perfectly in Coelogyne when a new definition of the genus consists of glabrous ovaries only, a lip with a saccate or flat base, and a hypochile with broad, erect lateral lobes; (2) removal of the species of Coelogyne sects. Coelogyne (in part), Cyathogyne, Tomentosae, Rigidiformes, Veitchiae, and Verrucosae; these species fit better in clade I because they share with other genera in this clade several synapomorphies, such as a relatively large number of simultaneously opening flowers with persistent floral bracts and hairy ovaries. Our phylogenetic analyses indicate that 160 species would be left in Coelogyne.
In contrast with the Coelogyne s.s. clade, a good morphological delimitation of clade I is still difficult. Many characters, although present in most taxa of clade I, do not map perfectly on the molecular cladograms due to convergent evolution in this group. In addition, generic boundaries within clade I are not yet clear, as most internal nodes have only low support. Additional sampling in clade I is needed to find the limits of more monophyletic groups and to justify formal proposals for nomenclatural changes.
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FOOTNOTES |
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6 Author for correspondence (e-mail: gravendeel{at}nhn.leidenuniv.nl
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LITERATURE CITED |
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