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Systematics |
2Natural Heritage Inventory, Oklahoma Biological Survey, University of Oklahoma, 111 E. Chesapeake St., Norman, Oklahoma 73019 USA; 3Department of Botany-Microbiology, University of Oklahoma, 223 Cross Hall, Norman, Oklahoma 73019 USA; 4Royal Botanic Garden, Mrs. Macquaries Road, Sydney, NSW 2000, Australia; 5Kings Park and Botanical Garden, Perth, Western Australia, Australia; 6Department of Conservation and Land Management, Perth, Western Australia, Australia; 7Cullman Molecular Systematics Laboratory, New York Botanical Gardens, Bronx, New York, USA; 8Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK
Received for publication September 12, 2000. Accepted for publication March 15, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: Acianthinae • Chloraeinae • Codonorchis • Diurideae • DNA sequences • matK • Megastylis • molecular systematics • monocots • Orchidaceae • phylogenetic relationships • trnL-F
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Mansfeld, 1937, 1955
; Lavarack, 1971, 1976
; Dressler, 1981, 1993
; Rasmussen, 1982, 1985
; Burns-Balogh and Funk, 1986
; Clements, 1995
; Szlachetko, 1995
). As delimited in the most widely accepted account (Dressler, 1993
), Diurideae include ten subtribes with 
43 genera containing >900 species. Nine of these subtribes (Acianthinae, Caladeniinae, Cryptostylidinae, Diuridinae, Drakaeinae, Prasophyllinae, Pterostylidinae, Rhizanthellinae, and Thelymitrinae) are predominantly from Australia (or Australia, New Zealand, and New Caledonia), whereas the tenth (Chloraeinae) is from South America and New Caledonia.
As characterized by Dressler (1993)
, Diurideae have prominent tuberoids, nonarticulate, convolute, chartaceous foliage, and acrotonic or pleurotonic anthers. However, none of these characters is unique to them, and some representatives of the tribe lack tuberoids or have scale-like leaves. As a result, Diurideae remain difficult to characterize morphologically, and their circumscription and possible affinities within the family have been problematic.
Historically, floral characters have been of the highest importance in orchids. Much of the work has focused on the column, specifically on the placement of the pollinia and associated structures with respect to the rostellum and stigma. Most orchid treatments make a distinction between acrotonic and basitonic anthers, but various authors have defined these terms differently. Dressler (1981, 1993)
stressed the spatial relationship between the anther and rostellum: acrotonic anthers were characterized as having the rostellum or viscidium associated with the apex of the anther, whereas basitonic anthers have the rostellum or viscidium associated with the base of the anther. Rasmussen (1982)
, on the other hand, used the point of attachment between the viscidium and pollinia: acrotonic anthers have the viscidium attached near the apex of the pollinia and basitonic anthers near the base. Orchids that lack a detachable viscidium cannot be categorized as either acrotonic or basitonic using the latter definition. Further confusing the issue, some authors recognize an intermediate condition termed mesotonic or pleurotonic (Mansfield, 1955
; Dressler, 1993
) in which the viscidium (or viscidia) are associated with the middle of the pollinia. In yet another definition of acrotony and basitony, Clements (1995)
emphasized the attachment of the anther to the column. According to Clements, the anther is attached to the column via a rudimentary anther filament in the acrotonic condition, whereas in the basitonic condition the individual locules of the anther are attached directly to the column for their entire length.
Given the different definitions of acrotony and basitony, characterizing anther types within Diurideae has been problematic. Dressler (1981, 1993)
viewed the tribe as having both acrotonic and pleurotonic anthers. Rasmussen (1982)
categorized the genera within the tribe with distinct viscidia as having either acrotonic or basitonic anthers, but Arthrochilus, Caladenia, Caleana, Chiloglottis, Cyanicula, Drakaea, Elythranthera, Glossodia, Leporella, Leptoceras, Lyperanthus, Paracaleana, Praecoxanthus, Pyrorchis, Rimacola, and Spiculea all lack a detachable viscidium and could not be categorized. Clements (1995)
considered all Diurideae to possess acrotonic anthers. In part because of these differing interpretations, acrotonic, pleurotonic, and basitonic anthers have been reported within Diurideae, leading different authors to interpret this condition as indicative of possible affinities to Spiranthoideae (sensu Dressler, 1981
), Neottioideae (sensu Rasmussen, 1982
) or both the latter and Epidendroideae (sensu Burns-Balogh and Funk, 1986
).
More recently, increasing emphasis has been placed on the use of vegetative and embryological characters in higher classification of the orchids (Dressler, 1981
; Clements, 1995
; Freudenstein and Rasmussen, 1999
). Unlike Spiranthoideae and Epidendroideae, but similar to terrestrial, basitonic orchidoid orchids, Diurideae have tubers. The similarity led Dressler (1981)
to group Diurideae, Orchideae, and Diseae in the subfamily Orchidoideae. Subsequent work (Kores, 1995
; Pridgeon and Chase, 1995
), however, has thrown enough doubt on the homology of the tubers in different lineages that these structures are now referred to as root-stem tuberoids (Dressler, 1993
). In addition, molecular studies (Cameron et al., 1999
; Kores et al., 2000
; Kores, in press
) have identified lineages basal to Orchidoideae with tuber-forming representatives, raising the possibility that their presence in Diurideae and Orchideae is plesiomorphic or convergent. Reliance on vegetative similarity links the acrotonic (or pleurotonic) Diurideae with the basitonic Orchideae rather than the acrotonic Spiranthoideae (Dressler, 1981, 1993
).
The mixed affinities of major floral and vegetative characters found in Diurideae led to a number of different placements of the tribe and subtribes. Lavarack (1971, 1976)
, Garay (1972)
, and Rasmussen (1982, 1985, 1986)
assigned Diurideae to Neottioideae. Dressler (1981, 1993)
included their constituents within Orchidoideae. Burns-Balogh and Funk (1986)
split Diurideae into three lineages that were dispersed among Spiranthoideae, Neottioideae, and Epidendroideae. Szlachetko (1991, 1995)
also divided the tribe, but he included the resulting groups within Orchidoideae and a new subfamily, Thelymitroideae. Clements (1995)
excluded two of the subtribes from Diurideae and placed the remainder of the tribe in Orchidoideae. Chloraeinae and Pterostylidinae, the two subtribes he excluded, were assigned to Spiranthoideae (sensu Dressler, 1993
). In addition, Clements also suggested that Diurideae and spiranthoid orchids were sister groups, which agreed with the findings of Kores et al. (1997)
and Cameron et al. (1999)
based on rbcL sequence data.
Recent work has used plastid matK sequences to increase the number of phylogenetically informative characters in an effort to better support the relationships of Diurideae (Kores et al., 2000
; Kores, in press
). This work has confirmed the sister relationship between Diurideae and Cranichideae and hence the need to subsume Spiranthoideae under Orchidoideae. It has also confirmed that the ten former subtribes do not all belong together. The members of Chloraeinae sampled at the time showed affinities to Cranichideae, not Diurideae, as did Pterostylidinae. Within Diurideae, a clade composed of Acianthinae and Prasophyllinae was sister to a lineage composed of two clades. One, the "core Caladeniinae," was sister to a weakly supported clade composed of a basal lineage of Diuris, Orthoceras, and Cryptostylis followed by members of Thelymitrinae, Drakaeinae, and a few genera formerly placed within Caladeniinae (sensu Dressler, 1993
).
The classification of the large Australasian genus Caladenia has been problematic. The formal classification by Bentham (1873)
remained the only comprehensive treatment until very recent research. Hopper and Brown in Hoffman and Brown (1992, 1998)
proposed the reinstatement of Leptoceras (R.Br.) Lindl. and the erection of three new genera: Cyanicula, Praecoxanthus, and Drakonorchis. Our data are discussed at greater length later in this paper and clearly support these proposals except for the status of Drakonorchis, which is embedded within Caladenia. Hopper and Brown (2000)
have recently formalized the description of Cyanicula and Praecoxanthus and erected a new subgeneric classification within Caladenia that includes the subgenera Caladenia, Calonema, Drakonorchis, Elevata, and Phlebochila.
In earlier work (Kores et al., 2000
), the strict consensus tree showed considerable resolution but bootstrap support for some lineages was low, especially so for relationships among major clades of Diurideae. As had happened repeatedly, this difficult tribe required more data to provide a well-supported hypothesis of generic relationships. Two regions, matK and trnL-F, were selected based on our previous work, as well as that of Jarrell and Clegg (1995)
for matK and Geilly and Taberlet (1994)
, Sheahan and Chase (2000)
, and Chase et al. (2000)
for trnL-F. The latter region encompasses the small 3' trnL exon (the only coding portion), the trnL intron, and the trnL-F intergenic spacer. The current work reports on results from multiple-gene matrices with increased taxon sampling.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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) but purified by ultracentrifugation using a CsCl2-ethidium-bromide density gradient (1.55 g/mL). Amplification was carried out in a Perkin-Elmer thermal cycler using 100-µL polymerase chain reactions (PCR). For matK, the protocol used 2.5 units of Taq polymerase (Promega, Madison, Wisconsin, USA), 2 µL 4% bovine serum albumin, 2.8 mmol/L MgCl2, and 100 ng of the two PCR primers, matK-19F (Molvray, Kores, and Chase, 2000
) and trnK2R (Johnson and Soltis, 1994, 1995
). The PCR profile included an initial premelt of 2 min 30 sec at 94°C, followed by 28 cycles of 1 min denaturation (94°C), 1 min annealing (52°C), and 2 min 30 sec elongation (72°C) with 8 sec added per cycle, followed by a 7 min final extension at 72°C. The two PCR primers for trnL-F were c and f (Geilly and Taberlet, 1994
). The PCR protocol differed from that of matK in that magnesium concentration was reduced to 2.5 mmol/L. The trnL-F PCR profile included an initial premelt of 2 min at 94°C, followed by 30 cycles of 1 min denaturation (94°C), 30 sec annealing (50°C), and 1 min elongation (72°C); and ending with a 7 min final extension at 72°C. The PCR products of both regions were purified using the Wizard PCR purification columns (Promega) following the manufacturer's protocols.
Cycle sequencing was carried out directly on the purified PCR product using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, California, USA), with 10 ng of primer, 3 µL of sequence dilution buffer, and 2 µL of cycle sequence mix in a 20-µL reaction volume. For trnL-F, primers c and f (Geilly and Taberlet, 1994
) were used for all taxa, supplemented by the internal forward and reverse primers (e and d) in templates with long homopolymer A or T regions. Six primers were used to sequence the complete matK–trnK region. These included the two PCR primers, three internal forward primers, and one internal reverse primer. The primer sequences are listed in Molvray, Kores, and Chase (2000)
. Cycle sequencing conditions for both regions were as follows: 26 cycles of 15 sec denaturation (96°C), 1 sec annealing (50°C), and 4 min elongation (60°C) using a Perkin-Elmer 9700 thermal cycler. Sequencing reactions were purified by ethanol precipitation and run on an ABI Prism 377 automated sequencer (Applied Biosystems). Electropherograms were assembled and edited with Sequencher 3.1 software (GeneCodes, Ann Arbor, Michigan, USA). Sequences were aligned manually. Two regions in noncoding trnL-F and two in the matK–trnK intergenic spacer have sufficiently high levels of variability between major clades that homology of the sequences at those positions cannot be assumed with confidence. The regions were excluded a priori from analysis. They were found in the aligned matrix at positions 1852–1895 and 2080–2115 in matK-trnK and 128–206, and 1478–1564 in trnL-F, for a total of 245 characters or 6.3% of the 3881 character data set. (Inclusion of these positions turns out not to affect topology, but does reduce bootstrap support slightly on some clades.) In addition, the trnL-F intron has an indel of highly variable length, which is absent in many Diurideae, but can be as long as 292 bp, for instance in Dendrobium. Alignment indicative of homology could not be established between subfamilies or orchidoid tribes. The indel was placed between positions 574 and 874, and the whole region was excluded both from analysis and totals of numbers of characters.
Phylogenetic analyses were performed on the two data matrices separately and in combination using PAUP* 4.02 (Swofford, 1998
). Starting trees were obtained using random sequence addition, searched using equally weighted maximum parsimony (Fitch, 1971
) with tree bisection-reconnection (TBR) branch swapping and MULPARS and Steepest Descent in effect. Tree limit was set at 5000. Positions where the gaps occurred were treated as missing data (Swofford, 1993
).
Internal support was assessed by bootstrapping (Felsenstein, 1985
), using equally weighted characters. Bootstrap percentages (BP) for each node were computed after resamplings followed by a maximum parsimony (MP) reconstruction (bootstrap option in PAUP* 4.02b, with 500 replicates of heuristic search, one random sequence addition per replicate, TBR branch swapping and maxtrees = 100).
The matK and trnL-F trees bootstrap consensus trees (Figs. 1 and 2) did not exhibit hard incongruence (Seelanan, Schnabel, and Wendel, 1997
), so the data sets were used in combined analyses. Increased resolution coupled with higher levels of bootstrap support were taken as another indication that phylogenetic signal was compatible between data sets and that direct combination was valid (Olmstead and Sweere, 1994
).
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; Cameron et al., 1999
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The matK region has a similar level of substitution for all three codon positions, stop codons are found within it, and indels occur that create reading frame shifts. Dissimilarities are that the trnL-F region appears to have more indels than matK, and some indels are much larger than in matK.
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The topology based on the combined matK and the trnL-F regions bears similarity to trees in Cameron et al. (1999)
based on the plastid gene rbcL and in Kores et al. (2000)
and Kores (in press)
based on matK. Subfamily Orchidoideae sensu lato forms a strongly supported clade (BP 100%) sister to a moderately supported Epidendroideae (BP 78%) (Fig. 3). Within Orchidoideae, three major lineages are evident. The first lineage includes Codonorchis, formerly considered a member of Chloraeinae and Diurideae, and its sister group, the basitonic orchids (BP 87%). The basitonic Orchideae form a strongly supported clade (BP 99%) that includes Diseae sensu Dressler (1993)
. The spiranthid orchids form the second major lineage (BP 99%), which includes Cranichideae, Megastylidinae, Pterostylidinae, and core Chloraeinae. The third major clade is composed of core Diurideae (BP 100%). Diurideae are sister to the spiranthid clade (BP 99%), and that combined clade is sister to Codonorchis–Orchideae.
Focusing on the spiranthid lineage (Fig. 3), three strongly supported suprageneric subclades are apparent. One of these includes the genera Pachyplectron through Gonatostylis and encompasses Goodyerinae and Pachyplectroninae (BP 100%). The second includes the genera Sacoila through Cranichis and represents Cranichidinae, Prescottiinae, and Spiranthinae (BP 100%). These two lineages, which include all subtribes of Cranichideae (except the unsampled Manniellinae), are sister to each other, although bootstrap support is currently lacking. Two other genera, Pterostylis and Megastylis pro parte, form a strongly supported group with the Cranichideae (BP 98%), but their precise relationship to each other or to the other members of Cranichideae is uncertain. Clements (1995)
suggested Pterostylis should be included among spiranthid orchids based on embryological characters, but otherwise both Pterostylis and Megastylis were formerly regarded as members of Diurideae. In our analysis, Megastylis pro parte is sister to Cranichideae, with Pterostylis sister to the combined cranichid–Megastylis clade, but there is no bootstrap support for either of these relationships. The third subclade within the spiranthid lineage, which is sister to all the other spiranthid orchids, includes the genera Chloraea and Gavilea (BP 100%), both South American representatives of the subtribe Chloraeinae, formerly in Diurideae.
In the core diurid lineage (Fig. 4), two subclades are apparent: one includes the genera from Adenochilus to Acianthus and represents the subtribes Acianthinae, Prasophyllinae, and core Caladeniinae (BP 80%); the other includes the genera from Orthoceras to Caleana representing the subtribes Drakaeinae, Thelymitrinae, Cryptostylidinae, Diuridinae, and portions of Chloraeinae (Megastylis rara) and Caladeniinae (Rimacola, Lyperanthus, Pyrorchis, Leporella) (BP 87%). Each of these subclades is considered in more detail below.
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Core Caladeniinae, sister to the Acianthinae–Prasophyllinae clade, lack bootstrap support and the most basal node collapses in a strict consensus of the eight trees obtained with equally weighted characters. However, the node above Adenochilus is strongly supported (BP 97%). Parenthetically, it is worth noting that Adenochilus is strongly supported (BP 88%) as sister to the Eriochilus–Caladenia clade in internal transcribed spacer (ITS) analysis of Caladeniinae alone (P. J. Kores and M. Molvray, unpublished data), and that this relationship is likely to be supported in further work. Within core Caladeniinae, Eriochilus branches off after Adenochilus, followed by Leptoceras and Praecoxanthus, the last of which is sister to a strongly supported clade comprising Caladenia, Cyanicula, Elythranthera, and Glossodia (BP 100%). Cyanicula, Elythranthera, and Glossodia form a weakly supported clade (BP 63%) that is sister to Caladenia (BP 100%).
The other major subclade within the core diurids includes representatives from five subtribes. Four of these subtribes constitute well-supported, monophyletic lineages: Diuridinae (Diuris and Orthoceras; BP 100%), Cryptostylidinae (Cryptostylis and Coilochilus; BP 100%), Thelymitrinae (Thelymitra and Calochilus; BP 100%), and Drakaeinae (Spiculaea to Caleana; BP 90%). Diuridinae are sister to a clade comprising Cryptostylidinae, Thelymitrinae, Drakaeinae, and several genera previously assigned to Caladeniinae. Within that clade, Cryptostylidinae are sister to the combined Thelymitrinae–Drakaeinae–Caladeniinae (pro parte) clade, but the relationship is weakly supported (BP 57%). The Thelymitrinae–Drakaeinae–Caladeniinae (pro parte) clade is well supported (BP 100%). Within this subclade, Thelymitrinae are sister to a well-supported lineage (BP 88%) made up of the genera Leporella, Pyrorchis, Lyperanthus, Rimacola, Megastylis (pro parte), and the representatives of subtribe Drakaeinae (Spiculaea to Caleana). The segregate genera Pyrorchis and Lyperanthus are sister to each other (BP 96%), as are the genera Paracaleana and Caleana (BP 100%). Also of interest is the placement of Megastylis rara near Drakaeinae and Rimacola elliptica with strong support (BP 93%).
The position of Rhizanthella could not be resolved in our studies. Unlike some other achlorophyllous orchids (M. Molvray, unpublished data), the plastid genome appears to be highly aberrant, and all attempts to amplify the plastid genes used in this study have failed. Recent work by P. J. Kores and M. Molvray (unpublished data) using nuclear 26S and 18S rDNA sequences place Rhizanthella in Diurideae (bootstrap support 93%), but its position within the tribe has not yet been resolved.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Kores et al., 2000;
Kores, in press
) are strongly supported in the current treatment, and some new relationships are reported here for the first time. Our study confirms that Diurideae are not monophyletic unless all of the representatives from the subtribes Chloraeinae (with the exception of Megastylis rara) and Pterostylidinae are excluded. The molecular data strongly support the transfer of the core Chloraeinae (Bipinnula, Chloraea, Gavilea, and Geoblasta) and Pterostylidinae to the spiranthid lineage, a placement supported by a number of morphological characters. All of the Diurideae sensu stricto, with the exception of Cryptostylis, Rimacola, Megastylis rara, Adenochilus, and Rhizanthella slateri, have tuberoids, but these structures are absent in all genera of core Chloraeinae except Geoblasta. In addition, most Diurideae have only one leaf per shoot, but core Chloraeinae, Megastylis, and Codonorchis have shoots with multiple leaves. Pterostylis also has multiple leaves per shoot, although, like Diurideae, the genus has prominent storage organs. The seed coat morphology in Pterostylis is typically spiranthid (Molvray and Kores, 1995
). Further, both Pterostylis and the representatives of the core Chloraeinae have a spiranthid embryo pattern (Clements, 1995, 1999
).
Another strongly supported finding is that subfamily Orchidoideae consists of three lineages, although the composition of these lineages differs somewhat from previous accounts based on single gene regions. The lineages are referred to in this discussion as the Codonorchis–Orchideae clade, the spiranthid clade, and the diurid clade. Only the last of these lineages closely conforms to the currently accepted circumscription for an existing tribe. Codonorchis has already been afforded subtribal status by Brieger (1974–1975)
, and in view of the molecular data Cribb and Kores (2000)
have proposed its elevation to the rank of tribe. However, we feel it would be premature to alter the delimitation of existing tribes within spiranthids or to propose new ones at this time because of our limited sampling within this clade.
The placement of Codonorchis sister to Orchideae has not been suggested in any previous classifications for the family. The genus includes from one (e.g., Hawkes, 1965
) to three (e.g., Mabberley, 1997
) species and is widely distributed in the temperate and subtropical regions of South America. The two specimens used in this study were obtained from Chile and the Falkland Islands, representing the eastern and westernmost limits of its range. Codonorchis has stalked subterranean storage organs similar to those found in some members of Orchideae and like most Orchideae it has several leaves per shoot. However, its floral structure is much more similar to the typical diurid rather than the basitonic orchidoid taxa. It has a long slender column without auricles, the anther is clearly acrotonic, the pollinia are not sectile, and only a single viscidium is present. Despite this obvious similarity in floral structure, embryological development in Codonorchis follows a pattern aberrant for a diurid (Clements, 1995, 1999
). Its placement sister to Orchideae is moderately well supported in the current treatment, which has a number of interesting implications. The position of Codonorchis sister to Orchideae offers further evidence that the basitonic anther is a derived condition, possibly a rapidly derived condition. Similarly, the presence of tuberoids in Codonorchis, Orchideae, and most Diurideae suggests their absence in most spiranthid orchids is a derived condition.
The other two lineages in Orchidoideae are Diurideae and the spiranthid orchids. The sister relationship between them, previously reported by Clements (1995)
, Kores et al. (1997)
, Cameron et al. (1999)
, Kores et al. (2000)
, and Kores (in press)
, is strongly confirmed by our molecular data, although there are few morphological characters that form unambiguous synapomorphies for this clade. Seed coats in both lineages have at least a few, sometimes many, intercellular gaps (Molvray and Kores, 1995
), but this character can be hard to interpret in some cases. Further, developmental studies have not yet been done, so there is no guarantee that gaps in different lineages are homologous. Clements (1995, 1999)
noted that the embryos of Orchideae have a multicelled suspensor, those of Cranichideae lack a suspensor, and those of Diurideae have a reduced, one- or two-celled suspensor. If reduction in the number of suspensor cells is considered a derived condition, then this multistate character could be ordered to constitute a synapomorphy for the diurid–spiranthid clade.
Despite the paucity of synapomorphies, the sister relationship between Diurideae and spiranthids has important implications for character phylogeny of underground storage organs and column morphology. The position of Chloraea and Gavilea, which lack tuberoids, as sister to other spiranthids, implies that the underground storage organs found in Pterostylis are either plesiomorphic or derived independently of those in core diurids. Further, the presence of tuberoids in both Orchideae and core diurids indicates that these organs are plesiomorphic and therefore unlikely to be phylogenetically informative until their homologies are subjected to detailed developmental studies.
Acrotony has been used to define relationships within the Orchidaceae, despite the fact that there are problems defining the term. If we accept Dressler's or Rasmussen's definition of acrotony, in which the viscdium or rostellum is associated with the apex of the anther or its pollinia, then the distribution of the character on the tree indicates symplesiomorphy or convergence. Acrotonic anthers are present in some epidendroids, the spiranthids, most diurids, and Codonorchis, whereas basitonic anthers, sensu Dressler, are restricted to core Orchideae or to core Orchideae and Diurideae sensu Rasmussen. However, floral developmental studies by Kurzweil (1987a, b, 1988)
show that all monandrous orchids are similar in early floral organogeny and that acrotony subsequently develops in a variety of different ways. In more-derived epidendroid orchids, acrotony is achieved by inward bending of the anther brought about by differential growth at its base. However, in the spiranthids and less-derived epidendroids, it is achieved by late growth of the rostellum. Within Diurideae, even among sister genera such as Thelymitra and Calochilus, acrotony is achieved via distinctly different developmental pathways (Andrew Perkins, Royal Botanic Gardens, Sydney, Australia, personal communication). In Thelymitra, the rostellum is pushed near the tip of the anther by the growth of the stigma as a whole, whereas in Calochilus it results from elongation of the central sublobe of the median stigma lobe between the anther loculi. Other Diurideae, notably Acianthinae, are considered to have an intermediate, pleurotonic anther. However, Acianthinae are sister to Prasophyllinae that are characterized by a typical acrotonic anther. Given the close and well-supported relationship between the two subtribes, the transition between the two states may not be as significant as previously thought. Our results are consistent with Rasmussen's (1982)
hypothesis that pleurotony results from a more or less symmetrical increase in the size of the pollinia in both the upper and lower halves of the locule during maturation. The sister relationship between acrotonic Codonorchis and basitonic Orchideae provides more evidence that column morphology may be more labile than previously hypothesized.
The lack of unambiguous morphological synapomorphies is also evident in the tribe Diurideae. All, except the suspensorless Townsonia, have a one- or two-celled suspensor, and the embryo develops entirely within the integuments. This type of development has been termed the diurid embryo pattern (Clements, 1995, 1999
). However, Megastylis "glandulifera" (fide Clements, 1995
) has a diurid embryo pattern, but in our analysis, Megastylis glandulosa is unequivocally a member of the spiranthid clade, making yet another exception to a diurid synapomorphy. In contrast, Megastylis rara is a true diurid, but its embryology has not been investigated. Most diurids have a one-leafed shoot, but there are some reversals (Diuris, Orthoceras, Arthrochilus, Chiloglottis, Aporostylis, Leporella, and Rimacola). Cryptostylis, Townsonia, Adenochilus, and Megastylis rara have one-leafed shoots connected by a rhizome, such that even though the whole plant has more than one leaf, the individual shoots are still one-leafed.
Morphological synapomorphies for the major lineages within Diurideae are similarly difficult to identify. One of the most striking aspects of Diurideae are their variously appendaged columns. They may be partially free from the column and variously shaped, or entirely united with the column and wing-like. Several authors have used these differences as characters (Dressler, 1993
; Clements, 1995
; Szlachetko, 1995
). However, there is little agreement between the plastid tree topology and what might be expected based on column appendages. Diuridinae, Thelymitra, and Prasophyllinae have partially free, vascularized, obliquely falcate column appendages, yet the latter is in one major clade of the tribe, the former two in another. Given that these processes often help to orient potential pollinators (Jones, 1970, 1974a, b, 1981
) and the widespread evidence of great morphological plasticity in response to pollinator selection, it appears likely that column appendages are yet another feature subject to considerable convergence.
At the subtribal level, morphological synapomorphies are sometimes present. Acianthinae have a broad, reniform leaf, a petiole fused with the peduncle, and lack secondary roots. Prasophyllinae have a cylindrical leaf, sectile pollen, and a hamulus. The latter two characters also occur outside Diurideae (Freudenstein and Rasmussen, 1997, 1999
). Caladeniinae have a recurved, often three-lobed labellum, clavate appendages on the labellum, and a distally winged column; the plants are almost always pubescent. Thelymitrinae have a column with prominent, vascularized column appendages united with a median filament forming a hood around the style. Multiple leaves per shoot, which may be a symplesiomorphy, are characteristic of Diuridinae, as well as a column with prominently vascularized column appendages equal to or surpassing the anther. Cryptostylidinae have rhizomes and nonresupinate flowers with a simple lip.
Considering relationships between subtribes within the Diurideae, obvious morphological synapomorphies appear rare. In the present analysis, Prasophyllinae are sister to Acianthinae, but the only character they appear to have in common is secretory tissue at the base of the labellum. Australian species of Acianthus and Cyrtostylis are reported to secrete nectar from paired glands at the base of the labellum (Jones, 1988
). Secretory tissue associated with the base of the labellum has also been reported within New Caledonian Acianthus by Kores (1995)
and for species of Genoplesium and Prasophyllum by Jones et al. (1999)
. Nectar production in Microtis has been suggested by Peakall and Beattie (1989)
. In their study of pollination in Microtis parviflora, they noted that ants forage persistently, visiting individual flowers and inflorescences repeatedly for nectar. They also observed that ants (Iridomyrmex gracilis) visited only newly opened flowers and that pollinia attachment and pollen transfer to the stigma occurred while they probed the base of the labellum. There are no reports of nectar production in Townsonia or Corybas, but the former is reported to be autogamic while the latter relies on pollination by deceit (Jones et al., 1999
). As a consequence, the secretory function of the labellum may have been secondarily lost in these genera. Other higher level relationships within Diurideae, such as the Diuridinae–Drakaeinae clade are not supported by any apparent nonmolecular synapomorphies.
Some authors have suggested that the mycorrhizal associations of terrestrial orchids may have phylogenetic significance (Warcup, 1981
; Clements, 1988
; Dressler, 1993
). The patterns of fungal associations are complex, judging by the trees presented here. Some well-supported subtribes are uniform with respect to symbionts. For instance, the Diuridinae–Drakaeinae clade has a preponderance of taxa that share association with Tulasnella. Burnettia will likely be a member of the Lyperanthus–Pyrorchis clade and also has the same fungus. Exceptions are Megastylis rara, Rimacola, Leporella, and Cryptostylis. All members of Caladeniinae clade share Sebacina, but other clades vary in their associations. Within Acianthinae, Acianthus and Corybas associate with Tulasnella, whereas Cyrtostylis has Sebacina. In Prasophyllinae, Microtis has Sebacina, but Genoplesium and Prasophyllum share Ceratobasidium. Repeated switches appear to have occurred among fungal symbionts and their hosts. Although it may be difficult to extract phylogenetic information from the patterns at higher taxonomic levels, fungal associations certainly bear examination and comparison based on a robust phylogenetic tree.
Our results indicate that Cryptostylidinae belong with other diurids, not with the spiranthoid orchids as postulated earlier (Dressler, 1981
; Rasmussen, 1985
; Burns-Balogh and Funk, 1986
). Stern et al. (1993)
, in their study of Spiranthoideae sensu Dressler (1981, 1993)
, noted that Cryptostylis does not share the anatomical synapomorphies he identified for the subfamily. In our analysis, Cryptostylidinae are embedded within a well-supported clade made up of four monophyletic lineages. Within this larger clade the subtribe is sister to the combined Thelymitrinae–Drakaeinae subclade, but this relationship has only weak bootstrap support. The molecular data do not support the elevation of Cryptostylidinae to tribal rank as proposed by Szlachetko (1995)
and Clements (1995)
.
The trees based on the molecular data presented here also indicate that the Chloraeinae are polyphyletic. One species, Megastylis rara, is placed with representatives of Drakaeinae (Diurideae), whereas the other two species, Megastylis gigas and M. glandulosa, are sister to Cranichideae in the spiranthid lineage. Gavilea and Chloraea (the core Chloraeinae) are also placed in the spiranthid lineage, but they are not sister to that part of Megastylis. As previously mentioned, Codonorchis, the other representative of Chloraeinae, is sister to Orchideae. In light of the molecular data, we have a possible explanation for the unusually high morphological diversity that has been noted within the subtribe by some authors (Ackermann and Williams, 1981
; Clements, 1995
; Molvray and Kores, 1995
). Once Codonorchis and Megastylis are removed from Chloraeinae, the remaining genera, which are all South American, form a well-supported group likely to remain monophyletic with increased sampling.
Polyphyly and paraphyly are also evident at the generic level. Megastylis has been mentioned above because it crosses tribal lines. Our trees indicate that Genoplesium is a grade leading to Prasophyllum, and its status as a segregate genus is doubtful. Acianthus is polyphyletic. The Australian clade, which contains the type species of the genus, is sister to a clade composed of one lineage of Corybas and Cyrtostylis and another lineage of Stigmatodactylus and New Caledonian Acianthus. Further sampling is needed within the latter lineage to resolve relationships between the two taxa. Townsonia is also a member of Acianthinae, based on our unpublished ITS data.
The DNA trees indicate that morphological plasticity in response to selective pressure is generally high in Diurideae. Kores et al. (2000)
have noted, for instance, that the rhizomatous habit has arisen at least four times in four separate lineages. Three of these rhizomatous genera lack root-stem tuberoids (Adenochilus, Cryptostylis, and Rimacola), and the fourth (Townsonia) has very reduced tuberoids. Most diurids form well-developed root-stem tuberoids, which give rise to deciduous leafy shoots, but the chlorophyllous, rhizomatous species are all evergreen. These evergreen diurids (with the exception of Australian Cryptostylis) live in habitats with abundant moisture throughout the year, hence it seems reasonable to speculate that the evergreen rhizomatous habit lacking tuberoids has evolved repeatedly in different lineages in response to increased water availability. In the case of Cryptostylis, many of the non-Australian species seem to grow in mesic habitats, but the Australian species are often found in drier areas as well. The genus is characterized by moderately fleshy roots, which some authors have interpreted as subterranean storage organs (Clements, 1995
), and these structures may facilitate survival in more xeric locations.
Pollination syndromes involving sexual deception of male hymenopterans, with all their highly derived floral modifications, have arisen at least six times: Cryptostylis–Ichneumonidae, Calochilus–Scolidae, Leporella–Formicidae, Spiculaea–Paracaleana–Tiphiidae. It is noteworthy that two of these independent innovations involve the same hymenopteran subfamily, Thynnidae. The current study provides one more example in the close relationship between the recently segregated Pyrorchis and Lyperanthus. The apparently large difference between Lyperanthus and Pyrorchis in leaf morphology, the latter having a fleshy, prostrate leaf, may be less important than it appears. Pyrorchis tend to prefer relatively exposed, sandy substrates, whereas Lyperanthus are found in more sheltered locations. Adaptations that reduce water loss may account for the difference in leaf morphology.
The morphological diversity of Diurideae and their lack of easily identifiable morphological synapomorphies have made them difficult to synthesize into an ordered framework that can be used to focus other studies. However, trees based on plastid DNA sequences are beginning to show the levels of support that promise to make them useful in this process. Molecular data have given us useful tools for studying phylogenies, but perhaps most interestingly, they facilitate the detection of phylogenetically informative morphological characters and suggest which characters may be most in need of further study to understand developmental processes and homologies.
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FOOTNOTES |
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9 Author for correspondence (e-mail: mmolvray{at}ou.edu
).
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