CSIRO PUBLISHING
www.publish.csiro.au/journals/ajb
Australian Journal of Botany, 2011, 59, 99–117
Phylogenetic relationships in Pterostylidinae (Cranichideae: Orchidaceae): combined evidence from nuclear ribomsomal and plastid DNA sequences Mark A. Clements A,C, J. Tupac Otero B and Joseph T. Miller A A
Centre for Australian National Biodiversity Research, GPO Box 1600, Canberra, ACT 2601, Australia. Departamento de Ciencias Biológicas and Instituto de Estudios Ambientales IDEA – Palmira, Universidad Nacional de Colombia Sede Palmira, Palmira, Valle, Colombia. C Corresponding author. Email:
[email protected] B
Abstract. A study to evaluate the relationships in subtribe Pterostylidinae (Cranichideae: Orchidaceae) was undertaken using DNA sequences from the nuclear ribosomal ITS region (256 taxa) and plastid matK (subset of 37 taxa). Parsimony analysis of nuclear, plastid and combined datasets revealed that there is strong support for the monophyly of Pterostylidinae, and three major groups therein. Clades A–C contain nine, possibly 10, identifiable groups supported by morphological synapomorphies. Clade A comprises the following two major, strongly supported groups that correlate with morphological synapomorphies: (1) Speculantha (including Petrorchis) and (2) Linguella and Eremorchis, sister to an unresolved polytomy containing Taurantha, a paraphyletic Crangonorchis and polyphyletic Diplodium. There is no support for continued recognition of Taurantha, Crangonorchis, Linguella and Eremorchis, all of which are embedded within the broader, strongly supported, monophyletic Diplodium. Clade B represents true Pterostylis. Clade C contains the morphologically disparate Bunochilus, Hymenochilus, Oligochaetochilus, Pharochilum, Plumatichilos, Stamnorchis and Urochilus (including Ranorchis) in a partially resolved tree. There is strong molecular and morphological synapomorphic internal support for the recognition of these taxa as genera. Our results revealed that none of the presently proposed classification systems for Pterostylidinae truly accounts for the underlying phylogenetic signal. A streamlined classification system, therefore, seems warranted, although further research based on a larger plastid DNA dataset is required to elucidate relationships in Clade C.
Introduction History of systematics of Pterostylidinae The tribe Cranichideae (Orchidaceae) contains nine subtribes (Achlydosinae, Chloraeinae, Cranichidinae, Galeottiellinae, Goodyerinae, Manniellinae, Prescottinae, Pterostylidinae and Spiranthinae; Salazar et al. 2009), with ~130 genera and >1500 species. It has a cosmopolitan distribution (Cribb, in Pridgeon et al. 2003). The greatest concentration of species is in the Americas, particularly South America, where representatives of six subtribes occur. The tribe Cranichideae is poorly represented in the Australasian region; the monospecific subtribe Achlydosinae is endemic to New Caledonia, and there are a few Spiranthinae species and 12 genera of Goodyerinae. However, the subtribe Pterostylidinae is endemic to Australasia and adjacent areas in Malesia. The subtribe Pterostylidinae currently comprises 17 genera with 216 named species (Jones and Clements 2002b; Jones 2006a), representing ~14% of taxa in the Cranichideae (Pridgeon et al. 2003). These are distributed mainly in Australia (181 species; Jones 2006a; Jones et al. 2006), but also in New Zealand (29 species; St George 1999, 2010), New Caledonia (4 species; Hallé 1977; Begaud et al. 1995), Papua CSIRO 2011
New Guinea (3 species; van Royen 1979), Indonesia (3 species; Schuiteman et al. 2008) and East Timor (1 species; Silveira et al. 2008). Only five species occur in more than one country. Species of Pterostylidinae grow in a wide range of habitats, from near sea level to subalpine and alpine regions up to ~3660-m altitude in New Guinea (van Royen 1979), and from coastal districts to far inland. Pterostylidinae contribute ~20% of the terrestrial-orchid diversity of the Australasian region (Jones et al. 2006). In its broadest and traditional circumscription the subtribe Pterostylidinae comprises a single genus, Pterostylis R.Br. (Chase, in Pridgeon et al. 2003; Jones, in Pridgeon et al. 2003). This was originally described in 1810 by Brown, based on plants collected in Australia during Captain Matthew Flinders’ voyage of 1801–1805 (Brown 1810; Stearn 1960). In that same year, Swartz (1810) also described Diplodium Sw., based on a specimen of Disperis alata (Pterostylis alata) collected at Recherche Bay, Tasmania, during d’Entrecasteaux’s 1791–1793 expedition. Pterostylis was adopted as a single genus by Lindley (1840a, 1840b); however, the following seven infrageneric classification systems have been proposed over the past 200 years: (1) Don (1830), (2) Reichenbach (1871), (3) Bentham (1873), (4) Rupp (1933), (5) Szlachetko (2001), (6) Jones and Clements (2002b) and (7) Janes and Duretto (2010). 10.1071/BT10190
0067-1924/11/020099
100
Australian Journal of Botany
Jones and Clements (2002a, 2002b) and Jones et al. (2002) proposed 17 genera in their combined morphology and molecular-based synopsis of Pterostylidinae. Jones and Clements (2002b) sequenced nuclear rDNA (ITS) from 38 representative species, and since the present paper was submitted, Janes et al. (2010) have published sequences for an additional 15 Tasmanian species. These are the most comprehensive analyses of the subtribe so far. Studies focusing on other subtribes have included only a few species of Pterostylidinae (Cameron et al. 1999; Kores et al. 2000, 2001; Salazar et al. 2003, 2009; Cameron 2004; AlvarezMolina and Cameron 2009). Few have recognised the 17 genera proposed by Jones and Clements (2002a, 2002b) and Jones et al. (2002) (see Entwistle 2003; Hopper and Brown 2004; Entwistle and Weston 2005). All Australian state and territory herbaria, via the Council of Heads of Australian Herbaria (CHAH), have rejected these proposed changes and supported the retention of the broader concept of a single genus, Pterostylis s.l., for the subtribe Pterostylidinae, as reflected in the Australian Plant Census (http://www.anbg.gov. au/chah/apc/index.html, last accessed 22 November 2009). Consequently, new species described within these genera are denied the status assigned to them by describing authors, and have been transferred into Pterostylis (Backhouse 2007a, 2007b, 2010; Thiele and Brown 2007; Barker and Bates 2008; Bostock 2008; Janes and Duretto 2010), with little discussion of supporting taxonomy. In contrast, the segregate genera within Pterostylidinae have been accepted and used by native orchid societies, including the New Zealand Native Orchid Society (St George 2010) and Native Orchid Society of South Australia (Hirst 2010), and have also permeated the popular and scientific literature. One of the main arguments used against the proposed segregation of Pterostylis and recognition of the 15 new genera, is a lack of phylogenetic evidence published in a peer-reviewed scientific journal (Hopper and Brown 2004). Another argument is that Pterostylis is a monophyletic group and therefore does not need subdivision (‘splitting’) into smaller groups (Chase, in Pridgeon et al. 2003; Hopper and Brown 2004; Hopper 2009). The dilemma of taxonomic rank confronts many botanists, especially those working on orchids. There has been much controversy about proposed generic changes resulting from molecular sequence analysis in many groups of orchids, including some European and Mesoamerican taxa. Some of these proposed changes are accepted because a genus has been shown by molecular data to be polyphyletic; for example, Orchis (Bateman et al. 1997a, 1997b, 2001, 2009) and Myoxanthus and Pleurothallis (Pridgeon and Chase 2001). Others, such as the inclusion of Barlia in Himantoglossum, have been more controversial (Bateman et al. 1997a, 1997b, 2001, 2009). Historically, the main characteristics used to delimit Pterostylidinae, and more specifically, Pterostylis, are a galea formed by the fusion of a hooded dorsal sepal with the margins of the petals, a mobile labellum, and an elongate column with terminal winged staminodia. Possession of a galea is not unique to the Pterostylidinae, and similar structures are present in several related genera such as Disa and Disperis (Linder and Kurweil 1999). A mobile labellum is not unique either and, furthermore, is absent in some Pterostylidinae species, such as P. (Diplodium)
M. A. Clements et al.
hians. It is the modification of the lateral petals to form an unequally obliquely falcate structure with a central rib and basal tear drop-like thickening that is the derived character for the Pterostylidinae (Jones and Clements 2002a). The winged column, which forms a tunnel through which pollinating insects must travel after triggering the labellum to close, is also a unique derived character. However, as pointed out by Jones and Clements (2002a), this does vary among major groups within the Pterostylidinae. The treatment of Pterostylidinae as a single genus is inconsistent with the treatments of subtribes Chloraeinae, Cranichidinae, Goodyerinae and Spiranthinae within the Cranichideae. With one exception (Achlydosinae), the original circumscription of genera within these subtribes is based on morphological characters only, but there are huge descrepancies with respect to the levels of morphological distinctiveness used for defining genera. The inconsistent approaches to subdividing subtribes in the Cranichideae into genera reinforce the need to revisit the process in the case of the Pterostylidinae to provide a sound, more objective classification. This, in turn, will provide a basis for parallel studies on co-evolution with associated mycorrhizal fungi (J. T. Otero, P. H. Thrall, M. A. Clements, J. Miller, J. J. Burdon, ‘Co-diversification of orchids (Pterostylidinae) and their associated mycorrhizal fungi’ submitted to Australian Journal of Botany) and of biogeographic and conservation issues. Here, we test all classification systems commencing from Brown (1810), but in particular those of Szlachetko (2001), Jones and Clements (2002b) and Janes and Duretto (2010), using a broad range of taxa with an analysis of both nuclear and plastid loci. Materials and methods Taxon sampling The ingroup comprised 247 DNA sequences, representing 152 species and including species from all traditional or recently recognised taxa within Pterostylidinae. Four non-Pterostylidinae sequences were included for outgroup comparison, including three Chloraeinae and one Achlydosinae. Outgroup taxa were chosen on the basis of results of previous studies by Cameron et al. (1999), Cameron (2004) and Salazar et al. (2003, 2009) (Appendix 1). Fresh leaf samples were collected either in the field or from cultivated plants of known provenance, and where no other material was available, from herbarium specimens or floral dissection cards. Reference vouchers for all fresh collections are housed either at CANB or CHR. DNA isolation, amplification and sequencing Genomic DNA was extracted from 10–100 mg of fresh or silica gel-dried leaf tissue, or from herbarium material, using either the Plant DNAZOL Reagent (GIBCOBRL)Life Technologies Inc, Grand Island, NY, USA) or a modification of the CTAB method of Thomson and Henry (1993) (Gibbs and Mackenzie 1997). The complete ITS region of the 18S-26S nrDNA was amplified by polymerase chain reaction (PCR), following methods outlined in Clements et al. (2002). The following primer pairs from Sun et al. (1994) were used: ITS4/ITS5, 17SE/26SE or 17SE/ITS4. A portion of the matK was
Phylogenetic relationships in Pterostylidinae
amplified for a subset of the samples, representing all genera recognised in Jones and Clements (2002b) (Appendix 1). The matK 5/6 primer set was used following Salazar et al. (2003). All amplification products were confirmed by size comparison using agarose gel electrophoresis. DNA extracted from some herbarium samples was degraded and so the ITS1 and ITS2 regions were amplified separately by using the primer pairs ITS5/ITS2 and ITS3/ITS4, respectively. For some herbarium samples, the specific amplified product was in very low concentration. These products were excised from an agarose gel, purified using the QIAquick gel extraction kit (QIAGEN, Melbourne, Vic., Australia) and reamplified. Specific amplification products were either purified directly by using the QIAquick PCR purification kit (QIAGEN), or after excision from an agarose gel, were purified as previously described. The purified doublestranded PCR product (10–50 ng) was sequenced from either end by using 3.2 pmol of the appropriate primer and the ABI Prism Big Dye terminator cycle sequencing kit (Applied Biosystems Melbourne, Vic., Australia), as outlined in Clements et al. (2002). Phylogenetic analyses Contiguous sequences were edited with Sequencher v. 3.0 (Gene Codes Corporation, Ann Arbor, MI, USA) and manually aligned in BioEdit sequence alignment editor v. 4.8.6 (Hall 1999). Sequence alignments and Nexus-formatted files are available from the authors on request, and all sequences are lodged in GenBank (see Appendix 1). Any uncertain base positions, generally located close to priming sites, and highly variable regions with uncertain sequence homology, were excluded from the phylogenetic analysis. Individual base positions were coded as unordered multistates and potentially informative insertions/deletions (indels) were coded as additional binary characters. Bayesian analyses were performed using MrBayes v. 3.1.2. (Ronquist and Huelsenbeck 2003). Two datasets were analysed. The first contained all the ITS sequences and the second contained
Australian Journal of Botany
101
37 taxa and included both ITS and matK sequences. The combined matK–ITS dataset was divided into separate partitions for ITS and matK. Modelltest v. 1.1 (Posada and Crandall 1998) determined that the GTR+I+gamma model was the best-fit model for both the ITS and matK datasets and was applied to each DNA sequence partition. Indel characters were included as a separate partition and a standard (morphology) discrete-state model with a gamma-shape parameter was applied to this partition. The ITS–matK dataset was run as a combined dataset as well as separately. The Markov-chain Monte Carlo search was run for 5 million generations, with trees sampled every 1000 generations. MrBayes performed two simultaneous analyses starting from different random trees (Nruns = 2), each with four Markov chains (Nchains = 4). The first 2000 trees were discarded from each run. A Bayesian consensus phylogram with posterior probability values plotted was calculated in MrBayes. Maximum parsimony analyses were performed with the heuristic search option (excluding uninformative characters) in PAUP* 4.02 (Swofford 1999). Following Olmstead and Palmer (1994), a four-step search method for multiple islands was performed with 10 000 random replicates. The fast bootstrap method with 10 000 replicates was used to measure support for internal branches (Felsenstein 1985). Results The ITS dataset for phylogenetic analysis comprised 247 DNA sequences with a matrix length of 723 nucleotides. There were, in total, 267 shared polymorphisms and 57 autapomorphic characters. All 10 indels >3 bp long were scored. In the combined ITS/matK dataset, eight indels were scored in the ITS portion. The matK section was 1982 bp in length and 12 indels were scored. The Bayesian and parsimony analysis of the ITS region resolved a phylogenetic tree with three main clades, designated A, B and C (Fig. 1). With respect to the genera of Jones and Clements (2002b), Clade A (Fig. 2) contains Diplodium, Taurantha, Crangonorchis, Linguella, Eremorchis,
Fig. 1. Bayesian tree showing the outgroups and three major clades. Numbers above branches indicate Bayesian posterior probabilities. See Figs 2–4 for details of the clades.
102
Australian Journal of Botany
M. A. Clements et al.
A
Fig. 2. Bayesian phylogram of ITS data showing Clade A of Fig. 1. Numbers above branches indicate Bayesian posterior probabilities (pp) and bold branches indicate pp > 0.95. Generic names are indicated at basal nodes. General localities are noted.
Speculantha and Petrorchis, and it is sister to Clade B (Fig. 3), which contains all samples of Pterostylis. Clade C (Fig. 4) contains Oligochaetochilus, Hymenochilus, Plumatichilos, Bunochilus, Stamnorchis, Pharochilum, Urochilus and Ranorchis. All three clades are highly supported, Bayesian posterior probability (pp) of 1.00; the node-joining Clades A and B are less well supported (pp = 0.94, 55% bootstrap). Bootstrap support is 92% for Clade A, 74% for Clade B and 79% for Clade C. Within Clade A (Fig. 2), there is substantial support (pp > 0.95, 82% bootstrap) for Speculantha and Petrorchis and their sister relationship, as well as for Linguella and Eremorchis and their sister relationship (pp > 0.98; 75% bootstrap). The Linguella–Eremorchis clade is sister to a clade containing a monophyletic Taurantha embedded within a poorly resolved clade (pp > 0.98, 53% bootstrap) containing Diplodium and Crangonorchis. In contrast, the eight genera represented in Clade C (Fig. 4) are all monophyletic and highly supported (pp = 1.0, 99–100%
bootstrap). The basal node contains Urochilus and Ranorchis, each with long branches. The only other strong support (pp > 0.95) for generic relationships is between Oligochaetochilus and Hymenochilus, although there is only moderate bootstrap support (71%) for this grouping. The matK analysis (not shown) of 37 taxa produced a similar topology to the combined ITS–matK Bayesian and parsimony analyses (Fig. 5) and resolved the same three major clades (pp = 1.00, 100% bootstrap support). However, the Pterostylis clade (Clade B) is now sister to Clade C rather than to Clade A (pp = 0.73, 59% bootstrap support). The smaller sampling in the combined dataset precludes extensive comparison between the plastid and the larger nuclear ITS DNA signals at the generic level but provides further support for some clades. Within Clade A (Figs 2, 5), the combined dataset still fails to resolve monophyly for Diplodium or Crangonorchis. Instead, the results show strong support (pp >1.00, 100% bootstrap) for Taurantha, although it is still embedded within a polyphyletic Diplodium.
Phylogenetic relationships in Pterostylidinae
Australian Journal of Botany
103
B
Fig. 3. Bayesian phylogram of ITS data showing the genus Pterostylis, Clade B of Fig. 1. Numbers above branches indicate Bayesian posterior probabilities (pp) and bold branches indicate pp > 0.95. General localities are noted.
Despite some polytomy within Clade C (Figs 4, 5), the combined dataset also resolves all genera as monophyletic. Stamnorchis is sister to Plumatichilos (pp = 0.99, 82% bootstrap support) in the combined dataset, but unresolved with ITS alone. Pharochilum is sister (pp = 0.91, 54% bootstrap support) to the strongly supported Ranorchis– Urochilus clade (pp = 1.00, 100% bootstrap support) in the combined dataset, with these clades now resolved as sister (pp = 1.00, 100% bootstrap support) to the Oligochaetochilus– Hymenochilus clade. Discussion Results of both the combined analysis (Fig. 5) and the ITS nuclear rDNA in isolation (Fig. 1) are highly congruent with those produced in our previous analyses of nuclear rDNA (Jones and Clements 2002a), and reaffirm the monophyly of Pterostylidinae. Traditionally, Pterostylidinae was part of tribe Diurideae, based on possession of root tubers, and it was linked to the Chloraeinae by a shared curved, cobra-like column (Dressler 1981, 1993). Placing subtribe Pterostylidinae within tribe Cranichideae, sister to Chloraea (Chloraeinae), Spiranthes (Spiranthinae) and Megastylis glandulosus (as part of Megastylidinae) was first proposed by Clements (1995, 1996, 1999) on the basis of a shared ‘spiranthoid’ embryo developmental pattern. Subsequent molecular data from matK
and trnL-F plastid genes (Kores et al. 1997, 2000, 2001; Salazar et al. 2003), rbcL (Cameron et al. 1999), psaB (Cameron 2004), ITS nuclear rDNA (Clements et al. 2002; Jones and Clements 2002a), and a low-copy nuclear code gene Xdh (Górniak et al. 2010) confirmed this alignment and implied a sister relationship between Pterostylidinae and Chloraeinae and/or M. glandulosa, and a more distant alignment with Spiranthinae. The Chloraeinae is an entirely South American group of species comprising four or possibly five genera, although a recent study based on plastid sequence analysis suggested that the group could be treated as a single genus (Chemisquy and Morrone 2010). This, coupled with the molecular data cited above, supports the use of Chloraeinae and Achlydosinae as outgroups for study of Pterostylidinae. Relationships within Pterostylidinae Our results identify three major lineages within the subtribe Pterostylidinae that partially correspond to previous classifications of Pterostylis (Brown 1810; Don 1830; Lindley 1840b; Reichenbach 1871; Bentham 1873; Pfitzer 1887, 1889; Rupp 1933; Szlachetko 2001; Jones and Clements 2002b; Jones et al. 2002; Janes and Duretto 2010). In some cases, there is incongruence between trees based on nuclear sequences (Figs 2–4) and those based on combined nuclear and plastid sequences (Fig. 5). Clade B (Pterostylis s.s.) is aligned with Clade C when using combined data, and with
104
Australian Journal of Botany
M. A. Clements et al.
C
Fig. 4. Bayesian phylogram of ITS data showing the Clade C of Fig. 1. Numbers above branches indicate Bayesian posterior probabilities (pp) and bold branches indicate pp > 0.95. Generic names are indicated at basal nodes. General localites are noted.
Clade A when the analysis is based on nuclear data alone. Plastid capture, driven by cytonuclear interactions, has been suggested as an explanation for incongruence between nuclear and plastid gene trees, possibly resulting from a hybridisation event during early evolution of a group, with no subsequent crossover among taxa (Tsitrone et al. 2003). Plastid capture is known to occur at both intra- and intergeneric levels in numerous taxa in both domesticated and wild species. Plastid capture has not been demonstrated in Orchidaceae, although Bellusci et al. (2008) suggested that this process might have occurred in Serapias (Orchideae). The possible occurrence of plastid capture within Pterostylidinae at a major-group level appears highly significant. Conditions for plastid capture include partial self-compatibility of one parent in a hybrid cross between related species (Tsitrone et al. 2003). The tendency for self-compatibility within some species of Pterostylis, such as P. venosa and P. humilis, which are basal in Clade B, meets this criterion for plastid capture. Phylogeny and taxonomy Although based only on a limited sample (152 of 214 described species with a single DNA sequence, 37 species with two
sequences), our results allow some insights into the taxonomy of the subtribe Pterostylidinae. In Pterostylis s.s. (Fig. 3), P. humilis and P. venosa appear distinct from each other, despite their overall similarity of structure. Likewise, the recognition of the Australian species P. falcata as distinct from the New Zealand P. micromega seems justified (St George 1999; Jones 2006a), as does the distinction between P. erecta and P. pedunculata (Jones 2006a). P. foliata has been interpreted as having a distribution that encompasses the Mount Lofty Range in South Australia (where originally described under the name P. vereenae), most of coastal and mountain Victoria (where originally described under the name P. gracile), Tasmania, some Bass Strait islands, and New Zealand (from where the type of P. foliata orginates) (Clements 1989). Our results show a polyphyletic species, with a collection from King Island in Bass Strait being allied to P. oreophila and well removed from sequences obtained from South Australian and New Zealand collections. The South Australian and New Zealand collections of P. foliata form a well supported grouping sister to a collection of the morphologically similar species P. alpina from near Canberra. The position and the long branch length of the King Island P. foliata relative to the other two collections is unusual. An
Phylogenetic relationships in Pterostylidinae
Australian Journal of Botany
105
C
B
A
Fig. 5. Bayesian phylogram of combined ITS and matK dataset. Numbers above branches indicate Bayesian posterior probabilities (pp) and bold branches indicate pp > 0.95. Major clades are labelled as in Fig. 1.
alternative explanation might be that the results represent an inadvertent analysis of paralogous ITS sequences, similar to that reported in other orchids such as Cypripedium (Cox et al. 1997). Further investigation using additional collections and other genes seems warranted for all species in this complex, and at the very least, the identity of the King Island collection should be reinvestigated because this may represent an example of a cryptic species within the subtribe. There is also still much work to be undertaken to unravel species delimitations in the P. banksii group from New Zealand, in which diversity is low. Genetic diversity among species is also low, at least in the ITS nuclear rDNA region, in several groups in Clade C (Fig. 4), such as Bunochilus, which has 26 described species (Jones 2006b), Plumatichilos, with ~15 (4 described) species (Jones et al. 2006), and Urochilus, with ~8 (3 described) species (Brown et al. 2008). Other groups exhibit high levels of genetic variability. Linguella, in Clade A (Fig. 2), displays high levels of diversity compared with most other groups in Pterostylidinae and this is possibly reflected in the high levels of morphological variability encountered therein (Hoffman and Brown 1992, 1998; Jeanes and Backhouse 2001; Jones et al. 2006). Brown et al. (2008), in the most recent account of the orchids of Western Australia, identified 19 species, entirely on the basis of morphological differences, only two of which (L. dilatatus and L. pyramidalis) are formally described. Our results, although only based on a single DNA
sequence, suggest this group is in need of extensive genetic study to unravel its complexities. At the broadest level, our results reaffirm the historical interpretation of a monophyletic subtribe Pterostylidinae. However, our results also show that Pterostylidinae is composed of three major strongly supported monophyletic lineages, Clades A–C (Figs 1, 5). Further, within Clades A and C, there are eight highly and one well supported, morphologically identifiable monophyletic groups. Clade A contains (1) Speculantha (including Petrorchis) (Fig. 6d) and (2) Diplodium (including Eremorchis, Linguella, Taurantha and Crangonorchis) (Fig. 6a–c). Clade C contains (3) Bunochilus, (4) Hymenochilus, (5) Oligochaetochilus (Fig. 6l), (6) Pharochilum, (7) Plumatichilos (Fig. 6i), (8) Stamnorchis (Fig. 6j) and (9) Urochilus (including Ranorchis) (Fig. 6h). In Clade C, Pharochilum is the one well rather than highly supported taxon in the combined analysis, although the arrangement of taxa therein has yet to be fully resolved (Fig. 5). The existence of these readily identifiable morphological groups therefore raises the issue of what rank should be assigned to these monophyletic taxa. In other words, is the historical treatment of the group as a single genus, Pterostylis, still valid in light of this new genetic data, or would a different treatment be more appropriate? The issue of what constitutes a genus is a perennial question among biologists. Recognising a genus and allocating species to it
106
Australian Journal of Botany
(a)
(c)
(b)
(d )
(f)
(e )
(h )
M. A. Clements et al.
(i)
(g )
(j ) (k)
(l )
Fig. 6. Representative species of Pterostylidinae from (a–d) Clades A, (e–g) B and (h–l) C. (a) Diplodium pulchellum, Fitzroy Falls, NSW; (b) Linguella nana, near Murray Bridge, SA; (c) Diplodium longicurvum, Warrumbungle Moutains, NSW; (d) Petrorchis bicornis, Mount Maroon, Qld; (e) Pterostylis nutans, Tidbinbilla, ACT; ( f ) Pterostylis erecta, Currowan State Forest, NSW; (g) Pterostylis baptistii, Fraser Island, Qld; (h) Urochilus sanguineus, near Perth, WA; (i) Plumatichilos turfosus, Bremer Bay, WA; ( j) Stamnorchis recurva, Jerramungup, WA; (k) Ranorchis sargentii, Gunapin Ridge Road, WA; and (l) Oligochaetochilus lepidus, Halbury, SA.
Phylogenetic relationships in Pterostylidinae
is a fundamental means of communication between scientists and the broader community and is therefore important ‘to get right’ (Oberwinkel, in Humphreys and Linder 2009). Historically, there have been many changing ideas about how genera should be delimited; however, all are conceptually founded (usually defined by morphological character differences; Humphreys and Linder 2009). Sometimes this conceptual basis for genera renders difficult the translation of phylogenies into useful, consistent and uniform classification systems. Nevertheless, many modern taxonomists agree that genera should be monophyletic (Hennig 1966; Funk 1985; Oberwinkler 1994; Backlund and Bremer 1998; Entwistle and Weston 2005; Pfeil and Crisp 2005; Stuessy 2009a, 2009b; all cited in Humphreys and Linder 2009), and ‘should be defined by robust clades, in order to maximise the chance of long-term stability’ (Pfeil and Crisp 2005). Species in these lineages should exhibit morphological characters that define the clades. With these criteria in mind we can turn to the Pterostylidinae to assess the best course of action with regard to their classification. Of the 10 major systems of classification of the group, i.e. (1) Brown (1810), (2) Don (1830), (3) Lindley (1840b), (4) Reichenbach (1871), (5) Bentham (1873), (6) Pfitzer (1887, 1889), (7) Rupp (1933), (8) Szlachetko (2001), (9) Jones and Clements (2002b); Jones et al. (2002) and (10) Janes and Duretto (2010), systems 1–7 and 10 use the large genus concept advocated by many authors (e.g. Chase, in Pridgeon et al. 2003; Hopper and Brown 2004; Chase et al. 2008; Hopper 2009; Humphreys and Linder 2009). Most recently, Janes and Duretto (2010) supported the opinion of Hopper and Brown (2004) that the segregation of Pterostylis either by Szlachetko (2001) into three genera, or by Jones and Clements (2002b) into 17 genera, represents excessive splitting and suggested that neither system has been widely accepted. Despite recognising strong support (100, 99 and 94 posterior probabilities in their Bayesian analysis) for three main clades in Pterostylidinae, and their strong morphological distinctiveness, Janes et al. (2010) concluded that there was no support for treating these clades at generic level. Recognition of a single all-encompassing genus, Pterostylis, with over 200 species, is inconsistent with the taxonomy of all other subtribes within tribe Cranichideae (see Pridgeon et al. 2003) or other closely related tribes, Orchideae and Diurideae, within the subfamily Orchidoideae (see Pridgeon et al. 2001). More importantly, these 200 species fall into distinct groups that have marked differences in habit, floral and vegetative morphology, habitat preferences and pollination mechanisms (Jones and Clements 2002a). Placing them all in a single genus fails to take advantage of available phylogenetic information that is associated with clear morphological synapomorphies. A consequence is that users will overgeneralise and are likely to miss significant information that might otherwise prove useful in ecological or conservation contexts. It would perhaps be more useful to consider the degree of morphological distinctness in assigning generic rank. Most of the genera detailed by Jones and Clements (2002a) are morphologically distinct and thus readily identified in the field, even by those unfamiliar with orchids (see examples in Fig. 6). The fact that many of these genera have historically been identified under colloquial or common names such as ‘bird’,
Australian Journal of Botany
107
‘jug’, ‘druid’s cap’ and ‘rufa group’ orchids, for example, is testimony to the insightfulness of our predecessors in recognising these genera as distinct, in some cases even before the species were formally described. This is never more apparent than with the genera in Clade C. For example, the ‘bird orchids’ (Plumatochilus) are easily recognised in the field (Fig. 6i), and Bentham (1873), Rupp (1933) and Szlachetko (2001) all treated these separately in their various classification systems. In fact, all the genera, with the exception of Diplodium and Pterostylis, are quite distinctive and easily recognisable. The single genus concept of Pterostylis s.l. fails to inform either biologists or laymen about the great structural, ecological and reproductive disparity displayed by these lineages. The recognition of a single all-encompassing genus, Pterostylis, may have historically served a function; however, with the information now available, this interpretation now seems better applied to the subtribe as a whole. Our new analyses show that neither the single genus concept of Pterostylis, nor the three genera of Szlachetko (2001) or the 17 genera of Jones and Clements (2002b) are satisfactory to classify the group. Our combined data support the separation and recognition of three major phylogenetic lineages within Pterostylidinae, each containing one or more distinct taxa, each with identifiable morphological synapomorphies. There are three possible taxonomic solutions. The first would be to recognise a single genus with three subgenera; however, as detailed below, this fails to properly account for the possible molecular origin of Clade B. The second option is to accept three genera reflective of Clades A–C, but this would likewise be difficult on morphological grounds (except for Clade B), where members of Clades A and C have conflicting morphological characters and states. The third is to recognise 9 or 10 genera reflecting the strongly supported monophyletic molecular groups with their morphological synapomorphies. This approach allows easy recognition in the field and discussion in the scientific literature, and provides useful ecological information. The third approach is also consistent with the treatment of other members of the tribe Cranichideae. Currently Chloraeinae comprises 70 species in four genera, Cranichidinae 213 species in 17 genera, Goodyerinae 581 species in 35 genera and Spiranthinae 480 species in 40 genera, whereas Pterostylidinae interpreted as a single genus has over 200 species (Pridgeon et al. 2003). It would appear to be merely an accident of history that, until 2002, only Pterostylis and Diplodium were recognised (although not accepted) as separate genera in a subtribe that appears more morphologically diverse than most of its counterparts in Cranichideae. Our studies make a strong case that Clade B, Pterostylis s.s., should be recognised and treated as a separate taxon. On the basis of a clear genetic separation of these species, with very high support levels in all analyses, coupled with morphological synapomorphies (Jones and Clements 2002a), this taxonomic group meets criteria for recognition at generic rank. As Clade B contains P. curta, the designated conserved type of the genus, the name Pterostylis would be applied to this group of species. The situation with species in Clade A is also fairly straightforward. It contains two major very strongly supported groups on comparably long branches. One branch contains species of Speculantha and Petrorchis and is supported by
108
Australian Journal of Botany
synapomorphic morphological characters, in particular, a multiflowered inflorescence with the flowers facing towards the stem axis (Fig. 6d). This is a readily identifiable and recognisable group of species, well supported genetically and morphologically. Using the criteria enunciated above and in particular by Chase et al. (2008), we suggest that Petrorchis be treated within the broader taxon Speculantha. Janes et al. (2010) came to the same conclusion in the case of Speculantha. Parallel to Speculantha is the branch containing the group of species recently placed or described in the genera Eremorchis, Linguella, Diplodium, Taurantha and Crangonorchis (Jones and Clements 2002a, 2002b; Jones et al. 2002, 2006; Jones 2006a). Although there is strong support for an internal division into two subgroups – Eremorchis and Linguella in one, and Diplodium, Taurantha and Crangonorchis in another – the branch lengths supporting these two subgroups are short, indicating that the molecular changes supporting the morphological characters are minimal. Moreover, Diplodium is paraphyletic within the second subgroup in the combined analysis (Fig. 5). In the more detailed analysis based just on the ITS data (Fig. 2), this branch is a complex polytomy where Crangonorchis is also paraphyletic, Taurantha is embedded within the group, and a polyphyletic Diplodium appears in four separate lineages. Morphological characters previously used to define groups within this complex, such as dimorphic flowering and non-flowering plants and bifid labellum apex, occur in species spanning both subbranches of this clade. All this makes a compelling case to treat this entire branch as a single genus, with two subgenera. The earliest name available is Diplodium. Janes et al. (2010) came to the same conclusion about Diplodium, although these authors did not recognise it at generic rank. The situation in Clade C is more complex. Although the group as a whole is clearly monophyletic with strong molecular support, its component parts are morphologically very disparate (although most have an exposed labellum sitting on reflexed lateral sepals). Lack of internal support for a definitive arrangement of major lineages identified in clade C is suggestive of a rapid morphological radiation or a slow rate of molecular evolution of the group at or near segregation from the other elements, Clades A and B. Szlachetko (2001) proposed that those species possessing an exposed labellum sitting on reflexed lateral sepals should be treated in the genus Oligochaetochilus, with P. rufa as the type. In the same paper, he also proposed that those species possessing the rather distinctive plumiform labellum should be treated in the genus Plumatichilos. Although both trees from the nrITS and combined analyses show an unresolved arrangement of taxa in this branch of Pterostylidinae, in both cases, Plumatichilos is embedded within those taxa Szlachetko assigned to Oligochaetochilus. This renders that genus concept paraphyletic. Jones and Clements (2002b) recognised Plumatichilos on the basis of 17 synapomorphies, including six apomorphic morphological characters, while proposing a much narrower concept for Oligochaetochilus and naming other monophyletic, morphologically distinct elements as separate genera. In the combined analysis, Clade C (Fig. 5) shows a basal trichotomy with strongly supported branches, representing seven major groups that all possess strong morphological
M. A. Clements et al.
synapomorphies. Of particular interest is the alignment of Stamnorchis with Plumatichilos and of Urochilus with Ranorchis, the latter pair forming a sister group to Pharochilum. Janes et al. (2010) found similar overall results, recognising the same close relationship of Urochilus with Ranorchis and suggesting that the two might be treated as one genus, but differed significantly with respect to relationship and support level for several other genera. Specifically, Hymenochilus in their study occupies a position sister to all other genera within Clade C, whereas our results have it deeply embedded within the clade and sister to Oligochaetochilus (Figs 1, 4, 5). They also found only weak support for Oligochaetochilus, whereas in all our analyses there was 100% support for this clade. Janes et al. (2010) also showed a possible close relationship between Bunochilus and Urochilus (including Ranorchis), whereas in our study these genera were never in near-juxtaposed positions. These discrepancies are puzzling, considering that for many species in their analyses Janes et al. (2010) used sequences from Jones and Clements (2002a) obtained from GenBank. Their classification is based on an ITS sequence alignment ~148 bases shorter (66 for ITS1, 82 for ITS2) than we used in our study. The excluded sequence regions contain phylogenetically informative data in the same or related species (Jones and Clements 2002a), and their absence possibly accounts for differences in the results. In summary, we would see Clade A as containing two genera, namely Speculantha (which includes Petrorchis) and Diplodium (with two subgenera – Eremorchis and Linguella in one, and Diplodium, Taurantha and Crangonorchis in the other). Clade B consists of Pterostylis s.s. Clade C, the most complex, contains seven of the Jones and Clements (2002b) genera, namely Bunochilus, Hymenochilus, Oligochaetochilus, Pharochilum, Plumatichilos, Stamnorchis and Urochilus (now including Ranorchis). There is strong molecular and morphological support for the recognition of these taxa as genera, although relationships within this clade remain incompletely resolved and need further research based on a larger plastid DNA dataset.
Acknowledgements This research was primarily funded from a grant by the Gatsby Charitable Trust and secondarily, for assistance with field work and collections, by the Australian Orchid Foundation, the Nell and Hermon Slade Trust, the Foundation for the Protection of Wild Orchids, Zürich, and the Foundation for Research, Science and Technology, New Zealand, and we are most grateful for their support. Plant material was collected with kind permission under Western Australian Department of Conservation and Land Management, Permit No. CE00418, New South Wales, Scientific Investigation Licence A1796, Tasmanian Department of Primary Industry, Water and Environment Permit No. FL 03121, and Nouvelle-Caledonie, Service de L’Environnement er de la Gestion des Parcs et Reserves, Autorisation No. 6024 765/ENV. We also thank our many colleagues who have aided in the collection or contributed plant material used in this study. DNA samples of some species were obtained from Mark Chase and Gerardo Salazar, Jodrell Laboratory, Royal Botanic Gardens, Kew, and for these we are most grateful. Technical assistance was provided by Kirsten Cowley, Kelli Gowland, Kristy Lam, Anne Mackenzie, Ish Sharma and Terri Weese. We also are most grateful to Catherine Busby who read, edited and provided many useful comments on various versions of the manuscript.
Phylogenetic relationships in Pterostylidinae
References Alvarez-Molina A, Cameron KM (2009) Molecular phylogenetics of Prescottiinae s.l. and their close allies (Orchidaceae, Cranichideae) inferred from plastid and nuclear ribosomal DNA sequences. American Journal of Botany 96, 1020–1040. doi:10.3732/ajb.0800219 Backhouse GH (2007a) New combinations in the terrestrial orchid genera Caladenia R.Br. and Pterostylis R.Br. (Orchidaceae) for Victoria, Australia. Victorian Naturalist 124, 123–124. Backhouse GH (2007b) A new name in the terrestrial orchid genus Pterostylis R.Br. (Orchidaceae) for Victoria, Australia. Victorian Naturalist 124, 341. Backhouse GH (2010) New combinations in the terrestrial orchid genera Caladenia R.Br., Corybas Salisb. and Pterostylis R.Br. (Orchidaceae) for south-eastern Australia. Victorian Naturalist 127, 55–57. Backlund A, Bremer K (1998) To be or not to be – the principles of classification and monotypic plant families. Taxon 47, 391–400. doi:10.2307/1223768 Barker R, Bates R (2008) New combinations in Pterostylis and Caladenia and other name changes in the Orchidaceae in South Australia. Journal of the Adelaide Botanic Gardens 22, 100–104. Bateman RM, Cox AV, Hapeman JR, Chase MW (1997a) Phylogenetics of subtribe Orchidinae (Orchidoideae, Orchidaceae) based on nuclear ITS sequences. I. Intergeneric relationships and polyphyly of Orchis sensu lato. Lindleyana 12, 89–109. Bateman RM, Pridgeon AM, Chase MW (1997b) Phylogenetics of subtribe Orchidinae (Orchidoideae, Orchidaceae) baed on nuclear ITS sequences. 2. Infrageneric relationships and reclassification to achieve monophyly of Orchis sensu stricto. Lindleyana 12, 113–141. Bateman RM, Hollingsworth PM, Preston J, Yi-Bo L, Pridgeon AM (2001) Phylogenetics. In ‘Genera Orchidacearum 2. Tribe Orchidineae’. (Eds AM Pridgeon, PJ Cribb, MW Chase, FN Rasmussen) pp. 224–232. (Oxford University Press: Oxford, UK) Bateman RM, James KE, Yi-Bo L, Lauri RK, Fulcher T, Cribb PJ, Chase MW (2009) Molecular phylogenetics and morphological reappraisal of the Platanthera clade (Orchidaceae: Orchidinae) prompts expansion of the generic limits of Galearis and Platanthera. Annals of Botany 104, 431–445. doi:10.1093/aob/mcp089 Begaud J, Laudereau C, Duchene P, Barnaire L (1995) ‘Native orchids of New Caledonia.’ (Société Néo-Calédonienne d’Orchidophilie: Noumea, New Caledonia) Bellusci F, Pellegrino G, Palermo AM, Musacchio A (2008) Phylogenetic relationships in the orchid genus Serapias L. based on noncoding regions of the chloroplast genome. Molecular Phylogenetics and Evolution 47, 986–991. doi:10.1016/j.ympev.2008.03.019 Bentham G (1873) ‘Flora Australiensis. Vol. 6.’ (L Reeve & Co: London) Bostock PD (2008) New combinations in Liparis Rich. and Pterostylis R.Br. for two species of Orchidaceae from Queensland. Austrobaileya 7, 737. Brown AP, Dundas P, Dixon K, Hopper SD (2008) ‘Orchids of Western Australia.’ (University of Western Australia Press: Perth) Brown R (1810) ‘Prodromus florae Novae Hollandiae, et insulae van Diemen.’ (J Johnson & Co: London) Cameron KM (2004) Utility of plastid psaB gene sequences for investigating intrafamilial relationships within Orchidaceae. Molecular Phylogenetics and Evolution 31, 1157–1180. doi:10.1016/j.ympev.2003.10.010 Cameron KM, Chase MW, Whitten WM, Kores PJ, Jarrell DC, Albert VA, Yukawa T, Hills HG, Goldman DH (1999) A phylogenetic analysis of the Orchidaceae: evidence from rbcL nucleotide sequences. American Journal of Botany 86, 208–224. doi:10.2307/2656938 Chase MW, Williams NH, Nueberg KM, Whitten WM (2008) Taxonomic transfer in Oncidiinae to accord with Genera Orchidacearum, Vol. 5. Lindleyana 21, 20–31. Chemisquy MA, Morrone O (2010) Phylogenetic analysis of the subtribe Chloraeinae (Orchidaceae): a preliminary approach based on three chloroplast markers. Australian Systematic Botany 23, 38–46. doi:10.1071/SB09026
Australian Journal of Botany
109
Clements MA (1989) Catalogue of Australian Orchidaceae. In ‘Australian Orchid Reseaerch 1’. (Ed. DL Jones) p. 122. (Australian Orchid Foundation, Reed Books Pty. Ltd: Frenchs Forest, New South Wales, Australia) Clements MA (1995) Reproductive biology in relation to phylogeny of the Orchidaceae, especially the tribe Diurideae. PhD Thesis, Australian National University, Canberra. Clements MA (1996) Reproductive biology in relation to phylogeny of the Orchidaceae, especially the tribe Diurideae. Journal and Proceedings of the Royal Society of New South Wales 129, 83. [Abstract] Clements MA (1999) Embryology. In ‘Genera Orchidacearum. 1’. (Eds AM Pridgeon, PJ Cribb, MW Chase, FN Rasmussen) pp. 38–58. (Oxford University Press: Oxford, UK) Clements MA, Jones DL, Sharma IK, Nightingale ME, Garratt MJ, FitzGerald KJ, Mackenzie AM, Molloy BPJ (2002) Phylogenetic systematics of the Diurideae (Orchidaceae) based on the ITS and 5.8S coding region of nuclear ribosomal DNA. Lindleyana 17, 135–171. Cox AV, Pridgeon AM, Albert VA, Chase MW (1997) Phylogenetics of the slipper orchids (Cypripedioideae, Orchidaceae): nuclear rDNA ITS sequences. Plant Systematics and Evolution 208, 197–223. Don G (1830) ‘Loudon’s Hortus Brittanicus: or a catalogue of the plants indigenous or cultivated in the gardens of Great Britian.’ 3rd edn. (James Ridgeway: London) Dressler RL (1981) ‘The orchids: natural history and classification.’ (Harvard University Press: Cambridge, MA) Dressler RL (1993) ‘Phylogeny and classification of the orchid family.’ (Dioscorides Press: Portland, OR) Entwistle TJ (2003) Report from CHAH. Australian Systematic Botany Society Newsletter 114, 25–26. Entwistle TJ, Weston PH (2005) Majority rules, when systematists disagree. Australian Systematic Botany 18, 1–6. doi:10.1071/SB04013 Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. doi:10.2307/2408678 Funk VA (1985) Cladistics and generic concepts in the Compositae. Taxon 34, 72–80. doi:10.2307/1221566 Gibbs A, Mackenzie A (1997) A primer pair for amplifying part of the genome for all potyvirids by RT–PCR. Journal of Virological Methods 63, 9–16. doi:10.1016/S0166-0934(96)02103-9 Górniak M, Paun O, Chase MW (2010) Phylogenetic relationships within Orchidaceae based on a low-copy nuclear coding gene, Xdh: congruence with organellar and nuclear ribosomal DNA results. Molecular Phylogenetics and Evolution 56, 784–795. doi:10.1016/j.ympev.2010.03.003 Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41, 95–98. Hallé N (1977) ‘Flore de la Nouvelle Calédonie et depéndances, 8. Orchidacées.’ (Museum National D’Histoire Naturelle: Paris) Hennig W (1966) ‘Phylogenetic systematics.’ (University of Illinois Press: Urbana, IL) Hirst D (2010) Plants benched May 2010. Journal of the Native Orchid Society of South Australia Inc 34, 50. Hoffman N, Brown AP (1992) ‘Orchids of South-west Australia.’ 2nd edn. (University of Western Australia Press: Perth) Hoffman N, Brown AP (1998) ‘Orchids of South-west Australia.’ 2nd edn, revised with supplement. (University of Western Australia Press: Perth) Hopper SD (2009) Taxonomic turmoil down-under: recent developments in Australian orchid systematics. Annals of Botany 104, 447–455. doi:10.1093/aob/mcp090 Hopper SD, Brown AP (2004) Robert Brown’s Caladenia and Pterostylis revisited. Orchadian 14, 366–371. Humphreys AM, Linder PH (2009) Concepts versus data in delimitation of plant genera. Taxon 58, 1054–1074. Janes JK, Duretto MF (2010) A new classification for subtribe Pterostylidinae (Orchidaceae), reaffirming Pterostylis in the broad sense. Australian Systematic Botany 23, 260–269. doi:10.1071/SB09052
110
Australian Journal of Botany
Janes JK, Steane DA, Vaillancourt RE, Duretto MF (2010) A molecular phylogeny of the subtribe Pterostylidinae (Orchidaceae): resolving the taxonomic confusion. Australian Systematic Botany 23, 248–259. doi:10.1071/SB10006 Jeanes J, Backhouse G (2001) ‘Pictorial guide to: wild orchids of Victoria, Australia.’ (Zoonetics, Seaford: Melbourne) Jones DL (2006a) ‘A complete guide to native orchids of Australia including the island territories.’ (Reed New Holland: Sydney) Jones DL (2006b) Towards a revision of Bunochilus D.L.Jones and M.A. Clem. Australian Orchid Review 5, 112–142. Jones DL, Clements MA (2002a) A reassessment of Pterostylis R.Br. (Orchidaceae). Australian Orchid Review 4, 3–63. Jones DL, Clements MA (2002b) A new classification of Pterostylis R.Br. (Orchidaceae). Australian Orchid Review 4, 64–124. Jones DL, Clements MA, Molloy BPH (2002) A synopsis of the subtribe Pterostylidinae. Australian Orchid Review 4, 125–144. Jones DL, Hopley T, Duffy SM, Richards KJ, Clements MA, Zhang X (2006) ‘Australian orchid genera and information and identification system.’ (CSIRO Publishing: Melbourne) Kores PJ, Cameron KM, Molvray M, Chase MW (1997) The phylogenetic relationships of Orchidoideae and Spiranthoideae (Orchidaceae) as inferred from rbcL plastid sequences. Lindleyana 12, 1–11. Kores PJ, Weston PH, Molvray M, Chase MW (2000) Phylogenetic relationships within the Diurideae (Orchidaceae): inferences from plastid matK DNA sequences. In ‘Monocots: systematics and evolution’. (Eds KL Wilson, DA Morrison) pp. 449–456. (CSIRO Publishing: Melbourne) Kores PJ, Molvray M, Weston PW, Hopper SD, Brown AP, Cameron KM, Chase MW (2001) A phylogenetic analysis of Diurideae (Orchidaceae) based on plastid DNA sequence data. American Journal of Botany 88, 1903–1914. doi:10.2307/3558366 Linder HP, Kurweil H (1999) ‘Orchids of Southern Africa.’ (A.A.Balkema: Rotterdam, The Netherlands) Lindley J (1840a) A sketch of the vegetation of the Swan River Colony. In ‘Edwards’s botanical register 1–23, Swan River, Appendix xlvi–lvii’. Ridgeway, Picadilly, London. Lindley J (1840b) ‘The genera and species of orchidaceous plants.’ (Ridgways: London) Oberwinkler F (1994) Genera in a monophyletic group: the Dacrymycetales. Mycologia Helvetica 6, 35–72. Olmstead RG, Palmer JD (1994) Chloroplast DNA and systematics: a review of methods and data analysis. American Journal of Botany 81, 1205–1224. doi:10.2307/2445483 Pfeil BE, Crisp MD (2005) What to do with Hibiscus? A proposed nomenclatural resolution for a large and well known genus of Malvaceae and comments on paraphyly. Australian Systematic Botany 18, 49–60. doi:10.1071/SB04024 Pfitzer EHH (1887) ‘Entwurf einer natürlichen Anordnung der Orchideen.’ (Carl Winter’s Universitätsbuchhandlung: Heidelberg, Germany) Pfitzer EHH (1889) Orchidaceae. In ‘Die natürlichen Pflanzenfamilien’. (Eds A Engler, K Prantl) pp. 52–218. (Wilhelm Engelmann: Leipzig, Germany) Posada D, Crandall KA (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817–818. doi:10.1093/bioinformatics/14.9.817 Pridgeon AM, Chase MW (2001) A phylogenetic reclassification of Pleurothallidinae (Orchidaceae). Lindleyana 16, 235–271. Pridgeon AM, Cribb PJ, Chase MW, Rasmussen FN (2001) ‘Genera Orchidacearum, 2. Orchidoideae (Part 1).’ ( Oxford University Press: Oxford, UK) Pridgeon AM, Cribb PJ, Chase MW, Rasmussen FN (2003) ‘Genera Orchidacearum, 3. Orchidoideae (Part 2), Vanilloideae.’ (Oxford University Press: Oxford, UK)
M. A. Clements et al.
Reichenbach HG (1871) Bemerkungen für eine Australische Orchideenkunde. In ‘Beiträge zur systematischen Pflanzenkunde’. pp. 53–70. (Druck von ThG Meissner: Hamburg, Germany) Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. doi:10.1093/bioinformatics/btg180 Rupp HMR (1933) The genus Pterostylis R.Br.: a new scheme of classification, with notes on the distribution of the Australian species. Proceedings of the Linnean Society of New South Wales 58, 421–428. Salazar GA, Chase MW, Soto Arenas MA, Ingrouille M (2003) Phylogenetics of Cranichideae with emphasis on Spiranthinae (Orchidaceae, Orchidoideae): evidence from plastid and nuclear DNA Sequences. American Journal of Botany 90, 777–795. doi:10.3732/ajb.90.5.777 Salazar GA, Cabrera LI, Madriñán S, Chase MW (2009) Phylogenetic relationships of Cranichidinae and Prescottiinae (Orchidaceae, Cranichideae) inferred from plastid and nuclear DNA sequences. Annals of Botany 104, 403–416. doi:10.1093/aob/mcn257 Schuiteman A, de Vogel E, Vermeulen JJ, Kessler P, Vogel A (2008) ‘Malesian orchid genera illustrated, Orchidaceae.’ (Nationaal Herbarium Nederland, Universiteit Leiden; Hortus Botanicus, Universiteit Leiden: Leiden, The Netherlands) Silveira P, Schuiteman A, Vermeulen JJ, Sousa AJ, Silva H, Paiva J, de Vogel E (2008) The orchids of Timor: checklist and conservation status. Botanical Journal of the Linnean Society 157, 197–215. doi:10.1111/j.1095-8339.2008.00796.x St George I (1999) ‘The nature guide to New Zealand native orchids.’ (Godwit: Auckland) St George I (2010) The NZ orchids: the editor’s annual list. New Zealand Native Orchid Journal 115, 31–40. Stearn WT (1960) An introduction to Robert Brown’s ‘Prodromus Florae Novae Hollandiae’. In ‘Historiae Naturalis Classica VI, Prodromus Florae Novae Hollandiae et Insulae van Diemen facsimile’. (Eds J Cramer, HK Swann) pp. i–lii. (HR Engelmann (J Cramer) and Wheldon & Wesley, Ltd, Hafner Publishing Co.: New York) Stuessy TF (2009a) Paradigms in biological classification (1707–2007): has anything really changed? Taxon 58, 68–76. Stuessy TF (2009b) ‘Plant taxonomy. The systematic evaluation of comparative data.’ (Columbia University Press: New York) Sun T, Skinner DZ, Liang GH, Hulbert SH (1994) Phylogenetic analysis of Sorghum and related taxa using internal transcribed spacers of the nuclear ribosomal DNA. Theoretical and Applied Genetics 89, 26–32. doi:10.1007/BF00226978 Swartz O (1810) Diplodium. Magazin für die Neuesten Entdeckungen in der Gesammten Naturkunde. Gesellschaft Naturforschender Freunde zu Berlin 4, 83–85. Szlachetko DL (2001) Genera et species Orchidalium, I. Polish Botanical Journal 46, 11–26. Swofford D (1999) ‘PAUP: phylogenetic analysis using parsimony, prerelease version 4.02.’ (Sinauer: Sunderland, MA) Thiele KR, Brown AP (2007) Recombinations in Western Australian Orchidaceae 1. Nuytsia 16, 473–474. Thomson D, Henry R (1993) Use of DNA from Dry Leaves for PCR and RAPD Analysis. Plant Molecular Biology Reporter 11, 202–206. Tsitrone A, Kirkpartick M, Levin DA (2003) A model for chloroplast capture. Evolution 57, 1776–1782. van Royen P (1979) ‘The alpine flora of New Guinea.’ (J Cramer: Vaduz, Liechtenstein)
Manuscript received 29 July 2010, accepted 13 February 2011
Phylogenetic relationships in Pterostylidinae
Australian Journal of Botany
111
Appendix 1. Taxa studied, voucher information and GenBank accessions H = herbarium specimen; K = floral dissection card; P = live plant; Can = Canberra; Ncc = New South Wales (NSW), Central Coast; Ncs = NSW, Central-west Slopes; Nct = NSW, Central Tablelands; Nnc = NSW, north coast; Nnt = NSW, Northern Tablelands; Nsc = NSW, south coast; Nst = NSW, Southern Tablelands; Qco = Queensland (Qld), Cook; Qdd = Qld, Darling Downs; Qkn = Qld, Kennedy north; Qle = Qld, Leichhardt; Qmo = Qld, Moreton; Qwb = Qld, Wide Bay; Ski = South Australia (SA), Kangaroo Island; Sls = SA, Lofty south; Sse = SA, south-east; Tas = Tasmania; Wav = Western Australia (WA), Avon; Wco = WA, Coolgardie; Wda = WA, Darling; Wey = WA, Eyre; AU = Australia; NC = New Caledonia; NZ = New Zealand Species Achlydosa glandulosa (Schltr.) M.A.Clem. et D.L.Jones Bunochilus littoralis D.L.Jones Bunochilus longifolius (R.Br.) D.L.Jones et M.A.Clem. Bunochilus melagrammus (D.L.Jones) D.L.Jones et M.A.Clem.
Bunochilus montanus D.L.Jones Bunochilus tenuis D.L.Jones Bunochilus umbrinus D.L.Jones Bunochilus viriosus D.L.Jones Bunochilus williamsonii (D.L.Jones) D.L.Jones et M.A.Clem. Crangonorchis depauperata (F.M.Bailey) D.L.Jones et M.A.Clem. Crangonorchis pedoglossa (Fitzg.) D.L.Jones et M.A.Clem.
Diplodium abruptum (D.L.Jones) D.L.Jones et M.A.Clem. Diplodium aestivum (D.L.Jones) D.L.Jones et M.A.Clem. Diplodium alatum (Labill.) D.L.Jones et M.A.Clem. Diplodium alobulum (Hatch) D.L.Jones et M.A.Clem. Diplodium alveatum (Garnet) D.L.Jones et M.A.Clem. Diplodium asperum (D.L.Jones et M.A.Clem.) D.L.Jones et M.A.Clem. Diplodium atrans (D.L.Jones) D.L.Jones et M.A.Clem. Diplodium brevichilum D.L.Jones MS Diplodium brumalis (L.B.Moore) D.L.Jones et M.A.Clem. Diplodium coccinum (Fitzg.) D.L.Jones et M.A.Clem. Diplodium decurvum (R.S.Rogers) D.L.Jones et M.A.Clem.
Provenance
Collector no.
Type of material
ITS
GenBank no.
NC: Mt Do
Clements 7806
P
AF348042
AU: Sse; Coorong AU: Ncc; Green Point
Jones 16461 Jones 15783
P P
GQ866440 AY134639
AU: Vgi; Yarram
Jones 16383
P
GQ866443
AU: Tas; Battery Point AU: Can; Gibraltar Falls AU: Nct; Cadia Res. AU: Can; Black Mountain Reserve AU: Sls; Upper Sturt AU: Tas; South Arm Rd
Clements 10797 Clements 10797a Clements 9736 Jones 13027a Otero1208 Clements 8276 Wapstra (ORG 746)
P P P P P P P
GQ866441 GQ866442 GQ866444 GQ866445 GQ866446 AY134652 AY134658
AU: Tas; Bicheno AU: Qco; Grey Range
Clements 10779a Broers 454
P P
GQ866447 GQ866310
AU: Qco; Mt White, Coen AU: Ncc; Bundeena-Jibbon
Jones 18908 Brinsley (ORG 2244)
P P
GQ866310 AY134644
AU: Tas; West Point Road AU: Tas; Labillardiere Peninsula AU: Tas; Rocky Cape NP
Clements 10827 Clements 10807 Clements 10835a Clements 10835b Jones 11373
P P P P P
GQ866307 GQ866306 GQ866308 GQ866309 GQ866269
Clements 9851
P
GQ866270
Whinray (ORG 1497) Richards 882 (Jones 9538) Parr 86/99 Jane 77/99
P
AY134620
P
GQ866271
P P
GQ866272 GQ866273
AU: Wda; Chaterhouse–Eaton road
French 1304
P
GQ866274
AU: Can; Brindabella Range
FitzGerald 112
P
GQ866275
AU: Wey; Kau Rocks
French 341
P
GQ866276
Cult. ex NZ: Bay of Islands
Thomas s.n.
K
GQ866278
NZ: Whangarei AU: Can; Tidbinbilla Nature Reserve (NR) AU: Can; Brindabella Range
Pullman 78/99 Clements 9856
P P
GQ866277 GQ866279
Clements 9739
P
AY134634
AU: Nnt; New England NP, Point Lookout Rd AU: Can; Brindabella Range AU: Tas; Flinders Island Cult. ex NZ: NZ: Northland NZ: Nelson
matK
GQ866261
GQ866260
GQ866241
GQ866242
(Continued )
112
Australian Journal of Botany
M. A. Clements et al.
Appendix 1. (continued ) Species
Diplodium dolichochilum (D.L.Jones et M.A.Clem.) D.L.Jones et M.A.Clem. Diplodium grandiflorum (R.Br.) D.L.Jones et M.A.Clem.
Diplodium laxum (Blackmore) D.L.Jones et M.A.Clem. Diplodium longicurvum (Rupp) D.L.Jones et M.A.Clem.
Diplodium longipetalum (Rupp) D.L.Jones et M.A.Clem. Diplodium aff. longipetalum (Rupp) D.L.Jones et M.A.Clem. Diplodium microchilum D.L.Jones MS Diplodium obtusum (R.Br.) D.L.Jones et M.A.Clem. Diplodium reflexum (R.Br.) D.L.Jones et M.A.Clem. Diplodium revolutum (R.Br.) D.L.Jones et M.A.Clem. Diplodium robustum (R.S.Rogers) D.L.Jones et M.A.Clem. Diplodium rogersii (E.Coleman) D.L.Jones et M.A.Clem. Diplodium scabrum (Lindl.) D.L.Jones et M.A.Clem. Diplodium tenuissimum (Nicholls) D.L.Jones et M.A.Clem. Diplodium torquatum (D.L.Jones) D.L.Jones et M.A.Clem. Diplodium trullifolium (Hook.f.) D.L.Jones et M.A.Clem. Eremorchis allantoidea (R.S.Rogers) D.L.Jones et M.A.Clem.
Hymenochilus bicolor (M.A.Clem. et D.L.Jones) D.L.Jones et M.A.Clem.
Hymenochilus clivicola D.L.Jones Hymenochilus cycnocephalus (Fitzg.) D.L.Jones et M.A.Clem.
Provenance
Collector no.
Type of material
ITS
GenBank no.
AU: Tas; St Patricks Head AU: Sse; Robe, Woakwine Conservation Park
Clements 10769 Murfet 3199
P P
GQ866280 GQ866281
AU: Tas; Freycinet NP
Clements 10790
P
GQ866282
AU: Nnt; Upper Wilson River, Jerusalem NP AU: Can; Tidbinbilla NR
ORG 4188
P
GQ866283
Clements 9855
P
AY134638
AU: Qdd; Texas State Forest
Crane 1626
P
GQ866285
AU: Nnt; Warrumbungle Mountains AU: Veh; Mt Raymond
Jones 6032
P
GQ866284
Richards 600
P
GQ866286
AU: Nnt; Doughboy Ck Crossing, Guyra–Ebor road AU: Wir; Zuytdorp Cliffs
Richards 1519 (Jones 14813) French 1393
P
GQ866287
P
GQ866288
AU: Qdd; Queen Mary Falls NP
Crane 2308
P
GQ866289
AU: Can; Tidbinbilla NR
Clements 9854
P
GQ866290
AU: Nns; Kings Plains, road to Emmerville AU: Can; Molongolo Gorge AU: Sml; Peake
Clements 8748
P
GQ866291
Otero 1221 Jones 16492
P P
GQ866292 GQ866293
French 1491
P
GQ866294
French 1377
P
GQ866295
AU: Wda; Minningup Rd, Bunbury AU: Wda; Moora AU: Vwp; Skull Creek, Peterborough AU: Nnt; 6.8 km N of New England NP turnoff NZ: Auckland
Richards (Jones 8647) Jones 12965
P
GQ866296
P
GQ866297
de Lange 81/99
P
GQ866299
NZ: S. Auckland AU: Wey; Raventhorpe Range
Molloy 183/00 Jones 12330
P H
GQ866298 AY134621
AU: Wey; Raventhorpe Range AU: Wey; Raventhorpe Range AU: Wco; Dundas Rock S of Norseman AU: Wco; Dundas Rock S of Norseman AU: Nst; Adelong
Clements 10928 Clements 10928b Clements 10891
P P P
GQ866332 GQ866333 GQ866331
Clements 10891a
P
GQ866334
Cunningham (ORG 1735)
P
AY134626
AU: Nnt; N of Cowra on Forbes Rd AU: Ncc; Albion Park AU: Can; Namadgi NP
Clements 9710 Clements 11041 Otero 1214
P P P
GQ866428 GQ866429 GQ866430
AU: Can; Namadgi NP AU: Ncs; Conimbla NP
Otero 1214 Clements 9707
P P
GQ866431 GQ866432
matK
GQ866237
GQ866238
GQ866245
GQ866257 GQ866258
(Continued )
Phylogenetic relationships in Pterostylidinae
Australian Journal of Botany
Appendix 1. Species
Hymenochilus aff. cycnocephalus. Hymenochilus muticus (R.Br.) D.L.Jones et M.A.Clem.
Hymenochilus rubenachii (D.L.Jones) D.L.Jones et M.A.Clem. Hymenochilus tanypoda (D.L.Jones, Molloy et M.A.Clem.) D.L.Jones et M.A.Clem. Linguella clavigera (Fitzg.) D.L.Jones et M.A.Clem. Linguella crispula D.L.Jones MS
Linguella dilatata (A.S.George) D.L.Jones et M.A.Clem.
Linguella erubescens D.L.Jones MS Linguella globosa D.L.Jones MS Linguella karri D.L.Jones MS Linguella nana (R.Br.) D.L.Jones et M.A.Clem. Linguella parva D.L.Jones MS Linguella puberula (Hook.f.) D.L.Jones et M.A.Clem. Linguella pyramidalis (Lindl.) D.L.Jones et M.A.Clem. Linguella setuosa D.L.Jones MS
Linguella timothyi MS
Oligochaetochilus aciculiformis (Nicholls) Szlach. Oligochaetochilus basalticus (D.L.Jones et M.A.Clem.) D.L.Jones et M.A.Clem. Oligochaetochilus bisetus (Blackmore et Clemesha) Szlach. Oligochaetochilus chaetophorus (M.A.Clem. et D.L.Jones) Szlach. Oligochaetochilus aff. excelsus (M.A.Clem.) Szlach.
Provenance
(continued ) Collector no.
Type of material
ITS
GenBank no.
AU: Can; Tharwa AU: Nst; Molongalo Gorge AU: Sle; Monarto AU: Sep; N of Cowell
Clements 9768 Otero 1222 Murfet 2200 Jones 14100
P P P P
AY134633 GQ866433 GQ866434 AY134640
AU: Wey; Ravensthorpe Range
Clements 10923a Clements 10923b French 1319 Wapstra (ORG 1010)
P P P P
GQ866437 GQ866435 GQ866436 GQ866438
NZ: Lake Coleridge
Molloy 012/98
P
GQ866439
AU: Ncs; Conimbla NP
Clements 9706
P
AY134629
AU: Wda; Murdoch Univ., Perth AU: Wda; corner of Baldivis Rd and Karnup Rd
French 1265 French 3656b
P P
GQ866312 GQ866313
French 3656d French 1427
P P
GQ866314 AY134635
AU: Wro; Frog Rock AU: Tas; Tiger Ck
AU: Wda; Fox’s Lair, Narrogin AU: Wda; D’Entrecasteaux NP, SW Hwy AU: Wda; E of Dardanup
Clements 10993a
P
GQ866315
French 1306
P
GQ866316
AU: Wda; NE of Australind AU: Wda; E of Dardanup AU: Tas; Mt William
French 1300 French 1305 Clements 10760
P P P
GQ866317 GQ866318 GQ866320
AU: Tas; West Point Rd AU: Wro; 9.8 km E of Karlgarin–Holt Rock road NZ: Coromandel
Clements 10828 Clements 10875a
P P
GQ866319 GQ866321
de Lange 141/99
P
GQ866322
AU: Wda; Preston Beach area
French 1283
P
AY134647
AU: Wro; 9.8 km E of Karlgarin–Holt Rock road
Clements 10879
P
GQ866323
Clements 10879a Clements 10879b Clements 10892
P P P
GQ866324 GQ866325 GQ866327
Clements 10926 Clements 10926a Clements 10926b Jones 15851
P P P P
GQ866328 GQ866329 GQ866330 GQ866405
AU: Vwp; Chatsworth Rd, Dundonnell
Jones 10877
P
GQ866406
AU: Sfr; Banycrooling Gorge
Jones (ORG 1214)
P
AY134628
AU: Nnc; Clarencetown
Jones 6738
P
GQ866407
AU: Sfr; Wilpena Pound, Loves Mine
Bates 45210
P
GQ866408
AU: Wco; Dundas Rock S of Norseman AU: Wey; Ravensthorpe Range
AU: Can; Mt Ainslie
113
matK
GQ866259
GQ866243
GQ866244
(Continued )
114
Australian Journal of Botany
M. A. Clements et al.
Appendix 1. (continued ) Species Oligochaetochilus exsertus D.L.Jones MS
Oligochaetochilus frenchii D.L.Jones Oligochaetochilus gibbosus (R.Br.) Szlach. Oligochaetochilus hamatus (Blackmore et Clemesha) Szlach. Oligochaetochilus leptochilus (M.A.Clem. et D.L.Jones) Szlach. Oligochaetochilus macrocalymmus (M.A.Clem. and D.L.Jones) Szlach. Oligochaetochilus mitchellii (Lindl.) Szlach. Oligochaetochilus mystacinus D.L.Jones MS Oligochaetochilus pusillus (R.S.Rogers) Szlach. Oligochaetochilus roensis (M.A.Clem. et D.L.Jones) Szlach. Oligochaetochilus aff. roensis (M.A.Clem. et D.L.Jones) Szlach. Oligochaetochilus rufus (R.Br.) Szlach.
Oligochaetochilus spathulatus (M.A.Clem.) Szlach. Oligochaetochilus squamatus (R.Br.) Szlach. Oligochaetochilus woollsii (Fitzg.) Szlach. Oligochaetochilus aff. xerophilus (M.A.Clem.) Szlach. Oligochaetochilus zebrinus D.L.Jones MS Petrorchis bicornis (D.L.Jones et M.A.Clem.) D.L.Jones et M.A.Clem. Pharochilum daintreanum (Benth.) D.L.Jones et M.A.Clem.
Plumatichilos barbatum (Lindl.) Szlach. Plumatichilos eurycolum D.L.Jones MS
Provenance AU: Wir; Moora
Collector no.
Type of material
ITS
GenBank no.
Jones 12073
P
AY134653
Jones 12073a Jones 12073b French 1290
P P P
GQ866409 GQ866410 GQ866411
matK
AU: Wda; Whittakers Mill, S of Yarloop AU: Ncc; Croom Sports Ground, Albion Park AU: Can; Tharwa
Clements 11039
P
GQ866412
Clements 9743
P
GQ866413
AU: Can; Pine Island Res. AU: Wey; Ravensthorpe Range
Otero 1210 Clements 10927b
P P
GQ866414 GQ866415
AU: Wir; Murchison River Bridge
French 2986
P
GQ866416
AU: Qdd; Texas State Forest
Crane 1425
P
GQ866417
AU: Qle; Mt Moffat Road
Crane 2049
P
GQ866418
AU: Sfr; St Mary’s Peak trail, Wilderness Park AU: Wro; Dundas Rocks, S of Norseman
Jones (ORG 1220)
P
GQ866419
Clements 10893b
P
GQ866420
AU: Weu; Madura
Murfet 1236a
P
GQ866421
AU: Qdd; Kogan
Crane 2214
P
AF348056
AU: Ncc; Yallah, Transgrid property entrance AU: Wir; Green Head Rd (Coorow–Green Head) AU: Can; Tharwa
Clements 11043
P
GQ866422
ORG 860
P
GQ866423
Clements 9788
P
GQ866424
AU: Nnt; Moonbi Range
Clements 8739
P
GQ866425
AU: Vma; Mallanbool Flora and Fauna Res AU: Wav; S of Lake Wallambin
Clements 9917
P
GQ866426
French 528
P
GQ866427
AU: Nnt; Glassy Mt, Woodenbong
Benwell (ORG 2629)
P
AY134627
AU: Qmo; Daves Creek Circuit AU: Qdd; Girraween NP
Dalyell (ORG 4177) Crane 1824
P P
GQ866344 AF348055
GQ866251
AU: Nnt; Upper Wilson River, Jerusalum NP AU: Nsc; Braidwood–Nerriga road, near Sassafras AU: Wda; Mundaring Weir Rd
Dalyell (ORG 4189)
P
GQ866460
GQ866265
Clements 10733
P
GQ866461
French 062
H
GQ866465
Clements 10990
P
GQ866466
AU: Wda; D’Entrecasteaux NP, along South-west Highway
GQ866252
GQ866254 GQ866253
GQ866256
GQ866255
GQ866263
(Continued )
Phylogenetic relationships in Pterostylidinae
Australian Journal of Botany
Appendix 1. Species
Plumatichilos galbulum D.L.Jones MS
Plumatichilos plumosum (Cady) Szlach. Plumatichilos serotinum D.L.Jones MS Plumatichilos tasmanicum (D.L.Jones) Szlach. Plumatichilos turfosum (Endl.) Szlach. Pterostylis acuminata R.Br. Pterostylis agathicola D.L.Jones, Molloy et M.A.Clem. Pterostylis alpina R.S.Rogers Pterostylis areolata Petrie Pterostylis auriculata Colenso
Pterostylis australis Hook.f. Pterostylis banksii A.Cunn.
Pterostylis aff. banksii A.Cunn.
Pterostylis baptistii Fitzg.
Pterostylis bureaviana Schltr. Pterostylis cardiostigma D.Cooper Pterostylis cernua D.L.Jones, Molloy et M.A.Clem. Pterostylis cucullata R.Br. Pterostylis curta R.Br.
Pterostylis erecta T.E.Hunt Pterostylis falcata R.S.Rogers Pterostylis foliata Hook.f.
Pterostylis graminea Hook.f. Pterostylis aff. graminea Hook.f. Pterostylis aff. graminea ‘Kauri’
Provenance
AU: Wda; D’Entrecasteaux NP, Mt Barnett carpark AU: Wda; S of New Norcia
115
(continued ) Collector no.
Type of material
ITS
GenBank no. matK
Clements 10990a Clements 10996
P P
GQ866467 GQ866468
GQ866264
French 1586
P
AY134625
AU: Wav; 9.8 km E of Kalgarin–Holt Rock road AU: Ncs; Parks–Wellington road
Clements 10877b
P
GQ866469
Jones 14329
P
AY134646
AU: Wda; E of Dardanup
French 1309
P
GQ866470
AU: Tas; Strzelecki Peaks
Whinray (ORG 1722)
P
GQ866471
NZ: Coromandel AU: Wey; Doubtful Island
Molloy 140/99 Heberle (ORG 1614)
P P
GQ866472 AY134655
AU: Ncc; West Pennant Hills NZ: Auckland
Clements 9976 Molloy 082/99
P P
GQ866345 GQ866346
AU: Can; Brindabella Range NZ: Port Hills, Christchurch NZ: Lake Wilkie NZ: Tautuku NZ: Stewart Island NZ: Craigieburn NZ: Tautuku Cult. ex NZ: Auckland NZ: Northland NZ: Chatham Islands NZ: Chatham Islands NZ: Wanganui NZ: Awaroa NZ: Taupo AU: Ncc; Green Point AU: Nnt; Upper Wilson River, Jerusalum NP NC: Yaté Road NZ: Auckland
Clements 9740 Molloy 011/98 Molloy 216/00 Molloy 206/00 Molloy 224/00 Molloy 041/98 Molloy 222/00 Thomas (DLJ 10576) Parr 87/99 Molloy 177/00 Molloy 223/00 Molloy 193/00 Molloy 192/00 Molloy 244/00 Jones 15781 Dalyell (ORG 4186)
P P P P P P P P P P P P P P P P
GQ866347 GQ866348 GQ866349 GQ866350 GQ866351 AY134622 GQ866352 GQ866353 AY134623 GQ866354 GQ866355 GQ866356 GQ866357 GQ866358 AY134624 GQ866246
Clements 7883 Molloy 187/00
K P
GQ866359 GQ866360
NZ: Westland
Molloy 138/99
P
GQ866361
AU: Tas; King Island AU: Sls; Belair AU: Can; Tidbinbilla AU: Ncc; Mona Vale AU: Ncc; E slopes of Wattagan Mountains AU: Ncc; Dharug NP, Mill Ck AU: Can; Smokers Trail, Namadgi NP NZ: Port Hills, Christchurch AU: Tas; King Island AU: Sls; Deep Creek Conservation Park NZ: Riccarton Bush NZ: Trotters Gorge NZ: Waipatiki NZ: Northland NZ: Northland
Jones 15940 Clements 9879 Clements 9734 Angus (ORG 1504) Jones15780
P P P P P
AY134632 GQ866362 AF348054 GQ866364 GQ866363
Clements 9756 Jones 16358
P P
GQ866365 GQ866366
Molloy 006/98 Jones 15952 Murfet 3350
P P P
AY134636 GQ866367 GQ866368
Molloy 001/98 Molloy 214/00 Banks (Jones 15898) Molloy 124/99 Forester 106/99
P P H P P
AY134637 GQ866369 GQ866371 GQ866370 GQ866372 (Continued )
116
Australian Journal of Botany
M. A. Clements et al.
Appendix 1. (continued ) Species Pterostylis hispidula Fitzg. Pterostylis humilis R.S.Rogers Pterostylis irsoniana Hatch Pterostylis irwinii D.L.Jones, Molloy et M.A.Clem. Pterostylis micromega Hook.f. Pterostylis montana Hatch
Pterostylis aff. montana Hatch
Pterostylis monticola D.L.Jones Pterostylis novoguineensis Ridley Pterostylis nutans R.Br.
Pterostylis oliveri Petrie Pterostylis oreophila Clemesha Pterostylis paludosa D.L.Jones, Molloy et M.A.Clem. Pterostylis papuana Rolfe Pterostylis patens Colenso Pterostylis pedunculata R.Br. Pterostylis porrecta D.L.Jones, Molloy et M.A.Clem. Pterostylis procera D.L.Jones et M.A.Clem. Pterostylis silvicultrix (F.Muell.) Molloy, D.L.Jones et M.A. Clem. Pterostylis ‘sphagnum’ Pterostylis stricta Clemesha et B.Gray Pterostylis venosa Colenso Ranorchis sargentii (C.R.P.Andrews) D.L.Jones et M.A.Clem.
Speculantha antennifera D.L.Jones MS Speculantha coarctata D.L.Jones MS Speculantha furva D.L.Jones MS Speculantha multiflora D.L.Jones Speculantha nigricans (D.L.Jones et M.A.Clem.) D.L.Jones et M.A.Clem.
Provenance
Collector no.
Type of material
ITS
GenBank no.
AU: Qdd; Girraween NP NZ: Mt Ruapehu NZ: Para Para Ridge NZ: Takaka Hill
Crane 1817 Molloy 233/00 Ducker 009/98 Ducker 008/98
P P P P
GQ866373 GQ866374 GQ866375 GQ866376
NZ: Erua NZ: Opuatia NZ: Stewart Island NZ: Para Para Ridge NZ: Chatham Island Cult. NZ: Christchurch NZ: Leith Hill NZ: Horopito NZ: Mt Ruapehu AU: Can; Brindabella Range Indonesia: Irian Jaya; Walabu AU: Can; Gibralta Falls NZ: Waihaha AU: Can; Black Mountain Reserve NZ: Pegleg Ck AU: Can; Brindabella Range NZ: Te Reo Arm
Molloy 238/00 de Lange 137/99 Molloy 213/00 Ducker 010/98 Molloy 178/00 Molloy 029/98 Molloy 210/00 Molloy 239/00 Molloy 236/00 Clements 9741 Rose 108 Clements 9735 Molloy 227/00 Otero 1207
P P P P P P P P P P P P P P
GQ866377 GQ866378 GQ866379 GQ866381 GQ866380 GQ866382 GQ866383 GQ866384 GQ866385 GQ866386 GQ866387 GQ866388 GQ866389 GQ866247
Molloy 042/98 Clements 9778 de Lange and Norton 049/98 Molloy 234/00 Clements 7199 Molloy 237/00 Molloy 228/00 Clements 9733 de Lange 149/99
P P P
GQ866390 GQ866391 GQ866392
P P H P P P
GQ866393 GQ866394 GQ866395 GQ866396 AY134645 GQ866397
NZ: Mt Ruapehu Papua New Guinea: Simbai NZ: Mt Ruapehu NZ: Waihaha AU: Can; Tidbinbilla NR NZ: Wellington Cult. ex AU: Qco; Moomin
P
GQ866399
AU: Qco; Moomin NZ: Chatham Island
Richards 1217 (Lawler12) Jones 18909 Molloy 167/00
P P
GQ866398 GQ866401
NZ: Chatham Island NZ: Taranaki roadside AU: Qkn; 7.4 km W of Paluma
Molloy 198/00 Dodunski 005/98 Mackenzie 0040/98
P P P
GQ866400 GQ866402 GQ866403
NZ: Arthurs Pass AU: Wav; Wubin
Molloy 039/98 Jones 12093
P H
GQ866404 AY134651
AU: Wro; Dandas Rocks, S of Norseman
Clements 10894
P
GQ866462
Clements 10894b Clements 10881a
P P
GQ866464 GQ866463
Crane 1882
P
GQ866335
FitzGerald 111
P
GQ866336
Tunstall (ORG 1408)
P
GQ866342
FitzGerald 102 Crane 2100
P P
GQ866337 AY134641
AU: Wav; 9.8 km E of Kalgarin–Holt Rock road AU: Qmo; Mt Coolum NP AU: Nct; Mt Wilson Rd, Blue Mountains AU: Nsc; Point Perpendicular Lighthouse AU: Can; Bendora Dam Rd AU: Qwb; Fraser Island
matK
GQ866248
GQ866268
(Continued )
Phylogenetic relationships in Pterostylidinae
Australian Journal of Botany
Appendix 1. Species Speculantha parviflora (R.Br.) D.L.Jones et M.A.Clem.
Speculantha rubescens D.L.Jones Speculantha vernalis D.L.Jones Stamnorchis recurva (Benth.) D.L.Jones et M.A.Clem.
Provenance
Taurantha splendens (D.L.Jones et M.A.Clem.) D.L.Jones et M.A.Clem. Taurantha taurus (M.A.Clem. et D.L.Jones) D.L.Jones et M.A.Clem. Taurantha aff. tenuicauda (Kraenzl.) D.L.Jones et M.A.Clem. Urochilus atrosanguineus D.L.Jones MS Urochilus oligantha D.L.Jones MS Urochilus sanguineus (D.L.Jones et M.A.Clem.) D.L.Jones et M.A.Clem.
GenBank no.
P
AY134643
AU: Qle; Carnarvon Gorge AU: Tas; Rocky Cape
Crane 2738 Clements 10843a Clements 10843b ORG 4185
P P P P
GQ866338 GQ866339 GQ866340 GQ866341
Otero 1203 Clements 9724 French 1266
P P P
GQ866343 AY134656 AY134648
Clements 10876a
P
GQ866456
Clements 10876b Clements 10995
P P
GQ866457 GQ866458
Clements 10995a Tunstall 296E
P P
GQ866459 AY134630
P
GQ866301
P
AY134631
AU: Tas; Bicheno AU: Qmo; Beachmere
Wapstra (ORG 744) Wapstra (ORG 748) Clements 10775a Crane 1540
P P
GQ866302 AY134642
AU: Qmo; Canungra NC: Mt Do
ORG 4200 Clements 7812
P P
GQ866303 GQ866304
AU: Qkn; Eungella Range
Jones 11637
P
AY134654
NC: Mt Koghis
Jones 15579
P
GQ866305
AU: Wda; Murdoch Univ., Perth
French 1262
P
To be submitted
AU: Wda; Murdoch Univ., Perth
French 1263
P
AY134650
AU: Wda; 10 km S of New Norcia AU: Sls; Stirling East
French 3653 Clements 8273
P P
GQ866449 AY134649
AU: Wav; D’Entreacasteaux NP; Mt Barnett AU: Wro; Dundas Rocks, S of Norseman
Clements 10992
P
GQ866267
Clements 10895
P
GQ866450
Clements 10895b French 3655
P P
GQ866451 GQ866452
French 3655a French 1308
P P
GQ866453 AY134657
French 1298 Clements 10994a
P P
GQ866454 GQ866266
AU: Nnt; Copeland AU: Tas; S. Arm Rd, near Clifton Beach turn off AU: Tas; Goats Bluff, S or Arm Rd
AU: Wda: Corner of Baldivis Rd and Karnup Rd Urochilus vittatus (Lindl.) D.L.Jones et M.A.Clem.
ITS
Jones 16376
AU: Wav; D’Entreacasteaux NP; Mt Barnett carpark
Taurantha ophioglossa (R.Br.) D.L.Jones et M.A.Clem.
Type of material
AU: Vgi; Tooradin
AU: Wav; 9.8 km E of Kalgarin–Holt Rock road
Taurantha collina (Rupp) D.L.Jones et M.A.Clem. Taurantha concinna (R.Br.) D.L.Jones et M.A.Clem.
(continued ) Collector no.
AU: Nnt; Upper Wilson River, Jerusalem NP AU: Can; Black Mountain Reserve AU: Nsc; Flat Rock Crk, Nowra AU: Wav; Murdoch Univ., Perth
AU: Wda; E of Dardanup AU: Wda; Marriot Rd, Perth AU: Wda; D’Entreacasteaux NP; Mt Barnett
117
http://www.publish.csiro.au/journals/ajb
matK
GQ866249
GQ866250
GQ866262
GQ866240
