THREE NEW SPECIES OF DENDROCHILUM ( ORCHIDACEAE ) AND THEIR PHYLOGENETIC POSITIONS ACCORDING TO PLASTID AND NUCLEAR RIBOSOMAL ITS SEQUENCES

Three new species of the Southeast Asian orchid genus Dendrochilum Blume are described. All three seem to belong to the phenetically defined subgenus Platyclinis (Benth.) Pfitzer. As indicated by the name, D. celebesense H.A. Pedersen & Gravend. originates from Sulawesi. The geographic origins of D. coccineum H.A. Pedersen & Gravend. and D. warrenii H.A. Pedersen & Gravend., on the other hand, are unknown, but parsimony analyses of sequences of the plastid accD-psaI intergenic spacer and nuclear ribosomal ITS1-5.8S-ITS2 region and morphological affinities suggest they should both be counted as members of the Philippine flora.


INTRODUCTION
In the latest complete survey of the Southeast Asian orchid genus Dendrochilum (Pedersen et al., 1997) 271 species were recognized.However, even after subsequent regional treatments covering the main diversity centres of this genus -viz.the Philippines (Pedersen, 1997a), Sumatra (Comber, 2001), and Borneo (Wood, 2001) -new species keep popping up (e.g., Pedersen, 1997bPedersen, , 2001)).In this paper, three further species are being added, which were all brought into cultivation for the first time over the past two years and of which only cultivated collections are known without precise locality data.
Morphologically, these three new species seem to belong to subgenus Platyclinis (Benth.)Pfitzer because of their synanthous inflorescences, column with apical wing or hood and entire, flat rostellum (Pedersen et al., 1997).To check whether molecular characters could be used to infer the geographic origin of the new species described here, DNA sequences were obtained from the plastid accD-psaI intergenic spacer and nuclear ribosomal ITS1-5.8S-ITS2region.These regions were used in previous studies of Dendrochilum (Barkman & Simpson, 2001, 2002), which enabled us to use already existing databases of sequences.A phylogenetic approach for inferring the geographic origin of cultivated plant material has been used before, i.e. by Wanntorp et al. (2002).

DNA extractions and amplifications
Total genomic DNA was extracted from 50 mg of silica gel dried leaf material using DNeasy extraction kits and protocols of QIAGEN (Leusden, The Netherlands).PCR products of the accD-psaI intergenic spacer and nrITS1-5.8S-ITS2regions of the three species here described were collected using the primers and PCR programs described in Barkman & Simpson (2002).DNA sequences were obtained on an ABI 377 automated sequencer (Applied Biosystems) using the protocols described in Gravendeel et al. (2001Gravendeel et al. ( , 2004) ) and submitted to the GenBank database under accession numbers AY534922-AY534927.

Phylogenetic analyses
The DNA sequences obtained for this study were added to the matrix compiled by Barkman & Simpson (2002).Maximum parsimony analyses were performed with PAUP*4.0b10(Swofford, 1999) using heuristic search, ten replicates of randomtaxon entry, and tree bisection reconnection (TBR) swapping.As outgroup, Pholidota clemensii Ames was used, just as in Barkman & Simpson (2002).All characters were assessed as independent, unordered, and equally weighted.The relative robustness of clades found was assessed by performing 1000 replicates of bootstrapping (Felsenstein, 1995) using simple stepwise additions, subtree pruning and regrafting (SPR) swapping, MULTREES on, and holding only ten trees per replicate.

Preparation of descriptions
The morphological terminology applied in the species descriptions generally follows Stearn's (1983) vocabulary and list of individual absolute terms.

Sequence divergence
The accD-psaI alignment consisted of 894 positions and contained 16 phylogenetically informative substitutions and 13 indels (of which 7 were synapomorphic) varying in size between 5 and 25 bp.Mean pairwise distances within the ingroup were generally low and varied between 0 and 2.6%.
The nrITS1-5.8S-ITS2alignment consisted of 656 positions and contained 93 phylogenetically informative substitutions and 12 indels (of which 6 were synapomorphic) varying in size between 1 and 8 bp.Mean pairwise distances within the ingroup varied between 0 and 11.5%.

Phylogenetic analyses
Maximum parsimony analysis of the nuclear sequences yielded 32 most parsimonious trees (MPTs; length 249 steps; CI = 0.71; RI = 0.80).The strict bootstrap consensus is shown in Fig. 1 (left-hand).Clades present in many of the separate MPTs consist of all Bornean (bootstrap support of 63%) and Sumatran (bootstrap support of 82%) species.Maximum parsimony analysis of the plastid sequences yielded > 10,000 MPTs (length 66 steps; CI = 0.99; RI = 0.98).The strict bootstrap consensus is shown in Fig. 1 (right-hand).Clades retrieved in many of the separate MPTs consist of three subsets of Bornean species (bootstrap support of 57%, 76% and 79%, respectively) and part of the species of the Philippines and Sulawesi (bootstrap support of 51%).Because of the presence of hybrids (Barkman & Simpson, 2002) and the different set of species sampled for both data sets, we refrained from performing a total evidence analysis.

DISCUSSION
Separate phylogenetic analyses of the sequences of the accD-psaI intergenic spacer and nrITS1-5.8S-ITS2regions indicate that Dendrochilum coccineum is most closely related to D. arachnites Rchb.f., D. celebesense, D. glumaceum Lindl., D. graciliscapum (Ames) Pfitzer, and D. warrenii and seems nested in 'the Philippine lineage' as described by Barkman & Simpson (2001).Dendrochilum celebesense and D. warrenii seem most closely related to the species of the 'Philippine lineage' in the accD-psaI tree and have a more isolated position in the nrITS1-5.8S-ITS2tree (Fig. 1).Summarising, the molecular results obtained firmly corroborate a Philippine origin of D. coccineum.Based on the molecular results obtained, it seems equally likely that the specimen analysed of D. warrenii originally came from Sulawesi or the Philippines, although it is morphologically most similar to Philippine species of Dendrochilum.It must be stressed, however, that the geographic origin of species of unknown provenance can only be inferred successfully with a phylogenetic approach when genotypic variability is not too large.

Dendrochilum coccineum
Distribution -Unknown.Notes -1.This species belongs in the phenetically defined sect.Eurybrachium (for sectional features, see Pedersen et al. 1997: 31, 32).Within this section it seems referable to a presumably natural group of Philippine endemics that also includes D. con- propinquum Ames, and D. ecallosum Ames (cf.Pedersen 1997a: 131) -none of which are included in the phylogenetic analysis above.Dendrochilum coccineum is particularly similar to D. convallariaeforme but differs from the latter species primarily by its leathery leaves and deep crimson flowers, the revolute margins of its sepals and petals, and the three long keels of its labellum.According to the nrITS sequence tree, D. coccineum is closely related to D. graciliscapum (Ames) Pfitzer, which is endemic to the Philippines and has a similarly shaped column and labellum.
2. The specific epithet is derived from the Latin coccineus, deep red, referring to the deep crimson flowers.
Distribution -Unknown.Notes -1.This species belongs to the phenetically defined sect.Platyclinis (for sectional features, see Pedersen et al. 1997: 42).Morphologically, it seems closest to the Philippine D. flexuosum H.A. Pedersen.It differs from the latter by its longer and narrower leaves, its linear-lanceolate, acute petals, its longer labellum which is widest below the middle, and its basal stelidia that are subequal to the column proper.
2. In March 2003, a live specimen of D. warrenii was exhibited by Blair Sibun at the European Orchid Conference in London under the horticultural name Dendrochilum sp.'Sherborne Star' (submitted as D. pictum, nom.nud.) -for a colour photograph, see Hermans & Hermans (2003: 209, centre right).Incidentally, the specimen received an RHS Certificate of Cultural Commendation.
3. The specific epithet honours Richard C. Warren who provided us with the material from which the type specimen was prepared.