Gardiner, Motes, Emerson, Roberts, Kocyan 1

GARDINER, L.M., MOTES, M., ROBERTS, D.L., KOCYAN, A., EMERSON, B.C; Phylogenetic Patterns in the

Phylogenetic Patterns in the Genus Vanda and Related Genera ()

Gardiner, L.M.1,*, Motes, M.2, Roberts, D.L.1,3, Kocyan, A.4, Emerson, B.C.5,6

1Herbarium, Royal Botanic Gardens Kew, Richmond, Surrey, TW9 3AE, UK

225000 162nd Street, Homestead, Florida, 33031, USA

3 Durrell Institute of Conservation and Ecology, School of Anthropology & Conservation, Marlowe Building,

University of Kent, Canterbury, Kent, CT2 7NR, UK

4 Biodiversity Research/Systematic Botany, Institute of Biochemistry and Biology, University of Potsdam,

Maulbeerallee 2a, D-14469 Potsdam, Germany

5Island Ecology and Evolution Research Group, IPNA-CSIC, C/Astrofísico Francisco Sánchez 3, 38206 La

Laguna, Tenerife, Canary Islands, Spain

6School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK

*Author for correspondence: ([email protected])

Gardiner, Motes, Emerson, Roberts, Kocyan 2 INTRODUCTION

The orchid genus Vanda Jones ex R.Br. comprises approximately 75-80 and is widely distributed throughout mainly South East ; from and , through southern to Korea and Japan, and then down through to the northern Australia and the Solomon Islands (Govaerts, 2012). Many species appear to be endemic within a limited geographically range, with a major centre of diversity in the South East

Asian archipelagos. Vanda species are epi- and lithophytes exhibiting monopodial growth, stiffly erect, praemorse tipped leaves, and range from small through to large, with often brightly coloured, sometimes fragrant, flowers. There is great diversity in floral shape, colour, and particularly labellum structure, and the genus is one of the five most horticulturally important orchid genera in the world, yet a number of species are vulnerable to extinction in the wild, being rare and geographically restricted in distribution (Motes, 1997).

The first species recorded in western literature were in the late 1600s by Rheede tot Draakenstein (1693), a governor of Malabar, and documented by Rumphius in his posthumously published magnum opus Herbarium

Amboinensis (1741-50). Linnaeus described two species of Vanda (as Epidendrum species) in the first and second editions of Species Plantarum (1753, 1762). The genus Vanda Jones ex R.Br. was first established in

1795 by William Jones, who based his concept on Epidendrum tessellatum Roxb. (now known as V. tessellata

(Roxb.) Hook. ex G.Don in J.C.Loudon), but the genus was not validly published until 1820 by Robert Brown.

By 1853 the genus comprised some 25 species, enough that John Lindley (1853) could re-circumscribe the genus – broadly with the same generic concept as Jones – but postulating five sections at the sub-generic level.

Two of Lindley’s sections have since been removed from Vanda and erected as genera in their own right - section Fieldia to Vandopsis Pfitzer in H.G.A.Engler & K.A.E.Prantl and Dimorphorchis Rolfe, and section

Anota to Rhynchostylis Blume.

Since Lindley’s landmark work, the basic concept of Vanda has remained broadly constant until today, with additional species being described in the genus throughout the 20th century and into the 21st century. Only in the last 25 years have questions about the generic limits of Vanda troubled the image of a previously seemingly taxonomically stable genus. Motes (1997) described the genus Vanda as a taxonomic ‘black pit’, and

Christenson (1987) stated that it required ‘complete taxonomic revision’. The placement of a number of small Gardiner, Motes, Emerson, Roberts, Kocyan 3 genera such as Ascocentrum Schltr., Euanthe Schltr., Trudelia Garay, and Christensonia Haager in relation to the genus Vanda has been debated and remains uncertain. Clarification as to the generic status of Vanda, and a number of the taxa within the genus, as well as a more systematic classification of Vanda species, has long been required. The presence of praemorse leaf tips and a three-lobed labellum are good characters for the genus

Vanda (Seidenfaden 1988), but the diversity in floral morphology seen across the taxa is extremely high – with a wide range of sizes, shapes, colours, and labellum structures (including presence or absence of a spur, foot, and mid-lobe ornamentation).

Relationships within the wider subtribe are similarly confused. Characterised by monopodial growth, members of the subtribe all have a highly developed velamen on the roots, an entire rostellum, a relatively small spur formed by the lip, and pollinaria with well-developed stipes and viscidia, two to four pollinia, and in several genera a prominent column foot (Chase et al. 2003; Topik et al. 2005). Attempts to clarify some of the relationships between genera within the Aeridinae have been made most notably by Christenson (1985, 1986,

1987a, 1987b, 1992, 1994) with a more ‘classical’ approach using floral, and to a lesser degree also non- reproductive characters. In contrast Senghas (1988), and Seidenfaden (1988) based their classification on pollinaria and column foot characters when publishing new species, combinations and genera, and writing floristic accounts. Christenson (1994) wrote, of the great range of morphologies exhibited by the subtribe, that there is ‘far too much research needed to understand the species and therefore the generic limits of the group’.

In the only subtribe-wide molecular systematic study based on matK and ITS data, Topik et al. (2005) stated that ‘morphological diversification and possible parallelism of vegetative and reproductive characters’, has probably been a significant contributor to the difficulty of resolving relationships within the group. In their study they included only Vanda coerulea – a species that is indisputably part of Vanda – but found a close relationship with , Christensonia, Trudelia, and Neofinetia. A number of recent molecular phylogenetic studies have been published on groups of species/genera in the Aeridinae: the genus Aerides (Kocyan et al.

2008), Holcoglossum (Fan et al. 2009), leafless vandoids (Carlsward et al. 2006), and (Padolina et al. 2005, Tsai et al. 2006, 2010) but none have to date concentrated specifically on Vanda.

Gardiner, Motes, Emerson, Roberts, Kocyan 4 The placement of a number of species in relation to Vanda remains a matter of debate, with authors disagreeing on their correct classification. The genus Ascocentrum contains short-stemmed species, often with brightly coloured flowers (reds, oranges, purples) with quite equal and , a long cylindrical nectar- filled spur, small side lobes and the epichile being ligulate. None of the species have ever been placed within the genus Vanda, although they are commonly described as a ‘sister’ genus to Vanda. Ascocentrum species hybridise readily in cultivation with Vanda species and Vanda hybrids (Christenson 1987a; Motes 1997; Topik et al. 2005).

Kamemoto (1962) studied the chromosomal relationships among many of the taxa within the Vanda-Aerides alliance (sensu Christenson 1987), which includes Aerides, Ascocentrum, Holcoglossum, ,

Seidenfadenia and Vanda, and closely related genera such as Euanthe, Neofinetia, Phalaenopsis, Renanthera,

Trichoglottis and Arachnis. This work found little support for the genus Euanthe as distinct from Vanda, due to the perfect chromosomal pairing of E. sanderiana with Vanda species and hybrids, and the resulting hybrids exhibiting high fertility. Holttum (1958), on the other hand, considered Euanthe as morphologically very distinct from Vanda, with its distinctive lip shape, broad flat petals and sepals, and very large lateral sepals. He, however, recognised the ease with which species of Euanthe hybridise with species of Vanda and stated that it

‘may be a practical step to reunite it to Vanda’. The karyotypes of the strap-leaved Vanda species (at the time,

Holcoglossum and Papilionanthe were still considered to be part of Vanda, as the terete-leaved species) and

Ascocentrum have been shown to be extremely similar (Kamemoto 1962), with chromosomes similar in size and morphology pairing well during meiosis, as were those of N. falcata. The chromosomes of the terete-leaved

Vanda species do not pair well with those of the strap-leaved species, lending support to the exclusion of these taxa from the genus. A single RAPD study of a few vandaceous species has shown the single Ascocentrum species included and Euanthe sanderiana nesting within a clade of strap-leaved Vanda species, with the terete- leaved V. teres (now Papilionanthe teres) and V. hookeriana (now P. hookeriana) separated from this clade

(Lim et al. 1999).

The species Aerides flabellata Rolfe ex Downie (: Vanda flabellata (Rolfe ex Downie) Christenson) has been a focus of much taxonomic disagreement, placed in Aerides on account of characters such as a long Gardiner, Motes, Emerson, Roberts, Kocyan 5 column foot and motile lip by some (Seidenfaden 1988, 1992), and Vanda by others due to the species’ short spur and broad lip (Garay 1972; Seidenfaden 1973; Christenson 1985, 1986; Motes 1997; Kocyan et al. 2008).

Christenson (1994) treated the species as nomina incertae sedis, until further study could be made. The more recently described species, Christensonia vietnamica Haager, with morphological similarities to Vanda and

Rhynchostylis Blume (Christenson, 1994), has been affiliated with A. flabellata, being described as ‘almost a yellow Aerides flabellata’. Recent molecular studies including A. flabellata and C. vietnamica have supported their placement in Vanda (Topik et al. 2005; Carlsward et al. 2006; Kocyan et al. 2008, Fan et al. 2009).

In summary, relationships within and around Vanda remain unclear, in spite of the recent molecular studies of other groups within the Aeridinae and, as Christenson (1987a) stated, the genus is a ‘poorly known, imprecisely defined group held together by symplesiomorphies. Vanda requires a complete taxonomic revision’.

The main questions that this molecular study aimed to address were: (1) Does the genus Vanda, as currently circumscribed, form a monophyletic group? (2) What are the phylogenetic relationships of Vanda with related genera in the Aeridinae? (3) Which morphology-based sectional classifications of Vanda are supported by molecular data? This study sequenced three chloroplast DNA regions from 35 species of Vanda and 22 non-

Vanda species of Aeridinae, in order to answer these questions.

MATERIALS AND METHODS

TAXON SAMPLING

In total, 101 individuals were sampled (table 2), representing 35 Vanda species and 22 non-Vanda species from

14 Aeridinae genera (Aerides, Ascocentrum, Christensonia, Dimorphorchis, Holcoglossum, Neofinetia,

Papilionanthe, Phalaenopsis, Renanthera, Rhynchostylis, Sedirea, Seidenfadenia, Taprobanea, Vandopsis).

Due to the various difficulties involved in collecting wild material, specimens were obtained from cultivated species material from Botanic Gardens and specialist growers of vandaceous species. Multiple accessions of a single species were included, where possible. Based on previous molecular studies of the Aeridinae,

Phalaenopsis (represented by P. cornu-cervi (Breda) Blume & Rchb.f.) was chosen as the outgroup for the rest Gardiner, Motes, Emerson, Roberts, Kocyan 6 of the Aeridinae (Topik et al. 2005; Carlsward et al. 2006; Kocyan et al. 2008, Fan et al. 2009). Taxon names and authorities are based on the World Checklist of with some modification (Govaerts 2012).

The authors experienced difficulties in obtaining good quality specimens for sampling with reliable identifications and provenance, with material in botanic garden and private collections often being sterile, unvouchered and of dubious ancestry. With a genus as horticulturally important as Vanda, the potential for accidental hybridisation and introgression between cultivated species is high, especially if the differences between species are not well understood. During this study a number of specimens from cultivated sources were rejected on the basis of dubious genetic origin. Supposed ‘species’ material was shown in several instances to be unlikely to be so, after sequencing and incorporation into preliminary phylogenetic trees.

AMPLIFICATION AND SEQUENCING

DNA was extracted from flower or leaf material dried in silica gel, according to the guidelines of Chase and

Hills (1991), using either of two methods, the modified CTAB-chloroform protocol of Doyle and Doyle (1987), and the DNeasy™ mini kit (Qiagen) following the manufacturer’s recommended protocol.

Three chloroplast DNA regions were amplified using polymerase chain reaction (PCR) and the primers listed in table 3. The primers 19F (Gravendeel et al. 2001), trnK-2R (Johnson & Soltis 1994) and OM (Topik et al.

2005) were used to amplify a region (referred to here as ‘matK’) including the coding matK gene and parts of the neighbouring 5’ and 3’ trnK intron regions. The primers psbA (Sang et al 1997) and trnH (Tate and

Simpson 2003) were used to amplify the psbA-trnH region (referred to here as ‘psbA’), encompassing the non- coding intergenic region between the two genes coding for the trnH tRNA and the gene psbA. Primers c and f

(Taberlet et al 1991) were used to amplify the region between the trnL (UAA) 5’ exon and trnF (GAA). The amplicon includes parts of the trnL intron the trnL (UAA)3’ exon and most of the spacer between trnL (UAA)

3’ and trnF (GAA) 5’. For the sake of easier readability we refer this region in the ongoing text as ‘trnL-F’. The

PCR amplifications were carried out in 50µl volumes, using 0.2 µM of each primer, 5 µl NH4 buffer (1x),

MgCl2 (3mM for matK, 2.5mM for psbA and trnL-F), 10mM dNTPs, 0.2 µl Taq polymerase (Bioline, 1 unit), and approximately 20-70 ng DNA template. PCR amplification was carried out in a DNA Engine Tetrad 2, Gardiner, Motes, Emerson, Roberts, Kocyan 7 Peltier Thermal Cycler (MJ Research) using the following reaction profile: 95°C (2 mins for matK, 5 mins for psbA, 1 min for trnL-F); 35 cycles of 95°C for 30 seconds, 1 min (50°C for matK and trnL-F, 48°C for psbA), and 72°C for 1 min 40 seconds; and 72°C for 5 min. PCR amplified products were purified using QIAquick™

PCR Purification Kit (Qiagen) following the manufacturer’s recommended protocol.

Cycle sequencing reactions were performed using Big Dye v.3.1 terminator chemistry (PE Biosystems) for all three regions with the same primers used for amplification plus an additional primer to sequence the central region of matK (Topik et al. 2005). Sequencing reactions were carried out in 10 µl volumes, with with 2 µl Big

Dye v.3.1, 1 µl Big Dye buffer (5x), 1 µl primer (3.2 µM for matK and trnL-F, 10 µM for psbA), and 1 µl DNA template (approximately 20-25 ng). A DNA Engine Tetrad 2, Peltier Thermal Cycler (MJ Research) was used with the following sequencing reaction profile: 96°C for 1 min; 25 cycles of 96°C for 10 seconds, 50°C for 5 seconds, and 60°C for 4 mins. Sequences were obtained using an automated ABI 3700 capillary sequencer (PE

Biosystems). Sequences were assembled and edited using the program Chromas (Technelysium Pty Ltd).

PHYLOGENETIC ANALYSES

Four data sets were analysed: (1) matK alone, (2) psbA alone, (3) trnL-F alone, and (4) all three regions combined. Sequences were aligned using the Clustal X algorithm implemented in the program BioEdit (Hall

1999), followed by manual optimisation.

Phylogenetic analyses were conducted under maximum parsimony (MP) and maximum likelihood (ML) optimality criteria using PAUP 4.0a123 for Macintosh (Swofford, 2002) for parsimony reconstruction and

GARLI v.2.0 (Zwickl, 2006) for ML inference. Settings for the heuristic analysis were as follows: TBR branch swapping and MulTrees activated with 100 replicates of random sequence addition, saving one tree for each replicate. All the analyses interpreted gaps as missing data. Internal support was assessed by non-parametric bootstrapping (Felsenstein, 1985). Ten thousand replicates were performed to calculate parsimony bootstrap percentages with the following heuristic settings (Müller, 2005): a simple-taxon-addition tree as the starting point, TBR swapping active, steepest descent not in effect, and one tree held in memory (Mul-Trees inactivated). Nodes with bootstraps of 85% are considered strongly supported here, whereas 75–84% is Gardiner, Motes, Emerson, Roberts, Kocyan 8 moderately and 50–74% is weakly supported (Chase et al. 2006). The most appropriate model of molecular evolution for each partition was found using the internal model find algorithm of GARLI v2.0, which selected a

GTR+I+C model. The number of generations for the ML analysis was set at five million, with allowance for an automated stop when no new and significantly better topology was detected. A ML heuristic bootstrap analysis

(BPML) was performed with 100 replicates to obtain statistical support for nodes. The partition homogeneity test (incongruence length difference test, Farris et al. 1994, 1995) has been shown to give misleading results

(Dolphin et al. 2000, Lee 2001, Reeves et al. 2001, Yoder et al. 2001, Barker and Lutzoni 2002, Darlu and

Lecointre 2002) and so was not used to assess congruence among the data sets. Instead, the results of each analysis were scrutinised manually to identify well-supported, conflicting relationships (Weins 1998).

RESULTS matK, psbA and trnL-F DNA sequences

Edited DNA sequences of matK, psbA and trnL-F were highly variable in length. matK sequences ranged from

1554 bp (Rhynchostylis gigantea) to 1754 bp (Papilionanthe uniflora), with a mean length of 1714 bp. psbA varied from 693 bp (Sedirea japonica) to 784 bp (Vanda cristata), with a mean length of 757 bp. The trnL-F region varied from 370 bp (Vanda flavobrunnea) to 807 bp (Vandopsis gigantea), with a mean length of 781 bp. Many indels were introduced to the aligned sequence matrices for each dataset in order to accommodate this length variation. In some regions, DNA sequences could not be unambiguously aligned, particularly due to short repeated sequences, and these regions were removed from the analyses of the matK, psbA, and trnL-F datasets (37 bp, 409 bp, and 597 bp), respectively. The matK alignment used for analysis comprised 1797 bp, containing 205 variable positions, 94 of which were parsimony-informative characters. The psbA alignment used for analysis comprised 839 bp, containing 46 variable positions, of which 22 were parsimony-informative characters. The trnL-F alignment used for analysis comprised 880 bp, containing 109 variable positions, of which 54 were parsimony-informative characters. The combined alignment, containing all three regions, comprised 3508 bp, containing 360 variable positions, 154 of which were parsimony-informative characters. In the matK and trnL-F sequences short (3 bp) and to medium sized (70 bp; Kocyan et al. 2008) inversions were Gardiner, Motes, Emerson, Roberts, Kocyan 9 detected. These were either excluded from the analyses, or reverse complemented for use in the tree calculations.

Phylogenetic analyses

The phylogenetic trees generated in the combined (total) evidence majority rule analysis showed a much higher level of resolution and bootstrap support values for clades than in the individual gene region. The trnL-F analyses are the most resolved of the separate datasets, with a number of well-supported clades in the majority rule phylogenetic trees produced. Those well-supported clades shown in the separate plastid majority rule trees were almost entirely congruent with those resolved in the combined total evidence trees (the few instances where additional or different clades were resolved with >50% bootstrap support are noted below, in the results section). Due to the overall congruence between analyses and the much higher resolution and bootstrap support values recovered in the combined evidence analyses, in accordance with Bone et al. (2012), we present only the majority rule phylogenetic trees resulting from the combined total evidence MP and ML analyses, annotated with bootstrap support values >50% consistent with these topologies,.

In the combined analysis of all three chloroplast regions there is excellent bootstrap support (93% MP, 98%

ML) for a Vanda sensu lato clade, to include Ascocentrum, Christensonia and Neofinetia. In the separate plastid analyses the Vanda s.l. clade was not resolved with bootstrap support >50% with either the matK or psbA datasets. With the trnL-F dataset, the clade was resolved with high support (91% and 90%) in the MP and ML analyses, respectively. The Vanda s.l. clade plus the three Holcoglossum species included in the analyses form a clade with 71% bootstrap support in the MP analysis, and 84% support in the ML analysis. The three

Holcoglossum species included in the analysis do not form a monophyletic group, but appear to be paraphyletic to the Vanda s.l. clade, as the branches separating them have no significant level of bootstrap support (i.e.

<50%).

Within the Vanda s.l. clade, resolution of the ‘backbone’ is extremely poor, with a large polytomy separating lower level clades. Most of these lower level clades receive moderate to high levels of bootstrap support and are considered individually here. The three Ascocentrum species included in the dataset do not form a Gardiner, Motes, Emerson, Roberts, Kocyan 10 monophyletic group in any of the analyses. A. ampullaceum and A. christensonianum group with high to good support of 80% and 74% in the MP and ML combined analyses (64% and 66% in the trnL-F analyses), but A. curvifolium forms a well-supported clade with the type species for Vanda, V. tessellata, and V. cristata and V. flavobrunnea (species formerly placed in their own genus, Trudelia) with 70% and 75% support (both

56% in the trnL-F analyses). These species, (V. tessellata, V. cristata and V. flavobrunnea, form a clade of their own with moderate support, 58% and 63%. A highly supported Philippines clade of V. lamellata, V. roeblingiana and V. sanderiana is resolved in the combined analyses with 98% and 97% support, and also in the trnL-F analyses (with 86% and 75% support) and 54% support in the psbA ML analysis. Christensonia vietnamica, V. flabellata, and V. lilacina form a moderately supported clade, with 63% and 59%, 62% and 61% in the trnL-F analyses (the first two form a clade with 71% and 57% support in the combined analyses, 62% and 54% in the trnL-F analyses). V. brunnea and V. denisoniana form a highly supported group with 96% support in both the MP and ML combined analyses, 96% and 91% in the matK analyses, 64% and 57% in the psbA analyses. V. coerulea and V. coerulescens form a moderately well supported group with 80% and 70% support in the combined analyses, rising to 96% and 95% in the matK analyses. V. bensonii, V. liouvillei and V. testacea form a moderately to highly supported clade with 82% and 89% support in the combined analyses,

62% and 52% in the matK analyses.

The combined analyses resolve a large, moderately to highly supported, ‘South East Asian archipelago’ clade with 82% and 89% support, comprising V. arcuata, V. celebica, V. dearei, V. devoogtii, V. furva, V. hastifera,

V. helvola, V. hindsii, V. insignis, V. jennae, V. limbata, V. lindenii, V. luzonica, V. merrillii, V. metusalae, V. perplexa (ined.), V. sumatrana, V. tricolor, and V. ustii. Within this clade, most relationships between taxa are unresolved, however several clades receive moderate to high support. V.limbata and V.perplexa form a highly supported clade with 95% and 98% support in the combined analyses, 63% and 67% in the matK analyses, 64% and 68% in the psbA analyses, 64% and 62% in the trnL-F analyses. V. celebica, V.furva and V. lindenii form a moderately supported clade with 63% and 68% support, 64% and 58% in the trnL-F analyses. V. tricolor and V. ustii received 63% and 69% support, 63% and 65% in the matK analyses. A moderately well supported smaller clade, comprising V. arcuata, V. dearei, V. devoogtii, V. helvola, V. hindsii, V. insignis, V. jennae, V. luzonica, Gardiner, Motes, Emerson, Roberts, Kocyan 11 V. merrillii, V. metusalae, V. sumatrana, V. tricolor and V. ustii is resolved in the combined analyses, with

63% and 59% support.

In the separate analyses, a few additional clades are resolved which are not seen in the combined analyses. In the psbA MP analysis, V. coerulea and one of the three V. coerulescens sequences group with V. ustii with 64% support. In the trnL-F MP analysis, A. ampullaceum, A. christensonianum and V. denisoniana, group with 54% support, and in this same analysis and in the trnL-F ML analysis, V. bensonii, two of the three V. coerulescens sequences, V. liouvillei, V. parviflora, and V. testacea form a group with 87% and 74% support. Outside of the

Vanda s.l. and Holcoglossum clade, the three Papilionanthe species form a clade with 100% support, the two

Aerides species with 88%, the two Renanthera species with 93%, and a clade of Aerides, Renanthera and

Taprobanea with 89%.

DISCUSSION

MOLECULAR CHARACTERISTICS AND INFORMATION CONTENT OF THE VANDA DATA

The molecular data obtained contained a relatively low content of phylogenetic information. Data from other

Aeridinae related studies included in matK 94 PICs (parsimony informative characters) in Aerides (Kocyan et al. 2008) whereas only 80 were found for Vanda (the aligned length of the compared data is shorter in the

Aerides study, hence the compared segment was shortened in Vanda in order to compare the two matrices therefore the number of PICs mentioned here is different from that mentioned in the results section of this paper) and in the overall Aeridinae study by Topik et al. (2006) more than 400 PICs were detected. In trnL-F almost double the information content was found in Aerides (98 versus 54 PICs). In addition, the often long and hypervariable P8 region in the trnL intron is inconspicuous in Vanda, which contrasts the situation of Aerides and other angiosperms which often exhibit extreme length variability in this region (Borsch et al. 2003, Kocyan et al. 2008). Another feature of the trnL intron is the relatively frequent occurrence of a 70 base pair long inversion in the P8 region. In Aeridinae this inversion appears in several unrelated genera and this region must either be reverse complemented or excluded from analysis otherwise tree search algorithms would result in Gardiner, Motes, Emerson, Roberts, Kocyan 12 wrong phylogenetic reconstructions (Kocyan et al. 2008; Kocyan unpubl.). However, in the dataset presented here, the inversion was only detected within the Seidenfadenia mitrata accession was, and within Vanda s.l. no such peculiarity was found.

RELATIONSHIPS OF VANDA AND ASCOCENTRUM TO OTHER AERIDINAE GENERA

Our analyses consistently resolve a Vanda s.l. clade with very high levels of support, to include Ascocentrum,

Neofinetia, and the monotypic Christensonia. Recently, in as yet unpublished analyses to be incorporated into volume 6 of the Genera Orchidacearum series (Pridgeon et al. in prep.), Kocyan has produced the most complete phylogeny of the Aeridinae to date, comprising 199 species in 98 genera, and using three plastid DNA regions and one nuclear region (Kocyan unpubl.). In this phylogeny, Kocyan has shown that Vanda,

Ascocentrum, and Christensonia form a highly supported clade (99% bootstrap support), and the larger grouping of this clade plus Ascocentropsis, Eparmatostigma and Neofinetia, similarly receives high support

(99% bootstrap support). In neither piece of research does Vanda s.l. clearly fall into well-supported clades corresponding with any of the existing genera with more than one species. As a result, we support the inclusion of the genera Ascocentrum and Neofinetia into Vanda. The retention of Ascocentropsis, Christensonia and/or

Eparmatostigma would require the splitting of Vanda. This cannot be done with any logic or consistency on the basis of these results. Instead, the most practical solution is to include these small genera into Vanda and publish the appropriate new combinations of names in Vanda (those of which have not already been made by previous authors, (Gardiner 2012, 2013)).

Although only three of eight species of the genus Ascocentrum were included in the analyses, the genus was not resolved as monophyletic in any of the analyses performed. Non-parametric bootstrapping of a smaller earlier

Vanda s.l. dataset (Gardiner 2008) could not provide support for the hypothesis of the monophyly of

Ascocentrum, and in spite of distinct morphological characters for these species, which supports their maintenance as a separate genus, it appears that the genus as it stands is paraphyletic and the species should be incorporated into the wider concept of the genus Vanda.

Gardiner, Motes, Emerson, Roberts, Kocyan 13 The genus Holcoglossum is shown here to be the most likely sister genus to Vanda s.l. although, with limited sampling of the genus, the three species included appear to be paraphyletic to the Vanda s.l. clade. Potentially the genus Holcoglossum should also be incorporated into Vanda s.l., however in the absence of additional taxa, we prefer to leave this morphologically distinctive genus excluded for the time being, with its semi-terete leaves and floral column foot, especially as it does not appear to nest within Vanda s.l. The unpublished results of Kocyan also reveal Holcoglossum to be the sister genus to Vanda s.l., although in these analyses the genus is resolved as a monophyletic sister group, rather than being paraphyletic to Vanda s.l.. Similarly, Fan et al.

(2009) found a sister relationship between Holcoglossum and a clade comprising four species of Vanda,

Aerides flabellata, Ascocentrum ampullaceum and Neofinetia falcata. The monotypic Taprobanea spathulata, previously placed in Vanda by Sprengel (1826), is shown in all analyses to fall well outside of the Vanda s.l. clade and is not considered to be a member of, or closely related to, the genus.

RELATIONSHIPS WITHIN VANDA AND BETWEEN SECTIONS

Within the genus Vanda s.l., although the main ‘backbone’ of the genus is unresolved, there are a number of lower level clades that receive moderate to high levels of bootstrap support. These clades are not congruent with existing morphological classifications of Vanda, but morphological characters can be attributed to most, in line with a proposed new classification of Vanda being prepared by Motes et al. (in prep.). The morphological sections proposed by Motes are shown in table 4 and are represented on the phylogenetic tree shown in figure

3. Furthermore, there is biogeographic signal in our data. The geographic distributions of the taxa represented in the phylogenetic tree are shown in Fig. 4, where we can see a well-supported group of species from mainly the Philippines that represent section Roeblingiana (V. barnesii, V. javierae, V. lamellata, V. roeblingiana, and

V. sanderiana), and another well-supported, but unresolved, group of many south-east Asian archipelago species, with short branch lengths separating the species (section Deltaglossa/Hastifera). The remaining groups of species are distributed in mainland Asia and Indochina.

POLLINATION AND SPECIATION AMONG VANDA S.L.

As in most epiphytic orchids there is an almost complete lack of pollination and pollinator information for the now extended concept of Vanda. Many Vanda species with large, showy, colourful flowers, often with open, Gardiner, Motes, Emerson, Roberts, Kocyan 14 spreading tepals, are visited by bees and butterflies in cultivation, and Holttum (1953) concluded that

‘typical’ Vanda species are pollinated by bees. Melitophily (bee pollination) and cantharophily (beetle pollination) seem to be the only pollination systems for which we have a few reports. Van der Pijl and Dodson

(1966) reported dead Xylocopa bees with pollinia attached to their heads in Vanda teres flowers and an odour bouquet that fits Xylocopa attraction. Pradhan (1983) observed beetles as efficient pollinators on Vanda cristata, and beetles may also pollinate V. denisoniana Benson & Rchb.f. (Christenson 1992). However, using the concept of floral syndromes on the now extended concept of the genus Vanda we may be able to distinguish four groups of animals as putative pollinators. Bees, in particular Xylocopa bees, may play an important role in pollination for most of what was previously considered to be Vanda s.s. and Christensonia; beetles for the species formerly separated into the genus Trudelia; bird pollination may be likely in Ascocentrum due to the red-orange colouring of the flowers and short, nectariferous spurs (one report exists of an Ascocentrum hybrid being pollinated by a honey-eater bird in cultivation (van der Cingel 2001)), and A. ampullaceum is seen to have violet coloured pollinia which is thought to be attractive to birds, although butterfly pollination is also possible; Ascocentropsis appears to exhibit the same pollination syndromes as Ascocentrum. The night-scented white flowers of Neofinetia species are likely visited by night-active long tongued moths searching for nectar in the long, narrow spurs of the flowers. Eparmatostigma also has small white flowers with distinct spurs that contain nectar (Kocyan pers. obs.) and pollination may be by small bees or possibly butterflies. The inclusion of the florally distinct genera Eparmatostigma and Neofinetia into Vanda s.l. supports the theory of pollinator- driven selection of floral forms allowing a relatively easy switch between pollination strategies (Kocyan et al.

2008, Sapir and Armbruster 2010). Moreover, it is unknown whether or not most taxa are nectariferous and it is possible that a substantial proportion of the species in Vanda s.l. are nectarless, as pollinator deceit seems to be one of the key factors in orchid speciation (Cozzolino and Widmer 2005).

Molecular data suggests that the taxa in Vanda s.l. exhibit remarkably low genetic divergence from each other, in spite of the dramatic morphological variation seen across the genus and the broad geographic range of Vanda s.l.. Low genetic diversity may be an indication for a relative recent origin of the group under investigation

(Bateman 1999), and as such Vanda may offer an interesting example of relatively rapid speciation and morphological diversification in response to pollination syndromes and allopatry within insular environments – Gardiner, Motes, Emerson, Roberts, Kocyan 15 particularly in the south-east Asian archipelago, where morphological diversity appears to be at its highest, and yet genetic divergence is at its lowest. The history of sea levels during the Pleistocene is well documented

(Voris 2000) and it is possible that during these relatively recent events some species became locally extinct with others produced through speciation processes. A series of Pleistocene low sea stands caused by glaciations resulted in a continuous landmass from Borneo to Malaysia- (Sundaland), and may help to explain the distributions patterns of certain taxa. For example V. helvola occurs in Borneo and Peninsular Malaysia, whereas other species from the same section (section Hastifera, e.g. V. dearei) occur exclusively on Borneo.

The first species may have its origin before the drought events whereas the latter could have originated in

Borneo after the isolation of the island.

This study used only plastid data from the taxa sampled, and unfortunately it was not possible to consistently isolate a nuclear marker for the taxa studied in this paper and resources did not allow for extensive cloning of

ITS paralogues. The few published studies on South East Asian plants that have utilised plastid and nuclear

DNA regions, and that were recovered tree topologies with substantial differences between the two sequence types, found a strong biogeographic signal in plastid data whereas nuclear data appeared to match morphological characters in the taxa more accurately (e.g. Nauheimer et al. 2012).

CONCLUSIONS AND FUTURE DIRECTIONS

It is clear from this study that Vanda s.l. remains an enigmatic genus, with unresolved relationships between its species, and a number of small genera having been incorporated from around its periphery. Plastid data alone results in relatively poor resolution within the genus, and is insufficiently informative to clearly resolve the backbone of the genus and be able to interpret the phylogeny along biogeographic lines. Nonetheless the plastid data is sufficiently powerful to show the monophyly of this expanded concept of Vanda, and to support the inclusion of Ascocentrum, Christensonia, Euanthe, Neofinetia and Trudelia into it, and to support

Holcoglossum as sister genus to Vanda. A reliably amplified, low-copy number, phylogenetically informative nuclear marker would be highly likely to add clarity to these results. Concerted efforts to amplify ITS sequences (the nuclear marker most commonly used in orchid phylogenetics) from Vanda species have been Gardiner, Motes, Emerson, Roberts, Kocyan 16 hampered in the past by the presence of multiple copies of the region, necessitating cloning of samples and complicating the interpretation of results obtained. The multiple copy problem of ITS may be due to the well- reported and discussed incomplete-concerted evolution tendency of the region (e.g. Xiao et al. 2010), but it also could be influenced by the exceptional variation of chromosome numbers among Vanda s.s. and therefore different ploidy levels; the basic chromosome number of Aeridinae is x = 19, which is relatively stable for the majority of the subtribe but in Vanda s.s. published reports are 19, 38 and 57 (Felix and Guerra 2010). Recent innovations in using nuclear regions such as the low-copy nuclear Xdh gene in orchid phylogenetics do offer promising prospects of furthering this research (Górniak et al. 2010, Kocyan unpubl.). Comparing the tree structure of plastid and nuclear data would also allow the search for topological conflicts between datasets and possibly answer questions on the reported hybrid origin of certain taxa and along with that raising questions about chloroplast capture and introgression in the genus Vanda as recently applied in the genus Alocasia,

Araceae (Nauheimer et al. 2012). In addition, with a more resolved phylogeny of this group, and with the recent re-evaluation of the age of the Orchidaceae as a family and increasingly reliable geological reconstructions of the main diversity centres in South East Asia, an estimation of the genus’ age should be possible and assessed in relation to the biogeographic distributions of the species and land changes at the time of their evolution (Hall 2001, Gustaffson et al. 2010).

Traditional sectional delimitation has influenced the sampling scheme of the research presented here, however our results do not support these sections, which has unfortunately had an effect on a rather uneven sampling strategy. More effort should be expended on ensuring that all of the newly proposed morphological sections are well sampled in any future molecular work on this group of taxa, as well including as many of the taxa formerly classified in separate genera as possible – Ascocentropsis, Ascocentrum, Eparmatostigma, and Neofinetia. It is hoped that future sampling will concentrate on incorporating more taxa from the under-represented groups in order to better elucidate their true relationships to each other.

ACKNOWLEDGEMENTS

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FIGURE LEGENDS

Figure 1: Maximum Parsimony majority rule tree of the combined analysis, showing bootstrap values over 50%

Figure 2: Maximum Likelihood majority rule tree of the combined analysis, showing bootstrap values over

50%

Figure 3: Simplified maximum likelihood tree of the combined analysis, showing the proposed morphological sections in Vanda

TABLES

Table 1: Genera closely related to Vanda within the Aeridinae, historically and determined in recent morphological (Christenson 1987a, 1987b, 1994) and molecular (Topik et al. 2005, and Kocyan et al. 2008) studies

Table 2: List of species names, geographic range, voucher specimens, and NCBI accession numbers for taxa used in this study Gardiner, Motes, Emerson, Roberts, Kocyan 23 Table 3: Primer sequences used to amplify and sequence matK, psbA, and trnL-F regions

Table 4: Overview of the morphological sections in Vanda proposed by Motes et al. (in prep.) Gardiner, Motes, Emerson, Roberts, Kocyan 24 Table 1: Genera closely related to Vanda within the Aeridinae, historically and determined in recent

morphological (Christenson 1987a, 1987b, 1994) and molecular (Topik et al. 2005, and Kocyan et al. 2008)

studies

Lindley (1853) Christenson (1994) Topik et al. (2005) Kocyan et al. (2008)

Vanda ‘Aerides-Vanda alliance’ ‘Aerides alliance’

Aerides Lour. Aerides Aerides

Ascocentrum Schltr. Ascocentrum Ascocentrum

Christensonia Haager Christensonia Christensonia

Holcoglossum Schltr. Holcoglossum

Neofinetia Hu Neofinetia Neofinetia

Papilionanthe Schltr. Papilionanthe

Rhynchostylis Blume Rhynchostylis included as Vanda section

Anota

Seidenfadenia Garay Seidenfadenia Seidenfadenia

Vanda Jones ex R.Br. Vanda Vanda Vanda

Trudelia Garay Trudelia included as Trudelia included as

Vanda section Cristatae Vanda section Cristatae

Vandopsis Pfitzer and

Dimorphorchis Rolfe included as Vanda section

Fieldia

Gardiner, Motes, Emerson, Roberts, Kocyan 25 Table 2: List of species names, geographic range, voucher specimens, and NCBI accession numbers for taxa used in this study

Species Geographic range Reference Voucher/Herbarium matK psbA trnL-F number in phylogeny Vanda arcuata J.J.Sm. 0-5406 (SBG)

Vanda bensonii Batem. Assam, Burma, Thailand 1 K LCD 12/2004-4

Vanda bensonii Batem. 2 Swansea Botanical Complex

S19920413

Vanda brunnea Rchb.f. China, Burma, Thailand, Luxembourg Botanic Garden,

Vietnam LUX2001082

Vanda celebica Rolfe Sulawesi Gardiner, 131 (K)

Vanda coerulea Griff. ex. Lindl. China, Assam, East 1 Gardiner, 121 (K)

Vanda coerulea Griff. ex. Lindl. , Burma, Thailand 2 Chase 22871 (K)

Vanda coerulea Griff. ex. Lindl. 3 Luxembourg Botanic Garden,

LUX2002050

Vanda coerulescens Griff. China, Assam, East 1 Gardiner, 120 (K)

Vanda coerulescens Griff. Himalayas, Burma, Thailand 2 OR-303-1999 (Salzburg)

Vanda coerulescens Griff. 3 Lyon Botanic Garden 900488

Vanda cristata Wall. ex. Lindl. China, Assam, Bangladesh, 1 Roberts, D.L., s.n. (K)

Vanda cristata Wall. ex. Lindl. India, Nepal, Burma, 2 Lyon Botanic Garden

Vanda dearei Rchb.f. Borneo, Gardiner, 112 (K)

Vanda denisoniana Benson & Rchb.f. , Burma, Thailand, 1 Gardiner, 10 (FLAS) (K)

Vanda denisoniana Benson & Rchb.f. Vietnam 2 Gardiner, 25 (FLAS) (K) Gardiner, Motes, Emerson, Roberts, Kocyan 26 Vanda denisoniana Benson & Rchb.f. 3 Gardiner, 27 (FLAS) (K)

Vanda denisoniana Benson & Rchb.f. 4 Lyon Botanic Garden 980277

Vanda denisoniana Benson & Rchb.f. 5 Luxembourg Botanic Garden 1995-01

Vanda devoogtii J.J.Sm. Sulawesi Gardiner, 135 (K)

Vanda flabellata (Rolfe ex Downie) , Laos, , 1 BG Basel 1039/97 (Z) EF655786 EF670410

Christenson Thailand

Vanda flabellata (Rolfe ex Downie) 2 K LCD 12/04

Christenson

Vanda flabellata (Rolfe ex Downie) 3 Roberts, 2004-739 (K)

Christenson

Vanda flavobrunnea Rchb.f. China, Assam, Nepal, check - s.n. (Z) EF655802 EF670416

Himalayas, Lao, Burma,

Thailand, Vietnam, Sumatra

V. furva (L.) Lindl. Moluccas Gardiner, 204 (K)

Vanda hastifera Rchb.f. Borneo Gardiner, 101 (K)

Vanda helvola Blume Borneo, Java, Malaysia, 1 Gardiner, 145 (K)

Vanda helvola Blume Sumatra, New Guinea 2 Gardiner, 206 (K)

Vanda helvola Blume 3 Gardiner, 116 (K)

Vanda hindsii Lindl. New Guinea, Solomon Islands, 1 Gardiner, 103 (K)

Vanda hindsii Lindl. Queensland 2 Gardiner, 53 (FLAS) (K)

Vanda hindsii Lindl. 3 Gardiner, 62 (FLAS) (K) Gardiner, Motes, Emerson, Roberts, Kocyan 27 Vanda hindsii Lindl. 4 Hortus Botanicus Leiden 20030985

Vanda insignis Blume Lesser Sunda Islands Gardiner, 180 (K)

Vanda jennae O’Bryne & Verm. Sulawesi 0-5665 (SBG) (K)

Vanda lamellata Lindl. Japan, Taiwan, Borneo, Gardiner, 59 (FLAS) (K)

Philippines, Marianas

Vanda lamellata var. remediosae Philippines 1 Gardiner, 6 (FLAS) (K)

Ames & Quisumb.

Vanda lamellata var. remediosae 2 Gardiner, 18 (FLAS) (K)

Ames & Quisumb.

Vanda lamellata var. boxallii Rchb.f. Philippines Gardiner, 44 (FLAS) (K)

Vanda lilacina Teijsm. & Binn. China, , Laos, 1 Gardiner, 60 (FLAS) (K)

Vanda lilacina Teijsm. & Binn. Burma, Thailand, Vietnam 2 Lyon Botanic Garden 900516

Vanda limbata Blume Java, Lesser Sunda Islands? 1 Gardiner, 161 (sterile photo) (K)

Vanda limbata Blume 2 Gardiner, 149 (K)

Vanda limbata Blume 3 Gardiner, 61 (K)

Vanda lindenii Blume New Guinea 1 Gardiner, 240 (K)

Vanda lindenii Blume 2 Gardiner, 110 (K)

Vanda liouvillei Finet. Assam, Laos, Burma, 1 Gardiner, 30 (FLAS) (K)

Vanda liouvillei Finet. Thailand, Vietnam 2 Gardiner, 31 (FLAS) (K)

Vanda luzonica Loher ex Rolfe Philippines 1 Gardiner, 107 (K)

Vanda luzonica Loher ex Rolfe 2 Gardiner, 9 (FLAS) (K)

Vanda merrillii Ames & Quisumb. Philippines 1 Gardiner, 126a (K) Gardiner, Motes, Emerson, Roberts, Kocyan 28 Vanda merrillii Ames & Quisumb. 2 Lyon Botanic Garden 012660

Vanda metusalae O'Byrne Sulawesi 0-4647 (SBG)

Vanda parviflora Lindl. Assam, India, Nepal, Sri Gardiner, 24 (FLAS) (K)

Lanka, Himalayas, Burma,

Thailand

Vanda perplexa Roberts, Gardiner & Lesser Sunda Islands Gardiner, 12 (FLAS)

Motes

Vanda roeblingiana Rolfe Philippines Gardiner, 124 (K)

Vanda sanderiana (Rchb.f.) Rchb.f. Philippines 1 Gardiner, 123 (K)

Vanda sanderiana (Rchb.f.) Rchb.f. 2 Gardiner, 115 (K)

Vanda sanderiana (Rchb.f.) Rchb.f. 3 K LCD 12/04

Vanda sanderiana (Rchb.f.) Rchb.f. 4 Lyon Botanic Garden 000604

Vanda sumatrana Schltr. Sumatra 1 Gardiner, 126b (K)

Vanda sumatrana Schltr. 2 Gardiner, 106 (K)

Vanda tessellata (Roxb.) W.J.Hook. ex China, Assam, Bangladesh, 1 Gardiner, 56 (FLAS) (K)

G.Don India, Nepal, Sri Lanka,

Vanda tessellata (Roxb.) W.J.Hook. ex Himalayas, Burma 2 Gardiner, 241 (K)

G.Don

Vanda tessellata (Roxb.) W.J.Hook. ex 3 Gardiner, 11 (K)

G.Don

Vanda tessellata (Roxb.) W.J.Hook. ex 4 Gardiner, 57 (K)

G.Don Gardiner, Motes, Emerson, Roberts, Kocyan 29 Vanda tessellata (Roxb.) W.J.Hook. ex 5 Gardiner, 43 (K)

G.Don

Vanda tessellata (Roxb.) W.J.Hook. ex 6 Chase, 17946 (K)

G.Don

Vanda tricolor Lindl. Java, Lesser Sunda Islands 1 913067 (L) EF655777 EF670422

Vanda tricolor Lindl. 2 Chase 17970 (K)

Vanda tricolor var. suavis (Lindl.) Java, Lesser Sunda Islands 1 OR-310-1999 (Salzburg)

Rchb.f. in W.G.Walpers

Vanda tricolor var. suavis (Lindl.) 2 Chase, 19069 (K)

Rchb.f. in W.G.Walpers

Vanda tricolor var. suavis (Lindl.) 3 Gardiner, 55 (FLAS) (K)

Rchb.f. in W.G.Walpers

Vanda tricolor var. suavis (Lindl.) 4 Chase, 17971 (K)

Rchb.f. in W.G.Walpers

Vanda ustii Golamco, Claustro & de Philippines Gardiner, 122 (K)

Mesa

Aerides odorata Lour. China, Assam, Bangladesh, Chase, 15081 (K)

India, Nepal, Himalayas,

Andaman Islands, Laos,

Burma, Thailand, Vietnam,

Borneo, Java, Lesser Sunda

Islands, Malaysia, Philippines, Gardiner, Motes, Emerson, Roberts, Kocyan 30 Sulawesi, Sumatra

Aerides rosea Lodd. ex Lindl. & China, Assam, East Gardiner, 32 (FLAS)

Paxton Himalayas, Laos, Burma,

Thailand, Vietnam

Ascocentrum ampullaceum (Roxb.) China, Assam, Bangladesh, Gardiner, 23 (FLAS)

Schltr. Nepal, Himalayas, Andaman

Islands, Laos, Burma,

Thailand, Vietnam

Ascocentrum christensonianum Vietnam Chase, 15795 (K)

Haager

Ascocentrum curvifolium (Lindl.) Laos, Burma, Thailand, 1 Gardiner, 20 (FLAS)

Schltr. Vietnam

Ascocentrum curvifolium (Lindl.) 2 BG Basel 1446/99WP (Z) EF655789 EF670423

Schltr.

Ascocentrum curvifolium (Lindl.) 3 Gardiner, 16 (FLAS)

Schltr.

Christensonia vietnamica Haager Vietnam 1 200 101 53 (L) EF655803 EF670413

Christensonia vietnamica Haager 2 Chase, 17943 (K)

Dimorphorchis graciliscapa (A.L. Lamb Borneo BG Heidelberg 122351

& Shim) P.J. Cribb

Holcoglossum flavescens (Schltr.) China Roberts, s.n. (K)

Z.H.Tsi Gardiner, Motes, Emerson, Roberts, Kocyan 31 Holcoglossum kimballianum (Rchb.f.) Yunnan, Laos, Burma, AK010825/1/01 (Z) EF655783 EF670419

Garay Thailand

Holcoglossum subulifolium (Rchb.f.) Hainan, Burma, Thailand, Munich, s.n. (M) EF670409

Christenson Vietnam

Neofinetia falcata (Thunb.) Hu China, Japan, Korea AK010824/1/01 (Z) EF655782 EF67021

Papilionanthe hookeriana (Rchb.f.) Thailand, Vietnam, Borneo, Gardiner, 54 (K)

Schltr. Malaysia, Sumatra

Papilionanthe teres (Rchb.f.) Schltr. China, Assam, Bangladesh, Gardiner, 52 (K)

East Himalayas, India, Nepal,

Andaman Islands, Laos,

Burma, Nicobar Islands,

Thailand, Vietnam

Papilionanthe uniflora (Lindl.) Garay Assam, East Himalayas, Nepal Chase, O-1489 (K)

Phalaenopsis cornu-cervi (Breda) Bangladesh, Laos, Burma, Chase, O-1356 (K)

Blume & Rchb.f. Nicobar Islands, Thailand,

Vietnam, Borneo, Java,

Malaysia, Philippines, Sumatra

Renanthera isosepala Holttum Thailand, Borneo Gardiner, 33 (K)

Renanthera storiei Rchb.f. Philippines Gardiner, 37 (K)

Rhynchostylis gigantea (Lindl.) Ridl. China, Cambodia, Laos, Leiden cult. 913013 (L)

Burma, Thailand, Vietnam,

Borneo, Malaysia, Philippines Gardiner, Motes, Emerson, Roberts, Kocyan 32 Sedirea japonica (Rchb.f.) Garay & Japan, Korea s.n. (Z) EF655794 EF670433

H.R.Sweet

Seidenfadenia mitrata (Lindl.) Holttum Burma Check with Alex EF655795 EF670414

Taprobanea spathulata (L.) India, Sri Lanka Gardiner, 108 (K)

Christenson

Vandopsis gigantea (Lindl.) Pfitzer China,Bangladesh, Laos, GT5512 (C)

Burma, Thailand, Vietnam,

Malaysia

Gardiner, Motes, Emerson, Roberts, Kocyan 33 Table 3: Primer sequences used to amplify and sequence matK, psbA, and trnL-F regions

Region Primer Sequence Author

matK 19F CGT TCT GAC CAT ATT GCA CTA TG Gravendeel et al. 2001

trnK-2R AAC TAG TCG GAT GGA GTA G Johnson & Soltis 1994

OM CAG AAT TTA CGA TCT ATT CAT Topik et al. 2005

psbA- psbA GTT ATG CAT GAA CGT AAT GCT C Sang et al. 1997

trnH trnH CGC GCA TGG TGG ATT CAC AAT CC Tate & Simpson 2003

trn L-F c CGA AAT CGG TAG ACG CTA CG Taberlet et al. 1991

f ATT TGA ACT GGT GAC ACG AG Taberlet et al. 1991

Table 4: Overview of the morphological sections in Vanda proposed by Motes et al. (in prep.)

Proposed Species Key morphological Geographic section/group characters distribution

Vanda Vanda bidupensis Aver. & Christenson • Small side lobes Mainland Asia and

9 species V. concolor Blume on labellum, adjacent islands, Sri

V. fuscoviridis Lindl. sometimes Lanka, Indian

V. motesiana Choltco pointed in shape subcontinent,

V. stangeana Rchb.f. • Fleshy labellum Himalayas, southern

V. subconcolor Tang & F.T.Wang • Rounded column China, Indochina

V. tessellate (Roxb.) W.J.Hook. ex G.Don

V. thwaitesii Hook.f. in H.Trimen

V. wightii Rchb.f. in W.G.Walpers

Cristatae V. alpina (Lindl.) Lindl. • Fleshy labellum Himalayas, one

6 species V. chlorosantha (Garay) Christenson • Labellum with species in Indochina

V. cristata Wall. ex. Lindl. minimal spur

V. flavobrunnea Rchb.f. • Tepals yellow- Gardiner, Motes, Emerson, Roberts, Kocyan 34 V. griffithii Lindl. green in colour

V.longitepala D.L.Roberts, L.M.Gardiner • Often with deep

& Motes red/purple

markings on

labellum

Coerulea V. coerulea Griff. ex. Lindl. • Blue colouration Himalayas, India,

2 species V.coerulescens Griff. to tepals and Nepal, , China,

labellum Indochina

• Long flower

spikes,

sometimes

branched

Testacea V. bensonii Batem. • Compact plants Indian subcontinent,

4 species V. liouvillei Finet. • Distinctly Indochina

V. parviflora Lindl. spurred labellum

V. testacea (Lindl.) Rchb.f.

Obtusiloba V. bicolor Griff. • Orbicular side Indochina and

4 species V. brunnea Rchb.f. lobes on adjacent Himalayas

V. denisoniana Benson & Rchb.f. labellum

V. vipanii Rchb.f. • Large, fleshy

labellum

• Labellum often

pandurate

• Minimal spur on

labellum

Flabellata V. flabellata (Rolfe ex Downie) • Labellum Indochina

3 species Christenson attached to Gardiner, Motes, Emerson, Roberts, Kocyan 35 V. lilacina Teijsm. & Binn. column foot

V. vietnamica (Haager) L.M.Gardiner

Deltoglossa V. arcuata J.J.Sm. • Cylindrical Indonesian and

17 species V. deareii Rchb.f. column with Philippine

V. devoogtii J.J.Sm. thickened base archipelagos

V. foetida J.J.Sm.

V. helvola Blume

V. hindsii Lindl.

V. insignis Blume

V. jennae O’Bryne & Verm.

V. limbata Blume

V. lombokensis J.J.Sm.

V. luzonica Loher ex Rolfe

V. merrillii Ames & Quisumb.

V. metusalae O'Byrne

V. perplexa Roberts, Gardiner & Motes

V. sumatrana Schltr.

V. tricolor Lindl.

V. ustii Golamco, Claustro & de Mesa

Hastifera V. celebica Rolfe • Distinct Island endemics in

8 species V. furva (L.) Lindl. appendages Indonesian

V. gibbsiae Rolfe (lobules) on archipelago

V. hastifera Rchb.f. labellum midlobe

V. lindenii Blume

V. frankieana Metusala and O'Byrne

V. scandens Holttum

Roeblingiana V. barnesii W.E.Higgins & Motes • Cylindrical Philippines and Gardiner, Motes, Emerson, Roberts, Kocyan 36 5 species V. javierae D.Tiu ex Fessel & Lückel column without adjacent islands

V. lamellata Lindl. thickened base

V. roeblingiana Rolfe • Usually

V. sanderiana (Rchb.f.) Rchb.f. modified/embelli

shed labellum

midlobe

Ascocentrum V. ampullacea (Roxb.) L.M.Gardiner • Diminutive size Himalayas, southern

11 species V. aurantiaca (Schltr.) L.M.Gardiner and compact China, Indochina,

V. aurea (J.J.Sm.) L.M.Gardiner growth habit through to Philippine

V. christensoniana (Haager) L.M.Gardiner • Small brightly and Indonesian

V. curvifolia (Lindl.) L.M.Gardiner coloured flowers archipelagos

V. garayi (Christenson) L.M.Gardiner • Nectar filled spur

V. himalaica (Deb, Sengupta & Malick) • Bird pollinated L.M.Gardiner

V. insularum (Christenson) L.M.Gardiner

V. miniata (Lindl.) L.M.Gardiner

V. rubra (Lindl.) L.M.Gardiner

V. semiteretifolia (Seidenf.) L.M.Gardiner

Ascocentropsis V. nana L.M.Gardiner Vietnam

1 species

Neofinetia V. falcata (Thunb.) Beer • White flowers China, Japan, eastern

3 species V. richardsiana (Christenson) • Long nectar Asia

L.M.Gardiner filled spur

V. xichangensis (Z.J.Liu & S.C.Chen) • Moth pollinated

L.M.Gardiner

Eparmatostigma Vanda dives (Rchb.f.) L.M.Gardiner • Compact plants Laos, Vietnam

1 species • Very numerous Gardiner, Motes, Emerson, Roberts, Kocyan 37 minute white

flowers on

arching raceme