Plant Syst. Evol. 228: 181±197 62001)

Evolution in Aeschynanthus Gesneriaceae) inferred from ITS sequences

J. Denduangboripant1,2, M. Mendum2, and Q. C. B. Cronk1,2

1Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, UK 2Royal Botanic Garden Edinburgh, Edinburgh, UK

Received February 8, 2001 Accepted June 8, 2001

Abstract. Aeschynanthus Jack, an epiphytic genus of the epiphytic ¯ora. The genus has some 160 with c.160 species, is widespread in SE Asia. We species distributed from Sri Lanka and the selected 50 species for ITS nrDNA sequencing, to Himalayas to New Guinea and the Solomon include all biogeographic areas and all infrageneric Islands. The genus therefore crosses Wallace's groupings, which are currently based on seed Line and it thus makes an interesting biogeo- morphology. Some species were sequenced directly graphical case study. A previous study based from PCR product; others cloned because of ITS on limited sampling 6Denduangboripant and length polymorphisms. The clone sequences were analysed individually and combined in an elision Cronk 2000) suggested that the genus was matrix. Results extend earlier ®ndings that Aeschy- divided into two main clades: one centred west nanthus is divided into two clades, one occurring and one centred east of Wallace's line but with primarily in mainland SE Asia and the other in extensive overlap in the Sunda shelf islands. Malesia. This pattern is interpreted as indicating an We have now more than doubled the sampling ancient vicariance event followed by dispersal and and can present a much more detailed analysis. plate fusion. Clade I has straight or clockwise spiral Most species appear to be bird-pollinated orientation of the testa cells and clade II anticlock- with scarlet tubular ¯owers 6but occasional wise spiral orientation. In clade I some species of green-¯owered species occur with unknown section Microtrichium form a basal group with pollination syndrome). Typically, after polli- other sections being polyphyletic or paraphyletic. nation the gynoecium elongates into a very In clade II the monophyletic section Aeschynanthus long 6up to 43 cm) thin capsule containing is nested within the paraphyletic basal Micro- trichium. large numbers of wind-dispersed seeds. Dis- persal is aided by the presence of one or more Key words: Aeschynanthus, biogeography, Gesner- usually hair-like seed appendages 6although in iaceae, internal transcribed spacers, molecular those species with short seed appendages, these phylogeny, seed morphology, nuclear ribosomal might be as important in substrate attachment DNA, Southeast Asia. as in dispersal). The appendages, one at the apical end of the seed and one or more at the In the forests of Southeast Asia the brightly hilar end, have been used to subdivide coloured 6usually red or orange) ¯owers of the genus into ®ve sections 6Bentham 1876, Aeschynanthus species are a characteristic part Clarke 1883, Burtt and Woods 1975). Wang 182 J. Denduangboripant et al.: Evolution in Aeschynanthus 6Gesneriaceae)

61984) added a sixth section based on corolla composites formed of microcontinents and characters. Recent SEM studies of seed and fragments of island arcs. The evolution and appendage morphology of 99 taxa 6Mendum geographical distribution of fauna and ¯ora et al. 2001) found that di€erences in testa cell has been greatly in¯uenced by the geological orientation and papilla and appendage struc- complexity, and this may go some way to- ture enable the genus to be divided into two wards explaining the high level of biodiversity groups, A and B, within which the existing in Southeast Asia 6Gaston et al. 1995, Taylor sections may be placed. Group A 6testa cell et al. 1999, Myers et al. 2000). The region has orientation spiral or rarely straight, papillae long been attractive to biogeographers. Wal- formed from single cells, hilar appendage one, lace's Line refers to the boundary proposed by short and smooth) contains sections Micro- Alfred Russel Wallace in 1860, separating the trichium, Aeschynanthus and Haplotrichium Asian from the Australasian faunistic region, s. str. Group B 6testa cell orientation always and marking the point at which the two biotas straight, papillae formed from the junction of collided after having been separated since the two cells, hilar appendages one or more, long break-up of Gondwanaland in the mid-Meso- and papillose) contains sections Polytrichium, zoic. This boundary divides Bali and Borneo Diplotrichium and a third assemblage of species from Lombok and Sulawesi, and passes south- with a single appendage at each end and east of the Philippines 6Wallace 1860). How- previously placed in section Haplotrichium. ever, many other biogeographic boundaries Seed morphology places the two members of are evident in Southeast Asia and other lines Wang's 61984) section Xanthanthos in group B have been proposed 6Wallace 1863, Huxley 6Mendum et al. 2001) although ¯oral charac- 1868, Weber 1904, Wallace 1910). The study of ters con¯ict with this placement 6however, the the phylogenetic patterns of widespread di€erences are no greater than those found in groups of organisms of di€erent evolutionary the very variable section Microtrichium). It ages should reveal patterns explicable as a may be that this assemblage will be placed in result of vicariance 6geotectonic separation of section Xanthanthos but until more material of land masses) and dispersal 6geotectonic fusion the latter becomes available for DNA sam- of land masses or inter-island dispersal) 6Nel- pling and further study, a decision cannot be son and Platnick 1980, Nelson and Platnick made. Therefore the assemblage is here referred 1981). In this context Aeschynanthus is a highly to as Section X. There is strong correlation suitable subject for study. between seed type and geographical distribu- As discussed in a previous paper 6Dendu- tion: group A species are essentially Malesian angboripant and Cronk 2000), many species of whereas group B species are largely con®ned to Aeschynanthus show a higher level of ITS mainland South and Southeast Asia, with the sequence polymorphism than is usual in the exception of the small section Polytrichium Old World Gesneriaceae. In some species the which is more widespread. sequence length polymorphism makes PCR Because the genus Aeschynanthus occurs consensus sequences unreadable and in these throughout Southeast Asia but shows a high species we have cloned the PCR product and degree of endemism, the phylogenetic relation- sequenced two clones. Two clones are su- ships of these species might be expected to cient as intra-individual clone variation is have some relevance to the geological evolu- slight 6consisting mainly of length polymor- tion of the area. Southeast Asia has a complex phism rather than base polymorphism) and the geological history, resulting from an intricate clones are all found to cluster together on the pattern of geotectonic movements 6De Boer tree 6Denduangboripant and Cronk 2000). No and Du€ells 1996, Hall 1998, Metcalfe 1998). problems of this kind were encountered during Many islands in the region, for instance ITS sequencing of Streptocarpus, an African Sulawesi and New Guinea, are geological genus also in the Gesneriaceae 6MoÈ ller and J. Denduangboripant et al.: Evolution in Aeschynanthus 6Gesneriaceae) 183

Cronk 1997a) 6except for S. saxorium with a Wall. and A. humilis Hemsl., respectively. The latter 4 bp insertion from duplication 6MoÈ ller, un- names are used here. published)). However, Denduangboripant and DNA extraction, PCR, cloning, and DNA Cronk 62000) reported that clone variation in sequencing. Details of DNA extraction, polymer- Aeschynanthus, although signi®cant, appeared ase chain reaction 6PCR) ampli®cation, PCR to postdate the origin of the species and thus cloning, and DNA sequencing strategies for reconstructing the Aeschynanthus phylogeny are cloned sequences could be used satisfactorily provided elsewhere 6MoÈ ller and Cronk 1997a, b; for phylogenetic reconstruction. The approach Denduangboripant and Cronk 2000). The genomic taken here is to combine cloned sequences with DNA of the newly added 27 Aeschynanthus taxa consensus PCR sequences where the latter are was prepared and used as template for PCR not problematic. ampli®cation, yielding the complete ITS region Sequence alignment is generally straight- 6both ITS1 and ITS2 and 5.8S ribosomal DNA). forward with one exception: a short region of The products were sequenced using either the ITS2 corresponding to arm 1 of the predicted Amplitaq-FS dye terminator cycle-sequencing kit ITS secondary structure. This arm 6stem-loop 6Perkin Elmer Biosystems Inc., Warrington, UK) structure) is notably long in Gesneriaceae and or Thermo Sequenase II 6Amersham Pharmacia the top of the arm appears to be redundant Biotech UK Limited, Bucks, England), and analysed and is sometimes deleted altogether 6Dendu- on an ABI 377 prism DNA sequencer 6Perkin Elmer, Applied Biosystem Inc., Foster City, CA, angboripant and Cronk 2001). This phenom- USA). When we found uninterpretable sequence enon also occurs in Streptocarpus and electropherograms caused by ITS length intraindi- corresponds to the 40 bp deletion found in vidual variation 6Denduangboripant and Cronk some species of that genus 6MoÈ ller and Cronk 2000), PCR cloning was then used for that species. 1997a). In the present study we have used Otherwise the consensus sequences from forward secondary structure analysis to guide the and reverse reactions were obtained without clon- alignment of this problematic area. Despite ing. For cloning, the PCR products were puri®ed these unusual features of ITS evolution in and ligated into plasmid vectors using the Topo TA Aeschynanthus, ITS appears to have robust Cloning kit 6Invitrogen Co., Carlsbad, CA, USA). phylogenetic utility in this genus. The subcloned plasmids were extracted from trans- formants. At least two transformed clones were sequenced. Materials and methods ITS sequence results were analysed and aligned with the previous DNA data matrix. A previous Plant materials. Fresh leaf material of one plant study 6Denduangboripant and Cronk 2001) found a representing each species was taken from the living problem in sequence alignment of a short region of collection held at the Royal Botanic Garden ITS2. This region corresponds to arm 1 6stem-loop Edinburgh, except for ®ve Sulawesi taxa which structure) of the predicted ITS secondary structure. were sequenced from wild-collected leaf samples. Therefore, we used minimum free energy secondary The allied genera Cyrtandra and Lysionotus were structure analysis to guide the alignment of this used as outgroups as in previous analyses. Voucher problematic area. RNA secondary structures of the herbarium specimens of all accessions analysed arm 1 of ITS2 region were generated by the program have been prepared and are lodged at E. For the RNAdraw version 1.1 6Matzura and Wennborg present study, 27 further species 6Table 1) were 1996). This method was also used to guide align- added to 23 species already sequenced in a ment in this study. However even with this aid, a preliminary study 6Denduangboripant and Cronk 20 bp region of the aligned matrix in this region was 2000) to give a total of 50 Aeschynanthus species, considered ambiguously aligned and was excluded about one-third of the genus, representing all from the analysis. Full details of the analysis can be morphological variation and geographical distri- found elsewhere 6Denduangboripant and Cronk bution of Aeschynanthus. Aeschynanthus mimetes 2001). All sequences and the alignment have been and A. hildebrandii in Denduangboripant and submitted to GenBank 6accession numbers Cronk 62000) have been re-identi®ed as A. fulgens AF349153-AF349312). The new alignment of all 184 J. Denduangboripant et al.: Evolution in Aeschynanthus 6Gesneriaceae)

Table 1. Accessions of 27 additional species of Aeschynanthus examined in this study

Taxon Locality Section RBGE Collected accession No.

61) Aeschynanthus acuminatus Taiwan Haplotrichium 19991496 Wall. Ex A. DC. 62) Aeschynanthus andersonii C. B. Clarke Yunnan 6China) Section X 19970465 63) Aeschynanthus arfakensis C. B. Clarke Irian Jaya Polytrichium 19972046 6Indonesia) 64) Aeschynanthus austroyunnanensis Yunnan Section X 19951561 W. T. Wang 6China) 65) Aeschynanthus batakiorum Palawan Polytrichium 19980285 Mendum & Madulid 6Philippines) 66) Aeschynanthus curtisii C. B. Clarke Sarawak 6Borneo) Aeschynanthus 19622237 67) Aeschynanthus ellipticus Papua New Guinea Microtrichium 19972009A Lauterb & K. Schum 68) Aeschynanthus garrettii Craib Thailand Microtrichium 19750205 69) Aeschynanthus irigaensis Luzon Microtrichium 19972532 6Merr.) Burtt & Woods 6Philippines) 610) Aeschynanthus javanicus Hook Cultivated Aeschynanthus 19971339 611) Aeschynanthus lineatus Craib Yunnan 6China) Diplotrichium 19970163 612) Aeschynanthus musaensis P. Woods Papua New Guinea Microtrichium 19750186 613) Aeschynanthus myrmecophilus Peninsular Polytrichium 19981953 P. Woods Malaysia 614) Aeschynanthus nummularius Papua New Guinea Microtrichium 19932365 6Burkill & S. Moore) K. Schum 615) Aeschynanthus obconicus Sarawak 6Borneo) Aeschynanthus 19622987 C. B. Clarke 616) Aeschynanthus oxychlamys Irian Jaya Microtrichium 19930953 Mendum 6Indonesia) 617) Aeschynanthus pachytrichus Yunnan 6China) Diplotrichium 19970171 W. T. Wang 618) Aeschynanthus philippinensis Mindoro Microtrichium 19972491 C. B. Clarke 6Philippines) 619) Aeschynanthus pseudohybridus Sarawak Section X 19971340 Mendum 6Borneo) 620) Aeschynanthus rhododendron Ridl. Peninsular Microtrichium 20001550 Malaysia 621) Aeschynanthus roseo¯orus Mendum Seram Microtrichium 19880263 622) Aeschynanthus sp. 6001) Sulawesi Microtrichium Mendum, Argent & Hendrian 001 623) Aeschynanthus sp. 60025) Sulawesi Microtrichium Mendum, Argent & Hendrian 0025 624) Aeschynanthus sp. 600171) Sulawesi Polytrichium Mendum, Argent & Hendrian 00171 625) Aeschynanthus sp. 600293) Sulawesi Microtrichium Mendum, Argent & Hendrian 00293 626) Aeschynanthus vinaceus P. Woods Sarawak 6Borneo) Microtrichium 19672118 627) Aeschynanthus cf. viridi¯orus Sulawesi Polytrichium Mendum, Argent Teijsm & Binn & Hendrian 00228 J. Denduangboripant et al.: Evolution in Aeschynanthus 6Gesneriaceae) 185 sequences used in this study is also available at frequency parameters and Gamma distribution http://www.icmb.ed.ac.uk/J_matrix2.pdf. shape parameter 61.0051) determined by Modeltest Phylogenetic analysis. Phylogenetic analyses were then used for the maximum likelihood by parsimony, branch support analyses, and other analysis in PAUP* with TBR swapping. sequence and tree statistics were performed as described previously 6Denduangboripant and Cronk 2000), with the program PAUP* 6Swo€ord Results 1998) version 4.0b4a and MacClade version 3.01 6Maddison and Maddison 1992). Heuristic searches ITS sequence characteristics. Of the 27 addi- were used to ®nd the most parsimonious trees by tional species sequenced here, six had to be using RANDOM sequence addition with TBR cloned 6for reasons discussed in Denduang- swapping for 10 000 replicates with Multrees and boripant and Cronk 2000). A further four Steepest Descent options. Decay Indices 6Bremer species showed ITS length polymorphism support values) were calculated using the program resulting from single 1±2 bp deletions between AutoDecay version 4.0 6Eriksson 1998). Three di€erent intraindividual ITS copies, but these methods of combining PCR consensus sequences could be interpreted satisfactorily by compar- and multiple clone sequences were used: 61) Clones ison of forward and reverse sequences, with the analysed as separate individual sequences, plus the indel bases coded as missing data. This sug- consensus PCR sequences; the problem here is the gests that nearly 40% of Aeschynanthus species relatively large number of terminal items for analysis 680 items representing 52 species). 62) show some evidence of signi®cant intrageno- Clones combined as a consensus sequence, plus mic polymorphism in their ITS sequences the consensus PCR sequences. Where clones di€er 622% with severe polymorphism, and 15% by substitutions, the new consensus sequence is with minor polymorphism). coded as both nucleotides 6e.g. A and G coded R When all the sequences are aligned, a etc.); where they di€er as an indel, the available matrix of 603 aligned positions results in 213 sequence is used 6gaps ignored). 63) Clones anal- 637.4%) potentially informative sites 6Table 2). ysed sequentially in an elision matrix. Both clone In addition, 92 indels were coded of which 64 sequences for each species are analysed in combi- are informative. The cause of the ITS poly- nation, while PCR consensus sequences are includ- morphism was usually evident from inspecting ed twice to give a matrix of uniform length. Elision the di€erences between the respective cloned matrices are commonly used in two gene studies to sequences. Most striking was A. pachytrichus combine data sets, and the method is used in an analogous way here. in which the clones di€er by a 9 bp indel event. A reweighting parsimony analysis was also The highest intraindividual clone divergence carried out by weighting characters according to yet recorded in Aeschynanthus is between the mean values of their rescaled consistency indices two highly divergent 67.68%) clones of Aes- 6RC). Successive reweighting was carried out four chynanthus sp. 60025) from Sulawesi. However, times, at which point no further topological when analysed separately, even these clones changes occurred. The results of parsimony anal- fall together on the tree. yses were compared to a maximum likelihood Phylogenetic analysis. Three di€erent 6ML) analysis. To ®nd the optimum model for the types of matrix were analysed 6see methods): likelihood analysis, the program Modeltest version 61) the full matrix with all clones analysed as 3.0 6Posada and Crandall 1998) was ®rstly used to separate entities 681 terminals); 62) the clones compare the likelihood score results and associated combined as a consensus matrix; and 63) P-values between 56 ML DNA-evolution models. The program then provides a choice of the model combined as an elision matrix. Strict consensus that best ®ts the data by nested likelihood ratio trees resulting from the three methods of matrix tests and the Akaike information criterion assembly have no con¯ict, being fully congru- 6minimum theoretical information criterion, AIC; ent at the species level. Minor di€erences in Akaike 1974). The model selected here was resolution are noticeable between trees. TrN + G. Appropriate substitution values, base Between two and three nodes supported in 186 J. Denduangboripant et al.: Evolution in Aeschynanthus 6Gesneriaceae)

Table 2. Sequence characteristics of ITS1 and ITS2 regions of 81 sequences 6representing 52 species) of Gesneriaceae. Characteristics of the aligned matrix excluding ambiguous sequence sites

Parameter ITS1 ITS2 ITS1 and ITS2

Length range 6bp) ± Ingroup + outgroup 217±237 206±254 430±491 ± Ingroup 217±233 206±247 430±477 ± Outgroup only 225±237 243±254 468±491 Length mean 6bp) ± Ingroup + outgroup 225.7 239.6 465.0 ± Ingroup 225.3 239.3 464.6 ± Outgroup only 231.0 248.5 479.5 Aligned length 6bp) 289 314 603 G + C content range 6%) 6complete matrix) 48.12±59.66 49.57±59.51 48.90±59.58 G + C content mean 6%) 6complete matrix) 54.71 55.24 54.98 Sequence divergence 6%) ± Ingroup to outgroup 13.87±22.36 16.00±24.38 15.96±23.38 ± Ingroup 6between spp.) 0.00±19.20 0.00±17.180.00±16.95 ± Ingroup 6within spp.)a 0.00±8.04 0.00±7.33 0.00±7.68 Number of indels ± Ingroup + outgroup 34 5892 ± Ingroup 6total) 29 46 75 ± Ingroup 6within spp.) 5 15 20 Size of indels 6bp) ± Ingroup + outgroup 1±4 1±9 1±9 ± Ingroup 6total) 1±4 1±9 1±9 ± Ingroup 6within spp.) 1, 2, 4 1, 2, 9 1, 2, 4, 9 Number of excluded sites 15 1833 Number of sites after exclusion 274 296 570 Number of variable sites 140 171 310 Number of constant sites 6%) 134 648.9 %) 125 642.2 %) 260 645.6 %) Number of potentially informative sites 6%) 94 634.3 %) 119 640.2 %) 213 637.4 %) Number of autapomorphic sites 6%) 46 617.1 %) 52 617.6 %) 97 617.0 %) Transitions on tree 6unambiguous) 124 173 314 Transversions on tree 6unambiguous) 59 106 166 Transitions/Transversions 2.10 1.63 1.89 a Divergence between clone pairs one tree collapse in the other and vice-versa. 697% clade I; 99% clade II) 6Fig. 1). The We found that the elision matrix was fastest to maximum likelihood tree 6Fig. 2) does not run and gave fewest trees 61440 trees), whereas show any incongruence with the maximum the full matrix and the consensus combination parsimony strict consensus tree, and provides matrix gave more than 37 600 trees 6trees further support for the two-clade division of exceeded a computer memory used by PAUP*) the genus. Many well-supported nodes on the and 10 073 trees respectively. The results MP tree allow us to conclude with a high presented here use the elision matrix, but none degree of certainty that the existing sections, of the conclusions reached is a€ected by the based largely on seed-appendage types, are type of analysis. paraphyletic or polyphyletic. An exception is All phylogenetic trees produced in this section Aeschynanthus which is monophyletic study, by whatever method, con®rm the divi- 6Bootstrap support ˆ 99%). In clade I, section sion of Aeschynanthus into two major clades Microtrichium is basal with the other sections both of which have high Bootstrap support polyphyletic or paraphyletic. In clade II most J. Denduangboripant et al.: Evolution in Aeschynanthus 6Gesneriaceae) 187

Fig. 1. Strict consensus of 1440 most parsimonious trees for 50 Aeschynanthus species and two outgroup Gesneriaceae taxa 61557 steps in length) based on parsimony analysis of an elision matrix of the combined ITS1 and ITS2 sequence data plus the alignment gap matrix. The ®rst values of upper numbers are full heuristic Bootstrap percentages of 100 replicates. The second values of upper numbers are 50% deletion Jackknife percentages 6``fast'' stepwise-addition) of 10 000 replicates. Lower numbers are decay indexes. The two arrows indicate branches that collapse when the gap matrix is excluded and the analysis rerun. The country of origin of the specimen is indicated. [CI ˆ 0.62, RI ˆ 0.79, RC ˆ 0.49] The two clones per species are designated A and B, except for Aeschynanthus sp. 0025 for which the four clones are designated 1234 188 J. Denduangboripant et al.: Evolution in Aeschynanthus 6Gesneriaceae)

Fig. 2. Maximum likelihood 6ML) tree for 50 Aeschynanthus species and two outgroup Gesneriaceae taxa 6)ln likelihood ˆ 8570.14) based on an analysis of the elision matrix of the combined ITS1 and ITS2 sequence data without the gap matrix. As the elision matrix is used, the branches are double their true length. Numbers along branches indicates the amount of character change 6branch length). Available chromosome numbers 6Rogers 1954, Eberle 1956, Ratter 1963, Ratter and Prentice 1967, Ratter and Milne 1970, Milne 1975, Hellmayr 1989, Kiehn and Weber 1997, Rashid et al. 2001) are given in brackets following the species names 6all as 2n for ease of comparison). Vertical lines denote major seed types and the main clades, which are congruent except for the four basal clade I species J. Denduangboripant et al.: Evolution in Aeschynanthus 6Gesneriaceae) 189 of the species belong to section Microtrichium tical vicariance patterns between the Asian with the monophyletic section Aeschynanthus mainland and eastern Malesia for two cicada nested within this. It is clear from inspection of groups some 20 million years ago. They Fig. 1 that there is considerable biogeographic suggested that the formation of a volcanic pattern in the phylogeny. The majority of island chain at the western Paci®c plate margin species in clade I occur in India, Indochina, allowed separation of mainland and island arc South China and a few on islands of the Sunda clades. The island arc no longer exists, having shelf. In contrast species in clade II occur in migrated west as discrete terranes which now New Guinea, Sulawesi, Philippines and Seram form parts of the Philippines, Sulawesi, and with some species also occurring on the Sunda New Guinea. The subsequent coming together shelf islands. of the Australasian and Asian plates would result in an overlap of clades along the Sunda shelf margins. This is entirely consistent with Discussion the observed geographic patterns in Aeschy- Aeschynanthus biogeography: ancient vicariance nanthus 6Fig. 6, more details presented in and recent dispersal. The extended analysis of Figs. 3 and 4), which would then be the result the phylogeny of Aeschynanthus con®rms the of ancient vicariance overlain by recent dis- division of the genus into two major clades persal and coalescence events, as summarised di€ering in geography. Of the 26 clade I diagrammatically in Fig. 7. species, 17 occur on mainland SE Asia 6in- An increase in seed appendage length and cluding Taiwan) and only nine are Malesian number 6providing a favourable surface area 6including Peninsular Malaysia). Six of the to mass ratio) appears to be a ``key innova- nine, including all clade I species east of tion'' allowing biogeographic transgression of Wallace's Line, are in section Polytrichium. the main clade areas. The Microtrichium type This wider distribution 6Fig. 3) is possibly due is basal in both clades and is probably the to the greater e€ectiveness of a coma 6hair- ancestral state. The only clade I species known tuft) of many hair-like appendages in wind to be present east of the dotted line in Fig. 6 dispersal. The long ®liform appendages pos- are in section Polytrichium, whose derived sessed by most clade I species are e€ective for morphology is extreme in appendage number. wind dispersal in dry conditions, and the Seed morphology re¯ects major clade struc- majority of these species occur in the seasonal ture in Aeschynanthus. The recent morpholog- climates of mainland Southeast Asia. The ical studies recognising two major groups in great majority of clade II species, with shorter Aeschynanthus align well with the molecular less elaborate appendages, occur in the more results. Clade I broadly corresponds to seed consistently wet forests of Malesia 6Fig. 3 and group B and clade II corresponds to seed Fig. 4). The 24 clade II species are all Male- group A 6Fig. 2). Those seed group A species sian. The species basal to clade I are from that do fall into clade I are those that are basal. Indo-China and Taiwan, implying a possible Aeschynanthus buxifolius and A. garrettii 6sec- ancestral area for clade I in this region. Clade tion Microtrichium) are two of the only three II 6Fig. 4) on the other hand has Philippine Microtrichium species to have ‹ straight testa and New Guinea species as basal with A. phi- cell orientation 6the others have an anticlock- lippinensis as most basal by ML and reweigh- wise spiral); they are also the only ones that are ting analyses 6Figs. 2 and 5). not Malesian. Aeschynanthus acuminatus and The geographical di€erence between the A. bracteatus are two members of the very two major clades implies an ancient vicariance small section Haplotrichium which is not event at the time of the origin of the genus known to occur in Malesia. Clade I and clade between Indo-China and the Philippines. II di€er in the orientation of the testa cells and De Boer and Du€ells 61996) postulated iden- can therefore be de®ned morphologically. 9 .Dnunbrpn ta. vlto in Evolution al.: et Denduangboripant J. 190 Aeschynanthus

Fig. 3. Distribution areas of 26 Aeschynanthus speciesfromcladeI60ˆ absence, 1 ˆ presence) 6Gesneriaceae) .Dnunbrpn ta. vlto in Evolution al.: et Denduangboripant J. Aeschynanthus Gseica)191 6Gesneriaceae)

Fig. 4. Distribution areas of 24 Aeschynanthus species from clade II 192 J. Denduangboripant et al.: Evolution in Aeschynanthus 6Gesneriaceae)

Fig. 5. Successive weighting strict consensus tree of 120 most parsimonious trees for 50 Aeschynanthus species and two outgroups 6733 steps in length) based on parsimony analysis of an ITS elision matrix plus the alignment gap matrix. The 1440 most parsimonious trees of the original elision matrix were used to weight characters by their mean RC value and the analysis rerun. The arrow indicates a branch that collapses when the gap matrix is excluded and the analysis rerun. The ®ve triangles show branches that collapse on the consensus tree of unweighted analysis. Aeschynanthus philippinensis isbasalincladeIinthisanalysisasintheMLanalysis 6Fig. 2). [CI ˆ 0.85, RI ˆ 0.92, RC ˆ 0.78] J. Denduangboripant et al.: Evolution in Aeschynanthus 6Gesneriaceae) 193

Fig. 6. Geographical distribution of sequenced species summarised from Fig. 3 and Fig. 4. The ®rst number in each ratio refers to the number of species from clade I. The second number refers to the number of species from clade II. The solid line shows the geographic distribution of the whole genus. The dashed line indicates an approximate western boundary of clade II species. Clade I species, with the exception of section Polytrichium, do not occur east of the dotted line. This line is similar to Huxley's line 6Huxley 1868) except that Palawan 6Philippines) is to the east of the line

Fig. 7. A tentative model of the geographic pattern of Aeschynanthus evolution, consistent with the ITS phylogeny, suggesting a combination of ancient vicariance, recent dispersal and coalescence events in the regions. The asterisk indicates a proposed ancient origin of Aeschynanthus clades I and II by a vicariance event

Clade I has straight or clockwise spiral orien- seed with a single hilar appendage, as do the tation and clade II anticlockwise spiral majority of Aeschynanthus species including orientation 6Fig. 8). those basal to clade I in this study. Prelim- Appendage numbers appear to be at least inary studies of Aeschynanthus seed ontogeny partly homoplastic. There is morphological by Saueregger and MuÈ hlbauer 6unpublished) evidence that the condition of more than one showed that, in section Polytrichium, one hilar appendage is derived. All other genera appendage develops a little before the others in tribe Trichosporeae are reported to have a and remains somewhat longer and stouter. 194 J. Denduangboripant et al.: Evolution in Aeschynanthus 6Gesneriaceae)

Fig. 8. Possible evolution of seed appendage types in Aeschynanthus as suggested by the phylogenetic analysis. The morphological types are represented by named exemplar species. The diagram illustrates suggested morphological transitions only, and does not imply transitions between these exemplar species J. Denduangboripant et al.: Evolution in Aeschynanthus 6Gesneriaceae) 195

Current studies by Christie and Mendum The authors thank M. MoÈ ller for advice on PCR 6unpublished) con®rm this and also show and comments on the manuscript; S. Scott for that in section Diplotrichium one appendage growing plant material; sta€ at the Royal Botanic develops a little before the other. A small Garden Edinburgh and the Institute of Cell and percentage of seeds of two Section X species Molecular Biology, University of Edinburgh, for show development of a second appendage, technical support and the use of research facilities. B. L. Burtt kindly commented on a ®rst draft of and a few seeds of one collection of this paper. We also thank W. Cherry 6RBG A. parasiticus 6section Diplotrichium) show de- Sydney) for supplying living material of A. bracte- velopment of a short third appendage. Thus atus and A. buxifolius; A. Weber 6Institut fuÈ r the clade I species with Type B seed Botanik, University of Vienna) for his helpful morphology do appear to be a natural interest in this work; and Maryjane Evans for seed group, notwithstanding the di€erences in of A. rhododendron. This research was partially appendage number. The short smooth ap- supported by the Development and Promotion of pendage type 6section Microtrichium) is basal Science and Technology Talents project 6DPST), in both clades, and is thus paraphyletic. the Royal Thai Government. Section Aeschynanthus, with ¯exuous trailing habit and tubular or obconic calyx with References abscission layer at the base, is a natural Akaike H. 61974) A new look at the statistical group. We propose a possible evolutionary model identi®cation. IEEE transactions on pattern of seed appendage types in Aeschy- automatic control. 19: 16±723. nanthus, suggested by the phylogenetic anal- Bentham G. 61876) Gesneriaceae. In: Bentham G., ysis, in Fig. 8. Hooker J. D. 6eds.) Genera Plantarum 2. Lon- Available chromosome numbers 6Rashid don, pp. 990±1025. et al. 2001) show a possible slight trend Burtt B. L., Woods P. J. B. 61975) Studies in the towards dysploid reduction in clade I species, Gesneriaceae of the Old World XXXIX: towards and polyploidy in clade II species. Counts are a revision of Aeschynanthus. Notes Roy. Bot. available for 23 of the sequenced species, 12 in Gard. Edinburgh 33: 417±489. clade I and 11 in clade II 6Fig. 2). The Clarke C. B. 61883) Cyrtandreae. In: De Candolle commonest number is 2n ˆ 32, but in clade I A., De Candolle C. 6eds.) Monographiae Phan- ®ve aneuploids occur 62n ˆ 30, 2n ˆ 28), but erogamarum 561). Paris, pp. 18±57. De Boer A. J., Du€els J. P. 61996) Historical only one polyploid, A. myrmecophilus with biogeography of the cicadas of Wallacea, New 2n ˆ 64. In clade II by contrast, only one Guinea and the West Paci®c: a geotectonic aneuploid occurs, but polyploids are more explanation. Palaeogeogr. Palaeoclimatol. Pal- common. More investigation of Aeschynanthus aeoecol. 124: 153±177. chromosome numbers is required to con®rm Denduangboripant J., Cronk Q. C. B. 62000) High or reject this possibility. intra-individual variation in ITS sequences in The present sectional classi®cation, based Aeschynanthus 6Gesneriaceae): implications for 6with the exception of section Xanthanthos) phylogenetics. Proc. Roy. Soc. London B 267: on easily observable appendage characters, 1407±1415. has proved to be of considerable practical Denduangboripant J., Cronk Q. C. B. 62001) taxonomic value and it is not our intention Evolution and alignment of the hypervariable to revise it here to re¯ect the ITS data. arm 1 of Aeschynanthus 6Gesneriaceae) ITS2 nuclear ribosomal DNA. Molec. Phylogeny However, the existence of two major clades Evolution 20: 163±172. in Aeschynanthus, di€ering in testa cell ori- Eberle P. 61956) Cytologische Untersuchungen an entation and in geographical distribution Gesneriaceae. I. Mitteilung. Die Struktur der patterns, raises the possibility of dividing PachytaÈ nchromosomen, sowie eine Reihe neu the genus into two clearly de®ned natural bestimmter Chromosomenzahlen. Chromosoma. subgenera. 8: 285±316. 196 J. Denduangboripant et al.: Evolution in Aeschynanthus 6Gesneriaceae)

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Addresses of the authors: Jessada Denduang- Kings Buildings, May®eld Road, Edinburgh EH9 boripant and Quentin C. B. Cronk 6E-mail: 3JH, UK. Mary Mendum and also Quentin C. B. [email protected]), Institute of Cell and Cronk, Royal Botanic Garden Edinburgh, 20A Molecular Biology, The University of Edinburgh, Inverleith Row, Edinburgh EH3 5LR, UK.