52 • February 2003: 33–50 Rapini & al. • Asclepiadeae classification Asclepiadeae classification: evaluating the phylogenetic relationships of New World Asclepiadoideae (Apocynaceae) Alessandro Rapini1, 2, Mark W. Chase 2, David J. Goyder2 & Jayne Griffiths 2 1 Departamento de Ciências Biológicas, Universidade Estadual de Feira de Santana, Av. Universitária s/n, CEP 44031-460, Feira de Santana, Bahia, Brazil. rapinibot@yahoo.com.br 2 Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3DS, U.K. m.chase@ rbgkew. org.uk; d.goyder@rbgkew.org.uk To provide an overview of New World Asclepiadoideae, we here evaluate Asclepiadeae classification by comparing the taxonom ic arrangement of subtribes with a topology obtained through analyses of two plastid DNA regions (trnL intron and trnL-F intergenic spacer) for 111 species of Asclepiadoideae representing the major lineages of the subfamily. Without Jobinia, Nephradenia and Barjonia, Asclepiadeae are not monophyletic. The monotypic African genus Eustegia, with pendent pollinia, m ay represent the sister clade of Marsdenieae Ceropegieae, the group composed of plants with erect pollinia. Metastelmatinae including African plants are also non-monophyletic, and the circumscription of the recently re-instated Cynanchinae should be studied further. Overall, Asclepiadeae are composed of three main clades. The Old World Astephaninae are the sister group of the other Asclepiadeae, which are divided into the ACTG (Asclepiadinae, Cynanchinae, Tylophorinae and Glossonematinae) and M OG (M etastelm atinae, Oxypetalinae and Gonolobinae) clades. According to this study, the New World Asclepiadoideae fall into just four clades: (1) M arsdenia (Marsdenieae), (2) Asclepias (Asclepiadinae, Asclepiadeae), (3) Cynanchum subgenus Mellichampia (Cynanchinae, Asclepiadeae), and (4) MOG, the clade comprising the majority of New World Asclepiadoideae. KEYWORDS: Apocynaceae, Asclepiadeae, Asclepiadoideae, classification, m olecular phylogeny, New World. INTRODUCTION Since the widespread acceptance of explicitly phylogenetic classification in botany in the 1990s, Apocynaceae Juss. have been subject of several phylogenetic studies. The pioneering work within Asclepiadoideae R. Br. ex Burnett was carried out in Microloma (Astephaninae Endl. ex Meisn.; Wanntorp, 1988). Generic relationships within Astephaninae were also investigated in morphological cladistic analyses (Liede, 1994, 1996a; Bruyns, 1999a) and recently re-investigated with molecular data (Liede, 2001). Other particular groups, such as Sarcostemma s.l. (Liede, 1996b; Liede & Täuber, 2000), Cynanchum (Liede, 1997a; Liede & Kunze, 2002; Liede & Täuber, 2002), stapeliads (Bruyns, 2000), Brachystelma Sims s.l. (Meve & Liede, 2001), Glossonematinae K. Schum. (Liede & al., 2002) and Asclepiadinae Endl. ex Meisn. (Goyder & al., unpubl.), have also been analysed phylogenetically (Table 1). Broader cladistic studies including members of Asclepiadoideae (Downie & Palmer, 1992; Chase & al., 1993; Olmstead & al., 1993; Judd & al., 1994; Struwe & al., 1994; Sennblad & Bremer, 1996, 2000, 2002; Civeyrel & al., 1998; Fishbein, 2001; Potgieter & Albert, 2001) have been concerned with relationships at higher taxonomic levels, providing little information within the subfamily. Sampling has been so sparse (Table 1) compared to the number of Asclepiadoideae taxa that these studies might be considered non-representative for statements regarding tribal circumscription (Endress & Bruyns, 2000). The broadly accepted amalgamation of Apocynaceae, Asclepiadaceae R. Br. and Periplocaceae Schltr. has emerged as result of these cladistic analyses. The taxonomic change of including Asclepiadaceae as subfamily of Apocynaceae s.l., however, was due to the adoption of a phylogenetic concept of classification, which avoids the recognition of non-monophyletic groups such as Apocynaceae s.str. (i.e., excluding members of Asclepiadaceae and/or Periplocaceae; Rapini, 2000). Within Asclepiadoideae, relationships are still far from consensus (Endress & Bruyns, 2000; Endress & Stevens, 2001). Due mainly to the artificial circumscription of many New World genera, which are still based on the variable corona and misinterpretations of inflorescence characters (Rapini, 2002), relationships among American groups are poorly understood. New World Asclepiadoideae (Fig. 1) have not yet been the focus of any phylogenetic study, and representation of these in analyses has been thin (Table 1); even large genera, such as Oxypetalum 33 Rapini & al. • Asclepiadeae classification 52 • February 2003: 33–50 Table 1. Main phylogenetic studies including Asclepiadoideae taxa, showing number of terminals, their representation of number of species and genera of Asclepiadoideae (Asclep.) and those in the New World (NW), as well as kind of data used in the studies. * including trnT-L intergenic spacer. Species Terminals (Asclep.) Genera Species Genera (Asclep.) (NW Asclep.) (NW Asclep.) Authors Year Wanntorp Downie & Palmer Chase & al. Olmstead & al. Judd & al. Liede Struwe & al. 1988 1992 1993 1993 1994 1994 1994 25 125 499 184 10 13 235 20 2 4 2 1 1 1 2 13 1 0 2 0 4 0 1 1 1 2 0 1 Liede Liede Sennblad & Bremer Liede Civeyrel & al. Bruyns Bruyns Liede & Täuber Sennblad & Bremer Fishbein Liede Potgieter & Albert 1996a 1996b 1996 1997 1998 1999a 2000 2000 2000/2002 2001 2001 2001 15 22 29 117 46 16 35 25 79 40 43 154 31 7 116 10 25 15 29 43 50 14 12 7 4 10 15 32 14 15 21 28 36 0 22 1 1 3 0 0 12 6 13 9 9 0 4 1 1 3 0 0 5 6 7 8 7 Meve & Liede Meve & Liede Liede & al. Sennblad & Bremer Liede & Kunze Liede & Kunze Liede & Kunze 2001 2001 2002 2002 2002 2002 2002 24 22 29 18 91 88 64 24 21 29 1 85 86 64 13 12 22 1 11 11 4 0 0 8 0 8 8 1 0 0 8 0 2 2 1 2002 this study 108 114 107 111 31 68 36 65 13 26 Liede & Täuber Rapini & al. (around 130 species), have not been considered. The study of Potgieter & Albert (2001), with 37 genera of Asclepiadoideae, includes the greatest generic diversity of the subfamily so far. However, that analysis has only seven New World genera of Asclepiadoideae, and five Asclepiadeae (R. Br.) Duby are not identified to species, which in some cases (e.g., Cynanchum) may represent completely unrelated taxa (Liede & Täuber, in press; see below). Since New World Asclepiadoideae are mainly composed of members of Asclepiadeae, the first step toward understanding the relationships of American groups is a good evaluation of the component subtribes. After Liede & Albers’ (1994) tribal arrangement of Asclepiadaceae (Asclepiadoideae, Secamonoideae Endl. and Periplocoideae R. Br. ex Endl.) genera, Liede (1997b) presented 34 Data Morphology Molecular (rbcL) Molecular (rbcL) Molecular (rbcL) Morphology Morphology Morphology, anatomy, chemistry, embryology Morphology Morphology Molecular (rbcL) Morphology Molecular (matK) Morphology Morphology Molecular (trnL-F*) Molecular (rbcL) Molecular (matK) Molecular (trnL-F*) Molecular (trnL-F), fruit morphology Molecular (trnL-F*) Molecular (ITS) Molecular (trnL-F*) Molecular (rbcL + ndhF) Molecular (trnL-F*) Molecular (ITS) Molecular (trnL-F*+ ITS), anatomy Molecular (trnL-F*) Molecular (trnL-F) a subtribal classification of Asclepiadeae. Despite that little phylogenetic information was available at that time and none was added in that paper, the subtribes were supposed to be defined by synapomorphies. An empirical morphological evaluation of Liede’s classification (Bruyns, 1999b) pointed out some inconsistencies of subtribes. Liede (1999) replied to the criticism, suggesting the exceptions outlined might represent reversals or parallelisms. Equally, in the absence of supporting evidence, one could argue that the similarities selected by Liede (1997b) as synapomorphies are actually homoplasies themselves. Moreover, omission of some New World genera (Rapini, 2002) has raised doubts concerning monophyly of the tribe itself, a point so far undisputed (Liede, 1999; Liede & al., 2002). Since Liede (1997b) based the subtribes of 52 • February 2003: 33–50 Rapini & al. • Asclepiadeae classification Fig. 1. Representative genera of Asclepiadeae. A, Ditassa cordeiroana (Brazil); B, Cynanchum roulinioides (Brazil); C, Oxypetalum insigne (Brazil); D, Blepharodon nitidum (Brazil); E, Marsdenia suberosa (Brazil); F, Asclepias curassavica (Brazil). Photos by A. Rapini. 35 Rapini & al. • Asclepiadeae classification Aidomene parvula Asclepias syriaca Aspidoglossum biflorum Aspidonepsis diploglossa Calotropis procera Cordylogyne globosa Fanninia caloglossa Glossostelma angolense Gomphocarpus fruticosus Kanahia laniflora Lagarinthus tenuis Mackenia Margaretta rosea Miraglossum pulchellum Odontostelma welwitschii Pachycarpus grandiflorus Parapodium costatum Pergularia tomentosa Schizoglossum atropurpureum Stathmostelma gigantiflorum Stenostelma capense Trachycalymma cristatum Woodia verruculosa Xysmalobium undulatum Asclepiadinae Ceropegia candelabrum Stapelia hirsuta CEROPEGIEAE Adelostemma gracillianum Ampelamus albidus Biondia chinensis Blepharodon lineare Cyathostelma Cynanchum acutum Ditassa banksii Folotsia sarcostemoides Funastrum Gonioanthela odorata Graphistemma chinensis Grisebachiella hieronymi Hemipogon acerosus Holostemma ada-kodien Karimbolea verrucosa Macroditassa adnata Mahawoa montana Merrillanthus hainanensis Metaplexis stauntonii Metastelma parviflorum Nautonia nummularia Orthosia congesta Oxystelma esculentum Platykeleba insignis Pentacyphus boliviensis Pentarrhinum insipidum Pentastelma auritum Peplonia nitida Petalostelma martianum Philibertia solanoides Raphistemma pulchellum Rhyssostelma nigricans Sarcostemma viminale Sichuania alterniloba Tassadia obovata Metastelmatinae 52 • February 2003: 33–50 Astephanus triflorus Blyttia arabica Diplostigma canescens Emicocarpus fissifolius Eustegia minuta Goydera somaliensis Microloma tenuifolium Oncinema roxburghii Pentatropis microphylla Pleurostelma grevei Ryncharrhena atropurpurea Schizostephanus alatus Seshagiria sahyadrica Tylophora flexuosa Vincetoxicum hirundinaria Glossonema boveanum Odontanthera reniformis Solenostemma arghel Astephaninae Fisheria scandens Gonolobus macrophyllus Gyrostelma oxypetaloides Hypolobus infractus Macroscepis obovata Matelea palustris Metalepsis cubensis Metoxypetalum retusum Pherotrichis balbisii Schubertia multiflora Stelmagonum hahnianum Trichosacme lanata Glossonematinae Gonolobinae Amblyopetalum coccineum Amblystigma hypoleucum Araujia sericifera Diplolepis meziesii Melinia candolleana Mitostigma tomentosum Morrenia odorata Oxypetalum banksii Schistogyne sylvestris Stenomeria decalepis Tweedia macrolepis Widgrenia corymbosa Oxypetalinae Fockea edulis FOCKEEAE ASCLEPIADEAE Secamone emetica Periploca graeca Periplocoideae Asclepiadoideae Barjonia racemosa Cionura erecta Dischidia nummularia Dregea floribunda Gymnema sylvestre Hoya carnosa Jobinia hernandifolia Marsdenia tinctoria Micholitzia obcordata Nephradenia acerosa Telosma odoratissima MARSDENIEAE Secamonoideae Fig. 2. Tribal arrangement of Asclepiadoideae with emphasis on subtribal classification of genera of Asclepiadeae sensu Liede (1997b; for complete generic list of other tribes, see Liede & Albers, 1994 and Liede’s homepage, www.unibayreuth.de/departments/planta2/triblist.html). Genera in bold are included in the analyses and when represented by type species the binomial is in bold. Asclepiadeae on supposed synapomorphies, they should, by implication, represent monophyletic groups (i.e., the hypothesis of phylogenetic patterns behind the classification). Our aim here is to evaluate the monophyly of Asclepiadeae sensu Liede (1997b) and its subtribes, summarising the phylogenetic information available for Marsdenieae Benth. and Asclepiadeae to establish an overview of New World Asclepiadoideae as a basis for further studies. We analysed DNA sequences of two plastid regions, the trnL intron and trnL-F intergenic spacer (hereafter trnL-F). Previous phylogenetic studies (Table 1) have often used trnL-F in molecular systematics of Asclepiadoideae, and to include a greater diversity of Asclepiadeae and Marsdenieae, the two tribes natively represented in New World, we selected representatives of most genera with sequences published, also adding new sequences (no published study has included all of these data). 36 MATERIALS AND METHODS We included sequences of 115 species in 69 genera of Asclepiadoideae; of these, 65 species of 26 genera represent New World taxa. Fifty-four species were sequenced for this study and the remaining 61 species retrieved from either GenBank (www.ncbi.nlm.nih.gov) or Liede’s homepage (www.uni-bayreuth.de/departments/planta2/ wgl/fsigrid2.html; Appendix 2). We have thus included the three genera of Glossonematinae (3), more than 50% of genera of Asclepiadinae (13), Astephaninae (11) and Metastelmatinae Endl. ex Meisn. (21), a third of genera of Oxypetalinae Decne. (4; we considered Mitostigma parviflorum Malme a member of Melinia and Tweedia coerulea D.Don ex Sweet a member of Oxypetalum), 25% of genera of Gonolobinae (G. Don.) Liede (3; we considered Metalepis albiflora Morillo a member of Cynanchum) and Marsdenieae (11), two genera of Ceropegieae Orb. (2) and Fockea edulis representing Fockeeae Kunze, Meve & Liede. Apocynum L. (Apocynoideae Burnett), Periploca L. (Periplocoideae), and Secamone R. Br. (Secamonoideae) were used as out- Rapini & al. • Asclepiadeae classification 52 • February 2003: 33–50 a 63 52 Stenostelma corniculatum b 55 Gomphocarpus fruticosus Asclepias curassavica Stathmostelma gigantiflorum Stenostelma corniculatum Calotropis procera Schizoglossum alpestre 55 Kanahia laniflora Pachycarpus spurius Stathmostelma gigantiflorum 62 Aspidoglossum ovalifolium Schizoglossum alpestre 87 Pachycarpus spurius Glossostelma spathulatum Xysmalobium undulatum Margaretta rosea 99 Aspidoglossum ovalifolium Gymnema sylvestre 55 95 Asclepias curassavica Glossostelma spathulatum Xysmalobium undulatum Kanahia laniflora 75 Calotropis procera Pergularia daemia Pergularia daemia Dregea sinensis 65 65 Marsdenia suberosa 58 Micholitzia obcordata 51 Telosma cordata Micholitzia obcordata 62 93 Hoya australis 100 100 Stapelia glanduliflora Ceropegia saxatilis 81 Dregea sinensis Dischidia bengalensis Stapelia glanduliflora Hoya australis Dischidia bengalensis 96 Gymnema sylvestre 62 Margaretta rosea Gomphocarpus fruticosus 62 Telosma cordata Ceropegia saxatilis Marsdenia suberosa Fockea edulis Fockea edulis Fig. 3. Incongruent results between the two pieces of trnL-F from Gymnema sylvestre. Numbers above branches show bootstrap percentages. a, position of G. sylvestre according to trnL intron. b, position of G. sylvestre according to trnLF intergenic spacer. Black bar: the Marsdenieae-Ceropegieae clade (with erect pollinia). White bar: Asclepiadeae (Asclepiadinae, with pendent pollinia). group (Civeyrel & al., 1998; Sennblad & Bremer, 2000; Potgieter & Albert, 2001). Type species of each genus were used when available (Fig. 2). Gymnema sylvestre was not included in general analyses because analyses of the general matrix raised suspicions about G. sylvestre sequences. To verify a possible disagreement between the two regions of trnL-F, we analysed each region independently. Fig. 3 shows bootstrap (gap coding included, no region excluded) trees of matrices (available upon request from MWC) including G. sylvestre, representatives of Ceropegieae and Asclepiadinae (Asclepiadeae); Fockea, sister group of the rest of Asclepiadoideae (see below), was used as outgroup (see Table 1 for accession numbers). According to trnL intron, G. sylvestre is nested in Marsdenieae– Ceropegieae clade (Fig. 3a), whereas using trnL-F intergenic spacer the species nests in Asclepiadinae (Asclepiadeae) (Fig 3b). In each case the position of G. sylvestre is supported by a high bootstrap percentage, indicating that its combined trnL-F may represent a hybrid sequence. Although trnL-F intergenic spacer represents less than 25% of the total sequence of these three regions (trnT-L intergenic spacer, trnL intron and trnL-F intergenic spacer), the use of this sequence of G. sylvestre as an outgroup may disturb analyses and might, for instance, explain the basal polytomy in Liede & Täuber (2000). DNA was extracted from either material dried in silica gel (Chase & Hills, 1991) or herbarium collections (Savolainen & al., 1995, and references therein) using 2X CTAB (Doyle & Doyle, 1987). Total DNA was cleaned with QIAquick columns (Qiagen, Inc.) using manufacturer’s protocol for PCR products. Primers “c” and “f” (forward and reverse, respectively) were used for PCR of DNA in good condition; in cases where the DNA was not intact, primers “c” and “d” as well as “e” and “f” (Taberlet & al., 1991) were combined (forward and reverse, respectively) to amplify trnL-F in two pieces. For amplification, 3 min. at 94°C preceded 28–32 cycles of denaturation at 94°C for 1 min., annealing at 48–50°C for 1 min. and extension at 72°C for 2 min., which was followed by more 2 min. of extension at 72°C. PCR products were purified with Concert Rapid PCR Purification System (GibcolBRL) and used as template for cycle sequence reaction with the same primers used for PCR. Sequences were processed on a 377 DNA sequencer (ABI Prism) following manufacturer’s protocols (Applied Biosystems, Inc.; ABI). Sequences were first edited in Sequence Navigator (ABI) and the two complementary strands overlain in AutoAssembler version 1.4 (ABI) for further edition. Alignment was carried out manually following the guidelines of Kelchner (2000), and we minimised the number of gaps at the expense of substitutions. Twenty 37 Rapini & al. • Asclepiadeae classification parsimony-informative gaps longer than two base pairs were coded according to the “simple indel coding” (presence/absence; Simmons & Ochoterena, 2000). Ambiguous regions were excluded from the analyses (matrix available upon request from MWC). Bayesian analysis was conducted in MrBayes (Huelsenbeck & Ronquist, 2001) using four Markov chains simultaneously started from random trees. Two evolutionary models were used: a simple two parameter (transition-transversion) substitution model (nst = 2, rate = equal and basefreq = equal) and the general timereversible model with gamma variation (nst = 6, rate = gamma and basefreq = estimate). Five hundred thousand cycles were performed with each model, sampling a tree at every 10 generations. Trees that preceded the stabilisation of the likelihood value (the burn in) were excluded, and the remaining trees were used to construct a consensus in PAUP (version 4.0; Swofford, 2001). Parsimony analyses were also carried out using PAUP. One thousand replicates of random taxon addition and tree-bisection-reconnection (TBR) branch swapping were performed, saving no more than 10 shortest trees per replicate to minimise swapping of suboptimal islands. Trees in memory were then used in another round of TBR swapping. To avoid an indefinite search, a limit of 20,000 trees was set. This procedure was carried out with (matrix 1) and without coding of the gaps (matrix 2). Support for clades was obtained with 1,000 bootstrap replicates with simple addition and TBR swapping, saving no more than 10 trees to minimise swapping on large islands. RESULTS The two models used for Bayesian analyses gave nearly identical results; changes in topology were restricted to clades with less than 50% probability, and variation in probabilities of clades with high confidence (above 94%) was up to 6%. We have opted for presentation of the consensus tree produced with the simplest model. Stabilisation of likelihood values was reached at about 100,000 replicates. Ten thousand trees were therefore discarded (the burn in) and the remaining 40,000 trees were used in estimating the probability of clades. Matrix 1, with 179 potentially parsimony-informative characters, generated most parsimonious trees of 594 steps, consistency index (CI) 0.72 and retention index (RI) 0.86, whereas matrix 2, with 159 potentially parsimony-informative characters, generated shortest trees with 563 steps, CI 0.72 and RI 0.85. The two matrixes each reached the limit of 20,000 trees. The strict consensus tree generated by matrix 1 differed from that generated by matrix 2 but did not contradict it. The analysis of matrix 1 gave less resolution within MOG (Metastelm38 52 • February 2003: 33–50 atinae, Oxypetalinae and Gonolobinae) clade, but was more informative concerning basal nodes, including the dichotomy between ACTG [Asclepiadinae, Cynanchinae K.Schum., Tylophorinae (K.Schum.) Liede and Glossonematinae] and MOG clades as well as the Glossonema–American Cynanchum clade. Gaps were important in providing evidence for particular groups, mainly in Asclepiadinae and the Glossonema–American Cynanchum clades (Figs. 4 and 5). In Asclepiadoideae (Figs. 4 and 5), Fockea edulis comes out as sister to the rest of Asclepiadoideae. Overall, the dichotomy between plants with erect pollinia, namely the Marsdenieae–Ceropegieae clade (100% posterior probability/88% bootstrap with matrix 1), and Asclepiadeae (99/61), with pendent pollinia, are present. Eustegia minuta, a species with pendent pollinia, however, appeared as sister to the plants with erect pollinia, a relationship with high posterior probability but weakly supported by the bootstrap (98/50). In Asclepiadeae, Astephaninae sensu Liede (2001) received 100/100 and appeared as sister to other Asclepiadeae, which are divided into two clades, ACTG (95/<50) and MOG (100/69). Although this latter dichotomy is not supported by the bootstrap, it is present in the strict consensus tree of matrix 1 and confirmed by Bayesian analysis. In the ACTG clade (Figs. 4 and 5), Asclepiadinae sensu Liede (1997b; 93/84) and Tylophorinae (100/83), as well as the American Cynanchum (100/78) and the clade composed of Glossonema, Odontanthera and Pentarrhinum (100/87), groups already observed in Liede & Täuber (2002), are confirmed. The clade composed of American Cynanchum, the Glossonema clade and Metaplexis (67/86), even though with low probability according to Bayesian analysis, had a great improvement in support with the inclusion of gap information (matrix 1). The clade comprising Sarcostemma and the stem-succulent, cynanchoid genera Folotsia, Karimbolea and Platykeleba (Liede & Meve, 2001) are supported (100/68). However, Cynanchinae, as circumscribed by Liede & Täuber (2002), are not supported, and the positions of Schizostephanus, Oxystelma and Solenostemma, as well as the relationship among subtribes are not resolved within the ACTG clade as a whole. In MOG (Figs. 4 and 5), the positions of the South American “Astephanus” geminiflorus and “Cynanchum” nummulariifolium, as well as the monotypic Grisebachiella, are not resolved. Metastelma scoparium, with a controversial placement (Rapini, 2002), is closely related to Orthosia urceolata (100/74). Jobinia is sister group of the Orthosia urceolata–Metastelma scoparium clade (100/71), and together they form a clade with “Cynanchum” morrenioides and “Ditassa” subtrivialis (87/<50). The main subtribal components are, however, 52 • February 2003: 33–50 included in the MOG core group (100/<50), a clade present in both parsimony analyses. Gonolobinae (100/96) are well supported, Oxypetalinae (95/<50), including Philibertia, are also present in both parsimony analyses, and Metastelmatinae (100/54) even though with high posterior probability are not present in the strict consensus tree of matrix 1. The erect, usually caespitose species of Ditassa (D. acerosa, D. decussata, D. ditassoides, D. grazielae and D. magisteriana) appear together (100/80), and D. cordeiroana is related to Petalostelma sarcostemma (93/65). Funastrum and Tassadia are not resolved in MOG core, and, as for ACTG, relationships among the component subtribes are unclear. DISCUSSION Fockeeae, including Cibirhiza Bruyns with two species and Fockea with around five species, are sister to other Asclepiadoideae, which agrees with previous phylogenetic studies (Civeyrel & al., 1998; Sennblad & Bremer, 2000; Fishbein, 2001; Potgieter & Albert, 2001) and also morphological evidence (Kunze, 1993; Kunze & al., 1994). Asclepiadoideae with erect pollinia form a monophyletic group. Due to the under-sampling of the Marsdenieae–Ceropegieae clade, however, the monophyly of Marsdenieae cannot be properly evaluated in this study (see Potgieter & Albert, 2001, for a broader molecular analysis of this group, and Bruyns & Forster, 1991, and Swarupanandan & al., 1996, for discussions of morphological characters). The rare, monotypic Eustegia, with a three-seriate corona and pendent pollinia (Bruyns, 1999a), has an unexpected position as sister to the taxa with erect pollinia (Fig. 4). Previously, pendent pollinia were treated as the ancestral state (Schumann, 1895). Currently, erect pollinia have been considered the plesiomorphic form and the pendent orientation an apomorphy of Asclepiadeae (Wanntorp, 1988; Kunze, 1993; Endress & Stevens, 2001). Few phylogenetic studies have been used to clarify this point, and studies on Asclepiadeae using members of Marsdenieae as outgroup (e.g., Liede, 2001) are unable to conclude anything concerning orientation of pollinia (contra Endress & Stevens, 2001). Some characters of Eustegia, e.g., the frail corpuscle with V-shaped profile floor and elaborate corona (Bruyns, 1999a), may represent plesiomorphic states shared with Fockeeae. Others, such as absence of a narrow neck between ovary and style head in mature flowers, are shared with taxa of Ceropegieae (Bruyns, 1999a). Although Decaisne (1844) included Eustegia and Fockea in the same “subtribe” (as division of a tribe), a relationship between Eustegia and taxa with erect pollinia has never been previously suggested due to the clearly pendent pollinia. Rapini & al. • Asclepiadeae classification If Eustegia minuta is confirmed as sister to the Marsdenieae–Ceropegieae clade, Asclepiadeae may then be divided into three main clades. Astephaninae, with 100% support, are sister to the clade that comprises the rest of the tribe, which is divided into the ACTG and MOG clades. Even though not supported by bootstrap in our analysis, the dichotomy between ACTG and MOG was supported by Bayesian inference and also the bootstrap when trnT-L intergenic spacer has been included (Liede, 2001; Liede & Täuber, 2002). MOG is restricted to the New World, whereas ACTG, despite having two New World groups, Asclepias and Cynanchum subgenus Mellichampia (A.Gray ex S.Wats.) Woodson, is centred in the Old World. Relationships among their components are still unresolved, but this dichotomy is significant for subtribal delimitation. Comparing the topology obtained from molecular data with that based on Asclepiadeae classification sensu Liede (1997b), disagreements are evident (Fig. 4; Table 2), and several changes have been made mainly by that author to improve the classification (Fig. 5). Astephaninae have been reduced to just three genera, Astephanus, Microloma and Oncinema, a group well supported by both morphological analysis (Liede, 1994, 1996a; but see Bruyns, 1999a) and molecular data (Liede, 2001). The remaining genera (Blyttia, Diplostigma, Goydera, Pentatropis, Pleurostelma Baill., Ryncharrhena F. Muell., Tylophora and Vincetoxicum) together with Biondia form a well-supported group now considered as Tylophorinae (Liede, 2001). The New World genera Barjonia, Nephradenia and Jobinia, previously included in Marsdeniinae K.Schum. (nom. superfl.) or Marsdenieae (e.g., Schumann, 1895, and Liede & Albers, 1994, respectively), have also been considered in Asclepiadeae (Endress & Bruyns, 2000; see also Rapini, 2002, and references therein), which is here supported by molecular data. Asclepiadinae sensu Liede (1997b) are confirmed, perhaps with Pergularia as sister to the rest (Fig. 5) and Asclepias nested within it. Metastelmatinae sensu Liede (1997b), on the other hand, are shown to be paraphyletic, and a division between members of New and Old World was found (Liede & Täuber, 2000; Liede, 2001), culminating in the re-instatement of Cynanchinae (Liede & Täuber, in press). Our study does support the splitting of Cynanchinae and Metastelmatinae, but the unresolved position of Cynachum acutum (the type of Cynanchum) in the ACTG clade prevents further consideration of their circumscription (Fig. 5). American Cynanchum appear to be more related to Metaplexis and the Glossonema clade than to the other Cynanchum, forming a group characterised by terminals with long branches (Fig. 5). Cynanchinae, as circumscribed by Liede & Täuber (2002), were not detected, which may be caused by the 39 Rapini & al. • Asclepiadeae classification 52 • February 2003: 33–50 100 96/92 100 86/86 99 63/63 Gonolobinae Metastelmatinae Oxypetalinae 95 --/-- 95 --/-100 --/-- 100 --/55 100 98/99 97 80/63 98 88/63 100 */61 100 80/73 100 69/66 100 --/ * 100 71/71 100 74/75 Schubertia grandiflora Gonolobus rostratus Gonolobus selloanus Matelea pedalis Schystogyne sylvestris Oxypetalum banksii Oxypetalum appendiculatum Oxypetalum coeruleum Oxypetalum capitatum Oxypetalum sublanatum Oxypetalum minarum Oxypetalum insigne Oxypetalum strictum Araujia sericifera Melinia parviflora Melinia discolor Melinia candolleana Philibertia vailiae Philibertia lysimachioides Funastrum clausum Funastrum odoratum Tassadia berteriana Tassadia obovata Metastelma parviflorum Petalostelma sarcostemma Metastelma linearifolium Metastelma schaffneri Blepharodon nitidum Blepharodon mucronatum Blepharodon lineare Nautonia nummularia Hemipogon acerosus Hemipogon luteus Hemipogon carassensis Ditassa cordeiroana Ditassa hastata Ditassa banksii Ditassa acerosa Ditassa decussata Ditassa grazielae Ditassa magisteriana Ditassa ditassoides Ditassa tomentosa Gonioanthela hilariana Nephradenia asparagoides Nephradenia acerosa Barjonia chloraeifolia Jobinia lindbergii Metastelma scoparium Orthosia urceolata Ditassa subtrivialis Cynanchum morrenioides Cynanchum nummulariifolium Astephanus geminiflorus Grisebachiella hieronymi Fig. 4. Consensus tree (posterior probability >94%) of Asclepiadoideae resulting from 40,000 trees generated from Bayesian analysis of trnL-F. The tribal (black bar, Marsdenieae; stippled bar, Ceropegieae; white bar, Asclepiadeae) and subtribal classification of Asclepiadoideae with emphasis on Asclepiadeae subtribes sensu Liede (1997b; depicted in Fig. 2) is mapped onto this topology. Numbers above branches indicate percentage of posterior probability for the clade. Numbers bellow branches indicate bootstrap percentage >49% from analyses of matrix 1 (considering gap information) and matrix 2 (not considering gap information), respectively. * = clades not present in the strict consensus tree; -- = clades <50% bootstrap present in the strict consensus tree. Upper portion of the consensus tree. 40 Rapini & al. • Asclepiadeae classification 52 • February 2003: 33–50 Asclepiadinae Astephaninae Glossonematinae Kanahia laniflora 100 78/86 Calotropis procera Stathmostelma gigantiflorum Metastelmatinae Stenostelma corniculatum Schizoglossum alpestre 99 72/61 98 72/53 Pachycarpus spurius 99 68/63 Margaretta rosea Gomphocarpus fruticosus Aspidoglossum ovalifolium 100 94/78 Glossostelma spathulatum Xysmalobium undulatum 98 66/64 100 87/85 Asclepias syriaca Asclepias curassavica Asclepias mellodora Pergularia daemia Solenostemma arghel 100 100/100 100 87/87 Glossonema boveanum Odontanthera radians Pentarrhinum insipidum Metaplexis japonica Oxystelma esculentum 100 --/ * Karimbolea verrucosa 99 61/54 Folotsia grandiflora 100 68/69 Platykeleba insignis Sarcostemma viminale Schizostephanus alatus Cynanchum acutum Cynanchum obovatum 98 82/70 100 78/78 Cynanchum montevidense Cynanchum albiflorum Cynanchum laeve Biondia henryi 100 88/85 100 74/70 Cynanchum roulinioides 100 99/99 100 83/79 Blyttia fruticulosa 100 64/65 Diplostigma canescens Goydera somaliensis Pentatropis nivalis Vincetoxicum hirundinaria Tylophora flexuosa Microloma tenuifolium 100 100/100 Oncinema lineare Astephanus triflorus Eustegia minuta Cionura erecta Dregea sinensis 98 50/ * 98 82/72 100 71/70 99 65/66 Marsdenia suberosa 98 62/60 Marsdenia zehntneri Marsdenia amorimii Telosma cordata 100 88/75 Micholitzia obcordata 100 99/98 100 100/100 Hoya australis Dischidia bengalensis 100 100/100 Stapelia leendertziae Stapelia glanduliflora Ceropegia saxatilis Fockea edulis Periploca graeca Apocynum androsaemifolium Secamone glaberrima Fig. 4 (continued). Lower portion of Asclepiadoideae consensus tree. 41 Rapini & al. • Asclepiadeae classification 52 • February 2003: 33–50 Oxypetalum coeruleum 2 Melinia parviflora 1 Melinia discolor 1 1 Melinia candolleana 94 Philibertia vailiae 1 1 * 1 Philibertia lysimachioides 3 1 *1 * Araujia sericifera Schystogyne sylvestris 2 Oxypetalinae Oxypetalum banksii Oxypetalum appendiculatum 2 Oxypetalum sublanatum Oxypetalum capitatum 1 2 Oxypetalum minarum Oxypetalum insigne 1 Oxypetalum strictum 2 2 *3 1 Blepharodon nitidum Blepharodon mucronatum Metastelma parviflorum 1 Metastelma schaffneri 6 Metastelma linearifolium Nephradenia acerosa 2 2 1 Core Group Nephradenia asparagoides Gonioanthela hilariana 3 Ditassa hastata 1 Hemipogon acerosus 2 Petalostelma sarcostemma 1 93/65 2 * 3 2 78 2 1 *1 Ditassa cordeiroana Nautonia nummularia 1 Hemipogon carassensis Ditassa banksii 1 6 Ditassa acerosa Ditassa grazielae 1 * Metastelmatinae Barjonia chloraeifolia Hemipogon luteus 2 * Ditassa magisteriana Ditassa ditassoides 2 Ditassa decussata Ditassa tomentosa 5 MOG 2 2 2 * 1 4 * Funastrum odoratum 4 3 2 * * Tassadia berteriana Cynanchum nummulariifolium Grisebachiella hieronymi 3 Ditassa subtrivialis 7 2 Cynanchum morrenioides 4 2 * Matelea pedalis Tassadia obovata Astephanus geminiflorus 7 1 * Gonolobinae 1 4 64 Gonolobus rostratus Gonolobus selloanus 2 *5 1 Schubertia grandiflora 2 3 5 1 Blepharodon lineare Funastrum clausum 2 * 2 3 Jobinia lindbergii Metastelma scoparium Orthosia urceolata 5 changes Fig. 5. Majority-rule consensus tree of 40,000 trees generated from Bayesian analysis of trnL-F, showing an updated classification after phylogenetic studies based on molecular data: Asclepiadinae sensu Liede (1997b), Astephaninae and Tylophorinae sensu Liede (2001), and Cynanchinae sensu Liede & Täuber (2002). Numbers of substitutions are indicated above branches. Percentage of posterior probabilities of clades >50% and <95% are under branches, with bootstrap percentage >50% from analysis of matrix 1 (containing gap information). * = clades >94% probability. 42 Rapini & al. • Asclepiadeae classification 52 • February 2003: 33–50 2 MOG * 1 Karimbolea verrucosa 1 Folotsia grandiflora Sarcostemma viminale 2 * 2 1 2 * 2 * 2 1 Cynanchinae Platykeleba insignis 2 Kanahia laniflora 2 5 Calotropis procera * Stathmostelma gigantiflorum 2 Stenostelma corniculatum 2 Pachycarpus spurius 1 Xysmalobium undulatum 3 Schizoglossum alpestre * Glossostelma spathulatum 1 Margaretta rosea 1 Asclepiadinae 6 7 1 Gomphocarpus fruticosus Aspidoglossum ovalifolium Asclepias syriaca 3 1 Asclepias curassavica 2 2 * Asclepias mellodora * Pergularia 7 daemia 1 Schizostephanus alatus Cynanchinae 1 Blyttia fruticulosa 2 Diplostigma canescens * 1 1 Goydera somaliensis * Tylophorinae 1 Tylophora flexuosa 3 * 3 Vincetoxicum hirundinaria Biondia henryi 6 Pentatropis nivalis * 93/84 6 1 2 1 * 21 1 * * * 11 * 4 1 * 1 1 * 8 3 * * 2 * 14 4 Fockea edulis Cynanchum albiflorum Cynanchum laeve Cynanchinae 4 2 4 * 4 86/52 * Pentarrhinum insipidum 4 7 Glossonema boveanum 9 * 1 Odontanthera radians * 18 Metaplexis japonica Solenostemma arghel 15 Oxystelma esculentum Microloma tenuifolium 2 Astephanus triflorus Astephaninae 2 Oncinema lineare 4 Cionura erecta 7 Telosma cordata 6 3 10 8 2 67/86 5 Cynanchum acutum 1 Cynanchum obovatum 57 6 Cynanchum roulinioides 2 12 Cynanchum montevidense 1 60 8 5 17 4 * ACTG Dregea sinensis Marsdenia suberosa Marsdenia amorimii Marsdenia zehntneri Marsdenieae 6 Micholitzia obcordata Hoya australis 6 Dischidia bengalensis 2 Stapelia leendertziae 6 2 Stapelia glandulifora 24 2 * Ceropegia saxatilis * Eustegia minuta Eustegiinae 3 2 Ceropegieae 21 Periploca graeca Apocynum androsaemifolium Secamone glaberrima 12 5 5 changes Fig. 5 (continued). Lower portion of Asclepiadoideae of the majority-rule consensus tree. ACTG-clade including Asclepiadinae, Cynanchinae, Tylophorinae and Glossonematinae. MOG-clade including Metastelmatinae, Oxypetalinae and Gonolobinae. Black bar, taxa with erect pollinia; white bar, taxa with pendent pollinia. New World clades are indicated by bold lines. 43 Rapini & al. • Asclepiadeae classification 52 • February 2003: 33–50 Table 2. Classification of genera treated in this study according to Schumann (1895), more recent general classifications (Fig. 2), and phylogenetic studies summarised in this paper (Figs. 4 and 5). Tribes are in all capital letters; groups are recognised in Asclepiadeae, unless mentioned. ACTG –group comprises members of Asclepiadinae, Cynanchinae, Tylophorinae, and Glossonematinae; MOG – group comprises members of Metastelmatinae, Oxypetalinae and Gonolobinae (Fig. 5). * denotes circumscription that contradicts the main patterns of relationship obtained with trnL-F. Liede & Albers, 1994; Phylogenetics Genus Schumann, 1895 Liede, 1997b (Fig. 2) (Figs. 4 and 5) Araujia Brot. Glossonematinae* Oxypetalinae Oxypetalinae Asclepias L. Asclepiadinae Asclepiadinae Asclepiadinae Aspidoglossum E. Mey. Asclepiadinae Asclepiadinae Asclepiadinae Astephanus R. Br. Astephaninae Astephaninae Astephaninae Barjonia Decne. TYLOPHOREAE (Marsdeniinae)* MARSDENIEAE* ASCLEPIADEAE (Metastelmatinae) Biondia Schltr. 1905 Metastelmatinae* Tylophorinae Blepharodon Decne. Asclepiadinae* Metastelmatinae Metastelmatinae Blyttia Arn. Cynanchinae* Astephanineae* Tylophorinae Calotropis R. Br. Asclepiadinae Asclepiadinae Asclepiadinae Ceropegia L. TYLOPHOREAE (Ceropegiinae) CEROPEGIEAE CEROPEGIEAE Cionura Griseb. TYLOPHOREAE (Marsdeniinae) MARSDENIEAE MARSDENIEAE Cynanchum L. Cynanchinae Metastelmatinae* Cynanchinae Diplostigma K.Schum. Astephaninae* Astephaninae* ACTG Dischidia R. Br. TYLOPHOREAE (Marsdeniinae) MARSDENIEAE MARSDENIEAE Ditassa R. Br. Asclepiadinae* Metastelmatinae Metastelmatinae Dregea E. Mey. TYLOPHOREAE (Marsdeniinae) MARSDENIEAE MARSDENIEAE Eustegia R. Br. Asclepiadinae* Astephanineae* Eustegiinae Fockea Endl. TYLOPHOREAE (Marsdeniiinae)* FOCKEEAE FOCKEEAE Folotsia Costantin & Bois 1908 Metastelmatinae* ACTG Funastrum E. Fourn. Asclepiadinae* Metastelmatinae MOG Glossonema Decne. Glossonematinae Glossonematinae ACTG Glossostelma Schltr. 1895 Asclepiadinae Asclepiadinae Gomphocarpus R. Br. Asclepiadinae Asclepiadinae Asclepiadinae Gonioanthela Malme 1927 Metastelmatinae Metastelmatinae Gonolobus Michx. GONOLOBEAE* ASCLEPIADEAE (Gonolobinae) ASCLEPIADEAE (Gonolobinae) Goydera Liede 1993 Astephaninae* ACTG Grisebachiella Lorentz ? Metastelmatinae* MOG (but outside core group) Hemipogon Decne. Astephaninae* Metastelmatinae Metastelmatinae Hoya R. Br. TYLOPHOREAE (Marsdeniinae) MARSDENIEAE MARSDENIEAE Jobinia E. Fourn. TYLOPHOREAE (Marsdeniinae)* MARSDENIEAE* ASCLEPIADEAE (MOG) Kanahia R. Br. Asclepiadinae Asclepiadinae Asclepiadinae Karimbolea Descoings 1960 Metastelmatinae* ACTG Margaretta Oliv. Asclepiadinae Asclepiadinae Asclepiadinae Marsdenia R. Br. TYLOPHOREAE (Marsdeniinae) MARSDENIEAE MARSDENIEAE Matelea Aubl. GONOLOBEAE* ASCLEPIADEAE (Gonolobinae) ASCLEPIADEAE (Gonolobinae) Melinia Decne. Asclepiadinae* Oxypetalinae Oxypetalinae Metaplexis R. Br. Cynanchinae Metastelmatinae* ACTG Metastelma R. Br. Asclepiadinae* Metastelmatinae Metastelmatinae Micholitzia N. E.Br. 1909 MARSDENIEAE MARSDENIEAE Microloma R. Br. Astephaninae Astephaninae Astephaninae Nautonia Decne. Astephaninae* Metastelmatinae Metastelmatinae Nephradenia Decne. TYLOPHOREAE* (Marsdeniinae) MARSDENIEAE* ASCLEPIADEAE (Metastelmatinae) OdontantheraWight Glossonematinae Glossonematinae ACTG Oncinema Arn. Cynanchinae?* Astephaninae Astephaninae Orthosia Decne. Cynanchinae* Metastelmatinae* MOG (but outside core group) Oxypetalum R. Br. Oxypetalinae Oxypetalinae Oxypetalinae Oxystelma R. Br. Glossonematinae Metastelmatinae* ACTG Pachycarpus E. Mey. Asclepiadinae Asclepiadinae Asclepiadinae 44 52 • February 2003: 33–50 Rapini & al. • Asclepiadeae classification Table 2. (continued). Liede & Albers, 1994; Phylogenetics Genus Schumann, 1895 Liede, 1997b (Fig. 2) (Figs. 4 and 5) Pentarrhynum E. Mey. Asclepiadinae Metastelmatinae* ACTG Pentatropis R. Br. Cynanchinae Astephaninae* ACTG Pergularia L. TYLOPHOREAE* (Marsdeniinae) ASCLEPIADEAE (Asclepiadinae) ASCLEPIADEAE (Asclepiadinae) Petalostelma E. Fourn. TYLOPHOREAE* (Marsdeniinae)ASCLEPIADEAE (Metastelmatinae) ASCLEPIADEAE (Metastelmatinae) Philibertia Kunth. Glossonematinae* Metastelmatinae* Oxypetalinae Platykeleba N.E. Br. 1895 Metastelmatinae* ACTG Sarcostemma R. Br. Cynanchinae Metastelmatinae* ACTG Schistogyne Hook. & Arn. Asclepiadinae* Oxypetalinae Oxypetalinae SchizoglossumE.Mey. Asclepiadinae Asclepiadinae ACTG SchizostephanusHochst. ex Benth. Cynanchinae Astephaninae* ACTG Schubertia Mart. Glossonematinae* Gonolobinae Gonolobinae Solenostemma Hayne Glossonematinae Glossonematinae ACTG Stapelia L. TYLOPHOREAE (Ceropegiinae) CEROPEGIEAE CEROPEGIEAE Stenostelma Schltr. Asclepiadinae Asclepiadinae Asclepiadinae Stathmostelma K. Schum. Asclepiadinae Asclepiadinae Asclepiadinae Tassadia Decne. Asclepiadinae* Metastelmatinae MOG Telosma Coville 1905 MARSDENIEAE MARSDENIEAE Tylophora R. Br. TYLOPHOREAE (Marsdeniinae)* ASCLEPIADEAE (Astephaninae)* ASCLEPIADEAE (ACTG) Vincetoxicum Wolf. Cynanchinae Astephaninae* ACTG Xysmalobium R. Br. Asclepiadinae Asclepiadinae Asclepiadinae smaller sequence as well as the inclusion of a greater diversity of ACTG taxa used in our study. According to their study, Cynanchinae would comprise Cynanchum and Sarcostemma, as well as the small genera Schizostephanus, Glossonema, Metaplexis, Pentarrhinum, Odontanthera, Folotsia, Karimbolea and Platykeleba. Cynanchum has had an unstable circumscription, and opinions on its delimitation have varied drastically. Nevertheless, Woodson’s (1941) concept of Cynanchum including many American genera of Metastelmatinae as well as Liede’s (1997c) infrageneric classification of the American Cynanchum may be discarded (see Liede & Täuber, 2002, for details on Cynanchum classification). The re-classification of several species of American Cynanchum (e.g., C. morrenioides and C. nummulariifolium) that are clearly unrelated to the C. acutum clade is required; others (e.g., C. montevidense and C. roulinioides; Fig. 5) might belong to Cynanchum (Liede & Täuber, 2002). These conclusions, however, are derived from the MOG–ACTG dichotomy (Fig. 5); the appropriate circumscription for Cynanchum and even Cynanchinae among ACTG groups is still unclear (Fig. 5). MOG represents the most diverse New World group, ranging from the showy, apocynoid-like flowers of Schubertia to the tiny flowers of Tassadia and from the minute sclerophyllous leaves of erect subshrubs of some Ditassa to the large leaves of long vines of Gonolobus. This huge morphological diversity, however, is not reflected in molecular diversity of trnL-F, contrasting with the morphologically less diverse American Cynanchum clade, which is characterised by a much greater level of molecular change. The three subtribes of MOG (Metastelmatinae, Oxypetalinae and Gonolobinae) can be recognised in the DNA tree, but the limited numbers of substitutions within the clades are insufficient to delineate generic patterns. Metastelmatinae, even reduced to American groups, are still unsatisfactory, and the exclusion of Orthosia and Jobinia as well as the transfer of Philibertia to Oxypetalinae should be considered. The position of the Orthosia-Jobinia clade and other species outside the MOG core group (Fig. 3b) indicates the need for new subtribes to accommodate these New World groups. Gonolobinae genera form a wellsupported group but of uncertain position in the MOG clade. Their inclusion in Asclepiadeae is appropriate, whereas their reinstatement at tribal level suggested by Endress & Stevens (2001) would be hierarchically incongruent. CONCLUSIONS These data, analysed by both Bayesian inference and parsimony methods, detected major patterns of relationships in Asclepiadoideae. Excluding the lack of evidence for Cynanchinae, the topology is congruent with those presented in studies that also included the trnT-L intergenic spacer, and confidence in clades estimated by bootstrap and posterior probability provides an evaluation of 45 Rapini & al. • Asclepiadeae classification current knowledge of phylogenetic relationships in Asclepiadeae. Since Eustegia might be sister to the Marsdenieae–Ceropegieae clade and treated as the only genus of the very reduced Eustegiinae Decne., Astephaninae may represent the sister group of the other Asclepiadeae. The rest of the tribe can then be divided into ACTG, including two American groups, and MOG, restricted to the New World. In the ACTG clade, Asclepiadinae and Tylophorinae are supported, but this is not true for Cynanchinae and Glossonematinae. Relationships among components of the ACTG clade are not clear, and the unresolved position of the nomenclaturally critical species Cynanchum acutum in a weakly supported clade of Cynanchinae permits several alternative hypotheses. Taxonomic changes (e.g., Liede & Meve, 2001) based exclusively on these molecular data, therefore, are premature. Conclusions about Glossonematinae (Liede & al., 2002) are also tied to the resolution of ACTG components and the consequent definition of Cynanchinae. Although not linked to any published phylogenetic analysis, Liede’s (1997b) arrangement of genera is closer to the one coming out of phylogenetic studies than is Schumann’s (1895) classification (Table 2). In only five years, however, several changes have been made, and many others are still needed to achieve a classification based on monophyletic groups. The phylogeny of New World Asclepiadoideae has not yet been well explored, and, until a better resolution of relationships indicates a reasonable generic rearrangement, taxonomic revisions of such groups are likely to produce ephemeral publications. Improvement in the understanding of Asclepiadoideae is evident, but it is too early to formalise taxonomic groups based on such weak patterns as those observed in our analyses of trnL-F. In this overview, four clades of New World Asclepiadoideae were detected (Fig. 3). (1) The American Marsdenia (Marsdenieae), comprising around 60 species, may form a monophyletic group, but analyses including a large diversity of the subtribe are needed to confirm this. (2) American Cynanchum seems to be restricted to the subgenus Mellichampia (Liede & Täuber, 2002), which may include C. roulinioides and the species classified in Metalepis Griseb., comprising a group with around 20 species. However, according to the molecular data presented here, this group seems to be more closely related to the Glossonema clade and Metaplexis than to other Cynanchum. (3) Asclepias with around 130 species, most of which are distributed in North and Central America, represents the only American group of Asclepiadinae and is nested within African genera. (4) MOG comprises members of the exclusively American Metastelmatinae (including species of “Cynanchum” and “Astephanus” that do not 46 52 • February 2003: 33–50 belong to the ACTG clade), Oxypetalinae, and Gonolobinae. With around 600 species, the MOG clade includes the great majority of American Asclepiadoideae, having their centre of diversity in South America. Based on these results, three main topics should be explored for an improved understanding of New World Asclepiadoideae: (1) the relationship between American Marsdenia and the rest of Marsdenieae, especially the Old World Marsdenia groups; (2) the position of Cynanchum subgenus Mellichampia in the ACTG clade for which the placement of C. acutum is crucial; and (3) a massive study of relationships within the large and morphologically diverse MOG clade. ACKNOWLEDGEMENTS This paper is part of the results of a postdoctoral fellowship of the first author (AR) at Royal Botanic Gardens, Kew, funded by the Mellon Foundation. Most of the material sequenced for this study was collected during the Ph.D. of AR, funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES). Renato de Mello-Silva, Rafaela C. Forzza, Tatiana Konno, and Maria Ana Farinaccio kindly provided material for DNA extraction, and Sigrid Liede gave access to unpublished manuscripts (and sequences used therein). We also thank Mary Endress for reading and encouraging this publication and an anonymous reviewer for criticisms and suggestions. LITERATURE CITED Bruyns, P. V. 1999a. The systematic position of Eustegia R. Br. (Apocynaceae–Asclepiadoideae). Bot. Jahrb. Syst. 121: 19–44. Bruyns, P. V. 1999b. Subtribes and genera of Asclepiadeae—a response to Liede. Taxon 48: 23–26. Bruyns, P. V. 2000. Phylogeny and biogeography of the stapeliads. 1 Phylogeny. Pl. Syst. Evol. 221: 199–226. Bruyns, P. V. & Forster, P. I. 1991. Recircumscription of Stapelieae (Asclepiadaceae). Taxon 40: 381–391. Chase, M. W. & Hills, H. G. 1991. Silica gel: an ideal material for field preservation of leaf samples for DNA studies. Taxon 40: 215–220. Chase, M. W., Soltis, D. E., Olmstead, R. 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Sequences not yet available on GenBank were obtained from Liede’s homepage (www.unibayreuth.de/departments/planta2/wgl/fsigrid2.html). i. s. = intergenic spacer; NW = New World; OW = Old World; ? = cultivated source and/or without stated provenance. Species Distribution Apocynum androsaemifolium L. U.S.A. Araujia sericifera Brot. Australia (native to NW) Asclepias curassavica L. Brazil A. mellodora A.St.-Hil. Brazil A. syriaca L. Germany (native to NW) Aspidoglossum ovalifolium (Schltr.) Kupicha South Africa Astephanus geminiflorus Decne. Chile A. triflorus R. Br. South Africa Barjonia chloraeifolia Decne. Brazil Biondia henryi (Warb. ex Schltr. & Diels) Tsiang & P.T. Li China Blepharodon lineare (Decne.) Decne. Argentina B. mucronatum (Schltdl.) Decne. Belize B. nitidum (Vell.) J.F. Macbr. Brazil Blyttia fruticulosa (Decne.) D.V. Field Kenya Calotropis procera (Aiton) W.T. Aiton Puerto Rico (native to OW) Ceropegia saxatilis Jum. & H. Perrier Madagascar Cionura erecta Griseb. Turkey Cynanchum acutum L. Egypt C. albiflorum (Urban) Liede Ecuador C. laeve (Michx.) Pers. U.S.A. C. montevidense Spreng. Argentina C. morrenioides Goyder Brazil C. nummulariifolium Hook. & Arn. Argentina C. obovatum Choux. Madagascar C. roulinioides (E. Fourn.) Rapini Brazil Diplostigma canescens K. Schum. Kenya Dischidia bengalensis Colebr. ? (OW) Ditassa acerosa Mart. Brazil D. banksii R.Br. ex Schult. Brazil D. cordeiroana Fontella Brazil D. decussata Mart. Brazil D. ditassoides (Silveira) Fontella Brazil D. grazielae Fontella & Marquete Brazil D. hastata Decne. Brazil D. magisteriana Rapini Brazil D. subtrivialis Griseb. Bolivia D. tomentosa (Vell.) Fontella Brazil 48 trnL intron trnL-F i. s. AF214308 AY163662 AY163664 AY163665 AF214311 AY163666 AJ410182 AJ410188 AY163667 AJ410191 AY163668 AJ290840 AY163669 AJ410194 AF214324 AJ410041 AJ410173 AY163670 AJ428775 AJ428652 AJ290849 AJ428685 AJ290852 AJ428802 AY163672 AJ410200 AF214343 AY163673 AY163674 AY163675 AY163677 AY163678 AY163679 AY163680 AY163681 AJ428755 AY163682 AF214154 AY163663 AY163664 AY163665 AJ410180 AY163666 AJ410183 AJ410189 AY163667 AJ410192 AY163668 AJ290841 AY163669 AJ410195 AF214170 AJ410042 AJ410174 AY163671 AJ428776 AJ428653 AJ290850 AJ428684 AJ290851 AJ428803 AY163672 AJ410201 AF214189 AY163673 AY163674 AY163676 AY163677 AY163678 AY163679 AY163680 AY163681 AJ428756 AY163682 Voucher Forster 7656 (K) Rapini 933 (SPF) Rapini 411 (SPF) Balkwill 10847 (K) Rapini 485 (SPF) Forzza 2027 (SPF) Rapini 938 (SPF) Boulos s.n. (K) Rapini 726 (SPF) Rapini 383 (SPF) Konno 754 (SPF) Rapini 347 (SPF) Rapini 556 (SPF) Rapini 557 (SPF) Rapini 379 (SPF) Rapini 777 (SPF) Rapini 597 (SPF) Rapini 616 (SPF) Rapini & al. • Asclepiadeae classification 52 • February 2003: 33–50 Appendix 1 (continued). Species Distribution Dregea sinensis Hemsl. ? (OW) Eustegia minuta (L.f.) N.E. Br. South Africa Fockea edulis K.Schum. ? (OW) Folotsia grandiflora (Jum. & H. Perrier) Madagascar Jum. & H. Perrier Funastrum clausum (Jacq.) Schltr. Argentina F. odoratum (Hemsl.) Schltr. Mexico Glossonema boveanum (Decne.) Decne. Yemen Glossostelma spathulatum (K. Schum.) Bullock Zimbabwe Gomphocarpus fruticosus (L.) W.T. Aiton Egypt Gonioanthela hilariana (E. Fourn.) Malme Brazil Gonolobus rostratus (Vahl) Schult. Ghana? (native NW) G. selloanus (E. Fourn.) Bacigalupo Brazil Goydera somaliensis Liede Somalia Grisebachiella hieronymi Lorentz Argentina Gymnema sylvestre (Retz.) R. Br. ex Schult. Cameroon Hemipogon acerosus Decne. Brazil H. carassensis (Malme) Rapini Brazil H. luteus E.Fourn. Brazil Hoya australis R. Br. ex J. Traill ? (OW) Jobinia lindbergii E. Fourn. Brazil Kanahia laniflora (Forssk.) R. Br. Tanzania Karimbolea verrucosa Desc. Madagascar Margaretta rosea Oliv. Tanzania Marsdenia amorimii Morillo Brazil M. suberosa (E. Fourn.) Malme Brazil M. zehntneri Fontella Brazil Matelea pedalis (E. Fourn.) Fontella & E.A. Schwarz Brazil Melinia candolleana (Hook. & Arn.) Decne. Argentina M. discolor Schltr. Argentina M. parviflora (Malme) Krap. & Cáceres Argentina Metaplexis japonica Makino Russia Metastelma linearifoliumA. Rich ? (NW) M. parviflorum (Sw.) R. Br. ex Schult. Puerto Rico M. schaffneri A. Gray Mexico M. scoparium (Nutt.) Vail Brazil Micholitzia obcordata N.E. Br. Thailand Microloma tenuifolium K. Schum. South Africa Nautonia nummularia Decne. Argentina Nephradenia acerosa Decne. Brazil N. asparagoides (Decne.) E. Fourn. Brazil Odontanthera radians (Forssk.) D.V. Field Yemen Oncinema lineare (L.f.) Bullock South Africa Orthosia urceolata E. Fourn. Brazil Oxypetalum appendiculatumMart. Brazil O. banksii R. Br. ex Schult. Brazil O. capitatum Mart. Argentina Oxypetalum coeruleum (D. Don ex Sweet) Decne. ? (NW) O. insigne (Decne.) Malme Brazil O. minarum E. Fourn. Brazil O. strictum Mart. Brazil O. sublanatum Malme Brazil trnL intron trnL-F i. s. AF214345 AJ410206 AF214353 AJ290855 AF214191 AJ410207 AF214199 AJ290856 AY163683 AJ290873 AY163684 AY163686 AY163687 AY163688 AF214362 AY163689 AJ410209 AJ410212 AJ402137 AY163690 AY163692 AY163693 AF214367 AY163694 AY163695 AJ290880 AY163696 AF214377 AY163697 AY163698 AY163699 AJ410176 AY163700 AJ410224 AJ428811 AY163683 AJ290874 AY163685 AY163686 AY163687 AY163688 AF214208 AY163689 AJ410210 AJ410213 AJ402142 AY163691 AY163692 AY163693 AF214213 AY163694 AY163695 AJ290879 AY163696 AF214223 AY163697 AY163698 AY163699 AJ410177 AY163700 AJ410225 AJ428812 AY163701 AJ410215 AY163703 AF214381 AJ410221 AJ410227 AY163704 AY163706 AJ428814 AJ410230 AY163708 AY163709 AY163710 AY163711 AF214443 AY163712 AY163713 AY163714 AY163715 AY163702 AJ410216 AY163703 AF214227 AJ410222 AJ410228 AY163705 AY163707 AJ428815 AJ410231 AY163708 AY163709 AY163710 AY163711 AF214289 AY163712 AY163713 AY163714 AY163715 Voucher Mello-Silva 1919 (SPF) Rowaished 3014 (K) Goyder 4108 (K) Chase 9370 (K) Rapini 710 (SPF) Rapini 609 (SPF) Forzza 599 (SPF) Rapini 778 (SPF) Forzza 610 (SPF) Farinaccio 194 (SPF) Goyder 3791 (K) Goyder 3931 (K) Rapini 384 (SPF) Pirani 4399 (SPF) Rapini 714 (SPF) Mello-Silva 1887 (SPF) Liede homepage Axelrod 8328 (K) Rapini 932 (SPF) Philcox 3303 (K) Irwin 13012 (K) Rapini 934 (SPF) Rapini 613 (SPF) Rapini 911 (SPF) Mello-Silva 1924 (SPF) Rapini 716 (SPF) Rapini 908 (SPF) Rapini 811 (SPF) Rapini 937 (SPF) 49 Rapini & al. • Asclepiadeae classification 52 • February 2003: 33–50 Appendix 1 (continued). Species Distribution trnL intron trnL-F i. s. Oxystelma esculentum (L.f.) Sm. Egypt Pachycarpus spurius (N.E. Br.) Bullock Tanzania Pentarrhinum insipidum E.Mey South Africa Pentatropis nivalis (J.F. Gmel.) D.V. Field & J.R.I. Wood Kenya Pergularia daemia (Forssk.) Chiov. Tanzania Periploca graeca L. ? (OW) Petalostelma sarcostemma (Lillo) Liede & Meve Argentina Philibertia lysimachioides (Wedd.) T. Mey. Argentina P. vailiae (Rusby) Liede Argentina Platykeleba insignis N.E.Br. Madagascar Sarcostemma viminale (L.) R. Br. Zimbabwe Schistogyne sylvestris Hook. & Arn. Argentina Schizoglossum alpestre K. Schum. Tanzania Schizostephanus alatus Hochst. ex K. Schum. Tanzania Schubertia grandiflora Mart. Argentina Secamone glaberrima K. Schum. Madagascar Solenostemma arghel (Delile) Hayne Egypt Stapelia glanduliflora Masson South Africa S. leendertziae N.E. Br. ? (OW) Stathmostelma gigantiflorum K.Schum. Kenya Stenostelma corniculatum (E. Mey.) Bullock South Africa Tassadia berteriana (Spreng.) W.D. Stevens Bolivia T. obovata Decne. Costa Rica Telosma cordata (Burm.f.) Merr. ? (OW) Tylophora flexuosa R. Br. Philippines Vincetoxicum hirundinaria Medic. Germany Xysmalobium undulatum (L.) W.T. Aiton South Africa AJ290885 AY163716 AJ410233 AJ410239 AJ290892 AF102468 AJ 428787 AY163717 AJ290904 AJ290907 AJ290913 AJ410245 AY163718 AJ410248 AJ428826 AF214420 AY163719 AJ402128 AF214424 AY163721 AY163722 AJ428790 AY163723 AF214280 AJ290916 AJ410275 AY163725 AJ290887 AY163716 AJ410234 AJ410240 AJ290893 AF214244 AJ428788 AY163717 AJ290905 AJ290906 AJ290912 AJ10246 AY163718 AJ410249 AJ428827 AF214266 AY163720 AJ402151 AF214270 AY163721 AY163722 AJ428791 AY163724 AF102493 AJ290917 AJ410276 AY163725 50 Voucher Goyder 3781 (K) Mello-Silva 1886 (SPF) Goyder 3892 (K) Boulos19142 (K) Harvey 64 (K) Balkwill 10908 (K) Angulo 300 (K) Balkwill 10846 (K)