Org. Divers. Evol. 2, 239–269 (2002) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ode Cynanchum and the Cynanchinae (Apocynaceae – Asclepiadoideae): a molecular, anatomical and latex triterpenoid study Sigrid Liede1,*, Henning Kunze2 1 2 Department of Plant Systematics, University of Bayreuth, Germany Minden, Germany Received 22 April 2002 · Accepted 29 July 2002 Abstract The phylogeny of the genus Cynanchum s. str. is studied using cpDNA spacers and ITS. Morphological, anatomical and latex triterpenoid data are interpreted in light of the molecular results, and discrepancies are discussed. Vegetative characters are better indicators of relationship than floral characters, especially corona characters. The monophyly of all Malagasy species and, nested within the latter, of all stem-succulent taxa is ascertained and the genera Folotsia, Karimbolea, Platykeleba and Sarcostemma are subsumed under Cynanchum. One African species, C. galgalense, is excluded from Cynanchum. Key words: Asclepiadoideae, cpDNA, ITS, latex triterpenoids, molecular phylogeny, stem anatomy Introduction The speciose genus Cynanchum L. has long been a “dustbin genus” for everything with a gynostegial, at least basally fused corona, C(is) sensu Liede & Kunze (1993). In the New World, the concept of Cynanchum was particularly confused since Woodson (1941) united a whole range of genera under Cynanchum, based mainly on North American material. But unlike Asclepias L., New World Cynanchum was never homogeneous. Sundell (1981) revised the most distinct subgenus Mellichampia (A.Gray ex S. Watson) Sundell, characterized by comparatively large flowers and heart-shaped leaves with “stipules” (extremely reduced short shoots with a pair of smaller and sometimes differently shaped leaves). Analysis of New World and Old World members of Cynanchum L. using cpDNA spacers (Liede & Täuber in press) has shown that in the New World only subgen. Mellichampia is correctly associated with Cynanchum s. str., whereas all other former members of the genus belong to an unrelated, exclusively American group of genera. Liede & Täuber (in press) restricted subtribe Metastelminae Endl. ex Meisn. to the American genera, and resurrected subtribe Cynanchinae K. Schum. for Cynanchum s. str. (incl. subgen. Mellichampia) and some smaller, related genera, exclusively of Old World origin. In the Old World, Vincetoxicum Wolf was suggested by Liede (1996a) to be a relative of Tylophora R. Br., rather than of Cynanchum, based on chemistry. This has been supported by various molecular studies (Civeyrel et al. 1998; Sennblad & Bremer 2000, 2002; Liede 2001). The cpDNA spacer analysis (Liede & Täuber in press), however, showed that even in the Old World the generic concept of Cynanchum is more complex than hitherto assumed, and that some of the small Asian genera (e.g., Metaplexis R. Br.) probably ought to be included in Cynanchum. In addition, this study indicated that the Old World stem succulent genera Folotsia Costantin & Bois, Karimbolea Desc., Platykeleba N. E. Br., and Sarcostemma R. Br. are nested within the Malagasy subclade of Cynanchum. However, Liede & Täuber (in press) refrained from nomenclatural consequences both in the New World and in the Old World, because a single dataset was not considered sufficient for the extensive nomenclatural changes necessary to reflect these new results. The present paper focuses on Cynanchum s. str., in particular on the status of the small succulent genera *Corresponding author: Sigrid Liede, Department of Plant Systematics, University of Bayreuth, D-95440 Bayreuth, Germany; e-mail: sigrid.liede@uni-bayreuth.de 1439-6092/02/02/03-239 $ 15.00/0 Org. Divers. Evol. (2002) 2, 239–269 240 Liede & Kunze (Folotsia, Karimbolea, Platykeleba, Sarcostemma), using evidence from a second molecular marker, ITS, as well as from stem anatomy and triterpenoid analysis. modified protocol based on Baldwin (1992) described in Meve & Liede (2001). ITS sequences of 87 species were obtained for the present study, two sequences have been used in a previous study of the senior author. Materials and methods Data analysis. cpDNA sequences were pre-aligned with Perkin Elmer Sequence Navigator Version 1.0.1, the alignment then adjusted manually. The alignment comprises 92 taxa and 2178 characters; 129 data cells and the whole trnT-L spacer of C. schistoglossum were unknown. In the trnL-F spacer, bp755–806 were excluded from all analyses, because alignment in this region is ambiguous. Indels were coded as “missing characters“; parsimony informative indels were coded separately as present/absent following the “simple gap coding” method of Simmons & Ochoterena (2000). Indels present or absent only in different accessions of the same species were not coded. Indels of doubtful homology (e.g., bp186–188, 186–189 in the trnT-L spacer of C. obtusifolium and C. madagascariense, respectively) were not coded, nor were indels caused by variable length of a chain of at least four repeats of the same base, because the length of these chains was found to vary even within the same species (Verhoeven et al. in press). In total, 48 indels were coded, 32 in the trnT-L spacer, 9 in the trnL intron and 7 in the trnL-F spacer. Sequences of rDNA were pre-aligned with Perkin Elmer Sequence Navigator Version 1.0.1, the alignment then adjusted manually. The alignment comprises 89 taxa and 827 characters, 27 data cells are unknown. Separate coding of indels was not conducted because of the heterogeneity of the dataset. All alignments are available from the senior author and can be viewed in TreeBase (Sanderson et al. 1994): study accession number = S776, matrix accession numbers = M1230, M1231. Sequence analysis, phylogenetic analysis and tests for clade support were performed using PAUP (Swofford 1998), version 4.0b5 (PPC), on a MacIntosh G3 Powerbook. Sequence divergence among taxa was calculated using the “Show Pairwise Distance” option for the trnTL spacer, the trnL intron and the trnL-F spacer, as well as for 5.8s, ITS1 and ITS2 (excluding the end of 18s and the beginning of 26s). Beginning and end of ITS1 and ITS2 were determined by comparison with Asclepiadoideae sequences submitted to EMBL (AJ402152–AJ402162, Meve & Liede 2001). As the dataset was too large for complete analysis, a search strategy in two steps was performed in all parsimony analyses in order to find as many tree islands as possible. First, starting trees were obtained (addition sequence random, 1000 replicates, TBR branch swapping, “MulTrees” off, “steepest descent” off). The resulting shortest starting trees were then subjected to TBR DNA sequence analysis Taxa. Following the results of Liede & Täuber (in press), Schizostephanus alatus was chosen as outgroup. More distantly related outgroups such as members of the Asclepiadinae (sensu Liede 1997b) or Tylophorinae (sensu Liede 2001) were not included in the analysis, because alignment of ITS sequences is problematic between members of different subtribes, and no other basal member of Cynanchinae has yet been identified among the 67 out of approx. 100 genera of Asclepiadeae for which cpDNA sequences are available (Liede 2001; Liede & Täuber 2000, in press; Liede et al. 2002, and unpubl.). The ingroup comprises 91 accessions and 85 species, because two different accessions were included for six species (Pentarrhinum somaliense, C. falcatum, C. galgalense, C. gerrardii, C. orangeanum, C. thesioides; Table 1, see there for authors of species). For C. chouxii, C. ligulatum, and one accession of C. galgalense, no ITS sequence could be obtained, for C. schistoglossum, the trnT-L spacer of cpDNA is missing. Eight species of the ingroup are members of the American Cynanchum subgen. Mellichampia and Metalepis Griseb., four species are Asian, including two members of Cynanchum sect. Rhodostegiella (Pobed.) Tsiang and one member of Metaplexis, one species (C. floribundum) is Australian. From the African mainland, twenty leafy species were included, constituting more than half of all known African Cynanchum species (Liede 1996b). From Madagascar, eighteen leafy species were included, again almost half of all known leafy Malagasy species. Of stem succulents, twenty Malagasy Cynanchum were included, as well as a collection of C. gerrardii from the African mainland. Eight species belonging to the small succulent genera Folotsia, Karimbolea, Platykeleba and Sarcostemma were also included, six of them from Madagascar. DNA extraction and PCR. DNA was isolated from fresh or dried leaf tissue according to Doyle & Doyle (1987). PCR primers and protocol for the chloroplast DNA (cpDNA) trnT-L and trnL-F spacers and the trnL intron correspond to Taberlet et al. (1991). cpDNA spacers of 19 species were sequenced for the present study, the remaining species have been used in previous studies of the senior author. The entire internal transcribed spacer region (ITS) including 5.8S of ribosomal DNA (rDNA) was amplified using the flanking primers ITS4 and ITS5 following a Org. Divers. Evol. (2002) 2, 239–269 Cynanchum and the Cynanchinae 241 Table 1. Materials used for DNA, anatomical, and triterpenoid studies. Taxa in boldface have been analyzed for stem anatomy, underlined taxa have been analyzed for latex triterpenoids. Taxon Country: Province Voucher EMBL Accession No. trnT-L spacer trnL intron trnL-F spacer EMBL Accession No. ITS OUTGROUP Schizostephanus alatus Hochst. ex K. Schum. Kenya: s. loc. Noltee s.n. sub IPPS 8111 (UBT) AJ410247 AJ410248 AJ410249 AJ329451 Madagascar: s. loc. BG Munich s.n. (UBT) AJ492801 Madagascar: s. loc. Rauh 68584 (HEID) AJ290854 AJ290855 AJ290856 – Madagascar: Toliara McPherson 14770 (MO) AJ492802 Madagascar: Toliara Liede et al. 2695 (UBT) – anat. Liede et al. 2667 (MO, P, UBT) – latex AJ428756 AJ428757 AJ428758 – – – AJ428804 AJ428805 AJ428806 AJ492803 INGROUP – Old World Cynanchum relatives Folotsia grandiflora (Jum. & H. Perrier) Jum. & H. Perrier (Cynanchum grandidieri Liede & Meve) Folotsia madagascariensis (Jum. & H. Perrier) Desc. (Cynanchum toliari Liede & Meve) Madagascar: Toliara Glossonema boveanum (Decne.) Decne. Kenya: Northern Frontier Liede & Newton 3239 (ULM) – – Karimbolea macrantha Madagascar: Toliara (Jum. & H. Perrier) Liede & Meve (Cynanchum macranthum Jum. & H. Perrier) Liede et al. 2808 (in cult. Bayreuth) AJ492372 AJ492373 AJ492374 AJ492804 Karimbolea mariensis Meve & Liede (Cynanchum mariense (Meve & Liede) Liede & Meve) Madagascar: Toliara Liede et al. 2825 (K, MSUN, UBT) AJ428768 AJ428769 AJ428770 AJ492805 Karimbolea verrucosa Desc. (Cynanchum verrucosum (Desc.) Liede & Meve) Madagascar: Toliara Liede et al. 2826 (MSUN, UBT) AJ290878 AJ290879 AJ290880 AJ492806 Metaplexis japonica Makino Japan: s. loc. ex BG Tartu (UBT) AJ428810 AJ428811 AJ428812 AJ492807 Odontanthera radians (Forssk.) D.V. Field North Yemen: Hodeidah Müller-Hohenstein & Deil 1967 (UBT) AJ428813 AJ428814 AJ428815 AJ492809 Pentarrhinum abyssinicum Decne. Bidgood et al. 2440 (K; MWC 8418) AJ428816 AJ428817 AJ428818 AJ492810 Liede & Newton 3157 (UBT) AJ428819 AJ428820 AJ428821 AJ492811 Tanzania: Ufipa Pentarrhinum gonoloboides (Schltr.) Liede Kenya: Naivasha Org. Divers. Evol. (2002) 2, 239–269 242 Liede & Kunze Table 1. (Continued). Taxon Country: Province Voucher EMBL Accession No. trnT-L spacer trnL intron trnL-F spacer EMBL Accession No. ITS Pentarrhinum insipidum E. Mey. South Africa: Orange Free State Liede 2940 (UBT) AJ410232 AJ410233 AJ410234 – AJ492812 Namibia: Karas Region Meve & Liede 584a (MO) Pentarrhinum somaliense (Schltr.) Liede Ethiopia: Sidamo Mesfin & Vollesen 4238 (UPS) Kenya: Northern Frontier Liede & Newton 3225 (UBT) Platykeleba insignis N.E. Br. (Cynanchum insigne (N.E. Br.) Liede & Meve) Madagascar: Antananarivo Rauh 68500 (HEID) Madagascar: Antananarivo Lavranos s.n. (UBT) Sarcostemma arabicum Bruyns & P. I. Forst. Yemen: Sana’a Sarcostemma australe R. Br. Sarcostemma pearsonii N.E.Br. AJ428822 AJ428823 AJ428824 AJ492376 AJ492377 AJ492378 – AJ492813 AJ492814 AJ290906 AJ290907 AJ290908 – – Radcliffe-Smith & Henchie 4624 (K, UBT) – – Australia: Queensland Kunze 205 (Minden) – – South Africa: Northern Cape Liede & Meve 582 (K, MSUN) AJ492816 South Africa: Northern Cape South Africa: Northern Cape Liede & Hammer 2518 (UBT) – anat. Liede & Hammer 2520 (UBT) – latex AJ290909 AJ290910 AJ290211 – – – Sarcostemma resiliens B. R. Adams & R. W. K. Holland Zimbabwe: Makoni Albers et al. 515 (UBT) – – Sarcostemma stolonifera B. R. Adams & R. W. K. Holland Tanzania: Arusha Noltee 199 (MSUN) – – Sarcostemma vanlessenii Lavranos Kenya: s. loc. Yemen: Dhamar Lavranos s.n. (UBT) Noltee 870 (MSUN) – – – – Sarcostemma viminale (L.) R.Br. ssp. viminale Madagascar: Toliara Liede et al. 2738 (MO, P, UBT) – latex Liede et al. 2706 (MO, P, UBT) – anat. Albers et al. 540 (MSUN, UBT) – – – – AJ290912 AJ290213 AJ290214 – AJ492817 – – – – – – ssp. suberosum Meve & Liede Zimbabwe: Bulawayo ssp. thunbergii (G. Don) Liede & Meve South Africa: Western Cape South Africa: Northern Cape South Africa: Northern Cape South Africa: Western Cape ssp. nov. Org. Divers. Evol. (2002) 2, 239–269 Liede & Hammer 2507 (UBT) – latex Liede & Hammer 2522 (UBT) – latex Liede & Hammer 2514 (UBT) – anat. Liede & Hammer AJ492815 – – Cynanchum and the Cynanchinae 243 Table 1. (Continued). Taxon Country: Province Voucher EMBL Accession No. trnT-L spacer trnL intron trnL-F spacer EMBL Accession No. ITS Cynanchum sect. Cynanchum (Old World) Cynanchum abyssinicum Decne. Tanzania: Arusha Liede & Meve 3373 (UBT) AJ428579 AJ428580 AJ428581 AJ492734 Cynanchum aculeatum (Desc.) Liede & Meve Madagascar: Toliara Liede et al. 2828 – – Cynanchum acutum L. Portugal: s. loc. BG Lisboa s.n. (UBT) AJ428582 AJ428583 AJ428584 AJ492735 Cynanchum adalinae K. Schum. ssp. adalinae Cameroon: Sud (Mt. Cameroon) Meve 902 (K, LBG) AJ428585 AJ428586 AJ428587 AJ492736 Cynanchum africanum Hoffsgg. South Africa: Western Cape Liede 2550 (MO) AJ492737 South Africa: Western Cape Meve & Liede 624 (UBT) AJ428588 AJ428589 AJ428590 – Cynanchum altiscandens K. Schum. Kenya: Kiambu Liede & Newton 2873 (UBT) AJ428591 AJ428592 AJ428593 AJ492738 Cynanchum ampanihense Jum. & H. Perrier Madagascar: Toliara Liede et al. 2817a (MSUN) AJ492739 Madagascar: Toliara Liede et al. 2824 (UBT) AJ428594 AJ428595 AJ428596 – Cynanchum angavokeliense Choux Madagascar: s. loc. Specks s.n.(UBT) AJ428597 AJ428598 AJ428599 AJ492733 Cynanchum appendiculatopsis Liede Madagascar: s. loc. Février s.n. (UBT) AJ492322 AJ492323 AJ492324 AJ492732 Cynanchum arenarium Jum. & H. Perrier Madagascar: Toliara Liede et al. 2686 (in cult. Bayreuth) AJ492740 Madagascar: Toliara Liede et al. 2713 (UBT) – anat. Liede et al. 2739 (MO, P, UBT) – latex AJ428600 AJ428601 AJ428602 – – Cynanchum bisinuatum Jum. & H. Perrier Madagascar: Toliara Hardy 2901 (PRE 14664) – – Cynanchum chouxii Liede & Meve Madagascar: Fianarantsoa Kotozafy 442 (MO, UBT) AJ492325 AJ492326 AJ492327 – – – – – Org. Divers. Evol. (2002) 2, 239–269 244 Liede & Kunze Table 1. (Continued). Taxon Country: Province EMBL Accession No. trnT-L spacer trnL intron trnL-F spacer EMBL Accession No. ITS Cynanchum clavidens N.E.Br. ssp. hastifolium (N.E.Br.) Liede Kenya: Northern Frontier Liede & Newton 3226 (UBT) AJ428609 AJ428610 AJ428611 AJ492743 Cynanchum comorense Choux Comores: Mayotte Pignal 1104 (P) AJ428612 AJ428613 AJ428614 AJ492744 Cynanchum compactum Choux ssp. compactum Madagascar: s. loc. Barad s.n. (UBT) AJ492745 Madagascar: s. loc. AJ290842 AJ290843 AJ290844 Lavranos & Newton 12161 (UBT) – – Cynanchum crassiantherae Liede Somalia: Balad Hedberg & Warfa 90 (UPS) AJ428615 AJ428616 AJ428617 AJ492746 Cynanchum crassipedicellatum Meve & Liede Madagascar: Toliara Hardy 2852 (K, MSUN) AJ492328 AJ492329 AJ492330 AJ492747 Cynanchum cucullatum N.E.Br. Madagascar: Antananarivo Liede et al. 2868 (MO, MSUN, P) AJ428618 AJ428619 AJ428620 AJ492748 Cynanchum danguyanum Choux Madagascar: Antsiranana Allorge 2026 (P) AJ428621 AJ428622 AJ428623 AJ492749 Cynanchum decaryi Choux Madagascar: Toliara Liede et al. 2691 (UBT) – – Cynanchum descoingsii Rauh Madagascar: Toliara Descoings 28244 (UBT) AJ428624 AJ428625 AJ428626 AJ492750 Cynanchum ellipticum R. Br. South Africa: Eastern Cape Liede 2933 (UBT) AJ290845 AJ290846 AJ290847 AJ320444 Liede 2916 (UBT) – – South Africa: Eastern Cape Voucher Cynanchum erythranthum Jum. & H. Perrier Madagascar: Antsiranana Rauh 74816 (HEID) AJ428627 AJ428628 AJ428629 AJ492751 Cynanchum falcatum Hutchinson & E.A. Bruce Ethiopia: Ogaden Kuchar & Abdirizak 21226 (UPS) AJ492753 Ethiopia: Sidamo Friis 3169 (K; MWC 8410) AJ492331 AJ492332 AJ492333 AJ428630 AJ428631 AJ428632 Org. Divers. Evol. (2002) 2, 239–269 AJ492752 Cynanchum and the Cynanchinae 245 Table 1. (Continued). Taxon Country: Province Voucher EMBL Accession No. trnT-L spacer trnL intron trnL-F spacer EMBL Accession No. ITS Cynanchum floribundum R.Br. Australia: Northern Territory Latz 12579 (MO) AJ428633 AJ428634 AJ428635 AJ492754 Cynanchum folotsioides Liede & Meve Madagascar: Toliara Rauh 21847 (MSUN) AJ492334 AJ492335 AJ492336 AJ492755 Cynanchum galgalense Liede Somalia: Bari Thulin & Warfa 6205 (K, UPS) AJ492756 Somalia: Bari Thulin et al. 9433 (UPS) AJ492337 AJ492338 AJ492339 AJ492341 AJ492342 AJ492343 Cynanchum gerrardii (Harvey) Liede Cynanchum hardyi Liede & Meve Yemen: Al Hudaydah Noltee 995 (MSUN) – latex Kenya: Northern Frontier Meve 962 (ULM) Madagascar: Toliara Liede et al. 2797 (MSUN) Madagascar: s. loc. Rauh s.n. (HEID, UBT) Madagascar: Mahajanga Mangelsdorff RMM 43 (UBT) Madagascar: Toliara Hardy & Jacobsen 3571 (PRE) – AJ428645 AJ428646 AJ428647 AJ428642 AJ428643 AJ428644 – AJ492343 AJ492344 AJ492345 – – – AJ492757 AJ492758 – AJ492759 – Cynanchum implicatum Jum. & H. Perrier Madagascar: Antsiranana Mangelsdorff 24 (UBT) AJ428648 AJ428649 AJ428650 AJ492760 Cynanchum itremense Liede Madagascar: Fianarantsoa Phillipson et al. 3857 (MO) AJ492346 AJ492347 AJ492348 AJ492761 Cynanchum juliani-marnieri Desc. Madagascar: Toliara Teissier s.n. (UBT) AJ492349 AJ492350 AJ492351 AJ492762 Cynanchum junciforme (Decne.) Liede Madagascar: Fianarantsoa Liede et al. 2864 (MO, P, UBT) – – Cynanchum leucanthum (K. Schum.) K. Schum. ssp. leucanthum Madagascar: Antsiranana Mangelsdorff 420 (UBT) AJ428654 AJ428655 AJ428656 AJ492764 Cynanchum lineare N.E.Br. ssp. lineare Madagascar: Fianarantsoa Röösli & Hoffmann 198 (UBT) AJ428660 AJ428661 AJ428662 AJ492765 Org. Divers. Evol. (2002) 2, 239–269 246 Liede & Kunze Table 1. (Continued). Taxon Country: Province Voucher EMBL Accession No. trnT-L spacer trnL intron trnL-F spacer EMBL Accession No. ITS Cynanchum luteifluens (Jum. & H. Perrier) Desc. var. luteifluens Madagascar: Toliara – – – – var. longicoronae Liede Madagascar: Fianarantsoa Liede et al. 2699 (MO, P, UBT) – anat. Liede et al. 2731 (MO, P, UBT) – latex Liede et al. 2624 (MO, P, UBT) – – Cynanchum longipes N.E.Br. Ghana: Brong-Ahafo Region Jongkind & Schmidt 1739 (MO) AJ428663 AJ428664 AJ428665 AJ492766 Cynanchum madagascariense K. Schum. Madagascar: Toliara Liede et al. 2756 (UBT) AJ428666 AJ428667 AJ428668 AJ492767 Cynanchum mahafalense Jum. & H. Perrier Madagascar: Toliara Liede et al. 2831 (UBT) AJ492768 Madagascar: Toliara Liede et al. 2649 (UBT) AJ428669 AJ428670 AJ428671 – Cynanchum marnieranum Rauh Madagascar: Toliara Rauh s.n. (MSUN) AJ492352 AJ492353 AJ492354 AJ492769 Cynanchum menarandrense Jum. & H. Perrier Madascar: Toliara Rauh 7593 (HEID, UBT) – – Cynanchum messeri (Buchenau) Jum. & H. Perrier Madagascar: Toliara Liede et al. 2721 (MO, P, UBT) AJ428672 AJ428273 AJ428274 AJ492770 Cynanchum mevei Liede Madagascar: Toliara Teissier 215 (UBT) AJ428675 AJ428676 AJ428677 – AJ492771 AJ428678 AJ428679 AJ428680 AJ492772 AJ492355 AJ492356 AJ492357 AJ492774 AJ428687 AJ428688 AJ428689 – AJ492775 AJ428801 AJ428802 AJ428803 – AJ492776 Liede et al. 2780 (MO, P, MSUN) Cynanchum meyeri Schltr. Namibia Cynanchum moramangense Choux Madagascar: Toamasina Rakotomalaza et al. 1202 (MO) Cynanchum natalitium Schltr. South Africa: s. loc. Nicholas 2583 (NH) South Africa: s. loc. Kunze 316 (Minden) Madagascar: Antsiranana Mangelsdorff M14 (UBT) Madagascar: Fianarantsoa Liede et al. 2859 (MO, P, UBT) Cynanchum obovatum Choux Org. Divers. Evol. (2002) 2, 239–269 Van Wyk 9030 (PRE) – – – – Cynanchum and the Cynanchinae 247 Table 1. (Continued). Taxon Country: Province Voucher EMBL Accession No. trnT-L spacer trnL intron trnL-F spacer EMBL Accession No. ITS Cynanchum obtusifolium L.f. South Africa: Eastern Cape Liede 2925 (UBT) AJ492777 South Africa: s. loc. Kunze 303 (Minden) AJ428690 AJ428691 AJ428692 – Botswana: Kgalagadi Cole 347 (PRE) South Africa: Northern Cape Van Rooyen 4537 (PRE) Cynanchum orangeanum N.E.Br. – AJ492358 AJ492359 AJ492360 AJ428693 AJ428694 AJ428695 AJ492778 AJ428696 AJ428697 AJ428698 AJ492780 AJ428699 AJ428700 AJ428701 – AJ492781 AJ428702 AJ428703 AJ428704 – AJ492782 AJ492779 Cynanchum ovalifolium Wight Philippines: Zamboanga Liede 3297 (ULM) Cynanchum pachycladon Choux Madagascar: Toliara Liede et al. 2741 (MO, P, UBT) Madagascar: Toliara Liede et al. 2663 (MO, P, UBT) Madagascar: Fianarantsoa Liede et al. 2862 (MSUN, UBT) Madagascar: Fianarantsoa Liede et al. 2622 (MO, TAN) Madagascar: s. loc. BG Berlin 027-03-74-80 (B) AJ428705 AJ428706 AJ428707 AJ492783 Madagascar: Fianarantsoa Liede et al. 2851 (UBT) – – Cynanchum papillatum Choux Cynanchum perrieri Choux – – Cynanchum phillipsonianum Liede & Meve Madagascar: Antsiranana Mangelsdorff M 25 (UBT) AJ428708 AJ428709 AJ428710 AJ492784 Cynanchum polyanthum K. Schum. Uganda: Buganda Synnott 688 (K; MWC 8413) AJ428711 AJ428712 AJ428713 AJ492785 Cynanchum praecox Schltr. ex S. Moore Tanzania: Ufipa Goyder et al. 3828 (PRE) AJ428714 AJ428715 AJ428716 AJ492786 Cynanchum pycnoneuroides Choux Madagascar: Fianarantsoa Service Forestier 26466 (P) AJ492787 Madagascar: Fianarantsoa Rauh 10605 (HEID) AJ428717 AJ428718 AJ428719 – Madagascar: Toliara Liede et al. 2744 (UBT) AJ492361 AJ492362 AJ492363 AJ492788 Cynanchum radiatum Jum. & H. Perrier – Org. Divers. Evol. (2002) 2, 239–269 248 Liede & Kunze Table 1. (Continued). Taxon Country: Province Voucher EMBL Accession No. trnT-L spacer trnL intron trnL-F spacer EMBL Accession No. ITS Cynanchum rauhianum Desc. Madagascar: Toliara Röösli s.n. sub Noltee 2662 (in cult. Bayreuth) AJ492789 Madagascar: Toliara Liede et al. 2630 (P, UBT) AJ428723 AJ428724 AJ428725 – Cynanchum repandum Choux Madagascar: Antananarivo Liede et al. 2867 (MO, P, UBT) AJ428726 AJ428727 AJ428728 AJ492791 Cynanchum rossii Rauh Madagascar: Toliara Singer 072478 (ZSS) AJ428729 AJ428730 AJ428731 AJ492792 Cynanchum rubricoronae Liede Somalia: Hiiraan Kuchar 16793 (K, MWC 8414) AJ428735 AJ428736 AJ428737 AJ492794 Cynanchum rungweense Bullock Tanzania: Mbeya Mwasumbi 16518 (MO, K, MWC 8415) AJ428738 AJ428739 AJ428740 AJ492795 Cynanchum schistoglossum Schltr. Burundi: Bujumbura Lewalle 5435 (MO) – AJ492364 AJ492365 AJ492796 Cynanchum sessiliflorum (Decne.) Liede Madagascar: Antsiranana Mangelsdorff M13 (UBT) AJ428741 AJ428742 AJ428743 AJ492797 Cynanchum sigridiae Meve & Teissier Madagascar: Toliara Teissier 135 (K, MSUN) AJ492366 AJ492367 AJ492368 AJ492798 – Cynanchum sect. Rhodostegiella (Old World) Cynanchum auriculatum Buch.-Ham. ex Wight China ex hort. Nanking s.n. (UBT) AJ410196 AJ410197 AJ419198 AJ492741 Cynanchum thesioides K. Schum. China Qingru 97-81 (MO) AJ492799 China: Gansu Wang et al. 93–414 (MO) AJ492369 AJ492370 AJ492371 AJ428747 AJ428748 AJ428749 AJ492800 Cynanchum subgen. Mellichampia (New World) Cynanchum blandum (Decne.) Sundell Org. Divers. Evol. (2002) 2, 239–269 Ecuador: Napo Burnham 1668 (MO) AJ428403 AJ428604 AJ428605 AJ492742 Cynanchum and the Cynanchinae 249 Table 1. (Continued). Taxon Country: Province Voucher EMBL Accession No. trnT-L spacer trnL intron trnL-F spacer EMBL Accession No. ITS Cynanchum foetidum Kunth Mexico: Oaxaca Campos 3956 (MO) AJ428636 AJ428637 AJ428638 – AJ492818 Liede s.n. (UBT) AJ428651 AJ428652 AJ428653 AJ492763 Cynanchum ligulatum (Benth.) Woodson Mexico: Sonora Martin & McWorther s.n. (MO) AJ428657 AJ428658 AJ428659 – Cynanchum montevidense Spreng. Argentina: Salta Liede & Conrad 3100 (ULM) AJ290848 AJ290849 AJ290850 AJ492773 Cynanchum cf. racemosum Jacq. Mexico: Tamaulipas Liede & Conrad 2609 (ULM) AJ428720 AJ428721 AJ428722 AJ492790 Cynanchum roulinioides (E. Fourn.) Rapini (C. contrapetalum Sundell) Bolivia: Chuquisaca Wood et al. 13300 (K, UBT) AJ428732 AJ428733 AJ428734 AJ492793 Metalepis albiflora Urban Ecuador: Napo Burnham 1611 (MO) AJ492808 Ecuador: Napo Gentry et al. 64103 (MO) AJ428774 AJ428775 AJ428776 – Kunze 624 (Minden) Cynanchum laeve (Michx.) Pers. USA: Missouri branch swapping (“MulTrees” on) to the limit of computer capacity (between 30,000 and 40,000 trees). Internal support was assessed using 1000 bootstrap replicates with random addition of taxa (10 addition sequence replicates), with a limit of 10 trees kept at each step. Decay analyses were performed with AutoDecay 4.0 (Eriksson 1998) in combination with the reverse constraint option of PAUP*. A partition homogeneity test (as implemented in PAUP, 1000 replicates) showed significant discordance between the cpDNA dataset and the rDNA dataset (p = 0.01), but if the partial cpDNA datasets were tested singly, no discordance was found (trnT-L spacer: p = 0.1, trnL intron: p = 0.99, trnL-F spacer: p = 0.59). With this result, the argument of Soltis et al. (2000) – that tests of incongruence are often too coarse and that the best way to detect true incongruence is to examine the internal support of nodes in separate and combined – – analysis – was followed, and a combination of the two datasets was analyzed. In addition, a Neighbor-Joining analysis of all sequence data (Saitou & Nei 1987) was conducted as well, employing Jukes-Cantor (Jukes & Cantor 1969) and Kimura 2-parameter (Kimura 1980) distance models. Finally, data from the morphological analysis of African Cynanchum (Liede 1997a) and the two molecular datasets were combined and analyzed, even though a partition homogeneity test showed highly significant discordance (p = 0.01) between the morphological data and both molecular datasets. Taxa for which either no molecular data or no detailed morphological data were available were omitted, as were double accessions of the same species, leaving 63 ingroup taxa. Liede (1997b) regarded Tylophora as a primitive Asclepiadeae genus and thus a suitable outgroup. Since then it has been established that Tylophora belongs to a different subtribe (TyOrg. Divers. Evol. (2002) 2, 239–269 250 Liede & Kunze lophorinae) in the Asclepiadeae (Liede 2001), and that ITS sequences of Tylophora and Cynanchum cannot be aligned unambiguously (Liede et al. in press). Therefore, Schizostephanus alatus was coded for morphological characters following Liede (1993b, as Cynanchum validum). To the morphological dataset of 87 characters (multistate characters treated as polymorphisms), the parsimony informative sequence characters of two molecular datasets (145 for ITS and 114 for cpDNA) were added. In addition to a parsimony analysis of the three combined datasets, an analysis of the molecular data alone was performed with the reduced number of taxa, and morphological characters were plotted on the resulting tree using MacClade 4.0 (Maddison & Maddison 2000). Stem anatomy Twenty-seven leafless, stem-succulent members of Cynanchum and related genera, three leafy Malagasy Cynanchum species, and a total of six non-succulent African, Asian and American species of Cynanchum s. l. were studied anatomically (Table 1). In addition, two American Funastrum E. Fourn. species, F. clausum Schltr. and F. pannosum Schltr., were studied. For the stem-succulent taxa, mature but not basal internodes were selected. Material was fixed in FAA, dehydrated via tertiary butanol, and embedded in paraffine. Sections were stained for 5 minutes each in 0.1% aqueous safranine and 0.5% Astra blue (Gerlach 1969). Sections were washed, deparaffinated with Roticlear® (Roth Chemicals), and coverslipped with Entellan®. The data were tabulated and plotted onto the tree resulting from analysis of the combined molecular data using MacClade 4.0 (Maddison & Maddison 2000) for the taxa for which both datasets were available. Latex triterpene analysis Latex samples collected in the field were dried overnight in low heat on top of the plant dryer. Latex samples from cultivated plants were dried overnight at 60 ºC. All samples were analyzed for their acetone-extracted profile by Paul G. Mahlberg at Indiana University. The latex samples were redissolved in acetone and the supernatant analyzed for triterpenoid components by gas-liquid chromatography (Mahlberg et al. 1988). Analyses were performed on a Hewlett-Packard 5710 chromatograph equipped with a flame ionization detector and the oven programmed from 240–290 ºC at 4 ºC/min. Nitrogen was the carrier gas, 20 ml/min. Injection port and detector temperatures were 250 and 350 ºC, respectively. Columns contained 3% OV-1 on 100/120 mesh Supelcoport. Individual compounds were quantified on a Hewlett Packard 3380. Org. Divers. Evol. (2002) 2, 239–269 Results DNA sequence analysis Sequence characteristics for both datasets are summarized in Table 2. Sequence divergences are higher in ITS1 with 0.0–19.4% than in ITS2 with 0.0–11.9%. In both cases, divergence between Glossonema boveanum and an American Cynanchum species (Metalepis albiflorum for ITS1, C. blandum for ITS2) is larger than between any ingroup taxon and the outgroup. For ITS1, the largest divergences are found between C. galgalense and Metalepis albiflora. For the cpDNA, the trnL intron has the lowest sequence divergence with 0.0–4.2% between ingroup taxa, and the trnL-T and trnL-F spacers are almost equally variable with 0.0–7.8 and 0.0–8.4%, respectively. Again, highest sequence divergence is not between any ingroup taxon and the outgroup, but between a New World and an Old World species: C. foetidum and C. acutum (trnT-L spacer), Metalepis albiflorum and Metaplexis japonica (trnL-F spacer), M. albiflorum and Glossonema boveanum (trnL intron). Pairs of the same species are identical in all regions studied in C. gerrardii and C. thesioides. Between the remaining species pairs, divergences of up to 1.2% occur in one or several regions. Analysis of the cpDNA dataset without C. schistoglossum (268 parsimony informative characters) resulted in more than 40,000 trees (l = 484 steps, CI = 0.677, RI = 0.84, RC = 0.562; strict consensus see Fig. 1). Adding the separately coded indels (312 parsimony informative characters) resulted in more than 44,000 most parsimonious trees (l = 575 steps, CI = 0.649, RI = 0.828, RC = 0.537; strict consensus see Fig. 1). Analysis of the rDNA dataset without C. ligulatum, C. chouxii and the second accession of C. galgalense (200 parsimony informative characters) resulted in more than 44,000 most parsimonious trees (l = 605 steps, CI = 0.514, RI = 0.774, RC = 0.40; strict consensus see Fig. 2) and retrieved largely the same main clades as the cpDNA dataset. However, the Malagasy clade is much better resolved, both for the leafy and the leafless taxa. The leafless taxa together with the stem-succulent but leafy C. pycnoneuroides form a reasonably well supported subclade of the Malagasy clade also including the leafless succulent taxa shared with mainland Africa (Sarcostemma and C. gerrardii). Analysis of the combined cpDNA–rDNA dataset did not change the well-supported clades of the single analyses. The combined analysis (505 parsimony informative characters) resulted in 2268 most parsimonious trees (l = 1175 steps, CI = 0.568, RI = 0.785, RC = 0.446; strict consensus tree see Fig. 3). Bootstrap values increased for all clades retrieved in the single analyses, Cynanchum and the Cynanchinae 251 Table 2. Sequence characteristics. trnT-L spacer Aligned total length (bp) 1179a trnL intron trnL-F spacer ITS1 ITS2 5.8s 578 421 337 299 162 Length range (bp) 685 (C. acutum) – 867 (Metalepis albiflora) 393 (C. longipes) – 533 (C. montevidense) 265 (C. longipes) –371 (Schizostephanus alatus) 207 (Pentarrhinum gonoloboides) –262 (Odontanthera radians) 221 159–162 (C. rubricoronae) -–259 (C. blandum) Length mean (bp) Number of parsimony informative indelsa 787.79 28 (indels coded 32) 502.84 9 361.35 7 232.63 – 244.04 – 159.76 – Sequence divergence (ingroup/outgroup) (%) 1.0–5.6 0.4–3.0 0.0–5.0 7.6–14.7 3.7–10.5 0.0–0.2 Sequence divergence (ingroup) (%) b 0.0–7.8 0.0–4.3 0.0–8.4 0.0–19.4 0.0–11.9 0.0–0.2 Sequence divergence (pairs of same species) (%) 0.0–1.2 0.0–0.4 0.0–0.5 0.0–1.0 0.0–1.2 0.0 a b 52 characters permanently excluded because of ambiguous alignment not taking into account different accessions of the same species indicating higher congruency of the two datasets than the partition homogeneity test. The Madagascar clade is supported by 100% bootstrap value, and the succulent clade, including the succulent but linear-leafed C. pycnoneuroides, by 79% bootstrap support. Interestingly, the likewise stem-succulent but large-leaved C. pachycladon is now basal to the succulent clade s. str., though only with 68% bootstrap support. Clades retrieved in the rDNA dataset but not in the cpDNA dataset were largely preserved in the combined analysis, but equally unsupported; nevertheless, some basal resolution is apparent. The results of the two separate Neighbor-Joining analyses based on Jukes-Cantor and Kimura 2-parameter distance models produced trees almost identical in topology (Fig. 4), the only difference concerning the C. floribundum-C. falcatum branch, which is unresolved in the Kimura-2-parameter analysis but neighbor to the Pentarrhinum/Mellichampia/C. acutum branch following the Jukes-Cantor model. The Neighbor-Joining tree is highly congruent with the parsimony-based strict consensus phylogeny (Fig. 3). It illustrates clearly the structure of Cynanchum with short distances between the groups and long distances between the taxa in a group in African, American and Asian taxa. The Malagasy taxa are separated by a comparatively long distance, but distances are very short within the Malagasy group, and even shorter within the group of succulent species. The two American sections – the North and Central American C. foetidum, C. laeve and C. racemosum (sect. Mellichampia) and the South American C. blandum, C. montevidense and C. roulinioides (sect. Roulinia) – together with Metalepis albiflorum form reasonably to well-supported clades in all analyses (Figs 1–3). The Central American C. ligulatum, for which no ITS sequence was available, falls into its expected place with C. foetidum and C. racemosum in the cpDNA analysis (Fig. 1). These two subclades form a well supported clade in the cpDNA and combined analyses (Figs 1, 3), whereas they are unresolved with subclades of the Pentarrhinum-clade (Liede et al. 2002) in the analysis resulting from ITS data. In the combined analysis, the Afro-Arabian Pentarrhinum-clade is sister to the American clade (Fig. 3). Most Asian taxa form a very well supported clade, including Metaplexis japonica (Figs 1–3). Cynanchum ovalifolium, a widespread coastal Australasian species, joins this clade with low support in the ITS and the combined datasets (Figs 2, 3); the Neighbor-Joining analysis (Fig. 4) shows very long branches both for the core Asian taxa and for C. ovalifolium. The only available Australian species, C. floribundum, is even more weakly joint to the Asian clade in the ITS and combined analyses (Figs 2, 3), but in the Neighbor-Joining analysis C. floribundum is next to the African C. falcatum. Org. Divers. Evol. (2002) 2, 239–269 252 Liede & Kunze African Cynanchum fall into three main groups. Stable and well supported by both datasets is the C. clavidens-clade, comprising the widespread C. clavidens, the two Somalian twiners C. crassiantherae and C. rubricoronae, as well as the geophytic C. orangeanum and C. praecox. In the combined analysis (Fig. 3), this clade is basal in Cynanchum. The South African C. africanum, C. ellipticum, C. meyeri and C. natalitium, and the East African C. altiscandens form a weakly supported clade for both datasets (Figs 1–3), but only the ITS dataset joins the South African C. obtusifolium without support (Fig. 2), and the East African endemic C. rungweense is added only in the combined analysis (Fig. 3). A tie to C. polyanthum is suggested by the Neighbor-Joining analysis (Fig. 4), but not by parsimony analysis. The association of C. polyanthum with the Australian C. floribundum in the cpDNA dataset without indels is most likely an artefact caused by long-branch attraction. The positions of the remaining species have less support. The Somalian C. galgalense shows no affinity to any clade in any dataset, even though a second specimen has been analyzed for cpDNA to ascertain the results obtained from the first specimen. The combined analysis (Fig. 3) suggests a clade of the remaining African Cynanchum species, but only the subclade of the West African C. adalinae and C. longipes is reasonably well supported. Very well supported by ITS data is the morphologically unsuspected sister species relationship between C. falcatum and C. schistoglossum (for which cpDNA data are incomplete). The members of this African clade – to which the type species of Cynanchum, the circum-Mediterranean C. acutum, belongs – all show very long branches (Fig. 4). Analysis of the combined morphological/molecular dataset (343 parsimony informative characters) results in 1232 most parsimonious trees (l = 1488 steps, CI = 0.337, RI = 0.587, RC = 0.198; strict consensus see Fig. 5). Homoplasy measures for the combined trees are considerably worse than for any of the molecular trees alone, but better than those found by Liede (1997a) for pure morphological trees. Support is improved by morphological characters only for the Folotsia-clade, sup- port for the other clades is either the same or worse than for the molecular data alone. In the molecularly unresolved stem-succulent C. arenarium-clade, morphological data strongly support respective sister group relationships between C. arenarium and C. hardyi, and C. crassipedicellatum and C. descoingsii. Plotting of the morphological characters defined in Liede (1993b) on the strict consensus tree resulting from analysis of the combined molecular datasets shows that almost all characters used are homoplasious. Only thickwalled, ornamented fruits are restricted to Pentarrhinum (and Glossonema/Odontanthera, see Liede et al. 2002), which forms a subclade of Cynanchum. Stem succulence (Fig. 6A) is almost restricted to the “stem-succulent” clade, which includes the linear-leafed C. pycnoneuroides. The large-leafed but succulent C. pachycladon is basal to this clade, though with low support. Outside this clade, weak stem succulence occurs only in C. phillipsonianum. Other characters, even conspicuous ones such as trichomes on the corolla, ligulate or constricted coronas, papillose connective appendages, etc., are found in several clades. Corona characters in particular are highly homoplasious, e.g. the degree of fusion of the gynostegial corona (Fig. 6B). Except for the Malagasy C. erythranthum-clade, all clades have members with coronas fused to various degrees. In the stem-succulent clade, possession of warts and striation of the stems can be used as a rough indicator for relationship in the leafless taxa (Fig. 7). All members of the C. arenarium polytomy (except for C. rauhianum) have warty stems, but the latter also occur in C. marnieranum (Fig. 7A). The wartiness of C. pachycladon is a feature of its corky bark and probably not homologous to the wartiness of the green stems of the remaining taxa. Striation is characteristic of the most derived subclade of the stem-succulent clade, but C. compactum and C. marnieranum are exceptions (Fig. 7B). Stem anatomy A total of 16 characters was studied (Table 3). All species follow the general pattern of Apocynaceae s.l., in which the pith is surrounded by an amphi- Fig. 1. Analysis of the cpDNA dataset without C. schistoglossum. Strict consensus of 44,000 most parsimonious trees resulting from analysis without indels (268 parsimony informative characters, l = 484 steps, CI = 0.677, RI = 0.84, RC = 0.562), and of more than 40,000 trees resulting from analysis with indels (312 parsimony informative characters, l = 575 steps, CI = 0.649, RI = 0.828, RC = 0.537). Clades only retrieved in the analysis without indels are represented by dashed lines, those retrieved only in the analysis with indels by dotted lines. Bootstrap percentages and decay indices are given for analysis without indels above branches, with indels below branches. The only conflicting arrangement of taxa, C. obovatum and C. repandum, is indicated at the right margin. Asterisks indicate African taxa in the Malagasy clade. Org. Divers. Evol. (2002) 2, 239–269 Fig. 2. Strict consensus of 44,000 most parsimonious trees resulting from analysis of the rDNA dataset (200 parsimony informative characters, l = 605 steps, CI = 0.514, RI = 0.774, RC = 0.40). For branch labelling, see Fig. 1. Fig. 3. Strict consensus of the 2268 most parsimonious trees resulting from analysis of all sequence data and cpDNA indels (505 parsimony informative characters, l = 1175 steps, CI = 0.568, RI = 0.785, RC = 0.446). For branch labelling, see Fig. 1. Fig. 4. Neighbor-Joining Tree resulting from analysis of all sequence data. Distance model: Kimura-2-parameter Fig. 5. Strict consensus of the 1232 most parsimonious trees resulting from analysis of the combined morphological/molecular dataset (343 parsimony informative characters; l = 1488 steps, CI = 0.337, RI = 0.587, RC = 0.198). For branch labelling, see Fig. 1. Fig. 6. Examples of distribution of morphological characters identified by Liede (1993b) on the molecular phylogeny. A. Shoots woody, herbaceous or succulent. B. Degree of corona fusion. Fig. 7. Examples of distribution of morphological characters identified by Liede (1993b) on the molecular phylogeny. A. Stems warty or smooth. B. Stems striate or uniform. 260 Xylem Endodermis Species Number stone cells loose ring nests: no nests (0); nests in one row (1); nests in 2–3 rows (2) reduced different cell sizes parenchyma undifferentiated (0); with sclerenchyma (1) palisade parenchyma number of layers (0: abssnt; 1: 1–2; 2:more than two) papillose number of layers (0: monolayered; 1: several layers) 0: normal position; 1: slightly sunken; 2: much sunken Stomata reduced ring Enpidermis single groups Hypodermis more than 10 cells Cortex 5–10 cells Sclerenchyma undifferentiated parenchyma (0); single lignified cells (1) lignified cells and sclerenchyma (2) Pith Funastrum clausum 2599 0 0 1 0 0 0 0 1 0 0 0 1 1 0 0 0 Funastrum pannosum 2600a 0 1 0 0 0 0 0 1 0 0 0 1 1 0 0 0 Metalepis albiflora Gentry 64103 – 0 1 0 0 1 0 2 0 0 0 0 2 0 0 – Cynanchum foetidum HK 624 0 1 0 0 0 0 0 1 0 1 0 0 1 0 1 0 Cynanchum ovalifolium 3279 0 1 0 0 0 0 0 1 0 0 0 0 1 0 1 – Pentarrhinum insipidum PVB s.n. 0 1 0 0 0 0 0 1 0 0 0 0 1 0 0 0 Cynanchum natalitium HK 316 0 0 1 0 0 0 0 1 0 0 0 0 1 0 0 0 Cynanchum obtusifolium HK 303 0 1 0 0 0 0 0 1 0 1 0 0 1 0 0 0 Cynanchum decaryi 2691 0 1 1 0 0 0 0 1 0 1 0 0 2 0 0 0 Cynanchum pachycladon 2663 0 0 1 0 0 0 0 1 0 1 0 0 1 – 1 – Cynanchum pycnoneuroides 10605 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 1 Cynanchum aculeatum 2828 0 1 0 0 0 0 1 0 0 1 1 0 1 1 0 2 Cynanchum ampanihense 2824 0 1 0 0 0 0 0 1 0 1 0 0 1 1 0 1 Liede & Kunze Org. Divers. Evol. (2002) 2, 239–269 Table 3. Stem anatomy. 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 2 Cynanchum bisinuatum 2901 0 1 0 0 0 0 0 2 0 1 0 0 1 0 0 2 Cynanchum compactum 12161 1 1 0 0 0 0 1 0 0 0 1 0 0 1 0 2 Cynanchum descoingsii 68638 0 1 0 0 0 0 1 0 0 0 0 0 1 0 0 0 Cynanchum gerrardii Rauh s.n. 1 1 0 0 0 0 1 0 0 0 0 0 1 1 0 1 Cynanchum hardyi 3571 0 1 0 0 0 0 1 0 0 01 0 0 1 1 0 2 Cynanchum juliani-marnieri Teissier s.n. 0 0 0 1 0 0 1 1 1 0 0 0 0 1 0 2 Cynanchum luteifluens var. luteifluens 2699 0 0 0 1 1 0 0 0 1 0 0 0 0 1 0 0 Cynanchum luteifluens var. longicoronae 2624 1 0 1 0 0 1 1 0 0 0 1 0 1 0 0 2 Cynanchum mahafalense 2649 2 0 1 0 0 1 1 0 0 0 1 0 2 1 0 2 Cynanchum marnieranum Rauh s.n. 1 1 0 0 0 0 1 0 0 0 0 0 1 0 0 1 Cynanchum menarandrense 7593 0 1 0 0 0 0 0 2 0 1 0 0 0 0 0 0 Cynanchum messeri 2721 2 1 0 0 0 0 1 0 0 0 1 0 1 1 0 2 Cynanchum mevei 2780 0 1 0 0 0 0 1 0 0 0 0 0 1 1 0 2 Cynanchum rauhianum 2630 0 1 0 0 0 0 0 2 0 1 0 0 1 0 0 2 Folotsia grandiflora 68584 0 0 0 1 0 0 0 1 0 01 0 0 1 1 0 2 Folotsia madagascariensis 2695 0 1 0 0 0 0 1 0 0 0 0 0 01 0 0 2 Karimbolea verrucosa 2826 0 0 1 0 0 0 1 1 0 0 0 0 0 0 0 1 Platykeleba insignis Lavranos s.n. 0 0 0 1 0 0 0 1 0 0 1 0 01 1 0 2 Sarcostemma arabicum R. & H. 4624 0 0 1 0 0 1 0 2 0 0 1 0 0 1 0 1 Sarcostemma australe HK 205 0 1 0 0 1 0 0 1 0 1 1 0 0 1 0 1 Sarcostemma pearsonii 2518 0 0 1 0 0 0 1 0 0 0 1 0 1 1 0 0 Sarcostemma viminale ssp. viminale 2706 1 1 0 0 0 0 0 1 0 0 1 0 1 1 0 1 Sarcostemma viminale ssp. thunbergii 2514 0 0 1 0 0 0 1 0 1 0 1 0 1 1 0 2 Sarcostemma viminale ssp. nov. 2552 0 0 1 0 0 1 0 1 1 0 1 0 1 1 0 0 261 2713 Cynanchum and the Cynanchinae Org. Divers. Evol. (2002) 2, 239–269 Cynanchum arenarium 262 Peak No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 14.0 14.5 14.8 15.3 15.5 15.7 16.3 17.2 18.0 19.3 20.4 21.9 16 29 26 19 29 52 6 2 7 1 1 9 t 13 24 19 38 10 87 62 68 37 84 12 9 2 2 3 7 6 2 1 2 2 1 6 20 20 14 17 14 20 17 17 21 73 58 59 74 79 73 77 81 54 2 5 12 7 5 3 2 1 9 4 4 3 4 1 4 3 6 3 1 4 18 21 22 17 24 19 18 15 16 23 73 76 54 79 63 73 62 56 68 63 2 2 16 3 5 2 5 25 15 3 2507 3 5 20 61 3 2522 5 5 30 59 3 2552 Thailand s.n. Singh s.n. 3 2 4 3 3 4 24 18 18 68 75 72 2 2 2 Retention time (min) 8.8 11.7 12.0 13.6 Species Pentarrhinum insipidum Cynanchum africanum Number 584a 624 Cynanchum cucullatum Cynanchum junciforme Cynanchum obovatum Cynanchum pachycladon Cynanchum papillatum 2868 2864 2859 2863 2862 Cynanchum aculeatum Cynanchum arenarium Cynanchum crassipedicellatum Cynanchum gerrardii 2828 2739 2852 995 Rauh s.n. 2731 2835 Rauh s.n. 2851 Cynanchum luteifluens var. luteifluens Cynanchum marnieranum Cynanchum perrieri Folotsia grandiflora Folotsia madagascariensis Karimbolea verrucosa Platykeleba insignis Sarcostemma pearsonii Sarcostemma resiliens Sarcostemma stolonifera Sarcostemma vanlessenii Sarcostemma viminale ssp. viminale Sarcostemma viminale ssp. thunbergii Sarcostemma viminale ssp. thunbergii Sarcostemma viminale ssp. nov. Sarcostemma viminale Sarcostemma viminale 68584 2667 2826 Lavranos s.n. 2520 515 199 Lavranos s.n. 870 2738 t t 4 t 1 13 1 t 1 t 1 1 t 2 1 1 6 7 1 1 1 1 t t 3 t t t t t 6 1 1 1 3 t 4 2 2 1 t t 1 4 1 t 1 Liede & Kunze Org. Divers. Evol. (2002) 2, 239–269 Table 4. Percentage composition of latex triterpenoids. Cynanchum and the Cynanchinae phloematic xylem. Adjacent to the outer phloem is a zone of sclerenchymatic fibres arranged either in a closed ring or in separate bundles with intermittent parenchyma. The cortex is formed by parenchymatic tissue. Usually, but not always, present are a hypodermis below the epidermis consisting of smaller cells of regular size, as well as a starch sheath on the inside of the cortex. Differences between species involve mainly specialized cells occurring in the pith or the cortex. 263 The American Funastrum species differ from all other analyzed species by a division of the outer cortex into an inner part of more or less isodiametric parenchyma cells and surrounding, radially elongated palisade cells (Fig. 9B). Special cells such as brachysclereids or single fibres are absent in Funastrum, but present in Sarcostemma; stomata are level with the surface in Funastrum, but sunken in Sarcostemma. Plotting of the characters on the cladogram derived from DNA characters shows that of the characters used Fig. 8. Stem anatomy. A. Cross section of a stem succulent Cynanchum (C. rauhianum) representing the basic pattern of stem anatomy. B. C. messeri, specialized pith with parenchyma interspersed with sclerenchyma fibres and lignified cells. C. Sarcostemma viminale ssp. nov. (SL 2552), cross section showing nests of stone cells between outer phloem and sclerenchyma of inner cortex. D. Sarcostemma pearsonii, outer cortex with stone cells, papillose epidermis and sunken stomata. H: hypodermis, iP: inner phloem, L: lignified cells, oP: outer phloem, S: sclerenchyma, St: stone cells, X: xylem. Org. Divers. Evol. (2002) 2, 239–269 264 Liede & Kunze the anatomical ones are no better indicators of relationship than are the morphological ones in succulent Cynanchum. Only a papillose epidermis coincides roughly with the main clades of succulent Cynanchum, but C. marnieranum with a smooth epidermis is found in the otherwise papillose clade, and C. hardyi with a papillose epidermis in the otherwise smooth clade. The two Folotsia species also differ in this character (Table 3). Only the C. mahafalense/C. messeri clade, which is morphologically otherwise well supported, is uniquely characterized by both lignified cells and sclerenchyma in the pith. Triterpene analysis Liede et al. (1993) used latex triterpenoid patterns to show that Karimbolea constitutes a true member of Asclepiadeae despite the erect pollinia of K. verrucosa, the only Karimbolea species known at the time of that analysis. In the course of that analysis (Liede et al. 1993), the very different triterpenoid composition of Funastrum and Old World Sarcostemma (Fig. 10A, E) – then both included in Sarcostemma sensu Holm (1950) – together with the striking uniformity of the stem-succulent Malagasy species with Sarcostemma s. str. (Fig. 10C, E) – then belonging to five different genera – led to a discontinuation of the study. Almost all stem succulents show the same general triterpenoid pattern with four major peaks, the largest one at a retention time of 18 min (Fig. 10C). A small fifth peak at 15.3 min is usually but not always present (Table 4). Exceptions are C. arena- rium and C. perrieri, which both show three peaks of approx. the same size at 14.8 min, 15.5 min and 16.3 min (Fig. 10F). Cynanchum crassipedicellatum with an exceptionally large peak at 15.3 min and 19.3 min, Folotsia madagascariensis with two late peaks, and Karimbolea verrucosa with an exceptionally large peak at 19.3 min also differ slightly. All Sarcostemma species – from Madagascar, mainland Africa or even India and Thailand – are remarkably uniform, except for S. vanlessenii for which the peak at 15.5 min is absent and the one at 19.3 min exceptionally large. Of the five leafy Malagasy species studied, C. pachycladon (Fig. 10D) shows the same peaks, but at different ratios, whereas C. papillatum (Fig. 10C) displays a different pattern (Table 4). The two African species, C. africanum and Pentarrhinum insipidum (Fig. 10B), show triterpenoid patterns different from each other and from the Malagasy species. Discussion The present results make a consistent treatment of Cynanchum L. difficult. The two extreme alternatives supported by the data and cladistic theory are either to recognize each clade as an independent genus, or to unite all clades to a very broadly circumscribed genus Cynanchum. As the first option would result in many small, morphologically poorly discernible genera, the second alternative is more practicable. On the other hand, this would mean that well established, easily recognizable Fig. 9. Stem anatomy. A. Cynanchum pycnoneuroides, cross-section of succulent stem of the only leafy species in the succulent clade. B. Funastrum clausum, palisade parenchyma. Abbreviations as in Fig. 8; P: palisade parenchyma. Org. Divers. Evol. (2002) 2, 239–269 Cynanchum and the Cynanchinae 265 Fig. 10. Latex triterpenoid patterns. A. Funastrum clausum (Liede & Conrad 2599, MO). B. Pentarrhinum insipidum (Meve & Liede 584, MO). C. Cynanchum papillatum (Liede et al. 2622, MO). D. Cynanchum pachycladon (Liede et al. 2663, MO). E. Sarcostemma pearsonii (Liede & Hammer 2520, UBT). F. Cynanchum arenarium (Liede et al. 2739, MO). Org. Divers. Evol. (2002) 2, 239–269 266 Liede & Kunze genera would have to be abandoned. Thus, the genera Glossonema, Odontanthera, Pentarrhinum, Metaplexis, Metalepis, Folotsia, Karimbolea, Platykeleba and Sarcostemma are all candidates for inclusion in a large Cynanchum. Some genera not analyzed here due to lack of material, e.g. the Asian Adelostemma Hook. f., might be likely candidates as well. However, especially for the Asian taxa and the American Metalepis, more species would need to be analyzed before such far-reaching conclusions can be drawn. Our results show that all leafless stem-succulent genera (Folotsia, Karimbolea, Platykeleba and Sarcostemma) are monophyletic with stem-succulent Cynanchum, and that this group is derived from the likewise monophyletic Malagasy subgroup of Cynanchum. Evidence comes from both a chloroplast and a nuclear molecular marker, as well as from stem anatomy and triterpenoid analysis. Therefore, these genera are included in Cynanchum here. While the former three genera are small and endemic to Madagascar, Sarcostemma comprises about 20 species even after the transfer of all American members to Funastrum, Philibertia Kunth and Tetraphysa Schltr. (Liede 1996b; Liede & Täuber 2000). The necessary name changes on species level have already been made for the Malagasy representatives (Liede & Meve 2001), and will be made for African and Australasian Sarcostemma following species-level revision of the group (Liede et al., in prep.). The hypothesis of Liede (1997a) and Meve & Liede (2002), that the colonization of mainland Africa by Cynanchum gerrardii and of the whole Old World Tropics by Sarcostemma originated in Madagascar, is supported independently by the cpDNA and the rDNA data. Both C. gerrardii and Sarcostemma are members of the succulent clade, which is nested in the well-supported clade of otherwise exclusively Malagasy species. The identity of all partial sequences for the African and Malagasy accessions of C. gerrardii points to a fairly recent event. In Sarcostemma, the identity of cpDNA sequences between S. viminale and the morphologically most distinctive S. pearsonii, along with only three base changes between the two species for ITS, also points to recent radiation and speciation of Sarcostemma on the African mainland. Preliminary RAPD data (Liede et al., unpubl.) agree with this conclusion. The short distances between the Malagasy species, in particular the stem-succulent ones, compared to the African species in both datasets (Fig. 4) also allows the speculation that the radiation of the Malagasy species is a fairly recent event, because it is unlikely that both ITS and cpDNA should undergo a parallel slowdown in modification rate in Madagascar. The phylogeny resulting from the molecular data explains both the triterpenoid and the stem anatomical data. Both studies were begun in the early 1990s to clarify the phylogeny of stem-succulent Malagasy Cynanchum relaOrg. Divers. Evol. (2002) 2, 239–269 tives, and were abandoned because of the then inexplicable similarity between then well-established genera on the one hand and the differences between species then believed to belong to one genus on the other hand (Sarcostemma sensu Holm; Liede 1996b, Liede & Täuber 2000). In the triterpenoid dataset, C. arenarium and C. perrieri share an apomorphic pattern (Fig. 10F), while all other stem-succulent species studied show slight variations of the basic pattern (Fig. 10E). In the anatomical dataset, the four basic patterns described by Puech (1912) can be retrieved: the basic pattern without any noticeable specialisations (e.g., C. rauhianum, Fig. 8A); the “Sarcostemma” pattern with single sclerenchymatic fibres dispersed in the parenchyma of the cortex (Fig. 8C, D); the C. gerrardii pattern with sclerenchymatic fibres in the pith; and, as a specialisation of the preceding one, the C. messeri–C. mahafalense pattern with the pith consisting almost exclusively of lignified cells and sclerenchymatic fibres (Fig. 8B). However, only the C. messeri–C. mahafalense pattern coincides with a clade retrieved by molecular analysis. The two Malagasy leafy stem-succulent species share the basic anatomical pattern (Fig. 9A). The large-leaved C. pachycladon is basal to the stem-succulent clade following ITS and combined data (Figs 2–4), whereas the linear-leaved C. pycnoneuroides is an undisputed member of the stem-succulent clade, so that its leaves must be understood as a secondary development. The leafy non-succulent Malagasy species fall in two unsupported clades. Well supported is the subclade C. erythranthum/C. sessiliflorum/C. papillatum/C. cucullatum. In this clade, the former three species have reddish flowers in which the gynostegium is entirely enclosed by the corolla throughout anthesis. C. sessiliflorum (and C. junciforme, which was not available for analysis) had been described under a different genus, Pycnoneurum, by Decaisne (1838), but were transferred to Cynanchum by Liede (1993a). The high support of the sister species pairs C. comorense/C. danguyanum and C. obovatum/ C. repandum is reflected in the morphological similarity of these species. It is surprising, though, that each of the two clades of leafy Malagasy species harbours a subclade with linear-leafed, tuberous species (C. angavokeliense, C. lineare, C. moramangense, and C. sessiliflorum, C. cucullatum, C. papillatum, respectively). Therefore, this conspicuous habit must have evolved more than once in Madagascar. The present results indicate that the small Afro-Arabian genera Pentarrhinum, Glossonema and Odontanthera are monophyletic, and together form a subclade within Cynanchum, as Liede et al. (2002) have demonstrated. This clade is characterized by thick-walled, ornamented fruits. However, as no further morphological or chemical evidence could be found, name changes are left to a species-level revision of the group. Cynanchum and the Cynanchinae The Asian members of subgen. Rhodostegiella (C. auriculatum and C. thesioides) form a clade together with Metaplexis, indicating that Metaplexis should be included in Cynanchum as well. Again, evidence is not considered sufficient to execute the necessary name changes. Affinities of subgen. Rhodostegiella, the remaining Asian species (e.g. C. ovalifolium) and the seven true Australian Cynanchum species (Forster 1991, Liede 1996a), of which only C. floribundum was available for the present study, need to be studied further as the ITS data (Fig. 2) indicate that these species might form a monophyletic subclade of Cynanchum. Likewise, Metalepis albiflora is member of the American subgen. Mellichampia following both molecular datasets. Again, only one species of the genus could be analyzed and there is no additional chemical or morphological evidence, so that no name changes are made here. The distances between the American species are as long as between the members of the C. acutum group and the Pentarrhinum group (Fig. 4), and hint at an old event. The exclusion of the Somalian C. galgalense from Cynanchum by both molecular datasets (Figs 1–4) is surprising considering its morphological characters (Liede 1993b). The only character unusual for Cynanchum concerns its long, bostrychoid, persistent inflorescences. Unfortunately, its latex color is still unknown. The highly fused corona of C. galgalense does not indicate a relationship to C. obtusifolium or another member of the C. africanum clade as suggested by Liede (1993b), but must be interpreted as parallelism. An attempt to align the cpDNA sequences of the two C. galgalense accessions to other published datasets (Liede 2001; Liede & Täuber in press) showed that the species occupies an isolated position in the Asclepiadeae and is not a member of any of the circumscribed subtribes (Liede, unpubl.). The African Cynanchum species fall into three clades. The problem that a morphological synapomorphy, or at least a unique character combination, could not be found even for well-supported clades is a frequent phenomenon in Asclepiadoideae (e.g., Liede 2001). In this subfamily, reticulate evolution and parallelisms are the normal condition and not the exception, both within and between genera (Liede & Täuber 2000; Meve & Liede 2001). The basal, predominantly East African/Somalian C. clavidens-clade is well supported. Morphologically, it consists of two subclades. The first comprises the rhizomataceous C. orangeanum and C. praecox, with linear and elliptical leaves, respectively. In the second subclade, leaves are triangular to hastate in outline. The clade is characterized by leaf bases that are never deeply cordate, and by a corona that is fused for about half of its length and possesses pronounced staminal and interstaminal 267 lobes. The following, mostly southern African C. africanum-clade (C. altiscandens and C. rungweense are East African species) is moderately supported, at least for the core species. Morphologically it is characterized by at the most shallowly cordate leaf bases and a highly fused corona (with the exception of C. meyeri, a shrubby Namibian endemic). The third clade, the C. adalinae-clade, comprises the large African twiners with normally deeply cordate leaf bases. However, C. falcatum and C. schistoglossum have much less pronouncedly cordate leaf bases. This clade is split into a West and an East African subclade (Fig. 3). Most members of this clade are unresolved in the cpDNA analysis, and only the C. longipes – C. adalinae sister species relationship is well supported. Corona shape is highly diverse in this clade, normally the staminal lobes are longer than the interstaminal ones, and ligules are present in some species (C. abyssinicum). This clade combines with the Asian, the American and the Pentarrhinum-clade to form a subclade of Cynanchum in sister-group position to the Malagasy subclade (Fig. 3). All these clades are constant and some even well supported, but basal resolution is unsupported. This pattern of speciation, in which several clades can be distinguished but no or unsupported basal resolution is found between them, seems to be rather frequent in Asclepiadoideae, as a similar situation has been encountered in Tylophora (Liede et al. in press) and in the stapeliads (Meve & Liede in press). One might speculate that a rather old group of taxa has undergone geographic isolation and is now evolving in different parts of the world at different speeds and reacting to different selection pressures, while at the same time hardly changed members of the genus are still extant in Africa, the center of origin of Asclepiadoideae (Kunze et al. 1994). Taxonomic treatment Cynanchum L. Cynanchum L., Sp. Pl.: 212. 1 May 1753. – Type: C. acutum L. ≡ Sarmasikia Bubani, Fl. Pyren. 1: 550. 1897, nom. illeg. = Bunburia Harv. Gen. S. Afr. Pl., ed. I: 416. Jul–Dec 1838. – Type: B. elliptica Harv. = Colostephanus Harv., Gen. S. Afr. Pl., ed. I: 417, in nota. 1838. – Type: C. capensis Harv. = Cyathella Decne. in Ann. Sci. Nat. Bot., sér. 2,9: 332. Jun 1838. ≡ Cynoctonum E. Mey., Comm. Pl. Afr. Austr.: 215. 1–8 Jan 1838 [non Cynoctonum J. F. Gmel., Syst. Nat. 2: 306, 443. Sep(sero)-Nov 1791. (Loganiaceae)]. – Type: non designatus. Org. Divers. Evol. (2002) 2, 239–269 268 Liede & Kunze = Decanema Decne. in Ann. Sci. Nat. Bot., sér. 2,9: 338. Jun 1838. – Type: D. bojerianum Decne. = Decanemopsis Costantin & Gall. in Bull. Mus. Hist. Nat. (Paris) 12: 418. 1906. – Type: D. aphylla Costantin & Gall. Syn. nov. = Diploglossum Meisn., Pl. Vasc. Gen. 1: 269; 2:176. 5–11 Apr 1840. – Type: non designatus. = Drepanostemma Jum. & H. Perrier in Rev. Gén. Bot. 23: 256. 1911. – Type: D. luteum Jum. & H. Perrier. Syn. nov. = Flanagania Schltr. in Bot. Jahrb. Syst. 18, Beibl. 45: 10. 22 Jun 1894. – Type: F. orangeana Schltr. = Folotsia Costantin & Bois in Compt. Rend. Hebd. Séances Acad. Sci. 147: 258. 1908. – Type: F. sarcostemmatoides Costantin & Bois. Syn. nov. = Karimbolea Desc. in Cactus 15: 77. Oct-Dec 1960. – Type: K. verrucosa Desc. Syn. nov. = Mahafalia Jum. & H. Perrier in Rev. Gén. Bot. 23: 255. 1911. – Type: M. nodosa Jum. & H. Perrier. = Mellichampia A. Gray ex S. Watson in Proc. Amer. Acad. Arts 22: 437. 25 Jun 1887. – Type: M. rubescens A. Gray ex S. Watson. = Monostemma Turcz. in Bjull. Moskovsk. Obsc. Isp. Prir., Otd. Biol. 21(1): 255. 1848. – Type: non designatus. Syn. nov. = Nematostemma Choux in Compt. Rend. Hebd. Séances Acad. Sci. 172: 1310. 1921. – Type: N. perrieri Choux. = Perianthostelma Baill., Hist. Pl. 10: 247. Jul–Aug 1890. – Type: non designatus. = Platykeleba N. E. Br., Bull. Misc. Inform. 1895: 250. Oct 1895. – Type: P. insignis N. E. Br. Syn. nov. = Pycnoneurum Decne. in Ann. Sci. Nat. Bot., sér. 2,9: 340. 1838. – Lectotype: P. junciforme Decne. = Rouliniella Vail in Bull. Torrey Bot. Club 29: 662. 30 Dec 1902. – Lectotype: R. corymbosa (Decne.) Bullock (Roulinia corymbosa Decne.) ≡ Roulinia Decne. in Candolle, Prodr. 8: 516. Mar(med.) 1844 (non Roulinia Brongn. in Ann. Sci. Nat. Bot., sér. 2,14: 320. Nov 1840, nom. illeg., [Liliaceae]). = Sarcocyphula Harv., Thes. Cap. 2: 58. 1863. – Type: S. gerrardii Harv. = Sarcostemma R. Br., Prodr.: 462. 27 Mar 1810. – Lectotype: S. viminale (L.) R. Br. ex R. W. Holm (Euphorbia viminalis L.). Syn. nov. = Symphyoglossum Turcz. in Bull. Soc. Imp. Naturalistes Moscou 21(1): 255. 1848. – Type: S. hastatum (Bunge) Turcz., nom. rej. [non Symphyglossum Schltr. in Orchis 13: 8. 15 Feb 1919. (Orchidaceae), nom. cons.]. = Voharanga Costantin & Bois in Compt. Rend. Hebd. Séances Acad. Sci. 147: 259. 1908. – Type: V. madagascariensis Costantin & Bois. = Vohemaria Buchenau in Abh. Naturwiss. Ver. Bremen 10: 372. 1889. – Type: V. messeri Buchenau. Org. Divers. Evol. (2002) 2, 239–269 At the species level, the necessary name changes for Malagasy species have been made in advance in order that the new names are available for the Flora of Madagascar treatment (Liede & Meve 2001). The remaining species to be renamed all belong to the former genus Sarcostemma, of which a RAPD study is well advanced (Liede, Gebauer & Meve, unpubl. data). To reduce name changes to a minimum, the African, Asian and Australian Sarcostemma species will only be renamed after completion of this study. Acknowledgements Studies in the genus Cynanchum have been sponsored for many years by the DFG (LI 496). M. Chase, Kew, extracted an extra set of Cynanchum specimens. A. Nicholas, Pietermaritzburg, R. Omlor, Mainz, L. Allorge, Paris, R. Mangelsdorff, Frankfurt, P. Morat, Paris, P. Raven, St. Louis, MO, M. Teissier, Les Cèdres, and M. 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