1 Zoologisches Forschungsinstitut und Museum Alexander Koenig, Germany; 2 Zoologisches Institut der Universita ¨t Mainz, Germany; 3 Zoologische Staatssammlung Mu ¨nchen, Germany Molecular phylogeny of hyperoliid treefrogs: biogeographic origin of Malagasy and Seychellean taxa and re-analysis of familial paraphyly M. Vences 1 , J. Kosuch 2 , F. Glaw 3 , W. Bo ¨ hme 1 and M. Veith 2 Abstract Treefrogs of the family Hyperoliidae are distributed in Africa, Madagascar and the Seychelles. In this study, their phylogeny was studied using sequences of fragments of the mitochondrial 16S and 12S rRNA and cytochrome b genes. The molecular data strongly confirmed monophyly of the subfamily Hyperoliinae but indicated that the genus Leptopelis (subfamily Leptopelinae) is more closely related to species of the African family Astylosternidae. The Seychellean genus Tachycnemis was the sister group of the Malagasy Heterixalus in all molecular analyses; this clade was deeply nested within the Hyperoliinae. A re-evaluation of the morphological data did not contradict the sister group relationships of these two genera. The subfamily Tachycneminae is therefore considered as junior synonym of the Hyperoliinae. In addition, the molecular analysis did not reveal justification for a subfamily Kassininae. Biogeographically, the origin of Malagasy hyperoliids may not be well explained by Mesozoic vicariance in the context of Gondwana breakup, as indicated by the low differentiation of Malagasy hyperoliids to their African and Seychellean relatives and by analysis of current distribution patterns. Key words: Amphibia – Anura – Hyperoliidae – Heterixalus – Tachycnemis – Madagascar – Seychelles – biogeography – phylogeny – 16S rRNA – 12S rRNA – cytochrome b – osteology Introduction The relationships and classification of the treefrogs of Africa, Asia and Madagascar have been controversial since the dawn of phylogenetic systematics. Old World treefrogs are defined by the presence of (cartilaginous or ossified) intercalary elements between ultimate and penultimate phalanges of fingers and toes. They belong to the superfamily Ranoidea based on their derived firmisternal shoulder girdle, as opposed to the super- family Hyloidea, which has a plesiomorphic arciferal condition of the shoulder girdle, is mainly distributed in the New World, and also contains treefrog families that convergently evolved intercalary elements (Duellman and Trueb 1986). For many years, all Old World treefrogs have been grouped in one family Rhacophoridae or Polypedatidae based on the presence of the intercalary element (e.g. Noble 1931). Laurent (1951) was the first to recognize that one large clade of Old World treefrogs was more closely related to a group of non- arboreal African frogs, today classified as family Astyloster- nidae. He considered this lineage as family Hyperoliidae. The separate status of the Hyperoliidae was later supported by the cladistic analyses of Liem (1970), Drewes (1984), Channing (1989) and Blommers-Schlo¨sser (1993). The monophyly of the family, however, has recently been questioned by molecular data (Emerson et al. 2000). The Hyperoliidae sensu lato include about 235 species (Glaw et al. 1998a) of mainly arboreal frogs which are characterized by combination of: (a) derived firmisternal condition of shoulder girdle; (b) derived presence of an intercalary element; and (c) plesiomorphic absence of a bony sternal style. These frogs are distributed in Africa, Madagascar and the Seychelles. The vast majority of genera and species occurs in Africa, whereas the monotypic Tachycnemis is endemic to the Seychelles, and Heterixalus (11 species; Vences et al. 2000b) is endemic to Madagascar. Heterixalus was placed by most workers close to African genera, mostly Hyperolius and Afrixalus, while Tachycnemis has often been considered as one of the most basal representative of the Hyperoliidae (Drewes 1984; Channing 1989). In contrast, the molecular study of Richards and Moore (1996), based on sequences of a fragment of the 12S rRNA gene in 14 hyperoliid taxa, arranged Heterixalus as the well-supported sister group of Tachycnemis. The Tachycnemis–Heterixalus clade was arranged as the sister group of Afrixalus, and Leptopelis was the sister group of all other included hyperoliid taxa. In a combined analysis of the available molecular and morpholo- gical data, Emerson et al. (2000) provided additional support for the Tachycnemis–Heterixalus relationships. By providing additional sequence data for a reduced set of taxa, these authors found indications of non-monophyly of hyperoliids, with Leptopelis being more closely related to the Arthrolept- idae than to the other Hyperoliidae. Because frogs are usually thought to be unable to disperse across the open sea (e.g. Bossuyt and Milinkovitch 2001), they appear to be a good model group to test plate tectonical events. The Heterixalus–Tachycnemis relationship appears to be congruent with the existence of a Madagascar–India continent in the Cretaceous, which also included the Seychelles (Richards and Moore 1996). In the present study, our aim was to test the assumed relationships between Malagasy and Seychellean hyperoliids by analysis of additional DNA sequences and by a re-assessment of morphological characters. Additionally, we investigated the possible non-monophyly of the family by inclusion, for the first time, of crucial outgroup taxa such as scaphiophrynine microhylids and astylosternids. Materials and methods DNA was extracted using QIAmp extraction kits (QIAgen) from tissue samples (hindleg muscle, either fresh or preserved in 98% ethanol). Voucher specimens were deposited in the Zoologisches Forschungsin- stitut und Museum Alexander Koenig, Bonn (ZFMK; see Table 1). Other museum acronyms used are BMNH (The Natural History J. Zool. Syst. Evol. Research 41 (2003) 205–215 Ó 2003 Blackwell Verlag, Berlin ISSN 0947–5745 Received on 7 March 2002 U.S. Copyright Clearance Center Code Statement: 0947–5745/03/4103–0205$15.00/0 www.blackwell.de/synergy