De´partement Syste´matique & Evolution, USM 602 Taxinomie & Collections, Reptiles & Amphibiens, Muse´um National d’Histoire Naturelle, Paris, France Evolution of reproduction in the Rhacophoridae (Amphibia, Anura) S. Grosjean, M. Delorme, A. Dubois and A. Ohler Abstract Rhacophorid treefrogs have different reproductive modes: some go through a tadpole stage and some have direct development, and the adults of some species produce foam nests. Philautus is the only genus characterized by direct development. The production of foam nests has been reported in the genera Polypedates, Rhacophorus, Chiromantis and Chirixalus. Recent molecular studies did not provide a robust hypothesis concerning the origin of these reproductive modes in the Rhacophoridae. In order to better understand the evolution of these reproductive modes, we tried to clarify relationships within this group, using DNA sequencing. Our data set consists in a large number of new sequences (1676 base pairs corresponding to threee genes) for five outgroup ranoids and 48 Rhacophoridae, including 16 undescribed species from Sri Lanka and southern India, and all homologous data available in Genbank. After the inclusion of Philautus from India, our data show that the separation of Philautus into clades does not coincide with their geographic distribution. Our data point to the existence of a clade, including the genera Rhacophorus, Polypedates, Chiromantis and Chirixalus, which confirms the results of Wilkinson et al. (2002) and suggests that the ability to produce foam nests has emerged only once in the Rhacophoridae, as already stated by these authors. Key words: Amphibia – Anura – Rhacophoridae – 12S – 16S – Rhodopsin – phylogeny – direct development – foam nests – reproductive modes Introduction Frogs are known to have a wide variety of reproductive modes that can also differ within families (Duellman and Trueb 1985). Rhacophorid treefrogs, that are either considered as a subfamily of the Ranidae Rafinesque, 1814 or as a family (option retained here), are a striking example in this respect. Whereas some species show a typical frog development through a free-feeding tadpole stage and metamorphosis, others have Ôdirect developmentÕ with lecithotrophy (Wourms 1981; Dubois 2004), in other words, feeding through the vitellus of the egg until hatching as a young frog similar to the adult. In addition, the adults of numerous species with a tadpole stage produce aerial foam nests into which they lay their eggs. Direct development with lecithotrophy is a remarkable feature that has evolved in several anuran families (Lamotte and Lescure 1977). Among the Ranoidea (sensu Frost et al. 2006), it is known in the Ceratobatrachidae Boulenger, 1884 (Boulenger 1886; Brown and Alcala 1983) and Dicroglossidae Anderson, 1871 (Taylor 1962; Ohler et al. 1999), in the Mantellidae Laurent, 1946 (Glaw and Vences 1994), and in the Rhacophoridae Hoffman, 1932 (Dring 1979; Brown and Alcala 1994; Bahir et al. 2005). Recent molecular phylogenetic studies (Richards and Moore 1998; Marmayou et al. 2000; Wilkinson et al. 2002; Delorme et al. 2004; Roelants et al. 2004) confirmed that this developmental mode appeared independently once or more times in each of these groups. Among rhacophorids, Philautus Gistel, 1848 is the only genus characterized by this mode of development. The geographical distribution of this genus is large, with some species occurring in Southeast Asia, others in northern India and others in southern India and Sri Lanka (Bossuyt and Dubois 2001). In recent years a great number of new species were discovered in Sri Lanka and southern India (Dutta and Manamendra Arachchi 1996; Pethiyagoda and Manamendra- Arachchi 1998; Biju 2001; Meegaskumbura et al. 2002a; Manamendra-Arachchi and Pethiyagoda 2005; Meegaskumb- ura and Manamendra-Arachchi 2005). The new species discovered by the researchers of the Wildlife Heritage Trust of Sri Lanka apparently all have direct development and are morphologically similar to other taxa in the genus Philautus. Although the results of Meegaskumbura et al. (2002a) showed a different origin of direct development in the Philautus from Sri Lanka from those of the Sunda Islands, these relationships have low or no support, then their data do not exclude close relationships between these two groups. The phylogenetic relationships of most of the new Indian rhacophorid species are unknown. The production of foam nests is another remarkable character apparently found in all species of the rhacophorid genera Polypedates Tschudi, 1838, Rhacophorus Kuhl and Van Hasselt, 1822, Chiromantis Peters, 1854 and in some species of Chirixalus Boulenger, 1893 (now in synonymy with Chiromantis according to Frost et al. 2006). The presence of foam nests in Theloderma Tschudi, 1838 has been reported only once by Boulenger (1903) for Theloderma horridum (Boulenger, 1903). Orlov (1997) did not find this behavior in Theloderma stellatum Taylor, 1962, Theloderma asperum (Boulenger, 1886), Thelo- derma gordoni Taylor, 1962 and Theloderma corticale (Bou- lenger, 1903). Recent molecular studies did not provide a conclusive hypothesis concerning the origin of foam nest production in the Rhacophoridae (Richards and Moore 1998; Meegaskumbura et al. 2002a; Wilkinson et al. 2002). In this paper, we attempt to summarize the current state of our phylogenetic knowledge on rhacophorids, in order to clarify the evolution of reproductive modes in this subfamily. We provide for the first time molecular data on all possible type-species of possible valid genera (eleven recognized by Delorme et al. 2005; Frost et al. 2006). Our data set consists of a large number of new sequences in 53 species, including 16 undescribed species from Sri Lanka and southern India, and all homologous data available in Genbank. The determination of species between Polypedates and Rhacophorus is very difficult. Wilkinson et al. (2002) tried to use molecular mark- ers, we show that the morphology of the tadpole is another system. Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Verlag, Berlin Accepted on 19 September 2007 J Zool Syst Evol Res doi: 10.1111/j.1439-0469.2007.00451.x J Zool Syst Evol Res (2008) 46(2), 169–176