Systematic study of the rhacophorid frogs in Vietnam

Backgound Rhacophorids (Anura: Rhacophoridae) are a large group of arboreal frogs and represent one of the most diverse anuran families in the Old World, containing over 360 species in 16 genera, and distributed throughout subsaharan Africa, China, India, Southeast Asia, Japan, Taiwan, the Philippines, and the Greater Sunda Islands (Frost, 2014). With more than 70 species in 11 genera recorded in Vietnam, rhacophorid frogs form a large group in the amphibian fauna of the country, but, like many other amphibian groups, systematic study of this family is still incomplete, and taxonomic assignment for many species is still doubtful. On the other hand, numerous new species have been discovered during the past decades, mainly based on the results of molecular phylogenetic studies. This resulted in the change in generic allocation in many species (Delorme et al., 2005, Li et al., 2008, 2009, Wilkinson et al., 2002; Yu et al., 2008, 2009, 2010, Rowley et al., 2011a, b), but the study of morphological synapomorphies to define new groups has been rarely done mainly due to their high level of diversification. Materials and Methods I studied Vietnamese rhacophorid frogs, with special attention to four genera: Liuixalus; Kurixalus; Rhacophorus, and Theloderma. Many species of these genera have restricted geographical distribution, hitherto known only from a single or very few specimens deposited in the museum collections. Thus, I tried in the present study to make new collections of specimens and relevant data from intensive fieldwork, so as to better and comprehensively investigate intra and interspecific variations of these rhacophorid frogs, and to fix their taxonomy. This approach could also elucidate the presence of possible undescribed and/or cryptic taxa. For these purposes, I first tried to elucidate phylogenetic relationships of rhacophorid frogs using molecular methods, and then made detailed examination of their morphology. Field surveys were conducted between 2008 and 2014 in 28 provinces of Vietnam. As a result, I accumulated a total 320 specimens, many of which were examined for partial DNA sequences of the mitochondrial 12S rRNA, tRNAval, and 16S rRNA genes. Using these newly obtained data and relevant sequence data available from GenBank, I tried to establish robust phylogenetic trees Results I obtained the following results: (1) Finding of a new species of the genus Liuixalus in Vietnam based on morphological and molecular data. (2) Updates of molecular and morphological data for the genus Kurixalus, which resulted in the descriptions of two new cryptic species, K. viridescens and K. motokawai, from central highland in the country. (3) Based on molecular analyses, Rhacophorus duboisi was considered a member of the R. dugritei group and R. pingbianensis was strongly suggested to have close relationship with R. omeimontis. The relationship between R. robertingeri and R. calcaneus agreed with a phylogenetic hypothesis previously suggested. Two genetic groups were recognized within R. rhodopus from Vietnam, although they were similar in their morphological characters. (4) I discovered and recorded the first occurrence of Philautus petilus in Vietnam based on comparative studies on morphological similarities with the holotype from Laos, and confirmed its placement in the genus Theloderma based on molecular data. Discussion (1) The record of a new Liuixalus is biogeographically significant, because it is new to the territory of Vietnam, and considerably expanded the range of the genus, which had been known only from China. (2) Descriptions of two new cryptic Kurixalus species made Vietnam to encompass eight species of the genus now, confirming the country as the center of speciation of the genus Kurixalus. (3) Clarifying phylogenetic relationships and taxonomic problems of Vietnam’s Rhacophorus is thought to be very important in understanding evolution of this genus, because Vietnam is the hotspot for the genus. For example, my study on R. robertingeri and R. calcaneus resulted in a robust taxonomic idea in this previously disputed problem. (4) My discovery and subsequent molecular analyses of P. petilus elucidated proper generic position of the species, and increased the species of the genus Theloderma in Vietnam to 15, making the country the center of speciation of this genus. Conclusion Due to the time limitation and difficulty in the collection of some species, the present study is still incomplete to clarify total diversity of Vietnamese rhacophorid frogs, and further researches on the following topics are now under study and left for future publications: (1) Further clarification of phylogenetic relationships using longer sequences among all species of the rhacophorid frogs, especially of the genus Rhacophorus in Vietnam; (2) Analyses of breeding behavior and advertisement calls that allow more rapid identification of species in the field, especially for species that are very similar in morphology (e.g., Kurixalus banaensis; K. viridescens and K. motokawai); and (3) Further clarification of diversity and biogeography of the rhacophorid frogs in Asia so as to determine biogeographic uniqueness of the Vietnamese fauna.