Aust. J. Zool., 1987, 35, 61-74 Phylogenetic Relationships among Australian Tree Frogs (Anura : Hylidae : Pelodryadinae): an Immunological Approach Mark N. HutchinsonAB and Linda R. MaxsonA A Department of Genetics and Development, University of Illinois, Urbana, Illinois 61801, U.S.A. Present address: Department of Zoology, La Trobe University, Bundoora, Vic. 3083. Abstract Phylogenetic relationships among hylid frogs of the Australian region were studied by micro-complement fixation comparisons of serum albumin. Although our data support current species-group arrangements, we do not find good agreement between our phylogenetic hypotheses and those derived from morphological and karyological studies. Immunological analyses provide data which allow the construction of a phylogeny for the Australian radiation of the speciose genus Litoria, and suggest dividing the species of Litoria examined into five major species-assemblages, each of which is probably monophyletic. The sister- group relationship between the Litoria aurea group and Cyclorana is confirmed, and the diphyletic origin of the terrestrial hylids is supported. The radiation of Australian hylids is monophyletic with respect to the outgroup taxon (Hyla) used in this study, and the origin of diversification within the genus correlates well with estimates of the final separation of Australia from Antarctica-South America. Preliminary data suggest that the endemic New Guinean taxa (Nyctimystes and the montane Litoria) are closely related to the Australian 'freycineti' assemblage within Litoria. Albumin from Litoria infrafrenata cross-reacted poorly with all available Australian antisera, suggesting that this species may have originated independently of the rest of the Australian hylids. Our data support the classification of Australian tree frogs as hylids, rather than as leptodactyloid offshoots. Introduction The tree frogs of Australia and New Guinea are a diverse group, including at least 135 species, and form one of the major anuran radiations in the Australian region. These frogs, traditionally regarded as members of the family Hylidae, have attracted considerable attention from workers interested in the higher relationships of anurans. This attention has resulted in a number of proposals which have conflicted with past interpretations and have also differed among themselves (e.g. Tyler 1971; Savage 1973; Bagnara and Ferris 1975; Duellman 1975; Laurent 1975). Most reappraisals have concerned family-level relationships. One major innovation was the suggestion that tree frogs of the Australian region were not hylids but represented a distinct family, the Pelodryadidae, whose closest relatives were leptodactyloid frogs of the Australian region (Savage 1973). This idea has been critically reviewed by Tyler (1979), who concluded that the two arboreal genera Litoria and Nyctimystes, together with the fossorial Cyclorana, formed a monophyletic taxon, the closest relatives of which were likely to be the hylid frogs. In Tyler's view, the Australian radiation was distinct only at the subfamily level and he recommended that the three Australopapuan genera be maintained in the family Hylidae as a separate subfamily, the Pelodryadinae. The largest of the three pelodryadine genera is Litoria; its 104 described species (Frost 1985) are divided roughly equally between Australia and New Guinea. This genus, although widely regarded as a polyphyletic assemblage (e.g. Tyler and Davies 1978; Cogger et al. 1983), has not yet been formally subdivided. Tyler and Davies (1978) identified a total of 37 species-