BackgroundMicrohylidae is a geographically widespread family of anurans. Although several extensive molecular analyses have attempted to elucidate their subfamilial relationships, and correlate these with Mesozoic and Cenozoic continental drifts, consensus has not been reached. Further, generic level relationships have not been well investigated in some microhylid subfamilies, and therefore subfamilial affiliations of some genera are still unclear. To elucidate the phylogenetic positions of two mysterious Asian genera, Gastrophrynoides and Phrynella, and to better understand the trans-continental distributions of microhylid taxa, we performed molecular phylogenetic and dating analyses using the largest molecular dataset applied to these taxa to date.ResultsSix nuclear and two mitochondrial genes (approx. 8 kbp) were sequenced from 22 microhylid frog species representing eight subfamilies. The maximum likelihood and Bayesian analyses could not fully elucidate the subfamilial relationships, suggesting a rapid radiation of these taxa between 85 and 66 million years ago. In contrast, generic relationships of Asian microhylines were generally well resolved.ConclusionOur results clearly showed that one of two problematic Asian genera, Phrynella, was nested in the clade of the Asian subfamily Microhylinae. By contrast, Gastrophrynoides occupied the most basal position of the Australian-New Guinean subfamily Asterophryinae. The estimated divergence of Gastrophrynoides from other asterophryine was unexpectedly around 48 million years ago. Although a colonization scenario via Antarctica to the Australian-New Guinean landmass has been suggested for Asterophryinae, our finding suggested a novel colonization route via Indo-Eurasia.

To survey the diversity of anuran species in Bangladesh, we compared mitochondrial 16S rRNA gene sequences (approximately 1.4 kbp) from 107 Bangladesh frog specimens. The results of genetic divergence and phylogenetic analyses incorporating data from related species revealed the occurrence of at least eight cryptic species. Hoplobatrachus tigerinus from two districts diverged considerably, indicating the involvement of a cryptic species. Two Fejervarya sp. (large and medium types) and Hylarana cf. taipehensis formed lineages distinct from related species and are probably new species. Microhyla cf. ornata differed from M. ornata with respect to type locality area and involved two distinct species. In addition, we found that Hylarana sp. and Microhyla sp. did not match congeners examined to date in either morphology or 16S rRNA sequence. The occurrence of M. fissipes was tentatively suggested. Consequently, at least, 19 species were found from Bangladesh in this study. These findings revealed a rich anuran biodiversity in Bangladesh, which is unexpected considering the rather simple topographic features of the country.

To elucidate the species composition, genetic divergence, evolutionary relationships, and divergence time of Hoplobatrachus and Euphlyctis frogs (subfamily Dicroglossinae, family Ranidae) in Bangladesh and other Asian countries, we analyzed the mitochondrial Cyt b, 12S, and 16S rRNA genes of 252 specimens. Our phylogenetic analyses showed 13 major clades corresponding to several cryptic species as well as to nominal species in the two genera. The results suggested monophyly of Asian Hoplobatrachus species, but the position of African Hoplobatrachus occipitalis was not clarified. Nucleotide divergence and phylogenetic data suggested the presence of allopatric cryptic species allied to Euphlyctis hexadactylus in Sundarban, Bangladesh and several parapatric cryptic species in the Western Ghats, India. The presence of at least two allopatric cryptic species among diverged Euphlyctis cyanophlyctis in Bangladesh, India, and Sri Lanka was also suggested. In some cases, our estimated divergence times matched the paleogeological events of South and Southeast Asian regions that may have led to the divergence of Hoplobatrachus and Euphlyctis taxa. Especially, land formation at Bangladesh (15-10Ma) may have allowed the spread of these frog taxa to Southeast Asian areas, and the aridification of central India (5.1-1.6Ma) might have affected the gene flow of widely distributed species. The present study revealed prior underestimation of the richness of the amphibian fauna in this region, indicating the possible occurrence of many cryptic species among these groups.

Consensus on the taxonomic system and phylogenetic relationships for the anuran genus Fejervarya has yet to be established. Morphological characters in this genus are generally unsuitable for species identification. To carry out molecular species identification and solve phylogenetic problems, we collected 67 Fejervarya specimens from 12 Asian countries and sequenced part of the mitochondrial (mt) Cytb gene. We also sequenced the mt 12S and 16S rRNA genes and seven nuclear genes (BDNF, CXCR4, NCX1, RAG-1, RAG-2, Rhod, and Tyr) for 25 Fejervarya taxa. These molecular markers appear to be adequate for the identification of species. We subjected the molecular data molecular to phylogenetic analyses. In the resulting trees, topotypic F. limnocharis and "F. multistriata" (from China) formed a clade. On the other hand, neither "F. limnocharis" from the Japan mainland nor "F. limnocharis" from eastern Taiwan formed a clade with the real F. limnocharis, and the genetic divergences were larger than the species threshold for frog taxa proposed in previous studies (> 3% for 16S). These results may suggest that "F. multistriata" is a junior synonym of F. limnocharis, or that only some of the populations now recognized as "F. multistriata" correspond to F. limnocharis. Our results also suggest that several cryptic species may be included among the widely distributed Fejervarya species. Finally, our datasets support paraphyly for the genus Fejervarya, although alternative phylogenetic topologies, including Fejervarya monophyly, were not rejected by KH and SH tests.

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