Morphological differences and geneic diversity of rice field frogs, Hoplobatrachus rugulosus (Wiegmann, 1835), from habitats in Thailand

Morphological differences in rice field frogs, Hoplobatrachus rugulosus, were examined using Principal Canonical Analysis (PCA) and cluster analysis while the genetic diversity was investigated using inter-simple sequence repeat (ISSR) and mitochondrial DNA sequence analysis from 18 localities (6 regions) in Thailand. Morphometric investigation in this species of 109 male and 91 female adult frogs showed a clear size sexual dimorphism. The dendrograms from the cluster analysis for separate male and female adult frogs grouped samples into six and seven groups for adult male and female, respectively. Nevertheless, these groups in both male and female did not correspond to any geographic region in Thailand. The genetic diversity based on ISSR to investigate population genetic structure of H. rugulosus from 18 localities in 6 regions of Thailand found that the highest genetic variability was found in the eastern region, whereas the lowest genetic variability was in the northern region. Moreover, the genetic distances and the geographic distances among populations from 6 regions were not correlated. The phylogenetic relationship using two mitochondrial DNA sequences, a 564 bp fragment of the cyt-b gene and a 813 bp combined fragment of the 12S and 16S rRNA genes, clearly revealed H. rugulosus into two distinct clades. The high sequence divergences between the two major clades suggest that H. rugulosus as currently recognized may contains two distinct species in Thailand. According to these results, the morphological data did not provide resolution sufficient to reveal any difference in morphological characters among 6 regions. On the other hand, the molecular data yielded better resolution and revealed at least one “hidden” entity within H. rugulosus. However, additional information including, but not limiting to, bioacoustic, physiological, ecological and behavioral characters, will be needed to further elucidate the species status of taxonomic of H. rugulosus.