Inter- and intraspecific evolutionary relationships of the rice frog Rana limnocharis and the allied species R. cancrivora inferred from crossing experiments and mitochondrial DNA sequences of the 12S and 16S rRNA genes

The rice frog Rana limnocharis is widely distributed in Southeast Asia and the rest of the Asian region extending from India to Japan. In Japan, the Sakishima-island populations of this species were regarded as a distinct species based on morphological and genetic divergences. The main purposes of this study were to confirm the presence of intraspecific reproductively isolating mechanisms in the Sakishima-island populations of R. limnocharis, and to clarify molecular inter- and intraspecific relationships of R. limnocharis and an allied species, Rana cancrivora. The hybridization experiments revealed that there were no reproductively isolating mechanisms between the Sakishima-island populations and other populations of R. limnocharis. The molecular evolutionary relationships were investigated by analyzing nucleotide sequences of the mitochondrial 12S and 16S rRNA genes using 12 populations of R. limnocharis from Japan and Taiwan, and two populations of R. cancrivora from Thailand and the Philippines. The phylogenetic trees constructed by the NJ method showed that the two populations of R. cancrivora were clearly separated from the 12 populations of R. limnocharis, and that the 12 populations of R. limnocharis were broadly divided into three clades; the first comprising eight populations from the main islands of Japan, the second comprising the Sakishima-island populations, and the third comprising the Okinawa-island and Taiwan populations. Interestingly, the Okinawa-island and Taiwan populations of R. limnocharis showed a close relationship that possibly reflected a secondary contact between the two populations. Based on the present crossing experiments and molecular data, it seems reasonable to regard the Sakishima-island populations as a single subspecies of R. limnocharis.