Evolutionary relationships among Japanese pond frogs inferred from mitochondrial DNA sequences of cytochrome b and 12S ribosomal RNA genes.

The evolutionary relationships among Japanese pond frogs (Rana nigromaculata, R.porosa porosa, and R. p. brevipoda) were investigated by analyzing nucleotide sequences of mitochondrial cytochrome b (cyt b) and 12S rRNA genes. The nucleotide sequences of 444-bp segment of the cyt b gene and 410-bp segment of 12S rRNA gene were determined by the PCR-direct sequencing method using 18 frogs from 13 populations of Japanese pond frogs, and phylogenetic trees were constructed by the neighbor-joining and maximum likelihood methods using R. catesbeiana as an outgroup. The sequenced 444-bp segment of cyt b gene provided 69 variables sites, and the sequenced 410-bp segment of 12S rRNA gene provided 21 variables sites. The numbers of nucleotide substitutions per site of the cyt b gene within ingroup were 0.0022-0.0205 at the populational level, 0.0368-0.0462 at the racial or subspecific level, and 0.1038-0.1244 at the specific level, whereas those of the 12S rRNA gene were 0-0.0074 at the populational or subspecific level, and 0.0378-0.0456 at the specific level. Most nucleotide substitutions within ingroup occurred at the third codon position of the cyt b gene and were silent mutations. High frequencies of transitions relative to transversions were shown at cyt b and 12S rRNA genes within ingroup. The phylogenetic trees constructed from the nucleotide sequences of the cyt b gene showed that after outgroup R. catesbeiana separated from ingroup frogs, ingroup Japanese pond frogs diverged into R.nigromaculata and R.porosa, then the latter diverged into R.p. porosa, R.p. brevipoda (the typical Okayama race), and the Nagoya race of R.p.porosa. The phylogenetic trees constructed from the nucleotide sequences of the 12S rRNA gene also showed distinct divergence between two species, but not any divergence within species.