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Intraspecific Differentiation in the Japanese Brown Frog Rana japonica Inferred from Mitochondrial DNA Sequences of the Cytochrome b Gene

Abstract Intraspecific differentiation of the Japanese brown frog Rana japonica was investigated by analyzing nucleotide sequences of the mitochondrial cytochrome b (cyt b) gene in order to clarify phylogenetic relationships among three population groups known to exist in this species. The nucleotide sequences of 447 base pair (bp) segments were determined by the PCR-direct sequencing method on 31 individuals from 14 populations of R. japonica from Honshu, and phylogenetic trees were constructed by the UPGMA and NJ methods using Rana catesbeiana as an outgroup. A sequence alignment provided 92 variable sites (15 corresponded to the first codon position, three to the second, and 74 to the third), and 19 haplotypes were identified from 31 frogs. The sequence divergences were 0.22∼2.50% (x̄ = 0.65%) within populations, 0.22∼12.02% (x̄ = 7.34%) between populations, and 23.59∼27.89% (x̄ = 26.19%) between R. japonica and R. catesbeiana. Although many nucleotide substitutions were silent mutations, 12 amino acid replacements were found to occur within R. japonica. A high frequency of transitions relative to transversions was observed within R. japonica. The present nucleotide sequence data showed that the eastern and western groups of R. japonica was considerably differentiated to each other, and that the Akita population of the northwestern group was evidently derived from the eastern group, but the Nakajo and Izumozaki populations of the northwestern group diverged considerably from each of the eastern and the western groups.