Phylogenetic relationships of four species of floating gobies (Gymnogobius) as inferred from partial mitochondrial cytochrome b gene sequences

To clarify the intra- and interspecific relationships of four Gymnogobius species, G. urotaenia, G. isaza, Gymnogobius sp. 1 (sumiukigori), and Gymnogobius sp. 2 (shimaukigori), partial mitochondrial cytochrome b gene sequences of 639 bp were obtained for a total of 31 specimens from Japan and Korea, plus 2 outgroup specimens. Twenty-nine haplotypes were identified in the ingroup, with a total of 122 variable sites (19.1%). The individuals regarded as the same species morphologically were monophyletic genetically. Sequence differences between amphidromous individuals of three species distributed in both Japan and Korea were relatively small (0.16-1.25%). The largest intraspecific sequence difference was observed between individuals of G. urotaenia from Lake Biwa and those from other localities (1.25-2.19%). Interspecific sequence differences ranged from 4.07% to 13.46%: neighbor-joining, maximum-parsimony, and maximum-likelihood methods indicated that Gymnogobius sp. 2 diverged first, followed by G. isaza, with Gymnogobius sp. I and G. urotaenia being monophyletic. The estimated divergence time of each species. based on estimated divergence rates for mitochondrial protein-coding genes already reported (0.8-2.8%/my), suggested that speciation occurred mainly in the Pliocene (possibly Miocene). with G. isaza (a Lake Biwa endemic) diverging significantly earlier (probably Lake Kouga stage) than estimated in previous studies. In contrast, according to the previous hypothesis, the substitution rates were highly overestimated to about 12-20%/my.