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To understand how geographical differentiation of gobioid fish species led to speciation, two populations of the Pacific Ocean and the Sea of Japan for each of the two gobioid species, Pterogobius elapoides and Pterogobius zonoleucus , were studied in both morphological and molecular features. Analyzing mitochondrial genes, Akihito et al. (2008) suggested that P. zonoleucus does not form a monophyletic clade relative to P. elapoides , indicating that “Sea of Japan P. zonoleucus ” and P. elapoides form a clade excluding “Pacific P. zonoleucus ” as an outgroup. Because morphological classification clearly distinguish these two species and a gene tree may differ from a population tree, we examined three nuclear genes, S7RP, RAG1, and TBR1, in this work, in order to determine whether nuclear and mitochondrial trees are concordant, thus shedding light on the evolutionary history of this group of fishes. Importantly, nuclear trees were based on exactly the same individuals that were used for the previously published mtDNA trees. The tree based on RAG1 exon sequences suggested a closer relationship of P. elapoides with “Sea of Japan P. zonoleucus ”, which was in agreement with the mitochondrial tree. In contrast, S7RP and TBR1 introns recovered a monophyletic P. zonoleucus . If the mitochondrial tree represents the population tree in which P. elapoides evolved from “Sea of Japan P. zonoleucus ”, the population size of P. elapoides is expected to be smaller than that of “Sea of Japan P. zonoleucus ”. This is because a smaller population of the new species is usually differentiated from a larger population of the ancestral species when the speciation occurred. However, we found no evidence of such a small population size during the evolution of P. elapoides . Therefore, we conclude that the monophyletic P. zonoleucus as suggested by S7RP and TBR1 most likely represents the population tree, which is consistent with the morphological classification. In this case, it is possible that the incongruent mitochondrial and RAG1 trees are either due to incomplete lineage sorting of ancestral polymorphisms or to introgression by hybridization. Because of a smaller effective population size of mitochondria compared with nuclear genes, the introgression might be a more likely scenario in explaining the incongruent mitochondrial tree than the incomplete lineage sorting. Because of smaller effective population size of “Sea of Japan P. zonoleucus ” than that of P. elapoides , the direction of the introgression was likely to be from the latter to the former. This evolutionary work of the two gobioid species highlights the need of analyzing multiple gene trees for both nuclear and mitochondrial genes as well as scrutinization of morphological characteristics to obtain a population tree representing the organismal evolutionary history. [ABSTRACT FROM AUTHOR]