Paleoenvironmental changes in marginal seas on the coast of the northwestern Pacific during the Quaternary have caused genetic differentiation and subsequent secondary contacts between populations of coastal organisms in this region. However, little is known about how geographical isolation and the subsequent connection events associated with these multiple marginal seas have shaped the population structure of coastal organisms in the boundary area. The coastal area of Kyushu Island of the Japanese Archipelago, where the East China Sea (ECS), the Sea of Japan (SJ), and the Pacific Ocean (PO) adjoin within a narrow region, would be a suitable field for elucidating the effect of multiple vicariances. Herein, we conducted phylogeographical analyses of the intertidal goby Chaenogobius annularis, focusing on populations around Kyushu Island, using partial mitochondrial (mt) DNA sequences and analysis of 8 microsatellite (ms) DNA loci. We discovered a new lineage distributed along the coast of the ECS distinct from the already-known SJ and PO lineages, which we termed the ECS lineage. This ECS lineage showed remarkable mito-nuclear discordance: mtDNA analyses indicated that the ECS lineage is a highly diverged lineage branched from the PO lineage, whereas msDNA analyses suggested that the ECS lineage is closely related to the SJ lineage. This discordance could have been due to ancient hybridization. In addition, we detected SJ-PO and ECS-SJ hybrid zones on the western Seto Inland Sea and on the northwestern coast of Kyushu, respectively. Such a complex population structure suggests that the secondary contacts after multiple isolation events among marginal seas around Kyushu have a great impact on coastal biodiversity in the area and emphasize the peculiarities of the region.
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