Dual expansion routes likely underlie the present-day population structure in a Parnassius butterfly across the Japanese Archipelago

The Japanese Archipelago, comprising a series of isolated yet interconnected islands, had been geographically separated from the Eurasian continent. The linear topography presents a unique biogeographic context for dispersing organisms from the continent. In this study, we utilized mitochondrial DNA (mtDNA) and single nucleotide polymorphism (SNP) variation to elucidate the dispersal history of the Japanese clouded butterfly Parnassius glacialis across the Japanese Archipelago, including North Island (Hokkaido), Main Island (Honshu) and Shikoku Island. Our analysis of mtDNA (COI, COII) at 1192 base pairs (bps) revealed 49 haplotypes and identified three distinct haplotype groups in the network. These groups correspond geographically to East Japan, West Japan, and Chugoku-Shikoku. The Chugoku-Shikoku group is the most ancient lineage. Interestingly, the Chugoku-Shikoku lineage showed a closer network connection to the East Japan lineage than the geographically proximate West Japan lineage. Divergence time estimates suggest that the Chugoku-Shikoku lineage diverged from the continental P. glacialis approximately 3.05 million years ago (MYA). Subsequently, from the Chugoku-Shikoku lineage, the East Japan and West Japan lineages diverged around 1.05 MYA, with the subsequent divergence between the East and West Japan lineages occurring at approximately 0.62 MYA. Based on the 3067 SNP genotypes, population structure analysis revealed five distinct genetic structures within the Japanese Archipelago, indicating geographical differentiation. From the analyses by mtDNA and SNP variations, four primary genetic barriers were identified: between Hokkaido and Honshu, between East and Central Japan, within the Kansai region, and within the Chugoku region. The former three lines corresponded to the Blakiston Line, the Itoigawa-Shizuoka Tectonic Line, and Lake Biwa, respectively. These findings suggest that P. glacialis diverged from the continental P. glacialis and expanded its range across the Japanese Archipelago via the North and South routes, establishing its current distribution.