Phylogenetic relationship and geographic structure
A Bayesian phylogenetic analysis classified the eDNA haplotypes into two distinct lineages (Fig. 1; Clade-A and Clade-B), each containing three genetic groups. The network analysis also confirmed their phylogenetic relationships (Fig. 2). Groups 1 and 6 revealed a large genetic divergence between the two distinct lineages, with the dominant haplotypes BTW-00 and BTO-00 occupying the centers of each group. Additionally, the haplotypes in other groups were connected in a star-like manner to Groups 1 and 6. Moreover, the distributions of these phylogenetic groups were regionally restricted in Hokkaido (Fig. 3). Clade-A and Clade-B were separately distributed in southern and northern Hokkaido, respectively. While the eDNA concentrations largely varied among rivers, the detected haplotype richness was not significantly correlated with eDNA concentration (copy/L) (R 2 = 0.04, p = 0.18; figs. S2, S3, and S4).
The phylogenetic analysis using Sanger-sequenced cyt-b sequences (868 bp) supported the phylogenetic relationship constructed using the eDNA-based analysis (fig. S5). In the haplotype derived from tissue-derived DNA of the Sakhalin specimen, a 221-bp sequence of the region targeted by the eDNA assay was identical to BTO-00 of Group-6, the major haplotype found in northern Hokkaido. While the reference sequences of B. toni and a closely-related species B. nudacollected from the continent (northern China and Amur Basin) were positioned at the ancestral outgroup, the B. toni haplotype obtained from the Sakhalin specimen was included within the northern lineage (Clade-B) in Hokkaido.
A molecular clock analysis (2.5%–2.8%/Myr (2325 )) informed that the two clades diverged in the early Pleistocene (1.59–1.78 Mya, table S2). During the early Pleistocene, the glacial and interglacial periods repeated in cycles and land bridges emerged at the Soya Strait due to sea-level drops during glaciation (26 ). Therefore, it is suggested that the ancestors of Clade-A first colonized Hokkaido from Sakhalin during a low-sea-level period in the early-Pleistocene glaciation. During an interglacial period, they were long-term isolated by the opening of the Soya Strait. In contrast, Clade-B includes the northern Hokkaido and Sakhalin populations, which diverged circa 0.16–0.18 Mya. This estimate indicates that the ancestors of Clade-B dispersed southward to Hokkaido from Sakhalin via land bridges formed around that period.