Spatial demography and population expansion
To examine the process of regional demography and the potential occurrence of genetic bottlenecks, we performed a mismatch distribution analysis using the spatial expansion model in Arlequin ver. 3.5.2.2 (27 , 28 ). Within each group except Group 1, the models did not reject the expected spatial expansion scenario with multimodal distributions of pairwise haplotypic differences (fig. S6 and table S3). In Group 1, the mismatch distribution exhibited a dominant frequency peak at one pairwise difference, indicating that the populations in this group had experienced a genetic bottleneck. The τ value, indicating the relative time span since a population spatially expanded through a region, was high in Group 2 (Clade-A) and also in Group 4 (Clade-B).
The southwestern part of Hokkaido, in which Group 1 is distributed with low genetic variation, had unique geological characteristics. For instance, the Ishikari Lowland (Fig. 3b) was submerged in seawater during the Mindel–Riss (0.18–0.23 Mya) and Riss–Würm (0.07–0.13 Mya) Interglacial periods (14 ). Moreover, results of the MiFish metabarcoding (fig. S1) also suggested the existence of many rivers in the west of the Ishikari Lowland where B. toni could not be detected using eDNA. This finding is consistent with the conventional view that the Ishikari Lowland prevented the southward dispersal of primary freshwater fishes from Siberia (13 ). In addition, the Shikotsu–Toya volcanic field have many active volcanoes, where large caldera-forming eruptions have occurred since approx. 0.10 Mya (15 ). Repeated seawater transgressions and large eruptions in this region have probably isolated the B. toni populations in southwestern Hokkaido, causing their genetic bottleneck. Several previous studies, mainly on terrestrial animals, have reported similar phylogeographies in this region. For example, the phylogeographic vicariance of the brown bear Ursus arctos , red fox Vulpes vulpes , and ezo salamander Hynobius retardatus in the Ishikari Lowland (2931 ) and low genetic variability among the southwestern populations of the dark red-backed vole Myodes rex(32 ) support the hypothesis that geological events affected the historical expansion and demography of terrestrial and freshwater organisms in southwestern Hokkaido.
Several rivers in our study were found to co-host populations of different lineages. In the Tokoro River system (No. 17 in Fig. 3b), phylogenetically distant genetic groups have seemingly and uniquely experienced secondary contact. The presence of genetically distinct individuals in this river was further confirmed through Sanger sequencing of DNA from captured individual fish samples (fig. S5). Examination of the evolutionary mechanism of this coexistence, such as reproductive isolation of the lineages, might reveal cryptic diversity in B. toni . Co-occurrences of phylogenetically distant groups were also found in other rivers, but whether such coexistence results from natural distribution or artificial translocations cannot be discerned. Although B. toni is not a target for aquaculture, human transmission through Hokkaido is possible through mixing with other fish resources such as salmon.