Comparative phylogeographic inference
The eDNA extracted from water scoops revealed the large-scale phylogeographic structure of B. toni . Two distinct lineages were found in the northern and southern regions of Hokkaido, which were not separated by obvious geographic barriers. The observed pattern differed from that of other aquatic species reported in Hokkaido. BesidesB. toni , two other species of primary freshwater fishes are also known to have colonized in Hokkaido from Sakhalin across the Soya Strait: the pond minnow Rhynchocypris percnurus sachalinensis and eight-barbel loach Lefua nikkonis (13 , 33 ). The lineages of R. p. sachalinensis are clearly differentiated on the east and west sides of the Hidaka Mountains at the center of Hokkaido (34 ). The Japanese crayfish Cambaroides japonicus(35 ) also exhibits the similar phylogeographic pattern. The earliest-branched lineage of L. nikkonis (belongs to the Nemacheilidae family, like B. toni ) is distributed along the Sea of Okhotsk (northeastern Hokkaido), while the late-branched lineage has wide-range distribution without either north–south or east–west divided structure (33 ). No prior study on aquatic organisms has demonstrated clear north–south phylogeographic vicariance in Hokkaido without current geographic barriers.
Based on the estimated divergence times, we concluded that B. toni immigrated multiple times to Hokkaido via land bridges during the ice age. The north–south phylogeographic structure of this species possibly arose from the long time lag (over one million years) between the independent colonization of Hokkaido by the ancestral populations of the two clades. The phylogeography of this species is a potentially important indicator of the historical footprints in regions of Hokkaido and the past connection between Hokkaido and Sakhalin. Through future studies on this species in Sakhalin and other Far East regions, we could understand the distribution expansion of B. toni and other species from the continent to the islands. Our findings highlight thatB. toni , which has been overlooked as a common species, is a valuable source of biogeographic information on past geology and environments.