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Phylogeography relates the formation of species distributions, ecological shifts, and evolutionary processes to geology (1 ). For example, one phylogeographic study has linked multiple glacial refugia in Europe to genetic diversity in plants (2 ). Other phylogeographic studies have revealed the common African origins of humans and multiple origins of domesticated livestock (35 ). In addition, environmental DNA (eDNA) analysis of sediment samples has unveiled a multimillion year old ecosystem (e.g., from the Kap København Formation in Greenland) (6 ). Nevertheless, the current phylogeographic approaches are largely limited by less number of samples. To comprehensively understand the ecology and evolution of a target species, hundreds or thousands of individual tissue samples are required within a large-scale geographic scheme. Small or unequal sample sizes leads to errors in statistical models, while low spatial coverage of the sampling points obscures phylogeographic patterns (79 ).
Advancements in bioinformatic processing methods, especially amplicon sequence variant (ASV) methods, can distinguish biological sequence variants from erroneous ones. These technologies have substantially improved eDNA-based population genetic studies while negating the need for sampling target species in the wild (1012 ). Because this technique minimizes the effort of sample collection (for instance, fish eDNA can be detected from water scooped from water bodies), it can innovatively reveal the fine-scale phylogeographic structure of a species without sampling biases.
In this study, we conducted an eDNA-based phylogeographic study of a primary freshwater fish, the Siberian stone loach Barbatula toni , in Hokkaido (the northernmost large island in Japan). Goto et al. proposed that B. toni colonized rivers in Hokkaido from Sakhalin, the northern continental Far East, via the Soya Strait, where land bridges were periodically formed during the ice age (13 ). Terrestrial and freshwater faunae in Hokkaido are particularly unique to the Japanese archipelago as they are segregated by a biogeographic boundary known as the Blakiston Line. In addition, the organisms on this island have been subjected to numerous large-scale volcanic and glacial events, leaving behind geological evidences, such as sedimentary layers (14 , 15 ). B. toni is among the most common primary freshwater fishes inhabiting the rivers in Hokkaido. Members of this primary freshwater fish species depend exclusively on their inhabited river system throughout their life history, often resulting in strong congruence between their geographic placements and phylogenetic breaks in their populations (16 ). Biologists have examined the history of colonization, expansion, and diversification of primary freshwater fishes in relation to geological and/or climatic changes. For example, European freshwater fishes (genus Sabanejewia ) expanded rapidly and became genetically homogenized during Pleistocene glaciations (17 ) and Mesoamerican fishes expanded and diverged from their South American origins via historical drainage connectivity (18 ). Thus, the intraspecific phylogeographic pattern of B. toni might provide insights into the paleogeological and climatic events that have shaped the distribution and diversity of living organisms on the Hokkaido island. Although eDNA analyses have captured large-scale intraspecific genetic diversity (11 , 12 ), no studies have addressed the novel histories of freshwater fishes over evolutionary timescales using eDNA till date.
Applying the eDNA technique, we detected the intraspecific genetic variations in the mitochondrial cytochrome b (cyt-b ) gene of B. toni and assessed their phylogeographic pattern throughout Hokkaido. After revealing the phylogeographic structures, we conducted molecular analyses of tissue-derived DNA to evaluate the potential scenarios of population expansion and vicariance of B. toni .