Environmental <scp>DNA</scp> unveils deep phylogeographic structure of a freshwater fish

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  • Tetsu Yatsuyanagi
    Graduate School of Agriculture Hokkaido University Sapporo Japan
  • Takashi Kanbe
    Research Faculty of Agriculture Hokkaido University Sapporo Japan
  • Kazuya Fujii
    Graduate School of Agriculture Hokkaido University Sapporo Japan
  • Shouko Inoue
    Research Faculty of Agriculture Hokkaido University Sapporo Japan
  • Hitoshi Araki
    Research Faculty of Agriculture Hokkaido University Sapporo Japan

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<jats:title>Abstract</jats:title><jats:p>Phylogeography bears an important part in ecology and evolution. However, current phylogeographic studies are largely constrained by limited numbers of individual samples. Using an environmental DNA (eDNA) assay for phylogeographic analyses, this study provides detailed information regarding the history of Siberian stone loach <jats:italic>Barbatula toni</jats:italic>, a primary freshwater fish across the whole range of Hokkaido, Japan. Based on an eDNA metabarcoding on 293 river water samples, we detected eDNA from <jats:italic>B. toni</jats:italic> in 189 rivers. A total of 51 samples, representing the entire island, were then selected from the <jats:italic>B. toni</jats:italic> eDNA‐positive sample set for the subsequent analyses. To elucidate the phylogeographic structure of <jats:italic>B. toni</jats:italic>, newly developed eDNA metabarcoding primers (Barba‐cytb‐F/R) were applied to these samples, specifically targeting their haplotypic variation in cytochrome <jats:italic>b</jats:italic>. After a bioinformatic processing to mitigate haplotypic false positives, a total of 50 eDNA haplotypes were identified. Two regionally restricted, genetically distinct lineages of the species were revealed as a result of phylogeographic analyses on the haplotypes and tissue‐derived DNA from <jats:italic>B. toni</jats:italic>. According to a molecular clock analysis, they have been genetically isolated for at least 1.5 million years, suggesting their ancient origin and colonisation of Hokkaido, presumably in the glacial periods. These results demonstrate how freshwater fishes can alter their distributions over evolutionary timescales and how eDNA assay can deepen our understanding of phylogeography.</jats:p>

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