Environmental DNA supports persistence of the mythical Japanese freshwater fish “Jindai Dojo” and its conspecificity with an Eastern Japanese relict species

Environmental DNA (eDNA) analysis provides a means of detecting cryptic and rare taxa and identifying their genetic lineages, even when they are no longer detected by conventional surveys. The freshwater dojo loach locally known as the “Jindai Dojo” was historically collected in Iga City, central Honshu, Japan, and morphological evidence has suggested that it is conspecific with Misgurnus sp. Type I, a cryptic relic species distributed in eastern Japan that is of high conservation concern. However, the “Jindai Dojo” has long been considered extinct, and neither its proposed taxonomic identity nor its persistence has been evaluated using genetic evidence. Therefore, we integrated a Misgurnus sp. Type I-specific eDNA assay with a phylogeographic framework and analyzed water samples collected from 33 sites in and around Iga City. Misgurnus sp. Type I eDNA was detected at six sites, including three sites within the historical range of the “Jindai Dojo” and three sites in nearby river basins with no previous collection records. Sequence analysis of the amplified mitochondrial DNA fragments showed that all detections belonged to subclade J of the Japan Sea-side clade of Misgurnus sp. Type I. These findings support the hypothesis that the “Jindai Dojo” is conspecific with Misgurnus sp. Type I and indicate that this lineage persists within its historical range and nearby river basins. Our study demonstrates the value of integrating species-specific eDNA assays with phylogeographic information to rediscover threatened lineages and clarify the taxonomic identities of organisms presumed to be extinct.