Multiplex real-time PCR enables the simultaneous detection of environmental DNA from freshwater fishes: a case study of three exotic and three threatened native fishes in Japan

Environmental DNA (eDNA) analysis has been used as a cost-efficient and non-invasive tool for monitoring invasive and threatened species. Previous studies typically involve two approaches; species-specific detection via PCR and multiple species detection via metabarcoding. However, the former could be costly when several species are targeted, and the latter could sometimes be insufficient to distinguish closely related species. Here, the simultaneous eDNA detection from multiple species via multiplex real-time PCR was applied to 99 ponds to evaluate the distribution of three exotic and three threatened native fish species over different seasons. We detected bluegill sunfish ( Lepomis macrochirus ) eDNA at 31 sites, largemouth bass ( Micropterus salmoides ) eDNA at 22 sites, smallmouth bass ( Micropterus dolomieu ) eDNA at one site, golden venus chub ( Hemigrammocypris rasborella ) eDNA at 11 sites, Japanese medaka ( Oryzias latipes ) eDNA at 26 sites, and weather loach ( Misgurnus anguillicaudatus ) eDNA at 41 sites. We found that eDNA detection rates were higher in early summer for all fish species. Moreover, exotic fish eDNA was detected more frequently in ponds which were easier to access by car and which have a larger surface area and higher pH. Furthermore, the detection rates of native fish eDNA were generally lower in the ponds where exotic fish eDNA was detected more frequently. Multiplex real-time PCR can help detect the distribution of exotic and threatened native species for conservation and ecosystem management. This method is expected to substantially contribute to the early detection of invasive species and the efficient protection of threatened species’ habitat.