An environmental DNA assay for the detection of the regionally endangered freshwater fish Alburnoides bipunctatus in Germany

The freshwater fish Alburnoides bipunctatus is a widespread species that suffered from river straightening and water pollution across Europe. The species is difficult to monitor, which hampers evaluation of current reintroduction action. We thus developed an environmental DNA (eDNA) based assay to allow for monitoring of A. bipunctatus in central Germany. A 63 bp cytochrome b-based mitochondrial marker proved highly sensitive and specific for A. bipunctatus . The assay was tested in the laboratory using different concentrations of reference samples from various local fish species. A real-life test using water samples from three reintroduction sites was used to validate the applicability of the assay.