Detecting Threatened Ichthyofauna in a Mediterranean Intensive Agricultural Landscape: From DNA Traces to Electrofishing

Intensive agriculture and farming activities, industry, as well as human settlement can seriously affect freshwater ecosystems and the species they host. These disturbances have been previously monitored using traditional catch surveys. New methods, such as environmental DNA (eDNA) metabarcoding, can significantly improve biodiversity mapping by accessing rarely visited areas and by enhancing detection of rare and invasive species. We conducted an eDNA survey in a complex river and tributary system at Tenagi, Philippi that hosts approximately 12.5% of Greek freshwater ichthyofauna. A primer pair targeting the 12S rRNA was used to analyse eDNA samples from 30 sites. A reference sequence database was generated to improve regional diversity monitoring of endemic, native and invasive fishes. Results were compared with data collected using electrofishing from stations that allowed access. Two endemic, three invasive, two translocated and 10 native species were detected in their current known distribution using both methods. Overall, eDNA metabarcoding being more sensitive, consistently detected more freshwater species than traditional methods on a single fieldwork expedition. Co-occurrence of species was also investigated for the first time, revealing minor variations in species composition within fish communities. Stations in intensive agricultural zones with potentially high accumulation of organic and inorganic pollutants (Tributary D), exhibited lower number of reads and species richness than other tributaries at Tenagi. Our results demonstrate the need for complementary use of both eDNA metabarcoding and electrofishing to assess fish assemblages in freshwater habitats, as a combined approach can address reliability issues and biases associated with each method.