Strategic eDNA metabarcoding sampling outperforms conventional monitoring methods and reveals spatial patterns of vertebrate biodiversity in a Mediterranean river basin

Effective monitoring is crucial for assessing biodiversity loss and detecting the early introduction of alien species, especially in freshwater ecosystems that are facing degradation at an alarming rate. However, traditional monitoring methods in lotic environments are often invasive, require specific expertise, and involve specimen handling that can lead to organism mortality. Emerging techniques, such as environmental DNA (eDNA) metabarcoding, offer a non-invasive alternative by generating biodiversity checklists from genetic material naturally shed by organisms. In this study, we applied eDNA metabarcoding to sample five strategic areas within the Serchio River basin, an understudied river system in Tuscany, central Italy, characterized by heterogeneous environments. Water samples were filtered, and eDNA was extracted from rivers of different sizes within each area. A total of 62 taxa were identified, encompassing both protected and invasive alien species representing the major vertebrate classes present in these rivers. Observed distribution patterns aligned with ecological expectations (i.e., known habitat preferences and longitudinal river zonation). Vertebrate biodiversity data obtained through eDNA were compared to efforts using traditional monitoring methods, including transect surveys conducted between March 2021 and October 2023 and electrofishing sessions. Across all areas, eDNA detected a higher number of species, including taxa not strictly associated with the river system, although both approaches identified some unique taxa. Taxa counts were generally higher in the mainstem than in smaller tributaries, suggesting that eDNA accumulates in main river branches. This finding underscores the potential of focusing on mainstem river courses for more efficient biodiversity monitoring at the basin scale. Through eDNA metabarcoding, we effectively characterized the Serchio River basin's biodiversity in a relatively short time compared to traditional methods, obtaining qualitative insights into the occurrences of both aquatic and terrestrial vertebrates, and capturing the gamma diversity of the area.