Contribution of eDNA Metabarcoding to Model Ecosystems: Case Study of Threatened Peri-Urban Mediterranean River

Freshwater ecosystems on Mediterranean islands are threatened refuges where conventional bioassessments often fail to capture the full spectrum of biotic assemblages. Here, environmental DNA (eDNA) metabarcoding targeting the mitochondrial 12S rRNA gene was applied across three representative sampling reaches (Upstream, Midstream, Downstream) of the Oreto River (Sicily, Italy) during a single sampling campaign to assess vertebrate composition. This targeted spatial snapshot captured aquatic, semi-aquatic, and terrestrial signals. Upstream reaches featured synanthropic taxa, while midstream sections showed high signals of livestock (e.g., Bos taurus, Ovis aries, Sus scrofa), likely reflecting agricultural land use. Downstream reaches exhibited high taxonomic heterogeneity, comprising native fauna-including the Critically Endangered European eel (Anguilla anguilla)-alongside wild birds, amphibians, and non-native species (e.g., Ameiurus melas, Micropterus salmoides). By recovering conservation-priority taxa, non-native species, and broader potential anthropogenic-associated signals (including dietary marine fish detections), this study demonstrates eDNA's effectiveness in capturing complex biological footprints. Although physicochemical conditions were not directly assessed, and despite the temporal limitations of a single snapshot, these findings highlight the promise of eDNA metabarcoding as a complementary tool for future environmental tracking, watershed management, and spatial conservation planning.