Assessing the conservation effectiveness of Natura 2000 areas for freshwater communities using environmental DNA

Although covering less than 1% of the Earth's surface, freshwater habitats host a high proportion of global animal biodiversity and provide essential ecosystem services. These habitats, however, are experiencing some of the fastest rates of biodiversity decline and are among the most threatened ecosystems worldwide. To evaluate the effectiveness of Natura 2000 (N2K) protected areas in conserving both freshwater and terrestrial biodiversity, we compared protected and adjacent unprotected streams of the Northern Apennines hydro-ecoregion (Central Italy) through an integrated monitoring approach. We combined multi-marker eDNA metabarcoding to simultaneously record vertebrate and invertebrate species across terrestrial and aquatic biomes with the analysis of physicochemical and geomorphological parameters of streams within and outside protected boundaries. We specifically assessed community richness and composition, taxonomic and phylogenetic diversity, environmental quality, and the occurrence of species of conservation concern. We detected over 1100 taxa and found that protected and unprotected sites exhibited comparable environmental conditions and did not differ significantly in overall species richness. However, protected streams hosted freshwater communities with significantly higher taxonomic diversity and a greater number of aquatic species of conservation interest. These results suggest that the targeted N2K sites effectively preserve freshwater-related communities. Furthermore, the good ecological status of adjacent unprotected sites highlights their role as functional buffer zones that bolster PA effectiveness. Finally, we discuss the application of eDNA metabarcoding and the use of taxonomically and phylogenetically weighted indices as robust, standardized ecological indicators which are critical for providing an in-depth evaluation of conservation effectiveness.