The Mediterranean Sea is a biodiversity hotspot that requires frequent monitoring to detect ecological changes and support conservation. Within this context, the Strait of Sicily (SoS), which forms the transition zone between the western and eastern Mediterranean sub-regions, is characterized by high biological diversity and complex oceanographic processes influencing species distribution. Although the area has been the subject of several studies, the available knowledge remains fragmented, as previous investigations have largely focused on limited portions of the territory or on specific taxonomic groups, without providing a comprehensive assessment of the overall metazoan diversity. Here, we used an environmental DNA (eDNA) metabarcoding approach to provide a comprehensive assessment of marine biodiversity across the region. Samples were collected across three distinct coastal areas, at different distances from the coast and at different depths. After taxonomic curation, metabarcoding analyses identified 362 genera and 227 unique species of marine metazoans across both markers (COI: 283 genera, 215 species; 18S: 79 genera, 15 species; 11 phyla, 22 classes, 30 orders and 3 species shared). Nearshore samples showed higher observed species richness (S obs) than offshore samples for both markers, similarly to the COI-based data for different depths. Beta-diversity analyses revealed significant differences in community composition between nearshore and offshore sites (PERMANOVA, p < 0.001). Both markers identified the westernmost site as markedly distinct, whereas central and eastern ones appeared more similar to each other. We also detected six non-indigenous species (NIS), and threatened species, such as the common bottlenose dolphin (Tursiops truncatus) and the dusky grouper (Epinephelus marginatus). Our study highlights the Strait of Sicily as a biogeographic crossroads with high biodiversity. The eDNA approach identified different communities along spatial and vertical gradients and provided relevant information for the monitoring of vulnerable marine fauna and alien species.
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