Comparative Multi‐Marker Environmental DNA Metabarcoding of Marine Metazoan Communities: Water vs. Sediment

This study investigates the metazoan biodiversity in the Southern Adriatic Sea using environmental DNA (eDNA) metabarcoding. Sediment and adjacent water samples were collected from three sites (one pristine, two impacted by human activities) at three distances from the coast across two seasons. The complex four-factor experimental design (576 samples) addresses key sources of eDNA variability and provides a valuable comparison of markers (COI and 18S) and sample types, which remain rare in the literature. Results showed differences in the number and type of taxa identified, taxonomic resolution, and number of amplicon sequence variants (ASV) per operational taxonomic unit (OTU) across markers. The obtained overall community structure (beta-diversity) was similar for both markers. Sediment samples had higher OTU richness, but lower diversity than water samples. The two sample types provided distinct and only partially overlapping views of biodiversity. Sediment samples were rich in benthic species, whereas water samples featured mostly planktonic and nektonic species. Biodiversity varied by site and season, with sediment samples showing less seasonal variability. The pristine site did not host higher biodiversity than impacted sites, likely because of the latter's habitat heterogeneity. This study confirms the effectiveness of eDNA metabarcoding for biodiversity assessment in coastal ecosystems and provides a foundational dataset for future monitoring. By highlighting the complementary nature of COI and 18S markers and the role of sample type, this research supports integrating eDNA metabarcoding into routine environmental monitoring programs while emphasising the need for further standardisation and improved reference databases.