Using marine environmental DNA for biodiversity monitoring and conservation planning

The Mediterranean Sea, a semi-enclosed basin at the intersection of three continents, represents one of the most distinctive and complex marine ecosystems on Earth, sustaining a remarkable fraction of global marine biodiversity. Being geographically isolated, the basin is acutely vulnerable to anthropogenic pressures. Within this dynamic and changing context, marine mammals play a fundamental ecological and functional role in the health of several biocenoses. As apex predators, nutrient vectors, and sentinels of marine conditions, their decline reflects not only individual species vulnerability but also the detriment of the entire marine trophic web and its associated ecosystem services. However, as these species mostly live and breed in the open sea, monitoring of marine mammals has been notoriously challenging, resulting in sparse or missing data on their conservation. Consequently, governance currently frames assessment targets that include marine mammal conservation status, underpinning the need for scalable, standardisable monitoring. The overarching goal of this doctoral research is to contribute to the understanding and conservation of Mediterranean marine mammal biodiversity through an integrated approach based on environmental DNA (eDNA) analysis. This work brings together ecological, genetic, and conservation perspectives to address both methodological and applied questions. The research addresses a core methodological limitation in marine biodiversity surveys, the under-detection of rare, low-density marine mammals’ eDNA, by developing a multiplexed metabarcoding workflow. Taking advantage of highly biodiverse habitats for cetaceans, such as the Maldivian Archipelago, new PCR protocols have been tested employing a combination of universal vertebrate primers and newly designed marine mammal-specific primers, demonstrating that primer multiplexing does not compromise sequencing depth while enhancing cetacean detection sensitivity. The obtained data allowed to highlight clear habitat-specific trends, confirming that eDNA metabarcoding can recover both diversity and trophic structure of entire marine communities. Building on these empirical results, the second component of the thesis integrates the molecular findings within the broader conservation goal of Conceptu Maris, a European-funded LIFE project aiming to fill the current lack of data on the distribution, habitat preferences and abundance of cetacean species in the Mediterranean Sea. Within the context of the project, offshore sampling of eDNA has been implemented by detailing a Standard Operating Procedure (SOP) for the collection and filtration of samples from commercial ferries. The protocol exploits maritime companies’ vessels, closing the accessibility gap in pelagic waters and turning ferries into “floating labs” that mainstream molecular monitoring in offshore conservation. The ferry-based protocol allowed for extensive sampling across the central-western Mediterranean Sea basin, resolving broad-scale biodiversity patterns for cetaceans, fishes, and invertebrates. Combining different eDNA molecular techniques, nine cetacean species were detected, including the first molecular evidence of the pygmy sperm whale (Kogia breviceps) in the basin. Spatial-seasonal analyses identified biodiversity hotspots, diel signals and prey associations via indicator and co-occurrence analyses. Ultimately, the goal of this research was to establish a validated molecular eDNA workflow for improving the detection of rare megafauna. Finally, applying eDNA to a basin-scale monitoring, we aimed at gathering conservation-grade information on species distributions, community structure, and diel/seasonal dynamics. This study provides validated tools for detecting and characterizing marine megafauna across spatial and ecological gradients, establishing a methodological basis for standardized, non-invasive monitoring of rare taxa.