Environmental DNA as a complementary tool for biodiversity monitoring: A multi-technique and multi-trophic approach to investigate cetacean distribution and feeding ecology

area and infer the ecological reasoning behind it by recurring to a universal approach to per- form a multi-trophic analysis. Furthermore, we compared the eDNA results on cetacean spe- cies detection with the data obtained by traditional visual monitoring techniques, in order to assess the true potential of eDNA as a complementary tool across the panoply of methods employed for cetacean monitoring. Methods Study area Surveys to collect eDNA samples were performed on the north coast of Continental Portugal. This subregion, located in the northwest of the Iberia Peninsula, is of particular ecological interest due to the upwelling phenomenon strongly present along the coastline, thus enhanc- ing primary production and providing great conditions for the development of complex and rich trophic chains [37–39]. In addition, the area is part of a particularly dynamic coastal region with several estuaries of rivers that flow into it [40]. Topographically, the study area is entirely placed on the continental shelf, with the presence of the Porto Canyon in the vicinity offshore, a relevant structure as submarine canyons are known to be impactful in hydrody- namic processes of coastal habitats, such as enhancing upwelling and subsequently organic productivity [41] (Fig 1). To survey the area, four seasonal monitoring campaigns were carried out between the sum- mer of 2021 and the winter of 2022/2023. Dates for all carried campaigns are described in Table 1.

Fig 1. Study area with surveyed transect of at-sea monitoring campaigns, conducted between the summer of 2021 and the winter of 2022/2023, on the north coast of Continental Portugal. Isobaths with bathymetry in meters. https://doi.org/10.1371/journal.pone.0300992.g001 PLOS ONE Environmental DNA as a complementary tool for biodiversity monitoring PLOS ONE | https://doi.org/10.1371/journal.pone.0300992 October 16, 2024 3 / 20