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

The use of environmental DNA (eDNA) to assess the presence of biological communities has emerged as a promising monitoring tool in the marine conservation landscape. Moreover, advances in Next-Generation Sequencing techniques, such as DNA metabarcoding, enable multi-species detection in mixed samples, allowing the study of complex ecosystems such as oceanic ones. We aimed at using these molecular-based techniques to characterize cetacean communities, as well as potential prey on the northern coast of Mainland Portugal. During four seasonal campaigns (summer 2021 to winter 2022/2023), seawater samples were collected along with visual records of cetacean occurrence. The eDNA isolated from 64 environmental samples was sequenced in an Illumina platform, with universal primers targeting marine vertebrates. Five cetacean species were identified by molecular detection: common dolphin (Delphinus delphis), bottlenose dolphin (Tursiops truncatus), Risso's dolphin (Grampus griseus), harbor porpoise (Phocoena phocoena) and fin whale (Balaenoptera physalus). Overall, except for the latter (not sighted during the campaigns), this cetacean community composition was similar to that obtained through visual monitoring, and the complementary results suggest their presence in the region all year round. In addition, the positive molecular detections of Balaenoptera physalus are of special relevance since there are no records of this species reported on scientific bibliography in the area. The detection of multiple known prey of the identified dolphins indicates an overlap between predator and prey in the study area, which suggests that these animals may use this coastal area for feeding purposes. While this methodological approach remains in a development stage, the present work highlights the benefits of using eDNA to study marine communities, with specific applications for research on cetacean distribution and feeding ecology.