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

value = 0.009). Pairwise tests highlight differences between the two summer campaigns and both winter campaigns with the summer of 2022 (S4B Table). With regard to distance from the shore, samples collected closer (2 nm) resulted in an average number of ASVs of 988.13 ± 2087.06 (Median = 0), while those further away (12 nm) resulted in 4,423.31 ± 17,126.03 ASVs (Median = 52.5). However, differences on obtained ASVs in rela- tion to the distance to the coast were not significant (p-value = 0.64). Of the 64 eDNA samples sequenced through metabarcoding, 33 (51.6%) had positive detec- tions of representative ASVs, from which 10 of the samples (15.6%) had positive detections of cetacean ASVs. By grouping the results by sampling station, we noticed that all sampling stations had positive detections for either Cetacea or Actinopteri. Relative abundances of all cetacean spe- cies identified, and Actinopteri taxa detected in 4 or more stations, are illustrated in Fig 2. Focusing on cetaceans, 5 different species were detected, with at least one of them identified in 7 out of the 16 stations (Fig 2 and S4 Table). The common dolphin (Delphinus delphis) was the most detected species, being identified in 6/16 stations, followed by the harbor porpoise (Phocoena phocoena) and the fin whale (Balaenoptera physalus), occurring in 2/16 sampling stations. The Risso’s dolphin (Grampus griseus) and the bottlenose dolphin (Tursiops trunca- tus) were detected in 1/16 stations. Regarding Actinopteri species, 19 species were identified, with at least one of them detected

in all sampling stations (Figs 2 and 3). Of all these detections, the most notable result was the Fig 2. Taxonomic assignment from environmental DNA samples collected in 16 sampling stations, in the north coast of Mainland Portugal, between 2021 and 2023. The plot represents the relative abundance (%) of sequences matched for each marine vertebrate species, considering pre-determined Cetacea, and Actinopteri species with positive detection in 4 or more stations (see methods). “Others” represents Actinopteri superclass species identified in 3 or fewer stations. https://doi.org/10.1371/journal.pone.0300992.g002 PLOS ONE Environmental DNA as a complementary tool for biodiversity monitoring PLOS ONE | https://doi.org/10.1371/journal.pone.0300992 October 16, 2024 7 / 20