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

widespread presence of the sardine (Sardina pilchardus)—in 14/16 sampling stations; the European seabass (Dicentrarchus labrax)—in 9/16 stations; and the European anchovy (Engraulis encrasicolus)—in 6/16 of the sampled sites (Figs 2 and 3). All the Actinopteri species identified, as well as the respective number of ASVs detected at each station, can be found in S4 Table. Additionally to the frequency of the detections across sampling stations, there is also a higher abundance of captured ASVs of the species S. pilchardus and D. labrax (Fig 3). In 5/16 sampling stations, 100% of the matched ASVs belonged to either S. pilchardus or D. labrax (Fig 2). The highest number of ASVs was obtained for S. pilchardus in Station S15 (off north of Viana do Castelo) (Fig 3). Station S01 (southwest of Porto, at ~2nm from the coast) had posi- tive detections for a higher number of selected Actinopteri species in both seasons (Fig 3). The Station S08 (near Po´voa de Varzim) only had one minor detection (9 BLAST hits for its respec- tive ASV, see S5 Table) for S. pilchardus across all monitoring campaigns. Cross-checking eDNA with visual monitoring data Overall, by cross-referencing the data obtained by the two sampling methodologies applied in this study, we can verify the occurrence of the following four species in both datasets (visual Fig 3. Number of Amplicon Sequence Variants (ASVs) detected for each species of the Actinopteri superclass molecularly identified in environmental

DNA samples, collected at the sampling stations surveyed in the summer (top) and winter (bottom) in the north coast of Mainland Portugal, between 2021 and 2023. The number of ASVs was logarithmised for scaling purposes. https://doi.org/10.1371/journal.pone.0300992.g003 PLOS ONE Environmental DNA as a complementary tool for biodiversity monitoring PLOS ONE | https://doi.org/10.1371/journal.pone.0300992 October 16, 2024 8 / 20