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

the monitoring effort is hampered by the inherent conditions of the sampled region (e.g., weather conditions), especially for less frequent species (possibly without existing visual rec- ords). Furthermore, by expanding the analysis to lower trophic levels, the overlap detection of dolphins and multiple known prey allows us to infer about their use of these coastal area for feeding purposes. In conclusion, this work has demonstrated the potential of an innovative monitoring meth- odology for studying complex marine biological communities, such as cetaceans, even allow- ing for a multi-trophic approach, essential for conservation efforts. Therefore, although the effectiveness of using eDNA as a tool in cetacean monitoring programmes remains under development, this work represents a step forward towards that goal. Supporting information S1 Fig. A—Cetacean occurrence records and spatial distribution registered during visual sur- veys in the North coast of Mainland Portugal, between 2021 and 2023, with reference in-land points. Coloured points represent cetacean sighting locations. B—Spatial representation of sampling stations surveyed in the North coast of Mainland Portugal, between 2021 and 2023. The map represents the positive molecular detection for cetacean species on environmental DNA samples (Phocoena phocoena, Delphinus delphis, Tursiops truncatus, Grampus griseus, Balaenoptera physalus). (TIF) S1 Table. Pre-defined eDNA sampling stations on the vertices of the monitoring transects, with distance from coastline and gegraphical coordinates (DD). (XLSX)

S2 Table. Sample log of all eDNA samples collected in pre-defined stations (see methods), with respective sampling date, DNA concentration quantified using Qubit™ dsDNA High Sensitivity (HS) assay kit (Invitrogen™) and total number of ASVs generated by Illumina MiSeq300 platform after read quality-filtering. (XLSX) S3 Table. Recorded sighintgs during the visual surveys on the season monitoring cam- paigns between the summer of 2021 and the winter of 2022/2023 in the north coast of con- tinental Portugal, sighing date, identified species and respective geographical coordinates (DD). Observed foraging behaviour was also recorded whenever it was possible to clearly determine it. (XLSX) S4 Table. Pairwise Wilcoxon test results between the collected eDNA samples originated data in the various monitoring campaigns carried out between the summer of 2021 and the winter of 2022/2023 in the north coast of Continental Portugal. A—DNA concentra- tions by monitoring campaign. B—Total number of target taxa ASVs (cetaceans and selected Actinopteri species with known occurrence in Continental Portugal). P-values of less than 0.05 were regarded as statistically significant. (XLSX) S5 Table. Grouped number of total ASVs detected for all identified species in the eDNA samples collected between the summer of 2021 and the winter of 2022/2023 in the north coast of continental Portugal. (XLSX) PLOS ONE Environmental DNA as a complementary tool for biodiversity monitoring PLOS ONE | https://doi.org/10.1371/journal.pone.0300992 October 16, 2024 15 / 20