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

Statistical tests (Kruskall-Wallis test and pairwise Wilcoxon tests, with the level of signifi- cance set to 0,05) were carried out to understand the variation in the total number of ASVs obtained in the samples at the different seasonal campaigns and the different distances from the coast (2 nm versus 12 nm). Results Visual surveys A total of 71 sightings of 4 different cetacean species were recorded during monitoring cam- paigns (S1A Fig and S3 Table). The majority of these records were from common dolphin (Delphinus delphis) with 45 sightings across the entire surveyed area, representing approxi- mately 63.4% of all records. As for the other 3 species—bottlenose dolphin (Tursiops trunca- tus), Risso’s dolphin (Grampus griseus) and harbor porpoise (Phocoena phocoena)–these were sighted on 3 different occasions each. Besides the identified records to the species level, there were also 13 sightings of unidentified Delphinidae (18,3% of total records) and, on 4 occasions, it was only possible to identify cetacean occurrence at the superorder level (Cetacea). All sight- ings recorded with the respective date and geographical coordinates can be found in S3 Table. eDNA sampling Among the campaigns conducted, the summer of 2021 showed much higher DNA concentra- tions when compared to the other seasonal campaigns, with an average quantification of 9.46 ± 9.05 ng/μL (Median = 9,22 ng/μL). This was followed by the winter of 2021/22 with 5.10 ± 6.11 (Median = 3.02 ng/μL), the winter of 2022/23 with 2.26 ± 3.50 ng/μL (Median = 0.8

ng/μL) and, finally, the summer of 2022 with 1.08 ± 2.38 ng/μL (Median = 0.67 ng/μL). Statisti- cal tests showed significant differences between the quantified concentrations of the different campaigns (p-value = 9.63e-06), these being explained by differences between summer 2021 and summer 2022, summer 2021 and winter 2022/23, and also between summer 2022 and win- ter 2022/23 (S4A Table). At the seasonal level, the summer shows higher concentrations, with 5.52 ± 8.18 ng/μL (Median = 3.51 ng/μL) compared to the 3.59 ± 5.02 ng/μL (Median = 1.49 ng/μL) obtained for the winter. However, this difference was not statistically significant (p- value = 0.79). Taking distance into account, the sampling stations near the coast (2 nm) had an average DNA concentration of 5.48 ± 8.18 ng/μL (Median = 3.2 ng/μL), while the DNA obtained in stations at 12 nm was quantified as 3.75 ± 5.21 ng/μL (Median = 1.26 ng/μL) on average. These differences were not statistically significant (p-value = 0.45). eDNA monitoring: Sequencing output and taxonomic identification A total of 5 876 226 reads were generated by the Illumina platform with an approximate aver- age of 90 403.48 ± 42 311.20. After quality-filtering steps, the final output was 3 735 272 total reads with an average of approximately 57 465.72 ± 40 520.33 (representing 65.6% of the input). There were 5 outlier samples that produced less than 50 reads. A total of 173 156 repre- sentative ASVs (of previously determined marine vertebrate species: cetaceans and selected

Actinopteri species with known occurrence in Continental Portugal). Among the different sea- sonal campaigns, winter 2022/23 obtained the highest number of resulting ASVs with an aver- age of 7 951.5 ± 23 991.69 (Median = 39), followed by winter 2022/23 with 2024 ± 2 980.45 ASVs (Median = 722.5), summer 2021 with 932.33 ± 1 716.23 ASVs (Median = 201.5), and finally summer 2022 with an average of 51.5 ± 178.56 ASVs (Median = 0). The statistical tests carried out indicate that there was a significant difference in the uptake of target eDNA taxa, both between seasons (p-value = 0.019) and between the different campaigns (p- PLOS ONE Environmental DNA as a complementary tool for biodiversity monitoring PLOS ONE | https://doi.org/10.1371/journal.pone.0300992 October 16, 2024 6 / 20