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

terms of composition of the cetacean community, in the summer campaigns, all the 4 species mentioned above were sighted; while in the winter campaigns, sightings of D. delphis accounted for all records, excepting one sighting of T. truncatus (considering sightings identi- fied at the species level) (Fig 4A). Through eDNA analysis though, all the species were molecu- larly detected in the winter campaigns (including also B. physalus), while in the summer season the only species identification through molecular detection of cetacean species was of D. delphis (Fig 4B). Disregarding seasonality and accounting for spatial distribution only, the location of molecular detections of each species at the sampling stations seems to align better with the visual records at the latitudinal level than in relation to distance to the coastline (i.e., longitudinally). In summary, it is worth highlighting that: i) in both datasets, D. delphis was found to be distributed across the entire area with higher frequency north of Po´voa de Varzim; ii) T. truncatus was distributed in the north of the study area, visually recorded at different dis- tances from the coast but only detected with molecular methods in the most coastal sampling station (S13); iii) P. phocoena was detected in both datasets south of Po´voa de Varzim, and mostly in stations near coast (with only one sighting further from the coast); iv) the molecular detections of B. physalus occurred in the stations closer to the coast, one in the north and another in the south of the study area (Fig 4B). Multi-trophic analysis

Across all campaigns, multiple Actinopteri detections coincided with the molecular presence of cetacean species (Table 2). D. delphis was detected by eDNA simultaneously to S. pilchardus Table 2. Coincident cetacea and Actinopteri species detections by environmental DNA at sampling stations surveyed across seasonal monitoring campaigns carried in the north coast of Mainland Portugal, between 2021 and 2023. Monitoring campaign Sampling station Cetacean Actinopteri Summer 2021 S11 Delphinus delphis Belone belone S13 Delphinus delphis Sardina pilchardus Engraulis encrasicolus Dicentrarchus labrax S15 Delphinus delphis Scomberesox saurus Winter 2021/22 S01 Phocoena phocoena Sardina pilchardus Dicentrarchus labrax Labrus bergylta Alosa alosa Pleuronectidae spp. Dicoglossa cuneata Hyperoplus lanceolatus Conger conger S04 Phocoena phocoena Sardina pilchardus Dicentrarchus labrax Alosa alosa Dicoglossa cuneata Scomber scombrus Sarpa salpa Winter 2022/23 S01 Delphinus delphis Labrus bergylta S02 Grampus griseus Sardina pilchardus S04 Delphinus delphis Sardina pilchardus Balaenoptera physalus https://doi.org/10.1371/journal.pone.0300992.t002 PLOS ONE Environmental DNA as a complementary tool for biodiversity monitoring PLOS ONE | https://doi.org/10.1371/journal.pone.0300992 October 16, 2024 10 / 20