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

interactions and prey/predator relationships. Universal primers also provide a deeper under- standing of unusual distribution patterns, as was the case in Zhang et al. [24] where the authors used data obtained by eDNA metabarcoding to assess the food resources of Eden’s whale (Balaenoptera edeni edeni), and in Visser et al. [72] where the cephalopod community compo- sition was studied to better understand G. griseus and Cuvier’s beaked whales (Ziphius caviros- tris) foraging behaviors. In this study, it was possible to detect numerous species from the Actinopteri superclass. In addition to S. pilchardus, highly abundant and dispersed in the study region, and reported as main target for the diet of D. delphis on the Iberian coast [73, 74], other species detected have also been found in its stomach contents in the portuguese continental coast, such as the afore- mentioned E. encrasicolus, the sole (Soleidae spp.), the needlefish (B. belone), the scads (Tra- churus spp.) and the Atlantic mackerel (Scomber spp.) [74]. Another species with abundant and dispersed detections in this study, D. labrax, was also found in the stomach contents of D. delphis in the English Channel [75]. This latter coastal fish species, of high economic interest, is of particular relevance from a cetacean conservation perspective since its feeding preferences overlap with those of the dolphins, with reported implications for their bycatch by promoting interactions with the fisheries sector [76]. S. pilchardus is also extremely relevant to this topic

due to its commercial importance in Portugal, being a main target of purse seine fisheries in the country [77]. In Continental Portugal, interactions between various dolphin species with this fishing method are frequent and can result in the bycatch of these animals [78, 79]. In the case of P. phocoena, many of the species identified simultaneously, such as Scomber scombrus and the ballan wrasse (Labrus bergylta), and others occurring in other stations, such as T. tra- churus, are also known prey for these animals in the ENA [80, 81]. Although not detected in coincidental stations, a wide variety of fish taxa identified in the studied area were previously found in T. truncatus stomach contents in Iberia, namely S. pilchardus, the European conger (Conger conger), E. encrasicolus, Trachurus spp. and Soleidae spp. [82]. Therefore, data obtained from metabarcoding techniques, in addition to occasional observed foraging behavior during dolphin sightings (S3 Table), suggest that this study area may act as a relevantfeeding ground for coastal cetaceans. It also highlights the prey species available to their populations, which may have relevant implications for management and con- servation strategies. Environmental DNA is thus proving to be a promising tool for multi- trophic assessments, not only to study biodiversity occurrence, but also to infer about ecological processes and investigate trophic relationships. Conclusion In summary, in this work we provide a proof-of-concept of the potential of metabarcoding

methods applied to eDNA samples, for biodiversity assessments, with special relevance as a cetacean monitoring technique for the study of cetacean distribution and feeding ecology. However, there are still obstacles and difficulties to overcome. More studies are necessary to better understand this novel sampling method, as there are still knowledge gaps in the applica- tion of the method, from environmental sampling to the analysis of the sequencing results. Positive detections of cetacean species in this work constitute important data, as it was pos- sible to characterize the northern coast of Continental Portugal in terms of cetacean occur- rence, not only reproducing similar results but also complementing the data obtained through visual monitoring. Here, eDNA monitoring allowed us to conclude that dolphin and porpoise species that are less often sighted during visual surveys, even with known occurrence in the study area, have a probable all-year-round presence in this subregion of the ENA. Addition- ally, this study also shows the potential of this molecular-based technique to collect data when PLOS ONE Environmental DNA as a complementary tool for biodiversity monitoring PLOS ONE | https://doi.org/10.1371/journal.pone.0300992 October 16, 2024 14 / 20