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

northern Portuguese coast was corroborated by molecular detection on eDNA samples. P. pho- coena was detected via eDNA in two stations located closer to the coastline. That result is in line with the frequent occurrence of the species at the mouth of the Douro River (located in proximity to the sampling sites), as described by Gil et al. [67] with occurrence records obtained through visual monitoring. As for T. truncatus and G. griseus, both species were only sighted on very few occasions. By eDNA, each of these was detected at one station, emphasiz- ing the less frequent occurrence in the north of Continental Portugal, in comparison with D. delphis and P. phocoena. T. truncatus was detected via eDNA in northern latitudes, where the sightings of the species were also registered. As for G. griseus, the DNA was detected in the southern quadrant of the study area, where the species was also recorded through visual moni- toring, where they were also recorded by molecular detection. On the other hand, T. truncatus is a frequent species in the northwest of the Iberian Peninsula [68], therefore detection through visual and molecular methods was anticipated, although more records were to be expected. Overall, the combination of the results from visual monitoring and eDNA suggests the year- round presence of the dolphin and porpoise species on the north coast of Continental Portu- gal. Given the difficult weather conditions for visual monitoring in the ENA for a large part of the year, mostly in winter months, and consequently low monitoring effort in the region, com-

plementary results from eDNA prove to be very useful to increase baseline knowledge of these coastal populations. Besides multi-species detection on eDNA samples, the application of the methodology for single-species detection may be of particular importance. This is the case given the need to monitor resident populations in decline, as shown by Ma et al. [14] where eDNA tools were applied for conservation efforts. In the north coast of Continental Portugal, we believe that this approach is extremely pertinent for species of conservation interest, such as P. phocoena, whose population in the mainland Portuguese coastline is predicted to be extinct in 20 years (Critically Endangered, [69]). Despite the aforementioned promising results, some challenges remain to be addressed for the future consistent application of this novel monitoring tool. In previous eDNA metabarcod- ing studies, it was usually not possible to distinguish between species of the same genus, as observed in Valsecchi et al. [22] for the Tursiops and Stenella genera, and even within the Del- phinidae family [4, 20]. In the present work, this was also a challenge during the assignment of ASVs, even though a conservative selection criterion was established. Therefore, this limitation represents a significant obstacle to meet monitoring objectives. In addition, although the use of universal primers may provide us with relevant information at the multi-trophic level, there is a major drawback when the technique is used to detect a certain taxon (e.g., monitoring cetacean

occurrence). Besides the likelihood that primers bind to non-target DNA in greater abundance at the time of amplification, which can result in false negative detections [70, 71], it is impossible to pre-select the samples with the target DNA before the sequencing step as the detections in the post-PCR electrophoresis may allude to different taxonomic groups. Thus, in the context of the applicability of eDNA for more efficient and accessible (i.e., cost-effective) taxon-specific monitoring, it is advisable to use taxon-specific primers. In the specific case of cetaceans, where, in addition, relative abundance may often be rather low (in comparison to other marine taxa), the development of cetacean-specific primers is one of the next key steps to address in the con- tinued emergence of the application of eDNA tools for cetacean monitoring. Monitoring multiple trophic levels The introduction of universal primers in the study of marine communities allows the collec- tion of data from multiple trophic levels, with posterior inference of possible ecological PLOS ONE Environmental DNA as a complementary tool for biodiversity monitoring PLOS ONE | https://doi.org/10.1371/journal.pone.0300992 October 16, 2024 13 / 20