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

is known to vary substantially interannually, with increasing extension into the ocean in winter months [49]. Overall, we consider that further studies are required in the future to monitor the influence of environmental conditions on eDNA uptake, and therefore evaluate sampling suc- cess in dynamic marine coastal ecosystems. Bioinformatic analysis criteria To compile the BLASTn databases, we established a strict criterion, comprising the amplified fragment of interest and the sequences of the forward and reverse primers for the aforemen- tioned species of interest. However, this type of approach for sequence selection has its pros and cons. On the positive side, it allows for greater certainty that a given sequence belongs to a respective species since smaller fragments can result in greater similarity between species (con- servative approach). However, this method can lead to a loss of information by possibly removing sequences of interest. Moreover, and particularly for assembling the database of sequences corresponding to the Actinopteri species, the approach was only focused on the spe- cies already recorded along the Portuguese coast, substantially reducing the size of the data- base. Such a decision was made since, in this context, the purpose for the detection of Actinopteri species was tied to the ecological role they play in the diet of cetaceans. Given the parameters applied in the BLASTn algorithm used for the taxonomic assignment (identity higher than 99%), there were only two cases in which multiple species matched to a

given ASV: one within Cetacea taxa, and another within Actinopteri. A specific example of multi-species assignment to an ASV was a case where the ASV was assigned to D. delphis with 100% identity, but other taxa were assigned with a percentage identity of 99.14% (the genera Tursiops spp. and Stenella spp.). This was to be expected given the genetic similarity of these species pertaining to the oceanic dolphin family (Delphinidae), as described by McGowen et al. [60]. Especially between D. delphis and the T. truncatus, there are only two Single Nucleo- tide Polymorphisms (SNPs) on the amplicon obtained with the MarVer3 primers. Besides this case, there was a double identification with 100% percent identity for the same ASV—7 sequences for the Atlantic horse mackerel (Trachurus trachurus) and 1 for the megrim (Lepi- dorhombus whiffiagonis) in the NCBI database. In this case, the species T. trachurus was selected as the correct identification. Such a decision was made because the matched sequence of L. whiffiagonis referred to samples collected in the Mediterranean Sea, in which case the authors of the work demonstrated a great variation in the mitochondrial 16S rRNA region between the Mediterranean and the Atlantic Ocean populations [61]. Cetacean detections: eDNA versus visual monitoring As described in the results, the species detected through visual monitoring were also detected by eDNA, with the additional record of the species B. physalus, which was only recorded through molecular methods in the environmental samples. This is a species with non-existent

sightings in scientific literature in this subregion of Continental Portugal, even though it has been recorded in other parts of the country’s coastline [62]. Additionally, in the Galician coastal waters, adjacent to the study area, the species occurs rather frequently [30, 31, 63, 64]. Offshore Galicia, there are relevant topographical structures, seamounts, that may act as hot- spots for pelagic biodiversity [65], such as the Galicia Bank [66]. The obtained detections of B. physalus DNA may be evidence of an occasional occurrence of the species in waters off north- ern Portugal, as it has been previously recorded in SCANS-IV surveys [31]. This showcases a likely case of eDNA displacement from its origin place of origin. As shown by the results, the common dolphin represented the vast majority of the sightings in the study area. The wide distribution and frequent presence of D. delphis along the entire PLOS ONE Environmental DNA as a complementary tool for biodiversity monitoring PLOS ONE | https://doi.org/10.1371/journal.pone.0300992 October 16, 2024 12 / 20