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

(on two occasions), B. belone, E. encrasicolus, D. labrax, S. saurus and L. bergylta. P. phocoena detections were coincident with a higher richness of Actinopteri species, including S. pilchar- dus, D. labrax, S. scombrus, L. bergylta, Pleuronectidae spp., H. lanceolatus and C. conger (Table 2). G. griseus and B. physalus were also detected at the same time as S. pilchardus (Table 2), while T. truncatus was solely detected without associated potential Actinopteri prey. Discussion Environmental DNA sampling and uptake Conducting the dedicated sampling surveys was highly constrained by weather conditions. This is particularly evident in the summer 2021 campaign, in which sampling was carried out with an interval of approximately 3 months at each end of the season (June and September, Table 1). Although this time interval between samplings from the same summer campaign is suboptimal, it still acts as a valid representation of the season when compared to winter. This is supported by the uniform oceanographic dynamics of the study region during the summer months [39, 48, 49]. The sampling techniques here employed were chosen due to their on-field practicability (collection of seawater and filtration) and lower risk of contamination (no direct handling of filters during DNA extraction). To date, there are no generalized standard proto- cols for the detection and identification of cetacean species through eDNA, with a wide varia- tion of methods currently being implemented [12]. The lack of a standardized, reliable, and

repeatable protocol for sample collection (and subsequent processing and analysis) still consti- tutes a major obstacle to the use of this monitoring tool for marine biodiversity monitoring [50]. Upwelling phenomena on the Iberian Peninsula tend to occur on a higher scale in the sum- mer months, with uniform upwelling-favorable conditions along the north coast [39]. There- fore, it was expected DNA concentrations to be uniformly higher in the samples collected during summer campaigns, which was not the case. However, while the 2021 summer cam- paign presented the highest values of DNA concentration, which could be explained by the mentioned oceanographic phenomena, the exact opposite was observed in 2022. This signifi- cant difference, although not ruling out possible human error in the different sampling steps, may be due to some unknown oceanographic event happening during sampling days or to other environmental variables with known impact on DNA preservation, such as temperature [51, 52], pH [53, 54] and/or UV radiation exposure [55]. The lack of inter-seasonal significant differences in the DNA concentration obtained from eDNA samples, in contrast to the signifi- cant inter-campaign differences observed, may be evidence that the seasonal (i.e., inter-annual cycles such as seasonal upwelling) influence is less relevant than the impact of less cyclic and more ephemeral phenomena on the DNA preservation and retrieval. Regarding the number of target ASVs, significant differences found between both seasons and monitoring campaigns,

suggest a higher target DNA uptake in winter months. Nevertheless, this result should be inter- preted with caution, since these differences are mainly justified by the low numbers evidenced in the summer 2022 campaign (for which a very low DNA concentration was obtained). As far as the proximity to the coast, although with some variation, there were no significant differ- ences in terms of DNA concentrations or the number of ASVs obtained. Researchers have reported a faster DNA degradation in inshore areas associated with intense anthropogenic pressures [56]. Nevertheless, coastal areas are also likely to have higher biomass, in comparison with offshore areas, due to the coastal upwelling phenomena [39, 50] and the input of the rivers [57, 58]. The coastal waters of the north coast of Continental Portugal are heavily influenced by the input of various fluvial water masses such as the Douro, Ca´vado, and Minho rivers [58, 59], which could also be interfering with sampling results. The river plume of the Douro River PLOS ONE Environmental DNA as a complementary tool for biodiversity monitoring PLOS ONE | https://doi.org/10.1371/journal.pone.0300992 October 16, 2024 11 / 20