Study area

Surveys to collect eDNA samples were performed on the north coast of Continental Portugal. This subregion, located in the northwest of the Iberia Peninsula, is of particular ecological interest due to the upwelling phenomenon strongly present along the coastline, thus enhancing primary production and providing great conditions for the development of complex and rich trophic chains [37–39]. In addition, the area is part of a particularly dynamic coastal region with several estuaries of rivers that flow into it [40]. Topographically, the study area is entirely placed on the continental shelf, with the presence of the Porto Canyon in the vicinity offshore, a relevant structure as submarine canyons are known to be impactful in hydrodynamic processes of coastal habitats, such as enhancing upwelling and subsequently organic productivity [41] (Fig 1).

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Fig 1. Study area with surveyed transect of at-sea monitoring campaigns, conducted between the summer of 2021 and the winter of 2022/2023, on the north coast of Continental Portugal.

Isobaths with bathymetry in meters.

https://doi.org/10.1371/journal.pone.0300992.g001

To survey the area, four seasonal monitoring campaigns were carried out between the summer of 2021 and the winter of 2022/2023. Dates for all carried campaigns are described in Table 1.

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Table 1. Seasonal monitoring campaign dates, conducted on the north coast of Mainland Portugal, between 2021 and 2023.

https://doi.org/10.1371/journal.pone.0300992.t001

Each campaign consisted of a survey transect with eight equidistant parallels, perpendicular to the coastline, spaced by approximately 6 nm, and covering distances of about 10 nm (approximately between 2 to 12 nm from the coastline), as shown in Fig 1. Permit for field access was given by the national maritime authority through the captaincy of the Leixões Port and of the Port of Douro. was For the collection of visual monitoring data, a previously established protocol [33] was followed to sample the occurrence of cetacean species sighted along the established transect (Fig 1). The transect was designed to have an equitable range of observation capacity at all its points, guaranteeing correct and complete visual monitoring of the study area. The collected visual monitoring data was imported into ArcGIS Pro for spatial analysis.

Water collection and filtration

The environmental samples were sampled at pre-defined stations at the vertices of the campaign transect (Fig 1 and S1 Table), using a bucket and a rope to collect 5 liters of water. Before water sampling, the samples were poured into 5L containers. All the materials used for sample collection were previously cleaned with 10% bleach, rinsed with MilliQ water, and washed with local seawater (i.e., seawater at the sampling station) just before sample collection. After collection, volumes ranging from 1 to 3 liters were filtered immediately on board through Sterivex units (0.22 μm) using a peristaltic pump. After filtration, the samples were kept in liquid nitrogen, and stored at -80°C upon arrival at the laboratory. In total, 64 samples were collected, 4 at each station (two summers and two winters), with few exceptions: (1) two additional collections were made at S09 and S10 during the first summer campaign (2021); (2) during the first winter campaign, it was not possible to collect samples at stations S07 and S08, due to adverse weather conditions (S2 Table).

DNA extraction

Total eDNA was isolated using the DNeasy® PowerWater® Sterivex™ Kit (QIAGEN), following the manufacturer’s instructions, with some adjustments to increase the DNA yield, namely: increased vortex times (10 minutes at all vortex steps) and 5 minutes rest time before the last centrifugation step. After extraction, DNA concentration for all samples was quantified using Qubit™ dsDNA High Sensitivity (HS) assay kit (Invitrogen™). Environmental DNA extraction was performed in a specifically dedicated laboratory for the extraction of genetic material, with the bench being always cleaned with ethanol before its use. All materials used were also sterilized in UV light before the start of the extraction process.

Library preparation, sequencing and bioinformatic analysis

All samples were sequenced in high throughput sequencing in an Illumina MiSeq300 platform, using MarVer3(A) primers [8] and the Superfi II Polymerase (Invitrogen™) (following the protocol from [42, 43]). For marine vertebrate library preparation, a fragment of the vertebrate mitochondrial 16S rRNA gene was amplified and reamplified. In the first amplification step, PCRs were carried out in triplicate in a final volume of 10 microliters (μL), containing 2 μL of template DNA, 0,5 μM of the primers, 1X Platinum SuperFi II DNA Polymerase (Invitrogen), 0,8 mM dNTPs, 1X SuperFi II Buffer, 1X CES [44], and ultrapure water up to 10 μL. The PCR protocol was the following: an initial denaturation step at 98°C for 30 seconds, followed by 35 cycles of 98°C for 10 seconds, 49°C for 10 seconds, 72°C for 30 seconds, and a final extension step at 72°C for 5 minutes. Triplicate PCR products were pooled together. The oligonucleotide indices that are required for multiplexing different libraries in the same sequencing pool were attached in a second amplification step with identical conditions but only 5 cycles and with an annealing temperature of 60°C. Libraries were then purified using the Mag-Bind RXNPure Plus magnetic beads (Omega Bio-tek), following the instructions provided by the manufacturer. Lastly, the pool was sequenced in a fraction (3/8) of a MiSeq PE300 flow cell (Illumina). DNA metabarcoding library preparation and sequencing were carried out by AllGenetics & Biology SL (www.allgenetics.eu).

Following that, sequencing adapters were removed using Cutadapt v3.5 (Martin, 2011) and the originated reads went through a DADA2 [45] tool pipeline to remove other non-biological DNA sequences (e.g., primers), filter the reads according to their quality, denoise, dereplicate, cluster the resulting sequences into Amplicon Sequence Variants (ASVs), merge corresponding forward and reverse reads, and remove chimeric sequences. The taxonomic assignment of each ASV (with a minimum of 5 reads) was performed by querying its representative sequence against a local instance of the NCBI’s Nucleotide database (last updated on 18/09/2023), using the algorithm BLASTn v2.13.0+ [46] with the following parameters: percent identity of 99%, e-value of 1e-05, and a minimum hit coverage of 80%. If, based on these parameters, a matched ASV represented multiple species, the one with 100% identity was considered. For this part of the work, the ASVs corresponding to all cetacean species and Actinopteri superclass species with known occurrence in Continental Portugal (as of the Ocean Biodiversity Information System [OBIS] up to 05/09/2023) were selected. For the construction of the databases, all available nucleotide sequences (containing the 16S rRNA gene) from selected taxa were retrieved from the NCBI nucleotide database. Then, using the Geneious® software (v.7.0.6), primer sequences (forward and reverse) were annotated (using the ‘Add Primers to the Sequence’ tool), sequences were cut (using the ‘Extract PCR product’ tool) so they only contain the fragments spanning (and including) the PCR primers, and all nucleotide sequences not containing the entire fragment of interest were excluded from the database. Positively matched ASVs of each sample were then grouped into the respective sampling stations and seasonal campaigns for a clear visual representation of the results. Representative stacked bar plots of identified taxa relative abundance and Actinopteri ASVs heatmaps for winter and summer monitoring campaigns were produced using the ggplot2 (v.3.4.3) package [47], in R.

Sampling statistical tests

To better understand whether there are significant variations in the concentrations obtained from samples taken in the different seasonal campaigns, Kruskal-Wallis chi-squared tests were carried out, and if significant differences were found (p-value < 0,05), we followed with a pairwise Wilcoxon test. The Wilcoxon test was also applied to compare the concentrations obtained from stations located at different distances from the coast (2 nm versus 12 nm). For this comparative analysis of DNA quantification results, samples that had DNA concentration below Qubit’s HS kit detection limit (0,005 ng/μL) were assigned a value of 0,001 ng/μL. A sample log of all collected and extracted samples can be found in S2 Table.

Statistical tests (Kruskall-Wallis test and pairwise Wilcoxon tests, with the level of significance set to 0,05) were carried out to understand the variation in the total number of ASVs obtained in the samples at the different seasonal campaigns and the different distances from the coast (2 nm versus 12 nm).

Visual surveys

A total of 71 sightings of 4 different cetacean species were recorded during monitoring campaigns (S1A Fig and S3 Table). The majority of these records were from common dolphin (Delphinus delphis) with 45 sightings across the entire surveyed area, representing approximately 63.4% of all records. As for the other 3 species—bottlenose dolphin (Tursiops truncatus), Risso’s dolphin (Grampus griseus) and harbor porpoise (Phocoena phocoena)–these were sighted on 3 different occasions each. Besides the identified records to the species level, there were also 13 sightings of unidentified Delphinidae (18,3% of total records) and, on 4 occasions, it was only possible to identify cetacean occurrence at the superorder level (Cetacea). All sightings recorded with the respective date and geographical coordinates can be found in S3 Table.

eDNA sampling

Among the campaigns conducted, the summer of 2021 showed much higher DNA concentrations when compared to the other seasonal campaigns, with an average quantification of 9.46 ± 9.05 ng/μL (Median = 9,22 ng/μL). This was followed by the winter of 2021/22 with 5.10 ± 6.11 (Median = 3.02 ng/μL), the winter of 2022/23 with 2.26 ± 3.50 ng/μL (Median = 0.8 ng/μL) and, finally, the summer of 2022 with 1.08 ± 2.38 ng/μL (Median = 0.67 ng/μL). Statistical tests showed significant differences between the quantified concentrations of the different campaigns (p-value = 9.63^e-06), these being explained by differences between summer 2021 and summer 2022, summer 2021 and winter 2022/23, and also between summer 2022 and winter 2022/23 (S4A Table). At the seasonal level, the summer shows higher concentrations, with 5.52 ± 8.18 ng/μL (Median = 3.51 ng/μL) compared to the 3.59 ± 5.02 ng/μL (Median = 1.49 ng/μL) obtained for the winter. However, this difference was not statistically significant (p-value = 0.79). Taking distance into account, the sampling stations near the coast (2 nm) had an average DNA concentration of 5.48 ± 8.18 ng/μL (Median = 3.2 ng/μL), while the DNA obtained in stations at 12 nm was quantified as 3.75 ± 5.21 ng/μL (Median = 1.26 ng/μL) on average. These differences were not statistically significant (p-value = 0.45).

eDNA monitoring: Sequencing output and taxonomic identification

A total of 5 876 226 reads were generated by the Illumina platform with an approximate average of 90 403.48 ± 42 311.20. After quality-filtering steps, the final output was 3 735 272 total reads with an average of approximately 57 465.72 ± 40 520.33 (representing 65.6% of the input). There were 5 outlier samples that produced less than 50 reads. A total of 173 156 representative ASVs (of previously determined marine vertebrate species: cetaceans and selected Actinopteri species with known occurrence in Continental Portugal). Among the different seasonal campaigns, winter 2022/23 obtained the highest number of resulting ASVs with an average of 7 951.5 ± 23 991.69 (Median = 39), followed by winter 2022/23 with 2024 ± 2 980.45 ASVs (Median = 722.5), summer 2021 with 932.33 ± 1 716.23 ASVs (Median = 201.5), and finally summer 2022 with an average of 51.5 ± 178.56 ASVs (Median = 0). The statistical tests carried out indicate that there was a significant difference in the uptake of target eDNA taxa, both between seasons (p-value = 0.019) and between the different campaigns (p-value = 0.009). Pairwise tests highlight differences between the two summer campaigns and both winter campaigns with the summer of 2022 (S4B Table). With regard to distance from the shore, samples collected closer (2 nm) resulted in an average number of ASVs of 988.13 ± 2087.06 (Median = 0), while those further away (12 nm) resulted in 4,423.31 ± 17,126.03 ASVs (Median = 52.5). However, differences on obtained ASVs in relation to the distance to the coast were not significant (p-value = 0.64).

Of the 64 eDNA samples sequenced through metabarcoding, 33 (51.6%) had positive detections of representative ASVs, from which 10 of the samples (15.6%) had positive detections of cetacean ASVs. By grouping the results by sampling station, we noticed that all sampling stations had positive detections for either Cetacea or Actinopteri. Relative abundances of all cetacean species identified, and Actinopteri taxa detected in 4 or more stations, are illustrated in Fig 2.

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Fig 2. Taxonomic assignment from environmental DNA samples collected in 16 sampling stations, in the north coast of Mainland Portugal, between 2021 and 2023.

The plot represents the relative abundance (%) of sequences matched for each marine vertebrate species, considering pre-determined Cetacea, and Actinopteri species with positive detection in 4 or more stations (see methods). “Others” represents Actinopteri superclass species identified in 3 or fewer stations.

https://doi.org/10.1371/journal.pone.0300992.g002

Focusing on cetaceans, 5 different species were detected, with at least one of them identified in 7 out of the 16 stations (Fig 2 and S4 Table). The common dolphin (Delphinus delphis) was the most detected species, being identified in 6/16 stations, followed by the harbor porpoise (Phocoena phocoena) and the fin whale (Balaenoptera physalus), occurring in 2/16 sampling stations. The Risso’s dolphin (Grampus griseus) and the bottlenose dolphin (Tursiops truncatus) were detected in 1/16 stations.

Regarding Actinopteri species, 19 species were identified, with at least one of them detected in all sampling stations (Figs 2 and 3). Of all these detections, the most notable result was the widespread presence of the sardine (Sardina pilchardus)—in 14/16 sampling stations; the European seabass (Dicentrarchus labrax)—in 9/16 stations; and the European anchovy (Engraulis encrasicolus)—in 6/16 of the sampled sites (Figs 2 and 3). All the Actinopteri species identified, as well as the respective number of ASVs detected at each station, can be found in S4 Table. Additionally to the frequency of the detections across sampling stations, there is also a higher abundance of captured ASVs of the species S. pilchardus and D. labrax (Fig 3). In 5/16 sampling stations, 100% of the matched ASVs belonged to either S. pilchardus or D. labrax (Fig 2). The highest number of ASVs was obtained for S. pilchardus in Station S15 (off north of Viana do Castelo) (Fig 3). Station S01 (southwest of Porto, at ~2nm from the coast) had positive detections for a higher number of selected Actinopteri species in both seasons (Fig 3). The Station S08 (near Póvoa de Varzim) only had one minor detection (9 BLAST hits for its respective ASV, see S5 Table) for S. pilchardus across all monitoring campaigns.

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Fig 3. Number of Amplicon Sequence Variants (ASVs) detected for each species of the Actinopteri superclass molecularly identified in environmental DNA samples, collected at the sampling stations surveyed in the summer (top) and winter (bottom) in the north coast of Mainland Portugal, between 2021 and 2023.

The number of ASVs was logarithmised for scaling purposes.

https://doi.org/10.1371/journal.pone.0300992.g003

Cross-checking eDNA with visual monitoring data

Overall, by cross-referencing the data obtained by the two sampling methodologies applied in this study, we can verify the occurrence of the following four species in both datasets (visual records, eDNA molecular detection): D. delphis, P. phocoena, T. truncatus and G. griseus. In addition to these, positive molecular detections of B. physalus were obtained through eDNA analysis. Regarding the spatial distribution of these records, it is clear that D. delphis was sighted not only in greater abundance but also with a dispersed distribution throughout the region studied, which is also in line with the data obtained by molecular detection. For the other identified species, their DNA abundance was lower and there were fewer visual records obtained as well.

Overall, no pattern of spatial or temporal correlation between molecular detections and visual records is clear. An inter-seasonal comparison of the spatial distribution of the records obtained by the visual surveys with the eDNA detections reveals contrasting results (Fig 4). In 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 identified at the species level) (Fig 4A). Through eDNA analysis though, all the species were molecularly 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 Póvoa de Varzim; ii) T. truncatus was distributed in the north of the study area, visually recorded at different distances 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 Póvoa 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).

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Fig 4.

Seasonality of cetacean records obtained by visual monitoring (A) and molecular analysis of environmental DNA (B), for summer (left) and winter (right) campaigns carried out in the North coast of Mainland Portugal, between 2021 and 2023.

https://doi.org/10.1371/journal.pone.0300992.g004

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 (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. pilchardus, 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.

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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.

https://doi.org/10.1371/journal.pone.0300992.t002

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 protocols for the detection and identification of cetacean species through eDNA, with a wide variation of methods currently being implemented [12]. The lack of a standardized, reliable, and repeatable protocol for sample collection (and subsequent processing and analysis) still constitutes 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 summer months, with uniform upwelling-favorable conditions along the north coast [39]. Therefore, 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 campaign presented the highest values of DNA concentration, which could be explained by the mentioned oceanographic phenomena, the exact opposite was observed in 2022. This significant 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 significant 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 interpreted 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 differences 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, Cávado, and Minho rivers [58, 59], which could also be interfering with sampling results. The river plume of the Douro River 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 success 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 aforementioned 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 (conservative 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 species already recorded along the Portuguese coast, substantially reducing the size of the database. 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 Nucleotide 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 (Lepidorhombus 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 hotspots 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 northern 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 northern Portuguese coast was corroborated by molecular detection on eDNA samples. P. phocoena 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, emphasizing 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 monitoring, 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 Portugal. 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, complementary 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 metabarcoding 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 Delphinidae 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 continued 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 collection of data from multiple trophic levels, with posterior inference of possible ecological interactions and prey/predator relationships. Universal primers also provide a deeper understanding 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 composition was studied to better understand G. griseus and Cuvier’s beaked whales (Ziphius cavirostris) 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 aforementioned E. encrasicolus, the sole (Soleidae spp.), the needlefish (B. belone), the scads (Trachurus 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. trachurus, 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 conservation strategies. Environmental DNA is thus proving to be a promising tool for multitrophic assessments, not only to study biodiversity occurrence, but also to infer about ecological processes and investigate trophic relationships.