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

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 manufac- turer. 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 correspond- ing 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 avail- able nucleotide sequences (containing the 16S rRNA gene) from selected taxa were retrieved from the NCBI nucleotide database. Then, using the Geneious1 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 frag- ments 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 pair- wise 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. PLOS ONE Environmental DNA as a complementary tool for biodiversity monitoring PLOS ONE | https://doi.org/10.1371/journal.pone.0300992 October 16, 2024 5 / 20