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

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 (approx- imately 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 proto- col [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 col- lected 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 cam- paign 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 Ster- ivex 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 col- lections 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 DNeasy1 PowerWater1 Sterivex™ Kit (QIAGEN), follow- ing 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 Table 1. Seasonal monitoring campaign dates, conducted on the north coast of Mainland Portugal, between 2021 and 2023. Monitoring campaign Dates S01 to S08 S09 to S16

Summer 2021 25th June - 1st July 2021 * 24th - 25th September 2021 Winter 2021/2022 2nd - 3rd February 2022 25th - 29th March 2022 Summer 2022 9th - 10th August 2022 30th - 31st August 2022 Winter 2022/2023 20th - 21st March 2023 27th - 28th March 2023 *In Summer 2021, S09 and S10 transects were also conducted between the 25th of June and 1st of July 2021. https://doi.org/10.1371/journal.pone.0300992.t001 PLOS ONE Environmental DNA as a complementary tool for biodiversity monitoring PLOS ONE | https://doi.org/10.1371/journal.pone.0300992 October 16, 2024 4 / 20