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

RESEARCH ARTICLE Environmental DNA as a complementary tool for biodiversity monitoring: A multi-technique and multi-trophic approach to investigate cetacean distribution and feeding ecology Luı´s AfonsoID1,2, Joana CostaID1, Ana Mafalda Correia1,3, Raul Valente1,3, Eva Lopes1,3, Maria Paola Tomasino1, A´ gatha Gil1,4,5, Cla´ udia Oliveira-Rodrigues1,3, Isabel Sousa Pino1,3, Alfredo Lo´ pez2,6,7, Paula Suarez-BreguaID8, Catarina Magalhães1,3* 1 CIIMAR–Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Matosinhos, Portugal, 2 Department of Biology, UA–University of Aveiro, Campus Universita´rio de Santiago, Aveiro, Portugal, 3 Department of Biology, FCUP–Faculty of Sciences of the University of Porto, Porto, Portugal, 4 Department of Biology and Environment, CITAB–Centre for the Research and Technology of Agro- Environmental and Biological Sciences, University of Tra´ s-os-Montes and Alto Douro (UTAD), Vila Real, Portugal, 5 IIM-CSIC–Institute of Marine Research of the Spanish National Research Council, Vigo, Pontevedra, Spain, 6 CESAM–Centre for Environmental and Marine Studies, University of Aveiro, Aveiro, Portugal, 7 CEMMA–Coordinator for the Study of Marine Mammals, Nigra´n, Spain, 8 IEO-CSIC–Spanish Institute of Oceanography of the Spanish National Research Council, Vigo, Pontevedra, Spain * cmagalhaes@ciimar.up.pt Abstract The use of environmental DNA (eDNA) to assess the presence of biological communities has emerged as a promising monitoring tool in the marine conservation landscape. More-

over, advances in Next-Generation Sequencing techniques, such as DNA metabarcoding, enable multi-species detection in mixed samples, allowing the study of complex ecosystems such as oceanic ones. We aimed at using these molecular-based techniques to characterize cetacean communities, as well as potential prey on the northern coast of Mainland Portugal. During four seasonal campaigns (summer 2021 to winter 2022/2023), seawater samples were collected along with visual records of cetacean occurrence. The eDNA isolated from 64 environmental samples was sequenced in an Illumina platform, with universal primers targeting marine vertebrates. Five cetacean species were identified by molecular detection: common dolphin (Delphinus delphis), bottlenose dolphin (Tursiops truncatus), Risso’s dol- phin (Grampus griseus), harbor porpoise (Phocoena phocoena) and fin whale (Balaenop- tera physalus). Overall, except for the latter (not sighted during the campaigns), this cetacean community composition was similar to that obtained through visual monitoring, and the complementary results suggest their presence in the region all year round. In addi- tion, the positive molecular detections of Balaenoptera physalus are of special relevance since there are no records of this species reported on scientific bibliography in the area. The detection of multiple known prey of the identified dolphins indicates an overlap between predator and prey in the study area, which suggests that these animals may use this coastal

area for feeding purposes. While this methodological approach remains in a development PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.0300992 October 16, 2024 1 / 20 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Afonso L, Costa J, Correia AM, Valente R, Lopes E, Tomasino MP, et al. (2024) Environmental DNA as a complementary tool for biodiversity monitoring: A multi-technique and multi-trophic approach to investigate cetacean distribution and feeding ecology. PLoS ONE 19(10): e0300992. https://doi.org/10.1371/journal. pone.0300992 Editor: Petr Heneberg, Charles University, CZECHIA Received: March 8, 2024 Accepted: July 29, 2024 Published: October 16, 2024 Copyright: © 2024 Afonso et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work is a result of the project ATLANTIDA (ref. NORTE-01-0145-FEDER-000040) supported by the Norte Portugal Regional Operational Programme (NORTE2020), under the PORTUGAL 2020 Partnership Agreement and through the European Regional Development Fund (ERDF). The funders had no role in study design,