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

Acknowledgments Ph.D. fellowships for authors RV (SFRH/BD/144786/2019) and AG (PD/BD/150603/2020) were granted by Fundac¸ão para a Ciência e Tecnologia (FCT, Portugal) under the auspices of Programa Operacional Regional Norte (PORN), supported by the European Social Fund (ESF) and Portuguese funds (MECTES). This work is a result of the project ATLANTIDA (ref. NORTE-01-0145-FEDER-000040) supported by the Norte Portugal Regional Operational Pro- gramme (NORTE2020), under the PORTUGAL 2020 Partnership Agreement and through the European Regional Development Fund (ERDF); and of the project EMPHATIC funded by Biodiversa+, the European Biodiversity Partnership, under the joint call 2022–2023 Biodiv- Mon for research proposals, co-funded by the European Commission and with the following funding organisations: Fundacio´n Biodiversidad (FB, Spain), Fundac¸ão para a Ciência e Tec- nologia (FCT, Portugal), Agence Nationale de la Recherche (ANR, France), and the Ministry of Universities and Research (MUR, Italy). Author Contributions Conceptualization: Luı´s Afonso, Joana Costa, Ana Mafalda Correia, Maria Paola Tomasino, Catarina Magalhães. Formal analysis: Luı´s Afonso, Joana Costa, Ana Mafalda Correia, Raul Valente. Funding acquisition: Isabel Sousa Pino, Catarina Magalhães. Investigation: Luı´s Afonso, Joana Costa, Ana Mafalda Correia. Methodology: Luı´s Afonso, Joana Costa, Ana Mafalda Correia, Raul Valente, Eva Lopes, Cla´u- dia Oliveira-Rodrigues, Paula Suarez-Bregua. Resources: Isabel Sousa Pino, Catarina Magalhães.

Supervision: Joana Costa, Ana Mafalda Correia, Alfredo Lo´pez, Catarina Magalhães. Visualization: Luı´s Afonso, A´ gatha Gil. Writing – original draft: Luı´s Afonso, Ana Mafalda Correia. Writing – review & editing: Luı´s Afonso, Joana Costa, Ana Mafalda Correia, Raul Valente, Eva Lopes, Maria Paola Tomasino, A´ gatha Gil, Cla´udia Oliveira-Rodrigues, Isabel Sousa Pino, Alfredo Lo´pez, Paula Suarez-Bregua, Catarina Magalhães. References 1. Takahashi M, Saccò M, Kestel JH, Nester G, Campbell MA, Van Der Heyde M, et al. Aquatic environ- mental DNA: A review of the macro-organismal biomonitoring revolution. Sci Total Environ. 2023; 873:162322. https://doi.org/10.1016/j.scitotenv.2023.162322 PMID: 36801404 2. Dı´az-Ferguson EE, Moyer GR. History, applications, methodological issues and perspectives for the use of environmental DNA (eDNA) in marine and freshwater environments. Revista de biologia tropical. 2014; 62(4):1273–1284. https://doi.org/10.15517/rbt.v62i4.13231 PMID: 25720166 3. Bohmann K, Evans A, Gilbert MTP, Carvalho GR, Creer S, Knapp M, et al. Environmental DNA for wild- life biology and biodiversity monitoring. Trends in Ecology and Evolution. 2014; 29(6):358–367. https:// doi.org/10.1016/j.tree.2014.04.003 PMID: 24821515 4. Andruszkiewicz EA, Starks HA, Chavez FP, Sassoubre LM, Block BA, Boehm AB. Biomonitoring of marine vertebrates in Monterey Bay using eDNA metabarcoding. PLoS One. 2017; 12(4). https://doi. org/10.1371/journal.pone.0176343 PMID: 28441466 5. Parsons KM, Everett M, Dahlheim M, Park L. Water, water everywhere: Environmental DNA can unlock

population structure in elusive marine species. Royal Society Open Science. 2018; 5(8). https://doi.org/ 10.1098/rsos.180537 PMID: 30225045 PLOS ONE Environmental DNA as a complementary tool for biodiversity monitoring PLOS ONE | https://doi.org/10.1371/journal.pone.0300992 October 16, 2024 16 / 20