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

stage, the present work highlights the benefits of using eDNA to study marine communities, with specific applications for research on cetacean distribution and feeding ecology. Introduction Environmental DNA (eDNA) is an emerging tool for biodiversity monitoring that has been gaining prominence in scientific research during the second half of the 21st century, with increasing numbers of scientific outputs being published every year [1]. For marine ecosys- tems, the application of eDNA detection methodologies is still in its infancy, although it has steadily evolved in the last decades given the interest in its potential [2]. Among the multiple applications, these allow verifying environmental health by studying microbial communities, characterizing and quantifying stocks of marine vertebrates, such as teleost fish which repre- sents vital knowledge for good management of the fisheries sector, and determining the pres- ence and abundance of elusive species, such as cetaceans, allowing greater effectiveness of the monitoring work [3–5]. Additionally, technological advances in Next-Generation Sequencing (NGS) techniques, via DNA metabarcoding, allow for simultaneous multi-species detections in environmental samples, permitting the study of multiple trophic levels within the same sam- ples [6–8]. Cetaceans are widely dispersed mammals that inhabit most marine environments, from coastal habitats to neritic waters [9], playing a key ecological role in maintaining the balance of these ecosystems [10]. As keystone species, cetacean conservation is often a top priority in

international agreements, especially considering the anthropogenic threats they are currently facing [11]. Addressing the impacting issues in cetacean ecology is crucial, however, obtaining data that provides a detailed understanding of these animals is rather complex. Cetaceans are elusive, spending the vast majority of their time underwater. Also, their distribution range is often very extensive, including areas where access for sampling is difficult due to logistical and financial limitations inherent to the marine wildlife monitoring work or even legal constraints [10]. Therefore, the development of new non-invasive methodologies, such as the metabarcod- ing analysis of eDNA samples, especially in complement to visual monitoring is a promising advance in the optimization of monitoring effectiveness towards a better understanding of these species [10]. Regarding the use of eDNA techniques for cetacean monitoring, few published studies have been focusing on marine mammals (see review on eDNA application for cetacean monitoring under [12]). Nevertheless, there are already successful case studies where it has been possible to identify a variety of cetacean species through environmental samples using both species-spe- cific [5, 13–16] and universal primers [4, 17–23]. The possibility of detecting multiple species within the same environmental samples enabled through metabarcoding, allows for a multi- trophic analysis that widens the utility of the samples for the monitoring of several taxa and

application to various fields of research, including the study of trophic chains and species feed- ing ecology [23, 24]. In the present study, a molecular-based detection methodology was developed, using eDNA samples as a tool for biodiversity monitoring, especially applied to cetacean species in a coastal region of the North of Portugal, located in the Eastern North Atlantic (ENA). The ENA region is an area of great interest regarding the diversity and abundance of cetaceans, with tens of different species of dolphins and whales being recurrently reported over the years [25–36]. Here, we sought to obtain additional and concrete data on the occurrence of cetaceans in this PLOS ONE Environmental DNA as a complementary tool for biodiversity monitoring PLOS ONE | https://doi.org/10.1371/journal.pone.0300992 October 16, 2024 2 / 20 data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.