Environmental DNA (eDNA) analyses have become a powerful tool for non-invasive biodiversity monitoring, yet the applicability of population-genetic approaches to environmental samples remains largely unexplored. Even when genetic traces originate from a single individual, low target DNA concentrations and amplification or sequencing artefacts can compromise downstream genetic inferences. Here, we present a novel approach for obtaining demographic insights and lineage-level mitogenomic information from aquatic eDNA samples collected near vertebrate individuals, while assessing the utility of paired tissue sampling for benchmarking eDNA-based population genetic analyses.

Paired eDNA and tissue samples were collected during sperm whale (Physeter macrocephalus) encounters in the Azores. Samples were screened for the presence of vertebrate eDNA and analysed with a novel molecular sex identification assay. Additionally, long-range PCR was used to amplify up to five mitochondrial DNA fragments (~3–4 k bp) before subsequent sequencing on an Oxford Nanopore Technologies platform. A stringent three-tier filtering framework capable of identifying true mitogenomic variation across eDNA samples was developed for maximum recovery of genetic diversity at the haplogroup level. By validating eDNA samples via their paired tissues, parameter values were optimized to maximize concordance and minimize spurious variant calls.

Sexing was successful for 50% of eDNA samples, with 96% concordance to paired tissues and marine vertebrate DNA concentration significantly predicted sexing success. Further, Medaka polishing produced high identity mitochondrial consensus sequences (>16 kb) from eDNA samples. Across filtering regimes in the framework, curated SNP panels comprising up to 453 high-confidence mitochondrial SNPs resolved 19 haplogroups, with 93% concordance between eDNA and tissue samples. An intermediate bioinformatics filtering strategy maximized biologically accurate haplogroup recovery while minimizing sequencing artefacts, providing the most reliable lineage-level inferences.

This integrative approach demonstrates that targeted nuclear assays combined with long-range mitochondrial sequencing can recover individual-level genetic information from aquatic eDNA. By defining analytical thresholds governing success and demonstrating how paired tissue benchmarking can calibrate eDNA-based population genetic insights for future applications, the framework advances non-invasive genetic monitoring of populations via eDNA and enables population-level monitoring and conservation of endangered and genetically-vulnerable species.

Monitoring cetaceans is essential for evaluating ecosystem health and informing the establishment of marine protected areas. Conventional cetacean monitoring techniques, such as photo-identification, acoustic surveys, and satellite tagging, are often resource-intensive, costly, and sometimes intrusive. Environmental DNA (eDNA)-based methods have emerged as non-invasive, cost-efficient complements based on the analysis of genetic material shed into the environment. However, eDNA research is still evolving, with ongoing efforts to optimize field sampling and laboratory protocols. Building on the challenges of conventional monitoring methods, this study sought to refine eDNA sampling parameters to offer a more efficient and scalable approach for cetacean research, leveraging citizen science platforms. From June to October 2023, eDNA samples were collected across three regions in the Northeast Atlantic Ocean and Mediterranean Sea aboard whale-watching vessels or monitoring platforms engaging citizen scientists. Samples were analyzed for total DNA concentration using Qubit fluorometry and target DNA concentration with quantitative polymerase chain reactions (qPCR). Key variables tested in the field included water volume (2, 5, and 10 L), sampling timing (immediately after a whale was present and at 5-, 10-, and 20-min intervals), and three filter types (pore sizes of 1.2, 0.8, and 0.45 μm). Our results illustrate that larger water volumes (10 L), sampling immediately after a whale breach or fluking behavior, and Smith-Root eDNA filters (1.2 μm pore size) significantly increased eDNA detection probability and signal strength. However, the combination of certain filter types with different water volumes had a significant impact on detection probability, with smaller pore sizes more effectively yielding detections with a lower water volume. These findings provide guidance for future cetacean research initiatives and highlight the potential of eDNA methods in enhancing research and conservation efforts through scalable citizen science-based initiatives.

Marine biodiversity faces challenges due to climate change, pollution, and human activities. Conservation efforts are often constrained by limited resources and public disengagement. This study evaluates the potential benefits of incorporating environmental DNA (eDNA) sampling and citizen science into whale-watching tours to raise environmental awareness and promote marine conservation. A Likert-scale questionnaire evaluating attitudes, knowledge, and willingness to contribute to conservation through financial support, behavioral changes, and volunteering was administered to 224 participants across the Pelagos Sanctuary (Italy), the Azores (Portugal), and Skjálfandi Bay (Iceland). Environmental awareness, interest in environmental DNA (eDNA), and higher education levels were identified as significant predictors of willingness to pay (WTP). Conversely, higher ticket prices negatively impacted financial commitment. The majority of respondents reported an increased awareness of marine biodiversity and eDNA, along with a strong sense of personal responsibility toward marine protection. The study outlined various participant profiles, indicating how age, education, and ecotourism experience influence conservation attitudes. Specifically, younger individuals who are highly educated and have prior whale-watching experience were more inclined to provide financial support and engage in citizen science activities. These findings demonstrate that integrating citizen science-based activities and environmental DNA (eDNA) sampling, into whale-watching tours is an effective way to foster meaningful pro-environmental engagement. This model presents a scalable and cost-effective approach to enhancing public participation in marine conservation and biodiversity monitoring, with potential applications extending to broader marine governance and ecotourism settings.

The rapid decline of marine biodiversity is a critical global challenge that requires immediate and sustained conservation action. Public support is essential for effective management and long-term conservation outcomes. Enhancing awareness of the ecological importance of marine biodiversity is key to fostering public engagement. Within the framework of the Biodiversa+ eWHALE project, this study assesses the effectiveness of combining non-invasive environmental DNA (eDNA) sampling, whale watching, and citizen science to promote environmental awareness among whale watching tourists. Participants from three European regions directly observed eDNA sampling during typical whale watching tours, facilitating hands-on engagement with conservation practices. Pre- and post-tour surveys (n=172 and n=106, respectively) were analyzed using multiple correspondence analysis and nonparametric tests. While most tourists demonstrated general awareness of biodiversity and conservation issues, nearly 80% were initially unaware of eDNA's role in marine monitoring. Results showed a significant increase in knowledge after the activity, regardless of tour duration. These results suggest that integrating eDNA sampling into citizen science–based ecotourism can effectively bridge knowledge gaps and encourage public participation in marine conservation. This participatory approach offers a scalable, cost-effective strategy that supports biodiversity monitoring and reinforces inclusive, adaptive ocean and coastal management frameworks.

The comparability of methods applied to environmental DNA (eDNA) samples across laboratories remains a significant challenge for biodiversity monitoring on a global scale. Performance differences between protocols can jeopardize effective conservation strategies across regions and focal species. To address potential discrepancies amongst four international partners within a collaborative eDNA initiative, an inter-laboratory comparison (i.e., ring test) was conducted to compare efficiencies of established DNA extraction methodologies based on 39 eDNA samples. Each laboratory contributed eight to eleven samples collected throughout the North-East Atlantic and the Mediterranean Sea near sperm whales, porbeagle sharks, basking sharks, bottlenose dolphins and common dolphins. After lysis, aliquots were exchanged between laboratories before subsequent DNA extraction using each facility’s preferred method. Extracts were returned to the lysates’ respective laboratories of origin for measurements of total DNA concentration, as well as quantitative PCR using three novel species-specific assays for marine megafauna. Our findings revealed similar concentrations of total DNA, yet a significant reduction in extraction performance for targeted qPCR reactions by one laboratory, who has therefore modified their extraction method to be used for the remainder of this project. Overall, detection success differed based on the target taxa with sharks being less often detected (and at lower concentrations) than marine mammals. Significant interaction effects were found between combinations of laboratories and species, suggesting a link between extraction protocols and variable environmental conditions. Our study serves as a foundational step towards establishing reproducible practices that are crucial for the success of multinational eDNA projects to enable comparable results.