Environmental DNA (eDNA) offers a non-invasive approach for monitoring cetaceans, yet its widespread application from operational platforms requires standardized and feasible sampling workflows. This study describes a protocol for collecting cetacean eDNA from whale-watching vessels, designed for implementation by researchers and trained citizen scientists under defined environmental and logistical conditions. The approach integrates flukeprint-targeted seawater collection, onboard filtration with self-preserving filters, and downstream molecular analyses to support operationally feasible data generation in real-world conditions. The protocol was implemented during coordinated field campaigns conducted in 2024 across three regions in the North Atlantic and Mediterranean Sea (Iceland, Portugal, and Italy), encompassing multiple cetacean taxa. Method performance was evaluated using a newly developed species-specific quantitative PCR assay targeting mitochondrial DNA of humpback whale (Megaptera novaeangliae), demonstrating consistent detection rates under variable environmental and logistical conditions. By providing practical guidance for field implementation and contamination control, this protocol supports reproducible eDNA-based cetacean monitoring and facilitates cross-regional comparability in marine biodiversity studies.
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