Sexing From Seawater: Application of Environmental DNA Beyond Species Detection for Cetaceans

Anthropogenic pressures on marine ecosystems are intensifying, highlighting the need to bridge knowledge gaps for species at risk. Data deficiencies, particularly for species recovering from historic declines, such as marine megafauna like whales, hinder effective management. Understanding long‐term population viability and identifying any sex‐biased threats is important for cetacean conservation. Killer whales ( Orcinus orca ) and humpback whales ( Megaptera novaeangliae ) are well‐studied in the Northeast Pacific, making them ideal for optimizing noninvasive environmental DNA (eDNA) techniques for sex determination. We collected eDNA flukeprint samples from killer whales ( n = 67) and humpback whales ( n = 18), analyzing ZFX/ZFY gene amplification using conventional PCR to compare results against known sexes. Samples from killer whales exhibited higher ZFX/ZFY PCR amplification success (53%) compared with humpback whales (44%). However, the close social structure of this species likely contributed to only 54% of samples matching the known sex of whales sampled. Conversely, humpback whale samples accurately matched the known sexes of individuals (100%). These findings demonstrate eDNA's potential to replace more invasive biopsies for sex determination but highlight the need for further optimization regarding sampling protocols and species‐specific ZFX/ZFY amplification approaches. Additionally, eDNA flukeprint sampling also shows promise for other solitary cetaceans such as large rorquals ( Balaenoptera spp.), which remain among the most data‐deficient species.