Validated Environmental <scp>DNA</scp> Assay for Detection of the Rare Rice's Whale (<i>Balaenoptera ricei</i>)

ABSTRACT The Rice's whale ( Balaenoptera ricei ) is a critically endangered baleen whale species that resides year‐round in the Gulf of America (formerly the U.S. Gulf of Mexico). The majority of whale sightings occur where the seafloor varies between 100 and 400 m in depth in a restricted region of the northeastern Gulf of America; however, historical whaling records suggest the species may have inhabited other areas and recent acoustic and visual observation data have detected their presence in the northwestern Gulf of America and southwestern Gulf of Mexico. A better understanding of their distribution and movements could aid in this species' recovery. In recent decades, non‐invasive genetic techniques have been developed to detect the presence or absence of species through the use of environmental DNA (eDNA). We developed a quantitative polymerase chain reaction (qPCR) assay targeting the mitochondrial DNA control region to detect eDNA shed by Rice's whales. A rigorous validation process was completed to determine the assay's sensitivity and specificity. The final assay detects low copy numbers of the target eDNA, is highly specific to Rice's whale, and was successfully used to detect the whale's presence in “flukeprint” samples ( n = 23). In addition, we tested eDNA collection methods such as filter pore size and the ability to detect Rice's whale eDNA at different time intervals after a whale surfaced and were able to detect eDNA as long as 10 min later. We also used eDNA methods to detect Rice's whale DNA obtained from Acousonde tags after being attached to a whale. Taken together, data from these sampling events will help to refine and improve eDNA collection techniques for many marine mammal species. Future research combining the newly developed eDNA assay with traditional survey methods could improve our understanding of the distribution and habitat use of this endangered and rare species.