An eDNA diagnostic test to detect a rare, secretive marsh bird

We describe a novel method to detect a rare, secretive marsh bird using environmental DNA (eDNA). The Black Rail (Laterallus jamaicensis) occurs in visually concealing habitats and is most commonly surveyed by auditory callback. This method does not detect unresponsive individuals, is constrained seasonally and temporally, and requires significant personnel effort. New minimally invasive detection methods are needed to determine distribution and habitat use of this threatened species. We developed a highly species-specific quantitative PCR assay. We conducted callback surveys targeting Black Rails at sites on the mid-Atlantic coastal plain to collect samples for validation of eDNA diagnostics. Our assay reliably produced a signal when sufficient copies of Black Rail template were present. We successfully amplified Black Rail eDNA from 47% of the environmental samples taken from locations with detections. We tested whether environmental factors (water depth, salinity, air temperature), or sampling and handling procedures (time between collection and DNA extraction, storage temperature before filtering, field detection method, time between detection and sample collection) affected eDNA detectability. Only water depth had a significant positive effect on amplification success, emphasizing the importance of small pools as reservoirs of eDNA for terrestrial vertebrates. Our technique can be used in combination with other conservation strategies such as measuring occupancy in conjunction with habitat restoration efforts and resurveying coastal marshes after extreme weather events. It is adaptable to other elusive species of concern.