qPCR‐Based Environmental DNA ( eDNA ) Detection of Cryptic Arboreal Species Interaction With Artificial Habitat Structures

Effective restoration of anthropogenically impacted sites requires continued monitoring of faunal species return, but restoration sites often lack mature habitat structures that many species rely on. Artificial habitat structures aim to supplement these to accelerate the return of target species and improve ecosystem function. Conventional monitoring techniques for artificial habitat structures are typically labor‐intensive and lack sensitivity. In this study, we designed and validated two species‐specific probe‐based qPCR assays targeting the cytochrome B gene region to detect two cryptic arboreal marsupials, the Red‐tailed Phascogale ( Phascogale calura ) and the Brush‐tailed Phascogale ( Phascogale tapoatafa ), from roller swabs of nest box surfaces. Sensitivity of environmental DNA monitoring was compared to conventional approaches (visual inspections and motion cameras) in a managed nature reserve and an active bauxite mine site in south‐western Australia. qPCR of target species eDNA collected from surfaces was successfully amplified and increased the detection rate of cryptic arboreal species interaction with artificial nest boxes. These results highlight the value of eDNA approaches for monitoring the use of artificial habitat structures by fauna, cryptic species in restoration landscapes, and arboreal species in general.