Both assays were highly sensitive and successfully detected the Louisiana Pinesnake under artificial and field conditions, including bedding samples collected from captive snake enclosures (100%), soil samples from Louisiana Pinesnake release sites (100%), and soil samples from sites where Louisiana Pinesnakes were documented via radio telemetry (45%).
We validated each assay in silico, in vitro, and in situ, and investigated the influence of eDNA extraction method and genetic marker on assay performance. We developed two quantitative polymerase chain reaction (qPCR) assays that target the mitochondrial cytochrome c oxidase subunit I (COI) and mitochondrially encoded ATP synthase membrane subunit 6 (ATP6) genes. We evaluated the efficacy of eDNA surveillance for the Louisiana Pinesnake (Pituophis ruthveni), an elusive, semi-fossorial, nonvenomous colubroid snake endemic to Louisiana and Texas, USA, that has dramatically declined in both distribution and abundance. Environmental DNA (eDNA) analysis may provide a rapid, cost-effective means of assessing RTE species presence/absence in viable habitat patches. However, assessments are challenging as RTE species typically exist as numerically small populations in often fragmented habitats and can possess complex natural histories. Successful conservation of rare, threatened, or endangered (RTE) species is dependent upon rapid and accurate assessment of their distribution and abundance.