title: Methodological considerations for detection of terrestrial small-body salamander eDNA and implications for biodiversity conservation.
abstract: Environmental DNA (eDNA) can be used as an assessment tool to detect populations of threatened species and provide fine-scale data required to make management decisions. The objectives of this project were to use quantitative PCR (qPCR) to: (i) detect spiked salamander DNA in soil, (ii) quantify eDNA degradation over time, (iii) determine detectability of salamander eDNA in a terrestrial environment using soil, faeces, and skin swabs, (iv) detect salamander eDNA in a mesocosm experiment. Salamander eDNA was positively detected in 100% of skin swabs and 66% of faecal samples and concentrations did not differ between the two sources. However, eDNA was not detected in soil samples collected from directly underneath wild-caught living salamanders. Salamander genomic DNA (gDNA) was detected in all qPCR reactions when spiked into soil at 10.0, 5.0, and 1.0 ng/g soil and spike concentration had a significant effect on detected concentrations. Only 33% of samples showed recoverable eDNA when spiked with 0.25 ng/g soil, which was the low end of eDNA detection. To determine the rate of eDNA degradation, gDNA (1 ng/g soil) was spiked into soil and quantified over seven days. Salamander eDNA concentrations decreased across days, but eDNA was still amplifiable at day 7. Salamander eDNA was detected in two of 182 mesocosm soil samples over 12 weeks (n = 52 control samples; n = 65 presence samples; n = 65 eviction samples). The discrepancy in detection success between experiments indicates the potential challenges for this method to be used as a monitoring technique for small-bodied wild terrestrial salamander populations.
authors.affiliation: Department of Biology, Tennessee Technological University, 1100N. Dixie Ave., Cookeville, TN, 38505, USA.
authors.forename: Donald M
authors.identifier: http://orcid.org/0000-0003-3119-8809
authors.initials: DM
authors.lastname: Walker
authors.affiliation: Department of Biology, Tennessee Technological University, 1100N. Dixie Ave., Cookeville, TN, 38505, USA.
authors.forename: Jacob E
authors.initials: JE
authors.lastname: Leys
authors.affiliation: Department of Biology, Tennessee Technological University, 1100N. Dixie Ave., Cookeville, TN, 38505, USA.
authors.forename: Kelly E
authors.initials: KE
authors.lastname: Dunham
authors.affiliation: Department of Biology, Tennessee Technological University, 1100N. Dixie Ave., Cookeville, TN, 38505, USA.
authors.forename: Joshua C
authors.initials: JC
authors.lastname: Oliver
authors.affiliation: Department of Biology, Tennessee Technological University, 1100N. Dixie Ave., Cookeville, TN, 38505, USA.
authors.forename: Emily E
authors.initials: EE
authors.lastname: Schiller
authors.affiliation: Department of Biology, Tennessee Technological University, 1100N. Dixie Ave., Cookeville, TN, 38505, USA.
authors.forename: Kelsey S
authors.initials: KS
authors.lastname: Stephenson
authors.affiliation: Department of Biology, Tennessee Technological University, 1100N. Dixie Ave., Cookeville, TN, 38505, USA.
authors.forename: John T
authors.initials: JT
authors.lastname: Kimrey
authors.affiliation: Department of Biology, Centre College, 600 W Walnut St., Danville, KY, 40422, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Wooten
authors.affiliation: U.S. Geological Survey, Tennessee Cooperative Fishery Research Unit, Tennessee Technological University, PO Box 5114, Cookeville, TN, 38505, USA.
authors.forename: Mark W
authors.initials: MW
authors.lastname: Rogers
mesh_terms: D000818:Animals; D044822:Biodiversity; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005243:Feces / Q000737:chemistry; D056186:Metagenomics / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction; D012867:Skin / Q000737:chemistry; D012987:Soil / Q000737:chemistry; D014562:Caudata / Q000145:classification* / Q000235:genetics*
chemical_list: D012987:Soil; D004247:DNA
keywords: 
  Plethodon cinereus
  ; eastern red-backed salamanders; environmental DNA; herpetofauna conservation; species declines; terrestrial eDNA