Development and validation of an environmental DNA assay to detect federally threatened groundwater salamanders in central Texas.

The molecular detection of DNA fragments that are shed into the environment (eDNA) has become an increasingly applied tool used to inventory biological communities and to perform targeted species surveys. This method is particularly useful in habitats where it is difficult or not practical to visually detect or trap the target organisms. Central Texas Eurycea salamanders inhabit both surface and subterranean aquatic environments. Subterranean surveys are challenging or infeasible, and the detection of salamander eDNA in water samples is an appealing survey technique for these situations. Here, we develop and validate an eDNA assay using quantitative PCR for E. chisholmensis, E. naufragia, and E. tonkawae. These three species are federally threatened and constitute the Septentriomolge clade that occurs in the northern segment of the Edwards Aquifer. First, we validated the specificity of the assay in silico and with DNA extracted from tissue samples of both target Septentriomolge and non-target amphibians that overlap in distribution. Then, we evaluated the sensitivity of the assay in two controls, one with salamander-positive water and one at field sites known to be occupied by Septentriomolge. For the salamander-positive control, the estimated probability of eDNA occurrence (ψ) was 0.981 (SE = 0.019), and the estimated probability of detecting eDNA in a qPCR replicate (p) was 0.981 (SE = 0.011). For the field control, the estimated probability of eDNA occurring at a site (ψ) was 0.938 (95% CRI: 0.714-0.998). The estimated probability of collecting eDNA in a water sample (θ) was positively correlated with salamander relative density and ranged from 0.371 (95% CRI: 0.201-0.561) to 0.999 (95% CRI: 0.850- > 0.999) among sampled sites. Therefore, sites with low salamander density require more water samples for eDNA evaluation, and we determined that our site with the lowest estimated θ would require seven water samples for the cumulative collection probability to exceed 0.95. The estimated probability of detecting eDNA in a qPCR replicate (p) was 0.882 (95% CRI: 0.807-0.936), and our assay required two qPCR replicates for the cumulative detection probability to exceed 0.95. In complementary visual encounter surveys, the estimated probability of salamanders occurring at a known-occupied site was 0.905 (SE = 0.096), and the estimated probability of detecting salamanders in a visual encounter survey was 0.925 (SE = 0.052). We additionally discuss future research needed to refine this method and understand its limitations before practical application and incorporation into formal survey protocols for these taxa.

Predicting surface abundance of federally threatened Jollyville Plateau Salamanders (Eurycea tonkawae) to inform management activities at a highly modified urban spring.

Urban expansion has contributed to the loss of habitat for range restricted species across the globe. Managing wildlife populations within these urban settings presents the challenge of balancing human and wildlife needs. Jollyville Plateau Salamanders (Eurycea tonkawae) are a range restricted, federally threatened, species of neotenic brook salamander endemic to central Texas. Almost the entire geographic range of E. tonkawae is embedded in the Austin, Cedar Park, and Round Rock metropolitan areas of Travis and Williamson counties, Texas. Among E. tonkawae occupied sites, Brushy Creek Spring has experienced some of the most extensive anthropogenic disturbance. Today the site consists of small groundwater outlets that emerge in the seams within a concrete culvert underlying a highway. Salamanders persist within this system though they are rarely detected. Here, we model the occurrence of salamanders within the surface habitat of Brushy Creek Spring using generalized linear models. In the absence of available data regarding the amount of water that is discharged from the spring, we use accumulated rainfall as a proxy for discharge to estimate salamander abundance. Additionally, we present evidence of reproduction, recruitment, and subterranean movement by E. tonkawae throughout this site. Infrastructure maintenance is inevitable at Brushy Creek Spring. We intend for our results to inform when maintenance should occur, i.e., during environmental conditions when salamanders are less likely to be observed in the surface habitat, to avoid unnecessary impacts to this federally threatened species.