Expansion of the known distribution of the coastal tailed frog, Ascaphus truei, in British Columbia, Canada, using robust eDNA detection methods

Abstract

The coastal tailed frog (Ascaphus truei) is endemic to the Pacific Northwest of North America and is listed as a species of Special Concern under the Canadian Species at Risk Act. Its range is limited to British Columbia where it occurs widely west of the Coast Mountain Ranges extending north almost to the Alaskan Panhandle. The present study focused on surveying within the Cayoosh, Bridge (Shulaps), Seton, Anderson, Carpenter, and Downton Lake drainages. Four years of previous inventory efforts using conventional time-constrained search (TCS) methods detected tailed frog at 23/292 discrete sites (7.9% detection rate) in seven watersheds. Non-invasive environmental DNA (eDNA) methods hold promise for cryptic and low-abundance species detection. We rigorously validated a quantitative real-time polymerase chain reaction (qPCR)-based tool for detecting coastal tailed frog eDNA in water samples. This eASTR4 test is highly specific and sensitive. We applied a two-step targeted eDNA analysis approach on duplicate filtered water samples from a total of 72 sites collected over five days. The first IntegritE-DNA step mitigates false negative results and tests all DNA samples for the ability to support amplification from endogenous plant chloroplast DNA as a measure of sample viability. Three DNA samples failed this step even after inhibitor clean up suggesting that these samples were poor quality and not reliable for targeted species’ DNA analyses. All other DNA samples were deemed viable and were then tested for species-specific DNA. Coastal tailed frog eDNA was detected in 55/72 (76%) discrete stream reaches; nine sites with historical known occurrence were all eDNA positive. The false negative rate for TCS compared to eDNA methods was 58%. The results expand known coastal tailed frog distribution to 24 watersheds effectively more than tripling extant occurrences and confirm a previously suspected, apparently isolated coastal tailed frog metapopulation in the Shulaps drainage.

The Effect of Silica Desiccation Under Different Storage Conditions on Filter-Immobilized Environmental DNA

“…As expected, the lters that were immediately processed ("None" Fig. 2) returned C t values between 20-23 (corresponding to ~ 50,000 copies/reaction) for intact samples consistent with previous observations with eld water samples (14,15). Filters that were stored in ethanol at 23 o C experienced a progressive decrease in eDNA sample quality (as demonstrated by the shift in C t value) across all time points compared to lter samples that were processed immediately.…”

“…The DNA from one quarter of each lter was isolated using the DNeasy Blood and Tissue kit (QIAGEN Inc., Mississauga, ON, Canada; Cat# 69506) following the procedure outlined in (13). The test for relatively abundant chloroplast signal using the IntegritE-DNA™ assay as described previously (13,14) and the tests for low abundance bullfrog DNA (eFrog3 and eLICA1) were previously validated and described (13). An additional validation step for these two assays was performed using gBlocks® synthetic DNA ordered from Integrated DNA Technologies (Coralville, Iowa, United States) following the method outlined previously (14).…”

Revising the range of Rocky Mountain tailed frog, Ascaphus montanus, in British Columbia, Canada, using environmental DNA methods

“…Ultraviolet rays, water temperature, pH, salinity, substrate type, and microbial community activity together affect degradation rates (Barnes et al, 2014;Strickler et al, 2015). Sample replication at the site (n = 2, for the present study) and technical levels (n = 3 in 2014; and n = 8 in (Deiner & Altermatt, 2014;Hobbs et al, 2019).…”

“…In these years, an IntegritE‐DNA™ test was applied to each sample preceding qPCR evaluation for eDNA from the focal taxa (i.e., tailed frog) (Hobbs et al, 2019; Veldhoen et al, 2016). The IntegritE‐DNA™ assay simultaneously tests for sample inhibition and degradation and is an effective means for mitigating false‐negative eDNA results (Hobbs et al, 2019).…”

“…The new drainages documented with Rocky Mountain tailed frog occurrences are indicated by the fuschia lines. Map source: OGC web map service https://www.openg eospa tial.org/stand ards/wms desiccant (Dry & Dry (Silica gel Factory) via Amazon.ca; Years 4-5)(Hobbs et al, 2015(Hobbs et al, , 2019.…”