Fig 1.
Attributes of coastal tailed frog tadpoles and frogs.
A) Coastal tailed frog tadpoles have an adhesive oral-disc, or mouth, to attach to rocks in stream habitats. B) Defining features include, the vertical pupils, lack of an external ‘ear’ membrane, and long outer hind toes. C) Male (right) and female (left) adult tailed frogs are sexually dimorphic–the ‘tail’ is visible on the adult male (white arrow). Photo credits: Jared Hobbs.
Table 1.
Detection frequencies of four previous coastal tailed frog TCS project results within the study area.
This is a reanalysis and correction of project details presented in [12].
Table 2.
Nucleotide sequences for the qPCR-based eASTR4 eDNA tool comprised of primers and a probe for coastal tailed frog detection.
The amplicon sequence for the creation of the synthetic DNA sequence is indicated.
Table 3.
Names and abbreviations of species used for coastal tailed frog eDNA test validation.
The number of technical replicates evaluated was n = 2 except for those indicated in bold where n = 25 technical replicates.
Fig 2.
Use of the IntegritE-DNA test leads to increased confidence of species-specific DNA (eTarget) results.
The IntegritE-DNA test evaluates the ability of the isolated DNA in a sample to amplify plant chloroplast DNA prior to testing for the target species. If the sample passes the IntegritE-DNA test, then it is then tested for the target species (eTarget). If a sample fails the IntegritE-DNA test, it undergoes clean-up to remove inhibitors before being retested. If the cleaned up sample fails the IntegritE-DNA test, it is deemed poor quality due to either degraded DNA or retention of inhibitors and the sample is not considered reliable. Photo credits: Jared Hobbs.
Fig 3.
Sample orientation on qPCR plates plays an important role in the success of the eDNA assay.
eDNA samples are randomized before DNA is isolated and the randomized samples are run in eight technical replicates for the eASTR4 test. Using the above layout spatially removes the positive control from the test samples to limit to chance of contaminating the test samples with pure target species total DNA. If a contamination event has occurred it will be detectable in the negative control right adjacent to the positive control.
Fig 4.
The eASTR4 test is highly specific for coastal tailed frog and is highly sensitive.
A) The sensitivity graph is displayed as mean abundance with standard error of the mean of n = 25 technical replicates of each species (refer to Table 1 for legend) and a no template control (NTC). Five μg/L total DNA were tested and the strength of detection (Abundance) was determined as the number of cycles (50) in the qPCR program minus the cycle threshold where detection occurred. B) The sensitivity graph was produced using a 5-fold dilution series of total DNA of the target species. The percent positive score is derived from the number of detected reactions divided by the number of technical replicates (n = 25). C) Percent binomial standard error determined at each dilution of total DNA for differing technical replicates: n = 3 (solid squares), n = 5 (solid circles), n = 8 (open circles), n = 12 (solid triangles), and n = 25 (open triangles).
Fig 5.
Sensitivity and determination of the limits of detection (LOD) and quantitation (LOQ) for the eASTR4 test using synthetic template DNA.
A) Five-fold serial dilutions of a synthetic template DNA were added to the eASTR4 reaction at copy numbers ranging from 62,500 to 0.032 copies per reaction and the mean transformed Ct values (50.001-Ct) determined for each of the eight or 24 technical replicates. The whiskers depict the standard error of the mean. The NTC controls never had any amplification detected. The proportion of technical replicates that passed the Ct cut off for detection (% Positive) and number of technical replicates (n) measured are indicated at the top of the graph for each dilution tested. B) Percent binomial standard error determined at each dilution of synthetic template DNA for differing technical replicates: n = 3 (solid squares), n = 5 (solid circles), n = 8 (open circles), n = 12 (solid triangles), and n = 25 (open triangles).
Fig 6.
Fifty-five of 72 sampling sites located in British Columbia’s southern coastal mountains were positive for coastal tailed frog using eDNA methods.
The sampling sites were located in Bridge River and Seton, Anderson, Carpenter and Downton lake drainages. Squares indicate sites that were assessed by previous time constrained surveys and circles indicate the eDNA results from the same sites. Squares, historical TCS results from the studies in Table 1; Circles, eDNA results; Red, positive detection; Yellow, negative detection. The map was created from open source material from the governments of Canada and British Columbia (https://www2.gov.bc.ca/gov/content/data/open-data/open-government-license-bc; https://open.canada.ca/en/open-government-licence-canada).
Table 4.
Relationship between coastal tailed frog historical TCS and eDNA results.
The number of positive and negative sites where both TCS and eDNA results were within a 2 km radius are shown.