Evaluating the Detection of Seasonally Present, Pond-Breeding Amphibians Using Environmental DNA: A Case Study with the Houston Toad (Bufo [=Anaxyrus] houstonensis)




Keitt, William W.

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Molecular survey techniques focusing on the detection environmental DNA (eDNA) are increasingly being implemented for a wide breadth of animal taxa in order to document occurrence across various habitats. Despite increasing instances of its use, relatively few studies have evaluated this method for seasonally present, pond-breeding amphibians. This study seeks to provide an evaluation of the efficacy of eDNA surveys for the detection of one such species, the Houston toad (Bufo [=Anaxyrus] houstonensis). The Griffith League Ranch (GLR), a primary recovery site in Bastrop county Texas, was sampled weekly during the Houston toad breeding season from February to June of 2016, and sporadically in the spring of 2017. Nine perennial ponds on the GLR were surveyed, and 557 water samples were collected for eDNA analysis, with 217 representing known positive captive controls, collected from buckets with each of the primary life stages of this amphibian. Samples were collected following a USGS approved protocol (Goldberg et al. 2011). Both PCR, and nested PCR assays were used to assess the utility of this technique in the detection of the Houston toad based on positive amplification of a diagnostic fragment of mitochondrial DNA. PCR assays successfully showed amplification of Houston toad eDNA in 82% of known positive captive controls, while only 1.1% of eDNA samples from the sampled ponds, and none of the eDNA samples of known positive pond controls showed amplification. Nested PCR assays proved more efficient, detecting Houston toad eDNA in 86% of all known positive captive controls, 7.4% of all pond samples, and 14% of the samples collected from known positive pond controls. Our results suggest that these PCR-based detection methodologies are unreliable in the detection of eDNA as they incur routine false negative detections, and therefore, are likely less reliable than current survey approaches. The inability of eDNA surveys to accurately detect species presence may be impacted by a variety of factors ranging from environmental inhibitors, to the limitations of the detection assay. Therefore, I offer many critical considerations for the effective implementation of this monitoring strategy, as a USGS approved protocol appears inadequate in the detection of elusive, seasonally present, pond-breeding amphibians, like the Houston toad.



eDNA, Houston Toad, Endangered species, Monitoring, Pond-breeding amphibian


Keitt, W. W. (2017). Evaluating the detection of seasonally present, pond-breeding amphibians using environmental DNA: A case study with the Houston Toad (Bufo [=Anaxyrus] houstonensis) (Unpublished thesis). Texas State University, San Marcos, Texas.


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