Development and Application of an eDNA Method to Detect the Critically Endangered Trinidad Golden Tree Frog (Phytotriades auratus) in Bromeliad Phytotelmata

Abstract

The use of environmental DNA (eDNA) to monitor rare and elusive species has great potential for conservation biology. Traditional surveying methods can be time-consuming, labour-intensive, subject to error or can be invasive and potentially damaging to habitat. The Trinidad golden treefrog (Phytotriades auratus) is one such species that would benefit from such an approach. This species inhabits the giant bromeliad (Glomeropitcairnia erectiflora) on two peaks on the Caribbean island of Trinidad. Traditional survey methods for this species have required the destruction of the giant bromeliad, which is the only known habitat of this frog. Here we described the development of an eDNA PCR-based assay that uses water drawn from the water-filled phytotelmata of the giant bromeliad along with the use of a synthetic DNA positive control that can be easily amplified in the bacterium Escherichia coli. The assay can detect to a DNA concentration of 1.4ng. Sampling of 142 bromeliads using this method revealed 9% were positive for P. auratus DNA. These data suggest that eDNA methods also have great potential for revealing the presence of elusive species in arboreal habitats.

Fig 1. Position of P. auratus sampling locations.

(A) Map of Trinidad showing the location of El Tucuche on the island of Trinidad, West Indies. The inset shows the location of the sample GPS coordinates along the summit (red points). The map was constructed from http://www.d-maps.com/carte.php?num_car=28037&lang=en and GPS coordinates plotted using HamsterMap. (B) Scatter plot of the samples as GPS coordinates plotted against height above ground of bromeliad phytotelmata sampled. Red points indicate those samples that were PCR positive for P. auratus DNA. (Supplementary Table 1: GTF Field and PCR raw data: https://dx.doi.org/10.6084/m9.figshare.4547338.v1).

Fig 2. Specificity and detection limits of the P. auratus PCR assay.

(A) Using P. auratus DNA as a template yields a single amplicon of 297 bp. Using Mannophryne trinitatis genomic DNA as a template yielded no amplicon. (B) Dilution series of pGTF-CytB DNA from 140 ng per reaction to 0.014 ng per reaction. Amplicons were apparent by agarose gel electrophoresis and ethidium bromide staining down to 1.4 ng of DNA per reaction.

Fig 3. Determination of P. auratus DNA in the phytotelmata of Glomeropitcairnia erectiflora.

(A) Electrophoretic analysis using DNA extracted from phytotelmata water samples as a template and Universal 16S rDNA primers (See Materials and Methods). + is a positive control consisting of Mannophryne trinitatis genomic DNA.–is a ‘No DNA’ negative control. (B) A representative electrophoretic analysis of a positive sample (Sample 86) from the phytotelmata water showing the presence of P. auratus DNA.