The use of eDNA as a novel method for the detection of elusive amphibians in tropical climates
By Eleanor Gourevitch, J. Roger Downie, Mark S. Greener, Sarah Brozio, Tom Burns and Paul A. Hoskisson
Affiliated with the University of Glasgow Trinidad Expedition 2015 and the University of Strathclyde
For those species which are elusive and hard to find there is an urgent need to develop new detection methods. Traditional methods can often be invasive and destructive, reducing the overall benefits that could be gained from surveying. The golden tree frog (Phytotriades auratus), for instance, lives its entire life cycle within the bromeliad (Glomeropitcairnia erectiflora) where surveying has previously consisted of total destruction of the plant to determine the frog’s presence or absence. Many amphibians could be considered elusive due to their sit-and-wait method of foraging, low metabolism and nocturnal behaviour (Duellman & Trueb 1994; Willson et al. 2011), but the golden tree frog is particularly elusive because of its restricted habitat and apparent lack of a call. In order to detect such animals, new rapid techniques must be developed, due to the threats facing many amphibians in response to climate change and emerging pathogens.
The use of environmental DNA (eDNA) as an indicator of presence is one such recent detection method. This method was first used to detect macroinvertebrates in ancient sediments (Willerslev et al. 2003). This led to further development of the method for the detection of many groups of freshwater organisms. Sampling involves collection of a small amount of water containing mucus, skin cells or faeces etc. of the organism in question. The extraction of total DNA from the sample is followed by PCR analysis. The specificity of the PCR reaction can then be used to confirm the presence or absence of the target species. This allows for non-invasive, non-destructive sampling which can be undertaken when the organism is not directly observed. However, there may be some limitations to this technique, such as the viability of this method when used within the tropics due to increased rates of DNA degradation and the higher level of biodiversity (Yoccoz et al. 2012).
In order to assess the application of this technique for surveying the critically endangered golden tree frog in the Northern Range of Trinidad and also the viability of this technique in tropical conditions, we sampled the phytotelma of the giant bromeliad, Glomeropitcairnia erectiflora – the only known habitat of this species. This bromeliad only grows above 600 m, which consequently means that this species of frog has only been found so far on the peaks of El Tucuche and El Cerro del Aripo in Trinidad and, recently a new population discovered on the Península de Paria, Venezuela (Rivas & De Freitas 2015).
In the summer of 2015, we sampled almost 200 bromeliads from the peak of El Tucuche over a 10 week period from June – August. The samples were fixed in ethanol in Trinidad and analysed back in the laboratory in Glasgow. DNA extraction and PCR was carried out on the samples, using primers designed to amplify golden tree frog DNA. We used a positive control based on the cytB for the golden tree frog sequence deposited in Genbank. This was made synthetically in a plasmid that replicates in the bacterium Escherichia coli, which allowed us to produce large amounts of high quality, easily quantified positive control PCR template. This allowed us to avoid collection of any new golden tree frog tissue samples. It was shown that P. auratus was detectable within many of the bromeliads sampled and that the use of eDNA is an effective method of detection, even in tropical conditions. These data will allow us to carry out a more detailed sampling regimen during our field season in 2016, which will result in population estimates of the golden tree frog.
We hope that this technique will be applied in future to surveying further elusive amphibian species. Accurate population estimates and the ability to rapidly determine population range are fundamental to the development of specific legislation and conservation efforts concerning the protection of critically endangered species.
Acknowledgements
PAH and EG would like to thank the Amphibian Survival Alliance (ASA) and Amphibian Specialist Group (ASG) for Seed Grant Funding to support this project (Amphibians.org). Thanks to Froglife for their help in our 2015 Crowdfunding campaign “Fighting for a golden future: Safeguarding the Golden Tree Frog”.
References
Duellman, W.E. & Trueb, L., 1994. Biology of Amphibians, JHU Press.
Rivas, G. & Freitas, M. De, 2015. Discovery of the Critically Endangered Golden Tree Frog, Phytotriades auratus (Boulenger, 1917), in Eastern Venezuela, with Comments on its Distribution, Conservation, and Biogeography. Herpetological Review, 46(2), pp.153–157.
Willerslev, E. et al., 2003. Diverse plant and animal genetic records from Holocene and Pleistocene sediments. Science (New York, N.Y.), 300(5620), pp.791–5.
Willson, J.D., Winne, C.T. & Todd, B.D., 2011. Ecological and methodological factors affecting detectability and population estimation in elusive species. Journal of Wildlife Management, 75(1), pp.36–45.
Yoccoz, N.G. et al., 2012. DNA from soil mirrors plant taxonomic and growth form diversity. Molecular Ecology, 21(15), pp.3647–3655.
