eDNA

eDNA for detecting great crested newts – a replacement for traditional survey techniques?

Environmental DNA, or eDNA, is released by most organisms as they occupy different habitats. Each species has a unique type of eDNA which can be recognised through laboratory analysis. This provides a novel tool for detecting species within the environment. Sources of eDNA may originate from sloughed skin or hair, eggs, faeces and saliva (Figure 1). Within aquatic habitats, most organisms release eDNA into the surrounding water. An increasing number of methods are now available for detecting the eDNA of a range of aquatic organisms including fish, damselfly nymphs, crustaceans and amphibians. In recent years, techniques have advanced to allow the detection of eDNA of great crested newts from their breeding ponds. This has proved particularly useful for ecologists and voluntary surveyors who need to determine the presence or absence of great crested newts for the purposes of conservation and mitigation. The eDNA technique, though expensive, is usually highly reliable and effective at detecting great crested newts. Research carried out by Biggs et al. (2015) of 35 ponds in Hampshire and Wales showed that eDNA could successfully detect the presence of great crested newts in 99.3% of ponds. This was significantly higher than the success rate of more traditional survey techniques.

Traditional survey techniques for detecting great crested newts such as egg-searching, night torching and bottle trapping are labour intensive, carry certain risks (e.g. potential suffocation of newts in traps) and are not always highly effective. Biggs et al. (2015) found that bottle trapping detected great crested newt presence in 76% of ponds and egg searching in only 44% of visits. Using traditional survey techniques, Natural England advice recommends four visits to a breeding pond in the newt breeding season (mid-March to mid-June) using a minimum of three traditional survey techniques (Natural England, 2015) (Figure 2). The eDNA technique, by contrast, can detect great crested newts on just one visit with minimal disturbance to breeding ponds (Biggs et al., 2015). Therefore, should eDNA replace traditional surveys techniques to detect great crested newts? In this article we highlight the relative advantages of disadvantages of eDNA and point out the limitations that ecologists and voluntary surveyors need to be aware of when using the technique.

One of the major advantages of eDNA is the relative ease that samples can be taken from a pond and the subsequent reduction in labour costs. For example, one fieldworker may be able to take samples for analysis within one survey visit, compared to the multiple sessions required using traditional survey techniques. However, the analysis costs associated with eDNA may be significant, especially if carrying out many samples. A second major advantage of eDNA is its overall reliability compared to other traditional methods. However, a range of studies have found variations in the success of eDNA, ranging from 60% to 99% (Buxton et al., 2017a).

Despite is apparent reliability and success in detecting great crested newts, there are a number of limitations of using eDNA to survey ponds. Six of the main limitations are outlined below:

1. Estimation of abundance: the level of eDNA detected from samples taken from a pond does not correlate well with the number of newts in the pond. Biggs et al. (2015) found that at low levels of detection, eDNA may reflect the number of newts but at higher levels, there was only a weak, non-significant correlation. Therefore, it is not currently possible to use eDNA to estimate the population size or relative abundance of great crested newts using ponds. Traditional survey techniques, such as night torching or bottle trapping, may provide an estimate of relative abundance. For example, if 120 newts are captured in bottle traps, this shows that at least this number of newts were present in the pond on this survey occasion.
2. Life stages: eDNA only records presence, or recent occupancy, of ponds by newts; it does not show which life stages are present e.g. adults, eggs or larvae, or when they were last in the pond (Rees et al., 2014a). Traditional survey techniques can identify actual animals which is useful for looking at life stage, sex ratio and potentially the body condition of individuals. Also, traditional techniques can record other species of newt which are present in ponds e.g. smooth or palmate newts, which is often important for survey records and provides an indication of the species diversity within a pond.
3. Breeding or non-breeding? It is often useful to determine whether great crested newts are breeding within a pond as this may show persistence of a viable, long-term population. However, the eDNA techniques is not able to detect if newts are laying eggs within a pond; it can only determine if they are present, or have recently been present. The traditional survey technique of egg-searching is able to detect the presence of breeding females demonstrating the value in using this technique.
4. False absences: a false absence is when a particular technique fails to determine the presence of the species, even though it is present. Although the level of false absences using eDNA was very low in one study carried out by Biggs et al. (2015), this was only carried out on 35 ponds in two counties in the UK. Similar research carried out by Rees et al. (2014) on 38 ponds showed that eDNA had a lower success rate of 89% in detecting great crested newts in ponds. Given the number of false absences in this study Rees et al. (2014) conclude: “Species detection by eDNA and field work is likely to be imperfect and may lead to an underestimation of the distribution of a species especially in the case of rare or threatened species”. False absences may come from a number of sources and are more likely to be a problem when they operate together (Biggs et al., 2015). Common sources of false absences include:

• when there are very few newts in a pond and the water samples fail to collect any eDNA;
• in ponds where surveyors can only access a small proportion of the pond perimeter due to logistical issues;
• at sites which have a very large area of shallow water, which prevents surveyors from reaching the deeper edges often favoured by newts (Biggs et al., 2015) (Figure 3).

5. False positives: detecting newts when they are not present is less commonly a problem with eDNA for great crested newts but can occur when there is contamination within the samples collected (Thomsen & Willerslev, 2015). Potential sources of contamination include: excrement from animals that prey on great crested newts; the eDNA of dead great crested newts being carried to the pond by other species (e.g. birds); or unsterilized equipment contaminating samples (Bohmann et al., 2014).

6. Reduction or loss of DNA in the water: There are three potential processes that can result in the loss or reduction in the quantity of DNA from within samples: inhibition, degradation, and binding to sediments. This can lead to increases in the likelihood of false absences occurring as less eDNA may be available to detect in analysis.

1. Inhibition: eDNA inhibition occurs when factors in the environment, such as decaying organic matter, interfere with the laboratory analysis, resulting in a failure to detect the eDNA (Buxton et al., 2017a).
2. Degradation: once eDNA is released into the environment it starts to decay and this rate depends on a range of factors such as UV radiation, temperature and microbes which occur in the pond (Buxton et al., 2017; Rees et al., 2017). Currently, our understanding of how quickly eDNA degrades under different conditions is limited (Rees et al., 2014a). This is of importance since if eDNA degrades faster in some ponds than others, this may limit the likely time window when surveys can be carried out. Although research has shown that eDNA may be present in ponds in all months of the year (Rees et al., 2017), the realistic time frame for detecting adults is March to the end of June (Rees et al., 2017) which is the same as for traditional survey techniques. However, if ponds contain breeding adults and subsequent larvae, these may be detected in ponds up until the end of August (Buxton et al., 2017).
3. Binding to sediments: research has also demonstrated that eDNA will bind to certain sediments in ponds. Buxton et al. (2017a) have shown that eDNA binds more effectively to clay and organic soils when compared to sand. This has important consequences for surveying since the eDNA will disappear much more quickly in ponds with clay sediments. This means that if great crested newts are present in two ponds, one with a clay sediment and one with a sand sediment, the available time frame for surveying may be shorter in the pond with clay lining. A late survey carried out may result in a false absence record in the pond with clay sediment, even though the newts had recently been present. Given the variation in occurrence of eDNA in water samples, the potential for false absences needs to be fully understood before using eDNA as a survey tool and it is important to realise that eDNA research is still an evolving discipline (Buxton et al., 2017a).

Overall, although using eDNA to detect the presence of great crested newts is highly effective and usually reliable, ecologists and surveyors must be aware of the potential limitations when carrying out surveys. Therefore, caution should be taken when analysing the results from eDNA; ecologists and surveyors should consider eDNA as an additional technique to complement traditional survey methods, rather than being viewed as replacing existing techniques. We conclude this article with a statement by Rees at al. (2014a): “Environmental DNA methodologies should not be used to replace or disregard the knowledge and expertise of experienced field ecologists and taxon specialists, but should become an important tool to enhance limited conservation resources”.

References

Biggs, J., Ewald, N., Valentini, A., Gaboriaud, C., Dejean, T., Griffiths, R.A., Foster, J., Wilkinson, J.W., Arnell, A., Brotherton, P., Williams, P. & Dunn, F. (2015) Using eDNA to develop a national citizen science-based monitoring programme for the great crested newt (Triturus cristatus). Biological Conservation, 183: 19–28.

Buxton, A.S., Groombridge, J.J., Zakaria, N.B. & Griffiths, R.A. (2017) Seasonal variation in environmental DNA in relation to population size and environmental factors. Science Reports, 7: 46294; doi: 10.1038/srep46294.

Buxton, A.S., Groombridge, J.J. & Griffiths, R.A. (2017a) Is the detection of aquatic environmental DNA influenced by substrate type? PLoS ONE, 12 (8): e0183371.

Bohmann, K., Evans, A., Thomas, M., Gilbert, P., Carvalho, R., Creer, S., Knapp, M., Yu, D.W. & de Bruyn, M. (2014) Environmental DNA for wildlife biology and biodiversity monitoring. Trends in Ecology & Evolution, 29 (6): 358-367.

Natural England (2015) Great crested newts: surveys and mitigation for development projects. https://www.gov.uk/guidance/great-crested-newts-surveys-and-mitigation-for-development-projects#when-to-survey. Accessed 20^th June 2018.

Rees, H.C., Bishop, K., Middleditch, D.J., Patmore, J.R.M., Maddison, B.C. & Gough, K.C. (2014) The application of eDNA for monitoring of the Great Crested Newt in the UK. Ecology and Evolution, 4 (21): 4023–4032.

Rees, H.C., Maddison, B.C., Middleditch, D.J., Patmore, J.R.M. & Gough, K.C. (2014a) The detection of aquatic animal species using environmental DNA – a review of eDNA as a survey tool in ecology. Journal of Applied Ecology, 51: 1450–1459.

Rees, H.C., Baker, C.A., Gardner, D.S., Maddison, B.C. & Gough, K. (2017) The detection of great crested newts year round via environmental DNA analysis. BMC Res Notes, 10: 327. DOI 10.1186/s13104-017-2657-y. 

Thomsen, P.F. & Willerslev, E. (2015) Environmental DNA – An emerging tool in conservation for monitoring past and present biodiversity. Biological Conservation, 183: 4–18.