Developing DNA-based Techniques in Natural England for Surveying and Monitoring Biodiversity

This article was originally published in the Bulletin of the Chartered Institute of Ecology and Environmental Management (CIEEM), inpractice, Issue 99, Genetic Techniques and Technologies, in March 2018.

Authors: Andy Nisbet MCIEEM and Dr Kat Bruce, NatureMetrics.

Natural England is funding a number of projects examining different applications of DNA technology for the survey and monitoring of terrestrial, freshwater and marine biodiversity. The benefits of using DNA are that we may be able to save money and time, detect species that are difficult to find, and develop new measures of ecosystem health. While there are still issues and challenges associated with this technology, we believe that it will produce significant and exciting improvements to how we monitor and assess biodiversity.

Figure 1. Sampling water from grazing marsh ditches in the Nene Washes SAC.
© Keith Porter/Natural England.

Introduction

Biodiversity survey and monitoring has often been limited to recording taxa that are easy to find, or that we have a legal obligation to report on. Identification of some groups relies on a small expert community and it can be a long time between sampling and making records accessible. DNA-based techniques have the potential to significantly change this by reducing costs, reducing sample-to-use times, improving our ability to detect species that are difficult to find or identify, and providing a tool for monitoring functional groups and ecosystem health.

Over the last five years, Natural England has supported research and innovation into the use of environmental DNA (eDNA) to detect the presence of great crested newts in ponds (Biggs et al. 2014). Since 2016, we have been funding a number of exploratory projects that are looking at species detection across a range of taxa in different ecosystems (standing and flowing freshwaters, saline lagoons, coastal waters and sediments, terrestrial invertebrate traps, deadwood mould, vegetation and soils). Table 1 lists these projects.

Table 1. Projects underway to explore the use of eDNA to detect species across a range of taxa and ecosystems.

Detecting species that are difficult to find

A number of projects have examined the potential for using eDNA to detect species that are difficult to survey using conventional methods (see Box 1 for work on detecting seahorses Hippocampus spp.).

Figure 1 shows a water sample being taken from a ditch on the Nene Washes Special Area of Conservation (SAC) to see if DNA from the spined loach Cobitis taenia could be detected. Spined loach is a small bottom-feeding fish listed under Annex 2 of the Habitats Directive, and the Nene Washes SAC was designated because of the high density of this species in one main drainage channel. However, we didn’t know if spined loach were present in the smaller ditches because of the difficulties in surveying them. Water samples were taken from five sites. Spined loach DNA was successfully detected from two of the three main drain samples but not in the samples from smaller ditches. This method was cheaper and easier than a conventional survey would have been and also detected a number of other fish species.

Our attempts to detect DNA from the violet click beetle Limoniscus violaceus were less successful. This species is also listed under Annex 2 of the Habitats Directive and is known from only three woods in England. It is saproxylic and depends on undisturbed, ancient and decaying beech Fagus sylvatica or ash Fraxinus excelsior trees where the larvae live and grow in decaying wood mould.

Surveying for the larvae is destructive and so we tested whether DNA from this species could be detected from samples of wood mould. We took samples from trees where the species was thought to occur using a soil corer. Unfortunately, no DNA of the violet click beetle was detected. This may be because no DNA was present, and the chance of DNA from these rare species being present in a small core is probably low. However, the results could also be false negatives because the target sequences were obscured by much more prevalent bacterial and fungal DNA.

Box 1. eDNA monitoring of seahorses

There are two species of seahorse in the UK: the short-snouted seahorse Hippocampus hippocampus and the spiny seahorse H. guttulatus. Both are elusive and live in shallow, weedy areas, particularly eel grass beds. Important populations of both species are present in Poole Harbour and Studland Bay in Dorset, southern England.

Seahorse surveys are difficult because seahorses are small, cryptic, chameleonic animals that live in an expansive, low clarity habitat among dense seagrass/seaweed beds. Diver surveys are the current standard but can only cover very small areas, making comprehensive survey very time consuming and expensive (Garrick-Maidment 2011).

In 2016, we trialled an eDNA metabarcoding approach for detecting seahorses on the basis that this has the potential to be a far cheaper and more efficient tool. We first analysed water samples from tanks housing both species at the Zoological Society of London, and then collected six samples from Poole Harbour and Studland Bay (see Figure 3). eDNA was captured using Sterivex filters (EMD Millipore, Burlington, MA) (three filters per sample), which were filled with ethanol to preserve the DNA during transport to the laboratory.

DNA was separately extracted from each filter and PCR amplified for a short region of the 12S rRNA gene using primers that target fish. Eight of the nine DNA samples (six natural and three tank samples) were successfully amplified for each of the replicates, but one sample from Poole harbour failed to amplify due to Polymerase Chain Reaction (PCR) inhibition despite dilution of the DNA and the addition of various PCR enhancers. Successful PCRs were sequenced on an Illumina MiSeq (Illumina, San Diego, CA) and raw sequence data were processed using a custom bioinformatics pipeline to generate a species-by-sample table.

Both species of seahorse were successfully detected in the aquarium tanks, with each detection supported by around 200,000 sequences. This confirmed that the primers used in this study do effectively amplify seahorse eDNA when it is present. No seahorses were detected in any of the Poole Harbour samples, although these did yield detections of other marine and estuarine fish species including starry flounder Platichthys stellatus, sea bass Dicentrarchus labrax, allis shad Alosa alosa, Atlantic herring Clupea harengus, a gurnard species Chelidonithyes sp., grayling Thymallus thymallus, minnow Phoxinus phoxinus, European bullhead Cottus gobio and brown trout Salmo trutta. All freshwater-associated species were detected in a single sample that was collected from within 1 km of the mouth of the river Frome, and all are typical inhabitants of this river. This provides a useful indication of the typical transport distance of environmental DNA in lowland river systems.

The failure to detect seahorses may have been a consequence of the sampling date, which was later in the year than is advisable for seahorse surveys. It is also possible that greater sampling effort and laboratory replication is required to detect rare species in a marine environment. This is now the focus of ongoing work in the 2017-18 season. Recent work has detected two species of pipefishes (Syngnathinae) from seagrass beds, so we are confident that seahorses can be detected using this approach.

Figure 3. Sampling water from Poole Harbour to test for seahorse DNA.
© Gavin Black/ Natural England.

A quicker and cheaper alternative

During surveys and monitoring, sampling effort can be limited by the available capacity to identify specimens collected as well as by costs. This identification bottleneck can mean that data are not made available quickly. DNA metabacoding of invertebrates captured in standard traps on Lampert Mosses SSSI in 2016 successfully identified a range of species and the time taken from sample collection to making data available was only six weeks. As well as being faster, this approach is also cheaper when scaled up and so DNA metabarcoding should make it possible to analyse larger numbers of samples efficiently.

We are also testing DNA analysis of soil samples to detect fungi of conservation importance (e.g. waxcaps Hygrocybe spp.). The current approach to establishing which fungi are present on a site involves repeated visits over a number of years in the right season. Fungi may not fruit if the sward is too tall but may still be present in the soil. DNA analysis can detect the presence of species (even if not fruiting) throughout the year.

A third area of investigation is the identification of invertebrates from intertidal and subtidal sediment samples. Conventional sampling and species identification is expensive and time consuming. Metabarcoding appears to generate similar species diversity information to morphological analysis across multiple species groups including molluscs, crustaceans and annelids. Ongoing work focuses on the ability of this approach to describe ecological gradients using both the macrofaunal and meiofaunal component of diversity.

Assessing ecosystem health and habitat restoration

Our knowledge of biodiversity outcomes is often constrained by our reliance on data from a small number of popular taxa, rather than on cross-taxa species assemblages or groups which are sensitive to environmental change. Other taxa or groups may tell us more about ecosystem health (e.g. soil biodiversity) and ecosystem services (e.g. pollinators). Defra have

recently funded work to generate and collate reference DNA barcode data for key pollinator species (Defra 2017). Figure 2 shows soil being taken from our Long-Term Monitoring Network and we are using DNA metabarcoding to identify soil mesofauna found in these samples (Nisbet et al. 2017).

Figure 2. Soil sampling on Natural England’s Long-Term Monitoring Network.
© Matthew Shepherd/Natural England.

Progress and challenges

Some of our projects have been straightforward and successful while others have presented more challenges. A full report on the first year’s projects is in preparation; some of the main conclusions are that:

• eDNA metabarcoding is effective for monitoring fish species in all aquatic habitats although eDNA transport distances may be significant in flowing water.
• Metabarcoding is effective and efficient for characterising terrestrial invertebrate assemblages.
• Plant species can be identified successfully from root material extracted from soil although the extraction process was time consuming.
• More work is needed on the laboratory processes for detecting invertebrates from water samples and substrates (sediment).

Some of the challenges and issues that need to be addressed include testing and developing field methodologies (especially for eDNA approaches); refining laboratory techniques and developing appropriate primers for particular species or groups; being able to estimate abundance or relative abundance; improving DNA databases; and being able to interpret and use this evidence to support site management. Despite these challenges, we expect that DNA applications will produce significant and exciting improvements to how we monitor and assess biodiversity.

References

Biggs, J., Ewald, N., Valentini, A., Gaboriand, C., Griffiths, R.A., Foster, J., Wilkinson, J., Arnett, A., Williams, P. and Dunn, F. (2014). Analytical and methodological development for improved surveillance of the Great Crested Newt. Defra Project WC1067. Freshwater Habitats Trust, Oxford.

Defra (2017). Taxonomic fellowship to support the National Pollinator Strategy. Evidence Project PH0521 Final Report. Department for Environment Food and Rural Affairs, London.

Garrick-Maidment, N. (2011). British Seahorse Survey 2011. The Seahorse Trust, Escott, Devon.

Nisbet, A., Smith, S. and Holdsworth, J. (eds) (2017). Taking the long view: An introduction to Natural England’s Long-Term Monitoring Network 2009 – 2016. Natural England Report NERR070. Available from here. Accessed 24 January 2017.

About the Authors

Andy is a Principal Adviser at Natural England and is responsible for managing their Evidence Programme. He is leading Natural England’s work to develop DNA-based applications.

Contact Andy at: Andy.Nisbet@naturalengland.org.uk

Kat is co-founder and CEO of NatureMetrics, a company that specialises in DNA-based Monitoring of biodiversity.

An ecologist by training, her PhD at the University of East Anglia centred on the use of invertebrate metabarcoding to inform environmental management activities and facilitate evidence-based decision-making.

Contact Kat at: kat@naturemetrics.co.uk