Environmental DNA

In this example, the fish leaves its eDNA behind as it moves through the water, but its eDNA slowly dissipates over time.

Environmental DNA or eDNA is DNA that is collected from a variety of environmental samples such as soil, seawater, snow or even air rather than directly sampled from an individual organism. As various organisms interact with the environment, DNA is expelled and accumulates in their surroundings. Example sources of eDNA include, but are not limited to, feces, mucus, gametes, shed skin, carcasses and hair. Such samples can be analyzed by high-throughput DNA sequencing methods, known as metagenomics, metabarcoding, and single-species detection, for rapid measurement and monitoring of biodiversity. In order to better differentiate between organisms within a sample, DNA metabarcoding is used in which the sample is analyzed and uses previously studied DNA libraries to determine what organisms are present (e.g. BLAST). The analysis of eDNA has great potential, not only for monitoring common species, but to genetically detect and identify other extant species that could influence conservation efforts. This method allows for biomonitoring without requiring collection of the living organism, creating the ability to study organisms that are invasive, elusive, or endangered without introducing anthropogenic stress on the organism. Access to this genetic information makes a critical contribution to the understanding of population size, species distribution, and population dynamics for species not well documented. The integrity of eDNA samples is dependent upon its preservation within the environment. Soil, permafrost, freshwater and seawater are well-studied macro environments from which eDNA samples have been extracted, each of which include many more conditioned subenvironments. Because of its versatility, eDNA is applied in many subenvironments such as freshwater sampling, seawater sampling, terrestrial soil sampling (tundra permafrost), aquatic soil sampling (river, lake, pond, and ocean sediment), or other environments where normal sampling procedures can become problematic.

Collection

Subglacial aquatic sediment continuous coring

The cylindrical platform can pass through the access borehole and penetrate the sediment. The lead ropes link between the surface winch and the underwater platform and the cable-suspended corer can repeatedly penetrate the same sediment borehole guided by the lead ropes.

Terrestrial sediments

The importance of eDNA analysis stemmed from the recognition of the limitations presented by culture-based studies. Organisms have adapted to thrive in the specific conditions of their natural environments. Although scientists work to mimic these environments, many microbial organisms can not be removed and cultured in a laboratory setting. The earliest version of this analysis began with ribosomal RNA (rRNA) in microbes to better understand microbes that live in hostile environments. The genetic makeup of some microbes is then only accessible through eDNA analysis. Analytical techniques of eDNA were first applied to terrestrial sediments yielding DNA from both extinct and extant mammals, birds, insects and plants. Samples extracted from these terrestrial sediments are commonly referenced as 'sedimentary ancient DNA' (sedaDNA or dirtDNA). The eDNA analysis can also be used to study current forest communities including everything from birds and mammals to fungi and worms.

Aquatic sediments

The sedaDNA was subsequently used to study ancient animal diversity and verified using known fossil records in aquatic sediments. The aquatic sediments are deprived of oxygen and are thus protect the DNA from degrading. Other than ancient studies, this approach can be used to understand current animal diversity with relatively high sensitivity. While typical water samples can have the DNA degrade relatively quickly, the aquatic sediment samples can have useful DNA two months after the species was present. One problem with aquatic sediments is that it is unknown where the organism deposited the eDNA as it could have moved in the water column.

Aquatic (water column)

Studying eDNA in the water column can indicate the community composition of a body of water. Before eDNA, the main ways to study open water diversity was to use fishing and trapping, which requires resources such as funding and skilled labour, whereas eDNA only needs samples of water. This method is effective as pH of the water does not affect the DNA as much as previously thought, and sensitivity can be increased relatively easily. Sensitivity is how likely the DNA marker will be present in the sampled water, and can be increased simply by taking more samples, having bigger samples, and increasing PCR. eDNA degrades relatively fast in the water column, which is very beneficial in short term conservation studies such as identifying what species are present.

Researchers at the Experimental Lakes Area in Ontario, Canada and McGill University have found that eDNA distribution reflects lake stratification. As seasons and water temperature change, water density also changes such that it forms distinct layers in small boreal lakes in the summer and winter. These layers mix during the spring and fall. Fish habitat use correlates to stratification (e.g. a cold-water fish like lake trout will stay in cold water) and so does eDNA distribution, as these researchers found.

Schematic of a drilling vessel recovering a sediment core for sedaDNA analysis and hypothetical past marine community composition. Schematic not to scale.

Schematic of different methodological approaches in modern and ancient marine genomics. (a) Metabarcoding is the amplification and analysis of equally sized DNA fragments from a total DNA extract. (b) Metagenomics is the extraction, amplification, and analysis of all DNA fragments independent of size. (c) Target-capture describes the enrichment and analysis of specific (chosen) DNA fragments independent of size from a total DNA extract.

In the diagram above on the left, the pink dashed line indicates the use of a chemical tracer for contamination tracking during coring. The white dashed line depicts the sediment core. Small yellow circles indicate theoretical sedaDNA sampling intervals, corresponding to pie charts on the right. Pie charts represent hypothetical paleo-communities detectable from sedaDNA shotgun analysis, where the majority (~75%) of the recovered sedaDNA sequences originate from bacteria, and where sedaDNA from fossilizing/cyst-forming taxa increases relative to non-fossilizing/non-cyst-forming taxa with subseafloor depth (assuming that sedaDNA of fossilizing/cyst-forming taxa preserves better than that of non-fossilizing/non-​cyst-forming taxa). The decreasing size of the pie charts with subseafloor depth indicates an expected decrease in sedaDNA.

Monitoring species

eDNA can be used to monitor species throughout the year and can be very useful in conservation monitoring. eDNA analysis has been successful at identifying many different taxa from aquatic plants, fishes, mussels, fungi and even parasites. eDNA has been used to study species while minimizing any stress inducing human interaction, allowing researchers to monitor species presence at larger spatial scales more efficiently. The most prevalent use in current research is using eDNA to study the locations of species at risk, invasive species, and keystone species across all environments. eDNA is especially useful for studying species with small populations because eDNA is sensitive enough to confirm the presence of a species with relatively little effort to collect data which can often be done with a soil sample or water sample. eDNA relies on the efficiency of genomic sequencing and analysis as well as the survey methods used which continue to become more efficient and cheaper. Some studies have shown that eDNA sampled from stream and inshore environment decayed to undetectable level at within about 48 hours.

Environmental DNA can be applied as a tool to detect low abundance organisms in both active and passive forms. Active eDNA surveys target individual species or groups of taxa for detection by using highly sensitive species-specific quantitative real-time PCR or digital droplet PCR markers. CRISPR-Cas methodology has also been applied to the detection of single species from eDNA; utilising the Cas12a enzyme and allowing greater specificity when detecting sympatric taxa. Passive eDNA surveys employ massively-parallel DNA sequencing to amplify all eDNA molecules in a sample with no a priori target in mind providing blanket DNA evidence of biotic community composition.

Commercial fisheries

The successful management of commercial fisheries relies on standardised surveys to estimate the quantity and distribution of fish stocks. Atlantic cod (Gadus morhua) is an iconic example that demonstrates how poorly constrained data and uninformed decision making can result in catastrophic stock decline and ensuing economic and social problems. Traditional stock assessments of demersal fish species have relied primarily on trawl surveys, which have provided a valuable stream of information to decision makers. However, there are some notable drawbacks of demersal trawl surveys including cost, gear selectivity/catchability, habitat destruction and restricted coverage (e.g. hard-substrate bottom environments, marine protected areas).

Environmental DNA (eDNA) has emerged as a potentially powerful alternative for studying ecosystem dynamics. The constant loss and shedding of genetic material from macrogranisms imparts a molecular footprint in environmental samples that can be analysed to determine either the presence of specific target species  or characterise biodiversity. The combination of next generation sequencing and eDNA sampling has been successfully applied in aquatic systems to document spatial and temporal patterns in the diversity of fish fauna. To further develop the utility of eDNA for fisheries management, understanding the ability of eDNA quantities to reflect fish biomass in the ocean is an important next step.

Positive relationships between eDNA quantities and fish biomass and abundance have been demonstrated in experimental systems. However, known variations between eDNA production  and degradation  rates is anticipated to complicate these relationships in natural systems. Furthermore, in oceanic systems, large habitat volumes and strong currents are likely to result in physical dispersal of DNA fragments away from target organisms. These confounding factors have been previously considered to restrict the application of quantitative eDNA monitoring in oceanic settings.

Despite these potential constraints, numerous studies in marine environments have found positive relationships between eDNA quantities and complimentary survey efforts including radio-tagging25, visual surveys13,26, echo-sounding27 and trawl surveys12,28. However, studies that quantify target eDNA concentrations of commercial fish species with standardised trawl surveys in marine environments are much scarcer28. In this context, direct comparisons of eDNA concentrations with biomass and stock assessment metrics, such as Catch Per Unit Effort (CPUE), are necessary to understand the applicability of eDNA monitoring to contribute to fisheries management efforts.

Terrestrial arthropods

Terrestrial arthropods are experiencing massive decline in Europe as well as globally, although only a fraction of the species have been assessed and the majority of insects are still undescribed to science. As one example, grassland ecosystems are home to diverse taxonomic and functional groups of terrestrial arthropods, such as pollinators, phytophagous insects, and predators, that use nectar and pollen for food sources, and stem and leaf tissue for food and development. These communities harbor endangered species, since many habitats have disappeared or are under significant threat. Therefore, extensive efforts are being conducted in order to restore European grassland ecosystems and conserve biodiversity. For instance, pollinators like bees and butterflies represent an important ecological group that has undergone severe decline in Europe, indicating a dramatic loss of grassland biodiversity. The vast majority of flowering plants are pollinated by insects and other animals both in temperate regions and the tropics. The majority of insect species are herbivores feeding on different parts of plants, and most of these are specialists, relying on one or a few plant species as their main food resource. However, given the gap in knowledge on existing insect species, and the fact that most species are still undescribed, it is clear that for the majority of plant species in the world, there is limited knowledge about the arthropod communities they harbor and interact with.

Terrestrial arthropod communities have traditionally been collected and studied using methods, such as Malaise traps and pitfall traps, which are very effective but somewhat cumbersome and potentially invasive methods. In some instances, these techniques fall short of performing efficient and standardized surveys, due to, for example, phenotypic plasticity, closely related species, and difficulties in identifying juvenile stages. Furthermore, morphological identification depends directly on taxonomic expertise, which is in decline. All such limitations of traditional biodiversity monitoring have created a demand for alternative approaches. Meanwhile, the advance in DNA sequencing technologies continuously provides new means of obtaining biological data. Hence, several new molecular approaches have recently been suggested for obtaining fast and efficient data on arthropod communities and their interactions through non‐invasive genetic techniques. This includes extracting DNA from sources such as bulk samples or insect soups, empty leaf mines, spider webs, pitcher plant fluid, environmental samples like soil and water (environmental DNA [eDNA]), host plant and predatory diet identification from insect DNA extracts, and predator scat from bats. Recently, also DNA from pollen attached to insects has been used for retrieving information on plant–pollinator interactions. Many of such recent studies rely on DNA metabarcoding—high‐throughput sequencing of PCR amplicons using generic primers.

Snow tracks

Wildlife researchers in snowy areas also use snow samples to gather and extract genetic information about species of interest. DNA from snow track samples has been used to confirm the presence of such elusive and rare species as polar bears, arctic fox, lynx, wolverines, and fishers.

See also

• Metagenomics
• DNA barcoding

External links

• BLAST
• Biomeme Guide to eDNA