Environmental DNA, or eDNA, is DNA collected from the environment, such as soil, water, air, or snow, instead of directly from living organisms. As animals and plants interact with their surroundings, they release DNA into the environment. This eDNA can be studied using special techniques to learn about the species present in an area, including tiny organisms that are hard to see. This method helps scientists create detailed records of biodiversity in a cost-effective and non-harmful way.

In recent years, eDNA has been used to find endangered animals that are hard to spot. In 2020, scientists began using eDNA methods to track the spread of the COVID-19 virus.

Examples of eDNA sources include feces, mucus, skin cells, hair, and dead tissue. Scientists use advanced DNA-reading tools, such as metagenomics, metabarcoding, and single-species detection, to quickly study biodiversity. To identify species in a sample, scientists compare the DNA to known databases, like BLAST, to match it with known organisms.

eDNA metabarcoding is a new way to study biodiversity. Scientists collect samples from water, soil, or air, extract the DNA, and use special tools to copy and read the DNA. This process creates thousands or millions of DNA readings, which help identify which species are present and how diverse an area is. This method combines field science with molecular biology and computer analysis.

Studying eDNA helps scientists learn about both common and rare species, which can guide conservation efforts. This method allows scientists to study organisms without harming them, making it easier to track invasive, hidden, or endangered species. eDNA is also cheaper than traditional methods. However, the quality of eDNA depends on how well it is preserved in the environment.

Soil, frozen ground, freshwater, and seawater are common places where eDNA is studied. Each of these areas has many smaller environments where eDNA can be found. Because eDNA is flexible, it is used in many settings, such as rivers, lakes, oceans, and places where regular sampling is difficult.

In December 2022, scientists found DNA that is two million years old in Greenland’s sediments, the oldest DNA ever studied.

eDNA is studied using many methods, and the accuracy of results depends on how well the methods are done. In water environments, collecting larger water samples increases the chance of finding DNA from species in the area. Since eDNA is unevenly spread, collecting water from multiple areas gives a better picture of biodiversity.

In the lab, repeating DNA copying processes, called PCR, improves results. Repeating the process multiple times helps avoid missing species due to random errors. For example, copying DNA 12 times reduces the chance of missing a species.

The number of DNA readings, called sequencing depth, also affects results. More readings increase the chance of finding rare species. In freshwater, reading hundreds of thousands of DNA pieces helps find species present in small numbers.

When scientists use strong sampling, repeated testing, and deep sequencing, eDNA methods are especially good at finding rare, hidden, or hard-to-find species, including those that are protected, endangered, or invasive.

Overview

Environmental DNA, or eDNA, is genetic material found in environmental samples such as water, soil, and air. This material can come from parts of organisms, such as skin, hair, waste, or even tiny pieces of dead organisms. To use eDNA, scientists first collect a sample from the environment. Then, they extract and clean the DNA from the sample. Next, they copy a specific part of the DNA so it can be read and compared to known genetic information. This process helps scientists identify which species are present in the sample.

eDNA can come from many sources, including skin, saliva, eggs, waste, and parts of plants and animals. The amount of eDNA produced depends on factors like the size of the organism, its activity, and how it interacts with its environment. While eDNA can help find species that are rare or hard to spot, it cannot provide details about the health or numbers of those species, such as how many males or females are present. However, eDNA is useful for finding invasive species, confirming the presence of species thought to be extinct, or detecting species that are hard to find using traditional methods.

Measuring the exact number of individuals in a species using eDNA is only possible in specific situations, such as in shallow rivers where other methods like electrofishing can be used. In most cases, both eDNA and traditional methods give estimates rather than exact numbers. eDNA methods often work better than traditional methods for showing whether a species is present or absent, how many are in an area, and how their numbers change over time. These data are important for studying biodiversity and tracking changes in ecosystems. eDNA also has practical benefits, such as being less harmful to animals, more cost-effective, and more reliable for finding rare or hidden species.

eDNA can break down quickly in the environment, especially in warm areas, which means only small pieces of genetic material may remain. This can make it hard to track how species move or change over time. Studies show that eDNA usually stays in water for only a few days to a week. Because of this, eDNA acts like a "snapshot" of which species were present at a specific time and place.

Even with these challenges, eDNA can help estimate how many individuals of a species are present, as some studies show it relates to the number of organisms. However, environmental factors make it hard to get exact numbers. eDNA is useful for conservation, monitoring ecosystems, and other research, but careful planning is needed to ensure samples are collected correctly for each study.

When comparing results from different samples or over time, it is important to standardize data so that differences in methods do not affect the results. This involves adjusting for how well DNA from different species is copied and read. Standardized data help scientists compare results from different places or times more accurately.

While eDNA seems simple to define, it can be hard to tell the difference between eDNA and other types of genetic material, such as community DNA, which comes from whole organisms. For example, DNA from feces is often called eDNA, but it may not always be clear whether it came from the same place or time as the organism it belongs to. This distinction is important because community DNA shows that an organism was present at a specific time and place, while eDNA may have come from a different location or even from the past.

The idea of selfDNA comes from research by scientists at the University of Naples Federico II, published in 2015. They found that DNA outside of cells in plants and other organisms can stop similar organisms from growing. This DNA, called selfDNA, is thought to come from dead plant material and other sources in different environments. Scientists have found that DNA pieces between 200 and 500 base pairs long can stop similar organisms from growing. This process may influence how species interact in ecosystems and could be used in new ways to control harmful organisms.

eDNA metabarcoding

By 2019, scientists had improved eDNA research methods to study entire communities from a single sample. This process, called metabarcoding, uses special tools called PCR primers to analyze mixed DNA samples from any source. After this step, scientists use advanced sequencing technology to identify which species are present in the sample. While this method has been widely used in microbiology for many years, it is now being applied to study larger organisms, such as plants and animals. Ecosystem-wide use of eDNA metabarcoding can help scientists understand community structures, biodiversity, and how species interact across large areas. However, this method may sometimes give incorrect results because of contamination or other errors. Overall, eDNA metabarcoding is faster, more accurate, and less expensive than traditional methods. It also improves species identification, but scientists need to develop standardized rules that combine taxonomy and molecular techniques for complete ecological studies.

eDNA metabarcoding can be used to monitor species diversity in all types of environments and for all types of organisms. It can also help scientists study ancient ecosystems, track plant-pollinator relationships, analyze animal diets, detect invasive species, study how pollution affects ecosystems, and monitor air quality. This method is still being developed and may change as technology improves and procedures become more consistent. As scientists refine the process and use it more widely, eDNA metabarcoding is expected to become a key tool for studying ecosystems and protecting the environment globally.

Extracellular and relic DNA

Relic DNA Dynamics

Extracellular DNA, also known as relic DNA, comes from dead microbes. Naked extracellular DNA (eDNA), mostly released when cells die, is found almost everywhere in the environment. In soil, its concentration can reach up to 2 micrograms per liter, and in natural water environments, it can reach as high as 88 micrograms per liter. Scientists have suggested several possible roles for eDNA: it may help transfer genes between organisms, provide nutrients, or help manage ions or antibiotics in the environment. In biofilms formed by some bacteria, eDNA acts as a key part of the structure that holds the biofilm together. It may help cells recognize each other, support biofilm growth, and increase the biofilm's strength and ability to withstand stress.

Environmental DNA, or eDNA, is increasingly used in scientific studies to track species in water, air, and land, and to measure biodiversity in an area.

In the diagram, the amount of relic DNA in a microbial environment depends on two factors: the release of DNA from living organisms that die, and the breakdown of relic DNA over time. If the variety of DNA sequences in relic DNA is very different from the DNA of living organisms, relic DNA might affect how scientists estimate the diversity of microbes in a sample (as shown by the colored boxes). To address this, a method called Standardised Data on Initiatives (STARDIT) has been proposed to help standardize data collection, analysis methods, and how different types of organisms are classified.

Collection

• Methods in modern and ancient marine genomics
• (a) Metabarcoding is the process of making many copies of DNA pieces of the same size from a total DNA sample and then studying them. (b) Metagenomics is the process of taking all DNA pieces from a sample, making many copies of them, and studying them without focusing on size. (c) Target-capture is the process of selecting and studying specific DNA pieces from a total DNA sample, regardless of their size.

The importance of eDNA analysis came from the challenges of studying organisms using methods that require growing them in a lab. Many organisms live in environments that are hard to copy in a lab, and some cannot be grown at all. Early eDNA studies used ribosomal RNA (rRNA) from microbes to learn about those living in harsh environments. For some microbes, their genetic information can only be studied using eDNA. Early eDNA techniques were first used on soil samples from Earth, where scientists found DNA from both ancient and living animals, plants, and insects. These soil samples are often called "sedimentary ancient DNA" (sedaDNA or dirt DNA). eDNA can also be used to study modern forests, including birds, mammals, fungi, and worms. Samples can be collected from soil, animal droppings, leaves bitten by animals, plants touched by animals, and blood meals from captured mosquitoes. Some methods use tools like hair traps and sandpaper in areas where target animals are likely to pass.

SedaDNA has been used to study ancient animal diversity and confirmed using known fossil records in underwater sediments. These underwater sediments lack oxygen, which helps protect DNA from breaking down. This method can also study modern animal diversity with high accuracy. While DNA in water samples often breaks down quickly, useful DNA can remain in underwater sediments for up to two months after an animal is present. A challenge with underwater sediments is that it is unclear where the eDNA came from, as it may have moved in the water.

• A drilling ship collecting sediment samples for sedaDNA analysis and a guess about what marine life lived there in the past
• Collecting continuous sediment samples from underwater areas covered by ice

Studying eDNA in water can show what animals live in a body of water. Before eDNA, scientists mainly used tools like electrofishing, nets, and traps to study life in open water. This method works well because water pH has less effect on DNA than previously thought, and sensitivity can be improved by taking more samples, using larger samples, or increasing PCR. eDNA breaks down quickly in water, which is helpful for short-term studies, such as identifying species in an area.

Researchers in Canada and at McGill University found that eDNA spreads in ways that match how lakes form layers of water. During summer and winter, small lakes in cold regions form layers based on temperature and water density. These layers mix during spring and fall. Fish often stay in specific layers (for example, cold-water fish like lake trout stay in cold water), and eDNA follows the same pattern, as these researchers discovered.

Monitoring species

Environmental DNA (eDNA) can help track species all year long and is useful for protecting wildlife. Scientists have used eDNA to identify many types of organisms, including aquatic plants, mammals, fish, mussels, fungi, and even parasites. eDNA allows researchers to study species without disturbing them, making it easier to monitor wildlife in large areas. Today, eDNA is often used to find species that are at risk, invasive species, and important species in ecosystems. It is especially helpful for species with small populations because it can detect them with little effort, often using soil or water samples. eDNA depends on DNA analysis techniques and survey methods that are becoming faster and less expensive. Some studies show that eDNA collected from streams and coastal areas can become undetectable within about 48 hours.

Environmental DNA can be used to find organisms that are not common, using both active and passive methods. Active eDNA surveys look for specific species or groups by using highly sensitive DNA tests. Scientists have also used CRISPR-Cas methods, such as the Cas12a enzyme, to detect single species more accurately. Passive eDNA surveys use DNA sequencing to analyze all DNA in a sample without focusing on specific targets, showing the full range of life in an area.

• How arthropod communities differ based on plant species
• A chart showing which plant species each arthropod family is found on. Plant names: Angeli (Angelica archangelica), Centau (Centaurea jacea), Daucus (Daucus carota), Echium (Echium vulgare), Eupato (Eupatorium cannabinum), Solida (Solidago canadensis), Tanace (Tanacetum vulgare).

Many land-based arthropods, such as insects, are declining in Europe and around the world. However, only a few species have been studied, and many insects are still unknown to science. Grassland areas are home to many types of arthropods, including pollinators, insects that eat plants, and predators. These groups rely on nectar, pollen, and plant parts for food and survival. Their habitats are disappearing or being threatened, so efforts are being made to restore grasslands and protect biodiversity. For example, pollinators like bees and butterflies have declined greatly in Europe, showing a loss of grassland diversity. Most flowering plants depend on insects and other animals for pollination. Many insects eat plants, and most are specialists that rely on one or a few plant species for food. However, because many species are still unknown, scientists know little about the arthropods that live on most plant species.

Traditionally, scientists have studied arthropods using methods like Malaise traps and pitfall traps, which are effective but can be difficult to use and may harm the environment. These methods sometimes fail to provide consistent results because of challenges like similar-looking species and the difficulty of identifying young stages. Also, identifying species based on physical traits requires specialized knowledge, which is becoming less common. These issues have led to the need for new methods. Advances in DNA sequencing have provided new ways to study arthropods and their interactions without harming them. Scientists now use DNA from sources like insect mixtures, leaf mines, spider webs, pitcher plants, soil, water, air, and flowers (eDNA). They also use DNA from plants attached to insects to study pollination. Many studies use DNA metabarcoding, a method that uses high-speed DNA sequencing to analyze many species at once.

• Canada lynx
• Tracks of a Canada lynx in snow

In snowy areas, scientists use snow samples to collect genetic information about wildlife. DNA from snow tracks has been used to confirm the presence of rare species like polar bears, arctic foxes, lynxes, wolverines, and fishers.

In 2021, researchers showed that eDNA can be collected from air to identify mammals. In 2023, scientists created a special tool and used aircraft to study biodiversity in the air, including mammals.

Managing commercial fisheries depends on surveys that estimate fish numbers and locations. Atlantic cod is an example of how poor data and bad decisions can lead to fish population crashes and economic problems. Traditional methods for studying fish rely on trawling, which has provided useful data but has drawbacks like high costs, damage to habitats, and limited coverage.

Environmental DNA (eDNA) is a promising alternative for studying ecosystems. Organisms constantly release genetic material, which can be found in environmental samples and used to identify species or study biodiversity. Combining DNA analysis with next-generation sequencing has helped scientists study fish diversity in water. To use eDNA for managing fisheries, scientists need to understand how eDNA levels relate to fish numbers in the ocean.

Studies have shown that eDNA levels often match fish numbers in controlled experiments. However, in natural environments, differences in how eDNA is produced and broken down may affect these relationships. In the ocean, large areas and strong currents may spread DNA away from fish, making it harder to track. These challenges have limited the use of eDNA in the ocean.

Despite these issues, studies in the ocean have found that eDNA levels often match fish numbers, showing that eDNA can still be useful for monitoring marine life.

Deep sea sediments

Extracellular DNA in surface deep-sea sediments is the largest source of DNA in the world’s oceans. This DNA comes mainly from the release of genetic material from dead benthic organisms, as well as from processes like cell breakdown caused by viruses, the release of substances from living cells, the breakdown of viruses, and material carried from the water column. Earlier research showed that a significant amount of extracellular DNA can avoid being broken down and remains stored in sediments. This DNA may act as a record of biological activity over time.

Recent studies found that DNA preserved in marine sediments contains many different gene sequences. This DNA has been used to study past diversity among prokaryotes (single-celled organisms) and eukaryotes (organisms with complex cells) in cold or permanently oxygen-free environments.

The diagram on the right shows an OTU (operational taxonomic unit) network of extracellular DNA from sediments of different continental margins. The size of each dot in the network reflects how many times the genetic sequences for each OTU appear. Red dots represent OTUs that are common to all samples, yellow dots show OTUs shared between two or more samples, and black dots are OTUs unique to one sample. Only OTUs appearing at least 20 times are shown. Numbers in parentheses indicate how many connections each OTU has: 1 for unique OTUs, 2–3 for shared OTUs, and 4 for common OTUs.

Earlier research suggested that DNA preservation may be more likely in benthic systems with high organic matter and sedimentation rates, such as continental margins. These areas, which cover about 15% of the global seafloor, are also rich in prokaryotic diversity and may be ideal for studying DNA preserved in extracellular samples.

The spatial distribution of prokaryotic diversity has been studied in deep-sea ecosystems using environmental DNA, which is genetic material collected directly from environmental samples without obvious biological sources. However, it is unclear how much extracellular DNA influences estimates of current prokaryotic diversity.

Sedimentary ancient DNA

Studies of ancient DNA found in different places have changed how scientists understand how species and ecosystems have changed over time. Earlier research focused on DNA from bones or frozen tissue, but new technology now allows scientists to study DNA from sediment layers, called sedaDNA. Scientists are actively researching how sedaDNA is collected and stored in land and lake sediments. However, studying DNA on the ocean floor is more complex because the DNA must travel through water for several kilometers. Unlike on land, where dead plant and animal material is often carried to sediment, most marine sedaDNA comes from plankton, which includes tiny marine microbes and protists. After these plankton die, their DNA travels through the water column, where much of the organic material is broken down and used by other organisms. This process can take between 3 and 12 days, depending on the size and shape of the plankton. Scientists are still unsure how plankton DNA survives this journey, whether it is sorted or carried sideways, or if the DNA that reaches the seafloor remains unchanged.

Even though DNA is exposed to breakdown in oxygen-rich water and the ocean floor has less organic material, evidence shows that plankton DNA can be preserved in marine sediments and may contain useful ecological information. Earlier studies found sedaDNA in sediments with very low oxygen and high organic material, but later research shows that sedaDNA can also be found in normal marine sediments made of minerals. The cold temperature of deep-sea water (0–4 °C) helps preserve sedaDNA. Scientists used planktonic foraminifera, tiny marine organisms, as a reference to compare modern DNA with fossil remains. In 2017, Morard and others showed that DNA from plankton that reaches the seafloor can record the ecological patterns of these organisms across large areas. This suggests that plankton DNA mixes with sinking material like shells and other plankton remains on the seafloor. If true, sedaDNA could reveal details about ocean conditions that affect plankton communities, similar to how fossil remains show these patterns. If plankton DNA sticks to minerals or particles during its journey, it might survive better, a process that also helps preserve DNA in sediments.

Planktonic foraminifera sedaDNA is a useful tool for studying past ocean conditions. It can show how ocean features changed across different locations and how these changes are recorded in sediment layers over time. The amount of planktonic foraminifera DNA should match the number of their fossil shells sinking to the seafloor, allowing scientists to compare DNA data with physical evidence. DNA is a powerful tool for studying ecosystems because it can identify all organisms in a sample, even those that are hard to see. Scientists match DNA sequences to known species using public databases. The classification of planktonic foraminifera is well understood, and their DNA can be matched to their physical remains. The types of planktonic foraminifera in the ocean are closely linked to ocean conditions, and this information is preserved in their fossil remains. Since foraminifera DNA found in ocean sediments can be studied, scientists can track changes in plankton and bottom-dwelling communities over time.

In 2022, scientists discovered and read DNA that is about 2 million years old in Greenland, which is the oldest DNA found so far.

Participatory research and citizen science

The ease of using eDNA sampling makes it a good choice for projects that want to include local communities in scientific research, such as collecting and studying DNA samples. This method allows local communities, including Indigenous peoples, to take an active role in checking which species live in an area. It also helps them make better choices through a type of research called participatory action. An example of this is the "Wild DNA" project by the charity Science for All.

Further references

• Schallenberg, Lena; Wood, Susie A.; Pochon, Xavier; Pearman, John K. (2020). "What Can DNA in the Environment Tell Us About an Ecosystem?" Frontiers for Young Minds. 7 150. doi: 10.3389/frym.2019.00150. hdl: 2292/50690. S2CID 210714520.