Environmental DNA, or eDNA, is DNA collected from places like soil, sediment, water, snow, or air, instead of directly from an organism. As organisms interact with their environment, DNA is released and builds up around them. This eDNA can be studied using scientific methods to learn about the species in an ecosystem, even those too small to see. This method helps scientists study biodiversity in a cost-effective, large-scale way without harming ecosystems.

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

Examples of eDNA sources include feces, mucus, eggs, shed skin, dead animals, and hair. Scientists use advanced DNA sequencing methods, such as metagenomics, metabarcoding, and single-species detection, to quickly study biodiversity. To better identify species in a sample, scientists use DNA metabarcoding, which compares the sample to known DNA databases, like BLAST, to determine which organisms are present.

eDNA metabarcoding is a new way to study biodiversity. Scientists collect samples from the environment, such as water, sediment, or air, extract DNA from them, and use universal tools to copy and read the DNA with next-generation sequencing. This process creates thousands to millions of data points, which help scientists identify species and measure overall biodiversity. This method combines traditional field ecology with modern molecular and computer tools.

Studying eDNA helps scientists not only track common species but also find and identify other species that may affect conservation efforts. This method allows scientists to study biodiversity without capturing living organisms, making it easier to study invasive, hidden, or endangered species without causing stress to them. This genetic information helps scientists understand population sizes, where species live, and how their numbers change over time. Importantly, eDNA methods are often less expensive than traditional sampling. The quality of eDNA samples depends on how well they are preserved in the environment.

Soil, permafrost, freshwater, and seawater are well-studied places where eDNA has been collected. These environments include many smaller areas with different conditions. Because eDNA methods are flexible, they can be used in many settings, such as freshwater, seawater, soil, or other places where regular sampling is difficult.

On December 7, 2022, a study in Nature reported finding eDNA that is two million years old in Greenland’s sediments, the oldest DNA sequenced so far.

eDNA methods use many different sampling and analysis steps, and the accuracy of results depends on how strong these methods are. In water environments, the way samples are collected is important. Filtering larger amounts of water usually gives more DNA, making it easier to find organisms at a site. Since eDNA is often spread unevenly, collecting water from multiple areas gives a more complete picture of biodiversity.

Laboratory steps also affect results, especially how many times scientists repeat the DNA copying process, called PCR. Repeating PCR helps find more species because some DNA might not copy correctly in one attempt. For example, repeating PCR 12 times means copying the DNA in 12 separate samples, reducing the chance of missing a species.

The number of DNA sequences analyzed, called sequencing depth, also affects results. More sequences increase the chance of finding rare or low-number species. In freshwater, analyzing hundreds of thousands of sequences (up to 500,000) or millions in marine samples improves detection, especially for 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-spot species, including those that are protected, endangered, or invasive.

Overview

Environmental DNA, or eDNA, is genetic material found in samples like water, soil, and air. This material can come from cells, DNA outside of cells, or even whole organisms. To study eDNA, scientists first collect a sample from the environment. They then extract and clean the DNA from the sample. Next, they copy a specific gene to prepare it for sequencing, which helps identify the species present. This process allows scientists to detect and classify species based on their genetic information.

eDNA can be found in many forms, such as skin, saliva, eggs, feces, and plant parts. It can also come from microorganisms. The amount of eDNA produced depends on factors like the size of the organism, its age, activity, and how it interacts with its environment.

eDNA can help scientists study biodiversity in ways that traditional methods cannot. It can detect rare or hard-to-find species, but it cannot provide details about population health, such as the number of males or females or the condition of individuals. This makes eDNA useful for supporting traditional studies. It is especially helpful for finding invasive species, confirming the presence of species thought to be extinct, or identifying species that are difficult to spot.

However, eDNA cannot always measure the exact number of organisms in an area. This is only possible in specific places, like shallow rivers, where other methods like electrofishing can be used. In most cases, both eDNA and traditional methods provide estimates of species abundance rather than exact numbers. eDNA methods are often better than traditional methods at showing whether a species is present, how many there are compared to others, and how they change over time and space. These details are important for studying biodiversity and tracking ecosystems over time.

eDNA has practical benefits, such as being able to detect rare species, being more consistent across different locations and times, costing less to use, and not harming the organisms being studied. However, eDNA can break down quickly in the environment, especially in warm areas, which limits how long it can be used to study species. DNA often stays in water for only a few days to a week, making eDNA a "snapshot" of which species were present at a specific time and place.

Even with these challenges, eDNA can still help estimate how common a species is compared to others. Some studies show that eDNA levels can relate to the number of organisms, though this is not always precise. While eDNA has many uses in conservation and monitoring, it is important to carefully plan studies to ensure accurate results.

When comparing data from different samples or over time, scientists need to standardize their methods to correct for differences in how DNA is collected and analyzed. This helps ensure results are reliable and can be compared across different studies.

eDNA is sometimes confused with community DNA, which refers to genetic material from whole organisms. It is unclear whether eDNA samples containing whole microorganisms should be classified as community DNA. Similarly, DNA from feces is often called eDNA, but it may not always indicate the presence of the organism at the time of collection. These differences are important for understanding how eDNA data should be interpreted.

The concept of selfDNA comes from research by scientists at the University of Naples Federico II, published in 2015. They found that extracellular DNA from plants and other organisms can stop the growth of similar organisms. This DNA is often found in plant litter and other environments, and its size can affect how it interacts with other organisms. Scientists believe this process, called selfDNA, may influence how species interact in ecosystems and could be used in new scientific applications.

eDNA metabarcoding

Environmental DNA (eDNA) metabarcoding is a scientific method used to study living things in water and land environments. By 2019, researchers could analyze entire communities from one sample using this technique. Metabarcoding works by using special tools called polymerase chain reaction (PCR) primers to examine mixed DNA samples from any source. These samples are then studied with advanced sequencing machines to identify which species are present. This method has been widely used in microbiology for many years but 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 over large areas. However, the method may sometimes give incorrect results due to contamination or other mistakes. Compared to older methods, eDNA metabarcoding is faster, more accurate, and less expensive. It also needs standard rules and consistent methods to combine species classification with molecular techniques for complete ecological studies.

This method can be used to monitor biodiversity in all habitats and for all types of living things. It can also help scientists study ancient ecosystems, track how plants and pollinators interact, analyze animal diets, detect invasive species, study how pollution affects life, and monitor air quality. eDNA metabarcoding is still being improved and may change as technology advances and procedures become more consistent. As the method becomes more reliable and widely used, it is expected to become an important tool for studying ecosystems and protecting the environment globally.

Extracellular and relic DNA

Relic DNA Dynamics

Extracellular DNA, also known as relic DNA, is DNA that 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 as high as 2 micrograms per liter, and in natural water environments, it can be as high as 88 micrograms per liter. Scientists have suggested several possible roles for eDNA: it may help transfer genes between microbes, provide nutrients, or help control the levels of certain chemicals or medicines. In biofilms formed by some bacteria, eDNA acts as a structural part of the biofilm. It may help microbes recognize each other, control how they attach to or separate from the biofilm, support biofilm growth, and increase the biofilm's strength and ability to survive difficult conditions.

Environmental DNA, or eDNA, is increasingly used in scientific research to study ecosystems. It helps scientists track the presence and movement of species in water, air, or on land, and measure the variety of life in an area.

In the diagram, the amount of relic DNA in a microbial environment depends on two factors: the amount added when living microbes with intact DNA die, and the amount lost as relic DNA breaks down. If the genetic diversity in relic DNA is very different from the genetic diversity in DNA from living microbes, relic DNA could affect estimates of microbial biodiversity when scientists study the total DNA from both living and dead microbes. The STARDIT initiative has been proposed as a way to standardize how data is collected, analyzed, and how species are classified and related to each other.

Collection

• Methods in modern and ancient marine genomics
• (a) Metabarcoding is a method that looks at equal-sized DNA pieces from a mix of DNA collected from a sample. (b) Metagenomics is the process of collecting and studying all DNA pieces from a sample, regardless of their size. (c) Target-capture is a technique that focuses on specific DNA pieces chosen for study, regardless of their size, from a mix of DNA collected from a sample.

The importance of eDNA analysis came from the challenges of studying microbes using traditional methods. Many microbes live in environments that are hard to recreate in labs, making it difficult to grow and study them. Early eDNA research used ribosomal RNA (rRNA) from microbes to learn more about species in extreme environments. For some microbes, eDNA is the only way to study their genetic information. Early eDNA methods were first used on soil samples to find 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 forest ecosystems, including birds, mammals, fungi, and worms. Samples can come from soil, animal waste, plant material where animals have fed, and blood meals from mosquitoes that have bitten animals. Some methods use tools like hair traps and sandpaper in areas where target species are likely to move.

sedaDNA was later used to study ancient animal life in water samples, and results were checked against known fossil records. Water sediments often lack oxygen, which helps protect DNA from breaking down. This method is useful for studying both ancient and modern animal life. While DNA in water can break down quickly, useful DNA can remain in water sediments for up to two months after an animal is present. One challenge with water sediments is that it is unclear where the eDNA came from, as it may have moved through the water.

• A drilling ship collecting sediment samples for sedaDNA analysis and a guess about past marine life
• Collecting continuous sediment samples from under glaciers

Studying eDNA in water can show what types of organisms live in a body of water. Before eDNA, scientists used methods like electrofishing, nets, and traps to study open water. eDNA is helpful 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 useful for short-term studies, like identifying species in an area.

Researchers in Canada and at McGill University found that eDNA spreads in lakes based on how water layers form. In small lakes, water layers change with seasons and temperature, creating distinct layers in summer and winter. These layers mix during spring and fall. Fish tend to stay in specific layers (for example, cold-water fish like lake trout live in colder water), and eDNA follows this pattern, as shown by the researchers.

Monitoring species

Environmental DNA (eDNA) is a tool used to track species throughout the year and helps scientists protect wildlife. eDNA analysis has successfully identified many types of organisms, including aquatic plants, fish, mammals, mussels, fungi, and even parasites. This method allows researchers to study species without disturbing them, making it easier to monitor wildlife in large areas. Today, eDNA is most often used to find species at risk, invasive species, and important species in all environments. It is especially helpful for species with small populations because eDNA can detect them with minimal effort, often using a soil or water sample. eDNA depends on DNA sequencing and survey methods, which are becoming faster and more affordable. Some studies show that eDNA collected from streams and coastal areas can become undetectable within about 48 hours.

Environmental DNA can detect organisms that are present in small numbers, both through active and passive methods. Active eDNA surveys focus on specific species or groups by using highly sensitive tests like PCR. CRISPR-Cas technology has also been used to detect single species from eDNA, improving accuracy when identifying similar species. Passive eDNA surveys use advanced DNA sequencing to analyze all DNA in a sample, providing information about the entire community of living things in an area.

• Differences in insect groups based on plant species
• A chart showing which plants each insect 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).

Insects and other land-dwelling arthropods are declining rapidly in Europe and worldwide, though many species remain unstudied. Grassland habitats support many types of arthropods, such as pollinators, plant-eating insects, and predators, which rely on plants for food and shelter. These communities include endangered species because many habitats have been lost or are threatened. Scientists are working to restore grasslands and protect biodiversity. For example, pollinators like bees and butterflies have declined sharply, showing a loss of grassland life. Most flowering plants are pollinated by insects, and many insect species eat specific parts of plants. However, because many insect species are still unknown, scientists know little about the relationships between plants and the arthropods they support.

Traditionally, scientists have used methods like Malaise traps and pitfall traps to study arthropods, but these methods can be time-consuming and may harm the environment. These techniques also struggle with identifying some species due to similarities in appearance or the difficulty of recognizing young stages. Since experts who identify species are becoming fewer, new methods are needed. Advances in DNA sequencing have led to new ways to study arthropods, such as using DNA from soil, water, air, spider webs, and even flowers. These methods include DNA metabarcoding, which uses high-speed sequencing to analyze DNA from many species at once.

• Canada lynx
• Footprints of a Canada lynx in snow

Scientists in snowy regions also use snow samples to collect genetic information about animals. DNA from snow tracks has helped 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 airplanes to study biodiversity in the air, including mammals.

Managing fish populations depends on surveys to estimate how many fish are present and where they live. Atlantic cod is an example of how poor data and bad decisions can lead to fish population crashes and problems for people who rely on fishing. Traditional methods, like trawling, have provided useful information but have issues like high costs, damage to the environment, and limited coverage.

Environmental DNA (eDNA) is a promising alternative for studying ecosystems. Animals constantly shed DNA into the environment, leaving a genetic trace that can be used to find species or study biodiversity. Combining eDNA with advanced DNA sequencing has helped scientists track fish diversity in water. To improve eDNA’s use in managing fisheries, scientists need to understand how eDNA levels relate to the number of fish in the ocean.

Studies have shown that higher eDNA levels often mean more fish, but natural factors like how quickly DNA breaks down may complicate this relationship. In the ocean, strong currents and large areas can spread DNA far from where it was released, making it harder to use eDNA for precise measurements. Despite these challenges, many studies in the ocean have found a link between eDNA levels and fish populations.

Deep sea sediments

• OTU (operational taxonomic unit) network of the extracellular DNA found in sediments from different continental margins.

Extracellular DNA in surface deep-sea sediments is the largest source of DNA in the world’s oceans. The main sources of this DNA include the release of DNA from dead benthic organisms, as well as processes such as cell breakdown caused by viruses, release of materials from living cells, virus decay, and inputs from the water column. Earlier research showed that a significant portion of this DNA can avoid being broken down and remains preserved in sediments. This DNA may act as a genetic record of biological processes that occurred over time.

Recent studies found that DNA preserved in marine sediments contains many highly diverse gene sequences. In particular, extracellular DNA has been used to study past diversity of prokaryotes and eukaryotes in cold or permanently oxygen-free benthic ecosystems.

The diagram on the right shows the OTU network of extracellular DNA from sediments of different continental margins. The size of each dot represents how many times each OTU was found. Dots surrounded by red lines show core OTUs (important OTUs shared by many samples), dots with yellow lines show OTUs shared by two or more samples, and dots with black lines show OTUs found only in one sample. Core OTUs that appear at least 20 times are shown. Numbers in parentheses indicate how many connections exist between OTUs and samples: 1 for OTUs found only in one sample, 2–3 for OTUs shared by two or more samples, and 4 for core OTUs.

Earlier studies suggested that DNA preservation may be more common in benthic systems with high organic matter and sedimentation rates, such as continental margins. These systems, which cover about 15% of the world’s ocean floor, are also rich in prokaryotic diversity and may be ideal places to study DNA preserved in extracellular DNA.

The spatial distribution of prokaryotic diversity has been studied in deep-sea ecosystems using "environmental DNA" (genetic material collected directly from environmental samples without clear biological sources). However, it is not yet known how much extracellular DNA affects estimates of current prokaryotic diversity.

Sedimentary ancient DNA

Studies of ancient DNA found in different archives have changed how scientists understand the evolution of species and ecosystems. Earlier research focused on DNA from bones and frozen tissue, but new methods now allow analysis of DNA from sediment samples, called sedaDNA. Scientists are actively studying how sedaDNA accumulates and is preserved in land and lake sediments. However, studying DNA on the ocean floor and its preservation in marine sediments is more complex because the DNA must travel through water for several kilometers. Unlike on land, where dead plant and animal material is often transported to sediments, most marine sedaDNA comes from plankton groups, which include tiny marine microbes and protists. After plankton die, their DNA travels through the water, where much of the organic matter is broken down by other organisms. This process can take 3 to 12 days, depending on the size and shape of the plankton. Scientists are still unsure how planktonic eDNA, the total DNA present in the environment after death, survives this journey. They are also studying whether DNA degradation or movement is linked to sorting or sideways movement in water, and whether eDNA that reaches the seafloor is preserved without changing its makeup.

Even though eDNA is exposed to breakdown in oxygen-rich water during its journey and the seafloor has less organic matter, evidence shows that planktonic eDNA can be preserved in marine sediments and may contain useful ecological information. Earlier studies found sedaDNA in marine sediments with unusually high organic matter, but newer research shows that sedaDNA can also be found in normal marine sediments made of minerals. The cold temperatures of deep-sea water (0–4 °C) help preserve sedaDNA. Scientists used planktonic foraminifera, tiny marine organisms, as a reference to compare sedaDNA with fossil remains of these organisms. In 2017, researchers showed that eDNA from plankton that reaches the seafloor preserves the ecological signal of these organisms across large areas. This suggests that planktonic eDNA, along with sinking material like aggregates and shells, is deposited on the seafloor. If true, sedaDNA could record changes in ocean conditions that affect plankton communities with the same detail as fossil remains. If eDNA attaches to mineral surfaces, such as those in plankton shells, it might survive the journey through water. This same process may explain how sedaDNA is preserved in sediments, with eDNA inside calcite shells being protected when buried.

Planktonic foraminifera sedaDNA is a useful tool for studying past ocean conditions both across large areas (horizontally) and through layers of sediment (vertically). The amount of foraminifera eDNA should match the number of dead foraminifera shells sinking to the seafloor, allowing scientists to compare eDNA with other data. eDNA is a powerful tool for studying ecosystems because it does not require knowing the exact species in a sample, allowing information about all organisms, even those that are hard to identify. Scientists match eDNA sequences to known species by comparing them to reference sequences stored in public databases. The classification of planktonic foraminifera is well understood, and their genetic markers (barcodes) allow most eDNA to be linked to their physical features. The makeup of planktonic foraminifera groups is closely tied to ocean conditions, and this information is preserved in fossil remains on the seafloor. Since foraminifera eDNA found in ocean sediments can be recovered, it can be used to study changes in plankton and seafloor communities over time.

In 2022, scientists discovered and sequenced genetic material from eDNA that is two million years old in Greenland. This is currently 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 include local communities in scientific research, such as collecting and studying DNA samples. This method allows local groups, including Indigenous peoples, to take an active role in tracking wildlife in their area and to help make decisions through a collaborative research approach. An example of this is the "Wild DNA" project, which was carried out 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.