On a warm, sunny day in April, biologists David Duffy and Jessica Farrell prepare to motor down the Matanzas River on a small boat to catalog the area’s aquatic life. Ripples signal the river’s lazy flow along Florida’s northeastern coast. Birds fly overhead, some settling onto mangroves occupying the river’s edge. The muddy-brown waters obscure most signs of any life in the river.
But then a pod of bottlenose dolphins appears. The sleek critters break through the water to take a breath as they swim against the current. The team will cross paths with many more of these cetaceans as the boat traverses the river and connecting waterways. A few dolphins toss a fish in the air, seemingly playing catch with their food. Others pass alongside the research vessel as underwater shadows.
Dolphins are hardly the river’s only inhabitants. It’s just that some species are harder to spot.
Sea turtles poke their heads above the water in occasional blink-and-you’ll-miss-it events. Diving manatees produce swirls on the surface that only a trained eye knows to look for.
But even the rarest, hardest-to-spot fauna — and flora — leave behind molecular traces of their presence. And that’s what Farrell and Duffy are searching for: DNA.
All living beings constantly shed bits of DNA, left behind from skin, scales, hair, urine, feces, pollen and more. This environmental DNA, or eDNA, has “changed everything” about how scientists study biodiversity and conservation, says molecular ecologist Elizabeth Clare of York University in Toronto.
Traditionally, conservation research and monitoring have required a physical presence, perhaps a person who keeps watch over monkeys, or a motion-sensitive camera that documents a passing mountain lion, or a light trap that collects moths. “These are excellent confirmations that something was there,” Clare says. But “if the animal walks behind your camera trap, you miss it. No record.”
That’s not a problem with eDNA. “eDNA is more like a footprint, and footprints last longer than the animal or the plant,” Clare says. “The marvelous thing about it is it widens your time window of detection.… It’s like having eyes on the back of your head.”
eDNA is particularly useful for biodiversity surveys, sleuthing out even elusive species that rarely cross paths with humans (SN: 10/28/24). Samples taken from remote areas and brought to the lab for analysis can help researchers track the spread of invasive species, say, or identify species thought to be extinct (SN: 3/20/24). But even as conservationists discover new ways to put eDNA to use or new places to look for it, they must overcome challenges in how to interpret the genetic material they find.
Getting a handle on uncertainties is worth it, says Melania Cristescu, an evolutionary biologist and ecologist at McGill University in Montreal. “These [genetic-monitoring] tools are going to make biodiversity programs possible at the global level, so that we have a way of monitoring biodiversity the way we monitor our weather, with consistency.”
Who’s there?
Farrell and Duffy, who both work at the University of Florida’s Whitney Laboratory for Marine Bioscience in St. Augustine, reach their first stop of the day, Dolphin Creek, an offshoot of the Matanzas.
Into the waist-high water, Farrell slips a long, metal pole with a 1-liter plastic bottle on the end and fills it with water. She pulls out the bottle and caps it with gloved hands — a precaution to avoid contaminating the sample — and plops it into a white cooler for transport. Back at the lab, the team will filter out any DNA for analysis and search genetic databases for potential matches to ID the species.
Duffy, meanwhile, is testing a method that could eliminate the need to lug around heavy bottles of water. On the boat’s deck, he sets up a portable pump and attaches it to a tube that pulls water through a pinkie-sized filter encased in plastic and submerged in the creek. After about five minutes, the once-white filter is now tinged light brown and hopefully loaded with DNA.
The idea of trawling water for genetic material goes back to the mid-1980s, when researchers used DNA to detect bacteria inhabiting marine sediments. Then in the early 2000s, scientists discovered that some sediments could preserve DNA from extinct animals. Woolly mammoth DNA was found deep in Siberian permafrost, and sediment from a cave in Arizona contained genetic material from an ancient giant ground sloth. These discoveries showed that DNA could persist for thousands of years.
Modern signs of life were present, too. The researchers working in Siberia, led by geneticist Eske Willerslev of the University of Copenhagen, pulled DNA belonging to present-day flowering plants and mosses from surface soil.
Clare credits Willerslev for inspiring conservationists to use such molecular traces to monitor biodiversity. His work with frozen sediments “really started the field,” she says, “and then it quickly went to the water.” In 2008, researchers in France used eDNA to detect invasive American bullfrogs (Lithobates catesbeianus) in natural ponds. Then in 2011, another team pointed to eDNA to suggest that two invasive Asian carp species were swimming in waters connected to Lake Michigan.
Back in Dolphin Creek, it’s not hard to guess one animal that will appear in Farrell and Duffy’s samples. “We do get dolphin DNA,” Duffy confirms.
With so many dolphins swimming by, some of the eDNA is guaranteed to be recent. But the Matanzas sampling illustrates one of the difficulties in interpreting eDNA. Unlike a time-stamped photo from a camera trap, it’s hard to know exactly when an animal left behind its genetic calling card.
Studies show that eDNA can persist for hours to weeks in the water column, says ecologist Kristy Deiner of ETH Zurich. After that, “you’ll find it sometimes and not other times.” Inconsistent detections in samples collected on the same day or over a few days provide a hint that an animal is long gone, or that the DNA traveled there from somewhere else.
How long eDNA sticks around in water depends on several factors. For instance, eDNA decays faster as temperatures increase above 20° Celsius, Deiner and colleagues reported in a 2022 meta-analysis. How DNA exists in the environment could also influence its staying power.
“We know DNA exists inside a nucleus, inside of a cell, inside of a big multicellular organism,” Deiner says. “But what are we actually detecting when we take a water sample? We don’t know if it’s the cell or coming from an organelle [a structure within a cell] or dissolved DNA floating around.” It’s unclear how these different states of eDNA might affect whether the molecule is detected or how it moves through a water system.
Also unknown is how water chemistry impacts eDNA degradation, or how the water it’s flowing in affects it. eDNA from a fast, glacier-fed river might behave differently than eDNA from the slowmoving Amazon River. “We haven’t done enough studies around the world to know that it’s context dependent or if there are universal equations that would predict [eDNA’s] behavior,” Deiner says.
But eDNA’s movement through the water might bring benefits. For instance, river water often ends up in lakes, which might act as a “biodiversity accumulator,” Deiner says. A single lake could be a repository of all the life in an entire watershed.
To test that idea, earlier this year in May, Deiner and colleagues asked citizen scientists globally to sample water from about 400 lakes, including in Africa and Southeast Asia, locations that are underrepresented in eDNA studies. By analyzing hundreds of samples, the team hopes to capture what organisms inhabit connecting waters, as well as the terrestrial species that live in the surrounding area.
Such a massive analysis of biodiversity wouldn’t be possible without eDNA, Deiner says. “It just allows you to think much bigger and much larger.”
Where’d you go?
In the moments before sunrise, the Florida sky is on fire. Red, orange and yellow hues blend into light blue as waves crash onto a sandy beach. Few people are out this early, but the steady growl of two utility task vehicles breaks the calm.
It’s the second day of the sea turtle patrol season, when volunteers come together at Mickler’s Landing, a beach north of St. Augustine, to comb for sea turtle nests. One task on this chilly April morning is to collect sand from nests to harvest DNA that turtles leave behind.
Unfortunately, it’s early in the season and there are no nests. But volunteer Lucas Meers explains the sampling process if a nest were present. He would kneel down, pull out a tube about the size of a toilet paper tube from his backpack and take up short, delicate scoops of sand. If turtle tracks were present, Meers says, he would also scoop samples from where the turtle’s flippers touched sand and where the turtle’s body scraped across the beach (and probably left behind secretions from laying eggs). Far away from the tracks, Meers would collect a control sample that should test negative for sea turtle DNA.
As with the Matanzas River samples, eDNA pulled out of the sand helps Duffy, Farrell and colleagues identify which turtle species are present, such as green turtles (Chelonia mydas), loggerheads (Caretta caretta) or leatherbacks (Dermochelys coriacea). The team also checks for the presence of a tumor-causing virus that’s infecting turtles around the globe, providing a window into sea turtle health.
Genetic analyses can also go beyond which species are present, Duffy says, and reveal where animals go.
By identifying the genetic fingerprint of a population and looking for that signature in eDNA, researchers may be able to pinpoint where groups of animals travel by gathering samples in multiple places. As a proof of concept, loggerhead turtle DNA found at Mickler’s Landing and other Florida beaches belonged to animals known to lay their nests in the southeastern United States, Duffy, Farrell and colleagues reported in 2022 in Molecular Ecology Resources. Using DNA to follow paths of travel could be helpful in making decisions about which habitats to protect.
“If you’re trying to conserve [a species] based on just protecting one portion of the habitat they need during life, that’s going to have limited success,” Duffy says. “If you can start to understand where those animals are coming from, then you get a much better idea of their range and what needs to be protected.”
Singling out the genetic signal of a single individual in a population might even be possible, allowing researchers to forgo tagging endangered species. In a 2023 study in eLife, for instance, scientists could discern some of the members of a highly studied kākāpō parrot population in New Zealand that left behind traces of DNA in soil samples. But it’s currently difficult to parse out specific individuals from eDNA unless their genetic material dominates the sample, giving researchers more material to work with to make a match. Most samples are a jumble of many individuals.
But an example from humans further demonstrates the possibility of tracking individuals. Duffy and colleagues have inadvertently collected snippets of human DNA in the environment that are intact enough to reveal genetic ancestry and even disease susceptibility. With targeted analyses, the collected material could be enough to identify people, the team reported in 2023 in Nature Ecology & Evolution.
While the possibility raises ethical concerns about privacy, conservationists typically take active steps to avoid analyzing human DNA from field samples in favor of other species.
Still, “if you translate [the human findings] in the future out to other species,” Duffy says, “that’s potentially a game changer in terms of the amount of information you can obtain.”
What’s in the air?
Like the innumerable chunks of DNA that float in water or settle in sand, researchers are discovering that with a vacuum in hand they can suck up eDNA from a new frontier: the air.
Wind gusts haul tree pollen and fungal spores the way people get swept up in a crowd. Fur and skin cells from animals can catch rides in dust. This immense repository of DNA is all around us, just as water surrounds aquatic creatures. Analyzing airborne eDNA faces many of the same interpretation challenges that water and soil samples do, and for now, just a handful of labs worldwide, including Clare’s at York University, are harvesting airborne eDNA to survey life’s gamut.
In the past, researchers focused on picking up DNA from microbes, spores and pollen — obvious targets in the air. On the animal side, Clare found very little, including a report from two Japanese high school students who picked up starling and owl DNA from the air for a science project (SNE: 5/16/19).
Then in 2022, Clare’s team and a separate group independently reported experiments pulling animal DNA out of thin air at zoos — confirmation that a whiff of air could reveal the area’s animal roster (SN: 1/18/22). Thanks to those demonstrations, interest in airborne eDNA is expanding. The abundance of such DNA, Duffy says, “opens up whole new ways of measuring biodiversity.”
That abundance helps scientists easily collect hundreds of air samples from the same area over and over again, says Clare, who studies both eDNA and bat ecology. “Me alone, I can sit there and watch one cave. But I can put 100 [air] samplers in 100 caves and do it every night for a week and suddenly understand neotropical roosting ecology.”
Scientists might even be able to collect genetic samples from existing infrastructure. Air pollution–monitoring stations, for instance, can be hidden warehouses for eDNA, Clare and colleagues reported in 2023 in Current Biology. Facilities worldwide collect daily or weekly samples to keep tabs on pollution. Some facilities store the samples for decades, meaning such stations could help researchers track biodiversity at a larger scale than ever before.
“Now we found a method that fits the surveillance system we already have,” Clare says.
But collecting genetic material from the air comes with uncertainties. Whether airborne DNA detects only local species or picks up ones that are many kilometers away is unclear. Also unknown is how long the signal lasts and what the overall abundance of DNA from one species in a sample means for how plentiful it is in nature.
The amount of eDNA in a sample of air — or soil or water — is roughly correlated with how many of that plant or animal are in the area, Clare says. “But there’s so much that can go wrong with that, that [abundance estimates are] difficult to use in any real way.”
For one, some organisms might shed more DNA than others, with furry animals perhaps popping up at higher rates than scaly critters by virtue of the “fluffiness factor.” And researchers themselves sometimes influence results without even knowing it.
While studying bats in Belize, Clare and her team harvested airborne eDNA from a classroom where they had brought bats into the room in cloth bags. The aim was to figure out how well vacuumed DNA identified different bat species and if it could pick up how many there were. While much of the genetic material from the room’s air matched the number of each kind of bat present, Clare says, “there were wild exceptions.”
Some bats were overrepresented and others underrepresented. “It took us ages to work out why,” she says. “And apparently I was quoting Taylor Swift every time I say this: The problem was me.”
Clare had to identify each bat. Some species are hard to classify, and because she spent time closely examining these bats’ bodies and teeth, they shed lots of DNA into the air. Vampire bats, on the other hand, made a distinct sound when someone touched the bag, so Clare didn’t need to open it to know what was inside. That may be why very little vampire bat DNA turned up in the experiment, Clare’s team reported in 2023 in Environmental DNA.
Still, airborne eDNA offers similar opportunities to monitor terrestrial species the way streams and oceans ferry DNA from water-living creatures. Depending on how far eDNA travels in the air, researchers could ask questions that encompass large swaths of land, especially compared with soil samples from many spots over a vast landscape. For example, “if we are installing solar panel farms in New Mexico,” Duffy asks, “what is the effect of that on the local biodiversity?”
What’s here right now?
Repeat collections give researchers more confidence in how to interpret eDNA findings. But new methods based on DNA’s molecular cousin, RNA, may help pinpoint which life-forms were most recently in a place.
RNA turns DNA’s genetic instructions into proteins. Compared with DNA, RNA offers a clearer sign that an animal was recently in an area because RNA breaks down much sooner. That fragility initially prompted scientists to assume that it would degrade too quickly for sampling, but emerging research shows there is a detection window. After an organism releases RNA into the environment, most of the RNA molecules are gone within three to five hours, Cristescu says, though studies suggest that eRNA may be detectable for up to 72 hours. Any eRNA found in a sample therefore implies that the organism that left it behind was in the area within the last few days.
The molecule offers other advantages, too. “What’s beautiful about RNA,” Cristescu says, “is that you can get a lot of ecological information that you cannot get with DNA.”
RNAs churned by individuals vary by factors like sex or environmental stress. By collecting eRNA, researchers can also largely distinguish between living and dead organisms, as well as adults and juveniles.
In a study of American bullfrog ponds in Idaho, water samples contained RNA that revealed when tadpoles were present, researchers reported in the May Molecular Ecology Resources.
Additional tests looking for long-toed salamander (Ambystoma macrodactylum) larvae came up negative in ponds when only adults were around. Adults lay their eggs in the spring and leave, so adults and offspring aren’t in the ponds at the same time. Just three months later, after the eggs had hatched, the team detected larva-specific RNA.
For now, samples must make it all the way back to the lab from the field, sometimes taking weeks, and undergo hours of processing before researchers can get a glimpse of the species detected in them. But biologist Ravi Nagarajan of the University of California, Davis says that eRNA might one day be analyzed in the field.
Such technology is already in development for eDNA. Using the molecular scissors CRISPR, scientists hope to design field tests that take less than an hour to detect DNA for a single or multiple species in water samples. This real-time sampling technique, dubbed SHERLOCK, could uncover preliminary signals for species of interest, pinpointing which sites require more thorough sampling, says Nagarajan, who has used SHERLOCK to scan an estuary in California for endangered fish. It could also help allocate a lab’s limited resources to spots with the most potential to answer questions.
SHERLOCK could be adapted to work for eRNA, Nagarajan says. By harvesting RNAs that become active in certain conditions from the environment, SHERLOCK could provide an immediate hint that an ecosystem is stressed. Such tests could also indicate the threat, whether organisms are imperiled because of chemical contaminants in the water or a massive heat wave, Nagarajan says. “There’s huge potential there.”
As species dwindle or go extinct amid stressors like climate change and other human influences, it has become all the more important to have a global approach to cataloging what’s out there, Cristescu says. “Not having a good understanding of biodiversity puts us in a very bad position of understanding what we are losing.”