Organisms constantly shed hair, saliva, scales, and other material, leaving detectable traces of their DNA in the environment they interact with. Conservation biologists are harnessing the power of this “environmental DNA (eDNA)” to determine whether or not species are present in a given ecosystem. Scientists collect samples from the environment rather than the organism (soil, water from streams, pollen swabs from flowers, and fecal matter, for example), isolate and sequence DNA from the samples, and compare the resulting DNA sequences to that of the species they’re trying to detect.

The Collaborative Ecological Genetics Lab (CEGL), led by Mark A. Davis, and the Urban Biotic Assessment Program (UBAP), led by Michael Dreslik, use eDNA for a variety of research and survey efforts, including documenting the presence of threatened and endangered species, mapping the sprawl of invasive species, monitoring the spread of harmful diseases, and measuring the biodiversity within an ecosystem.

Imperiled species

Bats

INHS scientists have extracted DNA from guano (bat feces) in order to determine what bats are eating, particularly what insect pests (like mosquitoes) bats are eating. They have also extracted DNA from what the mosquitoes feasted on before they were eaten by the bats. 

Read more about INHS mammal research.

Birds

Researchers at INHS are using eDNA to study the Illinois state endangered greater prairie chicken, state endangered American bittern, and state threatened least bittern.

The greater prairie chicken once dominated the American Midwest, but today the birds are suffering from a lack of habitat and declining genetic diversity. Between 1992 and 1998, teams imported more than 200 prairie chickens from other states to Illinois. To understand how well the birds were doing long after that first translocation, INHS researchers analyzed the DNA from feathers collected in the birds’ courtship grounds from 2010 to 2013. The study confirmed that the only two remaining populations of prairie chickens in Illinois—one in Marion County and the other in Jasper County—are genetically isolated from one another. Researchers conclude that a lack of habitat endangers prairie chickens’ long-term survival in Illinois, and without periodic human intervention—in the form of translocations of birds from other states—the population could die out.

INHS researchers plan to test whether soil or water samples contain more bird eDNA. Additionally, whether rail, heron, and bittern occupancy increases farther from urban centers and with an increased proportion of emergent wetlands is being investigated. The methods that are being developed can likely be used in any temperate wetlands to detect cryptic and rare species. 

Read more about INHS bird research.

Amphibians & reptiles 

INHS scientists are applying eDNA analysis to the state endangered alligator snapping turtle and the federally threatened Louisiana pinesnake and eastern massasauga rattlesnake.

Alligator snapping turtle populations are declining rangewide, and the United States Fish and Wildlife Service has recently proposed their listing as threatened under the U.S. Endangered Species act. However, the turtle species’ current range is poorly understood as traditional sampling is both labor and time-intensive, and researchers are limited by the number of traps that they can check in a day. With eDNA, researchers can cover a wide geographic area relatively rapidly by simply collecting a water sample. With less than a gallon of water taken from each sampling location, the eDNA method was able to detect radio-telemetered alligator snapping turtles up to two-thirds of a mile downstream. Using these methods, INHS scientists teamed up with the Indiana Department of Natural Resources to conduct eDNA surveys for the alligator snapping turtle throughout southern Indiana to update our understanding of its distribution in the northern extent of its range.

INHS scientists must take a more conservative approach when studying the federally protected Louisiana pinesnakes. These cryptic snakes spend most of their time underground, making them very difficult to sample. In fact, scientists currently don’t know when Louisiana pinesnakes mate, lay eggs, their population sizes, or genetic diversity. To find this otherwise undetectable organism, INHS researchers tested soil samples taken from pocket gopher burrows for pinesnake eDNA and successfully detected it. This approach gives researchers another tool to track where they think pinesnakes might occur.

The eastern massasauga rattlesnake is a small, cryptic, and federally threatened snake species that may benefit from eDNA detection methods using water samples collected from crayfish burrows at known occupied sites by the eastern massasauga in Clinton County, Illinois. If effective, eDNA sampling could facilitate more robust sampling efforts in detecting the fungus Ophidiomyces ophiodiicola, the causative agent of snake fungal disease, which can cause morbidity and mortality in the eastern massasauga.

Read more about INHS amphibian and reptile research.

Fish

Glacial lakes, also known as kettle lakes, are depressions in an outwash plain formed by retreating glaciers. Within Illinois, glacial lakes are only known from the northeastern portion of the state and contain a unique fish fauna that includes several state-listed species. The INHS Urban Biotic Assessment Program (UBAP) provides the Illinois State Toll Highway Authority with the ecological expertise needed to effectively and efficiently comply with state and federal environmental regulations, while also preserving habitats and species native to the Chicago metropolitan area. One way is by sampling for state-listed species. INHS scientists are using eDNA as another tool to document the presence of the six different state-listed fishes—pugnose shiner (Notropis anogenus), blackchin shiner (Notropis heterodon), blacknose shiner (Notropis heterolepis), western banded killifish (Fundulus diaphanus menona), starhead topminnow (Fundulus dispar), and Iowa darter (Etheostoma exile).

Read more about INHS fish research.

American brook lamprey

The American brook lamprey is a state-threatened species found in large clear creeks in northeastern Illinois. Its free-swimming adult stage is short—it spawns and dies shortly thereafter—while its larval stage can last more than five years, during which time the larva burrows in sandy, silty substrate with only its head exposed. The American brook lamprey’s cryptic life history makes it difficult to detect with traditional survey methods. For this reason, INHS’s early eDNA projects focused on developing a primer to detect this listed species. Now it can be more easily surveyed for by collecting water samples from suitable habitat and analyzing them for shed DNA.

Pollinators

Pollinators are incredibly important to Illinois’ agriculture economy. However, an increasingly large number of pollinator species are being considered for listing. In order to piece together what a pollinator community for a particular site looks like, INHS researchers are using very general primers to categorize a broad spectrum of DNA, using a metabarcode. Metabarcoding is the barcoding of DNA/RNA in a manner that allows for the simultaneous identification of many taxa within the same sample. 

INHS scientists can extract DNA samples left behind by pollinators on plants and use genetic sequencing to amplify the DNA and document what species has visited that plant.

Mollusks

The salamander mussel is state-endangered and has not been collected alive in Illinois in decades. Salamander mussels were recorded sporadically throughout the state, with recent dead shell records collected in Vermilion County. They are host-specific, and where most freshwater mussels release their glochidia (larval stage) onto fish species, the salamander mussel uses the common mudpuppy as a host. Salamander mussels and mudpuppies are difficult to detect during conventional-timed search surveys given their rarity, habitat association with large slabs or flat rocks, and diminutive size. Both species have poorly defined distributions within Illinois due to the lack of targeted surveys.

INHS scientists surveyed 12 sites in northern and east-central Illinois using eDNA to detect the salamander mussel presence in historical and likely extant locations in Illinois. Additionally, repeated sampling was conducted at two sites to determine if seasonality affected mudpuppy eDNA detection. Preliminary results have shown both salamander mussels and mudpuppies can be detected from water samples and have even successfully detected both species at one site. 

INHS scientists plan to expand on this pilot effort by conducting eDNA surveys for salamander mussels and mudpuppies in the Vermilion River basin (Wabash River drainage) in Illinois, and—together with the Minnesota Department of Natural Resources—salamander mussels in Minnesota and the Mississippi River.

Read more about INHS mollusk research and work on Endangered & Threatened Species.

Invasive species

Feral hogs

Feral hogs have been a big problem in Louisiana. They are incredibly destructive, especially to the wetland habitats of threatened and endangered amphibian species. Using eDNA, researchers are testing hog feces to document their diets. Evidence that the hogs are eating imperiled amphibian species might provide added and aggressive incentives to management practices. 

Invasive carp

Invasive carp are a massive problem for Illinois waterways, and many are concerned about where these fish are going next. One thing that’s proving difficult is the timing of reproduction—when and where they are reproducing. INHS scientists are ramping up and seeing if the number of copies of carp DNA is matching up with ichthyoplankton samples. Researchers can use this as a rapid response assay to better target, both in time and space, management interventions.

Virile crayfish

In a study of crayfish in the Current River in southeastern Missouri, INHS scientists and their colleagues set out to document the extent of the virile crayfish invasion by collecting and identifying mitochondrial DNA from environmental samples (eDNA). However, they discovered that some of the native spothanded crayfish (Faxonius punctimanus) had mitochondrial DNA sequences that were aligning with invasive virile crayfish, and virile crayfish had the mitochondrial DNA of spothanded crayfish. This meant that the two species were interbreeding, thus potentially altering the spothanded crayfish’s genetics, life history, and ecology.

The discovery should come as a warning to those using eDNA to look for an invasive species in an area with closely related native species. Currently, most eDNA detection markers use mitochondrial DNA, so the results of this research highlight the possibility of missed detections of invasive species if hybridization is occurring.

Corbicula fluminea

The effects of stream flow or discharge on eDNA have been minimally investigated in lotic environments. INHS researchers examined the role of stream flow on eDNA concentrations and detectability of an invasive clam (Corbicula fluminea), while also accounting for other abiotic and biotic variables. A longitudinal study was conducted over a year in two streams in central Illinois, as well as a seasonal study (summer, autumn) in eight streams, to investigate the effects of variable stream flow on eDNA concentrations and detectability. The study concluded that higher stream flows decreased eDNA concentrations, and floods produced false negatives or non-detections at locations where C. fluminea was relatively common. In addition, concentrations and detectability of C. fluminea eDNA tend to be higher in summer than in autumn.

For more information, see Invasive & Pest Species.

Methodology questions

Currently, eDNA has mostly been used for presence/absence research, but it can be used for population genetics within the right systems. Where traditional techniques for assessing population genetics required the physical capture of large numbers of individuals and collecting genetic samples from them, eDNA can provide a rapid, cost-effective, and non-invasive method of sampling. 

However, while eDNA is an emerging approach for detecting species, numerous methodological questions remain unanswered. INHS researchers are examining how various factors such as sample storage, light exposure, and seasonality can influence detection and measured eDNA concentration.

These factors have important implications for eDNA research where immediate access to refrigeration is not available, or for fieldwork that requires extended sampling time (e.g., canoeing a river). Furthermore, faster eDNA degradation times have been reported under ambient conditions than some previous aquaria or mesocosm studies, suggesting an ongoing need to study mechanisms related to eDNA persistence and sample storage.