Introduction

What is eDNA?

Organisms continually shed DNA from their skin cells, feathers, fur, feces,

and/or gametes into their surrounding environments. Environmental DNA, or

eDNA, can be extracted from freshwater, marine, and soil samples. eDNA can

trace a sample back to the species that produced the sample.

How is eDNA used?

Amphibians, mammals, fish, reptiles and arthropods have been detected with

eDNA. DNA of rare and cryptic species, such as Rocky Mountain tailed frogs,

Idaho giant salamanders, and Eastern Hellbenders, has been isolated from

eDNA samples in freshwater rivers and streams. Thomsen et al 2012

serendipitously detected migratory birds from marine water samples. Our

research focuses on using eDNA to quantify cryptic bird occupancy in wetland

patches in Cook and Lake County, Illinois.

Why focus on cryptic wetland birds in Illinois?

Wetland habitat has severely declined in Illinois over the past 200 years.

Wetland patches support over 100 breeding bird species, 15 of which are

either state threatened and endangered or globally imperiled. We have poor

distribution estimates of rare and secretive rail species. We cannot conserve

species unless we know which habitat patches they are using.

Why use eDNA if playback surveys can locate birds?

Birds do not always respond to playback, and eDNA sampling may cause less

disturbance and stress than playback to nesting birds.

Questions

(1) Can we use eDNA methods to detect cryptic wetland birds in emergent

wetland habitat?

(2) How do eDNA methods compare to traditional survey methods?

(3) Can we use eDNA sampling to track cryptic wetland bird migration?

Methods

In the field

• performed playback surveys using Black Rail, Yellow Rail, King Rail, Virginia

Rail, Sora, and Common Gallinule recordings at four sites in NE Illinois

• collected and filtered 1-L water samples at playback survey locations;

collected and processed 37 samples, 7 distilled water blanks

In the lab

• extracted eDNA from filters using a CTAB extraction protocol

• quantified eDNA extracts on a Qubit 3.0 fluorometer

• amplified 225 bp fragments of avian 16S in the mitochondrial genome;

degenerate primers were developed in June by Dalén et al 2017

• samples with band size matching Sora rail liver control were purified and

sent for sequencing.

Conclusions

• Amount of eDNA obtained from samples does not

significantly vary by site or over time during spring migration

and breeding, but more sampling is needed to confirm

preliminary results.

• Sample contamination can be reduced using adequate

levels of human blocking primer during PCR.

• eDNA sampling methods can increase occupancy estimates

obtained using playback recordings.

• Pending sequencing results, eDNA methods can likely be

used to identify multiple cryptic, declining rail species.

Open Questions and future directions

• On what timescale does bird DNA persist and degrade in

emergent wetlands? Do eDNA concentrations shift

significantly after storms?

• Can we use eDNA to estimate rail abundance in addition to

occupancy?

• How robust are eDNA diversity estimates compared to

passive point count methods?

Acknowledgements & ContactWe gratefully acknowledge the Illinois State Toll Highway Authority for funding this work, and the Forest

Preserve Districts of Cook and Lake Counties for land access. For fieldwork assistance, we thank Alison

Világ. For laboratory assistance, we thank Dr. Kurt Ash and Samantha Barratt. This research was

conducted by the Illinois Natural History Survey at the University of Illinois at Urbana-Champaign, as part of

the Urban Biotic Assessment Program.

Contact: Anastasia Rahlin (rahlin1@Illinois.edu)

Illinois Natural History Survey, 1101 W. Peabody Drive

University of Illinois at Urbana-Champaign, Urbana, IL 61801

ReferencesDalén, L., Lagerholm, V.K., Nylander, J.A.A., Barton, N., Bochenski, Z.M., Tomek, T., Rudling, D., Ericson, P.G.P., Irestedt, M., and Stewart, J.R.

2017. Identifying bird remains using ancient DNA barcoding. Genes 8(169): doi:10.3390/genes8060169.

Goldberg, C.S., Pilliod, D.S., Arkle, R.S., and Waits, L.P. 2011. Molecular detection of vertebrates in stream water: a demonstration using Rocky

Mountain tailed frogs and Idaho giant salamanders. PLoS ONE 6(7): e22746.

Olson, Z.H., Briggler, J.T., Williams, R.N. 2012. An eDNA approach to detect eastern hellbenders (Cryptobranchus a. alleganiensis) using samples

of water. Wildlife Research 39: 629-636.

Rees, H.C., Maddison, B.C., Middleditch, D.J., Patmore, J.R.M., Gough, K.C. Review: The detection of aquatic animal species using environmental

DNA – a review of eDNA as a survey tool in ecology. Journal of Applied Ecology 51(5): 1450-1459. DOI: 10.1111/1365-2664.12306.

Thomsen, P.F., Kielgast, J., Iversen, L.L., Wiuf, C., Rasmussen, M., Gilbert, M.T.P., Orlando, L., Willserslev, E. 2011. Monitoring endangered

freshwater biodiversity using environmental DNA. Molecular Ecology 21(11):2563-2573.

Thomsen, P.F., Kielgast, J., Iversen, L.L., Møller, P.R., Rasmussen, M., Willerslev, E. 2012. Detection of a diverse marine fish fauna using

environmental DNA from seawater samples. PLOS One 7(8):1-9.

Using environmental DNA sampling methods to determine

cryptic wetland bird occupancy in IllinoisAnastasia A. Rahlin1, Matthew A. Niemiller1, and Mark A Davis1

Illinois Natural History Survey, University of Illinois at Urbana-Champaign

Results

Figure 1. Filtered eDNA samples taken from different points within Orland Grassland show large variation in the amount of organic matter

intercepted by the filter. Variation in samples reflected large variations in wetland type within sites (e.g. emergent wetlands, ponds, drainage ditches).

Figure 2. Between and within-site

eDNA concentration differences

as measured by qubit fluorometric

quantification. a) No significant

differences in eDNA

concentrations were detected

between sites. Sample

concentrations ranged from levels

too low to be detected by the

fluorometer to 99.00 mg/ml. b) No

significant trends within sites were

detected in eDNA concentrations

vs. the date the sample was

collected. Additionally, we found

no significant trends when

aggregating eDNA concentration

data across sites.

a. b.

Figure 3. PCR results of eDNA samples, Sora rail controls, and PCR controls using AVES 16S 1AF primer, human blocking primer (HBP), and 100bp

ladder. Red arrows indicate likely presence of bird DNA; ~125bp fragments in marked samples match size of Sora liver control fragment. Red braces

bracket sample PIDU_05.31.17 contamination. a) 0µl HBP added to PCR mix. b) 1µl HBP added. c) 5µl HBP added. Bands in (b) and (c) can be

directly compared. Amplification of human-contaminated DNA was greatly reduced in PIDU_05.31.17 sample when HBP levels were increased.

La

dd

er 1 2

Sora

contr

ol 4 5 6 7

PC

R c

ontr

ol

La

dd

er 1 2 3 4 5 6

Sora

contr

ol

PC

R c

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La

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er 1 2 3 4 5 6

PC

R c

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a. b. c.

Sora

contr

ol

Site Name Collection dates Likely rail eDNA

detections

Rail playback

detections

Illinois Beach State

Park

05.31.17, 06.15.17 1/10 (10%) 0/10 (0%)

Pine Dunes 05.24.17, 05.31.17,

06.15.17

5/14 (35.7%) 4/20 (20%)

Paul Douglas

Forest Preserve

06.7.17 1/4 (25%) 1/8 (12.5%)

Orland Grassland 05.24.17, 06.08.17 4/9 (44.4%) 7/39 (17.9%)

Table 1. Sampling

localities where water

samples were collected

and playback surveys took

place for Black Rail,

Yellow Rail, King Rail,

Virginia Rail and Common

Gallinule. Percentages

indicate occupancy

estimates from eDNA PCR

results and field

observations.