Monitoring aquatic amphibian and
reptile populations
using environmental DNA
Katherine M. Strickler, Caren S. Goldberg, and
Alexander K. Fremier
Outline
 What is eDNA?
 When do we use it?
 How do we collect and detect eDNA?
 DoD projects: methods and preliminary results
 Conclusions
 Protocols
What is eDNA?
DNA in the aquatic environment
UV
endonucleases/
exonucleases
DNA of ~100 bp can persist 2 – 3 weeks (Dejean et al. 2011)
eDNA original papers
Bullfrog detection (Dejean et al. 2012)
Field surveys
7 ponds
14%
eDNA surveys
38 ponds
77%
Bullfrog detection (Dejean et al. 2012)
Field surveys
7 ponds
14%
eDNA surveys
38 ponds
77%
eDNA research
eDNA research
 Marine fish (Thomsen et al. 2012)
 Marine mammals (Foote et al. 2012)
 New Zealand mudsnails (Goldberg et al. 2013)
 Hellbenders (Olson et al. 2012, Spear et al. submitted)
 Burmese python (Piaggio et al. 2013)
 Brook trout, bull trout (Wilcox et al. 2013 , this study)
 Chinook salmon (Laramie 2013, this study)
 Bd (Schmidt et al. 2013 , this study)
 Ranavirus (this study)
Advantages of eDNA
 Non-destructive
 Highly sensitive – higher detection probabilities
 Multi-species detections (including pathogens)
 Reduced need for taxon-specific field training
 Reduced permitting requirements
When do we use eDNA?
 Under what circumstances is eDNA sampling
more efficient than standard field surveys?
‒ Likely will differ by species and system
When do we use eDNA?
 Under what circumstances is eDNA sampling
more efficient than standard field surveys?
Detection
Field
sampling
more costeffective
eDNA sampling
more cost effective
eDNA
sampling
High density
populations
Low density
populations
Effort
How do we collect eDNA?
Water sampling
How do we detect eDNA?
• DNA extraction
(DNeasy/Qiashredder)
• Quantitative PCR (qPCR)
eDNA projects - DoD
Fort Huachuca (AZ)
• Arizona treefrog
• Northern Mexican gartersnake
• Chiricahua leopard frog
• Sonora tiger salamander
• American bullfrog
• Ranavirus
• Bd
eDNA projects - DoD
Eglin Air Force Base (FL)
• Reticulated flatwoods salamander
• Ornate chorus frog
Yakima Training Center (WA)
• Bull trout, brook trout
• Spring and fall Chinook salmon
Developing species-specific guidance
 Collect 4 replicate water filter samples in
coordination with field surveys
 Compare detection probabilities of eDNA vs.
field surveys
Developing species-specific guidance
 Collect environmental covariates
• UV exposure
• Conductivity
• Water temperature
• pH
• Area
• Volume
 Use occupancy modeling to determine effects
of covariates on detection probabilities
Developing species-specific guidance
Arizona treefrog detection (1.0)
 15 sites sampled
 Detected at 4 sites
 1.0 detection probability
Chiricahua leopard frog detection (0.65)
• 20 sites sampled
• 1 site detected by field crews missed by eDNA
• 2 sites detected by eDNA missed by field crews
Chiricahua leopard frog detection probability
Model
Area
Volume
Grab sample
Null
Conductivity
pH
Canopy cover
Temperature
Sampling occasion
AIC
42.96
51.02
53.52
54.93
55.39
56.20
56.56
56.88
57.95
ΔAIC
0
8.06
10.56
11.97
12.43
13.24
13.60
13.92
14.99
Weight
0.970
0.017
0.005
0.002
0.002
0.001
0.001
0.001
0.001
Chiricahua leopard frog detection probability
Take samples at 2
locations
Take samples at 3
locations
American bullfrog detection (0.72)
• 50 sites sampled
• 1 site detected by field crews missed by eDNA
• 4 sites detected by eDNA missed by field crews
American bullfrog detection probability
Model
AIC
ΔAIC
Weight
Temperature
74.28
0
0.531
Null
78.05
3.77
0.081
Conductivity
78.78
4.50
0.056
Area
79.37
5.09
0.042
pH
79.95
5.67
0.031
Sample volume
80.02
5.74
0.030
Sampling replicate
83.74
9.46
0.005
American bullfrog detection probability
2 samples
3 samples
4 samples 5 samples
Sonora tiger salamander detection (0.73)
• 23 sites sampled
• 3 sites detected by field crews missed by eDNA
• 1 site detected by eDNA missed by field crews
Sonora tiger salamander detection probability
Model
Volume
Area
Null
AIC
38.31
57.73
60.07
Conductivity
Temperature
pH
Sampling occasion
ΔAIC
Weight
0
19.42
21.76
1.0
0
0
61.00
61.26
62.07
22.69
22.95
23.76
0
0
0
65.76
27.45
0
Sonora tiger salamander detection probability
Conclusions
 eDNA detection varies by species
 Sampling protocols need to maximize detection
• Season for sampling
• Number of replicates
• Spatial distribution of
replicates
•
•
•
•
Volume sampled
Preservation method
Extraction method
Analysis method
 Pilot study is critical
 eDNA sampling can complement field surveys
Protocols
• Field protocol
• Lab protocols
• Guidelines for eDNA sampling programs
Field protocol
• Materials
• Sample collection
• Filtration
• Contamination
prevention
Lab protocol
Guidelines for selecting a laboratory
to process eDNA samples
• Facilities (clean room)
• Techniques (qPCR or next-gen sequencing)
• Standard practices
• Positive and negative controls
Preliminary guidelines
Generalized guidelines
for designing eDNA sampling programs
• Determine the most appropriate season to conduct eDNA surveys
• Consider spatial sampling design
• Consider filter type
• Consider preservation method
• Conduct a pilot study
• Consider how eDNA sampling can complement existing field
methods
Thank you
Backup Slides
DNA barcoding
 All individuals within a species share particular
sequences
Thamnophis eques (mtDNA):
…GAAAGGCCCTAACCTGGTAGGACCAATA…
Thamnophis cyrtopsis (mtDNA):
…GAAAGGCCCCAACCTAGTAGGACCAATA…
Wood et al. 2011
www.barcodeoflife.org
Quantitative PCR (qPCR)
Blue – long-toed salamander test
Green – positive control
More DNA
Less DNA
qPCR negative – Idaho giant salamander
Blue – long-toed salamander test
Green – positive control
qPCR multiplex
Red – Arizona treefrog
Blue – Bd
Green – positive control
eDNA qPCR
Quantification as well as presence/absence
Pilliod et al. 2013
eDNA assay process
eDNA test development:
Identify target
species set
Collect DNA
sequence data
Create and
verify qPCR test
eDNA test application:
Analyze
detection data
Run qPCR test
Collect replicate
water samples
from DoD sites
Detection probabilities
Arizona treefrog
15
Detection
probability
1.00
N Mexican gartersnake
15
0.17
Chiricahua leopard frog
20
0.65
American bullfrog
50
0.72
Sonora tiger salamander
23
0.73
Ranavirus
23
0.88
Bd
45
0.80
Species
# sites
Northern Mexican gartersnake (0.17)
eDNA FAQs
Can we use eDNA for Species X?
Can we use eDNA to estimate abundance/density?
What are the chances of a false positive?
How much does it cost?
How far downstream can eDNA be detected in streams?