Molecular detection of vertebrates in stream water: a demonstration using Rocky Mountain tailed frogs and Idaho giant salamanders.

Abstract

Stream ecosystems harbor many secretive and imperiled species, and studies of vertebrates in these systems face the challenges of relatively low detection rates and high costs. Environmental DNA (eDNA) has recently been confirmed as a sensitive and efficient tool for documenting aquatic vertebrates in wetlands and in a large river and canal system. However, it was unclear whether this tool could be used to detect low-density vertebrates in fast-moving streams where shed cells may travel rapidly away from their source. To evaluate the potential utility of eDNA techniques in stream systems, we designed targeted primers to amplify a short, species-specific DNA fragment for two secretive stream amphibian species in the northwestern region of the United States (Rocky Mountain tailed frogs, Ascaphus montanus, and Idaho giant salamanders, Dicamptodon aterrimus). We tested three DNA extraction and five PCR protocols to determine whether we could detect eDNA of these species in filtered water samples from five streams with varying densities of these species in central Idaho, USA. We successfully amplified and sequenced the targeted DNA regions for both species from stream water filter samples. We detected Idaho giant salamanders in all samples and Rocky Mountain tailed frogs in four of five streams and found some indication that these species are more difficult to detect using eDNA in early spring than in early fall. While the sensitivity of this method across taxa remains to be determined, the use of eDNA could revolutionize surveys for rare and invasive stream species. With this study, the utility of eDNA techniques for detecting aquatic vertebrates has been demonstrated across the majority of freshwater systems, setting the stage for an innovative transformation in approaches for aquatic research.

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Molecular Detection of Vertebrates in Stream Water: A
Demonstration Using Rocky Mountain Tailed Frogs and
Idaho Giant Salamanders
Caren S. Goldberg1*, David S. Pilliod2, Robert S. Arkle2, Lisette P. Waits1
1Fish and Wildlife Resources, University of Idaho, Moscow, Idaho, United States of America, 2United States Geological Survey, Forest and Rangeland Ecosystem Science
Center, Boise, Idaho, United States of America
Abstract
Stream ecosystems harbor many secretive and imperiled species, and studies of vertebrates in these systems face the
challenges of relatively low detection rates and high costs. Environmental DNA (eDNA) has recently been confirmed as a
sensitive and efficient tool for documenting aquatic vertebrates in wetlands and in a large river and canal system. However,
it was unclear whether this tool could be used to detect low-density vertebrates in fast-moving streams where shed cells
may travel rapidly away from their source. To evaluate the potential utility of eDNA techniques in stream systems, we
designed targeted primers to amplify a short, species-specific DNA fragment for two secretive stream amphibian species in
the northwestern region of the United States (Rocky Mountain tailed frogs, Ascaphus montanus, and Idaho giant
salamanders, Dicamptodon aterrimus). We tested three DNA extraction and five PCR protocols to determine whether we
could detect eDNA of these species in filtered water samples from five streams with varying densities of these species in
central Idaho, USA. We successfully amplified and sequenced the targeted DNA regions for both species from stream water
filter samples. We detected Idaho giant salamanders in all samples and Rocky Mountain tailed frogs in four of five streams
and found some indication that these species are more difficult to detect using eDNA in early spring than in early fall. While
the sensitivity of this method across taxa remains to be determined, the use of eDNA could revolutionize surveys for rare
and invasive stream species. With this study, the utility of eDNA techniques for detecting aquatic vertebrates has been
demonstrated across the majority of freshwater systems, setting the stage for an innovative transformation in approaches
for aquatic research.
Citation: Goldberg CS, Pilliod DS, Arkle RS, Waits LP (2011) Molecular Detection of Vertebrates in Stream Water: A Demonstration Using Rocky Mountain Tailed
Frogs and Idaho Giant Salamanders. PLoS ONE 6(7): e22746. doi:10.1371/journal.pone.0022746
Editor: Brian Gratwicke, Smithsonian’s National Zoological Park, United States of America
Received May 15, 2011; Accepted July 4, 2011; Published July 26, 2011
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: Funding was provided by the United States Geological Survey Amphibian and Reptile Monitoring Initiative. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: cgoldberg@vandals.uidaho.edu
Introduction
Freshwater systems are hotspots for both biodiversity and
species endangerment [1], with freshwater fauna experiencing 123
documented extinctions in the 20thcentury [2]. Growing demand
for water resources indicates that threats to freshwater species will
further increase over the next century [3]. Stream species are
particularly vulnerable to cumulative changes in land cover [4,5],
climate [6], and biotic and abiotic inputs [7,8]. Migratory stream
salmonids (e.g. bull trout, Salvelinus confluentus, and Chinook
salmon, Oncorhynchus tshawytscha) are among the most imperiled
North American fishes [9] and the most catastrophic documented
amphibian population declines have been in streams [10].
Additionally, streams are increasingly being invaded, at great
ecological and economic costs, by exotic species, including
crayfish, aquatic mussels, and gastropods [11,12].
Investigations into the distribution and ecology of stream species
are often hindered by the challenges of working in these systems.
Stream species are difficult to inventory due to the complexity of
topography and vegetation in streambeds and riparian areas,
water turbidity and flow rate, low densities of individuals, cryptic
coloration, and the use of microhabitats. Due to these and other
factors, surveys for native and exotic species in streams can be
expensive and inaccurate [13,14]. For example, a major challenge
in amphibian decline research is that amphibians can be difficult
to detect, especially in streams [15]. Electrofishing techniques have
high success for detection of aquatic vertebrates in many cases
[16], but can be time consuming and difficult to apply in streams,
and may cause injury to target and non-target species [17].
Researchers have been using DNA from feces, urine, hair,
feathers, shed skin, and eggshells to detect terrestrial vertebrate
species for the past decade [18], and detection of microbial species
using environmental DNA (eDNA) found in soil and seawater is
revolutionizing species inventories [19] and enabling efficient
disease detection [20]. Recently, the reliable detection of aquatic
vertebrate species using eDNA in water was confirmed in wetlands
[21] and in a large river and canal system [22]. Using eDNA to
detect rare and secretive species in streams could increase
accuracy and decrease costs of surveys, increase the number of
sites sampled per unit effort, refine distribution and extinction
records, and provide early detection of invasive species in these
systems, without any risk to the species. However, the fast flow of
streams may move shed cells away from their source at a rate
prohibitive to eDNA collection. To evaluate the potential for using
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eDNA to survey for stream species, we collected water samples
from five small headwater streams in two seasons and tested them
for DNA of two amphibian species (Rocky Mountain tailed frogs,
Ascaphus montanus, and Idaho giant salamanders, Dicamptodon
aterrimus) known to be present at the sites. To achieve this, we
designed species-specific primers and tested multiple DNA
extraction and PCR protocols designed to amplify low quality
DNA templates.
Methods
In the first phase of the project, we collected one 10-L and two 5-
L water samples from a headwater stream (Table 1) with known
presence of two species of amphibians (Rocky Mountain tailed frogs
and Idaho giant salamanders) in late September of 2010 using a
flow-through filter with a peristaltic pump and 0.45 mm cellulose
nitrate filter paper (State of Idaho Wildlife Collection Permit
#030716 and Payette National Forest Research Permit #0105).
Each filter was preserved in 95% ethanol in a separate 2 mL tube.
We estimated the larval density of both species at this site using
standard kick-sampling methodology [23] in July and August 2010.
Density survey and water sample collections were made during base
flow, measured as 0.23 m3s21, in the study stream.
We designed a set of species-specific primers for each species
targeting a small region of the mitochondrial DNA (mtDNA)
cytochrome b gene (obtained from GenBank) [24,25] (Table 2).
The distribution of these two species is disjunct from their
congeners along the Pacific coast to the west; therefore, we
designed primers to be species-specific within our system (the
northern Rocky Mountains region) but also to detect the
congeners of each species for wider geographic applicability.
Target fragment length was 78 base pairs for Dicamptodon and 85
base pairs for Ascaphus. This test was designed to amplify
previously-published sequences characteristic of these species; no
new sequence data was generated that had not already been
published. All extractions and PCR set-up were done in a room
dedicated to low-quantity DNA sources; no DNA from amphib-
ians had previously been handled in this room.
In this first phase, we tested two DNA extraction and three PCR
protocols for the detection of Rocky Mountain tailed frogs and Idaho
giant salamanders using eDNA from these filter samples. First, we
removed the filters from the ethanol and air-dried them overnight.
Wethen divided each filter in halfand extracted each halfwith either
the DNeasy Tissue and Blood Kit (Qiagen, Inc.) or the UltraCleanH
Soil DNA isolation kit (MoBio Laboratories, Inc.). We then
attempted to amplify DNA from each sample using a standard
PCR protocol (PCR Protocol 1; Table S1). All reactions included a
negative extraction control, negative PCR control, and positive
controls for each of the target species. We ran PCR products on 3%
agarose gels to determine success. When this first protocol produced
no PCR products, we reran the reactions with a combination of each
DNA sample and a positive control in each tube to determine
whether PCR inhibitors were preventing amplification. For samples
extracted using the Qiagen DNeasy kit, we also tested three PCR
protocols (PCR Protocols 1, 2, and 3; Table S1) with a dilution series
of each sample (1X, 0.1X, 0.01X, and 0.001X).
We sequenced products of the most successful combination of
protocols using the BigDye system on a 3130xl capillary sequencer
(Applied Biosystems). To streamline the assay for large-scale
application, primers were labeled with fluorescent dyes and a PCR
multiplex was created using primer sets for both species with PCR
Protocol 3. We tested additional negative controls of DNA from
sympatric amphibian species (Ambystoma macrodactylum, Bufo boreas,
Pseudacris sierra, Rana luteiventris) with this multiplex, independently
(1 reaction/species; approximately 5 – 100 ng DNA/reaction) and
together with DNA from the target species, to verify the specificity
of our diagnostic test.
In the second phase of the project, we collected a 5-L water
filter sample from each of five headwater streams known to
contain populations of Rocky Mountain tailed frogs and Idaho
giant salamanders, including the original stream (Table 1).
Streams were sampled in late March and early April 2011 using
the same field collection techniques as above; density estimates for
these streams were obtained July and August 2010 (State of Idaho
Wildlife Collection Permit #030716 and Payette National Forest
Research Permit #0105). We used the DNeasy extraction method
and PCR Protocol 3 (Table S1) for one half of each filter and a
modified extraction, with the addition of the use of a QIAshredder
(Qiagen, Inc.) after overnight digestion with Proteinase K, and
PCR Protocol 4 (Table S1) for the other half of each filter. We also
tested the Qiagen Multiplex Plus PCR kit with this modified
protocol (PCR Protocol 5). These samples were only run at full
concentration. We tested whether field-estimated densities pre-
dicted PCR success for these samples using simple linear
regression in R 2.13.0 [26].
Results
In the first phase of the project, we recovered the targeted DNA
sequence from both species from all stream water filter samples
Table 1. Sampling sites, dates of sampling, PCR success for each species, and densities of Idaho giant salamanders (Dicamptodon
aterrimus; DIAT) and Rocky Mountain tailed frogs (Ascaphus montanus; ASMO) where stream filter samples were taken, estimated
using field methods in summer 2010.
Site LatitudeLongitude Date sampledDIAT per m2
DIAT PCR success (%) ASMO per m2
ASMO PCR success (%)
Phase 1
Nasty Creek44.877
2115.69625Sept100.032 1000.228100
Phase 2
Camp Creek 44.890
2115.706 27Mar11 0.0361000.09716.7
Deadman Creek 44.966
2115.66327Mar11 0.011 1000.1490
Goat Creek 44.759
2115.68427Mar110.029 1000.05233.3
Nasty Creek44.877
2115.696 03Apr11 0.0321000.22833.3
Reegan Creek44.949
2115.587 27Mar110.011 1000.33716.7
doi:10.1371/journal.pone.0022746.t001
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only when using the DNeasy extraction method and PCR Protocol
3. For two of the three samples (one 5-L, one 10-L) the correct
fragment was also detectable at 0.1X DNA concentration for
Rocky Mountain tailed frogs and down to 0.001X DNA
concentration for Idaho giant salamanders. Tests for inhibition
with PCR Protocol 1 showed that samples from the DNeasy
extraction method were inhibited (but the extraction negative
control was not), while samples from the MoBio extraction were
not inhibited, indicating the lack of target species DNA in the
results of these extractions. PCR multiplexing with fluorescently-
tagged primers provided clear and efficient detection of amplified
fragments (Fig. 1), with all samples and positive controls at .8000
fluorescent units, even when DNA from the target species was
mixed with DNA from non-target species. None of the negative
controls, including DNA from four sympatric amphibian species,
tested positive.
In the second phase of the project, where samples were collected
in the early spring, we detected Idaho giant salamanders in all
filter samples using PCR Protocol 3 but did not detect Rocky
Mountain tailed frogs. Amplifications for Idaho giant salamanders
were weak (x = 203, 95% C.I. 25 – 382 fluorescent units)
compared with samples collected in early fall. With the addition of
the QIAshredder kit and using PCR protocol 4, we detected both
species in all but one of the streams, with strong signal for Idaho
giant salamanders in all reactions (x = 5962, 95% C.I. 4555 –
7369 fluorescent units) and detection probability across 6 PCR
replicates for Rocky Mountain tailed frogs ranging from 0 to 33%
(Table 1; for successful amplifications, x = 7636, 95% C.I. 6176 –
9097 fluorescent units). Substitution of the Qiagen Multiplex Plus
PCR kit for the Qiagen Multiplex PCR kit in PCR Protocol 5 did
not improve performance (Table S1). There was no evidence that
the probability of detection of Rocky Mountain tailed frogs in a
PCR replicate was related to field-estimated density from the
previous summer (PCR Protocol 4; F1,3=0.036, P=0.86).
Discussion
Using filter samples taken from stream water, we developed an
efficient protocol for detecting targeted DNA sequences for two
secretive amphibian species, demonstrating that the recovery of
amphibian DNA from stream water is possible even when
amphibian populations are at low densities. The rapid field
collection protocol, relatively simple field equipment and low cost
(supply cost per sample with 6 PCR replicates = $10.11) make this
technique widely applicable to broad-scale inventory and
monitoring efforts. The probability of detection of eDNA across
densities likely varies with species, stream size, and discharge rate,
and by season, as suggested by this study. However, the potential
impact of this technique for inventorying species in stream systems
is far-reaching, including detection of rare or imperiled verte-
brates.
We found that only the DNeasy Blood & Tissue kit with the
Qiagen Multiplex PCR kit detected eDNA for both species in
water filter samples. We did not successfully extract DNA from the
filter samples using the UltraCleanH Soil DNA isolation kit (MoBio
Laboratories, Inc.); possibly the PowerWater DNA Isolation kit
(MoBio Laboratories, Inc.) used to detect eDNA of Asian carp [22]
would have yielded better results. Our results indicate that using
the Qiagen Multiplex PCR kit improves species detection in water
filter samples over a protocol using Amplitaq Gold DNA
polymerase and BSA; this latter combination was used to establish
that the detection of aquatic vertebrates using eDNA in water
samples was possible [21].
Although we only sampled one stream in the first phase of our
project, our results suggest that detection of Rocky Mountain tailed
frogs and Idaho giant salamanders using eDNA may be more difficult
whensamplesaretakeninearlyspringratherthanearlyfall.Thiscould
be due to decreased metabolism during cold weather or changes in
behavior of the target species, such as moving into the hyporheic zone.
For Idaho giant salamanders, we were able to compensate for this by
modifying protocols, but for Rocky Mountain tailed frogs, detectability
was still relatively low in early spring samples. This difference between
speciesmaybeduetospecies-specificseasonalchangesindensity;while
streams in the spring are likely to have one fewer Rocky Mountain
tailed frog tadpole cohort than in the early fall due to timing of
metamorphosis [27], the difference in overall population density is
likely less extreme for Idaho giant salamanders because they are
commonly neotonic [28]. This result demonstrates that sampling
design for eDNA needs to be informed by the ecology of target species
to maximize detection probabilities.
Our approach was to design species-specific primers to detect
species of interest; these kinds of targeted primers can be
multiplexed to test for many species in a single PCR reaction.
However, when the species list is large or inventory for unknown
species is the goal of sampling, universal primers and next-
generation sequencing techniques could be applied [19]. Using
these tools, researchers would sample a stream, river, or wetland,
use primers that work across taxa to amplify DNA from this sample,
and compare the sequences to those available in a reference library
[29]. If sequences are recovered that do not match any in the
library, sequences that are closest matches could be used to
determine the probable taxonomic group of the unknown species
and additional field surveys could be conducted to attempt to locate
the species. Next-generation sequencing is currently prohibitively
expensive for large survey efforts, but costs will likely be greatly
reduced in the near future as the technology improves [30].
The success of eDNA for detecting vertebrates efficiently across
freshwater systems indicates that this new tool has the potential to
revolutionize surveys for aquatic species with the techniques
currently available. The ability to survey for species across taxa
with a single water sample would greatly enhance data
Table 2. Primer sequences for species-specific amplification of short fragments of cytochrome b.
Species Primer name Primer sequence
Rocky Mountain tailed frog
(Ascaphus montanus)
ASMO F CGT CAA CTA TGG CTG GCT AA
ASMO R TCG GCC AAT GTG AAG ATA AA
Idaho giant salamander
(Dicamptodon aterrimus)
Dicamp F TCT GCA TCT TYC TAC ATA TYG GAC
Dicamp R ATC ACY CCG ACK TTT CAG GT
doi:10.1371/journal.pone.0022746.t002
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availability for aquatic species and benefit resource managers and
many fields of research, including community ecology, biogeog-
raphy, evolutionary biology, conservation biology, and invasion
biology. eDNA techniques could be used to form cost-efficient
multi-species inventory and monitoring programs for sensitive
species, in combination with occupancy models [31] to estimate
probabilities of detection. With next-generation sequencing,
DNA sequences of a community of aquatic vertebrates could
be analyzed simultaneously, exponentially increasing the data
available for analysis without disturbing sensitive species. Other
applications include early detection of invasive species [21,22],
determining whether invasive species have been successfully
removed through management actions, detecting rare individuals
surviving after catastrophic population declines, and discovering
new species in rapid bioassessement surveys. Sensitivity of these
techniques to density of individuals and covariates of detection
probability such as water temperature and discharge will need to
be determined for study systems individually; however, this
technique shows great potential for increasing our knowledge of
aquatic systems.
Supporting Information
Table S1
Rocky Mountain tailed frogs (Ascaphus montanus) and Idaho giant
salamanders (Dicamptodon aterrimus) from stream water.
(DOCX)
PCR protocols and results for amplifying DNA of
Acknowledgments
We thank David Tank for sequencing advice, Tomaz Skrbinsek for
suggesting the QIAshredder, Steve Arkle for assisting with sample
collection, and Steve Corn, Mark Miller, and an anonymous reviewer
for helpful comments on earlier versions of the manuscript. The use of any
trade, product or firm name is for descriptive purposes only and does not
Figure 1. Electropherograms of species-specific PCR amplification of DNA in positive controls and stream water. The blue peak
indicates the species-specific fragment for Idaho giant salamanders (Dicamptodon aterrimus) and the black peak indicates the species-specific
fragment for tailed frogs (Ascaphus montanus). All reactions were diluted to produce these images.
doi:10.1371/journal.pone.0022746.g001
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imply endorsement by the U.S. Government. This is contribution number
385 of the U.S. Geological Survey Amphibian Research and Monitoring
Initiative (ARMI) and contribution 1061 of the University of Idaho Forest,
Wildlife and Range Experiment Station.
Author Contributions
Conceived and designed the experiments: CSG DSP RSA LPW.
Performed the experiments: CSG RSA. Analyzed the data: CSG DSP
RSA LPW. Contributed reagents/materials/analysis tools: CSG DSP
LPW. Wrote the paper: CSG DSP RSA LPW.
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