An application of PCR-RFLP species

identification assay for environmental

DNA detection
Takeshi Igawa1, Teruhiko Takahara2,3, Quintin Lau4 and
Shohei Komaki5
1
Amphibian Research Center, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan
2
Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane, Japan
3
Estuary Research Center, Shimane University, Matsue, Shimane, Japan
4
Department of Evolutionary Studies of Biosystems, Sokendai (The Graduate University for
Advanced Studies), Hayama, Kanagawa, Japan
5
Division of Biomedical Information Analysis, Iwate Tohoku Medical Megabank Organization,
Disaster Reconstruction Center, Iwate Medical University, Shiwa, Iwate, Japan

ABSTRACT
Recent advancement of environmental DNA (eDNA) methods for surveying species
in aquatic ecosystems has been used for various organisms and contributed to
monitoring and conservation of species and environments. Amphibians are one of
the promising taxa which could be monitored efficiently by applying quantitative
PCR (qPCR) or next generation sequencing to eDNA. However, the cost of eDNA
detection using these approaches can be quite high and requires instruments that are
not usually installed in ecology laboratories. For aiding researchers in starting eDNA
studies of amphibians, especially those not specialized in molecular biology, we
developed a cost efficient protocol using PCR-RFLP method. We attempted to detect
eDNA of three Japanese Rana species (Rana japonica, Rana ornativentris, and Rana
tagoi tagoi) in various spatial scales including an area close to the Fukushima nuclear
power plant where the environment is recovering after the disaster in 2011. Our
Submitted 15 March 2019 PCR-RFLP protocol was successful in detecting Rana species in static water in both
Accepted 31 July 2019 laboratory and field; however, it could not detect Rana species in non-static water
Published 3 October 2019 samples from the field. Even a more sensitive detection method (standard qPCR) was
Corresponding authors unable to detect frogs in all non-static water samples. We speculate that our new
Takeshi Igawa,
protocol is effective for frogs living in lentic habitats, but not for lotic habitats which
[email protected]
Teruhiko Takahara, may still require the gold standard of field observation for detection approach.
[email protected]
Academic editor
Subjects Biodiversity, Conservation Biology, Ecology, Zoology, Freshwater Biology
Robert Toonen
Keywords Amphibian, Environmental DNA, Distribution range, Species identification,
Additional Information and Monitoring survey, Water sample, Frog species, Tadpole
Declarations can be found on
page 11
INTRODUCTION
DOI 10.7717/peerj.7597
Spatial distribution of organisms is one of the key components for understanding the
Copyright
ecology of the species and the ecosystem in which they inhabit. In addition, monitoring of
2019 Igawa et al.
species and population distribution are essential for their conservation. However,
Distributed under
Creative Commons CC-BY 4.0 traditional visual surveys and monitoring techniques can be problematic, related to
seasonal, ecological, and ethological differences within a target species, and difficulties in

How to cite this article Igawa T, Takahara T, Lau Q, Komaki S. 2019. An application of PCR-RFLP species identification assay for
environmental DNA detection. PeerJ 7:e7597 DOI 10.7717/peerj.7597
 distinguishing between closely related sympatric species and varied life stages. In recent
studies, environmental DNA (eDNA) methods, which involve the detection of species-
specific DNA fragments in the environment, have been used for surveying species in
aquatic ecosystems (Takahara, Minamoto & Doi, 2013; Rees et al., 2014; Goldberg,
Strickler & Pilliod, 2015). Although there are some issues related to field surveys, this is
a promising and non-invasive method, and thus has been used for various organisms
including both plants and animals in terrestrial and aquatic environments (Thomsen &
Willerslev, 2015).
However, such eDNA detection experiments usually require expensive instruments and
reagents for quantitative PCR (qPCR) (for single species) or next generation sequencing
(NGS) (for multiple species). This potentially limits the number of institutions able to
conduct eDNA studies. For sustainable monitoring activities and conservation of species
diversity, an efficient and easy method should be developed for many laboratories close to
a field site. Especially for amphibians, several studies support that detection is more cost-
effective with eDNA than with traditional monitoring approaches (Olson, Briggler &
Williams, 2012; Pilliod et al., 2013; Biggs et al., 2015; Buxton et al., 2017). Amphibians are
also intrinsic target species for eDNA studies in terms of their ecology because they
have both aquatic and terrestrial life cycles and inhabit different kinds of water bodies,
such as river (lotic) and pond (lentic) water systems, depending on species and life stages.
Although the sensitivity and cost of detection methods could be correlated, applying the
low cost PCR-RFLP for eDNA detection has already been successful in differentiating
several salmonid fish species (Clusa et al., 2017). However, to-date this PCR-RFLP
approach has not been applied to organisms that are not fully aquatic, including
amphibians. Therefore, it would be important to develop a cost efficient protocol using
PCR-RFLP method for detection of eDNA fragments, instead of qPCR, in amphibian
species.
In this study, we attempted to develop a protocol for detecting the presence of three true
frog species from the Rana genus (Rana japonica, Rana ornativentris and Rana tagoi
tagoi). These species are distributed across mainland Japan, including Honshu, Shikoku,
and Kyushu islands. Rana japonica and Rana ornativentris are often located near areas
of human development and thus could be threatened with population decline due to
human-related habitat loss. As a result, these two species have been listed as locally
threatened in some regions (Natural Environment Division & Bureau of Environment
TMG, 2010; Natural Environment Division & Department of Environmental Affairs FPG,
2014). In addition, these species are key indicator species for assessing the degree of
disturbance to the natural environment, especially in Japanese traditional rural areas called
“Satoyama” (Kitagawa, 2002). For example, agencies like the Biodiversity Center of
Japan, the Nature Conservation Bureau, and the Ministry of the Environment (e.g.,
Monitoring-Site 1000 project) have conducted ecological monitoring surveys and listed the
occurrence of Rana species in numerous reports. Habitat selection of the three species
differs to some extent, whereby each species inhabits a wide range of microhabitats. Rana
japonica and Rana ornativentris are mainly found in grasslands that range from lowlands
to hillsides; they migrate to marshes, ponds, or paddy fields before the breeding season

Igawa et al. (2019), PeerJ, DOI 10.7717/peerj.7597 2/13

 in winter (Maeda & Matsui, 1999), but Rana ornativentris tends to dwell at higher
elevations than Rana japonica (Kuramoto & Ishikawa, 2000; Osawa & Katsuno, 2001).
Another brown frog species, Rana tagoi tagoi, resembles Rana ornativentris in morphology
and is distributed in hilly areas, sometimes in sympatry with the other two species.
However, Rana tagoi tagoi lay their eggs under the ground on the edges of small mountain
streams during spring.
For development of our new protocol in this study, we adapted the PCR-RFLP method
that recognizes these three Rana species (Igawa et al., 2015) for eDNA detection. In this
method, mitochondrial 16S rRNA fragments were amplified and digested with two
restriction enzymes (SpeI and HphI) to identify species within the samples by
electrophoretic banding pattern of the digested fragments. To evaluate the performance of
our protocol, we attempted to detect eDNA diluted in water samples and compared the
results with visual observation and qPCR methods. To test applicability in various spatial
scales, various water samples were collected from regional levels to microhabitat in
addition to water tank experiments. In particular, we obtained regional samples covering
60 km from Fukushima nuclear power plant (FNPP) in spring and summer seasons
and assessed presence information of Rana tagoi tagoi in habitats recovering after the
disaster in 2011. Finally, we reported the effectiveness of the protocol for distribution
surveys in the field.

MATERIALS AND METHODS

Water tank experiment
To characterize basic performance of our PCR-RFLP assay for eDNA detection, we firstly
conducted model experiments by preparing eight different water tank culture setups with
the following animals: (1) 40 Rana japonica tadpoles for 2 days, (2) 40 Rana japonica
tadpoles for 5 days, (3) nine Rana japonica and one Rana ornativentris adults for 1 day,
(4) one Rana japonica and nine Rana ornativentris adults for 1 day, (5) five Rana japonica
and five Rana ornativentris adults for 1 day, (6) one Rana tagoi tagoi and nine Rana
japonica adults for 1 day, (7) one Rana tagoi tagoi and nine Rana ornativentris adults for
1 day, and (8) negative control (to check the specificity of our method)—no animals for
1 day (but water was used previously for other frog species (Rhacophorus arboreus)).
The tadpoles of Rana japonica were collected from a pond in Hiroshima University
campus in Higashi-Hiroshima city (Hiroshima Prefecture) in April 2016 just before
the experiment and were classified as growth stage 33 according to Tahara (1974)
(equivalent to stage 37 in Gosner (1960)). The adult frogs of Rana japonica, Rana
ornativentris, and Rana tagoi tagoi were collected from Higashi-Hiroshima City in 2014,
Kita-Hiroshima-cho (Hiroshima Prefecture) in 2015, and Kisa, Miyoshi City (Hiroshima
Prefecture) in 2011, respectively, and reared in the Amphibian Research Center,
Hiroshima University, until the start of experiments. We transferred and kept animals in
plastic tanks (450 × 650 ×150 mm) filled with 8 L aged tap water (water level about
30 mm which immersed half body of adults). The animals were not fed during the
experiment and were held at room temperature around 18  C under 12 h:12 h light-dark
cycle. We collected a 500 mL water sample from each tank after the specific periods

Igawa et al. (2019), PeerJ, DOI 10.7717/peerj.7597 3/13

 Figure 1 Sampling localities of field experiments. Sampling localities of field experiments: (A) regional
sampling in Fukushima, (B) local area sampling in Hiroshima University, (C) microhabitat level sam-
pling in Etajima Island. Results of field observation, qRCR and PCR-RFLP detection are indicated,
respectively (X/X/X). +, detected; −, not detected; NA, qPCR not conducted. Photo Credit: Takeshi Igawa.
Maps were projected by using ArcGIS 9.3 based on a 10-m grid digital elevation map, water line and
building outline provided by The Geospatial Information Authority of Japan.
Full-size  DOI: 10.7717/peerj.7597/fig-1

according to the treatments using DNA-free 500 mL bottles, and samples were immediately
transferred to a −30  C freezer. These experiments were conducted in April 2016 for tadpoles
(tanks 1 and 2) and September 2016 for adults (tanks 3–8); all animals were returned to
the original habitat or enclosures under healthy conditions soon after the experiments.
All procedures were approved by the Hiroshima University Animal Research
Committee (Approval number: G17-4) and were carried out in accordance with the
recommendations in the Guide for the Care and Use of Laboratory Animals of the
Hiroshima University Animal Research Committee.

Field survey and experiments

To check the performance and sensitivity of PCR-RFLP assay for eDNA from the field
environment, we collected water samples from ponds and rivers at 28 sites (Fig. 1). For
validation of applicability to monitoring studies in various spatial scales, we collected
samples in three hierarchical levels: (A) regional samples covering 60 km from FNPP
(sample no. 1–20), (B) local area samples from a single body of water in Hiroshima
University campus, Higashi-Hiroshima (sample no. 21–26), and (C) microhabitat level
samples from a single breeding site of Rana japonica in Etajima Island, Hiroshima
Prefecture (N 34.27067, E 132.47679) (sample no. 27 and 28). We collected 500 mL by
filling sodium hypochlorite-treated 500 mL bottles (i.e., DNA-free bottles). Field survey

Igawa et al. (2019), PeerJ, DOI 10.7717/peerj.7597 4/13

 was conducted in spring and summer because the true Rana species are active in these
seasons. Samples from Fukushima (A) were collected during two periods, May 12, 2015 to
May 14, 2015 and August 11, 2015 to August 13, 2015. Samples from Hiroshima
University (B) and Etajima (C) were collected on 11 April and 17 April in 2016,
respectively. To prevent contamination by a field sampler, we began collecting at
downstream sites, and moved upstream as subsequent samples were collected. Sampled
water bottles were transported on ice in a cooling box to the laboratory and stored at −30

C. The subsequent procedure of filtering water samples that were stored frozen was
performed within 3 weeks.
In addition, we recorded the presence and absence of frogs and/or tadpoles by visual
observation, exploring the vicinity of the field sampling sites. Basically, three or four
people conducted field observation for more than 20 min at each site. For sampling in
Fukushima, permission to enter the survey area was obtained from the local governments
(Iitate Vilage (approval number: 2705-01 and 2707-0) and Namine Town (approval
number: 755 and 1959)). Notification of the field survey was accepted by the Iwaki District
Forest Office, and permits were obtained from private landowners as required for each site.

DNA extraction and PCRs

To avoid contamination, we followed a unidirectional lab flow, whereby we performed all
PCR protocols, including preparation/addition of the standards and qPCR cycling, in two
separate rooms (rooms 1 and 2, respectively). To prevent carry-over contamination, no
equipment or samples were returned from room 2 to room 1. In both rooms, laboratory
benches were decontaminated using commercial bleach.
The water samples stored at −30  C were thawed and vacuum-filtered through glass
microfiber filters (GF/F; GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) with 0.7 µm
mesh size, which were used in other amphibian studies (Katano et al., 2017; Iwai,
Yasumiba & Takahara, 2019). Filter funnels and tweezers used in the filtration treatment
were sterilized with 10% commercial bleach (ca. 0.6% hypochlorous acid) for 5 min (i.e.,
sodium hypochlorite treatment), flushed with a large amount of tap water, and then rinsed
with DNA-free distilled water between samples to avoid cross contamination. Filtering
controls (i.e., 500 mL of distilled water) in laboratory experiment controls filtered on each
day of sample filtration. The filter papers were wrapped in new aluminum foil (i.e., DNA-
free), placed in plastic bags, and stored at −30  C. The eDNA was extracted from the filters
according to the methods of Uchii, Doi & Minamoto (2016), using a Salivette tube
(Sarstedt, Nümbrecht, Germany) and a DNeasy Blood and Tissue Kit for DNA purification
(Qiagen, Hilden, Germany). The filters were incubated by submersion in a mixed buffer
(400 µL buffer AL and 40 µL Proteinase K; Qiagen) using a Salivette tube in a dry oven at
56  C for 30 min. The tubes with filters were centrifuged at 5,000×g for 5 min at room
temperature. Then, 220 µL of TE (Tris–EDTA) buffer (pH: 8.0; 10 mM Tris–HCl and
one mM EDTA) was added to the filters, and tubes were centrifuged again at 5,000×g for
5 min. Buffer AL (200 µL) and 100% ethanol (600 µL) were then added to each filtrate
and mixed by pipetting. The mixture was applied to a DNeasy Mini spin column and
centrifuged at 6,000×g for 1 min. This step was repeated until the mixture was completely

Igawa et al. (2019), PeerJ, DOI 10.7717/peerj.7597 5/13

 processed. We followed the manufacturer’s instructions for further steps, and eDNA was
eluted from each sample solution with a final volume of 100 µL Buffer AE.
The eDNA samples were then used for PCR-RFLP assay to detect DNA from the three
target Japanese brown frog species based on Igawa et al. (2015). This method utilizes
species-specific restriction enzyme (SpeI and HphI) digestion sites in a partial nucleotide
fragment of mitochondrial 16S rRNA amplified by PCR. However, the oligonucleotide
primers used by Igawa et al. (2015) could amplify 16S rRNA fragments originating from
other vertebrate species including humans which showed similar banding patterns.
Therefore, we altered the primers to amplify a shorter region in which only the three target
frog species have restriction sites. Specifically, we modified the method of Igawa et al.
(2015) by changing the primers: F96 5′-GTCCAGCCTGCCCAGTGAYAAA-3′ and R19
5′-GTTGAACAAACGAACCATTGGT-3′. These new primers were designed from an
internal region of the previous study and amplify shorter fragments (Rana japonica:
514–517 bp, Rana ornativentris: 516–517 bp, and Rana tagoi tagoi: 514–516 bp).
For higher efficiency of PCR amplification, we also modified the PCR protocol described
by Igawa et al. (2015). In this study, PCR was conducted using KOD FX Neo (TOYOBO,
Osaka, Japan). The 20 µL total volume of reaction solution included 10 µL of 2 × PCR
Buffer for KOD FX Neo, 2 µL of 2 mM dNTPs, 10 pmole of each primer, 3 µL of eDNA
solution and 0.4 U of KOD FX Neo. Thermal cycling was performed using two-step PCR
cycling: 95  C for 3 min followed by 35 cycles of 98  C for 10 s and 65  C for 30 s.
Following PCR amplification, independent digestion using two restrictions enzymes
(SpeI and HphI) and electrophoresis were conducted in the same manner as Igawa
et al. (2015). Then, 5 µl of the original PCR product or 15 µl of each PCR product digested
with the two restriction enzymes were electrophoresed on a 2% agarose gel for 30 min at
100 V and visualized. We performed the experiment several times to confirm the
reproducibility.
For Rana japonica, the amplified fragments are expected to have different digestion
patterns between the western (including Hiroshima) and northern part (including
Fukushima) of the Japanese mainland (Igawa et al., 2015). In all Rana japonica
populations, amplicons digested with SpeI result in 255 and 259 bp subfragments.
However, after Hph1 digestion, Rana japonica frogs from the western part have no
subfragments, while those from the northern part have 301 and 220 bp subfragments
(Fig. 2A). For Rana ornativentris and Rana tagoi tagoi, amplified fragments are commonly
digested only with HphI, resulting in 231, 199, and 86 bp subfragments and 313 and 200 bp
subfragments, respectively (Fig. 2A). To confirm the specificity of eDNA detection by
our PCR-RFLP method, PCR products of sample no. 1, 10, 11, 14, 17, 19, and 20 were
directly sequenced using the same primers and BigDye Terminator ver 3.1 (Life
Technologies, Carlsbad, CA, USA) after PEG precipitation. The obtained nucleotide
sequences were annotated by NCBI blastn (https://blast.ncbi.nlm.nih.gov).

qPCR assay
To compare sensitivity between the methods, we conducted real-time qPCR assay for
samples from Fukushima to compare the sensitivity of eDNA detection for Rana tagoi

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 Figure 2 PCR-RFLP and gel electrophoresis expected banding patterns (A) and results of water tank
experiment (B) and field experiment (C). Full-size  DOI: 10.7717/peerj.7597/fig-2

Igawa et al. (2019), PeerJ, DOI 10.7717/peerj.7597 7/13

 tagoi. At first, we defined regions to design the primers and TaqMan MGB probes for the
qPCR assays for this species using PrimerExpress 3.0.1 (Thermo Fisher Scientific,
Carlsbad, CA, USA), for amplification of 106 bp fragments: Rtag16SrRNA-F, 5′-
AGAAGGAACTCGGCAAACCTT-3′; Rtag16SrRNA-R, 5′-CCGCGGCCGTTGAAT-3′;
Rtag16SrRNA-Pr, 5′-[FAM]-CCAGCCTGCCCAGTG-[NFQ]-[MGB]-3′. To prove that
the primers did not amplify other sympatric frog species, we confirmed no amplification
when the primers were applied to DNA extracted from Rana japonica and Rana
ornativentris tissues. However, in addition to Rana tagoi tagoi, three closely related species
(i.e., Rana sakuraii, Rana tagoi okiensis, and Rana tagoi yakushimensis) were also detected
in an in silico specificity screen, which was performed using Primer-BLAST, with the
setting “nr” for database and “vertebrates” for organisms parameters (http://www.ncbi.
nlm.nih.gov/tools/primer-blast/). Rana sakuraii may inhabit sympatrically with Rana
tagoi tagoi in our field survey. In contrast, Rana tagoi okiensis and Rana tagoi
yakushimensis never share distribution with our target species because they are only
found on Oki island in Shimane Prefecture and the Yakushima islands in Kagoshima
Prefecture, respectively. Thus, in this study, the qPCR primers we developed are specific
for Rana tagoi tagoi/Rana sakuraii.
Environmental DNA was quantified using a StepOnePlusTM Real-Time PCR system
(Life Technologies, Carlsbad, CA, USA). Each TaqMan reaction contained 10 µL of TaqManÒ
Environmental Master Mix 2.0 (Thermo Fisher Scientific, Carlsbad, CA, USA), 1 µL
of the primer (900 nM)/probe (125 nM) mix, 7 µL of distilled water, and 2 µL of eDNA
extract. The PCR cycles were as follows: 2 min at 50  C, 10 min at 95  C, then 55 cycles of
15 s at 95  C, and 60 s at 60  C. In order to produce standard DNA for the qPCR, a
target amplicon was inserted into a pMD20-T vector (Takara Bio, Shiga, Japan), and the
vector was digested with BamHI. A dilution series of the plasmid containing 1 × 101 to 1 ×
104 copies was amplified as standards in duplicates in all qPCR assays. The qPCR for
each sample was performed in eight wells, and the mean was used as the concentration
of eDNA (copies/L). If any of the eight replicates of each sample yielded a positive result,
the sample was designated as containing Rana tagoi tagoi/Rana sakuraii eDNA. As a
negative control, each qPCR assay included eight wells that contained no template (2 µL of
DNA-free water) (i.e., no template control (NTC)). In addition, using the primer-probe set
of this species, qPCR amplicons were sequenced directly from a positive PCR of the
field samples (N = 6) after treatment with ExoSAP-IT (USB Corporation, Cleveland, OH,
USA) or DNA fragment isolation with FastGeneTM Gel/PCR Extraction Kit (NIPPON
Genetics, Tokyo, Japan) following agarose gel electrophoresis. Products were sequenced by a
commercial sequencing service (Takara Bio, Shiga, Japan) or by our experiment using
ABI3130xl automated sequencer (Thermo Fisher Scientific, Carlsbad, CA, USA).
The range of qPCR efficiency across the entire study, calculated from the slope of
standard curves, was 91.937–94.716%, and the R2 value for standard curve was 0.997.
Since we could detect two copies of DNA in at least one of the three replicates, we defined
the limit of detection (LOD, the lowest concentration where all triplicate samples
registered a Ct value) for DNA from Rana tagoi tagoi using qPCR assay as two copies.

Igawa et al. (2019), PeerJ, DOI 10.7717/peerj.7597 8/13

 RESULTS
Water tank experiments
For the eDNA solutions from water tank experiments, we successfully amplified 16S rRNA
fragments from all samples, each showing a single band following gel electrophoresis
(Fig. 2B). The band patterns of digested fragments in each sample were completely
matched with the species that were held in each tank, except for the negative control tank
without animals (Fig. 2B). Notably, for the samples from water tanks in which two species
were cultured, we could simultaneously amplify eDNA from both species whereby both
band patterns specific to two species were displayed.

Field survey and experiments

In the survey in Fukushima (A), field observation of Rana tagoi tagoi adults were
recorded in 15 of 20 sites (Fig. 1; Table S1). Among the sites where Rana tagoi tagoi
adults were observed, adults of Rana ornativentris were also observed sympatrically in five
sites (Table S1). However, we could not identify presence of either Rana tagoi tagoi or
Rana ornativentris by PCR-RFLP analyses of eDNA from any of the samples from
Fukushima (Fig. S1). Subsequently, we applied the more sensitive Rana tagoi tagoi/
Rana sakuraii-specific qPCR which could still only confirm presence of Rana tagoi
tagoi in five of the 15 sites with field observations of adults of the species (Fig. 1;
Table S1). Our direct sequences of the qPCR amplicons (106 bp product size) were
almost identical to haplotypes of Rana tagoi tagoi deposited in DNA Data Bank of Japan
(DDBJ). In addition, no amplification was observed in any control samples (i.e., filtering
controls and NTC).
We could obtain clear sequence data of PCR products from site no. 1 and 21, while
the others showed mixed chromatograms that could not be further interpreted. In
Fukushima site no. 1, we also reported field observation of Bufo japonicus tadpoles, a
species outside of our target species. PCR amplification of eDNA from the same site
resulted in an approximately 600 bp fragment; subsequent sequencing and BLAST
search confirmed a match of nucleotide sequence with partial sequence of B. japonicus
(= B. j. formosus) 16S rRNA (AB159565, a haplotype from Atsumi, Yamagata Pref.
(Igawa et al., 2006)).
From field experiment (B) and (C), field observation of Rana japonica tadpoles was
recorded for three sites (no. 21, 22, and 27; Fig. 1; Table S1). This was consistent with our
PCR-RFLP which showed Rana japonica-specific banding patterns following SpeI
restriction digestion in the same samples (Fig. 2C). We also confirmed the sequence from
site no. 21 had BLAST identity with partial sequence of 16s rRNA from Rana japonica
(LC014155, a haplotype from Hiroshima University (Igawa et al., 2015)). In addition,
PCR-RFLP analyses of site no. 28 supports the presence of Rana japonica, despite no field
observation of tadpoles at this small drainage site. We also confirmed amplification of a
fragment that may be amplified from non-target species in site no. 21, 22, 27, and 28. These
fragments were digested with HphI and showed weak bands around 400 and/or 300 bp
that were different from the target species.

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 DISCUSSION
As shown in our water tank experiment, eDNA of Rana species was successfully amplified
and we could even simultaneously identify each pair of the three species using PCR-RFLP
method. Following success of this newly developed protocol, we applied it to a variety
of field collected water samples.
In field experiment (A) at Fukushima, despite frogs being identified in 15 of 20 sites by
field observation, we could not detect eDNA of Rana tagoi tagoi or Rana ornativentris
using our PCR-RFLP method. Even when we applied the more sensitive qPCR approach
for detecting Rana tagoi tagoi, this method only confirmed presence of the species in five of
the sites. There are differences in detection sensitivity across PCR-RFLP and qPCR
methods: qPCR is rather sensitive compared to conventional PCR in this case. In addition,
the more expensive approaches like digital PCR and NGS of eDNA could prove to be more
sensitive, despite the hurdles associated with cost.
The lack of detection by PCR-RFLP and limited sensitivity by qPCR could be attributed
to the water system of the environment. Such factors like water volume, water discharge
and flow rate could markedly impact the concentration of eDNA. It seems that flowing
waters of lotic habitats, such as in rivers or streams, could have lower eDNA
concentrations due to dilution from fresh water upstream. In particular, while only 500 mL
water samples were collected in this study, previous studies extracted eDNA from more
than one L of water (Pilliod et al., 2013; Baldigo et al., 2017). Such difference in water
sampling volume might be associated with the lack of eDNA detection. In the future,
increasing the volume of water collection might improve eDNA detection power.
In addition, Rana tagoi tagoi adults and their spawned tadpoles utilize small torrents or
subsoil water systems only in the breeding season. According to Maeda & Matsui (1999),
Rana tagoi tagoi breed during late June in colder regions of Japan like Fukushima; our
sample sessions occurred at both before and after this breeding period and thus could
result in low level of eDNA in the water. Overall, the detection of Rana species from river
water samples could potentially be challenging compared to static water samples, and
we need to select the appropriate time of sampling considering ecology and behavior of
the target species. Nevertheless, if future optimization can overcome these limitations,
there is potential to apply such approaches to effectively monitor distribution of frogs in
regenerating habitats in vicinity to the FNPP.
We also applied PCR-RFLP to static water samples collected in the field. From the eight
sites in field experiments (B) and (C), PCR-RFLP could successfully detect eDNA in all
three sites where Rana japonica tadpoles were visually observed. An additional site, site
no. 28, had no tadpoles observed in field study, but showed some evidence of presence
from PCR-RFLP analyses. This site is a small drainage site with a small volume and
water flowing upstream from a paddy field. Thus, it seems possible that PCR-RFLP can
detect presence of frogs that may inhabit upstream or in close proximity, even in the
absence of visual confirmation. When we compare this site of flowing water to that of
sites in Fukushima, the water volumes and flow rate are much smaller and slower,
respectively.

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 CONCLUSION
We have successfully developed a cost-effective method for detecting three common Rana
frog species from Japan. This approach works well in field-collected samples that originate
from static or slow-flowing water systems. However, our approach cannot be applicable
to lotic water systems. Further exploration of alternative cost-effective methods are
required, and could include collection of larger volumes of water and experimental
assessment of the impact of various factors like water volume and flow rate on eDNA
concentration. Nevertheless, this method has potential to be used to monitor frog
populations that are facing decline due to urbanization, especially Rana japonica which
inhabits static water bodies like rice paddy fields. Our approach also has potential to be
adapted and optimized for detection of other amphibian species from field sites.

ACKNOWLEDGEMENTS
We thank the Department of Molecular and Functional Genomics, Interdisciplinary
Center for Science Research, Organization for Research and Academic Information,
Shimane University for providing the DNA laboratory facilities. All methods adhered to
the laws of Japan.

ADDITIONAL INFORMATION AND DECLARATIONS

Funding
This work was supported by a Scientific Research Grant from the Kurita Water, Japan and
Environment Foundation Grant, by the Environment Research and Technology
Development Fund of the Ministry of the Environment (4RF-1302), and Environmental
Restoration and Conservation Agency (4-1503 and 4-1602), Japan, and the faculty of Life
and Environmental Science in Shimane University, Japan provided financial support
towards publishing this study. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.

Grant Disclosures
The following grant information was disclosed by the authors:
Scientific Research Grant from the Kurita Water, Japan.
The Environment Research and Technology Development Fund of the Ministry of the
Environment (4RF-1302), and Environmental Restoration and Conservation Agency
(4-1503 and 4-1602), Japan.
Faculty of Life and Environmental Science in Shimane University, Japan.

Competing Interests
The authors declare that they have no competing interests.

Author Contributions
 Takeshi Igawa conceived and designed the experiments, performed the experiments,
analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or
tables, authored or reviewed drafts of the paper, approved the final draft.

Igawa et al. (2019), PeerJ, DOI 10.7717/peerj.7597 11/13

  Teruhiko Takahara conceived and designed the experiments, performed the
experiments, analyzed the data, contributed reagents/materials/analysis tools, authored
or reviewed drafts of the paper, approved the final draft.
 Quintin Lau performed the experiments, authored or reviewed drafts of the paper,
approved the final draft.
 Shohei Komaki performed the experiments, approved the final draft.

Animal Ethics
The following information was supplied relating to ethical approvals (i.e., approving body
and any reference numbers):
Hiroshima University Animal Research Committee provided full approval for this
research (Approval number: G17-4).

Field Study Permissions

The following information was supplied relating to field study approvals (i.e., approving
body and any reference numbers):
Permission to enter the survey area was obtained from the Iitate Vilage (approval
number: 2705-01 and 2707-0) and Namine Town (approval number: 755 and 1959).

Data Availability
The following information was supplied regarding data availability:
The raw electrophoresis images are available in the Supplemental Files.

Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
peerj.7597#supplemental-information.

REFERENCES
Baldigo BP, Sporn LA, George SD, Ball JA. 2017. Efficacy of environmental DNA to detect and
quantify brook trout populations in headwater streams of the Adirondack Mountains,
New York. Transactions of the American Fisheries Society 146(1):99–111
DOI 10.1080/00028487.2016.1243578.
Biggs J, Ewald N, Valentini A, Gaboriaud C, Dejean T, Griffiths RA, Foster J, Wilkinson JW,
Arnell A, Brotherton P, Williams P, Dunn F. 2015. Using eDNA to develop a national citizen
science-based monitoring programme for the great crested newt (Triturus cristatus). Biological
Conservation 183:19–28 DOI 10.1016/j.biocon.2014.11.029.
Buxton AS, Groombridge JJ, Zakaria NB, Griffiths RA. 2017. Seasonal variation in
environmental DNA in relation to population size and environmental factors. Scientific Reports
7(1):1–9 DOI 10.1038/srep46294.
Clusa L, Ardura A, Fernández S, Roca AA, García-Vázquez E. 2017. An extremely sensitive
nested PCR-RFLP mitochondrial marker for detection and identification of salmonids in eDNA
from water samples. PeerJ 5(1):e3045 DOI 10.7717/peerj.3045.
Goldberg CS, Strickler KM, Pilliod DS. 2015. Moving environmental DNA methods from
concept to practice for monitoring aquatic macroorganisms. Biological Conservation 183:1–3
DOI 10.1016/j.biocon.2014.11.040.

Igawa et al. (2019), PeerJ, DOI 10.7717/peerj.7597 12/13

 Gosner KL. 1960. A simplified table for staging anuran embryos larvae with notes on identification.
Herpetologica 16:183–190.
Igawa T, Komaki S, Takahara T, Sumida M. 2015. Development and validation of PCR-RFLP
assay to identify three Japanese brown frogs of the true frog genus Rana. Current Herpetology
34(1):89–94 DOI 10.5358/hsj.34.89.
Igawa T, Kurabayashi A, Nishioka M, Sumida M. 2006. Molecular phylogenetic relationship of
toads distributed in the Far East and Europe inferred from the nucleotide sequences of
mitochondrial DNA genes. Molecular Phylogenetics and Evolution 38(1):250–260
DOI 10.1016/j.ympev.2005.09.003.
Iwai N, Yasumiba K, Takahara T. 2019. Efficacy of environmental DNA to detect and quantify
stream tadpoles of Odorrana splendida. Royal Society Open Science 6(1):181798
DOI 10.1098/rsos.181798.
Katano I, Harada K, Doi H, Souma R, Minamoto T. 2017. Environmental DNA method for
estimating salamander distribution in headwater streams, and a comparison of water sampling
methods. PLOS ONE 12(5):e0176541 DOI 10.1371/journal.pone.0176541.
Kitagawa Y. 2002. Regeneration of Satoyama landscapes. In: Takeuchi K, Brown RD, Washitani I,
Tsunekawa A, Yokohari M, eds. Satoyama: The Traditional Rural Landscape of Japan. Tokyo:
Springer-Verlag, 129–142.
Kuramoto M, Ishikawa H. 2000. Breeding ecology of brown frogs in Yamada park, Kitakyushu,
Japan. Bulletin of the Herpetological Society of Japan 2000:7–18.
Maeda N, Matsui M. 1999. Frogs and toads of Japan. Tokyo: Bunichi Sogo Shuppan.
Natural Environment Division, Bureau of Environment TMG. 2010. 2010 red list of threatened
species in Tokyo: 23-ward and Tama area version. Tokyo: Natural Environment Division, Bureau
of Environment, Tokyo Metropolitan Government.
Natural Environment Division, Department of Environmental Affairs FPG. 2014. Endangered
species in Fukuoka: red data book Fukuoka 2014. Fukuoka: Natural Environment Division,
Depertment of Environmental Affairs, Fukuoka Prefectural Government.
Olson ZH, Briggler JT, Williams RN. 2012. An eDNA approach to detect eastern hellbenders
(Cryptobranchus a. alleganiensis) using samples of water. Wildlife Research 39(7):629
DOI 10.1071/WR12114.
Osawa S, Katsuno T. 2001. Dispersal of brown frogs Rana japonica and R. ornativentris in the
forests of the Tama Hills. Current Herpetology 20(1):1–10 DOI 10.5358/hsj.20.1.
Pilliod DS, Goldberg CS, Arkle RS, Waits LP. 2013. Estimating occupancy and abundance of
stream amphibians using environmental DNA from filtered water samples. Canadian Journal of
Fisheries and Aquatic Sciences 70(8):1123–1130 DOI 10.1139/cjfas-2013-0047.
Rees HC, Maddison BC, Middleditch DJ, Patmore JRM, Gough KC. 2014. 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.
Tahara Y. 1974. Table of the normal developmental stages of the frog, Rana japonica. Memoirs of
Osaka Kyoiku University III, Natural Science and Applied Science 23:33–53.
Takahara T, Minamoto T, Doi H. 2013. Using environmental DNA to estimate the distribution of
an invasive fish species in ponds. PLOS ONE 8(2):e56584 DOI 10.1371/journal.pone.0056584.
Thomsen PF, Willerslev E. 2015. Environmental DNA—an emerging tool in conservation for
monitoring past and present biodiversity. Biological Conservation 183:4–18
DOI 10.1016/j.biocon.2014.11.019.
Uchii K, Doi H, Minamoto T. 2016. A novel environmental DNA approach to quantify the cryptic
invasion of non-native genotypes. Molecular Ecology Resources 16(2):415–422
DOI 10.1111/1755-0998.12460.

Igawa et al. (2019), PeerJ, DOI 10.7717/peerj.7597 13/13