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Abstract

Invasive species have a significant negative impact on the environment where they have been introduced, and amphibians are among some of worse invasives . All known amphibian introductions are linked to human activities, generally in relation with pest control or food provisioning. This is the case of all amphibians on Ulleung Island, Republic of Korea, which were originally and mistakenly thought to be “reintroduced” after extirpation, or introduced for specific but non-realised purposes such as food provisioning and pest-control. We conducted call and visual encounter surveys on all valleys of Ulleung Island, Republic of Korea, in April and May 2021 to detect the presence of amphibian species. The call surveys and subsequent call analyses revealed the presence of two independent populations of Pelophylax nigromaculatus, and encounter surveys resulted in the sampling of Rana tadpoles identified as Rana huanrenensis with molecular tools. These results highlight the presence of these two species at low density on this island, but do not provide data on the impact of their presence.
BioInvasions Records (2022) Volume 11, Issue 1: 278–2
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Bae et al. (2022), BioInvasions Records 11(1): 278286, https://doi.org/10.3391/bir.2022.11.1.28 278
CORRECTED PROOF
Rapid Communication
Record of invasive Rana huanrenensis Fei, Ye, and Huang, 1990 and
Pelophylax nigromaculatus (Hallowell, 1861) on Ulleung Island, Republic of Korea
Yoonhyuk Bae1, Jongsun Park2,3, Siti N. Othman1, Yikweon Jang4 and Amaël Borzée1,*
1Laboratory of Animal Behaviour and Conservation, College of Biology and the Environment, Nanjing Forestry University, Nanjing, Peoples
Republic of China.
2InfoBoss Inc., room 301, 670, Seolleung-ro, Gangnam-gu, Seoul, Republic of Korea
3InfoBoss Research Center, 301 room, 670, Seolleung-ro, Gangnam-gu, Seoul, Republic of Korea
4Department of Life Science and Division of EcoScience, Ewha Womans University, Seoul, Republic of Korea
*Corresponding author
E-mail: amaelborzee@gmail.com
Abstract
Non-native species have a significant negative impact on the environment where
they have been introduced, and amphibians are among some of worse invasives. All
known amphibian introductions are linked to human activities, generally in relation
with pest control or food provisioning. This is the case of all amphibians on Ulleung
Island, which were originally and mistakenly thought to be “reintroduced” after
extirpation, or introduced for unrealised purposes such as food provisioning and
pest-control. We conducted call and visual encounter surveys in all valleys of
Ulleung Island, Republic of Korea, in April and May 2021 to detect the presence of
amphibian species. The call surveys and subsequent call analyses revealed the
presence of two geographically independent populations of Pelophylax nigromaculatus,
and encounter surveys resulted in the sampling of Rana tadpoles identified as Rana
huanrenensis with molecular tools. These results highlight the presence of these
two species at low density on this island, but do not provide data on the impact of
their presence.
Key words: brown frog, pond frog, Northeast Asia, invasive species, amphibian
Introduction
Invasive species are one of the prime causes of biodiversity loss because of
their negative impact on native species (Kiesecker et al. 2001; Mayer et al.
2015). Within amphibians, the Cane toad (Rhinella marina (Linnaeus,
1758)), the African clawed frog (Xenopus laevis (Daudin, 1802)), the Asian
black-crested toad (Duttaphrynus melanostictus (Schneider, 1799)) and the
American bullfrog (Lithobates catesbeianus (Shaw, 1802)) are among the
worse, and most widespread, invasive species (Andersen et al. 2021). Their
presence threaten the survival of other species, as for instance L. catesbeianus
is threatening Rana draytonii Baird & Girard, 1852 (Lawler et al. 1999) in
North America and Dryophytes flaviventris Borzée & Min, 2020 (Borzée et
al. 2020b) in the Republic of Korea, among many species (Li et al. 2011; da
Silva Silveira and Guimarães 2021). The blame should however not be cast
Borzée A (2022) Record of invasive
Fei, Ye, and Huang,
and Pelophylax nigromaculatus
on Ulleung Island,
. BioInvasions Records
(1): 278286, https://doi.org/10.3391/bir.
2 September 2021
26 November 2021
24 January 2022
Yik Hei Sung
Stelios Katsanevakis
© Bae et al.
Attribution 4 .0 International - CC BY 4.0) .
OPEN ACCESS.
Invasive amphibians on Ulleung Island
Bae et al. (2022), BioInvasions Records 11(1): 278286, https://doi.org/10.3391/bir.2022.11.1.28 279
on species exploiting niches made available by human activities, but on the
decision made to introduce the species. These introduction are most
generally linked to pest control, for instance R. marina in Australia (Taylor
and Edwards 2005) and Glandirana rugosa (Temminck & Schlegel, 1838)
in Hawaii, USA, (Kraus et al. 1999), or food provisioning (Groffen et al. 2019).
In the Republic of Korea, the worse invasive amphibian is L. catesbeianus
(Groffen et al. 2019), although there are also records of D. melanostictus
(Othman et al. 2020a) and X. laevis, both linked to the trade (Borzée et al.
2021). In addition, the situation could get worse due to the presence of an
extremely large number of amphibian species in the pet trade (Koo et al.
2020). This is a possibly disastrous situation (Borzée et al. 2020a), although
potentially addressed by the request for updated regulations in the import
of non-native species (law case proposition number 7177).
Ulleung Island is a young volcanic island dating back to the late-
Pliocene and early Holocene (Kim and Lee 2008). No amphibian species is
native to the island, but this knowledge was not yet determined when
Pelophylax nigromaculatus (Hallowell, 1861) was erroneously “re”-introduced
in 1937 (Shin et al. 1996), and potentially introduced in 2013 with a batch
of Rana sp. as the species was still commonly referred to as “Rana
nigromaculata” at that time. In addition, other amphibians have been
introduced over time, and other species have been recorded but their origin
is unknown, such as Dryophytes japonicus (Günther, 1859) (Oh 2001).
The lack of knowledge on the non-native status of amphibians of
Ulleung Island is easily overcome through the grey literature, and for
instance Ulleung county office released around 4,500 Rana sp. individuals
in the Okcheon Stream for a restoration project in 2013 (Kim 2013). The
fate of the reintroduced individuals was however not monitored and most
frogs are expected to have been predated due to the high density of avian
predators (Shin et al. 1996). We conducted call and visual encounter
surveys on Ulleung Island in April and May 2021 to determine the
occurrence status of early breeding amphibian species on the island, and
employed acoustic and molecular analyses to identify the species.
Materials and methods
Field surveys
We conducted acoustic and visual encounter surveys between 28 April and
2 May 2021. We surveyed all streams and water bodies visible from satellite
views, and conducted at least one survey in each valley (Figure 1). Surveys
focused on habitat suitable for amphibians based on maps and satellite
views (google.com/maps; map.naver.com). We spent a minimum of 10
min listening for calling activity at each of the wetlands. We recorded all
calling individuals with a linear PCM recorder (Tascam DR-40; California,
Invasive amphibians on Ulleung Island
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Figure 2. Map of amphibian surveys conducted on Ulleung Island between 28 April and 2 May
2021. Surveys were conducted in the form or acoustic and visual encounters. Map created with
arcGIS online (www.arcgis.com). Imagery layer from Earthstar Geographics.
USA) linked to a unidirectional microphone (Unidirectional electret
condenser microphone HT-81, HTDZ; Xi’an, China). We could not collect
any of the individuals heard calling.
We conducted visual encounter surveys during both day time and night
time to maximise the chance of finding amphibians. We focused on
finding eggs or tadpoles during daytime, and adults during night time. We
did not find any adults, but collected three Rana tadpoles that we tail-
clipped following a least invasive method (Othman et al. 2020b). All
individuals were captured under the permit 2021-1 issued by Ulleung
county, allowing for capture of three Rana huanrenensis Fei, Ye & Huang,
1990 or Rana uenoi Matsui, 2014 for identification. All individuals were
released at the point of capture following the sampling procedure.
Call analyses
Prior to data extraction, we filtered out the background noises at 1 kHz,
and the set the spectrogram configuration at a 256-sample Hann window
size, 128-sample hop size with 50% frame overlap and 172-Hz frequency
grid spacing. We analysed each call for both temporal and spectral
domains (Raven Pro 1.4; Cornell Lab of Ornithology, New York, USA),
following the recommendations of Koehler et al. (2017).
Molecular analyses
Three Rana sp. tadpoles were collected on 29 April 2021 in Ulleung county
(37.472778 °N; 130.848611 °E). We extracted the total DNA from the tail
Invasive amphibians on Ulleung Island
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Table 1. Information of samples used for phylogenetic tree reconstruction.
Taxa
Locality
Accession number
Reference
Rana amurensis Liaoning, Zhangwu, China KU343216
Zhao et al. 2016
(direct submission to GenBank)
Rana coreana Republic of Korea KM590550
Jang and Hwang 2015
(direct submission to GenBank)
Rana chensinensis
Yellow Sea basin of North China
NC_023529
Li et al. (2014)
Rana kukunoris
Helan Mountains, Inner Mongolia, China
MN733918
Wang et al. 2020
Rana huanrenensis
(Ulleung Island)
Ulleung Island, Republic of Korea MZ779046 This study
Rana huanrenensis
(Northern China)
Huanren, Liaoning in China KT588071 Dong et al. (2016)
Rana omeimontis
Yucheng District, Sichuan, China
KU246050
Yang et al. 2018
Rana uenoi Republic of Korea MW009067
Suk and Min 2020
(direct submission to GenBank)
Rana dybowskii
Amur River basin of Northeast China
KF898355
Li et al. (2014)
clip of one individual (voucher number: 20RXV003) using the DNeasy
Blood & Tissue Kit (QIAGEN, Hilden, Germany) according to manufacturer
protocol to sequence the complete mitogenome of the individual. We then
constructed the sequencing library using Illumina TruSeq DNA PCR-Free
Library Preparation Kit (Illumina, San Diego, CA) following the manufacturer’s
recommendations with around 350-bp DNA fragments. We obtained a
total of 5.3 Gbp raw sequences from Illumina NovaSeq6000 at Macrogen
Inc., Korea, and we filtered the raw sequences using Trimmomatic v0.33
(Bolger et al. 2014). We then conducted the de novo assembly and confirmed
with Velvet v1.2.10 (Zerbino and Birney 2008), SOAPGapCloser v1.12
(Zhao et al. 2011), BWA v0.7.17 (Li et al. 2009), and SAMtools v1.9 (Song
and Liang 2013) under the environment of Genome Information System
(GeIS; http://geis.infoboss.co.kr) which has been used in previous studies
(Kim et al. 2021; Jung et al. 2021; Lee et al. 2020). We used Geneious R11
v11.1.5 (Biomatters Ltd, Auckland, New Zealand) to transfer the
annotation of the assembled mitogenome based on alignments with
another Rana huanrensis mitochondrial genome (NC_028521; Dong et al.
2016) and MITOS (Bernt et al. 2013). We deposited the sequence to the
GenBank database under the accession number MZ779046.
Phylogenetic analysis
Here, we determined the species identity using phylogenetic analyses. We
reconstructed a phylogenetic tree based on the complete mitochondrial
sequence of our Rana sample (19,146 bp; GenBank accession number:
MZ779046) with eight homologous sequences from Rana sp. distributed
across Northeast Asia (total n taxa = 9; Table 1). We aligned the
mitogenomes sequences using ClustalW2 (Larkin et al. 2007). To remove
the unnecessary gaps, we trimmed both ends of the aligned sequences and
obtained a final length of 13,546 kb for the aligned sequences. To infer the
phylogenetic relationship, we searched for the best-fit evolutionary model
using bModeltest v2.0 (Bouckaert and Drummond 2017). Here, we predicted
Invasive amphibians on Ulleung Island
Bae et al. (2022), BioInvasions Records 11(1): 278286, https://doi.org/10.3391/bir.2022.11.1.28 282
Figure 2. Call recording of Pelophylax nigromaculatus from Ulleung Island, Republic of Korea,
recorded in April 2021.
the model K80/HKY to be the best-fit evolutionary model for our full
length mtDNA dataset with a posterior probability of 74.15%. We
reconstructed a maximum clade credibility (MCC) phylogenetic tree using
BEAST v2.6.3 (Bouckaert et al. 2019). We then performed two independent
analyses of the tree following the Markov Chain Monte-Carlo (MCMC)
procedure for 20 million iterations. We ensured the adequacy of the
MCMC samplings by assessing the effective sample size (ESS) values of
each parameter (ESS > 200) using Tracer v1.7 (Rambaut et al. 2018). We
assembled all the generated trees in LogCombiner v2.6.1 (Bouckaert et al.
2019), and summarized the MCC tree after discarding 25% of the trees with
a posterior probability limit of 0.5 using Tree Annotator v2.6.3 (Bouckaert
et al. 2019). We visualised the MCC tree using Fig tree v.1.4.3 (Rambaut 2009).
Results
We assigned the calls we recorded (37.464155 °N; 130.869171 °E and
37.517096 °N; 130.815914 °E; Figure 1) to Pelophylax nigromaculatus based
on the analyses (Figure 2). Determining the identity of the species is
straight forward with call properties, but determining the population of
origin, potentially from Japan or the Korean mainland, is impossible at the
moment due to the absence of a database comparable to the one existing
for genetic information.
We collected three tadpoles (37.472778 °N; 130.848611 °E; Figure 1) that
were identified in the field as belonging to the Rana genus, in a habitat
similar to what would be expected for the genus within its native range
(Figure 3). Sequence similarity through BLAST showed a 98.58% match with
Invasive amphibians on Ulleung Island
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Figure 3. Pictures of the Rana huanrenensis individuals sampled in Ulleung Island, Republic of Korea in April 2021. (A) group
picture in situ; (B) in aquarium at the site; (C) individual in situ. Photographs by YB and AB.
Figure 4. Maximum clade credibility (MCC) tree showing the phylogenetic placement of Rana
huanrenensis from Ulleung Island, Republic of Korea (in red) inferred from the complete
mitochondrial genome. Node values indicate the Bayesian posterior probability (BPP).
Rana huanrenensis. Phylogenetic analyses the full mitogenome clustered
our sample with R. huanrenensis and showed a sister relationship between
our samples and the seven other Rana (Figure 4). The MCC tree strongly
supported the identity of the sample R. huanrenensis, within a monophyletic
clade (posterior probability (PP = 1.0) including the most closely related
northeast Asian Rana such as R. kukunoris and R. chensinensis (Figure 4).
The present of a shallow divergence between the two R. huanrenensis
(BPP = 1.0; Figure 4) indicated the recent introduction of the species.
Invasive amphibians on Ulleung Island
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Discussion
Our records of Rana huanrenensis tadpoles, the first time the species is
identified on the island, and the calls of Pelophylax nigromaculatus clarified
the presence of these two species on Ulleung Island. In addition, although
putatively in the case of P. nigromaculatus, the two species are breeding on
the island and therefore maintaining populations, which by their presence
have an impact on the local ecology of species. The extent of their influence
is however unknown and warrants additional research.
While originally laudable, the “re”-introduction of “extinct” Rana species
on Ulleung island instead resulted in the introduction of an non-native
species. The species was found to be restricted to small streams at
comparatively lower elevation, although not all streams could be surveyed on
their totality and the presence of populations at higher elevations is possible.
The case of P. nigromaculatus is different as the presence of the species
on the island was expected. The species was introduced for pest-control of
crops, and opportunistically to provide food. The absence of large scale
natural or agricultural wetlands should have been a predictor of the success
of the species’ introduction for pest control. The species is not currently
widespread, and we detected it in two small wetlands only, one of them
being a fish pond, and therefore sub-optimal for the species. There two
populations are likely to be independent as they are separated by landscape
barriers and a distance generally considered greater than what the species
is able to disperse. We recommend conducting a genetic analysis to
confirm the origin of the populations as they could originate from either
the Japanese archipelago or the Korean mainland.
Current available data lists 242 insect species on Ulleung Island, among
which 35 are endemics (Lim and Lee 2012). While the impact of the
presence of the two anuran species cannot be assessed from the data
available here, the species will clearly have access to niches that were not
occupied before their introduction, with unknown impacts on local
species. We recommend the implementation of a monitoring program to
ensure that the non-native but established species do not spread further
than their current presence, and potentially plan their eradication before
the populations extend beyond control. It is also possible that these are
remnant populations from the introduced individuals, and that their
numbers are slowly declining, a likely hypothesis for P. nigromaculatus.
Acknowledgements
Thank you to the anonymous reviewers for improving the quality of our manuscript.
Funding declaration
This work was supported by the Foreign Youth Talent Program (QN2021014013L) from the
Ministry of Science and Technology to AB and partially supported by a grant from the Korea
Environmental Industry & Technology Institute (KEITI 2021002270001) to YJ.
Invasive amphibians on Ulleung Island
Bae et al. (2022), BioInvasions Records 11(1): 278286, https://doi.org/10.3391/bir.2022.11.1.28 285
Ethical statement
The experiments conducted in this manuscript were approved by Nanjing Forestry University.
Authors’ contribution
Research conceptualization YB, AB; sample design and methodology YB, JP, SNO, AB;
investigation and data collection YB, SNO, AB; data analysis and interpretation YB, JP,
SNO, AB; funding provision YJ, AB; writing (original draft) YB, AB; writing (review and
editing) YB, JP, SNO, YJ, AB.
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