The origin of invasion of an alien frog species in
, Liqing FAN
, Conghui LIU
, Jiaqi LI
, and Yiming LI
Institute of Zoology, Chinese Academy of Sciences, Key Laboratory of Animal Ecology and Conservation Biology,
Beijing 100101, China,
Institute of Plateau Ecology, Tibet Agriculture and Animal Husbandry College, Bayi Town,
Linzhi County, Xizang Province 860000, China,
National Forest Ecosystem Observation & Research Station of Tibet
Linzhi, Linzhi 860000, China,
University of Chinese Academy of Sciences, Beijing 100049, China, and
Institute of Environmental Sciences under Ministry of Environmental Protection of China, Nanjing 210042, China
These authors contributed equally to this work.
*Address correspondence to Yiming Li. E-mail: email@example.com.
Received on 18 July 2016; accepted on 10 December 2016
Identifying the origins of alien species has important implications for effectively controlling the
spread of alien species. The black-spotted frog Pelophylax nigromaculatus, originally from East
Asia, has become an alien species on the Tibetan Plateau (TP). In this study, we collected 300 indi-
viduals of P. nigromaculatus from 13 native regions and 2 invasive regions (including Nyingchi and
Lhasa) on the TP. To identify the source region of the TP introductions, we sequenced portions of
the mitochondrial cyt b gene. We sequenced a 600-bp portion of the mitochondrial cyt b gene to
identify 69 haplotypes (124 polymorphic sites) in all sampled populations. According to the net-
work results, we suggest that the P. nigromaculatus found on the TP was most likely originated
from Chongqing by human introduction. Furthermore, we found that the genetic diversity was sig-
niﬁcantly lower for invasive than for native sites due to founder effects. Our study provides genetic
evidence that this alien species invaded the cold environment of high elevations and expanded the
distribution of P. nigromaculatus in China.
Key words: alien species, amphibians, chytridiomycosis, cold environment, invasion genetics, invasion route, Tibetan Plateau.
Invasive species are responsible for changes to native biological di-
versity, the extinction of many native species around the globe, and
the disruption of ecosystem functions (Lockwood et al. 2013). Their
presence can inflict huge economic costs (Mack et al. 2000). Several
methods for measuring how species invade, establish, and spread
have been proposed to provide information to prevent or manage in-
vasive species (Hulme et al. 2008). Molecular approaches are among
the most important methods and have become widely used to better
understand the invasion process and the relationship between inva-
sive and source populations (Bai et al. 2012;Liebl et al. 2015;
Moule et al. 2015;Rius et al. 2015). Exploring invasion pathways
and sources can contribute to identifying the ecological characteris-
tics and physiological tolerances of source populations (Ficetola
et al. 2008). Invasion pathways and source populations can be used
to simulate potential distributions or predict future expansion (Liebl
et al. 2015).
Previous studies have suggested that regions of high elevation are
viewed as resistant to biological invasions because of an extreme cli-
mate and limited accessibility (Bennett et al. 2015;Pauchard et al.
2016). However, the risk of biological invasions is increasing due to
land-use change, climate warming, and increasing globalization in
these regions (Li et al. 2013). Non-native species move into regions
of high elevation and may change the composition of the community
(Bennett et al. 2015;Pauchard et al. 2016). These changes can be
viewed as with both positive (increasing biodiversity) and negative
(decreasing biodiversity) consequences. In contrast to the large
CThe Author (2017). Published by Oxford University Press. 1
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number of studies conducted in low-elevation regions (Rollins et al.
2015;Sherwin et al. 2015;Wang et al. 2016), non-native species
invading regions of high elevation represent a problem in invasive
biology that remains to be addressed.
The Tibetan Plateau (TP) is the most extensive (covering an area
of 2.5 million km
) and highest (approximately 4500 m above sea
level on average) plateau in the world (Zhou et al. 2006). The TP is
a conservation priority because the plateau and its adjacent areas
cross 3 biodiversity hotspots: Indo-Burma, the Himalayas, and the
mountains of southwestern China (Mittermeier et al. 2011). The ex-
tensive variation in the topography and climate of the TP generates
a number of different habitats and supports abundant species diver-
sity (Mittermeier et al. 2011;Li et al. 2016). There are 56 amphibian
species on the TP, including 3 caudata species (Batrachuperus tibeta-
nus,Andrias davidianus, and Batrachuperus karlschmidti) and 53
anuran species (Amphibia China 2016). Furthermore, against the
background of global climate change, the TP is experiencing faster
warming than low-elevation regions at the same latitude (Liu and
Chen 2000;Qin et al. 2009;Wei and Fang 2013). This faster warm-
ing pattern may increase the risk of biological invasions and facili-
tate the rapid dispersal of disease vectors on the TP (Di Rosa et al.
2007;Liu et al. 2013).
The black-spotted frog Pelophylax nigromaculatus—native to
East Asia, including low-elevation regions of northern, eastern, cen-
tral, and southwestern mainland China; the Korean Peninsula;
Japan; and far-eastern Russia—has become an invasive species in
areas of western China, such as the Xinjiang Uyghur Autonomous
Region (Wang et al. 2016). Wang et al. (2016) showed that the inva-
sion of the alien P. nigromaculatus population in Yining originated
from the Beijing and Chongqing area based on an mtDNA analysis.
Prior to the present study, the black-spotted frog was never recorded
in the TP (Li et al. 2010). The TP is at a higher elevation than
Yining (average elevation 620 m); for example, the elevations of the
Chabalang Wetland and Nyingchi City are, respectively, 3,600 m
and 3,000 m. Such different environments may be the cause of dif-
ferent invasion processes.
In this study, we used an mtDNA marker to investigate the gen-
etic patterns underlying the expansion of P. nigromaculatus popula-
tion on the TP. We 1) compared the genetic diversity and examined
the genetic structure of P. nigromaculatus in its 13 native ranges in
China and in the 2 invaded territories of the TP, 2) identified the
source region(s) of the TP introductions, 3) discuss possible potential
damage caused by the invasion of P. nigromaculatus on the TP, and
4) discuss the application of our results to the planning of suitable
Materials and Methods
Surveying and sampling of P. Nigromaculatus
We estimated the introduction range of P. nigromaculatus by line
transect methods in the TP (Heyer et al. 1994;Li et al. 2011). We
suggested that P. nigromaculatus breeding populations had been es-
tablished in this site when both adult and sub-adult P. nigromacula-
tus (and tadpoles) were found at survey sites (Li et al. 2011). We
obtained information on the introduction history of P. nigromacula-
tus in the TP using a questionnaire survey (Li et al. 2006,2011). We
usually interviewed 2 or 3 residents living near the sampled water
bodies. The residence time was based on the time the first sighting
by the resident of tadpoles, eggs, or juvenile or adults of P. nigroma-
culatus or heard calls. If the interviewees gave different answers on
the residence time of P. nigromaculatus invasion for a water body,
we used the average value (year) of these answers. The longest value
for all surveyed sites in a region was defined as the residence time.
We collected 260 adult individuals from different locations (20
samples per locality) in 13 native ranges (Figure 1) in 2012. These
locations encompassed most of the distribution of this species in
northeastern, northern, central, northwestern, southeastern, and
southwestern China. We collected 40 frogs from the 2 different re-
gions of introduction (Nyingchi and Lhasa) on the TP (Figure 1) be-
tween 2014 and 2015. To determine whether a site has been
invaded successfully by P. nigromaculatus, we searched for tadpoles
of P. nigromaculatus using line transects that surveyed all accessible
water bodies at each site for 3 consecutive nights. The third toes of
individuals of P. nigromaculatus were collected, and then the tissue
samples were preserved separately in 95% ethanol and stored at
20C in the laboratory.
DNA extraction and amplification
Total genomic DNA was extracted from the toe tissue following the
standard method published previously (Wang et al. 2014;Shine
et al. 2016). A 695-bp segment of the mitochondrial cytochrome b
(cyt b) gene from all specimens was amplified using the primers
RanaLeuF5d (50-AA T MCC GWA AA T CTC ACCCCC T-30) and
RanacytbB1 (50-GCT GGT GTAAA T TGT CTG GGT C-30)(Yang
et al. 2003). The PCR protocol was initiated with an initial step of
denaturing of 95C for 5 min, followed by 35 cycles of 94C for
30 s, annealing of 56C for 30 s, extension of 72C for 30 s, and a
final extension step of 72C for 10 min. The PCR products were sub-
jected to electrophoresis on 2% agarose gels and directly sequenced
using the same forward and reverse primers used for amplification
(Beijing Genomics Institute, Beijing, China).
We used Clustal X in MEGA 6 (Tamura et al. 2013) to align and
edit the mitochondrial cyt b gene sequences. To identify unique
haplotypes in all sampling populations, we used DnaSP 5.10 to
define these sequences (Rozas et al. 2003). Genetic diversity was as-
sessed by calculating the number of haplotypes (Hn), haplotype di-
versity (Hd), and nucleotide diversity (p) within each sampling
population using ARLEQUIN ver3.5 (Excoffier and Lischer 2010).
A neighbor-joining tree of mtDNA was constructed from the
Kimura 2-parameter nucleotide distances using Mega 6 (Tamura
et al. 2013). Branch support was calculated by the bootstrap method
according to 1,000 replicates. To identify the origin of the TP popu-
lation, we utilized the software package TCS 1.21 (Clement et al.
2000) to construct cladogram networks of P. nigromaculatus cyt b
haplotypes by statistical parsimony. We compared differences in the
number of haplotypes (Hn) between native and invasive populations
using the independent samples t-test (R Development Core Team
We identified only 2 invasion sites (Nyingchi and Lhasa) for
P. nigromaculatus that has established breeding populations in the
TP. The residence time for P. nigromaculatus invasion is approxi-
mately 15 years (since the start of this century) in Nyingchi and 10
years (since 2005) in Lhasa. We determined that alien P. nigromacula-
tus in the TP originated from accidental introduction by fish farming.
In total, 300 individuals of P. nigromaculatus were collected
from the 2 invasive regions and 13 native regions (Figure 1) and
2Current Zoology, 2017, Vol. 0, No. 0
yielded a 622-bp DNA sequence for cyt b gene. All 69 haplotypes
(H1–69) were identified by the 124 polymorphic sites in all
sampled populations (Table 1). A list of their distributions is pro-
vided in the Appendix, and the phylogenetic relationships are
shown in Figure 2. Collectively, 49 unique haplotypes and 20
haplotypes are shared among sampled populations (Appendix).
Haplotypes H1 and H2 are not found in locations other than
Chongqing and the introduced populations. Hn,Hd, and pranged,
respectively, from 1 to 14, 0 to 0.963, and 0 to 0.01762 among
the sampled populations (Table 1). We found that the H1 and H2
haplotypes occurred in the Tibet (including Nyingchi and Lhasa)
and Chongqing populations, suggesting that the P. nigromaculatus
found in Tibet most likely originated from Chongqing (Figure 3).
The number of haplotypes (Hn) was significantly higher for native
than for invasive sites (native vs introduced populations: df ¼13,
Our results suggest that the alien P. nigromaculatus population on
the TP originated from a single native-range source region
(Chongqing population). Our data provide new evidence that low
genetic diversity does not impede successful amphibian invasion on
the TP. Our study also provides a new case of a non-native spe-
cies invading high-elevation environments due to human activities
and raises awareness of the growing importance of the expan-
sion of non-native species in high-elevation cold environments.
Furthermore, the new record from the TP extends the known distri-
bution range of P. nigromaculatus in Asia by approximately
1,000 km from its ancestral area (Fei et al. 1999).
Our study shows that the recently established populations of
P. nigromaculatus on the TP have reduced genetic variability in
Table 1. Sampling information and genetic diversity indices of
Population Abbreviation NHnHd p
Nyingchi LZ 20 2 0.526 0.01692
Lhasa LS 20 1 0 0
Chongqing CQ 20 4 0.742 0.01762
Xi’an XA 20 3 0.195 0.00032
Jiaxing JX 20 9 0.795 0.00748
Beijing BJ 20 10 0.863 0.00674
Dongying DY 20 14 0.963 0.00763
Ningbo NB 20 5 0.442 0.00127
Zhenjiang ZJ 20 12 0.926 0.00651
Qiqihar QQ 20 7 0.732 0.00162
Changchun CC 20 9 0.832 0.01631
Shenyang SY 20 6 0.621 0.00987
Xuzhou XZ 20 13 0.932 0.00721
Wenzhou WZ 20 6 0.579 0.00542
Fuzhou FZ 20 2 0.1 0.00016
Total 300 69 0.952 0.02461
Note: N, number of samples sequenced; hn, number of haplotypes; hd, haplo-
type diversity; p, nucleotide diversity.
Figure 1. Sampled areas for P. nigromaculatus in China. Backward diagonal areas indicate the Tibet Plateau. Diagonal cross areas indicate the distribution area in
Asia. Closed circles denote the sampling sites.
Origin of invasion of an alien frog 3
comparison to native populations. The haplotype diversity in the 2
recently established populations is significantly lower than that in
the area of origin, presumably due to founder effects during the
colonization of Nyingchi and Lhasa. Frankham (2005) suggested
that mechanisms (such as multiple introduction events, purging dele-
terious alleles, and high reproductive rates) can overcome the genetic
dilemma that causes invasive populations to often show low genetic
diversity and inbreeding in the invasive region (Frankham 2005).
Previous studies have shown that P. nigromaculatus has high repro-
ductive rates (Wang et al. 2008), which may be an important factor
in the successful invasion of P. nigromaculatus on the TP.
We found that P. nigromaculatus has successfully invaded the
high-elevation (>3,000 m) regions (Nyingchi and Lhasa) on the TP.
Furthermore, other studies have discovered that a number of non-
native species have successfully invaded these regions (Fan et al.
2016). For example, Fan et al. (2016) found that 13 non-native
fish species have successfully invaded the Lhasa River of Tibet.
These studies are not in accordance with previous hypotheses
that cold environments of high elevations are often regarded as
resistant to biological invasions due to an extreme climate and lim-
ited accessibility. Therefore, it is important that we conduct more re-
search on invasion biology in regions of high elevation, such as
Our study suggests that the alien P. nigromaculatus on the TP
stemmed from Chongqing. A previous study found that the amphib-
ian chytrid fungus Batrachochytrium dendrobatidis (Bd)(Zhu et al.
2014,2016), which is a lethal pathogen responsible for declines in
amphibians worldwide, was detected in P. nigromaculatus in
Chongqing. Furthermore, Bd has been found in other regions of
high elevations, such as the Andes (Seimon et al. 2007), the Rocky
Mountains (Pilliod et al. 2010), and the Sierra Nevada (Vredenburg
et al. 2010). Although some studies suggest that the cold tempera-
tures of high elevations can limit Bd (Muths et al. 2008;Pilliod et al.
2010), Knapp et al. (2011) suggest that the cold environments of
high elevations do not necessarily limit this pathogen (Knapp et al.
2011). Therefore, to prevent the introduction of Bd to native am-
phibians, we suggest that the government control the spread of P.
nigromaculatus from Chongqing to Tibet (such as developing a real-
time monitoring system).
Humans may facilitate the spread of alien species across biogeo-
graphical borders such as high elevations, which could generate
positive and negative conservation outcomes depending on these
species and the invaded community (Bennett et al. 2015). As global-
ization increases, there will not only be an intensification of biologi-
cal invasions, but the risk of pathogenic species being introduced as
contaminants of their hosts may rise (Pauchard et al. 2016). Based
on our study, schemes to prevent the invasion of P. nigromaculatus
Figure 2. Phylogenetic relationships among mtDNA haplotypes from P. nigro-
maculatus collected in both native and introduced regions. Only bootstraps
of 70 or greater are shown. The numbers correspond to the haplotype num-
bers in the Appendix.
Figure 3. Statistical parsimony cladogram network representing the relation-
ships among mtDNA haplotypes from P. nigromaculatus collected in both ori-
gin and introduced regions. Haplotype circle size is proportional to the
number of individuals, and the numbers correspond to the haplotype num-
bers in the Appendix. The backward diagonal represents the Nyingchi popu-
lation. The horizontal represents the Lhasa population. Black represents the
4Current Zoology, 2017, Vol. 0, No. 0
on the TP should be prioritized based on those likely to have the
greatest impact. Management should be more directed toward pre-
venting the arrival of this species or catch it in the early stages of in-
vasion. Other types of management could include developing early
detection and rapid response programs and increasing educational
outreach and public awareness.
We thank 3 anonymous referees for many valuable comments on an earlier
This study was funded by the National Natural Science Foundation of China
(31370545), the Beijing Natural Science Foundation (5164036), China
Postdoctoral Science Foundation (2016M601132), and Collaborative
Innovation Center for Research and Development of Tibetan Agricultural
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Population distribution of mtDNA haplotypes of P.nigromaculatus
Haplotypes LZ LS CQ XA JX BJ DY GJ ZJ QQ CC SY XZ WZ FZ
11 7 2 1 1
12 1 2 1
29 2 1
33 1 2
34 1 2
35 1 1
36 1 1
38 2 2
6Current Zoology, 2017, Vol. 0, No. 0
Haplotypes LZ LS CQ XA JX BJ DY GJ ZJ QQ CC SY XZ WZ FZ
50 2 1
Total 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20
Origin of invasion of an alien frog 7