Biological Journal of the Linnean Society, 2010, 99, 582–594. With 5 figures
Phylogeography of the Japanese pipistrelle bat,
Pipistrellus abramus, in China: the impact of ancient
and recent events on population genetic structure
LI WEI1, JON R. FLANDERS1,2, STEPHEN J. ROSSITER3,
CASSANDRA M. MILLER-BUTTERWORTH4, LI B. ZHANG5 and SHUYI Y. ZHANG1*
1
School of Life Sciences, East China Normal University, Shanghai 200062, China
School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK
3
School of Biological and Chemical Sciences, Queen Mary University of London, London E1 4NS,
UK
4
Penn State Beaver, 100 University Drive, Monaca, PA 15061, USA
5
Guangdong Entomological Institute, Guangzhou 510260, China
2
Received 6 August 2009; accepted for publication 29 September 2009
bij_1387
582..594
The influence of Pleistocene climatic oscillations on shaping the genetic structure of Asian biota is poorly known.
The Japanese pipistrelle bat occurs over a wide range in eastern Asia, from Siberia to Japan. To test the relative
impact of ancient and more recent events on genetic structure in this species, we combined mitochondrial
(cytochrome b) and microsatellite markers to reconstruct its phylogeographic and demographic history on continental China and its offshore islands, Hainan Island and the Zhoushan Archipelago. Our mitochondrial DNA tree
recovered two divergent geographical clades, indicating multiple glacial refugia in the region. The first clade was
mainly confined to Hainan Island, indicating that gene flow between this population and the continent has been
restricted, despite being repeatedly connected to the mainland during repeated glacial episodes. By contrast,
haplotypes sampled on the Zhoushan Archipelago were mixed with those from the mainland, suggesting a recent
shared history of expansion. Although microsatellite allele frequencies showed clear discontinuities across the
sampling range, supporting the current isolation of both Hainan Island and the Zhoushan Archipelago, we also
found clear evidence of more recent back colonization, probably via post-glacial expansion or, in the latter case,
occasional long distance dispersal. The results obtained highlight the importance of using multiple sets of markers
for teasing apart the roles of ancient and more recent events on population genetic structure.
© 2010 The
Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 582–594.
ADDITIONAL KEYWORDS: Chiroptera – cytochrome b – dispersal – microsatellites.
INTRODUCTION
Population genetics can provide powerful insights
into how historical and current processes have
influenced species’ dispersal and colonization routes,
as well as existing population structure (GarciaMudarra, Ibanez & Juste, 2009).
Previous studies have identified genetic discontinuities in numerous species over wide ranges (Hewitt,
1999; Salgueiro et al., 2004; Duvernell et al., 2008;
*Corresponding author. E-mail: [email protected]
582
Scandura et al., 2008). Such discontinuities can result
from barriers to recurrent gene flow, arising from
landscape processes such as habitat fragmentation
(Bergl & Vigilant, 2007), or geographical features
such as water bodies (Dobson & Wright, 2000) or
mountain ranges (Brown, Suarez & Pestano, 2002).
Alternatively, they can reflect suture zones resulting
from secondary contact of lineages that have diverged
in allopatry, even when physical isolation no longer
occurs (Rossiter et al., 2007; Duvernell et al., 2008).
The impact of Pleistocene climatic fluctuations on
taxon distributions in North America and Europe is
© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 582–594
PHYLOGEOGRAPHY OF THE JAPANESE PIPISTRELLE BAT
well documented (Hewitt, 1999; Avise, 2000). Less is
known about the effects of these events on Asian biota
(Xu et al., 2009). In South China, Hainan Island was
formed during the late Tertiary and early Quaternary
periods by tectonic activity and rising sea levels (Xing
et al., 1995). Subsequently, decreases in sea level as a
result of glacial cycles have caused Hainan Island to
be repeatedly connected to continental China, most
recently during the late Pleistocene. Hainan Island
last became separated from the mainland during the
Holocene approximately 10 000 years ago (Zhao et al.,
1999), when sea levels rose to form the Qiongzhou
Strait, currently spanning 20–40 km. Similarly,
the Zhoushan Archipelago (comprising 1339 small
islands) was also separated from the continent during
the Holocene. However, this archipelago was originally part of the Tiantai Mountains and, as a result of
its complex terrain, its separation took longer, becoming isolated from the continent approximately 7000–
9000 years ago (Wang & Wang, 1980).
Several genetic surveys have suggested that the
Qiongzhou Strait and Zhoushan Archipelago have
acted as effective barriers to gene flow subsequent to
the Pleistocene. For example, the tree fern, Alsophila
spinulosa occurs on both sides of the Qiongzhou
Strait, yet its spores rarely cross the ocean, thus
preventing effective gene flow leading to vicariance
(Su et al., 2005). Moreover, even though the Zhoushan
583
Archipelago is only separated from the continent by
just 4 km at its closest point, work on the plant
Neolitsea sericea (Wang et al., 2005) and ten amphibian species (Yiming, Niemela & Dianmo, 1998) have
failed to detect any gene flow between these two areas
subsequent to the archipelago’s formation.
The Japanese pipistrelle bat, Pipistrellus abramus
(Chiroptera: Vespertilionidae), is distributed from
Siberia to Japan and occurs throughout eastern continental China and its offshore islands (Simmons,
2005). This species is therefore a useful taxon for
assessing the relative importance of past sea level
changes and present isolation on population genetic
structure between continental China, Hainan Island
and the Zhoushan Archipelago. In the present study,
we address these issues by combining both mitochondrial (cytochrome b gene; cytb) and microsatellite
markers, which together can provide complimentary
insights at different geographical and temporal
scales.
MATERIAL AND METHODS
Pipistrellus abramus was sampled at 17 localities in
China from 2005 to 2007. Sampling localities covered
three main regions: the continent (eight localities),
the Zhoushan Archipelago (four), and Hainan Island
(five) (Fig. 1, Table 1). Bats were captured at dusk
Figure 1. Map of sampling locations for the 17 localities sampled and identification of the two major geographical
features tested (Qiongzhou Strait and Tiantai Mountains). Numbers correspond to sampling localities listed in Table 1.
© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 582–594
584
L. WEI ET AL.
Table 1. Names and locations of colonies sampled in the present study
Colony
Area
Locality
Easting
Northing
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Continental China
Dulesi
Shijiazhuang
Linyi
Wuhan
Yufeng
Xiangtang
Shanghai
Guilin
Daishan
Xiushan
Zhoushan
Cezi
Hongla village
Benhao
Lingshui
Shili
Shibian
40°03′091″N
38°02′310″N
35°15′500″N
30°34′263″N
30°32′093″N
28°25′211″N
31°13′410″N
25°16′005″N
30°14′322″N
30°10′12.22″N
30°04′457″N
30°05′041″N
18°56′171″N
18°37′247″N
18°42′015″N
18°38′415″N
18°38′757″N
117°24′109″E
114°29′550″E
117°58′150″E
114°22′286″E
106°26′150″E
115°55′723″E
121°24′317″E
110°19′566″E
122°12′164″E
122°9′44.17″E
121°59′807″E
121°56′275″E
109°53′992″E
109°57′308″E
109°56′357″E
109°40′120″E
109°39′278″E
Zhoushan Archipelago
Hainan Island
with mist nets set near to nursery roosts in buildings.
Tissue samples were obtained from wing-membrane
using a 3-mm diameter biopsy punch (Rossiter
et al., 2007) and stored in ethanol at -20 °C until
processing.
DNA
AMPLIFICATION AND SEQUENCING
Genomic DNA was isolated using DNeasy Tissue Kits
(Qiagen). Amplification and sequencing of the region
of the cytb were performed using the primers CY1
(5′-TAG AAT ATC AGC TTT GGG TG-3′) (Li et al.,
2006) and CY2 (5′-AAA TCA CCG TTG TAC TTC
AAC-3′) (Zhang et al., 2007). All sampled bats were
also genotyped at eight microsatellite loci (EU661775,
EU661776, EU661777, EU661779, EU661780,
EU661781, EU661782, and EU661783) (Wei et al.,
2009). Standard polymerase chain reactions, sequence alignment and genotyping were carried out
as outlined in the Supporting Information
(Appendix S1).
PHYLOGENETIC
ANALYSIS
Individual haplotypes were used to construct a
Neighbour-joining (NJ) and maximum parsimony tree
(MP) in PAUP*, version 4.0b10 (Swofford, 2002) and a
Bayesian inference (BI) tree in MRBAYES, version
3.1 (Huelsenbeck & Ronquist, 2001). The TVM+G
model (base frequencies: A, 0.2880; C, 0.2699; G,
0.1323; and T, 0.3098; transition/transversion
ratio = 46.9508; gamma distribution shape = 0.6820)
was selected as the most appropriate substitution
model using the Akaike information criteria imple-
mented in MODELTEST, version 3.7 (Posada & Crandall, 1998). Statistical support for branching patterns
was estimated by bootstrap replication (NJ, MP: 1000
replicates). BI was run with four simultaneous
chains, each of 1 ¥ 106 generations, sampled every 100
generations and the first 25% of trees were discarded
as ‘burn-in’. Pipistrellus cf. javanicus was chosen as
an outgroup (GenBank accession number: AJ504447).
A 95% minimum spanning haplotype network (MSN)
(Templeton, Crandall & Sing, 1992) was also constructed from the haplotypes using TCS, version 1.21
(Clement, Posada & Crandall, 2000).
MITOCHONDRIAL (MT) DNA
STATISTICAL ANALYSIS
Cytochrome b haplotype diversity (H) and nucleotide
diversity (p) were calculated for all samples ⱖ 2.
Values for polymorphic sites and the mean number of
pairwise differences were also estimated. All calculations were carried out in DNASP, version 4.10.6
(Rozas et al., 2003).
To test for genetic differentiation among samples
(N ⱖ 5), we calculated pairwise FST values, and tested
for significance by permutation (10 000). Analyses of
molecular variance (AMOVA) were undertaken to test
for hierarchical genetic structure, first including all
colonies (N ⱖ 5) and also on separate pairs of regions,
to evaluate the effect of two important geographical
features (the Qiongzhou Strait and the Tiantai
Mountains). Significance was again assessed using
permutation (10 000). Both analyses were conducted
in ARLEQUIN, version 3.1 (Excoffier, Laval &
Schneider, 2009). We also tested for isolation-bydistance (IBD) (Rousset, 1997) using log transformed
© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 582–594
PHYLOGEOGRAPHY OF THE JAPANESE PIPISTRELLE BAT
distances with linearized FST values [FST/(1 - FST)]
in a Mantel test (10 000 permutations) in IBDWS,
version 3.14 (Jensen, Bohonak & Kelley, 2005). Linear
Euclidean distances were derived from easting and
northing coordinates using the program GEOGRAPHIC DISTANCE MATRIX GENERATOR,
version 1.2.1 (Ersts Internet).
To assess population demographic history, we
examined sequence mismatch distributions, which
are typically ragged or multimodal for populations at
stationary demographic equilibrium, but smooth or
unimodal for populations that have undergone a
demographic expansion (Rogers & Harpending, 1992).
Goodness of fit tests for a model of population expansion were calculated from the sum of squared
deviation (SSD) and the raggedness index (r), and
significance was assessed by bootstrapping (10 000
replicates) in ARLEQUIN. Where evidence of population expansion was found, the expansion time in
generations (t) was derived following t = T/2u, where
T (tau) is a parameter of the time to expansion in
units of mutations, and u is the mutation rate per
generation for the DNA sequence. We used a mutation rate of 2% per Myr (Arbogast & Slowinski, 1998)
with a generation time of 2 years, based on age of first
breeding for most insectivorous bat species (Racey,
1982).
To infer the divergence time (t) of the two major
clades identified, we used the ‘isolation-withmigration’ coalescent model using the software IMA
(Hey & Nielsen, 2007). Multiple runs were performed
using a Hasegawa–Kishino–Yano model of sequence
evolution to estimate the run parameters (q1, q2, qA,
m1, m2, and t) by exploring the posterior probability
distributions. Run parameters were adjusted until
convergence was reached. Five independent runs of
ten chains and a geometric heating scheme were used
to sample 1 ¥ 106 genealogies of which the first 10%
were discarded as burn-in. We applied the mutation
rate of 2% per Myr.
MICROSATELLITE
STATISTICAL ANALYSIS
For microsatellite data, heterozygosity (observed and
expected), mean allelic richness (Rs), and tests for
linkage disequilibrium were calculated in GENEPOP,
version 3.3 (Raymond & Rousset, 1995). Deviations
from Hardy–Weinberg equilibrium were tested using
FSTAT, version 2.9.3 (Goudet, 1995) by calculating FIS
values for each population and locus, and significance
was assessed by randomization (1000 times) with
correction for multiple tests.
Population structure was quantified by estimating
genetic differentiation between colonies using both
FST (Weir & Cockerham, 1984) and RST (Slatkin,
1995). Differentiation between colonies was calcu-
585
lated using ARLEQUIN, and tested for significance
by permutation (1000). RST values were calculated
assuming a stepwise-mutation model using RST
CALC, version 2.2 (Goodman, 1997). To assess the
contribution of stepwise mutation in genetic differentiation, the observed RST values were compared to
expected values (pRST) based on 1000 permutations
of allele size using SPAGEDI, version 1.2g (Hardy &
Vekemans, 2002). When RST is significantly larger
than pRST, stepwise mutation has contributed to the
observed differentiation, whereas nonsignificant differences suggests that FST is the most appropriate
estimator (Hardy et al., 2003).
CLUSTERING
ANALYSIS
To reconstruct the hierarchical relationships among
colonies, Bayesian clustering of the microsatellite
data was implemented in STRUCTURE, version 2.2
(Pritchard, Stephens & Donnelly, 2000). Clustering
was undertaken on all samples combined as well as
the three main regions separately. For each group,
runs were undertaken for K = 2 upwards until no
population structure could be detected. We applied an
admixture model with a burn-in of 30 000 and a run
length of 106, and undertook ten replicate runs. To
compare runs of the same value of K, we derived
symmetric similarity coefficients (SSC) using the
Greedy algorithm in CLUMPP (Jakobsson & Rosenberg, 2007). Groups of runs with an SSC ⱖ 0.8 were
identified and combined. Bar plots were displayed
graphically using the software DISTRUCT (Rosenberg, 2004).
To describe further the pattern of genetic structure
between the different colonies, we undertook a factorial correspondence analysis (FCA) in the software
GENETIX, version 4.02 (Belkhir et al., 2004). Here,
axes are independent of one another with each axis
reporting the different levels of genetic variance they
explain.
RESULTS
MTDNA VARIABILITY
In total, 27 unique haplotypes based on 1122 bp of
cytb were identified from 102 individuals sampled
from 17 different locations (GenBank accession
numbers GQ332482–GQ332529). A total of 64 variable sites were recorded and no indels were observed.
Haplotype diversity (h) averaged 0.873 among the
three regions, ranging from 0.636 ± 0.18 (mean ± SD)
for the continental colonies, 0.639 ± 0.19 for the
Hainan Island colonies, to 0.855 ± 0.06 for the Zhoushan Archipelago colonies (Table 2). Fifteen haplotypes
were singletons (54%). Haplotype H1 (N = 29, 28%),
recorded in colonies 1 and 4–12, and haplotype H2
© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 582–594
7
5
6
7
9
5
5
6
7
5
5
5
8
5
6
5
6
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
3
1
3
2
4
2
3
3
4
1
2
4
5
3
2
2
1
Haplotypes
observed
3
–
2
1
3
1
2
41
4
–
1
3
9
2
2
1
–
Polymorphic
sites
1.524
–
0.867
0.286
1.278
–
1
21.067
1.429
–
–
1.4
2.714
0.8
1.2
0.4
–
Mean no. of
pairwise
differences
0.667
–
0.733
0.286
0.694
–
0.8
0.6
0.81
–
–
0.9
0.857
0.7
0.6
0.4
–
H*
0.00136
–
0.00077
0.00025
0.00114
–
0.00089
0.01878
0.00127
–
–
0.00125
0.00242
0.00071
0.00107
0.00036
–
p*
7
5
20
20
10
20
12
37
17
20
20
15
20
5
15
14
29
N
0.69
0.77
0.64
0.70
0.58
0.65
0.64
0.79
0.72
0.68
0.62
0.71
0.70
0.88
0.87
0.86
0.89
HO*
Microsatellite
0.67
0.67
0.70
0.77
0.70
0.78
0.77
0.81
0.77
0.78
0.79
0.80
0.82
0.71
0.82
0.80
0.82
HE *
4.88
4.13
7.88
9.00
6.38
8.63
7.38
9.00
8.00
7.88
8.38
8.38
10.13
4.75
8.75
8.00
10.36
A*
3.45
3.50
3.52
3.89
3.63
3.97
3.95
4.02
3.83
3.87
3.99
4.10
4.21
3.73
4.25
4.04
4.18
RS*
Values are averaged across loci. The locality numbers (1–17) are the same as in Table 1 and Fig. 1
*H, haplotype diversity; p, nucleotide diversity; A, mean number of alleles per locus; HO, observed heterozygosity; HE, expected heterozygosity; RS, allelic richness.
N
Colony
number
Mitchondrial DNA
Table 2. Genetic variability in ten populations of Pipistrellus abramus based on 1122 bp of cytb
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L. WEI ET AL.
© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 582–594
PHYLOGEOGRAPHY OF THE JAPANESE PIPISTRELLE BAT
GENEALOGICAL
(N = 17, 16%), recorded in colonies 1–3 and 9–12,
were the most common though neither were found on
Hainan Island. Haplotypes from Hainan Island were
not recorded elsewhere. Nucleotide diversity (p)
averaged over all colonies was 0.0165, ranging from
0.0011 ± 0.0009 on Hainan Island, 0.0012 ± 0.0001 on
Zhoushan Archipelago, to 0.0039 ± 0.007 on the continent (Table 2).
587
ANALYSIS
Our Bayesian phylogenetic tree recovered two divergent well supported clades that broadly corresponded
to distinct geographical regions (Fig. 2), which were
also evident in both our NJ and MP trees (data not
shown). Clade A contained all bats from the Zhoushan
Archipelago and continental China (except for two
individuals from Guilin: H13 and H14), whereas
H1
H2
Mainland China and
Zhoushan Archipelago
H3
Dulesi
H4
Linyi
H5
H6
Wuhan
H10
Xiangtang
H11
Shanghai
H12
Clade A
100
H15
Zhoushan Archipelago
H18
H19
H8
97
H7
Yufeng
H9
100
99
H16
H17
Zhoushan Archipelago
H13
Guilin
H14
H23
H24
66
H25
Clade B
H26
Hainan Island
H20
99
H21
H22
H27
Outgroup
0.05 substitutions/site
Figure 2. A 50% consensus tree from a Bayesian phylogenetic analysis. Pipistrellus cf. javanicus was used as the
outgroup.
© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 582–594
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L. WEI ET AL.
H9
H6
H10
H7
H12
H8
H1
H11
H2
H4
H15
H19
H5
H3
H17
H18
H16
25 steps
H14
H25
H13
H23
H24
H27
H21
H26
H20
H22
Figure 3. TCS haplotype network for Pipistrellus abramus. Colours represent different population regions: white,
continental China; grey, Zhoushan Archipelago; black, Hainan Island.
Clade B contained all haplotypes sampled on Hainan
Island plus two from Guilin.
In our MSN parsimony network, each clade formed
a separate sub-network separated by 35 mutational
steps (Fig. 3). Clade A showed a star-like topology
with a geographically widespread interior haplotype
(H1), whereas clade B showed a similar topology but
with an unsampled central haplotype.
POPULATION
STRUCTURE AND GENE FLOW
Global exact tests revealed significant genetic differentiation among all colonies sampled (P < 0.05). Pairwise values indicated that Hainan Island colonies
were consistently differentiated from the mainland
and Zhoushan Archipelago colonies (P < 0.05), but not
from each other, with the exception of Hongla.
Genetic differentiation was also detected among the
continental colonies in over half of pairwise comparisons (see Supporting Information, Table S1). AMOVA
identified significant genetic variance at all three
hierarchical levels tested (among regions, among colonies within regions, and within colonies) (P < 0.001)
(Table 3). An additional AMOVA for continental colonies and the Zhoushan Archipelago, conducted to test
the impact of the Tiantai Mountains and associated
sea crossing, revealed no significant variance among
regions (P > 0.05), although there was significant
© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 582–594
589
PHYLOGEOGRAPHY OF THE JAPANESE PIPISTRELLE BAT
Table 3. Hierarchical analysis of molecular variance with different geographical groups calculated from mtDNA sequence
data. The percentage of variation is provided for three hierarchical levels
Structure
Source of variation
Variation (%)
Fixation indices
P
Three main regions
(continent, Zhoushan Archipelago
and Hainan Island)
Continent haplotype diversity
Zhoushan Archipelago
Among
Among
Within
Among
Among
Within
Among
Among
Within
88.32
3.87
7.81
-3.70
28.79
74.90
90.59
3.15
6.26
FCT = 0.820
FSC = 0.309
FST = 0.876
FCT = -0.037
FSC = 0.278
FST = 0.251
FCT = 0.901
FSC = 0.334
FST = 0.937
< 0.001
< 0.001
< 0.001
0.797
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
Continent versus Hainan Island
regions
populations/within region
population
regions
populations/within region
population
regions
populations/within region
population
differentiation among colonies within regions and
within colonies (P < 0.001). However, a separate
AMOVA between the continental colonies and Hainan
Island showed significant genetic structure at both
hierarchical levels (P < 0.001) (Table 3). A significant
positive correlation between genetic and geographical distance (IBD) was detected across the study
area (r2 = 0.27, P < 0.001), with a sharp increase in
gradient corresponding to comparisons that included Hainan Island (see Supporting Information,
Fig. S1A).
DEMOGRAPHIC
ANALYSIS
Separate mismatch distribution analyses undertaken
for each clade in the phylogeny showed similar demographic histories with unimodal distributions that
failed to reject an expansion model (PSSD > 0.05 and
raggedness index PR > 0.05). The estimated timing
of expansion for the continent-Zhoushan clade was
approximately 70 000 years BP [95% confidence interval (CI) = 24 000 - 121 000 BP] and for the Hainan
clade was around 220 000 years BP (95% CI 12 000–
1870 000 BP).
The marginal posterior probability plots produced
in IMA for all five runs produced highly similar
results with posterior probability distributions of t
showing a sharp within a narrow range. The time of
divergence of the two clades was estimated to be
220 000 years BP (90% highest posterior density
interval = 121 000 - 800 000).
MICROSATELLITE
ANALYSIS
Genetic diversity
A total of 286 individuals were genotyped across eight
polymorphic loci from 16 different locations (Table 1).
No evidence of linkage disequilibrium was detected
between any loci, and no consistent deviation from
Hardy–Weinberg equilibrium was found for either
any populations or loci (N ⱖ 5) (Table 2).
Population structure and gene flow
Global FST revealed genetic differentiation among
sampling localities (P < 0.001), in line with the global
FST value. On the continent, 71% of the pairwise
population comparisons were significant, whereas, on
Hainan Island, two colonies (Hongla and Benhao)
were significantly different from each other, and also
from the three other island colonies sampled. A
smaller degree of genetic differentiation was seen on
the Zhoushan Archipelago, with only the Daishan
colony being significantly different from the others
(see Supporting Information, Table S1). IBD was
detected (r2 = 0.31, P < 0.001), and showed a similar
pattern to the mtDNA plot (see Supporting Information, Fig. S1B). Estimates of FST and RST were
strongly correlated; however, global RST was significantly larger than global pRST (P < 0.001), indicating
that stepwise mutation has contributed to the
observed pattern of differentiation. Indeed, corresponding pairwise values of RST and pRST had nonoverlapping jack-knifed 95% CI and were significantly
different from each other (P < 0.05, after Bonferroni
correction) in 50.7% of tests carried out, mostly as
a result of comparisons between the continent/
Zhoushan Archipelago group and Hainan Island
(see Supporting Information, Table S2).
Cluster analysis
Clustering of individuals based on their microsatellite
genotypes was run from K = 2 to K = 8. At K = 2, two
major clusters were identified, corresponding to the
clades of phylogenetic analyses (continent/Zhoushan
Archipelago and Hainan Island) with the Guilin
colony showing a mixture of these. At K = 3, the
Zhoushan Archipelago together with one continental
colony (Xiangtang) formed its own cluster, and Guilin
© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 582–594
590
L. WEI ET AL.
Continental China
Zhoushan Archipelago
Hainan Island
K=2
K=3
K=4
12
3
4
5
6
7
8
9
10
11
12
13 14 15 16
17
Colony Number
Figure 4. Clustering analysis for samples across the sampling range. The different colours represent the proportional
membership of individuals from each locality to a given cluster, undertaken for increasing numbers of clusters (K) using
STRUCTURE.
5 3
1
2
0.6
Axis 2 (12.7 %)
4
7
0.4
0.2
8
11 12
0.0
Mainland China
Zhoushan Archipelago
10
9
-0.2
6
Hainan Island
-0.4
16
17
-0.4
-0.2
13
0.0
15
14
Ax
is 1 0.2
(23
.0
0.4
%)
0.6
0.8
-0.8
-0.6
-0.4
-0.2
is
Ax
0.0
.4
3 (8
0.2
0.4
%)
Figure 5. Three-dimensional factorial correspondence analysis showing the mean position of each population. The
percentage variance explained by each axis is shown in parentheses.
showed a mixture of all three clusters (Fig. 4).
Forcing higher values of K did not reveal any new
clusters. Separate clustering analyses of the three
different regions showed no distinct clusters forming
between any of the populations on Hainan Island, and
some limited structuring among samples from the
Zhoushan Archipelago populations (see Supporting
Information, Fig. S2). Continental samples showed
no distinct clusters, although differences were seen
between both Xiangtang and Guilin and the other
samples.
FCA showed clear differences among samples of P.
abramus from across China (Fig. 5). The three major
axes explained 44.1% of the total inertia (23.0%,
12.7% and 8.4%, respectively). Axis 1 separated the
majority of the continental populations (excluding
Xiangtang and Guilin) from populations on the
Zhoushan Archipelago (including the colony from
Xiangtang) and the colonies on Hainan Island. Axis 1
also appears to separate the Guilin colony from the
rest of the continental colonies. Within Hainan
Island, the samples do not form a tight group, with
Hongla (13) showing separation along axis 3.
DISCUSSION
We applied both mtDNA and microsatellite analyses
to characterize the phylogeographic history of the
Japanese pipistrelle bat (P. abramus) across East
China. Our discovery of two divergent mtDNA lineages indicates that at least two populations
have evolved in isolation from each other for up
© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 582–594
PHYLOGEOGRAPHY OF THE JAPANESE PIPISTRELLE BAT
to approximately 220 000 years. Phylogenetic and
network analyses both showed that haplotypes from
clade B are mostly confined to Hainan Island, with
the exception of two on the neighbouring mainland
(see below). Demographic analyses suggest that this
population has undergone a rapid expansion in the
past. Although a lack of power in this sample precludes reliable dating of this event, it will have
occurred after divergence from clade A, possibly as
part of a post-glacial expansion during the warm
period of the Eemian which peaked approximately
125 000 years BP
By contrast, the populations sampled on the
Zhoushan Archipelago and continental China share
a common ancestral haplotype and appear to share a
common history. The star-like network topology
coupled with an inferred population expansion at
approximately 70 000 years BP (95% CI = 24 000 121 000 years BP) indicates that this lineage colonized both regions at the same time. In despite of the
wide confidence intervals, we can be reasonably
certain that this expansion predated the last glacial
maximum (LGM) (21 000–18 000 years BP). This
common ancestry also is reflected in a lack of detectable differentiation at mtDNA sequences.
Although relatively little is known about the impact
the LGM on temperate animal species in east Asia,
the results obtained in the present study support
those of recent studies that have found evidence of
multiple refugia in this region (Chen et al., 2006; Tian
et al., 2008; Flanders et al., 2009). We suggest that P.
abramus persisted continuously in the region that
includes Hainan Island for hundreds of thousands of
years.
Although our mtDNA haplotype data resolved
broad scale separation of Hainan Island from the
continent/Zhoushan Archipelago group, our microsatellite data provided insights into recent history and
contemporary gene flow. Significant differences were
found in comparisons of corresponding pairwise RST
and pRST values, indicating that the subdivision of the
Hainan Island population is at least partially a result
of the stepwise mutation of microsatellite loci,
reflecting a phylogeographic signal. Unsurprisingly,
however, F statistics and clustering analysis also
revealed strong differentiation between populations
on Hainan Island and others, thus corroborating our
mtDNA results and providing evidence of limited or
no current gene flow across the Qiongzhou Strait.
Similarly, colonies on the Zhoushan Archipelago
showed significant differences (pairwise FST values)
between all mainland colonies, with one exception
(see below). However, in this case, corresponding pairwise values of RST and pRST were not significantly
different from each other, indicating that genetic differentiation is likely a result of a lack of recurrent
591
gene flow across the sea and Tiantai Mountains
rather than an older signal, thus also supporting the
nonsignificant FST values obtained for the same
comparisons.
Several previous studies have also shown that gene
flow in bats can be hindered or prevented by water
bodies. For example, the narrow strait of Gibraltar
(approximately 14 km) acts as a barrier to dispersal
in Myotis myotis (Castella et al., 2000), whereas
the Taiwan Strait (131 km) and English Channel
(100 km) also appear to prevent gene flow in populations of Rhinolophus monoceros (Chen et al., 2006)
and Rhinolophus ferrumequinum (Rossiter et al.,
2007), respectively. In comparison, however, two Pipistrellus species in Europe (Pipistrellus pipistrellus
and Pipistrellus pygmaeus) do not exhibit marked
genetic differentiation between the mainland and offshore islands (Racey et al., 2007).
In the absence of sea barriers, gene flow among
P. abramus colonies appears to have homogenized
genetic structure following a stepping stone model, as
supported by our isolation by distance plots (Kimura
& Weiss, 1964). Indeed, pairwise values of FST and FST
among colonies tended to be much lower within
regions than between them (see Supporting Information, Table S1). Thus, gene exchange probably tends
to occur among neighbouring colonies via natal dispersal and temporary mating dispersal (Chen, Jones
& Rossiter, 2008), and will largely depend on the
distribution of colonies (Rossiter et al., 2000; Ruedi
et al., 2008), whereas differentiation over greater distances will also reflect vicariant forces, which are
more easily detected at larger geographical scales
(Bossart & Prowell, 1998; Chen et al., 2006).
At the same time, however, two notable exceptions
to this overall trend were evident. First, the continental colony of Xiangtang showed remarkable similarity with populations on the Zhoushan Archipelago,
as revealed by nonsignificant pairwise comparisons of
genetic distance, as well as both Bayesian clustering
of genotypes (K = 3; Fig. 4) and our factorial correspondence analysis (Fig. 5). Furthermore, this sample
displayed almost no genetic affiliation with two of its
direct neighbours (Wuhan and Shanghai). Therefore,
given that this sample represents an outlier in the
overall data, we suggest that this common ancestry is
best explained by one or more recent colonization
event(s) from the Zhoushan Archipelago to the Xiangtang area (approximately 600 km straight line
distance).
A second exception, also evident from our clustering
and factorial correspondence analyses, was seen in
the Guilin population. Here, the genotypes of several
bats were assigned membership to a cluster that
predominately comprised bats from Hainan Island. In
addition, the Guilin sample was also found to contain
© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 582–594
592
L. WEI ET AL.
two haplotypes (H14 and H15) that were most closely
related to those from Hainan Island. Taken together,
these two classes of markers strongly support further
mixing. One possibility is that the observed discontinuity represents a suture zone, formed by post-glacial
colonization of the mainland across the land bridge
that remained until 10 000 years ago. Alternatively,
mixing might have been more recent, via occasional
dispersal events across the Qiongzhou Strait, perhaps
assisted by the extreme winds (typhoons) that are
common in this region. It is also noteworthy that
other genotypes from the Guilin sample clustered
with those from either continental colonies or the
Zhoushan Archipelago, or had mixed membership.
Given its relative proximity to both Hainan Island
and Xiangtang, Guilin thus appears to have received
alleles from both island groups.
The present study is the first phylogeographic
assessment of P. abramus and reveals the importance
of combining different types of genetic marker in
teasing apart ancient and more recent events
(Flanders et al., 2009). Additional sampling would
help to resolve the extent of recent dispersal events
across the sea, which appears to have resulted in
mixing between the two ancient refugial lineages.
ACKNOWLEDGEMENTS
We thank Kailiang Hu, Huabin Zhao, Jie Cui,
Jinshuo Zhang, Wenchao Liu, Wei Zhang, Guangjian
Zhu, and Tiyu Hong for their assistance in the field
survey. We are grateful to two anonymous reviewers
for their comments on the manuscript. This work was
funded by grants awarded to S. Zhang under the Key
Construction Program of the National ‘985’ Project
and ‘211’ Project. J. Flanders was supported by a
Department for Innovation, Universities and Skills
Fellowship (UK) and S. Rossiter by a Royal Society
Fellowship (UK).
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article:
Table S1. Population pairwise FST (below diagonal) and FST (above diagonal) between 17 investigation localities
calculated from mtDNA and microsatellite data. Locality numbers are the same as Table 1. Bold indicates a
significant differentiation at P < 0.05.
Table S2. Comparison between observed RST (below diagonal) and expected RST (pRST) (above diagonal) values
for each of the 17 colonies studied in the microsatellite analysis. Locality numbers are the same as Table 1. Bold
indicates significant difference at P = 0.05.
Figure S1. Isolation-by-distance plots based on samples comprising at least five individuals for (a) FST values
and (b) FST values. Black squares represent comparisons on mainland China; black diamonds represent
comparisons within the Zhoushan Archipelago; white triangles represent comparisons between the continent/
Zhoushan Archipelago and Hainan Island; white circles represent comparisons comparisons within Hainan
Island.
Figure S2. Clustering analysis for samples (a) continental China, (b) Zhoushan Archipelago, and (c) Hainan
Island. The different colours represent the proportional membership of individuals from each locality according
to the number of clusters (K) that have been forced into using STRUCTURE.
Appendix S1. Polymerase chain reaction procedures.
Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials
supplied by the authors. Any queries (other than missing material) should be directed to the corresponding
author for the article.
© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 582–594