Journal of Heredity 2011:102(2):165–174
doi:10.1093/jhered/esq121
Ó The American Genetic Association. 2011. All rights reserved.
For permissions, please email: [email protected].
Genetic Status of Asiatic Black Bear
(Ursus thibetanus) Reintroduced into
South Korea Based on Mitochondrial
DNA and Microsatellite Loci Analysis
YUNG-KUN KIM*, YOON-JEE HONG*, MI-SOOK MIN*, KYUNG SEOK KIM*, YOUNG-JUN KIM,
INNA VOLOSHINA, ALEXANDER MYSLENKOV, GAVIN J. D. SMITH, NGUYEN DINH CUONG, HUYNH
HUU THO, SANG-HOON HAN, DOO-HA YANG, CHANG-BAE KIM, AND HANG LEE
From the College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul
151-742, Korea (Kim, Hong, K. S. Kim, Y-J. Kim, and Lee); the Conservation Genome Resource Bank for Korean Wildlife,
Seoul National University, Seoul, Korea (Y-K. Kim, Hong, Min, and Lee); the Lazovsky State Nature Reserve, Lazo, Primorsky
Krai, Russia (Voloshina and Myslenkov); the Program of Emerging Infectious Diseases, Duke-NUS Graduate Medical School,
Singapore (Smith); the Forest Protection Department of Ho Chi Minh City, Ho Chi Minh City, Vietnam (Cuong); the SubDepartment of Animal Health HCMC, Veterinary Diagnostic Laboratory Division, Ho Chi Minh City, Vietnam (Tho); the
Vertebrates Research Division, National Institute of Biological Resources, Incheon, Korea (Han); the Species Restoration
Center, Korea National Park Service, Gurae, Korea (Yang); and the Department of Green Life Science, Sangmyung
University, Seoul, Korea (C. B. Kim).
*These authors contributed equally to the work.
Address correspondence to Dr Kyung Seok Kim at the address above, or e-mail: [email protected].
Abstract
The Asiatic black bear is one of the most endangered mammals in South Korea owing to population declines resulting from
human exploitation and habitat fragmentation. To restore the black bear population in South Korea, 27 bear cubs from North
Korea and Russian Far East (Primorsky Krai) were imported and released into Jirisan National Park, a reservoir of the largest
wild population in South Korea, in 2004. To monitor the success of this reintroduction, the genetic diversity and population
structure of the reintroduced black bears were measured using both mitochondrial and nuclear DNA markers. Mitochondrial
D-loop region DNA sequences (615 bp) of 43 Japanese black bears from previous study and 14 Southeast Asian black bears in
this study were employed to obtain phylogenetic inference of the reintroduced black bears. The mitochondrial phylogeny
indicated Asiatic black bear populations from Russian Far East and North Korea form a single evolutionary unit distinct from
populations from Japan and Southeast Asia. Mean expected heterozygosity (HE) across 16 microsatellite loci was 0.648 for
Russian and 0.676 for North Korean populations. There was a moderate but significant level of microsatellite differentiation
(FST 5 0.063) between black bears from the 2 source areas. In addition, genetic evidences revealed that 2 populations are
represented as diverging groups, with lingering genetic admixture among individuals of 2 source populations. Relatedness
analysis based on genetic markers indicated several discrepancies with the pedigree records. Implication of the phylogenetic
and genetic evidences on long-term management of Asiatic black bears in South Korea is discussed.
Key words: Asiatic black bear, conservation, endangered species, genetic diversity, microsatellites, reintroduction, Ursus thibetanus
The Asiatic black bear (Ursus thibetanus) is threatened in
much of its native habitat. In South Korea, U. thibetanus has
been designated as an Endangered Species I (Ministry of
Environment of Korea 2005) and a Natural Monument
Species (No. 329; Cultural Heritage Administration Korea
1982, http://search.cha.go.kr/srch/jsp/search_top.jsp), and,
elsewhere, as a vulnerable (IUCN 2010) and noncommercial
trade species (IUCN 2010).
The Asiatic black bear has been of culturally and religiously
importance to Koreans for thousands of years. Despite this, the
species was systemically eradicated under the ‘‘Injurious Animal
Destruction’’ program during the Japanese occupation of Korean
165
Journal of Heredity 2011:102(2)
peninsula (Annual Reports of the Japanese Government-General
of Choson 1915–1924). In addition, much of their habitat
disappeared during the Korean War (1950–1953) and subsequent economic development. Overhunting and poaching
also continued until the Asiatic black bear was designated as
a ‘‘Natural Monument Species,’’ and all hunting was prohibited
in 1982. However, even this legal protection has not completely stopped the illegal hunting of the animals.
The Asiatic black bear of South Korea is estimated to
have been reduced to less than 20 individuals across a widely
scattered distribution. In Jirisan National Park (JNP),
considered as a reservoir of the largest wild population in
South Korea, fewer than 5 individuals are thought to survive
(Lee and Jeong 2009). A report from the Population and
Habitat Viability Assessment workshop, held in 2001 to
estimate the viability of Asiatic black bear population in
JNP, concluded that the Asiatic black bears in JNP could
not survive without supplementation from other populations (Lee and Jeong 2009). Based on the report, the Korea
Ministry of Environment initiated an ambitious reintroduction project to restore the black bear population in JNP.
Since 2004, 27 bear cubs from Russian Primorsky Krai
and North Korea had been imported and released into JNP
(Lee and Jeong 2009). North Korean and Russian black
bears have been considered as important source populations
for a reintroduction program in South Korea on the basis of
geographic proximity to South Korea and basic genetic
analysis of animals for reintroduction (Hong 2005).
However, genetic information on the populations from
Russia and North Korea remains scarce, and no information
about genetic structure of these populations is available.
Selection of appropriate population for reintroduction is
therefore of continuing concern, and efficacy of reintroduction program in South Korea needs to be evaluated.
In general, genetic variation is considered important for
a population to better adapt to a changing environment. It
has been shown that reduced genetic variation resulting
from population reductions, genetic drift, and founder
effects may impede the adaptation of a population to a new
environment and increase the likelihood of its extinction
(Frankham and Ralls 1998; Frankham et al. 2002; Allendorf
and Lundquist 2003). Therefore, genetic variation is
considered to be an important component of adaptability
and long-term sustainability of natural populations. This
situation is also true for newly introduced individuals in the
process of restoring threatened animal. Because reintroduction usually involves only a small number of founders, the
initial level of genetic diversity should be considered as an
important element to increase probability for successful
settlement and survival of these animals in a new habitat.
Moreover, individuals chosen for reintroduction programs
need to be screened for genetic variation to decrease the
chance of inbreeding depression by avoiding cointroduction
and subsequent mating of closely related individuals.
This study investigates the evolutionary status and the
extent of genetic diversity of source populations of black
bears employed in reintroduction program for restoring
Asiatic black bears in South Korea using both mitochondrial
166
DNA (mtDNA) and microsatellite markers. Our intent was
to assess effectiveness of the current Asiatic black bear
restoration program in South Korea and to evaluate genetic
health of the 2 resource populations employed for the
program.
Materials and Methods
Samples and DNA Extraction
Tissue samples were collected nonlethally from 24 Asiatic
black bears from Russian Primorsky Krai and North Korea
with help from the Species Restoration Center, Korea
National Park Service (Table 1). Most of Northeast Asiatic
black bear samples except for 3 specimens from Russian
Primorsky Krai were from the animals reintroduced into
JNP, South Korea from 2004 to 2007. The 3 samples
(Rus06, Rus08, and Rus12) were from animals that were
genetically analyzed, but not used for actual reintroduction.
Blood samples from an additional 14 Southeast Asiatic black
bears were collected from a local bear rescue center in
Vietnam during regular health surveys. In spite of limited
information on the exact original locality of the Southeast
Asian black bear in this study, it is certain that these animals
are native to Vietnam or adjacent area by collectors’
comments. Skin, hair, and blood samples stored in disodium
ethylenediaminetetraacetic acid were frozen at 70 °C.
Genomic DNA was extracted using Qiagen DNeasy blood
and tissue kit (Qiagen, Valencia, CA) following the
manufacturer’s instructions.
MtDNA Sequencing and Microsatellite Genotyping
The mtDNA control region was amplified with 4 pairs of
primers, L15775 and H651, URL2 and URH2, URL3 and
URH1, URL4 and H651, for D-loop region (Uchiyama
1998). Polymerase chain reaction (PCR) amplification was
performed in a 25 ll of reactions containing 25 ng total
genomic DNA, 1.5 mM MgCl2, 0.2 mM dNTP, 0.5 mM of
each primer, 0.5 unit of Taq polymerase using following
PCR condition: an initial denaturation for 5 min at 94 °C, 5
cycles of denaturation for 45 s at 94 °C, 35 cycles of primer
annealing for 45 s at 55 °C and an extension for 1 min 30 s
at 72 °C followed by a final extension of 5 min at 72 °C. The
PCR products of mtDNA were purified using QIAEX II
Extraction Kit (Qiagen, Cat. No. 20021, CA) and sequenced
in both direction on ABI PRISM 310 Genetic Analyzer
(Applied Biosystem, CA). The D-loop sequences used for
phylogenetic analysis have been deposited to GenBank
(EU264503–EU264527, HM135178–HM135193).
For microsatellite analysis, total of 16 primer sets from 3
different origins were used for genotyping determination.
Six primer pairs (MSUT2, MSUT3, MSUT4, MSUT5,
MSUT7, and MSUT8) were originally developed for
U. thibetanus (Kitahara et al. 2000). Five primer pairs
(G10B, G1D, G10L, G10P, and G10X) were originally
developed for the American black bear, U. americanus,
(Paetkau and Strobeck 1994). The remaining 5 primer pairs
Kim et al. Conservation Genetics of Northeast Asian black bears
Table 1 Sample information on Asiatic Black bears from Russian Primorsky Krai and North Korea analyzed in this study
Population
Locality
Sample ID
Sex
Tissue
Russia, Primorsky
Krai
Yakovlevsky District
Rus01
Rus02
Rus03
Rus04
Rus05
Rus06
$
#
#
$
#
#
Blood
Blood
Blood
Blood
Blood
Hair
Rus07
Rus08
Rus09
Rus10
Rus11
Rus12
NK01
NK02
NK03
NK04
NK05
NK06
NK07
NK08
NK09
NK10
NK11
NK12
$
#
$
$
$
$
$
#
#
$
#
$
$
#
#
#
$
$
Hair
Hair
Hair
Hair
Hair
Hair
Blood
Blood
Blood
Blood
Blood
Blood
Blood
Blood
Blood
Blood
Blood
Blood
Chuguevsky District
North Korea
Lazovsky Distict, Lazo Reserve,
Egerevka River
Yakovlevsky District
Primorsky
Yakovlevsky District
Yakovlevsky District
Chuguevsky District
Primorsky
North Korea
North Korea
North Korea
North Korea
North Korea
North Korea
North Korea
North Korea
North Korea
North Korea
North Korea
North Korea
Relationship by record
Genetic relatedness
—
Brothers
0.313
Brother and sister
0.547
—
—
—
Sisters
0.825
—
—
—
—
—
—
Brother and Sister
0.040
Brother and Sister
0.270
Brothers
0.399
Sisters
0.534
Genetic relatedness was represented by r value calculated by Relatedness version 5.0 software (Queller and Goodnight 1989). Pairwise relatedness among all
individuals is available from Supplementary Table 4.
(UarMU09, UarMU23, UarMU50, UarMU61, and UarMU64) were developed for the brown bear, U. arctos (Taberlet
et al. 1997).
PCR was carried out in a 12.5 ll reaction volume
containing 2 ll of DNA template, 1 PCR buffer
(iNtRON, Sungnam, Korea), 2 mM MgCl2, 0.2 mM of
each dNTP, 0.15 lM of each primer (only forward primers
were labeled with 3 different fluorescence dyes), 1 unit of
i-Star Taq polymerase (iNtRON). The PCR amplification
reactions were performed in a DICE PCR Thermal Cycler
(Takara Bio., Shiga, Japan) with the following conditions:
initial denaturation for 10 min at 94 °C, followed by 40
cycles (94 °C for 30 s, 44–52 °C for 30 s, and 72 °C for 60 s)
with a final extension for 10 min at 72 °C. PCR products
were resolved by electrophoresis on a 2% agarose gel, stained
by ethidium bromide, and visualized under ultraviolet
illumination. Amplified microsatellites were visualized using
an ABI PRISM 310 genetic analyzer (Applied Biosystems,
Foster City, CA). Three or 4 repeated genotypes were
conducted for each sample followed by the multitube
approach (Taberlet et al. 1996). Following PCR, products
were diluted 1:10–1:20, and 2 ll was mixed with an internal
standard according to the manufacturer’s instructions.
Data Analysis
mtDNA Sequence Analysis
MtDNA sequences (615 bp) were aligned using CLUSTAL_X (Thompson et al. 1997), with further modification
by eye. Haplotype (h) and nucleotide (p) diversities among
geographic locations were estimated in DnaSP version 4.10
(Rozas et al. 2003). A reduced median network (Bandelt
et al. 1999) was drawn using the program Network 4.5 to
investigate possible relationships among haplotypes of
Asiatic black bears. To define evolutionary significant units
(ESUs) of reintroduced black bears from Russian Primorsky
Krai and North Korea, we included 14 sequences from
Southeast Asian black bears in this study and 43 sequences
from Japanese black bears available on GenBank
(AB441772 to AB441814, Ohnishi et al. 2009). Neighborjoining tree among haplotypes of mtDNA D-loop was
reconstructed using the program MEGA version 3 (Kumar
et al. 2004). The stability of internal nodes was assessed by
1000 bootstrap replicates. The program GENALEX version
6.1 (Peakall and Smouse 2006) was used to carry out
a principal coordinate analysis and pairwise Upt value based
on mtDNA D-loop variation.
Microsatellites Analysis
The program Flexbin was used for automated binning of
microsatellite raw data (http://www.zoo.cam.ac.uk/zoostaff/amos). The numbers of different alleles per locus
and expected (HE) and observed (HO) heterozygosity were
calculated as indices of genetic diversity in each population
using CERVUS version 3.0 software (Marshall et al. 1998).
The program FSTAT version 2.9.3 (Goudet 1995; http://
www.unil.ch/izea/softwares/fstat.htm) was used to quantify
167
Journal of Heredity 2011:102(2)
the extent of genetic differentiation between the 2 populations using the FST and F statistics (Weir and Cockerham
1984) per locus across all populations and their respective P
values. The sequential Bonferroni correction was applied to
adjust significance levels for analyses involving multiple
comparisons (Rice 1989).
Tests for genotypic disequilibrium and deviation from
the Hardy–Weinberg equilibrium (HWE) were estimated for
each locus, following the heterozygote deficiency (Guo and
Thompson 1992), using GENEPOP version 3.3 software
(Raymond and Rousset 2003; http://genepop.curtin.edu.au/). STRUCTURE 2.3.3 software (Pritchard et al.
2000) was used to demonstrate the population structure for
24 individuals from the Russian Primorsky Krai and North
Korea. We tested for heterozygosity excess and shift in
allelic frequency distributions that would correlate with
a recent genetic bottleneck using the BOTTLENECK
version 1.2.02 software (Cornuet and Luikart 1996). The
Wilcoxon signed rank test was used to obtain probability
values for excess levels of heterozygosity due to the small
number of loci and small sample size. A 2-phased model of
mutation (TPM) was used to test for excess of heterozygosity. We also examined allele frequency distribution for
a mode-shift detected in a bottlenecked population. Lastly,
the M value of Garza and Williamson (2001) and its variance
across loci were calculated using the AGARST software
program (Harley 2001). M is the mean ratio of the number
of alleles to the range of allele size, which can be used to
detect reductions in both recent and historical population
sizes (Garza and Williamson 2001; Spear et al. 2006).
According to the characteristics of M-ratio by Garza and
Williamson, this shows relatively long-term bottleneck
events. On the other hand, stepwise mutation model
(SMM) and TPM determined by Wilcoxon sign-rank test
can detect relatively short-term bottleneck events.
Relatedness values were estimated using Relatedness
version 5.0 (Queller and Goodnight 1989). The program
calculates relatedness value (r) between individuals, which is
twice the coefficient of kinship (Fij) if 2 individuals are not
inbred. Principal component analysis (PCA) was performed
to visualize pairwise differentiation between individuals using
PCAGEN version 1.2 software (Goudet 1999). In total,
15 000 randomizations of genotypes were performed to test
for significance of axes. The statistical certainty of assignment
for individuals into their reference populations was evaluated
using GENECLASS version 2.0 software (Piry et al. 2004).
We followed the Bayesian approach developed by Rannala
Table 2
Results
Genetic Variability of Asiatic Black Bears
The mtDNA D-loop (615 bp in length) was sequenced for 38
Asiatic black bears, including 24 bears from Russian
Primorsky Krai or North Korea and 14 from Southeast
Asia. For the 4 major geographic regions studied, a total of 60
haplotypes were defined by 79 polymorphic sites (Table 2).
Most of polymorphic sites were accounted for by variation
in Japanese and Southeast Asiatic black bears as represented
by 53 haplotypes from 71 polymorphic sites. The overall
haplotype and nucleotide diversity from D-loop regions
were 0.974% and 2.706%, respectively. Relatively low
diversity was found for 2 source populations of reintroduction. There was no sharing haplotypes between Russian
(3 haplotypes) and North Korean (4 haplotypes) black
bears, with slightly higher haplotype (0.455 vs. 0.318) and
nucleotide (0.108 vs. 0.076) diversity in North Korean black
bears. GenBank accession numbers of mtDNA D-loop
sequence for all samples sequenced in this study are
provided in Supplementary materials(Supplementary Table 1).
In total, 24 bears from Russian Far East and North
Korea were genotyped for 16 microsatellite loci. Average
alleles per locus were 4.8 and 4.9 for Russian and North
Korean populations, respectively. In the Russian population,
MSUT3 and G10B were the least polymorphic with 2 alleles,
and UarMU09 was the most polymorphic with 8 alleles. In
the North Korean population, MSUT3 was the least
polymorphic with 2 alleles, and G10P was the most
polymorphic with 10 alleles. There were divergences of
allele frequencies and unique alleles between 2 populations.
Comparison of allelic data across the 2 populations showed
that 54 of the 102 total alleles (52.9%) were common to
both populations, and 25 (24.5%) and 23 (22.5%) alleles
were unique to Russia and North Korea, respectively.
Average expected heterozygosity for each population was
comparable, HE 5 0.648 for the Russian population and
0.676 for the North Korean population (Table 3). Departure
Mitochondrial DNA D-loop diversity of Asiatic black bears in this study (based on 615 bp)
North Korea
Russia
SE Asia
Japan
Total
168
and Mountain (1997) because the Bayesian method is
considered more accurate than frequency- and distance-based
methods (Cornuet et al. 1999; Koskinen 2003). Assignment
of each individual was tested using the ‘‘leave one out’’
procedure (Efron 1983), in which each individual was
excluded from the data set when performing its assignment.
N
No. of
Haplotype
No. of
Polymorphic site
Haplotype diversity
(SD)
% Nucleotide diversity
(standard deviation)
12
12
14
43
81
4
3
11
42
60
4
2
33
38
79
0.455
0.318
0.967
0.999
0.974
0.108
0.076
1.605
0.762
2.706
(±0.170)
(±0.164)
(±0.037)
(±0.005)
(±0.010)
(±0.049)
(±0.042)
(±0.309)
(±0.071)
(±0.077)
Kim et al. Conservation Genetics of Northeast Asian black bears
from HWE was found at 3 loci for the Russian population
and at 6 loci for the North Korea population (Table 3). All
observed biases were in the direction of heterozygote
deficiency. Linkage disequilibrium test showed 5 pairs of
significant linkage among all the possible locus pairs at
a global level as well as within the populations analyzed in
each of these 2 countries. The pair showing the lowest
significant P values at a global level was G10P-UarMU61
(P , 0.05). However, it was not significant after correction
for multiple tests.
Genetic Relationships and Population Structure of Asiatic
Black Bears
The neighbor-joining tree showed that mtDNA D-loop
sequences of Asiatic black bears form a number of regional
clades (up to 6) (Figure 1). Clades 1 and 2 are entirely
represented by Japanese black bears and Clades 3, 4, and 6 by
Southeast Asian black bears. Northeast Asian black bears,
that is, bears reintroduced into South Korea, were grouped
together with a high bootstrap support of 99% and formed
a distinct clade (Clade 5) from black bears from Japan,
displaying sister group to a cluster (Clade 6) of Southeast Asia.
It is noted that Southeast Asian black bears show complex
topology, suggesting complicated subpopulation structure of
this regional population. In addition, the Southeast Asian
black bear population had the highest level of genetic
diversity (Table 2). Reduced median network (Supplementary
Figure 1) based on mtDNA D-loop sequences (615 bp) for all
of black bears revealed 2 major groups separating Japanese
black bears from Northeast and Southeast Asian bears where
Southeast black bears formed more divergent group in
comparison with Northeast counterparts showing a single
cluster, which is consistent with the result of phylogenetic
tree (Figure 1) and other genetic evidences (Supplementary
Table 2 for pairwise Upt value of mtDNA sequences and
Supplementary Figure 2 for PCA).
Microsatellite loci showed a moderate degree of
differentiation between the Russian Primorsky Krai and
North Korea (FST value of 0.063, P , 0.05). PCA analysis
based on microsatellite allele frequencies of Northeast Asian
black bears from Russian Far East and North Korea
revealed 26.5% of the total variation in the first 2 axes and
showed evidence of a certain level of distinction between
sources of 2 bear groups despite admixture among a few of
individuals (Figure 2). Structure analysis showed the highest
posterior probability when population number (K) was set
to 2 (Figure 3) and suggested that Russian black bears
consisted of individuals with two distinct genetic backgrounds.
Population bottleneck events were tested for in the
reintroduced black bears (Table 4). SMM and TPM by
Wilcoxon sign-rank tests did not detect any significant
population reduction in the individuals from 2 source
populations. However, M-ratio of Garza and Williamson
showed a weak evidence for historical population reduction
in North Korean bears (M-ratio , 0.680).
Individual Assignment and Relatedness among Asiatic Black
Bears
Assignment test using the GENECLASS program showed
that individuals from Russian Far East and North Korea
displayed a tendency of differentiation consistent with
their population origin (Supplementary Table 3). When
assignment threshold of scores was set to 0.05, all
individuals of the Russian population were assigned to their
original population as the most likely source population.
However, for 3 Russian individuals, North Korean was not
excluded as the second possible source population at the
Table 3 Descriptive statistics for 24 samples of the 2 Asiatic black bear populations from North Korea and Russia
Russia Primorsky Population
North Korea Population
Total
Locus
N
A
HE
HO
P
N
A
HE
HO
P
N
A
HE
HO
P
FST
MSUT2
MSUT3
MSUT4
MSUT5
MSUT7
MSUT8
G1D
G10B
G10L
G10P
G10X
UarMU09
UarMU23
UarMU50
UarMU61
UarMU64
Mean
11
12
12
10
12
11
12
11
12
12
12
12
11
12
12
12
11.6
3
2
5
5
7
4
3
2
7
7
5
8
5
6
4
4
4.8
0.558
0.344
0.801
0.800
0.804
0.688
0.489
0.173
0.783
0.830
0.652
0.775
0.658
0.775
0.634
0.598
0.648
0.182
0.417
0.667
0.900
0.583
0.727
0.500
0.182
0.667
0.917
0.667
0.583
0.818
0.667
0.500
0.417
0.587
0.009*
1.000
0.131
0.712
0.060
0.485
0.706
1.000
0.463
0.916
0.573
0.009*
0.983
0.043*
0.236
0.154
—
10
12
12
12
12
10
12
12
12
12
12
12
10
12
12
12
11.6
4
2
8
5
6
5
3
4
6
10
4
6
3
4
4
5
4.9
0.363
0.431
0.848
0.775
0.641
0.789
0.638
0.656
0.812
0.888
0.612
0.703
0.626
0.757
0.627
0.656
0.676
0.300
0.583
0.833
0.667
0.750
1.000
0.500
0.917
0.500
0.750
0.250
0.583
0.300
0.917
0.167
0.417
0.590
0.307
1.000
0.415
0.153
0.965
1.000
0.107
1.000
0.011*
0.062
0.003*
0.022*
0.023*
0.964
0.027*
0.010*
—
21
24
24
22
24
21
24
23
24
24
24
24
21
24
24
24
23.3
5
2
8
5
9
6
4
4
9
12
6
8
5
7
5
7
6.4
0.517
0.383
0.859
0.794
0.762
0.776
0.598
0.469
0.815
0.893
0.626
0.759
0.700
0.785
0.624
0.638
0.687
0.238
0.500
0.750
0.773
0.667
0.857
0.500
0.565
0.583
0.833
0.458
0.583
0.571
0.792
0.333
0.417
0.589
0.033*
1.000
0.085
0.361
0.151
0.885
0.052
1.000
0.006*
0.148
0.013*
0.004*
0.062
0.039*
0.062
0.050
—
0.156
0.011
0.073
0.017
0.094
0.103
0.104
0.179
0.030
0.071
0.022
0.041
0.144
0.046
0.051
0.020
0.063
number of individuals (N), number of alleles (A), expected heterzoygosity (HE), observed heterozygosity (HO), P value for heterozygote deficit (P), and FST
value for each locus. Asterisks represent significant P value against HWE.
169
Journal of Heredity 2011:102(2)
Figure 1. Phylogenetic relationship among Asiatic black bears by the neighbor-joining tree of mitochondrial D-loop sequences.
Numbers at the major clades denote the bootstrap values. JAP: Japanese black bears, East (E), South (S), West (W), SEA:
Southeast Asiatic black bears, NK: North Korean, RUS: Russian, Number next to abbreviation denotes individual ID. Outgroup:
American black bear, Ursus americanus (AF303109).
threshold of 0.05. In the case of the North Korean
individuals, all were correctly assigned to their population of
origin as the most likely source, but 5 individuals including
NK08, for which assignment score was very close between
the 2 populations, were assigned to Russian population as
the second likely source population. There was slightly lower
assignment score for the North Korean population than for
the Russian population (Supplementary Table 3).
Pairwise relatedness test for 276 pairs among 24
individuals was estimated to verify the original pedigree
170
information of the reintroduced bears and to ascertain the
level of relatedness between individuals in the population
(Supplementary Table 4). Relatedness values for 7 bear pairs
with official pedigree record as sibling are shown in Table 1.
Five of 7 pairs showed value of r . 0.3, approximately
consistent with the expected value for siblings. However,
the remaining 2 pairs (NK05 vs. NK06 and NK07 vs.
NK08) displayed value of r , 0.05, far less than the
expected 0.5 for full sibs. From the remaining 269 individual
pairs, excepting the 7 pairs with official full-sib records, one
Kim et al. Conservation Genetics of Northeast Asian black bears
Figure 2. Scatter diagram of factor scores for 24 Asiatic black bears derived from PCA. NK: Black bears from North Korea,
Rus: Black bears from Russian Primorsky Krai.
case showed relatedness value greater than 0.5 (r 5 0.566
for Rus08 vs. Rus09), suggesting a first-order kinship not
recorded in the pedigree.
Discussion
ESUs designate populations or groups of populations with
long-term evolutionary isolation (Ryder 1986) and is
important for managing and establishing priority for
populations for conservation. The criteria for defining
ESUs are debatable (Moritz 1994) but can be conservatively
identified as sets of populations distinguished by strong
phylogenetic structuring of mtDNA variation and divergence in the frequencies of nuclear alleles. It has been
suggested previously that the Russian and North Korean
Asiatic black bear populations can be regarded as a single
ESU based on the result of mtDNA analysis (Hong 2005).
The present study based on mtDNA sequence comparison
among Asiatic black bears is in a good agreement with the
previous finding and those of Yoshiki et al. (2009), showing
a close relationship of mtDNA lineages. Therefore, previous
and present results support utilizing Russian/North Korean
black bears as the source population for reintroduction of
Asiatic black bears into JNP, South Korea.
Average expected heterozygosity was slightly higher in
North Korean Asiatic black bears than those from Russian
Primorsky Krai (0.676 vs. 0.648 in HE), and this tendency was
also found in mtDNA D-loop diversity. Relative comparison
of genetic diversity estimates among other black bear species/
populations would be informative to understanding of the
present status of genetic variability for 2 source populations
reintroduced to South Korea. Although different sets of
microsatellite loci were employed, level of average expected
heterozygosity ranged from 0.461 in U. thibetanus from East
Japan to 0.799 in U. americanus from Canada (Table 5). When
diversity comparison was made among U. thibetanus populations, heterozygosity of reintroduced populations in South
Korea was higher than that of most populations from Japan
(Table 5). This could imply that the reintroduced Asiatic black
bear in South Korea are unlikely to suffer from inbreeding
effects. However, because the different set of microsatellites
was employed in diversity comparison and this may cause an
inherent ascertainment bias that varies among primer pairs, it
should be interpreted with caution.
Figure 3. Bar plot (K 5 2) from population structure analysis for Asiatic black bears from Russian Primorsky Krai (Rus) and
North Korea (NK).
171
Journal of Heredity 2011:102(2)
Table 4
Results of tests to detect recent population reduction in the Korean and Russian black bear populations
Wilcoxon sign-rank testsa
Population
SMM
TPMb
Mode shift
M-ratio of Garza and Williamson
Russian Primorsky Krai
North Korea
0.874
0.570
0.550
0.248
Normal
Normal
0.680 (0.049)
0.656 (0.047)
c
a
One tail probability for observed heterozygosity excess relative to the expected equilibrium heterozygosity (Heq), which is computed from the observed
number of alleles under mutation-drift equilibrium. SMM, stepwise mutation model.
b
The test was conducted assuming a generalized stepwise mutation model (GSM) with a variance of 0.36 in geometric distribution of mutation lengths
(Estoup et al. 2001).
c
M value and its variance (in parenthesis) of Garza and Williamson’ (2001). M 5 the mean ratio of the number of alleles to the range of allele size.
The present study using nuclear microsatellite DNA
markers revealed a moderate degree of genetic differentiation between Asiatic black bears from Russian Primorsky
Krai and North Korea. This finding is in agreement with
mtDNA presented here and in a previous study (Hong
2005), showing that populations of Northeast Asia (Far East
Russia, North-East China, and Korean peninsula) cluster
into a single clade. Even though the microsatellite analysis
displays some signatures of genetic differentiation between
the 2 populations, we do not consider that this warrants
separate ESUs for the 2 populations. Instead, we consider it
more likely to reflect relatively recent isolation and loss of
diversity due to exploitation and habitat fragmentation.
There are 3 possible factors that might contribute to the
differentiation of the Russian population from the North
Korean population. The first is the natural geographic barrier
between the 2 populations. Tumen River is the most
prominent geographical barrier between Russia and North
Korea. Even though the size of the river is relatively small and
it is known that bears can swim and sometimes cross rivers
(Lance 2003), the downstream portion of the river that borders
Table 5
Microsatellite diversity for different species/populations of genus Ursus
Species
Ursus arctos
(Brown bear)
Ursus americanus
(American black bear)
Ursus thibetanus
(Asiatic black bear)
a
Russia and North Korea might be of sufficient size to cause
a certain level of reproductive isolation. More importantly,
wide stretch of low land between border of North Korea and
Russia may not be a good habitat for Asiatic black bears, and
this might function as a geographic barrier to migration
between 2 populations. The second factor might be artificial
barriers between the 2 regions, such as border fences and
barbed wire fences that have been erected, as well as human
development that has occurred over the last several decades
(Lee 2004; Norma 2007). The third factor, the systematic
eradication program against large carnivores that was carried
out in the early 20th century in the Korean peninsula almost
certainly resulted in some random loss of diversity (Endo
2009). Although the demographic bottleneck was not
evidenced by some bottleneck tests, M-ratio of Garza and
Williamson showed a signal of a recent historical population
reduction for North Korean bears (M-ratio , 0.680).
Accurate pedigree information is critical for the
maintenance of genetic health and diversity of a small
population under intensive management for conservation
(Ballou and Lacy 1995). Our results confirm the importance
Region of the
population
Sample
size
No. of
Loci
a
Mean number of
alleles per locus
HE
HO
Reference
6.8
7.38
4.38
9.50
0.71
0.758
0.551
0.799
0.66
—
—
—
Waits et al. (2000)
Paetkau et al. (1998a)
Naoki et al. (2007)
Scandinavia
Kluane, Canada
Yellowstone, USA
West Slope, Canada
377
50
57
116
19
6b
6
6
Western Chukogu, Japan
Eastern Chukogu, Japan
Western northern Kinki,
Japan
Eastern northern Kinki,
Japan
Central Honshu, Japan
Primorsky Krai, Russia
North Korea
72
46
50
10c
10
10
3.67
3.78
3.99
0.529
0.461
0.499
0.513
0.428
0.490
50
10
4.54
0.610
0.596
56
12
12
10
16d
16
6.55
4.81
4.94
0.703
0.648
0.676
0.643
0.587
0.590
Paetkau et al. (1998b)
This study
This study
19 loci (G1A, G1D, G10B, G10C, G10L, G10P, G10M, G10X, G10H, G10O, G10J, UarMU10, UarMU05, UarMU15, UarMU23, UarMU50, UarMU51,
UarMU59, and UarMU61).
b
6 loci (G1A, G10B, G1D, G10L, G10M, and G10X).
c
10 loci (G1A, G10B, G1D, G10L, G10M, G10X, MSUT1, MSUT2, MSUT6, and MSUT7).
d
16 loci (MSUT2, MSUT3, MSUT4, MSUT5, MSUT7, MSUT8, G1D, G10B, G10L, G10P, G10X, UarMU09, UarMU23, UarMU50, UarMU61, and
UarMU64).
172
Kim et al. Conservation Genetics of Northeast Asian black bears
of validating reintroduction records using genetic markers.
There was some discrepancy between the pedigree record in
the Species Restoration Center of Korean National Park
Service and result from the genetic relatedness test in this
study. Some of the individuals (e.g., Rus08-09) showed
estimates of relatedness consistent with full sibs, even
though they are recorded as unrelated by official record. In
contrast, some individuals that were recorded as full-sib
relationship showed a very low level of genetic relatedness
(Table 1). This might be caused by a low sensitivity of
analytical methods due to sampling error from small sample
size used for estimating population allele frequencies (Wang
2002; Blouin 2003). Alternatively, the pedigrees may have
been misrecorded. Because the reintroduced bears translocated from Russia were originally found in the wild as
orphaned bear cubs, it is possible that a litter of orphaned
cubs found individually was not recorded as being related.
In conclusion, the present study indicates that Asiatic
black bear populations in Korean peninsula and Russian Far
East belong to a single ESU, and the translocation of these
bears into JNP, South Korea will contribute to genetic
enrichment of the existing population. Accurate pedigree
information on a founder population of a reintroduction
program will help to manage subsequent generations in
a way to maximize the maintenance of genetic diversity that
is essential for long-term survival of the population. It is
highly recommended that the genetic status of the
reintroduced population should be closely monitored using
the molecular genetic methods in this study to confirm the
reproductive success of translocated individuals.
Supplementary Material
Supplementary material can be found at http://www.jhered.
oxfordjournals.org/.
Funding
This work was partially supported by the year 2005 grant
titled ‘‘Assessment of genetic information of Korean black
bears’’ funded by JNP Southern Office and the year-2009
grant titled ‘‘The genetic evaluation of important biological
resources’’ (No. 074-1800-1844-304) funded by The
National Institute of Biological Resources, Korean Government.
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Received September 10, 2010; Revised October 28, 2010;
Accepted November 3, 2010
Corresponding Editor: C. Scott Baker