Microevolution of the Mitochondrial DNA Control Region in the Japanese Brown Bear (Ursus arctos) Population
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Microevolution of the Mitochondrial DNA Control Region in the Japanese
Brown Bear (Ursus arctos) Population
Tamako Matsuhashi,* Ryuichi Masuda,*† Tsutomu Mano,‡ and Michihiro C. Yoshida*†
*Division of Bioscience, Graduate School of Environmental Earth Science, and †Chromosome Research Unit, Faculty of
Science, Hokkaido University, Sapporo, Japan; and ‡Hokkaido Institute of Environmental Sciences, Sapporo, Japan
We investigated nucleotide sequences of the mitochondrial DNA control region to describe natural genetic variations
and to assess the relationships between subpopulations of the brown bear Ursus arctos on Hokkaido Island, Japan.
Using the polymerase chain reaction product–direct sequencing technique, partial sequences (about 930 bases) of
the control region were determined for 56 brown bears sampled throughout Hokkaido Island. A sequence alignment
revealed that the brown bear control region included a variable sequence on the 59 side and a repetitive region on
the 39 side. Phylogenetic trees reconstructed from the 59 variable region (696–702 bases) exhibited 17 haplotypes,
which were clustered into three groups (Clusters A, B, and C). The distribution of each group did not overlap with
those of the others, and the three different areas were located in separate mountainous forests of Hokkaido Island.
Furthermore, most of the phylogenetically close haplotypes within each group were distributed geographically close
to each other. In addition, the 39 repetitive region (arrays of 10 bases) exhibited a much faster mutation rate than
the 59 variable region, resulting in heteroplasmy. Such mitochondrial DNA divergence in each group could have
occurred after the brown bears migrated from the continent to Hokkaido and became fixed in the different areas.
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Introduction
The brown bear Ursus arctos is widely distributed small and are threatened with extinction (Hokkaido
in the Holarctic region and has the largest habitat among Government 1996). A decrease in genetic variation due
the seven ursine species of the world. In the Japanese to habitat fragmentation could lead to extinction of the
islands, the brown bear occurs only on Hokkaido Island, population. A previous study of minisatellite DNA fin-
and this population is classified as a separate subspecies, gerprinting (Tsuruga et al. 1994) suggested low genetic
U. a. yesoensis Lydekker, 1897, based on morphological variability in the Hokkaido brown bear population com-
characteristics: a relatively small body size and a dark pared with those of other mammalian populations. It is
brown coat color. Since Hokkaido was connected with necessary to understand the genetic variation of the
Sakhalin and the Siberian continent until around 12,000 Hokkaido brown bear population for conservation and
years ago during the last glacial period (Ohshima 1991), management.
the Hokkaido brown bear population is generally con- In the present study, we investigated the mitochon-
sidered a glacial-period relict which has been isolated in drial DNA (mtDNA) control region sequences to de-
Hokkaido. Our preliminary study showed that the Hok- scribe natural genetic variation and to assess the rela-
kaido brown bears could be phylogenetically much clos- tionships between brown bear subpopulations on Hok-
er to the Russian brown bears, which are included in kaido Island. Since mtDNA control region sequences
the Eastern Europe lineage (Masuda et al. 1998). have high substitution rates, this region is an appropriate
Because Hokkaido Island belongs to a transition genetic marker for phylogenetic analysis of European
zone from the northern Asiatic temperate zone to the brown bears (Taberlet and Bouvet 1994; Kohn et al.
subarctic zone (Tatewaki 1958), vegetation of this island 1995) and North American brown bears (Waits et al.
is extremely variable. The forest vegetation mainly con- 1998). We also examined the mtDNA cytochrome b se-
sists of mixed conifer-hardwood forests, with forests of quences compared with geographic divergence of the
the Japanese beech in southwestern Hokkaido. Food control region. Based on these mtDNA data, we discuss
habits of the Hokkaido brown bear are different among the unique genetic structure of the brown bear popula-
areas, depending on available foods (Ohdachi and Aoi tion that has been isolated on Hokkaido Island.
1987). Moreover, morphological characteristics of the
brown bears differ between northeastern and southwest-
ern Hokkaido (Ohdachi et al. 1992). Thus, it is possible Materials and Methods
that there would be genetic variation in the brown bear DNA Extraction
population living on this small island.
The Hokkaido brown bear population likely de- Liver or muscle tissue samples were obtained from
creased rapidly after the late 19th century because of 56 brown bears collected at various regions on Hokkai-
deforestation and extermination. As a result, some sub- do Island. These bears were hunted legally from 1992
populations of the Hokkaido brown bears became very to 1997. Three Asiatic black bears, Selenarctos thibe-
tanus, of Japan were used as outgroup. Each tissue sam-
Key words: Ursus arctos, brown bear, mitochondrial DNA, con-
trol region, polymorphism, repetitive sequence. ple was preserved in 70% ethanol until analysis. Total
Address for correspondence and reprints: Ryuichi Masuda, Chro-
DNA was extracted from approximately 3 3 3 3 3 mm
mosome Research Unit, Faculty of Science, Hokkaido University, Sap- of the tissue using the conventional proteinase K/phenol/
poro 060-0810, Japan. E-mail: masudary@ees.hokudai.ac.jp. chloroform method (Sambrook, Fritsch, and Maniatis
Mol. Biol. Evol. 16(5):676–684. 1999 1989) with some modifications as described by Masuda
q 1999 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038 and Yoshida (1994).
676mtDNA Evolution of Brown Bears in Japan 677
FIG. 1.—A schematic diagram of the mtDNA cytochrome b and the control region of the brown bear. Arrows indicate positions of primers
(see text) for PCR and/or sequencing. ‘‘Tn’’ in the control region indicates the repeats of thymine. A dotted area shows the repetitive region
consisting of 10-base motifs. Abbreviations: E, glutamic acid–tRNA gene; T, threonine-tRNA gene; P, proline-tRNA gene; F, phenylalanine-
tRNA gene.
Polymerase Chain Reaction, Cloning, and Sequencing proportion (p)-distance method as implemented in the
The whole control region was amplified using poly- MEGA computer program, version 1.01 (Kumar, Ta-
merase chain reaction (PCR) with two primers: Cb-z, mura, and Nei 1993). Phylogenetic trees were recon-
59-ATGAATTGGAGGACAACCAGT-39; and D4, 59- structed by the neighbor-joining method (Saitou and Nei
AGGCATTTTCAGTGCCTTGCTTTG-39 (fig. 1). These 1987) and the unweighted pair-group method with arith-
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primers were designed with reference to the sequences metic mean (UPGMA) (Sneath and Sokal 1973) using
of the American black bear Ursus americanus (Shields MEGA.
and Kocher 1991) and the harbor seal Phoca vitulina
(Árnason and Johnsson 1992), respectively. Symmetric Results
PCR was performed with 35 cycles (948C for 1 min, mtDNA Control Region
608C for 30 s, and 728C for 2 min), using rTaq DNA
polymerase (TAKARA) with 1–10-ml DNA extracts in Using the PCR product–direct sequencing tech-
a total volume of 50 ml. PCR products were then puri- nique, partial fragments (926–932 bases) of the control
fied using the centrifugal dialysis kit QIAquick (QIA- region and the tRNA-Pro adjoining the 59 end of the
GEN) and used as template for direct sequencing. PCR control region were successfully sequenced for 56 Hok-
products of some selected individuals were cloned using kaido brown bears and 3 Asiatic black bears of Japan.
the TOPO TA Cloning Kit (Invitrogen). Five to eight A sequence alignment indicated that the control region
clones obtained from each individual were sequenced. sequences included a variable region on the 59 side and
The following four primers were used for sequenc- a more variable repetitive region consisting of tandem
ing (fig. 1): BED1, 59-AGCAACAGCTCCACTAC- 10-base motifs on the 39 side (fig. 1). The 59 variable-
CAG-39; BED2, 59-TCAGTCAATGGTCGCAGGAC- region sequences were used for construction of phylo-
39; BED3, 59-CGATTTAGTGGCGTTTATGTAC-39; genetic trees. The 39 repetitive sequences were analyzed
and D4. The first three primers were newly designed between individuals as described below. The present nu-
from sequences amplified with Cb-z and D4 in the pre- cleotide sequence data of the 59 variable region can be
sent study. Sequence reaction was done with the Thermo accessed in the DDBJ, EMBL, and GenBank nucleotide
Sequenase Pre-mixed Cycle Sequencing kit (Amer- sequence databases under accession numbers AB013040–
sham). Sequencing was performed using an automated AB013073.
sequencer (HITACHI SQ-5500).
Phylogenetic Relationships Based on the 59 Variable-
The whole cytochrome b sequences were amplified
Region Sequences
by PCR using the following two primers: CbM1, 59-
CTCACATGGAATCTAACCATGAC-39; and CbMR2, Seventeen haplotypes were identified from the par-
59-AGGGAATAGTTTAAATAGAATTTCAGC-39 (fig. tial sequences consisting of the tRNA-Pro (27 bases)
1). Symmetric PCR was performed with 35 cycles (948C and the first 59 sequences of the control region (669–
for 1 min, 508C for 30 s, and 728C for 1 min). PCR 675 bases) in 56 brown bears (table 1). Among the 56
products were purified and used as template for direct bears, nucleotide substitutions occurred at 38 sites out
sequencing. Sequencing was performed using the fol- of the 695 bases, exclusive of indels (table 1). Two of
lowing two primers: CbM2, 59-CCATGACTAATGA- the mutations were transversions, while the others were
TATGAAAAACC-39; and CbMR1, 59-TCTTCCTTG- transitions. Sequence differences among the 17 haplo-
AGTCTTAGGGAG-39 (fig. 1). types detected are shown in table 2.
Phylogenetic trees constructed using both the
Sequence Analysis and Phylogenetic Estimation neighbor-joining method (fig. 2) and the UPGMA (data
Sequences were aligned using the computer pro- not shown) indicated almost the same topology. The 17
gram GeneWorks, version 2.5.1 (IntelliGenetics). Posi- haplotypes were clustered into three groups (Clusters A,
tions of deletions or insertions (indels) were determined B, and C), with 90%–100% bootstrap values (fig. 2).
by observation by eye and excluded from estimation of Cluster A consisted of nine haplotypes (HB-01 to HB-
genetic distance. Genetic distance was estimated by Ki- 09), all of which were localized in central Hokkaido (fig.
mura’s (1980) two-parameter distance method and the 3). Cluster B (HB-10 to HB-13) and Cluster C (HB-14678
Table 1
Variable Positions in the Partial Sequences Consisting of tRNA-Pro (27 bp) and the 59 Sequences of the Control Region for 17 Haplotypes of Hokkaido Brown
Bears (669–675 bases) and Three Haplotypes of Asiatic Black Bears (683–685 bases)
Matsuhashi et al.
NOTE.—Positions of the sequences were numbered from the 59 end of the tRNA-Pro. Dots indicate identity with nucleotides of HB-01. Dashes indicate indel sites. Presence and absence of thymine (–⁄T) was considered
heteroplasmy.
Downloaded from http://mbe.oxfordjournals.org/ by guest on October 21, 2015Table 2
Proportion Distances (%) Obtained from Pairwise Comparisons of mtDNA Control Region and Cytochrome b Haplotypes
Haplotype HB-01 HB-02 HB-03 HB-04 HB-05 HB-06 HB-07 HB-08 HB-09 HB-10 HB-11 HB-12 HB-13 HB-14 HB-15 HB-16 HB-17 STH-02 STH-01 STH-03
HB-01. . . . . . 0.1 0.3 0.3 0.3 0.4 0.4 0.6 0.6 1.7 1.6 1.9 1.7 2.6 2.9 2.6 2.6 6.2 6.6 6.6
HB-02. . . . . . 0.1 0.1 0.1 0.3 0.3 0.4 0.4 1.9 1.7 2.0 1.9 2.7 3.0 2.7 2.7 6.3 6.8 6.8
HB-03. . . . . . 0.3 0.3 0.4 0.4 0.6 0.6 2.0 1.9 2.2 2.0 2.9 3.2 2.9 2.9 6.5 6.9 6.9
HB-04. . . . . . 0.0 0.3 0.4 0.4 0.6 0.6 2.0 1.9 2.2 2.0 2.6 2.9 2.6 2.6 6.5 6.6 6.9
HB-05. . . . . . 0.1 0.4 0.6 0.6 2.0 1.9 2.2 2.0 2.9 3.2 2.9 2.9 6.5 6.9 6.9
HB-06. . . . . . 0.6 0.4 0.7 1.9 2.0 2.3 2.2 3.0 3.3 3.0 3.0 6.6 7.1 7.1
HB-07. . . . . . 0.4 0.4 2.2 2.0 2.3 2.2 3.0 3.3 3.0 3.0 6.6 7.1 7.1
HB-08. . . . . . 0.3 2.0 2.2 2.2 2.3 3.2 3.5 3.2 3.2 6.5 6.9 6.9
HB-09. . . . . . 0.2 0.2 2.3 2.2 2.2 2.3 3.2 3.5 3.2 3.2 6.5 6.9 6.9
HB-10. . . . . . 1.2 1.2 1.1 0.1 0.4 0.3 3.7 4.0 3.7 3.7 7.1 7.5 7.5
HB-11. . . . . . 1.2 1.2 1.1 0.0 0.3 0.1 3.6 3.9 3.6 3.6 6.9 7.3 7.3
HB-12. . . . . . 0.1 3.9 4.2 3.6 3.6 6.9 7.3 7.3
HB-13. . . . . . 1.2 1.2 1.1 0.0 0.0 3.7 4.0 3.5 3.5 7.1 7.5 7.5
HB-14. . . . . . 0.6 0.9 0.9 7.6 7.8 8.1
HB-15. . . . . . 1.8 1.8 1.6 1.6 1.6 1.6 0.6 0.6 7.6 7.8 8.1
HB-16. . . . . . 1.9 1.9 1.7 1.7 1.7 1.7 0.1 0.3 7.3 7.5 7.8
HB-17. . . . . . 1.8 1.8 1.6 1.6 1.6 1.6 0.0 0.1 7.3 7.5 7.8
STH-02. . . . . 0.4 0.7
STH-01. . . . . 10.5 10.5 10.5 9.8 9.8 9.8 9.9 10.0 9.9 0.9
STH-03. . . . .
NOTE.—Control region values are shown above the diagonal, and cytochrome b values are shown below the diagonal. Indel sites were eliminated for the pairwise comparison of the control region. For cytochrome b, nine
brown bears and one Asiatic black bear were selected.
with those of the others.
these marks show the haplotype names.
are the Kimura’s (1980) two-parameter distances.
mtDNA Evolution of Brown Bears in Japan
three haplotypes were phylogenetically much closer to
cally closer haplotypes, HB-14 and HB-15, were found
bear of HB-08 (northern Hokkaido; fig. 3), while these
cally close to each other (figs. 2–4). For example, in the
each cluster showed similar relationships to those shown
m, Cluster A; V, Cluster B; M, Cluster C. Numbers to the right of
Hokkaido, respectively. Each cluster was separately dis-
on Hokkaido Island. The shaded island in the left map is Hokkaido.
located near the areas of one bear of HB-07 and one
to HB-17) were obtained from eastern and southwestern
in figure 2. Within each cluster, most of the phyloge-
679
distribution area of Cluster A, two bears of HB-09 were
Parsimonious trees (fig. 4) of the haplotypes within
of the Asiatic black bears (STH-01 to STH-03) as the outgroup. Num-
tributed, and its distribution area seldom overlapped
netically close haplotypes were distributed geographi-
each other (fig. 2). In Cluster C, the two phylogeneti-
FIG. 3.—Geographic distribution of the control region haplotypes
tions. Numbers of nucleotide substitution per site indicated by the scale
of the Hokkaido brown bears (HB-01 to HB-17), with three haplotypes
FIG. 2.—A control region neighbor-joining tree for 17 haplotypes
bers above internal branches are bootstrap values from 1,000 replica-
Downloaded from http://mbe.oxfordjournals.org/ by guest on October 21, 2015680 Matsuhashi et al.
gion in this repetitive sequence because of possible nat-
ural heteroplasmy and/or artificial replication slippage
by PCR.
Arrays of the repetitious 10-base motifs were var-
iable among individuals which shared the identical hap-
lotypes of the 59 phylogenetically informative region
(table 3). For example, the number and position of motif
c specific to Cluster B were different among four indi-
viduals (samples 811, 4162, 5010, and 6106) having
haplotype HB-13. In addition, the repeated numbers of
motif h and the Tn repeats found in the first half of the
control region were also variable in each cluster and
often identical between individuals of different clusters.
From the constitution of these two repetitive regions, the
FIG. 4.—Parsimonious networks among control region haplotypes 17 haplotypes found on 56 individuals were further clas-
in the three clusters. a, Cluster A. b, Cluster b. c, Cluster C. Solid sified into 48 types (table 3).
circles indicate identified haplotypes, and open circles indicate hypo- To determine sequences of the repetitive region
thetical haplotypes. Each bar on the branch means one nucleotide sub- without the effect of heteroplasmy, we cloned PCR
stitution at the position of that number (see table 1). * Nucleotide sub-
stitution at the same position. products of the control regions of five individuals (sam-
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ples 822, 2231, 3092, 4008, and 6107; table 4). Se-
quences of four to eight clones per individual had mul-
in neighboring areas. Other closer types, HB-16 and tiple fragments consisting of 10-base motifs (mostly
HB-17, were both localized in the southernmost area of types a and h; table 4). Repeated numbers of motif types
Hokkaido (fig. 3). Meanwhile, only one bear of HB-17 a and h were variable between fragments even in one
was found in the southern part of the Cluster A distri- bear, and some fragments had several other types (motifs
bution area. b, c, and e–g) at a few positions. The maximum number
The entire cytochrome b gene was sequenced for of tandem repeats among the cloned fragments was 36
comparison with the control region data. For the se- (sample 4008), while the minimum number was 13
quencing, three brown bears were selected from each of (sample 3092). Alignment of the cloned fragments clear-
three clusters of the control region phylogeny. Nucleo- ly showed that the heteroplasmic motifs (v, w, x, y, and
tide substitutions found at 26 sites of 1,140 bases were z) observed in PCR product–direct sequencing were pro-
all transitions among the nine brown bears. Sequence duced by mixtures of multiple sequences of fragments
differences among the haplotypes were smaller than (table 4).
those among the control regions (table 2). The phylo-
genetic relationships of cytochrome b between the nine Discussion
bears (data not shown) were the same as those of the Characteristic Distribution of the mtDNA Haplotypes
control region sequences (fig. 2).
The cytochrome b sequence data can be accessed The present study clearly showed that the brown
in the DDBJ, EMBL, and GenBank nucleotide sequence bear population on Hokkaido Island was divided into
databases under accession numbers AB020905– three lineages, and that each lineage was distributed in
AB020910. central, eastern, and southwestern Hokkaido, separately
(fig. 3). Ohdachi et al. (1992) reported that the skulls of
Higher Variation of the 39 Repetitive Region brown bears in southwestern Hokkaido were statistically
The 39 repetitive region, consisting of tandem 10- smaller than those of brown bears in northeastern Hok-
base motifs as well as the thymine repeats (Tn), was kaido. Their food habits were also different between
much more variable than the 59 phylogenetically infor- these two areas (Ohdachi and Aoi 1987). Our findings
mative region (table 3). At the sequences obtained with indicate that there are not only morphological and food
PCR product–direct sequencing, the 10-base motifs habit differences, but also genetic differences between
were classified into 11 types (a–d, h, i, and v–z in table southwestern and northeastern Hokkaido brown bear
3), all of which had transitional substitutions at one to populations.
three sites between them. Of the 11 identified types, 5 Before the late 19th century, since Hokkaido Island
were heteroplasmic at one (motifs v and y), two (motifs was not yet intensively cultivated, the brown bears could
w and z), and three (motif x) sites, and only motif i have freely migrated through the contact zones between
shared one indel site (table 3). Direct sequencing the genetically different lineages identified in the present
showed that each brown bear possessed at least 24 cop- study. In addition, the brown bears have an ability to
ies of the motifs repeated in tandem. The same sequenc- move relatively long distances (Mano 1994). Our results
es were reproducibly obtained from each specimen (fig. 3), however, indicate that the distributions of the
through independent direct sequencing experiments, in- three lineages did not almost overlap with each other,
dicating the reliability of these repetitive sequences. Us- similar to the genetic structure of the Scandinavian
ing the PCR product–direct sequence technique, how- brown bear population (Taberlet et al. 1995). We here
ever, it was impossible to determine more of the 39 re- propose some possible explanations for this HokkaidomtDNA Evolution of Brown Bears in Japan 681
Table 3
Constitution of the Repetitive Region and the Thymine Repeats (Tn) in the mtDNA Control Region
NOTE.—Motif: a, ACGCACGTGT; b, ACGCACGCGT; c, ACGCATGTGT; d, ACGCACGCAT; e, ACGCAGATGT; f, Downloaded from http://mbe.oxfordjournals.org/ by guest on October 21, 2015
ACGCGCGTGT; g, GCGCACGTGT; h, ACGCATACGT; i, ACG–ATACGT; v, ACGCAYGTGT; w, ACGCAYRTGT; x, ACGCAYRYGT;
y, ACGCATAYGT; z, ACGCATRYGT. When two peaks were observed at the same site in sequencing, the peak higher than the half of
the other was considered to be the nucleotide at that site.
* Number of thymine repeats in heteroplasmy.
brown bear population structure. The areas in which the for the distribution patterns of mtDNA haplotypes. It
three lineages were distributed almost overlap with sep- was reported that female bears disperse and establish
arate larger mountainous forests on Hokkaido Island. It territories near their mothers (Schwartz and Franzmann
is inferred that the brown bears seldom go down to the 1992; Rogers 1987). In fact, the same haplotype was
lowlands without forests and move into adjacent moun- plotted at the limited region (fig. 3), and the phyloge-
tainous areas. Another possibility is that the peculiar netically closer haplotypes were located in geographi-
movement pattern of female bears might be responsible cally closer areas (for instance, HB-07, HB-08, and HB-682
Table 4
Cloned Sequences and Direct Sequences of the Repetitive Region from Five Hokkaido Brown Bears
Matsuhashi et al.
ORDER IN TANDEM
SAMPLE NO. OF
HAPLOTYPE NO. CLONES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
HB-02. . . . 6107 1 a h h h h a a h h h a a a a a a a a a a a a a a a a a
1 a h h h a a h h h h a a a a a a a a a a a a a a a a a a a
1 a h h h a a h h h a a a a a a a a a a a a a a a a a a a a
1 a h h h a a h h h a a a a a a a a a a a a a a a a a a a a a a a a
1 a h h h h a a h h h h a a a a a a a a a a a a a a a a a a a a a a a a
Directa a h h h v a z h h w a a a a a a a a a a a a a a
2231 1 a h h h h a a a a a a a a a a a a a a a a a a a a a a
1 a h h h h h h a a a a a a a a a a a a a a a b a a a a
1 a h h h h h h a a a a a a a a e a a a a a a a a a a a a a a
1 a h h h h h h h h a a a a a a a a a a a a a f a a a a a a a a
1 a h h h h h h h h a a a a a a a a a a a a a a a a a a a a a a a
Directa a h h h h h y a a a a a a a a a a a a a a a a a
. . . . . . . . . 3092 2 a h h h a a a a a a a a a a a a a a a a a a a a a a a a
1 a h h h h a a a a a a a a a a a a a a a a a a a a a
1 a h h h h h h a a a a a a
1 a h h h h h h a a a a a a a
2 a h h h h h h a a a a a a a a a a a a a a a
1 a h h h h h h a a a a a a a a a a a a a a a a
Directa a h h h h y y a a a a a a a a a a a a a a a a a
HB-12. . . . 822 1 a h h a a a a c a a a a a a a a a a a a a a a a a
1 a h h a a a c c a a a a a c c a a a a a a a a a a a
1 a h h h h h a a a c c a a a a a a a a a a a a a a a a a
1 a h h h h h a a a c c a a a a a a a a a f a a a a a a a
1 a h h h h h a a a c c a a a a a a a a a a a a a a a a a a a a
1 a h h h h h h a a a c c a a c c a a a a a a a a a a a a a a
1 a h h h h h h a a a a a a a a a a a a a a a a a a a a a a a a
Directa a h h h x x v v a a v a a a a a a a a a a a a a
HB-16. . . . 4008 1 b h ib h h a a a a a a a a a a a a g a a a a a a a a a a a a a a a a a a
1 b h ib h h a a a a a a a a a a a a a a a a a a a a a a a a a a a a a
1 b h ib h h a a a a a a a a a a a a a a a a a a a a a a a a a a a
1 b h ib h a a a a a a a a a a a a a a a a a a a
Directa b h i h x a a a a a a a a a a a a a a a a a a a
NOTE.—Motifs are those in table 3.
a Sequences obtained by PCR product–direct sequencing.
b The motif i sequence was obtained with primer BED3, while the motif h sequence was obtained with primer D4.
Downloaded from http://mbe.oxfordjournals.org/ by guest on October 21, 2015mtDNA Evolution of Brown Bears in Japan 683
09; HB-14 and HB-15; HB-16 and HB-17; see fig. 3). Ohshima (1991) reported that Hokkaido Island and
These distribution patterns also suggest that the closer Sakhalin/Siberia were connected by a land bridge(s) un-
haplotypes could have evolved in and around those lim- til around 12,000 years ago. Our preliminary study (Ma-
ited areas after the brown bears migrated to and fixed suda et al. 1998) indicated that the Hokkaido brown
on Hokkaido Island. Compared with female bears, males bears are phylogenetically close to the Russian brown
have an ability to move in longer distances, disperse to bears in the Eastern Europe group reported by Taberlet
more remote places, and have larger habitats. In our and Bouvet (1994). Therefore, the present results sug-
study, however, few males intermixed with neighboring gest that the three genetic lineages of Hokkaido must
clusters (data not shown). One possible explanation of have diverged on the continent prior to their immigra-
this situation is that heavy extermination (hunting by tion to Hokkaido Island and that each lineage could have
humans) or habitat separation by town areas or agricul- been fixed in its respective area after the immigration,
tural farms may restrict the dispersion of males. Even if and then some haplotypes at each area could have di-
some males fix in the areas occupied by different genetic verged.
groups, haplotypes of these males’ mtDNA are not in- Variations of the Repetitive Sequence in the Control
herited by the next generation, because mtDNA is ma- Region
ternally transmitted. Our study revealed that the repetitive region on the
For the next step of our research, it is necessary to 39 side and the Tn repeat included in the control region
investigate in detail whether there are any hybrid zones evolved more rapidly than the 59 variable region. Even
between the three lineages, as demonstrated for voles among the brown bears sharing the common haplotypes
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(Tegelström 1987) and Scandinavian brown bears (Ta- of the 59 variable region, different repetitive patterns
berlet et al. 1995), by using a larger number of samples were found (table 3). From the variation of the repetitive
from possible boundaries. Then, we should study the region, the 17 haplotypes identified in the 59 region were
population structure using nuclear DNA markers such further divided into 48 types. This result indicates that
as microsatellites and confirm whether the lineages de- the mtDNA control region sequences could have
tected by the maternally transmitting mtDNA are com- evolved even within the limited generations of the
parable to those found by nuclear DNAs, which are bi- brown bears. Recently, several kinds of variable-number
parentally transmitted. If the lineages detected by the tandem repeats in mtDNA were reported for various an-
nuclear DNA are distinct from those detected by imals (Lunt, Whipple, and Hyman 1998).
mtDNA, the three lineages found in the present study Comparison between sequences by vector cloning
are probably maintained by the low dispersal of females. and direct sequencing indicated that sequence mixture
In contrast, if nuclear DNA haplotypes cluster as do the in direct sequencing could result from heteroplasmy (ta-
mtDNA haplotypes, there should be some impediments ble 4). Length variation of the repetitive region was ob-
that prevent mating between the three lineages. served between clones from a single individual, as re-
ported by Hoelzel et al. (1994). Even repeated numbers
Divergence Time of the Brown Bear in Hokkaido of motifs a and h, commonly found in all bears, were
The mean genetic distances of the mtDNA control variable in each sequence (table 4). The sequence data
regions were 2.0% between Clusters A and B, 3.0% be- identified by PCR product–direct sequencing were not
tween Clusters A and C, and 3.7% between Clusters B in discordance with mixture of vector cloning sequences
and C (calculated from table 2). These values were obtained from the individual in question. Heteroplasmic
much larger than those for populations of European motifs v, w, x, y, and z were all composed of motifs a
and h. Motifs b, c, e, f, and g could be derived from
brown bears reported by Taberlet and Bouvet (1994),
motif a with one point substitution.
although Hokkaido Island is comparable to just one
As an explanation for the length variation of tan-
sampling point in their European brown bear study. Us-
dem repeats of a single motif observed in mtDNA, DNA
ing the divergence time of about 3 Myr between U. arc- replication slippage is proposed (e.g., Hayasaka, Ishida,
tos and S. thibetanus (Wayne, Van Valkenburgh, and and Horai 1991; Wilkinson and Chapman 1991; Hoelzel
O’Brien 1991), the divergences of the three Hokkaido 1993; Casane et al. 1997). In fact, in the present study,
clusters were estimated to have occurred around 0.8 more of the 39 region of the repetitive sequences could
MYA (between Clusters A and B) and 1.4 MYA (be- not be determined by PCR product–direct sequencing.
tween Cluster C and the others). On the other hand, This probably resulted from length variation caused by
using the values calculated from the mean genetic dis- such natural DNA replication slippage in cells (hetero-
tance of cytochrome b (1.2% between clusters A and B, plasmy) and/or by artificial slippage via PCR (Madsen,
1.7% between Cluster C and the others, and 10.1% be- Ghivizzani, and Hauswirth 1993).
tween U. arctos and S. thibetanus; calculated from table The combination of sequence patterns in the 59 var-
2), the divergences of these three clusters were estimated iable region and the repetitive types in the 39 region
to have occurred around 0.3 MYA and 0.5 MYA, re- could provide a useful tool for investigating the popu-
spectively. Meanwhile, assuming 2.5%/Myr for the lation structure and for individual identification.
mean value of mammalian cytochrome b diversity
(Meyer et al. 1990; Irwin, Kocher, and Wilson 1991), Acknowledgments
each divergence date between clusters was roughly mid- We are grateful to many members of the Hunters’
way between the above values. Association of Hokkaido for providing brown bear spec-684 Matsuhashi et al.
imens. We thank K. Murata of Kobe Municipal Zoo and on comparison of mitochondrial cytochrome b nucleotide
S. Dakemoto for supplying Asiatic black bear samples. sequences. Zool. Sci. 11:605–612.
We thank N. Kurose for technical support. We are also MEYER, A., T. D. KOCHER, P. BASASIBWAKI, and A. C. WILSON.
grateful to Dr. S. Ohdachi of Hokkaido University for 1990. Monophyletic origin of Lake Victoria cichlid fishes
suggested by mitochondrial DNA sequences. Nature 347:
critical comments on the manuscript. This study was 550–553.
supported in part by Grants-in-Aid for Scientific Re- OHDACHI, S., and T. AOI. 1987. Food habits of brown bears in
search from the Ministry of Education, Science, Sports, Hokkaido, Japan. Int. Conf. Bear Res. Manage. 7:215–220.
and Culture, Japan, and by a second Toyota High-tech OHDACHI, S., T. AOI, T. MANO, and T. TSUBOTA. 1992. Growth,
Research Grant to R.M. sexual dimorphism, and geographical variation of skull di-
mensions of the brown bear Ursus arctos in Hokkaido. J.
Mamm. Soc. Jpn. 17:27–47.
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