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Journal of Heredity Advance Access published November 4, 2008 Journal of Heredity doi:10.1093/jhered/esn097

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Genetic Structure of the Asiatic Black Bear in Japan Using Mitochondrial DNA Analysis YOSHIKI YASUKOCHI, SHIN NISHIDA, SANG-HOON HAN, TOSHIFUMI KUROSAKI, MASAAKI YONEDA, HIROKO KOIKE

AND

From the Graduate School of Social and Cultural Studies, Kyushu University, Chuo-ku, Fukuoka city, Japan (Yasukochi, Nishida, and Koike); The Wildlife institute of Korea, Hwacheon-gun, Gangwon-do, Korea (Han); and the Japan Wildlife Research Center, Taitou-ku, Tokyo, Japan (Kurosaki and Yoneda). Address correspondence to Y. Yasukochi, Graduate School of Social and Cultural Studies, Kyushu University, 4-2-1 Ropponmatsu, Chuo-ku, Fukuoka city, Japan, or e-mail: [email protected].

Abstract The genetic structure of the Asiatic black bear (Ursus thibetanus) in Japan was studied to understand the events that occurred during its evolution. The left domain of the mitochondrial control region (about 240 bp) was sequenced, defining 27 haplotypes that consisted of 23 haplotypes from 333 bears in Japan and 22 bears in the Asian continent. The network tree of the control region indicated that the Japanese population formed a distinct clade from the continental population. The phylogeographic analysis of the haplotypes indicated that the Shikoku and Kii Hanto populations had diverged during the initial phase from the ancestral population. After the 3 dominant haplotypes were rapidly distributed throughout Japan in the early stage of the population dispersal, the Japanese population diverged into eastern and western populations. Using the entire mitochondrial cytochrome b sequence, divergence time between the Japanese and the Continental populations suggested that the Japanese population might have colonized into Japan through the land bridge from the Korean Peninsula around 500 ka, which is consistent with paleontological evidence. Our finding that bears in western Japan exhibit lower genetic diversity and higher levels of genetic differentiation than bears in eastern Japan provides a vital contribution to conservation policy for these isolated populations. Key words: Asiatic black bear, control region, cytochrome b, divergence time, genetic population structure, mitochondrial DNA, phylogeography, Ursus thibetanus

The Asiatic black bear Ursus thibetanus (G. Cuvier, 1823) is distributed throughout eastern Asia and the maritime territory of the Russian Far East (Servheen 1990). In Japan, the black bear is sometimes identified as the subspecies, Ursus thibetanus japonicus (Schlegel, 1857). Here, we refer to the black bear in Japan as the Japanese black bear. The black bear currently inhabits the Honshu to Shikoku region, with no bears observed in the Kyushu region for several decades (Japan Wildlife Research Center 1999) (Figure 1A). The Japanese black bear usually inhabits broadleaf forest. Conifer plantations started to restrict the habitat of black bears around 1950. However, in recent years, many farmlands and forestlands have been abandoned, after a decline in human populations in rural districts. In addition, bear hunting has decreased as the number of hunters has declined. These factors have widened the habitat of the Japanese black bear, leading to an increase in the number of

bears visiting farmlands and urban districts. Black bears often cause serious damage to agriculture, forestry, and beekeeping and have been known to even attack local people. Consequently, hunting was encouraged to prevent nuisance behavior, resulting in the culling of about 2000 black bears every year (Japan Wildlife Research Center 1999). The Ministry of the Environment in Japan has now implemented conservation and management programs for the Japanese black bear. Some populations in western Japan have been designated as ‘‘endangered’’ in the Red Data Book of Japan (Ministry of the Environment 2002) because their habitats have become fragmented and isolated. Mitochondrial DNA (mtDNA) has been widely used for studying the phylogeny of Ursidae. A previous study of the American black bear (Ursus americanus) in North America using mtDNA identified 2 distinct lineages (eastern and western) (Paetkau and Strocbeck 1996; Byun et al. 1997;

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Figure 1. (A) Upper left corner, a map depicting the Japanese Archipelago. The boxed area is shown in more detail in (B). (B) Fifteen local populations based on the conservation and management units for the Asiatic black bear (Yoneda 2001) are shown as bold circles. Numbers in each circle indicate the management units: 1, Northern Ouu; 2, Chokai Sanchi; 3, Gassan–Asahi–Iide; 4, Southern Ouu; 5, Echigo–Mikuni; 6, Kanto Sanchi; 7, Southern Alps; 8, north-central Alps; 9, Hakusan–Okumino; 10, Northern Kinki; 11, Eastern Chugoku; 12, Western Chugoku; 13, Kii Hanto; 14, Shikoku; and 15, Kyushu. Pie charts show the observed frequencies of the mtDNA haplotypes. The number of samples is depicted in the center of the chart, and numbers in parentheses show the number analyzed by Ishibashi and Saitoh (2004). Single asterisk indicates the number was taken from the result of an identification of individuals using microsatellite analysis (Nishida et al. 2002) because samples were obtained by hair trap. Double asterisk the haplotype was detected in an individual that was probably carried to its location by humans. (C) Network tree of the Asiatic black bear haplotypes (25 haplotypes in Japan and 15 haplotypes on the Asian continent) by the median-joining method (Bandelt et al. 1999). Colors on the network tree indicate the location at which each haplotype was detected (see Figure 1B). Closed dots indicate extant unsampled or extinct haplotypes. Numbers in parentheses are GenBank accession numbers. J node means the MRCA of the Japanese black bear. Single asterisk indicates the haplotype C1 detected from the offspring between male Japanese bear and female Chinese bear. Double asterisk indicates the haplotype R3/K1 detected in both Russia and the Korean Peninsula.

Wooding and Ward 1997; Stone and Cook 2000). Sequencing of the mtDNA control region and restriction fragment length polymorphism analysis of American black bears suggested that these bears had been separated into 2 eastern and western forest refugia in southern North America and that the 2 lineages had intercrossed twice in the past (Wooding and Ward 1997). Chu et al. (2000) analyzed the mtDNA control region and cytochrome b of the Asiatic black bear in a Taipei Zoo and in the Yushan area of Taiwan. Although most individuals could not be identified by the study, it was found that a few individuals in the zoo were derived from the Yushan area. The Japanese black bear is one of the species that are well studied using genetic techniques (Saitoh et al. 2001; Kitahara et al. 2002; Ishibashi and Saitoh 2004; Sato 2004). Using microsatellite DNA markers, Saitoh et al. (2001) studied some populations of black bear in western Japan and concluded that those in the Chugoku region were losing genetic diversity. A study using the mtDNA control region for black bear populations in the same area (Ishibashi and Saitoh 2004) suggested that these black bears had 2 lineages, with the apparent border between western and eastern parts in the northern Kinki region. Because no study of Asiatic black bear populations over the entire Japanese Archipelago has been published to date, we analyzed black bears in 15 local populations that

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encompass most of the bear habitats in Japan. We describe the genetic structure of these populations based on the sequence of the whole left domain (about 240 bp) of the mtDNA control region, from which we can infer evolutionary history and female gene flow. We also tried to estimate the divergence time from the Asiatic black bear population using the entire sequence of cytochrome b.

Materials and Methods In this study, we sequenced about 240 bp of the left domain of the mtDNA control region from 333 bear samples obtained from 15 management units in Japan (Figure 1B) together with 6 in the Korean Peninsula, 15 in Russia, and 1 from the offspring of a Japanese male bear and a Chinese female bear. Local populations of the black bear in Japan are divided into 19 conservation and management units (Yoneda 2001). These divisions were formed taking into account the factors assumed to prevent the movement of bears, such as rivers, roads, railways, and urban areas. The division of local populations followed the divisions of the bear management units. Samples consisted of 33 samples of muscle and liver tissue, 9 samples of blood, 34 samples of hair, 4 samples obtained from oral mucous membrane, 8 samples of feces, and 267 samples from tooth and mandibular bone. The

Yasukochi et al.  Genetic Structure of Asiatic Black Bears

samples from the Chokai Sanchi unit were obtained by a hair trap method rather than collected from individual bears. Nishida et al. (2002) analyzed 8 microsatellite loci and identified 21 individuals by the microsatellite analysis. Samples were collected during 1970–2006, except for one specimen that was captured at least 80 years ago in Kyushu Island. About 3 mg of tissue or 15–20 mg of tooth or mandibular bone was placed in 310 ll of RSB buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, and 25 mM EDTA  2Na), 15 ll of 10% sodium dodecyl sulfate, and 15 ll of 20 mg/ml proteinase K and incubated for 2 h at 55–60 °C on a rotator to facilitate protein digestion. About 20.5 ll of 0.5 M EDTA  2Na was added to decalcify the teeth and mandibular bone samples. Genomic DNA was extracted using an IsoQuick kit (ORCA Research Inc., Bothell, WA). Extracted DNA was amplified by polymerase chain reaction (PCR). Bear specific primers L15926.urs (5#-ATAGTAATTACCT-TGGTCTTGTAAGCC-3#) and URH5 (5#GGTATACGTACTCGCAAGGGTTGC-3#) and internal primers URL2 5#-CTGTTTAAACTATTCCCTGGTACAT3#) and URH2 (5#-GCCTGGTGATCAAGCTCCCGGACTA-3#) were used to amplify the whole left domain of the mtDNA control region. The URH5 primer was designed based on sequences of the Asiatic black bear, which were obtained using L15926.urs, URL2, and URH2 primers (Uchiyama 1998). The entire cytochrome b sequence was amplified by PCR using the 2 primers described in Matsuhashi et al. (1999): CbM1 (5#-CTCACATGGAATCTAACCATGAC-3#) and CbMR2 (5#-AGGGAATAGTTTAAATAGAATTTCAGC-3#) and the 2 following primers: URCbM1 (5#-TTCATCATCCTAGCACTAGCAGCAG-3#) and URCbMR2 (5#-TTTGTCCGAGTTGGATGGGATTCCAG-3#). The latter 2 primers were designed using reference sequences from the American black bear, the brown bear (Ursus arctos), the polar bear (Ursus maritimus) (Delisle and Strobeck 2002), and the Asiatic black bear (Matsuhashi et al. 1999). PCR amplification was carried out using a DNA Thermal Cycler in 25-ll reaction mixture containing 10 Ex Taq buffer, 2.5 mM deoxynucleoside triphosphate mixture, 2 pmol/ll of each primer and 0.625 units of Ex Taq Hot Start Version (TaKaRa Bio Inc., Shiga, Japan). After incubation at 94 °C for 1 min, 40 cycles were performed as follows: 30 s at 94 °C, 45 s at 55 °C, and 45 s at 72 °C, with a postcycling extension at 72 °C for 30 s. To confirm DNA amplification by PCR, 5 ll aliquots from each PCR product were run on a 1.5% agarose gel and visualized by ethidium bromide staining. PCR products used for sequencing were purified with a PCR Product Pre-Sequencing kit (United States Biochemical, Cleveland, OH), and cycle sequencing was performed with a DTCS Quick Start kit containing Dye Terminators (Beckman Coulter Inc., Fullerton, CA) using the above primers. Sequencing was conducted with a CEQ2000XL DNA auto-sequencer (Beckman Coulter Inc.). Sequences were aligned using GENETYX software, Ver. 6.1.0 (Software Development Co., Ltd., Tokyo, Japan). The positions of deletions or insertions were determined by eye and excluded from the estimation of genetic distance. For phylogenetic analysis, sequences were aligned with the 228bp fragment based on the sequence of the control region. A

network tree was constructed with NETWORK 4.1.1.2 (Fluxus Technology Ltd., Suffolk, UK) using the medianjoining method (Bandelt et al. 1999). The haplotypes from DDBJ/EMBJ/GenBank (accession numbers: AB101525 and AB101526 [Ishibashi and Saitoh 2004], AF319154– AF319164 [Chu et al. 2000]) were included in the network tree. We inferred the most recent common ancestor (MRCA) of the black bear in Japan (J node) by star contraction with optional preprocessing using NETWORK 4.1.1.2 software because multiple substitutions produced many cubes around the J node. The maximum likelihood (ML) tree for 1140 bp of the whole cytochrome b region of Ursidae was obtained by using PAUP* version 4.0b10 (Swofford 1998). The ML tree predominantly includes sequence data from Ursidae, with the exception of that from the giant panda (Ailuropoda melanoleuca) (Waits et al. 1999), which was obtained from GenBank (accession numbers: AB020905–AB020909 [Matsuhashi et al. 1999]; AY522429, AY522430, and X82307–X82309 [Arnason et al. 1995]; U18898 and U18899 [Talbot and Shields 1996b]; U23554, U23556, U23560, and U23562 [Talbot and Shields 1996a]; AF264047 [Loreille et al. 2001]; DQ349065 [Hou et al. 2007]). To identify the best model of nucleotide substitution for the data set, ML analysis was subjected to the Akaike Information Criterion (AIC; Akaike 1974) using Modeltest version 3.7 (Posada and Crandall 1998). The TIM þ C model was selected as the model of best fit by the AIC approach. ML heuristic searches were performed using tree-bisection-reconnection branch swapping with 100 random addition sequence replicates. Bootstrap analysis was performed using 100 replications of the ML tree using the fast stepwise addition method. Divergence times were estimated by the global rate minimum deformation method (GRMD) with the previously constructed ML tree using TREEFINDER (Jobb 2007). We also estimated the divergence times of the MRCA in Japan and the Asian continent by Bayesian analysis using BEAST version 1.4.7b (Drummond and Rambaut 2007), with the same sequences as the ML tree. The posterior distributions were obtained by Markov chain Monte Carlo analysis with 2.0  106 steps (with a burn-in of 2.0  106) sampling every 2000 steps under a relaxed clock model with an uncorrelated lognormal distribution. The relaxed clock method naturally incorporates the time-dependent nature of the evolutionary process without assuming a strict molecular clock (Drummond et al. 2006). Each sequence data set was partitioned in first and second codon positions together and third position, and the parameters of the substitution model Hasegawa–Kishino–Yano (HKY þ C ) were estimated independently for each partition. The results were viewed using Tracer v 1.4 (Rambaut and Drummond 2007). The Effective Sample Sizes (ESSs) for each parameter exceeded 200. A low ESS (,200) means that the parameter contained a lot of correlated samples and thus may not represent the posterior distribution well. Under the finite sites HKY model by Bayesian inference, the MDIV program (Nielsen and Wakeley 2001) was also used to estimate the divergence time between the Japanese population and the Continental population of the Asiatic black bear. MDIV program

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approximates the posterior distribution of 3 parameters; theta as h 5 2Nel, where Ne is effective population size and l is mutation rate per sequence per generation; the scaled migration rate as M 5 2Nem, where m is migration rate; and scaled divergence time as T 5 t/Ne, where t is the divergence time between populations in generations. We conducted 5  106 generations of the Markov chain (with burn-in 5  105 generations). Credibility intervals for the parameter estimates are taken as the interval that contains 95% of the posterior distribution. We analyzed the data with prior values of Mmax 5 0 and Tmax 5 30. The numbers of net nucleotide substitutions per site between populations (Da) (Nei 1987) with Jukes–Cantor correction (Jukes and Cantor 1969) were obtained using the DnaSP 4.10.9 program (Rozas et al. 2003). Tajima’s D (Tajima 1989) and Fu’s FS (Fu 1997) statistics were computed to test for recent population expansion using the Arlequin ver 3.01 program (Excoffier et al. 2005). Haplotype diversity (h) and nucleotide diversity (p) (Nei 1987) for each management unit and Wright’s F-statistics (FST: Wright 1951) between each of the management unit pairs were also calculated using Arlequin ver 3.01. A hierarchical analysis of molecular variance (AMOVA: Excoffier et al. 1992) was used to estimate the geographical structure of genetic variations using Arlequin ver 3.01. The variations were partitioned into 3 categories of population subdivision: among geographical regions, among populations within regions, and within populations. These analyses included the data set of Ishibashi and Saitoh (2004) (Northern Kinki unit, 64 individuals; Eastern Chugoku, 22; and Western Chugoku, 33).

Results Detection of Haplotypes in the mtDNA Control Region We sequenced the left domain (about 240 bp) of the mtDNA control region for the 333 bear samples obtained from 15 management units in Japan together with 22 from Eastern Asia. A total of 36 substitutions, consisting of 7 transversions and 29 transitions defined 27 haplotypes (J1– J23, R1, R2, R3/K1, and C1: GenBank accession numbers AB360915– AB360956). Haplotypes J1– J23 were detected in the Japanese black bears (Table 1). Haplotype C1 was detected from one offspring between a male Japanese bear and female Chinese bear. R1 and R2 were detected in the Russian bears. R3/K1 was detected in Russia, South Korea, and in a North Korean Zoo. A poly-T of 5–9 nucleotides defined subdivision into subhaplotypes a–e.

haplotypes simultaneously radiated from haplotype J11 and formed a bush-like tree. Geographical Distribution of Haplotypes Haplotypes J3, J10, and J11 were widely distributed throughout the management units, whereas the remaining haplotypes were detected within a specific geographical distribution (Table 2 and Figure 1B). Haplotypes J1– J8 and J21, J22, UtCR06, and UtCR07 were mainly observed in the Northern Kinki to Shikoku units. In particular, J1, J21, and J22 were only detected in the Shikoku and Kii Hanto units. Haplotypes J9–J20 and J23 were mainly observed in the Northern Ouu to Hakusan–Okumino units. Consequently, we defined the western population as the Northern Kinki to Shikoku units and the eastern population as the Northern Ouu to Hakusan–Okumino units (Figure 1B). The geographical distribution, including subhaplotypes, was more specific than that of the haplotypes alone. For example, J10d and J10e were only detected in the Chokai Sanchi unit; J5b*114 and J14d were only detected in the Hakusan–Okumino; J19d was detected only in the Southern Alps; and J3c, J5a, and J10c were detected only in the western Chugoku (Table 2). We analyzed 2 individuals in the Kyushu unit where bears were thought to be extinct. One of them had J14c, which was detected frequently in the Hakusan–Okumino unit (Table 2). It is highly possible that this individual was brought to this location by humans as the bear had teeth scars that could have resulted from biting an iron cage. We therefore excluded the haplotype of this individual from the phylogeographical analysis. Another individual was preserved in a museum of history and folklore in Kyushu and was thought to have inhabited Kyushu Island at least 80 years ago. This individual was identified as haplotype J11, which is widely distributed throughout Japan. Haplotype Diversity and Nucleotide Diversity in Management Units of Japan Haplotype diversity (h) (including subhaplotypes) and nucleotide diversity (p) were calculated for each management unit that contained more than 20 samples (Table 3). The h value in the north-central Alps unit was the highest and that in the Eastern Chugoku unit was the lowest among the populations. The p values of management units in western Japan tended to be lower than those in eastern Japan. The p value in the Eastern Chugoku unit was also the lowest of the populations analyzed.

Phylogenetic Relationship of Haplotypes

Genetic Differentiation of Local Populations

A median-joining network tree was generated using the 27 haplotypes detected in this study and 13 haplotypes from GenBank (Figure 1C). The network tree showed that all the Japanese haplotypes formed a single clade separated by 14 substitution distances from the MRCA of the Japanese black bear (J node) to haplotype B07 of the Continental node. Haplotypes J3 and J21 were closest to the J node. Some

The FST value represents the level of genetic differentiation between population pairs. We calculated the FST values between pairs of management units for black bears with subhaplotypes (Table 4). The FST values between unit pairs in the western population ranged from 0.03 to 0.76 (mean FST 5 0.43), unit pairs in the eastern population ranged from 0.33 to 0.64 (mean FST 5 0.12), and unit pairs

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Table 1.

The 27 haplotypes (including 42 subhaplotypes) detected from the left domain of the mtDNA control region in the Asiatic black bear

Haplotype

Mutation sites

9 C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

R1c R2b R2c R3c/K1c C1c

A A A . .

T T T T T

1 7 T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 9 C . . . . . . . . . . . . . . . . . . . . . . . . . . T T . . . . . . . .

2 2 C . . . . . . . . . . . . . . . . . . . . . . . . T T . . . . . . . . . .

2 6 C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 7 T . . . . . . . . . . . . C C C C C C C C C C C C C C C C C C C . . . C C

3 0 G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5 1 C . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . .

5 2 T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6 8 G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8 0 T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8 3 G 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 A A A A A A A

8 5 T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8 7 C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8 8 C . . . . . . . . . . . . . . . . . . . . . T T . . . . . . . . . . . . .

9 3 C . . . . . . . T A A A . . . . . . . . . . . . . . . . . . . . . . . . .

9 7 A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 0 3 T . . . . – . . . . . . . . . . . . . . . . . . . – . . . . . . . . . – .

1 0 4 T . – . . – – – – – . . – – . . . – . . . . . . – – . . . . . . – . – – –

1 0 5 – – – – – – – – – – – T – – – T T – – T – – – T – – – – – – T – – – – – –

1 0 6 – – – – – – – – – – – – – – – – T – – – – – – – – – – – – – – – – – – – –

1 1 4 T . . . . . . – . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 3 6 C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . .

1 6 5 T . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . .

1 6 6 C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 6 7 T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 7 4 G 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 7 5 T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 8 9 T C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C . . . C C

1 9 7 C . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . .

1 9 8 A . . . . . . . . G G G . . . . . . . . . . . . . . . . . G G G . . . . .

1 9 9 A . . . G G G G . . . . . . . . . . . . . G . . . . . . . . . . . . . G G

2 0 2 G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 0 4 T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 0 7 A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 0 9 T . . . . . . . . . . . . . . . . . . . . . . . . . . . . C C C . . . . .

2 1 1 C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 1 3 G . . . . . . . . . . . A A A A A A A A A A A A A A A A A A A A . . . . .

2 1 5 C . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . T . . . . .

2 1 6 T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C C C C C

. . . . .

T T T T T

T T T T T

C C C C C

A A A A A

. . . . .

C C C C C

A A A A A

C C C C C

A A A A A

A A A A A

. T T T T

. T T T T

. . . . .

G G G G G

. . . . .

. – . . .

– – – – –

– – – – –

. . . . .

. . . . .

C C C C .

T T T T T

G G G G G

. . . . .

C C C C C

. . . . .

. . . . .

G G G G .

. . . . .

A A A A A

. . . . C

. . . . G

. . . . .

. . . . T

. . . . .

. . . . .

. . . . C

5

Haplotype J was detected from Japan. Haplotype R was detected from Russia. Haplotype K was detected from Korean Peninsula. Haplotype C was detected from the individual, which was an offspring between a Japanese male bear and a Chinese female bear. The trailing lowercase letters of haplotype name indicate subtypes a to e which were subdivided by a poli-T with 5–9 nucleotides variation in sites 98– 106 (sites 103– 106 in the table). Dots indicate identity with the nucleotides of J1c. Dashes indicates deletions or insertions. Gray sites indicate transversions. *114 indicates deletion in site 114.

Yasukochi et al.  Genetic Structure of Asiatic Black Bears

J1c J2c J3b J3c J4c J5a J5b J5b*114 J6b J7b J7c J8d J9b J10b J10c J10d J10e J11b J11c J11d J12c J13c J14c J14d J15b J16a J16c J17c J18c J19c J19d J20c J21b J21c J22b J23a J23b

1 5 C T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T . . . T T

Journal of Heredity

6 Table 2.

The observed number of haplotypes detected from the conservation and management unit for the Asiatic black bear

Observed number of haplotypes Haplotypes subhaplotypes Conservation and management unit 1. Northern Ouu 2. Chokai Sanchi 3. Gassan–Asahi–Iide 4. Southern Ouu 5. Echigo–Mikuni 6. Kanto Sanchi 7. Southern Alps 8. North-central Alps 9. Hakusan–Okumino 10. Northern Kinki 11. Eastern Chugoku 12. Western Chugoku 13. Kii Hanto 14. Shikoku 15. Kyushu Total

J11

J10

J13 J23

J6 J15 J16

J9 J12 J20 J14

J18 J5

J19

J17 J7

J8 J4 J5

J3

J2

b

c c

J21

J22 J1

N b 2 21 6 2 16 2 8 23 74 32 9 121 8 7 1 333

c

d d e b c c

1 18a 1a 1a 1a 1 1 1 4 1 1 1 1 1 2 7 3 23 4

a b b b

a c b c

c

c

d c

b*114 c d c

c

b

d c

b

a

b c b

1

1 1

2 1 1 6

1 1

2 1 1

1

2 30 1 1

1 2 1 3 1

1

5 11 3 2 14 2 14 8 10 12 75 5 16

3 1 1 39 30 4 1 1 2 3 2

5 1 1 2 1 1 6

1 1 1 1

1

1b 33 1 1

5 1

4 2 1

N indicates the total of haplotypes detected from each unit. The horizontal order of haplotypes are aligned to associate with locations of each unit. a

The numbers follow the result of study using microsatellite analysis by Nishida et al. (2002) due to samples obtained by hair trap.

b

The number was detected from an individual which might be carried by human.

18 17 2 14 18 12 82 5 16 5 1 1 2

c

We generated the ML tree using the haplotypes detected in the entire mtDNA cytochrome b region (Cb-J1 to Cb-J6 in Japan, Cb-K1 and Cb-C1 on the continent: GenBank accession numbers AB360957–AB360964) and the sequence data obtained predominantly from Ursidae, with the exception of the giant panda (Figure 2A). The haplotypes of cytochrome b from our study were mainly detected in

0.52 0.44* 0.36 0.41* 0.03 0.44 0.51* 0.64* 0.70* 0.76 0.43* 0.35* 0.28* 0.33* 0.25 0.22* 0.46* 0.37* 0.28* 0.35* 0.24 0.15* 0.47* 0.33* 0.21* 0.29* 0.02 0.18* 0.54* 0.32* 0.20* 0.29* 0.06 0.20* 0.67* 0.37* 0.22 0.40* 0.00 0.22* 0.52* 0.29* 0.24* 0.26* 0.13 0.20* 0.68* 0.37* 0.27 0.40* 0.00 *P , 0.05.

Estimation of Divergence Time of the Japanese Population from the Continental Population Using Cytochrome b Region

0.18* 0.57* 0.33* 0.22* 0.31* 0.12

We performed Tajima’s D and Fu’s FS of selective neutrality test to estimate recent population expansion of 2 populations: the eastern population and the western population (Table 6). These analyses were computed using all variants of haplotypes including all subhaplotypes as it has been reported that the analyses should have used all variants of polymorphism data (Kreitman and Rienzo 2004; Soldevila et al. 2005, 2006). Tajima’s D values were negative but were neither significant for the eastern population (D 5 0.555, P 5 0.340) nor for the western population (D 5 0.183, P 5 0.471). Fu’s FS value for the western population was also nonsignificant (FS 5 6.002, P 5 0.055). In contrast, Fu’s FS value for the eastern population was negative and was significant (FS 5 12.97, P 5 0.002). The 2 neutrality tests indicated that the values obtained for the western population were lower than those obtained for the eastern population. In addition, Fu’s FS test suggested that the population expansion of the eastern population must be more recent.

Table 4.

Population Expansion

FST between unit pairs of the conservation and management for the Asiatic black bear

between the eastern population and the western population ranged from 0.90 to 0.74 (mean FST 5 0.27). FST values in the western population exhibited a higher trend than those in the eastern population. We evaluated genetic variances for each of the three categories (Table 5) on the basis of AMOVA analysis. The geographical partition was set as 2 regions: eastern population and western population. AMOVA analyses indicated that the most significant genetic variance occurred between the 2 regions (43.4%) and that the lowest value was among populations within regions (18.5%).

0.42* 0.75* 0.52* 0.60* 0.67* 0.90

Western population

N indicates the total of individuals in each unit. Numbers in parentheses represent the number of haplotype data analyzed by Ishibashi and Saitoh (2004).

Western 10. Northern Kinki 0.20* population 11. Eastern Chugoku 0.67* 12. Western Chugoku 0.37 13. Kii Hanto 0.22 14. Shikoku 0.40 15. Kyushu 1.00

0.007 0.006 0.007 0.002 0.004

0.06*

± ± ± ± ±

0.05 0.16*

0.011 0.001 0.011 0.003 0.005 —

0.17 0.05 0.07

0.05 0.03 0.03 0.10 0.04

0.01 0.10* 0.09* 0.17*

± ± ± ± ±

0.14 0.00 0.05 0.11 0.22*

0.85 0.72 0.75 0.25 0.56 —

0.15 0.13* 0.05 0.05 0.10* 0.20*

23 74 32 (þ64) 9 (þ22) 121 (þ33) 259 (þ119)

0.42* 0.64 0.45* 0.63 0.41* 0.36* 0.42*

North-central Alps Hakusan–Okumino Northern Kinki Eastern Chugoku Western Chugoku Total

p

0.26 0.04 0.00 0.03 0.33 0.09 0.14 0.03

h

Eastern 1. Northern Ouu population 2. Chokai–Sanchi 3. Gassan–Asahi–Iide 4. Southern Ouu 5. Echigo–Mikuni 6. Kanto Sanchi 7. Southern Alps 8. North-central Alps 9. Hakusan–Okumino

N

Eastern population

Conservation and management unit

Conservation and management unit

Table 3. Haplotype diversity (h) and nucleotide diversity (p) for each management unit

1. Northern 2. Chokai 3. Gassan– 4. Southern 5. Echigo– 6. Kanto 7. Southern 8. North- 9. Hakusan– 10. Northern 11. Eastern 12. Western 13. Kii 14. Shikoku Ouu Sanchi Asahi– Ouu Mikuni Sanchi Alps central Okumino Kinki Chugoku Chugoku Hanto Iide Alps

Yasukochi et al.  Genetic Structure of Asiatic Black Bears

7

Journal of Heredity Table 5.

A hierarchical AMOVA of mtDNA control region in the Asiatic black bear

Subdivision

Source

Variance components

Percentage of variation

u-statistics

Western population versus eastern population

Between regions Among populations within regions Within populations

0.79 0.34 0.69

43.4 % 18.5 % 38.1 %

0.43* 0.33* 0.62*

*P , 0.001. Population subdivision was set by 2 regions: eastern and western populations in Japan. Kimura 2 parameter was used as distance method. Significance tests of u-statistics performed 10 000 permutations.

individuals that had different haplotypes of the control region (J1–J23). The phylogenetic topology of the ML tree indicated that polar bears formed a sister taxon to the brown bear. Sun bears (Helarctos malayanus) formed a single clade with American black bears and Asiatic black bears. The Asiatic black bear formed a sister taxon with the American black bear. Haplotypes of the Japanese black bear formed an independent clade which was supported by a high bootstrap value that separates them from those of the black bear on the Asian continent. The evolutionary rate or divergence time within Ursidae (Wayne et al. 1991; Talbot and Shields 1996a; Loreille et al. 2001) has been estimated by several researchers; however, this estimate still remains a matter of debate. We applied the divergence time of about 1200 ka between the cave bear (Ursus spelaeus) and the brown bear (Loreille et al. 2001), which gave a relatively reasonable result. Based on 1200 ka (103 years ago) of divergence time between the cave bear and the brown bear (Loreille et al. 2001), the Asiatic black bear most probably diverged into the Japanese black bear at about 579 ka during the Middle Pleistocene, based on the GRMD with the constructed ML tree (Figure 2B). Bayesian analysis using BEAST program was used to estimate the MRCA of Asiatic black bears of Japan and the Asian continent. The MRCA of these bears was extrapolated from the calibration point derived from the divergence time of the cave bear and the brown bear. The result of Bayesian analysis suggested that the MRCA was around 667 ka (95% highest posterior density: 217–1210 ka). In addition, the divergence time was estimated using MDIV program. Although the suitable mutation rate (l) for the Asiatic black bear has been debated until now, we applied the estimated mutation rate of 6% per My for the cytochrome b region excluding first and second position of codons within Ursidae (Talbot and Shields 1996a). MDIV program estimated the divergence time between the Japanese population and the Continental population at about 475 ka (95% credibility intervals: 147–1295 ka). Table 6.

Estimation of Divergence Time between the Western and the Eastern Populations within Japan Using Control Region We estimated the divergence time between the western and eastern populations in Japan based on the genetic distance (mtDNA control region) using 579 and 667 ka as the divergence times of the Japanese and Asian black bears. Net nucleotide divergence (Da) is a measure of the extent of DNA divergence between populations and takes into account the effect of DNA polymorphism. Using the control region of mtDNA, we calculated that the Da between black bears in Japan and those on the continent was approximately 7.32%, and the Da between the western population and the eastern population in Japan was approximately 0.75%. This result suggested that the ancestral populations for western and eastern Japan might have split around 59 and 68 ka. We also attempted to estimate the divergence time using MDIV program, based on 475 ka estimated from cytochrome b region by the program. The divergence time was estimated about 58 ka (95% credibility intervals: 31–204 ka), when set prior values of Mmax was 0 and Tmax was 2 because the initial analysis suggested that M was close to 0 and that the divergence time was small. We applied the known mutation rate of 5.43  107 per substitution site per year in the brown bear for the control region (Ho et al. 2007; McFadden et al. 2008).

Discussion The ML tree of Ursidae, using the whole mtDNA cytochrome b region, indicated that haplotypes of the Japanese black bear formed an independent clade separating from that of the black bear on the Asian continent. It shows that the Japanese black bear is highly endemic. The Japanese black bear formed a sister clade with the continental Asiatic black bear. These results support the classification of the

Test for recent population expansion of black bears in Japan

Population

N

Tajima’s D

P

Fu’s FS

P

Western population Eastern population Entire Japanese population

179 (þ119) 154 333 (þ119)

0.183 0.555 0.233

0.471 0.340 0.455

6.002 12.97 21.49

0.055 0.002 0.000

N indicates the total of individuals in each unit. Numbers in parentheses represent the number of haplotype data analyzed by Ishibashi and Saitoh (2004).

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Yasukochi et al.  Genetic Structure of Asiatic Black Bears

Figure 2. (A) Phylogenetic relationship of 8 bears as determined by the ML method using the sequences of cytochrome b (the TIM þ C model, tree-bisection-reconnection branch swapping, fast stepwise search, and bootstrap trial; 100). Only bootstrap values more than 70% are shown in this figure. Numbers in parentheses are GenBank accession numbers. (B) The chronogram of the ML tree including the 8 bears by the GRMD method using TREEFINDER program. The black curvilinear triangle represents the date calibration point (1200 ka) of the postulated divergence time between the brown bear and the cave bear (Loreille et al. 2001). The 3 divergence times represented by arrows were estimated from different method: (a) divergence time from the GRMD method using TREEFINDER program, (b) divergence time from Bayesian coalescent model with Markov chain Monte Carlo (MCMC) method using MDIV, and (c) divergence time from Bayesian MCMC method using BEAST.

Japanese black bear, U. t. japonicus, as a subspecies of the Asiatic black bear. We estimated the divergence time between Japanese and Continental populations of the Asiatic black bear based on the divergence time of about 1200 ka between the cave bear and the brown bear (Loreille et al. 2001). Using the postulated divergence time as a calibration point, the Japanese population was estimated to have separated from the Asian continent during the Middle Pleistocene as 580 ka by GRMD method, 670 ka by BEAST program, and 480 ka by MDIV program. Fossil mammal fauna in Japan suggests that formation of land bridges between the Korean Peninsula and the Japanese Archipelago occurred twice, around 500 ka and around 300 ka during the Middle Pleistocene (Dobson and Kawamura 1998). Moreover, ancestral black bear fossils were found in Japan around 500 ka (Dobson and Kawamura 1998). Therefore, our results also support the hypothesis that the ancestral population might have migrated to the Japanese Archipelago

that occurred after the formation of land bridges around 500 ka. The whole left domain of the mtDNA control region was analyzed to estimate the genetic structure of the Japanese black bear population. In the median-joining network tree, the haplotypes of the Japanese black bear showed a relatively continuous connection. Therefore, we estimate that the migration of the Japanese bear from the Asian continent happened only once and resulted in the formation of the 3 ancestral haplotypes. The network tree and geographical distribution of haplotypes suggests that 3 haplotypes (J3, J10, and J11) near the estimated ancestral node (J node) were rapidly distributed throughout Japan in the initial stages of population dispersal. The phylogeographic approach showed haplotypes J1, J21, and J22, which were located near J node, were detected only in the Shikoku and Kii Hanto units in western Japan and might have diverged from the other bears at an early stage. Haplotypes that were close to J3 in the network tree were found from

9

Journal of Heredity

western Japan, suggesting that these western populations had expanded from J3 type population. On the other hand, haplotypes close to J11 were detected in eastern Japan. The geographic distribution of the haplotypes showed that the western and the eastern populations had different haplotype composition. FST value between the western and the eastern populations indicated significantly high genetic differentiation. The genetic variation between these populations also indicated a high value of 43.4% by AMOVA analysis. These results show that the eastern and western populations dispersed in different directions. We found evidence for a border between the eastern and western populations at the Northern Kinki unit and the Hakusan–Okumino unit. Ishibashi and Saitoh (2004) also analyzed the mtDNA control region of black bears from the Northern Kinki unit, Eastern Chugoku unit, and Western Chugoku unit in western Japan and suggested that the Japanese population had divided into 2 groups with an apparent border within the Northern Kinki unit. The result might indicate the existence of a genetic difference within the western population. We estimated the divergence time between the western and eastern populations in Japan by using the Da value of the populations based on sequences of the control region. The Da value suggested that the ancestral population of the Japanese black bear temporarily diverged into eastern and western populations about 60–70 ka in the Late Pleistocene. Existence of such different lineages of intraspecies is also seen in other Japanese mammals. The Sika deer (Cervus nippon) population has 2 distinct lineages of mtDNA (Tamate and Tsuchiya 1995; Tamate et al. 1998; Nagata et al. 1999; Nabata et al. 2004; Yamada et al. 2006), which migrated independently into Japan via different routes in the north and south (Nagata et al. 1999). The brown bear population in Hokkaido consists of 3 lineages of mtDNA, which separately colonized the region from Sakhalin/Siberia to Hokkaido Island during different periods (Masuda et al. 1998; Matsuhashi et al. 1999). We assume that the Japanese black bear colonized Japan from the southern part of the Asian continent (probably from the Korean Peninsula) in a single event. The Japanese macaque (Macaca fuscata) has 2 major haplotype groups (western and eastern populations), which constitute the basis of the border between the macaques of the Kinki region and Chugoku region (Kawamoto et al. 2007). The Japanese macaque was thought to have colonized into Japan from the Asian continent via a land bridge between the Korean Peninsula and the southern part of Japan (Kamei 1969; Iwamoto and Hasegawa 1972; Dobson and Kawamura 1998; Aimi 2002; Fooden and Aimi 2005). As mentioned above, the fossil record of the Japanese macaque suggests that its evolutionary history might be similar to that of the Japanese black bear with both species migrating to Japan during the same period via a similar pathway. However, the geographical distribution pattern of the 2 macaque lineages, which form a border between the populations of Kinki region and Chugoku region, is different from that of the black bear. This difference might be caused by the habitat preferences of each species.

10

Haplotypes J12–J18, which simultaneously radiated from haplotype J11 in the network, were mainly distributed in eastern Japan. This suggests that the ancestral eastern population, which was mainly derived from J11, rapidly expanded to the northern parts of Japan in recent times. The FS neutrality test, which was be more powerful than other tests in rejecting the hypothesis of neutrality of mutations (Fu 1997), showed that the eastern population may have experienced recent population expansion. The neutrality test in this study suggested that establishment of ancestral populations in western Japan occurred earlier than in eastern Japan. This type of simultaneous expansion has also been reported for the Japanese macaque (Kawamoto et al. 2007). Haplotype J5 and J7 were detected in the Hakusan–Okumino unit, and J7 and J8 were detected in the north-central Alps unit. As these haplotypes are derived from the western population, the results suggest that the western population of the northernmost region might have invaded the southernmost region of the eastern population in recent times. The hose n units pair the Tracer V distributed geographically close to each other. FST values between each of the management unit pairs indicated a high degree of genetic differentiation and tended to be higher between unit pairs in the western population than between those in the eastern population. Genetic diversity of each management unit was lower in the western population. These results support the fact that management units in western Japan are more isolated and fragmented than those in eastern Japan. This isolation and fragmentation might be due to destruction of bear habitat by human activity. In summary, we investigated the genetic structure and evolutionary event for the Asiatic black bear in Japan using mtDNA region sequences. The phylogenetic analysis using the cytochrome b region suggested that the Japanese population had been introduced into the Japanese Archipelago from the Korean Peninsula around 500 ka in the Middle Pleistocene, which was consistent with fossil evidence. The Japanese population formed a distinct clade from the Continental population, supporting the subspecies designation U. t. japonicus. The median-joining network using control region sequences indicated that haplotypes of the Japanese black bear were divided into western and eastern groups. The Shikoku and Kii Hanto haplotypes diverged from other Japanese haplotypes during the initial stage, and the 3 main haplotypes were rapidly distributed throughout Japan in the early stages of population dispersal. The other marginal haplotypes diverged into the eastern and western populations around the Late Pleistocene. We found that the western population had lower genetic diversity within each population and higher levels of genetic differentiation. These results are a vital contribution to the conservation policy for these isolated populations.

Acknowledgments The authors are grateful to Toshinao Okayama and Tomoyuki Uchiyama for their help in the experiments during this study. We are indebted to

Yasukochi et al.  Genetic Structure of Asiatic Black Bears institutes that helped in sample collection, including the Ministry of the Environment of Japan, the Japan Wildlife Research Center, the Species Restoration Center in the Korean National Park Research, and the Museum of History and Folklore in Kyushu.

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Received December 15, 2007; Revised September 25, 2008; Accepted October 6, 2008

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Corresponding Editor: James Womack

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