Heredity (2009) 102, 579–589 & 2009 Macmillan Publishers Limited All rights reserved 0018-067X/09 $32.00 www.nature.com/hdy ORIGINAL ARTICLE The influence of climatic oscillations during the Quaternary Era on the genetic structure of Asian black bears in Japan N Ohnishi1, R Uno2, Y Ishibashi3, HB Tamate4 and T Oi1 1Kansai Research Center, Forestry and Forest Products Research Institute, Nagaikyutaro, Kyoto, Japan; 2Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan; 3Hokkaido Research Center, Forestry and Forest Products Research Institute, Sapporo, Japan and 4Department of Biology, Faculty of Science, Yamagata University, Yamagata, Japan The Asian black bear (Ursus thibetanus) inhabits two of the Demographic tests rejected the expansion model in northern main islands, Honshu and Shikoku, in Japan. To determine populations. Haplotypes of the Western and Southern how climatic oscillations during the Quaternary Era affected clusters were unique to local populations. We conclude that the genetic structure of the black bear populations in Japan, the extant genetic structure of the Asian black bear we examined their phylogeographic relationships and com- populations arose as follows: first, populations became small pared their genetic structure. We analysed an approximately and genetic drift decreased genetic diversity in the northern 700-bp sequence in the D-loop region of mitochondrial area during the last glacial period, whereas large continuous DNA collected from 589 bears in this study with 108 bears populations existed in the southern part of central Japan. from a previous study. We observed a total of 57 haplotypes These patterns were essentially maintained until the present and categorized them into three clusters (Eastern, Western time. In western and southern Japan, the effects of climatic and Southern) based on the spatial distribution of the oscillations were smaller, and thus, local structure was haplotypes. All but 2 of the 41 haplotypes in the Eastern maintained. cluster were distributed locally. Genetic diversity was Heredity (2009) 102, 579–589; doi:10.1038/hdy.2009.28; generally low in northern Japan and high in central Japan. published online 25 March 2009 Keywords: female-biased philopatry; glacial periods; land bridge; phylogeography; vegetation shift Introduction phylogenetic studies. Specifically, the genetic diversity of species such as Japanese beech (Fagus crenata; Tomaru Climatic oscillations during the Quaternary Era led to et al., 1998; Fujii et al., 2002), an endemic deciduous shrub shifts in the distribution of animal and plant species. The (Stachyurus praecox; Ohi et al., 2003) and oak species extant species and their distributions are the result of (Quercus spp.; Kanno et al., 2004) is lower in the northern adaptations to specific environments, migration or both part of Honshu than in the southern part due to the factors (Hewitt, 2000). Phylogeographic studies based on founder effect that followed rapid postglacial expansion molecular markers have revealed the relationships of these species. between intraspecific genetic variation and the geo- It is possible that forest mammals shifted their graphic distributions of many plant and animal taxa, distributions according to the changes in vegetation, suggesting that glacial–interglacial oscillations have particularly for vegetation that was crucial for their altered these distributions. During the last glacial period, survival, and this would have affected their genetic broad-leaved trees were retained in refugia in the structure similarly to what occurred in the above- southern part of Honshu, the main island of Japan. In mentioned plant species. For example, Japanese addition, the majority of the area that is currently broad- macaques (Macaca fuscata) exhibit remarkably lower leaved forest was then covered by coniferous forest in variability in mitochondrial DNA (mtDNA) in northern northern Honshu (Tsukada, 1982). It was only after the than in southern Honshu (Kawamoto et al., 2007). This end of the last glacial period that the broad-leaved forest suggests that the macaques rapidly expanded their recovered and spread northward (Tsukada, 1982). The distribution into the northern area during the recovery effects of this shift on the genetic structure of some of the broad-leaved forests after the end of the last glacial broad-leaved tree populations have been revealed by period. Such a rapid expansion is also believed to have occurred in the Japanese giant flying squirrel (Petaurista leucogenys; Oshida et al., 2001). Correspondence. Current address: Dr N Ohnishi, Tohoku Research Center, The aim of this study was to determine the effect of Forestry and Forest Products Research Institute, Nabeyashiki, Morioka climatic oscillations during the Quaternary Era on the 020-0123, Japan. E-mail: [email protected] genetic structure of Asian black bear (Ursus thibetanus) Received 17 July 2008; revised 9 February 2009; accepted 18 populations in Japan. This species is a medium-sized February 2009; published online 25 March 2009 black bear that is distributed in southern and eastern Genetic structure of Asian black bears in Japan N Ohnishi et al 580 Asia (Servheen, 1990). The black bear currently inhabits occurred 0.5 and 0.3 My BP (Dobson and Kawamura, two of Japan’s main islands, Honshu and Shikoku 1998). During these glacial periods, the Korean Peninsula (Figure 1b), but it is thought to be extinct on Kyushu was continuous with Kyushu Island; thus, black bears Island (Biodiversity Center of Japan, 2004). The present could cross the Korea–Kyushu land bridge. Following distribution of the black bear is generally continuous this immigration, interglacial and glacial periods oc- from northern to central Honshu, except in the north- curred repeatedly; the last glacial period was between 80 ernmost peninsula, in contrast to the fragmented and and 12 ky BP. small populations that inhabit western Japan. When considering the effect of climatic oscillations on The black bear is thought to have migrated from the the distribution of this species, we should focus on Asian Continent to Japan during the glacial periods that changes in the distribution of the broad-leaved forest, Sakhalin N 140°E Asian continent Hokkaido B A Island Honshu Island Sea of Japan C Korean D Peninsula 38°N 136°E E Kyushu Pacific Island Ocean L H F I 132°E G M K J N Kanto region Lake Biwa 34°N O P Kyushu Yura Kinki region Island River Shikoku Island 0100 200 400 km E03 E22 E01 E04 E23 E02 E34 E09 E05 E24 E07 E35 E10 A E06 E25 E15 E36 E11 E08 E26 CR01 E12 E28 B E16 E31 E27 CR03 E13 A E17 E32 B B E14 A E29 W01 CR06 E18 E33 E30 C C C CR07 E19 E37 CR08 E20 E38 D E21 CR11 D E D F E H H E F H F L G I K I L J J I L J K G G K O O km O km km 0100 200 0100 200 0100 200 Figure 1 Maps of the study area and relevant adjacent regions: (a) altitudes and water depths of the Asian Continent near Japan, Japan and the areas between. The Korean Peninsula, the Sea of Japan, the Pacific Ocean and Sakhalin Island are indicated. (b) Distribution of the Asian black bear (Ursus thibetanus) in Japan (dark shading) and of the populations in this study (light shading). The Yura River, Lake Biwa, and the Kanto and Kinki regions are indicated: K, the eastern part of northern Kinki; L, the western part of northern Kinki; M, eastern Chugoku; N, the western Chugoku populations studied by Ishibashi and Saitoh (2004). (c–e) Points where black bears exhibiting the haplotypes in the A to L populations were captured: W01, E01 to E38 and CR01 to CR11 represent the UtCR-W01, UtCR-E and UtCR haplotypes, respectively. Heredity Genetic structure of Asian black bears in Japan N Ohnishi et al 581 because the black bear prefers these habitats (Hashimoto from the P population (Figure 1b). Tissue, blood and hair et al., 2003). At present, except for the mountainous areas samples were collected from bears that were hunted or that are covered by coniferous forests that were planted captured for pest control between 1991 and 2007. In the P for commercial use, the vegetation in western Japan is population, hairs were collected using hair traps between dominated by evergreen broad-leaved forest, which is 2003 and 2005. One sample of faeces was collected from thought to have replaced deciduous broad-leaved forests the O population in 2006. during glacial periods (Tsukada, 1982). Both forest types As the borders between populations are not known for are suitable for the black bear. Shikoku and Kyushu were the continuous populations east of Lake Biwa, we connected to Honshu, and the area that is currently predefined 10 populations (A–J; Figure 1) using Gene- underwater would also have been covered by deciduous land 2.0.12 (Guillot et al., 2005) with geographical broad-leaved forest during these glacial periods (Tsuka- information (that is, the locations at which individuals da, 1982). Conversely, in eastern Japan, palynological were sampled), as well as haplotype data that were studies of beech species (Minaki, 1996), on which the obtained in this study following the approach of Coulon black bear depends strongly in its present distribution in et al. (2006). northern Honshu (Oka et al., 2004), show that multiple We obtained data for 108 additional bears from refugia were sparsely located along the Pacific Ocean Ishibashi and Saitoh (2004): n ¼ 28 of 32 for the K and the Sea of Japan south of approximately 381N during population, n ¼ 25 of 32 for the L population and all data the last glacial maximum (25–15 ky BP). At approxi- for the M (n ¼ 22) and N (n ¼ 33) populations. The data mately 15 ky BP, beech forests began to expand rapidly from that previous study for the K and L populations towards the north and the interior from these coastal were selected based on the availability of location data refugia.