Mammal Study 31: 23–28 (2006) © the Mammalogical Society of Japan Distribution of two distinct lineages of sika deer (Cervus nippon) on Shikoku Island revealed by mitochondrial DNA analysis Masahiro Yamada1, Eiji Hosoi2,*, Hidetoshi B. Tamate3, Junco Nagata4, Shirow Tatsuzawa5, Hiroyuki Tado6 and Shinobu Ozawa2 1 The Course of Bioproduction Science, The United Graduate School of Agricultural Sciences, Tottori University, Tottori 680-8553, Japan 2 Department of Biological and Environmental Sciences, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan 3 Department of Biology, Faculty of Science, Yamagata University, Yamagata 990-8560, Japan 4 Department of Wildlife Biology, Forestry and Forest Products Research Institute, Tsukuba 305-8687, Japan 5 Department of Regional Science, Faculty of Letters, Hokkaido University, Sapporo 060-0810, Japan 6 Ymaguchi Prefectural Forestry Guidance Institute, Yamaguchi 753-0001, Japan Abstract. Nucleotide sequences of sika deer (Cervus nippon) collected from the eastern part of Shikoku Island were investigated & compared with those from other areas. Nucleotide sequence of the whole D-loop region of the mitochondrial DNA was determined by direct sequencing technique for each sample. The phylogenetic tree constructed by the sequences indicates that sika deer from Shikoku Island are divided into two distinct lineages: the northern Japan group and the southern Japan group. Proportion of the northern Japan lineage was higher in the northeastern part of the sampling area. There was no border between the distribution of the two lineages, rather it seemed that their distribution intermingled. Besides, there were locations where both lineages were found within a small area. These results indicate that two lineages might be hybridized in some areas of eastern Shikoku. Key words: Cervus nippon, phylogeny, Shikoku, sika deer. The sika deer, Cervus nippon, in east Asia has a wide westernmost part of Honshu Island, Tsushima Island, distribution from Vietnam to eastern China and in the Kyushu Island and southward (Fig. 1). Ussuri District of Siberian Russia. It is also distributed There seems to have been a boundary or a geographi- on islands such as Taiwan and the Japanese Archipelago cal barrier between the distribution of the two distinct (Whitehead 1993). It has been widely accepted that lineages, such as rivers, steep terrain, volcanic activity, there are six subspecies of the sika deer on the Japanese difference in suitable vegetation and so forth. Between Archipelago (Ohtaishi 1986). Studies of the phylogeny these distributions, however, there are areas where sika of the sika deer in Japan, however, revealed that its clas- deer populations exist but whose genetic statuses have sification on the level of subspecies is not applicable for yet to be investigated. One such area is Shikoku Island. some populations. The sika deer in Japan is divided into Shikoku Island has been known to have populations of two groups, the northern and southern groups, by mito- Kyushu Sika (C. n. nippon). All the Kyushu sika deer chondrial DNA (Nagata et al. 1995; Tamate et al. 1995; populations investigated so far are reported as belonging Tamate et al. 1998; Nagata et al. 1999). The northern to the southern Japan group. However, Shikoku Island is Japan group is distributed from Hokkaido Island to the very close to Honshu Island and the sika deer on Shodo Kinki region of Hyogo Prefecture in Honshu Island. The Island, which is part of Kagawa Prefecture, is classified southern Japan group is in Yamaguchi Prefecture, in the as Honshu sika deer and genetically belongs to the north- *To whom correspondence should be addressed. E-mail: [email protected] 24 Mammal Study 31 (2006) Table 1. D-loop haplotypes found in Shikoku Island and Kinki region. Number of DDBJ. Region Haplotype Locality N repeat units Acc. No Naka (2) 4 4Esk1 Kainan (4) 1 AB186349 Umaji (6) 10 4 4Esk2 Naka (2) 5 AB186350 4Esk3 Naka (2) 1 AB186351 4Esk4 Naka (2) 1 AB186352 Kainan (4) 1 66Esk1 AB186347 Umaji (6) 7 Naka (2) 6 77Esk1Kamikatsu (1) 1 AB186346 Shikoku Island Otoyo (7) 1 8 8Esk1 Umaji (6) 2 AB186345 Naka (2) 17 Hiwasa (3) 2 99Esk1 AB186344 Fig. 1. Location of Shikoku Island in Japanese Archipelago and Kaifu (5) 1 localities of the haplotypes reported by Nagata et al. (1999) and used Otoyo (7) 2 in the present study for comparison: white circles, black circles and a 6Kii1 Miyagawa (8) 1 AB248236 black square indicate the northern Japan types, the southern Japan types and the haplotype from China used as an outgroup. Miyagawa (8) 1 6 6Kii2 AB248237 Shimokitayama (9) 3 6Hyg1 Sasayama (11) 2 AB248238 ern Japan group (Yamada et al. 2003). Therefore, there 7Nra1 Nara Park (10) 2 AB248233 is a possibility that the northern Japan type is distributed Kinki Region 7 7Hyg1 Asago (12) 6 AB248234 on Shikoku Island, especially on its east side. The objec- 7Hyg2 Asago (12) 1 AB248235 tive of the present study is to identify the lineages of the sika populations in the eastern part of Shikoku Island. A number in parentesis next to each locality name corresponds to the number in Fig. 2. Materials and methods Extracted DNA was dissolved with TE buffer (10 µl) and Samples of tissues (muscle or liver) of sika deer which stocked at –20°C. were killed either during regular hunting season or in Polymerase Chain Reaction (PCR) (Mullis and pest control culling in eastern Shikoku Island were Faloona 1987) amplifications were performed in Gene collected from February 1997 to November 1999. We Amp PCR System 2400 (Applera Corporation: Perkin- collected a total of 62 specimens of sika deer from Elmer) in a 50 µl total volume containing 1.25 unit Taq Tokushima and Kochi prefectures. The localities are as DNA polymerase (Applied Biosystems), 5 µl of 10 × follows: Tokushima Prefecture: Naka Town, Kamikatsu PCR buffer, 0.2 mM each dNTP, 1.5 mM MgCl2, 0.5 µM Town, Kainan Town, Kaifu Town, and Hiwasa Town; each primer, and 0.05 µg of DNA template. PCR cycling Kochi Prefecture: Otoyo Town and Umaji Village. We parameters were as follows: forty cycles each consisted also collected samples from the Kinki region for com- of 95°C for 1 minute, 55°C for 1 minute, and 72°C for parison in the same manner. Sampling localities are 1 minute, with an initial hot start at 95°C for 5 minutes shown in Table 1 and Fig. 2. Samples were cut to about and a final extension at 72°C for 10 minutes. To 4–6 g pieces and they were stored in ethanol solution of amplify and sequence the D-loop regions of the sika 70–90%. deer, five primers (LD5, LD7, HD2, HD6, and HD8) DNA was extracted from ethanol-preserved tissue designed by Nagata et al. (1998) and three primers following standard procedures (Sambrook et al. 1989). (CervL1, CervL3 and CervH1) designed by Charles E. Yamada et al., Two lineages of sika deer on Shikoku Island 25 Fig. 2. Localities of the samples used in the present study. Cook were used. PCR products and 100 Base-Pair Yma1, Tma1 (C. n. centralis), Gto1, Mya2 (C. n. nippon), Ladder (Amersham Pharmacia Biotech) were run on 2% Yku1 (C. n. yakushimae), Kra1 (C. n. keramae), Chi1 agarose (Type II mediumEEO; Sigma chemical) gels (C. n. kopschi). Localities of these samples are shown in with Mupid-2 (Advance). We also ran the sample from Fig. 1. Although the sequence of the whole D-loop re- Miyazaki, Kyushu that had the same partial nucleotide gion was determined referring to Anderson et al. (1982), sequence as Mya2 (C. n. nippon) in Nagata et al. (1999). all the data were shortened to the same length as those Primer and unincorporated dNTPs were removed from from Nagata et al. (1999). PCR products using Micro Spin S-400HR Columns (Amersham Pharmacia Biotech). Dye terminator cycle Results sequencing was performed with BigDye Terminator Cycle Sequencing Ready Reaction Kit, 3.2 pmol primer, PCR products of various sizes were obtained. and 30–90 ng PCR products. Cycle sequencing param- Although PCR products of the samples from the Kinki eters were as follows: twenty-five cycles each consisted region, such as Hyogo, Nara and Mie, were all on the of 96°C for 10 seconds, 50°C for 5 seconds, and 60°C for larger side, those from Shikoku varied in size from the 4 minute, with an initial hot start at 96°C for 5 minutes. smallest to the largest (Fig. 3). Products of sequencing reactions were purified with It was figured out that this difference in size was CENTRI-SEP Columns (Applied Biosystems). due to the number of units in the tandemly repeated re- Sequencing analysis was performed using the com- gion, which were 4, 6, 7, 8 and 9 in the samples from puter program Gene Works (Intelligenetics). A phyloge- Shikoku. A total of eight haplotypes were found in netic tree was reconstructed with the neighbor-joining eastern Shikoku. Each haplotype was named according (NJ) method (Saitou and Nei 1987) in the computer to the number of units of tandemly repeated region and program Clustal W (Thompson et al. 1994). All align- the sampling locality. Thus, haplotypes from eastern ments were checked and edited manually. Tandemly re- Shikoku were named 4Esk1, 4Esk2 and so forth. peated regions were deleted for sequence analysis. The Schematic diagrams of the D-loop region of the differ- genetic distances were estimated using Kimura’s two- ent sizes are shown in Fig. 4. In Tokushima Prefecture 7 parameter method (Kimura 1980). The bootstrap analysis haplotypes were identified among 40 samples. In Kochi (Felsenstein 1985) consisted of 1000 replications for Prefecture 5 haplotypes were found among 22 samples; the NJ tree.