Distribution of Two Distinct Lineages of Sika Deer (Cervus Nippon) on Shikoku Island Revealed by Mitochondrial DNA Analysis

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. During sequencing analysis, we added the they are 4Esk1, 6Esk1, 7Esk1, 8Esk1 and 9Esk1. sequence data that appeared in Nagata et al. (1999) A phylogenetic tree was reconstructed by the neigh- such as Hka (C. n. yesoensis), Gyo1, Kmo1, Kna1, Wda1, bor-joining method including the data of Nagata et al. 26 Mammal Study 31 (2006)

Fig. 3. PCR products of D-loop region. Lane 1 and 2 are 100 bp ladder markers. Difference in size of the D-loop region among samples is due to the different number of units in the tandemly repeated region. Results of the sequence analysis shows that the northern Japan types had 6 or more repeated units (lane 4–7), whereas the southern Japan types had 4 or 5 of them without exception (lane 2 and 3).

Fig. 5. Phylogenetic tree of 547 base-pair region of the D-loop of the sika deer reconstructed by the neighbor-joining method. C. n. kopschi (Chi1) in Nagata et al. (1999) was used as an outgroup. Numbers near the internal branches are bootstrap probability values derived from 1000 replications. Values less than 50% were omitted. Gaps and the tandem repeat sequences were deleted for calculation of genetic distance.

Kochi Prefecture 4Esk1 had the highest proportion at 45.5%, but proportion of the northern Japan group was higher than the southern Japan group. It is notable that distribution of the two lineages seem to be intermingled. Umaji village of Kochi Prefecture is a fairly small village that is only 165.5 km2 in area. Not Fig. 4. Schematic diagram of mitochondrial DNA D-loop region. only were both the northern and the southern haplotypes found in the village, but they were also found in a limited area: Mt. Koishikawa in the Yanase area of the (1999) (Fig. 5). It was shown that the Sika population of village. Similarly, haplotypes of the two lineages were eastern Shikoku had both lineages, from the northern found in small areas of Naka Town and Kainan Town in Japan group and from the southern Japan group. All of Tokushima Prefecture and Otoyo Town in Kochi Pre- the samples from Mie, Nara and Hyogo belonged to the fecture. northern Japan group. Proportions of the haplotypes found in eastern Discussion Shikoku are shown in Fig. 6. In Tokushima Prefecture the proportion of 9Esk1 was highest, at 50%; the second As Tamate et al. (1995) and Nagata et al. (1995) highest was 7Esk1, at 17.5%. In the prefecture 70% of reported, sika deer in the Japanese Archipelago can be the samples belonged to the northern Japan group. In divided into two groups, the northern Japan group and Yamada et al., Two lineages of sika deer on Shikoku Island 27

tion is considered to be highly valuable in terms of bio- geography. In such a case artificial disturbance should be taken into consideration. However, it is reported that both of the two deer farms in this area have only exotic species, either Cervus unicolor or C. timorensis (Live- stock Industry Bureau, the Ministry of Agriculture, For- estry and Fisheries 1997). There is no sika deer farm of considerable size in eastern Shikoku. Besides, all the northern haplotypes of the Shikoku region identified in the present study are unique to this region and more closely related to the haplotypes from the Kinki region, such as Hyogo, Nara and Mie, than others from more distant places. Therefore, it should be considered that this is the result of natural expansion of the distribution of the northern sika deer. This could be supported by the ratio of the haplotypes. In the present study, samples of the northern type accounted for 64.5% in the Shikoku region. Such a huge genetic disturbance by small local deer farms would be impossible. The obtained result that the proportion of the northern haplotypes in Tokushima Prefecture, which is north of Kochi Prefecture, is higher than that in Kochi (Fig. 6) is in accordance with the probable invasion pattern of the northern lineage to Shikoku Island, since the northern Fig. 6. The ratios of the two lineages of the sika deer in eastern lineage is considered to have come from the northeast. Shikoku Island and in the two prefectures. It has been widely accepted that the sika deer on Shikoku Island is a Kyushu sika (C. n. nippon) based on the morphological study of the limited specimens the southern Japan group, based on analyses of mito- (Imaizumi 1949; Ohtaishi 1986; Miura 2005). It was chondrial DNA. According to analysis of the D-loop revealed by Tamate et al. (1995) and Nagata et al. (1995) region of mitochondrial DNA there is a clear difference that Kyushu sika distributed in the Kyushu region belong in nucleotide sequence between the two groups. The to the southern Japan lineage. In the present study the D-loop region of sika deer contains tandem repeats, in northern lineage was found in the Shikoku region. Sym- which each unit has 37 to 40 nucleotides. The number of patric existence of two genetically distinct lineages of the the repeated units is either 4 or 5 in the southern Japan same species has never been reported in cervid. There- group, and it is more than 6 in the northern Japan group. fore, the Kyushu sika population in this region should be In the present study as well as in Nagata et al. (1999), the recognized as a unique population. According to micro- repeated domain was excluded from construction of the satellite analysis by Goodman et al. (2001) there was no phylogenetic tree. Still the present result is consistent clear difference between the northern and the southern with the previous report that all samples included in the lineages. In explanation of this contradiction they argue northern Japan cluster have more units in the repeated the possibility of the “male-mediated gene flow” across domain. Although Nagata et al. (1999) did not clearly the border of the lineages. If that was the case, it is point out, the number of units in the repeated domain highly possible that the two lineages have been hybrid- might be used as a simple indicator in differentiating the ized at least in some places of eastern Shikoku because two lineages. the results of the present study show that both types were In the present study both northern and southern groups found in the same mountain at the same period. It must, were found in sampling localities in the Shikoku region. however, be verified by microsatellite analysis in order Since it has not been reported that two distinct lineages to prove this. inhabited the same area, the eastern Shikoku sika popula- 28 Mammal Study 31 (2006)

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