関東森林研究69-2(2018)

論 文

Identification of origin of sika deer (Cervus nippon) in recently expanded habitat
areas in Tokyo Metropolis based on mitochondrial D-loop sequences
東京都で生息域を拡大するニホンジカのミトコンドリア DNA(D-loop 領域)多型解析
Shoko HATA＊1, Sayaka KONISHI＊2, Sango YOSHIOKA＊3, Kazushi ARAI＊1, Yasushi MIZOGUCHI＊4
畑＊１

尚子＊1・小西清夏＊2・吉岡さんご＊3・新井一司＊1・溝口康＊4

Tokyo Metropolitan Agriculture and Forestry Research Center, Tachikawa, Tokyo 190-0013
東京都農林総合研究センター＊２

School of Agriculture, Meiji Univ., Kawasaki, Kanagawa 214-8571
明治大学大学院農学研究科＊３

Bureau of Environment, Tokyo Metropolitan Government, Shinjuku, Tokyo 163-8001
東京都環境局(元 東京都農林総合研究センター)＊４

School of Agriculture, Meiji Univ., Kawasaki, Kanagawa 214-8571
明治大学農学部

Abstract: Muscle tissue of 145 sika deer (Cervus nippon) collected in Tokyo Metropolis and surrounding regions insouthern Kanto were investigated based on mitochondrial D-loop sequences. Four (CN-1-CN-4) of the nine identified
haplotypes (CN-1-CN-9) accounted for 94.5% of the total study population. One (CN-1) of these four haplotypes waswidely distributed throughout the whole study region, but the other 3 haplotypes (CN-2, CN-3, CN-4) were locally
distributed in different areas of the region. Two (CN-2, 3) of these haplotypes were distributed around the Kanto
Mountains, whereas the other (CN-4) was around the Tanzawa Mountains. All 6 individuals sampled in Hachioji City,a new habitat area that sika deer in Tokyo Metropolis have recently entered, possessed the haplotype found locallyaround the Kanto Mountains. Thus, we concluded that the deer captured in Hachioji City have been originated fromthe Kanto Mountains.
Key-word: habitat expansion, D-loop region, haplotype, Kanto Mountains

要旨：東京都および周辺で捕獲された計 145 個体のニホンジカの筋肉片サンプルを用いてミトコンドリア
DNA の D-loop 領域の多型解析を行った。ハプロタイプは，9 タイプ(CN-1～CN-9)検出され，そのうち 4タイプが全体の 94.5％を占めていた。これら 4 タイプは，比較的広域に分布する CN-1 タイプと偏域的に分布する CN-2，CN-3，CN-4 タイプに二分された。このうち偏域分布する 3 タイプのうち，CN-2 およびCN-3 タイプは関東山地周辺に分布し，CN-4 タイプは丹沢山地周辺に分布していた。都内の生息拡大域にあたる八王子市において捕獲された 6 個体は，すべて関東山地のハプロタイプを有していた。このことから，八王子市に定着したニホンジカは，関東山地個体群がソースとなっていると考えられた。
キーワード：生息域拡大，D-loop 領域，ハプロタイプ，関東山地－1 －(受理日 2018 年 6 月 4 日)

Management Plan to control sika deer populations. Although

Ⅰ Introduction

the sika deer population density decreased and stabilized due tothe population control policy, the population has increased
again in recent years resulting in deer being sighted in Hachioji
City where they previously did not occur (Fig.1.). For example,in 2013, camera traps belonging to the Tokyo Metropolitan
Agriculture and Forestry Research Center photographed a sika
deer in Hachioji City that had been ear-tagged previously in theTanzawa Mountains in Kanagawa Prefecture (unpublished).
Thus, an understanding of sika deer migration routes isnecessary to mitigate further expansion of sika deer into areas
where they did not occur previously.
Therefore, in this study we sought to clarify the genotypes ofsika deer in Tokyo Metropolis and surrounding regions, andsought to determine the origin of individual deer captured inHachioji City.

Fig.1. Sample locations. Numbers indicate sample size ineach mesh (5×5km). The dashed line indicates therecently expanded habitat area in Hachioji City.
図－１．シカ肉片サンプル取得位置図。数字はメッシュ内(5×5km)で捕獲した頭数。破線は生息拡大域(八王子市)。

Ⅱ Materials and methods
We used 145 sika deer samples used in Konishi et al. (2017)(3). Muscle tissue samples were collected from 139 sika deer
between 2013 and 2014 in Tokyo and the surrounding regions;Sika deer (Cervus nippon) cause extensive damage to areas

namely Tokyo Metropolis, Saitama, Kanagawa and Yamanashi

that are being reforested. In Tokyo Metropolis, forest damage

prefectures. In addition, samples were also collected from 6

due to overpopulated sika deer has been a longstanding problem.

deer in Hachioji City, Tokyo Metropolis, in 2015 where sika

This overpopulation has serious ramifications, such as andeer have been expanding their habitats in recent years. Total

incident in Okutama Town in 2004, in which the sediment

DNA was extracted based on the phenol/proteinase K/sodium

blocked the drinking water supply brought about by a run-off

dodecyl sulfate method, with a slight modification. Then, thefrom denuded mountainsides undergoing reforestation. This

Mitochondrial D-loop regions (approximately 1,100 bp) wereincident prompted the metropolitan authorities to formulate

amplified by polymerase chain reaction (PCR). Three primers

management measures such as Sika Deer Conservation andTable1. Comparison of nucleotide sequences of sika deer D-loop haplotypes (polymorphic sites are shown)
表－１．ニホンジカの D-loop 領域のハプロタイプの塩基配列。多型のあった塩基部位を示す。
Nucleotide position of polymophic sites
Haplotype

N82CN-1
CN-2
CN-3
CN-4CN-5CN-6CN-7
CN-8CN-96730
211932111109119152 ≫ 93 . 2593594599600603606626642643676688703706736815850856859860960990997102010291030104910521057106710701074108010951102111111521173120512681269T - C C

C C C C C T C T T G A C A C G C C T T - C G A A A T A G G G A G A A C A T C T C A・ -・ ・・ T ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ - ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ -・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ C - ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ -・ ・・ T ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ - ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・・ -・ ・・ ・ ・ ・ ・ ・ ・ ・ C ・ ・ T ・ ・ ・ ・ ・ ・ ・ - ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・C C T T

T T T T T C T ・ C A G ・ G T A ・ T C ・ T ・ ・ G ・ ・ C G A ・ ・ ・ ・ G G ・ ・ C T C T G・ -・ T・ T T ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ T ・ ・ - T ・ ・ T T ・ ・ C C T C A ・ ・ T T ・ ・ ・ ・ ・・ -・ T・ T T ・ ・ ・ ・ ・ C ・ ・ ・ ・ ・ ・ ・ T ・ ・ - ・ A ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ G・ -・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ T ・ ・ ・ - ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・Site number starts from the same base as AB500007.
Dots denote the same nucleotide identical as haplotype CN-1.
The “-“sign indicates a single base gap.
Tandem repeat units “≫” are deleted in the analysis.

－2 －関東森林研究69-2(2018)

× CN-2△ CN-3● CN-4○ CN-1

Fig.2. Distribution of D-loop haplotypes in the sampled region.
Wide distributed type (left) and local distributed type (right). “○”, “×”, “△” and “●” signs indicate the presence of thehaplotype (CN-1-CN-4) in 5×5km square. Numbers in the left figure denotes haplotype CN-5, 6, 7, 8 or 9. The dashedline indicates the habitat area in Hachioji City into which sika deer is expanding its population.
図－２．D-loop ハプロタイプの地理的分布
左図が広域分布型，右図が偏域分布型のハプロタイプの分布を表す。四角の中の符号は，該当ハプロタイプが５×５km メッシュの中に存在していたことを示す。左図の数字はハプロタイプ CN-5,6,7,8,9 のいずれかを表す。破線は生息拡大域(八王子市)。
were used to amplify and sequence the D-loop regions; L15926

for 94.5% of the total study population, whereas haplotypes

and H597 designed by Nagata et al. (4) and a supplemental

CN-5 to CN-9 were each identified in only one to three deer.

primer we designed for this study (5'-ATGCTTGGACTCA

CN-1 was identical to sequences previously reported in deer inGCAATGG-3'). The PCR products were purified using ExoTochigi Prefecture (AB500007 and AB500008; Registered

SAP-IT (Affymetrix), and then these products were used asregion include tandem repeat region that is excluded in thistemplate for direct sequencing. The sequencing was carried outstudy). The CN-6 haplotype, identified in two deer in this study,

using an automated sequencer 3130xl Genetic Analyzer

was identical to a haplotype from Tanegashima Island,(Applied Biosystems), and the sequence alignment was

Kagoshima Prefecture (AB757723) (7). These two deer withperformed using MEGA 6.0 computer software (6). The D-loop

the CN-6 haplotype were considered to be immigrants, based

region of sika deer contains tandem repeats, each of which

on records of sika deer being imported and released inconsists of approximately 40 bp motifs. The repetition of these

Kanagawa Prefecture (2).

units differs greatly between populations (5), so these regions
were deleted for the present sequence analysis.

The geographical distribution of the nine haplotypes is shownin Figure 2. The CN-1 haplotype was widely distributed acrossthe study area, whereas the CN-2, CN-3, and CN-4 haplotypes

Ⅲ Results and discussion

were locally distributed in different regions. Specifically, theWe compared the nucleotide sequences of mtDNA D-loop

CN-2 and CN-3 haplotypes were distributed around the Kanto

regions from the 145 sika deer and identified nine haplotypes

Mountains; north of the Sagami River, whereas the CN-4(Table 1.). The most frequent haplotype was designated as CN-haplotype was distributed around the Tanzawa Mountains;

1 (46.2%), followed by CN-2 (20.7%), CN-3 (14.5%), CN-4

south of the Sagami River. Genetic divergence between deer(13.1%), CN-5 (2.1%), CN-6 (1.4%), CN-7 (0.7%), CN-8

from the Tanzawa Mountains and the Kanto Mountains have(0.7%), and CN-9 (0.7%). Haplotypes CN-1 to CN-4 accounted

been identified previously by short sequence repeat (SSR)－3 －analysis (3).
The CN-2 haplotype was found in four of the six sika deer

References

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(3) KONISHI, S., HATA, S., MATSUDA, S., ARAI, K.,

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Science Journal 88:1673-1677

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Ⅳ Conclusions

HAMASAKI, S., OCHAI, K., ASADA, M., TATSUZAWA,

1. Four of the nine D-loop haplotypes identified in the present

S., SUDA, K., TADO, H., YOSHIDA, MC. (1999) Two

study accounted for 94.5% of the analyzed sika deer population;

genetically distinct lineages of the sika deer, Cervus nippon, in

one haplotype was widely distributed (CN-1) and three wereJapanese islands: Comparison of mitochondrial D-loop region

locally distributed (CN-2, CN-3, and CN-4).

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2. The CN-2 and CN-3 haplotypes were locally distributed in519

the Kanto Mountains, and the CN-4 haplotype was distributed(6) TAMURA, K., STECHER, G., PETERSON, D., FILIPSKI,

in the Tanzawa Mountains.

A., KUMAR, S. (2013) MEGA6: molecular evolutionary

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genetics analysis version 6.0. Molecular biology and evolutionof sika deer has recently expanded into contained haplotypes of30, 2725-2729.

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(7) TERADA, C., YAMADA, T UNO, H., SAITOH, T. (2013)

captured in Hachioji City have been originated from the Kanto

New mtDNA haplotypes of the sika deer (Cervus nippon) found

Mountains.

in Hokkaido, Japan suggest human-mediated immigration.
Mammal Study 38: 123–129

Acknowledgments
We thank Dr. Hayato Iijima of the Yamanashi Forest
Research Institute, Dr. Toru Taniwaki of the Kanagawa
Prefecture Natural Environment Conservation Center, Mr.
Mutsumi Ohkawara of the Saitama Prefectural Agriculture andForestry Research Center, officials of the Water Resources
Administration Office (Bureau of Waterworks, Tokyo
Metropolitan Government), Dr. Takayuki Seto and the many
hunters for providing samples of sika deer. We also thank Dr.
Iain McTaggart for his editorial assistance. This research is
supported by The Tokyu Foundation for Better Environment.

－4 －