Table 1-1 Primers used for amplification and sequencing of the mitochondrial control region for the elephants.

(1)

九州大学学術情報リポジトリ

Kyushu University Institutional Repository

アフリカゾウ(Loxodonta africana)とアジアゾウ (Elephas maximus)のミトコンドリアDNA解析 : コントロール領域の遺伝的構造と個体群系統について

岡山, 俊直

九州大学比較社会文化研究科日本社会文化専攻

https://doi.org/10.11501/3159075

出版情報：Kyushu University, 1999, 博士（理学）, 課程博士バージョン：

権利関係：

(2)

Table 1-1 Primers used for amplification and sequencing of the mitochondrial control region for the elephants.

Name of primers L15775.Lox**

Ll5926.ele*

LoxLl LoxL2 LoxL3 LoxlA LoxHl LoxH2 LoxH3 H651*

Sequences

5'-ACATGAATTGGCAGCCAACCAGT-3' 5'-TAAGACATTACAATGGTCTTGTAAGCC-3' 5'-TCTAGTCACCATGCATATCACCTCCAA-3' 5'-GAATTGAGTATTAAGATCTGGCACGGT-3' 5'-GGGAAGAGGTCCACTTACCTCGCTATC-3' 5'-CCCCAAAAGCAGGACTATACTATAGAT-3' 5'-TGTCCTCCGAGCATTGACTGAATAATA-3' 5'-GGGACACGAAGATGGGCGGGTTGATGG-3' 5'-ATCTATAGTATAGTCCTGCTTTTGGGG-3' 5'-AAGGCTAGGACCAAACCT-3'

* Primers modified referring Ko

c

her

et

al. (1989)

* * Primers modified referring Irwin

et

al . (1991)

(3)

Table 2-1 Number of samples of L. africana analyzed in this study.

DNA source Countries n Appendix

Zoo in Japan Feces, saliva or hair Kenya or Tanzania 5 A-1

South Africa 16 A-1

Unknown 29 A-1

Imported ivory Ivory Congo 1 A-3

Gabon 1 A-3

Zaire 2 A-3

Kruger National Park Ivory or feces South Africa 38 A-5, A6

Addo Elephant Park Feces South Africa 24 A-7

Tembe National Park Feces South Africa 97 A-8

total 213

(4)

Table 2-2 Number of samples of E. maxim

us

analyzed in this study.

DNA source Countries n Appendix Zoo in Japan Feces, saliva or hair Thailand 20 A�2

Malaysia 1 A-2

Myanmar 3 A-2

India 7 A-2

Sri Lanka 3 A-2

Unknol-Vn 18 A-2

Pinnawala Orphanage Park Feces Sri Lanka 34 A-4

total 86

(5)

Table 2-3 Haplotypes detected from 351 bp of the mitochondrial control region for 213 L.

africana and 86 E. maximus .

Numbers indicate the nucleotide positions from the beginning of the control region.

Haplo

types

Ll L2 L3 L4 L5 L6 L7 L8 L9 LIO Lll Ll2 LIJ L14 LIS Ll6 Ll7 LIS

El E2 E3 E4 ES E7 E8 E9 ElO E12 Ell EIJ El4 El6 E17 El8 El9 E20 E21

nucleotide positions

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3

1 2 3 4 6 7 7 0 4 5 5 5 5 6 6 6 6 7 7 7 8 8 9 9 9 0 0 0 0 0 0 1 1 1 2 2 4 5 6 6 7 7 8 9 0 1 1 2 2 2

3 5 8 9 3 4 5 2 6 7 8 0 7 4 6 7 8 4 6 7 8 5 7 8 5 6 0 2 4 0 1 2 3 4 5 5 6 9 0 3 6 9 4 9 5 6 0 I 0 2 9 0 3 7

L.

africana

A C A C C T A C T A C T T T A C T a C A A T C A T T A 0 C T T A a T T T A T A T A T A A C a T C T C C a T C

a a G

T T T T T T T T T T T

c

C A

T C

T - C

T T T T T

T - - - C T

T T T

A a T

T C - T C

C A T C A

c c

c

A G A

E. maximus c - c c c - c -

G a

c a

C G

c a c c c c c c

- c - -

c c

c

c c A C C A C C

c

G C a c a c a c a c a c a c

c c c c c c c

c a a a a

G

G T

- A

- A -

A C A C A C c A C A A A A A A

T A

A

- A

C T

C - T

C T

C - T

C T

c

A T A T T C A C A A C T C T A T C a C A A T C C T T A a T T T A a T T T a C A T A T A a C a T C T C C G T C

c c c c c c

a a

T T

G

G G c a

T G

C T G C T G C T G

T T

a

T T T T a T G T T a T G T G T a T

T T T T T T T T T T T T G T G T G T a T a T G

c c c c c c

c - c c

- c

c c c

- c

c c c c c c c c c c

A A A A A A A A A A A A

a

A

A G A A A

T T T T T T

c

T T T T T T T T T T T T

(6)

Table 2-4 Haplotype distributions of L. africana .

Haplotypes

samples Regions (Countries) n

Ll

L2 l3

lA 1.5 L6 L7 L8 L9 LlO Lll L12 L13 L14 LIS L16 L17 L18 Zcxl

in Japan Feces, �diva or hair Eastern Mrica

(Kenya J Tanzania)

5 1 1 2 1

Southern Africa

(South Africa)

16 2 1 2 7 1 3

Unknown

29 1 2 2 1 3 7 1 3 6 2 1

Imported ivory Ivory Central Mrica

(Congo)

1 1

(Gabon)

1 1

(Zaire) 2 2

Kruger National Park Ivory or feces (South Africa)

38 2 19 16 1

Tern be Flephant Park Feces (South Africa)

24 5 12 7

Addo Elephant National Park Feces (South Africa)

97 77 20

total

213

(7)

Table 2-5 Haplotype distributions of E. maximus .

Haplotypes

samples Regions (Countries) n El F2 F3 FA

F5

E7 E8 E9 EIO Ell El2 E13 E14 El6 El7 E18 E19 E20 E21

Zoo

in Japan Feces, saliva or hair Southeast Asia

(Thailand) 21 1 1 2 1 1 1 7 1 2 4

(Malaysia) 1 1

(Myanmar) 3 1 1 1

South Asia

(India) 7 1 1 3 2

(Sri Lanka) 3 1 1 1

Unknown 17 1 1 2 2 1 2 2 1

4

1

Pinnawala Orphanage Park

Feces (Sri Lanka) 34 1 13 2 2 10 1

5

'···-

total 86

(8)

Table 2-6 Haplotype diversity (h )and nucleotide diversity (jt) for each population.

Number of Number of Haplotype Nucleotide samples haplotypes diversity diverdsity

(n (h ) (rr)

L. ajricana Zoo in Japan 50 13 0.87 2.14

South Africa 159 6 0.65 1.31

Kruger National Park 38 4 0.58 1.30

T em be Elephant Park 24 3 0.65 1.06

Addo Elephant National Park 97 2 0.33 0.88

E. maximus Zoo in Japan 52 11 0.91 2.17

Pinnawala Orphanage Park 34 7 0.76 0.65

h = n ( 1-2: xi 2) I ( n -1)

n=n 2:x, x1 nu I (n -1)

(9)

M f ^I

^•

M

800bp-

Fig. 1-1 An agarose gel electrophoresis of the extracted DNA from feces and ivory samples.

Lane f: feces sample; Lane i: ivory sample; Lane M: molecular weight marker.

(10)

mitochondrial control region

Left domain

I

Central conserved

domain

L15775 Lox Ll5926 ele Lox L3

_� _� _�

: ^{Lox Ll}

cytochrome-b

tRNA thr

tRNA pro

� I I

TAS

� _351bp -- _{Lox H2} ...

I 1

Lox L2

I �

Lox Hl ...

Right domain

repeat region

LoxL4

�

CSB2 CSB3 LoxH3 ...

lls rRNA

H651 ...

Fig. 1-2 Schematic diagram of the mitochondrial control region of the elephants.

Conserved sequence block or CSBs, and central conserved domains were specified according to 'Walberg and Clayton

(1981),

Doda et al.

(1981),

Foran et al.

(1988)

and Gemmell et al.

(19%).

Arrow heads indicate the locations and direction of the primers for PCR amplification and sequencing. Arrowbar indicates the analyzed region in this study.

(11)

M fl f2 ^I

^.

M

800bp-

Fig. 1-3 Amplification of mitochondrial control region using feces and ivory samples.

Lane fl: Feces sample amplified with L 15926. ele and H 651; Lane f2:

Feces sample amplified with L 15926. ele and Lox H2; Lane i: Ivory

sample amplified with L 15926. ele and Lox H2; Lane M: Molecular

weight marker.

(12)

DNA extraction

!

PCR

!

Cloning to T -vector

!

Transformation to E. coli '

� �

Culture of E. coli

!

Plasmid extraction

!

cycle sequencing reaction

! . sequencing

Insert check

Fig. 1-4 The flowchart of sequencing for the repeat region using

TA cloning.

(13)

-a

Pha.rrnacla

'-1' Biotech

File: D:\OK970910.ALF Run.

c 1

r-�---r- -'- - ' -, -�---,---,.-�-.-

AM V3.02 P(lQ6 *\ C:i\..,

Clone: 5, (Sh) (Sc) (R)

Time: 1 '40 • 3:1 7

[h:m]

Date: 09-11-97

T A C A C G T A C A C 0 T A C A C ₃ T A C A C G T A C A C G T A C A C G T

Comrnant: lan&15

130 uo 150 160

Operator.

Fig. 1-5 The sequence output of the repetitive region for L. africana.

(14)

L. africana E. maximus

CAGGCAA-TCAACCCGCTATGTATATCGTGCATTAAATGCTTGTCCCCATACATAATGAT .. A.T .. A .... T ... C

• • • • • • • • • • • • • • • ••

C

• • • • • • • • • • • •• • • • • •

ATATATTACTAACTATACTTAATCTTACATAGACCATACTATGTATAATCGTGCATCACATTATTTACCCCATG

• • • ••

A

• • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • •

CTTATAAGCAAGTACTGTTTAACTAATGTGTCAAGTCATATTCATGTAGATTCACAGGTCATGTTCTGGTTCAT

· · · · · .. . . . . . . . . .. . • . . .

TC

. • . . . . .

T ... T ... T ... T . A . C ...

^.

GGATATTATTTACCTACGATAAACCATAGTCTTACATAGCACATTAAAGCTCTTGGTCGTACATAGCACATCAC

• · ·· · · ·· · .

C

.· . • •. • • • . . • • . •• • • • • . . . • • . . . . . . . • . . • . . . . . . • .

A

• . • •

G

• . . • . •

G ... T

. •

Left domain _� ----+ CCD

�---�

TGAGAAATCTCTAGTCACCATGCATATCACCTCCAATGGTTGTACCTTAAC�ACCTACCTCCGAGAAACCATCA

• • • • • • • • • • • • •• • ••

T

• • • •• • •• • • • • • • • • • •

C

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

ACCCGCCCATCTTCGTGTCCCTCTTCTCGCTCCGGGCCCATCAATTGTGGGGGTTTCTATACTGGATCTATACC

TGGCATCTGGTTCTTTCTTCAGGACCATCTCACCTAAAATCGCCCATTCTTTCCTCTTAAATAAGACATCTCGA

• • • • • • • • • • • • • • •

A

• • • • • • • • • •• • • • • • • • • • •• • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

----+

Ril!ht domain

TGGATTAATGACTAATCAGCCCATGATCATAACATAACTGTGGTGTCATGCATTTGGTATCTT� � ^TAATTTTGG

GGATGCTGTGATTCAGCTATGGCCGTCTGAGGCCCTAACACAGTCAAGCAACTTGTAGCTGAGCTTGAATTGAG

• • • • • •• • • • • • • • • ••• • • ••• • • • • • • • • • • •

T

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

CSB-1

TATTAAGATCTGGCACGGTATATATGGGGTATTATTCAGTCAATGCTCGGAGGACATAAAATTTTTTAAACCTA

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

G

• • • • • • • • • • • • • • •

CSB-3

CCTTAGTTATCCTTGGCTCAATTTCTATAGGTATAATTGTGTTATCCTGTCAAACCCCAAAAGCAGGACTATAC

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

TATAGATGAAACCAAGAGTACCACACAACGGGAGGCATAACTAGTTACATAGGGGTGAAAGCAAATCATTTTGC

• • • • • • • •• • • • • • • • • • • •

G

• • •• • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

CATCGATACTTGGGTACTACTTAAAAAAATTTATTTTTTTTTCAAGAGCCATGTTCCTAGATTTGAAAAAAAAA

•

T

• • • • • • • • • • •• • • • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• • • • • • • • • • • • •

CCCCGGAGACGCGTTTCTTATAAAATTCTACCCCATCTC .

. .

.

. . . . . . . . . . .

. .

.

. .

. . . .

. .

.

. . . . . . . . . .

.

. .

Fig. 1-6 Sequences of the mitochondrial control region for L. africana (93EL50) and E. maximus (93EL25).

The orientation is from

5'

to 3' on the L strand. Dots indicate identity and dashes are proposed indels. The central conserved domain (CCD) is indicated by box. Conserved sequence block (CSB 1-3) are indicated by the underlines.

(15)

platypus opossum dasypus shrew human rabbit mouse cat Gseal FBwhale Wrhino cow

L.

africana E.maximus

platypus opossum dasypus shrew human rabbit mouse cat Gsela FBwhale Wrhino cow

L.

africana E. maximus

L.

africana E.maximus

L.

africana E.maximus

---GGTT CGAGAAACCAGCAATA CCT-AGACAAGGATTT-TCTAC-TCATGGGGTTCG ACCA-CTC CGAGAGATCATCATCC GCCATCTAAAGGCTTTACATCCTTCAGAGGAAACG ACCAACTC CGAGAAACCAGCAACC GCC-AGACAAGTATCCCTCTGC-TTGCTCTGAGCC ACCAACTC CGTGAAACCAGCAACC TTG-CGAGGCGGATCCCTCTTC-TCGCTCCGGGCC ACCATCCT CGTGAAATCAATATCC GCA-CAAGAGTGCTAC-TCTCC-TCGCTCCGGGCC ACCATCCT CGTGAAACCAACAACC GCC-CACCAAGGATCCCTCTTC-TCGCTCCGGGCC ACCATCCT CGTTAAACCAACAACC GCC-CACCAATGCCCC-TCTTC-TCGCTCCGGGCC ACCTGGCC CGAGAAACCAGCAATC TTGCTCGAACGTGTACCTCTTC-TCGCTCCGGGCC ACCATGCC CGGGAAATCAGCAACC TTG-TGAAACGTGTACCTAAACCTCGCTCCGGGCC AGCATGCC CGTGAAACCAGCAACC GCT-TGGCAGGGATCCCTCTTC-TCGCACCGGGCC ACCAAGCC GGGAAATCATCAATC TTCCACTTAA-TGCACTCGTTC-TCGCTCCGGGCC ACCATGCC CGTGAAACCAGCAACC GCT-AGGCAGGGATCCCTCTTC-TCGCTCCGGGCC ACCTACCT CGAGAAACCATCAACC GCCCATCTTCGTGTCCCTCTTC-TCGCTCCGGGCC ACCTACCT CGAGAAACCATCAACC GCCCATCTTCGTGTCCCTCTTC-TCGCTCCGGGCC

* * * ** * * * * *

Subse uence A

CGCAGCCATTGAAGCGTAGC-ATATCTTGCT-TTTTAAG GGCCTCTGGTTCCTTCTTCAG CATGAATT-GTGACG-TACC---TTGTTCCTTTGAT GGCTACTGGTTGTTACTTCAG CATAAACC-GTGGGGGTTTCTATA-CGTGAAACTTTATC GGCATCTGGTTCTTTCTTCAG CATAACTC-GTGGGGGTAGCTATA-TCTCACACTTTACC GGCATCTGGTTCTTACTTCAG CATAACAC-TTGGGGGTAGCTAA--AGTGAA-CTGTATC GACATCTGGTTCCTACTTCAG CATAAAAC-TTGGGGGTTTCTAA--TATGAAACTATAAC GGCAT-TGGTTCTTACCTCAG CATTAAAC-TTGGGG-TAGCTAA--ACTGAAACTTTATC GACATCTGGTTCTTACTTCAG CATTTCAACGTGGGGGTTTCTATA-ACGGAA-CTATACC GGCATCTGGTTCTTACTTCAG CATAACAT-GTGGGGGTTTCTATA-CTGGAA-CTATACC GGCATCTGGTTCTTACTTCAG CATCACTC-GTGGGGGTAGCTATTTAATGAT-CTTTAT GACATCTGGTTCTTACTTCAG CATAACAT-GTGGGGGTTTCTAGA-CCTGAAACTATACC GGCATCTGGTTCTTACCTCAG CATAAACC-GTGGGGGTCGCTATCCAATGAA-TTTTACC GGCATCTGGTTCTTTCTTCAG CATCAATT-GTGGGGGTTTCTAT--ACTGGATCTATACC GGCATCTGGTTCTTTCTTCAG CATCAATT-GTGGGGGTTTCTAT--ACTGGATCTATACC GGCATCTGGTTCTTACTTCAG

* * * * * * * ***** * * ****

GGACAT GGTCAT GGCCAT GGCCAT GGCCAT GGCCAT GGCCAT GGCCAT GGCCAT GACCAT GGCCAT GGCCAT GACCAT GACCAT

* ***

-CA---CTAAGAAATCATCATTAATTGAT -AAGTTTGTTCATTGCATC-CTAACTGCC TCA--CCTAAAACCGTC-CACTCATTCCC TCA--CCTAAAATCGCCCA-TTCATTCCC AAA--GCCTAAATAGCC-CACACGTTCCC GAA--CCTAAGATCGCC-CACACGTTCCT AAA--TGCGTTATCGCCTCATACGTTCCC -TCCTTGAAACCAATCCTTCAGTTCT AAAGTCCTAGAATCCAATCCTACTAACCC TTA--ACTTAAAATCGCCCACTCGTTCCC TCA--CCTAAAATCGCCTA-TTCTTTCCT TCA--TCT-AAAACGGTCCATTCTTTCCT TCA--CCTAAAATCGCC-CATTCTTTCCT TCA--CCTAAAATCGCC-CATTCTTTCCT

* *

Subsequence B

TTTAGGTAGGCATTTTCGAGG T TTAAATAAGGCATCACGATGT T TTAAATAAGACATCTCGATGG A TTAAATAAGACATCTCGATGG A TTAAATAAGACATCACGATGG T TTAAATAAGACATCTCGATGG A TTAAATAAGACATCTCGATGG T TCAAATGGGACATCTCGATGG A TCAAATGGGACATCTCGATGG A TTAAATAAGACATCTCGATGG G TTAAATAAGACATCTCGATGG A TTAAATAAGACATCTCGATGG A TTAAATAAGACATCTCGATGG A TTAAATAAGACATCTCGATGG A

* * * ***

Subse ence C

*

TTGAGTACACCCACGACCGTGATCGCGGCATAGCTGTCATGAAGA-ACTATTGGTATTTTT ACGATTACAGATCAGCCCAT-AACGCGGCATAACTGATTCTGACTGGCATGGGGTAAGATT CTTATGCCATATTAAACCGTGACCTTGCATCCCCTTATCTGTC-ATACATTTGGTACCTTT CTAATGACTAATCAGCCCATGCCGACA-CATAACTGTGGTGTC-ATACATTTGGTATCTTT CACAGGTCTA-TCACCCTATTAAC-CA-CTCACGGGAGCTCTCCATGCATTTGGTATTTTC CTAATGACTAATCAGCCCATGCTCACA-CATAACTGTGGATGTCATGCATTTG-TATTTTT ATCGGGTCTAATCAGCCCATGACC-AA-CATAACTGTGGTGTC-ATGCATTTGGTATCTTT CTAATGACTAATCAGCCCATGATCACA-CATAACTGTGGTGTC-ATGCATTTGGTATTTTT CTAATGACTAATCAGCCCATGATCACA-CATAACTGTGGTGTC-ATGCATTTGGTATCTTT TTAATTACTAATCAGCCCATGATCATAACATAACTGAGGTTTC-ATACATTTGGTATTTTT CTAATGACTAATCAGCCCATGATCACA-CATAACTGTGGTGTC-ATGCATTTGGTATCTTT CTAATGGCTAATCAGCCCATGCTCACA-CATAACTGTGCTGTC-ATACATTTGGTATTTTT TTAATGACTAATCAGCCCATGATCATAACATAACTGTGGTGTC-ATGCATTTGGTATCTTT TTAATGACTAATCAGCCCATGATCATAACATAACTGTGGTGTC-ATGCATTTGGTATCTTT

* * * * * *

Fig. ^1-7 Alignment of the central conserved domain (CCD) for ^L. africana,

E. maximus and other ¹² mammals.

The start and the end of

the CCD

_isfollowed by Gemmell et al. (1996) and accone et al. (1991), re pectively.

Subsequences

A-C

(Gemmell et

al. 1996) are boxed.

(16)

TAS-1

L. africana 1 CAGGCAA-TCAACCCGCTATGTATATCGTGCATTAAATGCTTGTCCCC �AcATAA ^TGAT � ATATTACTAACTATACTT 79 E. maximus 1 CAAGTAAATCAATCCGCTATGTACATCGTGCATTAAATGCTCGTCCCC � ^ACATAATGAT � ATAATACTAACTATACTT 80

+ + ++

TAS-2

+ + + + + ++

80 AATC � ACATAGACCAT � TATGTATAATCGTGCATCACATTATTTACCCCATGCTTATAAGCAAGTACTGTTTAACTAA 159 81 AATC � ACATAGACCAT AF TATGTATAATCGTGCATCACATTATTTACCCCATGCTTATAAGCAAGTACTGTTTAATCAA 160

+ + + +++

160 TGTGTCAAGTCATATTCATGTAGATTCACAGGTCATGTTCTGGTTCATGGATATTATTTACCTACGATAAACCATAGTCT 239 161 TGTGTTAAGTCATATTCTTGTAGATTCACAGGTTATGTTTTAGCTCATGGATATTATTCACCTACGATAAACCATAGTCT 240

+ +++ + ++ ++

TAS-3

+ + +

TAS-4

++++++ ++ ++ +

240 � ^ACATAGCACA TW AAAGCTCTTGGTC Qx ACATAGCACATC At TGAGAAATCTCTAGTCACCATGCATATCACCTCCAATG 319 241 g ^ACATAGCACA � AAAGCTCTTGATC � GCATAGCGCATT At TGAGAAATCTCTAGTCATCATGCATATCACCTCCAACG 320

+ + + + ++

start ^CCD 320 GTTGTACCTTAAC � CCTACCTCCGAGAAAC 350 321 GTTGTACCTTAAC � CCTACCTCCGAGAAAC 351

+ +

+ + + + ++

Fig. ^1-8 The location of the TAS (termination associated sequence) -like sequences in the left domain of the control region.

The T AS-like sequences starting with T ACA T are boxed. Short mirror symmetry sequences are indicated by italic

( TACA 1)

and underline (ATGT A). + indicate the variable sites within or between species.

(17)

L. africana Ll E. maximus El manatee

TAS-1

---CAAGCAAACCAACCC---GCTATGTATATCGTGCATTAAATGCTCGTCCCC ACATAATGAT ATATTA ---CAAGTAAATCAATCC---GCTATGTACATCGTGCATTAAATGCTCGTCCCC ACATAATGAT ATAATA AATTCTACTTAAACTACTCCCTGTACGGCTATGTAATTCGTGCATTATGTGCTCCTCCCCA---TATAGTA

* *** * **

TAS-2

******** ********** ***** ****** **** **

CTAACTATACTTAATC ACATAGACCAT TATGTATAATCGTGCATCACATTATTTACCCCATGCTTATAAGCAAGTAC CTAACTATACTTAATC ACATAGACCAT TATGTATAATCGTGCATCACATTATTTACCCCATGCTTATAAGCAAGCAC CTATCTATGTTTTATCTTACATACACCATCCTATGTATAATCGTACATTACATTATATACCCCATGCTTACAAGCAAGTAC

*** **** ** ********** ***** ************** *** ******* ************* ******* **

TGTTTAACTAATGTGTCAAGTCATATTCATGTAGATTC-ACAGGTCATGTTTTAGTTCATGGATATTATTTACCTACGATA TGTTTAATCAATGTGTCAAGTCATATTCCTGTAGATTC-ACAGGTTATGTTTTAGTTCATGGATATTGTTCACCTACGATA AGTATAAATAATGCTTCTAGACA---GTATATACCTTCCACTGCAAATTCCCAACCACATGGATATTCTTCAG-TCCATTC

** *** **** ** ** ** * ** *** ** *

TAS-3 TAS-4

** * ********** ** * * * *

�---�

AAC-CATAGTC ACATAGCACAT AAAGCTCTTGATC ACATAGCGCATTACT AGAAATC--TCTAGTCATCATGCAT AAC-CATAGTC ACATAGCACA AAAGCTCTTGATC GCATAGCGCATTACT AGAAATC--TCTAGTCATCATGCAT ACCTCTTAATATTGCATAATACATTACGCTCCTTAATCGTACATAGCACATCACTT-GAAATCATTCTCGCCAACATGCTT

* * * ** * ** **** ******

ATCACCTCCAACGGTTGTACCTTAAC ATCACCTCCAACGGTTGTACCTTAAC ATCACCTCCATTAGGCAGTCCCTATC

********** * ** ** *

*** ***** ****** *** *** ****** *** * ** ***** *

1 �

start CCD CCTACCTCCGAGAAAC CCTACCTCCGAGAAAC CCAAGCGCCGAGAAAC

Fig. 1-9 Alignment of left domain of the control region for L. africana haplotype Ll, E. 1naximus haplotype Eland manatee.

The manatee sequence is from Garcia-Rodriguez

et

al. 1998 (AF046158).

(18)

human (1,5,6,7) human ^{( 2)}

human ^{( 3)} human ^{( 4)} chimp gorilla mouse ( 1) mouse ^{( 2)} mouse ^{( 3)} mouse ^{( 4)} rat ^{(RE) (1)} rat ^{(RE) (2)} rat ^{(RE) (3)} rat ^{(RE) (4)} pig ( 1) pig ^{( 2)} cow ( 1) cow ^{( 2)}

COW

( 3)

L.

africana and

L.

africana and

E.maximus ( 1) E.maximus ( 2)

TAC-AT-AAAAACCC--A-AT TAC-AT-AAAAACCC--A-AC TAC-AT-AAAAATCC--A-AC TAC-AT-AGAAACCC--A-AT TAC-AT-AAAA-TCC--AC-T TACCAT-AAAA-CGCCCA-AT CAC-ATTAAA--CT---A-AT AAC-AT-AAA--TC---A-AT TAC-ATTAAA--TC---A-AT TAC-ATTAAA--CT---AT TAC-ATTAA---TTT----AT TAT-AT-ATAA-TT---A-AT AACTA--AAAA-TTT--AAAC TAC-ATTAAGA-T----A-AT TAC-AT-A----TT---AT TAC-AT-A----TC---AT TAC-AT-AA---C---AT AAC-AT-AA---T---AT TAC-ATTAAA--TT---AT TAC-AT-AA---TG---AT TAC-AT-AGA--CC---AT

L.

africana (3, 4-1) ,E.maximus (3-1) TAC-AT-AG---C----ACAT E. maximus (3-2) TAC-AT-GG---C----ACAT

L.

africana (4-2) TAC-AT-AG---T----GCAT

L.

africana (4-3) E. maximus (4-1) E. maximus (4-2)

TAC-AT-AG---C----GCAT TAC-AT-AG---C----GCAT TGC-AT-AG---C----GCAT

Fig. 1-10 Alignment ofT AS -like sequence for L. africana and E.

maxzmus.

TAS

sequences of other

⁷

mammals are from oran

^{et al.}

(1988).

(19)

A ^C ^A

C T

A A

G -c A - or

a-m ri-B 1-IJ rl-11

s6- a-m-1os

B

A T

A C

T G

m-n II·�

m-m

101-r!-l;-116 n-m

Fig.1-11 Putative secondary structures of short mirror symmetries.

(A) The secondary structure of short mirror symmetry associated with TAS-2

and

CSB-L2. 1r

indicates variable site within individuals of the elephant. (B) The secondary structure of short mirror

symmetry in CSB-L2.

(20)

CS-L1

L. africana 1 CAGGCAA-TCAACCCGC � TGTATATCGTGCATTAAATGCTTGTCCCCA � CATAATGATATATATTACTAACTATACTT 79 E. maximus 1 CAAGTAAATCAATCCGC � TGTACATCGTGCATTAAATGCTCGTCCCCA � CATAATGATATATAATACTAACTATACTT 80

+ + ++ + +

CS-L2

+ + + ++

80 AATCTTACATAGACCATAC �TGTATAATCGT GCATCACATTATTTACCCCA TG CTTATAAGCAAGTACTGTTTAACTAA 159 81 AATCTTACATAGACCATAC � TGTATAATCGTGCATCACATTATTTACCCCA TG CTTATAAGCAAGTACTGTTTAATCAA 160

+ + + +++

160 TGTGTCAAGTCATATTCATGTAGATTCACAGGTCATGTTCTGGTTCATGGATATTATTTACCTACGATAAACCATAGTCT 239 161 TGTGTTAAGTCATATTCTTGTAGATTCACAGGTTATGTTTTAGCTCATGGATATTATTCACCTACGATAAACCATAGTCT 240

+ + + + + ++ ++ + + + ++++++ ++ ++ +

240 TACATAGCACATTAAAGCTCTTGGTCGTACATAGCACATCACTGAGAAATCTCTAGTCACCATGCATATCACCTCCAATG 319 241 TACATAGCACATTAAAGCTCTTGATCGTGCATAGCGCATTACTGAGAAATCTCTAGTCATCATGCATATCACCTCCAACG 320

+ + -t + ++

start CCD 320 GTTGTACCTTAACT � CCTACCTCCGAGAAAC 350 321 GTTGTACCTTAACT � CCTACCTCCGAGAAAC 351

+ +

Fig. 1-12 The location of the CS- L.

+

The conserved sequence (CS)- Lis boxed. + indicate the variable sites within or between species.

+ + + ++

(21)

platypus oposswn dasypus 1 dasypus 2, 3 dasypus 4 shrew 1 shrew 2 hwnan rabbit Mouse cat 1 cat 2, 3 gray Seal 1 gray seal 2 finback whale 1 finback whale 2 manatee 1

manatee 2 white rhino cow

L.

africana

1 E.

maximus

¹ L.

africana

² E.

maximus

²

pheasant chicken partrige

ATGTATA-TAGTACATTAATTGCATGTCCGCAT ATGTATA-TAGTACATGGATTTATTTACCCCTA ATGTA-ATTCGTGCATACATTTATCTACCCCAT ATGTATAATCACACATACACTTATCTACCCCAT ATGTATAATCACACATACATTTATCTACCCCAT ATGTATA-TCGTACATTATATTCTCGTCCCCAT ATGTATA-TCGTACATTACATTCTTGACCTCAT ATGTATC-TCGTACATTAC-TGCCAGCCACCAT ATGTAATTCCGTGCATTAA-TGCTCGCCCCCAT ATGTATA-TCGTACATTAAACTATTTTCCCCAA ATGTACA-TCGTGCATTAATTGCTAGTCCCCAT ATGTATA-TCGTGCATTAATTGCTAGTCCCCAT ATGTACT-TCGTGCATTGCATGCTCCCCCCCAT ATGTATA-TCGTGCATTAA-TGGTTTGCCCCAT ATGTA-ACTCGTGCATGCATGTA-CTTCCACAT ATGTATAATTGTGCATTCAATTATTTTCACCA

ATGTAAT-TCGTGCATTATGTGCTCCTCCCCAT ATGTATAATCGTACATTACATTATATACCCCAT ATGTACA-TCGTGCATTAAATTGTTTGCCCCAT ATGTATA-TAGTACATTAAATTATATGCCCCAT ATGTATA-TCGTGCATTAAATGCTTGTCCCCAT CTGTATA-TCGTGCATTAAATGCTCGTCCCCAT ATGTATAATCGTGCATCACATTATTTACCCCAT ATGTATAATCGTGCATCACATTATTTACCCCAT

ATGTATAATCGTGCATACATTTATATACCACAT ATGCATAATCGTGCATACATTTATATACCACAT ATGTATAATCGTGCATACATTTATATACCACAT

Fig. 1-13 Alignment of CS-L for L. africana, E. maxim

^us

and 13 mammals.

Sequence sources are the same as Fig. 1-7 except for manatee, pheasant, chicken and

partrige.

(22)

>cat

tacataagacatactatgtacatcgtgcattaattgctagtccccatgaatattaagcatgtacaggagtttatatatat tacataagacatactatgtatatcgtgcattaattgctagtccccatgaatattaagcatgtacagtagtttatatatat tacataagacatactatgtatatcgtgcattaattgctagtccccatgaatattaagcatgtacagtagtttatatatat tacataagacata

>dasypus

ctatgtaattcgtgcatacatttatctaccccatacatatcattatatactaactattattaatcttacatagtacatt ctatgtataatcacacatacacttatctaccccatacatatcattatatactaactattattaatcttacatagtacatt ctatgtataatcacacatacacttatctaccccatacatatcattatatactaactattattaatcttacatagcacatt ctatgtataatcacacatacatttatctaccccat

>shrew

catataagcaagtactattaatctaattagtacattagacatactatgtatatcgtacattatattctcgtccccata catataagcaagtactattaatctaattagtacattagacatattatgtatatcgtacattacattcttgacctcatg

Fig. 1-14 The CS- Land repeat sequences of cat, dasypus and shrew.

/

The under lines show the CS-L. The cat sequence is from Lopez et al. (1996), the dasypus sequence is from Amason eta/. (1997) and the shrew

sequence is from Stewart and Baker ( 1994)

(23)

CSB-1 platypus opossum dasypus shrew human rabbit mouse cat

gray seal finback whale white rhino cow

Elephant

ATATAGATTAATGCTTGACGGACA-TA TATTTATTTAATATACGAAGGACAATA TATTCAGTCAATGGTTACAGGACA-TA ATATGGTTTAATGATCTGAAGACA-TA TAATTAATTAATGCTTGTAGGACA-TA CATTCTTTTAATGCTTGTCGGACA-TA TAATTA-TTCATGCTTGTTAGACA-TA TATTCAGTCAATGGTCACAGGACA-TA ATTTTCGTCAATGGTAGCGGGACA-TA AGTTAAATTAATGGTTACAGGACA-TA TATTCAGTCAATGGTTACAGGACA-TA ATTACAGTCAATGGTCACAGGACA-TA TATTCAGTCAATGCTCGGAGGACA-TA

TTTCCCCCCCCTTCCCCCC TAAACCCCC--TTACCCCC CAAACCCCCCCCCCTCCCC CAAACCCCC--TTACCCCC CAAACCCCCCCCCTCCCCC CAAACCCCCCC-TACCCCC CAAACCCCCC---ACCCCC CAAACCCCCC-TTACCCCC CAAACCCCCC-TTACCCCC CAAACCCCCCTCCTTCCAT CAAACCCCCCCACCCCCCA ---CCCCCCCTTC----

CAAACCCCCC-TTACCCCC

TCCGTCAAACCCCAAAACCG CTTGTCAAACCCCAAAAACA CTTGCCAAACCCCTAAAACA TCTGCCAAACCCCAAAAACA CCTGCCAAACCCCAAAAACA --TGCCAAACCCAAAAAACA CT�GCCAAACCCCAAAAACA TCTGCCAAACCCCAAAAACA

CTTGCCAAACCCCAAAAACA CCTGTCAAACCCCAAAAGCA

Fig. 1-15 Alignment of conserved sequence blocks (CSB) _1-3.

The conserved sequence blocks (CSB) 1-3 were referred from Gemmell el al. (1996). equence sources are the same as Fig. 1-7.

(24)

>L. ajricana (96LX79) clone 1

GCGTACACACATACGCATACGCATACGTACACGTACACGTACACGCGTACACACGTACACGTACACGTA CACGCGTACACACGTACACGTACACGTACACGTACACGTACACGTACACGTACACGTACACGTACACGT ACACGTACACGTACACGTACACGTACACGTACACGTACACGTACACGTACACGTACACGTACACGTACA CGCATACGCATACGCATACGCATACGCATACGCATACGCATACGCATACGCATACGCATACGCATACGC ATACGCATACGCATACGCATACGCATACGCATACGCATACGCATACGCATACGCATACGCATACGCATA CGCATACGCATACGCATACGTACACGTACACGTATACGTA

� AT=l, AC=2, GT=3, GC=4 432221241241232232232243222322322322432223223223223223223223223223223 223223223223223223223223223223223224124124124124124124124124124124124 124124124124124124124124124124124124124124123223223123

� 412=GCATAC=5, 322=GTACAC=6 462125566646266646266666666666666666666655555555555555555555555555663123

Fig. 1-16 An example of replacement for purine-pyrimidine altering region of L. africana

(96LX79, clone 1).

(25)

>E. maximus (93EL25) clone 1

GCGTACACATACGCATATGCATACGTACACACGTACACACACGTACACACGTACACACACGTACACACACGC ACACGTACACACACGCACACGTACACACACGCACACGTACACACACGTACACGCACACGCACACGTACACAC ACGTACACGCACACGCACACGTACGTACACGTATACACACACGTACACGCACACGCACACGTACGTACACGT ATACACGCACGTACACGCACGTACACGTACACGCGTACGCATACGCATACGCATACGCATACGCATACGCAT ACGCATACGCATACGCATACGCATACGCATACGCATACGCATACGCATACGCATACGCATACGCATACGCAT ACGCATACGCATACGCATACGCATACGCATACGCATACGCATACGCATACGCATACGCATACGCATACGCAT ACGTATACGTATACGTA

� AT=1, AC=2, GT=3, GC=4

432212411412322232222322232222322224223222242232222422322223224224223222 232242242232322312222322422422323223122423224232232243241241241241241241 241241241241241241241241241241241241241241241241241241241241241241241241 23123123

� I 412=GCATAC=5

322232222322232222322224223222242232222422322223224224223222232242242232 322312222322422422323223122423224232232243255555412555555555555555555555 554123123123

Fig. 1-17 An example of replacement for purine-pyrimidine altering region of E. nlaxilllUS (93EL25, clone 1).

(26)

93EL50

9810141 9810161 9810104 9810143 9810163 9810102 clone 3 clone 4 clone 1 clone 2

96LX79

clone 1 clone 2 clone 3

A (4621255)

4621255 4621255 4621255 4621255 4621255 4621255 4621255 4621255

B (6n462)

66646266666-462666--46 66646266666-462666--46 66646266666-462666--46 66646266666-462666--46 6664626666664626612246 666462666666462666--46 66646266666-462666--46 66646266666-462666--46

c

(6n)

666666666666666666--- 6666666666666666--- 666666666666666666666666 66666666666666666--- 6666666666666666--- 666666666666666666---

46212532

1

66646266666-462666--46

�

666666666666666666--- 4621255

�

66646266666-462666--46

2

6666666666666666---

4621255

j

666462666---462---

�

666666666666666666666--- 4621255 666462666---462--- 66666666666666666--- 4621255 666462666---462666--46 66666666666666---

D E

(5n)

55555555555555555555555555--- 555555555555555555555555555--- 5555555555555555555555--- 5555555555555555555555--- 5555555555555555555555555555555---- 555555555555555555---

5555555555555555555555555--- 5555555555555555555555555555---

F (663123)

55555555555555555555555555---

�

⁶⁶³¹²³

555555555555555555555555555555555-- 663123 555555555555555555555555555555555555663123

Fig. 1-18 Alignment of purine-pyrimidine altering region containing repeat sequence of L. africana (93EL50 and 96LX79).

This region could ^be divided into 6 domains, ^A to F. AT=1, AC=2, GT=3, GC=4, 412=GCATAC=5, 322=GTACAC=6. The domain ^B consist of 6n462,

the domain ^C consist of 6n and the domain ^E consist of 5n.

(27)

93EL2S

>26-7

>26-9

>26-3,5

>26-1

>28-1

>28-5

>981016-7

>981016-8

>981016-0,-9

>981010-6,41,42

A (43225411)

B

(322(32222)n) c

((3

^or

4)2n)

43225411532223222232223222232222--- ---42232222422322224223222232242242232222322422422323223AXT 43225411532223222232223222232222--- ---42232222422322224223222232242242232222322422422323223AXT 43225411532223222232222322223222232222422322224223222242232222---32242242232222322422422323223AXT 43225411532223222232222--- 4223222242232222422322224223222232242242232222322422422323223AXT 43225411532223222232222--- 422322224223222242232222---32242242232222322422422323223AXT 43225411532223222232222--- 422322224223222242232222---32242242232222322422422323223AXT 43225411532223222232222--- 422322224223222242232222---32242242232222322422422323223AXT 43225411532223222232222--- 422322224213222242232222---32242242232222322422422323223AXT 43225411532223222232222--- 422322224223222242232222---32242242232222322422422323223AXT 43225411532223222232222--- 422322224223222242232222---32242242232222322422422323223AXT

D E

(Sn)

2222322422422323223AXT224232242322322432

1

555555555555555555555555555555---

�

^3123123

2222322422422323223AXT224232242322322432555555555555555555555555555--- 3123123 ---224232242322322432555555555555555555555555555555555555553123123 ---22423224232232243255555555555555555555555555555555555--

---224232242322322432555555555555555555555555555555555---- ---3143 ---22423424AXT22---322432555555555555555555555555555555555----

242423224232232243241C2--- 5555555555555555555555555555555---

---224232242322322432555555555555555555555555555555555---- 3123123 ---2242322423223224325555555555555555555555555555555555--- 3123123 ---2242322423223224325555555555555555555555555555555555--- 3123123

Fig. 1-19 Alignment of purine-pyrimidine altering region containing repeat sequence of E. maximus (93EL25).

This region could be divided into 5 domains, A to E. AT=l, AC=2, GT=3, GC=4, 412=GCATAC=5. The domain Band C consist of (3 or 4)2n and the domain

D consist of 5n.

(28)

A c

T A T A

A c T A G-C

G C C-G G c c-G

c A G-C c A

A- T A C

A -T c A A c

A-T c-G c-G C A

T -A

A c T-A A- T It-T

C-G A c T-A T--A

G -c C-G G- C G-C

c A G-c c A c A

A -T c A A c A c

A-T c- G c-G

T -A

A c T-A A-T k-T

C-G A c T-A T--A

G -C C-G G-c G-C

c A G-c

A -T c A c A c A

T -A A-T A c A c

c- G c-G

A c T-A

C-G A c A-T It-T

c-G T- A T--A

G -c

A T A C G-C AT A C G- C A C G-C Ac

a b _c d

Fig. 1-20 Putative secondary structures of the repeat motives, 5 (GCATAC) _{and 6} (GTACAC).

a: odd number times (7 times) of repeat for motif 5, b: even number times (8 times) of repeat for 5,

^c:

Odd number

times (7 times) of repeat for motif 6, d: even number times (8 times) of repeat for motif 6.

(29)

Newly synthesized

C G G T

T-A A-T T G

G-c C-G G T

T-A A-T T G

G -c C-G G T

T-A A-T T G

daughter strand G -c

�

C-G

H 5 I -CGTATGCGTATGCGTATG TATGCGTATG •

L 3 I -GCATACGCATACGCATAC ATACGCATACGCATACGCATACGCATACGCATAC-5 ^I

/

^G-C

Template strand Ac -Gc T A

A

Newly synthesized A T

T G

daughter strand G-C

�

^C-G

H 51-CGTATGCGTATGCGTATG TATGCGTATG ..

L 3 I -GC

�

TACGCATACGCATAC ATACGCATACGCATACGCATAC-5 I

/

^{G- C}

Template strand C- G ^·

A C

T- A A- T

C A

G- C c-G

A C

T A

B

Fig. 1-21 Models of addition and loss for the repeat motif 5, during replication of the H strand.

Arrows indicate the direction of DNA synthesis. Template strand is composed of 10 repeat units. (A) An example of addition of repeat units ( 15) of the daughter strand, when the length of the hairpin loop of the daughter strand is longer than that of the template strand. (B)

An example of loss of repeat units (8) of the daughter strand, when the length of the hairpin loop of the daughter strand is shorter than that

of the template strand.

(30)

1500bp-

800bp-

M 1 2 3 4 5 6 7 8 9 10 M

Fig. 1-22 An electrophoresis image of amplified fragments for 10 clones of repetitive region for L. africana (93EL50).

l-10: Amplified fragment for 10 clones; M: Molecular weight marker.

(31)

A

B

(n=l) Congo (n=l)

I I I

I

^\

\

Kenya I Tanzania (n=S)

South Africa (n=16)

··"'·

�

I �

-Kruger National Park

I

·. · (n=38)

I ' �-

^. ^\^_.Tembe Elaphant Park

��

⁽ⁿ⁼²⁴⁾

\� /

��-�

Addo Elephant National Park (n=97)

Fig. 2-1 Present distribution of L. africana in Africa and samples

collected for this study . The exported countries of samples from

^zoo

in Japan are

indicated in (A). The sampling locations of ivory and feces samples in South Africa are

indicated in (B). (A) is modified from Hall-Martin, 1992. (B) is modified from Laur en

and Beckff, 1978.

(32)

A

B

Knysna Forest

/Kruger National

Park

/Tembe Elephant

Park

""- d .

A

do Elephant NatiOnal

Park

Fig. 2-2 Distribution of L. africana in South Africa during ca. 1650 (A)

and during ca. ¹⁹¹⁰ (B). (modified from Hall-Martin, 1992)

(33)

A

c

10000 Kruger

B

²⁰⁰ ^{Ad do}

8000

150

6000

100 4000

2000 50

0

[ I I

I

�I I

I

I I I

I

I I I I

⁰

1900 1920 1940 1960 1980 1993 1930 1940 1950 1960 1970

100 Tern be

D

^5o ^Knysna

80 40

60 30

40 20

20 10

0 0

1940 1950 1960 1970 1980 1990 1993 1900 1920 1940

Fig. 2-3 Population trends of L. africana in three parks and Knysna forest.

(All the data used in this figure were cited from Hall-Martin, 1992. )

1960

1980 1990 1994

1980 1994

(34)

· breeding individuals in Sk."Ukuza :

75, 76, 77:00

0

· sampling point unknown : 43: 0

0

: L9

(ivory)

0 : LlO

(ivory)

• : LlO

(feces, fresh)

� : L14

(ivory)

.& : L14

(feces, fresh)

X

: L18

(ivory)

0 SOkm

Skukuza

Male lane

Fig. 2-4 Haplotype distribution in Kruger National Park.

(35)

- ��._---�----�--�---. ..

Tembe Elephant Park

2.5km

e :

LlO (fresh)

o :

LlO (dry)

.& :

L14 (fresh)

.6 :

L14 (dry)

'Y:

L16 (fresh)

v:

L16 (dry)

Fig. 2-5 Haplotype distribution in Tembe Elephant Park.

(36)

Addo Elephant National Park

6. 66 109ll.Q 1111120�6.

671131H 11slli t1711811906.• ( UQ121ill123�IID 0•00 68 124 126

ill ill

06..6. ••

� ^.TI 129130131000

:z.B

0 1S3 143 133

1� 144 1340

15 1

^A

:

^L14

(fresh)

.6.

_:L14

(dry)

•:

^Lll

(fresh) D:

^Lll

(dry)

l.Okm

Fig. 2-6 Haplotype distribution in Addo Elephant National Park.

(37)

0.040 0.030

I I I I I I I I I I I I I I I I I I Ll

0.020 0.010 0.000

111111111111111111 IIIII

Cluster

LI 41 ._ _____________

Ll

E. maximus

----�o

--L4

83

LS

98

4

35

43

L14 Lll .. _____ L18

luster

Ell

---E2 ..----El

... --E16

97

Fig. _2-7 Phylogenetic tree constructed by neighbor-joining method

using ₁₈ haplotypes for _L. africana and ₁₉ haplotypes for _E. maximus.

(38)

0.030 0.020 0.010 0.000

I

^I ^I

I

^I ^I

I

^I

42

L3

Ll

Cluster LI

L2

L4

62

LS

L6

L. africana

₉₅

L7

LS

94

L9

LlO

Cluster LII

Lll

L12

43

L18

61

Ll3

L14 Ll6 LlS Ll7 E14

Cluster EI 79 84

El

E2

37

E3

E4

35

ES

E7

E. maximus

³⁶

ES

Ell E9 ElO El2 E13 El6

Cluster Ell 99

E17

El8 El9 E20 E21

Fig. 2-8 Phylogenetic tree constructed by UPGMA method using 18

haplotypes for L. africana and 19 haplotypes for E. maximus.

(39)

IJO

fl

1 0 1o'^t

10�

� 10�

ut

su

0.01

\

.

IJ•

J. ��

Fig. 2-9 Network using 18 haplotypes for L. africana and 19 haplotypes

for E. maximus.

(40)

Appendix A-1 Feces, saliva

or

hair samples of L africaoa collected in zoological gardens in Japan.

Gb.�o. Hiplotype Locatbns of zoo Places or ongen

&;x

93EL24 L16 Shizuoka female

93FL31 LlO Aichi female

93FL32 L14 Aichi female

93EL33 l5 Aichi South Africa male

93El.Al LlO Gunma male

93ELA2 L15 Gunma male

93ELA3 LlO Gunma female

93El.A4 L15 Gunma female

93EL45 L15 Gunma female

93EL46 L15 Gunma female

93FL50 LlO Hyogo South Africa female 93EL51 L9 ' Hyogo South Africa female 93EL52 L9 Hyogo South Africa female 93EL53 LlO Hyogo South Africa female

93:EL54 LlO Hyogo male

93EL66 LlO Shizuoka female

93EL67 Ll3 Shizuoka female

93EL68 Ll3 Shizuoka female

93EL75

L4

Miyagi female

93EL76 L8 Miyagi South Africa male 93EL77 L14 Miyagi South Africa female 93EL78 LlO Hirosihima South Africa female 93PL79 L6 Hirosihima eastern Africa female 93EL80 L17 Hirosihima eas tern Africa female

93EL83 L9 Hokkaido female

93FL89 Ll3 Tokyo Kenya I Tanzania. female 93EL90 L14 Tokyo Kenya I Tanzania. female 93EL91 L14 Tokyo Kenya I Tanzania. male 93EL92 LlO Akita South Africa male 93EL93 LlO Akita South Africa female

93EL94 LlO Hokkaido female

93EL97 L9 Kumamoto female

93EL98 L9 Kumamoto female

93EL99 L14 Kumamoto male

93PL102 l5 Tamaguchi male

93EL106

IA

Osaka South Africa female 93EL107

L4

Osaka South Africa female

93EL111

l3

Oki nawa male

93PL134 L14 Yamaguchi female

93EL135

L4

Hiroshima female

93EL137 L14 Oit:a female

93EL139 L12 Oit:a male

93PL140 Ll3 Oit:a female

93EL141 L14 Oit:a female

93EL142

L8

Oita female

93EL143 LlO Oit:a female

(41)

Appendix A-2 Feces, saliva or hair samples of E. maximus collected in zoological gardens in japan.

Lab.No. Haplotype Locations of zoo Places of origen Sex

93EL21 E21 Nagano female

93EL23 E14 Nagasaki Thailand female

93EL25 E4 Kanagawa India female

93EL26 E7 Hyogo Thailand male

93EL27 E19 Shizuoka female

93EL29 E8 Yamaguchi Thailand female

93EL34 E14 Aich female

93EL35 E4 Tokyo Thailand male

93EL36 E13 Tokyo Thailand female

93EL37 E20 Tokyo India female

93EL38 E17 Tokyo India female

93EL39 E19 Osaka Thailand female

93EL40 El Osaka Thailand female

93EL56 El Kyoto Malysia female

93EL57 E14 Kyoto female

93EL58 E20 Hokkaido India female

93EL59 Ell Hokkaido Thailand female

93EL60 E20 Ishikawa female

93EL61 E13 Kanagawa Thailand female 93EL62 E20 Kanagawa Thailand female

93EL73 E20 Miyagi female

93EL74 E13 Miyagi female

93EL81 E17 Kanagawa India male

93EL82 E17 Kanagawa India female

93EL84 E18 Hokkaido female

93EL85,119 E18 Hyogo female

93EL86 E20 Hokkaido female

93EL87 E20 Hokkaido Thailand female

93EL88 E5 Tokyo Sri Lanka male

93EL% E13 Shizuoka India female 93EL100 E19 Ibaragi Myanmar female

93EL101 E7 Ibaragi Myanmar female

93EL108 E17 Osaka female

93EL112 E4 Fukuoka Sri Lanka female 93EL113 E7 Fukuoka Sri Lanka female

93EL115 E21 chiba Myanmar female

93EL116 E13 Kagoshima Thailand male

93EL117 E13 Kagoshima Thailand female

93EL118 E20 Osaka female

93EL120 E9 Aichi male

93EL121 E20 Aichi Thailand female

93EL124 E13 Ehime Thailand male

93EL128 E16 Fukuoka female

93EL129 E17 Fukuoka female

93EL144 E4 Tochigi Thailand female

(42)

Appendix A-3 Ivoriy samples of L. africana .

Lab. No.

94EL05 94EL10 94EL17 94EL18

Country Congo Gabon Zaire Zaire

Haplotype

L8

L2

L7

(43)

Appendix A-4 Feces samples collected from Pinnawala Orphanage Park in Sri Lnka.

Sample

No.

Haplot}'Jle Captured place

Sex

95EL 1 ElO Hambantota male

95EL2 E9 Hambantota male

95EL3 El3 Polonnaruwa male

95EL4 E4 Anuradbapura male 95EL5 E7 Hambantota female 95EL6 ElO Anuradba pura female

95EL 7 ElO Puttalama male

95EL 9 E4 born in the orphanage female 95 EL 10 ElO born in the orphanage female

95 EL 11 E9 Anuradbapura male

95 EL 12 El3 Polonnaruwa male

95 EL 13 ElO Anuradbapura male

95 EL 14 E7 Vavl.llliya female

95 EL 15 E4 Put tal am a male

95 EL 16 E4 Anuradbapura female

95EL 1 7 E4 Maiyangana male

95 EL 1 8 ElO Anuradbapura male

95 EL 1 9 ElO Matale female

95 EL 20 El2 Puttalama female

95 EL 21 E4 Puttalama male

95 EL 22 El3 born in the orphanage female

95 EL 24 ElO Anuradbapura female

95 EL 25 E4 Puttalama male

95 EL 26 E4 Anuradbapura male

95 EL 27 El3 Sevanagala male

95 EL 28 E4 Monaragala female

95 EL 29 E4 Mahaweli female

95 EL30 E3 Anuradbapura female

95EL31 ElO Kirindi watta female

95 EL32 E4 Anuradbapura female

95 EL33 El3 Polonnaruwa male

95 EL34 E4 born in the orphanage male

95 EL35 E4 born in the orphanage female

95EL36 ElO Polonnaruwa female

(44)

Appendix A-5 Ivory samples collected in Kru2er National Park (1997/5/2-3).

Gb.�o. Iiaplotypes

Tusk No.

�ex Age Area round regwn

^Remarks

95LX14 L14 ZA232-91 5f llyrs Crocodile Bridge SOUTH

No.lOS-7/5/91

95LX16 LlO ZA334-91 5f 14yrs Crocodile Bridge SOUTH

No.163-20/5/91

95LX17 L14 ZA351-91 5f 8yrs Lower Sabie SOUTH

No.171-21/5/91

95LX20 L14 ZA054-92 (j't 23yrs Crocodile Bridge SOUTH

No.11-1013/92

95LX25 L14 ZA324-92 Shingwedzi NORTH

Found dead.

95LX29 L18 ZA517-92 (j't 18yrs Malelane SOUTH

No.2-2119/92

95LX32 L14 ZA552-92 Nwanetsi CENTRAL

Found dead.

95LX33 L14 ZA568-92 (j't 33yrs Skukuza. SOUTH

No.7-21110/92

95LX42 LlO ZA157-94

Found dead-8/6194

95LX47 L14 ZA436-94 (j't 13yrs Crocodile Bridge SO liTH

No. E170L-1317194

95LX49 LlO ZA501-94 (j't 17yrs Malelane SO liTH

No. E2.11 L-2217194

95LX50 LlO ZA522-94 (j't 13yrs Pretoriuskop SO liTH

No E2.24R-2517194

95LX51 L14 ZA535-94 5f Lower Sabie SO liTH

Found dead.

95LX54 LlO ZA571-94 5f 26yrs Lower Sabie SOUTH

No E2.45L-2717194

95LX55 LlO ZA572-94 5f 26yrs Lower Sabie SO liTH

No. E2.45R-2717194

95LX56 LlO ZA613-94 (j't 25yrs Shangoni FAR NORTH

No.E245L-8t8194

95LX57 LlO ZA614-94 (j't 25yrs Shangoni FAR NORTH

No E2.45R-8/8194

95LX59 L14 ZA654-94 (j't 16yrs Malelane SO liTH

No E2.85L-18/8194

95LX60 L14 ZA655-94 8' 16yrs Malelane SO liTH

No E2.85R-18/8194

95LX61 Ll4 ZA664-94 Hou tboschrand CENTRAL

Found dead

95LX65 LlO ZA688-94 16yrs Malelane SO liTH

No. EL.-2219/94

95LX66 L14 ZA690-94 18yrs Malelane SOUTH

No. ill-2219/94

95LX67 L14 ZA716-94 Letaba NORTH

Found dead

95LX73 LlO ZAT14-94 Woodland s NORTH

Found dead

95LX75 L9 ZA784-94 Died at Skukuza SO liTH

Calf used for research

95LX76 L9 ZA785-94 Died at Skukuza SO liTH

Calf used for research.

95LX77 LlO ZA795-94 Died at Skukuza SO liTH

Calf used for research.

95LX78 LlO ZA814-94 Tshokwane CENTRAL

Found dead

95LX82 L14 ZA009-95 8' ^Letaba ^NORTH

Found dead

(45)

Appendix A-6 Feces samples collected in Kruger National Park (1997/4/26-29).

Sample No. Ha Condition Sampling Date Location in Kruger Note

96LX1 LlO Fresh 1997/4/26 3.8km from Skukuza Gate Male 96LX2 LlO Fresh 1997/4/26 800m fr001 Kl Male

96LX3 L10 Fresh 1997/4/26 5.7km Male

96LX4 LlO Fresh 1997/4/26 1Om from

junction

for Malelane Male

96LXS LlO Fresh 1997/4/26 SOm fromK4 Male

96LX6 L10 Fresh 1997/4/26 12km Male

96LX7 LlO Fresh 1997/4126 27km, near Kuaggas Pan Male

96LX8 Ll3 Fresh 1997/4/26 37km Male

(46)

Appendix A-7 Feces sam pies collected in Tern be Elephant Park ( ^1997/4/27 -28).

Sample No. Haplotype Condition Sampling Date Locatlon in Tembe Note 96LX9 L14 Fresh 1997/4/27 55.06km Male 96LX10 L16 Fresh 1997/4/27 55.06km Male 96LX11 L14 Fresh 1997/4/27 56km Male 96LX12 L16 Fresh 1997/4/27 57.5km Male 96LX13 L14 Fresh 1997/4/27 57.7km Male 96LX14 L14 Fresh 1997/4/27 59.3km Male 96LX15 L14 Little dry 1997/4/27 62.8km Male 96LX16 L10 Little dry 1997/4/27 66.1km Male

96LX17 L14 Fresh 1997/4/28 near the Gate Male, small size 96LX18 L14 Fresh 1997/4/28 300m Male

96LX19 L14 Fresh 1997/4/28 1km Male

96LX20 L14 Fresh 1997/4/28 1.2km Male 96LX21 L10 Fresh 1997/4/28 1.6km Male 96LX23 L14 Very dry 1997/4/28 Mahlasela Pan Male 96LX24 L10 Very dry 1997/4/28 Mahlasela Pan Male 96LX25 L14 Very dry 1997/4/28 Mahlasela Pan Male 96LX31 L14 Very dry 1997/4/28 Mahlasela Pan Male 96LX32 L16 Very dry 1997/4/28 Mahlasela Pan Male 96LX37 L16 Very dry 1997/4/28 Mahlasela Pan Male

96LX38 L16 Fresh 1997/4/28 4km Male, large size 96LX39 L10 Little dry 1997/4/28 5.7km Male

96LX40 L10 Fresh 1997/4/28 5.8km Male 96LX41 L16 Fresh 1997/4/28 5.9km Male 96LX46 L16 Fresh 1997/4/28 near camp Male

Table 1-1 Primers used for amplification and sequencing of the mitochondrial control region for the elephants.

アフリカゾウ(Loxodonta africana)とアジアゾウ (Elephas maximus)のミトコンドリアDNA解析 : コン トロール領域の遺伝的構造と個体群系統について

Table 1-1 Primers used for amplification and sequencing of the mitochondrial control region for the elephants.

Name of primers L15775.Lox**

Ll5926.ele*

LoxLl LoxL2 LoxL3 LoxlA LoxHl LoxH2 LoxH3 H651*

Sequences

* Primers modified referring Ko

her

al. (1989)

* * Primers modified referring Irwin

al . (1991)

Table 2-1 Number of samples of L. africana analyzed in this study.

DNA source Countries n Appendix

Zoo in Japan Feces, saliva or hair Kenya or Tanzania 5 A-1

South Africa 16 A-1

Unknown 29 A-1

Imported ivory Ivory Congo 1 A-3

Gabon 1 A-3

Zaire 2 A-3

Kruger National Park Ivory or feces South Africa 38 A-5, A6

Addo Elephant Park Feces South Africa 24 A-7

Tembe National Park Feces South Africa 97 A-8

total 213

Table 2-2 Number of samples of E. maxim

analyzed in this study.

DNA source Countries n Appendix Zoo in Japan Feces, saliva or hair Thailand 20 A�2

Malaysia 1 A-2

Myanmar 3 A-2

India 7 A-2

Sri Lanka 3 A-2

Unknol-Vn 18 A-2

Pinnawala Orphanage Park Feces Sri Lanka 34 A-4

total 86

Table 2-3 Haplotypes detected from 351 bp of the mitochondrial control region for 213 L.

africana and 86 E. maximus .

Numbers indicate the nucleotide positions from the beginning of the control region.

africana

Table 2-4 Haplotype distributions of L. africana .

Ll

lA 1.5 L6 L7 L8 L9 LlO Lll L12 L13 L14 LIS L16 L17 L18 Zcxl

5 1 1 2 1

16 2 1 2 7 1 3

29 1 2 2 1 3 7 1 3 6 2 1

1 1

1 1

(Zaire) 2 2

38 2 19 16 1

24 5 12 7

97 77 20

213

Table 2-5 Haplotype distributions of E. maximus .

Haplotypes

samples Regions (Countries) n El F2 F3 FA

E7 E8 E9 EIO Ell El2 E13 E14 El6 El7 E18 E19 E20 E21

in Japan Feces, saliva or hair Southeast Asia

(Thailand) 21 1 1 2 1 1 1 7 1 2 4

(Malaysia) 1 1

(Myanmar) 3 1 1 1

South Asia

(India) 7 1 1 3 2

(Sri Lanka) 3 1 1 1

Unknown 17 1 1 2 2 1 2 2 1

1

Feces (Sri Lanka) 34 1 13 2 2 10 1

total 86

Table 2-6 Haplotype diversity (h )and nucleotide diversity (jt) for each population.

(n (h ) (rr)

L. ajricana Zoo in Japan 50 13 0.87 2.14

South Africa 159 6 0.65 1.31

E. maximus Zoo in Japan 52 11 0.91 2.17

Pinnawala Orphanage Park 34 7 0.76 0.65

h = n ( 1-2: xi 2) I ( n -1)

n=n 2:x, x1 nu I (n -1)

M f I

M

800bp-

Fig. 1-1 An agarose gel electrophoresis of the extracted DNA from feces and ivory samples.

Lane f: feces sample; Lane i: ivory sample; Lane M: molecular weight marker.

mitochondrial control region

アフリカゾウ(Loxodonta africana)とアジアゾウ (Elephas maximus)のミトコンドリアDNA解析 : コントロール領域の遺伝的構造と個体群系統について

M f ^I

: ^{Lox Ll}

� _351bp -- _{Lox H2} ...

M fl f2 ^I

TGGATTAATGACTAATCAGCCCATGATCATAACATAACTGTGGTGTCATGCATTTGGTATCTT� � ^TAATTTTGG