1. Molecular Phylogeography of the Japanese Weasel Mustela itatsi and the Siberean Weasel Mustela sibrica, revealed by complete mitochondiral DNA sequences Mohammed A. Shalabi (Hokkaido Univ.), S. Watanabe (Seian Univ.), Y. Kaneko (Tokyo Univ. of Agr. and Tech.), K. Yamazaki (Ibaraki Nat. M.), A.V. Abramov (Rus. Acad. of Sci.), P. A. Kosintsev (Rus. Acad. of Sci.), L-K Lin (Tunghai Univ., Taiwan) , S-H. Han (Nat. Inst. of Bio. Resources , Korea) , R. Masuda (Hokkaido Univ.) M

2. How Japan`s endemic mammals were evolved?

3. Speciation within continent
Introduction 1 of 5
Scenario 1
Species A
Speciation within continent
Species B
Migration
Species B

4. Introduction 2 of 5
Scenario 2
Species A
Migration to Northern Japan
Species A
Isolation, Land separation
Species A
Species B
Species A
Migration to Southern Japan
49 species of Japanese mammals (42%) follow either Sc. 1 or Sc.2 (Endemic)

5. Mustela sibrica (Species A Pattern) Mustela itatsi (Species B pattern)
Introduction 3 of 5
Target Species
Mustela sibrica (Species A Pattern)
H.Sasaki, (Sasaki, 2009), wild mammals of Japan
Mustela itatsi (Species B pattern)
Morioka Zoological Park, Masuda and Wantabe, 2009, Wild mammals of Japan)

6. Evidences for setting M.itatsi as a separate species from M.sibrica
Introduction 4 of 5
Evidences for setting M.itatsi as a separate species from M.sibrica
1- Ratio of tail length to head and body length T/HBL (Imaizumi,1960; Masuda et al. 2012).
2- Coat colour (Masuda and Wantabe 2009).
3- Seven skull measurments and several skull qualitative characters (Abramov 2000b).
4- Allometric skull differences (Suzuki et al. 2011).
5- MtDNA cytochrome b (Masuda & Yoshida, 1994a; Kurose, Abramov & Masuda, 2000a) and 12S rRNA (Kurose, Abramov & Masuda, 2008); large genetic distance between M.itatsi and M.sibrica.
6- Divergence time calculated;
A- 1.6–1.7 mya based on partial cytochrome b sequence differences (Masuda & Yoshida 1994b).
B- 1.7 mya from the whole sequence of cytochrome b data and 2.4 mya from nuclear (IRBP) data (Sato et al. 2003).
C mya using partial D-loop sequences (Masuda et al. 2012).

7. Tsushima, Russia and Korea
Introduction 5 of 5
Tsushima, Russia and Korea
Honshu Clade
Taiwan
Shikoku-Kyushu Clade
Clades of M.itatsi and M.sibrica, (Masuda et al. 2012)

8. Objectives
Determination of the sequences of the whole mitochondrial genome for both Mustela itatsi and Mustela sibrica

9. Materials and Methods 1 of 3
Russia (10)
China (1) (Yu et. al. 2011)
Aomori (1)
Morioka (1)
Iwate (2)
Ishikawa (1)
Gifu (1)
Okayama (1)
Ibaraki (4)
Korea (1)
Tshushima (4)
Nara (2)
Kita Kyushu (1)
Kochi (3)
Soo (2)
Yakushima Is. (4)
Wakayama (1)
Tanegashima Is. (2)
M.itatsi: 26 samples M.sibrica: 20 samples
Taiwan (5)

10. Primer Desgin:16 sets of primers of 1-1.8 kbp length were designed
Materials and Methods 2of 3
DNA Extration
Primer Desgin:16 sets of primers of kbp length were designed
PCR Amplification
Agarose Gel Electrophoresis of PCR products
Purification of PCR products
Sequencing by ABI 3730 DNA Analyzer
Sequences Alignment
Phylogenetic Analysis

11. Phylogenetic Analysis
Materials and Methods 3 of 3
Phylogenetic Analysis
A total of about bp were used;12 protein coding genes, 2 ribosomal RNA genes, and control region
The followings were excluded;
A- ND6 protein coding gene.
B- Ambigous sequences.
C- Insertions and deletions.
E- VTRs of D-loop.
F- tRNA sites.
Model Test; Kakusan 4 (Tanabe 2011).
Phylogenetic trees were reconstructed by maximum-likelihood (ML), using Akaike Information Criterion (AIC).
Maximum likelihood tree were reconstructed by Treefinder version March 2011 (Jobb et al. 2004).

12. Mustela itatsi Mustela sibrica
Results 1 of 2
Maximum likelihood Phylogenetic tree 83
East Honshu
Clade 1 Honshu
North Honshu
100
West central Honshu
Mustela itatsi
100
Kochi
Clade 2 Shikoku, Kyushu & Is.
Tanegashima Is.
100
Yakushima Is.
Kyushu
Clade 1 Russia & Tsushima
Russia
100
100
Tsushima
Clade 2 Korea, Taiwan & China
Mustela sibrica 98

13. Nucleotide Diversity (Pi)
Sample No.
Haplotype No.
Haplotype Diversity
(SD)
Polymorphic sites
Nucleotide Diversity (Pi)
M.itatsi
All Clades 26 19
0.972
(0.018)
264
( )
Clade 1 14 10
0.945
(0.045) 72
( )
Clade 2 12 9
0.939
(0.058) 93
( )
M.sibrica 20 15
0.0958
(0.033)
202
( ) 11
0.934
(0.061) 52
( ) 6 4
0.867
(0.129) 39
( )
Results 2 of 2

14. Migration History of M.itatsi Shikoku and Kyushu Colonization
Discussion 1 of 2
Honshu Clade
Shikoku and Kyushu Colonization
Scenario 1: A- Within continent speciation. B- Single migration event to Japan Is. C- Within Japan subsequent Migration.

15. Russia
Discussion 2 of 2
Clades of M.sibrica
China, Korea
Relict population of Tsushima from continental land
Land-bridge Hypothesis of Taiwanese M.sibrica

16. Summary
Mustela itatsi shows higher genetic differentiation than Mustela sibrica, either on the species or within-clades scale.
Geographical clustering of M.itatsi sub-clades.
Relict Tsushima population of M.sibrica.

17. Thank You