2. Definitive identification of all species of the genus Anguilla using the complete mitochondrial genome Yuki Minegishi 1, Jun Aoyama 1, Jun G. Inoue 1, Masaki Miya 2, Mutsumi Nishida 1, and Katsumi Tsukamoto 1 1 Ocean Research Institute, The University of Tokyo 2 Department of Zoology, Natural History Museum and Institute 1

3. Genetic methods for species identification Takagi and Taniguchi (1995) Tagliavini et al. (1995) Wakao et al. (1999) Zhang et al. (1999) Aoyama et al. (2000) Lehmann et al. (2000) Lin et al. (2002) Watanabe et al. (submitted) RAPD PCR-RFLP RAPD PCR-RFLP Real time-PCR Reference Method Introduction-1 2

4. A. reinhardtii / A. mossambica (Bastrop et al., 2000) A. marmorata / A. nebulosa labiata (Aoyama et al., 2001) - A. bicolor bicolor - A. malgumora (=A. borneensis) Same sequences Different sequences Introduction-2 Aoyama et al., 2001 / Lin et al., 2001 3

5. - To obtain the exact complete mitochondrial DNA sequences of all species of the genus Anguilla - To collect the basic data for population genetics, molecular phylogeny, DNA taxonomy, etc. Objective 4

6. Materials 1 3,8 2,16 4,14 5,6,7,10 9,11,15 12 13 17 1. A. anguilla 2. A. australis australis 3. A. australis schmidtii 4. A. bicolor bicolor 5. A. bicolor pacifica 6. A. borneensis 7. A. celebesensis 8. A. dieffenbachii 9. A. interioris 10. A. marmorata 11. A. megastoma 12. A. mossambica 13. A. nebulosa labiata 14. A. nebulosa nebulosa 15. A. obscura 16. A. reinhardtii 17. A. rostrata 18. A. japonica (Inoue et al., 2001) Watanabe S. (2003) Taxonomy of the freshwater eels, genus Anguilla, Schrank 1798 18 5

7. DNA extraction Long PCR Nested PCR Direct sequencing 154 fish-versatile primers 4 Anguilla-versatile primers 78 species-specific primers Methods 12S 16S ND1 ND2 CO1 CO2 ATP8 ATP6 CO3 ND3 ND4L ND4 ND5 ND6 cyb CR 6

8. 12S ribosomal RNA 16S ribosomal RNA tRNA-Phe tRNA-Val tRNA-Lue(UUR) tRNA-Ile tRNA-GlntRNA-Met tRNA-Trp tRNA-Ala tRNA-Asn tRNA-CystRNA-Tyr NADH dehydrogenase subunit I Cytochrome c oxidase I Results-1 The complete mitochondrial DNA sequences of the genus Anguilla (A part of the sequence of A. anguilla) NADH dehydrogenase subunit II 7

9. - Total length; 16,550~16,833 bp - Gene composition; 2 rRNA genes, 22 tRNA genes, 13 protein-coding genes, 1 control region - Base composition; A>C>T>G - Gene order General characters of mitochondrial DNA of Anguilla Identical to the mitochondrial DNA found in other vertebrates Results-2 Q A N S E P T HSLHSL R G KD W IMIM L V F C Y 12S 16S ND1 ND2 CO1 CO2 ATP8 ATP6 CO3 ND3 ND4L ND4 ND5 ND6 cyb CR mtDNA 8

10. Why have specimens of two different species been found to have the same sequence? Discussion-1 A. reinhardtii / A. mossambica (Bastrop et al., 2000) A. marmorata / A. nebulosa labiata (Aoyama et al., 2001) 9

11. A. reinhardtii / A. mossambica (Bastrop et al., 2000) Discussion-2 Bold line: distribution of Anguilla (Ege, 1939) A. mossambica A. reinhardtii Distinguishable 10

12. Discussion-3 Number of substitutions of 16S bic ang ros mar bor die mos aus cel int meg neb obs rei mos rei - 22 17 18 26 23 21 24 19 24 17 25 22 16 17 - 18 20 9 24 20 22 16 7 21 10 5 16 15 17 - 15 16 20 16 21 18 10 11 - 4 15 17 13 9 12 18 11 19 16 6 7 - 23 19 24 21 28 20 29 24 16 17 - 29 20 18 21 17 8 21 9 6 18 17 19 - 17 18 15 13 15 16 22 13 23 17 8 9 - 9 19 18 20 - 15 11 20 22 19 23 20 11 12 - 17 15 20 15 14 15 - 22 19 11 12 - 23 9 8 18 17 19 - 20 22 17 23 22 11 12 - 16 15 17 -- japbicmosangbormarrosnebdiecelmegintausobsreimos 0101 -1 jap Simple technical errors 11

13. Discussion-4 Why have specimens of the same species been found to have greater sequence differences than expected? Aoyama et al. (2001) / Lin et al. (2001) - A. bicolor bicolor - A. borneensis 12

14. 12S 16S Cytochrome b A. bicolor bicolor 0/860 site 11/860 sites Discussion-5 Amplification of the mitochondrial pseudogene 40/1140 sites GCGTTAGCTT AAAACCCAAA GGACTTGGCG GTGCCTCAAA CCCACCTAGA AGCCCCTTG AAACAGAACA CAACCTTATT CAGGAGGACA AGGATCATAT AGCCCCTTG AAACAGAACA CAACCTTACT CAGGAGGACA AGGATCATAT CCATATTTGT CGAGACGTTA ACTACGGATG ACTAATCCGC AACCTACATG CCACATCTGC CGAGATGTTA ACTATGGATG ATTAATCCGT AACCTACATG Sequence differences 13

15. Discussion-6 Number of substitutions of cytochrome b Differences of 12S: 38/967 sites bic ang ros mar bor die mos aus cel int meg neb obs rei bor* - 98 105 101 76 96 81 100 98 75 84 79 91 66 84 jap - 107 100 73 103 85 100 102 81 68 95 76 72 82 79 bic - 85 108 99 104 106 91 103 mos - 48 99 95 79 101 91 113 101 117 105 101 97 ang - 79 94 88 98 87 109 98 94 91 bor - 90 84 96 91 74 49 82 61 67 58 56 mar - 96 99 78 97 91 104 102 118 100 98 99 100 ros - 68 71 57 neb - 81 65 70 79 89 79 87 76 81 die - 75 70 75 86 74 84 cel - 87 97 86 98 meg - 85 57 64 71 41 int - 95 93 97 98 107 86 99 aus - 79 65 obs - 74 rei 92 * A. borneensis (=A. malgumora) jap - Amplification of pseudogenes - Introgression - Cryptic species 14

16. Conclusion - We obtained the exact complete mitochondrial DNA sequences of all species of the genus Anguilla. - Wrong sequences have appeared in published data. ex.) A. mossambica, A. bicolor bicolor - Definitive identification for all anguillid species will be possible. 15

17. Most parsimonious tree in Lin et al. (2001) (12S, cytochrome b)

18. Maximum-likelihood tree from the present data (total 15,243 sites, GTR+I+G))