Definitive identification of all species of the genus

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 2 Ocean Research Institute, The University of Tokyo Department of Zoology, Natural History Museum and Institute 1

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

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

Objective - 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. 4

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

Methods 12 S 16 S ND 6 ND 5 ND 1 ND 4 ND 2 4 L ND 3 ND CO 3 ATP Long PCR CR 6 ATP 8 DNA extraction cyb CO 1 CO 2 Nested PCR Direct sequencing 154 fish-versatile primers 4 Anguilla-versatile primers 78 species-specific primers 6

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

Results-2 General characters of mitochondrial DNA of Anguilla - Total length; 16, 550~16, 833 bp - Gene composition; 2 r. RNA genes, 22 t. RNA genes, 13 protein-coding genes, 1 control region - Base composition ; A>C>T>G - Gene order F T CR cyb ND 6 V 12 S P E 16 S ND 1 ND 5 mt. DNA H S L ND 4 ND 3 ND S 6 ATP CO 3 ATP 8 G K I M Q A N YC 4 L R L ND 2 W Identical to the mitochondrial DNA found in other vertebrates CO 1 CO 2 D 8

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

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

Discussion-3 Number of substitutions of 16 S jap bic ang ros mar bor die mos aus cel int meg neb obs rei mos jap bic ang ros mar bor die mos aus cel int meg neb obs rei 22 17 18 26 23 23 21 24 19 24 17 25 22 16 18 20 9 24 20 20 22 16 7 21 10 5 16 4 15 17 13 9 12 12 18 11 19 16 6 17 18 15 13 15 16 22 13 23 17 8 29 20 18 21 17 8 21 9 6 18 23 19 24 21 28 20 29 24 16 15 11 20 22 19 23 20 11 15 16 20 16 21 18 10 20 22 17 23 22 11 17 15 20 15 14 23 9 8 18 22 19 11 9 19 16 mos rei 17 17 15 17 6 7 8 9 17 19 16 17 11 12 10 11 11 12 14 15 17 19 11 12 18 20 15 17 Simple technical errors - - 0 - 1 1 11

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 - A. borneensis 12

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

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

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 - Definitive identification for all anguillid species will be possible. 15

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

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