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Definitive identification of all species of the genus Anguilla using the complete

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

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Definitive identification of all species of the genus Anguilla using the complete

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  1. Definitive identification of all species of the genus Anguilla using the complete mitochondrial genome Yuki Minegishi1, Jun Aoyama1, Jun G. Inoue1, Masaki Miya2, Mutsumi Nishida1, and Katsumi Tsukamoto1 1 Ocean Research Institute, The University of Tokyo 2 Department of Zoology, Natural History Museum and Institute 1

  2. Method Reference 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 PCR-RFLP PCR-RFLP PCR-RFLP RAPD PCR-RFLP Real time-PCR Introduction-1 Genetic methods for species identification 2

  3. 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 bicolor - A. malgumora (=A. borneensis) 3

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

  5. Materials 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) 1 18 17 4,14 5,6,7,10 13 9,11,15 2,16 12 3,8 Watanabe S. (2003) Taxonomy of the freshwater eels, genus Anguilla, Schrank 1798 5

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

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

  8. CR 12S cyb 16S ND6 ND1 ND5 ND2 ND4 ND4L ND3 CO1 CO3 ATP6 CO2 ATP8 Results-2 General characters of mitochondrial DNA of Anguilla - 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 F T V P Identical to the mitochondrial DNA found in other vertebrates E L mtDNA I M H S L Q A N W C Y R S G 8 K D

  9. 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

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

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

  12. 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

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

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

  15. Conclusion - We obtainedthe exactcomplete mitochondrial DNA sequences of all species of the genus Anguilla. -Wrong sequenceshave appeared in published data. ex.) A. mossambica, A. bicolor bicolor -Definitive identificationfor all anguillid species will be possible. 15

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

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

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