Definitive identification of all species

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
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2
Introduction-1
Genetic methods for species identification
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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)
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4
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.
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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)
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18
4,14
17
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
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Methods
DNA extraction
Long PCR
Nested PCR
Direct sequencing
154 fish-versatile primers 4 Anguilla-versatile
primers 78 species-specific primers
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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)
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Results-2
General characters of mitochondrial DNA of
Anguilla
- Total length 16,55016,833 bp
- Gene composition 2 rRNA genes, 22 tRNA
genes, 13 protein-coding genes, 1
control region
- Base composition ACTG
- 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
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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)
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Discussion-2
A. reinhardtii / A. mossambica (Bastrop et al.,
2000)
A. reinhardtii
A. mossambica
Bold line distribution of Anguilla (Ege, 1939)
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Discussion-3
Number of substitutions of 16S
jap
bic
mos
ang
bor
mar
ros
neb
die
cel
meg
int
aus
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 ob
s 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
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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
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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
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Discussion-6
Number of substitutions of cytochrome b
jap
bic
mos
ang
bor
mar
ros
neb
die
cel
meg
int
aus
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 ob
s 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)
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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.
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Most parsimonious tree in Lin et al. (2001) (12S,
cytochrome b)
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Maximum-likelihood tree from the present
data (total 15,243 sites, GTRIG))