Definitive identification of all speciesof 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 Tokyo2 Department of Zoology, Natural History Museum and Institute

1

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

PCR-RFLP

PCR-RFLP

PCR-RFLP

RAPD

PCR-RFLP

Real time-PCR

Reference Method

Introduction-1

2

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

- To obtain the exact complete mitochondrial DNA sequencesof all species of the genus Anguilla

- To collect the basic data forpopulation genetics, molecularphylogeny, DNA taxonomy, etc.

Objective

4

Materials

1

3,82,16

4,145,6,7,10

9,11,1512

13

17

1. A. anguilla2. A. australis australis3. A. australis schmidtii4. A. bicolor bicolor5. A. bicolor pacifica6. A. borneensis7. A. celebesensis8. A. dieffenbachii9. A. interioris10. A. marmorata11. A. megastoma12. A. mossambica13. A. nebulosa labiata14. A. nebulosa nebulosa15. A. obscura16. A. reinhardtii17. A. rostrata18. A. japonica (Inoue et al., 2001)Watanabe S. (2003)

Taxonomy ofthe freshwater eels, genus Anguilla, Schrank 1798

18

5

DNA extraction

Long PCR

Nested PCR

Direct sequencing

154 fish-versatile primers4 Anguilla-versatile primers78 species-specific primers

Methods

12S

16S

ND1

ND2

CO1

CO2ATP

8

ATP

6CO3ND3

ND4LND4

ND5

ND6

cybCR

6

12S ribosomal RNA

16S ribosomal RNA

tRNA-Phe

tRNA-Val

tRNA-Lue(UUR)

tRNA-IletRNA-Gln tRNA-Met

tRNA-Trp tRNA-AlatRNA-AsntRNA-Cys tRNA-Tyr

NADH dehydrogenase subunit I

Cytochrome c oxidase I

Results-1The complete mitochondrial DNA sequences of the genus Anguilla

(A part of the sequence of A. anguilla)

NADH dehydrogenase subunit II

7

- 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 DNAfound in other vertebrates

Results-2

Q

AN

S

EP

T

HSL

R

G

K D

W

IM

L

VF

CY

12S

16S

ND1

ND2

CO1

CO2

ATP

8

ATP

6CO3ND3

ND4L

ND4

ND5

ND6

cybCR

mtDNA

8

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

A. reinhardtii / A. mossambica (Bastrop et al., 2000)

Discussion-2

Bold line: distribution of Anguilla (Ege, 1939)

A. mossambica

A. reinhardtiiDistinguishable

10

Discussion-3Number of substitutions of 16S

bicangrosmarbordiemosauscelintmegnebobsrei

mosrei

-2217182623232124192417252216

1717

-1820

92420202216

72110

516

1517

-15162016211810

1011

-4

151713

9121218111916

6

67

-231924212820292416

1617

-2920182117

821

96

18

1719

-17181513151622132317

8

89

-9

19

1820

-1511202219232011

1112

-1715201514

1415

-221911

1112

-23

98

18

1719

-202217232211

1112

-16

1517

- -

jap bic mosang bormarros nebdie cel megintaus obs rei mos

01

-

1

jap

Simple technical errors

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 bicolor- A. borneensis

12

12S

16S

Cytochrome b

A. bicolor bicolor

0/860 site

11/860 sites

Discussion-5

Amplification ofthe mitochondrialpseudogene

40/1140 sites

GCGTTAGCTT AAAACCCAAA GGACTTGGCG GTGCCTCAAA CCCACCTAGAGCGTTAGCTT AAAACCCAAA GGACTTGGCG GTGCCTCAAA CCCACCTAGA

AGCCCCTTG AAACAGAACA CAACCTTATT CAGGAGGACA AGGATCATATAGCCCCTTG AAACAGAACA CAACCTTACT CAGGAGGACA AGGATCATAT

CCATATTTGT CGAGACGTTA ACTACGGATG ACTAATCCGC AACCTACATGCCACATCTGC CGAGATGTTA ACTATGGATG ATTAATCCGT AACCTACATG

Sequencedifferences

13

Discussion-6

Number of substitutions of cytochrome b

Differences of 12S:38/967 sites

bicangrosmarbordiemosauscelintmegnebobsrei

bor*

-98

105101769681

10098758484799166

84

jap

-10710073

10385

100102816895767282

79

bic

-85

10899

10410410691

103

mos

-48999579

10191

113101117105101101

97

ang

-7994889887

109989491

bor

-90849691744982616758

56

mar

-9699789791

1041021181009899

100

ros

-6871

57

neb

-8165707989798776

81

die

-7570758674

84

cel

-879786

98

meg

-85576471

41

int

-95939798

10786

99

aus

-79

65

obs

-

74

rei

92* A. borneensis (=A. malgumora)

jap

- Amplification of pseudogenes- Introgression- Cryptic species

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Conclusion

- We obtained the exact completemitochondrial DNA sequencesof all species of the genus Anguilla.

- Wrong sequences have appearedin published data.ex.) A. mossambica, A. bicolor bicolor

- Definitive identification forall anguillid species will be possible.

15

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

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