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Construction of A DNA Barcode System for Amphibians and Reptiles in Taiwan. SI-MIN LIN, H.-Y. Chao, H.-Y. Ho, Y.-R. Chen, W.-L. Kao Dept. of Life Science, Chinese Culture University. High percentage of endemic species . Lacertidae 86\% are endemic. Timaliidae 94\% are endemic.

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construction of a dna barcode system for amphibians and reptiles in taiwan

Construction of A DNA Barcode System for Amphibians and Reptiles in Taiwan

SI-MIN LIN,

H.-Y. Chao, H.-Y. Ho,

Y.-R. Chen, W.-L. Kao

Dept. of Life Science, Chinese Culture University

high percentage of endemic species
High percentage of endemic species

Lacertidae

86% are endemic

Timaliidae

94% are endemic

Rhacophorus

100% are endemic

Agamidae

100% are endemic

cryobanking of genetic materials of animals in taiwan since 2004
Cryobanking of Genetic Materials of Animals in Taiwan since 2004
  • Containing the following subprojects:
    • Fishes: K.-T. Shao
    • Mammals and birds: S.-H. Li and C.-T. Yao
    • Amphibians and reptiles: K.-Y. Lue and S.-M. Lin
    • Invertebrates: NMNS, Taichung
    • Barcode of Herps and birds: S.-M. Lin and S.-H. Li
  • Supported by
priority
Priority:
  • 1. endemic species and subspecies
  • 2. endangered and threatened species
  • 3. species with potential risks of competing with alien species or nonnative populations
  • 4. Herps:
    • Roughly 100 reptiles and 40 amphibian species
    • the first 2 taxa which could be accomplished within the shortest period
the cryobanking facility in the taipei zoo
The cryobanking facility in the Taipei Zoo
  • 95% amphibians and reptiles species are now available in the tissue bank
  • Most were took pictures when they were alive
slide10
Sampling and photographs

Frozen tissues

DNA samples

650 – 670 bp COI sequences

2-G01

slide11
Roughly 100 species
  • > 80% endemic sp.
  • > 60% endemic ssp.

Data provider: S.-H. Li

slide12
Colubridae

Elapidae

Viperidae

NJ tree, HKY

Typhlopidae

slide13
Gekkonidae

Lacertidae

Anguilidae

Scincidae

Agamidae

NJ tree, HKY

slide14
Microhylidae

Bufonidae

Rhacoporidae

Raniidae

NJ tree, HKY

difficulties of bol in herps the extremely high divergence in coi sequences
Difficulties of BOL in Herps: the Extremely High Divergence in COI Sequences
  • Data provider: S.-H. Li
sequence divergence within genus
Sequence divergence within genus

Takydromus (lizards) only

Lizards

Snakes

Frogs

slide19
High divergence between ancient and derived families

congeners

within family,

between genera

between families

difficulties in barcoding herps
Difficulties in barcoding Herps:
  • High divergence between species
  • No “really” university primers
what did we learn about herp biodiversity from herp bol
What did we learn about herp biodiversity from “herp-BOL”?
  • Case 1:
    • The “glass lizard”Ophisaurus
    • Polymorphism leads to synonym
  • Case 2:
    • The “grass lizard”Takydromus
    • Cryptic species
  • Case 3:
    • The skink Sphenomorphus
    • High intra-specific divergence
case 1
Case 1
  • Legless lizard
  • Anguidae

Ophisaurus harti

Ophisaurus formosensis

slide25
Synonym

Morphological polymorphism has misled for 70 years until DNA barcodes helped to solve the problem

=

case 2 takydromus formosanus
Case 2Takydromus formosanus

100

Northern pop.

n = 105

50

Eastern pop.

n = 80

78

100

T. heuehshanensis

n = 38

100

Western pop.

n = 100

100

0.02

slide28
Discriminate analysis

Northern pop.

Eastern pop.

-14

-16

-18

dim1

-20

-22

Western pop.

-24

Type

-26

10

12

14

16

18

dim2

slide29
T. viridipunctatus sp. nov

T. luyeanus sp. nov

T. formosanus

Reproductive isolation because of different secondary sex characters during breeding seasons

slide30
1

SEX102.seq

SEX101.seq

SEX105.seq

SEX107.seq

SEX103.seq

SEX104.seq

0212

0208

0217

0205

0213

R39.txt

R38.txt

R37.txt

0215

SEX106.seq

0201

0405-COI

0402-COI

0406-COI

0403-COI

0404-COI

0501

0502

0401-COI

0316-COI

0330-COI

0325-COI

0326-COI

0323-COI

0304-COI

SEXS0104.TXT

SEXS0105.TXT

SEXS0107.TXT

SEXS0102.TXT

SEXS0106.TXT

SEXS0101.TXT

Takydromus sexlineatus

species complex

Hongkong

100

100

100

Vietnam

86

94

96

100

100

100

100

100

100

Indonesia 1

100

100

100

70

91

91

100

100

100

Indonesia 2

ML tree, Tamura-Nei

case 3
Sphenomorphus taiwanensis

A high-mountain skink with poor dispersal ability

D

D

A*

A*

A*

100

E

B*

B*

A*

A*

B*

70

AD*

A*

D

AD*

AD*

J

J

J

J

J

J

J

J

100

I

I

I

I

100

J

H

I

HI

100

I

I

H

F

F

F

99

F

F

F

96

I

G

E

E

C*

C*

E

E

S. incognitus

S. indicus

0.01 substitutions/site

COI sequence

MP tree

Case 3

1

2

3

4

5

7

6

100

8

9

slide33
p-distance between lineages

1

2

3

4

5

6

7

8

2

Within-species divergence is possible to exceed 10% in reptiles!

3

4

5

6

7

8

9

no evidence to separate them into different species
No evidence to separate them into different species!
  • High intra-specific divergence
  • No morphological difference
  • No behavioral difference
  • Sympatric distribution
conclusion
Conclusion
  • Barcoding of all amphibian and reptile species in Taiwan will be accomplished within this year.
  • Almost impossible to develop “universal primers” for all taxa.
  • Difficult to amplify all species within a short period.
conclusion1
Conclusion
  • Effective tool in biodiversity evaluation.
  • Species delimitation - as the always problem in amphibians and reptiles.
  • Taxonomists and evolutionists are needed to involve in this issue before barcode could be used.
  • Call for international cooperation on

“Herp-BOL”, which is still not organized.

acknowledgements
Acknowledgements
  • a
  • Dr. Kwang-Tsao Shao
  • Dr. Shou-Hsien Li
  • Dr. Kuang-Yang Lue
  • Y.-J. Liang
  • K.-Y. Chung
  • W.-L. Kao
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