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

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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

Rhacophorus

100% are endemic

Agamidae

100% are endemic


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:

  • 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

  • 95% amphibians and reptiles species are now available in the tissue bank

  • Most were took pictures when they were alive


Sampling and photographs

Frozen tissues

DNA samples

650 – 670 bp COI sequences

2-G01


  • Roughly 100 species

  • > 80% endemic sp.

  • > 60% endemic ssp.

    Data provider: S.-H. Li


Colubridae

Elapidae

Viperidae

NJ tree, HKY

Typhlopidae


Gekkonidae

Lacertidae

Anguilidae

Scincidae

Agamidae

NJ tree, HKY


Microhylidae

Bufonidae

Rhacoporidae

Raniidae

NJ tree, HKY


Difficulties of BOL in Herps: the Extremely High Divergence in COI Sequences

  • Data provider: S.-H. Li


Between-species divergence of COI

p-distance


Sequence divergence within genus

Takydromus (lizards) only

Lizards

Snakes

Frogs


Generally speaking, divergence of congeners:

<

<

<<


High divergence between ancient and derived families

congeners

within family,

between genera

between families


Difficulties in barcoding Herps:

  • High divergence between species

  • No “really” university primers


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

  • Legless lizard

  • Anguidae

Ophisaurus harti

Ophisaurus formosensis


Synonym

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

=


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


Type location: Central Formosa (Taiwanfoo)

Collector: Mr. Holst

Type


Discriminate analysis

Northern pop.

Eastern pop.

-14

-16

-18

dim1

-20

-22

Western pop.

-24

Type

-26

10

12

14

16

18

dim2


T. viridipunctatus sp. nov

T. luyeanus sp. nov

T. formosanus

Reproductive isolation because of different secondary sex characters during breeding seasons


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


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


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!

  • High intra-specific divergence

  • No morphological difference

  • No behavioral difference

  • Sympatric distribution


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.


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

  • 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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