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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 . 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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  1. 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

  2. High percentage of endemic species Lacertidae 86% are endemic Timaliidae 94% are endemic Rhacophorus 100% are endemic Agamidae 100% are endemic

  3. 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

  4. 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

  5. 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

  6. Sampling and photographs Frozen tissues DNA samples 650 – 670 bp COI sequences 2-G01

  7. Roughly 100 species • > 80% endemic sp. • > 60% endemic ssp. Data provider: S.-H. Li

  8. Colubridae Elapidae Viperidae NJ tree, HKY Typhlopidae

  9. Gekkonidae Lacertidae Anguilidae Scincidae Agamidae NJ tree, HKY

  10. Microhylidae Bufonidae Rhacoporidae Raniidae NJ tree, HKY

  11. Difficulties of BOL in Herps: the Extremely High Divergence in COI Sequences • Data provider: S.-H. Li

  12. Between-species divergence of COI p-distance

  13. Sequence divergence within genus Takydromus (lizards) only Lizards Snakes Frogs

  14. Generally speaking, divergence of congeners: < < <<

  15. High divergence between ancient and derived families congeners within family, between genera between families

  16. Difficulties in barcoding Herps: • High divergence between species • No “really” university primers

  17. 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

  18. Case 1 • Legless lizard • Anguidae Ophisaurus harti Ophisaurus formosensis

  19. Synonym Morphological polymorphism has misled for 70 years until DNA barcodes helped to solve the problem =

  20. 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

  21. Type location: Central Formosa (Taiwanfoo) Collector: Mr. Holst Type

  22. Discriminate analysis Northern pop. Eastern pop. -14 -16 -18 dim1 -20 -22 Western pop. -24 Type -26 10 12 14 16 18 dim2

  23. T. viridipunctatus sp. nov T. luyeanus sp. nov T. formosanus Reproductive isolation because of different secondary sex characters during breeding seasons

  24. 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

  25. 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＊ Ｄ 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

  26. 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

  27. No evidence to separate them into different species! • High intra-specific divergence • No morphological difference • No behavioral difference • Sympatric distribution

  28. 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.

  29. 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.

  30. 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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