臺灣博碩士論文加值系統

目前臺灣陸生動物之族群遺傳結構研究多針對脊椎動物，且大多數研究皆顯示陸生物種有東西部分群之現象。這些研究結果顯示臺灣陸生脊椎動物間類似的族群分化模式，可能由下列因素所造成：（一）中央山脈隆起產生之隔離效應：將臺灣的物種區隔成中央山脈東西部兩大族群，族群間不易進行基因交流；（二）更新世冰河期的影響：海平面降低之後，臺灣與大陸相連使生物可以進行大規模遷移。但是，臺灣陸生節肢動物之族群分化模式為何，是否與脊椎動物相似，尚無人加以討論。本研究利用廣泛分佈於東亞地區之人面蜘蛛（Nephila maculata（Fabricius 1793）），以粒線體核酸之細胞色素氧化脢I部分序列，探討臺灣及鄰近地區人面蜘蛛族群之遺傳結構及其與地理分佈之關係。樣本收集自琉球、臺灣北、中、南、東、主要離島及中國大陸東南，共24個族群189個個體，以步足肌肉之粒線體DNA增幅出617 bp之COI序列進行分析，並以同屬之Nephila clavata Koch 1877及Nephila antipodiana (Walckenaer 1841)作為外群。根據目前已獲得之序列，利用Mega 和PAUP軟體以距離法（neighbor-joining, NJ method）及檢約法（maximum parsimony, MP method）所建構之系統發育樹（NJ tree之bootstrap values為99 %，而MP tree 為100 %）顯示，臺灣及離島地區人面蜘蛛之基因型（haplotype）可分成兩個主群（lineage）。第一群：包含23個族群的大部分個體；第二群：包含中國大陸廣東、廣西、雲南及臺灣西部火炎山、騎龍、東北部雙連埤族群的部分個體；第三群：則僅有臺灣東北部雙連埤族群之三隻個體。由於臺灣全島的人面蜘蛛都有EA基因型之分佈，顯示臺灣地區人面蜘蛛並無東西族群顯著分化的現象，不像其他陸生脊椎動物般受中央山脈的地理阻隔。中國大陸東南部與臺灣地區皆有EA基因型的個體，因此臺灣人面蜘蛛的祖先族群可能來自於大陸。另外，系統發育樹上分群的結果及各基因型之分佈模式顯示，現生臺灣地區人面蜘蛛族群可能多次由中國大陸遷入。

Abstract
Currently, most phylogeographic studies of terrestrial animals in Taiwan focus on vertebrates. Results from most studies showed that terrestrial vertebrates could be divided into eastern and western groups according to their population genetic structures. The common phylogeographic pattern exhibited by so many taxa was suggested to result from the upheaval of the Central Mountain Ridge, which led to isolation and differentiation of eastern and western populations. In this study, the widely-distributed giant wood spider Nephila maculata (Fabricius 1793) is used to investigate if population differentiation pattern of Taiwanese terrestrial arthropods resembles that of terrestrial vertebrates. Mitochondrial cytochrome oxidase I (COI) is used as genetic marker and its partial sequence is determined in 189 specimens collected from 24 localities in Ryukyu Islands, Taiwan and China. The 617 bases of COI gene are successfully amplified from DNA extracted from leg muscle and 11 haplotypes are identified from all specimens examined. Phylogenetic trees constructed by neighbor-joining (NJ) and maximum parsimony (MP) methods using Nephila clavata Koch 1877 and Nephila antipodiana (Walckenaer 1841) as the outgroups indicate that N. maculata populations can be separated into two major lineages, group A and group B, with high bootstrap values (99 % in NJ tree and 100 % in MP tree). Group A consists of most specimens from twenty-three localities. Group B consists of specimens from southeastern China and northwestern Taiwan. Another minor lineage is group C, which consists of 3 specimens from northeastern Taiwan. The wide distribution of the EA haplotype in western and eastern Taiwan indicats that the population differentiation pattern of N. maculata is different from those of terrestrial vertebrates examined so far. Central Mountain Ridge does not seem to be a major geographic barrier to N. maculata. The cooccurrence of specimens with EA haplotype in Taiwan and China suggests that some of the present populations in Taiwan might have splited from an ancestral population in mainland. In addition, judging from phylogenetic trees and the distribution patterns of haplotypes, entry of N. maculata from China to Taiwan might have occurred several times.

Contents
Chinese abstract….………………………….…………………………………….I
English abstract……………..…………………………………………………....III
Introduction
1. A review of phylogeographic studies of terrestrial animals in Taiwan…..….…1
2. The application of mitochondrial DNA for phylogeographic studies………….4
Materials and methods
1. Sampling localities……………………………………………………………..6
2. DNA extraction, amplification, and sequencing……………………………….7
3. Sequence alignment and phylogenetic analyses………………………………..8
4. Population genetic analysis………………………………….………………….9
Results
1. Divergence of the mitochondrial cytochrome oxidase I partial sequence…….………………………………………………………………...11
2. Characterization of the mitochondrial cytochrome oxidase I partial sequence in N. maculata.………………………………………………………………..12
3. Molecular phylogeny based on mitochondrial cytochrome oxidase I partial sequence…………………………………………………………………...… 13
4. Haplotype diversity and sequence diversity within and among regions………..….13
Discussion
1. Population genetic structure of N. maculata in Taiwan…….……………….…15
2. The Origin of N. maculata in Taiwan…………………………………………..20
References………………………………………………………………………...24
Table contents……………………………………………………………………..32
Figure contents…………………………………………………………………….33
Appendix contents………………………………………………………………..47
Author’s information…………………………………………………………….67
Table contents
Table 1. A review of population genetic studies of terrestrial animals
in Taiwan………………………………………………………………..34
Table 2. Specimen profiles of Nephila maculata examined in this study…………35
Table 3. Sequence variation of the mitochondrial cytochrome oxidase I partial sequence in Nephila maculata…..………………………………………37
Table 4. Pairwise comparison of mitochondrial cytochrome oxidase I partial sequences between 11 haplotypes………………………………………..38
Table 5. Sample size, haplotype diversity (h) and nucleotide diversity (π) of Nephila maculata in six regions of Taiwan……………………………..39
Table 6. The results of AMOVA test examining distribution of genetic variance among 14 populations of Nephila maculata from six regions of Taiwan……………………………………………………………….….39
Table 7. Pairwise FST and Nfm estimates between geographical regions of Nephila maculata based on sequence variation.……………………………………40
Table 8. Interpreting haplotype and nucleotide diversities for marine fishes……..40
Table 9. The comparison of haplotype and nucleotide diversities of three taxa in Taiwan………………………………………………………………….…41
Figure contents
Figure 1. Sampling localities of Nephila maculata examined in this study………………………….…………………………….……………42
Figure 2. Transitional plotted against genetic distance testing the occurrence of substitution saturation in partial COI gene……………………………..43
Figure 3. Phylogenetic relationships constructed by (a) neighbor-joining (NJ) and (b) maximum parsimony (MP) methods based on the mitochondrial cytochrome oxidase I partial sequence of Nephila maculata………………………………… ………………….…44
Figure 4. Relationship between genetic distance and geographic distance based on pair-wise comparisons between haplotypes……………………….…45
Figure 5. Geographical positions of sampling sites and distribution of mtDNA haplotypes in Taiwan……………………………………………………46

References
Avise, J. C. 1994. Molecular Markers, Natural History and Evolution. Chapman & Hall, Inc. New York.
Avise, J. C. 2000. Phylogeography: The History and Formation of Species. Harvard University Press. Cambridge, Massachusetts London, England.
Bond, J. E., Hedin, M. C., Ramirrez, M. G. & Opells, B. D. 2001. Deep molecular divergence in the absence of morphological and ecological change in the Californian coastal dune endemic trapdoor spider Aptostichus simus. Molecular Ecology. 10: 899-910.
Brader, L. 1972. Verspreiding van insekten door de lucht. Aerobiologie. Cent Landbouwpubl Landbouwdok, Wageningen, pp. 58-77.
Brown, W. M., George, M. & Wilson, A. C. 1979. Rapid evolution of animal mitochondrial DNA. Proceedings of the National Academy of Science of the USA. 76: 1967-1971.
Brown, W. M. 1985. The mitochondrial genome of animals. In: Molecular Evolutionary Genetics, Maclntyre, R. J. (ed.) New York: Plenum Press, pp.95-130.
Chen, S. H. 1996. The psechrid spiders of Taiwan (Araneae: Psechridae). Chinese Bioscience. 39: 23-56.
Chen, C. A. 1999. Analysis of scleractinian distribution in Taiwan indicating a pattern congruent with sea surface temperatures and currents: examples from Acropora and Faviidae Corals. Zoological Studies. 38: 119-129.
Chi, C. Y. 1999. Population phylogeny of Gekko hokouensis based on mitochondrial 12S rRNA and cytochrome b sequences. Master’s thesis, National Zhongshn University, Taipei, Taiwan. (in Chinese).
Coyle, F. A. 1983. Aerial dispersal by mygalomorph spiderlings (Araneae, Mygalomorphae). Journal of Arachnology. 11: 283-286.
Dean, D. A. & Sterling, W. L. 1985. Size and phenology of ballooning spiders in Texas. Journal of Arachnology. 13: 111-120.
Decae, A. E. 1987. Dispersal：Ballooning and Other Mechanisms. In: Ecophysiology of Spider (ed. Nentwig W). Springer-Verlag, Berlin.
Excoffier, L. Smouse, P. E. & Quattro, J. M. 1992. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA data. Genetics. 131:479-491.
Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 39: 783-791.
Felsenstein, J. 1993. PHYLIP (Phylogeny Inference Package). Seattle: Dept. of Genetics, University of Washington.
Folmer, O., Black, M., Hoeh, W., Lutz, R. & Vrijenhoek, R. 1994. DNA primers for amplification of mitochondrial cytochrome oxidase subunit from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology. 3: 294-299.
Funk, D. J., Futuyma, G. O. & Meyer, A. 1995. Mitochondrial DNA sequences and multiple data sets: a phylogenetic study of phylophagous beetles (Chrysomelidae: Ophraella). Molecular Biology and Evolution. 12:627-640.
Funk, D. J. 1999. Molecular systematics of cytochrome oxidase I and 16S from Neochlamisus leaf beetles and the importance of sampling. Molecular Biology and Evolution. 16: 67-82.
Gertsch, W. J. 1979. American Spiders, 2nd edu. Van Nostrand Reinhold, New York.
Grant, W. S. & Bowen, B. W. 1998. Shallow population histories in deep evolutionary lineages of marine fishes: Insights from sardines and anchovies and lessons for conservation. The Journal of Heredity. 89: 415-426.
Greenstone, M. H., Morgan, C. E., Hultsch, A. L., Farrow, R. A. & Dowse, J. E. 1987. Ballooning spiders in Missouri, USA and New South Wales, Australia: family landmass distribution. Journal of Arachnology. 15: 163-170.
Guiller, A., Coutellec-Vreto, M. A., Madec, L. & Deunff , J. 2001. Evolutionary history of the land snail Helix aspersa in the Western Mediterranean: preliminary results inferred from mitochondrial DNA sequences. Molecular Ecology. 10:81-87.
Hayes, J. P. & Harrison, R. G. 1992. Variation in mitochondrial DNA and the biogeography history of woodrats (Neotoma) of the eastern United States.
Systematic Biology. 41: 331-344.
Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41:95-98.
Hedin, M. C. 1997. Speciational history in a diverse clade of habitat-specialized
spiders (Araneae: Nesticidae: Nesticus): inferences from geographic-based
sampling. Evolution. 51: 1929-1945.
Hsu, Y. T. 1999. The evolutionary relationship of Chinese Bulbul (Pycnonotus sinensis) and Taiwan Bulbul (P. taivanus) revealed by their population genetic structure. Master’s thesis, National Taiwan University, Taipei, Taiwan. (in Chinese).
Hsu, F. H., Lin, F. J. & Lin. Y. S. 2000. Phylogeographic variation in mitochondrial DNA of formosan white-bellied rat Niviventer culturatus. Zoological Studies. 39:38-46.
Hsu, F. H., Lin, F. J. & Lin, Y. S. 2001. Phylogeographic structure of the formosan wood mouse, Apodemus semotus Thomas. Zoological Studies. 40: 91-102.
Hudson, R. R., Slatkin, M. & Maddison,W. P. 1992. Estimation of levels of gene flow from DNA sequence data. Genetics. 132: 583-589.
Irwin, D. M., Kocher, T. D. & Wilson, A. C. 1991. Evolution of the cytochrome b gene of mammals. Journal of Molecular Evolution. 32: 128-144.
Johnson, C. G. 1960. A basis for a general system of insect migration by flight. Nature. 186: 348-350.
Kayashima, I. 1962. About Taiwanese (Pscherus torvus) spiders. Atypus. 25: 9-11. (in Japanese)
Kimura, M. 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution. 16: 111-120.
Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. 2001. MEGA2: Molecular Evolutionary Genetics Analysis software, Bioinformatics.
Lee, C. L. 1964. The Taiwanese Spiders. Da-Jian Publishing Co. p84. (in Chinese)
Levi, H. W. 1982. The spider genera Pscherus and Fecenia (Araneae: Pschrus). Pac. Insects. 24: 114-138.
Lin, C. C. 1963. Quarternary in Taiwan. Reports on historiographical studies of Taiwan: Taiwan Wen Hsien. 14: 1-92. (in Chinese)
Lin, C. C. 1966. An outline of Taiwan’s quanternary geohistory with a special distribution of the relation between natural history and cultural history in Taiwan. Bulletin Department Archaeology Anthropology. 28: 7-44. (in Chinese).
Lin, C. H., Lee, C. N., Yang, C. C., Lue, B. W., Chen, S. H. & Fang, K. 1999. RAPD-estimated genetic relationship of Psechrid spiders (Araneae: Psechrus) in Taiwan. Biological Bulletin of National Taiwan Normal University. 34: 95-104.
Lin, J. Y. & Lin, L. K. 1983. The preliminary study of animal geography of mammal in Taiwan. Annual of Taiwan Museum. 26: 53-62.
Liu, K. C. 1995. Phylogeographic relationship of Japalura swinhonis based on analysis of mtDNA. Master’s thesis, National Zhongshn University, Taipei, Taiwan. (in Chinese).
Lue, K. Y. 1998. The study of evolution of terrestrial biology in Taiwan. Life Science News Letter, NSC. 12: 3-6.
Main, B. Y. 1982. Adaptations to arid habitats by mygalomorph spiders. In: Evolution of the Flora and Fauna of Arid Australia (eds Barker WR, Greensdale PJM). pp.273-284. SA Peacock Publishing Co., Frewville.
Moritz, C. C., Dowling, T. E. & Brown, W. M. 1987. Evolution of animal mitochondrial DNA: relevance for population biology and systematics. Annual Review of Ecology and Systematics. 18: 269-292.
Navajas, M., Lagnel, J., Gutierrez, J. & Boursot, P. 1998. Species-wide homogeneity of nuclear ribosomal ITS2 sequences in the spider mite Tetranychus urticae contrasts with extensive mitochondrial COI polymorphism. Heredity. 80:742-752.
Nei, M. 1987. Molecular Evolutionary Genetics. New York: Columbia University Press.
Nicholas, K. B., Nicholas, H. B. Jr. & Deerfied, D. W. II. 1997. GeneDoc: analysis and visualization of genetic variation. Embnew. News 4:14.
Page, R. D. M. & Holmes, E. C. 1998. Molecular evolution. Blackwell Science Ltd, Oxford.
Parker, P. G., Snow, A. A., Schug M. D., Booton, G. C. & Fuerst, P. A. 1998. What moleculaes can tell us about populations: choosing and using a molecular marker. Ecology. 79: 361-381.
Rozas, J. & Rozas, R. 1999. DNA SP version 3: an integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics. 15: 174-175.
Saitou, N. & Nei, M. 1987. The neighbor-joining method: A new method reconstructing phylogenetic trees. Molecular Biology and Evolution. 4: 406-425.
Sambrook, E., Fritsch, F. & Maniatis, T. 1989. Molecular Cloning: A Laboraatory Manual. 2nd ed, Cold Spring Harbor Laboratory, New York.
Schneider, S., David, R. & Excoffier, L. 2000. Arlequin ver. 2.000: A Software for Population Genetics Data Analysis. Genetics and Biometry Laboratory, University of Geneva, Switzerland.
Shen, C. F. 1996. The biogeography of Taiwan: 1. Background. Annual of Taiwan Museum. 39: 387-427.
Simon, C., Frati, F., Beckenbach, A., Crespi, B., Liu, H. & Flook, P. 1994. Evolution, weighting and phylogenetic utility of mitochondrial gene sequences and a compliation of conserved polymerase chain reaction primers. Annals of The Entomological Society of America. 87: 651-701.
Swofford, D. L. 1993. User Manual for PAUP Version 3.1: Phylogenetic Analysis Using Parsimony. Illinois Natural History Survey, Champaign, Illinois.
Thompson, J. D., Higgins, D. G. & Gibson, J. 1994. Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position- specific gap penalties and weight matrix choice. Nucleic Acids Research. 22: 4673-4680.
Toda, M., Nishida, M., Matsui, M., Lue, K. Y. & Ota, H. 1998. Genetic variation in the Indian rice frog Rana limocharis (Amphibia: anura) in Taiwan, as revealed by allozyme data. Herpetologica. 54: 73-82.
Tsukada, M. 1966. Late Pleistocene vegetation and climate in Taiwan. Proceedings of the National Academy of Science of the USA. 55: 543-548.
Tzeng, C. S. 1986. Distribution of the freshwater fishes of Taiwan. Journal of Taiwan Museum. 39: 127-146.
Veron, J. & Minchin, P. 1992. Correlations between sea surface temperature, circulation pattern and the distribution of hermatypic corals of Japan. Continental Shelf Res. 12: 835-857.
Wang, J. P., Hsu, K. C. & Chiang, T. Y. 2000. Mitochondrial DNA phylogeography of Acrossocheilus paradoxus (Cyprinidae) in Taiwan. Molecular Ecology. 9:1483-1494.
Wilson, A. C., Cann, R. L., Carr, S. M., Matthew, U. B., Gyllensten, K. M., Helm-Bychowski, R. G., Higuchi, S. R., Palumbi, E. M., Prager, R. D. S. & Stoneking, M. 1985. Mitochondrial DNA and two perspectives on evolutionary genetics. Biological Journal of the Linnean Society. 26: 275-400.
Wright, S. 1951. The genetical structure of populations. Annals of Eugenetics. 15: 323-354.
Yang, C. C. 1999. Molecular phylogenetic determination of Psechrus spiders in Taiwan. Master’s thesis, National Taiwan Normal University, Taipei, Taiwan. (in Chinese).
Yang, Y. J., Lin, Y. S., Wu, J. L. & Hui, C. F. 1994. Variation in mitochondrial DNA and population structure of the Taipei treefrog Rhacophorus taipeianus in Taiwan. Molecular Ecology. 3: 219-228.
Yeh, W. S. 1997. Phylogeographic structure of Rhacophorus moltrechti. Master’s thesis, National Taiwan University, Taipei, Taiwan. (in Chinese).