中文摘要
本研究藉由葉綠體DNA之atpB及rbcL基因間的非轉譯區間序列來探討四合木 (Tetraena mongolica Maxim.) 的親緣地理型式。四合木是蒺藜科中古老的孓遺單屬種植物，生長在中國內蒙古屬於瀕臨滅絕之特有物種。已列為內蒙古一級保育稀有植物，世界上僅存的四合木分布在內蒙古鄂爾多斯高原的荒漠區，此分布區是亞洲一批古老殘遺植物的避難所。四合木的生殖是以種子繁殖為主要方式，但在惡劣的環境下，則少數併行根部營養繁殖。本實驗共定序出8 個族群77個個體葉綠體DNA之atpB及rbcL基因間的非轉譯區間序列共計880鹼基對。利用葉綠體DNA資料估算族群間的基因交流值 (Nm) (Nm：0.04-2.01) 和族群間分化指數 (Fst) (Fst：0.199-0.895)，並將族群間地理距離對數值與分化指數做迴歸分析，結果呈現無顯著相關，顯示四合木其遺傳分化程度不符合「isolation by distance」 模式 (R2=0.0024)；根據由葉綠體DNA所構築的親緣網狀圖(minimum spanning network)，檢測地理形式與clade間的關係，顯示四合木族群地理區所呈現的地理分布模式，主要是受到過去棲地破碎化 (past fragmentation) 的影響，而黃河氾濫及千里山山脈的阻隔可能是造成四合木地理隔離的主因。

Abstract

Sequence variation of the atpB-rbcL noncoding spacer region of the chloroplast DNA was used to reconstruct the phylogeography of Tetraena mongolica.T. mongolica (Zogophylaceae) is a relict species of a monotypic genus of in Inner Mongolia of China. It has been listed as seriously endangered plants that only distributed on the Ordos plateau of Inner Mongolic. The area is a refuge of many relict Asian Plants. Reproduction of T. mongolica is mainly by seed but a reproduction is anther way under the stress. A total of 880 bp of the atpB-rbcL spacer of cpDNA was sequenced from 8 populations of T. Mongolic. According to the cpDNA data, the level of gene flow (Nm:0.04-2.01), and population differentiation (Fst:0.199-0.895) were indicated, and the “isolation by distance” model was not supported (R2 =0.0024). Based on minimum spanning network and nested clade analysis, the past fragmentation is the main phylogeographic factor affecting the population structure of T. mongolica .We suggested the deluges of Yellow River and isolation of Gianlishan Mountain cased the population fragmentation of T. mongolica.

Keywords: phylogeography, Tetraena mongolica, gene flow, population
differentiation, nested clade analysis, past fragmentation

目錄
誌謝................................................................................................. Ⅰ
中文摘要…………………………………………………………..Ⅱ
英文摘要…………………………………………………………..Ⅲ
目錄………………………………………………………………..Ⅳ
表目錄……………………………………………………………..Ⅵ
圖目錄……………………………………………………………..Ⅶ

第壹章 前言……………………………………………………….. 1
一、親緣地理學的研究………………………………………… 1
二、四合木的研究歷史………………………………………… 5
三、瀕危物種的保育與其遺傳變異…………………………...11
四、分子技術進步對於族群結構的應用……………………...15
五、研究目的 ………………………………………………….16
第貳材料與方法…………………………………………………...17
一、研究材料...............................................................................17
二、實驗方法 ........................................……………………….18
第參章 結果....…………………………………………………….24
一、葉綠體DNA atpB-rbcL noncoding spacer片段……….24
二、四合木的親緣地理………………………………………...26
第肆章 討論……………………………………………………….27
一、四合木的遺傳變異………………………………………...27
二、四合木的族群分化………………………………………...28
三、四合木的親緣地理模式…………………………………...29
四、四合木的保育……………………………………………...31
第伍章 結論……………………………………………………….32
第陸章 參考文獻…………………………………………………..33
表……………………………………………………………………40
圖……………………………………………………………………47
附錄…………………………………………………………………57

表目錄
表一、四合木之採樣地點、採集個體數目及族群代號.................40
表二、進行四合木DNA介於atpB及rbcL基因間的noncoding
spacer聚合酵素連鎖反應所使用之引子序列……………41
表三、根據葉綠體DNA資料估算四合木族群haplotype歧異
度、核柑酸歧異度………………………………………… 42
表四、根據葉綠體DNA資料所建構樹狀圖中各clade的地理
族群組成…………………………………………………….43
表五、根據葉綠體DNA資料估算四合木族群間基因交流值和
族群間分化指數…………………………………………….44
表六、估算葉綠體DNA親緣網狀圖各clade的Dc、Dn值……..45
表七、根據所估算之葉綠體DNA親緣網狀圖各clade的Dc、
Dn值，並配合檢索表推測四合木不同的clade所曾遭
遇的歷史事件……………………………………………….46

圖目錄
圖一、四合木植株照片....................................................................47
圖二、四合木之形態特徵…………………………………………48
圖三、四合木之取樣分布圖………………………………………49
圖四、四合木葉綠體DNA之atpB及rbcL 基因間的非轉譯區
間區列在八個族群的代表序列……………………………50
圖五、根據葉綠體DNA資料以聚分類分析法構築四合木的親
緣樹狀圖……………………………………………………54
圖六、根據葉綠體DNA資料構築的親緣網狀圖…………………55
圖七、四合木族群地理距離對數值與利用葉綠體DNA資料估
算的遺傳分化值之迴歸分析圖……………………………56

Avise, J. C., 2000. Phylogeography: the history and formation of species. Harvard University Press, Cambridge.
Avise, J. C., J. Arnold, R. M. Ball, E. Bermingham, T. Lamb, E. Neigel, C. A. Reeb and N. C. Saunder, 1987. Intraspecific phylogeography: the mitochondrial DNA bridge between population genetics and systematics. Ann. Rev. Ecol. Syst. 18: 489-522.
Bermingham, E., and A. P. Mortiz, 1998. Comparative mtDNA phylogeography of neotropical freshwater fishes: testing shared history to infer the evolutionary landscape of lower Central America. Mol. Ecol. 7: 499-518.
Borba, E. L., J. M. Felix, V. N. Solferini and J. Semir, 2001. Fly-pollinated Pleurothallis (Orchidaceae) species have high genetic variability: evidence from isozyme markers. Amer. J. Bot. 88: 419-428.
Broyles, S. B., and R. Wyatt, 1993. Allozyme diversity and genetic structure in southern Appalachian populations of poke milkweed, Asclepias exaliata. Syst. Bot. 18: 18-30.
Byrne, M., and G. f. Moran, 1994. Population divergence in chloroplast genome of Eucalyptus nitens. Heredity 73: 18-28.
Chiang, T. Y., 1998. The mosses of Taiwan: their conservation status. In C. I Peng and P. P. Lowry (eds.) Rare, Threatened and Endangered floras of Asia and the Pacific Rim. Institute of Botany, Academia Sinica Monograph Series No. 16. Academia Sinica, Taipei. p. 89-110.
Chiang, T. Y., 2000a. Molecular evolution of the atpB-rbcL noncoding spacer of chloroplast DNA in the moss family Hylocomiaceae. Botanical Bulletin of Academia Sinica 41: 85-92.
Chiang, T. Y., 2000b. Lineage sorting accounting for the disassociation between chloroplast and mitochondrial lineages in oaks of southern Trance. Genome 43: 1090-1094.
Chiang, T. Y., and B. A. Schaal, 1999. Phylogeography of North American populations of the moss species Hylocomium splendens based on the nucleotide sequence of internal transcribed spacer 2 of nuclear ribosomal DNA. Mol. Ecol. 8: 1037-1042.
Chiang, T. Y., B. A. Schaal and C. I. Peng, 1998. Universal primers for amplification and sequencing a noncoding spacer between atpB and rbcL genes of chloroplast DNA. Botanical Bulletin of Academia Sinica 39: 245-250.
Chiang, T. Y., and K. S. Hong, 1999. Genetic diversity of Quercus species during the postglacial periods. In: Lin, Y. S. (Ed.) Proceedings of 1999 Biodiversity Congress. The Council of Agriculture, Taipei. p. 431-443
Chiang, T. Y., Y. C. Chiang, Y. J. Chen, C. H. Chou, S. Havanond, T. N. Hong and S. Huang, 2001. Phylogeography of Kendelia candel in East Asiatic mangroves based on nucleotide variation of chloroplast and mitochondrial DNAs. Mol. Ecol. 11: 2669-2682.
Cole, C. T., and D. D. Biesboer, 1992. Monomorphism, reduced gene flow and cleistogamy in rare and common species of Lespedeza (Fabaceae). Amer. J. Bot. 79:567-575.
Cruzan, M. B., M. L. Arnold, S. E. Carney and K. R. Wollenberg, 1993. CpDNA inheritance in interspecific crosses and its effect on evolutionary inference in Louisiana irises. Amer. J. Bot. 80: 344.
Cox, C. B., and P. D. Moore. 2000. Biogeography: Anecological and Evolutionary
Approach. 6th ed. Blackwell Science Ltd. USA.
Doyle, J. L., and K. L. Doyle, 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19:11-15.
Dumolin-Lapègue, S., M. H. Pemonge and R. J. Petit, 1997a. An enlarged set of consensus primers for the study of organelle DNA in plants. Mol. Ecol. 6: 393-397.
Dumolin-Lapègue, S., M. H. Pemonge and R. J. Petit, 1998. Association between chloroplast and mitochondrial lineages in oaks. Mol. Biol. Evol. 15: 1321-1331.
Dvorak, J., and H. B. Zhang, 1992. Application of molecular tools for study of the phylogeny of diploid and polyploid taxa in Triticeae. Heredity 166: 37-42.
Excoffier, L., P. E. Smouse and J. M. Quattro, 1992. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA data. Genetics 131: 479-491.
Excoffier, L., and P. E. Smouse, 1994. Using allele frequencies and geographic subdivision to reconstruct gene trees within a species: Molecular variance parsimony. Genetics 136: 343-359.
Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791.
Ferris, C., R. P. Oliver, A. J. Davy, and G. M. Hewitt, 1995. Using chloroplast DNA to trace postglacial migration routes of oaks into Britain. Mol. Ecol. 4: 731-738.
Fleischer, R. C., E. A. Perry, K. Muralidharan, E. E. Stevens and C. M. Wemmer, 2001. Phylogeography of the Asian elephant (Elephas maximus) based on mitochondrial DNA. Evolution 55: 1882–1892.
Forcioli, D., P. Saumitou-Laprade, M. Valero, P. Vernet and J. Cuguen, 1998. Distribution of chloroplast DNA diversity within and among populations in gynodioecious Beta vulgaris ssp. maritima (Chenopodiaceae). Mol. Ecol. 7: 1183-1204.
Futuyma, D. J., 1986. Evolutionary Biology, 2nd. Edn, Sinauer, Sunderland, MA.
Futuyma, D. j., 1998 The geography of evolution . In: Evolution Biology, 3rd ed. Sinauer Associates, Inc., Sunderland, Massachusetts. U. S. A.
Ge, X. J., Y. Yan, N. X. Zhao, H. S. Chen and W. Q. Qi, 2003. Genetic variation in the endangered Inner Mongolia endemic shrub Tetraena mongolica Maxim. (Zygophyllaxeae). Biology conservation. 111:427-434.
Hamrick, J. L., and M. J. W. Godt, 1989. Allozyme diversity in plant species. In: Brown, A. H. D., M. T. Clegg, A. L. Kahler and B. S. Weir, (Eds.) Plant Population Genetics, Breeding and Genetic Resources. Sinauer, Sunderland, MA. p. 43-63.
Hayata, B., 1920. Icones Plantarum Formosanarum nec non et Contributiones ad Floram Formosanam. Vol.9. The Bureau of Forestry, Industries, Government of Formosa. p. 81-82.
Hedrick, P. W., 1983. Genetics of Populations. Science Book International, Inc. Boston, MA.
Hillis. D. M., and J. J. Bull, 1996. An empirical test of bootstrapping as a method assessing confidence in phylogenetic analysis. Syst. Biol. 41: 182-192.
Hsieh, C.F., and T. C. Huang, 1974. The Acanthaceous plants of Taiwan. Taiwania 19: 19-57.
Huang, J. C., and C. L. Wen, 1999. Propagation of Hygrophila pogonocalyx. Quart. Nat. Conserv. 26: 34-37.
Huang, S., Y. C. Chiang, B. A. Schaal, C. H. Chou and T. Y. Chiang, 2001. Organelle DNAs phylogeography of Cycas taitungensis, a relict species in Taiwan. Mol. Ecol. 11: 2669-2682.
King, R.A. and C. Ferris, 1998. Chloroplast DNA phylogeography of Alnus glutinosa (L.) Gaertn. Mol. Ecol. 7: 1151-1161.
Kumar, S., K. Tamura, I. B. Jakobsen and M. Nei, 2001. MEGA2: Molecular Evolutionary Genetics Analysis software, Bioinformatics.
Li, B. 1990. A study og natural resources and enbironment in Ordos Plateau, Inner Mongolia. Beijing : Scientific Press. 110-158.
Lu, S. Y., C. I Peng, Y. P. Cheng, K. H. Hong and T. Y. Chiang, 2001. Chloroplast DNA phylogeography of Cunninghamia konishii (Cupressaceae), an endemic conifer of Taiwan. Genome 44: 797-807.
MacHugh, D. E., M. D. Shriver, R. T. Loftus, P. Cunningham and D. G. Bradley, 1997. Microsatellite DNA variation and the evolution, domestication and phylogeography of taurine and zebu cattle (Bos taurus and Bos indicus) Genetics 146: 1071-1086.
Manual of Taiwan vascular plants. 1999. vol. 4. The Council of Agriculture. Taipei.
Michelia, M. R., and R. Bova, 1997. Fingerprinting methods based on arbitrarily primed PCR. Springer, Berlin.
Miller, M. P., 1997. Tools for population genetics analyses (TFPGA): A Windows program for the analysis of allozyme and molecular population genetic data. On-line: http://herb.bio.nau.edul~miller/tfpga.htm.
Miller, M. P., 1998. AMOVA-PREP 1.01: a program for the preparation of the AMOVA input files from dominant-marker raw data. Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ.
Moritz, C., 1994. Defining ‘evolutionary significant units’ for conservation. Tree. 9: 373-375.
Moritz, C., and P. Bermingham, 1998. Comparative phylogeography: concepts and applications. Mol. Ecol. 7: 367-370.
Mummenhoff, K. and H. Hurka, 1995. Allopolyploid origin of Arabidopsis suecica (Fries) norrlin: evidence from chloroplast and nuclear genome markers. Bot. Acta. 108: 449-456.
Nei, M. and F. Tajima, 1983. Maximum likelihood estimation of number of nucleotide substitution from restriction site data. Genetics 105: 207-217.
Nei, M. and W. H. Li, 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA. 76: 5269-5273.
O’Corry-Crowe, G. N., R. S. Suydwn, A. Rosenberg , K. J Frost and A. E. Dozon, 1997. Phylogeography, population structure and dispersal patterns of the beluga whale Delphinapterus leucas in the western Nearctic revealed by mitochondrial DNA. Mol. Ecol. 6: 955-970.
Olsen, K. M., and B. A. Schaal, 1999. Evidence on the origin of cassava: phylogeography of Manihot esculenta. Proc. Natl. Acad. Sci. USA. 96: 5586-5591.
Palmer, J. D. 1985. Evolution of chloroplast and mitochondria DNA in plants and algae. In R.J. MacIntyre, Molecular and Evolutionary Genetics.Plenum, New York. 131-240.
Petit, R. J., E. Pineau, B. Demesure, R. Bacilieri, A. Ducousso and A. Kremer, 1997. Chloroplast DNA footprints of postglacial recolonization by oaks. Proc. Natl. Acad. Sci. USA. 94: 9996-10001.
Peng, C. I, S. L. Liu and T. Y. Chiang, 1999. Conservation of Ludwigia