為確認節位突起（enation-structure）以辨識蔓澤蘭（Mikania cordata （Burm.f.）B.L.Robinson）和小花蔓澤蘭（Mikania micrantha H.B.K.）的有效性及了解兩者族群遺傳變異的情形，自台灣地區北、中、南、東、及離島，各設置2~3個採集點，依據節位突起辨識法進行採集工作，利用分子標誌技術直接定序（PCR- Sequencing）分析兩者的nuclear rDNA ITS region及 chloroplast trn L intron、trn L-trn F IGS等基因組中變異較快的區域，作為近緣種的種間分子標誌。取84個小花蔓澤蘭及48個蔓澤蘭樣品的nrDNA ITS region，以及自Gene Bank中取得第三種蔓澤蘭屬有害雜草（米甘草,M. scandens（L.）Willd.）的ITS序列進行分析，其結果顯示nrDNA ITS region 可有效區分三種蔓澤蘭屬的有害雜草，根據鄰接法獲得的樹狀圖指出蔓澤蘭和小花蔓澤蘭的親緣關係較近,其序列有97%的相似度。另外，蔓澤蘭和小花蔓澤蘭的葉綠體 trn L intron（436bp）及trn L-trn F IGS（345 bp）序列完全相同，因此無法作為種間分子標誌。nrITS region和ISSR (inter simple sequence repeat)分子指紋分析的結果支持節位突起辨識的有效性，因此建議農政單位利用節位突起的辨識方法推廣「除蔓活動」，如此可降低對本土弱勢的蔓澤蘭所造成的衝擊。進一步以ISSR的數據分析蔓澤蘭和小花蔓澤蘭的族群遺傳變異，結果顯示兩種蔓澤蘭屬植物種內族群間有很高的遺傳分化（Gst>0.55），其中小花蔓澤蘭的遺傳距離與地理距離無關（r=0.0053，p=0.47），推測小花蔓澤蘭入侵台灣時有具不同的遺傳變異，受到人為活動隨機的散佈，各地族群由少數個體（先驅者）所建立，由於此種族群結構易受遺傳漂變（genetic drift）的影響，使對偶基因隨機漂失或固定，造成族群分化；而本土蔓澤蘭則與地理距離相關（r=0.44，p=0.025＊），符合距離隔離模式（isolation by distance model），推測應是自然散佈的結果。整體來說，小花蔓澤蘭的族群結構呈現穩定的狀態，故建議防治研究應針對分化大的族群進行。

The objective of this study is to elucidate the efficiency of enation-structure (at node) recognition method at pre-flowing stage and to understand the population genetic variation of the Mikania weeds in Taiwan. The plant materials collected by recognizing enation-structure symptom method from North, Central, South and East Taiwan and off-shore islands. Using PCR– sequencing marker techniques, the sequencing revealed that nrDNA ITS region could identify three Mikania weeds and the 97% similarity of phylogenetic relationship between M.cordata and M.micrantha are more closer than that between M.cordata and M.scandens, whose ITS sequence is obtained from GeneBank. However ,the sequences of chloroplast DNA of M.cordata and M.micrantha at trnL intron（436bp）or trnL-trnF IGS（345 bp）are almost the same and could not be used as molecular markers. The recognizing techniques of enation-structure was supported by the nrDNA ITS region and ISSR results at the end, thus the finding can be recommended to the Council of Agriculture in order to eliminate the weed and to reduce the impact on M.cordata, which is native in Taiwan.
Moreover, the findings of ISSR analysis in the aspect of population genetic variation indicated that high genetic differentiation （Gst>0.5）was found among the M. cordata and M.micrantha populations. Based on the Mantle test, there was no relationship between genetic distance and geographic distance in M.micrantha（r=0.0053,p=0.47）.This phenomenon revealed that populations of M.micrantha had complex population variability within the short-term invaded into Taiwan that might be resulted from the random dispersion of human activities. The population of M.micrantha was established by few individuals (founders) and grown rapidly in Taiwan, resulting in population differentiation via genetic drift. In contrast to M.micrantha ,there was relationship between genetic distance and geographic distance in M.cordata （r=0.44,p=0.025＊）.It revealed that populations of M.cordata agreed to the concept of isolation by distance model, which might be evolved from the result of natural dispersion. In conclusion, the population structure of M.micrantha in Taiwan is stable , suggesting that control of Mikania population should be based on different populations where have large differentiation among them .

誌謝……………………………………………………………………I
中文摘要………………………………………………………………II
英文摘要………………………………………………………………III
圖表目次………………………………………………………………VI
前言……………………………………………………………………1
材料與方法……………………………………………………………12
結果……………………………………………………………………28
討論……………………………………………………………………52
結論……………………………………………………………………62
引用文獻………………………………………………………………64

孔國輝、吳七根、胡啟明.2000a.外來雜草（Mikania micrantha H.B.K.）在我國的出現.熱帶亞熱帶植物學報 8(1):27。
孔國輝、吳七根、胡啟明、葉萬輝.2000b.微甘菊（Mikania micrantha H.B.K.）的形態、分類與生態資料補記.熱帶亞熱帶植物學報 8(2):128-130。
王建波.2002.ISSR分子標誌及其在植物遺傳學研究中的應用.遺傳 24(5):613-616
王昭月、莊耿彰、范明仁.2001.以ISSR與RAPD分子標誌分析火鶴花栽培種間遺傳相似性.中華農業研究 50(1)54-67。
周昌弘 1990.植物生態學.聯經出版社 pp.252-264。
郭耀綸.2001.小花蔓澤蘭之個體生態學調查.行政院農業委員會林務局委託計畫成果報告.1:1-24。
陳富永、徐玲明、蔣慕琰 .2002.小花蔓澤蘭形態區別及RAPD-PCR分析.植物保護學會會刊 44:51-60。
陳滄海、陳仁昭.2001.小花蔓澤蘭之藥劑生物防治及天敵調查. 行政院農業委員會林務局委託計畫成果報告.3:1-28。
黃士穎、徐國凱.2001.應用葉綠體trn F-trn L核酸序列探討台灣八種杜鵑花之分子親緣關係.台灣林業科學 16(3):153-60。
黃忠良、曹洪麟、梁曉東、葉萬輝、馮惠玲、蔡楚雄.2000.不同生境和森林內微甘菊的生存與危害狀況. 熱帶亞熱帶植物學報 8(2):131-138。
蔣慕琰、徐玲明、陳富永.2002.入侵植物小花蔓澤蘭（Mikania micrantha Kunth）之確認.植物保護學會會刊 44:61-65。
蔡承憲.2001.應用ISSR研究台灣水柳與水社柳枝遺傳變異 國立中興大學植物學系碩士論文。
Ammiraju,J.S.S.,B.B.Dholakia,D.K.Santra,H.Singh,M.D.Lagu,S.A.Tamhankar,H.S. Dhaliwal,V.S.Rao,V.S.Gupta,and P.K.Ranjekar.2001.Identification of inter simple sequence repeat(ISSR) markers asspciated with seed size in wheat. Theor. Appl. Genet. 102:726-732.
Arcade,A.,F.Anselin,P.F.Rampant,M.C.Lesage,L.E.Pâques,and D.Prat.2000. Applicatin of AFLP,RAPD and ISSR markers to genetic mapping of European and Japanses Larch. Theor. Appl. Genet.100:299-307.
Barrett,S.C.B.1982.Genetic variation in weeds.R.Charudattan and H.L.Walker,eds. Biological Control of Weeds With Plant Pathogens,Jhon Wiley and Sons,New York.pp.78-112.
Blair,M.W.,O.Panaud,and S.R.MoCouch.1999.Inter-simple sequence repeat（ISSR）amplification for analysis of microsatellite motif frequence and fingerprinting in rice(Oryza sativa L.). Theor. Appl. Genet. 98:780-792.
Brown,A.H.D.and D.R.Marshall.1981.Evolutionary changes accompanying colonization in palnts. JL Revel,eds.Colonization,succession and stability.Blackwell Scientific,oxford.pp.351-363.
Burdon,J.J.and D.R.Marshall.1981.Biological Control and the reproductive mode of weeds. J. Appl. Ecol. 18:649-658.
Burdon,J.J.and A.H.D.Brown.1987.Population genetics of Echium plantagineum, a target weed for biological control. Aust. J. Biol. Sci. 39:369-378.
Bussell,J.D.1999.The distribution of random amplified polymorphic DNA（RAPD）diversity amoungst populations of Isotoma petraea（Lobeliaceae）.Molecular Ecology 8,775-789.
Casasoli,M.,C.Mattioni,M.Cherubini,and F.Villani.2001.A genetic linkage map of European chestnut（Castanea sativa Mill.） based on RAPD,ISSR and isozyme markers. Theor. Appl. Genet.102:1190-1199.
Cekic,C.,N.H.Battey,and M.J.Wilkinson.2001.The potential of ISSR-PCR primer-pair combinations for genetic linkage analysis using the seasonal flowering locus in Fragaria as a mode. Theor. Appl. Genet.103:540-546.
Chase,M.W.and H.H.Hills.1991.Silica gel:An ideal material for field preservation of leaf samples for DNA studies.Taxon 40:215-220.
Coblentz,B.E.1992.Exotic organisms:a dilemma for conservation biology. Conservation Biology 4(3):261-265.
Davila,J.A.,Y.Loarce,and E.Ferrer.1999.Molecular characterizatin and genetic mapping of random amplified microsatellite polymorphism in barley. Theor. Appl. Genet.98:265-273.
Dirlewanger,E.P.,V.Pronier,C.Parvery,C.Rothan,A.Guye,and R.Monet.1998.Genetic linkage map of peach(Prunus persica I.) using morphological and molecular markers. Theor. Appl. Genet.97:888-895.
Elton,C.1958.The ecology of invasions by animals and plants.Chap-man and Hall,London.pp.181.
Ellstrand,N.C.and D.R.Elam.1993.Population genetic consequences of small population size:implications for plant conservation. Annual Review of Ecology and Systematics 24:217-242.
Esselman,E.J.,L.Jianqiang,D.J.Crawford,J.L.Windus,and A.D.Wolfe.1999.Clonal diversity in the rare Calamagrostis porteri spp.insperata(Poaceae):comparative results for allozymes and random amplified polymorphuc DNA(RAPD) and intersimple sequence repeat(ISSR) markers.Mol.Ecol.8:443-451.
Evans,D.A.1999.How can technology feed the world safely and sustainably? Pesticide Chemistry and Bioscience:The Food-Environment Challenge ,eds.G.T.Brools and T.R.Roberts.Cambridge,UK:The Royal Society of Chemistry.pp.124.
Excoffier,L.1993.Analysis of Molecular Variance.Version 1.55.Genetics and Biometry Laboratory University of Geneva.
Fang,D.G.,R.R.Krueger,and M.I.Roose.1998.Phylogenetic relationships among selected Citrus germplasm accessions reveal by inter-simple sequence repeat(ISSR) markers. J. Amer. Soc. Horticul. Sci. 123:612-617.
Fernandez,M.E.,A.M.Figueiras,and C.Benito.2002.The use of ISSR and RAPD markers for detecting DNA polymorphism,genotype identification and genetic diversity among barley cultivars with known origin. Theor. Appl. Genet. 104:845-851.
Gaskin,J.F.and B.A.Schaal.2002.Hybrid Tamarix widespread in U.S.invasion and undetected in native Asian range.PNAS.99(17):11256-11259.
Ge Xue-Jun,Yan Yu,Nan-Xian Zhao,Hai-Shan Chen,and Wen-Qing Qi.2003.Genetic variation in the endangered Inner Mogolia endemic shrub Tetraena mongolica Maxim.(Zygophyllaceae).Biological Conservation 111:427-434.`
Gielly,L.and P.Taberlet.1996.A phylogeny of the European gentians inferred from chloroplast trn L（UAA）intron sequences.Botanical Journal of the Linnean Society.120:57-75.
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.
Hamada,H.and T.Kakunaga.1982.Potential Z-DNA formaing sequence are highly dispersed in the human genome.Nature 298:396-398.
Hamrick,J.E.and M.J.W.Godt.1989.Allozyme diversity in plants.In:Broen, A.H.D.,Clegg,M.T.,Kahier,A.L.,Weir,B.S.(Eds.),Plant Population Genetics,Breeding and Genetic Resource.Sinauer,Sunderland,Massachuset. pp.43-63.
Hao Gang,Dong-Hee Lee,June Seung Lee,and Nam Sook Lee.2002.A study of taxonomical relationships among species of Korean Allium sect.Sacculiferum（Alliaceae）and related species using inter-simple sequence repeat（ISSR） markers. Bot. Bull. Acad. Sin. 43:63-68.
Hashizume,T.,I.Shimamoto,and M.Hirai.2003. Construction of a linkage map and QTL analysis of horticultural traits for watermelon [Citrullus lanatus (THUNB.) MATSUM & NAKAI] using RAPD, RFLP and ISSR markers. Theor. Appl. Genet. 106(5) : 779-785.
Hills,L.A.1999. Mile-a-minute（Mikania micrantha）.Agnote pp.535. http://www.dpif.nt.gov.au/dpif/pubcat
Hollingsworth,M.L.,P.M.Hollingsworth,G.I.Jenkins,J.P.Bailey,and C.Ferris.1998. The use of molecular markers to study patterns of genotypic diversity in some invasive alien Fallopia spp.(Polygonaceae). Molecular Ecology 7:1681-1691.
Holt,J.1994.Genetic variation in life history traits in yellow nutsedge（Cyperus esculentus）from California.Weed Sci.42:385-389.
Horak,M.and J.S.Holt.1986.Isozyme variability and breeding systems in populations of yellow nutsedge（Cyperus esculentus）.Weed Sci.34:538-543.
Hou,Y.and T.M.Sterling.1995.Isozyme variation in broom snakeweed（Gutierrezia sarothrae）.Weed Sci.43:156-162.
Huang,S.1992.Patterns of genetic variation in northern red oak (Quercus rubra L.) populations in midwestern United Staes.Ph.D.dissertation,Univ.of Missouri,St.Louis.
Huffaker,C.B.1974.Fundamentals of biological weed control. P.DeBach ed. Biological Control of Insects Pests and Weeds Chapman and Hall Limited.London.pp.631-649.
Invaders Database System.2003. Taxon: Mikania cordata (Burm. f.) B. L. Rob. http://www.ars-grin.gov/cgi-bin/npgs/html/taxon.pl?316382
Ismail,B.S.and A.Kumar.1996.Effects of aqueous extracts and residues decomposition of Mikania micrantha H.B.K. on selected crops.Allelopathy Journal 3:195-206.
Ismail,B.S.and L.S.Mah.1993.Effects of Mikania micrantha H.B.K. on germination and growth of weed species. Plant and Soil 157:107-113.
IUCN.2001.Aliens. http://www.issg.org/aliens_newsletter/Aliens13.pdf
Joshi,S.P.,V.S.Gupta,R.K.Aggarwal,P.K.Ranjekar,and D.S.Brar.2000.Genetic diversity and phylogenetic relationship as revealed by inter simple sequence repeat(ISSR) polymorphism in the genus Oryza. Theor. Appl. Genet. 100:1311-1320.
Julien,M.H.1992.Biological Control of Weeds:A World Catalogue of Agents and their Target Weeds. C.A.B.International Wallingford,UK.pp.186.
Kantcty,R.V.,X.Zeng,and J.Bennetzen.1995.Assessment of genetic diversity in popcorn（Zea mays） inbred lines using inter-simple sequence repeat(ISSR) amplification. Mol. Breed 1:365-373.
Kimura,M.1980.A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol.Evo. 16:111-120.
Kojima,T.,Nagaoka T,and Noda K.1998.Genetic linkage map of ISSR and RAPD markers in Einkorn wheat in relation to that of RFLP markers.Theor. Appl. Genet. 96:37-45.
Kumar,S.,K.Tamura,I.B.Jakobsen,and M.Nei.2001.MEGA2:Molecular Evolutionary Genetics Analysis software,Bioinformatics.
Lai,J.A.,W.C.Yang,and J.Y.Hsiao.2001.An assessment of genetic relationships in cultivated tea clones and native wild tea in Taiwan using RAPD and ISSR markers. Bot. Bull. Acad. Sin. 42:93-100.
Loveless,M.D.and J.L.Harmrick.1984.Ecological determinants of genetic structure in plant populations. Ann. Rev. Ecol. Syst. 15:65-95.
Lym,R.,J.A.Kapaun,R.B.Carlson,and D.Mundal.1993.Effects of herbicide tretments and leafy spurge biotype on Spurgia esulae population.Proc.Leafy Spurge Symp.Colorado State Univ.pp.46.
Mantel,N.A.1967.The detection of disease clustering and a generalized regression approach. Cancer Res.27:209-220.
Meekins,J.F.,H.E.Ballard,Jr.,and B.C.McCaarthy.2001.Genetic variation and molecular biogeography of a north American incasive plant（Allaria petiolata,Brassicaceae）.Plant Science 162(1):161-169.
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.
Moodie,M.,R.P.Finch,and G.Marshall.1997.Analysis of genetic variation in wild mustard(Sinapis arvensis) using molecular markers.Weed Sci.45:102-107.
Moreno,S.,J.P.Martin,and M.Ortiz.1998.Inter-simple sequence repeats PCR for characterization of closely related grapevine germplasm.Euphytica 101:117-125.
Mulligan,G.A.and R.J.Moore.1961.Natural selection among hybrids between Carduus acanthoides and C.nutans in Ontario. Canadian J. Bot.39:269-279.
Nagaoka,T.and Y.Ogihara.1997.Applicability of inter-simple sequence repeat markers in wheat for use as DNA markers in comparison to RFLP and RAPD markers. Theor. Appl. Genet. 94:597-602.
Nei,M.,T.Maruyama,and R.Chakraborty.1975.The bottleneck effect and genetic variability in population.Evolution 29:1-10.
Nei,M.1973.Analysis of gene diversity in subdivided population. Proc. Natl. Acad. Sci.USA.70:3321-3323.
Nissen,S.J.,A.M.Robert,J.L.Donald,and L.R.Marthal.1995.DNA-based marker systems to determine genetic diversity of weedy species and their apllication to biocontrol.Weed Science 43:504-513.
Prevost,A.and M.J.Wilkinson.1998.A new system of comparing PCR primers applied to ISSR figerprinting of potato cultivars. Theor. Appl. Genet.98:107-112.
Pritchard,T.1960.Race formation in weedy species with special reference to Euphorbia cypressias L. and Hypericum perforatum L. J.L.Harper,ed.The Biology of weeds.pp.61-66.
Putievsky,E.,P.Weiss,and D.R.Marshall.1980.Interspecific hybridization between Emex australis and E.spinosa. Australian J.Bot.28:323-328.
Qian,W.,S.Ge,and D-Y.Hong.2001.Genetic variation within and among populations of a wild rice Oryza granulata from China detected by RAPD and ISSR markers. Theor. Appl. Genet.102:440-449.
Ratnaparkhe,M.B.,D.K.Santra,A.Tullu,and F.J.Muehlbauer.1998.Inheritance of inter-simpler- sequence-repeat polymorphism and linkage with a fusarium wilt resistance gene in chickpea. Theor. Appl. Genet. 96:348-353.
Rohlf,F.J.1997.NTSYSpc.Applied Biostatistics,Inc.New York.
Sankar,A.A.and G.A.Moore.2001.Evalution of inter-simple sequence repeat analysis for mapping in Citrus and extension of the genetic linkage flowering locus in Fragaria as a model. Theor. Appl. Genet. 103:540-546.
Scheiber,S.M.,R.L.Jarret,D.Robacker,and M.Newman.2000.Genetic relationships within Rhododendron L.section Pentanthera G.Don bases on sequence of the internal transcribed spacer(ITS) region. Sci. Hort. 85:123-35.
Schrag,S.and P.Wiener.1995.Emerging infectious disease:what are the relative roles of ecology and evolution? Trends Ecol. Evol. 10:319-324.
Sheppard,A.W.1992.Predicting biological weed control.Trends Ecol. Evol. 7:290-290
Slakin,M.and N.M.Barton.1989.A comparison of three indirect methods for estimating average levels of gene flow. Evolution 43:1349-1368.
Sun,M.1997.Population genetic structure of yellow starthistle（Centaurea solstitialis）,a colonizing weed in the western United States. Can. J. Bot. 75:1470-1478.
Swamy,P.S.and P.S.Ramakrishnan.1987.Growth and allocation patterns of Mikania micrantha in successional ecvironments after slash and burn agriculture. Can. J. Bot. 66:1465-1469.
Tikunov,Y.,L.Khrustaleva,and G.Karlov.2003. Application of ISSR markers in the genus Lycopersicon. Euphytica 131：71-81.
Vaughan,D.1989.The genus Oryza L.current status of taxonomy IRRI Research paper Series Number 138.
Warwick,S.I.1987.Isozyme variation in proso millet(Panicum miliaceum L.).J.Hered. 78:210-212.
Warwick,S.I.1990.Genetic variation in weeds-with particular reference to Canadian Agricultural weeds.S.Kawano,ed.Biological approaches and Evolutionary Trends in Plants. Academic Press. New York.pp.3-18.
Wolfe,A.D.and A.Liston.1998.Contributions of PCR-based methods to plant systematics and evolutionary biology. D.E.Soltis,P.S.Soltis,J.J.Doyle,eds. Plant molecular systematics. Vol2.Chapman Hall.New York.pp.43-86.
Wright,S.1943.Isolation by distance. Genetics 28:114-138.
Wright,S.1931.Evolution in Mendelian population. Genetics 16:97-159.
Zavaleta,E.2000. Valuing ecosystem services lost to Tarmarix invasion in the United States. In Mooney, H.A. and R.J. Hobbs (eds.).Ivasive Species in a Changing World, Island Press, Washington, D.C.
Zhang M,Ling B,Kong C,Zhao H,and Pang X.2002. Allelopathic potential of volatile oil from Mikania micrantha. Ying Yong Sheng Tai Xue Bao. 13(10):1300-2
Zietkiewicz,E.,A.Rafalski,and D.Labuda.1994.Genome fingerprinting by simple sequence repeat(SSR)-anchhored polymerase chain reaction amplication.Genome 20:176-183.