林讚標臺灣大學：植物科學研究所鄭育斌Cheng, Yu-PinYu-PinCheng2007-11-272018-07-062007-11-272018-07-062006http://ntur.lib.ntu.edu.tw//handle/246246/57990本研究利用母系遺傳的葉綠體DNA(cpDNA)非編碼區(noncoding region)序列和同功酵素電泳分析(allozyme electrophoresis analysis)，選擇原生並且廣泛分佈於台灣亞熱帶至溫帶的殼斗科植物長尾栲(Castanopsis carlesii) 為材料，研究其遺傳變異和探討親緣地理關係。 長尾栲葉綠體DNA的親緣地理研究，總計採集30個台灣的原生族群，包括201個單株，分析二段非編碼區序列(trnL intron 和trnV-trnM)。序列經排序後共有1663 bp，其中21個為polymorphic sites，共組成28個單套基因型(haplotype)，其中2個為原始型(ancestral type)，普遍分佈於大部分族群中。由核苷酸歧異度(π, nucleotide diversity)分析顯示，長尾栲在台灣地區有3個歧異度較高的族群，分別為北部的福山、中部蓮華池和東南部的利嘉一帶。單套型基因歧異度(h, haplotype diversity)較高的族群分別為，福山、鎮西堡、鞍馬山、蓮華池和藤枝。由π和h 顯示在台灣地區北、中、南，各有一歧異度較高的區域。分析所有族群和中央山脈(CMR, Central Mountain Range)二側的遺傳分化值 (NST - GST)，發現族群間具有高度的分化(GST=0.723)，但在中央山脈的二側族群間並沒有明顯的親緣地理結構。由葉綠體基因型的分佈情形顯示，中央山脈對台灣東、西部族群間的基因交流造成明顯阻礙，族群遷徙和基因交流僅能在中央山脈東西二側進行南北向的遷徙。根據親緣關係樹、稀有或獨特基因型的分佈、歧異度和族群間的分化(genetic divergence)等資料，推測在台灣北部雪山山脈北端和東南部各有一個冰河時期植物的可能避難所(potential refugia)。由星狀(star-like)的基因型演化關係樹、中性檢測(neutrality test)和mismatch等檢定，顯示長尾栲族群正處於族群擴張(demographic expansion)的階段。 同功酵素的電泳分析主要用來探討長尾栲的族群遺傳變異，共選擇22個族群。以水平式澱粉電泳分析法，利用9個酵素系統進行染色分析，共得到11個基因座(locus)，40個等位基因(allele)。由各個遺傳歧異度指數：等位基因平均數(the average number of alleles per locus, A= 2.5)、有效等位基因數(the effective number of alleles per locus, Ae = 1.38), 等位基因豐富度(the allelic richness, Ar = 2.38)、多型性基因座比率(the percentage of polymorphic loci, P= 69%)、異質結合度期望值(expected heterozygosity, He= 0.270)，顯示長尾栲族群具有高度的歧異度。廣泛的分佈範圍、配育系統(mating system)和其演化歷史，是影響歧異度的重要原因。而從各族群的歧異度(expected heterozygostiy)分析，發現中部的蓮華池和北部貢寮等二族群具有較高的歧異度。推測中部具有高度的歧異度是來自於後冰河期的族群拓展過程中 ，由不同的路徑所匯集而來。由各族群平均遺傳分化值(mean FST)顯示二個區域具有較高的分化值，一為位於中北部地區大約在24.80°N至24.20°N之間，包括鞍馬山(AM)、福山(FS)和東澳(TA)。另一區域位於東南部，約在22.40°N至23.10°N之間，包括新港山(HK)、多納(TN)和大武(TW)。這二個區域和許多利用葉綠體DNA研究的數種所提出的避難所位置吻合。顯示冰河期台灣植物可能的避難中心位於台灣中北部和東南部。The noncoding region sequence of cpDNA and the allozyme marker were used to conduct the phylogeographical study of Castanopsis carlesii, a subtropical and temperate tree species in Taiwan. C. carlesii, belonging to the Fagaceae, is one of the most common and dominant tree species which adapt to the subtropical and tempetate climate in Taiwan. In this study, we examined spatial patterns of chloroplast DNA (cpDNA) variation in a total of 30 populations of C. carlesii, including 201 individuals sampled throughout Taiwan. By sequencing two cpDNA fragments using universal primers (the trnL intron and the trnV-trnM intergenic spacer), we found a total of 1663 bp and 21 polymorphic sites. These gave rise to a total of 28 cpDNA haplotypes. The level of differentiation among the populations studied was relatively high (GST = 0.723). Two ancestral haplotypes are widely distributed. The Central Mountain Ridge (CMR) of Taiwan represents an insurmountable barrier to the east-west gene flow of C. carlesii. Among the populations studied, three separated populations, at Lienhuachih, Fushan and Lichia, have high nucleotide diversity. Estimates of NST - GST for populations on both sides of the CMR indicate that no phylogeographical structure exists. According to the genealogical tree, number of rare haplotype and population genetic divergence, this study suggests that two potential refugia existed during the last glaciation: the first refugium was located in a region to the north of Hsuehshan Range (HR) and west of the CMR; the second refugium was located in south, especially southeastern Taiwan. In fact, the second refugium happens to be the same as that reported for Cyclobalanopsis glauca. A “Star-like” genealogy is characteristic when all haplotypes rapidly coalesce and is a general outcome of population expansion. The neutrality test and mismatch distribution also suggest demographic expansion. Using genetic parameters to determine the heterozygosity and the mean FST value of each population, we inferred the evolutionary history of Castanopsis carlesii in Taiwan. In this study, 22 populations of C. carlesii throughout the island were sampled covering an elevational range of 50~2300 m. Starch gel electrophoresis was used to assay allozyme variations, and 11 loci from nine enzyme systems were detected. Average values of genetic parameters describing the within-population variation, the average number of alleles per locus (A = 2.5), the effective number of alleles per locus (Ae = 1.38), the allelic richness (Ar = 2.38), the percentage of polymorphic loci (P = 69%), and the expected heterozygosity (He = 0.270) were estimated. High levels of genetic diversity were found for C. carlesii compared with other local plant species. From the data of expected heterozygosity, one major diversity center was situated in central Taiwan corroborating previous reports for other plant species. According to the mean FST value of each population against the remaining populations, the most-divergent populations were situated in two places. One includes the populations of Anmashan, Fushan, and Tungao, and is located in north-central Taiwan between 24.80°N and 24.20°N. The other is located in southeastern Taiwan between 22.40°N and 23.10°N, and includes the populations of Hsinkangshan, Tona, and Tawu. These two regions are approximately convergent with the most divergent locations determined for several other plant species using chloroplastic DNA markers. In conclusion, the result of genetic differentiation study obtained from isozymes agrees well with that from chloroplastic DNA markers.中文摘要……………………………………………………………………………Ⅷ Abstract …………………………………………………………………………….Ⅹ 第一章 序論 一、親緣地理學(Phylogeography)………………………………………………..1 二、第四紀(Quaternary)冰河期對植物分佈和演化的影響……………………..1 三、分子技術於親緣地理學之應用………………………………………………2 四、台灣的植物親緣地理學研究…………………………………………………5 五、研究物種--長尾栲……………………………………………………………6 六、主要研究目的…………………………………………………………………7 七、參考文獻………………………………………………………………………9 第二章 利用長尾栲的親緣地理研究探討台灣在冰河期的潛在避難所 一、 前言………………………………………………………………………….14 二、 材料與方法………………………………………………………………….17 （一） 取樣………………………………………………………………………..17 （二） 基因體DNA之萃取及定量………………………………………………17 （三） 聚合酶連鎖反應(Polymerase Chain Reaction)和定序(Sequencing)……..18 1. 聚合酶連鎖反應（PCR, polymerase chain reaction）………………...18 2. 電泳分析(Electrophoresis analysis)…………………………………….18 3. DNA序列分析(DNA sequence analysis)………………………………20 （四） 資料分析(Data analysis)…………………………………………………..20 1. 序列比對和排序(alignment)……………………………………………20 2. 遺傳變異分析(Genetic variation)………………………………………21 3. 親緣分析(Phylogenetic analysis)……………………………………….22 4. 族群結構分析(analysis of genetic structure)…………………………...23 三、 結果………………………………………………………………………….24 (一) 葉綠體DNA的多形性和遺傳多樣性……………………………………24 (二) 單套基因型多樣性和分佈………………………………………………..27 (三) 單套型和族群間的親緣關係……………………………………………..30 (四) 中央山脈對基因型分佈的影響…………………………………………..34 (五) 中性假說檢定……………………………………………………………..36 (六) 族群結構和遺傳分化……………………………………………………..36 (七) 遺傳距離和地理距離間的相關…………………………………………..42 四、 討論………………………………………………………………………….45 (一) 葉綠體DNA的核苷酸歧異度……………………………………………45 (二) 歧異度中心(diversity center)……………………………………………...45 (三) 單套型分佈和中央山脈的阻隔效應……………………………………..46 (四) 親緣關係分析……………………………………………………………..47 (五) 冰河期避難所……………………………………………………………..49 (六) 族群匯集效應(Admixture effect)…………………………………………52 (七) 近期的族群擴張…………………………………………………………..54 (八) 後冰河期的族群拓殖……………………………………………………..54 五、 結論………………………………………………………………………….57 六、 參考文獻…………………………………………………………………….58 第三章 利用長尾栲族群的同功酵素變異探討歧異度中心和最大遺傳分化區域 一、 前言…………………………………………………………………………...67 二、 材料方法……………………………………………………………………...70 (一) 取樣…………………………………………………………………………70 (二) 電泳分析……………………………………………………………………70 (三) 資料分析……………………………………………………………………71 1. 遺傳歧異度指數…………………………………………………………71 2. 遺傳結構分析(Genetic structure analysis)………………………………74 3. Neighbor-Joining親緣關係和主成份分析(PCA)..……………………..74 4. 隔離分化(Isolation by distance)和瓶頸效應(Bottleneck)分析…..…….75 三、 結果…………………………………………………………………………..75 (一) 遺傳多樣性(genetic diversity)……………………………………………..75 (二) 族群間遺傳分化(genetic differentiation)和基因流傳(gene flow)………...77 (三) 隔離分化(Isolation by distance)檢測…………………………………..….78 (四) 瓶頸效應(Bottleneck)檢測………………………………………………...83 (五) 族群遺傳結構……………………………………………………………...83 (六) 族群間遺傳關係…………………………………………………………...83 (七) allozyme和葉綠體DNA遺傳分化比較………………………………….84 四、 討論…………………………………………………………………………..92 (一) 遺傳多樣性………………………………………………………………...92 (二) 歧異度中心(diversity center)………………………………………………93 (三) 群團分析與地理區域之相關分析…………………………………………94 (四) 族群遺傳結構和分化………………………………………………………96 (五) 遺傳分化中心………………………………………………………………98 五、 結論………………………………………..…………………………………99 六、 .參考文獻…………………………………………………………………...100 第四章 總結……………………………………………………………………...1088938742 bytesapplication/pdfen-US親緣地理學長尾栲殼斗科PhylogeographyCastanopsis carlesiiFagaceae[SDGs]SDG13[SDGs]SDG15otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/57990/1/ntu-95-D89226002-1.pdf