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研究生:洪龍華
研究生(外文):Lung-Hua Hung
論文名稱:利用細胞核核糖體DNA基因之ITS序列和轉譯延長因子重建台灣沼緣蜉蝣(CloeonmarginaleHagen)的親緣地理
論文名稱(外文):Phylogeography of Cloeon marginale Hagen (Ephemeroptera : Baetidae) in Taiwan based on nuclear ribosomal DNA and nuclear translation elongation factor 1α
指導教授:蔣鎮宇蔣鎮宇引用關係王建平王建平引用關係
指導教授(外文):Tzen-Yuh ChiangJiang-Ping Wang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物學系碩博士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:73
中文關鍵詞:基因交流關鍵族群相對年齡親系分配滅絕與再拓殖細胞核轉譯延長因子台灣沼緣蜉蝣親緣地理核糖體核酸ITS序列
外文關鍵詞:extinction-recolonization.phylogeographynuclear rDNA ITSgene flowlineage sortingrelated agemetapopulationmayfly Cloeon marginale Hagennuclear translation elongation factor (EF-1α)
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本研究係利用細胞核核糖體 DNA (rDNA) 之 ITS 序列和轉譯延長因子 (EF-1α) DNA 序列重建台灣沼緣蜉蝣的親緣地理型式,估算族群內及族群間的基因流轉,以測驗地理區域間族群遺傳分隔的假說。自全島北、中、南和東部 8 個代表性靜水型沼澤 13 個採樣區取得 130 個樣本,共選殖出 87 個 rDNA haplotypes和 53 個 EF-1α haplotypes,以 DnaSP 計算 haplotype diversity 和 nucleotide diversity,其中 rDNA haplotypes diversity 為 0.980,EF-1α haplotypes diversity 為 0.878,而 nucleotide diversity 分別是 0.133 與 0.114,全島以北部地區和南部地區的 nucleotide diversity 較高。由 neighbor-joining 和 network 親緣分析的結果均獲得 11 個 rDNA lineages (clade A-K) 和 7 個 EF-1α lineages (clade I-VII),不僅分佈最廣泛的 lineages 彼此相結合,稀有的 lineages 也有較高的比例和分佈最廣泛的 lineages 結合,卡方檢測顯示全島合一的大族群處於平衡,其中有一些稀有的 lineages 被侷限在少數地理區塊,推測應該是基因相對年輕或遺傳漂變的結果;而兩個基因譜系都建構出一致的親緣地理型式(族群間或地理區域間的遺傳分化低),顯示台灣沼緣蜉蝣發生頻繁的遷移。rDNA 和 EF-1α 都指出族群間或地理區域間有很高的基因交流,但基因交流的型式並不一致,推測應該是這兩個基因譜系處在 lineage sorting 所導致。從 haplotype 和 nucleotide 歧異度、neighbor-joining tree、network tree 及基因交流值結果均顯示台灣沼緣蜉蝣進行長距離散播,推測這個非預期的結果是由風和水鳥所導致。根據 rDNA 和 EF-1α 基因譜系推估出整個台灣地區的台灣沼緣蜉蝣是一個逢機配對的族群,所有採樣點是關鍵族群,根據生態資料和遺傳組成推測北部地區、南部地區和中部魚池樣點應該是台灣沼緣蜉蝣的根源地,而 sink 關鍵族群的遺傳組成深受滅絕和再拓殖的影響。
To reconstruct the phylogeographic pattern of Cloeon marginale Hagen, two DNA markers were sequenced. In total, 87 and 53 haplotypes of the nuclear ribosomal DNA internal transcribed spacer region (rDNA) and translation elongation factor 1α (EF-1α) were identified, respectively, from 130 individuals. Haplotype diversity of 0.980 and 0.878 was estimated, respectively, from rDNA and EF-1α. The nucleotide diversity of northern and southern regions was higher than that of other areas. Neighbor-joining trees and networks recovered eleven divergent rDNA lineages (clade A-K) and seven EF-1α divergent lineages (clade I-VII). A chi-square test revealed random associations between rDNA types and EF-1α types (X2=73.586, P=0.11176). Nevertheless, lock of clade-clade consistency between two gene genealogies was probably ascribed to effects of lineage sorting. Two data sets revealed consistent phylogeographic patterns, i.e., low levels of genetic differentiation among regions and among populations, indicating frequent long-distance gene-flow between populations. Typhoons or monsoons carrying mature mayflies across populations and geographical regions may be the mechanism explaining the low levels of population differentiation. Random associations between haplotype of two DNA markers suggested an equilibrium of populations of Taiwan as a whole, and a metapopulation structure that is consist of demes linked by different levels of gene flow. Ecological data and high level of intrapopulation variation implicated that northern and southern regions, as well as YU of central region may have been sources for migration into re-colonized populations. The sink demes were highly regulated by extinction-recolonization processes.
中文摘要 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥I
英文摘要‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥II
誌謝 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥III
目錄 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥V
圖目‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥VIII
表目次‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥IX
壹、前言
一、親緣地理學 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥1
二、蜉蝣研究文獻回顧 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥4
(一)蜉蝣的生物學 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥4
(二)台灣蜉蝣的文獻整理 ‥‥‥‥‥‥‥‥‥‥‥‥5
(三)台灣沼緣蜉蝣的研究近況 ‥‥‥‥‥‥‥‥‥‥5
(四)台灣沼緣蜉蝣是研究親緣地理的適當物種 ‥‥‥6
三、核糖體核酸序列的遺傳特性與在昆蟲上之應用 ‥‥7
四、細胞核轉譯延長因子的遺傳特性與在昆蟲上之應用 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥9
五、研究目的‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥11
貳、研究材料及方法
一、材料採集‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥12
(一)採集地點‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥12
(二)取樣方法與鑑定‥‥‥‥‥‥‥‥‥‥‥‥‥‥12
二、分子技術‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥12
(一)DNA萃取與定量 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥13
(二)聚合酵素連鎖反應‥‥‥‥‥‥‥‥‥‥‥‥‥13
1. 引子的設計 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥13
(1) 細胞核核糖體 DNA‥‥‥‥‥‥‥‥‥‥‥‥‥‥13
(2) 細胞核轉譯延長因子‥‥‥‥‥‥‥‥‥‥‥‥‥14
2. DNA 擴增反應 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥14
(1) 細胞核核糖體 DNA 片段增殖 ‥‥‥‥‥‥‥‥‥14
(2) 細胞核轉譯延長因子片段增殖片段增殖‥‥‥‥‥15
(三)純化‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥15
(四)T-A cloning ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥15
(五)轉型作用‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥15
1. 製備勝任細胞 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥15
2. 轉型作用 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥16
3. 塗碟 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥16
(六)微量製備質體DNA ‥‥‥‥‥‥‥‥‥‥‥‥‥16
1. 養菌 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥16
2. 微量抽取 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥16
(七)DNA定序 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥16
三、資料分析‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥17
(一)DNA序列分析與親緣分析 ‥‥‥‥‥‥‥‥‥‥17
1. 序列的整理與校對 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥17
2. 序列的排列 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥17
3. 序列特性分析 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥18
4. 親緣樹的建構 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥18
(二)族群遺傳分析‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥18
(三)哈溫平衡之檢測‥‥‥‥‥‥‥‥‥‥‥‥‥‥19
參、結果
一、定序 DNA 模版增殖結果 ‥‥‥‥‥‥‥‥‥‥‥20
二、DNA 序列特徵‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥20
(一)鹼基組成‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥20
1. 細胞核核糖體 DNA ‥‥‥‥‥‥‥‥‥‥‥‥‥‥20
2. 細胞核轉譯延長因子 ‥‥‥‥‥‥‥‥‥‥‥‥‥21
(二)haplotype diversity 和 nucleotide diversity ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥21
1. 細胞核核糖體 DNA ‥‥‥‥‥‥‥‥‥‥‥‥‥‥22
2. 細胞核轉譯延長因子 ‥‥‥‥‥‥‥‥‥‥‥‥‥22
(三)重組事件和中性測驗‥‥‥‥‥‥‥‥‥‥‥‥23
1. 細胞核核糖體 DNA ‥‥‥‥‥‥‥‥‥‥‥‥‥‥23
2. 細胞核轉譯延長因子 ‥‥‥‥‥‥‥‥‥‥‥‥‥23
三、基因譜系與 rDNA 和 EF-1α lineage 結合分析 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥23
1. 細胞核核糖體 DNA ‥‥‥‥‥‥‥‥‥‥‥‥‥‥24
2. 細胞核轉譯延長因子 ‥‥‥‥‥‥‥‥‥‥‥‥‥25
四、族群遺傳‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥26
1. 細胞核核糖體 DNA ‥‥‥‥‥‥‥‥‥‥‥‥‥‥26
2. 細胞核轉譯延長因子 ‥‥‥‥‥‥‥‥‥‥‥‥‥27
肆、討論
一、台灣沼緣蜉蝣的細胞核 ITSs DNA序列演化特徵 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥29
二、族群遺傳‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥30
三、基因譜系與 rDNA 和 EF-1α lineage 結合分析 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥33
四、族群動態‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥34
伍、結論‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥38
陸、參考文獻‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥39
柒、附錄‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥73


圖目次
Fig. 1 Structural features of the rDNA tandem repeat module (drawn to approximate scale) in insects. Black regions indicate internal transcribd spacers, which often differ in length. ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥48
Fig. 2 Cloeon marginale Hagen sample locations and distribution. Abbreviations of populations are given in Table 1. ‥‥‥‥‥‥‥‥‥‥‥‥‥49
Fig. 3 The region of rDNA used in the study and location of primers, arrow indicating the direction of PCR amplification. (Modify Whiting et al., 1997) ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥50
Fig. 4 Neighbor-joining tree representative sequences (haplotypes) of nuclear rDNA in Cloeon marginale Hagen. Numbers at notes indicate bootstrap values. rDNA types (A-K) are labeled on clades. ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥51
Fig. 5 Minimum spanning network generated using method of Excoffier and Smouse (1994) for types of nuclear rDNA of populations of Cloeon marginale Hagen. Mutational changes are indicated at nodes. ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥52
Fig. 6 Neighbor-joining tree representative sequences (haplotypes) of nuclear EF-1α in Cloeon marginale Hagen. Numbers at notes indicate bootstrap values. EF-1α types (I-VII) are labeled on clades.‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥53
Fig. 7 Minimum spanning network generated using method of Excoffier and Smouse (1994) for types of nuclear EF-1α of populations of Cloeon marginale Hagen. Mutational changes are indicated at nodes. ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥54
Fig. 8 Frequency of nuclear types (rDNA-EF-1α associations) in each population is indicated in pie diagrams. Abbreviations of populations are given in Table 1. ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥55
Fig. 9 Scatter plot of logarithmic scales of Nm and geographical distance between 13 populations of Cloeon marginale Hagen.‥‥‥‥‥‥‥‥‥‥‥57


表目次
Table 1. Materials of Cloeon marginale Hagen collected from different populations in Taiwan used for nuclear ribosomal DNA and elongation factor 1α (EF-1α) sequencing. Locality, area, sample size, nuclear types (rDNA type and EF-1α type associations), and aquatic profile of each population are indicated. ‥‥‥‥‥‥‥‥‥‥‥58
Table 2. Sequences and position of primers used for the PCR amplification of rDNA and EF-1α. ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥60
Table 3. Descriptive statistics for separate and combined rDNA partitions. ‥‥‥‥‥‥‥‥‥‥‥61
Table 4. Estimates of haplotype diversity (h) and nucleotide diversity (θ) with populations of Cloeon marginale Hagen based on rDNA sequences. Possible minimum recombination events are inferred using software DnaSP. Testing statistics for neutrality at rDNA. These symbols for populations see Table 1.‥‥‥‥‥‥‥‥‥‥‥‥62
Table 5. Estimates of haplotype diversity (h) and nucleotide diversity (θ) with populations of Cloeon marginale Hagen based on EF-1α sequences. Possible minimum recombination events are inferred using software DnaSP. Testing statistics for neutrality at EF-1α. These symbols for populations see Table 1. ‥‥‥‥‥‥‥‥‥‥‥64
Table 6. Distribution of rDNA types (A-K) among populations of Cloeon marginale Hagen. Regions are indicated: Northern region (N), Central region (C), Southern region (S), and Eastern region (E). ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥66
Table 7. Distribution of Elongation factor 1α (EF-1α) types (I-VII) among populations of Cloeon marginale Hagen. Regions are indicated: Northern region (N), Central region (C), Southern region (S), and Eastern region (E). ‥‥‥‥‥‥67
Table 8. Association Between rDNA types and EF-1α types of Cloeon marginale Hagen. Distribution region of each type is indicated in square brackets. Percentage of each complex type is indicated in parentheses. W: widespread. Other symbols see Table 1.‥‥‥‥‥‥‥‥‥‥‥‥‥‥68
Table 9. Pairwise FST/Nm estimates between populations based on genetic variation of rDNA.‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥69
Table 10.Pairwise FST/Nm estimates between populations based on genetic variation of EF-1α. ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥70
Table 11.Pairwise FST/ Nm estimates between geographical regions (N, E, S, C) based on genetic variation of nrDNA (below the diagonal) and EF-1α (above the diagonal).‥‥‥‥‥‥‥‥71
Table 12.Observed number (O) of genotype frequency is compared to expected value (E) based on Chi-Square analysis (X2=73.586, P=0.11176).‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥72
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