松科（Pinaceae）鐵杉屬（Tsuga）為亞高山的重要針葉樹種，化石證據推測其曾經廣布於整個北半球地區，如今卻呈現間斷分布，僅分布於北美、中國大陸與東亞島嶼。鐵杉屬依據Fajon（1990）認可的現存物種有9種，包括：南方鐵杉[Tsuga chinensis (Franchet) E. Pritzel]、雲南鐵杉[Tsuga dumosa (D. Don) Eichler]、麗江鐵杉[Tsuga chinensis var. forrestii (Downie) Silba]、北日本鐵杉[Tsuga diversifolia (Maxim.) Mast]、南日本鐵杉（Tsuga sieboldii Carrière）、加拿大鐵杉[Tsuga Canadensis (Linnaeus) Carrière]、科羅拉多鐵杉（Tsuga caroliniana Engelmann）、西方鐵杉[Tsuga heterophylla (Rafinesque) Sargent]和山地鐵杉[Tsuga mertensiana (Bongard) Carrière]。因分布於台灣的台灣鐵杉[Tsuga chinensis var. formosana (Hayata) H.L.Li & H.Keng]和韓國的鬱陵島鐵杉（Tsuga ulleungensis）呈現地理隔離分布型態，而包含在此研究中。本研究取得台灣鐵杉之功能性表現基因組，以此設計引子並成功篩選出18個細胞核基因座，另取得2組粒線體片段和4組葉綠體片段，以鐵杉屬11個物種共計89個樣本探討鐵杉屬的遺傳分化。
　　遺傳歧異度分析皆為低度歧異度，顯示鐵杉屬各分類群可能是由小族群繁衍而來，且因分歧時間不長而無法累積足夠的遺傳變異，在歸群分析中除了粒線體之外，北美地區的遺傳組成和中國、東亞島嶼物種的遺傳組成可以明顯分群。鐵杉屬各物種共同祖先的分歧時間約為9000萬年前，並以RASP推估共同祖先節點經歷擴散、隔離分化和滅絕事件，經實驗結果推測鐵杉屬可能是北美起源，並且由北美西部往東部擴散，再由北美東部往亞洲地區擴散，而在往亞洲地區擴散時分為二條路，一條往日本方向形成北日本鐵杉，一條往中國大陸地區形成雲南鐵杉／台灣鐵杉的共同祖先，接著再從中國大陸西部往東部地區擴散，甚至到達日本南部和台灣，而韓國的鬱陵島鐵杉則是由北日本鐵杉擴散的結果，在親緣關係與歸群分析中，都顯示二物種的關係密切。同時透過鐵杉屬分歧時間的分析協同化石證據，推論鐵杉屬可能是北美起源並且經由白令走廊進退於北美與亞洲地區，其中化石證據證明鐵杉屬在歐洲曾經廣泛分布，但因為後來溫度、濕度不適合鐵杉生存故而滅絕（180萬年前），但不可否認歐洲在過去不同時期皆是與東亞和北美有聯繫的重要地區。而鐵杉屬的種化模式因為其特殊的間斷分布方式認為可能多個種化模式造成而非單一模式種化。

Tsuga (Pinaceae) is an important coniferous taxon in the subalpine zone. The fossil record of Tsuga indicates that this genus was distributed throughout North America and Eurasia during the Late Cretaceous to the Plio-Pleistocene. However, currently, Tsuga exhibits a disjunctive distribution pattern across North America through East Asia island arcs and continental China. A total of 9 extant species recognized in Farjon (1990), including Tsuga chinensis (Franchet) E. Pritzel, Tsuga dumosa (D. Don) Eichler, Tsuga chinensis var. forrestii (Downie) Silba, Tsuga diversifolia (Maxim.) Mast, Tsuga sieboldii Carrière, Tsuga canadensis (Linnaeus) Carrière, Tsuga caroliniana Engelmann, Tsuga heterophylla (Rafinesque) Sargent, Tsuga mertensiana (Bongard) Carrière. In addition, we also sampled Tsuga chinensis var. formosana (Hayata) H.L.Li & H.Keng and Tsuga ulleungensis from Taiwan and Ullung Island, respectively; due to the unique distribution pattern such as vicariance. In the present study, we established a functional genomic database from Tsuga chinensis var. formosana then designed primers to amplified DNA sequences from 89 individuals which were sampled from 11 species. Eventually, we successfully conduct several amplification, including two mitochondria spacers, four chloroplast spacers and sixteen nuclear loci.
Our result demonstrated that Tsuga has low level of genetic diversity, indicated recent divergence of Tsuga and do not have enough time to accumulate mutations in the population. In STURCTURE assignment test based on chloroplast and nuclear loci show the best clustering at K=2 and K=7, North America, China, East Asia island have difference special genetic ingredient. The most recently common ancestor of Tsuga was at 90 mya, which is corresponding to the evidence of older Tsuga fossil (90 mya). Using RASP analysis to inferred events of dispersal, vicariance and extinction that from common ancestor nodes. The results suppose Tsuga may originate from North America that spread from Western North America to Eastern North America. Asia regional species common ancestors were from Eastern North America, that spread to two different path: one was proceed to North Japan and formatted Tsuga diversifolia , the other was proceed to China and formatted the Tsuga dumosa / Tsuga chinensis var. formosana common ancestor. The path in China spread from Eastern China to Western China even arrive at Taiwan and South Japan. The Tsuga ulleungensis was dispersal by Tsuga diversifolia from North Japan to Korea and they had closely relationship showed on the phylogenetic tree and assignment test.
Overview the results of divergence time analysis and fossil record, it can be inferred that Tsuga may have originated from North America and dispersed to Asia through the Bering Corridor. The fossil record proved the Tsuga widely distributed in Europe in the pass, years later the Tsuga became extinct cause by not suitable temperature and humidity for survival (1.8 mya). The disjunct distribution of Tsuga can be a result of speciation that can be resolved by multiple speciation models.

論文審定書................................................................................................................. i
誌謝............................................................................................................................. ii
中文摘要……………………………………………………………………………. iii
英文摘要……………………………………………………………………………. v
目錄…………………………………………………………………………………. viii
表目錄………………………………………………………………………………. xi
圖目錄………………………………………………………………………………. xii
第一章 前言
1.1　鐵杉屬的物種介紹與文獻回顧.................................................................... 1
1.2 第四紀冰河時期：物種的退縮與擴散........................................................ 5
1.3 生物地理學的概念與研究方法
1.3.1　概念介紹................................................................................................. 6
1.3.2 鐵杉屬的地質歷史................................................................................. 7
1.3.3　生物地理學的分析模型與方法............................................................. 8
1.4 物種的定義與種化機制................................................................................ 10
1.5 影響物種分歧的遺傳因素............................................................................ 12
1.6 天擇、基因漂變與中性假說........................................................................ 13
1.7 研究動機與目的............................................................................................ 15
第二章 材料與方法
2.1 研究樣本採集與製備.................................................................................... 17
2.2 以次世代定序取得鐵杉之功能性表現基因組............................................ 17
2.3 實驗方法
2.3.1 DNA濃度調整....................................................................................... 18
2.3.2 引子設計................................................................................................. 18
2.3.3 聚合酵素連鎖反應（PCR）................................................................. 19
2.3.4 產物的純化與定序................................................................................. 21
2.3.5 T&A clone............................................................................................... 21
2.4 資料分析
2.4.1 序列比對與排列..................................................................................... 22
2.4.2 遺傳多樣性分析..................................................................................... 22
2.4.3 親緣演化樹分析..................................................................................... 23
2.4.4 中性假說測試與天擇檢測..................................................................... 24
2.4.5 歸群分析................................................................................................. 26
2.4.6 分歧時間評估......................................................................................... 27
2.4.7 評估物種過去的歷史事件：Dispersal-Vicariance model..................... 28
第三章 結果
3.1 台灣鐵杉次世代定序之總轉錄本（transcriptome）.................................. 30
3.2 遺傳多樣性分析與中性檢測
3.2.1　遺傳多樣性分析..................................................................................... 30
3.2.2　中性檢測................................................................................................. 32
3.3 親緣關係之重建............................................................................................ 33
3.3.1 以粒線體片段重建親緣樹……………................................................. 34
3.3.2 以葉綠體片段重建親緣樹………………............................................. 35
3.3.3 以細胞核基因座重建親緣樹…………................................................. 36
3.4 天擇檢測........................................................................................................ 38
3.5 歸群分析........................................................................................................ 40
3.6 分歧時間估計................................................................................................ 44
3.7 物種過去的歷史事件.................................................................................... 46
第四章 討論
4.1台灣鐵杉之總轉錄本....................................................................................... 49
4.2鐵杉屬物種遺傳多樣性與親緣關係............................................................... 50
4.3親緣關係不一致性討論................................................................................... 53
4.4鐵杉屬分歧時間討論與過往歷史事件........................................................... 54
第五章 結論............................................................................................................. 58
參考文獻………………………………………………………………………......... 98

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