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研究生:林柏安
研究生(外文):Bo-An Lin
論文名稱:台灣產豆蘭屬植物系統分類研究
論文名稱(外文):Phylogenetics of Bulbophyllum (Orchidaceae) in Taiwan
指導教授:胡哲明胡哲明引用關係
指導教授(外文):Jer-Ming Hu
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生態學與演化生物學研究所
學門:生命科學學門
學類:生態學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:108
中文關鍵詞:豆蘭matKtrnC-rpoB intergenic spacertrnL-trnF intergenic spacer最大簡約法鄰近相接法貝葉氏導出式分析
外文關鍵詞:BulbophyllummatKtrnC-rpoB intergenic spacertrnL-trnF intergenic spacermaximum parsimonyneighbor-joiningBayesian inference
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蘭科 (Orchidaceae) 豆蘭屬 (Bulbophyllum Thouars) 植物為泛熱帶性分布的植物類群,同時也是全世界最大的屬,目前有超過2000個以上的物種被命名。台灣植物誌第2版所記載之台灣產豆蘭共有21個物種,後續數年的野外觀察又發表了數種新物種。至今日台灣的豆蘭屬植物目前共發表了30個分類群,其中共包括23個種和7個變種。本研究使用了28個台灣產豆蘭分類群資料,同時也加入非台灣產豆蘭之序列一併分析,分析方法為使用3種葉綠體分子標記:matK局部序列、trnC-rpoB intergenic spacer以及trnL-trnF intergenic spacer之局部序列進行PCR擴增並定序,再使用核酸排序後,進行最大簡約法、鄰近相接法 以及貝葉氏導出式分析來分析譜系關係,以期建立台灣產豆蘭屬內之親源關係,並佐以外部特徵來探究特徵演化之歷程。
三種葉綠體分子標記分析所得之演化樹雖不盡相同,但大致上之分群組成仍然相似;單花類型的物種在台灣產豆蘭屬中較靠近基群的位置,尤以無假球莖單花類型者最接近基群;而側萼片修長且相貼的捲瓣蘭節物種形成台灣產豆蘭核心的一群,而與側萼片較短的捲瓣蘭物種分開。而特徵演化的追蹤,也可發現捲瓣蘭類的重要特徵:側萼片形成修長狀是捲瓣蘭核心物種的重要特徵,在台灣的豆蘭中,這種特徵演化在3個分子標記建構的演化樹都一致。而針對3群種內變異的分析顯示產於南投的長萼片型白毛捲瓣蘭其核酸變異程度較大,而花蓮捲瓣蘭變異族群因為核酸差異非常小故仍以花蓮捲瓣蘭視之,長軸捲瓣蘭的天池變異族群,在核酸變異度上也未有非常明顯的變異。而本實驗對三種葉綠體分子標記的結果顯示,matK較不適合探討台灣產豆蘭屬的關係,因其變異度較低,對豆蘭屬譜系分析的解析度不高,相較之下,trnC-rpoB IGS與trnL-trnF IGS較適合。


The genus Bulbophyllum is the largest taxon in Orchidaceae and there are nearly 2000 species distributed in the pantropical region. There were 21 species documented in the Flora of Taiwan second edition, and several new Bulbophyllum taxa were described afterwards and currently there are 23 species and 7 varieties recorded in Taiwan. This study analyzed 28 Bulbophyllum taxa from Taiwan and 10 others from Southeast Asia. Three chloroplast DNA molecular markers were examined in this study: matK partial sequence, trnC-rpoB intergenic spacer and trnL-trnF intergenic spacer partial sequence. Phylogenetic analysis was perfomed using maximum parsimony, neighbor-joining and Bayesian inference. Morphological character evolution was examined on the molecular phylogenies.
Three different chloroplast DNA markers show similar pattern: solitary inflorescence group were at the basal position (especially the group without pseudobulb). Section Cirrhopetalum (lateral sepals longer than dorsal sepal, usually umbellate inflorescence) formed a core group, separated from those with shorter lateral sepals.
Intraspecific studies of molecular phylogenetics from B. albociliatum、B. hirundinis and B. elecrtinum were perfomed. The result show that a Nantou sample of B. albociliatum var. albociliatum with a larger variation in nucleotide sequence.
The result of three chloroplast markers used in this study show matK region is not a suitable marker in Bulbophyllum in Taiwan, because of its low sequence variation and problematic phylogenetic relationship of B. affine and B. macraei. New molecular markers are needed for phylogenetic studies of Bulbophyllum in the future.


英文摘要 X
壹、前言 1
貳、研究材料與方法 17
一、實驗材料 17
二、植物總DNA之萃取 17
三、植物總DNA之定量 20
四、聚合酶連鎖反應 20
五、PCR產物膠體電泳及定序 22
六、序列分析之軟體 23
七、序列與資料矩陣 24
八、譜系分析方法 25
1. 高度簡約原理 (Maximum Parsimony, MP) 25
2. 鄰近相接法 (Neighbor-Joining, NJ) 26
3. 貝葉氏導出氏分析 (Bayesian Inference, BI) 27
4. 最大似然性分析 (Maximum Likelihood, ML) 28
九、形態特徵 29
參、結果 35
一、序列資料分析 35
二、個別序列矩陣分析 36
1. 葉綠體matK局部序列資料矩陣分析 36
1.1 最大簡約法分析結果 36
1.2 鄰近相接法分析結果 37
1.3 貝葉氏導出氏分析結果 38
2. trnC-rpoB intergenic spacer 資料矩陣分析 40
2.1 最大簡約法分析結果 40
2.2 鄰近相接法分析結果 41
2.3 貝葉氏導出氏分析結果 41
3. trnL-trnF intergenic spacer 局部序列資料矩陣分析 43
3.1 最大簡約法分析結果 43
3.2 鄰近相接法分析結果 43
3.3 貝葉氏導出氏分析結果 44
三、基因間區塊同質性分析 (Partition homogeneity test) 46
四、核酸替換模型分析 49
五、合併序列超資料矩陣分析 (supermatrix analysis) 50
1. 最大簡約法分析結果 50
2. 最大似然性分析結果 52
3. 貝葉氏導出氏分析結果 52
六、特徵演化之分析 55
肆、討論 61
一、紋星蘭和烏來捲瓣蘭在葉綠體譜系中的矛盾 61
二、葉綠體matK基因區段 62
三、葉綠體trnC-rpoB IGS與trnL-trnF IGS基因區段 63
四、合併序列超矩陣分析 64
五、形態特徵 66
六、台灣豆蘭屬植物種以下的分類群 69
七、結論與未來展望 70
伍、參考文獻 79
陸、附錄 85


表次
表1-1 白毛捲瓣蘭與維明豆蘭之特徵差 9
表1-2 長軸捲瓣蘭與蘇氏長軸捲瓣蘭、無毛捲瓣蘭之特徵差異 9
表1-3 觀霧豆蘭與克森豆蘭、石仙桃豆蘭之特徵差異 9
表1-4 台灣產豆蘭屬物種分節之情形 12
表2-1 2X CTAB 緩衝液配方 19
表2-2 台灣產豆蘭屬植物之形態特徵資料矩陣表 30
表2-3 本研究主要使用之引子序列 34
表3-1 比較matK與trnC-rpoB IGS區塊同質性分析 47
表3-2 比較trnC-rpoB IGS與trnL-trnF IGS區塊同質性分析 47
表3-3 比較matK與trnL-trnF IGS區塊同質性分析 48
表3-4 各基因資料矩陣使用jModelTest軟體分析log likelihood score 49
表4-1 針對白毛捲瓣蘭變種與近緣分類群之距離矩陣 74
表4-2 針對花蓮捲瓣蘭變異族群與近緣分類群之距離矩陣 75
表4-3 長軸捲瓣蘭天池變異族群與其近緣分類群之距離矩陣 77
表6-1 用於序列分析之台灣產豆蘭採集紀錄 85
表6-2 用於序列分析之東南亞產豆蘭採集紀錄 87
表6-3 GenBank上所引用參考序列表 88

圖次
圖1-1 豆蘭屬世界分布圖 3
圖1-2 豆蘭形態圖 5
圖1-3 捲瓣蘭形態圖 6
圖2-1 鄰近相接法原理 26
圖2-2 matK序列擴增位置示意圖 33
圖2-3 trnC-rpoB intergenic spacer序列擴增位置示意圖 33
圖2-4 trnL-trnF intergenic spacer序列擴增位置示意圖 33
圖3-1 以matK 局部序列依貝葉氏導出式分析所建構之譜系樹合併統計支持度 39
圖3-2 以trnC-rpoB IGS序列依貝葉氏導出式分析所建構之譜系樹合併統計支持度 42
圖3-3 以trnL-trnF IGS序列之資料矩陣依貝葉氏導出式分析所建構之譜系樹合併統計支持度 45
圖3-4 以合併序列超資料矩陣依最大簡約法建構譜系樹之嚴格一致性樹 51
圖3-5 以合併序列超資料矩陣依貝葉氏導出式分析建構之合併統計支持度譜系樹 54
圖3-6 使用20個外部特徵依最大簡約法建立之嚴格一致性樹 57
圖3-7 綜合兩種特徵比較圖:萼片形狀與花序 58
圖3-8 綜合兩種特徵比較圖:側萼片長度與其貼合現象 59
圖3-9 綜合兩種特徵比較圖:假球莖與萼片被毛 60
圖4-1 trnL-trnF IGS去除GenBank上馬達加斯加島上所有豆蘭序列後,重新排序以貝葉氏導出式分析之譜系樹 72
圖4-2 trnL-trnF IGS去除GenBank上馬達加斯加島上所有豆蘭序列後,重新排序以貝葉氏導出式分析之譜系樹 (Phylogram) 73
圖4-3 白毛捲瓣蘭變異族群於trnC-rpoB IGS之變異位點 74
圖4-4 白毛捲瓣蘭變異族群於trnL-trnF IGS之變異位點 74
圖4-5 花蓮捲瓣蘭變異族群於trnC-rpoB IGS之變異位點 75
圖4-6 花蓮捲瓣蘭變異族群於trnL-trnF IGS之變異位點 75
圖4-7 長軸捲瓣蘭天池變異族群於matK之變異位點 76
圖4-8 長軸捲瓣蘭天池變異族群於trnL-trnF IGS之變異位點 76
圖4-9 長軸捲瓣蘭天池變異族群於trnL-trnF IGS之變異位點 76
圖6-1 以matK序列依最大簡約法所建構之嚴格一致性樹 90
圖6-2 以葉綠體matK局部序列資料矩陣依最大簡約法所建構同等長度之譜系樹 (phylogram) 之一 91
圖6-3 以葉綠體matK局部序列資料矩陣依鄰近連接法所建構之譜系樹 92
圖6-4 以葉綠體matK局部序列資料矩陣依鄰近相接法所建構之譜系樹 (Phylogram) 93
圖6-5 以matK 局部序列之資料矩陣依貝葉氏導出式分析所建構之譜系樹 94
圖6-6 以trnC-rpoB IGS序列依最大簡約法所建構之嚴格一致性樹 95
圖6-7 以葉綠體trnC-rpoB IGS序列資料矩陣依最大簡約法所建構同等長度之譜系樹 (phylogram) 之一 96
圖6-8 以葉綠體trnC-rpoB intergenic spacer序列資料矩陣依鄰近連接法所建構之譜系樹 97
圖6-9 以葉綠體trnC-rpoB intergenic spacer序列資料矩陣依鄰近相接法所建構之譜系樹 (phylogram) 98
圖6-10 以trnC-rpoB intergenic spacer序列之資料矩陣依貝葉氏導出式分析所建構之譜系樹 99
圖6-11 綠體trnL-trnF intergenic spacer局部序列資料矩陣依最大簡約法所建構之嚴格一致性樹 100
圖6-12 以trnL-trnF intergenic spacer 序列矩陣依最大簡約法所分析之同等長度譜系樹 (phylogram) 之一 101
圖6-13 以trnL-trnF intergenic spacer序列資料矩陣依鄰近連接法所建構之譜系樹 102
圖6-14 以trnL-trnF intergenic spacer序列資料矩陣依鄰近連接法所建構之譜系樹 (Phylogram) 103
圖6-15 trnL-trnF intergenic spacer序列之資料矩陣依貝葉氏導出式分析所建構之譜系樹 104
圖6-16 以合併序列超資料矩陣依最大簡約法建構同等長度譜系樹 (phylogram) 之一 105
圖6-17 以合併超序列資料矩陣依最大似然性分析建構之譜系樹 106
圖6-18 以合併序列超資料矩陣依最大似然性分析所建構之譜系樹(phylogram) 107
圖6-19 以合併序列超資料矩陣依貝葉氏導出式分析所建構之譜系樹(phylogram) 108


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