臺灣博碩士論文加值系統

本研究針對廣西地區廣布型的鹿寨秋海棠進行族群遺傳學與親緣地理學研究，共取樣10個族群，透過新開發的16組微衛星體以及葉綠體DNA片段trnC-ycf6，以不同的角度檢視中越邊境亞熱帶喀斯特地貌中，族群的分化模式及歷史的族群量變動，以檢驗從側膜組秋海棠演化樹所推論的種化機制假說，亦即在旺盛的喀斯特作用下，破碎地貌促成的距離隔離為喀斯特地貌中異域種化的主要機制。並進一步藉此特有於中越邊境石灰岩區域的代表性支序，作為研究喀斯特地景中生物多樣性起源的案例參考。
本研究首先以次世代定序技術，開發表現序列標幟微衛星體作為族群遺傳研究的分子標記。在44,368個組合序列中，搜尋出5,602個含有表現標幟微衛星體的基因座，並從中開發出16組具多型性的引子對。以此為工具，本研究進而分析鹿寨秋海棠的族群遺傳特性，其偏高的FST值顯示高度分化的狀況。另透過貝氏群聚分析，發現各種群集數量的分布皆具有顯著的地理結構；進而，在地理距離與族群遺傳距離的相關性檢測中發現高度的正相關，搭配trnC-ycf6序列的基因單型網狀圖以及巢狀支序分析，可證實在不同階層與時間尺度下距離隔離分化與族群擴張間歇出現，此在描述歷史族群量變動的差異分布分析、中性檢測以及貝氏天際線分析中均獲得不同時間尺度的驗證，顯示鹿寨秋海棠在起源之初曾隨著喀斯特地貌的旺盛發育而呈現族群擴張後距離隔離的分化歷史，而在近期有效族群量的緩慢下降，可能因亞洲大陸五萬年前受冰期影響所產生之乾燥氣候導致。由微演化的研究與巨觀的演化樹相對照，可推論喀斯特地貌中距離隔離的分化模式在更長的時間與空間尺度上很可能成為異域種化的重要成因，也很可能扮演著喀斯特生物多樣性起源的主要機制。
在廣西喀斯特石灰岩地區，族群的遺傳多樣性呈現在各處分化出的獨特基因型上，也表示著任何一地的局部滅絕均可能造成特定基因型的喪失，整體生物多樣性的調查與保育是當前重要的課題。

To investigate the origin of limestone karst biodiversity, this project takes Begonia sect. Coelocentrum as a model system. We incorporate the studies of population genetics and phylogeography of a widely distributed species Begonia luzhaiensis to test the hypothesis of allopatric speciation via isolation-by-distance triggered by extensive karstification. We sampled 10 populations from limestone karst area of Guangxi, China. Using next-generation sequencing (NGS) technique, 16 polymorphic EST-SSR markers was developed from 5,602 contigs, combine with chloroplast DNA sequence trnC-ycf6 as tool of the population genetics study. Significantly high FST suggests strong population differentiation. Based on Bayesian clustering analyses, all populations were restrictively distributed in isolated area. Mantel Tests indicate a significant positive correlation between genetic differentiation and geographic distances, which is also suggested by nested clade analyses of the haplotype network of trnC-ycf6. Historical demography was described by neutrality test, mismatch distribution analysis and Bayesian skyline plot analysis. In the higher level clades, a pattern of range expansion followed by isolation-by-distance was shown, as predicted based on the early developments of karst area. A recent decline of effective population size might have been caused by increased aridity of East Asia at ca. 50,000 years ago during the Pleistocene glacial period. The microevolution patterns of Begonia luzhaiensis suggest that the similar process might have caused allopatric speciation via isolation-by-distance in a larger time scale, and support strongly the importance of extensive karstification in shaping the species diversity on the Sino-Vietnamese limestone karsts. From a conservation prospective, geographically isolated populations on limestone karsts often harbor unique genetic variation that is threaten by rapid habitat destructions and their uniqueness deserves high conservation priority.

圖目錄 I
表目錄 II
一、前言 1
（一）喀斯特地貌簡介 1
（二）喀斯特地景生物多樣性之特色 2
（三）自巨演化角度看喀斯特地區秋海棠的起源 5
（四）以微演化角度看喀斯特物種的分化機制 9
（五）研究目標 12
二、材料與方法 14
（一）微衛星體開發 14
（二）取樣、定序與基因型鑑定 15
（三）族群遺傳參數統計 18
（四）族群遺傳結構 18
（五）分子遺傳變異數分析 19
（六）距離隔離模式檢驗 19
（七）基因單型網狀圖建立 20
（八）巢狀支序分析 20
（九）中性檢測 21
（十）差異分布分析 22
（十一）替換率估算與貝氏天際線分析 23
三、結果 24
（一）微衛星體開發結果 24
（二）鹿寨秋海棠的族群遺傳特徵 27
（三）族群遺傳結構 28
（四）族群分化模式 32
（五）親緣地理結構 36
（六）歷史族群變動 39
四、討論 41
（一）鹿寨秋海棠的族群分化歷史 41
（二）族群擴張事件與喀斯特地貌之演化 42
（三）近期的族群下降與保育上的啟示 42
（四）側膜組秋海棠種化機制之推論 43
五、結論 44
參考文獻 45
附錄：鹿寨秋海棠採集資訊一覽表 55

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