近年來，粒腺體基因分子標記被廣泛地使用在分析文昌魚（頭索動物亞門）之類緣關係，但卻鮮少使用同樣能提供演化訊息的核基因。本研究使用五個分子標記，包含粒腺體基因（12S rRNA, Cytochrome c oxidase I, Cytochrome b）和核基因 （Histone H3, 18S rRNA）來推論七種西太平洋與兩種大西洋文昌魚的類緣關係，涵蓋了文昌魚的三個屬，這是目前已知文昌魚類緣關係分析中種類最完整的。文昌魚與外群的關係中，文昌魚為脊索動物中的基群，尾索動物與脊椎動物為姐妹系群。類緣關係分析的結果中，最大簡約法（Maximum Parsimony）和最大似然法（Maximum Likelihood），但不包括貝葉氏譜系分析（Bayesian Inference），顯示偏殖文昌魚屬（Asymmetron）是基群，鰓口文昌魚屬（Branchiostoma）和側殖文昌魚屬（Epigonichthys）互為旁系群。文昌魚的最近共同祖先約起源於134.56 百萬年前的¬特提斯海，並於50百萬年間在白堊紀分化成三個屬，這段時間與東印度洋的開啟相吻合，這三個屬可能都起源於印度洋與西太平洋。本研究首次以核基因確認了偏殖文昌魚屬中兩個潛在隱蔽種各為單系群。鰓口文昌魚屬可分為兩個演化支，分別是大西洋演化支（B. floridae 與 B. lanceolatum）和太平洋演化支（B. japonicum、 B. belcheri 與 B. malayanum）。現生側殖文昌魚屬僅分佈在印度西太平洋，可能遷移路徑為從印度洋往西到西太平洋。另外，我們在南沙群島發現了短刀側殖文昌魚（E. cultellus）可能的隱蔽種，顯示南中國海文昌魚的多樣性比想像的更高。

Recently, the phylogeny of amphioxus has been widely investigated with molecular markers of mitochondrial gene, but rarely of nuclear gene which also provides evolutionary information. Our study inferred the phylogenetic relationships of seven amphioxus species from the West Pacific and two amphioxus species from the Atlantic covering the three genera with five molecular markers including both mitochondrial (12S rRNA, Cytochrome c oxidase I, Cytochrome b) and nuclear genes (Histone H3, 18S rRNA). This is the most comprehensive taxon sampling of amphioxus phylogeny to date. The phylogenetic relationships of cephalochordate and the outgroups show that cephalochordate is the basal chordate, and tunicate and vertebrate are sister clades. In our phylogenetic results, MP and ML but not BI approach, showed that Asymmetron is the basal clade, and Branchiostoma and Epigonichthys which are sisters diverged later. The most recent common ancestor of amphioxus probably originated at 134.56 million years ago in the Tethys Sea and diverged into three genera within 50 million years in Cretaceous. This period coincides with the opening of the eastern Indian Ocean. All the three genera probably originated in the Indo-West Pacific Ocean. Our phylogenetic trees confirmed the reciprocal monophylies of the two potential cryptic species in A. lucayanum with nuclear genes for the first time. Branchiostoma is divided into the Atlantic clade (B. floridae and B. lanceolatum) and the Indo-West Pacific clade (B. japonicum, B. belcheri and B. malayanum). Extant Epigonichthys species occurred only in the Indo-West Pacific Ocean. They probably migrated westward from the Indian Ocean into the Pacific Ocean. In addition, we found a potential cryptic species of E. cultellus in the Spratly Island which further increases the diversity of amphioxus in the South China Sea.

Thesis validation letter…………………………………………………….……………………..i
Acknowledgement………………………………………………………………………………ii
Abstract (Chinese)……………………………………………………………………………... iii
Abstract (English)…………………………………………………….……………………...... iv
Table of Contents…………………………………………………………………………….... vi
Table of Figures……………………………………………………………………………..…viii
Table of Tables…………………………………………………………………………….........ix
1 Introduction …………………………………………………………………………….......1
1.1 Introduction to amphioxus………………………………………………………..…1
1.2 Literature review of amphioxus phylogeny…………………………………………2
1.3 DNA markers…………………………………………………………………..……4
1.4 Goals in this study………………………………………………………………...…5
2 Materials and Methods……………………………………………………………………7
2.1 Sample collection……………………………………………………………………7
2.2 Species identification……………………………………………………………..…7
2.3 Molecular data………………………………………………………………….……7
2.3.1 DNA extraction……………………………………………………………7
2.3.2 PCR amplification and sequencing…………………………………..……8
2.4 Phylogenetic analyses………………………………………………………………10
2.4.1 Alignment…………………………………………………………...……10
2.4.2 Concatenation of genes………………………………………………...…10
2.4.3 Partitioning scheme and model selection…………………………………11
2.4.4 Phylogenetic analyses………………………………………………..……11
2.5 Estimation of divergence time………………………………………………………12
2.6 Ancestral biogeography reconstruction……………………………………………..13
3 Results………………………………………………………………….……………………14
3.1 Species identification………………………………………………………….……14
3.2 Molecular data, partitioning scheme and model selection…………………….……14
3.3 Phylogenetic analyses………………………………………………………………15
3.4 Estimation of divergence time………………………………….……………..……16
3.5 Ancestral biogeography reconstruction………………….……….…………………17
4 Discussion…………………………………………………………………..………………18
4.1 Phylogeny of amphioxus…………………………………………..…………..……18
4.2 Phylogenetic position of amphioxus as the basal chordate…………….……………20
4.3 Estimation of divergence time……………………………………………....………20
4.4 Dispersal route of amphioxus……………………………..………………...………22
5 Conclusions..………………………………………………………………..……….……...26
References ...……………………………………………………………………….…………27

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