Phylogeography and Population structure of Bigeye Tuna (Thunnus obesus) Inferred from Mitochondrial DNA sequences

大目鮪(Thunnus obesus) ，又稱短鮪，為大洋性中表層水域的高洄游性魚種，主要分布於大西洋、印度洋、太平洋各海域，適水溫域為17-29℃之間，為高經濟價值魚種，亦為世界漁業之主要漁獲對象，因此如能確實瞭解該魚種之族群結構與來源，將有助於吾人對全球大目鮪資源之評估、管理與利用。本研究係利用粒線體基因標記之生物技術方法，鑑定全球大目鮪之系群結構，並探討其親源關係。本研究採集了太平洋(五個採樣區)、印度洋(五個採樣區)與大西洋(三個採樣區)之大目鮪肌肉樣品，其粒線體DNA（mtDNA）經萃取後，利用特定的引子（primer），針對mtDNA控制區約860個鹼基對，以聚合酶鏈鎖反應（PCR）方法，將mtDNA控制區放大並定序，成功得到共695個大目鮪mtDNA控制區序列。所得控制區序列以Clustal X軟體進行分析比對，隨後，將此不同的單倍基因型以Tamura-Nei model 計算其遺傳距離，並利用neighbor-joining方法建構所得大目鮪單倍基因型之親緣關係樹。NJ樹形的可信度以1000次重複的bootstrap法進行統計檢測。結果顯示：親緣關係樹可將全球的大目鮪分為三個clades：Clade I 包含太平洋(約99%)、印度洋(約98%)、大西洋海域之大目鮪單倍基因型；Clade II為大西洋特有之族群(約72%)；Clade III則分布於太平洋與印度洋海域。此外，我們利用AMOVA 與F-statistics分析全球大目鮪的族群結構，結果顯示各洋域之內的族群遺傳結構分化情況並不顯著，即太平洋內、印度洋內與大西洋內為單一系群，印度洋與太平洋大目鮪有基因交流的現象，惟大西洋各採樣區與太平洋、印度洋各個採樣區有顯著的族群遺傳分化。最後，Tajima’s D test、Fu’s FS test以及 mismatch distribution的分析結果顯示，Clade I 與Clade II族群內的中性檢測為顯著負值，且其mismatch distribution為雙峰分布，推測該族群經歷過瓶頸效應然後再擴張其族群。此外，大目鮪族群的核苷酸歧異度與基因型歧異度皆高，推測，大目鮪在過去可能因冰河事件而遭受到地理隔離，在冰河期之後這些被隔離的族群又發生基因交流，並有族群擴張的現象。Phylogeography and population structure of bigeye tuna (Thunnus obesus), an important commercial species widely distributed throughout the Pacific Ocean, Indian Ocean and Atlantic Ocean were investigated using the first hypervariable region (HVR-1) of the mitochondrial control region sequence data. A total of 695 specimens were sampled from five regions in the Pacific Ocean (Western Pacific, Hawaii, Line, Marquesas and Eastern Pacific), five regions in the Indian Ocean (Cocos Islands, Southeastern Indian Ocean, Southwestern Indian Ocean, Seychelles and South Africa), and three regions in the Atlantic Ocean (North Guinea, South Guinea and Brazil). The reconstructed neighbor-joining phylogenetic tree showed that haplotypes from the Pacific Ocean and Indian Ocean could be grouped into two clades (Clade I and Clade III), whereas in the Atlantic Ocean, two divergent clades (Clade I and Clade II) coexisted. Each single stock of bigeye tuna in the Pacific, Indian and Atlantic Oceans was supported by hierarchical AMOVA tests and pairwise ΦST analyses. The Clade I phylogroup was a ubiquitous inter-oceanic clade which dominated the Pacific and Indian Oceans, making up 99% and 98% of these specimens. Clade II was a specific group exclusively restricted to the Atlantic Ocean which made up 72.3% of its specimens. A minor phylogroup, Clade III existed in both the Pacific and Indian Oceans with an exception in the Eastern Pacific Ocean. Significant negative Fu’s FS statistics and unimodal mismatch distributions indicated that the Clade I and Clade II phylogroups were from expanding populations. The low Harpending’s raggedness indexes further evidenced population expansion. The secondary contacts between previously differentiated allopatric lineages appeared to be a more possible explanation in this scenario.口試委員會審定書............................................................................................................i辭...................................................................................................................................ii文摘要..........................................................................................................................iiibstract..............................................................................................................................vhapter 1: Introduction......................................................................................................1.1 Bigeye tuna (Thunnus obesus) ..........................................................................1.2 Previous studies on bigeye tuna population genetics........................................2.3 Phylogeography ................................................................................................5.4 Aim of study......................................................................................................7hapter 2: Materials and Methods...................................................................................10.1 Sampling..........................................................................................................10.2 DNA extraction................................................................................................10.3 PCR amplification and automated sequencing………………………………122.4 Data Analyses..................................................................................................12 2.4.1 Alignment………………………………………..…………………..13 2.4.2 Genetic diversity and structure…………………..…………………..13 2.4.3 Analyses of population history…………………..…………………..15hapter 3: Results............................................................................................................19.1 Western Pacific Ocean.....................................................................................19 3.1.1 Sampling..............................................................................................19 3.1.2 Molecular characteristics.....................................................................19 3.1.3 Patterns of population structure...........................................................20.2 Indian Ocean....................................................................................................21 3.2.1 Sampling..............................................................................................21 3.2.2 Molecular characteristics.....................................................................22 3.2.3 Phylogeny and Patterns of population structure..................................23 3.2.4 Demographic pattern...........................................................................25.3 Global .............................................................................................................26.3.1 Sampling..............................................................................................26.3.2 Genetic Diversity and structure...........................................................26.3.3 Analyses of population history............................................................29hapter 4: Discussion......................................................................................................31eference.........................................................................................................................42igures.............................................................................................................................54ables...............................................................................................................................6