臺灣博碩士論文加值系統

絨螯蟹類屬於弓蟹科(Varunidae)，台灣共有2屬2種，台灣扁絨螯蟹(Platyeriocheir formosa)與日本絨螯蟹(Eriocheir japonica)。現今兩原生種棲地受天災的破壞、及過度捕捉，使兩族群量大幅減少。且國內業者未顧及對原生物種會造成威脅的危險，竟短視的將中華絨螯蟹引進做為養殖水產物。本研究將區別日本絨螯蟹；中華絨螯蟹與台灣扁絨螯蟹的形態差異。此外，使用粒線體細胞色素氧化酶I (COI)基因序列分析其分子分類學及歷史族群遺傳動態。這些結果可為兩原生種的保育提供更有用的訊息。日本絨螯蟹樣本(n = 40)取自新北市老梅溪與苗栗縣後龍溪，台灣扁絨螯蟹樣本(n = 35)取自台東縣金崙地區，中華絨螯蟹樣本(n = 45)則由屏東縣養殖場購得。以18個外部形質特徵進行主成分分析、聚類分析及單因子變異數分析。此外，兩原生種各隨機抽取20個樣本進行COI基因的定序及分析。結果顯示，在三物種間以外部形態差異可明顯將其分成三群。COI基因序列全長為1534 bp。台灣扁絨螯蟹與日本絨螯蟹種間平均遺傳距離為0.150 ± 0.0061，台灣扁絨螯蟹與中華絨螯蟹種間平均遺傳距離為0.150 ± 0.0027，日本絨螯蟹與中華絨螯蟹種間平均遺傳距離為0.045 ± 0.0027。台灣扁絨螯蟹與日本絨螯蟹種內序列變異位點觀測值分別為86及84個。台灣扁絨螯蟹與日本絨螯蟹的單型歧異度(Hd)和平均核苷酸歧異度(π)分別為1, 0.0063 ± 0.0032及1, 0.0074 ± 0.0037。以鄰近連接法(NJ)及最大簡約法(MP)建構親緣關係樹，能顯著將三物種明顯分群。將NCBI網站取得的參考序列加入分析，結果顯示台灣的日本絨螯蟹較相近於合浦絨螯蟹，與日本本島日本絨螯蟹歸類為不同群。Fu’s與Tajama測驗結果呈現為顯著的負值，表示兩族群歷史上曾經歷過族群擴張的事件。結論，外部形質與COI序列分析能明顯區別台灣扁絨螯蟹，日本絨螯蟹與中華絨螯蟹。台灣的日本絨螯蟹的學名在未來應需進行更名。
關鍵字: COI基因、種間遺傳距離、絨螯蟹、形質、親緣關係樹

The mitten crab group belongs to the Varunidae. Only two genera and species, Platyeriocheir formosa and Eriocheir japonica, are distributed in Taiwan. At present, population sizes of these two native species are dwindling due to habitat destruction by natural disasters and unrestricted over-harvesting. Moreover, E. sinensis was also introduced to Taiwan for short-term aquaculture which disregarded consequences of potential threatens to native species. The study attempted to distinguish morphological differences among E. japonica, E. sinensis, and P. formosa. Furthermore, molecular systematics and historical population dynamics were also analyzed using mitochondrial cytochrome oxidase subunit I (COI) gene sequences. These results can provide more-useful information for the conservation of these two native species. All E. japonica (n = 40) and P. formosa (n = 35) were collected from Lau-Mei Stream in New Taipei City, the Hou-Lung River in Miaoli County, and Jin-Luen in Taitung county. In total, 45 E. sinensis individuals were collected from an aquaculture farm in Pingtung County. Eighteen morphometric characters were measured and then analyzed using a primary component analysis, clustering, and one-way ANOVA. In addition, 20 individuals of each native species were randomly selected for sequencing and analysis of the COI gene. The results suggested that these three species have significant morphological differences and can be classified into three separate groups. The full-length COI gene is 1534 bp. The mean interspecific genetic distances were 0.150 ± 0.0061 for P. formosa vs. E. japonica, 0.150 ± 0.0027 for P. formosa vs. E. sinensis, and 0.045 ± 0.0027 for E. japonica vs. E. sinensis. In total, 86 and 84 variable sites were respectively observed within intraspecific sequences of P. formosa and E. japonica. Haplotype diversities (Hd) and mean nucleotide diversities (π) were 1 and 0.0063 ± 0.0032 for P. formosa and 1 and 0.0074 ± 0.0037 for E. japonica, respectively. Phylogenetic trees were constructed using Neighbor-joining (NJ) and Maximum parsimony (MP) methods. These trees presented significant clustering among the three species. When reference sequences from the NCBI were added to the analysis, results showed that E. japonica samples from Taiwan were more similar to E. hepuensis than to E. japonica samples from Japan. Fu’s and Tajama tests presented significantly minus values and suggested that historical population expansion had occurred for both E. japonica and P. formosa. In conclusion, P. formosa, E. japonica, and E. sinensis can clearly be distinguished by morphometric and COI sequence analyses. The scientific name of E. japonica on Taiwan should be corrected in the future.

Keywords: COI gene, interspecific genetic distance, mitten crab, morphometric, phylogenetic trees

摘 要 ..................................................................................................... I
Abstract ..................................................................................................... III
目錄 .......................................................................................................... VI
圖目錄 .................................................................................................... VIII
表目錄 ...................................................................................................... IX
第一章 前言............................................................................................. 1
1.1 物種介紹 ..................................................................................... 1 1.2 絨螯蟹類的生態學..................................................................... 3 1.3 絨螯蟹的分類學研究 ................................................................ 4 1.4 分子遺傳學 ................................................................................. 6 1.5 研究目的 ..................................................................................... 7
第二章 材料與方法 ................................................................................ 8
2.1 採樣 ............................................................................................. 8
2.2 外部形質量測 ............................................................................. 8 2.3 基因組DNA萃取 .................................................................... 10
2.4 粒線體COI基因選殖與定序 .................................................. 10
2.4.1 序列引子選擇 ................................................................. 10
2.4.2 聚合酶鏈鎖反應 (Polymerase chain reaction, PCR) .... 12
2.4.3 PCR產物純化 ................................................................ 12
2.4.4 TA選殖 (TA Cloning) .................................................. 13
2.4.5 勝任細胞製備 (competent cell preparation ) ................ 13
2.4.6 轉形作用(transformation) .............................................. 13 2.4.7 菌落聚合酶鏈鎖反應 ..................................................... 14
2.4.8 質體DNA萃取 (plasmid DNA extraction) .................. 14 2.4.9 DNA定序 (DNA sequencing) ....................................... 14 2.5 資料分析 ................................................................................... 16 第三章 結果........................................................................................... 17 3.1 形態學分析 ............................................................................... 17 3.2.1 核苷酸序列分析 ............................................................. 20
VII
3.2.2 核苷酸序列之鹼基組成 ................................................. 20 3.2.3 核苷酸序列間之變異及親緣關係樹建構 .................... 23 3.2.4 族群歷史動態分析 ......................................................... 37 第四章 討論........................................................................................... 42 4.1 形態學上的差異 ....................................................................... 42 4.2 台灣扁絨螯蟹的分類地位 ...................................................... 42 4.3 台灣地區絨螯蟹的遺傳歧異性 .............................................. 43 第五章 結論........................................................................................... 46 參考文獻 ................................................................................................... 47
作者簡介 ................................................................................................... 54

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