臺灣博碩士論文加值系統

本論文為探討東海海域浮游植物族群之分佈及貢獻量之研究。我們於2003年6月至2004年7月分別進行了三個東海夏季之航次，並以HPLC進行色素分析結合CHEMTAX化學分類運算軟體推估出各浮游植物族群的豐度及分佈比例。
由總葉綠素a之濃度結果得知，以OR1-686(2003年6月)的貢獻量為最高，其次為OR1-721(2004年6月)，而OR1-691(2003年8月)的貢獻量為最低，顯示長江沖淡水等環境因子對水體中浮游植物之豐度造成影響。
整體而言，大陸沿岸之優勢藻種組成呈現顯著的差異。在營養鹽較豐富的沿岸區域，以Bacillariaphyceae、Dinophytes及Cryptophytes對葉綠素a的貢獻量為較高；反之，在貧營養鹽的外洋區域，則是以Chrysophytes、Prasinophytes、Cyanobacteria及Prochlorophytes為較占優勢。
綜合以上，顯示HPLC色素分析技術應用於浮游植物之色素定量為一適當之分析工具，同時再結合化學分類CHEMTAX運算程式可有效的應用於東海大範圍海域浮游植物族群結構及豐度之探討。

海洋中逾五成以上的光合作用都是由超微浮游植物所進行，因此它們在基礎生產力及全球碳循環上提供了重要的角色。這些超微浮游植物主要為體型小於2 μM的原核綠藻（Prochlorococcus）以及聚球藻（Synechococcus）。本論文為探討台灣沿岸東海海域超微浮游植物族群之分佈及貢獻量之研究。
我們於2004年8月之OR2-1213航次在台灣沿岸東海海域進行採樣，並分別以藉由流式細胞儀進行超微浮細胞所發出的不同螢光來達到細胞計數，高效液體層析進行色素分析以及細胞之16S rDNA(長度1.2 Kb)保守序列的進行分析鑑定。綜合以上各實驗之結果發現，原核綠藻則只出現於外洋區，平均貢獻量為0.112 μg L-1；聚球藻分佈較廣，分佈於整條航次測線，並以陸棚區的分佈為最高，所提供之貢獻量平均值為0.291 μg L-1，而外洋區之平均貢獻量則為0.124 μg L-1。
結果顯示台灣沿岸東海海域超微浮游植物族群之多樣性，推測這些超微浮游植物族群在此區域可能是受到地理條件的阻隔並經演化造成遺傳組成上的差異。

This thesis discusses the community structure of phytoplankton was investigated from pigment composition of East China Sea during June 2003 to July 2004 on three summer cruises. Pigment concentration determination by high-performance liquid chromatography was interpreted using a matrix fastorization program “CHEMTAX” to obtain the Chlorophyll a biomass of phytoplankton groups at class level.
As for the northwestern part of the East China Sea, Especially the near-coastal of China Mainland, Chlorophyll a concentration were enriched all year-round, whilst the OR1-686(June, 2003) has the most highest abundance, followed by OR1-721(June, 2004) and OR1-691(August, 2004). The results indicating this might be the influence of the Changjiang fresh water discharge.
As a whole, the dominant phytoplankton near China Mainland coastal presented notable difference in these three cruises, cause of the hydrological factor in this area. However, we discovered that bacillariaphyceae, dinophytes and cryptophytes have high contribution in the nutrient-rich region. While the off-shelf region, chrysophytes were the most abundant taxa followed by prasinophytes, cyanobacteria and prochlorophytes.
Hence HPLC determination of the biomarker pigment is a useful tool for quantitative of each phytoplankton groups. At present, combined with CHEMTAX will be the most robust way to analyze dynamic of phytoplankton populations.

Over 50 % of photosynthesis were carried on picophytoplankton in the ocean, therefore they play an important role for the primary production and the global carbon cycling in the ocean. These tiny(2 μM), unicellular picophytoplankton were included Prochlorococcus and Synechococcus. The purpose of this thesis is to investigate the distribution and the contribution of picophytoplankton in the East China Sea near by Taiwan coastal.
Water samples were collected in August 2004 during the OR2-1213 cruise. Then we count the number of cells by Flow Cytometry, using HPLC to analyze the pigments and to determine the 16S rDNA sequences of these picophytoplankton. The results showed prochlorococcus occured only at the open ocean, and the average contribution were 0.112 μg L-1 ; the synechococcus have a ubiquitous distribution, and have a higher average contribution (0.291 μg L-1)at the continent shelf, where the open ocean were 0.124 μg L-1 .
We found the microdiversity of picophytoplankton in East China Sea, and suggested that the picophytoplankton genetic bases were evolution to different kinds, due to the biogeographical boundary in this site.

第一部份目錄
中文摘要 P1-ii
英文摘要 P1-iii
目錄 P1-iv
表目錄 P1-vi
圖目錄 P1-vii
第一章、前言 P1-1
1.1 浮游植物族群分佈研究的重要性 P1-1
1.2 浮游植物鑑定及量測方法 P1-2
1.3 研究背景 P1-4
1.4 研究目的 P1-6
第二章、材料與方法 . P1-8
2.1 HPLC分析系統 . P1-9
2.1.1 Barlow法分析流程 . P1-9
2.1.2 C8 管柱 . P1-9
2.1.3移動相試劑組成及梯度程式 . P1-10
2.2 色素標準品 . P1-10
2.2.1標準品的製備方式 P1-11
2.2.1.1 CHLa及CHLb標準品之濃度校正 P1-11
2.2.1.2色素標準品溶液之製備 P1-12
2.3 調查航次 . P1-13
2.4 樣本之採集及萃取方式 . P1-13
2.5 CHEMTAX之運算 . P1-14
2.5.1色素濃度比(Pigment Initial Ratio)之取得 . P1-14
2.5.2色素濃度(Data File)之取得 . P1-15
2.5.3 Ratio Limit File之取得 . P1-15
2.5.4結果之輸出 . P1-15
2.6其他水文資料與流式細胞儀資料之取得 . P1-16
第三章、HPLC色素分析與CHEMTAX運算結果 . P1-17
3.1 色素標準品及野外樣品HPLC層析圖 . P1-17
3.2 OR1-686航次表水樣品色素分析結果 . P1-17
3.3 OR1-691航次表水樣品色素分析結果 . P1-18
3.4 OR1-721航次表水樣品色素分析結果 . P1-20
3.5 OR1-686航次之CHEMTAX運算結果 . P1-21
3.6 OR1-691航次之CHEMTAX運算結果 . P1-21
3.7 OR1-721航次之CHEMTAX運算結果 P1-21
第四章、東海海域夏季之浮游植物組成 . P1-23
4.1 OR1-686航次表層水浮游植物之空間分佈 . P1-23
4.2 OR1-691航次表層水浮游植物之空間分佈 P1-25
4.3 OR1-721航次表層水浮游植物之空間分佈 P1-26
4.4 與流式細胞儀計數之比較 P1-28
4.5 低鹽區之CHEMTAX運算結果 P1-28
第五章、結論 . P1-31
參考文獻 . P1-34

表目錄
表1.1 浮游植物分類群、學名、俗名、中譯及縮寫對照表 P1-39
表1.2 浮游植物族群化學分類 P1-40
表2.1 移動相梯度時間組合表 P1-41
表2.2 色素標準品濃度、來源一覽表 P1-42
表2.3 色素標準品儲備溶液之配製 P1-43
表2.4 色素標準品之感應因子製作 P1-44
表2.5 色素濃度起始值(Pigment Initial Ratio ) P1-45
表3.1 航次之色素平均值 P1-46
表3.2 OR1-686航次之Output Ratio/Percent Change P1-47
表3.3 OR1-691航次之Output Ratio/Percent Change P1-48
表3.4 OR1-721航次之Output Ratio/Percent Change P1-49
表4.1 各航次浮植組成百分比平均值 P1-50
表4.2 各藻種對葉綠素a的貢獻量平均值 P1-51
表5.1 各航次之葉綠素a範圍及平均值 P1-52

圖目錄
圖1.1 浮游植物鑑定量測及海洋水色量測方法 P1-53
圖2.1 樣本之萃取方法 P1-54
圖2.2 航次之採樣測站圖 P1-55
圖3.1 色素標準品層析圖 P1-56
圖3.2 686航次第18站表水之野外樣品層析圖 P1-57
圖3.3 OR1-686航次CHL C3 (A)、CHL C2 (B)、
PERI (C)及BUT (D)色素濃度之水平分佈圖 P1-58
圖3.3 OR1-686航次FUCO (E)、HEX (F)、
PRAS (G)及DIAD (H)色素濃度之水平分佈圖 P1-59
圖3.3 OR1-686航次ALLO (I)、ZEA (J)、
CHL b (K)及DV CHL a (L)色素濃度之水平分佈圖 P1-60
圖3.3 OR1-686航次CHL a (M)及Fluometry-CHL a (N)
色素濃度之水平分佈圖 P1-61
圖3.4 OR1-691航次CHL C3 (A)、CHL C2 (B)、
PERI (C)及BUT (D)色素濃度之水平分佈圖 P1-62
圖3.4 OR1-691航次FUCO (E)、HEX (F)、
PRAS (G)及DIAD (H)色素濃度之水平分佈圖 P1-63
圖3.4 OR1-691航次ALLO (I)、DIAT (J)、
ZEA(K)及CHL b (L)色素濃度之水平分佈圖 P1-64
圖3.4 OR1-691航次DV CHL a (M)、CHL a (N)
及Fluometry-CHL a (O)色素濃度之水平分佈圖 P1-65
圖3.5 OR1-721航次CHL C3 (A)、CHL C2 (B)、
PERI (C)及BUT (D)色素濃度之水平分佈圖 P1-66
圖3.5 OR1-721航次FUCO (E)、HEX (F)、
PRAS (G)及DIAD (H)色素濃度之水平分佈圖 P1-67
圖3.5 OR1-721航次ALLO (I)、ZEA (J)、
CHL b (K)及DV CHL a (L)色素濃度之水平分佈圖 P1-68
圖3.5 OR1-721航次CHL a (M)及Fluometry-CHL a (N)
色素濃度之水平分佈圖 P1-69
圖4.1 OR1-686航次表層水Bacillariaphyceae(A)
及Dinoflagellates(B)之貢獻量 P1-70
圖4.1 OR1-686航次表層水Chrysophyceae(C)及
Prymnesiophyceae(D)之貢獻量 P1-71
圖4.1 OR1-686航次表層水Cryptophyceae(E)及
Chlorophytes(F)之貢獻量 P1-72
圖4.1 OR1-686航次表層水Prasinophyceae(G)
及Cyanobacteria(H)之貢獻量 P1-73
圖4.1 OR1-686航次表層水Prochlorophyceae(I)之之貢獻量 P1-74
圖4.2 OR1-691航次表層水Bacillariaphyceae(A)
及Dinoflagellates(B)之貢獻量 P1-75
圖4.2 OR1-691航次表層水Chrysophyceae(C)及Prymnesiophyceae(D)之貢獻量 P1-76
圖4.2 OR1-691航次表層水Cryptophyceae(E)
及Chlorophytes(F)之貢獻量 P1-77
圖4.2 OR1-691航次表層水Prasinophyceae(G)
及Cyanobacteria(H)之貢獻量 P1-78
圖4.2 OR1-691航次表層水Prochlorophyceae(I)之貢獻量 P1-79
圖4.3 OR1-721航次表層水Bacillariaphyceae(A)
及Dinoflagellates(B)之貢獻量 P1-80
圖4.3 OR1-721航次表層水Chrysophyceae(C)
及Prymnesiophyceae(D)之貢獻量 P1-81
圖4.3 OR1-721航次表層水Cryptophyceae(E)
及Chlorophytes(F)之貢獻量 P1-82
圖4.3 OR1-721航次表層水Prasinophyceae(G)
及Cyanobacteria(H)之貢獻量 P1-83
圖4.3 OR1-721航次表層水Prochlorophyceae(I)之貢獻量 P1-84
圖4.4 OR1-686航次Synechococcus(A)及 Prochlorococcus (B)
之流式細胞儀計數結果 P1-85
圖4.5 OR1-691航次Synechococcus(A)及 Prochlorococcus (B)
之流式細胞儀計數結果 P1-86
圖4.6 OR1-721航次Synechococcus(A)及 Prochlorococcus (B)
之流式細胞儀計數結果 P1-87
圖4.7 OR1-686航次之所有測站(A)與低鹽區(B)
之藻種種類組成之比較。 P1-88
圖4.8 OR1-691航次之所有測站(A)與低鹽區(B)
之藻種種類組成之比較。 P1-89
圖4.9 OR1-721航次之所有測站(A)與低鹽區(B)
之藻種種類組成之比較。 P1-90
圖5.1 OR1-686航次浮游植物貢獻量分佈樹狀圖 P1-91
圖5.2 OR1-691航次浮游植物貢獻量分佈樹狀圖 P1-92
圖5.3 OR1-721航次浮游植物貢獻量分佈樹狀圖 P1-93

第二部目錄
中文摘要 P2-ii
英文摘要 P2-iii
目錄 P2-iv
表目錄 P2-vi
圖目錄 P2-vii
第一章、前言 P2-1
第二章、材料與方法 .P2-6
2.1 HPLC色素分析 . P2-6
2.1.1樣本之取得及保存 . P2-6
2.1.2分析系統及步驟 . P2-6
2.2 流式細胞儀計數分析 . P2-7
2.2.1樣本之取得及保存 . P2-7
2.2.2分析儀器及步驟 . P2-7
2.3 16S rDNA序列分析 . P2-7
2.3.1樣本之取得 . P2-7
2.3.2材料製備及設備 . P2-7
2.3.3核酸樣本分離 . P2-8
2.3.4聚合酶連鎖反應 . P2-8
2.3.5勝任細胞的製備 . P2-9
2.3.6 PCR產物之選殖 . P2-10
2.3.7序列分析比對 . P2-11
第三章、研究結果 .P2-12
3.1 流式細胞儀細胞計數結果 . P2-12
3.2 HPLC色素分析結果 . P2-12
3.3 超微浮植細胞數與特徵色素分析結果比較 P2-15
3.4 16S rDNA基因序列之確認 . P2-16
3.5 CHEMTAX運算浮植貢獻量 . P2-19
第四章、討論 .P2-21
參考文獻 . P2-24

表目錄
表2.1 藥品製備及保存條件 P2-28
表3.1 各站序列比對結果 P2-29
表3.2 OR2-1213航次第1站至第10站之色素濃度起始值 P2-30
表3.3 OR2-1213航次第12站至第14站之色素濃度起始值 P2-31
表3.4 第1站至第10站之Output Ratio/Percent Change P2-32
表3.5 第12站至第14站之Output Ratio/Percent Change P2-33
表3.6 各站浮植之相對貢獻量平均值 P2-34

圖目錄
圖1.1 流式細胞儀辨識聚球藻及其他超微浮游植物分析圖 P2-35
圖2.1 OR2-1213航次採樣測站圖 P2-36
圖2.2 核酸樣本分離步驟流程 P2-37
圖2.3 PCR產物位置圖 P2-38
圖2.4 Colony PCR產物位置圖 P2-39
圖3.1 OR2-1213航次流式細胞儀分析聚球藻的分佈情形 P2-40
圖3.2 OR2-1213航次流式細胞儀分析原核綠藻的分佈情形 P2-41
圖3.3 OR2-1213航次CHL C3 (A)、CHL C2 (B)
色素濃度之剖面分佈圖 P2-42
圖3.3 OR2-1213航次ALLO (C)、BUT (D)
色素濃度之剖面分佈圖 P2-43
圖3.3 OR2-1213航次DIAD (E)、FUCO (F)
色素濃度之剖面分佈圖 P2-44
圖3.3 OR2-1213航次HEX(G)、ZEA (H)
色素濃度之剖面分佈圖 P2-45
圖3.3 OR2-1213航次CHLb(I)、DV CHL a (J)
色素濃度之剖面分佈圖 P2-46
圖3.3 OR2-1213航次CHLa(K)色素濃度之剖面分佈圖 P2-47
圖3.4 聚球藻細胞數與色素分析之比較
Station 4(A)及Station 6(B) P2-48
圖3.4 聚球藻細胞數與色素分析之比較
Station 8(A)及Station 10(B) P2-49
圖3.4 聚球藻細胞數與色素分析之比較
Station 12(A)及Station 14(B) P2-50
圖3.5 原核綠藻細胞數與色素分析之比較
Station 4(A)及Station 6(B) P2-51
圖3.5 原核綠藻細胞數與色素分析之比較
Station 8(A)及Station 10(B) P2-52
圖3.5 原核綠藻細胞數與色素分析之比較
Station 12(A)及Station 14(B) P2-53
圖3.6 原核綠藻之16S rDNA檢定比對 P2-54
圖3.7 聚球藻之16S rDNA檢定比對 P2-55
圖3.8 OR2-1213航次第1站至第10站之浮植貢獻量 P2-56
圖3.9 OR2-1213航次第12站至第14站之浮植貢獻量 P2-57

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