海洋生态与环境科学重点实验室知识库

球形棕囊藻（Phaeocystis globosa）是一种全球广布的有害藻华原因种。近年来，以囊体（colony）形成为特征的球形棕囊藻藻华在北部湾海域频频发生，对当地海洋生态环境、水产养殖和核电运行构成严重威胁，受到高度关注。在以往对球形棕囊藻藻华的研究中，常以19’-己酰氧基岩藻黄素（19’-hexanoyloxyfucoxanthin，Hex-fuco）作为特征色素，估算球形棕囊藻的生物量。但是，在对北部湾海域球形棕囊藻藻华的跟踪研究中发现，藻华形成时浮游植物样品中的Hex-fuco含量明显下降，而19’-丁酰氧基岩藻黄素（19'-butanoyloxyfucoxanthin，But-fuco）含量反而显著上升。北部湾海域的球形棕囊藻是否具有独特的色素组成特征？能否将其特征色素应用于现场调查，从而揭示北部湾球形棕囊藻藻华的分布和动态过程？针对这些问题，本研究以球形棕囊藻特征色素为切入点，对比分析了现有浮游植物色素提取与检测方法的有效性；在此基础上，探究了不同球形棕囊藻株系、形态（囊体或游离细胞）及环境因子对其色素组成的影响；进而将特征色素分析应用于2016–2017年和2018–2019年北部湾海域球形棕囊藻藻华的现场调查和研究，重点分析了北部湾海域球形棕囊藻藻华的早期来源、发展过程和年际变异，并探究了藻华过程中浮游植物群落结构的变化。

通过对比研究，分析评估了现有浮游植物色素提取与检测方法的有效性。比较了甲醇、95%甲醇水溶液、二甲基甲酰胺、90%丙酮水溶液等提取溶剂对球形棕囊藻（P. globosa）、三角褐指藻（Phaeodactylum tricornutum）、微绿球藻（Nannochloris sp.）、抑食金球藻（Aureococcus anophagefferens）、隐藻（Cryptomonas sp.）、东海原甲藻（Prorocentrum donghaiense）和自然海水样品中色素的提取效率，发现90%丙酮对叶绿素的提取效率较高，95%甲醇对类胡萝卜素的提取效率较高。但是，95%甲醇提取会促进叶绿素a衍生物的形成，尤其是在叶绿素酶活性较高的硅藻中，从而造成样品叶中绿素a含量的低估。在色素检测手段上，与高效液相色谱法（high performance liquid chromatography, HPLC）相比，荧光法会高估样品中的叶绿素a含量，可能是受到叶绿素a衍生物荧光信号的干扰。因此，现有的色素提取分析方法各有利弊，在应用色素分析手段开展浮游植物生态学研究时，应根据不同研究目的，选择合适的色素提取和检测方法。在北部湾海域针对球形棕囊藻特征色素和藻华过程开展研究时，以95%甲醇作为提取剂的HPLC法最为适用。

比较研究了不同球形棕囊藻株系、形态以及温度、光照和氮源等环境因子对球形棕囊藻特征色素组成的影响。研究发现，不同株系的球形棕囊藻特征色素存在明显差别；囊体和游离细胞的色素含量和比例虽有差异，但特征色素组成没有差别；环境因子会对球形棕囊藻的色素含量和比例产生一定影响，但不会改变其特征色素组成。因此，可以根据特征色素组成对球形棕囊藻的种下多态性状况进行解析。对球形棕囊藻不同藻株的特征色素分析结果表明，球形棕囊藻至少可以分成两个“色素型”，其中“色素B型”只含有特征色素But-fuco，“色素HB型”既含But-fuco，也含有Hex-fuco，且以Hex-fuco为主要特征色素。两种 “色素型”球形棕囊藻与囊体大小密切相关，“色素B型”能形成直径达厘米级的“巨型囊体（giant colony）”，而“色素HB型”只能形成直径 ≤ 3 mm的小囊体。两种“色素型”的球形棕囊藻均存在于北部湾海域。研究发现，北部湾海域的“色素B型”和“色素HB型”球形棕囊藻藻株对温度和营养盐的适应性存在差异，低温（18°C）有利于两种“色素型”球形棕囊藻形成囊体，高温（30°C）有利于其游离藻细胞的生长，但“色素B型”球形棕囊藻的比生长速率受温度影响较小，而“色素HB型”的比生长速率在高温下明显降低。对球形棕囊藻营养利用特性的研究发现，两种“色素型”的球形棕囊藻在不同氮源中均可生长，但“色素B型”球形棕囊藻在以尿素为氮源时能够形成直径更大的囊体。

以球形棕囊藻特征色素为标志物，研究了北部湾球形棕囊藻藻华原因种和藻华动态过程。研究发现，北部湾海域的大规模球形棕囊藻藻华主要由 “色素B型”球形棕囊藻形成。根据But-fuco在不同水团中的分布及其变化情况，反演了球形棕囊藻藻华的动态过程，推测北部湾海域的球形棕囊藻藻华存在两个不同的来源：广西近岸海域的藻华是由上升流自南部外海携带输入的球形棕囊藻形成，而雷州半岛西侧的藻华是由琼州海峡流携带输入的球形棕囊藻形成。东北季风的年际变化能通过改变琼州海峡流输入北部湾的营养盐和球形棕囊藻，影响球形棕囊藻藻华的分布和规模。球形棕囊藻藻华期间，硅藻、聚球藻等浮游植物类群及其特征色素分别与球形棕囊藻丰度呈现显著的正相关和负相关关系，其特征色素岩藻黄素（fucoxanthin）、玉米黄素（zeaxanthin）以及棕囊藻特征色素But-fuco的含量变化可以用作北部湾球形棕囊藻藻华的监测预警指标。

综上，本研究以球形棕囊藻为研究对象，以色素分析为研究手段，研究了北部湾海域球形棕囊藻的特征色素状况，分析了北部湾球形棕囊藻藻华过程及其影响因素。从特征色素入手揭示了球形棕囊藻的种下多态性，首次将球形棕囊藻划分为两个“色素型”，确认了北部湾海域的球形棕囊藻藻华主要由“色素B型”球形棕囊藻形成。发现北部湾海域的球形棕囊藻藻华具有两个不同来源，湾内藻华分布和规模受到东北季风年际变异的调控。相关研究成果丰富了对球形棕囊藻种下多态性的认识，为系统阐明球形棕囊藻藻华的发生机制提供了科学认识，并为球形棕囊藻藻华的监测预警提供了技术手段和科学依据。

Phaeocystis globosa is a globally distributed causative species of harmful algal bloom. In the Beibu Gulf, P. globosa blooms characterized by the formation of colonies have been recorded frequently during the last decade, posing serious threats to marine ecosystems, mariculture industry, and the operation of nuclear power plant. In previous studies on the P. globosa bloom, 19’-hexanoyloxyfucoxanthin (Hex-fuco) was often used as the diagnostic pigment to estimate the biomass of P. globosa. However, a sharply decrease in the content of Hex-fuco and a significant increase in the content of 19'-butanoyloxyfucoxanthin (But-fuco) in phytoplankton samples were observed during the occurrence of a P. globosa bloom in the Beibu Gulf. Does P. globosa in the Beibu Gulf has a unique profile of pigments? Can the diagnostic pigments be used as biomarkers to reveal the distribution and dynamics pattern of P. globosa blooms in the Beibu Gulf? Focusing on these questions, a series of researches were carried out to study P. globosa and its blooms in the Beibu Gulf, with a special attention to its diagnostic pigments. The feasibility of current phytoplankton pigment extraction and determination methods was analyzed. On this basis, the effects of different P. globosa strains, living forms (colony or solitary cell) and environmental factors on the pigment composition of P. globosa were studied. Then, the diagnostic pigment was applied in field investigations of P. globosa blooms in the Beibu Gulf from 2016 to 2017 and 2018 to 2019, focusing on their origin, dynamics, interannual variability, as well as the changes of phytoplankton communities associated with the blooms.

The feasibility of current phytoplankton pigment extraction and determination methods were evaluated. The efficiency of methanol, 95% methanol aqueous solution, dimethylformamide, and 90% acetone aqueous solution for pigment extraction from P. globosa, Phaeodactylum tricornutum, Nannochloris sp., Aureococcus anophagefferen, Cryptomonas sp., Prorocentrum donghaiense and a natural seawater sample were compared. It was found that 90% acetone had higher extraction efficiency for chlorophylls, while 95% methanol had higher extraction efficiency for carotenoids. However, 95% methanol promoted the formation of chlorophyll a (Chl a) derivatives, especially in diatoms with high chlorophyllase activity, which led to the underestimation of Chl a content. Compared with high performance liquid chromatography (HPLC), the fluorometric method overestimated Chl a content, probably due to the interference of co-occurred derivatives of Chl a. In view of the advantages and disadvantages of various pigment extraction and determination methods, appropriate pigment analysis protocol should be selected follow different purposes of phytoplankton ecology research. The HPLC method with 95% methanol as extractant is the most suitable for the study of diagnostic pigments and bloom dynamics of P. globosa in the Beibu Gulf.

The effects of different P. globosa strains, living forms and environmental factors such as temperature, light and nitrogen source on the pigment composition of P. globosa were analyzed. It was found that living forms and environmental factors could affect the content and ratio of pigment, but do not change the composition of diagnostic pigment. Therefore, the interspecific polymorphism of P. globosa can be analyzed based on the diagnostic pigment composition. The results showed that P. globosa can be divided into at least two “pigment type”, in which "pigment type B" only contained the diagnostic pigment But-fuco, while “pigment type HB” contains both But-fuco and Hex-fuco, and Hex-fuco is the main diagnostic pigment. The two pigment types are closely related to the size of P. globosa colony. “Pigment type B” can form “giant colony” with the diameter of centimeter-sized, while “pigment type HB” can only form small colonies with the diameter of less than 3 mm. These two pigment types of P. globosa coexisted in the Beibu Gulf. The study found that there were differences in the adaptability of the two pigment types of P. globosa to temperature and nutrients. Low temperature (18°C) promoted colony formation of both pigment types of P. globosa, while high temperature (30°C) promoted the growth of solitary cells. The specific growth rate of “pigment type B” was less affected by temperature, while that of “pigment type HB” decreased at high temperature. The two pigment types of P. globosa growth well in various nitrogen sources, but the “piment type B” could form larger colonies using urea as nitrogen source.

The causative species and dynamics of P. globosa bloom in the Beibu Gulf were studied using diagnostic pigment as biomarker. It was found that intense P. globosa blooms in the Beibu Gulf were mainly formed by “pigment type B” P. globosa. Based on the distribution of But-fuco in different water masses, the dynamics of P. globosa bloom were investigated, and it was suggested that P. globosa blooms in the Beibu Gulf could originate from two different origins. The bloom along the coast of Guangxi Zhuang Autonomous Region was formed by P. globosa imported from the South China Sea by upwelling, while the bloom in the region west to Leizhou Peninsula was formed by P. globosa transported into the Gulf through Qiongzhou Strait. The interannual variation of the northeast monsoon may affect the distribution and scale of P. globosa blooms by changing the flux of nutrients and P. globosa from the Qiongzhou Strait into the Beibu Gulf. During the P. globosa blooms, the abundance of diatoms and Synechococcus showed significant positive and negative correlation with P. globosa, respectively. The diagnostic pigments for colony-forming P. globosa (But-fuco), diatoms (fucoxanthin) and Synechococcus (zeaxanthin) could serve as potential early-warning indicators for the formation of P. globosa blooms in the Beibu Gulf.

In conclusion, this paper studied the diagnostic pigment signature of P. globosa in the Beibu Gulf, and the P. globosa bloom dynamics and its influencing factors from the aspect of pigments. The interspecific polymorphism of P. globosa was revealed in terms of diagnostic pigment, and two “pigment type” of P. globosa were defined for the first time. It was confirmed that the P. globosa blooms in the Beibu Gulf was mainly formed by “pigment type B”. Two origins of P. globosa blooms were proposed, and the distribution and scale of P. globosa blooms in the Beibu Gulf were regulated by the interannual variation of northeast monsoon. The results will contribute to the understanding of the interspecific polymorphism of P. globosa, and provide technical and scientific basis for research, monitoring and early-warning of P. globosa bloom in the future.

第1章 绪论

1.1 球形棕囊藻的生物学特征及藻华发生情况

1.1.1 球形棕囊藻的生物学特征

1.1.2 球形棕囊藻的种下多态性

1.1.3 球形棕囊藻藻华发生情况

1.2 球形棕囊藻藻华的特征色素研究

1.2.1 色素分析技术的发展及其在浮游植物研究中的应用

1.2.2 棕囊藻的特征色素及其在棕囊藻藻华研究中的应用

1.2.3 棕囊藻特征色素的影响因素

1.3 北部湾海域环境特征及球形棕囊藻藻华问题

1.4 研究目的及意义

第2章 浮游植物色素提取与检测方法研究

2.1 前言

2.2 材料与方法

2.2.1 材料与试剂

2.2.2 藻株培养和样品收集

2.2.3 荧光法测定叶绿素a

2.2.4 高效液相色谱法色素分析

2.2.5 实验设计

2.2.6 统计分析

2.3 结果

2.3.1 不同有机试剂对叶绿素a及其衍生物的提取效率

2.3.2 不同有机试剂对其他叶绿素类和类胡萝卜素的提取效率

2.3.3 荧光法与高效液相色谱法对叶绿素a含量的测定结果对比

2.4 讨论

2.4.1 浮游植物生理特征对色素提取效率的影响

2.4.2 有机试剂和色素的化学性质对色素提取效率的影响

2.4.3 浮游植物生态学研究中色素提取与检测方法的选择

2.5 小结

第3章 球形棕囊藻色素组成与生长状况研究

3.1 前言

3.2 材料与方法

3.2.1 实验藻株

3.2.2 球形棕囊藻囊体细胞计数方法建立

3.2.3 实验设计

3.2.4 统计分析

3.3 结果

3.3.1 球形棕囊藻囊体细胞计数方法

3.3.2 不同球形棕囊藻株系色素组成和囊体大小差异

3.3.3 球形棕囊藻囊体与游离细胞的色素组成差异

3.3.4 环境因子对球形棕囊藻色素组成和生长状况的影响

3.4 讨论

3.4.1 球形棕囊藻的特征色素及其影响因素

3.4.2 环境因子对不同“色素型”球形棕囊藻生长和囊体形成的影响

3.5 小结

第4章 北部湾球形棕囊藻藻华动态过程研究

4.1 前言

4.2 材料与方法

4.2.1 调查区域和航次设计

4.2.2 样品采集

4.2.3 色素分析

4.2.4 CHEMTAX分析

4.2.5 水团分析

4.2.6 风场和表层流数据获取

4.2.7 统计分析

4.3 结果

4.3.1 2016–2017年北部湾海域球形棕囊藻藻华的动态变化

4.3.2 2018–2019年北部湾海域球形棕囊藻藻华的动态变化

4.3.3 北部湾球形棕囊藻藻华与海域环境的年际变化状况

4.4 讨论

4.4.1 特征色素But-fuco对北部湾球形棕囊藻藻华的指示作用

4.4.2 北部湾球形棕囊藻藻华的早期来源

4.4.3 北部湾球形棕囊藻藻华年际差异及其原因分析

4.4.4 北部湾浮游植物群落结构动态变化及其指示意义

4.5 小结

第5章 结论与展望

5.1 结论

5.2 创新点

5.3 不足与展望

参考文献

附录Ⅰ  浮游植物色素名称列表

附录Ⅱ  球形棕囊藻藻株信息

附录Ⅲ  缩略语表

致  谢

作者简历及攻读学位期间发表的学术论文与研究成果