近二十余年，球形棕囊藻和抑食金球藻在我国近海频发藻华，对海域生态系统、海水养殖业和沿岸工程等造成巨大的威胁。前人研究发现包括我国近海在内的世界沿海的形棕囊藻不仅存在遗传分化，而且出现囊体大小、特征色素组成等生物性状分化。我国球形棕囊藻藻华过程常形成厘米级大小囊体以及特征色素转化等独特现象。但目前对我国球形棕囊藻遗传多样性、以及其遗传分化与生物性状分化之间的关系认识不清楚。无法为这些独特现象形成机理的阐明提供科学依据，也限制了对我国球形棕囊藻藻华发生机制的深入认识。抑食金球藻褐潮仅先后发生于美大西洋沿岸、南非海域以及我国渤、黄海海域。目前对于我国抑食金球藻是入侵种还是本地种依然存在争议。究其原因是与对抑食金球藻种下遗传分化认识不清楚有关，限制了对我国近海抑食金球藻遗传多样性以及中美抑食金球藻遗传关系的认识。本研究通过研发球形棕囊藻和抑食金球藻的特异性分子标记，对我国近海这两种藻藻华频发海域和大面海域中球形棕囊藻和抑食金球藻种下遗传多样性开展研究，分析其分布特征，及其与环境因子的相关性。通过研究得到如下认识：

1）基于棕囊藻叶绿体基因间隔区开发了三对棕囊藻特异性的高分辨率分子标记，其中rbcS-rpl27分子标记分辨率最高，可以将20余株世界不同地理株系的球形棕囊藻分为4个基因分型（Ⅰ~Ⅳ型），厘清了分型与囊体大小、特征色素组成之间的关系，其中Ⅰ、Ⅱ和Ⅲ型只能形成直径小于3 mm小囊体，以19’-hexanoyloxyfucoxanthin（Hex-fuco）和19’-butanoyloxyfucoxanthin（But-fuco）为特征色素，而Ⅳ型可形成厘米级大小的囊体，以But-fuco为特征色素。基于抑食金球藻线粒体基因间隔区研发一对抑食金球藻特异性分子标记trnD-dam1。基于该分子标记，发现抑食金球藻不同地理株系间具有遗传分化。

2）基于开发的分子标记开展我国近海大面海域球形棕囊藻和抑食金球藻种下遗传多样性研究，发现这两种藻种下遗传多样性复杂。球形棕囊藻存在7个分型，除了纯培养株系的Ⅰ~Ⅳ型外，还有另外3个无培养株系的分型（Ⅴ~Ⅶ型）。不同海域球形棕囊藻分型组成和相对丰度存在明显差异。渤海绝对优势分型为Ⅵ型，黄海优势分型为Ⅲ和Ⅵ型，东海绝对优势为Ⅱ型，南海优势分型为Ⅰ~Ⅳ型和Ⅵ型。抑食金球藻存在包括纯培养株系的Ⅰ和Ⅱ型在内的三个分型。其中，Ⅱ和Ⅲ型是广布分型，Ⅰ型仅在少数站位检出。Ⅲ型为三个海域的绝对优势分型。我国近海球形棕囊藻和抑食金球藻的不同分型的分布特征与环境因子的相关性也有差异，可能体现了不同分型的环境适应性。

3）基于开发的分子标记开展我国近海典型海域藻华过程中球形棕囊藻和抑食金球藻种下遗传多样性研究。发现南海海域浮游植物样品中游离球形棕囊藻细胞遗传多样性复杂，优势分型为Ⅰ和Ⅳ型，特征色素为Hex-fuco和But-fuco；囊体样品中Ⅳ型占绝对优势，以But-fuco为特征色素。2021年冬季青岛近岸发生球形棕囊藻藻华，为南黄海海域首次暴发。藻华过程出现大量厘米级囊体，其基因分型也为Ⅳ型，以But-fuco为特征色素；提出了我国近海球形棕囊藻藻华主要是由独特“巨囊型”生态型引发的科学认识。发现2013年和2014年秦皇岛褐潮暴发过程分型相对丰度呈现动态变化，褐潮前期和后期样品中均检出Ⅰ和Ⅱ型，但高潮样品中仅检出Ⅰ型。

综上所述，本研究通过研发特异性分子标记对我国近海的球形棕囊藻和抑食金球藻种下遗传多样性进行研究，对球形棕囊藻遗传分型与性状分化进行关联，提出我国近海能形成厘米级囊体的球形棕囊藻藻华由独特的“巨囊型”生态型引发的认识；初步发现了中国近海抑食金球藻存在种下遗传分化，褐潮暴发时分型的动态变化过程。本研究为球形棕囊藻和抑食金球藻的种下遗传多样性研究提供有力的分子工具，对解析我国近海的球形棕囊藻和抑食金球藻藻华形成机制具有重要意义。

Over the past 20 years, the frequent occurrence of harmful algal blooms (HABs) caused by Phaeocystis globosa and Aureococcus anophagefferens in the coastal waters of China have posed a great threat to the marine ecosystem, mariculture and coastal engineering. Biological traits such as colony size and composition of marker pigments are differentiated among geographical strains of P. globosa. During the P. globosa blooms in the coastal waters of China, the colony formation of with centimeter size and the transformation of marker pigments are still not well understood. At present, there is a poor understand of the genetic diversity of P. globosa in the coastal waters of China, which prevent to bulild the association between biological traits and genetic diversity. The blooms of A. anophagefferens occurred successively along the coasts of the United States, and the Bohai Sea and Yellow Sea in China. At present, the genetic diversity of A. anophagefferens are not uncovered, which makes it is controversial whether A. anophagefferens is an invasive species or a native species in China. In this study, we firstly developed intraspecies-specific molecular markers for P. globosa and A. anophagefferens; and then, we investigated the intraspecies genetic diversity of P. globosa and A. anophagefferens in the coastal waters of China with high frequency blooms of these two species to determine which genetic type is the dominant type triggering the blooms; Lastly, we analyzed the genetic diversity and distribution of these two species and their correlations with environmental factors in the coastal waters of China .The results were as followed:

1) Three pairs of high-resolution molecular markers of Phaeocystis were developed based on the chloroplast intergenic spacer region of Phaeocystis. Among which, a molecular marker rbcS-rpl27 has the highest resolution and can classify more than 20 strains of P. globosa from different geographical regions worldwide into four genetic types. There is a good correlation between the genetic types and colony size and the composition of marker pigments of P. globosa. The Types I, Ⅱ, and Ⅲ could form small colonies with diameter less than 3 mm and characterized by marker pigment of 19’-hexanoyloxyfucoxanthin (Hex-fuco) and 19’-butanoyloxyfucoxanthin (But-fuco), whereas the Type Ⅳ formed giant colonies with diameter more than 1 cm and characterized by a unique marker pigment of But-fuco.

2) During the period 2015 to 2021, 16 colony samples of P. globosa and 18 phytoplankton samples collected in the Beibu Gulf and Guangdong coastal waters in of the South China Sea (SCS) where P. globosa frequently outbreak the blooms. Using the developed molecular marker rbcS-rpl27, the gene cloning libraries of these samples were built and sequenced, and a total of more than 1000 sequences of P. globosa were obtained from these samples. Five genetic types of P. globosa were found in the SCS. The solitary cells of P. globosa in phytoplankton communities were dominated by Types I and Ⅳ with relative abundances of 45% and 40%, respectively; Type Ⅳ, with relative abundances of more than 95%, dominated the colony samples. And the Type Ⅳ is dominant type in the colonies during the P. globosa in coastal waters of Qingdao in the winter of 2021. Based on the physiological and ecological characteristics and genetic information of P. globosa, it was proposed that the blooms of P. globosa featuring giant colonies in the coastal waters of China was mainly caused by a unique "giant colony" ecotype, which was belonging to the Type Ⅳ, and can form centimeter sized colonies with But-fuco as marker pigment.

3) Using the molecular marker rbcS-rpl27-high-throughput sequencing assay, a total of 72 OTUs of P. globosa were obtained from 49 phytoplankton samples collected from the coastal waters of China. It was found the complex genetic diversity of P. globosa in these field samples. In addition to the four types I ~ Ⅳ of the P. globosa isolates, three new types in the coastal waters of China. There were obvious variation of the composition of genetic diversity and relative abundance of P. globosa in the different regions. Type Ⅵ was a unique absolutely dominant type in the Bohai Sea (BS) with the relative abundance of 95.4%. The mainly dominant types were Type Ⅲ (38.9%) and Type Ⅵ (32.7%) in the Yellow Sea (YS); meanwhile, the relative abundance of Type Ⅳ and Type Ⅱ was more than 10%. The dominant types were Type Ⅱ (69.1%) and Type I (10.4%) in the East China Sea (ECS). Except for the low abundance of Type V and Ⅶ, the relative abundances of the other five types are between 9.3% and 29.2% in the South China Sea (SCS). The correlation of different types with environmental factors differed. Types I and Ⅶ were not significantly correlated with temperature, salinity and dissolved inorganic nitrogen, phosphorus, and silicate. Type Ⅱ had an extremely significant positive correlation (p < 0.01) with inorganic phosphate and silicates. Type Ⅲ had a significant positive association with ammonium (p < 0.05). Type Ⅳ had an extremely significant positive correlation with temperature (p < 0.01), but an extremely significant and significant negative correlation with inorganic phosphate and ammonium (p < 0.01 and p < 0.05). Type Ⅵ had extremely significant and significant negative correlations with temperature and silicate (p < 0.01 and p < 0.05).

 4) An intraspecies-specific molecular marker trnD-dam1 was developed based on the mitochondrial intergenic region of A. anophagefferens. Based on this molecular marker, it was found that there was genetic differentiation among different geographical strains of A. anophagefferens.

5) Using the molecular marker trnD-dam1, the gene libraries of six phytoplankton samples collected during the brown tide in the coastal waters of Qinhuangdao in 2013 and 2014, were built and sequenced. A total of 281 sequences were obtained from these field samples, and included 173 sequences of Type Ⅰ with the relative abundance of 61.6%, and 108 sequences of Type Ⅱ with the relative abundance of 38.4%. Among them, the sequences of A. anophagefferens at bloom peaks of brown tides blooms were dominated by Type Ⅰ.

6) Using the molecular marker trnD-dam1, more than 1500 sequences were obtained from 45 environmental samples collected from 30 stations in the BS, YS and ECS, respectively. It was found that A. anophagefferens could be divided into three types, of which Types Ⅱ and Ⅲ were widespread in the BS, YS and ECS, but Type Ⅰ was detected from a few sites in the YS and ECS. Type Ⅲ was absolutely dominant type in these samples, with the relative abundance ranging from 76.0% to 95.3%. There were obvious differences on the correlation between different types of A. anophagefferens and the environmental factors. Type Ⅱ had significant or extremely significant negative correlation with temperature, nitrite, and silicate (p < 0.05 and p < 0.01), respectively. In contrast, Type Ⅲ had significant or extremely significant negative correlations with temperature, nitrite, and silicate (p < 0.05 and p < 0.01).

In summary, this study developed specific molecular markers to study the intraspecies genetic diversity of P. globosa and A. anophagefferens in the coastal waters of China, and to correlate the differentiation traits and genetic typing of P. globosa. A hypothesis was proposed that the P. globosa blooms featuring centimeter sized colonies in the coastal waters of China, are triggered by a unique "giant colony" ecotype. Intraspecies genetic differentiation was found in A. anophagefferens communities. These results will help to further understand the intraspecies genetic diversity and distribution of P. globosa and A. anophagefferens in the coastal waters of China, which is of great significance for understanding of the formation mechanism of P. globosa and A. anophagefferens blooms in the coastal waters of China.

第1章  绪论... 1

1.1 近海有害藻华发展趋势... 1

1.2 球形棕囊藻藻华发生情况及其生物学特征... 2

1.2.1 球形棕囊藻藻华发生情况及其危害... 2

1.2.2 球形棕囊藻生物学特征... 4

1.2.3 开展球形棕囊藻种下遗传多样性研究的必要性... 6

1.3 抑食金球藻褐潮发生情况及其生物学特征... 7

1.3.1 抑食金球藻褐潮发生情况及危害效应... 7

1.3.2 抑食金球藻生物学特征... 9

1.3.3 开展抑食金球藻种下遗传多样性研究的必要性... 10

1.4 球形棕囊藻和抑食金球藻种下遗传多样性... 11

1.4.1 遗传多样性的定义... 11

1.4.2 遗传多样性研究方法... 11

1.4.3 有害藻华种下遗传多样性研究进展... 13

1.4.4 球形棕囊藻和抑食金球藻种下遗传多样性研究进展... 14

1.5 研究目的及意义... 15

第2章  我国近海球形棕囊藻遗传多样性... 17

2.1 前言... 17

2.2 材料与方法... 17

2.2.1 藻类培养... 17

2.2.2 样品获取... 19

2.2.3 DNA的提取... 26

2.2.4 PCR扩增以及测序... 26

2.2.5 引物设计与选择... 26

2.2.6 克隆文库构建... 27

2.2.7 高通量测序... 27

2.2.8 数据分析... 28

2.3 结果... 29

2.3.1 球形棕囊藻高分辨率分子标记的开发... 29

2.3.2 球形棕囊藻性状分化与遗传特征关联... 42

2.3.3 我国近海不同分型球形棕囊藻地理分布特征... 44

2.3.4 球形棕囊藻不同分型地理分布特征与环境因子相关性分析... 55

2.3.5 我国典型海域球形棕囊藻遗传特征... 58

2.4 讨论... 61

2.4.1 基于叶绿体基因组开发的球形棕囊藻种下遗传多样性分子标记的适用性... 61

2.4.2 我国近海球形棕囊藻藻华主要由“巨囊”生态型引发... 62

2.4.3 我国近海球形棕囊藻种下分型地理分布特征及其与环境因子的关系... 64

2.5 小结... 66

第3章  我国近海抑食金球藻种下遗传多样性... 69

3.1 前言... 69

3.2 材料与方法... 69

3.2.1 藻类培养... 69

3.2.2 样品获取... 71

3.2.3 DNA的提取... 74

3.2.4 PCR扩增以及测序... 75

3.2.5 抑食金球藻特异性分子标记研发... 75

3.2.6 克隆文库构建... 76

3.2.7 数据分析... 76

3.3 结果... 76

3.3.1 抑食金球藻高分辨率分子标记的开发... 76

3.3.2 我国近海抑食金球藻种下遗传多样性和地理分布特征... 79

3.3.3 我国近海抑食金球藻不同分型地理分布特征与环境因子相关性分析... 84

3.3.4 2013年和2014年秦皇岛海域褐潮暴发期间抑食金球藻的组成... 88

3.4 讨论... 90

3.4.1 抑食金球藻高分辨分子标记的适用性... 90

3.4.2 我国近海抑食金球藻种下多样性、分布特征及其与环境因子的关系... 91

3.5 小结... 93

第4章  结论、创新性和展望... 95

4.1 结论... 95

4.2 创新性... 95

4.3 展望... 96

参考文献... 97

致  谢... 109

作者简历及攻读学位期间发表的学术论文与研究成果    111