海洋生态与环境科学重点实验室知识库

球形棕囊藻（Phaeocystis globosa）常以囊体的形式在全球温带至热带海域中形成有害藻华。2011 年以来，我国南海北部湾海域频发球形棕囊藻藻华，对海洋生态系统、旅游业、养殖业和沿岸的核电安全运行造成严重危害，备受关注。近期研究发现球形棕囊藻的遗传多样性与特征色素组成、囊体大小等性状分化可关联，能将其分为多个基因型；至少 4 个基因型球形棕囊藻共存于北部湾海域，其中 I 型和 IV 型是浮游植物群落中球形棕囊藻种群的优势基因型。但是，对北部湾球形棕囊藻优势基因型藻华动态过程及其与环境因子的相关、I 型和 IV 型球形棕囊藻生物学特征差异及其在藻华形成和维持中的作用认识有限。因此，本研究建立 I 型和 IV 型球形棕囊藻特异性 qPCR 定量方法，研究北部湾 2016 年和 2018 年冬季两次藻华中 I 型和 IV 型球形棕囊藻时空分布特征，分析其与环境因子的相关性，针对关键环境因子开展室内生物学特征对比实验。通过研究得到如下主要认识：

基于球形棕囊藻线粒体 atp8 基因，分别建立 I 型和 IV 型球形棕囊藻高灵敏的特异性 qPCR 定量检测方法，具备良好的野外应用潜力。研究发现球形棕囊藻线粒体 atp8 基因具有种内分辨能力，对球形棕囊藻分型认识与前人研究结果一致，可作为基因型特异性 qPCR 检测方法的靶区。针对 I 型和 IV 型球形棕囊藻分别设计特异性引物和探针，建立 qPCR 定量反应体系；两种方法均具有良好的特异性，仅能特异性扩增目标基因型球形棕囊藻；两种方法的稳定性高，方法的重复性和重现性均大于 98%；两种方法的定量检出范围广，可达 6~8 个数量 级，检出下限低于 1.0 cells/qPCR 反应，可对藻华动态过程进行检测。

应用建立的 qPCR 定量检测方法，研究北部湾球形棕囊藻藻华动态，发现 I 型和 IV 型球形棕囊藻空间分布特征相似，但 IV 型丰度远高于 I 型，确认藻华优势基因型是 IV 型，发现了三个早期藻种可能来源，揭示了硝酸盐、温度和盐度等关键环境因子在藻华发生过程的作用。发现北部湾球形棕囊藻藻华维持时间长（4~5 个月）；在 2016 年和 2018 年冬季两次藻华过程中，I 型和 IV 型球形棕囊藻分布特征相似，主要分布于钦州湾邻近海域和雷州半岛西侧海域；两次藻华高潮期，IV 型藻细胞丰度均超过 10⁵ cells/L，显著高于 I 型，是藻华的优势基因型；藻华初期，湾南部外海的南海底层逆风流会将球形棕囊藻输入到钦州湾邻近海域，雷州半岛东侧海域球形棕囊藻可通过琼州海峡西向流进入到雷州半岛西侧海域，海南岛西北部上升流也会携带球形棕囊藻补充至雷州半岛西侧海域。藻华动态主要受到硝酸盐、温度和盐度的调控，硝酸盐在藻华过程中被大量消耗，低温（<24℃）为藻华发生提供时间窗口，盐度影响球形棕囊藻空间分布；2016 年冬季藻华规模大、持续时间长与海域高硝酸盐和长低温期密切相关。

通过室内实验对比I型和IV型球形棕囊藻的盐度耐受、生长（细胞和囊体）、 囊体功能等基本生物学特征差异，并辅以转录组学数据，发现 I 型和 IV 型球形棕囊藻生存策略有鲜明差异，分别倾向于 r-策略者和 K-策略者。Ⅳ型的最适盐度范围（25~40）比 I 型（30~35）广；在生长过程中，I 型的生长速率快，最高细胞密度是 IV 型的 3~5 倍；I 型生长周期短（8~14 天），IV 型则超过 20 天；I 型形成高丰度（>1000 个/mL）小囊体（直径<1 mm），IV 型仅能形成低丰度（<100 个囊体/mL）大囊体（直径>5 mm）；I 型主要以无性繁殖为主，IV 型既能无性繁 殖，又能有性繁殖；IV 型囊体内高浓度的营养盐以及囊体的保护作用为其内部高丰度的单倍体游离细胞的生长提供了稳定环境。结果表明 I 型和 IV 型的生存策略不同，I 型倾向于 r-策略者，IV 型倾向于 K-策略者，有助于解释 IV 型在北部湾海域环境易取得竞争优势，形成长时间的藻华。

综上，本研究针对北部湾两种主要基因型球形棕囊藻分别建立了高灵敏的特异性 qPCR 定量检测方法，解析北部湾球形棕囊藻藻华动态，发现 IV 型是藻华优势基因型，揭示了硝酸盐、温度等关键环境因子对藻华动态和年际变异的影响；通过室内实验，发现 I 型和 IV 型的生存策略存在差异，IV 型倾向于 K-策略者，有助于其在北部湾藻华过程取得竞争优势，维持长时间藻华。本研究结果深化了对南海北部湾球形棕囊藻藻华的认识，为阐明藻华暴发机制提供了重要的科学依据，为藻华监测预警提供了技术手段。

Phaeocystis globosa is a common causative species of harmful algal blooms (HABs) in the form of colonies from temperate to tropical waters worldwide. Since 2011, the HABs of P. globosa have been of great concern because of their serious hazards on the coastal ecosystem, tourism, aquaculture, and the safe operation of nuclear power along the coastline. Recent studies have found that genetic differentiation of P. globosa is associated with traits such as the composition of marker pigments and colony size, implying that P. globosa can be divided into multiple genotypes. There are more than four genotypes of P. globosa coexist in the Beibu Gulf, South China Sea (SCS) and the Type I and Type IV are proved to be predominant in the phytoplankton community. In order to reveal the dynamics of the dominant genotypes during the P. globosa blooms and the role of key environmental factors in the Beibu Gulf, and differences in biological characteristics between Type I and Type IV P. globosa and its role in the formation and maintenance of P. globosa blooms, this study developed two genotype-specific qPCR methods to analyze the spatial and temporal distribution patterns of Type I and Type IV P. globosa in two blooms in winters of 2016 and 2018, explored their distribution patterns and the environmental influences, and conducted comparative experiments on the biological characteristics of two genotypes of P. globosa for key environmental factors. The results were as followed:

Based on the mitochondrial gene atp8 of P. globosa, two highly sensitive and specific qPCR assays for P. globosa Type I and Type IV were established, respectively, which have good potential for field application. It was found that the mitochondrial gene atp8 of P. globosa has enough intraspecific resolution to distinguish the different genotypes of P. globosa, which is consistent with the results of previous studies on the typing of P. globosa, and could be used as the sequence target for genotype-specific qPCR assays. Specific primers and probes were designed for Type I and Type IV P. globosa to establish qPCR assays for their quantitative detection. Both methods have good specificity and can only specifically amplify the target genotype of P. globosa. Both developed methods had good repeatability and reproducibility (>98%). They had low detection limits (<1.0 cells/qPCR reaction) and wide detection range (6~8 order of magnitudes). The results suggested both developed qPCR methods have the potential to determine dynamics of P. globosa blooms.

The newly developed two genotype-specific qPCR assays were applied to determine the dynamics of two P. globosa blooms in the Beibu Gulf. It was found that the spatial distribution patterns of Type I and Type IV were similar, but the abundance of Type IV was much higher than that of Type I. The dominant genotype of P. globosa blooms was confirmed as Type IV, and three possible seed sources were found, revealing the role of key environmental factors such as nitrate, temperature and salinity in the P. globosa blooms. It was noted that the P. globosa blooms in the Beibu Gulf was maintained for a long time (4~5 months). During the two P. globosa blooms in the gulf, the distribution patterns of the two genotypes of P. globosa were similar, mainly distributed in the adjacent waters of Qinzhou Bay and the western waters of Leizhou Peninsula. At climax of the blooms, the abundance of Type IV exceeded 10⁵ cells/L, which was greater than that of Type I, indicating that Type IV dominated the blooms in the Beibu Gulf. At the beginning of the P. globosa blooms, P. globosa in the SCS Counter-wind deep current off the southern of Beibu Gulf was imported to the adjacent waters of Qinzhou Bay, the algal cells in the Guangdong coastal waters entered the western waters of Leizhou peninsula through the westward flow of Qiongzhou Strait, and the algal cells in the northwestern upwelling of Hainan Island was supplemented to the peninsula western waters. The dynamics of P. globosa blooms were primarily regulated by some environmental factors, including nitrate, temperature, and salinity. Of which, nitrate was consumed by P. globosa blooms in large amounts, low temperature (<24℃) provided a suitable time window for algal blooms, and salinity affected the spatial distribution of P. globosa. Lastly, it was found that the higher concentration of nitrate and longer low temperature period was resulted in the larger-scale bloom in the winter of 2016 in the term of bloom intensity and duration than that in the winter of 2018.

Experiments comparing the differences in biological characteristics such as salinity tolerance, growth (cell and colony), and colony function between Type I and Type IV P. globosa, complemented by transcriptomic data, revealed distinct differences in survival strategies between Type I and Type IV P. globosa, favoring r-strategists and K-strategists, respectively. The optimal growth salinity of Type IV P. globosa (25~40) was wider than that of Type I (30~35). The growth of Type Ⅰ was faster, and its highest cell density was 3~5 times greater than that of Type Ⅳ; the growth cycle of Type Ⅰ was shorter (ranging from 8 to 14 days) than that of Type Ⅳ (>20 days). Type I formed high abundance (>1000 colonies/mL) of small colonies (<1 mm), but Type IV could form low abundance (<100 colonies/mL) of large colonies (>5 mm). Type I was mainly asexual and Type IV was capable of both asexual and sexual reproduction during their growth. High nutrient content in the colonies of Type IV and the protective effects of these colonies provided more suitable survival environment for its internal haploid free-living cells. The results implied that the Type I cells tend to be r- strategists, and the Type IV cells tended to be K-strategists, which helped the Type IV cells achieve a competitive advantage during the blooms and prolonged the bloom duration in the Beibu Gulf.

In the present study, the dynamics of P. globosa blooms in the Beibu Gulf were studied by newly developed genotype-specific qPCR assays with highly sensitive for two dominate genotypes (Type I and Type IV). It was founded that the Type IV dominated the blooms and the key environmental factors including nitrate and temperature affected the bloom dynamics and interannual variability in the gulf. The survival strategies Type I and Type IV differed: Types I and IV tended to be r- and K-strategists, respectively. These results will profound the understanding of P. globosa blooms in the Beibu Gulf, furnish an important scientific basis for the mechanism of P. globosa bloom, and provide effective technical assays to monitor and early-warning of P. globosa blooms.