实验海洋生物学重点实验室知识库

金潮和绿潮分别是由马尾藻属（Sargassum）大型褐藻或石莼属（Ulva）大型绿藻暴发性增殖引发的海洋生态现象。在世界范围内，金潮和绿潮近年来呈现显著的高发趋势，在中国近海表现尤为明显。自2007年起，黄海绿潮连年发生；2016年底，黄海金潮首次大规模暴发；2017年黄海绿潮与金潮同时发生。遗传学分析表明，黄海金潮与绿潮均由单一致因种的特定基因型引发，提示两种大型海藻的暴发具有明确的生物学成因。藻类共附生细菌与宿主间具有广泛的相互作用，密切参与宿主的生长发育、形态建成等重要生物学过程，揭示暴发性海藻共附生细菌的群落结构与功能，将为阐明藻华暴发的“生物学”机制提供新认知。

本文选择2017年不同站位采集的漂浮铜藻样本、以及不同年份与站位采集的漂浮浒苔样本，通过石英砂涡旋震荡法分别制备了漂浮海藻的外生菌和内生菌，筛选出可有效避免藻类细胞器16S rDNA干扰的测序引物，基于Illumina Miseq高通量测序，分析了两种暴发性海藻共附生细菌的群落结构特征，利用PICRUSt预测了菌群功能，取得以下研究结果：

1）漂浮铜藻共附生细菌的群落结构与功能

分子鉴定结果显示，黄海金潮藻由单一物种铜藻（Sargassum horneri）组成。2017年分别采集相对高营养海区的褐色藻体与相对低营养海区的黄色藻体，测定前者的叶绿素含量显著高于后者，提示黄色藻体处于营养胁迫状态。菌群结构与功能分析结果表明，与环境背景相比，两种状态的漂浮铜藻共附生细菌都明显不同于海水细菌，说明漂浮铜藻对其共附生细菌均具有明显的选择性，但是，褐色与黄色藻体的菌群组成却存在显著差异。在褐色藻体中，漂浮铜藻共附生细菌表现出明显的组织特异性分布趋势，但这种趋势在黄色藻体中不显著，提示藻体生理状态影响其共附生细菌群落的组装。研究发现，褐色和黄色铜藻菌群的优势功能均为氮、铁转运，提示漂浮铜藻共附生细菌的群落组装是基于功能而不是分类群。研究还首次揭示了漂浮铜藻内生菌与外生菌的组成特征，通过比较两种状态的藻体，发现优势内生菌组成稳定，均来自Bacillus和Propionibacterium；而优势外生菌变化明显，黄色藻体外生菌主要来自Flavobacterium、Paracoccus，褐色藻体外生菌主要来自Kocuria、Bacteroides与Pseudomonas。结合PICRUSt功能预测，发现优势功能蛋白主要来自外生菌，与褐色藻体相比，黄色藻体外生菌中含有更高丰度的氮、铁转运蛋白。因此我们推测，受环境影响，黄色藻体生理状态不良，将通过提出不同强度的功能需求，最终决定其共附生细菌群落的组装。

2）漂浮浒苔共附生细菌的群落结构与功能

选择采自2007、2016、2017、2018不同年份并且来自相对高营养与相对低营养不同海区的漂浮生态型浒苔（Ulva prolifera）作为样本。菌群结构与功能分析的结果表明，与环境背景相比，漂浮浒苔对其共附生细菌同样具有明显的选择性。但是，菌群组成总体上随年际和站位而显著变化，研究发现，菌群的优势功能蛋白保持一致，均为氮转运，提示漂浮浒苔共附生细菌的群落组装同样是基于功能而不是分类群。研究还首次揭示了漂浮浒苔内生菌与外生菌的组成特征，发现Cupriavidus属单一OTU在历年漂浮浒苔共附生细菌中稳定存在。PICRUSt功能预测结果显示，氮转运功能主要来自漂浮浒苔内生菌。

3）漂浮铜藻与漂浮浒苔共附生细菌群落的比较分析

与环境样本中细菌的组成相比，漂浮铜藻和漂浮浒苔都对其共附生细菌具有明显的选择性，受环境条件和宿主生理状态影响，菌群的组成均会发生明显变化，但优势功能均保持不变，说明两种漂浮海藻对其共附生细菌的组装均是基于功能而非分类群，其中，氮转运功能在两种海藻的共附生细菌中均占优势，提示共附生细菌可能参与了宿主的暴发；此外，本研究在漂浮浒苔和漂浮铜藻中均检出稳定存在的内生菌株，甚至检出同一Loktanella属优势内生菌OTU在两种海藻间共同存在。但是，二者绝大部分优势菌的组成不同；菌群优势功能在漂浮铜藻中主要来自外生菌，在漂浮浒苔中主要来自内生菌；另外，与漂浮浒苔相比，漂浮铜藻共附生细菌还含有高丰度的铁转运功能蛋白。

本研究初步揭示了黄海两种暴发性大型海藻共附生细菌的群落结构、功能及其动态变化特征，并开展了比较分析。上述科学认识将为在藻华现场开展细菌功能验证提供重要线索，同时，也将指导重要菌株的分离培养与后续藻菌互作试验体系的构建，从生物学角度为大型海藻藻华的暴发机制研究提供新认知。

Golden tides and green tides are marine ecological phenomena mainly caused by fulminant proliferation of brown seaweeds Sargassum or green seaweeds Ulva respectively, which have shown a significant uptrend worldwide in recent years, especially in coastal China. Since 2007, green tide has occurred in the Yellow Sea for successive years; at the end of 2016, large scale golden tide firstly broke out in the Yellow Sea; in 2017, the green tide and the golden tide even co-occurred in this sea area. Genetic analysis showed that, either the golden tide or the green tide in the Yellow Sea were dominated by a specific genotype of single species, clearly suggesting that the outbreaks of these two seaweeds have biological causes. Algae-associated bacteria were extensively interacted with their host, and were closely involved in those important biological processes such as growth and morphogenesis of the host. Revealing the structure and function of the bacterial communities associated with the fulminating seaweed, which would provide new cognition for elucidation of the “biological” mechanism of seaweed blooms.

In this paper, the samples of floating Sargassum horneri were collected at different sites in 2017, and the samples of floating Ulva prolifera were collected in different years and sites. Both the epiphytic and endophytic bacteria from two kinds of floating seaweeds were prepared by silica sand vortex oscillating method. In order to effectively avoid the interference generated from the 16S rDNA encoded in genomes of algal organelles, several pairs of primers were analyzed. Based on Illumina Miseq high-throughput sequencing, the community structure characteristics of bacteria associated with two blooming seaweeds were analyzed, and the PICRUSt was used to predict flora function. The results were as following:

1) Community structures and functions of the bacteria associated with the floating S. horneri.

Molecular identification results showed that the golden tide seaweed in the Yellow Sea was consisted by a single species of S. horneri. In 2017, both the brown thalli in the relatively high-nutrition sea area and the yellow thalli in the relatively low-nutrition sea area were collected, respectively. The chlorophyll contents of the former were significantly higher than that of latter, suggesting that the yellow thalli were under nutritional stress. Analysis of the structure and functions of bacterial communities showed that, compared with the environmental background, bacterial communities of S. horneri were differed from that of seawater regardless of host status, indicating the floating S. horneri in two status all had obvious selectivity to their associated bacteria, but the flora compositions were extremely significant differed between brown and yellow thalli. The bacterial communities of brown S. horneri exhibited clearly tissue specific distribution tendency, which in yellow thalli was unconspicuous, indicating that host status affected the assemblage of associated bacteria. Studies found that the dominant functions of bacterial communities in brown and yellow thalli both were nitrogen and iron transport, suggesting that the community assembly of bacteria associated with floating S. horneri was based on function rather than taxonomy. Moreover, this study firstly both revealed the composition of endophytic and epiphytic bacteria in the floating S. horneri. The composition of dominant endophytic bacteria was relatively stable both come from Bacillus and Propionibacterium regardless of host status, whereas the dominant epiphytic bacteria varied obviously. The epiphytic bacteria of yellow thalli were mainly from Flavobacterium and Paracoccus, and that of brown thalli were mainly from Kocuria, Bacteroides and Pseudomonas. The results of PICRUSt functional prediction found that the dominant functional proteins were mainly derived from epiphytic bacteria. Compared with the brown thalli, the epiphytic bacteria of the yellow thalli contained much higher abundance of nitrogen and iron transporters. Therefore, we speculated the yellow thalli in a worse physiological state under the influence of environmental conditions，was the key factor to ultimately determine the assembly of the associated bacterial communities through affect the intensity of functional requirements .

2) Community structures and functions of the bacteria associated with the floating U. prolifera

The drifting Ulva prolifera were collected in 2007, 2016, 2017, 2018, and from relatively high-nutrition or relatively low-nutrition sea areas. The results of flora structure and function analysis showed that, compared with the environmental background, the floating U. prolifera samples also have obvious selectivity to its associated bacteria, but the composition of the bacterial community was significantly varied with interannual and sampling sites. Studies found that the dominant functions of the bacterial community were consistent, i.e. nitrogen transport, suggesting that the assemblage of bacterial communities associated with floating U. prolifera was also based on functions rather than taxonomy. Moreover, this study revealed the composition characteristics of endophytic and epiphytic bacteria of floating U. prolifera for the first time, and found a single OTU from Cupriavidus was stable in floating U. prolifera over the years. The result of PICRUSt functional prediction showed that the nitrogen transport function was mainly from the endophytes of floating U. prolifera.

3) Comparative analysis of the bacterial communities associated with the floating S. horneri and floating U. prolifera.

Compared with the bacterial composition of environmental samples, floating S. horneri and floating U. prolifera both has obvious selectivity for its-associated bacteria, under the influence of environmental conditions and physiological status of host, the composition of associated bacteria were changed obviously, but the dominant functions remain consistent, indicating that the associated bacterial community assemblage of two kinds of blooming seaweeds were based on function rather than taxonomy. Nitrogen transport was predicted as the dominant function in floating S. horneri and floating U. prolifera, suggesting that the associated bacteria participated to host outbreak. Additionally, both the S. horneri and the U. prolifera were detected to harbor stable endophytic strains, and even consistent in the same dominant OTU of Loktanella. However, we found a vast majority of dominant bacterial composition was differed between two seaweed; the dominant functions of bacterial communities associated with floating S. horneri were mainly from epiphytic bacteria, while in the floating U. prolifera were from endophytes; meanwhile, compared with floating U. prolifera, the iron transport function was also dominant in the floating S. horneri.

This study preliminary revealed the community structure, function and dynamics of bacterial communities associated with two blooming seaweeds in the Yellow Sea, and carried out a comparative analysis. The above scientific knowledge will provide important clues for the verification of bacterial function during the seaweed bloom in the field, and also will guide the isolation and culture of important strains, and will construct an algae-bacteria interaction test system subsequently, which would provide new insights for studying the outbreak mechanism of seaweed blooms from a biological perspective.