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黄渤海日本鳗草沉积物三域微生物的分布特征、生态功能及驱动因素
Alternative TitleStudies on the microorganisms of three domains associated with seagrass (Zostera japonica) meadows: distributions, ecological functions, and environmental drivers
刘鹏远
Subtype博士
Thesis Advisor胡晓珂
2022-05-12
Training institution中科院烟台海岸带研究所
Degree Grantor中国科学院大学
Degree Name理学博士
Degree Discipline海洋生物学
Keyword海草 三域微生物 环境驱动力 暖化 生物地理学
Abstract海草床作为最富生产力的近海生态系统之一,发挥着海岸防护、蓝碳封存、鱼类产卵场、水质净化等重要的生态价值。海草作为唯一能在海水中生存的被子植物,孕育着丰富多样的微生物。这些微生物一方面与海草的生长及健康休戚与共,另一方面为整个海草生态系统的养分循环发挥着重要的调节作用。在人类生产活动加剧和全球暖化的时代背景下,我们关于微生物活动对海草健康产生的直接或间接影响仍缺乏系统性认知;尽管目前细菌在全球海草床系统的地理分布特征已被较好地揭示,但对于古菌和微型真核生物,这两类重要的微生物群落在海草床系统中有着怎样的分布规律和生态重要性,我们的认知仍非常有限。本研究选择分布于东营、威海、大连的三个典型温带海草床(日本鳗草,Zostera japonica)作为研究区域,利用扩增子测序、宏基因组学、荧光定量PCR、室内模拟实验等技术和方法,(1)在群落层面,调查沉积物中三域微生物的多样性、季节动态、生境偏好性及海草定植的影响;(2)在功能层面,揭示草区与裸露区沉积物中参与氮、硫循环的功能类群多样性和代谢途径,及其在海草不同生长阶段的变化规律;(3)基于野外调查结果,我们提出“升温导致硫循环类群间的失衡构成海草的潜在威胁”的假设,并构建微宇宙模拟实验进行验证。主要研究结果如下: (1) 海草表层沉积物中,真核微生物主要由硅藻(Diatomea;21.8%)和甲藻(Dinoflagellata;20.4%)等单细胞藻类组成。在古菌群落中,氨氧化古菌(Ca. Nitrosopumilus和Ca. Nitrocosmicus)是主导类群(58.1%)。硫卵菌属(Sulfurovum)、伍斯菌属(Woeseia)、Sva0081等硫循环类群在细菌群落中丰度较高(18.8%)。共现网络显示,真核生物与古菌间存较高的共现性,可能具有紧密的互作关系。相比于海草定植作用,生境异质性及季节变化是影响三域微生物α-多样性的重要因素。这可能是因为沉积物理化因素在大尺度空间上的差异,掩盖了海草定植的影响。季节上,草区真核微生物α-多样性呈现出春、夏季高于秋、冬季,而细菌与古菌多样性略有推迟,在夏、秋季高于春、冬季节。三域微生物多样性对环境响应的灵敏度为:细菌 > 真核微生物 > 古菌。 (2) 无论海草定植还是季节变化都能显著改变细菌、古菌和真核微生物群落结构,并且海草定植影响(R > 0.08,P < 0.001)大于季节变化(R > 0.07,P < 0.011),表明根际效应确实能明显影响微生物群落分布且不受季节影响。草区的SO42−、有机碳(Total organic carbon,TOC)和有机氮(Total organic nitrogen,TON)含量明显高于裸露区,因而促进了硫酸盐还原菌(Sulfate-reducing bacteria,SRB)、固氮菌、纤维降解菌(深古菌及MBG-D)、产甲烷古菌(甲烷叶菌属)等功能类群在海草定植区中富集。另外,海草定植能显著降低单细胞藻类及病原菌的多样性,这可能与海草表面富集的溶藻细菌及海草分泌的代谢产物对藻类孢子及病原菌的抑制作用有关。海草在不同季节也具有不同的特征微生物类群,其中溶氧(Dissolved oxygen,DO)、营养盐(SO42−、NH4+和NO3–)和重金属(Cr、Cd和As)是导致三域微生物群落结构季节差异的主要驱动因子。 (3) 硫循环功能分析显示,δ-变形菌纲和γ-变形菌纲是海草底栖SRB的重要组成,而硫化物氧化过程主要由γ-变形菌纲和α-变形菌纲完成。硫化物氧化基因fccA在海草沉积物硫循环基因库中占主导地位。草区沉积物显著富集了硫代硫酸盐还原基因,而裸露区中具有更多异化硫酸盐还原基因、连四硫酸盐还原基因及硫氧化基因。温度和DO是影响硫循环基因多样性和丰度的关键因素。氮循环功能分析发现,参与氮循环的主要类群为γ-变形菌纲和α-变形菌纲。羟胺氧化基因(hao)及硝酸盐还原基因(narI/narV和nrfA)在氮循环基因库中占据优势地位。草区富集了更多羟胺氧化基因(hao)和固氮基因(nifK),而非草区中反硝化基因(nosZ、nirS)更为丰富。在季节上,反硝化基因(nrfA、narGH、nosZ和nirS)的相对丰度在冬季低于夏季,而固氮基因(nifDHK)呈冬高夏低的分布规律,因此微生物通过增加氮固定并减少氮素流失可能是海草越冬的关键。随机森林分析显示温度和NH4+是影响氮循环基因多样性和丰度的主要环境因子。SRB是耦联氮、硫循环的关键类群。此外,在日本鳗草沉积物中检测到较高丰度的金属抗性基因、单碳代谢 (C1)基因以及无机磷酸盐转运基因。 (4) 微宇宙培养实验,证实温度在塑造海草底栖细菌(尤其是硫代谢相关类群)群落结构中发挥着关键作用。绝对定量结果显示细菌、SRB、硫氧化菌(Sulfur-oxidizing bacteria, SOB)对温度的响应规律一致:绝对丰度都随温度先升后降;然而SRB:SOB值却随着温度的升高而增加,表明高温胁迫对SOB的抑制作用明显高于SRB。SRB和SOB的失衡可能导致硫化物不能被SOB充分氧化而毒害海草健康,初步验证了我们的猜想。 综上,本研究系统地探究了海草生境中细菌、古菌和真核微生物的多样性、丰度和功能在不同生态位、区域和季节间的分布规律,阐明三域微生物间的网络关系以及环境因素(如营养盐、重金属污染和升温)的调控规律,并提出微生物介导的“升温-硫循环-海草退化”机制,有助于我们更好地理解三域微生物与海草之间的互作机理,并为海草床健康监测和生态修复提供新见解。
Other AbstractSeagrass meadow is one of the most valuable coastal ecosystems, serving significant ecological functions in coastal protection, carbon sequestration, fish nursery, and water purification. As the only angiosperm that can live in seawater, seagrass host abundant and diverse communities of microorganisms. These microbes fundamentally influence the seagrass physiology and health, and also regulate the biogeochemical dynamics of entire seagrass meadows. Under the scenario of intensified anthropogenic activities and global warming, a systematic understanding of the direct or indirect effects of microbial activities on seagrass health and growth is still lacking. Although the biogeographical distribution of bacteria associated with seagrass has been well depicted, knowledge of ecologically important archaea and microeukaryotes on how to interact with seagrass remain scarce. In this study, three typical temperate seagrass meadows (Zostera japonica) distributed in northern China (Dongying, Weihai, and Dalian) were investigated. With amplicon sequencing, metagenomics, qPCR, and microcosm approaches, we aimed to reveal the followings: (1) the effects of season variations, habitat heterogeneity, and seagrass colonization on the distributions of three-domain microorganisms at the community level; (2) the dynamic of functional microbial guilds involved in nitrogen and sulfur cycles with seagrass growth, and their metabolic differences between vegetated and bare areas at the gene level; (3) the hypothesis that “warming threatens seagrass health by broking the balance of benthic sulfur-cycling microorganisms”, and then verified by a microcosm experiment. The main findings are listed as follows: (1) The microeukaryotic assemblages associated with surficial sediments of Z. japonica were mainly composed of diatoms (21.8%) and dinoflagellates (20.4%). Ammonia-oxidizing archaea (Ca. Nitrosopumilus and Ca. Nitrocosmicus) was the dominant archaeal group (58.1%). Sulfur-cycling taxa Sulfurovum, Woeseia, and Sva0081 were frequently detected (18.8%) in benthic bacterial communities. The network analysis showed a relatively high co-occurrence between archaea and eukaryotes, indicating their close interkingdom interactions. In contrast with seagrass-vegetated effects, the α-diversity of the three-domain microorganisms was more affected by habitat heterogeneity and seasonal variations. A possible reason is that environmental factors changed evidently on a large spatial scale, which masked the effects of seagrass vegetation. The microeukaryotic community exhibited higher α-diversity estimators in spring and summer than those in autumn and winter. For the bacterial and archaeal assemblages, however, α-diversity estimators were higher in summer and autumn than those in spring and winter. The highest sensitivity of microbial diversity to environmental shifts was bacteria, followed by microeukaryotes, and archaea the last. (2) Community structures of the three-domain microorganisms were significantly affected by both seagrass colonization and seasonal changes, and the effect of the former (R > 0.08, P < 0.001) appears to be greater than the latter (R > 0.07, P < 0.011) within a given meadows. It indicated that the rhizosphere effect was a major driving force for the microbial distribution. Compared with bare areas, higher contents of SO42−, total organic carbon (TOC), and total organic nitrogen (TON) were detected in the seagrass-vegetated sediments where enriched several functional groups, such as sulfate-reducing bacteria (SRB), diazotrophic bacteria, fiber-degrading microorganisms (Bathyarchaeota and MBG-D), methanogenic archaea (Methanolobus), etc. However, seagrass-vegetated sediments had lower diversity of microalgae and pathogens, which may be suppressed by algicidal bacteria inhabiting in seagrass surface and metabolites secreted by seagrass. Furthermore, seagrass meadows possessed distinct microbial taxa in different seasons, which were mainly driven by DO, nutrients (SO42−, NH4+, NO3−), and heavy metals (Cr, Cd, and As). (3) Shotgun sequencing revealed that δ- and γ-proteobacteria were the most prevalent SRB members, while γ- and α-proteobacteria were the major sulfur oxidizers. Sulfide oxidizing gene fccA dominated in the sulfur-cycling gene pool. Thiosulfate reduction genes were enriched in seagrass-vegetated sediments, and genes encoding dissimilatory sulfate reductase, tetrathionate reductase, and sulfur-oxidizing protein were detected with higher abundance in the bare sediments. Temperature and DO were the major influential environmental factors on the diversty and the relative abundance of sulfur-cycling genes. The main participants in nitrogen cycling were composed of γ- and α-proteobacteria. Gene hao encoding hydroxylamine dehydrogenase and gene narI/narV and nrfA encoding nitrate reductase were the most abundant in the nitrogen-cycling gene pool of Z. japonica system. The relative abundances of genes hao and nifK were more frequently detected in the vegetated areas, and more abundant nosZ and nirS genes were found in unvegetated sediments. The distribution of denitrifying genes (nrfA, narGH, nosZ, nirS) presented lower abundances in the winter but higher in the summer, whereas an opposite pattern was found in genes nifDHK that related to nitrogen fixation. Therefore, these nitrogen fixators may play a key role in seagrass overwintering by providing nitrogen nutrients. Random forest analysis showed that the temperature and NH4+ were the crucial environmental drivers on the diversity and abundance of nitrogen-cycling genes. SRB was a key group coupling the transformations of nitrogen and sulfur elements. In addition, genes involved in heavy metal resistance, single-carbon (C1) metabolic pathways, and inorganic phosphate transportation were highly detected in seagrass ecosystems. (4) The microcosm assays validated that the temperature plays a key role in shaping benthic bacterial community structure in seagrass sediments. The qPCR results showed that bacteria, SRB, and sulfur-oxidizing bacteria (SOB) had consistent patterns in response to warming, and their abundances increased firstly and then decreased from 26°C to 35°C. In addition, SRB:SOB ratio increased with warming, indicating that SOB is more readily to be suppressed by high temperature than SRB. The imbalance between SRB and SOB might lead to the accumulation of sulfide, which is harmful to seagrass health. These findings basically verified our assumption. In conclusion, we comprehensively investigated the dynamics of diversity, abundance, and functions of bacteria, archaea, and microeukaryotes among the three seagrass meadows across four seasons. The complex network relationships among the three-domain microorganisms and the regulation mechanisms of environmental drivers (such as nutrient concentrations, heavy metals, and temperature) were unveiled at large, and we proposed a microbial-mediated frame of "warming-sulfur cycling-seagrass degradation". These findings improved our understanding of the interactions between three-domain microorganisms and seagrass, and provided new insights into the health monitoring and ecological restoration of seagrass meadows.
Pages202
Language中文
Document Type学位论文
Identifierhttp://ir.yic.ac.cn/handle/133337/30823
Collection中科院烟台海岸带研究所知识产出
Recommended Citation
GB/T 7714
刘鹏远. 黄渤海日本鳗草沉积物三域微生物的分布特征、生态功能及驱动因素[D]. 中国科学院大学,2022.
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