尽管潮间带存在着温度、光照、水淹等剧烈的环境变化，但潮滩最表层的底栖微生物膜在自养固碳及有机碳沿微食物网传递的过程中发挥重要且相对稳定的服务功能。目前，对底栖生物膜中真核微生物多样性与垂直迁移行为的研究大多基于显微镜观察，对其中数量巨大、多样性高的其它真核微生物的物种构成、类群间的相互作用、垂直迁移及有机碳的周转的了解却极少，也缺乏大空间尺度分布规律的认识。本项目拟在我国珠江口、长江口、黄河口的典型潮滩采取生物膜样品，主要利用PMA法提取DNA、高通量测序、定量PCR等分子生态学技术，分析生物膜中活的真核微生物类群的多样性与数量，验证底栖生物膜真核微生物群落的纬度梯度分布规律，探索真菌与硅藻的共存关系，解析表层沉积物中真核微生物群落随着光照与潮汐周期在垂直尺度上的变化，利用RNA-SIP法评估胞外多聚物的碳流传递到何种原生动物，为理解潮间带微生物对剧烈环境变化的适应与响应机制提供基础资料。

Although many environmental factors are quickly and sharply changing in intertidal zones, the thin benthic biofilm (1-2 mm thick) comprising microalgae, fungi, protozoa and bacteria on the sediment surface contributes enormously, and in a relative stable manner, to the ecosystem functions as such carbon fixation and channeling to higher trophic levels. At present, most studies of the biodiversity in this important biofilm system are based on morphological observations, and mostly focused on the autotrophs, or the microphytobenthos. Very few studies have been carried out using molecular tools that may reveal hidden diversity not recognized using traditional methodologies. Consequently, the identification of microbial eukaryotic species might be observer-dependent, and the community structure datasets of distant geographic estuarine tidal flats are hardly comparable to each other, rendering the difficulties in uniting a geographic pattern of microeukaryotes in benthic biofilms on a large scale. Furthermore, little is known about the interactions between microeukaryotes, and what protozoan species in the biofilm receive the carbon flow from diatom-derived extracellular polymeric substances (EPS). This project proposes to decipher the hidden diversity and function of microeukaryotes (especially heterotrophic components in the community, such as fungi and protozoa) in tidal benthic biofilms. We will use the propidium monoazide (PMA) assay to extract DNA from live microbial cells for downstream applications including 18S rDNA high throughput sequencing to reveal the alpha and beta diversities of microeukaryotes. Fungal and diatom reads will be retrieved, and co-occurrence of the species from these two groups will be analyzed. Different fungal clades will also be quantified using quantitative PCR targeting. We will test the follow hypotheses: there are significant differences between three large estuaries (the Pear River, the Yangtz River, and the Yellow River) in China along a latitude gradient, between muddy flat vs. sandy flat, and between morphology-based vs. molecular-based results. By sequencing and quantifying the slices from the top 1-cm layers collected from different time in a day, we can reconstruct the dynamics of microeukaryotes that could migrate vertically as often observed from benthic diatoms. Also, what protozoan species consuming particulate carbon derived from EPS will investigated using RNA stable isotope probing (RNA-SIP). The outcome of this project will be crucial to our understanding the assembly mechanisms of benthic biofilm and functional redundancy of this ecologically important system.