河口是河流、陆地和海洋交汇处，存在着淡水和海水的混合，具有明显的物理、化学梯度，是研究微生物多样性、微生物驱动的生物地球化学循环与环境条件相互关系天然的实验场。环境中真菌通常是有机物的分解者，在碳循环中发挥重要作用；真菌在水环境中也可能侵染浮游植物，从而影响初级生产。近来的研究发现环境中存在一类未可培养的真菌---- 隐真菌门。现已知隐真菌在淡水、土壤、海洋环境中普遍存在且具有较高的多样性，但它们在河口环境的多样性、分布与环境条件的关系等还不清楚。另外，在环境因子对细菌多样性影响研究方面，盐度与季节性被认为是两个最重要的环境因子，但它们的相对重要性仍有待研究，古菌与真核微生物多样性对环境变化的响应模式是否一致也为未可知。基于以上背景，主要开展两个方面的工作：1）河口滩涂沉积物中真菌的多样性、垂直分布及与沉积物及水质的关系；2）河口强烈盐度梯度下细菌、古菌、真核微生物群落结构、多样性的周年变化及对环境因子的响应。

    于 2010 年 11 月、2011 年 8 月采集了莱州湾胶莱河、白浪河、堤河下潮区沉积物芯样，比较了 3 个河口沉积物表层 2 个深度（0-2 cm，2-5 cm）、3 个重复、2 个季节真菌的群落群落结构。通过真菌 ITS-LSU rDNA 克隆文库的构建与系统进化分析，检测到真菌界中的担子菌门、壶菌门、子囊菌门、芽枝霉门、隐真菌门以及处于真菌系统发育树基部的系统地位暂时不明的真菌类群。这些基部类群的序列在样品中相对丰度均较高，极可能属于隐真菌的另一支系，但还需进一步证据支持。基于末端限制性酶切片段长度多态性（T-RFLP）的群落结构比较及非参数多维尺度定标（nMDS）显示，36 个沉积物样品中真菌群落结构无明显的深度或地点区分，但冬夏差别明显(p=0.04)；典型对应相关分析（CCA）表明，沉积物中真菌群落结构只与铜离子浓度显著相关（p<0.05），与地点、深度、温度及其他环境因子（上覆水盐度、pH、叶绿素含量、溶氧量；沉积物中亚硝酸盐、硝酸盐、活性磷酸盐、铵离子、溶解有机氮、总有机碳）均无显著相关。

    从 2012 年 1 月起连续 12 个月在烟台逛荡河低、中、高三个盐度梯度站点采集水样36 份，通过特异性引物及 T-RFLP 分别分析真核微生物、细菌、古菌群落结构与盐度、季节变化的关系。nMDS 与 CCA 分析表明烟台逛荡河水体中真核微生物群落结构与温度、溶氧极显著相关性，与 pH 有显著相关性；细菌群落结构与盐度、温度、铵离子显著性相关；古菌的群落结构与温度、溶解氧、总氮及 pH 有显著性相关。

    本研究给出了河口沉积物中隐真菌或低等基部真菌普遍存在的证据；研究还表明盐度（而非季节性或温度）是影响河口环境中细菌群落最大的环境因子；温度则是对真核微生物、古菌群落结构影响最大的环境因子。该工作为海洋真菌的多样性及生物地理、河口微生物对环境变化的响应研究提供了基础资料。

Estuaries are important ecosystems where freshwater and seawater mixes. The steep physical and chemical gradients in estuaries present a natural laboratory for studying the microbial diversity, microbially drving biogeochemical cycles, and their relationships with environmental conditions. Fungi play an important role in carbon cycle in estuaries as decomposers of organic matters, and in regulating primary production as parasites to phytoplankton. In 2011, a novel fungal phylum Cryptomycota was established, with diversity being estimated to be high and ubiquitously distributed. However, little is known for the diversity and distribiton of Cryptomycota in estuarine environments. Regarding the effect of environmental condition on microbial diversity, it has been demonstrated that salinity and seasonality, are two most important factors structuring bacterial community, whereas which factor is more influential is conserversial. It is also unclear whether or not the response 

patterns of Archaea and Eukarya to environmental stresses are similar to Bacteria. This work focused on two aspects: 1) the diversity, vertical distribution of fungi (especially Cryptomycota) in estuarine sediments and their relationships with environmental conditions; 2) comparison of the annual cycles of bacterial, archaeal and eukaryotic microbes along a salinity gradient. 

For the fungal study, surface sediment samples were collected from three different estuaries off Laizhou Bay, Jiaolai River, Bailang River, and Di River, in November (winter) 2010 and August (summer) 2011. The top two layers (0-2 cm, 2-5 cm) at 9 sites in two seasons were investigated. Clone library and sequencing of PCR amplified fungal ITS-LSU rDNA showed that there was six fungal phyla namely Basidiomycota, Chytridiomycota and Ascomycota, Blastocladiomycota and Cryptomycota were detected. Interestingly, a large number of environmental sequences were phylogenetically placed in the basal position of the fungal tree of life, indicating they are putatively Cryptomycota. Terminal restriction fragment length polymorphism (T-RFLP) analysis of rDNA was carried out to assess the fungal community changes among samples. Non-metric multidimensional scaling (nMDS) and ANOSIM analyses indicated that there was no significant difference of benthic fungal 

assemblages between different locations and vertical layers, except for seasons (p<0.05). Canonical correspondence analysis (CCA) showed there were no significant relationships between the fungal community and environmental factors, except for copper concentration in sediments. 

For comparison of distribution pattern of three domains in estuaries, a total of 36 water samples were monthly collected through 2012, at three sites of the estuary of Guangdong River Yantai city. The three sites were categorized into freshwater, brackish, and marine. Eukaryotic microorganisms, bacteria, and archaea communities were analysed by using domain-specific primers and PCR-T-RFLP. nMDS and ANOSIM indicated that there were significant differences of bacterial community structures along the salinity gradient, whereas communities of archaea and microbial eukaryotes are significantly different by season. CCA indicated that the eukaryotic community had significant correlations with temperature,dissolved oxygen and pH. Archaeal community varied significantly with temperature, dissolved oxygen, dissolved inorganic nitrogen, and pH. Bacterial community was significantly affected by temperature, salinity, and ammonium.

In summary, this study highlights that there could be high and yet unknown diversity of Cryptomycota in estuarine sediments, supports that fungi may disperse in estuaries in random, and indicates that bacteria, archaea, and microbial eukaryotes response differently to environmental gradients.