基于高通量测序的不同环境因子对网柄细胞状黏菌个体发育及多样性影响研究

Dictyostelid cellular slime molds (dictyostelids) are the main group of slime molds which could feed, carry and seed bacteria. This kind of convergent evolution between dictyostelids and bacteria form an important model for the evolutionary researches of eukaryotes. Dictyostelids are the main consumer of bacteria in soil which have an important effects on the health and nutritional cycle of soil. So, dictyostelids are the main group in soil ecosystem. Nowadays, information on possible roles of dictyostelid in soil is extremely limited. As a consequence, we know very little about the behavior of dictyostelids in the wild or their ecological relationships with other taxa. There is no research about the the effects of macroscopic and microscopic environment on the ontogeny and diversity of dictyostelids. Based on the diversity researches of dictyostelids for more than 10 years, in this stiudy, the effects of macroscopic environmental facters (such as forest type, elevation and latitude) and soil microecology on the ontogeny and diversity of dictyostelids will be analyzed by the methods combined with next-generation sequencing (amplicon and metagenome sequencing) and traditional isolation. The relationships of soil mircroorganism and diversity of dictyostelids diversity will be discussed here. The essential environmental factors which affected the ontogeny and diversity of dictyostelids will be clarified. The convergent evolution between bacteria and dictyostelids will be discussed in further. This study have significances for the clearance of the distribution and resource of dictyostelids in China. The present study will provide basic information for the determination of characteristics of circulation in soil ecosystem.

网柄细胞状黏菌（即网柄菌）是广义“黏菌”的主要类群，它可摄食、携带并播种细菌，二者间的这种协同进化关系，使其成为真核生物演化研究的重要模式生物。网柄菌是土壤细菌的主要“消费者”，对土壤的健康状况及营养循环起重要作用，进而成为土壤生态系统的主要成员。目前，网柄菌在土壤生态循环中的具体功能、与其它微生物群体间的生态关系尚不清楚，缺少宏观、微观环境因子对网柄菌发育及多样性影响的相关研究。本项目在十余年网柄菌多样性研究的基础上，采用扩增子和宏基因组高通量测序技术，辅以传统分离方法，综合比较不同植被、海拔、纬度的宏观环境因子及土壤微生态特征对网柄菌的个体发育和多样性差异产生的影响，研究土壤微生物群体与网柄菌多样性的关系，阐明影响网柄菌个体发育和多样性的关键环境因子，进一步探讨网柄菌与细菌的协同进化关系。这对明确我国网柄菌的分布及资源蕴藏量具有重要意义，为阐明土壤生态系统的物质循环特征提供基础数据。

项目组在2019-2022年度期间，采集吉林省长白山国家级自然保护区、湖北省西部的神农架林区、及海南省尖峰岭国家级自然保护区的200份土壤样品，从中分离得到网柄菌2目3科4属20种，基于传统形态分类学、个体发育和分子系统学研究，确定其分类地位，共包括已知种8种，新种12种。通过自主设计的适于网柄菌18S扩增子测序的引物，对环境样品进行自然状态下网柄菌的多样性分析，获得151个ASVs（共239288条），注释到网柄菌已知种26个，61个ASVs为未知种。表明自然界中大量潜在的网柄菌物种未曾被发现或分离，并验证了网柄菌的分布具有一定的生物地理模式。通过CCA方法进行环境因子的关联分析，得到本研究中影响不同环境条件下网柄菌群落变化的主要环境因子是pH和海拔。得出不同种类网柄菌的最适环境因子，由此预测网柄菌的物种分布特征及稀有种分布信息。通过对来自不同环境条件的网柄菌菌株不同发育阶段共生细菌的16S扩增子测序，发现不同环境下的网柄菌的生活史阶段携带细菌的相对丰度变化趋势不同，且不同环境中分离得到的网柄菌携带的共生细菌种类存在差异。结合其土壤生境中细菌微生态特征，进行环境因子的关联分析，确定影响网柄菌个体发育的关键环境因子为植被类型和经度。