Collaborative research: Diversity and dynamics of planktonic ciliates - what can next-generation sequencing technologies tell us?

合作研究：浮游纤毛虫的多样性和动态——下一代测序技术能告诉我们什么？

• 批准号: 1130033
• 负责人: George McManus
• 金额: $ 40.88万
• 依托单位: University of Connecticut
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1130033&HistoricalAwards=false
• 关键词: Collaborative; research; Diversity; dynamics; planktonic

The Ocean's biomass and diversity are predominantly microbial, yet this aspect of diversity remains underexplored. Efforts in recent years have begun to document microbial diversity in marine systems, and to elucidate the processes that structure assemblages across space and time. This project focuses on two important sister clades of microbial eukaryotes, the oligotrich and choreotrich ciliates. These organisms comprise a major component of planktonic food webs as they graze on phytoplankton, and are in turn eaten by zooplankton and larval fish.Earlier molecular work on ciliate diversity relied on light microscopy, construction of clone libraries and Sanger sequencing. This revealed a high degree of cryptic diversity (similar species that are genetically distinct), which is surprising, given the long-held idea that all microbes are globally distributed and that few species exist, at least as compared to animals and plants. This past work also showed that ciliate assemblages contain a few highly abundant forms and many rare ones, consistent with the concept of a "rare biosphere". However, these methods are limited by high costs of both labor and materials, so that efforts to sample any local assemblage comprehensively usually resulted in undersaturation (repeated sampling continued to uncover new species). Next generation approaches are needed to truly assess the depths of biodiversity in planktonic ciliates.This project brings together investigators with strengths in ecology, taxonomy and oceanography (PI McManus) and in molecular evolution, systematics and bioinformatics (PI Katz). Pyrosequencing will be used to sample the oligotrichs and choreotrichs "to exhaustion" in coastal environments. Denaturing gradient gel electrophoresis (DGGE), a technique that generates a fingerprint of the diversity in a sample, will be used to pre-select samples for pyrosequencing based on where strong gradients are observed in the composition of assemblages in relation to environmental factors (density fronts, thermoclines, etc.). Using these approaches, combined with an informatics pipeline already in place, this project addresses three specific objectives:Objective 1. Determine the spatial scale of variability in ciliate diversity by measuring how ciliate assemblages change over meter, kilometer, 100 km, and basin scales.Objective 2. Assess the contributions of different size classes of ciliates to overall assemblage diversity.Objective 3. Experimentally evaluate factors that control the temporal shift of individual species from rarity to commonness in a natural assemblage, and vice versa.BROADER IMPACTS: A program to involve K-12 teachers in research, initiated under a previous award, will be continued. For the past three years, teachers have been hosted at the UConn marine labs, where they conducted independent research, learned to sample and enumerate plankton, and developed teaching resources for subsequent use in the classroom. In this project the program will be adapted to accommodate the shorter, more intense experiences most teachers prefer, and to incorporate an instructional component in broader marine sciences. In addition to the teacher program, this project will involve training of postdoctoral fellows and undergraduates. This will include cross-disciplinary work through exchanges between the PI laboratories, with a focus on experimental design, data collection/analysis, and manuscript preparation. Both PIs are committed to involving students from underrepresented groups in their research, and PI Katz maintains intensive recruiting efforts in this area. Hence, the project will further increase the diversity of researchers studying biological oceanography.

海洋的生物量和多样性主要是微生物，但这方面的多样性仍未得到充分探索。近年来，人们开始努力记录海洋系统中的微生物多样性，并阐明跨空间和时间构建组合的过程。该项目重点关注微生物真核生物的两个重要的姐妹进化枝，寡毛纤毛虫和杂毛纤毛虫。这些生物体以浮游植物为食，构成浮游食物网的主要组成部分，进而被浮游动物和幼鱼吃掉。早期关于纤毛虫多样性的分子研究依赖于光学显微镜、克隆文库的构建和桑格测序。这揭示了高度的神秘多样性（基因上不同的相似物种），这是令人惊讶的，因为长期以来人们一直认为所有微生物都分布在全球，并且存在很少的物种，至少与动物和植物相比是这样。过去的工作还表明，纤毛虫组合包含一些高度丰富的形式和许多稀有的形式，与“稀有生物圈”的概念一致。然而，这些方法受到劳动力和材料成本高昂的限制，因此对任何当地组合进行全面采样的努力通常会导致饱和度不足（重复采样不断发现新物种）。需要下一代方法来真正评估浮游纤毛虫生物多样性的深度。该项目汇集了生态学、分类学和海洋学 (PI McManus) 以及分子进化、系统学和生物信息学 (PI Katz) 方面具有优势的研究人员。焦磷酸测序将用于对沿海环境中“耗尽”的寡毛虫和杂毛虫进行采样。变性梯度凝胶电泳 (DGGE) 是一种生成样品多样性指纹的技术，将用于根据在与环境因素（密度前沿、温跃层等）相关的组合组成中观察到强梯度的位置来预选用于焦磷酸测序的样品。利用这些方法，结合已有的信息学流程，该项目实现了三个具体目标： 目标 1. 通过测量纤毛虫组合在米、公里、100 公里和盆地尺度上的变化来确定纤毛虫多样性变异的空间尺度。目标 2. 评估不同大小类别的纤毛虫对整体组合多样性的贡献。目标 3. 通过实验评估控制单个物种时间变化的因素 更广泛的影响：一项让 K-12 教师参与研究的计划将继续进行，这项计划是根据先前的奖项发起的。在过去的三年里，教师们一直在康涅狄格大学海洋实验室进行独立研究，学习对浮游生物进行采样和计数，并开发了随后在课堂上使用的教学资源。在这个项目中，该项目将进行调整，以适应大多数教师喜欢的更短、更密集的体验，并纳入更广泛的海洋科学的教学内容。 除了教师计划外，该项目还将涉及博士后研究员和本科生的培训。这将包括通过 PI 实验室之间的交流进行跨学科工作，重点是实验设计、数据收集/分析和手稿准备。两位 PI 都致力于让来自代表性不足群体的学生参与他们的研究，PI Katz 在这一领域保持着密集的招募工作。因此，该项目将进一步增加生物海洋学研究人员的多样性。