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

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开发。近年来的努力已经开始记录海洋系统中的微生物多样性，并阐明跨越空间和时间的组合结构的过程。该项目的重点是微生物真核生物的两个重要姐妹分支--寡毛类和舞毛类纤毛虫。这些生物以浮游植物为食，构成浮游食物网的主要组成部分，然后被浮游动物和幼鱼吃掉。早期关于纤毛虫多样性的分子工作依赖于光学显微镜、克隆图书馆的构建和桑格测序。这揭示了高度的神秘多样性(遗传上截然不同的相似物种)，这是令人惊讶的，因为长期以来人们认为所有的微生物都是全球分布的，几乎没有物种存在，至少与动物和植物相比是这样。这项过去的工作还表明，纤毛虫组合包含几种高度丰富的形式和许多稀有形式，这与“稀有生物圈”的概念是一致的。然而，这些方法受到高昂的人力和材料成本的限制，因此对任何局部组合进行全面采样的努力通常会导致不饱和(重复采样继续发现新物种)。这个项目汇集了在生态学、分类学和海洋学(Pi McManus)以及在分子进化、分类学和生物信息学(Pi Katz)方面具有优势的研究人员。焦磷酸测序将被用来对沿海环境中的寡毛虫和编毛虫进行采样，直到精疲力竭。变性梯度凝胶电泳法(DGGE)是一种在样品中生成多样性指纹的技术，它将被用于预先选择样品进行焦磷酸测序，其基础是与环境因素(密度前锋、温跃层等)相关的组合组成中观察到强烈梯度的地方。使用这些方法，结合已经到位的信息学管道，这个项目解决了三个具体的目标：目标1.通过测量纤毛虫群落如何随米、公里、100公里和盆地尺度的变化来确定纤毛虫多样性的空间尺度。目标2.评估不同大小类别的纤毛虫对总体群落多样性的贡献。目标3.实验评估控制自然群落中个别物种从稀有到常见以及反之亦然的因素。BROADER影响：将继续实施一项由K-12教师参与研究的计划，该计划是在先前的奖项下发起的。在过去的三年里，老师们一直被招待在康涅狄格州大学的海洋实验室，在那里他们进行独立的研究，学习采样和计数浮游生物，并开发教学资源供随后的课堂使用。在这个项目中，该计划将进行调整，以适应大多数教师喜欢的更短、更激烈的体验，并在更广泛的海洋科学中纳入教学部分。除了教师计划，该项目还将包括博士后研究员和本科生的培训。这将包括通过国际和平研究所实验室之间的交流开展跨学科工作，重点是实验设计、数据收集/分析和手稿准备。这两家私人投资机构都致力于让来自代表性不足群体的学生参与他们的研究，皮卡茨在这一领域保持着密集的招聘努力。因此，该项目将进一步增加研究生物海洋学的研究人员的多样性。

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.