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然没有得到充分探索。近年来的努力已经开始记录海洋系统中的微生物多样性，并阐明在空间和时间上构建组合的过程。这个项目的重点是两个重要的姐妹分支的微生物真核生物，寡毛类和舞毛类纤毛虫。这些生物是浮游生物食物网的主要组成部分，因为它们吃浮游植物，然后又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，克隆文库的构建和桑格测序。这揭示了高度的神秘多样性（遗传上不同的相似物种），这是令人惊讶的，因为长期以来的想法是所有微生物都分布在全球，并且很少有物种存在，至少与动物和植物相比。这项过去的工作还表明，纤毛虫组合包含一些高度丰富的形式和许多罕见的，符合“稀有生物圈”的概念。然而，这些方法受到劳动力和材料成本高的限制，因此对任何当地组合进行全面采样的努力通常会导致不饱和（重复采样不断发现新物种）。该项目汇集了在生态学、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。焦磷酸测序将被用于对沿海环境中的寡毛藻和舞毛藻进行采样，直到它们“耗尽”。变性梯度凝胶电泳（DGGE），一种产生样品多样性指纹的技术，将用于预先选择样品进行焦磷酸测序，这是基于在与环境因素（密度前沿，温跃层等）相关的组合物组成中观察到的强梯度。利用这些方法，结合已经到位的信息学管道，该项目解决了三个具体目标：目标1。通过测量纤毛虫群落在米、公里、100公里和流域尺度上的变化，确定纤毛虫多样性变化的空间尺度。评估纤毛虫不同大小级对总体群聚多样性的贡献。实验评估的因素，控制个别物种从稀有到常见的自然组合的时间转移，反之亦然。更广泛的IMPLEMENTARY：一个计划，涉及K-12教师的研究，根据以前的奖项发起，将继续下去。在过去的三年里，教师们一直在康州大学海洋实验室，在那里他们进行了独立的研究，学会了采样和计数浮游生物，并开发了教学资源，供随后在课堂上使用。在这个项目中，该计划将进行调整，以适应更短，更激烈的经验，大多数教师喜欢，并纳入更广泛的海洋科学的教学组成部分。 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。两个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在这方面保持着密集的招聘工作。因此，该项目将进一步增加生物海洋学研究人员的多样性。

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.