Collaborative Research: Combining single-cell and community 'omics' to test hypotheses about diversity and function of planktonic ciliates

浮游纤毛虫是海洋食物网中的关键成员，它们在海洋食物网中扮演着不同的角色，包括作为较小微生物和较大浮游生物之间的食物链纽带。由于纤毛虫体型小，难以识别和培养，我们对海洋中纤毛虫多样性和分布的了解比对鱼类和无脊椎动物等大型生物的了解要少。该团队之前的工作测量了沿海水域的纤毛虫多样性，发现不同的遗传变异在时间和空间上是分开的，这种方式可能与海洋温度、盐度和深度梯度等因素有关。许多问题仍未得到解答，重要的是要了解控制浮游生物多样性和分布的环境因素，如纤毛虫，以预测这些生物在未来几十年可能如何应对不断变化的环境。这个项目的重点是：1)如何利用单细胞基因组学和转录组学来描述纤毛虫物种；2)基于DNA的研究，将提供生态背景的所有纤毛虫和其他浮游动物的SAR支系(Stramenopila，Alveolata，Rhizaria)；3)单细胞和转录组学的原位基因表达；以及4)培育的纤毛虫物种的基因表达的实验室研究。该项目涉及博士后学者、研究生和本科生的培养。研究人员致力于创建多样化和包容性的研究实验室；参与者的招募将通过与我们校园内的适当团体合作完成。该项目与史密斯学院和康涅狄格州大学(包括康涅狄格州大学/神秘水族馆联合REU)的本科生暑期研究经验(REU)活动相结合，特别关注代表人数不足的学生。该项目还加强了与康涅狄格州大学诺伊斯学者计划合作扩大对生物多样性的了解的努力，该计划帮助转行的STEM专业人员成为服务不足的中学的教师。该项目将评估生殖隔离物种的分布，使用一种新的方法来表征纤毛虫生殖系基因组的区域。此外，它还将利用系统基因组学方法，从陆架环境中的后转录中确定纤毛虫的分支特异转录物(例如，纤毛虫的转录物)，并通过实地收集的细胞的单细胞转录组来扩大纤毛虫功能的知识。这些方法将是对基于培养的方法的重大改进，这些方法可能偏向海洋中的“杂草”物种。明确的物种鉴定与通过单细胞和转录组分进行功能评估相结合，有望在海洋浮游生物生态学方面取得重大进展。这项研究集中在两个广泛的问题上：1)观察到的系统发育信息基因的高度多样性是否反映了浮游纤毛虫的生殖隔离和功能分化？2)不同种类的浮游纤毛虫是否表现出与海洋中不同生态位相对应的实质性功能差异？该项目通过分析浮游纤毛虫形态物种胚线微核的模式来评估物种边界(即生殖隔离)；描述海洋中密切相关的纤毛虫跨越生态梯度的转变；并通过转录本的分析来检查物种内和物种之间以及群落中的功能差异。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

Planktonic ciliates are key members of marine food webs where they serve diverse roles, including as food chain links between smaller microbes and larger plankton. Due to their small size and difficulties in identifying and cultivating them, we know less about ciliate diversity and distributions in the ocean than we do about larger organisms such as fish and invertebrates. Previous work from this team measured ciliate diversity in coastal waters and found that distinct genetic variants were separated in time and space in a way that could be related to factors such as ocean temperature, salinity, and depth gradients. Many questions remained unanswered, and it is important to understand the environmental factors that control the diversity and distribution of plankton such as ciliates to predict how these organisms may respond to a changing enviroment in the coming decades. This project focuses on: 1) how ciliate species are delineated using single-cell genomics and transcriptomics; 2) DNA-based studies of all ciliates and other planktonic members of the SAR clade (Stramenopila, Alveolata, Rhizaria), which will provide ecological context; 3) in situ gene expression by single-cell and meta- transcriptomics; and 4) laboratory studies of gene expression in cultivated ciliate species. This project involves training of postdoctoral scholars, graduate students, and undergraduates. The researchers are committed to creating diverse and inclusive research labs; recruitment of participants will be done through partnership with appropriate groups on our campuses. The project integrates with summer Research Experiences for Undergraduates (REU) activities at both Smith College and UCONN (including the UCONN/Mystic Aquarium joint REU), which are especially focused on underrepresented students. This project also enhances efforts to broaden understanding of biodiversity in partnership with the UCONN Noyce Scholars Program, which facilitates career-changing STEM professionals to become teachers in underserved secondary schools.This project will assess distributions of reproductively-isolated species, determined using a new method to characterize regions of the ciliate germline genome. Furthermore, it will use phylogenomic methods to identify clade-specific transcripts (e.g. those of spirotrich ciliates) within metatranscriptomes from the shelf environment and to expand knowledge of ciliate function with single-cell transcriptomics of field-collected cells. These approaches will be a substantial improvement over the culture-based methods that are potentially biased towards "weedy" species in the ocean. The combination of definitive species identification with assessment of function via single-cell and meta- transcriptomics promises to provide significant advances in marine plankton ecology. The research focuses on two broad questions: 1) does the observed high diversity in phylogenetically-informative genes reflect reproductive isolation and functional differentiation in planktonic ciliates? and 2) do different co-occurring species of planktonic ciliates show substantial functional differences that correspond to different niches in the ocean? The project assesses species boundaries (i.e. reproductive isolation) through analyses of patterns in the germline micronuclei of planktonic ciliate morphospecies; characterizes transitions of closely-related ciliates across ecological gradients in the ocean; and examines functional differences within and between species, and in communities, through analyses of transcriptomics.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.