DISSERTATION RESEARCH: Temporal Sampling and DNA Metabarcoding to Test the Climate Variability Hypothesis

论文研究：时间采样和 DNA 元条形码来检验气候变异假说

• 批准号: 1502008
• 负责人: Chris Funk
• 金额: $ 1.93万
• 依托单位: Colorado State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1502008&HistoricalAwards=false
• 关键词: DISSERTATION; RESEARCH; Temporal; Sampling; DNA

The Climate Variability Hypothesis (CVH) proposes that the range of temperatures species can tolerate increases with the annual range of temperatures they have experienced over evolutionary time. Climate variability increases with latitude and thus it is expected that temperate species will have wider thermal tolerances, higher dispersal ability, and wider distributions than tropical species. Researchers previously asked if temperate mountain stream insects from Colorado have larger elevation range sizes than tropical species from Ecuador; however, range sizes could only be estimated for common species and specimens could only be collected once. To strengthen this work, stream insects will now be sampled over time (temporal sampling) and a DNA approach will be used to identify all species (common and rare). Temporal sampling will allow researchers to track species' elevational positions as they drift from higher to lower elevations over time. Because the flow of water constantly pushes larvae downstream and temperate larvae spend more time in stream, temperate zone larvae are expected to drift farther downstream during development than tropical species. This work will allow researchers to detect previously overlooked rare species. These advances strengthen an already extensive test of the CVH by incorporation of species movement and rare species into estimates of distributions of elevation range sizes. Results of this work will provide information about how natural selection resulting from climate variability influences species dispersal ability and range sizes with general implications for understanding speciation and the evolution of biodiversity. Larval aquatic insects will be collected 6 times over a yearlong period from streams located along transects from ~1500-3500m in the Colorado Rocky Mountains and the Ecuadorian Andes (11 sites at each latitude). On each sampling date at each site, insects will be collected for 5 minutes from all microhabitats within a 100m stream reach using a D-frame net. Samples will be preserved in ethanol and insects will be separated from sediment and detritus. For each sample, all insect tissue will be homogenized and DNA will be extracted and purified using commercially available kits. The animal DNA barcoding gene, Cytochrome c Oxidase subunit I (COI), will be PCR amplified. Full-length DNA barcodes will be generated by shotgun sequencing and assembly of COI amplicons using next-generation sequencing on an Illumina platform. DNA barcodes will be queried against a regional reference library to determine the composition of samples, re-estimate elevation range sizes for taxa, and compare range sizes and dispersal ability among latitudes.

气候变化假设（CVH）提出，温度范围可以随着它们在进化时期所经历的年度温度范围而忍受的范围。气候变异性随纬度的增加而增加，因此可以预期温带物种将具有比热带物种更大的热公差，更高的分散能力和更大的分布。研究人员以前曾询问，来自科罗拉多州的温带山流昆虫是否比厄瓜多尔的热带物种具有更大的海拔范围。但是，只能估计公共物种的范围尺寸，只能收集一次标本。为了加强这项工作，现在将随着时间的推移采样流昆虫（时间采样），并且将使用DNA方法来识别所有物种（常见和稀有）。时间抽样将使研究人员随着时间的流逝从较高的海拔升至较低的海拔时，可以跟踪物种的海拔位置。由于水的流动不断将幼虫推向下游，温带幼虫在溪流上的花费更多，因此预计在发育过程中，温带幼虫在下游时会比热带物种更远。这项工作将使研究人员能够检测到以前被忽视的稀有物种。这些进步通过将物种运动和稀有物种纳入高度范围尺寸分布的估计值来增强对CVH的广泛测试。这项工作的结果将提供有关气候变异性产生的自然选择如何影响物种分散能力和范围大小的信息，对理解物种形成和生物多样性的演变具有一般影响。幼虫水生昆虫将在一年中从位于科罗拉多洛矶山脉和厄瓜多尔安第斯山脉（每个纬度的11个地点）的横断面的河流中收集6次。在每个地点的每个采样日期中，使用D型框架网中从100m流到达的所有微生境中收集昆虫5分钟。样品将保存在乙醇中，昆虫将与沉积物和碎屑分离。对于每个样品，所有昆虫组织都将被匀浆，并使用市售试剂盒提取和纯化DNA。动物DNA条形码基因，细胞色素C氧化酶亚基I（COI）将被放大。通过在Illumina平台上使用下一代测序的COI扩增子的shot弹枪测序和组装，将生成全长DNA条形码。 DNA条形码将与区域参考文献进行查询，以确定样品的组成，重新估计分类单元的高度范围，并比较纬度之间的范围大小和分散能力。