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

气候变异性假说(CVH)提出，物种可以容忍的温度范围随着它们在进化过程中经历的年度温度范围的增加而增加。气候变异性随着纬度的增加而增加，因此预计温带物种将比热带物种具有更宽的耐热性、更高的扩散能力和更广泛的分布。研究人员此前询问，科罗拉多州的温带山地溪流昆虫是否比厄瓜多尔的热带物种具有更大的海拔范围；然而，只能估计常见物种的范围大小，而且只能收集一次标本。为了加强这项工作，现在将随着时间的推移对溪流昆虫进行采样(时间采样)，并将使用DNA方法来识别所有物种(常见和罕见)。时间采样将允许研究人员跟踪物种随着时间的推移从较高海拔漂移到较低海拔的海拔位置。由于水流不断将幼虫推向下游，而温带幼虫在溪流中停留的时间更长，因此在发育过程中，温带幼虫预计会比热带物种漂移到下游更远的地方。这项工作将使研究人员能够发现以前被忽视的稀有物种。这些进展通过将物种移动和稀有物种纳入海拔范围大小分布的估计中，加强了对CVH的广泛测试。这项工作的结果将提供有关气候变化导致的自然选择如何影响物种扩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进化具有普遍意义。在一年的时间里，将从科罗拉多州落基山脉和厄瓜多尔安第斯山脉(每个纬度11个地点)约1500-3500米横断面上的溪流中采集6次水生昆虫幼虫。在每个地点的每个采样日期，将使用D-Frame网从100米溪流范围内的所有微生境收集昆虫5分钟。样品将保存在乙醇中，昆虫将从沉积物和碎屑中分离出来。对于每个样本，所有昆虫组织都将被均质，DNA将使用商业试剂盒进行提取和纯化。动物DNA条形码基因，细胞色素c氧化酶亚基I(COI)，将被聚合酶链式反应(PCR)扩增。全长DNA条形码将通过鸟枪测序和使用Illumina平台上的下一代测序组装COI扩增来生成。将根据区域参考库查询DNA条形码，以确定样本的组成，重新估计分类群的海拔范围大小，并比较范围大小和在纬度之间的扩散能力。

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.