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

气候变率假说（CVH）提出，物种可以忍受的温度范围随着它们在进化过程中所经历的年度温度范围的增加而增加。气候变化随纬度的增加而增加，因此，温带物种将具有更宽的耐热性，更高的扩散能力，比热带物种分布更广。研究人员先前询问来自科罗拉多的温带山溪昆虫是否比来自厄瓜多尔的热带物种具有更大的海拔范围大小;然而，只能估计常见物种的范围大小，并且标本只能收集一次。为了加强这项工作，现在将对溪流昆虫进行长期采样（时间采样），并将使用DNA方法来识别所有物种（常见和罕见）。时间采样将使研究人员能够跟踪物种的海拔位置，因为它们随着时间的推移从高海拔漂移到低海拔。由于水流不断地将幼虫推向下游，温带幼虫在溪流中停留的时间更长，因此温带幼虫在发育过程中预计会比热带物种向下游漂得更远。这项工作将使研究人员能够发现以前被忽视的稀有物种。这些进展加强了已经广泛的测试CVH纳入物种运动和稀有物种的海拔范围大小的分布估计。这项工作的结果将提供有关气候变异性导致的自然选择如何影响物种的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演变具有普遍意义。在为期一年的时间内，将从科罗拉多落基山脉和厄瓜多尔安第斯山脉约1500- 3500 m处的沿着的溪流中采集6次水生昆虫幼虫（每个纬度11个地点）。在每个采样日期，在每个地点，将使用D形框架网从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，昆虫将与沉积物和碎屑分离。对于每份样本，将所有昆虫组织均质化，并使用市售试剂盒提取和纯化DNA。将对动物DNA条形码基因细胞色素c氧化酶亚基I（COI）进行PCR扩增。全长DNA条形码将在Illumina平台上通过鸟枪测序和使用下一代测序组装COI扩增子来生成。将对照区域参考图书馆查询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，并比较不同纬度的范围大小和扩散能力。

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.