Flow cytometric techniques in Quaternary research: Improving radiocarbon age determination and evaluating potentials of stable isotope and aDNA analyses based on high-purity fossil pollen samples

化石花粉组合分析（即花粉分析= PA）是第四纪研究中最常用的方法之一，对我们了解过去的环境变化、人类与环境的相互作用、土地利用历史以及农业的起源和传播有着重要的贡献。现有的基于花粉的重建方法，包括定性和定量方法，考虑了沉积物样品中指标分类群的缺失和/或分类群的百分比。最近的研究表明，花粉来源的稳定同位素和古DNA （aDNA）记录具有第四纪研究的潜力。陆生花粉稳定同位素信号已被提出作为一种新的古气候指标，可以克服气候变化与植被响应之间的时间差。后者是PA的一个严重限制，特别是在高分辨率研究中。花粉aDNA已被证明能够在物种水平上识别来源植物，这是传统PA难以做到的，并且可以精确识别生态/气候指示分类群、古代驯化动物和作物的传播。花粉化石在放射性碳定年中的应用，有助于克服大多数沉积档案由于储层效应而导致的年龄测定误差。上述所有研究方法都需要有效地提取包含整个光谱或选定分类群的纯花粉浓缩物。我自己的初步调查和同事的实验表明，流式细胞术是一种很有前途的方法来完成这项任务。在本项目中，我将首先重点从日本北部Kushu湖（Lake Kushu） RK12沉积物岩心的年代久远且无储层的上部（距今0-6000年）中，基于流式细胞术，使用不同的技术方法和设备提取清洁、无污染的花粉组分。在第二步中，我将测量从RK12岩心中提取的花粉分类群（如冷杉和橡树）的稳定同位素组成（δ13C和δ18O）。DNA分析将用于检测选定的生态指标物种和驯化作物，包括大麦和小米。我还会对花粉浓缩液进行放射性碳测年。结果将与使用常规技术在RK12核上获得的结果进行比较。如果这些测试成功完成，在第三步中，这些方法将应用于来自东亚和东南亚的不同沉积物部分，包括RK12的较低、年代较不确定的部分，以及来自日本中部和越南北部的部分。这些应用程序旨在优化工作流程，并深入了解东亚夏季风的演变和晚冰期-全新世期间的短期气候变化。此外，我打算检测不同驯化植物（大麦，小米，水稻）的种植/传播。

The analysis of fossil pollen assemblages (i.e. pollen analysis = PA) is one of the most frequently used methods in Quaternary research and has significantly contributed to our understanding of past environmental changes, human–environment interactions, land use history and the origin and spread of agriculture. The existing pollen-based reconstructions, including qualitative and quantitative approaches, take into account the absence/presence of indicator-taxa and/or the taxa percentages in the examined sediment samples. Recent studies have indicated potential of pollen-derived stable isotope and ancient DNA (aDNA) records for Quaternary research. Stable isotope signals of terrestrial pollen have been suggested as a new palaeoclimate proxy that would overcome the time lag between climate change and vegetation response. The latter is a serious constraint in PA, particularly in high-resolution studies. Pollen aDNA has shown to be capable for identifying source plants to species level, which is seldom possible by traditional PA and would allow precise identification of ecological/climatic indicator taxa, ancient domesticates, and spread of crops. The application of fossil pollen in radiocarbon dating helps to overcome errors in age determination due to reservoir effects, which most sedimentary archives suffer from. All mentioned research methods require effective extraction of pure pollen concentrates comprising entire spectra or selected taxa. My own preliminary investigations and the experiments of colleagues have shown that flow cytometry is a promising approach to accomplish this task. In this project, I will first focus on extracting clean, contamination-free pollen fractions from the well-dated and reservoir-free upper part (0–6000 years before present) of the Lake Kushu (northern Japan) RK12 sediment core based on flow cytometry using different technical approaches and devices. In a second step, I will measure stable isotope compositions (δ13C and δ18O) of selected pollen taxa (e.g. fir and oak) fractions extracted from the RK12 core. DNA analysis will be tested for detection of selected ecological indicator species and domesticated crops including barley and millets. I will also perform radiocarbon dating on pollen concentrates. The results will be compared with those obtained using conventional techniques on the RK12 core. If these tests are completed successfully, in a third step, the methods will be applied to different sediment sections from East and Southeast Asia comprising the lower, less well dated part of RK12 and one each from central Japan and northern Vietnam. These applications aim at optimizing workflows and gaining deeper insights into the evolution of the East Asian summer monsoon and short-term climate variations during the late-glacial–Holocene interval. Moreover, I intend to detect the cultivation/spread of different domesticated plants (barley, millets, rice).