Measuring biological diversity is a crucial but difficult undertaking, as exemplified in oaks where complex patterns of morphological, ecological, biogeographical and genetic differentiation collide with traditional taxonomy, which measures biodiversity in number of species (or higher taxa). In this pilot study, we generated high‐throughput sequencing amplicon data of the intergenic spacer of the 5S nuclear ribosomal DNA cistron (5S‐IGS) in oaks, using six mock samples that differ in geographical origin, species composition and pool complexity. The potential of the marker for automated genotaxonomy applications was assessed using a reference data set of 1,770 5S‐IGS cloned sequences, covering the entire taxonomic breadth and distribution range of western Eurasian Quercus, and applying similarity (blast) and evolutionary approaches (maximum‐likelihood trees and Evolutionary Placement Algorithm). Both methods performed equally well, allowing correct identification of species in sections Ilex and Cerris in the pure and mixed samples, and main lineages shared by species of sect. Quercus. Application of different cut‐off thresholds revealed that medium‐ to high‐abundance (>10 or 25) sequences suffice for a net species identification of samples containing one or a few individuals. Lower thresholds identify phylogenetic correspondence with all target species in highly mixed samples (analogous to environmental bulk samples) and include rare variants pointing towards reticulation, incomplete lineage sorting, pseudogenic 5S units and in situ (natural) contamination. Our pipeline is highly promising for future assessments of intraspecific and interpopulation diversity, and of the genetic resources of natural ecosystems, which are fundamental to empower fast and solid biodiversity conservation programmes worldwide.

中文翻译：

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橡树中 5S-IGS 的高通量测序：探索基因组内变异和算法以识别纯样品和混合样品中的目标物种

衡量生物多样性是一项至关重要但困难的工作，例如在橡树中，形态、生态、生物地理和遗传分化的复杂模式与传统分类法相冲突，传统分类法以物种（或更高分类群）的数量衡量生物多样性。在这项初步研究中，我们使用地理起源、物种组成和池复杂性不同的六个模拟样本，生成了橡树中 5S 核核糖体 DNA 顺反子 (5S-IGS) 基因间间隔区的高通量测序扩增子数据。使用 1,770 个 5S-IGS 克隆序列的参考数据集评估了该标记在自动化基因分类学应用中的潜力，涵盖了欧亚西部栎属的整个分类学广度和分布范围，并应用了相似性（blast）和进化方法（最大似然树和进化放置算法）。两种方法都表现得同样好，可以正确识别纯样品和混合样品中冬青和樱桃部分中的物种，以及 sect 物种共有的主要谱系。栎属. 应用不同的截止阈值表明，中高丰度（>10 或 25）序列足以对包含一个或几个个体的样本进行净物种鉴定。较低的阈值确定了与高度混合样本（类似于环境批量样本）中所有目标物种的系统发育对应关系，并包括指向网状、不完整谱系分类、假基因 5S 单位和原位（自然）污染的罕见变异。我们的管道对于未来对种内和种群间多样性以及自然生态系统遗传资源的评估非常有希望，这对于在全球范围内实施快速而可靠的生物多样性保护计划至关重要。