Species delimitation in the apomictic polyploid Ranunculus auricomus complex using an integrative TaxonOMICs approach

在无融合植物中，物种划分仍然是一个挑战。通过种子进行无性繁殖。无融合多倍体复合体通常包括少数性祖先物种和许多无融合杂交衍生物。兼性可以导致形成众多的，局部分布的形态表型。特别是在北欧，许多这样的形态在没有任何进化或遗传背景的情况下被传统地描述为无性生殖。为了克服这种对缺乏根据的实体的纯粹描述性分类，该项目的目标是基于在空间和时间上具有长期持久性的进化谱系进行物种划分。我们将采用综合方法完成对北欧欧亚毛茛（euranunculus auricomus）复合物的分析（已描述的形态种约600种）：(1)系统基因组学，结合两种新一代测序方法，即；将核基因富集与质体基因组扫描相结合，以聚结为基础进行物种划分和限制性内切位点相关DNA测序（RAD-seq），作为评估物种内和物种间多样性的精细基因组标记。对约600个核基因亚群进行靶富集，可解析类群的系统发育框架，并可通过生物地理分析和分子定年来研究其时空多样性。RAD-seq将用于分析后起源进化，基因流动和多样性内部和之间的无杂交谱系。(2)流式细胞术将通过流式细胞术种子筛选（FCSS）来确定倍性水平和兼性程度。(3)利用植物数字成像、几何形态计量学和多元统计分析等方法，客观评价植物类群间的形态分化和类群内的多样性。分类将以综合结果为基础，正式承认只有已确定的进化单位为物种。与其他项目的合作包括为无性生物和植物多倍体复合体的分类发展一个全面的物种概念，以及在方法方面。

Species delimitation is still a challenge in plants with apomixis, ie. the asexual reproduction via seeds. Apomictic polyploid complexes usually comprise a few sexual progenitor species, and numerous apomictic hybrid derivatives. Facultative sexuality can result in the formation of numerous, locally distributed morphological phenotypes. Particularly in Northern Europe, numerous such morphotypes have been traditionally described as agamospecies without regarding any evolutionary or genetic background. To overcome this solely descriptive classification of ill-founded entities, the project is aiming at a species delimitation based on evolutionary lineages that have a long-term persistence in space and time. We will complete the analysis of the Eurasian Ranunculus auricomus complex in Northern Europe (>600 described morphospecies) by using an integrative approach combining established methods: (1) Phylogenomics, with a combination of two next-generation sequencing methods, ie. target enrichment of nuclear genes combined to plastid genome skimming for coalescence-based species delimitation and restriction site-associated DNA sequencing (RAD-seq) as a fine-scale genomic marker to assess diversity within and among species. Target enrichment of a subset of c. 600 genes nuclear genes will resolve the phylogenetic framework of taxa, and will be used to study spatial and temporal diversification with biogeographical analysis and molecular dating. RAD-seq will be used to analyze post-origin evolution, gene flow and diversity within and among apomictic lineages. (2) Flow cytometry will be used to determine ploidy levels and degree of facultative sexuality via flow cytometric seed screening (FCSS). (3) Digital imaging of plants, geometric morphometrics and multivariate statistical analysis will be used to objectively assess morphological differentiation among taxa and diversity within taxa. Classification will be based on a combination of results to recognize formally only established evolutionary units as species. Collaborations with other projects include the development of a comprehensive species concept for classification of asexual organisms and for plant polyploid complexes, and on methodical aspects.