Hybridization and polyploidization play important roles in plant evolution but it is still not fully clarified how these evolutionary forces contribute to the establishment of apomicts. Apomixis, the asexual reproduction via seed formation, comprises several essential alterations in development compared to the sexual pathway. Furthermore, most natural apomicts were found to be polyploids and/or hybrids. The Ranunculus auricomus complex comprises diploid sexual and polyploid apomictic species and represents an excellent model system to gain knowledge on origin and evolution of apomixis in natural plant populations. In this study, the second generation of synthetically produced homoploid (2x) and heteroploid (3x) hybrids derived from sexual R. auricomus species was analyzed for aposporous initial cell formation by DIC microscopy. Complete manifestation of apomixis was determined by measuring single mature seeds by flow cytometric seed screen. Microscopic analysis of the female gametophyte formation indicated spontaneous occurrence of aposporous initial cells and several developmental irregularities. The frequency of apospory was found to depend on dosage effects since a significant increase in apospory was observed, when both F1 parents, rather than just one, were aposporous. Other than in the F1 generation, diploid Ranunculus F2 hybrids formed BIII seeds and fully apomictic seeds. The results indicate that hybridization rather than polyploidization seems to be the functional activator of apomictic reproduction in the synthetic Ranunculus hybrids. In turn, at least two hybrid generations are required to establish apomictic seed formation.

Species are the basic units of biodiversity and evolution. Nowadays, they are widely considered as ancestor-descendant lineages. Their definition remains a persistent challenge for taxonomists due to lineage evolutionary role and circumscription, i.e., persistence in time and space, ecological niche, or a shared phenotype. Recognizing and delimiting species is particularly methodically challenging in fast-evolving, evolutionary young species complexes often characterized by low genetic divergence, hybrid origin, introgression, and incomplete lineage sorting. Ranunculus auricomus is a large Eurasian apomictic polyploid complex that probably has arisen from the hybridization of a few sexual progenitor species. However, even delimitation of and relationships among diploid sexual progenitors are unclear, ranging from 2 to 12 species. Here, we present an innovative workflow combining phylogenomic methods based on 86,782 parameter-optimized RADseq loci and target enrichment of 663 nuclear genes accompanied by geometric morphometrics to delimit sexual species in this evolutionary young complex (<1 Mya). For the first time, we revealed a fully resolved and well-supported maximum likelihood tree phylogeny congruent to neighbor-net network and STRUCTURE results based on RADseq data. In a few clades, we found evidence of discordant patterns indicated by quartet sampling, and reticulation events in the neighbor-net network probably caused by introgression and incomplete lineage sorting. Together with coalescent-based species delimitation approaches based on target enrichment data, we found five main genetic lineages, with an allopatric distribution in central and southern Europe. A concatenated geometric morphometric dataset including data from basal and stem leaves, as well as receptacles, revealed the same five main clusters. We accept those five morphologically differentiated, geographically isolated, genetic main lineages as species: R.

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