Phylogenomics supported by geometric morphometrics reveals delimitation of sexual species within the polyploid apomictic Ranunculus auricomus complex (Ranunculaceae)

Abstract 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 of a lineage. 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 (ILS). 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 and relationships of diploid sexual progenitors have been unclearly ranging from two to twelve 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 together with 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 (ML) 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 (QS) and reticulation events in the neighbor-net network probably caused by introgression and ILS. 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 data set including 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. cassubicifolius s.l. (incl. R. carpaticola ), R. flabellifolius , R. envalirensis s.l. (incl. R. cebennensis) , R. marsicus and R. notabilis s.l. (incl. R. austroslovenicus , R. calapius , R. mediocompositus, R. peracris and R. subcarniolicus ). Our comprehensive workflow combing phylogenomic methods supported by geometric morphometrics proved to be successful in delimiting closely related sexual taxa and applying an evolutionary species concept, which is also transferable to other evolutionarily young species complexes.