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.

The time frame and geographical patterns of diversification processes in European temperate‐montane herbs are still not well understood. We used the sexual species of the Ranunculus auricomus complex as a model system to understand how vicariance versus dispersal processes in the context of Pleistocene climatic fluctuations have triggered speciation in temperate‐montane plant species. We used target enrichment sequence data from about 600 nuclear genes and coalescent‐based species tree inference methods to resolve phylogenetic relationships among the sexual taxa of the complex. We estimated absolute divergence times and, using ancestral range reconstruction, we tested if speciation was enhanced by vicariance or by dispersal processes. Phylogenetic relationships among taxa were fully resolved with some incongruence in the position of the tetraploid R. marsicus. Speciation events took place in a very short time at the end of the Mid‐Pleistocene Transition (830–580 thousand years ago [ka]). A second wave of intraspecific geographical differentiation occurred at the end of the Riss glaciation or during the Eemian interglacial between 200 and 100 ka. Ancestral range reconstruction suggests a widespread European ancestor of the R. auricomus complex. Vicariance has triggered allopatric speciation in temperate‐montane plant species during the climatic deterioration that occurred during the last phase of the Mid‐Pleistocene Transition. Vegetation restructuring from forest into tundra could have confined these forest species into isolated glacial macro‐ and microrefugia. During subsequent warming periods, range expansions of these species could have been hampered by apomictic derivatives and by other congeneric competitors in the same habitat.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Speciation via hybridization and polyploidization is a major evolutionary force in plant evolution but is still poorly understood for neopolyploid groups. Challenges are attributed to high heterozygosity, low genetic divergence, and missing information on progenitors, ploidy, and reproduction. We study the large Eurasian Ranunculus auricomus species complex and use a comprehensive workflow integrating reduced-representation sequencing (RRS) genomic data to unravel reticulate evolution, genome diversity and composition of polyploids.We rely on 97 312 restriction site-associated DNA sequencing (RAD-Seq) loci, 576 targeted nuclear genes (48 phased), and 71 plastid regions derived from 78 polyploid apomictic taxa and four diploid and one tetraploid putative sexual progenitor species. We applied (phylo)genomic structure, network, and single nucleotide polymorphism (SNP)-origin analyses.Results consistently showed only 3-5 supported and geographically structured polyploid genetic groups, each containing extant sexual and one unknown progenitor species. Combined analyses demonstrated predominantly allopolyploid origins, each involving 2-3 different diploid sexual progenitor species. Young allotetraploids were characterized by subgenome dominance and nonhybrid SNPs, suggesting substantial post-origin but little lineage-specific evolution.The biodiversity of neopolyploid complexes can result from multiple hybrid origins involving different progenitors and substantial post-origin evolution (e.g. homoeologous exchanges, hybrid segregation, gene flow). Reduced-representation sequencing genomic data including multi-approach information is efficient to delimit shallow reticulate relationships.

Intraspecific trait variation (ITV), based on available genetic diversity, is one of the major means plant populations can respond to environmental variability. The study of functional trait variation and diversity has become popular in ecological research, for example, as a proxy for plant performance influencing fitness. Up to now, it is unclear which aspects of intraspecific functional trait variation (iFDCV) can be attributed to the environment or genetics under natural conditions. Here, we examined 260 individuals from 13 locations of the rare (semi‐)dry calcareous grassland species Trifolium montanum L. in terms of iFDCV, within‐habitat heterogeneity, and genetic diversity. The iFDCV was assessed by measuring functional traits (releasing height, biomass, leaf area, specific leaf area, leaf dry matter content, Fv/Fm, performance index, stomatal pore surface, and stomatal pore area index). Abiotic within‐habitat heterogeneity was derived from altitude, slope exposure, slope, leaf area index, soil depth, and further soil factors. Based on microsatellites, we calculated expected heterozygosity (He) because it best‐explained, among other indices, iFDCV. We performed multiple linear regression models quantifying relationships among iFDCV, abiotic within‐habitat heterogeneity and genetic diversity, and also between separate functional traits and abiotic within‐habitat heterogeneity or genetic diversity. We found that abiotic within‐habitat heterogeneity influenced iFDCV twice as strong compared to genetic diversity. Both aspects together explained 77% of variation in iFDCV (Radj2 = .77, F2, 10 = 21.66, p < .001). The majority of functional traits (releasing height, biomass, specific leaf area, leaf dry matter content, Fv/Fm, and performance index) were related to abiotic habitat conditions indicating responses to environmental heterogeneity. In contrast, only morphology‐related functional traits (releasing height, biomass, and leaf area) were related to genetics. Our results suggest that both within‐habitat heterogeneity and genetic diversity affect iFDCV and are thus crucial to consider when aiming to understand or predict changes of plant species performance under changing environmental conditions.