Although species are central units for biological research, recent findings in genomics are raising awareness that what we call species can be ill-founded entities due to solely morphology-based, regional species descriptions. This particularly applies to groups characterized by intricate evolutionary processes such as hybridization, polyploidy, or asexuality. Here, challenges of current integrative taxonomy (genetics/genomics + morphology + ecology, etc.) become apparent: different favored species concepts, lack of universal characters/markers, missing appropriate analytical tools for intricate evolutionary processes, and highly subjective ranking and fusion of datasets. Now, integrative taxonomy combined with artificial intelligence under a unified species concept can enable automated feature learning and data integration, and thus reduce subjectivity in species delimitation. This approach will likely accelerate revising and unraveling eukaryotic biodiversity.

Polyploidy is common in the genus Salix. However, little is known about the origin, parentage and genomic composition of polyploid species because of a lack of suitable molecular markers and analysis tools. We established a phylogenomic framework including species of all described sections of Eurasian shrub willows. We analyzed the genomic composition of seven polyploid willow species in comparison to putative diploid parental species to draw conclusions on their origin and the effects of backcrossing and post-origin evolution. We applied recently developed programs like SNAPP, HyDe, and SNiPloid to establish a bioinformatic pipeline for unravelling the complexity of polyploid genomes. RAD sequencing revealed 23,393 loci and 320,010 high quality SNPs for the analysis of relationships of 35 species of Eurasian shrub willows (Salix subg. Chamaetia/Vetrix). Polyploid willow species appear to be predominantly of allopolyploid origin. More ancient allopolyploidization events were observed for two hexaploid and one octoploid species, while our data suggested a more recent allopolyploid origin for the included tetraploids and identified putative parental taxa. SNiPloid analyses disentangled the different genomic signatures resulting from hybrid origin, backcrossing, and secondary post-origin evolution in the polyploid species. Our RAD sequencing data demonstrate that willow genomes are shaped by ancient and recent reticulate evolution, polyploidization, and post-origin divergence of species.

Summary: 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.

Hybridization of species with porous genomes can eventually lead to introgression via repeated backcrossing. The potential for introgression between species is reflected by the extent of segregation distortion in later generation hybrids. Here we studied a population of hybrids between Salix purpurea and S. helvetica that has emerged within the last 30 years on a glacier forefield in the European Alps due to secondary contact of the parental species. We used 5758 biallelic SNPs produced by RAD sequencing with the aim to ascertain the predominance of backcrosses (F1 hybrid x parent) or F2 hybrids (F1 hybrid x F1 hybrid) among hybrid offspring. Further, the SNPs were used to study segregation distortion in the second hybrid generation. The analyses in structure and NewHybrids revealed that the population consisted of parents and F1 hybrids, whereas hybrid offspring consisted mainly of backcrosses to either parental species, but also some F2 hybrids. Although there was a clear genetic differentiation between S. purpurea and S. helvetica (FST = 0.24), there was no significant segregation distortion in the backcrosses or the F2 hybrids. Plant height of the backcrosses resembled the respective parental species, whereas F2 hybrids were more similar to the subalpine S. helvetica. The co-occurrence of the parental species and the hybrids on the glacier forefield, the high frequency of backcrossing, and the low resistance to gene flow via backcrossing make a scenario of introgression in this young hybrid population highly likely, potentially leading to the transfer of adaptive traits. We further suggest that this willow hybrid population may serve as a model for the evolutionary processes initiated by recent global warming.

The large and diverse genus Salix L. is of particular interest for decades of biological research. However, despite the morphological plasticity, the reconstruction of phylogenetic relationships was so far hampered by the lack of informative molecular markers. Infrageneric classification based on morphology separates dwarf shrubs (subg. Chamaetia) and taller shrubs (subg. Vetrix), while previous phylogenetic studies placed species of these two subgenera just in one largely unresolved clade. Here we want to test the utility of genomic RAD sequencing markers for resolving relationships at different levels of divergence in Salix. Based on a sampling of 15 European species representing 13 sections of the two subgenera, we used five different RAD sequencing datasets generated by Ipyrad to conduct phylogenetic analyses. Additionally we reconstructed the evolution of growth form and analyzed the genetic composition of the whole clade. The results showed fully resolved trees in both ML and BI analysis with high statistical support. The two subgenera Chamaetia and Vetrix were recognized as nonmonophyletic, which suggests that they should be merged. Within the Vetrix/Chamaetia clade, a division into three major subclades could be observed. All species were confirmed to be monophyletic. Based on our data, arctic‐alpine dwarf shrubs evolved four times independently. The structure analysis showed five mainly uniform genetic clusters which are congruent in sister relationships observed in the phylogenies. Our study confirmed RAD sequencing as a useful genomic tool for the reconstruction of relationships on different taxonomic levels in the genus Salix.

Sex determination systems in plants can involve either female or male heterogamety (ZW or XY, respectively). Here we used Illumina short reads, Oxford Nanopore Technologies (ONT) long reads and Hi‐C reads to assemble the first chromosome‐scale genome of a female willow tree (Salix dunnii), and to predict genes using transcriptome sequences and available databases. The final genome sequence of 328 Mb in total was assembled in 29 scaffolds, and includes 31,501 predicted genes. Analyses of short‐read sequence data that included female and male plants suggested a male heterogametic sex‐determining factor on chromosome 7, implying that, unlike the female heterogamety of most species in the genus Salix, male heterogamety evolved in the subgenus Salix. The S. dunnii sex‐linked region occupies about 3.21 Mb of chromosome 7 in females (representing its position in the X chromosome), probably within a pericentromeric region. Our data suggest that this region is enriched for transposable element insertions, and about one‐third of its 124 protein‐coding genes were gained via duplications from other genome regions. We detect purifying selection on the genes that were ancestrally present in the region, though some have been lost. Transcriptome data from female and male individuals show more male‐ than female‐biased genes in catkin and leaf tissues, and indicate enrichment for male‐biased genes in the pseudo‐autosomal regions. Our study provides valuable genomic resources for further studies of sex‐determining regions in the family Salicaceae, and sex chromosome evolution.