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.

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.

Chromosome-scale assembly of the genome of Salix dunnii reveals a male-heterogametic sex determination system on chromosome 7

Plants produce an astonishing diversity of specialized metabolites as defences against herbivores, pathogens or detrimental abiotic conditions. Plants growing at different elevations are exposed to different biotic and abiotic conditions and typically show pronounced differences in their chemistry. Understanding how these differences arise through changes in various measures of chemical diversity can inform us concerning factors that contribute to the variety of metabolites found among plants. We focused on elevational changes in concentration, richness and intra‐ and interspecific variation in specialized chemistry in willows (Salix, Salicaceae) and compare them among metabolite classes with different functions. We aim to show how these various measures of chemical diversity change with elevation to reveal trends contributing to changes in plant chemistry along major ecological gradients. We studied chemistry, herbivory and fungal pathogen damage in an assemblage of seven willow species along an elevational gradient in the Alps (800–2600 m a.s.l.). We examined trends in chemical diversity using untargeted metabolomics, and further quantified trends in three specific classes: proanthocyanidins and salicinoids involved in biotic interactions, and flavonoids involved mainly in abiotic protection. We use measures of willow chemistry that take structural relatedness of metabolites into account to show if the roles of structurally distinct metabolites change with elevation. Willows from low elevations exhibited greater proanthocyanidin concentration and structural richness of flavonoids. In contrast, willows from high elevations showed greater structural richness of salicinoids and greater variation in total metabolite composition at both the intra‐ and interspecific levels. The trends in salicinoid richness and proanthocyanidin concentration were explained by elevational changes in temperature. Our results show how elevational differences in plant chemistry arise through trends in various aspects of their chemical diversity. Willows at high elevations showed reduced structural richness of metabolites involved in abiotic protection. This may reflect focused investment in metabolites with the highest ecological benefit relative to their concentration in high‐elevation willows. At the same time, they possessed greater richness of metabolites involved in biotic interactions, while variation in microhabitat preferences among high‐elevation species likely contributed to the high variation in their total metabolite pool. Read the free Plain Language Summary for this article on the Journal blog.

14Despite the general progress in using next generation sequencing techniques for 15 evolutionary research questions, the analysis of polyploid species is still hampered by the lack 16 of suitable analytical tools and the statistical difficulties of dealing with more than two alleles 17 per locus. Polyploidization and especially allopolyploidy leads to new combinations of traits 18 by combining genomes of two or more parental species. This enhances the adaptive potential 19 and often results in speciation. However, multiple origins of polyploids, backcrossing to the 20 parental species and post-origin evolution can strongly influence the genome composition of 21 polyploid species. Here, we used RAD sequencing, which revealed 23,393 loci and 320,010 22 high quality SNPs, to analyze the relationships and origin of seven polyploid species of the 23 diverse genus Salix by utilizing a phylogenomic and a network approach, as well as analyzing 24 the genetic structure and composition of the polyploid genome in comparison to putative 25 parental species. We adapted the SNiPloid pipeline that was originally developed to analyse 26 SNP composition of recently established allotetraploid crop lineages to RAD sequencing data 27 by using concatenated RAD loci as reference. Our results revealed a well-resolved phylogeny 28 of 35 species of Eurasian shrub willows (Salix subg. Chamaetia/Vetrix), including 28 diploid 29 and 7 polyploid species. Polyploidization in willows appears to be predominantly connected 30 to hybridization, i.e. to an allopolyploid origin of species. More ancient allopolyploidization 31 events involving hybridization of more distantly related, ancestral lineages were observed for 32 two hexaploid and one octoploid species. Our data suggested a more recent allopolyploid 33 origin for the included tetraploids within the major subclades and identified putative parental 34 taxa that appear to be plausible in the context of geographical, morphological and ecological 35 patterns. SNiPloid and HyDe analyses disentangled the different genomic signatures resulting 36 from hybrid origin, backcrossing, and secondary post-origin evolution in the polyploid 37 species. All tetraploids showed a considerable post-origin, species-specific proportion of 38 Origin of polyploid Salix species 3 SNPs. The amount of extant hybridization appears to be related to the degree of geographical 39 and ecological isolation of species. Our data demonstrate that high-quality RAD sequencing 40 data are suitable and highly informative for the analysis of the origin and relationships of 41 polyploid species. The combination of the traditional tools RAxML, STRUCTURE, 42 SplitsTree and recently developed programs like SNAPP, HyDe and SNiPloid established a 43 bioinformatic pipeline for unraveling the complexity of polyploid genomes.44

AbstractThe Hengduan Mountains (HDM) in South West China are an important hotspot of plant diversity and endemism and considered to be a secondary diversification center for the woody plant genus Salix (Salicaceae). This study aimed to reconstruct the spatio-temporal evolution of the Salix Chamaetia-Vetrix clade in the HDM and to test for the occurrence of a radiation. We inferred phylogenetic relationships based on more than 34,000 RAD loci of 27 species. Phylogenetic analyses recovered a well-resolved tree topology with two major clades, the Eurasian and the HDM clade and a divergence time of c. 23.9 Ma. The HDM clade comprises two subclades. The species of the HDM clade originated in north HDM and adjacent areas and then dispersed into the south HDM, westwards to the Himalayas and eastwards to the Qinling Mountains. Niche modelling analyses revealed that during the last glacial maximum, range contractions were observed in the northern areas, while southward expansions resulted in range overlaps. The reconstruction of putative adaptive character evolution of plant height, inflorescence and flower morphology indicate that adaptations to altitudinal distribution contributed to the diversification of the HDM willows. Our data indicate that a radiation occurred in HDM within the Salix Chamaetia-Vetrix clade. Dispersal within the mountain system and to adjacent regions as well as survival in glacial refugia have shaped the biogeographical history of the clade. Differentiation along altitudinal zonation concomitant to morphological adaptations to colder climates may be important ecological factors for the high species diversity of Salix in this area.

Diverse specialised metabolites contributed to the success of vascular plants in colonising most terrestrial habitats. Understanding how distinct aspects of chemical diversity arise through heterogeneous environmental pressures can help us understand the effects of abiotic and biotic stress on plant evolution and community assembly. We examined highland and lowland willow species within a phylogenetic framework to test for trends in their chemical α‐diversity (richness) and β‐diversity (variation among species sympatric in elevation). We show that differences in chemistry among willows growing at different elevations occur mainly through shifts in chemical β‐diversity and due to convergence or divergence among species sharing their elevation level. We also detect contrasting phylogenetic trends in concentration and α‐diversity of metabolites in highland and lowland willow species. The resulting elevational patterns contribute to the chemical diversity of willows and suggest that variable selective pressure across ecological gradients may, more generally, underpin complex changes in plant chemistry.

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