Universal target enrichment kits maximize utility across wide evolutionary breadth while minimizing the number of baits required to create a cost-efficient kit. The Angiosperms353 kit has been successfully used to capture loci throughout the angiosperms, but the default target reference file includes sequence information from only 6-18 taxa per locus. Consequently, reads sequenced from on-target DNA molecules may fail to map to references, resulting in fewer on-target reads for assembly, and reducing locus recovery. METHODS:We expanded the Angiosperms353 target file, incorporating sequences from 566 transcriptomes to produce a 'mega353' target file, with each locus represented by 17-373 taxa. This mega353 file is a drop-in replacement for the original Angiosperms353 file in HybPiper analyses. We provide tools to subsample the file based on user-selected taxon groups, and to incorporate other transcriptome or protein-coding gene data sets. RESULTS:Compared to the default Angiosperms353 file, the mega353 file increased the percentage of on-target reads by an average of 32%, increased locus recovery at 75% length by 49%, and increased the total length of the concatenated loci by 29%.DISCUSSION: Increasing the phylogenetic density of the target reference file results in improved recovery of target capture loci. The mega353 file and associated scripts are available at: https://github.com/chris jacks on-pelli cle/NewTa rgets.

Universal target enrichment kits maximise utility across wide evolutionary breadth while minimising the number of baits required to create a cost-efficient kit. Locus assembly requires a target reference, but the taxonomic breadth of the kit means that target references files can be phylogenetically sparse. The Angiosperms353 kit has been successfully used to capture loci throughout angiosperms but includes sequence information from 6−18 taxa per locus. Consequently, reads sequenced from on-target DNA molecules may fail to map to references, resulting in fewer on-target reads for assembly, reducing locus recovery. We expanded the Angiosperms353 target file, incorporating sequences from 566 transcriptomes to produce a mega353 target file, with each gene represented by 17−373 taxa. This mega353 file is a drop-in replacement for the original Angiosperms353 file in HybPiper analyses. We provide tools to subsample the file based on user-selected taxon groups, and to incorporate other transcriptome or protein-coding gene datasets. Compared to the default Angiosperms353 file, the mega353 file increased the percentage of on-target reads by an average of 31%, increased loci recovery at 75% length by 61.9%, and increased the total length of the concatenated loci by 30%. The mega353 file and associated scripts are available at: https://github.com/chrisjackson−pellicle/NewTargets

Understanding what factors influence plastic and genetic variation is valuable for predicting how organisms respond to changes in the selective environment. Here, using gene expression and DNA methylation as molecular phenotypes, we study environment-induced variation among Arabidopsis lyrata plants grown at lowland and alpine field sites. Our results show that gene expression is highly plastic, as many more genes are differentially expressed between the field sites than between populations. The environmentally responsive genes evolve under strong selective constraint — the strength of purifying selection on the coding sequence is high, while the rate of adaptive evolution is low. We find, however, that positive selection on cis-regulatory variants has likely contributed to genetically variable environment-responses, but such variants segregate only between distantly related populations. In contrast to gene expression, DNA methylation at genic regions is largely insensitive to the environment, and plastic methylation changes are not associated with differential gene expression. Besides genes, we detect environmental effects at transposable elements (TEs): TEs at the high-altitude field site have higher expression and methylation levels, suggestive of a broad-scale TE activation. Compared to the lowland population, plants native to the alpine environment harbor and excess of recent TE insertions, and we observe that specific TE families are enriched within environmentally responsive genes. In sum, although our results suggest that strong evolutionary constraint at environmentally responsive genes may limit the species' adaptive potential, plastic responses at TEs could rapidly create novel heritable variation that is primed towards environmental adaptation.

Understanding what factors influence plastic and genetic variation is valuable for predicting how organisms respond to changes in the selective environment. Here, using gene expression and DNA methylation as molecular phenotypes, we study environmentally induced variation among Arabidopsis lyrata plants grown at lowland and alpine field sites. Our results show that gene expression is highly plastic, as many more genes are differentially expressed between the field sites than between populations. These environmentally responsive genes evolve under strong selective constraint – the strength of purifying selection on the coding sequence is high, while the rate of adaptive evolution is low. We find, however, that positive selection on cis-regulatory variants has likely contributed to the maintenance of genetically variable environmental responses, but such variants segregate only between distantly related populations. In contrast to gene expression, DNA methylation at genic regions is largely insensitive to the environment, and plastic methylation changes are not associated with differential gene expression. Besides genes, we detect environmental effects at transposable elements (TEs): TEs at the high-altitude field site have higher expression and methylation levels, suggestive of a broad-scale TE activation. Compared to the lowland population, plants native to the alpine environment harbor an excess of recent TE insertions, and we observe that specific TE families are enriched within environmentally responsive genes. Our findings provide insight into selective forces shaping plastic and genetic variation. We also highlight how plastic responses at TEs can rapidly create novel heritable variation in stressful conditions.

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