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Intraspecific genetic variation in foundation species such as aspen (Populus tremuloides Michx.) shapes their impact on forest structure and function. Identifying genes underlying ecologically important traits is key to understanding that impact. Previous studies, using single-locus genome-wide association (GWA) analyses to identify candidate genes, have identified fewer genes than anticipated for highly heritable quantitative traits. Mounting evidence suggests that polygenic control of quantitative traits is largely responsible for this “missing heritability” phenomenon. Our research characterized the genetic architecture of 30 ecologically important traits using a common garden of aspen through genomic and transcriptomic analyses. A multilocus association model revealed that most traits displayed a highly polygenic architecture, with most variation explained by loci with small effects (likely below the detection levels of single-locus GWA methods). Consistent with a polygenic architecture, our single-locus GWA analyses found only 38 significant SNPs in 22 genes across 15 traits. Next, we used differential expression analysis on a subset of aspen genets with divergent concentrations of salicinoid phenolic glycosides (key defense traits). This complementary method to traditional GWA discovered 1243 differentially expressed genes for a polygenic trait. Soft clustering analysis revealed three gene clusters (241 candidate genes) involved in secondary metabolite biosynthesis and regulation. Our work reveals that ecologically important traits governing higher-order community- and ecosystem-level attributes of a foundation forest tree species have complex underlying genetic structures and will require methods beyond traditional GWA analyses to unravel.

Many ecological interactions of aspens and their relatives (Populus spp.) are affected by products of the phenylpropanoid pathway synthesizing condensed tannins (CTs), whose production involves trade-offs with other ecologically important compounds and with growth. Genes of this pathway are candidates for investigating the role of selection on ecologically important, polygenic traits. We analysed sequences from 25 genes representing 10 steps of the CT synthesis pathway, which produces CTs used in defence and lignins used for growth, in 12 individuals of European aspen (Populus tremula). We compared these to homologs from P.trichocarpa, to a control set of 77 P. tremula genes, to genome-wide resequencing data and to RNA-seq expression levels, in order to identify signatures of selection distinct from those of demography. In Populus, pathway position exerts a strong influence on the evolution of these genes. Nonsynonymous diversity, divergence and allele frequency shifts (Tajima's D) were much lower than for synonymous measures. Expression levels were higher, and the direction of selection more negative, for upstream genes than for those downstream. Selective constraints act with increasing intensity on upstream genes, despite the presence of multiple paralogs in most gene families. Pleiotropy, expression level, flux control and codon bias appear to interact in determining levels and patterns of variation in genes of this pathway, whose products mediate a wide array of ecological interactions for this widely distributed species.

Condensed tannin (CT) contents of European aspen (Populus tremula L.) vary among genotypes, and increases in nitrogen (N) availability generally reduce plants’ tannin production in favor of growth, through poorly understood mechanisms. We hypothesized that intrinsic tannin production rates may co-vary with gene expression responses to soil N and resource allocation within the phenylpropanoid pathway (PPP). Thus, we examined correlations between soil N levels and both expression patterns of eight PPP genes (measured by quantitative-reverse transcription PCR) and foliar phenolic compounds (measured by liquid chromatography–mass spectrometry) in young aspen genets with intrinsically extreme CT levels. Monitored phenolics included salicinoids, lignins, flavones, flavonols, CT precursors and CTs. The PPP genes were consistently expressed more strongly in high-CT trees. Low N supplements reduced expression of genes throughout the PPP in all genets, while high N doses restored expression of genes at the beginning and end of the pathway. These PPP changes were not reflected in pools of tannin precursors, but varying correlations between gene expression and foliar phenolic pools were detected in young and mature leaves, suggesting that processes linking gene expression and the resulting phenolics vary spatially and temporally. Precursor fluxes suggested that CT-related metabolic rate or sink controls are linked to intrinsic carbon allocation strategies associated with N responses. Overall, we found more negative correlations (indicative of allocation trade-offs) between PPP gene expression and phenolic products following N additions in low-CT plants than in high-CT plants. The tannin-related expression dynamics suggest that, in addition to defense, relative tannin levels may also be indicative of intraspecific variations in the way aspen genets respond to soil fertility.