Signatures of selection and constraint from natural populations

We calculated gene-level summary statistics for signatures of selection and constraint in the following way. Synonymous and non-synonymous polymorphism among natural A. thaliana accessions and divergence from A. lyrata (Pn, Ps, Dn and Ds, respectively) were calculated using mkTest.rb (https://github.com/kr-colab). The alpha test statistic for evidence of selection, which is a derivative of the McDonald-Kreitman test^76–78, was calculated from these values for each gene where data were available (not all genes have orthologues assigned in A. lyrata) as 1 − (Ds × Pn)/(Dn × Ps). Positive values of alpha are conventionally interpreted as evidence of positive selection because non-synonymous variants in genes with such values tend to become fixed. For each decile of genes classified according to mutation probability, we calculated the proportion for which alpha is positive. Enrichment of non-synonymous variants compared to genome-wide average were confirmed by independent calculation of Waterson’s diversity estimate (θ) of non-synonymous variation. The frequency of loss-of-function mutations was calculated as before^79,80, where loss of function was defined as premature stop codons and frameshifts disrupting at least 10% of the coding region of the canonical gene model. Genes experiencing purifying selection should exhibit lower levels of natural polymorphism than what would be predicted by mutation rate alone. To test this, we built a linear model of coding region polymorphisms as a function of predicted mutation rates. We calculated scaled residuals for each gene and tested whether they are more negative in genes expected to be under purifying selection. To estimate constraints on gene regulatory function, we looked at average expression across diverse genotypes. We also tested for relationships between predicted mutation rates and the coefficient of variation in gene expression, additive genetic variance for gene expression across diverse genotypes, and environmental variance in gene expression⁷¹.