Neutrality test (dN/dS and H statistic)

To look for NLR clusters under positive selection, possibly in response to coevolution with the pathogen, we analysed dN/dS, the ratio between non-synonymous (amino acid changing) to synonymous mutations (Figure 5a, outmost ring). None of the NLR transcript clusters had an ω value greater than one, which would indicate positive selection pressure. However, site-wise analysis of dN/dS revealed that 23 of the clusters contained a varying number of one to 12 amino acid positions under positive selection, based on Bayes Empirical Bayes (BEB) analysis (P>95%). The cluster with the highest number of loci under selection returned a BLAST hit to an Arabidopsis NLR protein responsible for resistance against Lettuce mosaic virus (Revers et al., 2003).
In order to investigate potential selection pressure by a complementary method, considering the shortcomings of within population dN/dS analysis (Kryazhimskiy & Plotkin, 2008), we also calculated Fay & Wu H statistics on the whole NLRs using putative snapdragon orthologs (Li, Zhang, et al., 2019) as the ancestral state. A positive value of H is an indication of balancing or purifying selection, suggesting the tendency to preserve the current protein sequence, whereas high negative values are an indication of diversifying positive selection, selective sweeps, or drift, for example from population bottlenecks.
We identified 23 NLR transcripts with regions having H statistics less than -3.5 (Figure 5a; Supplementary Figure 4; Supplementary Table 5). This set included two genes from the cluster with homology to RPP13, and the sites under selection in all but one were located in the conserved NB-ARC domain. Since this domain is the activation domain (Van Ooijen et al., 2008), the mutations may affect the strength of the activation. Additionally, we identified selection in the LRR domains of three genes. Since they are involved in pathogen perception and induced in our experiment, these domains may have been under positive selection to detect the P. plantaginis pathogen.