Unusual signatures of highly adaptable R-loci in closely-related Arabidopsis species
Introduction
Confronted with a vast array of pathogens including fungi, bacteria, viruses, and nematodes, plants have developed an array of resistance (R) genes that provide a toolbox to identify and survive pathogen invasions (Dangl and Jones, 2001). R-genes encode specific receptors that recognize avirulence pathogen factors in a gene-for-gene manner (Flor, 1971). R-genes proliferate in number extensively in plant genomes, and most are members of a family characterized by specific nucleotide binding sites plus a leucine-rich repeat (the NBS-LRR family). There are currently 149 predicted NBS-LRR genes in Arabidopsis thaliana, but only a handful have been directly characterized for specific functional interactions (Meyers et al., 2003).
Among R-genes, only a subset has been examined for patterns of molecular polymorphism in A. thaliana. As reported elsewhere, some R-genes are conserved with low Ka/Ks ratios, such as RPM1 and RPS5, and have substitution patterns consistent with strong selective constraint (Shen et al., 2006). Hypervariable R-genes have also been detected, such as the highly diverse gene RPP13 (Rose et al., 2004). Strong positive selection resulting in high Ka/Ks ratios was revealed in most R-loci between A. thaliana and Arabidopsis lyrata (Chen et al., 2010). A genome-wide survey of polymorphisms in R-genes revealed diverse patterns in A. thaliana and Oryza sativa, suggesting that alternative modes of natural selection shape genetic variation among R-genes (Bakker et al., 2006, Yang et al., 2006).
The presence of alternative patterns of genetic polymorphism and divergence in R-genes indicates that different mechanisms generate and maintain standing genetic variation across genes in this family and suggests that persistence of adaptive alleles across R-genes varies. The resistant response to pathogens can potentially arise either through the evolution of novel resistant alleles or by recycling existing ancestral alleles (Holub, 2001). Comparative investigation of R-genes in recently-derived closely related species provides the possibility to reveal persistent genetic variation retained from a common ancestor as well as derived mutations that have accumulated independently since lineage divergence (Ramos-Onsins et al., 2004). One expected pattern of ancestral and derived allele partitioning in pathogen response genes is trans-specific polymorphism. Trans-specific polymorphism is the dual conservation of ancestral polymorphisms by two species and provides strong evidence of long-term balancing selection transcending species boundaries, because it is unlikely to be under neutrality (Charlesworth et al., 2006). When these polymorphisms segregate with different alleles, frequency-dependent selection could favor rarer genotypes (Muirhead et al., 2002). This phenomenon has been documented in genes with significantly important functions coupled with high overall genetic variation such as the major histocompatibility complex (MHC) genes of jawed vertebrates and self-incompatibility (SI) genes of flowering plants (Cai et al., 1997, Clark, 1997), and is therefore expected to also shape pathogen response genes such as R-genes.
A. thaliana and A. lyrata are closely related species that diverged about 5 million years ago (Koch et al., 2000). In contrast to the inbreeding annual species A. thaliana, A. lyrata is self-incompatible, out-crossing, and perennial. A. lyrata comprises three subspecies: Arabidopsis lyrata ssp. lyrata (A. l. lyrata), Arabidopsis lyrata ssp. petraea (A. l. petraea) and Arabidopsis lyrata ssp. Kamchatica (Brueggeman et al., 2002). High inbreeding rates in A. thaliana are assumed to cause a strong reduction of both sequence variation and effective recombination in comparison with outcrossing in A. lyrata (Ramos-Onsins et al., 2004).
What are the expected patterns of genetic variation following rapid adaptation? Given that R-genes are highly adaptive to respond to a changed environment following recent speciation, rapidly evolved genes should accumulate detectable ‘signatures’ in at least a subset of genes. Some examples of expected patterns include a significant difference in the level of within-species polymorphism for the same gene, a divergent rate of mutational accumulation between species at different genes, a presence/absence polymorphism for the same gene, and interspecific differences in the rates and patterns of trans-specific polymorphisms and synonymous and non-synonymous substitutions. Loci maintaining highly adaptive alleles should not simply follow the typical evolutionary pattern of reduced polymorphisms observed in other genes in the selfing species. To test if R-genes have substitutions that have accumulated in patterns significantly following expectations under alternative models of molecular evolution, we selected five well-characterized R-genes (RPS2, RPM1, RPS5, RPP13 and RPP8) and investigated the patterns of diversity and divergence in the two closely-related Arabidopsis species, A. thaliana and A. lyrata. These genes represent a range of diversity levels estimated in this gene family, from the highly polymorphic RPP13 to the strongly conserved RPM1. Most of the selected R-genes are single copy genes; however, RPP8 is present in multiple copy numbers in A. thaliana. Previous reports suggest that RPP8 is derived from a single-copy progenitor gene by translocation, duplication and unequal crossover (Kuang et al., 2008). All RPP8 homologs used in this study are in the same gene family with high nucleotide sequence similarity, ranging from 0 to 0.166. In this study, we found that some R-genes (RPS5 and RPP13) are shaped by different evolutionary processes following short divergence times, possibly resulting in rapid adaptation to different pathogens.
Section snippets
Plant materials and orthologous sequences
A. lyrata ssp. petraea has a patchy distribution from Central Europe to Northern Europe (Muller et al., 2008). We collected 15 individuals from 5 populations of Central European A. l. petraea, 3 individuals per population, from Bavaria, Germany (Plech, Haselbrunn, and Neutras), central Germany (Stolberg) and Austria (Schaerftal). All A. l. petraea samples except from the Schaerftal population were collected by A.L.R. at the Max Planck Institute for Chemical Ecology. The Schaerftal population is
Patterns of polymorphism in R-genes between two closely-related species
In order to test whether there was a genome-wide departure from the neutral model in the populations we studied, tests for neutral equilibrium were performed considering allele and haplotype frequency at both R-genes and reference loci (Table S3 and Table S4). Tajima's D and Fay and Wu's H tests reveal no genome-wide departure from equilibrium expectations within each species, although there is a significant negative value (P < 0.05) at RPS2 within A. l. petraea for both tests. The only one locus
Signatures of adaptive evolution in R-genes between closely-related species
The maintenance and rates of nucleotide substitutions were examined for R-genes and the reference genes using estimates of nucleotide diversity, Ka/Ks ratios, levels of trans-specific polymorphism, and presence/absence polymorphism. The ‘signatures’ were observed at R-genes in comparison to the reference genes: a significant difference in the level of within-species polymorphism, a higher rate of mutational accumulation between species and variable selection pressures between species at R
Acknowledgments
This work was supported by funding from the National Natural Science Foundation of China (31071062 and 30930049) to D.T. and Clemson University to A.L.R. Sampling was partially sponsored by the Max Planck Institute for Chemical Ecology to A.L.R. Collaboration between the laboratories of D.T. and A.L.R. was facilitated by the Fifth Okazaki Biology Conference of the National Institute for Basic Biology, Japan.
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The first two authors contributed equally.