Evolution of virulence of a plant RNA virus in age-diverse host populations

Natural host populations are intrinsically age-structured, and developmental stages differ in susceptibility and within-host dynamics, meaning age compositions can impose distinct selective pressure on pathogens. However, how host age structure shapes viral evolution remains largely untested. We experimentally evolved turnip mosaic virus (TuMV) for five passages in Arabidopsis thaliana populations spanning seven demographic regimes (from juvenile-dominated to mature-dominated cohorts). We quantified disease progression, symptom severity and viral load, cross-inoculated evolved lineages across host stages to build quantitative infection matrices, and performed whole-population sequencing at passages 1 and 5 to infer selection coefficients and test for parallelism. Disease traits changed strongly with passage, demography, and their interaction. Disease progression evolved faster in older host populations, whereas severity showed no detectable dependence on median age, implying demographic reweighting of virulence components. Viral load increased through passages and positively correlated with severity, linking within-host fitness to symptoms. Cross-assays revealed a non-nested but modular infection network: juvenile-evolved lineages specialized on pre-bolting plants, whereas lineages from intermediate/older demographies were more stage-generalist. Genomically, both parallel and demography-specific solutions emerged, with a dense cluster of recurrent changes in VPg and several synonymous variants showing consistent or sign-flipping selection across demographies. In conclusion, host age structure is a primary ecological driver of virulence evolution, determining whether viruses evolve faster disease timing versus stronger severity, and whether they specialize or generalize across host stages. These results integrate phenotypes with genome-level responses and suggest that manipulating crop age pyramids could steer virus evolution toward less damaging outcomes.