Phylogenomic signatures of repeat-induced point mutations across the fungal kingdom

Fungal genome sizes exhibit more than a 100-fold variation, largely driven by the expansion of repetitive sequences such as transposable elements (TEs). Silencing mechanisms targeting TEs at the epigenetic or transcript level have independently evolved in many lineages. In fungi, repeat-induced point mutation (RIP) targets TEs by recognizing repetitive sequences and inducing mutagenesis. However, the prevalence of RIP across the fungal kingdom and the fidelity of the canonical C-to-T mutation signatures remain unclear. In this study, we address these gaps by tracking shifts in genome architecture across the fungal kingdom. We find that a striking approximately 30-fold increase in genome size within a clade of leotiomycetes is associated with the absence of several RIP-related genes, suggesting a relaxation of genome defense mechanisms during this expansion. To track the impact of genome defenses, we designed a quantitative screen for RIP-like mutation signatures. The phylum of ascomycetes was unique in showing enrichment in mutation signatures in non-coding and repetitive sequences, consistent with a phylogenetically restricted occurrence of RIP-like genome defense systems. Then, we performed a phylogeny-aware association study to identify gene functions associated with RIP-like mutation signatures. We identified a zinc-finger protein as the strongest candidate underpinning a novel mechanism of genome defenses. Our findings reveal the multifaceted drivers of genome defense systems and their close ties to genome size evolution in fungi, particularly in lineages with evidence for recent RIP activity, highlighting how proximate molecular mechanisms can shape genome evolution on deep phylogenetic scales.