Ecological genomics of saprotrophy to biotrophy transitions in the genus <i>Clitopilus</i> (Fr. ex Rabenh.) P. Kumm. (<i>Agaricales</i>, <i>Entolomataceae</i>)

Transitions between saprotrophic and biotrophic lifestyles represent pivotal evolutionary events in fungal ecology; however, the genomic and physiological mechanisms underlying such shifts remain poorly understood. The agaric genus <i>Clitopilus</i> (<i>Basidiomycota</i>, <i>Entolomataceae</i>) offers a valuable model system, with most species being soil saprotrophs. <i>Clitopilus cf. baronii</i> Consiglio & Setti exhibits genomic signatures suggesting incipient biotrophic capacity. Here, we investigated the genomic and eco-physiological properties of seven strains representing five <i>Clitopilus</i> species to identify traits associated with lifestyle transitions. ITS-based phylogeny combined with ecological metadata revealed potential facultative biotrophy in multiple taxa from the section <i>Scyphoides</i>. Physiological profiling showed that all strains utilized mannitol and sucrose poorly, preferred organic nitrogen compounds, and produced variable amounts of indole-3-acetic acid (IAA) <i>in vitro</i> in a strictly tryptophan-dependent manner. Enzymatic assays revealed substantial variations in the nitrogen and phosphorus acquisition capabilities among the strains. Comparative genomics of high-quality assemblies identified a pleuromutilin biosynthetic gene cluster (BGC) across all strains, although synteny analysis revealed considerable structural variation and putative gene loss, indicating that genomic plasticity potentially affects antibiotic production. Principal component analysis of carbohydrate-active enzymes (CAZymes) across 25 fungal genomes partitioned <i>Clitopilus</i> strains into two distinct groups: one resembling saprotrophic white-rot basidiomycetes, the other matching biotrophic ectomycorrhizal and endophytic taxa. This first comprehensive genomic analysis of <i>Clitopilus</i> revealed that nutritional specialization, phytohormone production, and CAZyme repertoire remodeling collectively signal an ongoing evolutionary transition from saprotrophy to plant-associated lifestyles in multiple lineages. These findings provide a rare genomic window into the early stages of symbiosis evolution, offering insights into how free-living fungi acquire the molecular toolkit for mutualistic partnerships.