Disentangling taxonomic chaos in Agaricaceae s.l.: an integrative phylogenomic framework with divergence dating reconstructs classification

The family Agaricaceae sensu lato (s.l.) represents a highly diverse and ecologically significant group of Basidiomycota, yet its taxonomic framework has long been contentious due to morphological convergence and conflicting phylogenetic hypotheses. Here, we present a comprehensive phylogenomic and taxonomic revision of Agaricaceae s.l. integrating multi-locus phylogenetics (ITS, nrLSU, rpb2, tef1), including 522 newly generated sequences, totaled 996 sequences from 334 species across 60 genera, genome-scale data (1764 single-copy orthologs from 118 genomes including 12 newly generated for this study), divergence time estimation, and detailed morphological analyses. Our analyses resolve Agaricaceae s.l. as a monophyletic lineage comprising five ancient, robustly supported clades that diverged during the Cretaceous. Based on this integrative evidence, we propose a revised classification recognizing five families: the re-circumscribed Agaricaceae sensu stricto and Lycoperdaceae; the reinstated Battarreaceae and Coprinaceae; and corroborating the family rank of Verrucosporaceae. Within the redefined Agaricaceae, we establish four subfamilies (Agaricoideae, Leucocoprinoideae, Macrolepioideae, and Podaxioideae) to provide a stable internal framework. Additionally, two new genera (Furfuragaricus, Conioexocarpus) and 43 novel species are described, along with 10 new combinations. This new classification provides unprecedented stability by resolving the contentious family-level status of major gasteroid lineages and clarifying the boundaries of historically problematic genera (Leucoagaricus, Leucocoprinus, Lepiota). This integrative framework combines molecular, morphological, and temporal evidence, resolving long-standing taxonomic ambiguities and providing a robust foundation for future studies on fungal diversity, evolution, and ecology. Our results underscore the critical role of phylogenomics in disentangling complex taxonomic groups and highlight the underestimated diversity within Agaricaceae s.l.