Abstract: Ectomycorrhizal (ECM) fungi form symbiotic relationships with woody plants, completing their life cyclesthrough mutualistic associations. The evolution of this symbiosis involves genomic adaptations including gene gain andloss. However, how these genomic characteristics reflect speciation and adaptation throughout the evolutionaryhistory of ECM fungi remains unclear. In the present study, we explored speciation and host adaptation in Tricholoma species, an ecologically relevant clade of ECM basidiomycetes. We compared the genomes of three species, Tricholoma matsutake, T. populinum, and T. bakamatsutake, which despite their close phylogenetic relationships, have different tree hosts. A phylogenetic tree constructed using single‐copy orthologous genes estimated the divergence time of T. populinum to be approximately 28.48 Mya, coinciding with the diversification of subg. Eupopulus in East Asia. The split between T. matsutake and T. bakamatsutake was estimated at around 8.08 Mya, corresponding to the diversification period of evergreen broadleaved forests in East Asia. In this study, we identified 19, 13, and 13 positively selected genes in the genomes of T. bakamatsutake, T. matsutake, and T. populinum, respectively. Additionally, 2983, 2783, and 1548 genes have undergone rapid evolution in their genomes. Gene ontology enrichment analysis revealed the functions of these rapidly evolving genes, including those associated with cell cycle, cytoplasmic components, and GTPase mediation. Gene flow analysis indicated unidirectional migration from the ancestor of T. populinum to T. matsutake and T. bakamatsutake, whereas bidirectional gene flow was observed in the ancestors of T. matsutake and T. bakamatsutake. This study suggested that host‐induced immigrant unviability in symbiotic fungi is the primary causeof prezygotic isolation. The combination of ecology‐based genomic evidence and gene flow analysis offers new insightinto the speciation and evolutionary mechanisms of symbiotic fungi.

Key words: ecological speciation, ectomycorrhizal fungi, gene ?ow, host speci?city, transposable elements