Summary: Forest trees rely on ectomycorrhizal (ECM) fungi for acquiring scarce resources such as water and nutrients. However, the molecular mechanisms governing ECM traits remain inadequately understood, particularly the role of intraspecific fungal variation in root‐tip colonisation and trophic interactions. This study examined six ECM traits using Pisolithus microcarpus, an ECM fungus capable of forming ECM rootlets in poplar. A collection of 40 sibling monokaryons and their parental dikaryon was analysed through genome and transcriptome sequencing to examine quantitative trait loci, gene expression and mating‐type loci. These findings revealed a pronounced phenotypic continuum in poplar root colonisation by sibling monokaryons, ranging from incompatible to fully compatible strains. Genetic recombination among monokaryons was demonstrated, and genomic regions potentially involved in ECM fungal traits were identified. Transcriptomic analysis revealed greater differentiation in transcriptomic profiles between fungal strains than between fungal tissues, and uncovered tissue‐specific functional responses for ECM and free‐living mycelia. Poplar exhibited distinct transcriptomic responses when interacting with different sibling monokaryons and the parental dikaryon. Allele sorting at 11 mating‐type loci confirmed the species' heterothallic tetrapolar system. This study advances understanding of the genetic and transcriptomic mechanisms underlying ECM symbioses, highlighting intraspecific fungal diversity's role in forest ecosystem functioning.

Transposable elements (TEs) play crucial roles in genome evolution and ecological adaptation in fungi, yet their dynamics in ectomycorrhizal species remain poorly understood. Cenococcum geophilum, the most widespread ectomycorrhizal fungus in boreal and temperate forests with its large, repeat-rich genome, represents an ideal system to investigate TE-mediated adaptation to the physical environment and symbiotic lifestyle. However, previous studies have been limited by fragmented genome assemblies that prevented the resolution of repeat-rich regions. We assembled a telomere-to-telomere reference genome of C. geophilum strain 1.58 using PacBio HiFi and Hi-C datasets, resulting in a 178.54 Mbp genome with seven contiguous chromosomes. We identified 14,145 genes and over 78% of the genome consists of transposable elements (TEs). Of these, 94% are affected by repeat-induced point mutations (RIP), a genome defense mechanism that acts during the sexual reproduction phase, indicating cryptic or ancient sexual reproduction in this putatively asexual fungus. Long terminal repeat retrotransposons, LINEs, and DNA transposons dominate, with three TE families (Ty3, Ty1, and Tad1) contributing over 60% of the genome size, indicating recent transposition bursts. Screening of 15 additional C. geophilum strains revealed recent and lineage-specific TE expansions, implying that several TEs escaped the RIP machinery and retained potential activity. Supporting TE activity in the context of symbiosis, we found 56 TEs differentially transcribed between ectomycorrhizal and free-living mycelium tissues. An even higher number (n = 66) of TEs were differentially expressed between stress resistance morphology (i.e. sclerotia) and free-living mycelium. This supports that TEs are differentially regulated as a response to symbiotic and stress-related conditions. Our results demonstrate that the C. geophilum genome expansion was driven by a few lineage-specific TE families in recent history, with high RIP activity attesting to sexual reproduction. We also provide insights how TEs could respond to lifestyle transitions and traits associated with desiccation resistance.