Virtually all terrestrial plants depend on symbiotic interactions with fungi. Arbuscular mycorrhizal (AM) fungi evolved over 450 million years ago, are obligate biotrophs and cannot complete their lifecycle without obtaining carbon from host roots. Mediating nutrient uptake and sequestering carbon in soil this symbiosis lie at the core of all terrestrial ecosystems. Plants on the other hand are facultative mycotrophs but under natural conditions all host roots are colonized as a result of multiple beneficial effects of AM fungi. In the symbiosis, both plants and fungi are promiscuous, forming interactions across individuals and species. In the absence of host-symbiont specificity and given their inability to discriminate among partners prior to interaction, evolutionary theory predicts that “free riders” would evolve and spread. Yet AM fungi remain evolutionary and ecologically successful. I propose that this is thanks to their unique genomic organization, a temporally dynamic heterokaryosis.Unlike other eukaryotes, AM fungi have no single nucleate stage in their life cycle, instead they reproduce asexually by forming large multinucleate spores. Genetic variation is high and nuclei can migrate and mix within extensive mycelial networks. My group has recently established a single nucleus genomics method to study genetic variation among nuclei within AM fungi. With this method I can resolve the extent of heterokaryosis in AM fungi and its temporal dynamics. I will study the emergence of “free riders” upon intra organismal segregation of genetically distinct nuclei during AM fungal adaptation to host. Further I will study how hyphal fusion and nuclear mixing counteract segregation to stabilize the symbiosis. The research program has great potential for novel discoveries of fundamental importance to evolutionary and environmental biology and will also contribute to agricultural practice and management of terrestrial ecosystems.