Biogeographic and Genomic Signatures of Thermal Adaptation in Facultative Symbionts

Many plant endosymbionts are facultative, switching between host-associated and free-living stages. Extensive genomic and experimental studies suggest that adaptation during the saprophytic, off-host phase, rather than adaptation to hosts, primarily constrains the biogeographic distribution of these microbes. To test this hypothesis, we analyzed the growth capacities and genomic features of 38 Sinorhizobium and Ensifer strains isolated from the nodules of Medicago lupulina (black medic), collected from two regions with distinct thermal environments. The warmer region is predominantly inhabited by S. meliloti, while S. medicae and Ensifer strains are more common in the cooler region. Laboratory assays demonstrated that at 40 °C, the upper temperature limit of their region of origin, S. meliloti remained viable, albeit with reduced growth, whereas S. medicae and Ensifer strains failed to grow under heat stress. Comparative genomics revealed isolation-by-distance in both the core and accessory genomes, particularly in S. meliloti in the warmer region, which exhibits less within-region thermal variation. This is consistent with an isolation-by-distance model where population divergence is governed by restricted gene flow. These findings suggest that metabolic constraints shape the regional distribution of this facultative microbial symbiont, while limited gene flow influences local population structure.