Molecular clock dating under mixture substitution models indicates contemporaneous emergence of Bradyrhizobium nodulation and legumes anchored by non-symbiotic relatives

The transition from free-living bacteria to mutualistic symbionts is a major evolutionary innovation, but its timing and stepwise nature remain unresolved because non-symbiotic ancestors have rarely been sampled. Here we isolated 10 Bradyrhizobium strains from non-legume plants and soils, and discovered three basal clades within the B. japonicum supergroup that completely lack nodulation (nod) genes, which are required for Nod factor synthesis and legume symbiosis. To date the emergence of symbiosis, we employed a mitochondrial endosymbiosis-based molecular clock dating framework that jointly estimates the divergence times of bacterial symbionts and their hosts in a single analysis. Using these non-nodulating lineages as evolutionary anchors, we show that nodulating B. japonicum arose contemporaneously with nodulating legumes most likely around 90-80 million years ago. Excluding these basal clades makes rhizobial nodulation appear to predate nodulating legumes, while the use of the poorly-fitting homogeneous models produces a similar temporal distortion. The inferred patterns are robust to alternative calibration strategies, gene sets, and mixture models. Together, these findings highlight the importance of comprehensive taxon sampling and appropriate substitution models in relaxed molecular clock dating, and support a co-emergence model in the origin of legume-rhizobia mutualism.