A staircase model for the co‑evolution of Bradyrhizobium and legumes dated 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 unclear because non‑symbiotic ancestors have rarely been sampled. Here we isolated 75 Bradyrhizobium strains from non‑legume plants and soils and discovered three basal clades within the B. japonicum supergroup that completely lack nod genes. Using these non‑nodulating lineages as evolutionary anchors in molecular clock analyses, we show that nodulating B. japonicum arose contemporaneously with nodulating legumes around 90 million years ago. Excluding these basal clades erroneously makes rhizobial nodulation appear to predate nodulating legumes, highlighting the critical importance of taxon sampling. Quantitative comparative genomics reveals a staircase model: the three basal non‑nodulating clades show progressively higher similarity to nodulating lineages in their plant‑association gene repertoires. Ecological capacities such as nitrogen fixation, denitrification, and hydrogen recycling were already present in the earliest ancestors, whereas nod genes and type III secretion systems were added only in the branch leading to nodulating B. japonicum. Other Bradyrhizobium supergroups later acquired nodulation through horizontal transfer. This staircase trajectory, precisely dated by non‑symbiotic relatives, demonstrates that co‑evolution with hosts, not deep pre‑adaptation, triggered the final assembly of symbiosis machinery.