Estimated timeline for the evolution of symbiotic nitrogen fixing Paraburkholderia

The nitrogen-fixing and nodule-forming symbionts of legumes, which belong to the class Betaproteobacteria, are informally known as beta-rhizobia. Thus far, members of this group have only been found in the genera Paraburkholderia, Trinickia and Cupriavidus. In this study, we investigate the poorly characterized evolutionary history of this trait in the predominant beta-rhizobial genus, Paraburkholderia. This was determined in the context of the current evolutionary theories and date estimates of rhizobia, the genus Paraburkholderia and the earth. Evolutionary divergence dates of rhizobial Paraburkholderia as well as their ancestral nodulation states were estimated using over 800 diverse proteobacterial genomes. Molecular dating was carried out using the software BEAST (Bayesian Evolutionary Analysis Sampling Trees) and APE (using the 'chronopl' function). Our results showed that the most recent common ancestor (MRCA) of the extant beta-rhizobial species emerged between 2744 and 1752 million years ago (Ma) and later (2135-514 Ma) diverged into the lineages Cupriavidus, Trinickia and Paraburkholderia. However, major diversifications of rhizobial Paraburkholderia occurred in three phases: (i) during the Permian and Triassic periods (400-200 Ma) when Pangaea was fully assembled and its landmass filling up with flora and fauna, (ii) during the Jurassic period (200-150 Ma) when fauna and flora were flourishing in Pangaea, and (iii) during the Cretaceous and Paleogene periods (150-23 Ma) when Gondwana was breaking up. Furthermore, Paraburkholderia were estimated to have acquired their precursor nodulation loci that evolved into their current nodulation loci from different sources between 103 and 48 Ma. Accordingly, our study describes the evolutionary history of rhizobial Paraburkholderia, thus enabling us to understand the past environmental factors that shaped the current geographical distribution of these agriculturally important bacteria, and to identify locations potentially rich in beta-rhizobia.