giardinii or R. gallicum[66]. In contrast we were unable to transfer R. grahamii ERs to other rhizobia. It is worth noting that tropici symbiotic plasmids are more conserved than phaseoli ones, and both are more conserved than the grahamii group pSyms. It is tempting to suggest that genome conservation among distinct species is related to transferability. On the other hand, transfer of plasmids to novel hosts can also detonate their evolution by picking up new genetic information (that would affect the genomic content) from other genomic backgrounds. We do not know if in natural habitats or in the presence of a microbial community, the lack

of transferability of R. grahamii ERs holds true. Besides, the limited conservation of pSyms among R. grahamii and R. mesoamericanum suggests that they are not frequently

interchanged among these species. Transfer of the R. grahamii symbiotic plasmid BYL719 to Agrobacterium was dependent on quorum sensing, a mechanism that regulates transfer of plasmids in rhizobia [25, 67] and agrobacteria [68, 69]. This lack of ER flow and existence of a genetic barrier could be due to different mechanisms, such as DNA restriction/methylation systems or to surface or entry exclusion systems. Surface exclusion at the level of formation of stable mating aggregates and entry exclusion seem to inhibit conjugation in a later step of the mating aggregate [70, 71]. Limited transfer may be due to a system similar to CRISPR/Cas, an adaptive immunity system found in Archaea and bacteria that eliminates virus or plasmids in a new host [72, 73]. These possibilities deserve further research. Putative chromids FDA approved Drug Library screening (megaplasmids) in the grahamii group have a lower percentage of gene content conservation than the chromosomes and symbiotic plasmids, in spite of their fairly high ANI values (Figure 1B and C). Considering the conserved genomic content in chromosomes, symbiotic MG-132 solubility dmso plasmids and putative chromids in the grahamii group, there clearly are three different degrees of conservation

(Figure 1C). We suggest a layout where the rhizobial genome is a 3 gear genome with different rates of change in each of the replicon types. In animals and plants, different regions of the genome exhibit variable levels of genetic divergence between populations (reviewed in Nosil et al.[74]). The extrachromosomal replicons of R. grahamii CCGE502 were related to those from R. mesoamericanum. An exception is the plasmid integrated in the R. grahamii chromosome for which no equivalent plasmid was found in R. mesoamericanum or in other rhizobia. However some common genes were found in the R. grahamii integrated replicon and in other Rhizobium species. ER organization plasticity was reported previously in rhizobia with the integration of plasmids or megaplasmids into the chromosome [75, 76]. This seems to have occurred in R. grahamii CCGE502 as we report here. It is noteworthy that some of the genes highly expressed in R.