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The symbioses between legumes and nitrogen-fixing rhizobia make the greatest contribution to the global nitrogen input via the process of biological nitrogen fixation (BNF). Bradyrhizobium stands out as the main genus nodulating basal Caesalpinioideae. We performed a polyphasic study with 11 strains isolated from root nodules of Chamaecristafasciculata , an annual multi-functional native legume of the USA. In the 16S rRNA gene phylogeny the strains were clustered in the Bradyrhizobium japonicum superclade. The results of analysis of the intergenic transcribed spacer (ITS) indicated less than 89.9 % similarity to other Bradyrhizobium species. Multilocus sequence analysis (MLSA) with four housekeeping genes ( glnII , gyrB , recA and rpoB ) confirmed the new group, sharing less than 95.2 % nucleotide identity with other species. The MLSA with 10 housekeeping genes ( atpD , dnaK , gap , glnII , gltA , gyrB , pnp , recA , rpoB and thrC ) indicated Bradyrhizobium daqingense as the closest species. Noteworthy, high genetic diversity among the strains was confirmed in the analyses of ITS, MLSA and BOX-PCR. Average nucleotide identity and digital DNA-DNA hybridization values were below the threshold of described Bradyrhizobium species, of 89.7 and 40 %, respectively. In the nifH and nodC phylogenies, the strains were grouped together, but with an indication of horizontal gene transfer, showing higher similarity to Bradyrhizobium arachidis and Bradyrhizobium forestalis . Other phenotypic, genotypic and symbiotic properties were evaluated, and the results altogether support the description of the CNPSo strains as representatives of the new species Bradyrhizobiumfrederickii sp. nov., with CNPSo 3426 T (=USDA 10052 T =U686 T =CL 20 T ) as the type strain.

Information about the symbionts of legumes of the Caesalpinioideae subfamily is still limited, and we performed a polyphasic approach with three Bradyrhizobium strains-CNPSo 3448 T , CNPSo 3394 and CNPSo 3442-isolated from Chamaecrista fasciculata , a native legume broadly distributed in the USA. In the phylogenetic analysis of both the 16S rRNA gene and the intergenic transcribed spacer, the CNPSo strains were clustered within the Bradyrhizobium japonicum superclade. Multilocus sequence analysis with six housekeeping genes- glnII , gyrB , recA , rpoB , atpD and dnaK -indicated that Bradyrhizobium diazoefficiens is the closest species, with 83 % of nucleotide identity. In the genome analyses of CNPSo 3448 T , average nucleotide identity and digital DNA-DNA hybridization results confirmed higher similarity with B. diazoefficiens , with values estimated of 93.35 and 51.50 %, respectively, both below the threshold of the same species, confirming that the CNPSo strains represent a new lineage. BOX-PCR profiles indicated high intraspecific genetic diversity between the CNPSo strains. In the analyses of the symbiotic genes nodC and nifH the CNPSo strains were clustered with Bradyrhizobium arachidis , Bradyrhizobium forestalis , Bradyrhizobium cajani , Bradyrhizobium kavangense and Bradyrhizobium vignae , indicating a different phylogenetic history compared to the conserved core genes. Other physiological (C utilization, tolerance to antibiotics and abiotic stresses), chemical (fatty acid profile) and symbiotic (nodulation host range) properties were evaluated and are described. The data from our study support the description of the CNPSo strains as the novel species Bradyrhizobiumniftali sp. nov., with CNPSo 3448 T (=USDA 10051 T =U687 T =CL 40 T ) designated as the type strain.