Argyrolobium legumes from an African centre of endemism associate with novel Bradyrhizobium species harbouring unique sets of symbiosis genes

Given that several, mainly endemic South African Genisteae genera occupy basal positions in legume phylogenetic trees, this region of Africa is considered a primaeval centre of diversification of this legume tribe. Despite the importance of South Africa in Genisteae evolution, almost all studies have focused on rhizobia nodulating Genisteae in their centres of diversity in either the Mediterranean Basin or the Americas. Therefore, this study aimed to identify and characterize rhizobial strains associated with Argyrolobium species native to areas of the Grassland biome associated with the Great Escarpment, which dominates the subcontinent’s eastern landscape, and compare these to bradyrhizobia nodulating Genisteae in other centres of diversity. Phylogenetic analyses of five housekeeping genes (dnaK, glnII, gyrB, recA, and rpoB) separated the 18 Bradyrhizobium strains examined into five well-supported groups. Three of these were conspecific with B. arachidis, B. brasilense/B. australafricanum and B. ivorense, while the remaining two appeared to be new to science. After confirming their novelty using Average Nucleotide Identity, a metric for genome relatedness, and certain phenotypic traits, we recognized them as novel species for which we proposed the names B. spitzkopense sp. nov. (Arg816^Ts) and B. mpumalangense sp. nov. (Arg237L^Ts). Phylogenetic analyses of nodA gene sequences showed that about half of the strains examined, irrespective of their species identity, harboured alleles known only from the Grassland biome along the Great Escarpment that were previously detected in Bradyrhizobium strains nodulating Crotalarieae endemic to this region. Genome-based analyses of data from this and previous studies further showed that strains with these unique nodA alleles typically encode the nodH gene, the product of which adds a sulfate moiety to the Nod factor (the signalling molecule for establishing the nitrogen-fixing symbiosis). The remaining strains had nodA alleles commonly encountered elsewhere in South Africa and other tropical regions of the world. Also, the genomes of these other strains lacked nodH but encoded nodZ, the gene involved in the fucosylation of the Nod factor. Our findings, therefore, showed that the root nodules of Genisteae (and its sister tribe Crotalarieae) native to the Grassland biome along the Great Escarpment are often related Bradyrhizobium strains that are distinct from bradyrhizobia nodulating Genisteae in the Mediterranean and the Americas.