Phylogenetic multilocus sequence analysis identifies seven novel Ensifer genospecies isolated from a less-well-explored biogeographical region in East Africa

The diversity of 71 rhizobial strains belonging to the genus Ensifer , isolated from root nodules of woody legumes growing in southern Ethiopia, was studied using multilocus sequence analysis (MLSA) and phenotypic approaches. Phylogenetic analyses based on core genes revealed that 43 strains were clustered in seven distinct and consistent positions (genospecies I–VII), while another 25 strains were also distinct but were discrepant in their placement on the different gene trees. The remaining three strains occupied the same phylogenetic branches as defined Ensifer species and thus were not distinct. Irrespective of their chromosomal background, the majority of the test strains were highly related with respect to their nifH and nodC gene sequences, suggesting that these symbionts might have acquired these genes recently from a common origin. On the nifH phylogenetic tree, the branch containing the test strains and reference species isolated from woody legumes in Africa was clearly separate from those isolated outside the continent, suggesting that these symbionts have a long history of separate evolution within Ensifer for this gene. A cross-inoculation study showed that our strains were capable of eliciting effective nodulation on the homologous host and on other host species. This suggests a potential to improve nitrogen fixation by selecting for broad-host-range inoculants. Our study confirms the presence of a wide diversity of Ensifer in East Africa and, while contributing to the general knowledge of the biodiversity within the genus, also highlights the need to focus on previously less-well-explored biogeographical regions to unravel as-yet-unidentified rhizobial resources.