sp. nov., efficient nodulating rhizobia isolated from roots in Tunisia.

Three bacterial strains, 1AS14I, 1AS12I and 6AS6, isolated from root nodules of , were characterized using a polyphasic approach. Phylogenetic analysis based on sequences placed all three strains within the complex. Further phylogeny, based on 1 756 bp sequences of four concatenated housekeeping genes (, , and ), revealed their distinction from known rhizobia species of the complex (Rlc), forming a distinct clade. The closest related species, identified as , with a sequence identity of 96.4% based on concatenated --- sequences. The type strain, 1AS14I, showed average nucleotide identity (ANI) values of 94.9, 94.3 and 94.1% and DNA-DNA hybridization values of 56.1, 57.4 and 60.0% with the type strains of closest known species: , and respectively. Phylogenomic analyses using 81 up-to-date bacteria core genes and the Type (Strain) Genome Server pipeline further supported the uniqueness of strains 1AS14I, 1AS12I and 6AS6. The relatedness of the novel strains to NCBI unclassified sp. (396 genomes) and metagenome-derived genomes showed ANI values from 76.7 to 94.8% with a species-level cut-off of 96%, suggesting that strains 1AS14I, 1AS12I and 6AS6 are a distinct lineage. Additionally, differentiation of strains 1AS14I, 1AS12I and 6AS6 from their closest phylogenetic neighbours was achieved using phenotypic, physiological and fatty acid content analyses. Based on the genomic, phenotypic and biochemical data, we propose the establishment of a novel rhizobial species, sp. nov., with strain 1AS14I designated as the type strain (=DSM 113914=LMG 33206). This study contributes to the understanding of microbial diversity in nitrogen-fixing symbioses, specifically within ecosystems in Tunisia.