Bayesian Phylogenetic Analysis of Rhizobia Isolated From Root-Nodules of Three Tunisian Wild Legume Species of the Genus Sulla

Abstract

We used a relaxed-clock program (Multidivtime) to estimate the phylogenetic relatedness and substitution rates by comparative analyses of 16S rRNA gene and ITS region sequences. Four rhizobial strains isolated from three Tunisian wild Sulla legumes were used together with reference strains to obtain the 16S rRNA gene and ITS region alignments. Bayesian inferred trees were congruent, and showed a clear split between Agrobacterium and Rhizobium species. Among the four Tunisian isolates, Hsc01 belonged to Rhizobium species and formed a monophyletic clade with Rhizobium sullae. The strain Hc04 was placed into the Agrobacterium group, but it belonged to Rhizobium species. Two remaining strains (Hc01 and Hcar01) belonged to typically Agrobacterium species. Divergence times between these four strains were earlier the existence of legumes. Using sequence alignments of the 16S rRNA genes and ITS regions, we inferred different rates of nucleotide substitutions across these two molecular markers. The ITS region evolutionary rate was 15-fold higher than the 16S rRNA gene rate, suggesting that the ITS region represented an appropriate molecular marker for inferring phylogenies and divergence times in bacteria.