Aeschynomene indica-Nodulating Rhizobia Lacking Nod Factor Synthesis Genes: Diversity and Evolution in Shandong Peninsula, China

Aeschynomene indica rhizobia (AIRs) can form both root and stem nodules via Nod factor-independent processes, which distinguishes them from other rhizobia. This study systematically uncovered the diversity and community composition of A. indica rhizobia distributed in eastern China. Our results reclassified all the A. indica rhizobia across the world and represent a useful contribution to evaluating the diversity and distribution of the symbiont. The presence of novel genospecies specifically distributed in China enriched the A. indica rhizobia resources and provided insight into the geographic distribution of rhizobia. The phylogenetic relationship between nifH and pufLM of A. indica rhizobia across the world provides insight into the evolution of their nitrogen fixation and photosynthetic abilities. ABSTRACT Aeschynomene indica is a semiaquatic legume that forms both stem and root nodules with rhizobia. Some A. indica rhizobia (AIRs) have been reported to nodulate the host using a Nod factor-independent pathway and possess photosynthetic abilities. To investigate the diversity and community structure of AIRs in China, a total of 300 rhizobial isolates were acquired from the root and stem nodules of A. indica grown at 4 sites in Shandong Peninsula, China. Nineteen representative strains were selected according to their recA phylogeny. With further classification in comparison with reference strains, 10 Bradyrhizobium genospecies were defined based on the 16S rRNA gene phylogeny and multilocus sequence analysis (MLSA) of housekeeping genes (HKGs) recA, atpD, glnII, dnaK, gyrB, and rpoB. In addition, 6 genospecies were found only in China. No nodulation gene (nodA, nodB, nodC, or nodZ) was detected in the AIRs isolates by PCR amplification and Southern blotting. Phylogenetic analysis of nifH and the photosynthesis-related gene pufLM revealed their common origins. All representative strains formed root nodules, but only 9 representative strains for 4 genospecies formed stem nodules on A. indica, indicating that the stem nodulation process of A. indica is limited to some strains. The nucleotide diversity and recombination events of the HKGs, as well as nifH and pufLM genes, showed that mutation contributes more than recombination in evolution. The distribution of dominant AIR genospecies was mainly affected by available nitrogen, organic carbon, total nitrogen, and pH. Our study helps to characterize the diversity and evolution of AIRs. IMPORTANCE Aeschynomene indica rhizobia (AIRs) can form both root and stem nodules via Nod factor-independent processes, which distinguishes them from other rhizobia. This study systematically uncovered the diversity and community composition of A. indica rhizobia distributed in eastern China. Our results reclassified all the A. indica rhizobia across the world and represent a useful contribution to evaluating the diversity and distribution of the symbiont. The presence of novel genospecies specifically distributed in China enriched the A. indica rhizobia resources and provided insight into the geographic distribution of rhizobia. The phylogenetic relationship between nifH and pufLM of A. indica rhizobia across the world provides insight into the evolution of their nitrogen fixation and photosynthetic abilities.