Genetic composition of symbiotic and free-living Frankia communities in Alnus–Frankia networks
We obtained a total of 352 ASVs from 138 samples consisting of riparian soil, rhizosphere soil, and root nodules (Fig. S1). Of the total ASVs, 90.34 % were unique singleton variants, which occurred once in 138 samples. We found profoundly diverse ASVs from rhizosphere and riparian soils (rhizosphere: 228 ASVs, n = 54; riparian: 103 ASVs,n = 12). In contrast, in root nodules, the total number of ASVs was quite limited (38 ASVs, n = 72). These results suggest that host individuals establish symbiosis only with limited Frankiagenotypes in soil.
To classify the Frankia genetic variants, we used OTU methods based on the genetic similarity of the nif D-K loci. A total of 19 OTUs were obtained at a 97% similarity threshold. Consequently, most ASVs from root nodules and riparian soils converged to three OTUs (OTU01, 02, and 03), while a variety of OTUs were found in the rhizosphere soil (Fig. 2). Consistently, the sample-based rarefaction curves showed that the number of Frankia OTUs did not completely reach saturation but were significantly greater in number in rhizospheres than in root nodules after standardization of the sample size (Fig. S3). In addition, the number of Frankia OTUs was lower in riparian soils than in rhizosphere soils (Fig. S3). These findings suggest that the occurrence of host plants promote the local assembly of nitrogen-fixing bacterial symbionts in soil rather than homogeneity dispersion of diverse bacterial strains in a natural forest.
Moreover, the phylogenetic trees indicated that the Frankia OTUs detected in our study covered a wide variety of Alnus -infecting strains (Fig. S2). The most abundant strains (i.e., OTU01, 02, and 03) belonged to phylogenetically distant clades (Fig. S2), indicating that the Alnus host population accepts genetically diverse bacterial symbionts.