Impacts of Natural Selection on Evolution of Core and Symbiotically Specialized (sym) Genes in the Polytypic Species Neorhizobium galegae.

Nodule bacteria (rhizobia) represent a suitable model to address a range of fundamental genetic problems, including the impacts of natural selection on the evolution of symbiotic microorganisms. Rhizobia possess multipartite genomes in which symbiotically specialized (sym) genes differ from core genes in their natural histories. Diversification of sym genes is responsible for rhizobia microevolution, which depends on host-induced natural selection. By contrast, diversification of core genes is responsible for rhizobia speciation, which occurs under the impacts of still unknown selective factors. In this paper, we demonstrate that in goat's rue rhizobia (Neorhizobium galegae) populations collected at North Caucasus, representing two host-specific biovars orientalis and officianalis (N2-fixing symbionts of Galega orientalis and G. officinalis), the evolutionary mechanisms are different for core and sym genes. In both N. galegae biovars, core genes are more polymorphic than sym genes. In bv. orientalis, the evolution of core genes occurs under the impacts of driving selection (dN/dS > 1), while the evolution of sym genes is close to neutral (dN/dS ≈ 1). In bv. officinalis, the evolution of core genes is neutral, while for sym genes, it is dependent on purifying selection (dN/dS < 1). A marked phylogenetic congruence of core and sym genes revealed using ANI analysis may be due to a low intensity of gene transfer within and between N. galegae biovars. Polymorphism in both gene groups and the impacts of driving selection on core gene evolution are more pronounced in bv. orientalis than in bv. officianalis, reflecting the diversities of their respective host plant species. In bv. orientalis, a highly significant (P0 < 0.001) positive correlation is revealed between the p-distance and dN/dS values for core genes, while in bv. officinalis, this correlation is of low significance (0.05 < P0 < 0.10). For sym genes, the correlation between p-distance and dN/dS values is negative in bv. officinalis but is not revealed in bv. orientalis. These data, along with the functional annotation of core genes implemented using Gene Ontology tools, suggest that the evolution of bv. officinalis is based mostly on adaptation for in planta niches while in bv. orientalis, evolution presumably depends on adaptation for soil niches. New insights into the tradeoff between natural selection and genetic diversity are presented, suggesting that gene nucleotide polymorphism may be extended by driving selection only in ecologically versatile organisms capable of supporting a broad spectrum of gene alleles in their gene pools.

Dynamic of the Soil Microbiota in Short-Term Crop Rotation.

Crop rotation is one of the oldest and most effective methods of restoring soil fertility, which declines when the same plant is grown repeatedly. One of the reasons for a reduction in fertility is the accumulation of pathogenic and unfavorable microbiota. The modern crop rotation schemes (a set of plant species and their order in the crop rotation) are highly effective but are designed without considering soil microbiota dynamics. The main goal of this study was to perform a short-term experiment with multiple plant combinations to access the microbiological effects of crop rotation. It could be useful for the design of long-term crop rotation schemes that take the microbiological effects of the crop rotation into account. For the analysis, five plants (legumes: vetch, clover, and cereals: oats, wheat, and barley) were used. These five plants were separately grown in pots with soil. After the first phase of vegetation, the plants were removed from the soil and a new crop was planted. Soil samples from all 25 possible combinations of primary and secondary crops were investigated using v4-16S rDNA gene sequencing. It was shown that the short-term experiments (up to 40 days of growing) are effective enough to find microbial shifts in bulk soil from different plants. Both primary and secondary cultures are significant factors for the microbial composition of microbial soil communities. Changes are the most significant in the microbial communities of vetch soils, especially in the case of vetch monoculture. Growing clover also leads to changes in microbiota, especially according to beta-diversity. Data obtained can be used to develop new crop rotation schemes that take into account the microbiological effects of various crops.

Microevolution, speciation and macroevolution in rhizobia: Genomic mechanisms and selective patterns.

Nodule bacteria (rhizobia), N2-fixing symbionts of leguminous plants, represent an excellent model to study the fundamental issues of evolutionary biology, including the tradeoff between microevolution, speciation, and macroevolution, which remains poorly understood for free-living organisms. Taxonomically, rhizobia are extremely diverse: they are represented by nearly a dozen families of α-proteobacteria (Rhizobiales) and by some ß-proteobacteria. Their genomes are composed of core parts, including house-keeping genes (hkg), and of accessory parts, including symbiotically specialized (sym) genes. In multipartite genomes of evolutionary advanced fast-growing species (Rhizobiaceae), sym genes are clustered on extra-chromosomal replicons (megaplasmids, chromids), facilitating gene transfer in plant-associated microbial communities. In this review, we demonstrate that in rhizobia, microevolution and speciation involve different genomic and ecological mechanisms: the first one is based on the diversification of sym genes occurring under the impacts of host-induced natural selection (including its disruptive, frequency-dependent and group forms); the second one-on the diversification of hkgs under the impacts of unknown factors. By contrast, macroevolution represents the polyphyletic origin of super-species taxa, which are dependent on the transfer of sym genes from rhizobia to various soil-borne bacteria. Since the expression of newly acquired sym genes on foreign genomic backgrounds is usually restricted, conversion of resulted recombinants into the novel rhizobia species involves post-transfer genetic changes. They are presumably supported by host-induced selective processes resulting in the sequential derepression of nod genes responsible for nodulation and of nif/fix genes responsible for symbiotic N2 fixation.

Rhizobia Isolated from the Relict Legume Vavilovia formosa Represent a Genetically Specific Group within Rhizobium leguminosarum biovar viciae.

Twenty-two rhizobia strains isolated from three distinct populations (North Ossetia, Dagestan, and Armenia) of a relict legume Vavilovia formosa were analysed to determine their position within Rhizobium leguminosarum biovar viciae (Rlv). These bacteria are described as symbionts of four plant genera Pisum, Vicia, Lathyrus, and Lens from the Fabeae tribe, of which Vavilovia is considered to be closest to its last common ancestor (LCA). In contrast to biovar viciae, bacteria from Rhizobium leguminosarum biovar trifolii (Rlt) inoculate plants from the Trifolieae tribe. Comparison of house-keeping (hkg: 16S rRNA, glnII, gltA, and dnaK) and symbiotic (sym: nodA, nodC, nodD, and nifH) genes of the symbionts of V. formosa with those of other Rlv and Rlt strains reveals a significant group separation, which was most pronounced for sym genes. A remarkable feature of the strains isolated from V. formosa was the presence of the nodX gene, which was commonly found in Rlv strains isolated from Afghanistan pea genotypes. Tube testing of different strains on nine plant species, including all genera from the Fabeae tribe, demonstrated that the strains from V. formosa nodulated the same cross inoculation group as the other Rlv strains. Comparison of nucleotide similarity in sym genes suggested that their diversification within sym-biotypes of Rlv was elicited by host plants. Contrariwise, that of hkg genes could be caused by either local adaptation to soil niches or by genetic drift. Long-term ecological isolation, genetic separation, and the ancestral position of V. formosa suggested that symbionts of V. formosa could be responsible for preserving ancestral genotypes of the Rlv biovar.

Microvirga ossetica sp. nov., a species of rhizobia isolated from root nodules of the legume species Vicia alpestris Steven.

Gram-stain-negative strains V5/3MT, V5/5K, V5/5M and V5/13 were isolated from root nodules of Vicia alpestris plants growing in the North Ossetia region (Caucasus). Sequencing of the partial 16S rRNA gene (rrs) and four housekeeping genes (dnaK, gyrB, recA and rpoB) showed that the isolates from V. alpestris were most closely related to the species Microvirga zambiensis (order Rhizobiales, family Methylobacteriaceae) which was described for the single isolate from root nodule of Listia angolensis growing in Zambia. Sequence similarities between the Microvirga-related isolates and M. zambiensis WSM3693T ranged from 98.5 to 98.7 % for rrs and from 79.7 to 95.8 % for housekeeping genes. Cellular fatty acids of the isolates V5/3MT, V5/5K, V5/5M and V5/13 included important amounts of C18 : 1ω7c (54.0-67.2 %), C16 : 0 (6.0-7.8 %), C19 : 0 cyclo ω8c (3.1-10.2 %), summed feature 2 (comprising one or more of iso-C16 : 1 I, C14 : 0 3-OH and unknown ECL 10.938, 5.8-22.5 %) and summed feature 3 (comprising C16 : 1ω7c and/or iso-C15 : 02-OH, 2.9-4.0 %). DNA-DNA hybridization between the isolate V5/3MT and M. zambiensis WSM3693T revealed DNA-DNA relatedness of 35.3 %. Analysis of morphological and physiological features of the novel isolates demonstrated their unique phenotypic profile in comparison with reference strains from closely related species of the genus Microvirga. On the basis of genotypic and phenotypic analysis, a novel species named Microvirga ossetica sp. nov. is proposed. The type strain is V5/3MT (=LMG 29787T=RCAM 02728T). Three additional strains of the species are V5/5K, V5/5M and V5/13.