Vavilovia formosa rhizobia symbionts belong to Rhizobium leguminosarum bv. viciae species, but form a separate group within it

Ecological isolation, group separation of hkg and sym genes, along with the results of the sterile tube test demonstrate that symbionts of V. formosa belong to R. leguminosarum bv. viciae species, but form a separate group within it.

The Vavilov Institute’s (VIR) contribution to the survey and study of Vavilovia formosa (Fabaceae)

In the 1960s–1980s, the Vavilov Institute of Plant Industry, known worldwide as VIR, organized a substantial series of studies dedicated to a Caucasian endemic, the Pliocene relic Vavilovia formosa (Fabeae: Fabaceae). Those investigations are little known to the scientific community, although, in fact, the priority right for integrated research into this plant belongs to VIR. As a result of the cycle of studies, the first ideas about the biology and ecology of the species, the manner of its reproduction, its intraspecific diversity, the degree of crossability with other members of the tribe, its karyotype and anatomy were developed. The plants of V. formosa are not reproduced ex situ and their germplasm is absent in the world’s gene banks. Therefore, collecting V. formosa plants in their hard-to-reach habitats in the mountains is always crucial for research purposes, so a number of collecting expeditions were launched to the Greater Caucasus. The data thus obtained are discussed in the context of modern achievements in world science regarding this species.

The plastid and mitochondrial genomes of Vavilovia Formosa (Stev.) Fed. and the phylogeny of related legume genera

The plastid and mitochondrial genomes of Vavilovia formosa (Stev.) Fed. were assembled on the base of the data of high-throughput sequencing of DNA isolated from a sample from North Osetia, Russia, using Illumina and PacBio platforms. The long PacBio reads were sufficient for reliable assembling organellar genomes while the short Illumina reads obtained from total DNA were unacceptable for this purpose because of substantial contamination by nuclear sequences. The organellar genomes were circular DNA molecules; the genome of mitochondria was represented by two circular chromosomes. A phylogenetic analysis on the basis of plastid genomes available in public databases was performed for some representatives of the tribes Fabeae, Trifolieae and Cicereae. As was expected, the V. formosa branch proved to be sister to the Pisum branch, and the tribe Fabeae was monophyletic. The position of Trifolium L. appeared sensitive to the phylogeny reconstruction method, either clustering with Fabeae or with the genera Medicago L., Trigonella L. and Melilotus Mill., but the internodes between successive divergences were short in all cases, suggesting that the radiation of Trifolium, other Trifolieae and Fabeae was fast, occurring within a small time interval as compared to further evolution of these lineages. The data on the relatedness of the plastid genomes of Trifolium and Fabeae correlate with the similarity of N2-fixing symbionts in these legumes represented by Rhizobium leguminosarum biovars trifolii and viciae, while the symbionts of Medicago, Melilotus and Trigonella belong to the Sinorhizobium meliloti and S. medicae species, which are distant from Rhizobium.

Search for Ancestral Features in Genomes of Rhizobium leguminosarum bv. viciae Strains Isolated from the Relict Legume Vavilovia formosa

Vavilovia formosa is a relict leguminous plant growing in hard-to-reach habitats in the rocky highlands of the Caucasus and Middle East, and it is considered as the putative closest living relative of the last common ancestor (LCA) of the Fabeae tribe. Symbionts of Vavilovia belonging to Rhizobium leguminosarum bv. viciae compose a discrete group that differs from the other strains, especially in the nucleotide sequences of the symbiotically specialised (sym) genes. Comparison of the genomes of Vavilovia strains with the reference group composed of R. leguminosarum bv. viciae strains isolated from Pisum and Vicia demonstrated that the vavilovia strains have a set of genomic features, probably indicating the important stages of microevolution of the symbiotic system. Specifically, symbionts of Vavilovia (considered as an ancestral group) demonstrated a scattered arrangement of sym genes (>90 kb cluster on pSym), with the location of nodT gene outside of the other nod operons, the presence of nodX and fixW, and the absence of chromosomal fixNOPQ copies. In contrast, the reference (derived) group harboured sym genes as a compact cluster (<60 kb) on a single pSym, lacking nodX and fixW, with nodT between nodN and nodO, and possessing chromosomal fixNOPQ copies. The TOM strain, obtained from nodules of the primitive “Afghan” peas, occupied an intermediate position because it has the chromosomal fixNOPQ copy, while the other features, the most important of which is presence of nodX and fixW, were similar to the Vavilovia strains. We suggest that genome evolution from the ancestral to the derived R. leguminosarum bv. viciae groups follows the “gain-and-loss of sym genes” and the “compaction of sym cluster” strategies, which are common for the macro-evolutionary and micro-evolutionary processes. The revealed genomic features are in concordance with a relict status of the vavilovia strains, indicating that V. formosa coexists with ancestral microsymbionts, which are presumably close to the LCA of R. leguminosarum bv. viciae.

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.