The article suggests the original method for ecological suitability evaluation of the habitat for local populations of Vavilovia formosa (Steven) Al.Fed. in the Republic of Dagestan (Northern Caucasus). Particular attention is paid to the development of methodological issues of studying the state of rare species local populations. The improvement of the methodology for constructing predictive ranges and the expert evaluation procedure of the obtained results is illustrated by the example of the eastern range fragment detailed analysis of V. formosa in the North Caucasus. The research has identified the limiting factors complexes that determine the patterns of local populations spatial distribution on slopes of different exposure, steepness and absolute height in models of different spatial resolution. For the first time, the translation of the initial probability values into a new habitat suitability index has been proposed and implemented. This index allows working with probability values in the form of natural numbers, which greatly facilitated their comparison. Evaluation of predictors by their contribution to predictive models of different spatial resolution convincingly shows the need to build a full-term series of models that will include the entire range of available spatial resolutions – from 1/6 to 1/120 degrees. Only such an approach will allow the researcher to obtain the most meaningful information base for a correct assessment of the contribution of predictors, taking into account the levels of spatial climate data generalization. A promising direction for further research can be considered dis-cretization of predictive values of the habitat suitability index by dividing small climatic areas into even smaller ones, for example, with a dimension of 1/360 degrees in longitude and latitude.

The high mountain perennial pea Vavilovia formosa (Steven) Fed., commonly known as beautiful vavilovia, is a rare species of the high alpine flora of Armenia bound to scree habitats. Populations of V. formosa in Armenia, as well as in other regions of the world, are endangered and need to be studied and preserved both in natural and ex situ conditions. Studies of the relic alpine pea V. formosa, unique in its bioecological features and beauty, were initiated in Armenia in the20-30s of the last century and continue at the present time. Thanks to numerous expeditions tothe hard-to-reach habitats of Vavilovia in the Gegham and Syunik highlands, Zangezur ridge, manyresearchers of the Armenian flora collected herbarium material, conducted ecological and cоenoticstudies, analyzed karyology on the basis of local populations, repeatedly conducted experimentson growing Vavilovia in laboratory conditions and in botanical gardens of Armenia. As a result of fieldobservations, information was obtained on the status of Vavilovia populations in different regionsof the republic, in natural conditions, floristic and coenotic composition of habitats at the Aknasarand Sevkatar (Sevsar) mountain peaks, on slopes near the Aknalich lake, of the Mets Ishkhansarmountain, and near the Ughtasar mountain peak. One of the main natural threats leading to thechange in and shrinking of Vavilovia populations is the overgrowth of mobile scree habitats with turf-forming grasses, which creates conditions for the development of mountain-meadow vegetationon screes. The ex situ conservation strategy for V. formosa is one of the possible ways to evaluateits adaptive capacity to environmental changes, as well as to conserve it and use in basic andapplied research. An experiment on the introduction and cultivation of Vavilovia conducted by theYerevan and Sevan Botanical Gardens of Armenia has yielded some data on biomorphological andecophysiological features in ex situ conditions.

Priming of seeds increases uniformly germination rate in cultivated and wild species as well, it also shortens germination and emergence time. Beautiful vavilovia, Vavilovia formasa (Stev.) Al. Fed. is a relict and endangered legume crop which is the closest relatives of the genus Pisum L. In beautiful vavilovia, seed priming has ignored due to insufficient seed production of the plant. In the present report, three seed priming including hydro-priming, glycerol (%1) and potassium chloride (KCl) of 5% were compared to control (no-priming application). Findings have shown that seed priming with glycerol significantly accelerated germination time in beautiful vavilovia. Despite better germination time with hydro-priming after glycerol, hydro-priming had almost similar to control. Germination was stalled off at KCl treatment indicating that beautiful vavilovia could be salt susceptible. Priming with glycerol can be suggested as the fast and reliable germination of seeds of relict beautiful vavilovia.

Spatial Monitoring of the Modern Environmental Situation in Localities of Vavilovia formosa (Fabaceae) Based on Predictive Climatic Modeling

Background. Plant chloroplast genome have conservative structure, but its nucleotide sequence is polymorphous due to which cpDNA fragments are often used in taxonomic and phylogenetic studies. Despite the widespread distribution and use of Fabeae species, mainly peas (Pisum), data on the intraspecific diversity of cpDNA fragments are almost absent. The aim of the work was to analyze the intraspecific variability of three cpDNA spacers in Pisum.
 Materials and methods. As a result of the work, intergenic spacers trnYtrnT, trnHpsbA and rpoBtrnC in 38 accessions of the Pisum and related Fabeae species were sequenced. Despite the fact that the selected chloroplast fragments are generally considered to be sufficiently variable in plants and are often used for phylogenetic studies, Pisum accessions have been found to have no intraspecific differences in two of the three spacers sequences analyzed.
 Results and conclusion. A total 97 SNPs were detected in Pisum accessions, seven of them distinguished P. sativum from P. fulvum. The most variable of the analyzed fragments was the intergenic spacer rpoBtrnC. Based on rpoBtrnC sequence 17 haplotypes in P. sativum and four haplotypes in P. fulvum were revealed. The cpDNA sequencing data were used for a phylogenetic analysis. On the obtained tree Vavilovia formosa accession formed a separate branch from pea accessions. All Pisum accessions fall in one cluster, split into distinct P. sativum and P. fulvum subclusters (BI = 99%).

The natural conditions of highlands of the northern part of the Central and Eastern Caucasus are described. Three high-rise belts – alpine, subniveal, niveal – were identified within the studied territory. Features of vegetation cover of these belts are emphasized: fragmented vegetation in the form of separate rock-talus groups and microgroups; sparseness and simplification of its structure, participation of mosses and lichens and huge amounts of types of underlying high-rise belts. According to the high-rise distribution in the subniveal belt, the following groups of species were identified: 1) found at 3100 m above sea level, involved in creating fragmentary carpet-like alpine meadows at the bottom of the belt; 2) found mainly at 3000-3300 m above sea level; 3) living mainly in the range from 3000 to 3600-3700 m above sea level. There is a prevalence of petrophytes, high percentage of endemicity in the subniveal belt (Pseudobetckea, Pseudovesicaria, Didymophysa, Trigonocarium, Eunomia, Vavilovia, etc.). The paper lists the most characteristic families of monocotyledones (Poaceae, Cyperaceae, Juncaceae and Liliaceae), the most characteristic families of dycotyledonae (Asteraceae, Brassicaceae, Caryophyllaceae, Fabaceae, Saxifragaceae, Rosaceae, Scrophulariaceae, Campanulaceae) and species growing on different substrates – taluses of clay shale, moving taluses, rocks, rocky and crushed slopes, moraines, alpine lawns, common and rare species (Galium rugosum, Jurinea filicifolia, Jurinella moschus, Vavilovia formosa), as well as Veronica bogosensis, Cerastium multiflorum Delphinium caucasicum endemic for the eastern part of the studied area – Erysimum subnivale, Ranunculus tebulossicus, Pyrettrum aromatucum, Pseudobetckea caucasicqa, Sedum stevenianum, etc.

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.

Jedan usputni ali i podrobni pogled na genetiku hibridnog potomstva vrsta Pisum sativum i 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.

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.

Wild perennial pea Vavilovia formosa is a relic and an endangered species of the upper alpine flora of Armenia, specialized to scree habitats. The populations of V. formosa in Armenia, as well as in other sites of the world, are in danger of extinction and need both in situ and ex situ complementary investigation and conservation. Present article contains information on V. formosa Aknasar mountain population studies. Data on natural conditions, flora composition, ecological and coenotic characteristic of the habitat and bio-ecological features of V. formosa are given. The present state of Vavilovia population of mountain Aknasar is satisfactory. The activation of seed reproduction along with vegetative propagation is observed. One of the natural threats leading to the change and reduction of Vavilovia population was estimated to be the overgrowing of mobile scree habitats with turf-forming grasses, which creates conditions for the mountain-meadow vegetation development. V. formosa ex situ conservation strategy is one of the possible ways of its adaptive capacity estimation to environment changes as well as for its saving and using in basic and applied researches. An introductory experiment on the cultivation of Vavilovia in the Yerevan and Sevan Botanical Gardens has started, and some data on bio-morphological and eco-physiological peculiarities under ex situ conditions were obtained.

Comparative sequence analysis of symbiotic genes (nodA, nodC, nodD, nifH), which are elements of accessory component of the rhizobial genome, demonstrated that the strains of Rhizobium leguminosarum bv. viciae, isolated from the nodules of a relic legume, Vavilovia formosa, the closest relative of hypothetical common ancestor of the tribe Fabeae, represented a group separated from the strains of R. leguminosarum bv. viciae, isolated from other representatives of this tribe (Vicia, Lathyrus, Pisum, Lens). No isolation was observed relative to the genes representing the core component of the rhizobial genome (16S rDNA, ITS, glnII) or relative to host specificity of the rhizobia. The data obtained suggest that sequence divergence of symbiotic genes marks the initial stage of sympatric speciation, which can be classified as the isolation of the relic “vaviloviae” symbiotype, a possible evolutionary precursor of the “viciae” biotype.

Many bacteria belonging to the order Rhizobiales have fixNOQP genes which encode cytochrome oxidase with high affinity to oxygen required for oxidative phosphorylation in microaerophilic conditions. There is one copy of the identified fixNOQP operon in ancestral forms of rhizobia (Bradyrhizobium), as well as in their putative evolutionary predecessors (bacteria related to Rhodopseudomonas). At the same time, forms deeply specialized in symbiosis (Rhizobium leguminosarum, Sinorhizobium meliloti) have multiple (2–3) copies, some of them have a high similarity (>90%) to fixNOQP genes of Bradyrhizobium and Rhodopseudomonas, and others have only 30–50% similarity. Two divergent copies fixNOQP are detected in Tardiphaga, which is a representative of the Bradyrhizobiaceae family, lacking the ability to fix N2 (lack of nif genes encoding the synthesis of nitrogenase) and to induce the formation of nodules on legumes roots (lack of nod genes encoding the synthesis of signal Nod factors activating symbiosis development). The presence of Tardiphaga in nodule bacterial communities from a range of legumes, including Vavilovia formosa (relic representative of the tribe Fabeae, for which R. leguminosarum bv. viciae is the main microsymbiont), suggests that the ancestral gene duplication and subsequent divergence of fixNOQP operon in bacteria related to Tardiphaga opened the possibility of wide dissemination of functionally different copies of this cluster among symbiotically active forms of Rhizobiales. It is possible that the acquisition of fixNOQP genes determines adaptation of bacteria to microaerophilic niches not only in plants nodules but also in their environment (the rhizosphere, rhizoplane, internal portions of soil aggregates).

Vavilovia formosa is one of five genera in tribe Fabeae, (Fabaceae, Leguminosae) with close phylogenetic relationships to Pisum. It grows in subalpine and alpine levels in Armenia, Azerbaijan, Georgia, Iran, Iraq, Lebanon, Russia and Turkey and is recognized as an endangered and protected plant. This study was conducted to reveal its intraspecific variability, as well as to predict the past, extant and future species distribution range. We analysed 51 accessions with common phylogenetic markers (trnF-trnL, trnS-trnG, matK, rbcL, psbA-trnH and ITS). These represent in total up to 2551 bp of chloroplast and 664 bp of nuclear sequences per sample. Two populations from Turkey and Armenia were analysed for genetic diversity by AFLP. Leaf morphometry was conducted on 1457 leaflets from 43 specimens. Extracted bioclimatic parameters were used for niche-modelling approach. Analysis of cpDNA revealed two haplotypes, 12 samples from Armenia, Daghestan, Nakhichevan and Iran belonged to H1 group, while 39 samples of all Turkish and part of Armenian were in H2 group. The mean intrapopulation diversity based on AFLP was low (H E = 0.088) indicating limited outcrossing rate. A significantly positive correlation between geographical latitude and leaf area ( $$\rho$$ = 0.527, p < 0.05) was found. Niche modelling has shown temporal variation of predicted occurrence across the projected time periods. Vavilovia formosa has suffered a range reduction following climate warming after last glacial maximum, which classify this species as cold-adapted among the Fabeae species as well as a glacial relict.

Beautiful vavilovia, the closest species to the common now extinct ancestor of the whole tribe Fabeae holds significant taxonomical interest and also for breeding within this group of species, which includes the most cultivated leguminous pulses in the world. In spite of this, vavilovia has attracted very scarce research to date and is in danger of complete extinction. Thus, as a part of the research carried out by an informal international group of researchers from various countries, we report here various experiments for the development and exploitation of a range of biotechnology tools for vavilovia, ranging from standard in vitro propagation, to plant regeneration from explant-derived callus, and also from protoplasts. Plants were successfully recovered following propagation from nodes, and by regeneration through organogenesis from callus derived from internodes (which provided the best responses) and leaves. Also, protoplasts were isolated from leaves and stems from in vitro shoots and from callus derived from these two explants, with the latter undergoing sustained division. Subsequently, protoplasts isolated from internode callus proliferated and also underwent organogenesis coupled with whole plant recovery at a low frequency, while protoplasts from leaf callus origin followed both organogenesis and embryogenesis simultaneously but failed to yield viable plants. Flow cytometry assessments permitted to ascertain the genetic fidelity of both propagated and regenerated plants irrespectively of the source tissue from which they were derived (i.e., either callus from explants or from protoplasts). Finally, flow cytometry also permitted us to provide the first record on the relative nuclear DNA content and genome size for Vavilovia formosa.

Developing biotechnology tools for ‘beautiful’ vavilovia (Vavilovia formosa), a legume crop wild relative with taxonomic and agronomic potential

Beautiful Vavilovia [Vavilovia formosa (Stev.) Fed.] is a perennial legume of great importance for the evolution and taxonomy of the tribe Fabeae Rchb. The abundant data on its collection sites on the Caucasus Mountains, made during the past two centuries, were collated and analysed. The studied area covers the mountain ranges of both the Greater and Lesser Caucasus in Russian Federation, Georgia, Armenia and Azerbaijan, except the ranges in north-eastern and eastern Turkey and north-western Iran. The presented data are the results of an analysis of the herbarium collections of various institutions and available literature resources, providing novel information about 110 collection points. The analysed data allowed to specify the borders of the area in the region, as well as describe the features of the high-altitude distribution of V. formosa within the studied area, revealing the lower and upper altitude ranges as between 2021 and 3500 m above sea level. The results also indicate a reduction of the number of its populations, as well as the necessity and urgency of monitoring and protecting all the known locations of this highly endangered species.

Eleven extra-slow-growing strains were isolated from nodules of the relict legume Vavilovia formosa growing in North Ossetia (Caucasus) and Armenia. All isolates formed a single rrs cluster together with the type strain Tardiphaga robiniae LMG 26467(T), while the sequencing of the 16S-23S rDNA intergenic region (ITS) and housekeeping genes glnII, atpD, dnaK, gyrB, recA and rpoB divided them into three groups. North Ossetian isolates (in contrast to the Armenian ones) were clustered separately from the type strain LMG 26467(T). However, all isolates were classified as T. robiniae because the DNA-DNA relatedness between them and the type strain LMG 26467(T) was 69.6% minimum. Two symbiosis-related genes (nodM and nodT) were amplified in all isolated Tardiphaga strains. It was shown that the nodM gene phylogeny is similar to that of ITS and housekeeping genes. The presence of the other symbiosis-related genes in described Tardiphaga strains, which is recently described genus of rhizobia, as well as their ability to form nodules on any plants are under investigation.

The Gram-negative, rod-shaped slow-growing strains Vaf-17, Vaf-18(T) and Vaf-43 were isolated from the nodules of Vavilovia formosa plants growing in the hard-to-reach mountainous region of the North Ossetian State Natural Reserve (north Caucasus, Russian Federation). The sequencing of 16S rDNA (rrs), ITS region and five housekeeping genes (atpD, dnaK, recA, gyrB and rpoB) showed that the isolated strains were most closely related to the species Bosea lathyri (class Alphaproteobacteria, family Bradyrhizobiaceae) which was described for isolates from root nodules of Lathyrus latifolius. However the sequence similarity between the isolated strains and the type strain B. lathyri LMG 26379(T) for the ITS region was 90% and for the housekeeping genes it was ranged from 92 to 95%. All phylogenetic trees, except for the rrs-dendrogram showed that the isolates from V. formosa formed well-separated clusters within the Bosea group. Differences in phenotypic properties of the B. lathyri type strain and the isolates from V. formosa were studied using the microassay system GENIII MicroPlate BioLog. Whole-cell fatty acid analysis showed that the strains Vaf-17, Vaf-18(T) and Vaf-43 had notable amounts of C16:0 (4.8-6.0%), C16:0 3-OH (6.4-6.6%), C16:1 ω5c (8.8-9.0%), C17:0 cyclo (13.5-13.9%), C18:1 ω7c (43.4-45.4%), C19:0 cyclo ω8c (10.5-12.6%) and Summed Feature (SF) 3 (6.4-8.0%). The DNA-DNA relatedness between the strains Vaf-18(T) and B. lathyri LMG 26379(T) was 24.0%. On the basis of genotypic and phenotypic analysis a new species Bosea vaviloviae sp. nov. (type strain RCAM 02129(T)=LMG 28367(T)=Vaf-18(T)) is proposed.

Erratum to: Beauty will save the world, but will the world save beauty? The case of the highly endangered Vavilovia formosa (Stev.) Fed.