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Based on 20 autosomal microsatellite markers, the results of analysis of the genetic structure and spatial autocorrelation in the populations of grey wolf (Canis lupus Linnaeus, 1758) on the territory of the Russian Federation and the Republic of Kazakhstan are described. Using molecular markers, hidden genealogical patterns are revealed, which have the most pronounced effect on distances up to 150 km but may be traced over much wider spaces, up to 700–1600 km. Our research has shown that identification and exclusion of closely related genotypes has limited effect on the results of the analysis of intrapopulation genetic diversity. Meanwhile, this procedure is recommended for a more adequate assessment of the population structure since it allows for streamlining some statistical approaches. Thus, the obtained data demonstrate the integrating effect of natal migration, which works against the differential effect of philopatry. Finally, it is also shown that the exclusion of closely related individuals can lead to lower values of genetic distances between populations. [ABSTRACT FROM AUTHOR]

Two species of East Asian five-needle pines of the section Strobus, Korean pine, Pinus koraiensis Sieb. et Zucc. and Armand's pine, Pinus armandii Franch are traditionally placed in two different subsections, Cembrae and Strobi, respectively.However, data on similarity of these pines in needle anatomy and pollen structure are reported. By means of starch gel electrophoresis we studied allozyme variation within populations of these species and between the two species. Among 30 studied loci we selected 17 polymorphic loci with reliable interpretation of allele correspondencebetween species. Allozyme patterns were similar in both species with respect to number of loci and predominant alleles at most loci. Intra-specific differentiation among 12 populations of Pinus koraiensis from Russia, Northeast China and South Korea was low, FST (proportion of among population variation in total variation) was as low as 0.019, DN (Nei's genetic distance) varied between 0.001 and 0.006. This genetic uniformity can be explained by a common gene pool of this species within virtually continuous range under similar ecological conditions. Common vectors of selection, natural gene flow through pollen and seed dispersal as well as mixing of genetic material between regions by humans (at least in Russian forestry practice) are putative factors responsible for low differentiation in Korean stone pine. We estimated the same parameters in Pinus armandii from continental China (Guizhou, Shaanxi, Shenyan) where its range is highly fragmented, and found differentiation in this species to be much more pronounced (FST = 0.067, DN = 0.011-0.029). By adding a sample from Taiwan both FST (0.338) and DN (up to 0.232) were substantially higher indicating high level of differences between continental and Taiwanese P. armandii, putatively due to longer isolation time. Maximal contribution was fromloci Adh-1, Got-3, Mdh-1, 6-Pgd-3, Pgm-2 and Skdh-2. Differences were mainly caused by allelic frequencies but not by presence/absence of particular alleles, therefore observed allozyme patterns are not evidence for full species' status of Taiwan variety. In the two species, P.koraiensis and P. armandii, electrophoresis showed the same number of activity zones. In a combined set of P. koraiensis and P. armandiisamples, the level of interspecific differences was high (FST =0.477, DN =0.407÷0.457). Species had no common alleles or very little overlapping in allelic profiles at loci Adh-1, Got-2, and Pgm-1. Out of 61 alleles 35 were shared between species and 26 were found in only one of them. Cluster (UPGMA) and multivariate (PCA) analyses of allozyme variation showed good differentiation within P.armandii, and poor differentiation within P. koraiensis.

We examine the diversity of six microsatellite loci and partial RAG1 exon of “barabensis” and “pseudogriseus” karyoforms in Cricetulus barabensis sensu lato species complex. A total of 435 specimens from 68 localities ranging from Altai to the Far East are investigated. The results of the population structure analysis (factor analysis and NJ tree based on Nei genetic distances) support subdivision into two well-differentiated clusters corresponding to the two karyoforms. These karyoforms are also well differentiated by the level of microsatellite variability. In several “barabensis” specimens, we found microsatellite alleles that are common in “pseudogriseus” populations but are otherwise absent in “barabensis.” Most of these specimens originate from a single population in one of the zones of potential contact between karyoforms, Kharkhorin in Central Mongolia. These molecular results are consistent with previously published karyological data in suggesting that rare hybridization events between the two chromosomal races occur in nature.

Aspen Populus tremula L. (Salicaceae) is the fast-growing tree species of environmental and economic value. Aspen is capable of reproduction by both seeds and vegetative means, forming root sprouts. In an adult stand, identification of ramets of one clone among the trees of seed origin based on their morphology is difficult. A panel of 14 microsatellite loci developed for individual identification of aspen was applied for the clonal structure analysis in four natural aspen stands of the European part of Russia: Moscow and Voronezh oblasts, the Mari-El Republic, and the Republic of Tatarstan. In 52 trees from the Moscow sample, 41 multilocus genotypes were identified; in the Voronezh sample, among 30 individuals, 25 different genotypes were detected; and in the sample from Mari-El, 32 trees were represented by 13 genotypes. In the stand from Sabinsky Forestry, Tatarstan, all of the examined 29 trees were represented by a single genotype. The ancestral tree carrier of this genotype which was the most heterozygous (0.929) among all studied aspen individuals (sample mean, 0.598) obviously has spread over a large territory during several cutting and reproduction cycles, currently occupying the area of 2.2 ha. For aspen, usually suffering from Aspen trunk rot, such high viability is evidence of resistance to the main pathogens. The revealed superclone deserves further study with karyological methods and flow cytometry to determine ploidy level and analysis of the growth rate and the quality of wood for possible use in plantation forest production.

The Siberian stone pine (Pinus sibirica Du Tour) is one of the main forest-forming coniferous species of the boreal ecosystems of Western Siberia. We used the isozyme method to analyze 11 ecotypes representing the latitudinal and longitudinal profiles within the species range, including samples from the geographic boundaries of the distribution. The genetic structure of the ecotypes is described on the basis of the variability for 26 isozyme loci encoding for 16 enzyme systems. The greatest genetic diversity was observed in the taiga ecotypes in the central part of the studied area, while the ecotypes along the species range boundaries were shown to be genetically depauperized. Approximately 8.1% of the observed genetic diversity is attributed to differences between the studied ecotypes. We detected high levels of genetic diversity for the Fest_2, Pgm_1, Sod_4, and a few otherloci, as well as a correlation between allele frequencies and geographical locations of the populations. The results of multivariate analysis of allelic frequencies as well as cluster analysis allowed us to discriminate three major groups of ecotypes: north-eastern, central and south-western. In view of our results, we compare two hypotheses: one which attributes the spatial distribution of genetic variations to the selectivity for some of the polymorphic allozyme loci, and the other based in the history of the formation of the range of the Siberian stone pine.

The genetic diversity of SSR markers was studied in six populations of Siberian larch, two populations of Gmelin larch, and four populations of Cajander larch. Seven pairs of nuclear microsatellite loci were used for this analysis. 103 allelic variants were detected in 365 individuals of three species of larch. According to the AMOVA results, the variability proportion that characterizes the differences between three Larix species was twice as higher (15%) than the proportion that accounts for among-population differences within the species (6%). The differentiation of the populations of three species of larch based on SSR markers exceeded 12% (FsT = 0.121). A significant correlation of the genetic distances with the geographic distances between populations was found (r = 0.835, P = 0.01).

Siberian dwarf pine, or Japanese stone pine, Pinus pumila (Pall.) Regel is widespread in eastern Siberia and the Russian Far East; the species is bird-dispersed and has a unique spreading or shrub living form. A mixed mating system (predominant outcrossing with self-pollination and matings of close relatives) leads to the formation of partly inbred progenies in P. pumila, as in the majority of other conifers. The question arises as to whether inbred individuals persist in the reproductive part of a population, which can have negative genetic consequences. The ADH, FDH, FEST, GDH, GOT, IDH, LAP, MNR, MDH, PEPCA, 6-PGD, PGI, PGM, SKDH, and SOD isozyme systems were analyzed to study the dynamics of heterozygosity in four P. pumila natural populations from the Pacific region, optimal for the species. Samples were collected in northern Koryakia, southern Kamchatka (two samples), and the Kunashir Island (Kurils). Wright's fixation index was used to estimate the level of inbreeding in embryos from dormant seeds resulting from free pollination and in maternal plants. A substantial level of inbreeding (F(IS) = 0.124-0.342) was observed in the embryo samples but not in three out of the four adult samples. The inbreeding level at the reproductive age was high only in the sample from Koryakia, which was explained by a relatively young age of plants in the population frequently affected by fires. A general increase in heterozygosity, characteristic of other conifers as well, was attributed to elimination of inbred progenies and by balancing selection for heterozygotes, which is a key factor maintaining allozyme polymorphism in populations.

A system of urban ecological genetic monitoring has been developed for the first time in Russia and in the world. As a model, two species of brown frogs, Rana arvalis Nilss. and R. temporaria L. (16 populations, seven isozyme loci) were used. An evaluation of the gene pool state in urban frog populations has shown that the diversity in the Moscow frog populations was lower than that in natural populations (up to 80 and 50% in respectively R. anvalis and R. temporaria). Mean heterozygosities per locus were higher in large natural populations than in small urban isolates: in R. arvalis, these values were 0.16 and 0.06; in R. temporaria, 0.34 and 0.18, respectively. The number of polymorphic loci was also higher in natural populations than in the urban ones: 4 versus 2 in R. arvalis and 5 versus 4 in R. temporaria. Using superoxiddismutase as an example, fixation of different alleles of the same locus in different small isolates was shown. The gene pool condition of all but one urban populations of brown frog was evaluated as unsatisfactory, and that of the R. arvalis populations, as critical. These changes of the gene pool are explained mainly by gene drift accompanied by inbreeding, which was caused by human-induced fragmentation of the range and a decrease in population size of the species. The results of this study was employed in the development of the Moscow governmental program on restoration of the gene pools of vanishing animal species on specially protected natural urban territories. The series of works on long-term monitoring and assessment of the state of natural populations of model species in anthropogenic landscapes of Moscow and Moscow district has laid a foundation for a new branch of science, gene urbanology.

Swiss stone pine Pinus cembra L. is a species with fragmented range, occurring in the Alpine-East Carpathian mountain system. Seeds of P. cembra are dispersed by nutcrackers, which offers potential possibilities for gene exchange among populations. Using isozyme analysis, we have examined five samples from two parts of the Swiss stone pine range: the Alps (Switzerland and Austria) and the Carpathians (two samples from the northern macroslope of the Gorgany Ridge, Eastern Carpathians, Ivano-Frankovsk oblast and one sample from Trans-Carpathian oblast of Ukraine). The allele frequencies of 30 isozyme loci, coding for enzymes ADH, FDH, FEST, GDH, GOT, IDH, LAP, MNR, MDH, PEPCA, 6PGD, PGI, PGM, SDH, SKDH, SOD, were analyzed using cluster analysis and methods of principal components. Two clusters, corresponding to the isolated Alpine and Carpathian parts of the range, were found. The main contribution to these differences were made by loci Adh-1, Adh-2, Fest-2, Lap-3, Mdh-4, and Sod-4. The interpopulation differentiation proved to be somewhat higher than that typical for pines (F(ST) = 7.4%), but within the limits characteristic for taxonomically close species. Thus, isolation of the populations did not lead to their marked differentiation, which may be explained by gene flow and balancing selection, which equalizes gene frequencies across the fragmented species area. Interlocus F(ST) heterogeneity (from 0.003 to 0.173) suggests adaptive significance of some of the allozyme polymorphisms or linkage of some loci with adaptive genes. The Carpathian populations were shown to have higher gene diversity than the Alpine ones (expected heterozygosities 0.095-0.114 and 0.060-0.080, respectively). A deficit of heterozygotes (as compared to the Hardy-Weinberg proportions), observed in the embryo sample, was probably explained by inbreeding. The reduction in the area of Carpathian pine forests in Holocene, caused by the global climatic changes and the anthropogenic impact, is hazardous for the gene pool of the species. The maintenance of genetic uniqueness of both Carpathian populations of P. cembra in general, and individual stands in particular, requires special measures for protection of Swiss stone pine in the Eastern Carpathians.

Using shrub snail Bradybaena fruticum Müll (20 populations) as a model, we were the first in Russia and in the world to develop a system of urban ecological genetic monitoring of the gene pool of an invertebrate species. The results of isozyme polymorphism studies in shrub snail populations showed a dramatic (up to 70%) reduction in genetic diversity in small isolates from the urbanized environment as compared to large natural populations. In urban populations, genetic diversity parameters were demonstrated to be lower than in natural ones: the mean heterozygosity per locus was reduced up to 0.08 (0.15-0.20 in natural populations); the mean allele number, to 1.9 (2.7 in natural populations); and the number of polymorphic loci, to four, i.e., 2.2-fold (nine in natural populations). In Moscow district, the number of polymorphic loci also decreased to five in the population subjected to anthropogenic pressure. The changes in the population gene pool (as shown by the number of polymorphic loci) were different in Moscow and Moscow district. The percentage of populations with the number of polymorphic loci as low as four to six was 76.9 in Moscow and 23.1% in Moscow district. The gene pool quality of 80% of the urban snail populations was estimated as unsatisfactory, and in half of them, as critical. The main reason for these changes seems to be genetic drift accompanied by inbreeding, caused by fragmentation of the range and reduction in the abundance of populations of the species, due to the anthropogenic pressure. The results of the study were employed in the program of the Moscow government for restoring the gene pools of endangered animals species on the preserved territories of the city.

Allozyme variation in natural populations of basidiomycetes fungus Pleurotus ostreatus (88 individuals) from three regions of central Russia was studied. The species was shown to have 92.86% of polymorphic allozyme loci and expected heterozygosity He = 0.49. The mean number of alleles per locus was 3.5. The genetic differences among populations were supported by F-statistics (FST = 0.750). The low level of inbreeding (FIS = 0.018) suggests that the P. ostreatus populations are panmictic, and the main reproduction mode involves basidiospores dispersing at long distances. Using cluster analysis, geographically isolated populations and intersterile groups were differentiated within the complex P. ostreatus species.

Analysis of commercial strains of two edible mushrooms, Pleurotus ostreatus and Agaricus bisporus, using PCR and isozyme electrophoresis techniques allowed us to differentiate groups of genetically similar and distant strains. Among the commercial strains of P. ostreatus, the level of genetic variation was higher suggesting a broader genetic basis employed in breeding of this mushroom. The cultivars and hybrids of, A. bisporus, showed a higher level of homology. The isozyme markers (nonspecific esterase, leucinaminopeptidase, and phosphoglucoisomerase) are recommended for identification of the commercial strains of edible mushrooms.