A. A. Vergun

Nucleotide sequences of the microsatellite locus Du215 (arm) allelic variants in the parthenospecies Darevskia armeniaca (Lacertidae)

Using monolocus PCR analysis with the pairs of primers designed for the Du215 locus of Darevskia unisexualis, allelic polymorphism at the orthologous locus in the populations of the related parthenospecies D. armeniaca was investigated. It was demonstrated that Du215 (arm) locus was polymorphic and in the populations of parthenospecies D. armeniaca (n = 127) represented by at least three allelic variants, differing from each other by the size and composition of microsatellite cluster, and by single nucleotide substitutions in flanking DNA. Unlike the Du215 locus, Du215 (arm) was shown contain not only GATA, but also (GACA) repeats, which were absent in D. unisexualis. Thus, in this study, the data on the molecular nature of allelic polymorphism at one of the microsatellite loci of the parthenospecies D. armeniaca were reported.

Clonal Diversity and Clone Formation in the Parthenogenetic Caucasian Rock Lizard Darevskia dahli

The all-female Caucasian rock lizard species Darevskia dahli and other parthenogenetic species of this genus reproduce normally via true parthenogenesis. Previously, the genetic diversity of this species was analyzed using allozymes, mitochondrial DNA, and DNA fingerprint markers. In the present study, variation at three microsatellite loci was studied in 111 specimens of D. dahli from five populations from Armenia, and new information regarding clonal diversity and clone formation in D. dahli was obtained that suggests a multiple hybridization origin. All individuals but one were heterozygous at the loci studied. Based on specific allele combinations, 11 genotypes were identified among the individuals studied. Individuals with the same genotypes formed distinct clonal lineages: one major clone was represented by 72 individuals, an intermediate clone was represented by 21 individuals, and nine other clones were rare and represented by one or several individuals. A new approach based on the detection and comparison of genotype-specific markers formed by combinations of parental-specific markers was developed and used to identify at least three hybridization founder events that resulted in the initial formation of one major and two rare clones. All other clones, including the intermediate and seven rare clones, probably arose through postformation microsatellite mutations of the major clone. This approach can be used to identify hybridization founder events and to study clone formation in other unisexual taxa.

Molecular structure of allelic variants of microsatellite loci Du281 and Du47 in unisexual and bisexual lizard species of the genus Darevskia

Experimental data on the molecular structure and variability of microsatellite loci in unisexual and bisexual lizard species of the genus Darevskia were analyzed. The allelic variants of Du281 and Du47 were found to differ in the number of monomers, the structure of microsatellite clusters, and point mutations in these clusters and flanking DNA. Interspecific comparison of alleles of these loci revealed both variable regions in the microsatellite clusters and allele-specific evolutionarily conserved nucleotide groups. In general, the results of comparative structural analysis of allelic variants testify to a high genetic similarity of the unisexual and bisexual lizard species studied and reveals the characteristic features of their interspecies variability.

Molecular genetic characteristics of the allelic variants of microsatellite loci Du281, Du215, and Du323 in parthenogenetic lizards Darevskia rostombekovi (Fam. Lacertidae)

Assessment of genetic diversity of unisexual (parthenogenetic) species of vertebrates is among the major objectives of research in these species. Various nuclear or mitochondrial genome markers can be used for such an assessment. Microsatellite DNAs are among the most efficient genetic markers, since the mutation rate in these fragments is high. Identification and characterization of such markers are the basic stages of genetic research in parthenogenetic species. Allelic polymorphism of three microsatellite loci in populations of the parthenogenetic species Darevskia rostombekovi (n = 42) and bisexual parent species D. raddei (n = 6) and D. portschinskii (n = 6) has been assessed by locus-specific PCR for the first time. All representatives of the parthenogenetic species D. rostombekovi used in the present study turned out to be heterozygous. The number of alleles of the different loci ranged from two to five in the populations investigated. The nucleotide sequence of the allelic variants of the loci investigated has been determined. The differences between the alleles were apparently related to variation in the structure of microsatellite clusters and single-nucleotide substitutions in DNA fragments located in the vicinity of the clusters at fixed distances from the latter. Structural variants of the alleles formed allele-specific haplotype markers that were inherited from the bisexual parent species. The origin (inheritance from the maternal or paternal species) has been determined for each allele of the parthenogenetic species. The distribution, frequency of occurrence, and pattern of combination of the alleles of microsatellite loci in D. rostombekovi populations have been characterized; these features determined the identity of each population. The data obtained can be used for assessment of the clonal diversity of the parthenogenetic species D. rostombekovi and the identification of a possible scenario of the emergence of the diversity in the populations.

Molecular and genetic characterization of the allelic variants of Du215, Du281, Du323, and Du47G microsatellite loci in parthenogenetic lizard Darevskia armeniaca (Lacertidae)

A key issue in the study of unisexual (parthenogenetic) vertebrate species is the determination of their genetic and clonal diversity. In pursuing this aim, various markers of nuclear and mitochondrial genomes can be used. The most effective genetic markers include microsatellite DNA, characterized by high variability. The development and characterization of such markers is a necessary step in the genetic studies of parthenogenetic species. In the present study, using locus-specific PCR, for the first time, an analysis of allelic polymorphism of four microsatellite loci is performed in the populations of parthenogenetic species Darevskia armeniaca. In the studied populations, allelic variants of each locus are identified, and the nucleotide sequences of each allele are determined. It is demonstrated that allele differences are associated with the variation in the structure of microsatellite clusters and single nucleotide substitutions at fixed distances in flanking DNA regions. Structural allele variations form haplotype markers that are specific to each allele and are inherited from their parental bisexual species. It is established which of the parental alleles of each locus were inherited by the parthenogenetic species. The characteristics of the distribution and frequency of the alleles of microsatellite loci in the populations of D. armeniaca determining specific features of each population are obtained. The observed heterozygosity of the populations at the studied loci and the mutation rates in genome regions, as well as Nei’s genetic distances between the studied populations, are determined, and the phylogenetic relationships between them are established.

The origin of multiple clones in the parthenogenetic lizard species Darevskia rostombekowi

The all-female Caucasian rock lizard Darevskia rostombekowi and other unisexual species of this genus reproduce normally via true parthenogenesis. Typically, diploid parthenogenetic reptiles exhibit some amount of clonal diversity. However, allozyme data from D. rostombekowi have suggested that this species consists of a single clone. Herein, we test this hypothesis by evaluating variation at three variable microsatellite loci for 42 specimens of D. rostombekowi from four populations in Armenia. Analyses based on single nucleotide polymorphisms of each locus reveal five genotypes or presumptive clones in this species. All individuals are heterozygous at the loci. The major clone occurs in 24 individuals and involves three populations. Four rare clones involve one or several individuals from one or two populations. Most variation owes to parent-specific single nucleotide polymorphisms, which occur as heterozygotes. This result fails to reject the hypothesis of a single hybridization founder event that resulted in the initial formation of one major clone. The other clones appear to have originated via post-formation microsatellite mutations of the major clone.

Genetic Differentiation among Natural Populations of the Lizard Complex Darevskia raddei as Inferred from Genome Microsatellite Marking

The article presents the genetic parameters of the populations of lizards of the Darevskia raddei complex (D. raddei nairensis and D. raddei raddei) and the populations of D. valentini calculated on the basis of the analysis of variability of 50 allelic variants of the three nuclear genome microsatellite-containing loci of 83 individuals. It was demonstrated that the Fst genetic distances between the populations of D. raddei nairensis and D. raddei raddei were not statistically significantly different from the Fst genetic distances between the populations of different species, D. raddei and D. valentini. At the same time, these distances were statistically significantly higher than the Fst distances between the populations belonging to one species within the genus Darevskia. These data suggest deep divergence between the populations of D. raddei raddei and D. raddei nairensis of the D. raddei complex and there arises the question on considering them as separate species.

Detection of genotypic changes in parthenogenetic lizards (Darevskia armeniaca (Mehely)) introduced from Armenia to Ukraine

The article provides data on allelic and genotypic diversity of Ukrainian and Armenian populations of a parthenogenetic lizard of Darevskia armeniaca. The individual genotypes of studied specimens were established due to combination of alleles of three microsatellite loci. It is supposed that in the introduced Ukrainian population after the invasion two new genotypes appeared. Molecular mechanisms of the emergence of these new genotypes are suggested. Values of observed heterozygosity and genetic Fst-distances for the introduced Ukrainian population, native Armenian population and group of other Armenian populations are provided.

The complete mitochondrial genome of the parthenogenetic Caucasian rock lizard Darevskia unisexualis (Squamata: lacertidae) contains long tandem repeat formed by 59 bp monomer

Abstract The first complete mitochondrial genome sequence of parthenogenetic Caucasian rock lizard Darevskia unisexualis (Lacertidae family) is determined by hybrid assembly with Illumina HiSeq and PacBio RS II platforms. The circular 21.4 kbp mitogenome contains 13 protein-coding genes, 12S and 16S rRNA genes, 20 tRNAs, two pseudogenized tRNAs, and one long tandem repeats with 4.1 kbp length formed by 59 bp monomer repeated x70.6 times located before control region. This finding represents a new example of mitogenome variation in lizards of hybrid origin, providing fundamental data for following study of a unique hybridization system formed by parthenogenetic and bisexual species in the mountain steppe of central Armenia.

[Intra- and interspecific polymorphism of (AAT)n in microsatellite locus du47D in parthenogenetic species of the genus Darevskia].

The molecular structure of the allelic variants of (AAT)n of the Du47D microsatellite locus was determined in parthenogenetic lizards Darevskia dahli, D. armeniaca, and D. rostombekovi. Comparative analysis of these alleles showed that they were characterized by perfect structure of microsatellite cluster, and were different in the number of (AAT) monomeric units, as well as in the combinations of species-specific substitutions and deletions in the microsatellite flanking regions. Molecular structure of microsatellite cluster, species-specific single nucleotide polymorphism (SNP), and different representation of alleles Du47 in the samples of parthenogenetic species examined point to the origin of the alleles from different bisexual species, which is consistent with the hybrid nature of unisexual species of the genus Darevskia. In addition, these data reflect different combination patterns of interspecific hybridization events with the participation of the same bisexual species upon the formation of hybrid genomes of parthenogenetic species. Possible application of the allelic variants of microsatellite loci of parthenogenetic lizards as the genetic markers for the analysis of the genomes of parthenogenetic species in the light of evolution, ecology, and parthenogenetic type of reproduction in vertebrates is discussed.