[The problems of molecular phylogenetics on the example of squamata reptiles. Mitochondrial DNA markers].

The review is devoted to the recent problems of molecular phylogenetics based on the mitochondrial and chromosomal DNA sequences. The main subjects of the paper are the most widely used mtDNA markers though their application is not well enough (of critically considered) supported from the point of physiology and biochemistry of these organelles. Besides some well known aspects embarrassing the mt-markers phylogeny interpretations (hybridization and introgression events, ancestral polymorphism and nuclear paralogs of mtDNA) the attention is concentrated on non-neutrality and disproportional mutability of mt-genes and their fragments, on uniparental inheritance violations, recombinational events and heteroplasmy. These factors may influence the congruence of phylogenetic inferences and trees based on different mt-markers and when they compared with nuclear markers. Many authors suppose that the sequence comparisons of mt-genes and their fragments alone are not adequate to the studies of evolution. The notion is put forward that the influence of external conditions on metabolism and physiology of mitochondria could not be fully taken into account and modelled to be applied for phylogenetic tasks. The values of mt-markers could be revealed by the comparison of the whole mtDNA sequences of different taxa and searching the informative syn- and apomorphic properties and nuclear paralogs.

[Molecular genetic relationships and some issues of systematics of rock lizards of the genus Darevskia (Squamata: Lacertidae) based on locus analysis of SINE-type repeats (Squam1)].

To study the molecular genetic relationships and correlate them with the taxonomy within the complex of lacertid lizards of the genus Darevskia, the locus analysis of the copies of the SINE-type repeat (Squaml) typical of the order Squamata was used. It was demonstrated that one of the loci (No. 34) contained the Squam1 copy insert in all species and subspecies of the examined genus. SINE allelic copies in some of the loci contained large indels and specific sets of mutations. The allelic variant M (medium, about 340 bp) was found most frequently; it was detected in all subspecies of D. saxicola (saxicola, darevskii, szczerbaki, lindholmi) and in most of the other species of the genus. Two species, D. derjugini and D. praticola, differed from the other species in the presence of long (L) and short (S) alleles. The longest allele was characteristic of the D. derjugini population from the Northern Caucasus (L, 379 bp, ssp. silvatica), while the shortest allele (97 bp) united the derjugini and barani subspecies. The second allele S (279 bp) characterizes the subspecies D. praticola praticola; some individuals of which also carry allele M. The second subspecies, D. p. pontica, contains allele L2, which differs from all other medium alleles in the presence of strictly specific short indel. In addition to apomorphic indels, the specificity and mutation distribution patterns among the Squam1 alleles were also examined. An analysis of the NJ tree indicated the concordance between morphological and molecular genetic characters of the species derjugini, praticola, and saxicola. Furthermore, four subspecies of D. saxicola were much closer to each other than the subspecies within the first two species; D. d. silvatica and the group of D. d. derjugini (+) + barani were clearly separated. It cannot be excluded that populations from Azerbaijan and Serbia can be treated as the independent subspecies of D. praticola.

[Repeated DNA sequences as an engine of biological diversification].

Several aspects of the functional role of non-protein-coding DNA elements in the cell life and taxa evolution were discussed in connection with modern views on three-dimensional regulatory network as a basis for life support and evolution. In accordance with this problem, the leading role of duplication and multiplication of any DNA fragments (from single nucleotides to segmental duplications) in origination of genome structure and reshaping were considered in relationships with chromatin proteins. By this way the diversities in genome and chromosome architectonics leads to diversity of morphogenesis. The regulation and plasticity of the genome is determined by the structure, plasticity and evolution of genomic satellite and dispersed repetitive elements, which, being tightly bound with life phylogeny, result in somatic and inherited changes. Repeated sequences take part and perform some epigenetic regulation via repeats themselves, their modifications, or via their RNA transcripts. There is a growing evidence that the usage of methaphoric designations of protein-noncoding sequences as "egoistic, junk or parasitic" are senseless and useless. To the contrary, the hypothesis is formulated that the repetitive non-coding DNA part could be considered as a main substrate of life, something like a "carcass" serving as a basis for protein-coding sequences. The own molecular evolution of this carcass defines the pattern of DNA transcription and then evolution of structural proteins and morphological differentiation. Genes involved in main energetic, "house-keeping" and metabolic processes are forming some kind of the protecting envelope for the hereditary material, and also provides the enzymatic pathway of replication, transcription and epigenetic regulation.

[A family of short retroposons (Squaml) from squamate reptiles (Reptilia: Squamata): structure, evolution and correlation with phylogeny].

We have isolated and characterised sequences of a SINE family specific for squamate reptiles from a genome of lacertid lizard that we called Squam1. Copies are 360-390 bp in length and share a significant similarity with tRNA gene sequence on its 5'-end. This family was also detected by us in DNA of representatives of varanids, iguanids (anolis), gekkonids, and snakes. No signs of it were found in DNA of mammals, birds, amphibians, and crocodiles. Detailed analysis of primary structure of the retroposons obtained by us from genomic libraries or GenBank sequences was carried out. Most taxa possess 2-3 subfamilies of the SINE in their genomes with specific diagnostic features in their primary structure. Individual variability of copies in different families is about 85% and is just slightly lower on the genera level. Comparison of consensus sequences on family level reveals a high degree of structural similarity with a number of specific apomorphic features which makes it a useful marker of phylogeny for this group of reptiles. Snakes do not show specific affinity to varanids when compared to other lizards, as it was suggested earlier.

[Molecular-genetic diversification of lizard complex D. raddei (Sauria: Lacertidae): early stages of speciation].

A number of populations of genus Darevskia lizards were studied using inter-SINE-PCR (IS-PCR). The number and size of PCR-amplified spacers of genomic DNA flanked by SINE-type repeats were compared in 17 populations of the D. raddei species complex along with several other species from the same genus. Nei and Li's (D(NL)) genetic distances between populations were unequivocal. Individual differences among D. r. nairensis sample specimens and between the samples fall into a range of less than 0.1. Individual variability intra each of the D. r. raddei samples--into a range of 0.1-0.2, but the inter samples differences in this subspecies depends on its geographic localizations. Thus the differences between 10 samples from Armenia and Karabach fall into a range of 0.2-0.3, but between all these and two samples from Talysh (Azerbajdzhan)--of 0.3-0.4. At the same time, the variability between both known subspecies (D. r. raddei and D. r. nairensis) reaches the same values. The differences between "good" species D. raddei and D. rudis were about 0.60-0.7. It may be supposed that D. r. raddei is currently undergoing the speciation process. The population of lizards from Turkey which is considered to be close to D. raddei by some researchers and to D. rudis by others is, according to our molecular data, more closely related to the latter species. The difference of homogeneity between other populations was revealed by comparison of the DNL values. Possible phylogeographic pathways of Darevskia species distribution based on the molecular data were proposed.

[Molecular markers of nuclear DNA in the study of evolution and speciation process in an example of "Lacerta agilis complex" (Sauria: Lacertidae)].

In order to elucidate the molecular-genetic relations of some Lacerta s. str. lizard populations, subspecies and species in comparison with some other genera we used methods revealing four types of nuclear DNA markers. Among these were taxonprint, RAPD, Inter-MIR-PCR markers and also satellite DNA monomer sequences. The aim was to compare the phylogeny and systematics of this reptilian group based on morphological and molecular criteria. This problem has a general importance for understanding a speciation process. Our results show a good correlation between both approaches when genera and species levels were studied. Systematic status of five subspecies of L. agilis were supported but not in all cases, some subspecies have no meaningful genetic differences by three types of molecular markers, but all of them were differed by RAPD markers. The data confirm the subdivision of L. agilis populations into west and east clades proposed by other authors earlier on the basis of mitochondrial DNA and morphology. The population structure of one of the subspecies--L. agilis exigua, was studied on a number of populations distributed from Ural region up to Kabardino-Balkaria by IMP method. There were no significant differences among these 14 populations investigated. The data testify the rapid distribution of the species after the end of Pleistocene glaciation.

[Molecular evolution of satellite DNA CLsat in lizards of the Darevskia species (Sauria: Lacertidae): correlation with species diversity]. / Molekuliarnaia évoliutsiia satellitnoi DNK CLsat iashcherits roda Darevskia (Sauria: Lacertidae): korreliatsiia s vidovym raznoobraziem.

The structure and evolution of a satellite DNA family was examined in lizards from the genus Darevskia (family Lacertidae). Comparison of tandem units of repeated DNA (satDNA), CLsat, in all species from the genus Darevskia has shown that their variability is largely based on single-nucleotide substitutions, which constitute about 50 diagnostic positions underlying classification of the family into three subfamilies. Maximum differences between the subfamilies reached 25%. At this level of tandem unit divergence between the subfamilies, no cross-hybridization between them was observed (at 65 degrees C). The individual variability of one subfamily within the species was on average 5% while the variability between species consensuses within a subfamily was 10%. The presence of highly conserved regions in all monomers and some features of their organization show that satellites of all Darevskia species belong to one satDNA family. The organization of unit sequences of satellites CLsat and Agi also detected by us in another lizard genus, Lacerts s. str. was compared. Similarity that was found between these satellites suggests their relatedness and common origin. A possible pathway of evolution of these two satDNA families is proposed. The distribution and content of CLsat repeat subfamilies in all species of the genus was examined by Southern blotting hybridization. Seven species had mainly CLsatI (83 to 96%); three species, approximately equal amounts of CLsatI and CLsatIII (the admixture of CLsatII was 2-3%); and five species, a combination of all three subfamilies in highly varying proportions. Based on these results as well as on zoogeographic views on phylogeny and taxonomy of the Darevskia species, hypotheses on the evolution of molecular-genetic relationships within this genus are advanced.

[Using molecular DNA markers in phylogeny and systematics]. / Molekuliarnye markery DNK v uzuchenii filogenii i sistematiki.

The review considers data on the use of the main evolutionary markers (ribosomal, mitochondrial, and RAPD markers; dispersed and tandem repeats). Some circumstances impending analysis of these data are discussed.

[Satellite DNA restriction site variability as a molecular basis of taxonoprint method: evidence from the study of Caucasian rock lizards]. / Variabel'nost' saitov restriktsii satellitnoi DNK kak molekuliarnaia osnova metoda taksonoprinta (na primere skal'nykh iashcherits Kavkaza).

The restriction site distribution in satellite DNA of 17 Caucasian rock lizard species of the genus Lacerta (Darevskia gen. nov.), (Squamata, Lacertidae) was analyzed. The distribution patterns were shown to reflect the degree of satellite DNA evolutionary divergence, which could be revealed by taxonprint method, i.e., through the analysis of genomic DNA with a set of restriction endonucleases and subsequent computer-aided treatment. Thus, the taxonprint method offers an opportunity to examine the satellite DNA divergence in closely related species and infer their phylogeny of the species studied without reserting to costly and labor-consuming procedures. This is the advantage of using this technique at the early stages of genomic DNA phylogenetic analysis for rapid and effective estimation of relationships between closely related species as well as in the cases when DNA cloning and sequencing are too expensive or not feasible.

[Reticulate evolution of parthenogenetic species of the Lacertidae rock lizards: inheritance of CLsat tandem repeats and anonymous RAPD markers]. / Setchataia évoliutsiia partenovidov skal'nykh iashcherits sem. Lacertidae: nasledovanie tandemnogo povtora CLsat i anonimnykh markerov metoda RAPD (RAPID).

The genetic relatedness of several bisexual and of four unisexual "Lacerta saxicola complex" lizards was studied, using monomer sequences of the complex-specific CLsat tandem repeats and anonymous RAPD markers. Genomes of parthenospecies were shown to include different satellite monomers. The structure of each such monomer is specific for a certain pair of bisexual species. This fact might be interpreted in favor of co-dominant inheritance of these markers in bisexual species hybridogenesis. This idea is supported by the results obtained with RAPD markers; i.e., unisexual species genomes include only the loci characteristic of certain bisexual species. At the same time, in neither case parthenospecies possess specific, autoapomorphic loci that were not present in this or that bisexual species.

Genetic variation in parthenogenetic Caucasian rock lizards of the genus Lacerta (L. dahli, L. armeniaca, L. unisexualis) analyzed by DNA fingerprinting.

Multilocus DNA fingerprinting has been used to study the variability of some mini- and microsatellite sequences in parthenogenetic species of Caucasian rock lizards of the genus Lacerta (L. dahli, L. armeniaca and L. unisexualis). We demonstrate that these clonally reproducing lizards possess species-specific DNA fingerprints with a low degree of intra- and interpopulation variation. Mean indices of similarity obtained using M13 DNA, (GACA)4 and (TCC)50 as probes were 0.962 and 0.966 in L. dahli and L. armeniaca, respectively. The mean index of similarity obtained using M 13 and GATA probes in L. unisexualis was estimated to be 0.95. However, despite the high degree of band-sharing, variable DNA fragments were revealed in all populations with the microsatellite probes. An particularly high level of variability was observed for (TCC)n microsatellites in populations of L. unisexualis. In fact TCC-derived DNA fingerprints were close to being individual-specific, with a mean index of similarity of 0.824. Fingerprint analysis of parthenogenetic families of L. armeniaca showed that all maternal fragments were inherited together by the progeny, and no differences in fingerprint patterns were observed. On the other hand, while identical DNA fingerprints were obtained from L. unisexualis families with M13 and (GATA)4 probes, use of the (TCC)50 probe revealed remarkable intrafamily variation in this species. It is assumed that the genetic heterogeneity observed in parthenogenetic populations may be explained, at least in part, by the existence of genetically unstable microsatellite loci. Our data serve to illustrate processes of spontaneous mutagenesis and the initial stages of clonal differentiation in natural populations of the lizard species studied.

Variable and invariable DNA repeat characters revealed by taxonprint approach are useful for molecular systematics.

A specially optimized restriction analysis of highly repetitive DNA elements, called DNA taxonprint, was applied for phylogenetic study of primates and lizards. It was shown that electrophoretic bands of DNA repeats revealed by the taxonprint technique have valuable properties for molecular systematics. Approximately half of taxonprint bands (TB) are invariable and do not disappear from the genomes during evolution or change spontaneously. Presumably these invariable bands are restriction fragments of dispersed DNA repeats. Another group represents variable taxonprint bands that differ even between closely related species. These variable bands are probably represented by tandem DNA repeats and could be used as species-specific markers. It was shown that taxonprint bands are independent characters since the appearance of a new taxonprint band does not change the previous band pattern. Phylogenetic reconstruction carried out on taxonprint data demonstrated that this approach could be of general utility for molecular systematics and species identification.

Restriction endonuclease analysis of highly repetitive DNA as a phylogenetic tool.

Multiple band patterns of DNA repeats in the 20-500-nucleotide range can be detected by digesting genomic DNA with short-cutting restriction endonucleases, followed by end labeling of the restriction fragments and fractionation in nondenaturing polyacrylamide gels. We call such band patterns obtained from genomic DNA "taxonprints" (Fedorov et al. 1992). Here we show that taxonprints for the taxonomic groups studied (mammals, reptiles, fish, insects-altogether more than 50 species) have the following properties: (1) All individuals from the same species have identical taxonprints. (2) Taxonprint bands can be subdivided into those specific for a single species and those specific for groups of closely related species, genera, and even families. (3) Each restriction endonuclease produces unique band patterns; thus, five to ten restriction enzymes (about 100 bands) may be sufficient for a statistical treatment of phylogenetic relationships based on polymorphisms of restriction endinuclease sites. We demonstrate that taxonprint analysis allows one to distinguish closely related species and to establish the degree of similarity among species and among genera. These characteristics make taxonprint analysis a valuable tool for taxonomic and phylogenetic studies.

[Comparison of repeat DNA sequences in the Erinaceidae mammal family by restriction analysis]. / Sravnenie povtoriaiushchikhsia posledovatel'nostei DNK mlekopitaiushchikh semeistva Erinaceidae metodom restriktaznogo analiza.

Hedgehog (Erinaceidae) DNA was digested with teh Sau 96 I, Bsp 143 I, Csp 6 I, Taq I, Hinf I, Msp I, Eco 130I, Bcn I, BsuR I restriction endonucleases. The obtained products were end-labeled and electrophoretically separated in polyacrylamide gel. DNA fragments consisting of highly repetitive genomic sequences were detected as a set of bands corresponding to fragments between 30 and 500 bp in length. Comparison of DNA restriction patterns of the species analyzed revealed the presence of species-specific bands as well as common bands. Phylogenetic trees were constructed by means of the maximum parsimony method and the bootstrap procedure. Our data suggest that hedgehog species from arid areas are clearly distinguished from forest species.