V. V. Grechko

Molecular DNA Markers in Phylogeny and Systematics

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.

Variable and Invariable DNA Repeat Characters Revealed by Taxonprint Approach are Useful for Molecular Systematics

Abstract. 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.

Molecular Evolution of Satellite DNA CLsat in Lizards from the Genus Darevskia (Sauria: Lacertidae): Correlation with Species Diversity

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 Darevskiahas shown that their variability is largely explained by single-nucleotide substitutions, which form 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 in the subfamilies, no cross-hybridization between them was observed (at 65°C). The individual variability within 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 Agi160 also detected by us in another lizard genus, Lacerta 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 hybridization. Seven species had mainly CLsatI (83 to 96%); three species, approximately equal amounts of CLsatI and CLsatIII (the admixture of CLsatII was 2–5%); and five species, a combination of all three subfamilies in highly varying proportions. Based on these results as well as on zoogeographic views on the taxonomy and phylogeny of theDarevskia species, hypotheses on the evolution of molecular-genetic relationships within this genus are advanced.

Molecular genetic diversification of the lizard complex Darevskia raddei (Sauria: Lacertidae): Early stages of speciation

To characterize the molecular genetic diversity of the genus Darevskia, several populations were examined by the inter-SINE-PCR method, reporting the number and sizes of the spacers between individual copies of SINE-like interspersed repeats. Examination of 17 D. raddei geographical populations and several reference species revealed unequal genetic differences, measured as Nei and Li’s genetic distances (DNL), for different groups of samples. The highest homogeneity was observed for the apparently panmictic D. raddei nairensis population from the basin of the Hrazdan River: genetic differences within each of the five samples and between them were similarly low (less than 0.1). The difference between ten samples of D. raddei raddei from Armenia and Karabakh (0.2–0.3) was somewhat higher than the interindividual difference within each sample (0.1–0.2), indicating that the samples belonged to different populations. The assumption was supported by the phylogenetic tree topology and multidimensional scaling. The differences between samples from the morphological subspecies D. raddei raddei and D. raddei nairensis ranged 0.3–0.4. The difference of two D. raddei raddei samples of Talysh (Azerbaijan) from other samples of the same subspecies corresponded to the subspecific level. The genetic distances between the good species D. raddei and D. rudis was 0.6–0.7. In terms of DNL, a questionable population from northwestern Turkey (“D. tristis”) was closer to D. rudis (DNL = 0.45), probably representing its subspecies. The phylogeography of D. raddei is discussed.

Reticulate Evolution of Parthenospecies of the Lacertidae Rock Lizards: Inheritance of CLsat Tandem Repeats and Anonymous RAPD Markers

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.

Variability of Restriction Sites in Satellite DNA as a Molecular Basis of Taxonprint Method: Evidence from the Study of Caucasian Rock Lizards

The distribution of restriction sites 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 analysis. Thus, the taxonprint method offers an opportunity to examine the satellite DNA divergence in closely related species and infer the 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 phylogenetic analysis of genomic DNA 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.

Short interspersed elements (SINEs) of squamate reptiles (Squam1 and Squam2): Structure and phylogenetic significance

Short interspersed elements (SINEs) are important nuclear molecular markers of the evolution of many eukaryotes. However, the SINEs of squamate reptile genomes have been little studied. We first identified two families of SINEs, termed Squam1 and Squam2, in the DNA of meadow lizard Darevskia praticola (Lacertidae) by performing DNA hybridization and PCR. Later, the same families of retrotransposons were found using the same methods in members of another 25 lizard families (from Iguania, Scincomorpha, Gekkota, Varanoidea, and Diploglossa infraorders) and two snake families, but their abundances in these taxa varied greatly. Both SINEs were Squamata-specific and were absent from mammals, birds, crocodiles, turtles, amphibians, and fish. Squam1 possessed some characteristics common to tRNA-related SINEs from fish and mammals, while Squam2 belonged to the tRNA(Ala) group of SINEs and had a more unusual and divergent structure. Squam2-related sequences were found in several unannotated GenBank sequences of squamate reptiles. Squam1 abundance in the Polychrotidae, Agamidae, Leiolepididae, Chamaeleonidae, Scincidae, Lacertidae, Gekkonidae, Varanidae, Helodermatidae, and two snake families were 10(2) -10(4) times higher than those in other taxa (Corytophanidae, Iguanidae, Anguidae, Cordylidae, Gerrhosauridae, Pygopodidae, and Eublepharidae). A less dramatic degree of copy number variation was observed for Squam2 in different taxa. Several Squam1 copies from Lacertidae, Chamaeleonidae, Gekkonidae, Varanidae, and Colubridae were sequenced and found to have evident orthologous features, as well as taxa-specific autapomorphies. Squam1 from Lacertidae and Chamaeleonidae could be divided into several subgroups based on sequence differences. Possible applications of these SINEs as Squamata phylogeny markers are discussed.

A New Subfamily of the Satellite DNA, CLsatIV, of the Lizard Darevskia lindholmi (Sauria, Laceridae): Structure and Evolution

A new subfamily (CLsatIV) of the tandem repeats CLsat, specific of the genus Darevskia , was discovered and sequenced only in the genomic DNA of the lizard Darevskia lindholmi (this endemic Crimean species until recently was regarded as the subspecies Lacerta saxicola lindholmi [1]). The CLsat repeated sequences were found in Caucasian representatives of the genus. They are characterized by a high level of variability and a sufficient rate of homogenization, which resulted in the formation of subfamilies [2‐4]. The orthologs of the three subfamilies of CLsat of the Caucasian lizards were also found in the D. lindholmi DNA using the method of hybridization, and two of them (CLsatI and CLsatIII) were sequenced. The sequences and organization of CLsatIV, CLsatI, and CLsatIII of the Crimean species, as well as the two last sequences and similar satellites of the Caucasian species were subjected to comparative analysis. The CLsatIV subfamily is characterized by a 30 bp decrease in length and a higher degree of divergence from the general consensus of the family. It was concluded that the data on the presence of the specific CLsatIV repeats, together with some other molecular genetic data [5, 6], allows regarding the Crimean lizards as a separate species of D. lindholmi , differing from all studied populations of the species D. saxicola , with which it has been classified until recently. An endemic Crimean lizard of the genus Darevskia (until recent time, the genus Lacerta [7, 8]) was attributed earlier to the lindholmi subspecies of the Caucasian rock lizard Lacerta saxicola [9]. On the basis of certain new findings (including our results [10]), this group, as well as other species attributed earlier to the so-called complex L. saxicola , are currently regarded as