Cytogenetic mechanisms of unisexuality in rock lizards

Darevskia rock lizards is a unique complex taxa, including more than thirty species, seven of which are parthenogenetic. In mixed populations of Darevskia lizards, tri- and tetraploid forms can be found. The most important issues in the theory of reticulate evolution of Darevskia lizards are the origin of parthenogenetic species and their taxonomic position. However, there is little data on how meiosis proceeds in these species. The present work reports the complex results of cytogenetics in a diploid parthenogenetic species – D. unisexualis. Here we detail the meiotic prophase I progression and the specific features оf mitotic chromosomes organization. The stages of meiosis prophase I were investigated by immunocytochemical analysis of preparations obtained from isolated primary oocytes of D. unisexualis in comparison with maternal species D. raddei nairensis. It has been shown that in D. unisexualis at the leptotene-zygotene stages the axial elements and the synaptonemal complex (SC) form typical “bouquets”. At the pachytene-diplotene stage, 18 autosomal SC-bivalents and thickened asynapted sex Z and w univalents were observed. The presence of SYCP1 protein between the lateral elements of autosomal chromosomes proved the formation of assembled SCs. Comparative genomic hybridization (CGH) on the mitotic metaphase chromosomes of D. unisexualis was carried out using the genomic DNA isolated from the parental species D. raddei nairensis and D. valentini. In the pericentromeric regions of half of the mitotic chromosomes of D. unisexualis, specific regions inherited from maternal species have been found. Following our results, we suggest a model for diploid germ cells formation from diploid oocytes without premeiotic duplication of chromosomes in the oogenesis of diploid parthenogenetic lizards D. unisexualis. Taken as a whole, our findings confirm the hybrid nature of D. unisexualis and shed light on heterozygosity and automixis in diploid parthenogenetic forms.

Synaptonemal complex (SC) karyotypes of D. valentini and D. raddei males comprise 19 acrocentric bivalents. Minor differences in chromosome pairing during the zygotene stage and certain differences in the average number of crossing-over sites have been described for these species 39 . Later, a species-specific SC-karyotype feature was found in one of the parental species -D. raddei, namely, the formation of additional centromere proteins signals localized near the native centromeres. Then, 3-9 dicentric chromosomes were found in the D. raddei spermatocytes I pachytene nuclei and the hypothesis of the epigenetic-based mechanism of additional neocentromeres formation has been proposed as a result 40 .
Sex chromosome identification in lacertids is highly important due to active evolution processes described for the ZW system in this group 41,42 and the reptiles in general. Most works describe mitotic metaphase chromosomes [41][42][43][44][45][46] . Z and W lampbrush chromosomes were described for Eremias velox 47 . Studies on the sex chromosomes' homology in the wide phylogenetic spectrum of lacertids and their outgroups revealed long-term evolutional stability of the ZW sex chromosome system across the lacertids 48 .
Previous cytogenetic studies of somatic karyotypes of four parental species of genus Darevskia revealed interspecific differences in the W chromosomes structure. Particularly, in two paternal species D. valentini and D. portschinskii, W chromosomes are heterochromatin-enriched macrochromosomes. At the same time, w chromosomes of the two maternal species D. raddei nairensis and D. mixta are shorter in length but contain large heterochromatic pericentromeric regions 49 . It was suggested by the authors that deletion in the euchromatic region of the W macrochromosome could lead to the formation of the w microchromosome in the maternal species, assuming that W macrochromosome is the ancestral, and w chromosome is the progressive type of sex chromosome. According to Kupriyanova (1997), D. unisexualis inherited the reduced w microchromosome from the maternal species D. raddei nairensis 50 .
The current work aimed to characterize the somatic karyotype (mitotic metaphase) as well as chromosome behavior during meiosis in the parthenogenetic rock lizard species D. unisexualis. Comparative genomic hybridization (CGH) on mitotic metaphase chromosomes of the parthenogenetic species D. unisexualis was performed. The study of meiotic cells was carried out to reveal the number of bivalents during all substages of prophase I, to study the specific features of homeologous chromosomes synapsis, as well as to identify Z and W sex chromosomes and investigate their behavior in meiosis. Towards this end, we performed the following procedures: • Fluorescent labelling of the whole genomic DNA of D. raddei nairensis and D. valentini parental species and comparative genomic hybridization (CGH) on D. unisexualis mitotic metaphase chromosomes. • Immunocytochemical study of the spread and squashed nuclei preparations of parthenogenetic D. unisexualis primary oocytes at the meiotic prophase I stage.
Results comparative genomic hybridization (cGH) on the metaphase plates of the parthenogenetic species D. unisexualis using the genomic fluorescence in situ hybridization (fiSH) DnA probes corresponding to the parental species D. raddei nairensis and D. valentini. One-half of the chromosomes (19 of the 38) of the parthenogenetic species D. unisexualis with diploid karyotype contains relatively large pericentromeric DNA fragments specific for the parental species D. raddei nairensis (chromatin fragments are shown in red) (Fig. 2). Despite the fact they share some repetitive DNA content in the same chromosome, the exclusive presence of highly accumulated repeated DNA (red and green signals) on 19 distinct chromosomes provides additional cytogenetic confirmation of the hybrid origin of the parthenogenetic species under study.
immunocytochemical study of the spread and squashed nuclei of bisexual species D. raddei nairensis primary oocytes. We traced the assembly of synaptonemal complexes in the female of the maternal species D. raddei nairensis from zygotene ( Fig. 3a) to pachytene (Fig. 3b,c). 18 autosomal bivalents and Z and w sex chromosomes were determined at the pachytene stage (Fig. 3c). Heteromorphic sex chromosomes have thickened axial elements and smaller centromere foci. We detected both variants of sex chromosomes location in the spread preparation of synaptonemal complexes: sex chromosomes located closely (Fig. 3b), and asynaptic w and Z univalents (Fig. 3c).
immunocytochemical study of the spread and squashed nuclei of diploid parthenogenetic species D. unisexualis primary oocytes. Here, for the first time, we were able to obtain the preparations of oocyte nuclei from the parthenogenetic species D. unisexualis, squashed or spread at different stages of meiotic prophase I: from leptotene to mid-diplotene. Immunocytochemical studies of 103 primary oocyte nuclei were performed (Fig. 4). Formation of the axial elements of meiotic chromosomes started at the late leptotene stage near one of the cell poles and was detected in 19 out of 103 analyzed nuclei (Fig. 4a). At the early zygotene stage, the assembly of the chromosome axial elements continues, and the formation of the "chromosome bouquet" structure begins (Fig. 4b).
The "chromosome bouquet" (U-shaped chromosomes) is associated with the initiation of chromosome synapsis and is the hallmark of presynaptic stages of prophase I (Fig. 4a-c) 51,52 . At the mid-zygotene stage in D. unisexualis, the synapsis of the axial elements is elongated while maintaining the configuration of the "chromosome bouquet" (Fig. 4c-e). DNA double-strand breaks (DSB) repair loci were detected associated with the assembling axial elements of chromosomes at the early zygotene stage (Fig. 5d). Twenty-two primary oocyte nuclei at the early and mid-zygotene stages were studied. www.nature.com/scientificreports www.nature.com/scientificreports/ To examine the synapsis of homeologous (inherited from the two parental species D. raddei nairensis and D. valentini) chromosomes and the assembly of synaptonemal complexes, we performed a detailed immunocytochemical study of D. unisexualis leptotene, zygotene, and pachytene stages (Fig. 5). For the immunocytochemical study, we used two antibodies combination which reactivity in reptiles has been experimentally proved by us in this study (see control antibodies test in the Supplementary Materials). Immunostaining with the anti-SYCP1 protein antibodies revealed the central element of the assembled synaptonemal complexes in all late zygotene (Fig. 5a) and pachytene (Fig. 5b) nuclei studied, but not in the nuclei at the leptotene stage (Fig. 5b). Subsequent immunostaining of the same nuclei with the anti-SYCP3 antibodies revealed asynapsed axial elements of the chromosomes at the leptotene stage (Fig. 5b') and partial asynaptic zones at the zygotene stage (Fig. 5a').
The pachytene stage in D. unisexualis (17 nuclei studied) is characterized by the complete synapsis of autosomal chromosomes (Fig. 4e,f). The number of bivalents, as well as merged centromere signals, did not exceed 20  in the pachytene nuclei, which corresponds to the centromere signals on 18 autosomal bivalents and one smaller sized signal on each of Z and w chromosomes, respectively. The diplotene stage (45 nuclei studied) begins with the formation of short desynapsed zones at the ends of SC-bivalents (Fig. 4g). Intensive staining of thickened asynapted axial elements of Z and w sex chromosomes was obtained with the anti-SYCP3 protein antibodies in early diplotene (Fig. 4g). Then, there was a gradual fragmentation of the axial structures associated with SYCP3 protein unloading from the axial elements (Fig. 4h,i), which was also a diplotene stage-specific event in the males of both parental species 39 . At the diplotene stage, 18 desynapting autosomal bivalents and two completely asynaptic heteromorphic Z and w chromosomes are clearly visible, lying at a considerable distance from each other (Fig. 4h,i). These two chromosomes are characterized by a much stronger signal in the case of SYCP3 protein immunostaining (Figs. 4h,i and 6a) and by smaller centromere foci (Fig. 6b). FISH with the D. raddei nairensis gDNA probe revealed a strong specific signal in the pericentromeric region of the short w chromosome, but not of the Z chromosome (Fig. 6c). All the facts supporting the identity of these two chromosomes as Z and w sex chromosomes are listed in the DISCUSSION section below. Therefore, the number of centromeres signals at the diplotene stage is 20, taking into account the initiation of desynapsis at the bivalent ends of some autosomes ( Fig. 4g-i). Thus, the number of autosomal SC-bivalents detected in the parthenogenetic D. unisexualis is 18, which is typical for the meiotic prophase I of the parental bisexual rock lizard species. But unlike in males (ZZ), the heteromorphic asynapted axial elements of the Z and w chromosomes are clearly visible. None of the nuclei we studied at the diplotene stage showed signs of an increase in the number of chromosomes. This indicates the absence of the pre-meiotic chromosome doubling stage.

Discussion comparative genomic hybridization (cGH) on mitotic chromosomes of parthenogenetic D. unisexualis.
The CGH method has been successfully implemented for the identification of parental genomes in hybrids/allopolyploids [53][54][55][56][57][58] . In the present work, the CGH results proved to be informative and allowed us to precisely characterize the hybridization process that led to the emergence of the parthenogenetic D. unisexualis species.  www.nature.com/scientificreports www.nature.com/scientificreports/ As a result of the CGH experiments performed on the parthenogenetic D. unisexualis mitotic metaphase chromosomes, we have found that one-half of the chromosomes (19 out of 38) of this species carry relatively large fragments of pericentromeric chromatin which is specific to the D. raddei nairensis parental species (Fig. 2). This result apart from being the clear confirmation of the hybrid origin of the species under study also points to the conservation of heterozygosity for these chromosomal fragments in many generations (age of the unisexual form) of the parthenogenetic D. unisexualis. The maternal status of D. raddei nairensis for the parthenogenetic D. unisexualis has previously been confirmed by mitochondrial DNA analysis 14,15,20 . D. valentini was proposed as the paternal species in several studies 10,14,20 , although this should be further confirmed by comparative cytogenetic analysis such as for example multicolor FISH.
Additionally, it seems reasonable to assume that the pericentromeric chromatin regions found on 19 chromosomes, annealed mainly with the red-labeled D. raddei nairensis gDNA, may represent D. raddei nairensis-specific pericentromeric repetitive DNAs. The clearly visible separation of parental genomes, as highlighted in Fig. 2, is a well-known feature in vertebrates 59 .
In our previous study, we had detected double centromere signals in several meiotic chromosomes in the D. raddei male karyotype 40 . Additional centromeres in the chromosomes may be associated with the formation of the so-called centromere environment, usually consisting of satellite tandemly repeated DNA elements 60 . Species-specific centromere organization in the D. raddei karyotype may be connected with the red pericentromeric signals detected on 19 chromosomes of D. unisexualis in our current CGH experiments. On the other hand, the mechanism of double centromere formation in D. raddei may involve epigenetic chromatin modification as far as a high degree of variability was shown for the formation of additional immunostaining centromere signals on the certain chromosomes 40 .
An important conclusion that can be made based on our CGH data is that no considerable exchange of DNA loci within the identified DNA fragments (red signal colors) between two homeologous (parental) sets of chromosomes seems to have occurred in the D. unisexualis hybrid karyotype. It is known, that centromere has crossing-over interference activity in meiosis, reducing the probability of crossing-over in the regions close to centromeres 61,62 . This is consistent with our results of CGH in the pericentromeric regions on half of the chromosomes in the D. unisexualis somatic karyotype (Fig. 2).
Therefore, at present time we may detect only the «residual traces» of the differences between the two chromosome sets belonging to the parental species, which are preserved explicitly in the pericentromeric chromosome regions that were not involved in the process of possible (see below) crossing-over between the homeologous chromosomes.

Meiotic prophase I stages in maternal bisexual species D. raddei nairensis and parthenogenetic species D. unisexualis.
It is known that meiosis constitutes a serious obstacle to the production of fertile progeny by interspecific hybrids. The studies of meiotic prophase I in mammalian interspecies hybrids have demonstrated that when chromosome synapsis is disturbed, cells undergo selection at the pachytene stage 63 and are removed by apoptosis. Synapsis between the heteromorphic sex chromosomes is of especial value with the defects in synapsis or at least abnormalities in common sex body formation between heteromorphic sex chromosomes leading to aneuploidy in the germ cells 63,64 .
Studies of oogenesis in reptiles reported so far were mainly performed using the histological sections of ovarian tissue 34,35,65,66 . In some of them, squashed preparations were analyzed, or ovarian tissue was examined directly using confocal microscopy 24,25,67,68 . Such studies allow to assess the main parameters of oogenesis, however, it appears practically impossible to understand the peculiarities of the individual chromosomes behavior in meiotic prophase I. Using the preparations of this type, it is challenging to obtain any detailed information about the synapsis of homeologous chromosomes and the formation of chiasmata.
Our study of oogenesis in reptiles is the first to be performed using immunostaining on the preparations of spread primary oocyte nuclei.We traced the key stages of prophase I in D. raddei nairensis female (Fig. 3) and in D. unisexualis (Figs. 4 and 5). To determine every stage we used a combination of stage-specific morphological criteria similar to our previous studies in Darevskia 39,40 . Synapsis of homeologous chromosomes and assembly of synaptonemal complexes in the D. unisexualis hybrid karyotype. When we examined the micrographs of the successive stages of meiotic prophase I in D. unisexualis (Fig. 4a-i), we could observe no obvious disturbance of the synaptic dynamics in the 18 autosomal pairs, while the pair of sex chromosomes (Z and w) did not synapse. We also confirmed the fact that the synapsis of chromosomes at the zygotene stage in D. unisexualis is associated with DSB repair (Fig. 5d), similar to the results obtained in our previous study performed in the parental species males 39 .
Thus, synapsing chromosomes of D. unisexualis, which are homeologous, demonstrate not only the limited (partial) peritelomeric synapsis, described previously in the diploid parthenogenetic species of the genus Aspidoscelis (Cnemidophorus) 25 , but also the full-length synapsis. In contrast, in the diploid parthenogenetic reptile Aspidoscelis neomexicana, the synapsis of homeologous chromosomes during the early meiotic prophase I is possible only in small peritelomeric chromosome regions (possibly due to the large degree of karyotype divergence in the parental species). As a result, the development of primary oocytes after the late zygotene stage is blocked in A. neomexicana. Both divisions of meiosis in A. neomexicana may be completed only by those cells in which duplication of chromosomes and their synapsis (non-homeologous) occur at the early stages of meiosis 25 .
It has been demonstrated that the SC-karyotypes of the two parental species for D. unisexualis (D. valentini and D. raddei nairensis) have similar characteristics, consisting of 19 acrocentric SC-bivalents. However, certain differences in the dynamics of chromosome synapsis and the average number of crossing-over sites have www.nature.com/scientificreports www.nature.com/scientificreports/ been observed in these two parental species 39 including the presence of additional centromeric signals on some SC-bivalents of D. raddei 40 as well (Fig. 3c).
It may be assumed that after the primary act of interspecific hybridization and the production of hybrid individuals, germ cells enter meiotic prophase I and here possible problems associated with the synapsis of homeologous chromosomes, as well as problems in chiasmata formation may emerge.
However, we have shown earlier that during meiosis in the allotriploid hybrid D. unisexualis x D. valentini males, extended asynaptic zones in the structure of SC trivalents formed by homeologous chromosomes as well as incomplete double-strand breaks (DSBs) repair did not interfere with cells completing both meiotic divisions 39 . In this regard, it would seem reasonable to assume that in a hybrid individual formed via the cross between D. valentini male and D. raddei nairensis female, which once gave rise to parthenogenetic population, the germ cells entering meiosis were able to overcome the above-mentioned problems of synapsis and incomplete DSB repair problems. This is confirmed by the large proportion of oocytes we have observed at the diplotene stage, in contrast to the case with the parthenogenetic A. neomexicana, where only the cells with chromosomal duplication and «pseudobivalent» formation can reach the diplotene stage 25 .
The completion of prophase I without premeiotic chromosome duplication is facilitated by the absence of strict meiotic checkpoints, previously described in rock lizards 39 and birds 69 . In rock lizards, this is confirmed not only by the identification of cells at all meiotic prophase I stages, but also by the formation of a large number of mature spermatozoa in triploid male hybrids (3n = 57, 54 A + ZZw), albeit with morphological abnormalities 39 .
Therefore, we may speculate that the above-mentioned characteristics of meiosis in rock lizard, the reduction or complete absence of checkpoints, and the similarity of parental karyotypes, represent the basis for the formation of the D. unisexualis hybrid karyotype.
During subsequent generations, the processes associated with overcoming the so-called "genomic shock" 70 , also called "genomic stress" in many of publications 70-72 , may have followed. This term was suggested to describe the mechanisms that «forced the genome to restructure itself in order to overcome a threat to its survival» 70 . Most works are devoted to describing this phenomenon in allopolyploid plants 73,74 or animals 72,[75][76][77][78] . However, several works on genomic shock (or stress) in diploids of hybrid origin have also been performed 71,72,79,80 . Genomic shock refers to events that constitute the early stages of interaction between two different genomes and are associated with the high heterozygosity of the newly formed hybrid karyotype 76 . Several studies have shown that the genomic shock condition caused by the formation of a new hybrid karyotype may be accompanied by multiple genetic and epigenetic changes: elimination of chromatin 74 , changes in gene expression 81 , epigenetic destabilization of genomic repeats 75,82 , and the expansion of transposons 83 . The next stage following hybridization, the hybrid genome stabilization, occurs in several subsequent generations 83,84 .

Sex chromosomes of maternal bisexual species D. raddei nairensis and parthenogenetic species D. unisexualis.
We identified Z and w sex chromosomes in D. raddei nairensis and D. unisexualis based on the following observations: 1. Identification of two asynapted axial elements with different lengths, following the data on the reduced type of w chromosome inherited from the maternal D. raddei species 49,50 . 2. The axial elements of these two chromosomes are thickened (Figs. 3b,c and 4g-i), which makes them distinguishable from SC-bivalents by immunostaining with the anti-SYCP3 protein antibodies and which is characteristic of the sex chromosomes of many animal species 85-88 . 3. Immunostaining with antibodies against centromere proteins (ACA) revealed that the ACA-foci on this asynapted chromosome pair look smaller than those formed by synapsed autosomal SC-bivalents, where closely located centromeres give doubled or brighter signal 85 (Figs. 3a,c, 4h,i and 5b). The total number of ACA foci per nucleus in both species is 20, which corresponds to the signals on 18 autosomal chromosomes and one small signal on each of Z and w chromosomes, respectively. 4. FISH with D. raddei nairensis gDNA at the diplotene stage in D. unisexualis revealed a strong signal on the W chromosome pericentromeric region, but not on Z chromosome (Fig. 6c). This result corresponds with the fact that the heterochromatin-enriched w chromosome was inherited from the maternal species D. raddei nairensis 89,90 .
In D. raddei nairensis two variants of sex chromosomes location on the synaptonemal complexes spread preparation were observed: sex chromosomes located closely (Fig. 3b), and asynaptic w and Z univalents (Fig. 3c). In D. unisexualis we found the absence of synapsis between the Z and w chromosomes at the pachytene and diplotene stages (Fig. 4g-i).
It is known that synapsis and chiasma formation between heteromorphic sex chromosomes is important for balanced gamete production. Disruption of synapsis and the absence of chiasma between sex chromosomes can lead to the formation of aneuploid germ cells. The question that arises is: 'How is asynapsis of sex chromosomes compensated for in the parthenogenetic species analyzed in this study?' In most organisms studied so far, a chiasma is observed between the sex Z and W chromosomes, which often have a small zone of homology (pseudoautosomal region, PAR). The formation of a single recombination nodule in the ZW bivalent in chicken and quail has been described in the PAR region very close to the telomeric end 91 . Recent studies of oogenesis in chicken have revealed that asynapsis of the Z and W chromosomes and incomplete repair of DSBs normally occurs in 20% of primary oocytes. Oocytes carrying these asynapsed bivalents are, however, able to pass to the diplotene stage 69 . The authors, hence, suggested the lower efficiency or complete absence of a checkpoint (possibly its secondary loss during evolution) in meiotic prophase I in bird oogenesis. The absence of strict checkpoints in meiotic prophase I was also suggested by us in triploid hybrids (3n = 57, 54 A + ZZw) of D. unisexualis x D. valentini rock lizards 39 .
It is possible that synaptic Z and w chromosomes were not detected because of the differences in the PARs acquired during the evolution of the rock lizard parental species. As it is known that the w chromosome of the maternal species D. raddei has been significantly shortened during evolution, and it is referred to as the progressive w chromosome type 90 , in contrast to the ancestral type of W chromosome found in other bisexual species of rock lizards studied to date (D. valentini, D. portschinskii). the mechanism of ploidy restoration during oogenesis in diploid parthenogenetic D. unisexualis species. Based on our analysis of chromosome synapsis during meiotic prophase I (Fig. 4), we concluded that unlike previously demonstrated by Newton et al. 25 for the diploid parthenogenetic species A. neomexicana, premeiotic chromosome duplication in the rock lizard D. unisexualis does not occur. This is consistent with the data of Darevsky & Kulikova (1962;1964) 34,35 , who showed that the most closely related parthenogenetic rock lizard species D. armeniaca has normal meiosis (similar to the females of the bisexual species D. raddei nairensis). In such parthenogenetic females, the first polar body and the metaphase plate of the second division are formed during the first reduction division. However, the authors were unable to detect the second polar body 34,35 . Based on these observations, it was suggested that diploidy during oogenesis in D. armeniaca is restored during the subsequent stages of meiosis. Terminal fusion and gamete duplication mechanisms could not support high levels of heterozygosity between generations 92 . As the most probable mechanism for diploidy restoration, in the absence of fertilization is the fusion of haploid pronucleus with one of the haploid descendants of the first polar body was proposed 36 , the so-called "central fusion".
"Central fusion" implies the reunification of Z and w sex chromosomes, which previously segregated in the meiotic anaphase I, in the same nucleus 93 . We cannot also exclude the variant where problems with Z and w segregation may arise during anaphase I because of sex chromosomes asynapsis. For example, both sex chromosomes may remain in the pronucleus resulting in polar body lacking sex chromosomes. But in the case of the "central fusion", it is possible to restore the full set of chromosomes, even with the temporary cell aneuploidy at the intermediate stages of this process.
Moreover, diploidy restoration through the "central fusion" explains the preservation of a heteromorphic autosomal chromosome pair in one of the parthenogenetic species -D. rostombekowi 36 . Our results are overall similar to what had been found for parthenogenetic species D. armeniaca [33][34][35] .
We present below the schematic representation of the possible automictic process variants which may take place during oogenesis in the parthenogenetic species D. unisexualis considering our new data: specifics of the pericentromeric regions of homeologous chromosomes and asynaptic Z and w sex chromosomes of D. unisexualis (Fig. 7). the possibility of meiotic recombination in parthenogenetic species D. unisexualis. Evolutionary processes in automictic populations are studied in the context of crossing-over and its effects on heterozygosity levels in offsprings 92 . As described above, the diploid parthenogenetic reptile Aspidoscelis neomexicana overcomes meiosis by chromosome duplication and synapsis of "pseudo-bivalents". This mechanism excludes recombination between homeologs. Surprisingly, the level of fertility in such animals is comparable with that in their bisexual relatives 25 . This is believed to be necessary to maintain the heterozygous state of the organism through the generations 12,80 . Comparative studies of allozyme data in bisexual and parthenogenetic www.nature.com/scientificreports www.nature.com/scientificreports/ species of rock lizards revealed high heterozygosity levels in the latter, which may indicate the suppression of meiotic recombination 7 .
We have shown that in the D. unisexualis parthenogenetic rock lizard species, oocytes enter the prophase I of meiosis in the normal way (without ploidy duplication). Moreover, no obvious disturbance of the axial elements assembly in the autosomes were revealed (Fig. 4). At the pachytene stage, we did not detect any asynaptic zones (Fig. 4f).
The most intriguing question obviously is the question whether chiasmata form between the homeologous chromosomes of D. unisexualis. If it is so, what is the pattern of the distribution of the crossing-over sites? Earlier, we have demonstrated that the studied parental species populations differ in the average number of crossing-over sites per spermatocyte nucleus 39 . It was also shown that in back-cross triploid hybrid rock lizard males, 1-2 foci of MLH1 protein, the crossing-over marker, could be found in the open trivalent homeologous chromosomes 39 .
If we assume that in every generation of the parthenogenetic form, normal crossing-over occurs between pairs of homeologous chromosomes, then during the lifetime of this form, heterozygosity could be maintained longer at those loci which are nearer to the centromere 37,94 . Perhaps it was this phenomenon (residual traces of heterozygosity) that we were able to identify in the somatic karyotype of D. unisexualis using CGH (Fig. 2). Otherwise, in the parthenogenetic species with preserved synapsis of homeologous chromosomes, a new mechanism of meiotic recombination suppression should exist and its study being of great interest.
Our further studies will focus on the immunostaining patterns of DSBs repair markers at the late prophase I and crossing-over markers in parthenogenetic rock lizard species. These studies will help us understand the relationships between unisexual reproduction and the levels of heterozygosity in parthenogenetic rock lizards.
The results of this study are extremely important for understanding the mechanisms of heterozygosity maintenance in hybrid karyotypes of diploid parthenogenetic species, and in general, to explain the genetic basis of the widespread distribution of unisexual animal populations. Total genomic DNA (gDNA) was extracted from the liver tissue of the D. valentini female (Sepasar population, ZMMU R-11920/12) and D. raddei nairensis female samples (Arzni population, ZMMU R-16042) from the collection of the Zoological Museum of the Lomonosov Moscow State University (ZMMU) using the DNA-extran-2 kit (EX-511, Syntol, Russia). The gDNA of D. raddei nairensis was directly labeled with Atto550 (red) using the Nick-translation labeling kit (Jena Bioscience), while the gDNA of D. valentini was labeled with Atto488 (green) using the Nick-translation labeling kit (Jena Bioscience) as well. Both gDNA were hybridized against the chromosomal background of D. unisexualis following the CGH protocol described in Symonová et al. 96 . It should be emphasized that unlabelled Cot-1 DNA (DNA fraction enriched with DNA repeats) was intentionally not used in the experiments since the alternative CGH variant performed with the presence of Cot-1 DNA in the hybridization mixture appeared to be unable to reveal significant differences in the hybridization patterns.
Germinal beds were isolated and disaggregated in the Eagle medium (C-160, Paneco, Russia). Squashed primary oocytes were prepared and fixed using the technique developed by Page et al. 97 , the residual cell suspension was used for the spread oocytes nuclei preparations according to Navarro et al. 98 . Poly-l-lysine-coated slides were used in all immunofluorescence studies. The slides were washed with phosphate-buffered saline (PBS) and incubated overnight at 4 °C with primary antibodies diluted in the antibody dilution buffer (ADB: 3% bovine serum