Tendency towards clonality: deviations of meiosis in parthenogenetic Caucasian rock lizards

doi:10.21203/rs.3.rs-3936576/v1

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Tendency towards clonality: deviations of meiosis in parthenogenetic Caucasian rock lizards

https://doi.org/10.21203/rs.3.rs-3936576/v1

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Cytogenetic mechanisms of unisexuality in diploid parthenogenetic species of genus Darevskia remained debatable until recently. The mechanism that allows the unisexual form to maintain its heterozygosity in a number of generations is important for long-term existence in nature. In this work, for the first time for a parthenogenetic species of genus Darevskia, in addition to primary oocytes with the usual ploidy (18 + ZW bivalents in the meiotic prophase I) oocytes that underwent premeiotic genome endoduplication and carry a doubled number of bivalents (36 + ZZ + WW) were found.

Here we present a detailed comparative analysis of total preparation of synaptonemal complexes in the oocyte nuclei without and with genome endoduplication, and the behavior of sex Z and W chromosomes. We show the details of assembly of bivalents in the pachytene nuclei, where either homeologues or doubled identical copies of chromosomes compete for synapsis and form multivalents. For the first time, the WW sex pseudobivalent has been visualized in parthenogenetic reptiles. We show the reverse side of meiotic deviations in obligate parthenogenesis - cases of non-viable embryos with specific abnormalities.

Biological sciences/Evolution/Evolutionary genetics

Biological sciences/Developmental biology/Experimental organisms

Biological sciences/Genetics/Animal breeding

Biological sciences/Genetics/Development

Biological sciences/Evolution/Experimental evolution

Studies of reticulate evolution and parthenogenesis in reptiles over the past decades have revealed several important characteristics of the biology of this large and highly polymorphous taxonomic group.

Firstly, it is intensive interspecific hybridization in reptiles, which can lead to the formation of parthenogenetic forms (Darevsky 1958; Darevsky et al., 1985; Borkin, Darevsky 1980, Neaves et al., 2011). It is assumed that a certain level of divergence and, as a consequence, the incompatibility of parental karyotypes in interspecific hybrids can lead to the start of a unisexual mode of reproduction, i.e. a new parthenogenetic lineage (Murphy et al., 2000). The combination of two divergent genomes in one hybrid karyotype of a parthenogenetic species is associated with a number of deviations at the key stage of the life cycle - meiosis. Figuratively speaking, parthenogenesis is supposed to be a way to avoid hybrid sterility (Dedukh et al., 2020).

Secondly, the existence of obligate parthenogenetic forms of reptiles of different ploidy levels: diploids (Darevsky et al., 1985), most common triploids (Newton et al., 2016), and tetraploids (Cole et al., 2014; 2023). Within the group of Caucasian rock lizards, only diploid parthenogenetic forms, which initially appeared as hybrids, have been preserved as independent species (Arakelyan et al., 2023).

Thirdly, сytogenetic studies of interspecific hybrids revealed an important feature of gametogenesis in reptiles - weak checkpoints of meiosis. In particular, the germ cells of hybrid triploid males of genus Darevskia, which successfully pass through the both divisions of meiosis (Spangenberg et al., 2017). These triploid hybrids result from the mating of parthenogenetic species with their sexual relatives in hybrid zones. Numerous defects in synapsis of chromosomes and incomplete DNA double strand breaks (DSB) repair did not lead to arrest meiosis or spermatogenesis. An assumption was made about a weak selection of meiocytes with abnormalities in prophase I (Spangenberg et al., 2017).

Fourth, molecular biological studies of distinct populations of parthenogenetic species of hybrid origin indicate the possibility of a de novo appearance of minor differences between them (Ryskov et al., 2017, Osipov et al., 2021, Omelchenko et al., 2016). In addition, we identified evolutionary changes in the karyotypes of parthenogenetic forms (Spangenberg et al., 2020b; Spangenberg et al., 2021). Overall, it appears that it is precisely due to the plasticity of meiosis and its weak checkpoints that a large variety of obligate unisexual forms of hybrid origin can arise in reptiles.

From the point of view of the cytological mechanisms of unisexual reproduction, several variants of automixis are considered (Neiman et al., 2014). Each variant suggests different consequences for the offspring (Engelstädter 2017). Two key preconditions for the formation and long-term existence of the unisexual form are (a) a successful combination of parental genomes and (b) the presence of a reliable mechanism for maintaining the primary heterozygous state of the F1 hybrid in a number of generations (Grebelnyi, 2008).

The most striсt conditions for maintaining parental karyotypes in the parthenogenetic form in an unchanged (original) state can be provided by the mechanism of pre-meiotic genome endoduplication. In this case, in a hybrid karyotype consisting of pairs of homeologous chromosomes, each chromosome is duplicated before meiosis. During the early stages of meiotic prophase I, in the leptotene and zygotene stages, synapsis occurs not between homeologs, but between just doubled identical chromosomes (Lutes et al., 2010). The assembled synaptonemal complexes in this case are called “pseudobivalents”. Thus, the number of pachytene “pseudobivalents” in the nucleus after genomic endoduplication will be 2 times more than usual. This mechanism allows both divisions of meiosis to undergo, avoiding the problems of chromosome incompatibility from two divergent parental species (Lutes et al., 2010, Newton et al., 2016, Dedukh et al., 2022).

Despite the well-known general scheme of this cytological mechanism in reptiles, some details of prophase I of meiosis in nuclei with genomic endoduplication remained behind the scenes. This is due to the difficulties in obtaining rare endoduplicated oocytes I on pachytene and early diplotene stages. These stages allow to visualize competitive interactions between chromosomes and deviations from the canonical scheme of meiotic prophase I.

Previous studies of hybrid diploid karyotypes of Darevskia parthenogenetic species showed the preservation of the species-specific composition of pericentromeric chromosome regions (Spangenberg et al., 2020, 2021). That is, half of the chromosomes bear pericentromeric sat-DNA of the paternal species, whereas the second half, including the W-chromosome, has the maternal composition of pericentromeric sat-DNA. However, a more detailed study using CGH approach of the somatic karyotype of the parthenogenetic species D. rostombekowi revealed species-specific differences not only in the pericentromeric regions, but also in the interstitial regions of homeologous chromosomes (Spangenberg 2020a, 2021). These data indicated the possibility of maintaining the original state of the chromosomal sets of parental species in Darevskia unisexual species in a number of generations. Therefore, the clarification of the mechanism of ploidy restoration during meiosis and maintenance of heterozygosity in the group of Darevskia species remained debatable and required a detailed study (Arakelyan et al., 2023).

Previously, we suggested that the restoration of ploidy in unisexual species of Darevskia occurs by automixis according to the central fusion variant. That is, as a result of a specific post-meiotic fusion of products of meiosis (Spangenberg et al., 2020a). The assumption was made on the basis that oocytes I with normal ploidy form a successful synapsis of homeologous (but not homologous) chromosomes. Thus, samples, obtained from two populations of D. unisexualis, demonstrated numerous pachytene and early diplotene nuclei of oocytes I with normal ploidy and assembled autosomal bivalents (Spangenberg et al., 2020, 2021). No significant deviations in assembly of homeologous bivalents were detected. These results contrasted strongly with other studied parthenogenetic reptiles, where homeologous synapsis is fragmentary only: peritelomeric synapsis, partial synaptic zones, formation of multivalents. Bivalents assembly in such oocytes terminates at the zygotene stage (Newton et al., 2016). This is apparently due to significant differences in parental chromosomal sets or because of more stringent synaptic checkpoint (Newton et al., 2016, Dedukh et al., 2022).

Many studies pay special attention to reptilian ZW sex chromosomes in connection with the switch to unisexual reproduction (Odierna et al., 1993, Kupriyanova 2010). The W chromosome in Lacertids is recognized as one of the most evolutionary dynamic parts of the genomes (Suwala et al., 2020). Most often, the changes are characterized as: reduction in size, accumulation of heterochromatin, tandem repeats (telomeric or satellite DNA) (Matsubara et al., 2015, Suwala et al., 2020, Kupriyanova 2010). In Lacertidae a gradual transformation into the heterochromatic w sex microchromosome (Zw type) was suggested as an evolutionary trend (Odierna et al., 1993, Kupriyanova 2010). Recent bioinformatic analysis of Darevskia species showed enrichment of the CLsat family of tandem DNA repeats in the pericentromeric regions of autosomes and especially in the W sex chromosome (Ciobanu et al., 2004; Nikitin et al., 2023 in print). On the other hand, a comparative study of the evolution of the W chromosome in Lacertids revealed rather independent and species-specific changes (Suwala et al., 2020).

In this work, we conducted a detailed study of meiosis in oocytes I of the unisexual species D. armeniaca in order to elucidate the characteristics of meiotic prophase I that allow the long-term existence of the unisexual form.

Animals studied

D. armeniaca is a diploid parthenogenetic species of hybrid origin, the result of ancestral cross between the paternal species D. valentini and the maternal species D. mixta (Uzzell, Darevsky 1975). Three juvenile individuals and one adult were used in the study. All experiments were approved by the Ministry of Environment Republic of Armenia, permission № 3/29.7/1043 in accordance with the Regulations for Laboratory Practice.

Isolation of primary oocytes

Primary oocytes were obtained from two juvenile D. armeniaca, Tsahkadzor population (specimen VSj0401; specimen VSj0405) 10 days after hatching. Total preparations of synaptonemal complexes from oocytes I were obtained according to the protocol (Spangenberg et al., 2022). No colchicine treatment was applied before obtaining the preparations of primary oocytes.

Immunostaining

Slides were washed with phosphate-buffered saline (PBS) and were incubated with primary antibodies diluted in the antibody dilution buffer (ADB: 3% bovine serum albumin (BSA), 0.05% Triton X-100 in PBS) at 4°C overnight. Axial elements of meiotic chromosomes were detected using rabbit polyclonal antibodies against protein SYCP3 (ab15093, 1:250, Abcam, Cambridge, UK), centromeres were detected using human anti-centromere antibodies ACA (1:500; Antibodies Incorporated 15–234, Davis, CA, USA). After washing, the corresponding secondary antibodies that had been diluted in ADB were used: goat anti-rabbit Immunoglobulin (Ig) G, Alexa Fluor 488 (ab150077, 1:500, Abcam, Cambridge, UK), goat anti-human Alexa Fluor 555 (A21433, ThermoFischer, Waltham, MA USA). Secondary antibody incubations were performed in a humid chamber at 37°C for 3 h.

All preparations were mounted with the Vectashield antifade mounting medium with DAPI, 4′,6-diamidino‐2‐phenylindole (Vector Laboratories, Burlingame, CA, USA).

Mitotic Chromosome Preparation, C-banding and Telomere repeats FISH mapping

Mitotic chromosome preparations were obtained from one juvenile and one adult D. armeniaca, Tsahkadzor population (specimen VSj0404; specimen VS404) from bone marrow and all somatic abdominal organs using a direct suspension technique (Spangenberg et al., 2022a). C-banding was performed according to Salvadori et al. (2015). In situ hybridization with telomeric sequences was performed using the 5′-TAMRA-(CCCTAA)4 (Syntol, Moscow, Russia) following standard protocol (Spangenberg et al., 2020).

Microscopy

Meiotic preparation were examined in October 2022 using the Axio Imager D1 microscope (Carl Zeiss, Germany) equipped with the Axiocam HRm CCD camera (Carl Zeiss, Germany), Carl Zeiss filter sets (FS01, FS38HE, and FS43HE), and the image-processing AxioVision Release 4.8. software (Carl Zeiss, Germany). Meiotic bivalent lengths were measured using ImageJ software (https://imagej.nih.gov/ij, accessed on 30 January 2024). The mitotic chromosome preparation, stained with DAPI, were examined using the Leica DM microscope equipped with the Axiocam HRm CCD camera and filter set A, and processed with AxioVision Release 4.8. software (Carl Zeiss, Germany). The mitotic chromosome slides, conventionally stained with Giemsa, were examined using an Axioplan 2 Imaging microscope (Carl Zeiss, Germany) equipped with a CV-M4 + CL camera (JAI, Kanagawa, Japan) and the Ikaros software (MetaSystems, Altlussheim, Germany).

Primary oocytes with normal ploidy in D. armeniaca.

From the primary oocytes of D. armeniaca, we obtained 118 pachytene and early diplotene total spread preparations of synaptonemal complexes (SCs). Most of the oocytes I nuclei analyzed by us had a normal number of bivalents 18A + Z + W, where A - autosomal bivalents. This is the usual number of bivalents for 2n = 38 diploid karyotypes in unisexual and bisexual Darevskia species (Fig. 1A). We specifically separated the sex chromosomes in the record (Z + W), since the Z and W chromosomes in 53% of the studied nuclei (among 81 nuclei with detectable sex univalents) did not form normal sex bivalent (without a region of local synapsis - pseudoautosomal region, PAR) and were not even located nearby (Fig. 1B). On the contrary, contacting Z and W sex chromosomes were found in only 47% of oocytes I nuclei. The region of close proximity between Z and W was located in their centromeric regions (Fig. 1A; Figure SM1).

An important characteristic of the W sex chromosome in D. armeniaca is the interstitial block of heterochromatin, which is clearly visible on the DAPI and Giemza staining (Fig. 1B,C; Figure SM1A). We confirmed this interstitial chromatin block using C-banding approach (Supplementary materials Figure SM2B), telomere FISH mapping revealed no enrichment of telomeric repeats on the W chromosome (Figure SM2С). This marker on the W sex chromosome is an important characteristic that distinguishes it from autosomes (in particular from the nearest pair of microchromosomes m1 and m2).

We performed detailed measurements of total preparation of synaptonemal complexes (SC) obtained from the normal ploidy and endoduplicated nuclei. Idiograms of the SC-karyotypes demonstrate length of bivalents of homeologs (Fig. 3A) and pseudobivalents (Fig. 3B).

Detection of primary oocytes with duplicated number of bivalents in D. armeniaca.

Apart from numerous oocytes nuclei with normal ploidy, we found 19 endoduplicated nuclei with a doubled number of bivalents 36A + ZZ + WW, i.e. 38 pseudo bivalents - bivalents between pairs of newly duplicated chromosomes (Fig. 1C-D, Figure SM3). Such nuclei were detected at the pachytene (complete synapsis) and early diplotene (with desynaptic forks at the ends of bivalents) stages. We detected important characteristics of the endoduplicated nuclei listed below.

Identification of the WW bivalent in the endoduplicated nuclei. WW bivalent was for the first time visualized in the endoduplicated pachytene nuclei as the only bivalent carrying well-defined interstitial heterochromatin block, well seen on the DAPI-staining (Fig. 1C-D). In all analyzed nuclei with endoduplication, we found this single bivalent with a heterochromatin block. This fact proves the assembly of the WW bivalent, namely from the newly duplicated identical W chromosomes. In other words, the formation of WW “pseudobivalent”.
Ectopic synapsis and formation of synaptonemal complexes tetravalents. In five oocyte nuclei with endoduplication, we found the formation of SC-tetravalents (Fig. 1d, indicated “T”, Fig. 2, Figure SM3). Different configurations and details of such structures are presented in Fig. 2: pronounced synapsis in the distal ends of acrocentric chromosomes (Fig. 3A-A`), in the centromeric regions (Fig. 3B-B`), or equal ectopic assembly in the distal and centromeric regions (Fig. 3C-C`). Total preparation of synaptonemal complexes can be found in the Supplementary Materials Figure SM3.

Meiotic prophase I in the oocytes without chromosome duplication.

Most of the pachytene and early diplotene oocytes I previously studied by us (Spangenberg et al., 2020; 2021), had a surprising feature for hybrid karyotypes; the synapsis of all autosomal bivalents, composed of pairs of homeologs but not homologous chromosomes. The fact that in oocytes without chromosome duplication we detect definitely a homeologous but not homologous synapsis was proved by us earlier (Spangenberg et al., 2021). Visualization of somatic SC-trivalent in the unisexual form with an odd number of chromosomes D. unisexualis 2n = 37 (Keti population) confirmed the synapsis of homeologs (Spangenberg et al., 2021). Additional evidence was the visualization of significant divergence of centromeric DNA in the chromosomes involved in meiotic trivalent assembly performed by comparative genomic hybridization (CGH) (Spangenberg et al., 2021).

Interestingly, in studies of diploid parthenogenetic species of genus Aspidoscelis (Lutes et al., 2010; Newton et al., 2016) Lepidodactylus, Hemiphyllodactylus and Heteronotia (Dedukh et al., 2022), the authors noted that oocytes without genome duplication necessarily contain unpaired chromosomes - univalents. This pattern is typical for all studied diploid parthenogenetic species, except for unisexual species of the genus Darevskia. The authors justifiably assume that such oocytes with long regions of asynapsis are doomed to apoptosis due to the zygotene or pachytene checkpoints (Newton et al., 2016; Dedukh et al., 2022). Thus, cells with genome duplication are the only cells that are able to reach the diplotene stage (Newton et al., 2016).

In contrast, in two Darevskia parthenogenetic species we have studied to date (D. unisexualis and D. armeniaca), in addition to nuclei at the pachytene stage without visible synaptic disturbances, we also find a large number of normal diplotene nuclei (Spangenberg et al., 2020, 2021; this study Fig. 1A-B). This is an important feature of the unisexual Darevskia species. Such cells with homeologous synapsis only, theoretically could overcome the synaptic checkpoints of meiosis I similar to the triploid hybrids D.unisexualis х D.valentini we studied before (Spangenberg et al., 2017).

It remains unclear whether such primary oocytes without endoduplication can undergo post-meiotic fusion processes suggested by us previously (Spangenberg et al., 2020, 21). If so, what contribution do they make to the offspring of unisexual species? Perhaps such oocytes are eliminated from the germ cell line and undergo apoptosis. It is important to recall previous studies and our own observations of the incubation of egg clutches in parthenogenetic species. It was shown that some parthenogenetic Darevskia lizards do not hatch from eggs. Studies of such embryos have shown multiple developmental anomalies (Darevsky 1960; Darevsky and Kulikova 1961, Danielyan 1970). It cannot be ruled out that some of such abnormal embryos are the result of the development of oocytes along the path of postmeiotic fusion of nuclei or a result of unequal segregation during meiotic I anaphase stage leading to aneuploidy (See below section “Developmental deviation in parthenogenetic species of the genus Darevskia”).

Z and W sex chromosomes in oocytes without genome duplication.

Analysis of the behavior of sex Z and W chromosomes in the hybrid karyotype of D. armeniaca showed two variants of their localization during pachytene and diplotene stages - joint and separate localization on spread preparation of synaptonemal complexes (Fig. 1A,B). Nuclei with ZW joint localization showed that the centromeric regions of the sex chromosomes were the most contiguous regions (Figure SM1). Thus, it is likely that a pseudoautosomal region (PAR) is located in pericentromeric regions (Figure SM1). Nevertheless, the question of the truth synapsis or Z-W association in PAR remains open for unisexual forms of Darevskia (Spangenberg et al., 2020a).

Regardless of whether the Z and W sex chromosomes are located separately or together, Z and W univalents often formed curved or even circular axial structures (Fig. 1A,B). In general, the curved structure of sex univalents is characteristic of elongated asynaptic regions of sex chromosomes in different animals, especially in late prophase I, in diplotene

(Bogdanov et al., 2011; Spangenberg et al., 2022a; Surov and Feoktistova 2023). Thus, the lengths of sex chromosomes in the oocytes nuclei without endoduplication do not reflect the real lengths of univalents, and do not allow us to accurately determine the position (according length) of sex chromosomes in the karyotype. On the other hand, we were able to measure the length of the WW sex pseudobivalent (see the section below “de novo formed WW bivalent in the nuclei of oocytes after genomic duplication”).

Meiotic prophase I in the oocytes with genome endoduplication.

The nuclei with genomic duplication found by us contain 38 bivalents (twice more than usual) at the pachytene and early diplotene stages. These are 36 autosomal pseudobivalents and two sex chromosome bivalents: ZZ and WW. All pseudobivalents are composed of identical copies of chromosomes due to premeiotic endoduplication.

The mechanism of premeiotic endoreplication, described for some unisexual animals of hybrid origin, is considered as a way to avoid hybrid sterility (Dedukh et al., 2020). Thus, in particular unisexual reptile species, can restore and maintain their 2n or 3n ploidy in many generations. It is important that, due to the identity of the chromosomes that are synapsing in the endoduplicated nuclei, crossing over does not lead to the emergence of new genetic combinations. We can talk about the stop of recombination (Grebelnij 2008; Grebelnij 2009), a kind of "freezing” of the initial heterozygous state of the interspecific F1 hybrid.

An important marker in Darevskia karyotypes is a clearly visible heterochromatin block on the W chromosome (Fig. 1C-D, D`), previously described in mitotic metaphase plates (Odierna et al., 1993; Kupriyanova, 2010). Since such a heterochromatin block is present only on one of the 38 bivalents, this is another confirmation of assembly of the pseudobivalents but not homeologous bivalents like in the non-duplicated nuclei.

Interestingly, the m1 and m2 microchromosomes in the D. armeniaca hybrid karyotype inherited from two parental species have different centromere positions clearly visible on the Immunostained SC preparation (Fig. 1D) and on the idiogram (Fig. 2B). In the oocytes after endoduplication, the corresponding SCs (m1m1 and m2m2) are formed without any shifting the centromeres in bivalents, in addition confirming assembly of the pseudobivalents.

On the other hand in the non-duplicated oocyte nuclei we detected different variants: formation of the m1m2 homeologous SC (Figure SM1A) possibly after synaptic adjustment (Maguire et al., 1984; Lisachov et al., 2014; Stundlova et al., 2022), as well as located nearby or completely asynaptiс m1 and m2 univalents (Figure SM1B).

De novo formed WW bivalent in the nuclei of oocytes after genomic endoduplication.

The temporary appearance of a second copy of the W chromosome in a germ cell line is, of course, an extraordinary fact. However, the ultrastructure of the synaptonemal complex allows normal assembly and synapsis of the newly emerged WW bivalent (Fig. 1D`).

It should be noted that during the evolutionary period of existence of the bisexual maternal species (D. mixta), the W-chromosome obviously did not have the possibility to synapse with its homologue, to form a WW bivalent. This is a normal situation for W and Y chromosomes in bisexual animals. Rare cases of polysomy on X and Y chromosomes are known in human: 47, XXY; 47, XYY; 48, XXXY; 48, XYYY; 48 XXYY; 49 XXXXY; 49 XXXYY (Visootsak and Tartaglia 2013). On the other hand, rare cases of Y polisomies lead to severe developmental abnormalities and do not represent reproductive strategies.

We should separately note an interesting feature of the morphology of sex chromosomes (especially W chromosome) in the nuclei of oocytes after premeiotic genomic endoduplication. Usually, in animals with heteromorphic pairs of sex chromosomes, in meiotic prophase I, the sex chromosomes Z and W (or X and Y) have lengths that do not correspond to their chromosome numbers in the somatic karyotype. That is, the Z and W axial elements in meiotic prophase I are usually atypically elongated, curved, deformed, or coiled (Fig. 1A, B). That is why a heteromorphic pair of sex chromosomes is often taken out separately from autosomal pairs on karyotype diagrams (Gil-Fernandes et al., 2021; Surov and Feoktistova 2023). In contrast, in the homogametic sex, such as male reptiles, the sex Z chromosomes form a ZZ bivalent that is difficult to distinguish from autosomes (Spangenberg et al., 2022a).

In the case of premeiotic duplication of chromosomes, sex W chromosome receives its copy and can form a normal bivalent. We do not observe any deformations of axial elements in the structure of the WW bivalent in D. armeniaca (Fig. 1D-D`). That is, in this case, the WW-bivalent has a relevant length, since a normal synaptonemal complex is formed, similar to the usual ZZ-bivalent in male reptiles. According to the constructed idiogram of the SC-karyotype of endoduplicated nucleus, the WW bivalent has number 36 in the karyotype of D. armeniaca (Fig. 2B).

SC-tetravalents consisting of sister and homeologous chromosomes in the SC-karyotypes of D. armeniaca.

An interesting finding we made in the endoduplicated nuclei of D. armeniaca is the SC-tetravalents. We found a fairly extensive ectopic synapsis at both: distal (Fig. 3A) and centromeric (Fig. 3B) ends of chromosomes leading to formation of the SC-tetravalents in nuclei after genome endoduplication. It is likely that such configurations arise between corresponding homeologous. Apparently, during the assembly of SCs in the endoduplicated nucleus of the D. armeniaca oocyte, active processes of correction of ectopic synapsis occur, which were previously described for many polyploids (Holm and Rasmussen 1979, Jenkins and Jimenez 1995; Martinez-Perez et al., 2001; Moore 2002; Vasil’ev et al., 2022) or for the competitive synapsis between unpaired regions (univalents) of XY sex bivalents and autosomes in male mammals (Spangenberg et al., 2021a). These studies show that non-homologous partial synapsis often occurs in zygotene in the pericentromeric/peritelomeric regions of chromosomes and can be corrected in subsequent stages of meiotic prophase I. Indeed, it is known that such synaptic associations can be resolved before metaphase I and do not affect normal chromosome segregation (Holm, Rasmussen 1979, Spangenberg et al., 2021).

Premeiotic genome endoduplication and postmeiotic central fusion pathways.

An important question is, after the discovery of pre-meiotic genome endoduplication in D. armeniaca, can we reject the possibility of a post-meiotic automictic mechanism through the central fusion - the fusion of the oocyte and the first polar body (Spangenberg et al., 2020, Ho et al., 2023)? Of course, the mechanism of premeiotic endoduplication ensures long-term preservation of heterozygosity of the unisexual form. On the other hand, in numerous nuclei without chromosome duplications, we showed homoeologous synapsis for both unisexual species examined: D. unisexualis (Spangenberg et al., 2020, 2021)d armeniaca (Fig. 1A,B; Figure SM1A,B). Moreover, we showed loading of the MLH1 protein associated with crossing over into bivalents of homeologues (Spangenberg et al., 2021). And finally, we previously revealed weak meiotic checkpoints in Darevskia hybrid individuals, allowing even triploid males to produce numerous mature but aneuploid spermatids (Spangenberg et al., 2017).

Thus, there is a fairly high probability that oocytes without endoduplication are able to overcome the synaptic checkpoints of meiosis in parthenogenetic Darevskia. What happens next to these oocytes is unknown. If natural populations of unisexual Darevskia species contain individuals born as a result of a central fusion mechanism, then we should expect deviations from strict clonality and some polymorphism in the populations. According to some studies, all unisexual species of Darevskia and especially D. armeniaca demonstrate some diversity in their morphology. This issue requires detailed analysis in future. In other words, if the appearance of several clones within one parthenogenetic species could be the result of rare cases of automixis through the central fusion (fusion of meiotic products), and not pre-meiotic endoduplication?

Developmental deviation in parthenogenetic species of the genus Darevskia.

Numerous findings of embryonic abnormalities have been documented for parthenogenetic Darevskia rock lizards (Darevsky 1960; Darevsky and Kulikova 1961, Danielyan 1970). Data from previous studies indicate that these developmental deviations (up to 10 different types) occur quite often 3-6.8% in unisexual species of genus Darevskia (4.5–4.9% for D. armeniaca) compared to bisexual species (1.1–1.8%) (Danielyan 1970).

Also, during the eggs incubation in our current work, out of 14 clutches studied, several of the eggs stopped developing, and two cases were associated with the inability of the embryo to hatch from the egg. This is consistent with previous findings of developmental embryonic abnormalities, previously described in the classification suggested by I.S. Darevsky: unclosed body cavities, complete absence of the lower jaw, disproportion and curvature of the jaws (Darevsky, 1960; Danielyan 1970). We have documented two cases of the same abnormalities in late embryos (Figure SM4А,B). These data may indicate a high risk of errors in oogenesis during the process of endoduplication. Further comparative studies of the karyotypes of such individuals may help in understanding which of the mechanisms of ploidy restoration or incorrect synapsis between duplicated or homeologous chromosomes took place in such cases.

In general, one thing is clear: the mechanisms of ploidy restoration in parthenogenetic Darevskia also carry increased risks of the formation of non-viable embryos (Figure SM4А,B). Whether they are associated with the formation of aneuploid karyotypes or loss of heterozygosity are questions for further studies.

Competing interests.

The authors declare no competing interests.

Funding.

This research was funded by the State Contract of VIGG RAS 0092-2022-0002 (cytogenetic experiments), the Science Committee of RA, in the frames of the research project № 23IRF-1F06 (microscopy), Russian National Foundation under Grant Agreement No 22-14-00227 (expeditions and keeping the animals).

Author contributions.

V.S. and A.M. conceived the study. A.M. performed collecting of animals. V.S. performed keeping of parthenogenetic lizards, incubation of eggs, obtaining of meiotic preparations, microscopy, cytogenetic analysis of primary oocytes. S.A.S and Y.D. performed obtaining of mitotic chromosomes preparations, FISH experiments and cytogenetic analysis. V.S. wrote the paper, with contributions from and S.A.S., O.K., A.M., E.K. who helped finalize the text.

Acknowledgments.

The authors would like to thank Sargis Aghayan, Konstantin Milto, Eduard Galoyan, Oleg Nikolaev, Eugene Iryshkov, Natalia Sopilko, Eugene Krysanov. We thank the Genetic Polymorphism Core Facility of the Vavilov Institute of General Genetics of the Russian Academy of Sciences, Moscow.

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Tendency towards clonality: deviations of meiosis in parthenogenetic Caucasian rock lizards

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Introduction

Materials and methods

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Isolation of primary oocytes

Immunostaining

Mitotic Chromosome Preparation, C-banding and Telomere repeats FISH mapping

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