Rb translocations affect the genome by modifying gene positions and altering recombination during meiosis. Overall, our findings present experimental evidence supporting assumptions that chromosomal rearrangements redesign a genome and may contribute to speciation due to meiotic alterations in hybrids. We have demonstrated that hybrids with the same diploid number and identical chromosome combination could be sterile (interspecific) or have reduced fertility (intraspecific), a condition that is in line with the behavior of chromosomes in meiosis. Despite the fact that both types of hybrids had similar irregularities during prophase I, there were different synaptic patterns in SC trivalents, especially specifics of centromeric segments. We hypothesize that these differences originated due to altered meiotic architecture and could to be responsible for the species divergence.
3.1. Chromosome Synapsis Instability and Reduced Recombination in Hybrids
There is a huge amount of data and knowledge that allows us to unambiguously state that hybrids possess distinct patterns of chromosomal synapsis and recombination compared with their parents, which have a significant evolutionary output [
35,
63,
64]. Intra- and interspecific mole vole hybrids with 10 trivalents showed variation in fertility, as has been shown for heterozygous lemurs. Indeed, the first known case of hybrids with numerous Rb chromosomes was described for lemurs [
65]. At that time, researchers believed that hybridization occurred between lemur subspecies [
66]. According to the modern taxonomy [
67,
68], interspecific lemur hybrids were studied in 1988. Lemurs with 3–6 trivalents had fertility similar to their parents, while lemurs with eight trivalents had reduced fertility [
65,
66].
The first group of lemurs did not have any associations of bivalents and trivalents with each other and with XY. The second group regularly demonstrated chains of SC trivalents, combined by heterosynapsis of the short arms of acrocentrics, and association with the sex bivalent [
66]. A large number of trivalents were likely unable to complete synapsis in time, and this deficit could lead to aberrant chromosome segregation, arrest of cells at M1 or M2 stages, germ cell aneuploidy, and decreased fertility. We observed the synapsis delay for SC trivalents in intraspecific mole vole hybrids; this finding is consistent with previous studies [
45,
53]. However, in contrast to lemur hybrids, in intraspecific mole vole hybrids, there were often gaps in the axial elements of the metacentrics in open SC trivalents [
45,
53]. Based on immunostaining, now we interpret such specific gaps as stretching of the centromeric regions.
Mus musculus domesticus that were heterozygous for Rb metacentrics were also highly variable in their fertility [
57,
69]. Thus, in some intraspecific mice hybrids, for example, heterozygous for three Rb translocations, there was no decrease in fertility, although there were “XY–trivalent” and “trivalent–trivalent” associations [
70]. In other cases, comparative analysis revealed differences in fertility level due to distinct karyotype structures: a slight decrease (four SC trivalents) and a significant decline in fertility (seven SC trivalents) [
71]. Possibly, the difference was due to higher associations of SC trivalents with sex XY bivalent and associations of trivalents with each other in animals of the second group.
In other experiments, fertile hybrids heterozygous for eight Rb translocations had open and closed SC trivalents [
72]. The authors suggested that the meiotic progression of cells with an asynaptic area of SC trivalents was due to the insignificant genetic value of the unsynapsed chromatin regions, the inactivation of which did not lead to the activation of the pachytene arrest program.
Hybridization of different forms of the musk shrew (
Suncus murinus) could lead to the formation of five SC trivalents in meiocytes. Some of the F1 hybrids were sterile, while others were fertile, depending on different parental combinations. Researchers have suggested that genetic factors play a crucial role in determining fertility/sterility [
73,
74]. In the case of mole vole hybrids, animals regularly had impaired fertility (intraspecific) or were sterile (interspecific), although this fact does not exclude the effect of unknown genetic factors in determining the fertility level.
Alterations in recombination might be crucial for species evolution [
75,
76]. Physical problems in the synapsis of rearranged homologs can restrict the formation of recombination sites [
77,
78], which can lead to univalence, unbalanced chromosome segregation and selection of germ cells [
79,
80,
81]. Comparison of distinct mole vole hybrids reliably demonstrated that recombination was reduced in hybrids, a phenomenon that was most likely correlated with a delay in synapsis of SC trivalents or their physical stretching. It is likely that some of the achiasmatic chromosomes in trivalents can be incorrectly segregated, and such cells will be eliminated in meiotic checkpoints [
82].
3.2. Centromere Identity and Stretched Centromeres of the SC Trivalents
Centromeres are unique chromosomal regions that organize the assembly of the kinetochore, a large multiprotein complex that allows chromosomes to attach to spindle microtubules and move during mitotic and meiotic cell division [
83,
84]. There is no universal DNA sequence responsible for centromere formation. This fact, and the emergence of new centromeres, led to the hypothesis that centromeres are determined by accumulation of tandem repeats (satellite DNA) [
85,
86,
87] and retrotransposons [
88,
89] alongside epigenetic modifications such as histone variants [
90,
91]. The centromere-specific variant of histone H3, CENPA, is a platform for protein assembly at the kinetochore [
92,
93]. The rapid diversification of centromeres has been suspected to lead to reproductive isolation between species [
85]. The position of the centromere is easily identified by routine staining; in immunostaining centromeres typically appear as one-spotted signals within chromosomes.
In mole voles, closed SC trivalents developed three contact points with the nuclear envelope: (1) the attachment point of the proximal ends of acrocentrics, formed by their short arms, and (2 and 3) two attachment points of the distal telomeres of metacentrics and acrocentrics (see
Figure 3F). As prophase I progresses, the attachment points move away from each other; therefore, SC trivalents can have different spatial configurations [
94]. However, open SC trivalents must be associated with the nuclear envelope at four points. In addition, acrocentrics were ectopically connected by short SCs with neighboring acrocentrics. There were multiple interlockings in interspecific mole vole hybrids. All these specifics caused the strongest tension of chromosomes involved in SC trivalents, stretching of the centromeric regions of the Rb metacentrics, and in some cases, the centromeric regions of the acrocentrics.
We used different antibodies against kinetochore proteins to identify centromeric regions of meiotic chromosomes in mole vole parental species and hybrids (
Figure S4). As noted above, we observed 3 types of SC trivalents in hybrid spermatocytes (
Figure 4). Thus, the distance between the attachment points determined the ultrastructural organization of SC trivalents. These attachment points were most clearly manifested in closed free SC trivalents (
Figure 4C). If the distance between the attachment points of the SC trivalent to the nuclear envelope was markedly greater than the metacentric length, then the metacentric axis undergoes strong stretching, and this did not allow the formation of a continuous axial element. Therefore, electron microscopic examination revealed a gap in the structure of the stretched axial element of the metacentric (
Figure 4B). However, when immunostaining with antibodies to kinetochore proteins (ACA, CREST, and CENPA), there were no gaps, and a centromeric linear structure was visible in this area (
Figure 4B). If the attachment points were located very far from each other (on different sides of the nucleus), then the centromeric region was hyperstretched up to gaps (
Figure 4A).
Such stretched centromeres are intriguing because no similar regions have been found within SC trivalents before the present study. It is possible that these centromeric stretches are associated with the structural specifics of the pericentromeric chromatin. Chromatin in the centromeric region has the classic epigenetic marks of constitutive heterochromatin: H3K9me2, H3K9me3, H3K27me3, and H4K20me3 [
95,
96]. For example, in the study of unfolded pericentromeric heterochromatin (prekinetochores) in interphase, chromosomes subjected to stretching by TEEN buffer, there was alternation of the CENPA and H3K9me3 subdomains with the gaps [
97]. If any natural or artificial extension does not lead to structural breaks, it likely entails a linear spatial unfolding of the structural components of pericentromeric heterochromatin. This phenomenon probably explains why we saw an alternating change of centromeric points, centromeric lines, and gaps in the metacentrics of SC trivalents. This feature might indicate high plasticity of mole vole pericentromeric heterochromatin.
Stretching of the centromeric regions of acrocentrics between SC trivalents is even more mysterious. This phenomenon was rarer than centromeric stretching of metacentrics. We suppose that this may also be due to a special stretching of the pericentromeric heterochromatin of the acrocentric short arms associated in chains of several SC trivalents. Moreover, heterochromatin was involved in the nonhomologous synapsis of the short arms of the acrocentrics of SC trivalents and in binding to the nuclear envelope in intraspecific hybrids (
Figure S9). The centromeric associations of SC trivalents are of particular interest (see “Nuclear architecture: simulated chromosome configurations in mole vole pachytene spermatocytes” below).
In general, the presence of stretched centromeric regions may indicate specific properties of centromeres in the genus
Ellobius. Stretched centromeres in bivalents as a likely result of Rb chromosome fusion have been identified in African pygmy mouse [
98]. Centromeric satellites in animals and plants undergo rapid evolution [
85], and they may differ even in closely related species [
99,
100,
101]. These differences have been explained by the “library hypothesis” [
102]: an ancestral form has an initial satellite pool (“library” of satellites), which in different evolutionary lineages manifests in various patterns (in quantity and quality), thus forming species-specific satellite profiles [
103]. The hypothesis is supported by some examples [
104]. In addition, it has been established that closely related species may differ in their retrotransposons. A classic example is a well-studied kangaroo. In interspecific kangaroo hybrids, there was centromere destabilization, which was caused by the activation of resident retroelements (in this case, kangaroo endogenous retrovirus (KERV)) [
88,
105,
106].
It would seem that such important chromosome elements like centromeres should be conserved, but they exhibit incredible structural and functional variability and dynamic evolution and are hotspots for chromosomal rearrangements [
20,
107]. The main remaining issues include the question of the true reasons for the centromeric region stretching in
Ellobius hybrids as well as the molecular mechanisms of their striking plasticity. The study of centromeric satellites and retrotransposons in the
Ellobius genus will be promising. It is obvious that additional detailed study of centromeric regions in
Ellobius, including stretched centromeres and different centromeric localization of the heteromorphic chromosome #7 pair in interspecific hybrids, will be necessary. The centromere features established here and data from previous works [
50,
52,
60,
61] may suggest that the centromeres in mole voles, if not a driver of chromosomal evolution, are essential for karyotypic divergence.
3.3. Chromosome #7: Implications from the Hybrid Meiotic Nuclei Studies
As mentioned above, the parental 54-chromosome karyotypes of
E. talpinus and
E. tancrei differ in only one pair of chromosomes (#7). In these species, chromosome #7 pairs were identical in G-bands, but in
E. talpinus it is an acrocentric, and in
E. tancrei it is a submetacentric. For a long time, it was assumed that the submetacentric emergence was due to pericentric inversion [
108]. However, the SC study in interspecific hybrids clearly showed that this chromosome pair is completely synapsed without inversion loops, forming a full-length SC along the entire chromosome at the early-to-mid pachytene stage. We previously suggested that the submetacentric in
E. tancrei emerged through the neocentromere formation [
60,
61]. This assumption was confirmed by additional Zoo-FISH data [
50], and later by immuno-identification of the central element of SCs and recombination nodules [
52].
The chromosome #7 pair was used as a marker bivalent in the SC analysis. In interspecific hybrids, this was the only heteromorphic SC bivalent with two centromeres located at a distance from each other. Of note, we discovered another remarkable property of the SC #7. In both hybrids, bivalent #7 practically does not participate in chromosome associations: There was only one association of this chromosome with the open SC trivalent (
Figure S4E). We assume that the inertness of chromosome #7 in meiosis may be due to the absence or extremely low content of constitutive heterochromatin. This was confirmed using antibodies to histone H3K9me3, a marker of constitutive heterochromatin, both in bivalent #7 in
E. tancrei (unpublished) and in a sibling species
E. alaicus [
109].
Thus, the ancestors of modern E. tancrei and E. alaicus, concomitant with the emergence of the neocentromeric submetacentric chromosome #7 pair, probably acquired chromosomal instability with the formation of various karyotypic forms, which may indicate some causal link between these events. Perhaps one of them could become an evolutionary trigger, which entails a chain of genetic and/or cytogenetic changes in the mole vole karyotype.
3.4. Nuclear Architecture: Simulated Chromosome Configurations in Mole Vole Pachytene Spermatocytes
The organization of the internal contents is not random in interphase [
110,
111] and meiotic [
112] nuclei; therefore, it is customary to speak of nuclear architecture [
113] or intranuclear landscape [
114]. The “chromosome territory” concept is considered to be generally accepted [
115]. Chromosome territories are spatial domains of different sizes that specifically occupy a certain volume in the nucleus [
116]. The position of chromosomes in the nucleus can be preserved in closely related taxa [
117], a phenomenon called “phylogenetic memory” [
118]. Heterochromatic compartments play a significant role in the nucleus content [
119]. However, gene mutations and chromatin and chromosomal rearrangements can change the intranuclear organization, which can lead to diseases and the appearance of various abnormal manifestations [
120,
121,
122,
123,
124,
125].
There is also a point of view, according to which the correct formation of chromosome territories diminishes the translocation potential of the cells [
126]. The organization of interphase and meiotic nuclei has significant differences [
127]. An SC forms a specific chromatin pattern [
128] and interacts with the nuclear envelope through a special Sun–KASH system [
129]. The presence of asynaptic chromosome regions in prophase I initiates a meiotic silencing of unsynapsed chromatin (MSUC) [
130,
131]. Nuclear architecture of prophase I meiocytes is specific for each species [
132,
133,
134].
As a result, the nuclear architecture in meiotic prophase I is determined by the SC structure and dynamics, types of chromosomes (single-armed or bi-armed), their length, the heterochromatin amount, the specificity of centromeric regions, the “chromosome–nuclear envelop” interactions, the ability to form chromocenters and nucleoli, and sex chromosome organization and behavior [
134]. It should be emphasized that if the parental genomes differ significantly, then complex chromosome compounds are formed in hybrid and mutant meiotic nuclei (for example, [
21,
135,
136,
137]), and the processes of repair, recombination, and meiotic silencing are disrupted (for example, [
24,
138,
139,
140]), which can cause an imbalance in the nuclear architecture.
In two hybrid mole vole groups, a different number of free closed SC trivalents formed. Pericentromeric heterochromatin likely played an important role in the formation of closed trivalent configurations, as described in heterozygous mice [
94]. Thus, on the three-dimensional nuclei of interspecific mole vole hybrids, there were closed SC trivalents, which were attached by the short arms of acrocentrics to the nuclear envelope, and open SC trivalents with stretched centromeric regions (CREST cloud around) (
Figure S8). CREST antibodies can non-specifically immunostain heterochromatic regions and heterochromatin-like structures, for example, ChBs (
Figure 2H and
Figure 3D). The same specificity was revealed during the electron microscopic examination of the intraspecific heterozygous spermatocytes. At the attachment site of the short arms of the SC trivalent, a cloud of electron-dense material formed, associated with the nuclear envelope, which was usually interpreted as heterochromatin mass (
Figure S9, schemes of hemispheres in
Figure 3F).
SC trivalent chains were demonstrated in intraspecific mole vole hybrids [
45,
53]. Such ectopic associations formed due to the heterochromatic contacts of the short arms of acrocentrics of two different open SC trivalents (
Figure S7), as had been noted earlier [
46]. Chromosome associations can be determined by H3K9me3 immunodetection [
94]. This protein is believed to mark DAPI-positive chromocenters (for example, [
141]). However, chromocenters in all mole voles were usually not detected by DAPI staining. Nevertheless, we found that acrocentric chromosomes were grouped by their pericentromeric regions around the H3K9me3 domains in the sibling species
E. alaicus [
109]. We saw a similar grouped position of acrocentrics in
E. talpinus (
Figure 2A). In
E. tancrei (2
n = 34), the centromeric sites of the acrocentrics were also located around the H3K9me3 clouds, while these sites of the Rb metacentrics had a more linear form of the H3K9me3 signals, as seen in the micrographs (
Figure S9A,A′).
Combining the results on the ultrastructure and behavior of SCs and SC trivalents in spreads and squashes, we present these data as three-dimensional simulations of chromosome configurations in the meiotic nuclei of mole vole parental species and hybrids (
Figure 6).