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1 Meiotic nuclear architecture in distinct mole vole hybrids with 2 Robertsonian translocations: chromosome chains, stretched 3 centromeres, and distorted recombination 4 5 Sergey Matveevsky 1*, Artemii Tretiakov 1, Irina Bakloushinskaya 2, Anna Kashintsova 1, Oxana 6 Kolomiets 1* 7 8 1 Department of Cytogenetics, Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia, 9 2 Department of Genome Evolution and Mechanisms of Speciation, Koltzov Institute of Developmental Biology, Russian 10 Academy of Sciences, Moscow, Russia 11 12 * [email protected] (SM), [email protected] (OK) 13 14 Abstract 15 16 Genome functioning in hybrids faces inconsistency. This mismatch is manifested clearly in meiosis during 17 chromosome synapsis and recombination. Species with chromosomal variability can be a model for exploring 18 genomic battles with high visibility due to the use of advanced immunocytochemical methods. We studied 19 synaptonemal complexes (SC) and prophase I processes in 44-chromosome intraspecific (Ellobius tancrei × E. 20 tancrei) and interspecific (Ellobius talpinus × E. tancrei) hybrid mole voles heterozygous for 10 Robertsonian 21 translocations. The same pachytene failures were found for both types of hybrids. In the intraspecific hybrid, the 22 chains were visible in the pachytene stage, then 10 closed SC trivalents formed in the late pachytene and diplotene 23 stage. In the interspecific hybrid, as a rule, SC trivalents composed the SC chains and rarely could form closed 24 configurations. Metacentrics involved with SC trivalents had stretched centromeres in interspecific hybrids. Linkage 25 between neighboring SC trivalents was maintained by stretched centromeric regions of acrocentrics. This 26 centromeric plasticity in structure and dynamics of SC trivalents was found for the first time. We assume that 27 stretched centromeres were a marker of altered nuclear architecture in heterozygotes due to differences in the 28 ancestral chromosomal territories of the parental species. Restructuring of the intranuclear organization and meiotic 29 disturbances can contribute to the sterility of interspecific hybrids, and lead to the reproductive isolation of studied 30 species. 31 32 Author summary 33 34 Meiosis is essential for sexual reproduction to produce haploid gametes. Prophase I represents a crucial meiotic 35 stage because key processes such as chromosomal pairing, synapsis and desynapsis, recombination, and 36 transcriptional silencing occur at this time. Alterations in each of these processes can activate meiotic checkpoints 37 and lead to the elimination of meiocytes. Here we have shown that two groups of experimental hybrids, intraspecific 38 and interspecific—which were heterozygous for 10 identical Robertsonian translocations—had pachytene 39 irregularities and reduced recombination. However, intraspecific and interspecific hybrids exhibited different 40 patterns of synaptonemal complex (SC) trivalent behavior. In the former, open SC trivalents comprised SC chains 41 due to heterosynapsis of short arms of acrocentrics in early and mid-pachytene and were then able to form 2–4 and 42 even 7 and 10 closed SC trivalents in the late pachytene and diplotene stages. In the second mole voles, SC 43 trivalents had stretched centromeres of the metacentrics, and chains of SC trivalents were formed due to stretched 44 centromeres of acrocentrics. Such compounds could not lead to the formation of separate closed SC trivalents. The 45 distant ancestral points of chromosome attachment with a nuclear envelope in the heterozygous nuclei probably lead 46 to stretching of SC trivalents and their centromeric regions, which can be regarded as an indicator of the 47 reorganization of the intranuclear chromatin landscape. These abnormalities, which were revealed in in prophase I, 48 contribute to a decrease the fertility of intraspecific mole voles and promote the sterility of interspecific mole voles. 49 50 Introduction 51 52 Genome integrity is crucial for a species; its specificity is supported by reproductive isolation. 53 Hybrid sterility may develop between species or genetically differentiated populations, as a primary or 54 secondary feature of reproductive isolation (Dobzhansky, 1937, Benirschke, 1967, Basrur, 1969, White, 55 1977, Pavlova and Searle, 2018). Two genetic materials in heterozygous admixed genome interact in 56 various compositions (Runemark et al., 2019) and these hybrid states are often referred to as "genomic 57 conflict" (Johnson, 2010, Crespi and Nosil, 2013), "genomic shock" (McClintock, 1984, Ha et al., 2009), 58 "genomic stress" (McClintock 1984, Petrov et al., 1995), or "nucleus at war" (Jones and Langdon, 2013). 59 60 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.301473; this version posted September 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

61 62 Fig 1. Scheme of the experimental hybridization of Ellobius species and hybrids. (A–C) Karyotypes for E 63 talpinus, 2n = 54, NF = 54 (A); E. tancrei, 2n = 54, NF = 56 (B); and E. tancrei, 2n = 34, NF = 56 (C). (D, E) F1 64 hybrid karyotypes for E. tancrei × E. tancrei, 2n = 44, NF = 56 (D) and E. talpinus × E. tancrei, 2n = 44, NF = 55 65 (E). (F, G) Chromosome heteromorphic configurations in meiotic prophase I of F1 hybrids: 10 trivalents (F), and 10 66 trivalents and the heteromorphic chromosome #7 (G). 67 68 Hybrid incompatibility (Watkins, 1932, Dobzhansky, 1937, Muller, 1942) is expressed either by 69 visible or cryptic changes in the phenotype (Hale et al., 1993, Foreit, 1996, Ishishita et al., 2015) or 70 disturbances in chromosome sets and gene expression and alterations in the gene networks (Haerty and 71 Singh, 2006, Landry et al., 2007). Epistatic interactions between chromosomal regions (heterochromatin 72 blocks, centromeres, and telomeres), satellite DNA, small RNA, and epigenetic chromatin modifications 73 (Brown, O'Neill, 2010) are essential for the emergence of Dobzhansky–Muller incompatibility. Hybrid 74 and heterozygous animals are excellent models for studying the effect of chromosomal rearrangements on 75 the development of the organism, cellular function, intracellular structures, germ cell formation— 76 including the most important stage, meiosis—and, in general, chromosomal evolution. Chromosome 77 differences in hybrid meiocytes can manifest as various irregularities in chromosome synapsis, 78 recombination, chromatin landscape, and transcriptional inactivation (Johannisson and Winking, 1994, 79 1998, Sharma et al., 2003, Bhattacharyya et al., 2013). 80 Chromosome heterozygosity can be studied either in laboratory hybrids (Forejt et al., 1980, Gill, 81 1980, Matsuda et al., 1992, Matveevsky et al., 2014, Gureeva et al., 2016) or in natural hybrids, for 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.301473; this version posted September 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

82 example, from hybrid zones (Ivanitskaya et al., 2010, Matveevsky et al., 2012, Lin et al., 2018). 83 Experimental hybridization allows researchers to obtain offspring from parental forms with known 84 karyotypic data, which facilitates a more accurate assessment of the effect of structural hybridity on the 85 processes of cell division, morphology, and fertility (Chang et al., 1969, King, 1993). 86 Mole voles are gratifying sources for experimental hybridization. One of the three cryptic species 87 has a stable karyotype, namely Ellobius talpinus (2n = 54, NF = 54), and no natural hybrids are known 88 (Lebedev et al., 2020). Two other species have Robertsonian (Rb) chromosome variability: Ellobius 89 tancrei (2n = 54–30, NF = 56) and Ellobius alaicus (2n = 52–48, NF = 56) (Vorontsov et al., 1969; 90 Lyapunova et al., 1984, 2010, Bakloushinskaya et al., 2013, 2019). These species demonstrate intra- and 91 interspecific hybridization (Bakloushinskaya et al., 2019, Romanenko et al., 2019). The 54-chromosome 92 karyotypes of E. talpinus and E. tancrei are identical and differ only in chromosome #7 due to centromere 93 repositioning (Romanenko et al., 2007). Ten pairs of bi-armed (Rb) chromosomes are a feature of the 34- 94 chromosome karyotype of E. tancrei (Romanenko et al., 2019) (Fig 1). Karyotypic variability allows 95 researchers to simulate different natural chromosomal combinations in experimental hybrids (Kolomiets 96 et al., 1983, 1985, 1986, Lyapunova and Yakimenko, 1985, Lyapunova et al., 1990, Bakloushinskaya et 97 al., 2010, 2012, Matveevsky et al., 2015, 2017). The first description of chromosome chains was made for 98 an intraspecific E. tancrei hybrid heterozygous for 10 Rb translocations (Kolomiets et al., 1985, 99 Bogdanov et al., 1986). It would be extremely interesting to compare the heterozygotes with the same 100 diploid numbers obtained from crossing E. tancrei chromosomal forms between themselves and between 101 two species, E. talpinus and E. tancrei. 102 In this study, we first compared chromosome synapsis and recombination in intra- and 103 interspecific hybrids with the same chromosome number and with numerous translocations. We analyzed 104 44-chromosome F1 hybrids, intraspecific E. tancrei × E. tancrei, and interspecific E. talpinus × E. 105 tancrei, heterozygous for 10 identical Rb translocations (Fig 1, compare D and E). Each of the hybrids is 106 expected to form 10 identical SC trivalents. A comparative analysis of chromosome synapsis, 107 recombination, and meiotic silencing in the fertile and sterile hybrids allowed us to assess the effect of 108 chromosome rearrangements on these processes, as well as to draw conclusions about the degree of 109 species divergence. 110 111 112 Results 113 114 Experimental hybridization 115 116 We studied parental mole vole species—E. talpinus (2n = 54, NF = 54), E. tancrei (2n = 54, NF = 117 56), and another form of E. tancrei (2n = 34, NF = 56)—and F1 hybrids—intraspecific E. tancrei × E. 118 tancrei (2n = 44, NF = 56) and interspecific E. talpinus × E. tancrei (2n = 44, NF = 55) (Fig 1). 119 The karyotypes of interspecific and intraspecific hybrids differ little from each other. They 120 include 5 pairs of acrocentrics, 10 metacentrics and 20 acrocentrics, which are homologous to the arms of 121 metacentrics, pair #7 and a pair of isomorphic sex (XX) chromosomes. Two types of hybrids differ only 122 by the chromosome #7 pair. In the interspecific hybrid, this pair is heteromorphic (Fig 1), while in the 123 intraspecific hybrid, it is represented by a pair of homologous submetacentrics. In total, we detected 10 124 SC trivalents, 6 SC bivalents, including a heteromorphic SC bivalent in the interspecific hybrid, and a sex 125 XX bivalent in meiotic prophase I in spermatocyte spreads and squashes of both hybrids (Fig 1). 126 The gonadosomatic index (GSI) can be used as an indicator of the state of the reproductive 127 system. It is calculated as the ratio of the weight of the testes to the weight of the body (Adebayo et al., 128 2009). This parameter is species specific; varies depending on age, stage of development, sex, and 129 breeding season (Pochron et al., 2002); and may reflect the rates of sperm production as well as sperm 130 function (Gomendio et al., 2006). Comparison of the parameter in closely related forms, species, and 131 hybrids can be informative. Hence, we calculated the GSIs in adult mole voles, parental species, and 132 hybrids. GSI (mean [M] ± standard deviation [SD]) in the interspecific hybrid (0.11 ± 0.04, n = 3) was 133 approximately half of the E. talpinus GSI (0.20 ± 0.02, n = 3) and approximately one third of the E. 134 tancrei (2n = 34) GSI (0.31 ± 0.10, n = 3). Species and hybrids significantly differ from each other in this 135 parameter (p < 0.05). This indicator suggests that interspecific hybrids have reproductive dysfunction. 136 Unfortunately, we do not have data on GSI of intraspecific hybrids. 137 138

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139 140 Fig 2. Chromosome synapsis and irregularities in pachytene spermatocytes of Ellobius species and hybrids. 141 The green numbers in the micrographs and diagrams correspond to parental species and hybrids (see captions under 142 diagram K). Chromosome #7 was clearly identified in all nuclei (A–H). (A) Ellobius talpinus: 26 synaptonemal 143 complexes (SCs) and 1 XX bivalent. (B) E. tancrei (2n = 54): 26 SCs and 1 XX bivalent. (C) E. tancrei (2n = 34): 144 16 SCs and 1 XX bivalent. (D–F) Intraspecific hybrid E. tancrei × E. tancrei (2n = 44): No nucleus had the 145 expected chromosome formula (6 SCs, 1 XX bivalent, 10 SC trivalents). There were several SCs, 3–5 free SC 146 trivalents, a XX bivalent, and SC trivalents in chains. (G, H) Interspecific hybrid E. talpinus × E. tancrei (2n = 44): 147 Expected chromosome formula: 6 SCs, 1 XX bivalent, 10 SC trivalents. One nucleus contained 3 SCs, 1 XX 148 bivalent, 1 SC trivalent with a stretched centromere (white star), and SC trivalents in a chain (G). The other nucleus 149 had 6 SCs, 1 XX bivalent, and SC trivalents in a chain (no free SC trivalents) (H). Some metacentrics in the SC 150 trivalents had a stretched centromere (blue arrowheads). (I) The number of cells in which the XX bivalent moved to 151 the periphery of the nuclei: Hybrids had significantly lower rates compared with the parent species (see Table S1). 152 (J) The number of cells with an open XX bivalent: This parameter was much higher in hybrids than in parents 153 (Table S1 indicates significant differences). This parameter increased from intraspecific hybrids to interspecific 154 hybrids (differences between them were not significant). For examples of open XX bivalent, see D, E, and H. (K) 155 The number of cells with associations of XX with autosomes and trivalents: There were significant differences 156 between parents and hybrids, and this parameter was slightly higher in the intraspecific hybrid (2n = 44) than in the 157 other hybrid (see Table S1). An open sex bivalent associated with trivalents are shown in D, E, and H. Scale bars 158 represent 5 μm. 159

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160 There were numerous spermatocytes and mature spermatids in the testicular cell suspension of the 161 parental species and the intraspecific hybrids (Fig S1A, S1B, S1C, and S1D). In the interspecific hybrids, 162 the number of spermatocytes was significantly lower, and mature spermatids and spermatozoa were not 163 detected at all, which was confirmed by histological examination of testicular tissue sections (Fig S1B, 164 S1C, and S1D). Testicles of the interspecific hybrids were significantly smaller than in 34-chromosome 165 parental forms (Fig S1E). Testis weights (and most likely testis volumes) < 55% of normal values 166 indicate sterility (de Boer and de Jong, 1989). None of the 93 interspecific F1 E. talpinus × E. tancrei 167 hybrids produced offspring (shown graphically in Fig S1F, compare all species and hybrids). The fertility 168 data of mole voles in this work are in good agreement with the previous data (Lyapunova and 169 Yakimenko, 1985). Moreover, for the intraspecific hybrids, there was an increased time interval from the 170 moment of coupling to the first litter and an increased period between births compared with the parental 171 forms (Lyapunova and Yakimenko, 1985). Thus, the intraspecific hybrid had slightly reduced fertility, 172 and the interspecific hybrid was sterile. 173 174 Synaptic behavior of chromosomes 175 176 We first described the chromosome behavior in pachytene spermatocytes of parental forms. In E. 177 talpinus, there were 26 acrocentric bivalents and a sex (XX) bivalent, covered by a γH2AFX cloud, that 178 moved to the periphery of the nucleus (Fig 2A and S2A, S2B, and S2C). In 54-chromosome E. tancrei, 179 there were 25 acrocentric SC bivalents and 1 submetacentric SC bivalent, and a XX bivalent, covered by 180 a γH2AFX cloud, that moved to the periphery of the nucleus (Fig 2B S2D, S2E, and S2F). In 34- 181 chromosome E. tancrei, there were 10 metacentric SC bivalents, 1 submetacentric SC bivalent #7, and 5 182 acrocentric bivalents, and a XX bivalent, covered by a γH2AFX cloud, that moved to the nuclear 183 periphery (Fig 2C S2G, S2H, and S2I). 184 In an intraspecific hybrid, there were 10 closed and open SC trivalents, 5 acrocentric bivalents, 1 185 submetacentric bivalent #7, and a XX bivalent (Fig 2D, 2E and 2F S3A–, S3B, and S3C). The γH2AFX 186 cloud completely covered the XX bivalent and asynaptic regions of the open SC trivalent (Fig S2J, S2K, 187 and S2L). In the interspecific hybrid, there were 10 closed and open SC trivalents, 5 acrocentric bivalents, 188 1 heteromorphic chromosome #7 bivalent, and a XX bivalent (Fig 2G and 2H and S4A). The γH2AFX 189 cloud completely covered the XX bivalent and some asynaptiс regions of open SC trivalents (Fig S2M, 190 S2N, and S2O). 191 The XX bivalent, as a rule, had two telomeric synaptic sites and a large central asynaptic area 192 (closed configuration). A chromatin-dense body (ChB) was formed in one of the axial elements of the 193 XX' extended area of asynapsis (Fig S3D and S4C) (Kolomiets et al., 1991, Matveevsky et al., 2016). 194 One of the synaptic sites may have been absent when the axis or axes were associated with SC trivalents 195 or bivalents (open configuration of XX bivalent). 196 In mammals, sex bivalents are usually moved to the periphery of meiotic nuclei and covered in a 197 cloud of protein inactivators of asynaptic chromatin. In hybrids, the XX bivalent was significantly less 198 likely to move to the nucleus periphery than in the parents. However, the difference between intra- and 199 interspecific hybrids was not statistically significant (Fig 2I, Table S1[2]). The number of open XX 200 bivalents in hybrids was higher than in parents; the difference between parents and hybrids was 201 significant, but not significant within each of the groups (Fig 2E, 2H, and 2J, S1(3) Table, S3C, S3E and 202 S4F). 203 204 Synapsis defects and associations of the XX and SC trivalents in hybrids 205 206 Chromosome synapsis defects included atypical lengthening of the short arms of acrocentrics of 207 the SC trivalent in both hybrids (Fig 3H, S3B, and S4H), as well as atypical lengthening of one of the 208 synaptic regions of the sex bivalent in the intraspecific hybrid (Fig 3H). This lengthening occurred due to 209 a slight stretch at the centromeric region. 210 The interspecific hybrid also exhibited atypically large XX bivalents (Fig 2G) and ring univalents 211 at the pachytene stage (Fig S4I). The intraspecific hybrid showed a triple synapsis in the region of the 212 short arms of the SC trivalent (Fig S3G). 213 Associations of the XX bivalent with autosomes were rare and only observed in two forms of E. 214 tancrei nuclei: 5.9% (2n = 54) and 2.25% (2n = 34). In both F1 hybrids, associations of the sex bivalent 215 with autosomes/trivalents were much more common: 50.37% (intraspecific hybrid) and 45.34% 216 (interspecific hybrid) of the nuclei had these features (Fig 2K, Table S1[4]). Examples of XX bivalent

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217 association with SC trivalents are presented in several of the figures (intraspecific: Fig 2D, 2E and 2F, 218 S3A, S3B, S3C, and S3E; interspecific: Fig 2H, S4A, and S4B). It should be emphasized that in 219 interspecific hybrids, there were associations of one of the X axes with an autosome or trivalent by a thin 220 CREST-positive linear link (Fig S4B and S4D), as well as an association through a ChB (Fig 3C). 221 222 Free closed SC trivalents and open SC trivalents in chains 223 224 The intra- and interspecific hybrids differed in the number of formed free closed SC trivalents. 225 Based on the karyotypes, we assumed that at the pachytene stage of both hybrids, 10 metacentrics and 20 226 acrocentrics should have formed 10 SC trivalents. However, in intraspecific hybrids, 1–4 free closed SC 227 trivalents were most often found (Fig 3A, 3B, 3G, and S3F). It should be emphasized that in single nuclei, 228 we found 7 and 10 SC trivalents (Fig 3E, 3F, S3C and S3H). In one of the animals, there were closed SC 229 trivalents with SYCP3- and AgNO3-positive dense material in the region of the short arms of the 230 acrocentrics (Fig S3H, S3H', S3H", S3I, S3J, S3J', and S3K) or in the pericentromeric region of SC 231 trivalents (Fig S3L and S3L'). Open SC trivalents formed SC chains due to heterosynapsis between the 232 short arms of acrocentrics (Fig S3A and S3B). 233 Free closed SC trivalents, as a rule, were not detected in spermatocytes of interspecific hybrids 234 (Fig 3E, 3F, 3K, S4A, S4J, S4K, and S4L, Table S1[5]), i.e., they remained open and embedded in SC 235 chains. There was 1 closed SC trivalent in 21% of nuclei (Fig 3H), as well as in squashes (Fig S8). There 236 were 2 (Fig 3C) or 4 (Fig 3D) SC trivalents in single cells (Fig 3F). In the chains of the SC trivalents, the 237 centromeric sites of the metacentrics were strongly stretched (see next section). 238 239 Stretched centromeres in SC trivalents and their participation in SC trivalents chains 240 241 In the nuclei of cells from both hybrids, there was stretching of the centromeric regions of the 242 metacentrics of the SC trivalents (intraspecific: Fig 3A and 3G; interspecific: Fig 3C, 3D, 3H, and 3K). 243 However, we only observed this phenomenon in 16% of pachytene cells from intraspecific hybrids, and 244 their length was less than 3 µm (Fig 3I and 3J). By contrast, in interspecific hybrids, we observed 245 centromere stretching in 70% of spermatocytes. In most cases the length was > 3 µm (Fig 3I and 3J, 246 Table S1[6]), and in some nuclei it reached ≥ 10 µm (for example, Fig 3K). In interspecific hybrids, the 247 centromeric region of the metacentrics was strongly stretched (Fig 3H, 3K, S4B, and S4F). There was 248 trivalent centromere stretching in zygotene spreads (Fig S5) and pachytene squashes (Fig S8). Squashes 249 with preserved three-dimensional nuclear space confirmed that stretched centromeres were not an artifact 250 of the spreading technique (Fig S8). 251 Of note, we detected centromere stretching in the acrocentrics of the SC trivalents, although less 252 frequently than in metacentrics (Fig S6). This feature was shown in SC trivalents, which were part of SC 253 chains (Fig. S6A, S6B, S6C, S6D, S6E, S6F, S6G, and S6I). 254 Thus, a comparison of the chains of SC trivalents allowed us to establish some differences between 255 hybrids. In intraspecific hybrids, the connection between trivalents occurred due to the short arms of 256 acrocentrics (heterosynapsis). In interspecific hybrids, the formation of such SC chains was accompanied 257 by a strong stretching of the centromeric regions of the metacentrics and acrocentrics in SC trivalents, and 258 neighboring SC trivalents can join due to the stretched centromeric regions of the acrocentrics (shown 259 schematically in Fig S7 for both hybrids). 260 In general, three main types of SC trivalents can be distinguished in mole vole hybrids (Fig 4). It 261 should be noted that we identified centromere stretching by immunostaining with both polyclonal and 262 monoclonal antibodies to centromere proteins obtained from different manufacturers (Fig S4A, S4J, S4K 263 and S4L).

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264 265 Fig 3. SC trivalents and stretched centromeres in Ellobius hybrids. (A–F) SC trivalents, free and in chains, in 266 44-chromosome intraspecific (A, B) and interspecific (C, D) hybrids. Both hybrids had variations in the number of 267 free closed trivalents (see A–D). Fewer free closed SC trivalents in the nuclei indicated that more open SC trivalents 268 were in the chains. The number of free trivalents in hybrid nuclei was significantly different: 90.6% in intraspecific 269 (n = 99) and 22% in interspecific (n = 102) hybrids (see ring diagrams in E and see Table S1). There were typically 270 1–4 free SC trivalents identified in most nuclei of intraspecific hybrids (see the light orange columns of the bar chart 271 in F). As a rule, there were no free SC trivalents in most nuclei of interspecific hybrids (see the dark-red columns of 272 the bar chart in F). Free closed SC trivalents simulated in the hemisphere (F): Black stars show proximal telomeres 273 of acrocentrics, and red stars show distal telomeres of metacentrics and acrocentrics of SC trivalents. (G–K) 274 Stretched centromeres in SC trivalents (blue arrowheads in G, H, K). The prevalence of the stretched centromeres in 275 hybrid nuclei was variable: 16% (15 nuclei from n = 96) in intraspecific and 70% (73 from n = 105) in interspecific 276 hybrid (see bar chart in I; Table S1 indicates significant differences). The length of the stretched centromeres was 277 different; there were two notable groups: (1) < 3 µm and (2) > 3 µm. Group (1) prevailed in the intraspecific hybrid, 278 while group (2) was more prevalent in the interspecific hybrid (see ring diagrams in J). (L) Prevalence of 279 chromosome #7 detected in the parent (2n = 34) and hybrids. Chromosome #7 was clearly identified in all nuclei 280 (see A–D, G, H, and K). Parents (2n = 34) always had chromosome #7 (without chromosome associations). Both 281 types of hybrids had chromosome #7 in the vast majority of pachytene nuclei (see the columns of the bar chart in L). 282 Pink arrows show associations of the sex (XX) bivalents (C, K). The white arrow shows atypical lengthening of one 283 of the XX bivalent synaptic sites (H). The yellow arrow shows atypical prolonging of short arms of acrocentrics of 284 the SC trivalent (H). The nucleus in K is presented in Fig S6A–G). Scale bars represent 5 μm. 285

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286 287 Fig 4. SC trivalent types of the Ellobius hybrids and their simulated location on a plane and a sphere. Light 288 micrographs after immunostaining (1, 2, 3, 6, 8, and 11): SCs were immunostained with antibodies against SYCP3 289 (green) and centromeres—with an antibody to kinetochores (CREST, red). Electron micrographs after AgNO3- 290 staining (4, 5, 7, 9, and 10): Blue arrowheads show centromeric regions of metacentrics in SC trivalents. M indicates 291 metacentric and A indicates acrocentric. (A) SC trivalents elongated by centromeric region stretching in 292 spermatocytes of the E talpinus × E tancrei interspecific hybrid. The length of the centromeric region of 293 metacentrics in such SC trivalents is > 3 µm. (1) SC trivalents in which short arms were not visible: If there were no 294 immunostained centromeric regions, then the SC trivalents would be identified as two pseudobivalents. (2) SC 295 trivalents with short arms of acrocentrics. (3) SC trivalents with very stretched centromere. As a rule, such SC 296 trivalents stretch across the length of the entire nucleus (from one side to the other). (B) SC trivalents with short 297 stretched centromeric regions (< 3 µm in length). This type of SC trivalent was found in all hybrids. (4) Two 298 pseudobivalents that are part of the SC trivalent. (5) The metacentric in the SC trivalent has a gap, in which there is 299 probably a stretched centromeric region. (6) SC trivalents like in 5; the stretched centromere of the metacentric are 300 visible. (7 and 8) Short SC trivalents with faintly distinguished short arms of acrocentrics and not very stretched 301 centromeres. (C) Closed SC trivalents with clearly visible short arms of acrocentrics (blue stars) and dot-like 302 centromeres (9–11): This type of SC trivalent is found in all hybrids. (D) Simulation of SC trivalents on a plane and 303 a sphere. SC trivalents of A type (see A) have far-reaching attachment points to the nuclear envelope and long 304 stretched centromeric region with gaps. SC trivalents of B and C types (see panels B and C) are more compact and 305 do not have long stretched centromeres. Scale bars represent 5 μm. 306 307 Bivalent #7 behavior 308 309 Chromosome #7 is an excellent karyotypic marker, and it can be used to identify both intra- and 310 interspecific hybrids. Based on previous research, the submetacentric in E. tancrei is formed due to the 311 neocentromere formation established by SCs analyses (Matveevsky, 2011a, 2011b) and then 312 supplemented by Zoo-FISH data (Bakloushinskaya et al., 2012). In interspecific hybrids, this 313 chromosome pair appears as an SC bivalent with two centromeres, because an acrocentric homolog (E.

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314 talpinus) and a submetacentric homolog (E. tancrei) entered into synapsis. In the vast majority of nuclei 315 in both hybrids, SC #7 was easily identified (Fig 2 and 3, Table S1[7]). There was only a clear association 316 of the SC chromosome #7 bivalent, or rather its acrocentric homolog, with the SC trivalent (Fig S4E). 317 318 Chromosome recombination 319 320 We studied meiotic recombination in Ellobius parental species and hybrids using the MLH1 321 protein, a marker of crossovers. The average number of MLH1 foci per nucleus is an important 322 recombination parameter. It can be used to compare closely related species, in homozygous and 323 heterozygous forms (Bhattacharyya et al., 2013), as well as to examine recombination in larger taxa 324 (Segura et al., 2013). In the latter case, the haploid number of autosomes (NFha) and the haploid number 325 of autosomal arms (NFha.a) are considered. 326 The number of MLH1 foci per nucleus (M ± SD) was 23.48 ± 3.3 in E. talpinus (2n = 54, NF = 327 54; NFha = 26, NHha.a = 26), 23.1 ± 3.1 in E. tancrei (2n = 54, NF = 56; NFha = 26, NHha.a = 27), and 328 22.8 ± 3.7 in E. tancrei (2n = 34, NF = 56; NFha = 16, NHha.a = 27) (Fig 5A–C). These rates were not 329 significantly different among species (Table S1). The number of MLH1 foci in all species was slightly 330 lower than the NFha.a values. Thus, not every chromosome arm has at least one MLH1 signal. We 331 previously described detection of MLH1 signals in the XX bivalent; the rate was 46% for E. talpinus and 332 65% for E. tancrei (2n = 54) (Matveevsky et al., 2016). 333 The average number of MLH1 signals was 15.4 ± 4.4 for the intraspecific hybrid (2n = 44, NF = 56; 334 NFha = 21, NHha.a = 27) and 14.8 ± 4.7 for the interspecific hybrid (2n = 44, NF = 55; NFha = 21, 335 NHha.a = 26.5) (Fig 5D and 5E). These rates differed significantly between the hybrids and compared 336 with the parents (Table S1) and were significantly lower than the NHha.a number. A decrease in the 337 recombination level in hybrid spermatocytes was due to the formation of chains of SC trivalents, where 338 the arms of acrocentrics did not have a complete synapse with the arms of metacentrics. 339 340 Discussion 341 342 Rb translocations affect the genome by modifying gene positions and altering recombination 343 during meiosis. Overall, our findings present experimental evidence supporting assumptions that 344 chromosomal rearrangements redesign a genome and may contribute to speciation due to meiotic 345 alterations in hybrids. We have demonstrated that hybrids with the same diploid number and identical 346 chromosome combination could be sterile (interspecific) or have reduced fertility (intraspecific), a 347 condition that is in line with the behavior of chromosomes in meiosis. Despite the fact that both types of 348 hybrids had similar irregularities during prophase I, there were different synaptic patterns in SC trivalents, 349 especially specifics of centromeric segments. We hypothesize that these differences originated due to 350 altered meiotic architecture and could to be responsible for the species divergence. 351 352 Сhromosome synapsis instability and reduced recombination in hybrids 353 354 There is a huge amount of data and knowledge that allows us to unambiguously state that hybrids 355 possess distinct patterns of chromosomal synapsis and recombination compared with their parents, which 356 have a significant evolutionary output (King, 1993, Arnold, 1997, Abbott et al., 2016). Intra- and 357 interspecific mole vole hybrids with 10 trivalents showed variation in fertility, as has been shown for 358 heterozygous lemurs. Indeed, the first known case of hybrids with numerous Rb chromosomes was 359 described for lemurs (Moses et al., 1979). At that time, researchers believed that hybridization occurred 360 between lemur subspecies (Ratomponirina et al., 1988). According to the modern taxonomy (Wilson and 361 Reeader, 2005, Schwitzer et al., 2013), interspecific lemur hybrids were studied in 1988. Lemurs with 3–6 362 trivalents had fertility similar to their parents, while lemurs with 8 trivalents had reduced fertility (Moses 363 et al., 1979; Ratomponirina et al., 1988). 364

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365 366 Fig 5. Recombination in Ellobius species and hybrid males. Axial elements were identified using anti-SYCP3 367 antibodies (green), kinetochores using CREST antibodies (red), and anti-MLH1 (white) was used as a marker of 368 recombination nodules. (A) E talpinus (2n = 54, NF = 54); (B) E. tancrei (2n = 54, NF = 56); (C) E. tancrei (2n = 369 34, NF = 54); (D) F1 E. tancrei × E. tancrei hybrid; (E) F1 hybrid E. talpinus × E. tancrei hybrid. (F) Comparison 370 of MLH1 foci per nucleus in mole voles: The numbers above the dot diagrams correspond to the number of counted 371 cells. White numbers in a gray frame correspond to the average number of MLH1 foci. Table S1 presents details on 372 significant statistical differences between species and hybrids. Scale bars represent 5 μm. 373 374 The first group of lemurs did not have any associations of bivalents and trivalents with each other 375 and with XY. The second group regularly demonstrated chains of SC trivalents, combined by 376 heterosynapsis of the short arms of acrocentrics, and association with the sex bivalent (Ratomponirina et 377 al., 1988). A large number of trivalents were likely unable to complete synapsis in time, and this deficit 378 could lead to aberrant chromosome segregation, arrest of cells at M1 or M2 stages, germ cell aneuploidy, 379 and decreased fertility. We observed the synapsis delay for SC trivalents in intraspecific mole vole 380 hybrids; this finding is consistent with previous studies (Kolomiets et al., 1985, Bogdanov et al., 1986). 381 However, in contrast to lemur hybrids, in intraspecific mole vole hybrids, there were often gaps in the 382 axial elements of the metacentrics in open SC trivalents (Kolomiets et al., 1985; Bogdanov et al., 1986). 383 Based on immunostaining, now we interpret such specific gaps as stretching of the centromeric regions. 384 Mus musculus domesticus that were heterozygous for Rb metacentrics were also highly variable 385 in their fertility (de Boer and de Jong, 1989; Hauffe and Searle, 1998). Thus, in some intraspecific mice 386 hybrids, for example, heterozygous for three Rb translocations, there was no decrease of fertility,

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387 although there were "XY–trivalent" and "trivalent–trivalent" associations (Grao et al., 1989). In other 388 cases, comparative analysis revealed differences in fertility level due to distinct karyotype structures: a 389 slight decrease (4 SC trivalents) and a significant decline in fertility (7 SC trivalents) (Wallace et al., 390 2002). Possibly, the difference was due to higher associations of SC trivalents with sex XY bivalent and 391 associations of trivalents with each other in animals of the second group. 392 In other experiments, fertile hybrids heterozygous for 8 Rb translocations had open and closed SC 393 trivalents (Manterola et al., 2009). The authors suggested that the meiotic progression of cells with an 394 asynaptic area of SC trivalents was due to the insignificant genetic value of the unsynapsed chromatin 395 regions, the inactivation of which did not lead to the activation of the pachytene arrest program. 396 Hybridization of different forms of the musk shrew (Suncus murinus) could lead to the formation 397 of 5 SC trivalents in meiocytes. Some of the F1 hybrids were sterile, while others were fertile, depending 398 on different parental combinations. Researchers have suggested that genetic factors play a crucial role in 399 determining fertility/sterility (Borodin et al., 1998; Rogatcheva et al., 2000). In the case of mole vole 400 hybrids, animals regularly had impaired fertility (intraspecific) or were sterile (interspecific), although 401 this fact does not exclude the effect of unknown genetic factors in determining the fertility level. 402 Alterations in recombination might be crucial for species evolution (Rieseberg, 2001, Livingstone and 403 Rieseberg, 2004). Physical problems in the synapsis of rearranged homologs can restrict the formation of 404 recombination sites (Cattanach and Moseley, 1973, Davisson and Akeson, 1993), which can lead to 405 univalence, unbalanced chromosome segregation and selection of germ cells (Baker et al., 1996, Eaker et 406 al., 2001, 2002). Comparison of distinct mole vole hybrids reliably demonstrated that recombination was 407 reduced in hybrids, a phenomenon that was most likely correlated with a delay in synapsis of SC 408 trivalents or their physical stretching. It is likely that some of the achiasmatic chromosomes in trivalents 409 can be incorrectly segregated, and such cells will be eliminated in meiotic checkpoints (Morelli and 410 Cohen, 2005). 411 412 Centromere identity and stretched centromeres of the SC trivalents 413 414 Centromeres are unique chromosomal regions that organize the assembly of the kinetochore, a large 415 multiprotein complex that allows chromosomes to attach to spindle microtubules and move during mitotic 416 and meiotic cell division (Cheeseman, 2014, Talbert and Henikoff, 2020). There is no universal DNA 417 sequence responsible for centromere formation. This fact, and the emergence of new centromeres, led to 418 the hypothesis that centromeres are determined by accumulation of tandem repeats (satellite DNA) 419 (Henikoff et al., 2001, Garagna et al., 2002, Plohl et al., 2008) and retrotransposons (Ferreri et al., 2011, 420 Chang et al., 2019) alongside with epigenetic modifications such as histone variants (Amor and Choo, 421 2002, Dalal, 2009). The centromere-specific variant of histone H3, CENPA, is a platform for protein 422 assembly at the kinetochore (Bernard et al., 2009, Musacchio and Desai, 2017). The rapid diversification 423 of centromeres has been suspected to lead to reproductive isolation between species (Henikoff et al., 424 2001). The position of the centromere is easily identified by routine staining; in immunostaining 425 centromeres typically appear as one-spotted signals within chromosomes. 426 In mole voles, closed SC trivalents developed three contact points with the nuclear envelope: (1) 427 the attachment point of the proximal ends of acrocentrics, formed by their short arms, and (2 and 3) two 428 attachment points of the distal telomeres of metacentrics and acrocentrics (see Fig 3F). As prophase I 429 progresses, the attachment points move away from each other; therefore, SC trivalents can have different 430 spatial configurations (Berrios et al., 2014). However, open SC trivalents must be associated with the 431 nuclear envelope at four points. In addition, acrocentrics were ectopically connected by short SCs with 432 neighboring acrocentrics. There were multiple interlockings in interspecific mole vole hybrids. All these 433 specifics caused the strongest tension of chromosomes involved in SC trivalents, stretching of the 434 centromeric regions of the Rb metacentrics, and in some cases, the centromeric regions of the 435 acrocentrics. 436 We used different antibodies against kinetochore proteins to identify centromeric regions of 437 meiotic chromosomes in mole vole parental species and hybrids (Fig S4). As noted above, we observed 3 438 types of SC trivalents in hybrid spermatocytes (Fig 4). Thus, the distance between the attachment points 439 determined the ultrastructural organization of SC trivalents. These attachment points were most clearly 440 manifested in closed free SC trivalents (Fig 4C). If the distance between the attachment points of the SC 441 trivalent to the nuclear envelope was markedly greater than the metacentric length, then the metacentric 442 axis undergoes strong stretching, and this did not allow the formation of a continuous axial element. 443 Therefore, electron microscopic examination revealed a gap in the structure of the stretched axial element

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444 of the metacentric (Fig 4B). However, when immunostaining with antibodies to kinetochore proteins 445 (ACA, CREST, and CENPA), there were no gaps, and a centromeric linear structure was visible in this 446 area (Fig 4B). If the attachment points were located very far from each other (on different sides of the 447 nucleus), then the centromeric region was hyperstretched up to gaps (Fig 4A). 448 Such stretched centromeres are intriguing because no similar regions have been found within SC 449 trivalents before the present study. It is possible that these centromeric stretches are associated with the 450 structural specifics of the pericentromeric chromatin. Chromatin in the centromeric region has the classic 451 epigenetic marks of constitutive heterochromatin: H3K9me2, H3K9me3, H3K27me3, and H4K20me3 452 (Richards and Elgin, 2002; Nishibuchi and Dejardin, 2017). For example, in the study of unfolded 453 pericentromeric heterochromatin (prekinetochores) in interphase, chromosomes subjected to stretching by 454 TEEN buffer, there was alternation of the CENPA and H3K9me3 subdomains with the gaps (Ribeiro et 455 al., 2010). If any natural or artificial extension does not lead to structural breaks, it likely entails a linear 456 spatial unfolding of the structural components of pericentromeric heterochromatin. This phenomenon 457 probably explains why we saw an alternating change of centromeric points, centromeric lines, and gaps in 458 the metacentrics of SC trivalents. This feature might indicate high plasticity of mole vole pericentromeric 459 heterochromatin. 460 Stretching of the centromeric regions of acrocentrics between SC trivalents is even more 461 mysterious. This phenomenon was rarer than centromeric stretching of metacentrics. We suppose that this 462 may also be due to a special stretching of the pericentromeric heterochromatin of the acrocentric short 463 arms associated in chains of several SC trivalents. Moreover, heterochromatin was involved in the 464 nonhomologous synapsis of the short arms of the acrocentrics of SC trivalents and in binding to the 465 nuclear envelope in intraspecific hybrids (Fig S9B and S9C). The centromeric associations of SC 466 trivalents are of particular interest (see "Nuclear architecture: simulated chromosome configurations in 467 mole vole pachytene spermatocytes " below). 468 In general, the presence of stretched centromeric regions may indicate specific properties of 469 centromeres in the genus Ellobius. Stretched centromeres in bivalents as a likely result of Rb 470 chromosome fusion have been identified in African pygmy mouse (Gil-Fernández et al., 2020). 471 Centromeric satellites in animals and plants undergo rapid evolution (Henikoff et al., 2001), and they may 472 differ even in closely related species (Mestrović et al., 1998, Lee et al., 2005, Talbert et al., 2018). These 473 differences have been explained by the "library hypothesis" (Salser et al. 1976): An ancestral form has an 474 initial satellite pool ("library" of satellites), which in different evolutionary lineages manifests in various 475 patterns (in quantity and quality), thus forming species-specific satellite profiles (Miklos et al., 1985). The 476 hypothesis is supported by some examples (see Smalec et al., 2019). In addition, it has been established 477 that closely related species may differ in their retrotransposons. A classic example is a well-studied 478 kangaroo. In interspecific kangaroo hybrids, there was centromere destabilization, which was caused by 479 the activation of resident retroelements (in this case, kangaroo endogenous retrovirus [KERV]) (O'Neill et 480 al., 1998, Metcalfe et al., 2007, Ferreri et al., 2011). 481 It would seem that such important chromosome elements like centromeres should be conserved, 482 but they exhibit incredible structural and functional variability and dynamic evolution and are hotspots for 483 chromosomal rearrangements (Brown and O'Neill, 2010, Brown et al., 2012). The main remaining issues 484 include the question of the true reasons for the centromeric region stretching in Ellobius hybrids as well 485 as the molecular mechanisms of their striking plasticity. The study of centromeric satellites and 486 retrotransposons in the Ellobius genus will be promising. It is obvious that additional detailed study of 487 centromeric regions in Ellobius, including stretched centromeres and different centromeric localization of 488 the heteromorphic chromosome #7 pair in interspecific hybrids, will be necessary. The centromere 489 features established here and data from previous works (Matveevsky, 2011a, 2011b, Bakloushinskaya et 490 al., 2012, Matveevsky et al., 2017) may suggest that the centromeres in mole voles, if not a driver of 491 chromosomal evolution, are essential for karyotypic divergence. 492 493 Chromosome #7: implications from the hybrid meiotic nuclei studies 494 495 As mentioned above, the parental 54-chromosome karyotypes of E. talpinus and E. tancrei differ 496 in only one pair of chromosomes (#7). In these species, chromosome #7 pairs were identical in G-bands, 497 but in E. talpinus it is an acrocentric, and in E. tancrei it is a submetacentric. For a long time, it was 498 assumed that the submetacentric emergence was due to pericentric inversion (Lyapunova and Vorontsov, 499 1978). However, the SC study in interspecific hybrids clearly showed that this chromosome pair is 500 completely synapsed without inversion loops, forming a full-length SC along the entire chromosome at

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501 the early-to-mid pachytene stage. We previously suggested that the submetacentric in E. tancrei emerged 502 through the neocentromere formation (Matveevsky, 2011a, 2011b). This assumption was confirmed by 503 additional Zoo-FISH data (Bakloushinskaya et al., 2012), and later by immuno-identification of the 504 central element of SCs and recombination nodules (Matveevsky et al., 2017). 505 The chromosome #7 pair was used as a marker bivalent in the SC analysis. In interspecific 506 hybrids, this was the only heteromorphic SC bivalent with two centromeres located at a distance from 507 each other. Of note, we discovered another remarkable property of the SC #7. In both hybrids, bivalent #7 508 practically does not participate in chromosome associations: There was only one association of this 509 chromosome with the open SC trivalent (Fig S4E). We assume that the inertness of chromosome #7 in 510 meiosis may be due to the absence or extremely low content of constitutive heterochromatin. This was 511 confirmed using antibodies to histone H3K9me3, a marker of constitutive heterochromatin, both in 512 bivalent #7 in E. tancrei (unpublished) and in a sibling species E. alaicus (Matveevsky et al., 2020). 513 Thus, the ancestors of modern E. tancrei and E. alaicus, concomitant with the emergence of the 514 neocentromeric submetacentric chromosome #7 pair, probably acquired chromosomal instability with the 515 formation of various karyotypic forms, which may indicate some causal link between these events. 516 Perhaps one of them could become an evolutionary trigger, which entailed a chain of genetic and/or 517 cytogenetic changes in the mole vole karyotype. 518 519 Nuclear architecture: simulated chromosome configurations in mole vole pachytene spermatocytes 520 521 The organization of the internal contents is not random in interphase (Cremer et al., 1993, Parada 522 and Misteli, 2002) and meiotic (Foster et al., 2005) nuclei; therefore, it is customary to speak of nuclear 523 architecture (Cremer and Cremer, 2010) or intranuclear landscape (Cremer et al., 2006). The 524 "chromosome territory" concept is considered to be generally accepted (Cremer and Cremer, 2001). 525 Chromosome territories are spatial domains of different sizes that specifically occupy a certain volume in 526 the nucleus (Misteli, 2008). The position of chromosomes in the nucleus can be preserved in closely 527 related taxa (Tanabe et al., 2002), a phenomenon called "phylogenetic memory" (Mora et al., 2006). 528 Heterochromatic compartments play a significant role in the nucleus content (Politz et al., 2016). 529 However, gene mutations and chromatin and chromosomal rearrangements can change the intranuclear 530 organization, which can lead to diseases and the appearance of various abnormal manifestations (Garagna 531 et al., 2001, Oberdoerffer and Sinclair, 2007, Lammerding, 2011, Fournier et al., 2010, Berrios et al., 532 2018, Abdelhedi et al., 2019). 533 There is also a point of view, according to which the correct formation of chromosome territories 534 diminishes the translocation potential of the cells (Rosin et al., 2019). The organization of interphase and 535 meiotic nuclei has significant differences (Alsheimer et al., 1999). An SC forms a specific chromatin 536 pattern (Hernandez-Hernandez et al., 2009) and interacts with the nuclear envelope through a special 537 Sun–KASH system (Alsheimer, 2009). The presence of asynaptic chromosome regions in prophase I 538 initiates a meiotic silencing of unsynapsed chromatin (MSUC) (Schimenti, 2005; Turner et al., 2005). 539 Nuclear architecture of prophase I meiocytes is specific for each species (Berrios et al., 1999, 2004, 540 Berrios, 2017). 541 As a result, the nuclear architecture in meiotic prophase I is determined by the SC structure and 542 dynamics, types of chromosomes (single-armed or bi-armed), their length, the heterochromatin amount, 543 the specificity of centromeric regions, the "chromosome–nuclear envelop" interactions, the ability to form 544 chromocenters and nucleoli, and sex chromosome organization and behavior (Berrios, 2017). It should be 545 emphasized that if the parental genomes differ significantly, then complex chromosome compounds are 546 formed in hybrid and mutant meiotic nuclei (for example, Johannisson and Winking, 1994, Ribagorda et 547 al., 2019, Matveevsky and Kolomiets, 2011, 2016), and the processes of repair, recombination, and 548 meiotic silencing are disrupted (for example, Bhattacharyya et al., 2013, Turner, 2007, Burgoyne et al., 549 2009, Homolka et al., 2007), which can cause an imbalance in the nuclear architecture. 550 In two hybrid mole vole groups, a different number of free closed SC trivalents formed. 551 Pericentromeric heterochromatin likely played an important role in the formation of closed trivalent 552 configurations, as described in heterozygous mice (Berrios et al., 2014). Thus, on the three-dimensional 553 nuclei of interspecific mole vole hybrids, there were closed SC trivalents, which were attached by the 554 short arms of acrocentrics to the nuclear envelope, and open SC trivalents with stretched centromeric 555 regions (CREST cloud around) (Fig S8). CREST antibodies can non-specifically immunostain 556 heterochromatic regions and heterochromatin-like structures, for example, ChBs (Fig 2H and 3D). The 557 same specificity was revealed during the electron microscopic examination of the intraspecific

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558 heterozygous spermatocytes. At the attachment site of the short arms of the SC trivalent, a cloud of 559 electron-dense material formed, associated with the nuclear envelope, which was usually interpreted as 560 heterochromatin mass (Fig S9, schemes of hemispheres in 3F). 561 SC trivalent chains were demonstrated in intraspecific mole vole hybrids (Kolomiets et al., 1985; 562 Bogdanov et al., 1986). Such ectopic associations formed due to the heterochromatic contacts of the short 563 arms of acrocentrics of two different open SC trivalents (Fig S7), as had been noted earlier (Kolomiets et 564 al., 1986). Chromosome associations can be determined by H3K9me3 immunodetection (Berrios et al., 565 2014). This protein is believed to mark DAPI-positive chromocenters (for example, Berrios et al., 2010). 566 However, chromocenters in all mole voles were usually not detected by DAPI staining. Nevertheless, we 567 found that acrocentric chromosomes were grouped by their pericentromeric regions around the H3K9me3 568 domains in the sibling species E. alaicus (Matveevsky et al., 2020). We saw a similar grouped position of 569 acrocentrics in E. talpinus (Fig 2A). In E. tancrei (2n = 34), the centromeric sites of the acrocentrics were 570 also located around the H3K9me3 clouds, while these sites of the Rb metacentrics had a more linear form 571 of the H3K9me3 signals, as seen in the micrographs (Fig S9A, S9A'). 572 Combining the results on the ultrastructure and behavior of SCs and SC trivalents in spreads and 573 squashes, we present these data as three-dimensional simulations of chromosome configurations in the 574 meiotic nuclei of mole vole parental species and hybrids (Fig 6). 575 576 Concluding remarks 577 578 Hybridization manifests a wide range of selective mechanisms, including decreased fertility or 579 even sterility, due to defective synapsis and recombination (Miklos, 1974, de Boer, 1986, Davisson and 580 Akeson, 1993), meiotic silencing failure (Homolka et al., 2007, Burgoyne et al., 2009), unbalanced 581 chromosome segregation (Eaker et al., 2001), and alteration of nuclear architecture (Garagna et al., 2001). 582 Modification of the nuclear architecture can be demonstrated by comparing native (parental) and 583 experimental hybrids. The 34-chromosome mole voles differ in the positions of the chromosomal 584 territories compared with the native 54-chromosome form due to 10 Rb pairs. To assess the differences in 585 the location of chromosome territories, we performed experimental hybridization of 54- and 34- 586 chromosome mole voles. We obtained two types of heterozygous mole voles with the same diploid 587 number and identical chromosomal combinations. These two models are different in the manifestation of 588 their fertility: intraspecific hybrids have slightly reduced fertility while interspecific hybrids are sterile. 589 Forming the same number of SC trivalents, germ cells of the heterozygotes showed a different behavioral 590 spectrum of chromosome combinations. In both hybrids, SC trivalents formed SC chains. However, in 591 intraspecific hybrids, SC trivalents were able to leave such associations and form closed configurations, 592 while in interspecific hybrids, only a few (usually 1–2) trivalents could complete the synapsis. The 593 remaining SC trivalents could not dissociate from the SC chains, probably due to the peculiarities of the 594 organization of centromeric regions in SC trivalents. The degree of nuclear architecture reorganization in 595 the two hybrids was different, although the chromosome combinations were identical. These results 596 suggest that alterations of nuclear architecture depend not only on the chromosome composition but also 597 on other genetic or/and epigenetic factors ones. 598 Given that the behavior of the centromeric regions of SC trivalents was different in the two types of 599 mole vole hybrids, it was logical to assume that this specificity could at least powerfully contribute to 600 reproductive breakdown or be considered a cause of the sterility of interspecific hybrids. Because 601 centromeres can be associated with patterns of gene expression and chromatin modifications (Zhao et al., 602 2016), physically stretched centromeric regions (as an indicator of nuclear architecture reorganization) 603 could indirectly modify or destabilize these processes. Thus, we hypothesize that the shift in the 604 intranuclear organization and the centromere stretching could change genetically significant regions of 605 the genome through chromatin reformatting or altering the patterns of gene expression, which, in turn, 606 could provoke the activation of meiotic checkpoints.

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607 608 Fig 6. Simulation of chromosome synapsis and interactions of SC trivalents in pachytene nuclei of Ellobius 609 species and hybrids. A partial set of chromosomes and lower—inside the spheres—features of the configuration of 610 the axial elements of these chromosomes during synapsis at the pachytene stage are presented in each part of the 611 figure. White dots at chromosome ends mark the attachment points of the chromosomes to the nuclear envelope. (A, 612 B) Chromosomes: Seven pairs of autosomes and a pair of isomorphic sex (XX) chromosomes in A and B. The 613 chromosome sets differ from each other only in the chromosome 7 pair: acrocentric in Ellobius talpinus (2n = 54), 614 neocentromeric submetacentric in Ellobius tancrei (2n = 54). Spheres: The positions of SCs over the nuclear 615 envelope in the pachytene nuclei in A and B are different, because it is assumed that chromosome territories can be 616 distinguished between two species (Berrios et al., 2014, 2017). (C) Chromosomes: Conditional karyotype of E. 617 tancrei (2n = 34) consists of three pairs of homologous Robertsonian (Rb) metacentrics (1.2, 3.4 and 5.6), a 618 submetacentric chromosome #7 pair and sex (XX) chromosomes. Sphere: The chromosomal arms of metacentrics 619 do not always occupy the ancestral position (compare spheres C, B, and A), because Rb metacentric formation 620 causes a shift in the chromosome territories in the meiotic nucleus (see Garagna et al., 2001, Berrios et al., 2014). 621 (D) Chromosomes: Karyotype of the intraspecific hybrid E. tancrei obtained from crossing two forms (2n = 54 and 622 2n = 34). The scheme of partial karyotype shows three Rb metacentrics (1.2, 3.4, 5.6) and six acrocentrics (1–6). 623 Sphere: Three Rb metacentrics and acrocentrics form three SC trivalents. They are attached to the nuclear envelope 624 and part of them remains open for a long time at the mid pachytene. The synapsis of chromosomes in all SC 625 trivalents is gradually completed to the diplotene, which will ensure their correct segregation in the future. Open and 626 closed SC trivalents are presented on the left. (E) Chromosomes: Karyotype of the sterile interspecific hybrid 627 obtained from crossing two species E. talpinus (2n = 54) and E. tancrei (2n = 34). The scheme of partial karyotype 628 shows three Rb metacentrics (1.2, 3.4, and 5.6) and six acrocentrics (1–6) like the intraspecific hybrid (D). Sphere: 629 The main difference in the chromosome synapsis in SC trivalents is that the centromeric regions of the metacentrics 630 are very stretched. The centromeric regions of acrocentrics are also stretched in some cases. Centromere stretching 631 is associated with the difference in chromosome territories of two parent species. SC trivalents with stretched 632 centromeres are presented on the right. Associations of sex chromosomes with SC trivalents (yellow stars) are often 633 observed in both hybrids. Chromatin dense bodies (ChBs) of XX bivalents are a white ball on one of the X axes. 634

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635 636 We suppose that pachytene irregularities and decreased recombination could synergistically, 637 along with centromere instability and reorganization of intranuclear contents, contribute to the complete 638 sterility of interspecific mole vole hybrids. In intraspecific hybrids, synaptic irregularities and a reduced 639 crossover number could reduce fertility, but only in the F1 generation; future generations demonstrated 640 ordinary fertility (Lyapunova and Yakimenko, 1985). The role of genetic factors in the development of 641 sterility cannot be ruled out, because "hybrid sterility genes directly or indirectly modulate the sensitivity 642 of synapsis to the sequence divergence between heterospecific chromosomes, either enhancing or 643 suppressing it" (Bhattacharyya et al., 2013). 644 Summarizing our findings and previous studies, we conclude that a unique group of subterranean 645 rodents, mole voles from the genus Ellobius, exhibits a wide range of interesting chromosomal 646 phenomena, such as different systems of sex chromosomes, heterology of two isomorphic male X 647 chromosomes, expressed in asynchronous chromatin inactivation at prophase I, multiple Rb translocations 648 and monobrachial homologous metacentrics, neocentromere formation, stretched centromeres, acrocentric 649 interactions leading to the formation of dicentric chromosomes and new Rb chromosome emergence. 650 Each of the events could be considered as a driver or trigger of the karyotypic evolution of the rodents. 651 652 Materials and Methods 653 654 Animals 655 656 All Ellobius males were obtained from a mole vole collection of the IDB RAS. For meiotic studies, 657 the parental Ellobius forms: three E. talpinus, two E. tancrei (2n=54), five E. tancrei (2n=34) and two 658 groups of F1 hybrids: three intraspecific hybrids E. tancrei X E. tancrei, five interspecific hybrids E. 659 talpinus X E. tancrei were used in this work. 660 The fertility of parental forms and hybrids was determined based on relevant data: 66 pups and 23 661 litters in E. talpinus, 168 pups and 71 litters in E. tancrei (2n=54), 197 pups and 82 litters in E. tancrei 662 (2n=34), 41 pups and 20 litters in the intraspecific hybrid F1 E. tancrei X E. tancrei, 93 pups and 24 663 litters in the interspecific hybrid F1 E. talpinus X E. tancrei. 664 665 Ethics statement 666 667 Experimental procedures were performed in strict accordance with the international, national, and 668 institutional guidelines for animal care. Studies were approved by the Ethics Committee for Animal 669 Research of the Vavilov Institute of General Genetics RAS (order №3 of November 10, 2016) and the 670 Koltzov Institute of Developmental Biology RAS (Annual permissions, 2013-2017). 671 672 Spermatocytes suspensions 673 674 The removed testes exempted from tunics, large blood vessels and fat. A piece of testis tissue was placed 675 on cavity slide in 1 ml of Eagle's medium (without glutamine), 37°С (Paneco, Russia). The testis tissue 676 was crushed with razor blades, then the cell suspension was homogenized using an automatic pipette for 677 10-15 minutes. The degree of homogenization of the suspension and its cell composition was controlled 678 under a light microscope. 679 680 Spermatocytes spreads 681 682 Spermatocyte spreads were obtained in accordance the procedure of Kolomiets et al. (1986, 2010). 5 µl of 683 testis cell suspension in Eagle's medium was applied to the surface of the convex meniscus of a drop (20 684 µl) of 0.2 M sucrose solution (hypophase). The cells swelled up and then spread over the surface of the 685 hypophase. After 1 min, a slide with a polylysine or coated with an electronically transparent chemically 686 resistant film is touched to the surface of the hypophase (a 0.5% solution of the fragments of Falcon dish 687 in chloroform). Cells adhering to the film surface were fixed with a 4% solution of paraformaldehyde, pH 688 8.0-8.4, washed with a 0.4% solution of Photoflo (Kodak), pH 8.0, and dried in air. 689 690

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691 Spermatocytes squashes 692 693 As emphasized by Berrios et al., 2010, squashes allow to preserve chromatin condensation and 694 organization (3D space) of nuclei. Spermatocyte squashes were prepared in accordance the procedure of 695 Page et al. (1998) (personal training from Jesus Page and Ana Gil-Fernandes in 2017). Removed testes 696 were fixed in 2% formaldehyde in PBS with 0.05% Triton X-100 during 10 min. Then, several pieces of 697 the seminiferous tubules were placed on a slide and squashed by exerting pressure on the coverslip. The 698 slides were immersed in liquid nitrogen and the coverslips were removed with a knife. The slides were 699 washed in PBS for 15 minutes and were immunostained. 700 701 Antibodies and immunocytochemistry 702 703 The slides were washed in PBS. Spreads and squashes were blocked with HB (holding buffer: PBS, 0.3% 704 BSA, 0.005% Triton X-100). The slides were incubated overnight at 4°C with primary antibodies: rabbit 705 polyclonal antibodies SCP1 (Abcam, Cambridge, UK), rabbit polyclonal antibodies SCP3 (Abcam, 706 Cambridge, UK) both diluted to a concentration of 1:500 in ADB (Antibody Dilution Buffer: PBS, 3% 707 BSA, 0.05% Triton X-100), mouse antibodies MLH1 (1:50, Abcam, Cambridge, UK), mouse anti- 708 phospho-histone H2AX (1:1000, Abcam, Cambridge, UK), human anti-centromere antibodies CREST 709 (Fitzgerald Industries International, Massachusetts, USA), ACA (Antibody Incorporated, California, 710 USA) or monoclonal CENPA (Abcam, Cambridge, UK), all diluted to a concentration from 1:200 to 711 1:400 in ADB. The slides were washed in PBS and incubated with goat anti-rabbit Alexa Fluore 488 712 conjugated antibodies (1:500, Abcam, Cambridge, UK), goat anti-human Alexa Fluore 546 conjugated 713 antibodies (1:200 – 1:400) and goat anti-human Alexa Fluore 546 conjugated antibodies (1:500) at 37°C 714 for two hours. The slides were washed with PBS, rinsed briefly with distilled water, dried and mounted in 715 Vectashield with DAPI (Vector Laboratories). Some details of immunocytochemystry procedure see in 716 Matveevsky et al., 2016, 2018. The slides were analyzed with an Axioimager D1 microscope CHROMA 717 filter sets (Carl Zeiss, Jena, Germany) equipped with Axiocam HRm CCD camera (Carl Zeiss), and 718 image-processing AxioVision Release 4.6.3. software (Carl Zeiss, Germany). Images were processed by 719 Adobe Photoshop CS5 Extended 720 721 AgNO3-staining and electron microscopy 722 723 The slides were stained with 50% AgNO3 solution in a humid chamber at 56°C for 3 hours. The slides 724 were washed in four changes of distilled water and air-dried. The stained slides were observed in a light 725 microscope, suitably spread cells were selected, and plastic (Falcon film) circles were cut out with a 726 diamond tap and transferred onto grids. The slides were examined under JEM 100B or JEM 1011 electron 727 microscope. 728 729 Histological analysis of testis sections 730 731 Testes were removed, fixed overnight in Bouin’s solution, and then stored in 70% ethanol until use. The 732 fixed testes were dehydrated in a graded ethanol series, immersed sequentially in ethanol / xylene and 733 xylene, and then embedded in paraffin. The testes were sectioned at a thickness of 6 mm and mounted on 734 glass slides. The sections were deparaffinized and stained with hematoxylin and eosin. 735 736 Statistical analysis 737 738 The statistical analysis of all data was performed using GraphPad Prism 8 software (San Diego, CA, 739 USA). Mean values (M) and standard deviation (SD) were calculated by descriptive option. P-values 740 reported in Table S1 were calculated by Mann–Whitney two-sided non-parametric test. Bar charts, ring 741 and dots diagrams were created by graph options of the software. 742 743 ======744 745 Funding: This research was supported by the research grant of the Russian Foundation for Basic Research No 20- 746 34-70027 (SM, AT, AK) and VIGG RAS State Assignment Contract (SM, OK); and by the Russian Foundation for 747 Basic Research No 20-04-00618 (IB) and IDB RAS State Assignment for Basic Research (IB). Each of two RFBR

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748 grants supported only those parts of the work for which each of the co-authors was contribute. The funders had no 749 role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 750 751 Competing interests: The authors have declared that no competing intersts exist. 752 753 Data Availability Statement: All relevant data are within the manuscript and its supporting information file. 754 755 Acknowledgments 756 757 We thank the Genetic Polymorphisms Core Facility of the VIGG RAS, Moscow, for the possibility to use their 758 microscopes. We thank S. Kapustina and V. Tambovtseva from IDB RAS for their help in maintaining the mole 759 vole collection. We are grateful to GN Davidovich and AG Bogdanov of the Electron Microscopy Laboratory of 760 Biological Faculty of Moscow State University for the technical assistance. 761 762 Author Contributions 763 764 Maintenance of mole vole collection, hybridization, fertility data and karyotyping of Ellobius species and 765 hybrids were performed by IB. All meiotic studies and calculation of gonadosomatic indices were carried out by 766 SM, AT, AK and OK. Conceptualization of all meiotic researches of mole voles and its performing were supervised 767 by OK. The visualization and presentation of all data and images design were done by SM. The primary original 768 draft of the manuscript, including Supplementary materials, was written by SM and OK. All the authors read, 769 discussed, modified and approved the final text of the submitted manuscript and the supporting information. All 770 authors have read and agreed to the published version of the manuscript. 771 772 References 773 1. Abbott RJ, Barton NH, Good JM. Genomics of hybridization and its evolutionary consequences. Mol Ecol. 774 2016; 25(11):2325-2332. https://doi.org/10.1111/mec.13685 PMID: 27145128 775 2. Abdelhedi F, Chalas C, Petit JM, Abid N, Mokadem E, Hizem S, et al. Altered three-dimensional 776 organization of sperm genome in DPY19L2-deficient globozoospermic patients. J Assist Reprod Genet. 777 2019; 36(1): 69-77. https://doi.org/10.1007/s10815-018-1342-y PMID: 30362053 778 3. Adebayo AO, Oke BO, Akinloye AK. Characterizing the gonadosomatic index and its relationship with age 779 in greater cane rat (Thryonomys swinderianus, Temminck). J Vet Anat. 2009; 2(2), 53-59. 780 https://doi.org/10.21608/JVA.2009.42311 781 4. Alsheimer M. The dance floor of meiosis: evolutionary conservation of nuclear envelope attachment and 782 dynamics of meiotic telomeres. In Benavente R, Wolf J-N., editors. Meiosis. Genome Dynamics. Basel: 783 Karger Publishers; 2009. p. 81-93. https://doi.org/10.1159/000166621 PMID: 18948709 784 5. Alsheimer M, von Glasenapp E, Hock R, Benavente R. Architecture of the nuclear periphery of rat 785 pachytene spermatocytes: distribution of nuclear envelope proteins in relation to synaptonemal complex 786 attachment sites. Mol Biol Cell. 1999; 10(4):1235-1245. https://doi.org/10.1091/mbc.10.4.1235 PMID: 787 10198069 788 6. Amor DJ, Choo KA. Neocentromeres: role in human disease, evolution, and centromere study. Am J Hum 789 Genet. 2002; 71(4):695-714. https://doi.org/10.1086/342730 PMID: 12196915 790 7. Arnold ML. Natural hybridization and evolution. New York: Oxford University Press; 1993; P.214 791 8. Baker SM, Plug AW, Prolla TA, Bronner CE, Harris AC, Yao X, et al. Involvement of mouse Mlh1 in 792 DNA mismatch repair and meiotic crossing over. Nat Genet. 1996; 13(3):336-342. 793 https://doi.org/10.1038/ng0796-336 PMID: 8673133 794 9. Bakloushinskaya IY, Matveevsky SN, Romanenko SA, Serdukova NA, Kolomiets OL, Spangenberg VE, et 795 al. A comparative analysis of the mole vole sibling species Ellobius tancrei and E. talpinus (Cricetidae, 796 Rodentia) through chromosome painting and examination of synaptonemal complex structures in hybrids. 797 Cytogenet Genome Res. 2012; 136(3):199-207. https://doi.org/10.1159/000336459 PMID: 22343488 798 10. Bakloushinskaya IY, Romanenko SA, Graphodatsky AS, Matveevsky SN, Lyapunova EA, Kolomiets OL. 799 The role of chromosome rearrangements in the evolution of mole voles of the genus Ellobius (Rodentia, 800 Mammalia). Rus J Genet 2010; 46(9):1143-1145. https://doi.org/10.1134/S1022795410090346

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Supplementary materials

Meiotic nuclear architecture in distinct mole vole hybrids with Robertsonian translocations: chromosome chains, stretched centromeres, and distorted recombination

Sergey Matveevsky 1*, Artemii Tretiakov 1, Irina Bakloushinskaya 2, Anna Kashintsova 1, Oxana Kolomiets 1*

1 Department of Cytogenetics, Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia, 2 Department of Genome Evolution and Mechanisms of Speciation, Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia

* [email protected] (SM), [email protected] (OK) bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.301473; this version posted September 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Table S1. Total meiotic data of Ellobius species and hybrids bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.301473; this version posted September 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Fig S1. Fertility parameters of Ellobius species and hybrids. (A) DAPI-stained spermatocytes. (B, C) Spermatozoa: light (B) and electron (C) micrographs. Spermatozoa in B were detected by nonspecific immunostaining (SYCP3 antibody, green) and DAPI-staining (blue). (D) Suspension of spermatocytes. (E) Comparison of histological sections of testes and the size of testes in Ellobius tancrei (on the left) and the interspecific hybrid (on the right). (F) The average number of pups per litter in parents and hybrids: 1. Ellobius talpinus; 2. E. tancrei (2n = 54); 3. E. tancrei (2n = 34); 4. E. tancrei × E. tancrei hybrid; 5. E. talpinus × E. tancrei hybrid. bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.301473; this version posted September 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Fig S2. Pachytene spermatocytes of Ellobius species and hybrids. Synaptonemal complexes (SC)s were immunostained with antibodies against SYCP3 (green) and centromeres—with an antibody to kinetochores (CREST, red). An anti-γH2AFX (violet) antibody was used as a marker of chromatin inactivation. XXs were moved to the periphery of the nuclei and were covered by γH2AFX-cloud in parent species (A–C, D–F, and G–I). The γH2AFX-cloud covered the XX bivalent and spread to some asynaptic segments in both 44-chromosome hybrids (J–L and M–O). Scale bars represent 5 μm. bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.301473; this version posted September 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Fig S3. Synaptic behavior of chromosomes in intraspecific E. tancrei × E. tancrei hybrid. Light micrographs after immunostaining (A, H, J, and L). SCs were immunostained with antibodies against SYCP3 (green) and centromeres— with an antibody to kinetochores (CREST, red). An anti-γH2AFX (violet) antibody was used as a marker of chromatin inactivation. Electron micrographs after AgNO3-staining (B-G, I, and K). The numbers of autosomes (B, C, and H) correspond to the chromosome number of the karyotype (see Fig 1E). Orange arrowheads show synaptic sites of the sex (XX) bivalent. Black arrows show chromatin dense body (ChB) of the XX (B, D, and E). Tr indicates trivalent, M indicates metacentric, and A indicates acrocentric. (A, B) SC trivalents (A/M/A) chains joined together by short arms of nonhomologous acrocentrics at the early-to-mid pachytene stage (blue stars). XX bivalents have been associated with SC trivalents (Trs) or bivalents (see the violet signal in A and pink arrow in B). Both nuclei had two free Trs. The light orange arrow shows atypical prolonging of short arms of acrocentrics of the SC trivalent (B). (C) Seven free and closed SC Trs were formed at the late pachytene stage. XX bivalents were associated with SC chains (pink arrow). (D) Closed sex bivalent with two synaptic sites at the ends and a central unpaired region at the periphery of the nucleus. A long ChB was observed along one of the axes (black arrow). (E) One of the X chromosomes axes was in contact with the SC trivalent (pink arrow). (F) A closed SC trivalent with well-structured chromatin. (G) Triple synapsis in the region of short arms of a SC trivalent (green star). (H, J, K, and L) The only case of the nucleus with all 10 trivalents (H). SYCP3- and AgNO3-positive dense material was revealed in the region of short arms of some SC trivalents (yellow stars; white "Tr" in H; I–K). Some trivalents had a stretched metacentric, in the stretch segment of which SYCP3- positive dense material was also observed. Most nuclei had no such features in hybrid spermatocytes (L, L'). Scale bars represent 5 μm. bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.301473; this version posted September 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Fig S4. Synaptic behavior of chromosomes in interspecific E. talpinus × E. tancrei hybrid. SCs were immunostained with antibodies against SYCP3 (green) and centromeres—with antibodies to kinetochores (ACA, CREST, or CENP-A, red). The numbers of autosomes (A) correspond to the chromosome number of the karyotype (Fig 1D). Orange arrowheads show synaptic sites of the sex (XX) bivalent (B, C, D, and F). White arrows show chromatin dense body (ChB) of the XX (b, c, d, and k). Tr –indicates trivalent, M indicates metacentric, and A indicates acrocentric. (A) There were no free SC trivalents in the pachytene nucleus; all SC trivalents were in chains. The XX bivalent is not visible. (B) Double association of the XX bivalent with SC trivalents. An X univalent was associated with a SC trivalent by a thin CREST-positive line. (C) The XX bivalent with two synaptic sites and ChB. (D) An open XX bivalent with ChB. One of X univalent was associated with SC trivalent by a thin CREST-positive line. (E) The only case of the association of chromosome #7 with a SC trivalent. An acrocentric homolog of chromosome #7 involved in an association (blue arrow). (F) An open XX bivalent and SC trivalent with a stretched centromere. (G) A free closed SC trivalent. (H) The yellow arrow shows atypical lengthening of short arms of acrocentrics of the SC trivalent (H). (I) A ring-like univalent. (J–L) CENP-A-positive (J) and ACA-positive (K and L) centromeric region of metacentrics in some SC trivalents were stretched. There were no free SC trivalents. Scale bars represent 5 μm. bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.301473; this version posted September 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Fig S5. Synaptic behavior of chromosomes in interspecific E. talpinus × E. tancrei hybrid at zygotene nucleus. (A–D) SCs were immunostained with antibodies against SYCP3 (green) and centromeres—with an antibody to kinetochores (CREST, red). DAPI stained chromatin (blue). M indicates metacentric and A indicates acrocentric. Acrocentrics and metacentrics in SC trivalents adjusted together. Some acrocentrics and some arms of metacentrics had already entered synapsis, and some were just starting the synapsis process (yellow stars). Some metacentrics of SC trivalents had stretched centromeres (blue arrowheads). Sex chromosomes did not enter synapse and were located as X univalents. Their identification was possible based on length and a similar scenario of the behavior of axial elements, DAPI-positive signals, chromatin-dense body (ChB), and comparison with other micrographs (see Fig 2H). Scale bars represent 5 μm.

bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.301473; this version posted September 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Fig S6. SC trivalents and stretched centromeres in interspecific E. talpinus × E. tancrei hybrid. SCs were immunostained with antibodies against SYCP3 (green) and centromeres—with an antibody to kinetochores (CREST, red). Blue arrowheads show centromeric regions of metacentrics in SC trivalents. Violet arrowheads show centromeric regions of acrocentric in SC trivalents. The white arrow shows a chromatin dense body (ChB) of the XX bivalent. Green stars show interlocking points. Tr indicates trivalent, M indicates metacentric, and A indicates acrocentric. (A–C) Pachytene nucleus: SYCP3-fragments (pseudobivalents) of SC trivalents were distant from each other (A). These fragments were connected by extensive stretched centromeric regions (B, C). (D-G) Part of the nucleus in A–C: Tree SC trivalents were linked by stretched centromeres of metacentrics and acrocentrics. SYCP3-segment of axial elements (green star) was an interlocking point. The scheme of the SC trivalent chains (E) and simulation of this chain in the nucleus (F) are presented. White dots at the chromosome ends mark the attachment points of the chromosomes to the nuclear envelope (F). Two SC trivalents A1/M1/A2 and A3/M2/A4 are connected by stretched centromeres of acrocentrics (pink stars) (D–G). (H–J) Examples of SC trivalents with stretched centromeres of metacentrics and acrocentrics. Scale bars represent 5 μm .

bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.301473; this version posted September 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Fig S7. Types of SC trivalent chains in Ellobius hybrids. Red dashed inserts correspond to centromere stretching. Green lines correspond to the axial elements. See explanations in the text.

bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.301473; this version posted September 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Fig S8. Spermatocyte squashes of interspecific E. talpinus × E. tancrei hybrid. Axial elements were identified using anti‐SYCP3 antibodies (green), a central element using anti‐SYCP1 (magenta), and an anti‐CREST antibody for kinetochores (red). An anti-γH2AFX (violet) antibody was used as a marker of chromatin inactivation. DAPI stained chromatin (blue). Blue arrowheads show stretched centromeric regions in SC trivalents. One of the SC trivalent had a stretched centromere. (A–E) The short arms of the acrocentrics in the SC trivalents were attached to the nuclear envelope. The central element (SYCP1) was formed in the SC trivalent. White dots at the chromosome ends mark the attachment points of the chromosomes to the nuclear envelope (E). (F, G) Three squashes with some stretched centromeres of a SC trivalent. One of the squashes had a closed XX bivalent covered by a γH2AFX cloud. White stars show synaptic sites of the XX bivalent (enlarged in inset). Some open SC trivalents were also covered by a γH2AFX cloud (G). Some SC trivalents had a centromeric link in interlocking point (green stars) (F). (H) Zygotene-like stage: Chromosome–nuclear envelope interactions were visible. Scale bars represent 5 μm. bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.301473; this version posted September 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Fig S9. Heterochromatic links of chromosomes and nuclear envelope. Pink arrows show heterochromatin mass of chromosomes and SC trivalents. (A, A') Part of the nucleus of E. tancrei (2n = 34) at the early pachytene stage. The linear heterochromatin link between two metacentrics (M) and cloud-like heterochromatic mass is visible. (Insets) SCs were immunostained by antibodies against SYCP3 (green), heterochromatin—by an H3K9me3 antibody (yellow)—and centromeres—by an antibody to kinetochores (CREST, red). H3K9me3 foci are cloud-like signals near the centromeres of acrocentrics (top inset) and linear signal near the centromere of the submetacentric (bottom inset). (B, C) Pachytene nuclei of the E. tancrei × E. tancrei hybrid. Some closed SC trivalents have a heterochromatic spot in the short arms of acrocentrics linked with the nuclear envelope (B', C'). Heterochromatic spots of some acrocentrics have links with the nuclear envelope too (B', C').