From asexuality to sexual reproduction: cyclical switch of gametogenic pathways in hybrids depends on ploidy level

The cellular and molecular mechanisms governing sexual reproduction is highly conserved across eukaryotes. Nevertheless, hybridization can disrupt such machinery leading to asexual reproduction. To investigate how hybridization and polyploidization affect gametogenesis and reproductive outcomes of asexual hybrids, we conducted a comprehensive study on diploid and triploid hybrids along with their sexual parental species from the freshwater fish family Cobitidae. In diploid and triploid hybrids, most gonocytes maintain their original ploidy level. During meiosis, such gonocytes experience abnormal chromosome pairing preventing progression beyond pachytene. Diploid hybrid females regain fertility through premeiotic genome endoreplication, resulting in the rare emergence of tetraploid gonocytes. Tetraploid gonocytes bypass meiosis and lead to clonal diploid gametes. In contrast, triploid hybrids lack genome endoreplication but utilize premeiotic genome elimination of a single-copy parental genome forming diploid gonocytes that undergo meiosis and produce haploid gametes. Therefore, the interplay of parental genomes leads to diverse gametogenic outcomes in hybrids dependent on their ploidy and genome dosage. These alterations in gametogenic pathways can persist across generations, potentially enabling the cyclic maintenance of asexual/polyploid hybrids in natural populations.

. Hypothetically, the reductional 70 division is skipped, but the equational division is normal again resulting in the same type of 71 clonal progeny (Dedukh et al., 2022b;Monaco et al., 1984). 72 After forming clonal eggs, asexuals have to avoid fertilization to prevent ploidy elevation, as 73 polyploid animals are usually sterile. In gynogenetic organisms, sperm is required to activate the 74 egg but not incorporate its genetic material into the egg (Beukeboom and Vrijenhoek, 1998; 75 Zhang et al., 2015). To maintain clonal lineages in natural populations, gynogens thus have to 76 exploit males of sexual species, whose sperm is "wasted", acting as so-called "sexual parasites"    Our study investigates particular cellular mechanisms in natural diploid and triploid hybrids with 121 different reproductive outcomes. We examined oocytes during the pachytene and diplotene 122 stages of meiosis and the gonocyte's genome composition and ploidy level. Additionally, we 123 analysed the distribution of meiocytes throughout the ovaria of asexual diploid and triploid 124 hybrid biotypes. Finally, we investigated the mechanisms of hybrid sterility in triploid hybrid 125 males.

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Sexual species exhibit normal pairing of chromosomes 128 We found that the somatic cells of C. hankugensis and I. longicorpa have 2n = 48 and 2n = 129 50 chromosomes, respectively, identical to the previous finding (Kim and Lee, 1990). Thus, in 130 the somatic cells of diploid HL hybrids, we observed 2n = 49 chromosomes; in the somatic cells 131 of triploid HHL hybrids, we detected 3n = 73, which was also the same as the previous finding 132 ( Kim and Lee, 1990). FISH with distinguished satellite DNA SatCE1 and SatCE5 repeat markers  Figure S1C). 138 We investigated pachytene chromosomes in three males and one female of C. hankugensis 139 and one male and three females of I. longicorpa (Supplementary Table 1). To confirm bivalent 140 formation during the pachytene stage of the sexual species, we stained the axial (SYCP1) and  In addition, we investigated gonadal microanatomy and revealed the distribution of 157 gonocytes, meiocytes and gametes in both males and females of the studied sexual species 158 (Supplementary Figure S6A-D). In C. hankugensis and I. longicorpa females, we observed gonocytes and pachytene clusters between pre-vitellogenic and vitellogenic oocytes. In C.   In contrast to diplotene oocytes, in pachytene, we observed oocytes only with unduplicated 186 genomes ( Figure 3F). In total, we found 13 oocytes during pachytene from two hybrid females.  can proceed beyond pachytene ( Figure 5E). 203 We tested whether incidences of cells with endoreplicated genomes differ among diploid and 204 triploid hybrids. To perform such analysis, we used the generalized linear model with binomial 205 error distribution to compare the counts of gonocytes with initial ploidy level and duplicated 206 ones. As a result, we found highly significant differences (p < 10 -4 ), whereby diploids possesses 207 ~ 6% of duplicated cells, while HHL triploids had none.   Figure 6B). The partial pairing was also sometimes observed between individual univalents 226 ( Figure 2B). Using FISH with SatCE1 DNA marker, we distinguished signal on one bivalent and 227 one univalent (Supplementary Figure S8A). We also confirmed crossing over loci only on 228 bivalents, while no crossing over loci was found on univalents ( Figure 3B). Second population of 229 pachytenic oocytes included diploid oocytes with 24 bivalents (n = 28) possibly represented by 230 C. hankugensis chromosomes ( Figure 2C; type III in Figure 6B). Such bivalents always 231 exhibited at least one crossing over locus ( Figure 3C). In addition, we detected one signal of  Table S1; Figure 5D; Supplementary Figure S7F). 246 We assume that diploid gonocytes emerged after premeiotic genome elimination. Among  Figure S7D). In 22 diplotene oocytes, we detected one signal, and in eight oocytes, we observed 258 three signals. It may suggest methodological difficulties with merging two signals into one or 259 that a small portion of oocytes with univalents and both with bivalents and univalents can 260 proceed beyond pachytene. In any case, our results suggest that there is no elimination between 261 pachytene and diplotene, and genome elimination occurs premeiotically.

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Triploid hybrid males are sterile due to aberrant pairing of chromosomes 263 We analysed 24 spermatocytes during pachytene from two triploid hybrid HHL males. The 264 analysis of synaptonemal complexes using antibodies against their lateral (SYCP3) and axial 265 (SYCP1) components revealed incomplete SCs with 6-13 properly formed bivalents ( Figure 2E).

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In some bivalents, SYCP3 was usually localized to subtelomeric regions, while inner fragments 267 of chromosomes lacked the SYCP3 signals ( Figure 2E). The analysis of crossing over loci 268 revealed 4-10 MLH1 loci per bivalent ( Figure 3E). We conclude that hybrid males have aberrant 269 pairing, with only a few chromosomes being able to form bivalents.

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To further investigate the ability of spermatocyte I to proceed beyond metaphase I (MI) 271 and check whether they can form spermatids and spermatozoa, we performed the analysis of 272 gonadal microanatomy using 3D analysis. In the gonads of triploid hybrid males, we detected The present study investigated gametogenesis in diploid and triploid hybrids from C.  Triploid hybrids, however, seem unable to perform such a premeiotic genome 303 endoreplication pathway and their fertility relies on different gametogenic alteration ( Figure 6B).

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Specifically, investigated meiocytes of females with HHL genome composition eliminated a 305 single-copied (I. longicorpa) genome, and formed bivalents between double-copied genomes (C.  hybrids also seems to be restricted to only minor population of gonocytes. Moreover, we found 356 that, at least in some gonocytes, the genome elimination occurs before meiosis, which is evident 357 from our observation of individual diploid pachytene oocytes and diploid gonocytes ( Figure 6B).

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In other asexuals, genome elimination may be partial or even absent during gametogenesis   interactions between hybrids and sexual species and relies on specific modifications of 483 gametogenic program which vary between hybrids with different ploidy levels (Figure 1, 6A, B).

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Samples studied and preparation of specimens for cytogenetic examination 486 The fish samples of Cobitis hankugensis and Iksookimia longicorpa and their diploid and 487 triploid hybrids were separately collected from three different places along the Lam Stream in 488 the province of Unbong-eup Namwon-si Jeollabuk-do in Korea according to previous 489 distributional reports (Lee, 1992;Lee, 1995;Ko, 2009). The ethical review for the fish collection