Parthenogenesis as a Solution to Hybrid Sterility: The Mechanistic Basis of Meiotic Distortions in Clonal and Sterile Hybrids

Abstract

Hybrid sterility is a hallmark of speciation, but the underlying molecular mechanisms remain poorly understood. Here, we report that speciation may regularly proceed through a stage at which gene flow is completely interrupted, but hybrid sterility occurs only in male hybrids whereas female hybrids reproduce asexually. We analyzed gametogenic pathways in hybrids between the fish species Cobitis elongatoides and C. taenia, and revealed that male hybrids were sterile owing to extensive asynapsis and crossover reduction among heterospecific chromosomal pairs in their gametes, which was subsequently followed by apoptosis. We found that polyploidization allowed pairing between homologous chromosomes and therefore partially rescued the bivalent formation and crossover rates in triploid hybrid males. However, it was not sufficient to overcome sterility. In contrast, both diploid and triploid hybrid females exhibited premeiotic genome endoreplication, thereby ensuring proper bivalent formation between identical chromosomal copies. This endoreplication ultimately restored female fertility but it simultaneously resulted in the obligate production of clonal gametes, preventing any interspecific gene flow. In conclusion, we demonstrate that the emergence of asexuality can remedy hybrid sterility in a sex-specific manner and contributes to the speciation process.

Keywords: Cobitis; clonality; endoreplication; gynogenesis; hybridization; meiosis; polyploidy; speciation.

Figure 1

Schematic overview of gametogenic pathways and aberrations in parental species and hybrids of both sexes. (A) Scheme of reproductive interactions among studied genomotypes and the types of resulting progeny. (B) Hypothetical gametogenic pathways before meiosis. (C) Observed stages of meiosis. Note that for methodological reasons, we studied the pachytene stage in both sexes, but that diplotene and metaphase I stages could be observed only in females and males, respectively.

Figure 2

Female meiotic spreads at diplotene and pachytene stages. (A–D) The spreads of lampbrush chromosomes from diplotene oocytes of C. taenia with 24 bivalents (A), C. elongatoides with 25 bivalents (B), diploid ET hybrid with 49 bivalents (C), and triploid ETT hybrid with 73 bivalents (D). Lampbrush chromosomes are numbered numerically according to their size and morphology (see Figure S1 for a detailed map of lampbrush chromosomes). Subscripts in italics indicate the distinguishable lampbrush chromosomes unequivocally corresponding to C. elongatoides “e” and C. taenia “t,” respectively. Bar, 50 µm. (E–G) Spread pachytene oocytes of C. elongatoides (E), diploid ET (F), and triploid ETT (G) hybrid females stained with DAPI (blue); synaptonemal complexes were immunolabeled with antibodies against SYCP3 protein (green) and MLH1 protein (red). Bar, 10 µm.

Figure 3

Meiotic spreads at pachytene stages. (A) Male C. elongatoides, (B) triploid ETT hybrid female, (C) diploid ET hybrid male and (D) triploid ETT hybrid male. Synaptonemal complexes visualized by immunolabeling with antibodies against SYCP3 protein (green) and SYCP1 (red), stained with DAPI (blue). Synapsed chromosomes show both SYCP3 and SYCP1 localization (indicated by arrows), while asynapsed chromosomes exhibit only SYCP3 staining (arrowheads). Bar, 10 mm.

Figure 4

Male meiotic spreads at metaphase I and pachytene stages. (A, B, and D) Giemsa-stained chromosomes (gray) with FISH-labeled telomeres (blue) in C. elongatoides, ET and ETT hybrid males, respectively. (C and E) The same metaphases as in (B and D) of hybrids after comparative genomic hybridization revealing the origins of individual chromosomes (red colour chromosomes correspond to C. elongatoides and green ones to C. taenia). Thin arrows indicate exemplary cases of bivalents, arrowheads exemplary univalents, and thick arrows exemplary cases of multivalents. (F–H) Meiotic spreads at pachytene stage of C. elongatoides (F), diploid ET hybrid (G), and triploid ETT hybrid (H) males stained with DAPI (blue). Synaptonemal complexes were immunolabeled with antibodies against SYCP3 protein (green) and MLH1 protein (red). Arrows indicate exemplary bivalents, arrowheads show examples of abnormal pairing and failures of bivalent formation. Bar, 10 mm. (I–K) Flow cytometry results of testes of C. elongatoides (I), diploid ET hybrid males (J), and triploid ETT hybrid males (K). (L) Diagram shows the average frequencies of crossovers (COs) per cell in studied genomotypes (males and females indicated in blue and red, respectively).

Figure 5

Comparison of spermatogenesis between male representatives of sexual diploid species and hybrid genomotypes. As the variation between two species and between two hybrid genomotypes is negligible, for simplicity we selected C. elongatoides and ETT males as representatives of both groups. (A and A’) Semithin sections show spermatogonia (arrow), spermatocyte in zygotene/leptotene (white firm line) and pachytene (black dashed line) of prophase I, spermatocyte in metaphase I (white dotted line), spermatids (white dashed line), and spermatozoa (black firm line), while testes of hybrid displays defective development with only a few single spermatozoa and germ cells of one cyst at different stages (black dotted line). (B and B’) Spermatogonia A with nucleus (N), nucleolus (black arrow), Golgi apparatus (asterisk), mitochondria (black circle), nuage (white arrow), and Sertoli cell (Se). (C and C’) Spermatocyte in zygotene/leptotene of prophase I with nucleus (N), mitochondria (black circle), nuage (white arrow), and synaptonemal complexes (white asterisk). (D and D’) Spermatocytes in metaphase I show compact chromatin (Ch) in equatorial position with spindle fibers (black asterisk) and mitochondria (black circle) (in contrast, hybrid exhibits irregular distribution of chromatin and no spindle fiber formation). (E and E’) Spermatids in sexual diploid display nucleus (N), mitochondria (black circle), basal body (white arrow), and flagellum (white circle), while spermatids of hybrids are usually fragmented and contain numerous axonemes/flagella. (F and F’) spermatozoa are composed of nucleus (N), mitochondria (black circle), basal body (white arrow), and flagellum (white asterisk) (hybrids exhibit very rare occurrence of spermatozoa with generally bigger nucleus).