Spermatogenesis is a complex process where spermatogonia develop into haploid, mobile sperm cells. The genes guiding this process are subject to an evolutionary trade-off between preserving basic functions of sperm while acquiring new traits ensuring advantages in competition over fertilization of female gametes. In species with XY sex chromosomes, the outcome of this trade-off is found to vary across the stages of spermatogenesis but remains unexplored for species with ZW sex chromosomes. Here we characterize avian spermatogenesis at single cell resolution from testis of collared and pied flycatchers. We find evidence for relaxed evolutionary constraint of genes expressed in spermatocyte cells going through meiosis. An overrepresentation of Z-linked differentially expressed genes between the two species at this stage suggests that this relaxed constraint is associated with the lack of sex-chromosome silencing during meiosis. We conclude that the high throughput of bird spermatogenesis, at least partly, is explained by relaxed developmental constraint.

Identifying genes involved in genetic incompatibilities causing hybrid sterility or inviability is a long-standing challenge in speciation research, especially in studies based on natural hybrid zones. Here we present the first high-probability candidate genes for hybrid male sterility in birds by using a combination of whole genome sequence data, histology sections of testis and single cell transcriptomics of testis samples from male pied-, collared-, and hybrid flycatchers. We reveal failure of meiosis in hybrid males and propose candidate genes involved in genetic incompatibilities causing this failure. Based on identification of genes with non-synonymous fixed differences between the two species and revealing miss-expression patterns of these genes across the various stages of hybrid male spermatogenesis we conclude aberrant chromosome segregation and/or faulty chromatin packing. A lower proportion of spermatids produced by hybrid males implies that a proportion of the aberrant spermatids undergo apoptosis. Finally, we report an overrepresentation of Z-linkage of the revealed candidate incompatibility genes. Our results challenge the assumption that speciation processes are driven by fast evolving genes by showing that a few changes in genes with highly conserved and central functions may quickly ensure reproductive isolation through post-zygotic isolation.