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Although the theory behind the mechanisms generating intrinsic post-zygotic isolation is well established, very few concrete examples of genetic incompatibilities have been described, especially in vertebrates. Consequently, our understanding of the evolutionary forces shaping the appearance of genetic incompatibilities between natural populations and the overall role of genetic incompatibilities in the speciation process is limited. In this doctoral thesis I will contribute to filling this gap in knowledge by using different approaches to investigate the causes and genetic basis of male hybrid sterility in a natural Ficedula flycatcher hybrid zone. I started by analyzing hybrid inviability patterns using 17 years of long-term monitoring data and found evidence for hybrid inviability at different life stages (Paper I). Early developmental failure of hybrids as revealed by the lower hatching success of mixed-species pairs suggesting emerging severe but non-fixed incompatibilities between the two species. Subtler differences in terms of lower growth potential and shorter lifespan indicate mito-nuclear incompatibilities as elevated metabolic rate can cause accumulation of toxic by-products in the form of Reactive Oxygen Species (ROS). Because previous evidence indicated problems during spermatogenesis in male hybrids, I characterized collared and pied flycatcher spermatogenesis at a single-cell level (Paper II). Since this was the first single-cell study investigating avian spermatogenesis, I identified the three main stages of spermatogenesis and described expression patterns of autosomes and Z-linked genes. By analyzing differential gene expression and estimates of protein evolution, I found that meiosis appears to be less evolutionary constraint in birds than in mammals. I propose that this fundamental difference is caused by the lack of MSCI in the spermatogenesis of ZW systems. Using the spermatogenesis characterization as a baseline, I then explored hybrid spermatogenesis to detect the stage of failure and associated genes (Paper III). By using a combination of histology sections, single-cell RNA sequencing and whole genome re-sequencing data, I found strong evidence of meiosis failure in hybrid spermatogenesis. I identified genes with non-synonymous fixed differences between the two species that were also DE during spermatogenesis. This enabled me to identify candidate genes causing genetic incompatibilities leading to meiosis failure in hybrid flycatchers. Finally, I explored the role of the enigmatic Germline restricted chromosome (GRC) in flycatcher spermatogenesis (Paper IV). I sequenced the GRC and revealed the gene contents for both species of flycatchers. Then we verified the transcription of the contents of the GRC and identified testis cell clusters containing GRC transcripts to reveal at what developmental stages of spermatogenesis the GRC linked genes are transcribed. I found big differences in the patterns of expression of GRC-linked genes between the two species, adding support for the notion that GRC evolution is very fast. Among the transcribed GRC genes, I found three relevant genes for spermatogenesis, sex-determination and germline maintenance shared by both species, suggesting a possible role of the GRC in those processes. The main conclusion from my work is that, in contrast to expectations, incompatibilities causing hybrid sterility can be found in genes with conserved functions. This is because a few changes in these genes may disrupt important networks of genes and quickly cause post-zygotic isolation at secondary contact.