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Hybrid inviability is an important post-zygotic reproductive barrier between species, but emerging signs of reduced viability can be difficult to study across the lifespan of natural hybrids. We use a combination of long-term monitoring, extra-pair paternity, and mitochondrial DNA identification in a natural hybrid zone of Ficedula flycatchers to detect emerging signs of intrinsic hybrid inviability across their entire lifespan. We evaluate possible evidence of Darwin's corollary to Haldane's rule, predicting asymmetries in inviability between hybrids resulting from reciprocal crosses, due to incompatible genetic factors with sex-specific inheritance patterns. We found higher hatching failure among mixed-species pairs, possibly indicating early developmental impairments associated with specific parental genetic combinations. Adult hybrids had a higher basal mortality rate than both parental species and different age-specific mortality trajectories. There were signs of differences in age-independent mortality rates between the reciprocal hybrid crosses: hybrids with a pied flycatcher mother experienced slightly increased mortality later in life. Using an exceptional dataset with many natural hybrids tracked across life stages, we provide evidence for several emerging signs of reduced hybrid viability. Incompatibilities between alleles located on autosomes and uniparentally inherited factors such as Z-linked and/or mitochondrial genes are strong candidates underlying intrinsic hybrid dysfunction in this system.

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. 

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.

Whether females should prefer to mate with old males is controversial. Old males may sire offspring of low quality because of an aging germline, but their proven ability to reach an old age can also be an excellent indicator of superior genetic quality, especially in natural populations. These genetic effects are, however, hard to study in nature, because they are often confounded with direct benefits offered by old males to the female, such as experience and high territory quality. We, therefore, used naturally occurring extra-pair young to disentangle different aspects of male age on female fitness in a natural population of collared flycatchers because any difference between within- and extra-pair young within a nest should be caused by paternal genetic effects only. Based on 18 years of long-term data, we found that females paired with older males as social partners experienced an overall reproductive advantage. However, offspring sired by old males were of lower quality as compared to their extra-pair half-siblings, whereas the opposite was found in nests attended by young males. These results imply a negative genetic effect of old paternal age, given that extra-pair males are competitive middle-age males. Thus, offspring may benefit from being sired by young males but raised by old males, to maximize both genetic and direct effects. Our results show that direct and genetic benefits from pairing with old males may act in opposing directions and that the quality of the germline may deteriorate before other signs of senescence become obvious.

Identifying the traits causing reproductive isolation and the order in which they evolve is fundamental to understanding speciation. Here, we quantify prezygotic and intrinsic postzygotic isolation among allopatric, parapatric, and sympatric populations of the butterflies Heliconius elevatus and Heliconius pardalinus. Sympatric populations from the Amazon (H. elevatus and H. p. butleri) exhibit strong prezygotic isolation and rarely mate in captivity; however, hybrids are fertile. Allopatric populations from the Amazon (H. p. butleri) and Andes (H. p. sergestus) mate freely when brought together in captivity, but the female F1 hybrids are sterile. Parapatric populations (H. elevatus and H. p. sergestus) exhibit both assortative mating and sterility of female F1s. Assortative mating in sympatric populations is consistent with reinforcement in the face of gene flow, where the driving force, selection against hybrids, is due to disruption of mimicry and other ecological traits rather than hybrid sterility. In contrast, the lack of assortative mating and hybrid sterility observed in allopatric populations suggests that geographic isolation enables the evolution of intrinsic postzygotic reproductive isolation. Our results show how the types of reproductive barriers that evolve between species may depend on geography.

Population divergence in sexual signals may lead to speciation through prezygotic isolation. Sexual signals can change solely due to variation in the level of natural selection acting against conspicuousness. However, directional mate choice (i.e., favoring conspicuousness) across different environments may lead to gene flow between populations, thereby delaying or even preventing the evolution of reproductive barriers and speciation. In this study, we test whether natural selection through predation upon mate-choosing females can favor corresponding changes in mate preferences. Our study system, Oophaga pumilio, is an extremely color polymorphic neotropical frog with two distinctive antipredator strategies: aposematism and crypsis. The conspicuous coloration and calling behavior of aposematic males may attract both cryptic and aposematic females, but predation may select against cryptic females choosing aposematic males. We used an experimental approach where domestic fowl were encouraged to find digitized images of cryptic frogs at different distances from aposematic partners. We found that the estimated survival time of a cryptic frog was reduced when associating with an aposematic partner. Hence, predation may act as a direct selective force on female choice, favoring evolution of color assortative mating that, in turn, may strengthen the divergence in coloration that natural selection has generated.

Hybrid inviability is an important post-zygotic reproductive barrier between species but emerging signs of intrinsic dysfunction can be difficult to study across the lifespan of natural hybrids. Here, we use a combination of long-term monitoring of individuals in a natural hybrid zone of Ficedula flycatchers together with information on extra-pair paternity and mitochondrial DNA identification with the main goal of detecting emerging signs of intrinsic hybrid inviability across the entire lifespan of these naturally hybridizing birds. We also evaluate possible evidence of Darwin’s corollary to Haldane’s rule, which predicts asymmetries in the degree of inviability between hybrids resulting from reciprocal crosses, due to incompatible genetic factors with sex-specific inheritance patterns. We found higher hatching failure among mixed-species pairs, which could indicate early developmental impairments associated with specific parental genetic combinations. Adult hybrids had a higher basal mortality rate than both parental species, and different age-specific mortality trajectories. There were some signs of differences in age-independent mortality rates between the reciprocal hybrid crosses, with hybrid individuals with a pied flycatcher mother experiencing slightly increased mortality rates later in life. Clashes between the maternally inherited mitochondrial genome and part of the paternally inherited nuclear genome in these hybrids, leading to reduced efficiency of the cellular energy metabolism, are good candidates underlying intrinsic postzygotic isolation in this system.

Since the discovery of the maternally inherited Germline Restricted Chromosome (GRC) in the songbird clade, the function of this chromosome has been highly debated. Whether it is a parasitic element or has an essential adaptive role needs to be unveiled. Here, we use a combination of linked reads DNA sequencing and single cell RNA sequencing to examine the gene content of the GRC in pied and collared flycatchers and the expression pattern of these genes during spermatogenesis. We found 157 paralogs on the GRC of which only 46 were shared between the two species. We also found that the identities of the genes that were expressed during spermatogenesis differed between these species. We found important genes for meiosis, germline maintenance and sex-determination. These finding suggest a possible essential role of the GRC during spermatogenesis. This evidence together with the rapid gene content and gene expression evolution suggests that GRCs could also contribute to the appearance of genetic incompatibilities between closely related species.

Structural variants, typically defined as mutations affecting more than 50bp, have been shown to encompass a significant portion of the genome and can have large phenotypic effects. Additionally, increasing empirical evidence demonstrates that structural variants may play a substantial role in speciation, which could previously have been overlooked because of difficulties in identifying them with short-read data. However, with the increased availability of long-read sequencing technology we are now equipped better than ever to address this limitation and study the contribution of different types of structural variants to genetic variation within and genetic differentiation between closely related species. Here, we follow this approach and combine PacBio HiFi and HiC sequencing for two closely related passerine birds, the collared flycatcher and the pied flycatcher. This enables us to generate a chromosome-level genome assembly for both species, and identify structural variants between the two species. Based on population-level HiFi sequencing for both species, we then investigate patterns of single nucleotide diversity and differentiation within and between species and their association with different types of structural variation. We find widespread structural variation between the two species, where both the sex chromosomes show a disproportionate number of structural variants, which may help explain the suspected role of the Z-chromosome in contributing to genetic incompatibilities. We also find that genomic differentiation peaks are enriched in both translocations and inversions, which supports a mechanistic role of structural variation in population differentiation and speciation.

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 missexpression 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.