Unravelling the genomic landscape of divergence between lineages is key to understanding speciation. The naturally hybridizing collared flycatcher and pied flycatcher are important avian speciation models that show pre- as well as postzygotic isolation. We sequenced and assembled the 1.1-Gb flycatcher genome, physically mapped the assembly to chromosomes using a low-density linkage map and re-sequenced population samples of each species. Here we show that the genomic landscape of species differentiation is highly heterogeneous with approximately 50 /`divergence islands/' showing up to 50-fold higher sequence divergence than the genomic background. These non-randomly distributed islands, with between one and three regions of elevated divergence per chromosome irrespective of chromosome size, are characterized by reduced levels of nucleotide diversity, skewed allele-frequency spectra, elevated levels of linkage disequilibrium and reduced proportions of shared polymorphisms in both species, indicative of parallel episodes of selection. Proximity of divergence peaks to genomic regions resistant to sequence assembly, potentially including centromeres and telomeres, indicate that complex repeat structures may drive species divergence. A much higher background level of species divergence of the Z chromosome, and a lower proportion of shared polymorphisms, indicate that sex chromosomes and autosomes are at different stages of speciation. This study provides a roadmap to the emerging field of speciation genomics.

The typically repetitive nature of the sex-limited chromosome means that it is often excluded from or poorly covered in genome assemblies, hindering studies of evolutionary and population genomic processes in non-recombining chromosomes. Here, we present a draft assembly of the non-recombining region of the collared flycatcher W chromosome, containing 46 genes without evidence of female-specific functional differentiation. Survival of genes during W chromosome degeneration has been highly non-random and expression data suggest that this can be attributed to selection for maintaining gene dose and ancestral expression levels of essential genes. Re-sequencing of large population samples revealed dramatically reduced levels of within-species diversity and elevated rates of between-species differentiation (lineage sorting), consistent with low effective population size. Concordance between W chromosome and mitochondrial DNA phylogenetic trees demonstrates evolutionary stable matrilineal inheritance of this nuclear–cytonuclear pair of chromosomes. Our results show both commonalities and differences between W chromosome and Y chromosome evolution.

Significance: Uniquely human biology is the result of genetic differences between humans and other primates, but identifying the critical changes still poses a major challenge. We screened >32,000 human-specific substitutions in two classes of putative transcriptional enhancers implicated in human evolution for their effects on enhancer activity in human neural stem cells, a cell type fundamental for cortical development and expansion. We identify hundreds of substitutions that modify enhancer activity either alone or in combination with other variants. We find different patterns of substitution effects in different enhancer types. Our data support substitutions often interacting by modifying the same transcription factor binding sites. The functional substitutions we identify provide a rich set of candidate loci for studies of human-specific regulatory evolution.

Relatively little is known about the character of gene expression evolution as species diverge. It is for instance unclear if gene expression generally evolves in a clock‐like manner (by stabilizing selection or neutral evolution) or if there are frequent episodes of directional selection. To gain insights into the evolutionary divergence of gene expression, we sequenced and compared the transcriptomes of multiple organs from population samples of collared (Ficedula albicollis) and pied flycatchers (F. hypoleuca), two species which diverged less than one million years ago. Ordination analysis separated samples by organ rather than by species. Organs differed in their degrees of expression variance within species and expression divergence between species. Variance was negatively correlated with expression breadth and protein interactivity, suggesting that pleiotropic constraints reduce gene expression variance within species. Variance was correlated with between‐species divergence, consistent with a pattern expected from stabilizing selection and neutral evolution. Using an expression P_ST approach, we identified genes differentially expressed between species and found 16 genes uniquely expressed in one of the species. For one of these, DPP7, uniquely expressed in collared flycatcher, the absence of expression in pied flycatcher could be associated with a ≈20‐kb deletion including 11 of 13 exons. This study of a young vertebrate speciation model system expands our knowledge of how gene expression evolves as natural populations become reproductively isolated.

Sex chromosome divergence, which follows the cessation of recombination and degeneration of the sex-limited chromosome, can cause a reduction in expression level for sex-linked genes in the heterozygous sex, unless some mechanisms of dosage compensation develops to counter the reduction in gene dose. Because large-scale perturbations in expression levels arising from changes in gene dose might have strong deleterious effects, the evolutionary response should be strong. However, in birds and in at least some other female heterogametic organisms, wholesale sex chromosome dosage compensation does not seem to occur. Using RNA-seq of multiple tissues and individuals, we investigated male and female expression levels of Z-linked and autosomal genes in the collared flycatcher, a bird for which a draft genome sequence recently has been reported. We found that male expression of Z-linked genes was on average 50% higher than female expression, although there was considerable variation in the male-to-female ratio among genes. The ratio for individual genes was well correlated among tissues and there was also a correlation in the extent of compensation between flycatcher and chicken orthologs. The relative excess of male expression was positively correlated with expression breadth, expression level, and number of interacting proteins (protein connectivity), and negatively correlated with variance in expression. These observations lead to a model of compensation occurring on a gene-by-gene basis, supported by an absence of clustering of genes on the Z chromosome with respect to the extent of compensation. Equal mean expression level of autosomal and Z-linked genes in males, and 50% higher expression of autosomal than Z-linked genes in females, is compatible with that partial compensation is achieved by hypertranscription from females’ single Z chromosome. A comparison with male-to-female expression ratios in orthologous Z-linked genes of ostriches, where Z–W recombination still occurs, suggests that male-biased expression of Z-linked genes is a derived trait after avian sex chromosome divergence.

A detailed knowledge about spatial and temporal gene expression is important for understanding both the function of genes and their evolution. For the vast majority of species, transcriptomes are still largely uncharacterized and even in those where substantial information is available it is often in the form of partially sequenced transcriptomes. With the development of next generation sequencing, a single experiment can now simultaneously identify the transcribed part of a species genome and estimate levels of gene expression.