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.

Speciation is a continuous process during which genetic changes gradually accumulate in the genomes of diverging species. Recent studies have documented highly heterogeneous differentiation landscapes, with distinct regions of elevated differentiation (“differentiation islands”) widespread across genomes. However, it remains unclear which processes drive the evolution of differentiation islands; how the differentiation landscape evolves as speciation advances; and ultimately, how differentiation islands are related to speciation. Here, we addressed these questions based on population genetic analyses of 200 resequenced genomes from 10 populations of four Ficedula flycatcher sister species. We show that a heterogeneous differentiation landscape starts emerging among populations within species, and differentiation islands evolve recurrently in the very same genomic regions among independent lineages. Contrary to expectations from models that interpret differentiation islands as genomic regions involved in reproductive isolation that are shielded from gene flow, patterns of sequence divergence (d_xy and relative node depth) do not support a major role of gene flow in the evolution of the differentiation landscape in these species. Instead, as predicted by models of linked selection, genome-wide variation in diversity and differentiation can be explained by variation in recombination rate and the density of targets for selection. We thus conclude that the heterogeneous landscape of differentiation in Ficedula flycatchers evolves mainly as the result of background selection and selective sweeps in genomic regions of low recombination. Our results emphasize the necessity of incorporating linked selection as a null model to identify genome regions involved in adaptation and speciation.

Detailed linkage and recombination rate maps are necessary to use the full potential of genome sequencing and population genomic analyses. We used a custom collared flycatcher 50 K SNP array to develop a high‐density linkage map with 37 262 markers assigned to 34 linkage groups in 33 autosomes and the Z chromosome. The best‐order map contained 4215 markers, with a total distance of 3132 cM and a mean genetic distance between markers of 0.12 cM. Facilitated by the array being designed to include markers from most scaffolds, we obtained a second‐generation assembly of the flycatcher genome that approaches full chromosome sequences (N50 super‐scaffold size 20.2 Mb and with 1.042 Gb (of 1.116 Gb) anchored to and mostly ordered and oriented along chromosomes). We found that flycatcher and zebra finch chromosomes are entirely syntenic but that inversions at mean rates of 1.5–2.0 event (6.6–7.5 Mb) per My have changed the organization within chromosomes, rates high enough for inversions to potentially have been involved with many speciation events during avian evolution. The mean recombination rate was 3.1 cM/Mb and correlated closely with chromosome size, from 2 cM/Mb for chromosomes >100 Mb to >10 cM/Mb for chromosomes <10 Mb. This size dependence seemed entirely due to an obligate recombination event per chromosome; if 50 cM was subtracted from the genetic lengths of chromosomes, the rate per physical unit DNA was constant across chromosomes. Flycatcher recombination rate showed similar variation along chromosomes as chicken but lacked the large interior recombination deserts characteristic of zebra finch chromosomes.

Profound knowledge of demographic history is a prerequisite for the understanding and inference of processes involved in the evolution of population differentiation and speciation. Together with new coalescent-based methods, the recent availability of genome-wide data enables investigation of differentiation and divergence processes at unprecedented depth. We combined two powerful approaches, full Approximate Bayesian Computation analysis (ABC) and pairwise sequentially Markovian coalescent modeling (PSMC), to reconstruct the demographic history of the split between two avian speciation model species, the pied flycatcher and collared flycatcher. Using whole-genome re-sequencing data from 20 individuals, we investigated 15 demographic models including different levels and patterns of gene flow, and changes in effective population size over time. ABC provided high support for recent (mode 0.3 my, range <0.7 my) species divergence, declines in effective population size of both species since their initial divergence, and unidirectional recent gene flow from pied flycatcher into collared flycatcher. The estimated divergence time and population size changes, supported by PSMC results, suggest that the ancestral species persisted through one of the glacial periods of middle Pleistocene and then split into two large populations that first increased in size before going through severe bottlenecks and expanding into their current ranges. Secondary contact appears to have been established after the last glacial maximum. The severity of the bottlenecks at the last glacial maximum is indicated by the discrepancy between current effective population sizes (20,000–80,000) and census sizes (5–50 million birds) of the two species. The recent divergence time challenges the supposition that avian speciation is a relatively slow process with extended times for intrinsic postzygotic reproductive barriers to evolve. Our study emphasizes the importance of using genome-wide data to unravel tangled demographic histories. Moreover, it constitutes one of the first examples of the inference of divergence history from genome-wide data in non-model species. Demographic processes leave specific and detectable signatures within species genomes. Analysis of patterns of variation within and between closely related species can be used to unravel their divergence history and is crucial for understanding evolutionary processes such as speciation. We applied a set of novel population-genomic tools to investigate patterns of natural variation and infer demographic history of two avian speciation model species: pied flycatcher and collared flycatcher. The analysis supported a scenario consistent with allopatric speciation with recent, postglacial secondary contact. Most likely the ancestral species persisted through one of the glacial periods of the middle Pleistocene and then split into two large descendent populations that appear to have increased in size before experiencing severe bottlenecks during expansion into their current ranges. The two species established secondary contact after the last glacial maximum. This resulted in unidirectional gene flow from pied flycatcher to collared flycatcher. The results are consistent with a scenario where pied flycatcher recolonized northern Europe more rapidly than collared flycatcher. Our study increases the knowledge about the dynamics of the speciation process and constitutes one of the first examples of the inference of complex demographic history using information from genome-wide data in non-model species.

Recombination rate is heterogeneous across the genome of various species and so are genetic diversity and differentiation as a consequence of linked selection. However, we still lack a clear picture of the underlying mechanisms for regulating recombination. Here we estimated fine‐scale population recombination rate based on the patterns of linkage disequilibrium across the genomes of multiple populations of two closely related flycatcher species (Ficedula albicollis and F. hypoleuca). This revealed an overall conservation of the recombination landscape between these species at the scale of 200 kb, but we also identified differences in the local rate of recombination despite their recent divergence (<1 million years). Genetic diversity and differentiation were associated with recombination rate in a lineage‐specific manner, indicating differences in the extent of linked selection between species. We detected 400–3,085 recombination hotspots per population. Location of hotspots was conserved between species, but the intensity of hotspot activity varied between species. Recombination hotspots were primarily associated with CpG islands (CGIs), regardless of whether CGIs were at promoter regions or away from genes. Recombination hotspots were also associated with specific transposable elements (TEs), but this association appears indirect due to shared preferences of the transposition machinery and the recombination machinery for accessible open chromatin regions. Our results suggest that CGIs are a major determinant of the localization of recombination hotspots, and we propose that both the distribution of TEs and fine‐scale variation in recombination rate may be associated with the evolution of the epigenetic landscape.

Using genetic data to resolve the evolutionary relationships of species is of major interest in evolutionary and systematic biology. However, reconstructing the sequence of speciation events, the so-called species tree, in closely related and potentially hybridizing species is very challenging. Processes such as incomplete lineage sorting and interspecific gene flow result in local gene genealogies that differ in their topology from the species tree, and analyses of few loci with a single sequence per species are likely to produce conflicting or even misleading results. To study these phenomena on a full phylogenomic scale, we use whole-genome sequence data from 200 individuals of four black-and-white flycatcher species with so far unresolved phylogenetic relationships to infer gene tree topologies and visualize genome-wide patterns of gene tree incongruence. Using phylogenetic analysis in nonoverlapping 10-kb windows, we show that gene tree topologies are extremely diverse and change on a very small physical scale. Moreover, we find strong evidence for gene flow among flycatcher species, with distinct patterns of reduced introgression on the Z chromosome. To resolve species relationships on the background of widespread gene tree incongruence, we used four complementary coalescent-based methods for species tree reconstruction, including complex modeling approaches that incorporate post-divergence gene flow among species. This allowed us to infer the most likely species tree with high confidence. Based on this finding, we show that regions of reduced effective population size, which have been suggested as particularly useful for species tree inference, can produce positively misleading species tree topologies. Our findings disclose the pitfalls of using loci potentially under selection as phylogenetic markers and highlight the potential of modeling approaches to disentangle species relationships in systems with large effective population sizes and post-divergence gene flow.