The fine-scale recombination rate variation and associations with genomic features in a butterfly.

Recombination is a key molecular mechanism that has profound implications on both micro- and macroevolutionary processes. However, the determinants of recombination rate variation in holocentric organisms are poorly understood, in particular in Lepidoptera (moths and butterflies). The wood white butterfly (Leptidea sinapis) shows considerable intraspecific variation in chromosome numbers and is a suitable system for studying regional recombination rate variation and its potential molecular underpinnings. Here, we developed a large whole-genome resequencing data set from a population of wood whites to obtain high-resolution recombination maps using linkage disequilibrium information. The analyses revealed that larger chromosomes had a bimodal recombination landscape, potentially caused by interference between simultaneous chiasmata. The recombination rate was significantly lower in subtelomeric regions, with exceptions associated with segregating chromosome rearrangements, showing that fissions and fusions can have considerable effects on the recombination landscape. There was no association between the inferred recombination rate and base composition, supporting a limited influence of GC-biased gene conversion in butterflies. We found significant but variable associations between the recombination rate and the density of different classes of transposable elements, most notably a significant enrichment of short interspersed nucleotide elements in genomic regions with higher recombination rate. Finally, the analyses unveiled significant enrichment of genes involved in farnesyltranstransferase activity in recombination coldspots, potentially indicating that expression of transferases can inhibit formation of chiasmata during meiotic division. Our results provide novel information about recombination rate variation in holocentric organisms and have particular implications for forthcoming research in population genetics, molecular/genome evolution, and speciation.

Nascent evolution of recombination rate differences as a consequence of chromosomal rearrangements.

Reshuffling of genetic variation occurs both by independent assortment of chromosomes and by homologous recombination. Such reshuffling can generate novel allele combinations and break linkage between advantageous and deleterious variants which increases both the potential and the efficacy of natural selection. Here we used high-density linkage maps to characterize global and regional recombination rate variation in two populations of the wood white butterfly (Leptidea sinapis) that differ considerably in their karyotype as a consequence of at least 27 chromosome fissions and fusions. The recombination data were compared to estimates of genetic diversity and measures of selection to assess the relationship between chromosomal rearrangements, crossing over, maintenance of genetic diversity and adaptation. Our data show that the recombination rate is influenced by both chromosome size and number, but that the difference in the number of crossovers between karyotypes is reduced as a consequence of a higher frequency of double crossovers in larger chromosomes. As expected from effects of selection on linked sites, we observed an overall positive association between recombination rate and genetic diversity in both populations. Our results also revealed a significant effect of chromosomal rearrangements on the rate of intergenic diversity change between populations, but limited effects on polymorphisms in coding sequence. We conclude that chromosomal rearrangements can have considerable effects on the recombination landscape and consequently influence both maintenance of genetic diversity and efficiency of selection in natural populations.

Meiotic drive against chromosome fusions in butterfly hybrids.

Species frequently differ in the number and structure of chromosomes they harbor, but individuals that are heterozygous for chromosomal rearrangements may suffer from reduced fitness. Chromosomal rearrangements like fissions and fusions can hence serve as a mechanism for speciation between incipient lineages, but their evolution poses a paradox. How can rearrangements get fixed between populations if heterozygotes have reduced fitness? One solution is that this process predominantly occurs in small and isolated populations, where genetic drift can override natural selection. However, fixation is also more likely if a novel rearrangement is favored by a transmission bias, such as meiotic drive. Here, we investigate chromosomal transmission distortion in hybrids between two wood white (Leptidea sinapis) butterfly populations with extensive karyotype differences. Using data from two different crossing experiments, we uncover that there is a transmission bias favoring the ancestral chromosomal state for derived fusions, a result that shows that chromosome fusions actually can fix in populations despite being counteracted by meiotic drive. This means that meiotic drive not only can promote runaway chromosome number evolution and speciation, but also that it can be a conservative force acting against karyotypic change and the evolution of reproductive isolation. Based on our results, we suggest a mechanistic model for why chromosome fusion mutations may be opposed by meiotic drive and discuss factors contributing to karyotype evolution in Lepidoptera.

Tissue-specific patterns of regulatory changes underlying gene expression differences among Ficedula flycatchers and their naturally occurring F1 hybrids.

Changes in interacting cis- and trans-regulatory elements are important candidates for Dobzhansky-Muller hybrid incompatibilities and may contribute to hybrid dysfunction by giving rise to misexpression in hybrids. To gain insight into the molecular mechanisms and determinants of gene expression evolution in natural populations, we analyzed the transcriptome from multiple tissues of two recently diverged Ficedula flycatcher species and their naturally occurring F1 hybrids. Differential gene expression analysis revealed that the extent of differentiation between species and the set of differentially expressed genes varied across tissues. Common to all tissues, a higher proportion of Z-linked genes than autosomal genes showed differential expression, providing evidence for a fast-Z effect. We further found clear signatures of hybrid misexpression in brain, heart, kidney, and liver. However, while testis showed the highest divergence of gene expression among tissues, it showed no clear signature of misexpression in F1 hybrids, even though these hybrids were found to be sterile. It is therefore unlikely that incompatibilities between cis-trans regulatory changes explain the observed sterility. Instead, we found evidence that cis-regulatory changes play a significant role in the evolution of gene expression in testis, which illustrates the tissue-specific nature of cis-regulatory evolution bypassing constraints associated with pleiotropic effects of genes.

Environmental stress during larval development induces DNA methylation shifts in the migratory painted lady butterfly (Vanessa cardui).

Seasonal environmental fluctuations provide formidable challenges for living organisms, especially small ectotherms such as butterflies. A common strategy to cope with harsh environments is to enter diapause, but some species avoid unsuitable conditions by migrating. Despite a growing understanding of migration in the life cycles of some butterfly species, it remains unknown how individuals register and store environmental cues to determine whether and where to migrate. Here, we explored how competition and host plant availability during larval development affect patterns of DNA methylation in the migratory painted lady (Vanessa cardui) butterfly. We identify a set of potentially functional methylome shifts associated with differences in the environment, indicating that DNA methylation is involved in the response to different conditions during larval development. By analysing the transcriptome for the same samples used for methylation profiling, we also uncovered a non-monotonic relationship between gene body methylation and gene expression. Our results provide a starting point for understanding the interplay between DNA methylation and gene expression in butterflies in general and how differences in environmental conditions during development can trigger unique epigenetic marks that might be important for behavioural decisions in the adult stage.

Migratory behaviour is positively associated with genetic diversity in butterflies.

Migration is typically associated with risk and uncertainty at the population level, but little is known about its cost-benefit trade-offs at the species level. Migratory insects in particular often exhibit strong demographic fluctuations due to local bottlenecks and outbreaks. Here, we use genomic data to investigate levels of heterozygosity and long-term population size dynamics in migratory insects, as an alternative to classical local and short-term approaches such as regional field monitoring. We analyse whole-genome sequences from 97 Lepidoptera species and show that individuals of migratory species have significantly higher levels of genome-wide heterozygosity, a proxy for effective population size, than do nonmigratory species. Also, we contribute whole-genome data for one of the most emblematic insect migratory species, the painted lady butterfly (Vanessa cardui), sampled across its worldwide distributional range. This species exhibits one of the highest levels of genomic heterozygosity described in Lepidoptera (2.95 ± 0.15%). Coalescent modelling (PSMC) shows historical demographic stability in V. cardui, and high effective population size estimates of 2-20 million individuals 10,000 years ago. The study reveals that the high risks associated with migration and local environmental fluctuations do not seem to decrease overall genetic diversity and demographic stability in migratory Lepidoptera. We propose a "compensatory" demographic model for migratory r-strategist organisms in which local bottlenecks are counterbalanced by reproductive success elsewhere within their typically large distributional ranges. Our findings highlight that the boundaries of populations are substantially different for sedentary and migratory insects, and that, in the latter, local and even regional field monitoring results may not reflect whole population dynamics. Genomic diversity patterns may elucidate key aspects of an insect's migratory nature and population dynamics at large spatiotemporal scales.

Linkage mapping and genome annotation give novel insights into gene family expansions and regional recombination rate variation in the painted lady (Vanessa cardui) butterfly.

Characterization of gene family expansions and crossing over is crucial for understanding how organisms adapt to the environment. Here, we develop a high-density linkage map and detailed genome annotation of the painted lady butterfly (Vanessa cardui) - a non-diapausing, highly polyphagous species famous for its long-distance migratory behavior and almost cosmopolitan distribution. Our results reveal a complex interplay between regional recombination rate variation, gene duplications and transposable element activity shaping the genome structure of the painted lady. We identify several lineage specific gene family expansions. Their functions are mainly associated with protein and fat metabolism, detoxification, and defense against infection - critical processes for the painted lady's unique life-history. Furthermore, the detailed recombination maps allow us to characterize the regional recombination landscape, data that reveal a strong effect of chromosome size on the recombination rate, a limited impact of GC-biased gene conversion and a positive association between recombination and short interspersed elements.

Host plant diet affects growth and induces altered gene expression and microbiome composition in the wood white (Leptidea sinapis) butterfly.

In a time with decreasing biodiversity, especially among insects, a detailed understanding about specific resource utilization strategies is crucial. The physiological and behavioural responses to host switches in phytophagous insects are poorly understood. Earlier studies indicate that a host plant switch might be associated with distinctive molecular and physiological responses in different lineages. Expanding the assessment of such associations across Lepidoptera will reveal if there are general patterns in adaptive responses, or if each switch event is more of a unique character. We investigated host plant preference, fitness consequences, effects on expression profiles and gut microbiome composition in two common wood white (Leptidea sinapis) populations with different host plant preferences from the extremes of the species distribution area (Sweden and Catalonia). Our results show that female Catalonian wood whites lack preference for either host plant (Lotus corniculatus or L. dorycnium), while Swedish females laid significantly more eggs on L. corniculatus. Individuals from both populations reared on L. dorycnium had longer developmental times and smaller body size as adults. This indicates that both environmental and genetic factors determine the choice to use a specific host plant. Gene expression analysis revealed a more pronounced response to host plant in the Catalonian compared to the Swedish population. In addition, host plant treatment resulted in a significant shift in microbiome community structure in the Catalonian population. Together, this suggests that population specific plasticity associated with local conditions underlies host plant utilisation in wood whites.

Lack of gene flow: Narrow and dispersed differentiation islands in a triplet of Leptidea butterfly species.

Genome scans in recently separated species can inform on molecular mechanisms and evolutionary processes driving divergence. Large-scale polymorphism data from multiple species pairs are also key to investigate the repeatability of divergence-whether radiations tend to show parallel responses to similar selection pressures and/or underlying molecular forces. Here, we used whole-genome resequencing data from six wood white (Leptidea sp.) butterfly populations, representing three closely related species with karyomorph variation, to infer the species' demographic history and characterize patterns of genomic diversity and differentiation. The analyses supported previously established species relationships, and there was no evidence for postdivergence gene flow. We identified significant intraspecific genetic structure, in particular between karyomorph extremes in the wood white (L. sinapis)-a species with a remarkable chromosome number cline across the distribution range. The genomic landscapes of differentiation were erratic, and outlier regions were narrow and dispersed. Highly differentiated (FST ) regions generally had low genetic diversity (θπ ), but increased absolute divergence (DXY ) and excess of rare frequency variants (low Tajima's D). A minority of differentiation peaks were shared across species and population comparisons. However, highly differentiated regions contained genes with overrepresented functions related to metabolism, response to stimulus and cellular processes, indicating recurrent directional selection on a specific set of traits in all comparisons. In contrast to the majority of genome scans in recently diverged lineages, our data suggest that divergence landscapes in Leptidea have been shaped by directional selection and genetic drift rather than stable recombination landscapes and/or introgression.

Gene expression profiling across ontogenetic stages in the wood white (Leptidea sinapis) reveals pathways linked to butterfly diapause regulation.

In temperate latitudes, many insects enter diapause (dormancy) during the cold season, a period during which developmental processes come to a standstill. The wood white (Leptidea sinapis) is a butterfly species distributed across western Eurasia that shows photoperiod-induced diapause with variation in critical day-length across populations at different latitudes. We assembled transcriptomes and estimated gene expression levels at different developmental stages in experimentally induced directly developing and diapausing cohorts of a single Swedish population of L. sinapis to investigate the regulatory mechanisms underpinning diapause initiation. Different day lengths resulted in expression changes of developmental genes and affected the rate of accumulation of signal molecules, suggesting that diapause induction might be controlled by increased activity of monoamine neurotransmitters in larvae reared under short-day light conditions. Expression differences between light treatment groups of two monoamine regulator genes (DDC and ST) were observed already in instar III larvae. Once developmental pathways were irreversibly set at instar V, a handful of genes related to dopamine production were differentially expressed leading to a significant decrease in expression of global metabolic genes and increase in expression of genes related to fatty acid synthesis and sequestration. This is in line with a time-dependent (hour-glass) model of diapause regulation where a gradual shift in the concentration of monoamine neurotransmitters and their metabolites during development of larvae under short-day conditions leads to increased storage of fat, decreased energy expenditures, and ultimately developmental stasis at the pupal stage.

The genomic landscape of species divergence in Ficedula flycatchers.

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.

Association mapping of morphological traits in wild and captive zebra finches: reliable within, but not between populations.

Identifying causal genetic variants underlying heritable phenotypic variation is a long-standing goal in evolutionary genetics. We previously identified several quantitative trait loci (QTL) for five morphological traits in a captive population of zebra finches (Taeniopygia guttata) by whole-genome linkage mapping. We here follow up on these studies with the aim to narrow down on the quantitative trait variants (QTN) in one wild and three captive populations. First, we performed an association study using 672 single nucleotide polymorphisms (SNPs) within candidate genes located in the previously identified QTL regions in a sample of 939 wild-caught zebra finches. Then, we validated the most promising SNP-phenotype associations (n = 25 SNPs) in 5228 birds from four populations. Genotype-phenotype associations were generally weak in the wild population, where linkage disequilibrium (LD) spans only short genomic distances. In contrast, in captive populations, where LD blocks are large, apparent SNP effects on morphological traits (i.e. associations) were highly repeatable with independent data from the same population. Most of those SNPs also showed significant associations with the same trait in other captive populations, but the direction and magnitude of these effects varied among populations. This suggests that the tested SNPs are not the causal QTN but rather physically linked to them, and that LD between SNPs and causal variants differs between populations due to founder effects. While the identification of QTN remains challenging in nonmodel organisms, we illustrate that it is indeed possible to confirm the location and magnitude of QTL in a population with stable linkage between markers and causal variants.

Quantitative Mass Spectrometry Reveals Partial Translational Regulation for Dosage Compensation in Chicken.

There is increasing evidence that dosage compensation is not a ubiquitous feature following sex chromosome evolution, especially not in organisms where females are the heterogametic sex, like in birds. Even when it occurs, compensation can be incomplete and limited to dosage-sensitive genes. However, previous work has mainly studied transcriptional regulation of sex-linked genes, which may not reflect expression at the protein level. Here, we used liquid chromatography-tandem mass spectrometry to detect and quantify expressed levels of more than 2,400 proteins in ten different tissues of male and female chicken embryos. For comparison, transcriptome sequencing was performed in the same individuals, five of each sex. The proteomic analysis revealed that dosage compensation was incomplete, with a mean male-to-female (M:F) expression ratio of Z-linked genes of 1.32 across tissues, similar to that at the RNA level (1.29). The mean Z chromosome-to-autosome expression ratio was close to 1 in males and lower than 1 in females, consistent with partly reduced Z chromosome expression in females. Although our results exclude a general mechanism for chromosome-wide dosage compensation at translation, 30% of all proteins encoded from Z-linked genes showed a significant change in the M:F ratio compared with the corresponding ratio at the RNA level. This resulted in a pattern where some genes showed balanced expression between sexes and some close to 2-fold higher expression in males. This suggests that proteomic analyses will be necessary to reveal a more complete picture of gene regulation and sex chromosome evolution.

Divergence in gene expression within and between two closely related flycatcher species.

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 PST 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.

The genome of a songbird.

The zebra finch is an important model organism in several fields with unique relevance to human neuroscience. Like other songbirds, the zebra finch communicates through learned vocalizations, an ability otherwise documented only in humans and a few other animals and lacking in the chicken-the only bird with a sequenced genome until now. Here we present a structural, functional and comparative analysis of the genome sequence of the zebra finch (Taeniopygia guttata), which is a songbird belonging to the large avian order Passeriformes. We find that the overall structures of the genomes are similar in zebra finch and chicken, but they differ in many intrachromosomal rearrangements, lineage-specific gene family expansions, the number of long-terminal-repeat-based retrotransposons, and mechanisms of sex chromosome dosage compensation. We show that song behaviour engages gene regulatory networks in the zebra finch brain, altering the expression of long non-coding RNAs, microRNAs, transcription factors and their targets. We also show evidence for rapid molecular evolution in the songbird lineage of genes that are regulated during song experience. These results indicate an active involvement of the genome in neural processes underlying vocal communication and identify potential genetic substrates for the evolution and regulation of this behaviour.

Assembly errors cause false tandem duplicate regions in the chicken (Gallus gallus) genome sequence.

The complexity of eukaryote genomes makes assembly errors inevitable in the process of constructing reference genomes. Next-generation sequencing (NGS) could provide an efficient way to validate previously assembled genomes. Here, we exploited NGS data to interrogate the chicken reference genome and identified 35 pairs of nearly identical regions with >99.5 % sequence similarity and a median size of 109 kb. Several lines of evidence, including read depth, the composition of junction sequences, and sequence similarity, suggest that these regions present genome assembly errors and should be excluded from forthcoming genomic studies.

A house finch (Haemorhous mexicanus) spleen transcriptome reveals intra- and interspecific patterns of gene expression, alternative splicing and genetic diversity in passerines.

BACKGROUND: With its plumage color dimorphism and unique history in North America, including a recent population expansion and an epizootic of Mycoplasma gallisepticum (MG), the house finch (Haemorhous mexicanus) is a model species for studying sexual selection, plumage coloration and host-parasite interactions. As part of our ongoing efforts to make available genomic resources for this species, here we report a transcriptome assembly derived from genes expressed in spleen. RESULTS: We characterize transcriptomes from two populations with different histories of demography and disease exposure: a recently founded population in the eastern US that has been exposed to MG for over a decade and a native population from the western range that has never been exposed to MG. We utilize this resource to quantify conservation in gene expression in passerine birds over approximately 50 MY by comparing splenic expression profiles for 9,646 house finch transcripts and those from zebra finch and find that less than half of all genes expressed in spleen in either species are expressed in both species. Comparative gene annotations from several vertebrate species suggest that the house finch transcriptomes contain ~15 genes not yet found in previously sequenced vertebrate genomes. The house finch transcriptomes harbour ~85,000 SNPs, ~20,000 of which are non-synonymous. Although not yet validated by biological or technical replication, we identify a set of genes exhibiting differences between populations in gene expression (n = 182; 2% of all transcripts), allele frequencies (76 FST ouliers) and alternative splicing as well as genes with several fixed non-synonymous substitutions; this set includes genes with functions related to double-strand break repair and immune response. CONCLUSIONS: The two house finch spleen transcriptome profiles will add to the increasing data on genome and transcriptome sequence information from natural populations. Differences in splenic expression between house finch and zebra finch imply either significant evolutionary turnover of splenic expression patterns or different physiological states of the individuals examined. The transcriptome resource will enhance the potential to annotate an eventual house finch genome, and the set of gene-based high-quality SNPs will help clarify the genetic underpinnings of host-pathogen interactions and sexual selection.

Reconstruction of gross avian genome structure, organization and evolution suggests that the chicken lineage most closely resembles the dinosaur avian ancestor.

BACKGROUND: The availability of multiple avian genome sequence assemblies greatly improves our ability to define overall genome organization and reconstruct evolutionary changes. In birds, this has previously been impeded by a near intractable karyotype and relied almost exclusively on comparative molecular cytogenetics of only the largest chromosomes. Here, novel whole genome sequence information from 21 avian genome sequences (most newly assembled) made available on an interactive browser (Evolution Highway) was analyzed. RESULTS: Focusing on the six best-assembled genomes allowed us to assemble a putative karyotype of the dinosaur ancestor for each chromosome. Reconstructing evolutionary events that led to each species' genome organization, we determined that the fastest rate of change occurred in the zebra finch and budgerigar, consistent with rapid speciation events in the Passeriformes and Psittaciformes. Intra- and interchromosomal changes were explained most parsimoniously by a series of inversions and translocations respectively, with breakpoint reuse being commonplace. Analyzing chicken and zebra finch, we found little evidence to support the hypothesis of an association of evolutionary breakpoint regions with recombination hotspots but some evidence to support the hypothesis that microchromosomes largely represent conserved blocks of synteny in the majority of the 21 species analyzed. All but one species showed the expected number of microchromosomal rearrangements predicted by the haploid chromosome count. Ostrich, however, appeared to retain an overall karyotype structure of 2n=80 despite undergoing a large number (26) of hitherto un-described interchromosomal changes. CONCLUSIONS: Results suggest that mechanisms exist to preserve a static overall avian karyotype/genomic structure, including the microchromosomes, with widespread interchromosomal change occurring rarely (e.g., in ostrich and budgerigar lineages). Of the species analyzed, the chicken lineage appeared to have undergone the fewest changes compared to the dinosaur ancestor.

Evidence from a house finch (Haemorhous mexicanus) spleen transcriptome for adaptive evolution and biased gene conversion in passerine birds.

Identifying genes influenced by natural selection can provide information about lineage-specific adaptations, and transcriptomes generated by next-generation sequencing are a useful resource for identifying such genes. Here, we utilize a spleen transcriptome for the house finch (Haemorhous mexicanus), an emerging model for sexual selection and disease ecology, together with previously sequenced avian genomes (chicken, turkey, and zebra finch), to investigate lineage-specific adaptations within birds. An analysis of 4,398 orthologous genes revealed a significantly higher ratio of nonsynonymous to synonymous substitutions and significantly higher GC content in passerines than in galliforms, an observation deviating from strictly neutral expectations but consistent with an effect of biased gene conversion on the evolutionary rate in passerines. These data also showed that genes exhibiting signs of positive selection and fast evolution in passerines have functional roles related to fat metabolism, neurodevelopment, and ion binding.

A high-density linkage map enables a second-generation collared flycatcher genome assembly and reveals the patterns of avian recombination rate variation and chromosomal evolution.

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.