An annotated chromosome-scale reference genome for Eastern black-eared wheatear (Oenanthe melanoleuca).

Pervasive convergent evolution and in part high incidences of hybridization distinguish wheatears (songbirds of the genus Oenanthe) as a versatile system to address questions at the forefront of research on the molecular bases of phenotypic and species diversification. To prepare the genomic resources for this venture, we here generated and annotated a chromosome-scale assembly of the Eastern black-eared wheatear (Oenanthe melanoleuca). This species is part of the Oenanthe hispanica complex that is characterized by convergent evolution of plumage coloration and high rates of hybridization. The long-read-based male nuclear genome assembly comprises 1.04 Gb in 32 autosomes, the Z chromosome, and the mitogenome. The assembly is highly contiguous (contig N50, 12.6 Mb; scaffold N50, 70 Mb), with 96% of the genome assembled at the chromosome level and 95.5% benchmarking universal single-copy orthologs (BUSCO) completeness. The nuclear genome was annotated with 18,143 protein-coding genes and 31,333 mRNAs (annotation BUSCO completeness, 98.0%), and about 10% of the genome consists of repetitive DNA. The annotated chromosome-scale reference genome of Eastern black-eared wheatear provides a crucial resource for research into the genomics of adaptation and speciation in an intriguing group of passerines.

A Phylogenomic Assessment of Processes Underpinning Convergent Evolution in Open-Habitat Chats.

Insights into the processes underpinning convergent evolution advance our understanding of the contributions of ancestral, introgressed, and novel genetic variation to phenotypic evolution. Phylogenomic analyses characterizing genome-wide gene tree heterogeneity can provide first clues about the extent of ILS and of introgression and thereby into the potential of these processes or (in their absence) the need to invoke novel mutations to underpin convergent evolution. Here, we were interested in understanding the processes involved in convergent evolution in open-habitat chats (wheatears of the genus Oenanthe and their relatives). To this end, based on whole-genome resequencing data from 50 taxa of 44 species, we established the species tree, characterized gene tree heterogeneity, and investigated the footprints of ILS and introgression within the latter. The species tree corroborates the pattern of abundant convergent evolution, especially in wheatears. The high levels of gene tree heterogeneity in wheatears are explained by ILS alone only for 30% of internal branches. For multiple branches with high gene tree heterogeneity, D-statistics and phylogenetic networks identified footprints of introgression. Finally, long branches without extensive ILS between clades sporting similar phenotypes provide suggestive evidence for the role of novel mutations in the evolution of these phenotypes. Together, our results suggest that convergent evolution in open-habitat chats involved diverse processes and highlight that phenotypic diversification is often complex and best depicted as a network of interacting lineages.

Endosomal disentanglement of a transducible artificial transcription factor targeting endothelin receptor A.

Cell-penetrating peptides (CPPs) hold great promise for intracellular delivery of therapeutic proteins. However, endosomal entrapment of transduced cargo is a major bottleneck hampering their successful application. While developing a transducible zinc finger protein-based artificial transcription factor targeting the expression of endothelin receptor A, we identified interaction between the CPP and the endosomal membrane or endosomal entanglement as a main culprit for endosomal entrapment. To achieve endosomal disentanglement, we utilized endosome-resident proteases to sever the artificial transcription factor from its CPP upon arrival inside the endosome. Using this approach, we greatly enhanced the correct subcellular localization of the disentangled artificial transcription factor, significantly increasing its biological activity and distribution in vivo. With rational engineering of proteolytic sensitivity, we propose a new design principle for transducible therapeutic proteins, helping CPPs attain their full potential as delivery vectors for therapeutic proteins.

Seasonal migration patterns and the maintenance of evolutionary diversity in a cryptic bird radiation.

Morphological differentiation associated with evolutionary diversification is often explained with adaptive benefits but the processes and mechanisms maintaining cryptic diversity are still poorly understood. Using genome-wide data, we show here that the pale sand martin Riparia diluta in Central and East Asia consists of three genetically deeply differentiated lineages which vary only gradually in morphology but broadly reflect traditional taxonomy. We detected no signs of gene flow along the eastern edge of the Qinghai-Tibetan plateau between lowland south-eastern Chinese R. d. fohkienensis and high-altitude R. d. tibetana. Largely different breeding and migration timing between these low and high altitude populations as indicated by phenology data suggests that allochrony might act as prezygotic isolation mechanism in the area where their ranges abut. Mongolian populations of R. d. tibetana, however, displayed signs of limited mixed ancestries with Central Asian R. d. diluta. Their ranges meet in the area of a well-known avian migratory divide, where western lineages take a western migration route around the Qinghai-Tibetan plateau to winter quarters in South Asia, and eastern lineages take an eastern route to Southeast Asia. This might also be the case between western R. d. diluta and eastern R. d. tibetana as indicated by differing wintering grounds. We hypothesize that hybrids might have nonoptimal intermediate migration routes and selection against them might restrict gene flow. Although further potential isolation mechanisms might exist in the pale sand martin, our study points towards contrasting migration behaviour as an important factor in maintaining evolutionary diversity under morphological stasis.

Evolution: Small populations, low recombination, big trouble?

Small populations harbour less genetic diversity and more harmful mutations. They thus adapt more slowly. A new study supports these notions and suggests that reduced recombination exacerbates these effects, highlighting the impact of genome architecture on adaptability.

Identifying the causes and consequences of assembly gaps using a multiplatform genome assembly of a bird-of-paradise.

Genome assemblies are currently being produced at an impressive rate by consortia and individual laboratories. The low costs and increasing efficiency of sequencing technologies now enable assembling genomes at unprecedented quality and contiguity. However, the difficulty in assembling repeat-rich and GC-rich regions (genomic "dark matter") limits insights into the evolution of genome structure and regulatory networks. Here, we compare the efficiency of currently available sequencing technologies (short/linked/long reads and proximity ligation maps) and combinations thereof in assembling genomic dark matter. By adopting different de novo assembly strategies, we compare individual draft assemblies to a curated multiplatform reference assembly and identify the genomic features that cause gaps within each assembly. We show that a multiplatform assembly implementing long-read, linked-read and proximity sequencing technologies performs best at recovering transposable elements, multicopy MHC genes, GC-rich microchromosomes and the repeat-rich W chromosome. Telomere-to-telomere assemblies are not a reality yet for most organisms, but by leveraging technology choice it is now possible to minimize genome assembly gaps for downstream analysis. We provide a roadmap to tailor sequencing projects for optimized completeness of both the coding and noncoding parts of nonmodel genomes.

Linked-read sequencing enables haplotype-resolved resequencing at population scale.

The feasibility to sequence entire genomes of virtually any organism provides unprecedented insights into the evolutionary history of populations and species. Nevertheless, many population genomic inferences - including the quantification and dating of admixture, introgression and demographic events, and inference of selective sweeps - are still limited by the lack of high-quality haplotype information. The newest generation of sequencing technology now promises significant progress. To establish the feasibility of haplotype-resolved genome resequencing at population scale, we investigated properties of linked-read sequencing data of songbirds of the genus Oenanthe across a range of sequencing depths. Our results based on the comparison of downsampled (25×, 20×, 15×, 10×, 7×, and 5×) with high-coverage data (46-68×) of seven bird genomes mapped to a reference suggest that phasing contiguities and accuracies adequate for most population genomic analyses can be reached already with moderate sequencing effort. At 15× coverage, phased haplotypes span about 90% of the genome assembly, with 50% and 90% of phased sequences located in phase blocks longer than 1.25-4.6 Mb (N50) and 0.27-0.72 Mb (N90). Phasing accuracy reaches beyond 99% starting from 15× coverage. Higher coverages yielded higher contiguities (up to about 7 Mb/1 Mb [N50/N90] at 25× coverage), but only marginally improved phasing accuracy. Phase block contiguity improved with input DNA molecule length; thus, higher-quality DNA may help keeping sequencing costs at bay. In conclusion, even for organisms with gigabase-sized genomes like birds, linked-read sequencing at moderate depth opens an affordable avenue towards haplotype-resolved genome resequencing at population scale.

Lack of statistical power as a major limitation in understanding MHC-mediated immunocompetence in wild vertebrate populations.

Disentangling the sources of variation in developing an effective immune response against pathogens is of major interest to immunoecology and evolutionary biology. To date, the link between immunocompetence and genetic variation at the major histocompatibility complex (MHC) has received little attention in wild animals, despite the key role of MHC genes in activating the adaptive immune system. Although several studies point to a link between MHC and immunocompetence, negative findings have also been reported. Such disparate findings suggest that limited statistical power might be affecting studies on this topic, owing to insufficient sample sizes and/or a generally small effect of MHC on the immunocompetence of wild vertebrates. To clarify this issue, we investigated the link between MHC variation and seven immunocompetence proxies in a large sample of barn owls and estimated the effect sizes and statistical power of this and published studies on this topic. We found that MHC poorly explained variation in immunocompetence of barn owls, with small-to-moderate associations between MHC and immunocompetence in owls (effect size: .1 ≥ r ≤ .3) similar to other vertebrates studied to date. Such small-to-moderate effects were largely associated with insufficient power, which was only sufficient (>0.8) to detect moderate-to-large effect sizes (r ≥ .3). Thus, studies linking MHC variation with immunocompetence in wild populations are underpowered to detect MHC effects, which are likely to be of generally small magnitude. Larger sample sizes (>200) will be required to achieve sufficient power in future studies aiming to robustly test for a link between MHC variation and immunocompetence.

Avian MHC Evolution in the Era of Genomics: Phase 1.0.

Birds are a wonderfully diverse and accessible clade with an exceptional range of ecologies and behaviors, making the study of the avian major histocompatibility complex (MHC) of great interest. In the last 20 years, particularly with the advent of high-throughput sequencing, the avian MHC has been explored in great depth in several dimensions: its ability to explain ecological patterns in nature, such as mating preferences; its correlation with parasite resistance; and its structural evolution across the avian tree of life. Here, we review the latest pulse of avian MHC studies spurred by high-throughput sequencing. Despite high-throughput approaches to MHC studies, substantial areas remain in need of improvement with regard to our understanding of MHC structure, diversity, and evolution. Recent studies of the avian MHC have nonetheless revealed intriguing connections between MHC structure and life history traits, and highlight the advantages of long-term ecological studies for understanding the patterns of MHC variation in the wild. Given the exceptional diversity of birds, their accessibility, and the ease of sequencing their genomes, studies of avian MHC promise to improve our understanding of the many dimensions and consequences of MHC variation in nature. However, significant improvements in assembling complete MHC regions with long-read sequencing will be required for truly transformative studies.

Genome-wide evidence supports mitochondrial relationships and pervasive parallel phenotypic evolution in open-habitat chats.

In wheatears and related species ('open-habitat chats'), molecular phylogenetics has led to a comprehensively revised understanding of species relationships and species diversity. Phylogenetic analyses have suggested that, in many cases, phenotypic similarities do not reflect species' relationships, revealing traditionally defined genera as non-monophyletic. This led to the suggestion of pervasive parallel evolution of open-habitat chats' plumage coloration and ecological phenotypes. However, to date, the molecular evidence for the phylogenetic relationships among open-habitat chats is mainly limited to mitochondrial DNA. Here, we assessed whether the mitochondrial relationships are supported by genome-wide data. To this end, we reconstructed the species tree among 14 open-habitat chat taxa using multi-species coalescent analyses based on ~1'300 SNPs. Our results confirm previous ones based chiefly on mitochondrial DNA; notably the paraphyly of the Oenanthe lugens complex and the clustering of individual species formerly placed in the genera Cercomela and Myrmecocichla within Oenanthe. Since several variable morphological and ecological characteristics occur in multiple places across the open-habitat chat phylogeny, our study consolidates the evidence for pervasive parallel evolution in the plumage coloration and ecology of open-habitat chats.

Footprints of adaptive evolution revealed by whole Z chromosomes haplotypes in flycatchers.

Detecting positive selection using genomic data is critical to understanding the role of adaptive evolution. Of particular interest in this context is sex chromosomes since they are thought to play a special role in local adaptation and speciation. We sought to circumvent the challenges associated with statistical phasing when using haplotype-based statistics in sweep scans by benefitting from that whole chromosome haplotypes of the sex chromosomes can be obtained by resequencing of individuals of the hemizygous sex. We analyzed whole Z chromosome haplotypes from 100 females from several populations of four black and white flycatcher species (in birds, females are ZW and males ZZ). Based on integrated haplotype score (iHS) and number of segregating sites by length (nSL) statistics, we found strong and frequent haplotype structure in several regions of the Z chromosome in each species. Most of these sweep signals were population-specific, with essentially no evidence for regions under selection shared among species. Some completed sweeps were revealed by the cross-population extended haplotype homozygosity (XP-EHH) statistic. Importantly, by using statistically phased Z chromosome data from resequencing of males, we failed to recover the signals of selection detected in analyses based on whole chromosome haplotypes from females; instead, what likely represent false signals of selection were frequently seen. This highlights the power issues in statistical phasing and cautions against conclusions from selection scans using such data. The detection of frequent selective sweeps on the avian Z chromosome supports a large role of sex chromosomes in adaptive evolution.

Parallel plumage colour evolution and introgressive hybridization in wheatears.

Genetic and phenotypic mosaics, in which various phenotypes and different genomic regions show discordant patterns of species or population divergence, offer unique opportunities to study the role of ancestral and introgressed genetic variation in phenotypic evolution. Here, we investigated the evolution of discordant phenotypic and genetic divergence in a monophyletic clade of four songbird taxa-pied wheatear (O. pleschanka), Cyprus wheatear (Oenanthe cypriaca), and western and eastern subspecies of black-eared wheatear (O. h. hispanica and O. h. melanoleuca). Phenotypically, black back and neck sides distinguish pied and Cyprus wheatears from the white-backed/necked black-eared wheatears. Meanwhile, mitochondrial variation only distinguishes western black-eared wheatear. In the absence of nuclear genetic data, and given frequent hybridization among eastern black-eared and pied wheatear, it remains unclear whether introgression is responsible for discordance between mitochondrial divergence patterns and phenotypic similarities, or whether plumage coloration evolved in parallel. Multispecies coalescent analyses of about 20,000 SNPs obtained from RAD data mapped to a draft genome assembly resolve the species tree, provide evidence for the parallel evolution of colour phenotypes and establish western and eastern black-eared wheatears as independent taxa that should be recognized as full species. The presence of the entire admixture spectrum in the Iranian hybrid zone and the detection of footprints of introgression from pied into eastern black-eared wheatear beyond the hybrid zone despite strong geographic structure of ancestry proportions furthermore suggest a potential role for introgression in parallel plumage colour evolution. Our results support the importance of standing heterospecific and/or ancestral variation in phenotypic evolution.

Success and failure in replication of genotype-phenotype associations: How does replication help in understanding the genetic basis of phenotypic variation in outbred populations?

Recent developments in sequencing technologies have facilitated genomewide mapping of phenotypic variation in natural populations. Such mapping efforts face a number of challenges potentially leading to low reproducibility. However, reproducible research forms the basis of scientific progress. We here discuss the options for replication and the reasons for potential nonreproducibility. We then review the evidence for reproducible quantitative trait loci (QTL) with a focus on natural animal populations. Existing case studies of replication fall into three categories: (i) traits that have been mapped to major effect loci (including chromosomal inversion and supergenes) by independent research teams; (ii) QTL fine-mapped in discovery populations; and (iii) attempts to replicate QTL across multiple populations. Major effect loci, in particular those associated with inversions, have been successfully replicated in several cases within and across populations. Beyond such major effect variants, replication has been more successful within than across populations, suggesting that QTL discovered in natural populations may often be population-specific. This suggests that biological causes (differences in linkage patterns, allele frequencies or context-dependencies of QTL) contribute to nonreproducibility. Evidence from other fields, notably animal breeding and QTL mapping in humans, suggests that a significant fraction of QTL is indeed reproducible in direction and magnitude at least within populations. However, there is also a large number of QTL that cannot be easily reproduced. We put forward that more studies should explicitly address the causes and context-dependencies of QTL signals, in particular to disentangle linkage differences, allele frequency differences and gene-by-environment interactions as biological causes of nonreproducibility of QTL, especially between populations.

Lack of evidence for selection favouring MHC haplotypes that combine high functional diversity.

High rates of gene duplication and the highest levels of functional allelic diversity in vertebrate genomes are the main hallmarks of the major histocompatibility complex (MHC), a multigene family with a primordial role in pathogen recognition. The usual tight linkage among MHC gene duplicates may provide an opportunity for the evolution of haplotypes that associate functionally divergent alleles and thus grant the transmission of optimal levels of diversity to coming generations. Even though such associations may be a crucial component of disease resistance, this hypothesis has been given little attention in wild populations. Here, we leveraged pedigree data from a barn owl (Tyto alba) population to characterize MHC haplotype structure across two MHC class I (MHC-I) and two MHC class IIB (MHC-IIB) duplicates, in order to test the hypothesis that haplotypes' genetic diversity is higher than expected from randomly associated alleles. After showing that MHC loci are tightly linked within classes, we found limited evidence for shifts towards MHC haplotypes combining high diversity. Neither amino acid nor functional within-haplotype diversity were significantly higher than in random sets of haplotypes, regardless of MHC class. Our results therefore provide no evidence for selection towards high-diversity MHC haplotypes in barn owls. Rather, high rates of concerted evolution may constrain the evolution of high-diversity haplotypes at MHC-I, while, in contrast, for MHC-IIB, fixed differences among loci may provide barn owls with already optimized functional diversity. This suggests that at the MHC-I and MHC-IIB respectively, different evolutionary dynamics may govern the evolution of within-haplotype diversity.

Linked selection, demography and the evolution of correlated genomic landscapes in birds and beyond.

Selection has a deep impact on the distribution of genetic diversity and population differentiation along the genome (the genomic landscapes of diversity and differentiation), reducing diversity and elevating differentiation not only at the sites it targets, but also at linked neutral sites. Fuelled by the high-throughput sequencing revolution, these genomic footprints of selection have been extensively exploited over the past decade with the aim to identify genomic regions involved in adaptation and speciation. However, while this research has shown that the genomic landscapes of diversity and differentiation are usually highly heterogeneous, it has also led to the increasing realization that this heterogeneity may evolve under processes other than adaptation or speciation. In particular, instead of being an effect of selective sweeps or barriers to gene flow, accentuated differentiation can evolve by any process reducing genetic diversity locally within the genome (Charlesworth, ), including purifying selection at linked sites (background selection). In particular, in genomic regions where recombination is infrequent, accentuated differentiation can evolve as a by-product of diversity reductions unrelated to adaptation or speciation (Cruickshank & Hahn, ; Nachman & Payseur, ; Noor & Bennett, ). In such genomic regions, linkage extends over physically larger genome stretches, and selection affects a particularly high number of linked neutral sites. Even though the effects of selection on linked neutral diversity (linked selection) within populations are well documented (Cutter & Payseur, ), recent observations of diversity and differentiation landscapes that are highly correlated even among independent lineages suggest that the effects of long-term linked selection may have a deeper impact on the evolution of the genomic landscapes of diversity and differentiation than previously anticipated. The study on Saxicola stonechats by Van Doren et al. () reported in the current issue of Molecular Ecology lines in with a rapidly expanding body of evidence in this direction. Correlations of genomic landscapes extending from within stonechats to comparisons with Ficedula flycatchers add to recent insights into the timescales across which the effects of linked selection persist. Absent and inverted correlations of genomic landscapes in comparisons involving an island taxon, on the other hand, provide important empirical clues about the role of demographic constraints in the evolution of the genomic landscapes of diversity and differentiation.

Genomewide patterns of variation in genetic diversity are shared among populations, species and higher-order taxa.

Genomewide screens of genetic variation within and between populations can reveal signatures of selection implicated in adaptation and speciation. Genomic regions with low genetic diversity and elevated differentiation reflective of locally reduced effective population sizes (Ne ) are candidates for barrier loci contributing to population divergence. Yet, such candidate genomic regions need not arise as a result of selection promoting adaptation or advancing reproductive isolation. Linked selection unrelated to lineage-specific adaptation or population divergence can generate comparable signatures. It is challenging to distinguish between these processes, particularly when diverging populations share ancestral genetic variation. In this study, we took a comparative approach using population assemblages from distant clades assessing genomic parallelism of variation in Ne . Utilizing population-level polymorphism data from 444 resequenced genomes of three avian clades spanning 50 million years of evolution, we tested whether population genetic summary statistics reflecting genomewide variation in Ne would covary among populations within clades, and importantly, also among clades where lineage sorting has been completed. All statistics including population-scaled recombination rate (ρ), nucleotide diversity (π) and measures of genetic differentiation between populations (FST , PBS, dxy ) were significantly correlated across all phylogenetic distances. Moreover, genomic regions with elevated levels of genetic differentiation were associated with inferred pericentromeric and subtelomeric regions. The phylogenetic stability of diversity landscapes and stable association with genomic features support a role of linked selection not necessarily associated with adaptation and speciation in shaping patterns of genomewide heterogeneity in genetic diversity.

100 million years of multigene family evolution: origin and evolution of the avian MHC class IIB.

BACKGROUND: Gene duplication has led to a most remarkable adaptation involved in vertebrates' host-pathogen arms-race, the major histocompatibility complex (MHC). However, MHC duplication history is as yet poorly understood in non-mammalian vertebrates, including birds. RESULTS: Here, we provide evidence for the evolution of two ancient avian MHC class IIB (MHCIIB) lineages by a duplication event prior to the radiation of all extant birds >100 million years ago, and document the role of concerted evolution in eroding the footprints of the avian MHCIIB duplication history. CONCLUSIONS: Our results suggest that eroded footprints of gene duplication histories may mimic birth-death evolution and that in the avian MHC the presence of the two lineages may have been masked by elevated rates of concerted evolution in several taxa. Through the presence of a range of intermediate evolutionary stages along the homogenizing process of concerted evolution, the avian MHCIIB provides a remarkable illustration of the erosion of multigene family duplication history.

Whole-genome patterns of linkage disequilibrium across flycatcher populations clarify the causes and consequences of fine-scale recombination rate variation in birds.

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.

Covariation in levels of nucleotide diversity in homologous regions of the avian genome long after completion of lineage sorting.

Closely related species may show similar levels of genetic diversity in homologous regions of the genome owing to shared ancestral variation still segregating in the extant species. However, after completion of lineage sorting, such covariation is not necessarily expected. On the other hand, if the processes that govern genetic diversity are conserved, diversity may potentially covary even among distantly related species. We mapped regions of conserved synteny between the genomes of two divergent bird species-collared flycatcher and hooded crow-and identified more than 600 Mb of homologous regions (66% of the genome). From analyses of whole-genome resequencing data in large population samples of both species we found nucleotide diversity in 200 kb windows to be well correlated (Spearman's ρ = 0.407). The correlation remained highly similar after excluding coding sequences. To explain this covariation, we suggest that a stable avian karyotype and a conserved landscape of recombination rate variation render the diversity-reducing effects of linked selection similar in divergent bird lineages. Principal component regression analysis of several potential explanatory variables driving heterogeneity in flycatcher diversity levels revealed the strongest effects from recombination rate variation and density of coding sequence targets for selection, consistent with linked selection. It is also possible that a stable karyotype is associated with a conserved genomic mutation environment contributing to covariation in diversity levels between lineages. Our observations imply that genetic diversity is to some extent predictable.

Genomic distribution and estimation of nucleotide diversity in natural populations: perspectives from the collared flycatcher (Ficedula albicollis) genome.

Properly estimating genetic diversity in populations of nonmodel species requires a basic understanding of how diversity is distributed across the genome and among individuals. To this end, we analysed whole-genome resequencing data from 20 collared flycatchers (genome size ≈1.1 Gb; 10.13 million single nucleotide polymorphisms detected). Genomewide nucleotide diversity was almost identical among individuals (mean = 0.00394, range = 0.00384-0.00401), but diversity levels varied extensively across the genome (95% confidence interval for 200-kb windows = 0.0013-0.0053). Diversity was related to selective constraint such that in comparison with intergenic DNA, diversity at fourfold degenerate sites was reduced to 85%, 3' UTRs to 82%, 5' UTRs to 70% and nondegenerate sites to 12%. There was a strong positive correlation between diversity and chromosome size, probably driven by a higher density of targets for selection on smaller chromosomes increasing the diversity-reducing effect of linked selection. Simulations exploring the ability of sequence data from a small number of genetic markers to capture the observed diversity clearly demonstrated that diversity estimation from finite sampling of such data is bound to be associated with large confidence intervals. Nevertheless, we show that precision in diversity estimation in large outbred population benefits from increasing the number of loci rather than the number of individuals. Simulations mimicking RAD sequencing showed that this approach gives accurate estimates of genomewide diversity. Based on the patterns of observed diversity and the performed simulations, we provide broad recommendations for how genetic diversity should be estimated in natural populations.