Whole-genome sequencing projects are increasingly populating the tree of life and characterizing biodiversity1–4. Sparse taxon sampling has previously been proposed to confound phylogenetic inference5, and captures only a fraction of the genomic diversity. Here we report a substantial step towards the dense representation of avian phylogenetic and molecular diversity, by analysing 363 genomes from 92.4% of bird families—including 267 newly sequenced genomes produced for phase II of the Bird 10,000 Genomes (B10K) Project. We use this comparative genome dataset in combination with a pipeline that leverages a reference-free whole-genome alignment to identify orthologous regions in greater numbers than has previously been possible and to recognize genomic novelties in particular bird lineages. The densely sampled alignment provides a single-base-pair map of selection, has more than doubled the fraction of bases that are confidently predicted to be under conservation and reveals extensive patterns of weak selection in predominantly non-coding DNA. Our results demonstrate that increasing the diversity of genomes used in comparative studies can reveal more shared and lineage-specific variation, and improve the investigation of genomic characteristics. We anticipate that this genomic resource will offer new perspectives on evolutionary processes in cross-species comparative analyses and assist in efforts to conserve species.

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Sex chromosomes have evolved from the same autosomes multiple times across vertebrates, suggesting that selection for recombination suppression has acted repeatedly and independently on certain genetic backgrounds. Here, we perform comparative genomics of a bird clade (larks and their sister lineage; Alaudidae and Panuridae) where multiple autosome–sex chromosome fusions appear to have formed expanded sex chromosomes. We detected the largest known avian sex chromosome (195.3 Mbp) and show that it originates from fusions between parts of four avian chromosomes: Z, 3, 4A and 5. Within these four chromosomes, we found evidence of five evolutionary strata where recombination had been suppressed at different time points, and show that stratum age explained the divergence rate of Z–W gametologs. Next, we analysed chromosome content and found that chromosome 3 was significantly enriched for genes with predicted sex-related functions. Finally, we demonstrate extensive homology to sex chromosomes in other vertebrate lineages: chromosomes Z, 3, 4A and 5 have independently evolved into sex chromosomes in fish (Z), turtles (Z, 5), lizards (Z, 4A), mammals (Z, 4A) and frogs (Z, 3, 4A, 5). Our results provide insights into and support for repeated evolution of sex chromosomes in vertebrates.

How the avian sex chromosomes first evolved from autosomes remains elusive as 100 million years (Myr) of divergence and degeneration obscure their evolutionary history. The Sylvioidea group of songbirds is interesting for understanding avian sex chromosome evolution because a chromosome fusion event ∼24 Myr ago formed “neo-sex chromosomes” consisting of an added (new) and an ancestral (old) part. Here, we report the complete female genome (ZW) of one Sylvioidea species, the great reed warbler (Acrocephalus arundinaceus). Our long-read assembly shows that the added region has been translocated to both Z and W, and while the added-Z has remained its gene order the added-W part has been heavily rearranged. Phylogenetic analyses show that recombination between the homologous added-Z and -W regions continued after the fusion event, and that recombination suppression across this region took several million years to be completed. Moreover, recombination suppression was initiated across multiple positions over the added-Z, which is not consistent with a simple linear progression starting from the fusion point. As expected following recombination suppression, the added-W show signs of degeneration including repeat accumulation and gene loss. Finally, we present evidence for non-random maintenance of slowly evolving and dosage-sensitive genes on both ancestral- and added-W, a process causing correlated evolution among orthologous genes across broad taxonomic groups, regardless of sex-linkage.

Sex chromosomes in birds have long been considered to be extremely stable. However, this notion has lately been challenged by findings of independent autosome–sex chromosome fusions within songbirds, several of which occur within a single clade, the superfamily Sylvioidea. To understand what ecological and evolutionary processes drive changes in sex chromosome systems, we need complete descriptions of sex chromosome diversity across taxonomic groups. Here, we characterize the sex chromosome systems across Sylvioidea using whole-genome data of species representatives of 10 different families, including two published and eight new genomes. We describe a novel fusion in the family Cisticolidae (represented by Cisticola juncidis ) involving a part of chromosome 4. We also confirm the previously identified fusion between chromosome Z and a part of chromosome 4A in all 10 families and show that fusions involving parts of chromosomes 3 and 5 are not found outside the families where they were first discovered (Alaudidae and Panuridae). These findings add to the complexity of the sex chromosome system in Sylvioidea, where four independent autosome–sex chromosome fusions have now been identified.

The evolution of suppressed recombination between sex chromosomes is widely hypothesized to be driven by sexually antagonistic selection (SA), where tighter linkage between the sex‐determining gene(s) and nearby SA loci is favored when it couples male‐beneficial alleles to the proto‐Y chromosome, and female‐beneficial alleles to the proto‐X. Despite limited empirical evidence, the SA selection hypothesis overshadows several alternatives, including an incomplete but often‐repeated “sheltering hypothesis” that suggests that expansion of the sex‐linked region (SLR) reduces homozygous expression of partially recessive deleterious mutations at selected loci. Here, we use population genetic models to evaluate the consequences of deleterious mutational variation for the evolution of neutral chromosomal inversions expanding the SLR on proto‐Y chromosomes. We find that SLR‐expanding inversions face a race against time: lightly loaded inversions are initially beneficial, but eventually become deleterious as they accumulate new mutations, and must fix before this window of opportunity closes. The outcome of this race is strongly influenced by inversion size, the mutation rate, and the dominance coefficient of deleterious mutations. Yet, small inversions have elevated fixation probabilities relative to neutral expectations for biologically plausible parameter values. Our results demonstrate that deleterious genetic variation can plausibly drive recombination suppression in small steps and would be most consistent with empirical patterns of small evolutionary strata or gradual recombination arrest.

A major goal in evolutionary biology is to understand the genetic basis of adaptive traits. In migratory birds, wing morphology is such a trait. Our previous work on the great reed warbler (Acrocephalus arundinaceus) shows that wing length is highly heritable and under sexually antagonistic selection. Moreover, a quantitative trait locus (QTL) mapping analysis detected a pronounced QTL for wing length on chromosome 2, suggesting that wing morphology is partly controlled by genes with large effects. Here, we re-evaluate the genetic basis of wing length in great reed warblers using a genomewide association study (GWAS) approach based on restriction site-associated DNA sequencing (RADseq) data. We use GWAS models that account for relatedness between individuals and include covariates (sex, age and tarsus length). The resulting association landscape was flat with no peaks on chromosome 2 or elsewhere, which is in line with expectations for polygenic traits. Analysis of the distribution of p-values did not reveal biases, and the inflation factor was low. Effect sizes were however not uniformly distributed on some chromosomes, and the Z chromosome had weaker associations than autosomes. The level of linkage disequilibrium (LD) in the population decayed to background levels within c. 1 kbp. There could be several reasons to why our QTL study and GWAS gave contrasting results including differences in how associations are modelled (cosegregation in pedigree vs. LD associations), how covariates are accounted for in the models, type of marker used (multi- vs. biallelic), difference in power or a combination of these. Our study highlights that the genetic architecture even of highly heritable traits is difficult to characterize in wild populations.

In the Iberian Peninsula, populations of two subspecies of the reed bunting have become 2 increasingly fragmented during the last decades when suitable habitats have been lost and/or the 3 populations have gone extinct. Presently, both subspecies are endangered. We estimated the amount 4 of genetic variation and population structure in order to define conservation units and management 5 practices for these populations. We found that the subspecies lusitanica has clearly reduced genetic 6 variation in nuclear and mitochondrial markers, has a drastically small effective population size and 7 no genetic differentiation between populations. In contrast, the subspecies witherbyi is significantly 8 structured, but the populations still hold large amounts of variation even though the effective 9 population sizes are smaller than in the non-endangered subspecies schoeniclus. We suggest several 10 management units for the Iberian populations. One unit includes subspecies lusitanica as a whole; 11 the other three units are based on genetically differentiated populations of witherbyi. The most 12 important genetic conservation measure in the case of lusitanica is to preserve the remaining 13 habitats in order to at least maintain the present levels of gene flow. In the case of the three 14 management units within witherbyi, the most urgent conservation measure is to improve the habitat 15 quality to increase the population sizes.