Structural variation (SV) constitutes an important type of genetic mutations providing the raw material for evolution. Here, we uncover the genome-wide spectrum of intra-and interspecific SV segregating in natural populations of seven songbird species in the genus Corvus. Combining short-read (N = 127) and long-read re-sequencing (N = 31), as well as optical mapping (N = 16), we apply both assembly-and read mapping approaches to detect SV and characterize a total of 220,452 insertions, deletions and inversions. We exploit sampling across wide phylogenetic timescales to validate SV genotypes and assess the contribution of SV to evolutionary processes in an avian model of incipient speciation. We reveal an evolutionary young (~530,000 years) cis-acting 2.25-kb LTR retrotransposon insertion reducing expression of the NDP gene with consequences for premating isolation. Our results attest to the wealth and evolutionary significance of SV segregating in natural populations and highlight the need for reliable SV genotyping.

The tuatara (Sphenodon punctatus)-the only living member of the reptilian order Rhynchocephalia (Sphenodontia), once widespread across Gondwana 1,2-is an iconic species that is endemic to New Zealand 2,3. A key link to the now-extinct stem reptiles (from which dinosaurs, modern reptiles, birds and mammals evolved), the tuatara provides key insights into the ancestral amniotes 2,4. Here we analyse the genome of the tuatara, which-at approximately 5 Gb-is among the largest of the vertebrate genomes yet assembled. Our analyses of this genome, along with comparisons with other vertebrate genomes, reinforce the uniqueness of the tuatara. Phylogenetic analyses indicate that the tuatara lineage diverged from that of snakes and lizards around 250 million years ago. This lineage also shows moderate rates of molecular evolution, with instances of punctuated evolution. Our genome sequence analysis identifies expansions of proteins, non-protein-coding RNA families and repeat elements, the latter of which show an amalgam of reptilian and mammalian features. The sequencing of the tuatara genome provides a valuable resource for deep comparative analyses of tetrapods, as well as for tuatara biology and conservation. Our study also provides important insights into both the technical challenges and the cultural obligations that are associated with genome sequencing.

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.