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.

Secondary contact between species often results in the formation of a hybrid zone, with the eventual fates of the hybridizing species dependent on evolutionary and ecological forces. We examine this process in the Amazon Basin by conducting the first genomic and phenotypic characterization of the hybrid zone formed after secondary contact between two obligate army-antfollowers: the White-breasted Antbird (Rhegmatorhina hoffmannsi) and the Harlequin Antbird (Rhegmatorhina berlepschi). We found a major geographic displacement (∼120 km) between the mitochondrial and nuclear clines, and we explore potential hypotheses for the displacement, including sampling error, genetic drift, and asymmetric cytonuclear incompatibilities. We cannot exclude roles for sampling error and genetic drift in contributing to the discordance; however, the data suggest expansion and unidirectional introgression of hoffmannsi into the distribution of berlepschi.

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