Whole-genome sequencing projects are increasingly populating the tree of life and characterizing biodiversity^1,2,3,4. 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.

Insertion/deletion (indel) mutations, which are represented by gaps in multiple sequence alignments, have been used to examine phylogenetic hypotheses for some time. However, most analyses combine gap data with the nucleotide sequences in which they are embedded, probably because most phylogenetic datasets include few gap characters. Here, we report analyses of 12,030 gap characters from an alignment of avian nuclear genes using maximum parsimony (MP) and a simple maximum likelihood (ML) framework. Both trees were similar, and they exhibited almost all of the strongly supported relationships in the nucleotide tree, although neither gap tree supported many relationships that have proven difficult to recover in previous studies. Moreover, independent lines of evidence typically corroborated the nucleotide topology instead of the gap topology when they disagreed, although the number of conflicting nodes with high bootstrap support was limited. Filtering to remove short indels did not substantially reduce homoplasy or reduce conflict. Combined analyses of nucleotides and gaps resulted in the nucleotide topology, but with increased support, suggesting that gap data may prove most useful when analyzed in combination with nucleotide substitutions.

It has long been appreciated that analyses of genomic data (e.g., whole genome sequencing or sequence capture) have the potential to reveal the tree of life, but it remains challenging to move from sequence data to a clear understanding of evolutionary history, in part due to the computational challenges of phylogenetic estimation using genome-scale data. Supertree methods solve that challenge because they facilitate a divide-and-conquer approach for large-scale phylogeny inference by integrating smaller subtrees in a computationally efficient manner. Here, we combined information from sequence capture and whole-genome phylogenies using supertree methods. However, the available phylogenomic trees had limited overlap so we used taxon-rich (but not phylogenomic) megaphylogenies to weave them together. This allowed us to construct a phylogenomic supertree, with support values, that included 707 bird species (~7% of avian species diversity). We estimated branch lengths using mitochondrial sequence data and we used these branch lengths to estimate divergence times. Our time-calibrated supertree supports radiation of all three major avian clades (Palaeognathae, Galloanseres, and Neoaves) near the Cretaceous-Paleogene (K-Pg) boundary. The approach we used will permit the continued addition of taxa to this supertree as new phylogenomic data are published, and it could be applied to other taxa as well.

Strisores is a clade of neoavian birds that include diurnal aerial specialists such as swifts and hummingbirds, as well as several predominantly nocturnal lineages such as nightjars and potoos. Despite the use of genome-scale molecular datasets, the phylogenetic interrelationships among major strisorean groups remain controversial. Given the availability of next-generation sequence data for Strisores and the clade’s rich fossil record, we reassessed the phylogeny of Strisores by incorporating a large-scale sequence dataset with anatomical data from living and fossil strisoreans within a Bayesian total-evidence framework. Combined analyses of molecular and morphological data resulted in a phylogenetic topology for Strisores that is congruent with the findings of two recent molecular phylogenomic studies, supporting nightjars (Caprimulgidae) as the extant sister group of the remainder of Strisores. This total-evidence framework allowed us to identify morphological synapomorphies for strisorean clades previously recovered using molecular-only datasets. However, a combined analysis of molecular and morphological data highlighted strong signal conflict between sequence and anatomical data in Strisores. Furthermore, simultaneous analysis of molecular and morphological data recovered differing placements for some fossil taxa compared with analyses of morphological data under a molecular scaffold, highlighting the importance of analytical decisions when conducting morphological phylogenetic analyses of taxa with molecular phylogenetic data. We suggest that multiple strisorean lineages have experienced convergent evolution across the skeleton, obfuscating the phylogenetic position of certain fossils, and that many distinctive specializations of strisorean subclades were acquired early in their evolutionary history. Despite this apparent complexity in the evolutionary history of Strisores, our results provide fossil support for aerial foraging as the ancestral ecological strategy of Strisores, as implied by recent phylogenetic topologies derived from molecular data.

Microinversions are cytologically undetectable inversions of DNA sequences that accumulate slowly in genomes. Like many other rare genomic changes (RGCs), microinversions are thought to be virtually homoplasy-free evolutionary characters, suggesting that they may be very useful for difficult phylogenetic problems such as the avian tree of life. However, few detailed surveys of these genomic rearrangements have been conducted, making it difficult to assess this hypothesis or understand the impact of microinversions upon genome evolution.