With a standard set of primers directed toward conserved regions, we have used the polymerase chain reaction to amplify homologous segments of mtDNA from more than 100 animal species, including mammals, birds, amphibians, fishes, and some invertebrates. Amplification and direct sequencing were possible using unpurified mtDNA from nanogram samples of fresh specimens and microgram amounts of tissues preserved for months in alcohol or decades in the dry state. The bird and fish sequences evolve with the same strong bias toward transitions that holds for mammals. However, because the light strand of birds is deficient in thymine, thymine to cytosine transitions are less common than in other taxa. Amino acid replacement in a segment of the cytochrome b gene is faster in mammals and birds than in fishes and the pattern of replacements fits the structural hypothesis for cytochrome b. The unexpectedly wide taxonomic utility ofthese primers offers opportunities for phylogenetic and population research.During the past decade, geneticists and taxonomists have used restriction endonucleases rather than sequencing to examine variation within and between species in specific segments of DNA (1-7). Although the indirect assessment of sequence variation obtained with the restriction endonuclease method is known to have many drawbacks, § sequence data have been difficult to obtain. The construction and screening of clone libraries has been too tedious and have demanded too much expertise for routine use by those geneticists and taxonomists who must analyze many individuals.Dependence on restriction analysis has limited our understanding of the dynamics of DNA sequence evolution. The presence or absence of a restriction site reveals little about the kinds of nucleotide substitutions that have occurred. Thus, although restriction analysis of mtDNA from closely related mammals first showed that these genomes have a higher rate of evolutionary substitution than does nuclear DNA, the demonstration that this acceleration results mainly from an increase in the number of transitions relative to transversions came only from conventional cloning and sequencing (1, 3). Because most studies of animal mtDNA have used restriction analysis, it has been difficult to determine whether a high rate of evolution and a transition bias are characteristic of all animal mtDNAs (8-10). There has been a need for simple methods of sequencing mtDNA to examine the pattern of evolutionary substitution in other animal groups.

To better determine the history of modern birds, we performed a genome-scale phylogenetic analysis of 48 species representing all orders of Neoaves using phylogenomic methods created to handle genome-scale data. We recovered a highly resolved tree that confirms previously controversial sister or close relationships. We identified the first divergence in Neoaves, two groups we named Passerea and Columbea, representing independent lineages of diverse and convergently evolved land and water bird species. Among Passerea, we infer the common ancestor of core landbirds to have been an apex predator and confirm independent gains of vocal learning. Among Columbea, we identify pigeons and flamingoes as belonging to sister clades. Even with whole genomes, some of the earliest branches in Neoaves proved challenging to resolve, which was best explained by massive protein-coding sequence convergence and high levels of incomplete lineage sorting that occurred during a rapid radiation after the Cretaceous-Paleogene mass extinction event about 66 million years ago.

Birds are the most species-rich class of tetrapod vertebrates and have wide relevance across many research fields. We explored bird macroevolution using full genomes from 48 avian species representing all major extant clades. The avian genome is principally characterized by its constrained size, which predominantly arose because of lineage-specific erosion of repetitive elements, large segmental deletions, and gene loss. Avian genomes furthermore show a remarkably high degree of evolutionary stasis at the levels of nucleotide sequence, gene synteny, and chromosomal structure. Despite this pattern of conservation, we detected many non-neutral evolutionary changes in protein-coding genes and noncoding regions. These analyses reveal that pan-avian genomic diversity covaries with adaptations to different lifestyles and convergent evolution of traits.

The zebra finch is an important model organism in several fields1,2 with unique relevance to human neuroscience3,4. Like other songbirds, the zebra finch communicates through learned vocalizations, an ability otherwise documented only in humans and a few other animals and lacking in the chicken5—the only bird with a sequenced genome until now6. Here we present a structural, functional and comparative analysis of the genome sequence of the zebra finch (Taeniopygia guttata), which is a songbird belonging to the large avian order Passeriformes7. We find that the overall structures of the genomes are similar in zebra finch and chicken, but they differ in many intrachromosomal rearrangements, lineage-specific gene family expansions, the number of long-terminal-repeat-based retrotransposons, and mechanisms of sex chromosome dosage compensation. We show that song behaviour engages gene regulatory networks in the zebra finch brain, altering the expression of long non-coding RNAs, microRNAs, transcription factors and their targets. We also show evidence for rapid molecular evolution in the songbird lineage of genes that are regulated during song experience. These results indicate an active involvement of the genome in neural processes underlying vocal communication and identify potential genetic substrates for the evolution and regulation of this behaviour.

The reconstruction of the Tree of Life has relied almost entirely on concatenation methods, which do not accommodate gene tree heterogeneity, a property that simulations and theory have identified as a likely cause of incongruent phylogenies. However, this incongruence has not yet been demonstrated in empirical studies. Several key relationships among eutherian mammals remain controversial and conflicting among previous studies, including the root of eutherian tree and the relationships within Euarchontoglires and Laurasiatheria. Both Bayesian and maximum-likelihood analysis of genome-wide data of 447 nuclear genes from 37 species show that concatenation methods indeed yield strong incongruence in the phylogeny of eutherian mammals, as revealed by subsampling analyses of loci and taxa, which produced strongly conflicting topologies. In contrast, the coalescent methods, which accommodate gene tree heterogeneity, yield a phylogeny that is robust to variable gene and taxon sampling and is congruent with geographic data. The data also demonstrate that incomplete lineage sorting, a major source of gene tree heterogeneity, is relevant to deep-level phylogenies, such as those among eutherian mammals. Our results firmly place the eutherian root between Atlantogenata and Boreoeutheria and support ungulate polyphyly and a sister-group relationship between Scandentia and Primates. This study demonstrates that the incongruence introduced by concatenation methods is a major cause of long-standing uncertainty in the phylogeny of eutherian mammals, and the same may apply to other clades. Our analyses suggest that such incongruence can be resolved using phylogenomic data and coalescent methods that deal explicitly with gene tree heterogeneity.