Jason T. Howard

Whole-genome analyses resolve early branches in the tree of life of modern birds

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.

Hybrid error correction and de novo assembly of single-molecule sequencing reads

Single-molecule sequencing instruments can generate multikilobase sequences with the potential to greatly improve genome and transcriptome assembly. However, the error rates of single-molecule reads are high, which has limited their use thus far to resequencing bacteria. To address this limitation, we introduce a correction algorithm and assembly strategy that uses short, high-fidelity sequences to correct the error in single-molecule sequences. We demonstrate the utility of this approach on reads generated by a PacBio RS instrument from phage, prokaryotic and eukaryotic whole genomes, including the previously unsequenced genome of the parrot Melopsittacus undulatus, as well as for RNA-Seq reads of the corn (Zea mays) transcriptome. Our long-read correction achieves >99.9% base-call accuracy, leading to substantially better assemblies than current sequencing strategies: in the best example, the median contig size was quintupled relative to high-coverage, second-generation assemblies. Greater gains are predicted if read lengths continue to increase, including the prospect of single-contig bacterial chromosome assembly.

The genome of a songbird.

The zebra finch is an important model organism in several fields with unique relevance to human neuroscience. 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 chicken—the only bird with a sequenced genome until now. 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 Passeriformes. 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.

Assemblathon 2: evaluating de novo methods of genome assembly in three vertebrate species

BackgroundThe process of generating raw genome sequence data continues to become cheaper, faster, and more accurate. However, assembly of such data into high-quality, finished genome sequences remains challenging. Many genome assembly tools are available, but they differ greatly in terms of their performance (speed, scalability, hardware requirements, acceptance of newer read technologies) and in their final output (composition of assembled sequence). More importantly, it remains largely unclear how to best assess the quality of assembled genome sequences. The Assemblathon competitions are intended to assess current state-of-the-art methods in genome assembly.ResultsIn Assemblathon 2, we provided a variety of sequence data to be assembled for three vertebrate species (a bird, a fish, and snake). This resulted in a total of 43 submitted assemblies from 21 participating teams. We evaluated these assemblies using a combination of optical map data, Fosmid sequences, and several statistical methods. From over 100 different metrics, we chose ten key measures by which to assess the overall quality of the assemblies.ConclusionsMany current genome assemblers produced useful assemblies, containing a significant representation of their genes and overall genome structure. However, the high degree of variability between the entries suggests that there is still much room for improvement in the field of genome assembly and that approaches which work well in assembling the genome of one species may not necessarily work well for another.

Comparative genomics reveals insights into avian genome evolution and adaptation

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.

Convergent transcriptional specializations in the brains of humans and song-learning birds.

INTRODUCTION Vocal learning, the ability to imitate sounds, is a trait that has undergone convergent evolution in several lineages of birds and mammals, including song-learning birds and humans. This behavior requires cortical and striatal vocal brain regions, which form unique connections in vocal-learning species. These regions have been found to have specialized gene expression within some species, but the patterns of specialization across vocal-learning bird and mammal species have not been systematically explored. Identifying molecular brain similarities across species. Brain region gene expression specializations were hierarchically organized into specialization trees of each species (blue lines), including for circuits that control learned vocalizations (highlighted green, purple, and orange regions). A set of comparative genomic algorithms found the most similarly specialized regions between songbird and human (orange lines), some of which are convergently evolved. RATIONALE The sequencing of genomes representing all major vocal-learning and vocal-nonlearning avian lineages has allowed us to develop the genomic tools to measure anatomical gene expression across species. Here, we asked whether behavioral and anatomical convergence is associated with gene expression convergence in the brains of vocal-learning birds and humans. RESULTS We developed a computational approach that discovers homologous and convergent specialized anatomical gene expression profiles. This includes generating hierarchically organized gene expression specialization trees for each species and a dynamic programming algorithm that finds the optimal alignment between species brain trees. We applied this approach to brain region gene expression databases of thousands of samples and genes that we and others generated from multiple species, including humans and song-learning birds (songbird, parrot, and hummingbird) as well as vocal-nonlearning nonhuman primates (macaque) and birds (dove and quail). Our results confirmed the recently revised understanding of the relationships between avian and mammalian brains. We further found that songbird Area X, a striatal region necessary for vocal learning, was most similar to a part of the human striatum activated during speech production. The RA (robust nucleus of the arcopallium) analog of song-learning birds, necessary for song production, was most similar to laryngeal motor cortex regions in humans that control speech production. More than 50 genes contributed to their convergent specialization and were enriched in motor control and neural connectivity functions. These patterns were not found in vocal nonlearners, but songbird RA was similar to layer 5 of primate motor cortex for another set of genes, supporting previous hypotheses about the similarity of these cell types between bird and mammal brains. CONCLUSION Our approach can accurately and quantitatively identify functionally and molecularly analogous brain regions between species separated by as much as 310 million years from a common ancestor. We were able to identify analogous brain regions for song and speech between birds and humans, and broader homologous brain regions in which these specialized song and speech regions are located, for tens to hundreds of genes. These genes now serve as candidates involved in developing and maintaining the unique connectivity and functional properties of vocal-learning brain circuits shared across species. The finding that convergent neural circuits for vocal learning are accompanied by convergent molecular changes of multiple genes in species separated by millions of years from a common ancestor indicates that brain circuits for complex traits may have limited ways in which they could have evolved from that ancestor. Song-learning birds and humans share independently evolved similarities in brain pathways for vocal learning that are essential for song and speech and are not found in most other species. Comparisons of brain transcriptomes of song-learning birds and humans relative to vocal nonlearners identified convergent gene expression specializations in specific song and speech brain regions of avian vocal learners and humans. The strongest shared profiles relate bird motor and striatal song-learning nuclei, respectively, with human laryngeal motor cortex and parts of the striatum that control speech production and learning. Most of the associated genes function in motor control and brain connectivity. Thus, convergent behavior and neural connectivity for a complex trait are associated with convergent specialized expression of multiple genes.

A molecular neuroethological approach for identifying and characterizing a cascade of behaviorally regulated genes

Songbirds have one of the most accessible neural systems for the study of brain mechanisms of behavior. However, neuroethological studies in songbirds have been limited by the lack of high-throughput molecular resources and gene-manipulation tools. To overcome these limitations, we constructed 21 regular, normalized, and subtracted full-length cDNA libraries from brains of zebra finches in 57 developmental and behavioral conditions in an attempt to clone as much of the brain transcriptome as possible. From these libraries, ≈14,000 transcripts were isolated, representing an estimated 4,738 genes. With the cDNAs, we created a hierarchically organized transcriptome database and a large-scale songbird brain cDNA microarray. We used the arrays to reveal a set of 33 genes that are regulated in forebrain vocal nuclei by singing behavior. These genes clustered into four anatomical and six temporal expression patterns. Their functions spanned a large range of cellular and molecular categories, from signal transduction, trafficking, and structural, to synaptically released molecules. With the full-length cDNAs and a lentiviral vector system, we were able to overexpress, in vocal nuclei, proteins of representative singing-regulated genes in the absence of singing. This publicly accessible resource http://songbirdtranscriptome.net can now be used to study molecular neuroethological mechanisms of behavior.

Three crocodilian genomes reveal ancestral patterns of evolution among archosaurs

INTRODUCTION Crocodilians and birds are the two extant clades of archosaurs, a group that includes the extinct dinosaurs and pterosaurs. Fossils suggest that living crocodilians (alligators, crocodiles, and gharials) have a most recent common ancestor 80 to 100 million years ago. Extant crocodilians are notable for their distinct morphology, limited intraspecific variation, and slow karyotype evolution. Despite their unique biology and phylogenetic position, little is known about genome evolution within crocodilians. Evolutionary rates of tetrapods inferred from DNA sequences anchored by ultraconserved elements. Evolutionary rates among reptiles vary, with especially low rates among extant crocodilians but high rates among squamates. We have reconstructed the genomes of the common ancestor of birds and of all archosaurs (shown in gray silhouette, although the morphology of these species is uncertain). RATIONALE Genome sequences for the American alligator, saltwater crocodile, and Indian gharial—representatives of all three extant crocodilian families—were obtained to facilitate better understanding of the unique biology of this group and provide a context for studying avian genome evolution. Sequence data from these three crocodilians and birds also allow reconstruction of the ancestral archosaurian genome. RESULTS We sequenced shotgun genomic libraries from each species and used a variety of assembly strategies to obtain draft genomes for these three crocodilians. The assembled scaffold N50 was highest for the alligator (508 kilobases). Using a panel of reptile genome sequences, we generated phylogenies that confirm the sister relationship between crocodiles and gharials, the relationship with birds as members of extant Archosauria, and the outgroup status of turtles relative to birds and crocodilians. We also estimated evolutionary rates along branches of the tetrapod phylogeny using two approaches: ultraconserved element–anchored sequences and fourfold degenerate sites within stringently filtered orthologous gene alignments. Both analyses indicate that the rates of base substitution along the crocodilian and turtle lineages are extremely low. Supporting observations were made for transposable element content and for gene family evolution. Analysis of whole-genome alignments across a panel of reptiles and mammals showed that the rate of accumulation of micro-insertions and microdeletions is proportionally lower in crocodilians, consistent with a single underlying cause of a reduced rate of evolutionary change rather than intrinsic differences in base repair machinery. We hypothesize that this single cause may be a consistently longer generation time over the evolutionary history of Crocodylia. Low heterozygosity was observed in each genome, consistent with previous analyses, including the Chinese alligator. Pairwise sequential Markov chain analysis of regional heterozygosity indicates that during glacial cycles of the Pleistocene, each species suffered reductions in effective population size. The reduction was especially strong for the American alligator, whose current range extends farthest into regions of temperate climates. CONCLUSION We used crocodilian, avian, and outgroup genomes to reconstruct 584 megabases of the archosaurian common ancestor genome and the genomes of key ancestral nodes. The estimated accuracy of the archosaurian genome reconstruction is 91% and is higher for conserved regions such as genes. The reconstructed genome can be improved by adding more crocodilian and avian genome assemblies and may provide a unique window to the genomes of extinct organisms such as dinosaurs and pterosaurs. To provide context for the diversification of archosaurs—the group that includes crocodilians, dinosaurs, and birds—we generated draft genomes of three crocodilians: Alligator mississippiensis (the American alligator), Crocodylus porosus (the saltwater crocodile), and Gavialis gangeticus (the Indian gharial). We observed an exceptionally slow rate of genome evolution within crocodilians at all levels, including nucleotide substitutions, indels, transposable element content and movement, gene family evolution, and chromosomal synteny. When placed within the context of related taxa including birds and turtles, this suggests that the common ancestor of all of these taxa also exhibited slow genome evolution and that the comparatively rapid evolution is derived in birds. The data also provided the opportunity to analyze heterozygosity in crocodilians, which indicates a likely reduction in population size for all three taxa through the Pleistocene. Finally, these data combined with newly published bird genomes allowed us to reconstruct the partial genome of the common ancestor of archosaurs, thereby providing a tool to investigate the genetic starting material of crocodilians, birds, and dinosaurs.

Global view of the functional molecular organization of the avian cerebrum: mirror images and functional columns.

Based on quantitative cluster analyses of 52 constitutively expressed or behaviorally regulated genes in 23 brain regions, we present a global view of telencephalic organization of birds. The patterns of constitutively expressed genes revealed a partial mirror image organization of three major cell populations that wrap above, around, and below the ventricle and adjacent lamina through the mesopallium. The patterns of behaviorally regulated genes revealed functional columns of activation across boundaries of these cell populations, reminiscent of columns through layers of the mammalian cortex. The avian functionally regulated columns were of two types: those above the ventricle and associated mesopallial lamina, formed by our revised dorsal mesopallium, hyperpallium, and intercalated hyperpallium; and those below the ventricle, formed by our revised ventral mesopallium, nidopallium, and intercalated nidopallium. Based on these findings and known connectivity, we propose that the avian pallium has four major cell populations similar to those in mammalian cortex and some parts of the amygdala: 1) a primary sensory input population (intercalated pallium); 2) a secondary intrapallial population (nidopallium/hyperpallium); 3) a tertiary intrapallial population (mesopallium); and 4) a quaternary output population (the arcopallium). Each population contributes portions to columns that control different sensory or motor systems. We suggest that this organization of cell groups forms by expansion of contiguous developmental cell domains that wrap around the lateral ventricle and its extension through the middle of the mesopallium. We believe that the position of the lateral ventricle and its associated mesopallium lamina has resulted in a conceptual barrier to recognizing related cell groups across its border, thereby confounding our understanding of homologies with mammals. J. Comp. Neurol. 521:3614–3665, 2013.

Sequencing three crocodilian genomes to illuminate the evolution of archosaurs and amniotes

The International Crocodilian Genomes Working Group (ICGWG) will sequence and assemble the American alligator (Alligator mississippiensis), saltwater crocodile (Crocodylus porosus) and Indian gharial (Gavialis gangeticus) genomes. The status of these projects and our planned analyses are described.