Complexity of avian evolution revealed by family-level genomes.

Despite tremendous efforts in the past decades, relationships among main avian lineages remain heavily debated without a clear resolution. Discrepancies have been attributed to diversity of species sampled, phylogenetic method and the choice of genomic regions1-3. Here we address these issues by analysing the genomes of 363 bird species4 (218 taxonomic families, 92% of total). Using intergenic regions and coalescent methods, we present a well-supported tree but also a marked degree of discordance. The tree confirms that Neoaves experienced rapid radiation at or near the Cretaceous-Palaeogene boundary. Sufficient loci rather than extensive taxon sampling were more effective in resolving difficult nodes. Remaining recalcitrant nodes involve species that are a challenge to model due to either extreme DNA composition, variable substitution rates, incomplete lineage sorting or complex evolutionary events such as ancient hybridization. Assessment of the effects of different genomic partitions showed high heterogeneity across the genome. We discovered sharp increases in effective population size, substitution rates and relative brain size following the Cretaceous-Palaeogene extinction event, supporting the hypothesis that emerging ecological opportunities catalysed the diversification of modern birds. The resulting phylogenetic estimate offers fresh insights into the rapid radiation of modern birds and provides a taxon-rich backbone tree for future comparative studies.

Complete vertebrate mitogenomes reveal widespread repeats and gene duplications.

BACKGROUND: Modern sequencing technologies should make the assembly of the relatively small mitochondrial genomes an easy undertaking. However, few tools exist that address mitochondrial assembly directly. RESULTS: As part of the Vertebrate Genomes Project (VGP) we develop mitoVGP, a fully automated pipeline for similarity-based identification of mitochondrial reads and de novo assembly of mitochondrial genomes that incorporates both long (> 10 kbp, PacBio or Nanopore) and short (100-300 bp, Illumina) reads. Our pipeline leads to successful complete mitogenome assemblies of 100 vertebrate species of the VGP. We observe that tissue type and library size selection have considerable impact on mitogenome sequencing and assembly. Comparing our assemblies to purportedly complete reference mitogenomes based on short-read sequencing, we identify errors, missing sequences, and incomplete genes in those references, particularly in repetitive regions. Our assemblies also identify novel gene region duplications. The presence of repeats and duplications in over half of the species herein assembled indicates that their occurrence is a principle of mitochondrial structure rather than an exception, shedding new light on mitochondrial genome evolution and organization. CONCLUSIONS: Our results indicate that even in the "simple" case of vertebrate mitogenomes the completeness of many currently available reference sequences can be further improved, and caution should be exercised before claiming the complete assembly of a mitogenome, particularly from short reads alone.

Response to Comment on "Whole-genome analyses resolve early branches in the tree of life of modern birds".

Mitchell et al. argue that divergence-time estimates for our avian phylogeny were too young because of an "inappropriate" maximum age constraint for the most recent common ancestor of modern birds and that, as a result, most modern bird orders diverged before the Cretaceous-Paleogene mass extinction event 66 million years ago instead of after. However, their interpretations of the fossil record and timetrees are incorrect.

Phylogenomic analyses data of the avian phylogenomics project.

BACKGROUND: Determining the evolutionary relationships among the major lineages of extant birds has been one of the biggest challenges in systematic biology. To address this challenge, we assembled or collected the genomes of 48 avian species spanning most orders of birds, including all Neognathae and two of the five Palaeognathae orders. We used these genomes to construct a genome-scale avian phylogenetic tree and perform comparative genomic analyses. FINDINGS: Here we present the datasets associated with the phylogenomic analyses, which include sequence alignment files consisting of nucleotides, amino acids, indels, and transposable elements, as well as tree files containing gene trees and species trees. Inferring an accurate phylogeny required generating: 1) A well annotated data set across species based on genome synteny; 2) Alignments with unaligned or incorrectly overaligned sequences filtered out; and 3) Diverse data sets, including genes and their inferred trees, indels, and transposable elements. Our total evidence nucleotide tree (TENT) data set (consisting of exons, introns, and UCEs) gave what we consider our most reliable species tree when using the concatenation-based ExaML algorithm or when using statistical binning with the coalescence-based MP-EST algorithm (which we refer to as MP-EST*). Other data sets, such as the coding sequence of some exons, revealed other properties of genome evolution, namely convergence. CONCLUSIONS: The Avian Phylogenomics Project is the largest vertebrate phylogenomics project to date that we are aware of. The sequence, alignment, and tree data are expected to accelerate analyses in phylogenomics and other related areas.

Transcriptome analysis of the white body of the squid Euprymna tasmanica with emphasis on immune and hematopoietic gene discovery.

In the mutualistic relationship between the squid Euprymna tasmanica and the bioluminescent bacterium Vibrio fischeri, several host factors, including immune-related proteins, are known to interact and respond specifically and exclusively to the presence of the symbiont. In squid and octopus, the white body is considered to be an immune organ mainly due to the fact that blood cells, or hemocytes, are known to be present in high numbers and in different developmental stages. Hence, the white body has been described as the site of hematopoiesis in cephalopods. However, to our knowledge, there are no studies showing any molecular evidence of such functions. In this study, we performed a transcriptomic analysis of white body tissue of the Southern dumpling squid, E. tasmanica. Our primary goal was to gain insights into the functions of this tissue and to test for the presence of gene transcripts associated with hematopoietic and immune processes. Several hematopoiesis genes including CPSF1, GATA 2, TFIID, and FGFR2 were found to be expressed in the white body. In addition, transcripts associated with immune-related signal transduction pathways, such as the toll-like receptor/NF-κß, and MAPK pathways were also found, as well as other immune genes previously identified in E. tasmanica's sister species, E. scolopes. This study is the first to analyze an immune organ within cephalopods, and to provide gene expression data supporting the white body as a hematopoietic tissue.

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.

Reconstruction of gross avian genome structure, organization and evolution suggests that the chicken lineage most closely resembles the dinosaur avian ancestor.

BACKGROUND: The availability of multiple avian genome sequence assemblies greatly improves our ability to define overall genome organization and reconstruct evolutionary changes. In birds, this has previously been impeded by a near intractable karyotype and relied almost exclusively on comparative molecular cytogenetics of only the largest chromosomes. Here, novel whole genome sequence information from 21 avian genome sequences (most newly assembled) made available on an interactive browser (Evolution Highway) was analyzed. RESULTS: Focusing on the six best-assembled genomes allowed us to assemble a putative karyotype of the dinosaur ancestor for each chromosome. Reconstructing evolutionary events that led to each species' genome organization, we determined that the fastest rate of change occurred in the zebra finch and budgerigar, consistent with rapid speciation events in the Passeriformes and Psittaciformes. Intra- and interchromosomal changes were explained most parsimoniously by a series of inversions and translocations respectively, with breakpoint reuse being commonplace. Analyzing chicken and zebra finch, we found little evidence to support the hypothesis of an association of evolutionary breakpoint regions with recombination hotspots but some evidence to support the hypothesis that microchromosomes largely represent conserved blocks of synteny in the majority of the 21 species analyzed. All but one species showed the expected number of microchromosomal rearrangements predicted by the haploid chromosome count. Ostrich, however, appeared to retain an overall karyotype structure of 2n=80 despite undergoing a large number (26) of hitherto un-described interchromosomal changes. CONCLUSIONS: Results suggest that mechanisms exist to preserve a static overall avian karyotype/genomic structure, including the microchromosomes, with widespread interchromosomal change occurring rarely (e.g., in ostrich and budgerigar lineages). Of the species analyzed, the chicken lineage appeared to have undergone the fewest changes compared to the dinosaur ancestor.

Global patterns of apparent copy number variation in birds revealed by cross-species comparative genomic hybridization.

There is a growing interest in copy number variation (CNV) and the recognition of its importance in phenotype, disease, adaptation and speciation. CNV data is usually ascertained by array-CGH within-species, but similar inter-species comparisons have also been made in primates, mice and domestic mammals. Here, we conducted a broad appraisal of putative cross-species CNVs in birds, 16 species in all, using the standard array-CGH approach. Using a chicken oligonucleotide microarray, we detected 790 apparent CNVs within 135 unique regions and developed a bioinformatic tool 'CNV Analyser' for analysing and visualising cross-species data sets. We successfully addressed four hypotheses as follows: (a) Cross-species CNVs (compared to chicken) are, as suggested from preliminary evidence, smaller and fewer in number than in mammals; this 'dogma' was rejected in the light of the new evidence. (b) CNVs in birds are likely to have a functional effect through an association with genes; a large proportion of detected regions (70 %) were indeed associated with genes (suggesting functional significance), however, not necessarily more so than in mammals. (c) There are more CNVs in birds with more rearranged karyotypes; this hypothesis was rejected. Indeed, Falco species contained fewer than most with relatively standard (chicken-like) karyotypes. (d) There are more CNVs per megabase on micro-chromosomes than macrochromosomes; this hypothesis was accepted. Indeed, in species with rearranged karyotypes characterised by chromosomal fusions, the fused former microchromosomes still 'behaved' as though they were their microchromosomal ancestors. Gene ontology analysis of CNVRs revealed enrichment in immune response and antigen presentation genes and five CNVRs were perfectly correlated with the unique loss of sexual dichromatism in one Galliformes species.

HighSSR: high-throughput SSR characterization and locus development from next-gen sequencing data.

MOTIVATION: Microsatellites are among the most useful genetic markers in population biology. High-throughput sequencing of microsatellite-enriched libraries dramatically expedites the traditional process of screening recombinant libraries for microsatellite markers. However, sorting through millions of reads to distill high-quality polymorphic markers requires special algorithms tailored to tolerate sequencing errors in locus reconstruction, distinguish paralogous loci, rarify raw reads originating from the same amplicon and sort out various artificial fragments resulting from recombination or concatenation of auxiliary adapters. Existing programs warrant improvement. RESULTS: We describe a microsatellite prediction framework named HighSSR for microsatellite genotyping based on high-throughput sequencing. We demonstrate the utility of HighSSR in comparison to Roche gsAssembler on two Roche 454 GS FLX runs. The majority of the HighSSR-assembled loci were reliably mapped against model organism reference genomes. HighSSR demultiplexes pooled libraries, assesses locus polymorphism and implements Primer3 for the design of PCR primers flanking polymorphic microsatellite loci. As sequencing costs drop and permit the analysis of all project samples on next-generation platforms, this framework can also be used for direct simple sequence repeats genotyping. AVAILABILITY: http://code.google.com/p/highssr/

The fat body transcriptomes of the yellow fever mosquito Aedes aegypti, pre- and post- blood meal.

BACKGROUND: The fat body is the main organ of intermediary metabolism in insects and the principal source of hemolymph proteins. As part of our ongoing efforts to understand mosquito fat body physiology and to identify novel targets for insect control, we have conducted a transcriptome analysis of the fat body of Aedes aegypti before and in response to blood feeding. RESULTS: We created two fat body non-normalized EST libraries, one from mosquito fat bodies non-blood fed (NBF) and another from mosquitoes 24 hrs post-blood meal (PBM). 454 pyrosequencing of the non-normalized libraries resulted in 204,578 useable reads from the NBF sample and 323,474 useable reads from the PBM sample. Alignment of reads to the existing reference Ae. aegypti transcript libraries for analysis of differential expression between NBF and PBM samples revealed 116,912 and 115,051 matches, respectively. De novo assembly of the reads from the NBF sample resulted in 15,456 contigs, and assembly of the reads from the PBM sample resulted in 15,010 contigs. Collectively, 123 novel transcripts were identified within these contigs. Prominently expressed transcripts in the NBF fat body library were represented by transcripts encoding ribosomal proteins. Thirty-five point four percent of all reads in the PBM library were represented by transcripts that encode yolk proteins. The most highly expressed were transcripts encoding members of the cathepsin b, vitellogenin, vitellogenic carboxypeptidase, and vitelline membrane protein families. CONCLUSION: The two fat body transcriptomes were considerably different from each other in terms of transcript expression in terms of abundances of transcripts and genes expressed. They reflect the physiological shift of the pre-feeding fat body from a resting state to vitellogenic gene expression after feeding.

Cochlear labyrinth volume in euarchontoglirans: implications for the evolution of hearing in primates.

Using high resolution X-ray computed tomography data we examined the relationship between cochlear labyrinth volume and body mass in extant, non-primate euarchontoglirans, and in two fossils, to allow for comparison with the results of Kirk and Gosselin-Ildari (2009). Modern primates have significantly higher cochlear labyrinth volumes relative to body mass than other euarchontoglirans, which may be related to a downward shift in the highest and lowest audible frequencies over the course of primate evolution, and to the relative increase in brain size observed in Euprimates.

Phylogeny of "core Gruiformes" (Aves: Grues) and resolution of the Limpkin-Sungrebe problem.

Opinions on the systematic relationships of birds in the avian order Gruiformes have been as diverse as the families included within it. Despite ongoing debate over monophyly of the order and relationships among its various members, recent opinion has converged on the monophyly of a "core" group of five families classified as the suborder Grues: the rails (Rallidae), the cranes (Gruidae), the Limpkin (Aramidae), the trumpeters (Psophiidae), and the finfoots (Heliornithidae). We present DNA sequence data from four mitochondrial (cytochrome b, 12S rRNA, Valine tRNA, and 16S rRNA) and three nuclear loci (intron 7 of beta-fibrinogen, intron 5 of alcohol dehydrogenase-I, and introns 3 through 5 of glyceraldehyde-3-phosphate dehydrogenase) to test previous hypotheses of interfamilial relationships within Grues, with particular attention to the enigmatic family Heliornithidae. Separate and combined analyses of these gene sequences confirm the monophyly of Grues as a whole, and of the five families individually, including all three species of Heliornithidae. The preferred topology unambiguously supports relationships among four of the five families, with only the position of Psophiidae remaining equivocal. Bayesian "relaxed-clock" dating methods suggest that the divergences of the three heliornithid species occurred in the mid-Tertiary, suggesting that their present disjunct pantropical distribution is a result of early- to mid-Tertiary dispersal.

Multilocus perspectives on the monophyly and phylogeny of the order Charadriiformes (Aves).

BACKGROUND: The phylogeny of shorebirds (Aves: Charadriiformes) and their putative sister groups was reconstructed using approximately 5 kilobases of data from three nuclear loci and two mitochondrial genes, and compared to that based on two other nuclear loci. RESULTS: Charadriiformes represent a monophyletic group that consists of three monophyletic suborders Lari (i.e., Laridae [including Sternidae and Rynchopidae], Stercorariidae, Alcidae, Glareolidae, Dromadidae, and Turnicidae), Scolopaci (i.e., Scolopacidae [including Phalaropidae], Jacanidae, Rostratulidae, Thinocoridae, Pedionomidae), and Charadrii (i.e., Burhinidae, Chionididae, Charadriidae, Haematopodidae, Recurvirostridae, and presumably Ibidorhynchidae). The position of purported "gruiform" buttonquails within Charadriiformes is confirmed. Skimmers are most likely sister to terns alone, and plovers may be paraphyletic with respect to oystercatchers and stilts. The Egyptian Plover is not a member of the Glareolidae, but is instead relatively basal among Charadrii. None of the putative sisters of Charadriiformes were recovered as such. CONCLUSION: Hypotheses of non-monophyly and sister relationships of shorebirds are tested by multilocus analysis. The monophyly of and interfamilial relationships among shorebirds are confirmed and refined. Lineage-specific differences in evolutionary rates are more consistent across loci in shorebirds than other birds and may contribute to the congruence of locus-specific phylogenetic estimates in shorebirds.

Parallel radiations in the primary clades of birds.

Knowledge of avian phylogeny is prerequisite to understanding the circumstances and timing of the diversification of birds and the evolution of morphological, behavioral, and life-history traits. Recent molecular datasets have helped to elucidate the three most basal clades in the tree of living birds, but relationships among neoavian orders (the vast majority of birds) remain frustratingly vexing. Here, we examine intron 7 of the beta-fibrinogen gene in the most taxonomically inclusive survey of DNA sequences of nonpasserine bird families and orders to date. These data suggest that Neoaves consist of two sister clades with ecological parallelisms comparable to those found between marsupial and placental mammals. Some members of the putative respective clades have long been recognized as examples of convergent evolution, but it was not appreciated that they might be parts of diverse parallel radiations. In contrast, some traditional orders of birds are suggested by these data to be polyphyletic, with representative families in both radiations.

Ostrich ancestors found in the Northern Hemisphere suggest new hypothesis of ratite origins.

Modern ratites (ostriches, rheas, cassowaries, emus, and kiwis) are flightless birds which have a palatal structure termed 'palaeog-nathous'1 and are found on daughter-landmasses of the Mesozoic supercontinent Gondwanaland. It has been suggested2-4 that a single flightless ancestor, widely distributed in Gondwanaland, gave rise to the various lineages of ratite birds. The temporal calibration of the DNA molecular clock is primarily based on the divergence of ratites, and depends on the validity of this hypothesis. Newly studied fossils suggest that the ancestors of ostriches are instead among a group of North American and European birds, the 'Lithornis-cohort', that had the potential of flight and from which the kiwis may have arisen separately.