The compositional patterns of the avian genomes and their evolutionary implications

The compositional distributions of large (main-band) DNA fragments from eight birds belonging to eight different orders (including both paleognathous and neognathous species) are very broad and extremely close to each other. These findings, which are paralleled by the compositional similarity of homologous coding sequences and their codon positions, support the idea that birds are a monophyletic group.The compositional distribution of third-codon positions of genes from chicken, the only avian species for which a relatively large number of coding sequences is known, is very broad and bimodal, the minor GC-richer peak reaching 100% GC. The very high compositional heterogeneity of avian genomes is accompanied (as in the case of mammalian genomes) by a very high speciation rate compared to cold-blooded vertebrates which are characterized by genomes that are much less heterogeneous. The higher GC levels attained by avian compared to mammalian genomes might be correlated with the higher body temperature (41–43°C) of birds compared to mammals (37°C).A comparison of GC levels of coding sequences and codon positions from man and chicken revealed very close average GC levels and standard deviations. Homologous coding sequences and codon positions from man and chicken showed a surprisingly high degree of compositional similarity which was, however, higher for GC-poor than for GC-rich sequences. This indicates that GC-poor isochores of warm-blooded vertebrates reflect the composition of the isochores of the genome of the common reptilian ancestor of mammals and birds, which underwent only a small compositional change at the transition from cold- to warm-blooded vertebrates. In contrast, the GC-rich isochores of birds and mammals are the result of large compositional changes at the same evolutionary transition, where were in part different in the two classes of warm-blooded vertebrates.Correspondence to: G. Bernaadi     

Origin and evolution of avian microchromosomes

The origin of avian microchromosomes has long been the subject of much speculation and debate. Microchromosomes are a universal characteristic of all avian species and many reptilian karyotypes. The typical avian karyotype contains about 40 pairs of chromosomes and usually 30 pairs of small to tiny microchromosomes. This characteristic karyotype probably evolved 100-250 million years ago. Once the microchromosomes were thought to be a non-essential component of the avian genome. Recent work has shown that even though these chromosomes represent only 25% of the genome; they encode 50% of the genes. Contrary to popular belief, microchromosomes are present in a wide range of vertebrate classes, spanning 400-450 million years of evolutionary history. In this paper, comparative gene mapping between the genomes of chicken, human, mouse and zebrafish, has been used to investigate the origin and evolution of avian microchromosomes during this period. This analysis reveals evidence for four ancient syntenies conserved in fish, birds and mammals for over 400 million years. More than half, if not all, microchromosomes may represent ancestral syntenies and at least ten avian microchromosomes are the product of chromosome fission. Birds have one of the smallest genomes of any terrestrial vertebrate. This is likely to be the product of an evolutionary process that minimizes the DNA content (mostly through the number of repeats) and maximizes the recombination rate of microchromosomes. Through this process the properties (GC content, DNA and repeat content, gene density and recombination rate) of microchromosomes and macrochromosomes have diverged to create distinct chromosome types. An ancestral genome for birds likely had a small genome, low in repeats and a karyotype with microchromosomes. A "Fission-Fusion Model" of microchromosome evolution based on chromosome rearrangement and minimization of repeat content is discussed.     

A gene-based genetic linkage map of the collared flycatcher (Ficedula albicollis) reveals extensive synteny and gene-order conservation during 100 million years of avian evolution

By taking advantage of a recently developed reference marker set for avian genome analysis we have constructed a gene-based genetic map of the collared flycatcher, an important "ecological model" for studies of life-history evolution, sexual selection, speciation, and quantitative genetics. A pedigree of 322 birds from a natural population was genotyped for 384 single nucleotide polymorphisms (SNPs) from 170 protein-coding genes and 71 microsatellites. Altogether, 147 gene markers and 64 microsatellites form 33 linkage groups with a total genetic distance of 1787 cM. Male recombination rates are, on average, 22% higher than female rates (total distance 1982 vs. 1627 cM). The ability to anchor the collared flycatcher map with the chicken genome via the gene-based SNPs revealed an extraordinary degree of both synteny and gene-order conservation during avian evolution. The great majority of chicken chromosomes correspond to a single linkage group in collared flycatchers, with only a few cases of inter- and intrachromosomal rearrangements. The rate of chromosomal diversification, fissions/fusions, and inversions combined is thus considerably lower in birds (0.05/MY) than in mammals (0.6-2.0/MY). A dearth of repeat elements, known to promote chromosomal breakage, in avian genomes may contribute to their stability. The degree of genome stability is likely to have important consequences for general evolutionary patterns and may explain, for example, the comparatively slow rate by which genetic incompatibility among lineages of birds evolves.     

Conservation of chromosomes syntenic with avian autosomes in squamate reptiles revealed by comparative chromosome painting

In contrast to mammals, birds exhibit a slow rate of chromosomal evolution. It is not clear whether high chromosome conservation is an evolutionary novelty of birds or was inherited from an earlier avian ancestor. The evolutionary conservatism of macrochromosomes between birds and turtles supports the latter possibility; however, the rate of chromosomal evolution is largely unknown in other sauropsids. In squamates, we previously reported strong conservatism of the chromosomes syntenic with the avian Z, which could reflect a peculiarity of this part of the genome. The chromosome 1 of iguanians and snakes is largely syntenic with chromosomes 3, 5 and 7 of the avian ancestral karyotype. In this project, we used comparative chromosome painting to determine how widely this synteny is conserved across nine families covering most of the main lineages of Squamata. The results suggest that the association of the avian ancestral chromosomes 3, 5 and 7 can be dated back to at least the early Jurassic and could be an ancestral characteristic for Unidentata (Serpentes, Iguania, Anguimorpha, Laterata and Scinciformata). In Squamata chromosome conservatism therefore also holds for the parts of the genome which are homologous to bird autosomes, and following on from this, a slow rate of chromosomal evolution could be a common characteristic of all sauropsids. The large evolutionary stasis in chromosome organization in birds therefore seems to be inherited from their ancestors, and it is particularly striking in comparison with mammals, probably the only major tetrapod lineage with an increased rate of chromosomal rearrangements as a whole.     

Molecular evolutionary genomics of birds

Insight into the molecular evolution of birds has been offered by the steady accumulation of avian DNA sequence data, recently culminating in the first draft sequence of an avian genome, that of chicken. By studying avian molecular evolution we can learn about adaptations and phenotypic evolution in birds, and also gain an understanding of the similarities and differences between mammalian and avian genomes. In both these lineages, there is pronounced isochore structure with highly variable GC content. However, while mammalian isochores are decaying, they are maintained in the chicken lineage, which is consistent with a biased gene conversion model where the high and variable recombination rate of birds reinforces heterogeneity in GC. In Galliformes, GC is positively correlated with the rate of nucleotide substitution; the mean neutral mutation rate is 0.12-0.15% at each site per million years but this estimate comes with significant local variation in the rate of mutation. Comparative genomics reveals lower d(N)/d(S) ratios on micro- compared to macrochromosomes, possibly due to population genetic effects or a non-random distribution of genes with respect to chromosome size. A non-random genomic distribution is shown by genes with sex-biased expression, with male-biased genes over-represented and female-biased genes under-represented on the Z chromosome. A strong effect of selection is evident on the non-recombining W chromosome with high d(N)/d(S) ratios and limited polymorphism. Nucleotide diversity in chicken is estimated at 4-5 x 10(-3) which might be seen as surprisingly high given presumed bottlenecks during domestication, but is lower than that recently observed in several natural populations of other species. Several important aspects of the molecular evolutionary process of birds remain to be understood and it can be anticipated that the upcoming genome sequence of a second bird species, the zebra finch, as well as the integration of data on gene expression, shall further advance our knowledge of avian evolution.     

The evolutionary relationship of avian and mammalian myosin heavy-chain genes

Summary Sequence comparisons of avian and mammalian skeletal and cardiac myosin heavy-chain isoforms are used to examine the evolutionary relationships of sarcomeric myosin multigene families. Mammalian fast-myosin heavy-chain isoforms forms from different species, with comparable developmental expression, are more similar to each other than they are to other fast isoforms within the same genome. In contrast, the developmentally regulated chicken fast isoforms are more similar to each other than they are to myosin heavy-chain isoforms in other species. Extensive regions of nucleotide identity among the chicken fast myosin heavy chains and in the mouse and rat α- and β-cardiac myosin heavy-chain sequences suggest that geneconversion-like mechanisms have played a major role in the concerted evolution of these gene families. We also conclude that the chicken fast myosin heavy-chain multigene family has undergone recent expansion subsequent to the divergence of birds and mammals and that both the developmental regulation and the specialization of myosin isoforms have likely developed independently in birds and mammals.     

Comparative metal binding and genomic analysis of the avian (chicken) and mammalian metallothionein

Chicken metallothionein (ckMT) is the paradigm for the study of metallothioneins (MTs) in the Aves class of vertebrates. Available literature data depict ckMT as a one-copy gene, encoding an MT protein highly similar to mammalian MT1. In contrast, the MT system in mammals consists of a four-member family exhibiting functional differentiation. This scenario prompted us to analyse the apparently distinct evolutionary patterns followed by MTs in birds and mammals, at both the functional and structural levels. Thus, in this work, the ckMT metal binding abilities towards Zn(II), Cd(II) and Cu(I) have been thoroughly revisited and then compared with those of the mammalian MT1 and MT4 isoforms, identified as zinc- and copper-thioneins, respectively. Interestingly, a new mechanism of MT dimerization is reported, on the basis of the coordinating capacity of the ckMT C-terminal histidine. Furthermore, an evolutionary study has been performed by means of in silico analyses of avian MT genes and proteins. The joint consideration of the functional and genomic data obtained questions the two features until now defining the avian MT system. Overall, in vivo and in vitro metal-binding results reveal that the Zn(II), Cd(II) and Cu(I) binding abilities of ckMT lay between those of mammalian MT1 and MT4, being closer to those of MT1 for the divalent metal ions but more similar to those of MT4 for Cu(I). This is consistent with a strong functional constraint operating on low-copy number genes that must cope with differentiating functional limitation. Finally, a second MT gene has been identified in silico in the chicken genome, ckMT2, exhibiting all the features to be considered an active coding region. The results presented here allow a new insight into the metal binding abilities of warm blooded vertebrate MTs and their evolutionary relationships.     

The chicken genome contains no HMG1 retropseudogenes but a functional HMG1 gene with long introns

We have cloned the genomic sequence coding for the high mobility group 1 (HMG1) protein in chickens. Multiple sequence alignment shows that the chicken HMG1 gene is highly homologous to the human and the mouse HMG1 genes. The gene structure of chicken HMG1 is similar to that of the mouse and the human HMG1 genes, with the same exon-intron boundaries. However, in contrast to other avian genes that have shorter introns, the chicken HMG1 gene has introns that are twice as long as their mammalian homologues. In addition to the functional, intron-containing HMG1 gene, all mammalian genomes contain more than 50 copies of HMG1 retropseudogenes each, while in the chicken genome there are no HMG1 retropseudogenes. This finding suggests that the HMG1 retropseudogenes arose in mammals after their divergence away from the birds.     

Molecular evolution of genes in avian genomes

Background

Obtaining a draft genome sequence of the zebra finch (Taeniopygia guttata), the second bird genome to be sequenced, provides the necessary resource for whole-genome comparative analysis of gene sequence evolution in a non-mammalian vertebrate lineage. To analyze basic molecular evolutionary processes during avian evolution, and to contrast these with the situation in mammals, we aligned the protein-coding sequences of 8,384 1:1 orthologs of chicken, zebra finch, a lizard and three mammalian species.

Results

We found clear differences in the substitution rate at fourfold degenerate sites, being lowest in the ancestral bird lineage, intermediate in the chicken lineage and highest in the zebra finch lineage, possibly reflecting differences in generation time. We identified positively selected and/or rapidly evolving genes in avian lineages and found an over-representation of several functional classes, including anion transporter activity, calcium ion binding, cell adhesion and microtubule cytoskeleton.

Conclusions

Focusing specifically on genes of neurological interest and genes differentially expressed in the unique vocal control nuclei of the songbird brain, we find a number of positively selected genes, including synaptic receptors. We found no evidence that selection for beneficial alleles is more efficient in regions of high recombination; in fact, there was a weak yet significant negative correlation between ω and recombination rate, which is in the direction predicted by the Hill-Robertson effect if slightly deleterious mutations contribute to protein evolution. These findings set the stage for studies of functional genetics of avian genes.     

The compositional transition between the genomes of cold- and warm-blooded vertebrates: codon frequencies in orthologous genes

The genomes of the ancestors of mammals and birds underwent a compositional change in which the gene-richest regions increased their GC levels. Here we investigated this compositional transition by analyzing the levels of G and C in third codon positions, as well as the codon frequencies of orthologous genes from human, chicken and Xenopus. The results may be summed up as follows: (i) GC-poor genes, that did not undergo the compositional transition, showed only minor differences in orthologous sets from Xenopus, human and chicken; this is remarkable in view of the very many nucleotide substitutions that occurred over the long evolutionary times separating these species; (ii) GC-rich genes, that underwent the compositional transition, showed large differences between Xenopus and warm-blooded vertebrates, but not between chicken and human. In other words, the independent changes that occurred in avian and mammalian genes, on the average, were the same.     

Avian UCP: The Killjoy in the Evolution of the Mitochondrial Uncoupling Proteins

The understanding of mitochondrial functioning is of prime importance since it combines the production of energy as adenosine triphosphate (ATP) with an efficient chain of redox reactions, but also with the unavoidable production of reactive oxygen species (ROS) involved in aging. Mitochondrial respiration may be uncoupled from ATP synthesis by a proton leak induced by the thermogenic uncoupling protein 1 (UCP1). Mild uncoupling activity, as proposed for UCP2, UCP3, and avian UCP could theoretically control ROS production, but the nature of their transport activities is far from being definitively understood. The recent discovery of a UCP1 gene in fish has balanced the evolutionary view of uncoupling protein history. The thermogenic proton transport of mammalian UCP1 seems now to be a late evolutionary characteristic and the hypothesis that ancestral UCPs may carry other substrates is tempting. Using in silico genome analyses among taxa and a biochemical approach, we present a detailed phylogenetic analysis of UCPs and investigate whether avian UCP is a good candidate for pleiotropic mitochondrial activities, knowing that only one UCP has been characterized in the avian genome, unlike all other vertebrates. We show, here, that the avian class seems to be the only vertebrate lineage lacking two of the UCP1/2/3 homologues present in fish and mammals. We suggest, based on phylogenetic evidence and synteny of the UCP genes, that birds have lost UCP1 and UCP2. The phylogeny also supports the history of two rounds of duplication during vertebrate evolution. The avian uncoupling protein then represents a unique opportunity to explore how UCPs' activities are controlled, but also to understand why birds exhibit such a particular relationship between high metabolism and slow rate of aging.     

The effects of macrophage-derived cytokines on lipid metabolism in chicken (Gallus domesticus) hepatocytes and adipocytes

1. The effects of avian and mammalian cytokines on avian lipid metabolism were compared using cultured chicken hepatocytes and adipocytes. 2. Conditioned medium from an endotoxin-stimulated chicken macrophage cell line was used as a source of chicken cytokines. Incubation of chicken adipocytes with conditioned medium greatly decreased their lipoprotein lipase activity. 3. Inhibition of lipoprotein lipase synthesis in similar experiments in mammals has been attributed to the effects of TNF-alpha and/or IL-1, but recombinant human TNF-alpha and IL-1 had no effect on lipoprotein lipase activity in chicken adipocytes. 4. Conditioned medium from chicken macrophages produced a 2-fold increase in lipogenesis in chicken adipocytes but had no effect on lipogenesis in chicken hepatocytes. 5. The results point to major differences between mammals and birds in the way that lipid metabolism responds to cytokines and provide further evidence that mammalian cytokines are ineffective in birds.     

Deep ancestry of mammalian X chromosome revealed by comparison with the basal tetrapod Xenopus tropicalis

ABSTRACT: BACKGROUND: The X and Y sex chromosomes are conspicuous features of placental mammal genomes. Mammalian sex chromosomes arose from an ordinary pair of autosomes after the proto-Y acquired a male-determining gene and degenerated due to suppression of X-Y recombination. Analysis of earlier steps in X chromosome evolution has been hampered by the long interval between the origins of teleost and amniote lineages as well as scarcity of X chromosome orthologs in incomplete avian genome assemblies. RESULTS: This study clarifies the genesis and remodelling of the X chromosome by using a combination of sequence analysis, meiotic map information, and cytogenetic localization to compare amniote genome organization with that of the amphibian Xenopus tropicalis. Nearly all orthologs of human X genes localize to X. tropicalis chromosomes 2 and 8, consistent with an ancestral X-conserved region and a single X-added region precursor. This finding contradicts a previous hypothesis of three evolutionary strata in this region. Homologies between human, opossum, chicken and frog chromosomes suggest a single X-added region predecessor in therian mammals, corresponding to opossum chromosomes 4 and 7. A more ancient X-added ancestral region, currently extant as a major part of chicken chromosome 1, is likely to have been present in the progenitor of synapsids and sauropsids. Analysis of X chromosome gene content emphasizes conservation of single protein coding genes and the role of tandem arrays in formation of novel genes. CONCLUSIONS: Chromosomal regions orthologous to Therian X chromosomes have been located in the genome of the frog X. tropicalis. These ancestral components experienced a series of fusion and breakage events to give rise to avian autosomes and mammalian sex chromosomes. The early branching tetrapod X. tropicalis' simple diploid genome and robust synteny to amniotes greatly enhances studies of vertebrate chromosome evolution.     

Genetic mapping in a natural population of collared flycatchers (Ficedula albicollis): conserved synteny but gene order rearrangements on the avian Z chromosome

Data from completely sequenced genomes are likely to open the way for novel studies of the genetics of nonmodel organisms, in particular when it comes to the identification and analysis of genes responsible for traits that are under selection in natural populations. Here we use the draft sequence of the chicken genome as a starting point for linkage mapping in a wild bird species, the collared flycatcher - one of the most well-studied avian species in ecological and evolutionary research. A pedigree of 365 flycatchers was established and genotyped for single nucleotide polymorphisms in 23 genes selected from (and spread over most of) the chicken Z chromosome. All genes were also found to be located on the Z chromosome in the collared flycatcher, confirming conserved synteny at the level of gene content across distantly related avian lineages. This high degree of conservation mimics the situation seen for the mammalian X chromosome and may thus be a general feature in sex chromosome evolution, irrespective of whether there is male or female heterogamety. Alternatively, such unprecedented chromosomal conservation may be characteristic of most chromosomes in avian genome evolution. However, several internal rearrangements were observed, meaning that the transfer of map information from chicken to nonmodel bird species cannot always assume conserved gene orders. Interestingly, the rate of recombination on the Z chromosome of collared flycatchers was only approximately 50% that of chicken, challenging the widely held view that birds generally have high recombination rates.     

Comparative mapping of Z-orthologous genes in vertebrates: implications for the evolution of avian sex chromosomes

Sex chromosomes of birds and mammals are highly differentiated and share several cytological features. However, comparative gene mapping reveals extensive conserved synteny between the chicken Z sex chromosome and human chromosome 9 but not the human X sex chromosome, implying an independent origin of avian and mammalian sex chromosomes. To better understand the evolution of the avian Z chromosome we analysed the synteny of chicken Z-linked genes in zebrafish, which is the best-mapped teleost genome so far. Existing zebrafish maps do not support the existence of an ancestral Z linkage group in the zebrafish genome, whereas mammalian X-linked genes show at least some degree of synteny conservation. This is consistent with in situ hybridisation mapping data in the freshwater pufferfish, Tetraodon nigroviridis where mammalian X-linked genes show a much higher degree of conserved synteny than human chromosome 9 or the avian Z chromosome. Collectively, these data argue in favour of a more recent evolution of the avian Z chromosome, compared with the mammalian X.     

Low diversity,activity, and density of transposable elements in five avian genomes

In this study, we conducted the activity, diversity, and density analysis of transposable elements (TEs) across five avian genomes (budgerigar, chicken, turkey, medium ground finch, and zebra finch) to explore the potential reason of small genome sizes of birds. We found that these avian genomes exhibited low density of TEs by about 10% of genome coverages and low diversity of TEs with the TE landscapes dominated by CR1 and ERV elements, and contrasting proliferation dynamics both between TE types and between species were observed across the five avian genomes. Phylogenetic analysis revealed that CR1 clade was more diverse in the family structure compared with R2 clade in birds; avian ERVs were classified into four clades (alpha, beta, gamma, and ERV-L) and belonged to three classes of ERV with an uneven distributed in these lineages. The activities of DNA and SINE TEs were very low in the evolution history of avian genomes; most LINEs and LTRs were ancient copies with a substantial decrease of activity in recent, with only LTRs and LINEs in chicken and zebra finch exhibiting weak activity in very recent, and very few TEs were intact; however, the recent activity may be underestimated due to the sequencing/assembly technologies in some species. Overall, this study demonstrates low diversity, activity, and density of TEs in the five avian species; highlights the differences of TEs in these lineages; and suggests that the current and recent activity of TEs in avian genomes is very limited, which may be one of the reasons of small genome sizes in birds.     

Characterization of chicken Mda5 activity: regulation of IFN-β in the absence of RIG-I functionality

In mammals, Mda5 and RIG-I are members of the evolutionary conserved RIG-like helicase family that play critical roles in the outcome of RNA virus infections. Resolving influenza infection in mammals has been shown to require RIG-I; however, the apparent absence of a RIG-I homolog in chickens raises intriguing questions regarding how this species deals with influenza virus infection. Although chickens are able to resolve certain strains of influenza, they are highly susceptible to others, such as highly pathogenic avian influenza H5N1. Understanding RIG-like helicases in the chicken is of critical importance, especially for developing new therapeutics that may use these systems. With this in mind, we investigated the RIG-like helicase Mda5 in the chicken. We have identified a chicken Mda5 homolog (ChMda5) and assessed its functional activities that relate to antiviral responses. Like mammalian Mda5, ChMda5 expression is upregulated in response to dsRNA stimulation and following IFN activation of cells. Furthermore, RNA interference-mediated knockdown of ChMda5 showed that ChMda5 plays an important role in the IFN response of chicken cells to dsRNA. Intriguingly, although ChMda5 levels are highly upregulated during influenza infection, knockdown of ChMda5 expression does not appear to impact influenza proliferation. Collectively, although Mda5 is functionally active in the chicken, the absence of an apparent RIG-I-like function may contribute to the chicken's susceptibility to highly pathogenic influenza.