Genomic Variation, Population History, and Long-Term Genetic Adaptation to High Altitudes in Tibetan Partridge (Perdix hodgsoniae).

Species residing across elevational gradients display adaptations in response to environmental changes such as oxygen availability, ultraviolet radiation, and temperature. Here, we study genomic variation, gene expression, and long-term adaptation in Tibetan Partridge (Perdix hodgsoniae) populations residing across the elevational gradient of the Tibetan Plateau. We generated a high-quality draft genome and used it to carry out downstream population genomic and transcriptomic analysis. The P. hodgsoniae populations residing across various elevations were genetically distinct, and their phylogenetic clustering was consistent with their geographic distribution. We identified possible evidence of gene flow between populations residing in <3,000 and >4,200 m elevation that is consistent with known habitat expansion of high-altitude populations of P. hodgsoniae to a lower elevation. We identified a 60 kb haplotype encompassing the Estrogen Receptor 1 (ESR1) gene, showing strong genetic divergence between populations of P. hodgsoniae. We identified six single nucleotide polymorphisms within the ESR1 gene fixed for derived alleles in high-altitude populations that are strongly conserved across vertebrates. We also compared blood transcriptome profiles and identified differentially expressed genes (such as GAPDH, LDHA, and ALDOC) that correlated with differences in altitude among populations of P. hodgsoniae. These candidate genes from population genomics and transcriptomics analysis were enriched for neutrophil degranulation and glycolysis pathways, which are known to respond to hypoxia and hence may contribute to long-term adaptation to high altitudes in P. hodgsoniae. Our results highlight Tibetan Partridges as a useful model to study molecular mechanisms underlying long-term adaptation to high altitudes.

Independent loss events of a functional tetherin gene in galliform birds.

IMPORTANCE: Birds represent important hosts for numerous viruses, including zoonotic viruses and pathogens with the potential to cause major economic losses to the poultry industry. Viral replication and transmission can be inhibited or blocked by the action of antiviral restriction factors (RFs) encoded by the host. One well-characterized RF is tetherin, a protein that directly blocks the release of newly formed viral particles from infected cells. Here, we describe the evolutionary loss of a functional tetherin gene in two galliform birds, turkey (Meleagris gallopavo) and Mikado pheasant (Syrmaticus mikado). Moreover, we demonstrate that the structurally related protein TMCC(aT) exerts antiviral activity in several birds, albeit by a mechanism different from that of tetherin. The evolutionary scenario described here represents the first documented loss-of-tetherin cases in vertebrates.

Antiviral Activity and Adaptive Evolution of Avian Tetherins.

Tetherin/BST-2 is an antiviral protein that blocks the release of enveloped viral particles by linking them to the membrane of producing cells. At first, BST-2 genes were described only in humans and other mammals. Recent work identified BST-2 orthologs in nonmammalian vertebrates, including birds. Here, we identify the BST-2 sequence in domestic chicken (Gallus gallus) for the first time and demonstrate its activity against avian sarcoma and leukosis virus (ASLV). We generated a BST-2 knockout in chicken cells and showed that BST-2 is a major determinant of an interferon-induced block of ASLV release. Ectopic expression of chicken BST-2 blocks the release of ASLV in chicken cells and of human immunodeficiency virus type 1 (HIV-1) in human cells. Using metabolic labeling and pulse-chase analysis of HIV-1 Gag proteins, we verified that chicken BST-2 blocks the virus at the release stage. Furthermore, we describe BST-2 orthologs in multiple avian species from 12 avian orders. Previously, some of these species were reported to lack BST-2, highlighting the difficulty of identifying sequences of this extremely variable gene. We analyzed BST-2 genes in the avian orders Galliformes and Passeriformes and showed that they evolve under positive selection. This indicates that avian BST-2 is involved in host-virus evolutionary arms races and suggests that BST-2 antagonists exist in some avian viruses. In summary, we show that chicken BST-2 has the potential to act as a restriction factor against ASLV. Characterizing the interaction of avian BST-2 with avian viruses is important in understanding innate antiviral defenses in birds.IMPORTANCE Birds are important hosts of viruses that have the potential to cause zoonotic infections in humans. However, only a few antiviral genes (called viral restriction factors) have been described in birds, mostly because birds lack counterparts of highly studied mammalian restriction factors. Tetherin/BST-2 is a restriction factor, originally described in humans, that blocks the release of newly formed virus particles from infected cells. Recent work identified BST-2 in nonmammalian vertebrate species, including birds. Here, we report the BST-2 sequence in domestic chicken and describe its antiviral activity against a prototypical avian retrovirus, avian sarcoma and leukosis virus (ASLV). We also identify BST-2 genes in multiple avian species and show that they evolve rapidly in birds, which is an important indication of their relevance for antiviral defense. Analysis of avian BST-2 genes will shed light on defense mechanisms against avian viral pathogens.

Evidence of adaptive evolution of alpine pheasants to high-altitude environment from mitogenomic perspective.

Adaptive evolutions to high-altitude adaptation have been intensively studied in mammals. However, considering the additional vertebrate groups, new perception regarding selection challenged by high-altitude stress on mitochondrial genome can be gained. To test this hypothesis, we compiled and analyzed the mitochondrial genomes of 5 alpine pheasants and 12 low-altitude species in Phasianidae. The results that evolutionary rates of ATP6 and ND6 showing significant fluctuation among branches when involved with five alpine pheasants revealed both genes might have implications with adapting to highland environment. The radical physico-chemical property changes identified by the modified MM01 model, including composition (C) and equilibrium constant (ionization of COOH) (Pk') in ATP6 and beta-structure tendencies (Pß), Pk', and long-range non-bonded energy (El) in ND6, suggested that minor overall adjustments in size, protein conformation and relative orientation of reaction interfaces have been optimized to provide the ideal environments for electron transfer, proton translocation and generation of adenosine triphosphate (ATP). Additionally, three unique substitution sites were identified under selection in ND6, which could be potentially important adaptive changes contributing to cellular energy production. Our findings suggested that adaptive evolution may occur in alpine pheasants, which are an important complement to the knowledge of genetic mechanisms against the high-altitude environment in non-mammal animals.

Complete mitochondrial genome of Palawan peacock-pheasant Polyplectron napoleonis (Galliformes, Phasianidae).

The complete mitochondrial genome of the Palawan peacock-pheasant Polyplectron napoleonis is 16,710 bp and contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a control-region. All protein-coding genes use the standard ATG start codon, except for cox1 which has GTG start codon. Seven out of 13 PCGs have TAA stop codons, two have AGG (cox1 and nd6), and three PCGs (nd2, cox2 and nd4) have incomplete stop codon of just T- - nucleotide.

Functional characteristics of the growth factor receptor family and some ligands in the oropharyngeal cavity of the Chukar partridge (Alectoris chukar).

1. The aim of the present study is to describe, immunohistochemically, the expression and cell type localisation of growth factor receptors and some of their ligands in the oropharyngeal organs of the Chukar partridge. 2. The tissue samples from 10 healthy adult partridges were dissected under ether anaesthesia and then embedded in paraffin following routine histological procedures. The immunoreaction for receptors and ligands of the epidermal growth factor receptor (EGFR)/ligand system was localised in the cell membrane, nucleus and cytoplasm of the luminal and glandular epithelial cells, stromal and striated muscle cells, and vascular endothelial and smooth muscle cells. 3. Variations were observed in the avian oropharyngeal organs. The immunostaining for the erbB1/HER1 (human epidermal growth factor receptor 1) and the EGF (epidermal growth factor) and AREG (Amphiregulin) ligands in the luminal epithelial cells was higher than in the glandular epithelial, stromal and striated muscle cells. However, the immunostaining for erbB3/HER3 (human epidermal growth factor receptor 3) and erbB4/HER4 (human epidermal growth factor receptor 4) were similar in the luminal epithelium, stromal and striated muscle cells. 4. Growth factor receptors and some of their ligands were localised in different cell types in the oropharyngeal organs. We suggest that erbB/HERs (human epidermal growth factor receptors) and their ligands play an important role in proliferation, differentiation, growth, survival and migration of the cells.

Complete mitochondrial genome of Germain's Peacock-Pheasant Polyplectron germaini (Aves, Galliformes, Phasianidae).

The Germain's Peacock-Pheasant Polyplectron germaini (Aves, Galliformes, Phasianidae) is classified as Near Threatened on the IUCN Red List. The complete mitochondrial genome of P. germaini is 16,699 bp, consisting of 13 protein-coding genes, 2 rRNA, 22 tRNA genes and 1 control region. All of the 13 protein-coding genes have ATG as start codon. Eight of the 13 protein-coding genes have TAA as stop codon.

The evolution of peafowl and other taxa with ocelli (eyespots): a phylogenomic approach.

The most striking feature of peafowl (Pavo) is the males' elaborate train, which exhibits ocelli (ornamental eyespots) that are under sexual selection. Two additional genera within the Phasianidae (Polyplectron and Argusianus) exhibit ocelli, but the appearance and location of these ornamental eyespots exhibit substantial variation among these genera, raising the question of whether ocelli are homologous. Within Polyplectron, ocelli are ancestral, suggesting ocelli may have evolved even earlier, prior to the divergence among genera. However, it remains unclear whether Pavo, Polyplectron and Argusianus form a monophyletic clade in which ocelli evolved once. We estimated the phylogeny of the ocellated species using sequences from 1966 ultraconserved elements (UCEs) and three mitochondrial regions. The three ocellated genera did form a strongly supported clade, but each ocellated genus was sister to at least one genus without ocelli. Indeed, Polyplectron and Galloperdix, a genus not previously suggested to be related to any ocellated taxon, were sister genera. The close relationship between taxa with and without ocelli suggests multiple gains or losses. Independent gains, possibly reflecting a pre-existing bias for eye-like structures among females and/or the existence of a simple mutational pathway for the origin of ocelli, appears to be the most likely explanation.

A mitogenomic perspective on the ancient, rapid radiation in the Galliformes with an emphasis on the Phasianidae.

BACKGROUND: The Galliformes is a well-known and widely distributed Order in Aves. The phylogenetic relationships of galliform birds, especially the turkeys, grouse, chickens, quails, and pheasants, have been studied intensively, likely because of their close association with humans. Despite extensive studies, convergent morphological evolution and rapid radiation have resulted in conflicting hypotheses of phylogenetic relationships. Many internal nodes have remained ambiguous. RESULTS: We analyzed the complete mitochondrial (mt) genomes from 34 galliform species, including 14 new mt genomes and 20 published mt genomes, and obtained a single, robust tree. Most of the internal branches were relatively short and the terminal branches long suggesting an ancient, rapid radiation. The Megapodiidae formed the sister group to all other galliforms, followed in sequence by the Cracidae, Odontophoridae and Numididae. The remaining clade included the Phasianidae, Tetraonidae and Meleagrididae. The genus Arborophila was the sister group of the remaining taxa followed by Polyplectron. This was followed by two major clades: ((((Gallus, Bambusicola) Francolinus) (Coturnix, Alectoris)) Pavo) and (((((((Chrysolophus, Phasianus) Lophura) Syrmaticus) Perdix) Pucrasia) (Meleagris, Bonasa)) ((Lophophorus, Tetraophasis) Tragopan))). CONCLUSIONS: The traditional hypothesis of monophyletic lineages of pheasants, partridges, peafowls and tragopans was not supported in this study. Mitogenomic analyses recovered robust phylogenetic relationships and suggested that the Galliformes formed a model group for the study of morphological and behavioral evolution.

Mhc class II diversity and balancing selection in greater prairie-chickens.

The major histocompatibility complex (Mhc) of domestic chickens has been characterized as small and relatively simple compared with that of mammals. However, there is growing evidence that the Mhc of many bird lineages may be more complex, even within the Order Galliformes. In this study, we measured genetic variation and balancing selection at Mhc loci in another galliform, the greater prairie-chicken. We cloned and sequenced a 239 bp fragment of Mhc Class II beta-chain (BLB) exon 2 in 14 individuals. There was a total of 10 unique sequences and a minimum of four BLB loci. The d(N)/d(S) ratio at peptide-binding codons was significantly greater than one, suggesting balancing selection is acting on the BLB. We also recovered two YLB sequences, which clustered tightly with YLB sequences from three other species: domestic chicken, black grouse and common quail. The relatively large number of loci revealed in our study suggests that even closely related galliforms differ in the level of Mhc variation and structure.

Molecular evidence for species status of the endangered Hainan peacock pheasant.

The Hainan peacock pheasant is an endangered taxon found only on Hainan Island of China. Due to lack of detailed taxonomic studies, whether it is a subspecies of the grey peacock pheasant (Polyplectron bicalcaratum katsumatae) or a full species (Polyplectron katsumatae) remained unclear. We used molecular markers, including the complete mitochondrial cytochrome b gene and intron G of the nuclear ovomucoid gene, to reevaluate the taxonomy of the Hainan peacock pheasant. The results showed phylogeographic monophyly and large genetic distance between the Hainan peacock pheasant and the grey peacock pheasant. Sequence differences corroborated the species-level distinction between these two peacock pheasants, which were inferred to have diverged about 1.4+/-0.3 million years ago, near the time Hainan Island became separated from mainland China. Because the population density of the Hainan peacock pheasant is very low in its tropical forest on the island and the wild population is declining, it is now becoming severely endangered and should be ranked as the rarest species in the Order Galliformes in China. Our results increase the urgency of getting more morphological data to support the classification of the Hainan peacock pheasant as a distinct species and taking more conservation action immediately to protect this endangered island species.

Insertion events of CR1 retrotransposable elements elucidate the phylogenetic branching order in galliform birds.

Using standard phylogenetic methods, it can be hard to resolve the order in which speciation events took place when new lineages evolved in the distant past and within a short time frame. As an example, phylogenies of galliform birds (including well-known species such as chicken, turkey, and quail) usually show low bootstrap support values at short internal branches, reflecting the rapid diversification of these birds in the Eocene. However, given the key role of chicken and related poultry species in agricultural, evolutionary, general biological and disease studies, it is important to know their internal relationships. Recently, insertion patterns of transposable elements such as long and short interspersed nuclear element markers have proved powerful in revealing branching orders of difficult phylogenies. Here we decipher the order of speciation events in a group of 27 galliform species based on insertion events of chicken repeat 1 (CR1) transposable elements. Forty-four CR1 marker loci were identified from the draft sequence of the chicken genome, and from turkey BAC clone sequence, and the presence or absence of markers across species was investigated via electrophoretic size separation of amplification products and subsequent confirmation by DNA sequencing. Thirty markers proved possible to type with electrophoresis of which 20 were phylogenetically informative. The distribution of these repeat elements supported a single homoplasy-free cladogram, which confirmed that megapodes, cracids, New World quail, and guinea fowl form outgroups to Phasianidae and that quails, pheasants, and partridges are each polyphyletic groups. Importantly, we show that chicken is an outgroup to turkey and quail, an observation which does not have significant support from previous DNA sequence- and DNA-DNA hybridization-based trees and has important implications for evolutionary studies based on sequence or karyotype data from galliforms. We discuss the potential and limitations of using a genome-based retrotransposon approach in resolving problematic phylogenies among birds.

Phagocytotic removal of apoptotic endocrine cells by folliculostellate cells and its functional implications in clusterin accumulation in pituitary colloids in helmeted guinea fowl (Numida meleagris).

The different cell types in the anterior pituitary behave as dynamic populations. The gland maintains a continuous renewal of cells to ensure a dynamic balance between cell division, differentiation, growth arrest and apoptosis. Apoptosis is a frequent event in the anterior pituitary in which unwanted cells are eliminated without affecting neighboring cells. We examined the link between apoptosis and the occurrence of colloids in the guinea fowl (Numida meleagris) pituitary gland and the relationship of clusterin accumulation in the colloids. S-100 positive folliculostellate (FS) cells were found surrounding colloids. Apoptotic cells detected by single stranded DNA (ssDNA) immunohistochemistry were observed in the whole anterior pituitary and preferentially near colloid masses. Clusterin protein was detected in endocrine cells, FS cells and in the colloids. In situ hybridization showed clusterin mRNA in endocrine cells and FS cells. Simultaneous localization was performed to determine whether clusterin mRNA and ssDNA within anterior pituitary was present within the same cell. Clusterin mRNA was not detected in apoptotic cells but was present in neighboring surviving cells. At the ultrastructural level, numerous endocrine cells at different stages of apoptosis were found phagocytosed by FS cells. Our results suggest that clusterin is produced by endocrine cells for cytoprotection before death. Apoptotic endocrine cells are phagocytosed by FS cells and digested by their lysosomal enzymes. In FS cells, clusterin interacts and aggregates with by-products of digestion that subsequently become stored in colloid as a residual body.

Experimental infection of Attwater's/greater prairie chicken hybrids with the reticuloendotheliosis virus.

Reticuloendotheliosis virus (REV), a common pathogen of poultry, has been associated with runting and neoplasia in an endangered subspecies of grouse, the Attwater's prairie chicken. The pathogenesis of REV infection was examined in experimentally infected prairie chickens. Three groups of four Attwater's/greater prairie chicken hybrids were infected intravenously with varying doses (tissue culture infective dose [TCID50], 200, 1000, and 5000) of a prairie chicken-isolated REV. A fourth group of four birds was not infected. Blood was collected prior to infection, and at various times up to 37 wk following infection. Peripheral blood mononuclear cells were examined for integrated proviral DNA by a single-amplification polymerase chain reaction (PCR) and nested PCR of a region within the pol gene. The nested PCR identified REV proviral DNA in all REV-inoculated birds by 2 wk postinfection and confirmed chronic infection throughout the study. With the exception of a bird that died from bacterial pneumonia 8 wk postinfection, neoplasia, resembling that seen in naturally occurring infections, was observed in all birds, even those receiving as little as 200 TCID50 of virus.

[Molecular cloning of activin betaA subunit mature peptide from peafowl and its application in taxonomy and phylogeny].

The sequences of activin gene betaA subunit mature peptide have been amplified from white peafowl, blue peafowl (pavo cristatus) and green peafowl (pavo muticus) genomic DNA by polymerase chain reaction (PCR) with a pair of degenerate primers. The target fragments were cloned into the vector pMD18-T and sequenced. The length of activin gene betaA subunit mature peptide is 345bp, which encoded a peptide of 115 amino acid residues. Sequence analysis of activin gene betaA subunit mature peptide demonstrated that the identity of nucleotide is 98.0% between blue peaflowl and green peafowl, and the identity of that is 98.8% between blue peaflowl and white peafow. Sequences comparison in NCBI revealed that the sequences of activin gene betaA subunit mature peptides of different species are highly conserved during evolution process. In addition, the restriction enzyme map of activins is high similar between white peafowl and blue peafowl. Phylogenetic tree was constructed with Mega 2 and Clustalxldx software. The result showed that white peafowl has a closer relationship to blue peafowl than to green peafowl. Considered the nucleotide differences of peafowls' activin gene betaA subunit mature peptides, a highly conserved region, we supported that white peafowl was derived from blue peafowl, and it is more possible the hybrid but just the product of color mutation, or maybe as a subspecies of Pavo genus.

[The use of c-mos nuclear gene as a phylogenetic marker in tetraonidae birds]. / Ispol'zovanie c-mos-gena iadernogo genoma v kachestve filogeneticheskogo markera u tetereviykh ptits.

A 480-bp fragment of nuclear c-mos gene was sequenced in nine bird species representing four genera of the family Tetraonidae. It was demonstrated that nuclear genome region examined was highly conservative. The data were used to construct phylogenetic relationships among the c-mos gene sequences in Tetraonidae. The data obtained point to a paraphyletic origin of hazel grouse (Bonasa bonasia) and ruffed grouse (Bonasa umbellus).

Species identification of poultry egg products.

Species-specific primers for duck were deduced from the mitochondrial ATPase8 gene sequence. Species-specific PCR for turkeys and ducks showed no cross reaction with mixtures from chicken and guinea eggs and detection was possible to a concentration of 0.1% of homogenized duck egg and 5% of homogenized turkey egg when a PCR of 35 cycles was applied. A PCR of 30 cycles detected 10% of homogenized duck egg. The same sensitivities were obtained in dilutions of homogenized egg yolk; however, no PCR signals were obtained in egg white. Analysis of 13 industrial egg product samples showed the practical relevance of the species-specific PCR tests described herein.