17 research outputs found

    Bone-Associated Gene Evolution and the Origin of Flight in Birds

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    Background Bones have been subjected to considerable selective pressure throughout vertebrate evolution, such as occurred during the adaptations associated with the development of powered flight. Powered flight evolved independently in two extant clades of vertebrates, birds and bats. While this trait provided advantages such as in aerial foraging habits, escape from predators or long-distance travels, it also imposed great challenges, namely in the bone structure. Results We performed comparative genomic analyses of 89 bone-associated genes from 47 avian genomes (including 45 new), 39 mammalian, and 20 reptilian genomes, and demonstrate that birds, after correcting for multiple testing, have an almost two-fold increase in the number of bone-associated genes with evidence of positive selection (~52.8 %) compared with mammals (~30.3 %). Most of the positive-selected genes in birds are linked with bone regulation and remodeling and thirteen have been linked with functional pathways relevant to powered flight, including bone metabolism, bone fusion, muscle development and hyperglycemia levels. Genes encoding proteins involved in bone resorption, such as TPP1, had a high number of sites under Darwinian selection in birds. Conclusions Patterns of positive selection observed in bird ossification genes suggest that there was a period of intense selective pressure to improve flight efficiency that was closely linked with constraints on body size

    The Genetic Origins of Dragons

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    Dragons are popular creatures in fantasy that are large winged lizards that can be the size of a house. This report looks at the basic genetic requirements to produce Dragons. It finds that HOXD and HOXc-6 genes are necessary requirements for the formation of the dragon’s wings. It also investigates the size limits for birds and finds that due to the current bird size being limited by feather size dragons will likely lack them, and be closer to the ancient creatures that flew, such as the Quetzalcoatlus

    Intraskeletal histovariability, allometric growth patterns, and their functional implications in bird-like dinosaurs

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    With their elongated forelimbs and variable aerial skills, paravian dinosaurs, a clade also comprising modern birds, are in the hotspot of vertebrate evolutionary research. Inferences on the early evolution of flight largely rely on bone and feather morphology, while osteohistological traits are usually studied to explore life-history characteristics. By sampling and comparing multiple homologous fore-and hind limb elements, we integrate for the first time qualitative and quantitative osteohistological approaches to get insight into the intraskeletal growth dynamics and their functional implications in five paravian dinosaur taxa, Anchiornis, Aurornis, Eosinopteryx, Serikornis, and Jeholornis. Our qualitative assessment implies a considerable diversity in allometric/isometric growth patterns among these paravians. Quantitative analyses show that neither taxa nor homologous elements have characteristic histology, and that ontogenetic stage, element size and the newly introduced relative element precocity only partially explain the diaphyseal histovariability. Still, Jeholornis, the only avialan studied here, is histologically distinct from all other specimens in the multivariate visualizations raising the hypothesis that its bone tissue characteristics may be related to its superior aerial capabilities compared to the non-avialan paravians. Our results warrant further research on the osteohistological correlates of flight and developmental strategies in birds and bird-like dinosaurs

    Genomic adaptations to a high sugar diet in birds

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    While excessive consumption of glucose- and fructose-sweetened soft drinks is a major risk factor for type 2 diabetes and metabolic syndrome in humans, several nectarivorous bird lin- eages have adapted their metabolism to rely mostly on simple sugars obtained from flower nectar. These lineages are spread around the world and include hummingbirds (Americas), honeyeaters and lorikeets (Australasia), and sunbirds (Africa and Asia). All these nectarivores have evolved distinct phenotypic traits allowing them to rely mostly on nectar as a source of nutrients. However, the genomic underpinnings of these natural adaptations to nectarivory are largely unknown. In order to identify genomic changes underlying metabolic adaptations of nectarivorous birds, we produced new genomic and transcriptomic data, and combining them with publically avail- able data, we ran a number of comprehensive comparative screens. To confirm our theoretical findings, we complemented them with experimental validation. The genome-wide screen for hummingbird-specific gene losses identified the loss of FBP2, a gene encoding a key gluconeogenic enzyme that is normally active in muscles of all tetrapods. Loss of FBP2 occurred around a time where energy-demanding hovering flight is thought to have evolved in hummingbirds. We hypothesized that FBP2 loss could have contributed to the evolution of their metabolic adaptations in muscles. To test this, we downregulated the gene in a bird muscle cell line. Even a partial knockout of FBP2 significantly upregulated glycolysis and mitochondrial respiration in cells. In our experiments, we also show that the latter is likely happening due to the increased number of mitochondria. Together, these results suggest that FBP2 loss contributed to metabolic adaptations that likely enhanced hummingbirds’ ability to immediately process newly ingested sugars thus providing energy for the hovering flight. To study the convergence of adaptations to nectarivory, we ran a number of screens search- ing for convergent and lineage-specific genomic changes. A screen for rapid adaptive evolution identified that a rate-limiting glycolytic enzyme (hexokinase 3) evolved under strong positive se- lection in the stem honeyeaters, potentially underlying similar metabolic changes to what FBP2 loss has introduced in hummingbirds. These results provide a deep insight into the genomic basis of adaptations to high-sugar ‘soft drink-like’ diets in birds. These findings have the potential not only to answer important evolu- tionary questions but also to teach us lessons concerning type 2 diabetes, metabolic syndrome, and obesity in humans.:Zusammenfassung 4 Abstract 6 Chapter I. Introduction 8 Diet diversity in birds 8 Nectarivory and its challenges 8 Adaptations to nectarivory 11 Hummingbirds 13 Other nectar-feeding birds 14 Genomic changes underlying phenotypic variation 15 Gene copy-number variation 16 Gene duplication mechanisms and its evolutionary fates 17 Gene duplications contribute to adaptive evolution 17 Detection of gene duplications 19 Gene loss 19 The use-it-or-lose-it hypothesis 20 The less-is-more hypothesis 20 Detection of gene losses 20 Selection pressure 21 Positive selection can drive adaptive evolution 21 Detection of selection 22 Amino acid substitutions 23 Previous research on adaptations to nectarivory 23 Outline of the thesis 24 Chapter II. Genomic basis of metabolic adaptations in hummingbirds 26 Section I: Screen for hummingbird-specific gene losses 26 Overview 26 Results 26 Assembly of the long-tailed hermit genome 26 Identification of hummingbird-specific gene losses 27 Hummingbird muscle expresses no FBPases 29 Discussion 30 Section II: Experimental exploration of the metabolic role of the FBP2 loss 31 Overview 31 Results 31 Expression of FBPase encoding genes in cell lines 31 Inhibition of FBPases in QM7 32 Testing the FBPase inhibitor with glycogen assays 33 Testing the FBPase inhibitor with Seahorse Glycolytic Rate Assay 37 Generation of FBP2 knockout in QM7 38 Infection - transfection strategy 38 Electroporation strategy 40 Detection of FBP2-encoded protein with immunostaining 42 Knockout of FBP2 upregulates glycolysis in avian myoblast cells 44 FBP2 downregulation upregulates OXPHOS 44 FBP2 knockout increases the number of mitochondria 44 Genes important for mitochondrial biosynthesis and function are upregulated in hummingbirds 46 FBP2 knockout is associated with increased lipid deposition 49 Discussion 49 Section III: Hummingbird tissues analysis 50 Overview 50 Results 51 Glycogen content in hummingbirds tissues 51 Histological comparison 51 Biochemical quantification of total glycogen 52 Lipid content in hummingbird tissues 53 Discussion 54 Section IV: Evolution of glucose metabolism genes in hummingbirds 55 Overview 55 Results 55 Positive selection in glycolytic genes in hummingbirds 55 Copy number of glucose transporters in hummingbirds 57 Expression of glucose transporters in hummingbirds 58 Discussion 59 Chapter III. Convergence in the evolution of nectarivory 60 Overview 60 Results 61 Sequencing and assembly of new genomes of nectarivorous birds 61 Annotation of protein-coding genes 62 Gene families expansion-contraction 64 Signatures of positive selection related to nectarivory 65 The rate-controlling glycolytic enzyme evolved faster in honeyeaters 70 Discussion 72 Chapter IV. General discussion and outlook 72 Future work 75 Chapter V. Methods 77 Methods I: Screen for hummingbird-specific gene losses 77 Genome assembly 77 Modeling and masking repeats 77 Generating pairwise genome alignments 77 Detecting gene losses 77 Gene loss dating 77 Methods II: Experimental exploration of the metabolic role of the FBP2 loss 78 Cell culture 78 Guide RNA design 78 Infection and transfection of QM7 myoblasts 78 Electroporation of QM7 myoblasts 79 Genotyping of QM7 cell pools 79 RNA isolation 80 Real-time qPCR 80 Seahorse glycolytic rate assay 80 Cell number quantification 80 Mitochondrial number estimation 80 Biochemical glycogen qualification in cells 81 Fluorescent glycogen qualification in cells 81 Lipid quantification in cells 81 Western blot 82 Homology modeling 82 RNA sequencing 82 Analysis of transcriptomic data 83 Methods III: Hummingbird tissues analysis 83 Tissue preparation 83 Paraffin embedding and sectioning 83 Glycogen staining tissues 84 Freezing tissues and cryosectioning 84 Lipid staining tissues 84 Imaging histological sections 84 Biochemical glycogen qualification in tissues 84 Methods IV: Changes in glucose metabolism genes in hummingbirds 85 Positive selection analysis 85 Methods: Convergence in the evolution of nectarivory 85 Protein-coding gene annotation 85 Positive selection analysis 86 Estimating time-calibrated phylogeny 87 Gene families expansion-contraction analysis 87 Homology modeling 87 Appendix 8

    Data for "Bone-associated gene evolution and the origin of flight in birds"

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    <div><div>The data contained in them pretains to the manuscript: </div><div><br></div><div>Machado, JP, Johnson, WE, Gilbert, MTP, Zhang, G, Jarvis, ED, O’Brien, SJ, & Antunes, A (2016). </div><div>Bone-associated gene evolution and the origin of flight in birds. BMC genomics, 17(1), 1.</div><div><br></div><div>4 zip files:</div><div><br></div><div><b>1) Codeml site models:</b></div><div>    gene alignments,</div><div>    tree,</div><div>    parameters template.</div><div><br></div><div><b>2) Codeml branch models:</b></div><div>    gene alignments,</div><div>    tree,</div><div>    parameters template,</div><div>    results from Codeml.</div><div><br></div><div><b>3) Codeml branch-site models:</b></div><div>    gene alignments,</div><div>    tree,</div><div>    parameters template,</div><div>    results from Codeml. </div><div><br></div><div><b>4) CoEvol:</b></div><div>   input files and results.</div></div><div><br></div

    Additional file 19: Table S13. of Bone-associated gene evolution and the origin of flight in birds

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    Estimation of dN and dS for each branch under Model 0. For each branch, average of dN and dS and the corresponding standard deviation. (DOC 165 kb

    Additional file 6: Table S4. of Bone-associated gene evolution and the origin of flight in birds

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    Positively selected sites of bone-associated genes in Reptilian dataset after multiple testing correction. The alignment length is on Amino acids (aa). Bold represents statistical significance (p < 0.05). Q-value estimations for multiple testing are represented as positive selected (1) and negative selected (0). (DOC 158 kb

    Additional file 16: Table S12. of Bone-associated gene evolution and the origin of flight in birds

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    Covariance between dS, ω (dN/dS), gc content, and the three body mass measures (minimum, maximum and average) in 39 mammalian genomes using gene-based tree. The upper triangle shows the values obtained for all mammals and the lower triangle excluding bats. Each cell represent the covariance values and posterior probability are the bracketed values, posterior probability (** - < = 0.025 or > =0.975; * - < =0.05 or > =0.95) are highlighted in bold for the statistically significant correlations. (DOC 35 kb
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