Morphology and distribution of scales, dermal ossifications, and other non-feather integumentary structures in non-avialan theropod dinosaurs.

Modern birds are typified by the presence of feathers, complex evolutionary innovations that were already widespread in the group of theropod dinosaurs (Maniraptoriformes) that include crown Aves. Squamous or scaly reptilian-like skin is, however, considered the plesiomorphic condition for theropods and dinosaurs more broadly. Here, we review the morphology and distribution of non-feathered integumentary structures in non-avialan theropods, covering squamous skin and naked skin as well as dermal ossifications. The integumentary record of non-averostran theropods is limited to tracks, which ubiquitously show a covering of tiny reticulate scales on the plantar surface of the pes. This is consistent also with younger averostran body fossils, which confirm an arthral arrangement of the digital pads. Among averostrans, squamous skin is confirmed in Ceratosauria (Carnotaurus), Allosauroidea (Allosaurus, Concavenator, Lourinhanosaurus), Compsognathidae (Juravenator), and Tyrannosauroidea (Santanaraptor, Albertosaurus, Daspletosaurus, Gorgosaurus, Tarbosaurus, Tyrannosaurus), whereas dermal ossifications consisting of sagittate and mosaic osteoderms are restricted to Ceratosaurus. Naked, non-scale bearing skin is found in the contentious tetanuran Sciurumimus, ornithomimosaurians (Ornithomimus) and possibly tyrannosauroids (Santanaraptor), and also on the patagia of scansoriopterygids (Ambopteryx, Yi). Scales are surprisingly conservative among non-avialan theropods compared to some dinosaurian groups (e.g. hadrosaurids); however, the limited preservation of tegument on most specimens hinders further interrogation. Scale patterns vary among and/or within body regions in Carnotaurus, Concavenator and Juravenator, and include polarised, snake-like ventral scales on the tail of the latter two genera. Unusual but more uniformly distributed patterning also occurs in Tyrannosaurus, whereas feature scales are present only in Albertosaurus and Carnotaurus. Few theropods currently show compelling evidence for the co-occurrence of scales and feathers (e.g. Juravenator, Sinornithosaurus), although reticulate scales were probably retained on the mani and pedes of many theropods with a heavy plumage. Feathers and filamentous structures appear to have replaced widespread scaly integuments in maniraptorans. Theropod skin, and that of dinosaurs more broadly, remains a virtually untapped area of study and the appropriation of commonly used techniques in other palaeontological fields to the study of skin holds great promise for future insights into the biology, taphonomy and relationships of these extinct animals.

Direct delivery of adenoviral CRISPR/Cas9 vector into the blastoderm for generation of targeted gene knockout in quail.

Zygotes at the 1-cell stage have been genetically modified by microinjecting the CRISPR/Cas9 components for the generation of targeted gene knockout in mammals. In the avian species, genetic modification of the zygote is difficult because its unique reproductive system limits the accessibility of the zygote at the 1-cell stage. To date, only a few CRISPR/Cas9-mediated gene knockouts have been reported using the chicken as a model among avian species, which requires 3 major processes: isolation and culture of primordial germ cells (PGCs), modification of the genome of PGCs in vitro, and injection of the PGCs into the extraembryonic blood vessel at the early embryonic stages when endogenous PGCs migrate through circulation to the genital ridge. In the present study, the adenoviral CRISPR/Cas9 vector was directly injected into the quail blastoderm in newly laid eggs. The resulting chimeras generated offspring with targeted mutations in the melanophilin (MLPH) gene, which is involved in melanosome transportation and feather pigmentation. MLPH homozygous mutant quail exhibited gray plumage, whereas MLPH heterozygous mutants and wild-type quail exhibited dark brown plumage. In addition, the adenoviral vector was not integrated into the genome of knockout quail, and no mutations were detected in potential off-target regions. This method of generating genome-edited poultry is expected to accelerate avian research and has potential applications for developing superior genetic lines for poultry production in the industry.

An intercross population study reveals genes associated with body size and plumage color in ducks.

Comparative population genomics offers an opportunity to discover the signatures of artificial selection during animal domestication, however, their function cannot be directly revealed. We discover the selection signatures using genome-wide comparisons among 40 mallards, 36 indigenous-breed ducks, and 30 Pekin ducks. Then, the phenotypes are fine-mapped based on resequencing of 1026 ducks from an F2 segregating population generated by wild × domestic crosses. Interestingly, the two key economic traits of Pekin duck are associated with two selective sweeps with fixed mutations. A novel intronic insertion most possibly leads to a splicing change in MITF accounted for white duck down feathers. And a putative long-distance regulatory mutation causes continuous expression of the IGF2BP1 gene after birth which increases body size by 15% and feed efficiency by 6%. This study provides new insights into genotype-phenotype associations in animal research and constitutes a promising resource on economically important genes in fowl.

Diverse feather shape evolution enabled by coupling anisotropic signalling modules with self-organizing branching programme.

Adaptation of feathered dinosaurs and Mesozoic birds to new ecological niches was potentiated by rapid diversification of feather vane shapes. The molecular mechanism driving this spectacular process remains unclear. Here, through morphology analysis, transcriptome profiling, functional perturbations and mathematical simulations, we find that mesenchyme-derived GDF10 and GREM1 are major controllers for the topologies of rachidial and barb generative zones (setting vane boundaries), respectively, by tuning the periodic-branching programme of epithelial progenitors. Their interactions with the anterior-posterior WNT gradient establish the bilateral-symmetric vane configuration. Additionally, combinatory effects of CYP26B1, CRABP1 and RALDH3 establish dynamic retinoic acid (RA) landscapes in feather mesenchyme, which modulate GREM1 expression and epithelial cell shapes. Incremental changes of RA gradient slopes establish a continuum of asymmetric flight feathers along the wing, while switch-like modulation of RA signalling confers distinct vane shapes between feather tracts. Therefore, the co-option of anisotropic signalling modules introduced new dimensions of feather shape diversification.

Phylogeography, inter-specific limits and diversification of Turdus ignobilis (Aves: Turdidae).

Turdus ignobilis is a widely distributed thrush species throughout northern South America and the Amazon, inhabiting a diverse set of habitats ranging from floodplain forests, white sand "campinas", to highland forests (Andes and Tepuis). There are currently six known subspecies of T. ignobilis, which vary extensively phenotypically and also ecologically, but whose interspecific limits and evolutionary history have never been investigated before. In this study, we used molecular data and plumage characters to review the taxonomy and uncover the evolutionary relationships of the six T. ignobilis subspecies described to date. We estimated gene and species trees based on both mitochondrial (ND2 and COI) and nuclear (TGFB2 and G3PDH) genes, which recovered, with high statistical support, the polyphyly of Turdus ignobilis, as currently defined. Therefore, based on our results, we propose a new taxonomic treatment that splits T. ignobilis into at least three separate species based on both molecular data and plumage characters. Each newly recognized species inhabit a distinct habitat type, with "true" T. ignobilis occurring in highland habitats of the Tepuis and the Andes, while T. arthuri and T. debilis are tied to "white-sand forest" and várzea floodplain forests in lowland Amazonia, respectively.

A second look at the colors of the dinosaurs.

In earlier work, I predicted that we would probably not be able to determine the colors of the dinosaurs. I lost this epistemic bet against science in dramatic fashion when scientists discovered that it is possible to draw inferences about dinosaur coloration based on the microstructure of fossil feathers (Vinther et al., 2008). This paper is an exercise in philosophical error analysis. I examine this episode with two questions in mind. First, does this case lend any support to epistemic optimism about historical science? Second, under what conditions is it rational to make predictions about what questions scientists will or will not be able answer? In reply to the first question, I argue that the recent work on the colors of the dinosaurs matters less to the debate about the epistemology of historical science than it might seem. In reply to the second question, I argue that it is difficult to specify a policy that would rule out the failed bet without also being too conservative.

Histological and histochemical study of the uropygial gland of chimango caracara (Milvago chimango vieillot, 1816).

The uropygial glands of birds are sebaceous organs that contribute to the water-repellent properties of the feather coat. We studied the histological and histochemical characteristics of the uropygial gland of chimango caracara using hematoxylin and eosin (H & E), Gomori´s trichrome, orcein, Gomori´s reticulin, periodic acid-Schiff (PAS), Alcian blue (AB) and a variety of lectins. The gland is composed of two lobes and a papilla with 20 downy feathers. It is surrounded by a capsule of dense connective tissue that contains elastic, reticular and smooth muscle fibers. The papilla is delicate and has two excretory ducts. The gland mass relative to body mass was 0.143%. Both adenomer cells and their secretions were stained with Sudan IV, PAS and AB, and were positive for numerous lectins that indicated the presence of lipids and carbohydrates. Immunohistochemical techniques to detect PCNA confirmed cell proliferation in the basal stratum of the adenomer cells. The lipids and glycoconjugates secreted by the uropygial gland serve numerous functions including protection against microorganisms.

Fish oil disrupts seabird feather microstructure and waterproofing.

Seabirds and other aquatic avifauna are highly sensitive to exposure to petroleum oils. A small amount of oil is sufficient to break down the feather barrier that is necessary to prevent water penetration and hypothermia. Far less attention has been paid to potential effects on aquatic birds of so called 'edible oils', non-petroleum oils such as vegetable and fish oils. In response to a sardine oil discharge by a vessel off the coast of British Columbia, we conducted an experiment to assess if feather exposure to sheens of sardine oil (ranging from 0.04 to 3 µm in thickness) resulted in measurable oil and water uptake and significant feather microstructure disruption. We designed the experiment based on a previous experiment on effects of petroleum oils on seabird feathers. Feathers exposed to the thinnest fish oil sheens (0.04 µm) resulted in measurable feather weight gain (from oil and water uptake) and significant feather microstructure disruption. Both feather weight gain and microstructure disruption increased with increasing fish oil thickness. Because of the absence of primary research on effects of edible oils on sea birds, we conducted interviews with wildlife rehabilitation professionals with experience rehabilitating sea birds after edible oil exposure. The consensus from interviews and our experiment indicated that physical contact with fish and other 'edible oils' in the marine environment is at least as harmful to seabirds as petroleum oils.

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.

Evidencia morfológica de hibridación entre las subespecies de Ramphocelus flammigerus (Passeriformes: Thraupidae) en Colombia / Morphological evidence of hybridization between Ramphocelus flammigerus subspecies (Passeriformes: Thraupidae) in Colombia

Morphological evidence of hybridization between Ramphocelus flammigerus subspecies (Passeriformes: Thraupidae) in Colombia. Habitat modifications such as deforestation and the increase of agricultural activities, have led to uncommon faunal interactions. In Colombia, this condition have caused the secondary contact of subspecies of Ramphocelus flammigerus populations from Cauca valley and the Pacific coast; and some specimens with rumps of intermediate colors of the subspecies have been found and are thought as hybrids. The objective of this study was to assess the presence of morphological evidence that may suggest hybridization and may explain the origin of individuals with intermediate coloration. We predict that if subspecies hybridize, they will be more similar in morphology when coexisting than when separated. Alternatively, coexisting subspecies might diverge in sympatry, because of selection to reduce competition for resources (character displacement). For this, a survey in 15 localities was undertaken: 10 allopatric areas (five for each subspecies), and five sympatric areas. Mist nets were used to capture individuals and a total of seven morphological characters were measured. To identify the patterns of morphological variation, we compared morphology of subspecies, sympatric and allopatric populations and individuals of intermediate colors. Consequently, we performed discriminant analysis and test for differences between groups by using 95% confidence intervals for log-ratio tests. A total of 112 individuals were captured (46 intermediate-colored individuals, 20 R. f. flammigerus, and 46 R. f. icteronotus. Discriminant analyses showed that subspecies were well differentiated, and intermediate individuals overlapped with them. Log-ratio test, based on Mahalanobis distances, showed that intermediate individuals were morphologically more similar to both subspecies than subspecies themselves. In addition, log-ratio tests showed that subspecies sympatric populations were similar but allopatric ones were different, and that individuals of intermediate colors were more similar to sympatric than to allopatric populations of the two subspecies. Therefore, morphological evidence supports the predictions of a hybridization hypothesis among the subspecies of R. flammigerus. In conclusion, the analysis of morphological variation in R. flammigerus suggests that hybridization between subspecies is occurring and that a process of genetic introgression is probably in progress.

Las modificaciones de los hábitats naturales, tales como la deforestación y el incremento de las actividades agrícolas, han conducido a interacciones faunísticas inusuales. En Colombia, esta situación ha generado el contacto secundario entre las poblaciones de las subespecies de Ramphocelus flammigerus del Valle del Cauca y de la costa Pacífica; y se han encontrado individuos con rabadillas de colores intermedios entre las subespecies que se han catalogado como híbridos. El objetivo del presente estudio fue evaluar si existe evidencia morfológica que sugiera hibridación y que pueda explicar el origen de los individuos de coloración intermedia. Con este fin, se obtuvieron muestras de 15 localidades; 10 zonas alopátricas (cinco por cada subespecie) y cinco zonas simpátricas. Para la captura de individuos se utilizaron redes de niebla y fueron tomados siete caracteres morfológicos. Asimismo, se predijo que si las subespecies están hibridando, las mismas, podrían ser morfológicamente más similares cuando coexisten que cuando se encuentran separadas. Alternativamente, cuando las subespecies coexisten, éstas pueden divergir en simpatría debido a presiones selectivas para reducir la competencia por recursos (desplazamiento de caracteres). Para identificar los patrones de variación morfológica, se comparó la morfología de las subespecies, de poblaciones simpátricas y alopátricas de ambas subespecies y de los individuos de cloración intermedia. Consecuentemente, se realizó un análisis discriminante y se evaluaron las diferencias entre los grupos con la utilización de intervalos de confianza del 95% para las relaciones logarítmicas. Y se capturaron un total de 112 individuos (46 de coloraciones intermedias, 20 R. f. flammigerus y 46 R. f. icteronotus). Los análisis discriminantes mostraron que las subespecies se diferencian entre ellas y que los individuos de coloraciones intermedias se traslapan con estas. Las relaciones logarítmicas, basadas en las distancias cuadradas de Mahalanobis, mostraron que los individuos intermedios fueron más similares morfológicamente a ambas subespecies que las subespecies entre ellas. Adicionalmente, se encontró que las poblaciones simpátricas de ambas subespecies son similares pero las poblaciones alopátricas son diferentes, y que los individuos de coloraciones intermedias son más similares a las poblaciones simpátricas de las dos subespecies que a las alopátricas. Por lo tanto, la evidencia morfológica evidencia las predicciones derivadas de la hipótesis de hibridación entre las subespecies de R. flammigerus. En conclusión, el análisis morfológico de la variación en R. flammigerus sugiere que se presenta hibridación entre las subespecies y que probablemente está en progreso un proceso de introgresión genética.

The roles of time and ecology in the continental radiation of the Old World leaf warblers (Phylloscopus and Seicercus).

Many continental sister species are allopatric or parapatric, ecologically similar and long separated, of the order of millions of years. Sympatric, ecologically differentiated, species, are often even older. This raises the question of whether build-up of sympatric diversity generally follows a slow process of divergence in allopatry, initially without much ecological change. I review patterns of speciation among birds belonging to the continental Eurasian Old World leaf warblers (Phylloscopus and Seicercus). I consider speciation to be a three-stage process (range expansions, barriers to gene flow, reproductive isolation) and ask how ecological factors at each stage have contributed to speciation, both among allopatric/parapatric sister species and among those lineages that eventually led to currently sympatric species. I suggest that time is probably the critical factor that leads to reproductive isolation between sympatric species and that a strong connection between ecological divergence and reproductive isolation remains to be established. Besides reproductive isolation, ecological factors can affect range expansions (e.g. habitat tracking) and the formation of barriers (e.g. treeless areas are effective barriers for warblers). Ecological factors may often limit speciation on continents because range expansions are difficult in 'ecologically full' environments.

Effects of sheens associated with offshore oil and gas development on the feather microstructure of pelagic seabirds.

Operational discharges of hydrocarbons from maritime activities can have major cumulative impacts on marine ecosystems. Small quantities of oil (i.e., 10 ml) results in often lethally reduced thermoregulation in seabirds. Thin sheens of oil and drilling fluids form around offshore petroleum production structures from currently permissible operational discharges of hydrocarbons. Methodology was developed to measure feather microstructure impacts (amalgamation index or AI) associated with sheen exposure. We collected feather samples from two common North Atlantic species of seabirds; Common Murres (Uria aalge) and Dovekies (Alle alle). Impacts were compared after feather exposure to crude oil and synthetic lubricant sheens of varying thicknesses. Feather weight and microstructure changed significantly for both species after exposure to thin sheens of crude oil and synthetic drilling fluids. Thus, seabirds may be impacted by thin sheens forming around offshore petroleum production facilities from discharged produced water containing currently admissible concentrations of hydrocarbons.

The expression of melanin-based plumage is separately modulated by exogenous oxidative stress and a melanocortin.

Melanin-based traits involved in animal communication have been traditionally viewed as occurring under strict genetic control. However, it is generally accepted that both genetic and environmental factors influence melanin production. Medical studies suggest that, among environmental factors influencing melanization, oxidative stress could play a relevant role. On the other hand, genetic control would be exerted by the melanocortin system, and particularly by the alpha-melanocyte-stimulating hormone (alpha-MSH), which triggers the production of eumelanins (black pigments). To determine how the melanocortin system and an exogenous source of oxidative stress interact in the expression of melanin-based plumage, developing red-legged partridges (Alectoris rufa) were manipulated. Some partridges were injected with alpha-MSH, while other birds received a pro-oxidant molecule (diquat) in drinking water. Controls and birds receiving both treatments were also studied. Both alpha-MSH- and diquat-treated individuals presented larger eumelanin-based traits than controls, but alpha-MSH+diquat-treated birds showed the largest traits, suggesting that oxidative stress and melanocortins promote additive but independent effects. Diquat also induced a decline in the level of a key intracellular antioxidant (glutathione), which is associated with high expression of eumelanin-based signals in other bird species. Some scenarios for the evolution of melanin-based traits in relation to oxidative stress are proposed.

Wnt3a gradient converts radial to bilateral feather symmetry via topological arrangement of epithelia.

The evolution of bilaterally symmetric feathers is a fundamental process leading toward flight. One major unsolved mystery is how the feathers of a single bird can form radially symmetric downy feathers and bilaterally symmetric flight feathers. In developing downy feather follicles, barb ridges are organized parallel to the long axis of the feather follicle. In developing flight-feather follicles, the barb ridges are organized helically toward the anterior region, leading to the fusion and creation of a rachis. Here we discover an anterior-posterior molecular gradient of wingless int (Wnt3)a in flight but not downy feathers. Global inhibition of the Wnt gradient transforms bilaterally symmetric feathers into radially symmetric feathers. Production of an ectopic local Wnt3a gradient reoriented barb ridges toward the source and created an ectopic rachis. We further show that the orientation of the Wnt3a gradient is dictated by the dermal papilla (DP). Swapping DPs between wing covert and breast downy feathers demonstrates that both feather symmetry and molecular gradients are in accord with the origin of the DP. Thus the fates of feather epidermal cells are not predetermined through some molecular codes but can be modulated. Together, our data suggest feathers are shaped by a DP--> Wnt gradient-->helical barb ridge organization-->creation of rachis-->bilateral symmetry sequence. We speculate diverse feather forms can be achieved by adjusting the orientation and slope of molecular gradients, which then shape the topological arrangements of feather epithelia, thus linking molecular activities to organ forms and novel functions.

The morphogenesis of feathers.

Feathers are highly ordered, hierarchical branched structures that confer birds with the ability of flight. Discoveries of fossilized dinosaurs in China bearing 'feather-like' structures have prompted interest in the origin and evolution of feathers. However, there is uncertainty about whether the irregularly branched integumentary fibres on dinosaurs such as Sinornithosaurus are truly feathers, and whether an integumentary appendage with a major central shaft and notched edges is a non-avian feather or a proto-feather. Here, we use a developmental approach to analyse molecular mechanisms in feather-branching morphogenesis. We have used the replication-competent avian sarcoma retrovirus to deliver exogenous genes to regenerating flight feather follicles of chickens. We show that the antagonistic balance between noggin and bone morphogenetic protein 4 (BMP4) has a critical role in feather branching, with BMP4 promoting rachis formation and barb fusion, and noggin enhancing rachis and barb branching. Furthermore, we show that sonic hedgehog (Shh) is essential for inducing apoptosis of the marginal plate epithelia, which results in spaces between barbs. Our analyses identify the molecular pathways underlying the topological transformation of feathers from cylindrical epithelia to the hierarchical branched structures, and provide insights on the possible developmental mechanisms in the evolution of feather forms.

Involvement of the Sonic hedgehog gene in chick feather formation.

To elucidate the molecular mechanisms of chick feather formation, we observed expression patterns of the Sonic hedgehog (Shh) gene, which is one of the vertebrate homologs of the Drosophila segment polarity gene, hedgehog, and encodes a signaling molecule functioning in limb pattern formation and motor neuron induction. We found that the Shh gene is also expressed in the apical region of the feather placodes and then in nine to eleven longitudinal stripes along feather filaments. The stripe was found to correspond to one of the outer marginal zones of each barb ridge, termed the zone of Shh expression. No significant expression signal was detected in the scale bud of developing legs. Thus, Shh is likely to function as an epithelial signaling molecule in epithelio-mesenchymal interaction during feather formation. Furthermore, since genes of bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-4 (FGF-4) are coexpressed with Shh during feather formation as observed in limb morphogenesis, interactions among FGF-4, Shh and BMP-2 may be involved in formation of feather filaments and barbs in a similar fashion as elucidated in limb pattern formation.

Assessment of softness properties of feathers.

An instrument to be called the "Crimp Meter" was designed and used with a conventional balance to enable the plotting of a force-displacement curve for individual feathers. A comparison of the curves described by different feathers was made by regression analysis. The type of curve described by a feather is characteristic of its tensile properties and its degree of softness. This procedure was employed to objectively confirm the observation that chicks fed toxic levels of methionine (1.45% added) in corn-soybean diets had significantly softer (P less than .001) feathers than those fed nontoxic levels of cysteine (.203% added). The basal diet was analyzed and found to contain .35% methionine and .37% cystine. This device had potential for the down-feather, wool, and fabric industries.