Concordant evolution of plumage colour, feather microstructure and a melanocortin receptor gene between mainland and island populations of a fairy-wren

Studies of the patterns of diversification of birds on islands have contributed a great deal to the development of evolutionary theory. In white-winged fairy-wrens, Malurus leucopterus, mainland males develop a striking blue nuptial plumage whereas those on nearby islands develop black nuptial plumage. We explore the proximate basis for this divergence by combining microstructural feather analysis with an investigation of genetic variation at the melanocortin-1 receptor locus (MC1R). Fourier analysis revealed that the medullary keratin matrix (spongy layer) of the feather barbs of blue males was ordered at the appropriate nanoscale to produce the observed blue colour by coherent light scattering. Surprisingly, the feather barbs of black males also contained a spongy layer that could produce a similar blue colour. However, black males had more melanin in their barbs than blue males, and this melanin may effectively mask any structural colour produced by the spongy layer. Moreover, the presence of this spongy layer suggests that black island males evolved from a blue-plumaged ancestor. We also document concordant patterns of variation at the MC1R locus, as five amino acid substitutions were perfectly associated with the divergent blue and black plumage phenotypes. Thus, with the possible involvement of a melanocortin receptor locus, increased melanin density may mask the blue-producing microstructure in black island males, resulting in the divergence of plumage coloration between mainland and island white-winged fairy-wrens. Such mechanisms may also be responsible for plumage colour diversity across broader geographical and evolutionary scales.     

The anatomical basis of sexual dichromatism in non-iridescent ultraviolet-blue structural coloration of feathers

Despite extensive research on the evolution of avian dichromatism, the anatomical bases for differences between the sexes in species with structurally coloured plumage remain largely unknown. Using full‐spectrum spectrometry and transmission electron microscopy, we compared the colour and morphology of rump feathers of male and female eastern bluebirds (Sialia sialis). The ultraviolet (UV)‐blue feather colour in this species is caused by coherent scattering of light within the medullary ‘spongy layer’ of feather barbs. This spongy layer lies beneath a keratin cortex and on top of a layer of melanin granules that surround a hollow central vacuole. Irregularly shaped electron‐dense regions are present within the cortex. Male and female S. sialis differed substantially in their plumage colour and feather structure. A backwards logistic regression predicted sex with 100% accuracy using the colour variables brightness, UV‐violet (UV‐V) chroma and spectral saturation. A second backwards logistical regression predicted sex with 100% accuracy using relative cortex area and size of air spaces. Thus, S. sialis are dimorphic both in colour and in the structures causing this colour. Multiple regression analyses using data pooled from both sexes indicated that multiple features of feather barb structure contributed to colour variation in complex ways. Brightness was negatively related to the relative surface area of cortex in barb cross‐sections. Hue was positively related and UV‐V chroma was negatively related to the distance between scattering elements (i.e. keratin rods and air spaces) in the spongy layer. In contrast, hue was negatively related and UV‐V chroma was positively related to the thickness of the spongy layer. UV‐V chroma was also negatively related to the relative area of electron‐dense regions in the cortex. Spectral saturation was negatively related to the distance between scatterers and the standard error of the size of air spaces. These results suggest that the dimensions of spongy‐layer elements are critical to colour production, but that UV‐blue coloration can also be modified by the cortex and the thickness of the spongy layer. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84 , 259–271.     

Nanostructure predicts intraspecific variation in ultraviolet-blue plumage colour

Evidence suggests that structural plumage colour can be an honest signal of individual quality, but the mechanisms responsible for the variation in expression of structural coloration within a species have not been identified. We used full-spectrum spectrometry and transmission electron microscopy to investigate the effect of variation in the nanostructure of the spongy layer on expression of structural ultraviolet (UV)-blue coloration in eastern bluebird (Sialia sialis) feathers. Fourier analysis revealed that feather nanostructure was highly organized but did not accurately predict variation in hue. Within the spongy layer of feather barbs, the number of circular keratin rods significantly predicted UV-violet chroma, whereas the standard error of the diameter of these rods significantly predicted spectral saturation. These observations show that the precision of nanostructural arrangement determines some colour variation in feathers.     

Feather iridescence of Coeligena hummingbird species varies due to differently organized barbs and barbules

Hummingbirds are perhaps the most exquisite bird species because of their prominent iridescence, created by stacks of melanosomes in the feather barbules. The feather colours crucially depend on the nanoscopic dimensions of the melanosome, and the displayed iridescence can distinctly vary, dependent on the spatial organization of the barbs and barbules. We have taken the genus Coeligena as a model group, with species having feathers that strongly vary in their spatial reflection properties. We studied the feather morphology and the optical characteristics. We found that the coloration of Coeligena hummingbirds depends on both the Venetian-blind-like arrangement of the barbules and the V-shaped, angular arrangement of the barbules at opposite sides of the barbs. Both the nanoscopic and microscopic organization of the hummingbird feather components determine the bird''s macroscopic appearance.     

A fourier tool for the analysis of coherent light scattering by bio-optical nanostructures

The fundamental dichotomy between incoherent (phase independent) and coherent (phase dependent) light scattering provides the best criterion for a classification of biological structural color production mechanisms. Incoherent scattering includes Rayleigh, Tyndall, and Mie scattering. Coherent scattering encompasses interference, reinforcement, thin-film reflection, and diffraction. There are three main classes of coherently scattering nanostructures-laminar, crystal-like, and quasi-ordered. Laminar and crystal-like nanostructures commonly produce iridescence, which is absent or less conspicuous in quasi-ordered nanostructures. Laminar and crystal-like arrays have been analyzed with methods from thin-film optics and Bragg's Law, respectively, but no traditional methods were available for the analysis of color production by quasi-ordered arrays. We have developed a tool using two-dimensional (2D) Fourier analysis of transmission electron micrographs (TEMs) that analyzes the spatial variation in refractive index (available from the authors). This Fourier tool can examine whether light scatterers are spatially independent, and test whether light scattering can be characterized as predominantly incoherent or coherent. The tool also provides a coherent scattering prediction of the back scattering reflectance spectrum of a biological nanostructure. Our applications of the Fourier tool have falsified the century old hypothesis that the non-iridescent structural colors of avian feather barbs and skin are produced by incoherent Rayleigh or Tyndall scattering. 2D Fourier analysis of these quasi-ordered arrays in bird feathers and skin demonstrate that these non-iridescent colors are produced by coherent scattering. No other previous examples of biological structural color production by incoherent scattering have been tested critically with either analysis of scatterer spatial independence or spectrophotometry. The Fourier tool is applied here for the first time to coherent scattering by a laminar array from iridescent bird feather barbules (Nectarinia) to demonstrate the efficacy of the technique on thin films. Unlike previous physical methods, the Fourier tool provides a single method for the analysis of coherent scattering by a diversity of nanostructural classes. This advance will facilitate the study of the evolution of nanostructural classes from one another and the evolution of nanostructure itself. The article concludes with comments on the emerging role of photonics in research on biological structural colors, and the future directions in development of the tool.     

Structure and colour-production of the blue barbs of Agapornis roseicollis and Cotinga maynana

Summary The spongy structure in medullary cells responsible for the colour of blue barbs in rump feathers of Agapornis roseicollis and back feathers of Cotinga maynana is studied with the scanning and the transmission electron microscope. The Agapornis structure is an irregular three-dimensional network of connected keratin rods which in many places form rings with outer diameters 0.25–0.3 . The air-filled space likewise consists of an irregular network of connected channels. The Cotinga structure consists of spherical cavities fairly evenly distributed in a keratin matrix.Earlier spectrophotometric measurements on the Agapornis structure have shown that the colour production has to be ascribed to the interference of light, not to Rayleigh (Tyndall) scattering as previously held. The optical path lengths corresponding to the outer diameter of the Agapornis rings and to the Cotinga cavities including their horny walls both are c. 0.4 . It is suggested that the colours are produced by the interference of light reflected from the front and rear surfaces of the rings, respectively the spherical cavities.I wish to thank Miss Annelise Nørgaard Jensen, Institute of Historical Geology and Palaeontology, University of Copenhagen, for operating the scanning electron microscope for me.     

How are feathers digested by raptors?

In order to determine the cause of the evident degradation of feathers from ingested prey in pellets regurgitated by raptors, in vitro digestions of whole feather barbs by pellet extracts, pepsin or trypsin were carried out. The material was analysed by using biochemical and electron microscopic methods. The results show that the changes in the feathers which occur in the stomach of the Falconidae do not arise from digestion of keratin but from hydrolysis of protein acting as a cement matter in the feather.     

What makes a feather shine? A nanostructural basis for glossy black colours in feathers

Colours in feathers are produced by pigments or by nanostructurally organized tissues that interact with light. One of the simplest nanostructures is a single layer of keratin overlying a linearly organized layer of melanosomes that create iridescent colours of feather barbules through thin-film interference. Recently, it has been hypothesized that glossy (i.e. high specular reflectance) black feathers may be evolutionarily intermediate between matte black and iridescent feathers, and thus have a smooth keratin layer that produces gloss, but not the layered organization of melanosomes needed for iridescence. However, the morphological bases of glossiness remain unknown. Here, we use a theoretical approach to generate predictions about morphological differences between matte and glossy feathers that we then empirically test. Thin-film models predicted that glossy spectra would result from a keratin layer 110-180 nm thick and a melanin layer greater than 115 nm thick. Transmission electron microscopy data show that nanostructure of glossy barbules falls well within that range, but that of matte barbules does not. Further, glossy barbules had a thinner and more regular keratin cortex, as well as a more continuous underlying melanin layer, than matte barbules. Thus, their quasi-ordered nanostructures are morphologically intermediate between matte black and iridescent feathers, and perceived gloss may be a form of weakly chromatic iridescence.     

Measuring Spatially- and Directionally-varying Light Scattering from Biological Material

Light interacts with an organism''s integument on a variety of spatial scales. For example in an iridescent bird: nano-scale structures produce color; the milli-scale structure of barbs and barbules largely determines the directional pattern of reflected light; and through the macro-scale spatial structure of overlapping, curved feathers, these directional effects create the visual texture. Milli-scale and macro-scale effects determine where on the organism''s body, and from what viewpoints and under what illumination, the iridescent colors are seen. Thus, the highly directional flash of brilliant color from the iridescent throat of a hummingbird is inadequately explained by its nano-scale structure alone and questions remain. From a given observation point, which milli-scale elements of the feather are oriented to reflect strongly? Do some species produce broader "windows" for observation of iridescence than others? These and similar questions may be asked about any organisms that have evolved a particular surface appearance for signaling, camouflage, or other reasons.In order to study the directional patterns of light scattering from feathers, and their relationship to the bird''s milli-scale morphology, we developed a protocol for measuring light scattered from biological materials using many high-resolution photographs taken with varying illumination and viewing directions. Since we measure scattered light as a function of direction, we can observe the characteristic features in the directional distribution of light scattered from that particular feather, and because barbs and barbules are resolved in our images, we can clearly attribute the directional features to these different milli-scale structures. Keeping the specimen intact preserves the gross-scale scattering behavior seen in nature. The method described here presents a generalized protocol for analyzing spatially- and directionally-varying light scattering from complex biological materials at multiple structural scales.     

A New Helical Crossed-Fibre Structure of β-Keratin in Flight Feathers and Its Biomechanical Implications

The feather aerofoil is unequalled in nature. It is comprised of a central rachis, serial paired branches or barbs, from which arise further branches, the barbules. Barbs and barbules arise from the significantly thinner lateral walls (the epicortex) of the rachis and barbs respectively, as opposed to the thicker dorsal and ventral walls (the cortex). We hypothesized a microstructural design of the epicortex that would resist the vertical or shearing stresses. The microstructures of the cortex and epicortex of the rachis and barbs were investigated in several bird species by microbe-assisted selective disassembly and conventional methods via scanning electron microscopy. We report, preeminent of the finds, a novel system of crossed fibres (ranging from ∼100–800 nm in diameter), oppositely oriented in alternate layers of the epicortex in the rachis and barbs. It represents the first cross-fibre microstructure, not only for the feather but in keratin per se. The cortex of the barbs is comprised of syncitial barbule cells, definitive structural units shown in the rachidial cortex in a related study. The structural connection between the cortex of the rachis and barbs appears uninterrupted. A new model on feather microstructure incorporating the findings here and in the related study is presented. The helical fibre system found in the integument of a diverse range of invertebrates and vertebrates has been implicated in profound functional strategies, perhaps none more so potentially than in the aerofoil microstructure of the feather here, which is central to one of the marvels of nature, bird flight.     

Retinoic acid induction of featherlike structures from reticulate scales

Retinoic acid-induced transformation of reticulate scales to feather-like structures (Dhouailly and Hardy, '78) provides a useful model to study biochemical differentiation in avian skin. In this study, immunofluorescent analysis of reticulate scale-feathers (RSFs) indicates that they contain beta keratin in feather barbs and, thus, are true feathers, biochemically. Epidermal cells that would otherwise produce only alpha keratin in reticulate scales are induced to reorganize and differentiate into barb ridge cells that accumulate feather beta keratins. The mechanism for these dramatic morphological and biosynthetic responses to retinoic acid is unknown.     

Thermal behavior of fowl feather keratin

Differential scanning calorimetry (DSC) was applied to elucidate the thermal behavior of fowl feather keratins (barbs, rachis, and calamus) with different morphological features. The DSC curves exhibited a clear and relatively large endothermic peak at about 110-160 degrees C in the wet condition. A considerable decrease in transition temperature with urea and its helical structure content estimated by Fourier transform infrared spectroscopy (FT-IR), and the disappearance of one of the diffraction peaks with heating at 160 degrees C for 30 min, indicated that DSC could be used to evaluate the thermal behavior of keratin. Barbs showed a lower denaturation temperature than rachis and calamus. The pulverized samples showed a slightly higher denaturation temperature than the native samples. In the dry condition, thermal transition occurred in a markedly higher temperature region close to 170-200 degrees C. It is hence concluded that fowl feather keratins have very high thermal stability, and that the elimination of water brings about even greater thermal stability.     

Cell organization of barb ridges in regenerating feathers of the quail: implications of the elongation of barb ridges for the evolution and diversification of feathers

This ultrastructural study on the regenerating feathers of quail describes the cellular organization of the barb ridges responsible for the ramification of adult feathers. Bilateral symmetry of the barb ridges determines the organization of feather cells into feather branching. The length of the barb ridges, derived from the number of cells associated to form the barbule plates, determines the length of the barbule branching. Long chains of barb cells form long barbs that branch from the rachis with an increase of feather size. Supportive cells function as spacers between the barbule cells. New cells derive from stem cells localized in the collar region of the feather follicle, as indicated from the re‐organization of collar cells into barb ridges (a morphogenetic process inherited from that of embryonic feathers), production of an embryonic type of keratin (feather keratin), permanence of periderm granules (typical embryonic organelles) in barb vane ridge cells. Variations in the process of barb ridge morphogenesis allow the fusion of ridges into a rachis. The differentiation of hooklets contributes to the origin of planar feathers. Separation between rachis and merging barb ridges is by supportive cells, derived from the marginal plates of the barb ridges. Speculations on the evolution and diversification of feathers are presented.     

An ultrastructural study of the regenerating breast feather of the fowl

The developmental morphology of regenerating male breast feathers of the jungle fowl was studied at the ultrastructural level. The process of keratinization was observed in the three types of cells which form feather barbs: barbule cells, cortical cells, and medulla cells. Keratinization first became evident in the barbule cells and resembled the process of keratinization as observed in hair cortical cells and embryonic down feathers. Eventually the whole cytoplasmic area of the barbule cell was occupied by keratin. The barb cortex cells became keratinized in a similar fashion as the barbule cells but not until they were developmentally twice as old as the barbule cells. When keratinization was complete in these cells, the keratin was in the form of large agglomerates scattered in the cytoplasm. The barb medulla cells showed no obvious signs of keratinization until they were developmentally three times as old as the barbule cells. Keratin filament bundles were first seen near the plasma membranes of the medulla cells. Large empty vacuoles appeared in the cytoplasm which also contained moderate amounts of glycogen.     

Theory of the development of curved barbs and their effects on feather morphology

Feathers exhibit an extraordinary diversity of shapes, which are used by birds to accomplish a diverse set of functions. Pennaceous feathers have a double branched morphology that develops from a tube of epidermis, and variation in branch geometry determines feather shape. Feather development is both complex (i.e., a simple developmental modification can have multiple effects on mature feather shape), and redundant (i.e., different developmental modifications can create the same shape). Due to this, it is not readily apparent how different feather shapes develop. In many feathers, barbs are not straight, but instead curve in toward, or away, from the feather tip. Barb curvature can affect the shape of mature feathers but the development of curved barbs is unknown. Previous research has hypothesized that barb curvature could develop either during the helical growth of barb ridges in the tubular feather germ, or during barb angle expansion as the feather unfurls from the sheath. To better understand the development of curved barbs and their effects on mature feathers we present a theoretical model of curved barb development and test the model with empirical investigations of feathers. We find that curved barbs affect many aspects of feather morphology including vane width, barb length, and barb spacing. In real feathers, curved barbs can develop both during helical barb ridge growth and during barb angle expansion, with most of the observed curvature due to barb angle expansion. Our results demonstrate that barb angle expansion as a feather unfurls from the sheath is a complex and dynamic process that plays an important role in determining the shape and structure of mature feathers. Curved barbs create heterogeneity in barb geometry within the feather vane, which could have important implications for aerodynamic function and the development of within feather pigmentation patterns. J. Morphol. 277:995–1013, 2016. © 2016 Wiley Periodicals, Inc.     

Sexual dichromatism,size dimorphism,and microscale anatomy of white wing stripe in blue tits

Achromatic patches are a common element of plumage patterns in many bird species and there is growing body of evidence that in many avian taxa they can play a signaling role in mate choice. Although the blue tit Cyanistes caeruleus is a well-established model species in the studies on coloration, its white wing patch has never been examined in the context of sex-specific trait expression. In this exploratory study, we examined sexual size dimorphism and dichromatism of greater covert’s dots creating white wing patch and analyzed its correlations with current body condition and crown coloration—a trait with established role in sexual selection. Further, we qualitatively analyzed microstructural barb morphology underlying covert’s coloration. We found significant sexual dimorphism in the dot size independent of covert size and sexual dichromatism in both white dot and blue outer covert’s vane spectral characteristics. Internal structure of covert barbs within the white dot was similar to the one found in barbs from the blue part that is, with a medullary area consisting of dead keratinocytes containing channel-type ß-keratin spongy nanostructure and centrally located air cavities. However, it lacked melanosomes which was the main observed difference. Importantly, UV chroma of covert’s blue vane was positively correlated with crown UV chroma and current condition (the latter only in males), which should be a premise for further research on the signal function of the wing stripe.     

Lack of melanized keratin and barbs that fall off: how the racketed tail of the turquoise-browed motmot Eumomota superciliosa is formed

The racket-tipped tail of the motmots is uniquely shaped and its formation has attracted much attention. Barbs that grow along the wire of the motmot's two central tail feathers are weakly attached and shed soon after development. The cause of the weak attachment of these barbs is unclear. I induced feather growth by plucking the central tail feathers from seven turquoise-browed motmots Eumomota superciliosa and then collected the regrown feathers before the barbs along the wire had fully shed. I compared the barb-rachis junction (petiole of the ramus) along the distal flag (the racket-tip of the tail) where barbs are not shed, to the barb-rachis junction along the wire where barbs would later be shed. In these two regions, I examined the size and structure of the attachment of the barb to the rachis with a scanning electron microscope (SEM). I also used a light microscope to score the grayness of the proximal rami of these two regions to estimate the amount of melanized keratin. SEM imaging showed that the barbs are attached to the rachis with a larger supporting flange along the distal flag compared to along the wire. Images from a light microscope showed that the rami along the distal flag were black, whereas rami along the wire were translucent or gray. The lower gray-scale color score of the rami along the wire is likely due to reduced melanized keratin. These data suggest that that the barbs along the wire are weakly attached due to a combination of a reduced structural attachment and a lack of structurally enhancing melanin.     

Diffuse reflectance spectroscopy of fibrous proteins

UV-visible diffuse reflectance (DR) spectra of the fibrous proteins wool and feather keratin, silk fibroin and bovine skin collagen are presented. Natural wool contains much higher levels of visible chromophores across the whole visible range (700-400?nm) than the other proteins and only those above 450?nm are effectively removed by bleaching. Both oxidative and reductive bleaching are inefficient for removing yellow chromophores (450-400?nm absorbers) from wool. The DR spectra of the four UV-absorbing amino acids tryptophan, tyrosine, cystine and phenylalanine were recorded as finely ground powders. In contrast to their UV-visible spectra in aqueous solution where tryptophan and tyrosine are the major UV absorbing species, surprisingly the disulphide chromophore of solid cystine has the strongest UV absorbance measured using the DR remission function F(R)(∞). The DR spectra of unpigmented feather and wool keratin appear to be dominated by cystine absorption near 290?nm, whereas silk fibroin appears similar to tyrosine. Because cystine has a flat reflectance spectrum in the visible region from 700 to 400?nm and the powder therefore appears white, cystine absorption does not contribute to the cream colour of wool despite the high concentration of cystine residues near the cuticle surface. The disulphide absorption of solid L: -cystine in the DR spectrum at 290?nm is significantly red shifted by ~40?nm relative to its wavelength in solution, whereas homocystine and lipoic acid showed smaller red shifts of 20?nm. The large red shift observed for cystine and the large difference in intensity of absorption in its UV-visible and DR spectra may be due to differences in the dihedral angle between the crystalline solid and the solvated molecules in solution.     

Morphological basis of glossy red plumage colours

Brightly coloured feathers, including the brilliant reds produced by carotenoids, are sometimes shiny in appearance. Gloss is a common property of materials and usually arises through specular reflection from smooth, flat surfaces. However, the production of gloss on red feathers has never been examined. In the present study, we compared the optical and structural properties of glossy and matte carotenoid‐based red feathers of multiple species to identify the proximate basis for their glossiness. Although specular reflectance did not differ between glossy and matte feathers, diffuse reflectance was lower in glossy than in matte feathers, leading to a higher contrast gloss. Compared to matte feathers, glossy red feathers had thicker barbs with a flatter and more homogeneous morphology, consistent with expectations, as well as thicker outer keratin cortices. Moreover, glossiness was predicted by a principal component regression using these same morphological traits. We demonstrate that the gloss of carotenoid‐based red feathers is produced at least in part by a smooth, flattened barb microstructure and an enhanced nanostructure, illustrating a novel colour‐producing interaction that neither pigment, nor microstructure could alone attain. How the ecology and evolution of species with glossy red feather differ from those with typical matte red feathers represent rich areas for future study.