Colour-producing β-keratin nanofibres in blue penguin (Eudyptula minor) feathers

The colours of living organisms are produced by the differential absorption of light by pigments (e.g. carotenoids, melanins) and/or by the physical interactions of light with biological nanostructures, referred to as structural colours. Only two fundamental morphologies of non-iridescent nanostructures are known in feathers, and recent work has proposed that they self-assemble by intracellular phase separation processes. Here, we report a new biophotonic nanostructure in the non-iridescent blue feather barbs of blue penguins (Eudyptula minor) composed of parallel β-keratin nanofibres organized into densely packed bundles. Synchrotron small angle X-ray scattering and two-dimensional Fourier analysis of electron micrographs of the barb nanostructure revealed short-range order in the organization of fibres at the appropriate size scale needed to produce the observed colour by coherent scattering. These two-dimensional quasi-ordered penguin nanostructures are convergent with similar arrays of parallel collagen fibres in avian and mammalian skin, but constitute a novel morphology for feathers. The identification of a new class of β-keratin nanostructures adds significantly to the known mechanisms of colour production in birds and suggests additional complexity in their self-assembly.     

Modular color evolution facilitated by a complex nanostructure in birds

The way in which a complex trait varies, and thus evolves, is critically affected by the independence, or modularity, of its subunits. How modular designs facilitate phenotypic diversification is well studied in nonornamental (e.g., cichlid jaws), but not ornamental traits. Diverse feather colors in birds are produced by light absorption by pigments and/or light scattering by nanostructures. Such structural colors are deterministically related to the nanostructures that produce them and are therefore excellent systems to study modularity and diversity of ornamental traits. Elucidating if and how these nanostructures facilitate color diversity relies on understanding how nanostructural traits covary, and how these traits map to color. Both of these remain unknown in an evolutionary context. Most dabbling ducks (Anatidae) have a conspicuous wing patch with iridescent color caused by a two‐dimensional photonic crystal of small (100–200 nm) melanosomes. Here, we ask how this complex nanostructure affects modularity of color attributes. Using a combination of electron microscopy, spectrophotometry, and comparative methods, we show that nanostructural complexity causes functional decoupling and enables independent evolution of different color traits. These results demonstrate that color diversity is facilitated by how nanostructures function and may explain why some birds are more color‐diverse than others.     

On the blue coloration of vertebrates

Although the various vertebrate classes, from fishes to mammals are each distinctive, they possess many common features making it important to understand their comparative biology. One general feature that has long commanded interest is the integumental pigmentary system. Thus, much is known about particular pigment cells; however, the basis for some specific colors, such as blue, has escaped the scrutiny of the comparative approach. Regardless of Class, blue is almost always a structural color based upon incoherent or coherent scatter of blue wavelengths from the animal surface. The source of scatter may be intracellular or extra-cellular. A main intracellular scatterer is the surface of reflecting platelets of iridophores of lower vertebrates. Extra-cellular scatter is widespread and thought to occur from ordered dermal collagen arrays in primitive fishes, birds and mammals including humans. Among birds, feather structures provide major means for extra-cellular light scatter. There is only one known example of blue color deriving from a blue pigment found within a pigment cell. For amphibians, reptiles and birds, the scatter of blue wavelengths, together with the presence of yellow pigmentation, is fundamental for the expression of green coloration.     

On the blue coloration of vertebrates†

Although the various vertebrate classes, from fishes to mammals are each distinctive, they possess many common features making it important to understand their comparative biology. One general feature that has long commanded interest is the integumental pigmentary system. Thus, much is known about particular pigment cells; however, the basis for some specific colors, such as blue, has escaped the scrutiny of the comparative approach. Regardless of Class, blue is almost always a structural color based upon incoherent or coherent scatter of blue wavelengths from the animal surface. The source of scatter may be intracellular or extra‐cellular. A main intracellular scatterer is the surface of reflecting platelets of iridophores of lower vertebrates. Extra‐cellular scatter is widespread and thought to occur from ordered dermal collagen arrays in primitive fishes, birds and mammals including humans. Among birds, feather structures provide major means for extra‐cellular light scatter. There is only one known example of blue color deriving from a blue pigment found within a pigment cell. For amphibians, reptiles and birds, the scatter of blue wavelengths, together with the presence of yellow pigmentation, is fundamental for the expression of green coloration.     

Physiological color change in squid iridophores

Evidence is presented that changes in the optical properties of active iridophores in the dermis of the squid Lolliguncula brevis are the result of changes in the ultrastructure of these cells. At least two mechanisms may be involved when active cells change from non-iridescent to iridescent or change iridescent color. One is the reversible change of labile, detergent-resistant proteinaceous material within the iridophore platelets, from a contracted gel state (non-iridescent) to an expanded fluid or sol state when the cells become iridescent. The other is a change in the thickness of the platelets, with platelets becoming significantly thinner as the optical properties of the iridophores change from non-iridescent to iridescent red, and progressively thinner still as the observed iridescent colors become those of shorter wavelengths. Optical change from Rayleigh scattering (non-iridescent) to structural reflection (iridescent) may be due to the viscosity change in the platelet material, with the variations in observed iridescent colors due to changes in the dimensions of the iridophore platelets.     

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.     

Structural color change following hydration and dehydration of iridescent mourning dove (Zenaida macroura) feathers

Dynamic changes in integumentary color occur in cases as diverse as the neurologically controlled iridiphores of cephalopod skin and the humidity-responsive cuticles of longhorn beetles. By contrast, feather colors are generally assumed to be relatively static, changing by small amounts only over periods of months. However, this assumption has rarely been tested even though structural colors of feathers are produced by ordered nanostructures that are analogous to those in the aforementioned dynamic systems. Feathers are neither innervated nor vascularized and therefore any color change must be caused by external stimuli. Thus, we here explore how feathers of iridescent mourning doves Zenaida macroura respond to a simple stimulus: addition and evaporation of water. After three rounds of experimental wetting and subsequent evaporation, iridescent feather color changed hue, became more chromatic and increased in overall reflectance by almost 50%. To understand the mechanistic basis of this change, we used electron microscopy to examine macro- and nanostructures before and after treatment. Transmission electron microscopy and transfer matrix thin-film models revealed that color is produced by thin-film interference from a single (∼335 nm) layer of keratin around the edge of feather barbules, beneath which lies a layer of air and melanosomes. After treatment, the most striking morphological difference was a twisting of colored barbules that exposed more of their surface area for reflection, explaining the observed increase in brightness. These results suggest that some plumage colors may be more malleable than previously thought, leading to new avenues for research on dynamic plumage color.     

Two-dimensional Fourier analysis of the spongy medullary keratin of structurally coloured feather barbs

We conducted two-dimensional (2D) discrete Fourier analyses of the spatial variation in refractive index of the spongy medullary keratin from four different colours of structurally coloured feather barbs from three species of bird: the rose-faced lovebird, Agapornis roseicollis (Psittacidae), the budgerigar, Melopsittacus undulatus (Psittacidae), and the Gouldian finch, Poephila guttata (Estrildidae). These results indicate that the spongy medullary keratin is a nanostructured tissue that functions as an array of coherent scatterers. The nanostructure of the medullary keratin is nearly uniform in all directions. The largest Fourier components of spatial variation in refractive index in the tissue are of the appropriate size to produce the observed colours by constructive interference alone. The peaks of the predicted reflectance spectra calculated from the 2D Fourier power spectra are congruent with the reflectance spectra measured by using microspectrophotometry. The alternative physical models for the production of these colours, the Rayleigh and Mie theories, hypothesize that medullary keratin is an incoherent array and that scattered waves are independent in phase. This assumption is falsified by the ring-like Fourier power spectra of these feathers, and the spacing of the scattering air vacuoles in the medullary keratin. Structural colours of avian feather barbs are produced by constructive interference of coherently scattered light waves from the optically heterogeneous matrix of keratin and air in the spongy medullary layer.     

Divisionistic generation of skin hue and the change of shade in the scalycheek damselfish, Pomacentrus lepidogenys

In addition to melanophores and xanthophores, there existed two types of iridophore in the dermis of the scalycheek damselfish, Pomacentrus lepidogenys. There are dendritic iridophores which reflect white light-rays by Tyndall scattering, and the round or somewhat ellipsoidal iridophores which reflect rays with a relatively narrow spectral peak from blue to green through the non-ideal thin-film interference. Most of the dendritic iridophores were covered with xanthophores and were situated over melanophores, thus constituting a kind of chromatophore unit which produces a yellow or yellowish-green color. The characteristic yellowish-green hue of the integument results from a compound effect of small contributions by more elementary colors. During color changes of the skin, the position of the spectral peak does not shift. Unlike the iridophores of the blue damselfish, both types of iridophore of the scalycheek damselfish were found to be inactive. It appears, therefore, that the aggregation and dispersion of pigment within the melanophores is the primary mechanism responsible for the changes in color of this species.     

Structural coloration in a fossil feather

Investigation of feathers from the famous Middle Eocene Messel Oil Shale near Darmstadt, Germany shows that they are preserved as arrays of fossilized melanosomes, the surrounding beta-keratin having degraded. The majority of feathers are preserved as aligned rod-shaped eumelanosomes. In some, however, the barbules of the open pennaceous, distal portion of the feather vane are preserved as a continuous external layer of closely packed melanosomes enclosing loosely aligned melanosomes. This arrangement is similar to the single thin-film nanostructure that generates an iridescent, structurally coloured sheen on the surface of black feathers in many lineages of living birds. This is, to our knowledge, the first evidence of preservation of a colour-producing nanostructure in a fossil feather and confirms the potential for determining colour differences in ancient birds and other dinosaurs.     

Feathers with Ocular Architecture: Implications for Functional and Evolutionary Similarities of Visual Signals and Receptors

Studies of visual receptors typically assume that only functionally similar structures are relevant to the evolution of complex eyes. This approach ignores growing evidence that different functional classes of organs often share structural and developmental patterns that pertain to biological sameness (deep homology). However, the potential relevance of non-receptor structures to eye evolution remains largely unexplored. An “ocular” feather color mechanism is described whose structural and optical features resemble those of chambered, image-forming eyes to a remarkable degree. These similarities include a laterally expanded, domed light receiving surface similar to that of an eye, an encapsulated spongy tissue mass whose coherent light scattering properties in the human-visible (destructive) and ultraviolet (constructive) wavelength ranges resemble those of cornea and lens, intervening spaces such as those with humors, and a laminar pigmented shelf whose structure and optics resemble a mirrored tapetum lucidum found behind many retinas. Fourier analysis and optical principles indicate that ocular structures adhere to the same light-handling properties regardless of higher function (receptor or signal). The extent to which chambered eyes and ocular feathers have evolved independently is surprisingly equivocal. On the one hand, broad differences in the location, composition, and development of chambered eyes and ocular feather signals suggest convergent evolution on an ocular organization. However, some level of evolutionary parallelism (generative homology) between chambered eyes and ocular feathers is implicated by similarities in constructional materials, tissue development, and signal transduction cascades. Structural, optical, and developmental similarities also occur between more primitive eyes and the colored dermal papillae responsible for avian skin ornamentation. Functional constraints on light-handling requirements, coupled with developmental constraints in high-stress environments on the body surface, may enhance the similar evolutionary outcomes in the different functional setting. Regardless of the mechanistic details, repeated evolution of eye-like structures in different functional settings reveals a biological potential to produce such organs that is much greater than would be inferred from a survey of receptor structures alone.     

FINE STRUCTURAL OBSERVATIONS RELATING TO THE PRODUCTION OF COLOR BY THE IRIDOPHORES OF A LIZARD, ANOLIS CAROLINENSIS

This paper presents the results of light and electron microscopy done on iridophores in the dorsal skin of the lizard Anolis carolinensis. New fine-structural details are revealed, and their importance is discussed. Of some interest is the complex of filaments between crystalline sheets in the cell. It is proposed that this complex is involved in the arrangement of crystals into crystalline sheets, and that the crystal arrangement and spacing are critical for the production of the cells' blue-green color. Tyndall scattering and thin-film interference are discussed as possible explanations for iridophore color production in relation to the fine-structural data obtained.     

Exceptional three-dimensional preservation and coloration of an originally iridescent fossil feather from the Middle Eocene Messel Oil Shale

A feather from the Eocene Messel Formation, Germany, has been demonstrated to have been originally structurally colored by densely packed sheets of melanosomes similar to modern iridescent feathers exhibiting thin-film diffraction. The fossil itself currently exhibits a silvery sheen, but the mechanism for generating this optical effect was not fully understood. Here we use scanning electron microscopy, electron probe microanalysis, and dual-beam focused ion beam scanning electron microscopy to investigate the source of the silvery sheen that occurs in the apical feather barbules. Focused ion beam scanning electron microscopy provides a powerful tool for studying three-dimensionality of nanostructures in fossils. Use of the method reveals that the flattened apical barbules are preserved almost perfectly, including smooth structural melanosome sheets on the obverse surface of the fossil feather that are identical to those that cause iridescence in modern bird feathers. Most of each apical barbule is preserved beneath a thin layer of sediment. The silvery sheen is generated by incoherent light diffraction between this sediment layer and melanosomes and, although related to the original iridescence of the feather, is not a feature of the feather itself. The reddish and greenish hues frequently exhibited by fossil feathers from the Messel Formation appear to be due to precipitates on the surface of individual melanosomes.     

Evolution of plumage coloration in the crow family (Corvidae) with a focus on the color-producing microstructures in the feathers: a comparison of eight species

Plumage coloration has been the subject for a variety of questions that comprise the center of modern evolutionary biology. Unlike carotenoids that the concentration directly influences the intensity of the color, melanin, in addition to produce brown or black colors, is often involved in producing the structural coloration such as glossiness or iridescence. As the melanin granules can be located in the barbs or the barbules, we aim to (i) discern if the colors observed at macro scale comes from the barbs, the barbules or both in a series of related species and (ii) estimate the evolutionary history of the color-producing mechanisms in the family Corvidae that are known to have melanin-based coloration. From a preliminary comparative analysis on eight representative species, we found three coloration schemes in Corvidae; (1) matte colors of brown or black that were produced in barbs and barbules; (2) non-iridescent structural colors such as blue, bluish gray and white, that were produced in the barbs and (3) iridescent structural colors that were produced only in distal barbules. Comparative character analysis of these coloration schemes suggests that the ancestral state among these species were the colors produced in the barbs and that the color produced in the distal barbules is a derived character. The evolution of iridescence seems tightly linked to the evolution of the colors produced in the distal barbules. Data from more species should be incorporated in order to grasp a full picture on the evolutionary history of plumage coloration in this group of birds.     

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.     

Proximate bases of silver color in anhinga (Anhinga anhinga) feathers

Colors of living organisms are produced by selective light absorption from pigments and/or by light scattering from highly ordered nanostructures (i.e., structural color). While the physical bases of metallic colors of arthropods and fish are fairly well‐known, those of birds are not. Here we examine structurally based silver color and its production in feathers of the waterbird species Anhinga. This achromatic color is distinguished from grey by high specular reflectance, from white by low diffuse reflectance, and from both by high gloss. Light and electron microscopy revealed three modifications of feathers likely leading to silver color. First, proximal barbules were highly elongated and contained glossy black color at their base and white color at their pennulum. Second, this glossy black portion contained a single outer layer of keratin weakly bounded by melanosomes. Finally, the white portion contained a disordered amorphous matrix of keratin and air. Optical analyzes suggest that these structures produce, respectively, glossy black color through thin‐film interference and white color through incoherent light scattering. Silver color likely results from the combined reflectance of these adjacent structures. This represents a distinct mechanism for attaining silver colors that may have been partially derived through selection for display, thermoregulation or decreased hydrophobicity. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.     

Fossilized biophotonic nanostructures reveal the original colors of 47-million-year-old moths

Structural colors are generated by scattering of light by variations in tissue nanostructure. They are widespread among animals and have been studied most extensively in butterflies and moths (Lepidoptera), which exhibit the widest diversity of photonic nanostructures, resultant colors, and visual effects of any extant organism. The evolution of structural coloration in lepidopterans, however, is poorly understood. Existing hypotheses based on phylogenetic and/or structural data are controversial and do not incorporate data from fossils. Here we report the first example of structurally colored scales in fossil lepidopterans; specimens are from the 47-million-year-old Messel oil shale (Germany). The preserved colors are generated by a multilayer reflector comprised of a stack of perforated laminae in the scale lumen; differently colored scales differ in their ultrastructure. The original colors were altered during fossilization but are reconstructed based upon preserved ultrastructural detail. The dorsal surface of the forewings was a yellow-green color that probably served as a dual-purpose defensive signal, i.e. aposematic during feeding and cryptic at rest. This visual signal was enhanced by suppression of iridescence (change in hue with viewing angle) achieved via two separate optical mechanisms: extensive perforation, and concave distortion, of the multilayer reflector. The fossils provide the first evidence, to our knowledge, for the function of structural color in fossils and demonstrate the feasibility of reconstructing color in non-metallic lepidopteran fossils. Plastic scale developmental processes and complex optical mechanisms for interspecific signaling had clearly evolved in lepidopterans by the mid-Eocene.     

Ontogeny of an iridescent nanostructure composed of hollow melanosomes

Iridescent colors in feathers are some of the brightest in nature, and are produced by coherent light scattering from periodic arrangements of melanosomes (melanin‐containing organelles). Hollow melanosomes, an evolutionary innovation largely restricted to birds, contain an optically powerful combination of high and low refractive indices (from the melanin and air, respectively) that enables production of brighter and more saturated colors than solid melanosomes. However, despite their significance to avian color and potential utility as optical biomaterials, little is known about the ontogeny of either the melanosomes themselves or the nanostructures they comprise. We used light and electron microscopy to characterize nanostructural development in regenerating feathers of wild turkeys, a species with iridescent color produced by a hexagonally close‐packed array of hollow melanosomes. We found that melanosomes form as solid bodies in melanocytes. Later in development, largely after placement in developing barbules, their interiors dissolve and leave hollow cores. These now hollow melanosomes are initially disorganized in the barbule, but become close‐packed as they are pulled to the edge of the barbule, likely through a combination of forces including depletion–attraction. These data suggest that these structurally colored tissues are self‐assembled and represent novel pathways of development. J. Morphol. 276:378–384, 2015. © 2014 Wiley Periodicals, Inc.     

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.     

Dual structural color mechanisms in a scarab beetle

The cuticle of a Mycterophallus cetoniine scarab species displays both red iridescence due to a multilayer reflector mechanism and rainbow iridescence due to a superimposed diffraction grating mechanism. This is the first reported example of an animal possessing two independent classes of structural colors arising from interference at the wavelengths of visible light. In this work, the Mycterophallus cuticle is characterized by light microscopy, spectrophotometry, scanning electron microscopy, and transmission electron microscopy. We compare the cuticle of the Mycterophallus species to two closely related Lomaptera scarab species, one with only a multilayer reflector and the second with only a diffraction grating. We calculate the correspondence between the nanostructural parameters and the optical properties of the Mycterophallus cuticle to determine the relative optical contributions of the two color mechanisms and the interactions between them. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.