Evolutionary shifts in the melanin‐based color system of birds

Melanin pigments contained in organelles (melanosomes) impart earthy colors to feathers. Such melanin‐based colors are distributed across birds and thought to be the ancestral color‐producing mechanism in birds. However, we have had limited data on melanin‐based color and melanosome diversity in Palaeognathae, which includes the flighted tinamous and large‐bodied, flightless ratites and is the sister taxon to all other extant birds. Here, we use scanning electron microscopy and spectrophotometry to assess melanosome morphology and quantify reflected color for 19 species within this clade. We find that brown colors in ratites are uniquely associated with elongated melanosomes nearly identical in shape to those associated with black colors. Melanosome and color diversity in large‐bodied ratites is limited relative to other birds (including flightless penguins) and smaller bodied basal maniraptoran dinosaur outgroups of Aves, whereas tinamous show a wider range of melanosome forms similar to neognaths. The repeated occurrence of novel melanosome forms in the nonmonophyletic ratites suggests that melanin‐based color tracks changes in body size, physiology, or other life history traits associated with flight loss, but not feather morphology. We further anticipate these findings will be useful for future color reconstructions in extinct species, as variation in melanosome shape may potentially be linked to a more nuanced palette of melanin‐based colors.     

Melanosome diversity and convergence in the evolution of iridescent avian feathers—Implications for paleocolor reconstruction

Some of the most varied colors in the natural world are created by iridescent nanostructures in bird feathers, formed by layers of melanin‐containing melanosomes. The morphology of melanosomes in iridescent feathers is known to vary, but the extent of this diversity, and when it evolved, is unknown. We use scanning electron microscopy to quantify the diversity of melanosome morphology in iridescent feathers from 97 extant bird species, covering 11 orders. In addition, we assess melanosome morphology in two Eocene birds, which are the stem lineages of groups that respectively exhibit hollow and flat melanosomes today. We find that iridescent feathers contain the most varied melanosome morphologies of all types of bird coloration sampled to date. Using our extended dataset, we predict iridescence in an early Eocene trogon (cf. Primotrogon) but not in the early Eocene swift Scaniacypselus, and neither exhibit the derived melanosome morphologies seen in their modern relatives. Our findings confirm that iridescence is a labile trait that has evolved convergently in several lineages extending down to paravian theropods. The dataset provides a framework to detect iridescence with more confidence in fossil taxa based on melanosome morphology.     

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.     

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.     

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.     

How hollow melanosomes affect iridescent colour production in birds

Developmental constraints and trade-offs can limit diversity, but organisms have repeatedly evolved morphological innovations that overcome these limits by expanding the range and functionality of traits. Iridescent colours in birds are commonly produced by melanin-containing organelles (melanosomes) organized into nanostructured arrays within feather barbules. Variation in array type (e.g. multilayers and photonic crystals, PCs) is known to have remarkable effects on plumage colour, but the optical consequences of variation in melanosome shape remain poorly understood. Here, we used a combination of spectrophotometric, experimental and theoretical methods to test how melanosome hollowness—a morphological innovation largely restricted to birds—affects feather colour. Optical analyses of hexagonal close-packed arrays of hollow melanosomes in two species, wild turkeys (Meleagris gallopavo) and violet-backed starlings (Cinnyricinclus leucogaster), indicated that they function as two-dimensional PCs. Incorporation of a larger dataset and optical modelling showed that, compared with solid melanosomes, hollow melanosomes allow birds to produce distinct colours with the same energetically favourable, close-packed configurations. These data suggest that a morphological novelty has, at least in part, allowed birds to achieve their vast morphological and colour diversity.     

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.     

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.     

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.     

Morphological changes in hair melanosomes by aging

Various changes appear in hair by aging, and graying is the most remarkable one. Changes in melanocytes have been well studied as the cause; however, little is known about the change in melanosomes which have a role of carrying melanin pigments into hair shafts. Using pigmented hairs of Japanese females from their age of 4–75, I isolated melanosomes and observed them. As a result, I found a significant change in the morphology of hair melanosomes with age. They were ellipsoidal on the whole and there was no age dependence in the major axis, while the minor axis significantly increased and its frequency distribution broadened with age. The anticipated volume of the melanosome of the oldest person hairs was about twice larger than that of child hairs. This enlargement of melanosome seems to be a cause of the age‐related color change in pigmented hairs from brown to black.     

Warning Signal Efficacy: Assessing the Effects of Color,Iridescence, and Time of Day in the Field

Warning coloration deters predators from attacking distasteful or toxic prey. Signal features that influence warning color effectiveness are not well understood, and in particular, we know very little about how effective short‐wavelength and iridescent colors are as warning color elements in nature and how warning signal effectiveness changes throughout the day. We tested the effect of these factors on predation risk in nature using specimens of the distasteful pipevine swallowtail butterfly, Battus philenor. B. philenor adults display both iridescent blue and diffusely reflecting orange components in their warning signal. We painted B. philenor wings to create five different model types: all‐black, only‐iridescent‐blue, only‐orange, iridescent‐blue‐and‐orange (intact signal), and matte‐blue‐and‐orange. We placed 25 models in each of 14 replicate field sites for 72 h and checked for attacks three times each day. Model type affected the likelihood of attack; only‐orange models were, the only model attacked significantly less than the all‐black model. Iridescence did not enhance or decrease warning signal effectiveness in our experiment because matte‐blue‐and‐orange models were attacked at the same rate as iridescent‐blue‐and‐orange models. Time of day did not differentially affect model type. Video recordings of attacks revealed that insectivorous birds were responsible. The results of this experiment, when taken with previous work, indicate that the response to blue warning coloration is likely dependent on predator experience and context, but that iridescence per se does not affect warning signals in a natural context.     

How are proliferation and differentiation of melanocytes regulated?

Coat colors are determined by melanin (eumelanin and pheomelanin). Melanin is synthesized in melanocytes and accumulates in special organelles, melanosomes, which upon maturation are transferred to keratinocytes. Melanocytes differentiate from undifferentiated precursors, called melanoblasts, which are derived from neural crest cells. Melanoblast/melanocyte proliferation and differentiation are regulated by the tissue environment, especially by keratinocytes, which synthesize endothelins, steel factor, hepatocyte growth factor, leukemia inhibitory factor and granulocyte-macrophage colony-stimulating factor. Melanocyte differentiation is also stimulated by alpha-melanocyte stimulating hormone; in the mouse, however, this hormone is likely carried through the bloodstream and not produced locally in the skin. Melanoblast migration, proliferation and differentiation are also regulated by many coat color genes otherwise known for their ability to regulate melanosome formation and maturation, pigment type switching and melanosome distribution and transfer. Thus, melanocyte proliferation and differentiation are not only regulated by genes encoding typical growth factors and their receptors but also by genes classically known for their role in pigment formation.     

Keratinocyte-melanocyte interactions during melanosome transfer

The epidermal-melanin unit is composed of one melanocyte and approximately 36 neighboring keratinocytes, working in synchrony to produce and distribute melanin. Melanin is synthesized in melanosomes, transferred to the dendrite tips, and translocated into keratinocytes, forming caps over the keratinocyte nuclei. The molecular and cellular mechanisms involved in melanosome transfer and the keratinocyte-melanocyte interactions required for this process are not yet completely understood. Suggested mechanisms of melanosome transfer include melanosome release and endocytosis, direct inoculation ('injection'), keratinocyte-melanocyte membrane fusion, and phagocytosis. Studies of the keratinocyte receptor protease-activated receptor-2 (PAR-2) support the phagocytosis theory. PAR-2 controls melanosome ingestion and phagocytosis by keratinocytes and exerts a regulatory role in skin pigmentation. Modulation of PAR-2 activity can enhance or decrease melanosome transfer and affects pigmentation only when there is keratinocyte-melanocyte contact. Moreover, PAR-2 is induced by UV irradiation and inhibition of PAR-2 activation results in the prevention of UVB-induced tanning. The role of PAR-2 in mediating UV-induced responses remains to be elucidated.     

Variation in human hair ultrastructure among three biogeographic populations

Human scalp hairs are often examined microscopically to study the variation and diversity among a range of visible morphological traits. In this study, we focused on the ultrastructure of human scalp hair within its keratinized matrix, emphasizing, the density and distribution of melanosomes, variation in cuticle thickness within populations, and the relationship of hair fiber ultrastructure with biogeographic ancestry. We used transmission electron microscopy (TEM) to visualize hair cross-sections and generate micron-scale resolution images for analysis of particle morphology and the layered hair matrix. Our results revealed considerable variation in all parameters examined, including the relationship of ultrastructure to biogeographic ancestry. Among the three metapopulations studied (European, African, and East Asian), we identified hair cross-sectional shape, cuticle dimensions, and melanosome distribution as traits that reveal statistically significant ancestry-related patterns. This study establishes trait patterns in hair morphology and ultrastructure among three biogeographically defined metapopulations to improve the current understanding of human variation in hair form and establish a foundation for future studies on the genetic and developmental bases of phenotypic variation in hair ultrastructure related to genotype.     

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.     

Keratinocyte–Melanocyte Interactions During Melanosome Transfer

The epidermal–melanin unit is composed of one melanocyte and approximately 36 neighboring keratinocytes, working in synchrony to produce and distribute melanin. Melanin is synthesized in melanosomes, transferred to the dendrite tips, and translocated into keratinocytes, forming caps over the keratinocyte nuclei. The molecular and cellular mechanisms involved in melanosome transfer and the keratinocyte–melanocyte interactions required for this process are not yet completely understood. Suggested mechanisms of melanosome transfer include melanosome release and endocytosis, direct inoculation (‘injection’), keratinocyte–melanocyte membrane fusion, and phagocytosis. Studies of the keratinocyte receptor protease‐activated receptor‐2 (PAR‐2) support the phagocytosis theory. PAR‐2 controls melanosome ingestion and phagocytosis by keratinocytes and exerts a regulatory role in skin pigmentation. Modulation of PAR‐2 activity can enhance or decrease melanosome transfer and affects pigmentation only when there is keratinocyte–melanocyte contact. Moreover, PAR‐2 is induced by UV irradiation and inhibition of PAR‐2 activation results in the prevention of UVB‐induced tanning. The role of PAR‐2 in mediating UV‐induced responses remains to be elucidated.     

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 method for quantifying melanosome transfer efficacy from melanocytes to keratinocytes in vitro

Several different in vivo and in vitro bioassays are used to evaluate melanosome transfer efficacy from melanocytes to keratinocytes. However, these methods are complicated and time consuming. Here, we report on a simple, rapid, direct, and reliable in vitro method for observing the process of melanosome transfer from melanocytes to keratinocytes. First, we selected and tested a melanoma cell line RPMI-7951 that can normally synthesize melanin and transfer from mature melanosomes to keratinocytes in vitro. We cocultured these cells with a human ovarian teratoma transformed epidermal carcinoma cell line, which is also capable of accepting melanosomes transferred from melanocytes, as in normal keratinocytes. The cells were cocultured for 24-72 h and double labeled with FITC-conjugated antibody against the melanosome-associated protein TRP-1, and with Cy5-conjugated antibody against the keratinocyte-specific marker keratin 14. The cells were examined by fluorescence microscope and flow cytometry. Melanosome transfer from melanocytes to keratinocytes increased in a time-dependent manner. To verify the accessibility of this method, the melanosome transfer inhibitor, a serine protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, and a melanosome transfer stimulator, alpha-melanocyte-stimulating hormone, were added. The serine protease inhibitor decreased melanosome transfer, and alpha-melanocyte-stimulating hormone increased melanosome transfer, in a dose-dependent manner. In conclusion, this is a simple, rapid, and effective model system to quantify the melanosome transfer efficacy from melanocytes to keratinocytes in vitro.     

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.