Nanoscale millefeuilles produce iridescent bill ornaments in birds.

Colors are well studied in bird plumage but not in other integumentary structures. In particular, iridescent colors from structures other than plumage are undescribed in birds. Here, we show that a multilayer of keratin and lipids is sufficient to produce the iridescent bill of Spermophaga haematina. Furthermore, that the male bill is presented to the female under different angles during display provides support for the hypothesis that iridescence evolved in response to sexual selection. This is the first report of an iridescent bill, and only the second instance of iridescence in birds in which melanosomes are not involved. Furthermore, an investigation of museum specimens of an additional 98 species, showed that this evolved once, possibly twice. These results are promising, as they suggest that birds utilize a wider array of physical phenomena to produce coloration and should further stimulate research on nonplumage integumentary colors.

The evolution of multiple colour mechanisms is correlated with diversification in sunbirds (Nectariniidae).

How and why certain groups become speciose is a key question in evolutionary biology. Novel traits that enable diversification by opening new ecological niches are likely important mechanisms. However, ornamental traits can also promote diversification by opening up novel sensory niches and thereby creating novel inter-specific interactions. More specifically, ornamental colours may enable more precise and/or easier species recognition, and may act as key innovations by increasing the number of species-specific patterns and promoting diversification. While the influence of colouration on diversification is well-studied, the influence of the mechanisms that produce those colours (e.g. pigmentary, nanostructural) is less so, even though the ontogeny and evolution of these mechanisms differ. We estimated a new phylogenetic tree for 121 sunbird species and combined colour data of 106 species with a range of phylogenetic tools to test the hypothesis that the evolution of novel colour mechanisms increases diversification in sunbirds, one of the most colourful bird clades. Results suggest that (1) the evolution of novel colour mechanisms expands the visual sensory niche, increasing the number of achievable colours. (2) Structural colouration diverges more readily across the body than pigment-based colouration, enabling an increase in colour complexity. (3) Novel colour mechanisms might minimize trade-offs between natural and sexual selection such that colour can function both as camouflage and conspicuous signal. (4) Despite structural colours being more colourful and mobile, only melanin-based colouration is positively correlated with net diversification. Together, these findings explain why colour distances increase with increasing number of sympatric species, even though packing of colour space would predict otherwise.

Back in black: melanin-rich skin colour associated with increased net diversification rates in birds.

Evolutionary biologists have long been interested in understanding the factors that promote diversification in organisms, often focussing on distinct and/or conspicuous phenotypes with direct effects on natural or sexual selection such as body size and plumage coloration. However, multiple traits that potentially influence net diversification are not conspicuous and/or might be concealed. One such trait, the dark, melanin-rich skin concealed beneath the feathers, evolved more than 100 times during avian evolution, frequently in association with white feathers on the crown and UV-rich environments, suggesting that it is a UV-photoprotective adaptation. Furthermore, multiple species are polymorphic, having both light and dark skin potentially aiding occupation in different UV radiation environments. As such these polymorphisms are predicted to occur in species with large latitudinal variation in their distribution. Furthermore, by alleviating evolutionary constraints on feather colour, the evolution of dark skin may promote net diversification. Here, using an expanded dataset on bird skin coloration of 3033 species we found that more than 19% of species had dark skin. In contrast to our prediction, dark skinned birds have smaller distribution ranges. Furthermore, both dark skin and polymorphism in skin coloration promote net diversification. These results suggest that even concealed traits can influence large scale evolutionary events such as diversification in birds.

Rapid and reversible humidity-dependent colour change by water film formation in a scaled springtail.

Colour is often not a static trait but can change over time either through biotic or abiotic factors. Humidity-dependent colour change can occur through either morphological change (e.g. to feather barbules in birds) or by the replacement of air by water causing a shift in refractive index, as seen in arthropod multi-layer cuticles or scales. The scaled springtail Lepidocyrtus cyaneus has scales that produce colour largely via thin film interference from their lamina. We observed a marked colour change from golden to violet/purple coloration in humid conditions. Light microscopy, micro-spectrophotometry, contact angle goniometry and optical modelling indicate that the formation of a thin film of water on top of the hydrophilic scales increases their laminar thin film thickness, causing a shift towards violet/purple colour. Evaporation of the water film causes the metallic golden colour to return. This constitutes a remarkably rapid colour change (in the order of seconds), only limited by the speed of water film condensation and evaporation, that may serve as inspiration for new dynamically coloured materials and sensors.

Body size and substrate use affect ventral, but not dorsal, brightness evolution in lizards.

Substrate properties can affect the thermal balance of organisms, and the colored integument, alongside other factors, may influence heat transfer via differential absorption and reflection. Dark coloration may lead to higher heat absorption and could be advantageous when substrates are cool (and vice versa for bright coloration), but these effects are rarely investigated. Here, we examined the effect of substrate reflectance, specific heat capacity (cp), and body size on the dorso-ventral brightness using 276 samples from 12 species of cordylid lizards distributed across 26 sites in South Africa. We predicted, and found, that bright ventral colors occur more frequently in low cp (i.e., drier, with little energy needed for temperature change) substrates, especially in larger body-sized individuals, possibly to better modulate heat transfer with the surrounding environment. By contrast, dorsal brightness was not associated with body size nor any substrate thermal property, suggesting selection pressures other than thermoregulation. Ancestral estimation and evolutionary rate analyses suggest that ventral brightness rapidly differentiated within the Cordylinae starting 25 Mya, coinciding with an aridification period, further hinting at a thermoregulatory role for ventral colors. Our study indicates that substrate properties can have a direct role in shaping the evolution of ventral brightness in ectotherms.

How woodcocks produce the most brilliant white plumage patches among the birds.

Until recently, and when compared with diurnal birds that use contrasting plumage patches and complex feather structures to convey visual information, communication in nocturnal and crepuscular species was considered to follow acoustic and chemical channels. However, many birds that are active in low-light environments have evolved intensely white plumage patches within otherwise inconspicuous plumages. We used spectrophotometry, electron microscopy, and optical modelling to explain the mechanisms producing bright white tail feather tips of the Eurasian woodcock Scolopax rusticola. Their diffuse reflectance was approximately 30% higher than any previously measured feather. This intense reflectance is the result of incoherent light scattering from a disordered nanostructure composed of keratin and air within the barb rami. In addition, the flattening, thickening and arrangement of those barbs create a Venetian-blind-like macrostructure that enhances the surface area for light reflection. We suggest that the woodcocks have evolved these bright white feather patches for long-range visual communication in dimly lit environments.

How keratin cortex thickness affects iridescent feather colours.

The bright, saturated iridescent colours of feathers are commonly produced by single and multi-layers of nanostructured melanin granules (melanosomes), air and keratin matrices, surrounded by an outer keratin cortex of varying thicknesses. The role of the keratin cortex in colour production remains unclear, despite its potential to act as a thin film or absorbing layer. We use electron microscopy, optical simulations and oxygen plasma-mediated experimental cortex removal to show that differences in keratin cortex thickness play a significant role in producing colours. The results indicate that keratin cortex thickness determines the position of the major reflectance peak (hue) from nanostructured melanosomes of common pheasant (Phasianus colchicus) feathers. Specifically, the common pheasant has appropriate keratin cortex thickness to produce blue and green structural colours. This finding identifies a general principle of structural colour production and sheds light on the processes that shaped the evolution of brilliant iridescent colours in the common pheasant.

Pterosaur melanosomes support signalling functions for early feathers.

Remarkably well-preserved soft tissues in Mesozoic fossils have yielded substantial insights into the evolution of feathers1. New evidence of branched feathers in pterosaurs suggests that feathers originated in the avemetatarsalian ancestor of pterosaurs and dinosaurs in the Early Triassic2, but the homology of these pterosaur structures with feathers is controversial3,4. Reports of pterosaur feathers with homogeneous ovoid melanosome geometries2,5 suggest that they exhibited limited variation in colour, supporting hypotheses that early feathers functioned primarily in thermoregulation6. Here we report the presence of diverse melanosome geometries in the skin and simple and branched feathers of a tapejarid pterosaur from the Early Cretaceous found in Brazil. The melanosomes form distinct populations in different feather types and the skin, a feature previously known only in theropod dinosaurs, including birds. These tissue-specific melanosome geometries in pterosaurs indicate that manipulation of feather colour-and thus functions of feathers in visual communication-has deep evolutionary origins. These features show that genetic regulation of melanosome chemistry and shape7-9 was active early in feather evolution.

Properties, Genetics and Innate Immune Function of the Cuticle in Egg-Laying Species.

Cleidoic eggs possess very efficient and orchestrated systems to protect the embryo from external microbes until hatch. The cuticle is a proteinaceous layer on the shell surface in many bird and some reptile species. An intact cuticle forms a pore plug to occlude respiratory pores and is an effective physical and chemical barrier against microbial penetration. The interior of the egg is assumed to be normally sterile, while the outer eggshell cuticle hosts microbes. The diversity of the eggshell microbiome is derived from both maternal microbiota and those of the nesting environment. The surface characteristics of the egg, outer moisture layer and the presence of antimicrobial molecules composing the cuticle dictate constituents of the microbial communities on the eggshell surface. The avian cuticle affects eggshell wettability, water vapor conductance and regulates ultraviolet reflectance in various ground-nesting species; moreover, its composition, thickness and degree of coverage are dependent on species, hen age, and physiological stressors. Studies in domestic avian species have demonstrated that changes in the cuticle affect the food safety of eggs with respect to the risk of contamination by bacterial pathogens such as Salmonella and Escherichia coli. Moreover, preventing contamination of internal egg components is crucial to optimize hatching success in bird species. In chickens there is moderate heritability (38%) of cuticle deposition with a potential for genetic improvement. However, much less is known about other bird or reptile cuticles. This review synthesizes current knowledge of eggshell cuticle and provides insight into its evolution in the clade reptilia. The origin, composition and regulation of the eggshell microbiome and the potential function of the cuticle as the first barrier of egg defense are discussed in detail. We evaluate how changes in the cuticle affect the food safety of table eggs and vertical transmission of pathogens in the production chain with respect to the risk of contamination. Thus, this review provides insight into the physiological and microbiological characteristics of eggshell cuticle in relation to its protective function (innate immunity) in egg-laying birds and reptiles.

Enhanced photothermal absorption in iridescent feathers.

The diverse colours of bird feathers are produced by both pigments and nanostructures, and can have substantial thermal consequences. This is because reflectance, transmittance and absorption of differently coloured tissues affect the heat loads acquired from solar radiation. Using reflectance measurements and heating experiments on sunbird museum specimens, we tested the hypothesis that colour and their colour producing mechanisms affect feather surface heating and the heat transferred to skin level. As predicted, we found that surface temperatures were strongly correlated with plumage reflectivity when exposed to a radiative heat source and, likewise, temperatures reached at skin level decreased with increasing reflectivity. Indeed, nanostructured melanin-based iridescent feathers (green, purple, blue) reflected less light and heated more than unstructured melanin-based colours (grey, brown, black), as well as olives, carotenoid-based colours (yellow, orange, red) and non-pigmented whites. We used optical and heat modelling to test if differences in nanostructuring of melanin, or the bulk melanin content itself, better explains the differences between melanin-based feathers. These models showed that the greater melanin content and, to a lesser extent, the shape of the melanosomes explain the greater photothermal absorption in iridescent feathers. Our results suggest that iridescence can increase heat loads, and potentially alter birds' thermal balance.

The evolution of darker wings in seabirds in relation to temperature-dependent flight efficiency.

Seabirds have evolved numerous adaptations that allow them to thrive under hostile conditions. Many seabirds share similar colour patterns, often with dark wings, suggesting that their coloration might be adaptive. Interestingly, these darker wings become hotter when birds fly under high solar irradiance, and previous studies on aerofoils have provided evidence that aerofoil surface heating can affect the ratio between lift and drag, i.e. flight efficiency. However, whether this effect benefits birds remains unknown. Here, we first used phylogenetic analyses to show that strictly oceanic seabirds with a higher glide performance (optimized by reduced sink rates, i.e. the altitude lost over time) have evolved darker wings, potentially as an additional adaptation to improve flight. Using wind tunnel experiments, we then showed that radiative heating of bird wings indeed improves their flight efficiency. These results illustrate that seabirds may have evolved wing pigmentation in part through selection for flight performance under extreme ocean conditions. We suggest that other bird clades, particularly long-distance migrants, might also benefit from this effect and therefore might show similar evolutionary trajectories. These findings may also serve as a guide for bioinspired innovations in aerospace and aviation, especially in low-speed regimes.

Springtail coloration at a finer scale: mechanisms behind vibrant collembolan metallic colours.

The mechanisms and evolution of metallic structural colours are of both fundamental and applied interest, yet most work in arthropods has focused on derived butterflies and beetles with distinct hues. In particular, basal hexapods-groups with many scaled, metallic representatives-are currently poorly studied and controversial, with some recent studies suggesting either that thin-film (lamina thickness) or diffraction grating (longitudinal ridges, cross-ribs) elements produce these colours in early Lepidoptera and one springtail (Collembola) species. Especially the collembolan basal scale design, consisting of a single lamina and longitudinal ridges with smooth valleys lacking cross-ribs, makes them an interesting group to explore the mechanisms of metallic coloration. Using microspectroscopy, Raman spectroscopy, electron microscopy and finite-difference time-domain optical modelling, we investigated scale colour in seven springtail species that show clear metallic coloration. Reflectance spectra are largely uniform and exhibit a broadband metallic/golden coloration with peaks in the violet/blue region. Our simulations confirm the role of the longitudinal ridges, working in conjunction with thin-film effects to produce a broadband metallic coloration. Broadband coloration occurs through spatial colour mixing, which probably results from nanoscale variation in scale thickness and ridge height and distance. These results provide crucial insights into the colour production mechanisms in a basal scale design and highlight the need for further investigation of scaled, basal arthropods.

Morphogenesis of Iridescent Feathers in Anna's Hummingbird Calypte anna.

Color is a phenotypic trait of utmost importance, particularly in birds, which are known for their diverse color signals and color-producing mechanisms including pigment-based colors, light scattering from nanostructured feather tissues and combinations thereof. Bright iridescent plumage colors of hummingbirds are caused by light scattering by an organized array of flattened, pigment organelles, containing air-filled vesicles, called melanosomes. These hollow platelets are organized in multilayer arrays that contain numerous sharp air/melanin refractive index interfaces, producing brilliant iridescent colors. Despite their ecological significance and potential for inspiration of new optical materials, how platelets form and spatially arrange in nanostructures in growing feathers remains unknown. Here, we tested the hypothesis that melanosome formation and organization occurs mostly through passive self-assembly processes by assembling a developmental time series of growing hummingbird feathers using optical and electron microscopy. We show that hummingbird platelets contain air bubbles or vesicles upon their formation in pigment-producing cells, melanocytes. When melanosomes are transferred to neighboring keratinocytes (the cells shaping barbule structure) they drastically expand in size; and variation in this enlargement appears to be driven by physical constraints caused by the placement of the melanosomes within the barbule plate and their proximity to other melanosomes. As the barbule elongates and narrows, polymerizing feather corneous beta-protein orients melanosomes unilaterally, forcing them into a stacked configuration. These results reveal potentially novel forces driving the self-assembly of the nanostructures producing some of the brightest colors in nature.

Anisotropic Synthetic Allomelanin Materials via Solid-State Polymerization of Self-Assembled 1,8-Dihydroxynaphthalene Dimers.

Melanosomes in nature have diverse morphologies, including spheres, rods, and platelets. By contrast, shapes of synthetic melanins have been almost entirely limited to spherical nanoparticles with few exceptions produced by complex templated synthetic methods. Here, we report a non-templated method to access synthetic melanins with a variety of architectures including spheres, sheets, and platelets. Three 1,8-dihydroxynaphthalene dimers (4-4', 2-4' and 2-2') were used as self-assembling synthons. These dimers pack to form well-defined structures of varying morphologies depending on the isomer. Specifically, distinctive ellipsoidal platelets can be obtained using 4-4' dimers. Solid-state polymerization of the preorganized dimers generates polymeric synthetic melanins while maintaining the initial particle morphologies. This work provides a new route to anisotropic synthetic melanins, where the building blocks are preorganized into specific shapes, followed by solid-state polymerization.

Untangling the structural and molecular mechanisms underlying colour and rapid colour change in a lizard, Agama atra.

With functions as diverse as communication, protection and thermoregulation, coloration is one of the most important traits in lizards. The ability to change colour as a function of varying social and environmental conditions is thus an important innovation. While colour change is present in animals ranging from squids, to fish and reptiles, not much is known about the mechanisms behind it. Traditionally, colour change was attributed to migration of pigments, in particular melanin. More recent work has shown that the changes in nanostructural configuration inside iridophores are able to produce a wide palette of colours. However, the genetic mechanisms underlying colour, and colour change in particular, remain unstudied. Here we use a combination of transcriptomic and microscopic data to show that melanin, iridophores and pteridines are the main colour-producing mechanisms in Agama atra, and provide molecular and structural data suggesting that rapid colour change is achieved via melanin dispersal in combination with iridophore organization. This work demonstrates the power of combining genotypic (gene expression) and phenotypic (microscopy) information for addressing physiological questions, providing a basis for future studies of colour change.

Evolution of eggshell structure in relation to nesting ecology in non-avian reptiles.

Amniotic eggs are multifunctional structures that enabled early tetrapods to colonize the land millions of years ago, and are now the reproductive mode of over 70% of all terrestrial amniotes. Eggshell morphology is at the core of animal survival, mediating the interactions between embryos and their environment, and has evolved into a massive diversity of forms and functions in modern reptiles. These functions are critical to embryonic survival and may serve as models for new antimicrobial and/or breathable membranes. However, we still lack critical data on the basic structural and functional properties of eggs, particularly of reptiles. Here, we first characterized egg shape, shell thickness, porosity, and mineralization of eggs from 91 reptile species using optical images, scanning electron microscopy, and micro computed tomography, and collected data on nesting ecology from the literature. We then used comparative analyses to test hypotheses on the selective pressures driving their evolution. We hypothesized that eggshell morphology has evolved to protect shells from physical damage and desiccation, and, in support, found a positive relationship between thickness and precipitation, and a negative relationship between porosity and temperature. Although mineralization varied extensively, it was not correlated with nesting ecology variables. Ancestral state reconstructions show thinning and increased porosity over evolutionary time in squamates, but the opposite in turtles and crocodilians. Egg shape, size, porosity and calcification were correlated, suggesting potential structural or developmental tradeoffs. This study provides new data and insights into the morphology and evolution of reptile eggs, and raises numerous questions for additional research.

Thermoregulation and heat exchange in ospreys (Pandion haliaetus).

The osprey (Pandion haliaetus) is a cosmopolitan and long-distant migrant, found at all thermal extremes ranging from polar to tropical climates. Since ospreys may have an unusually flexible thermal physiology due to their migration over, and use of, a wide range of habitats, they represent an interesting study system to explore thermoregulatory adaptations in a raptor. In this study, we investigated the efficiency of heat exchange between body and environment in ospreys using micro-computed tomography (µ-CT), infrared thermography and behavioral observations. µ-CT revealed that the osprey bill has its largest potential for heat exchange at the proximal bill region, where arteries are situated most closely under the surface. However, thermal images of 10 juvenile ospreys showed that the bill contributes to only 0.3% of the bird's total heat exchange. The long legs and protruding claws played a more prominent role as heat dissipation areas with a contribution of 6% and 7%, respectively. Operative thresholds, i.e. the ambient temperature below which heat is lost, were high (>38.5 °C) in these body parts. However, we found no indication of active regulation of heat exchange. Instead we observed multiple behavioral adaptations starting at relatively low ambient temperatures. At 26.3 °C ospreys had a 50% probability of showing panting behavior and above 27.9 °C they additionally spread their wings to enable heat dissipation from the less insulated ventral side. The thermal images revealed that at an ambient temperature of 32.1 °C ospreys had a 50% probability of developing a ≥2 °C and up to 7.5 °C colder stripe on the head, which was likely caused by cutaneous evaporation. Our observations suggest that ospreys more strongly rely on behavioral mechanisms than on active thermal windows to cope with heat stress. This study not only improves our understanding of the role of different body parts in ospreys' total heat exchange with the environment but further provides an insight about additional adaptations of this raptor to cope with heat stress.

Substrate thermal properties influence ventral brightness evolution in ectotherms.

The thermal environment can affect the evolution of morpho-behavioral adaptations of ectotherms. Heat is transferred from substrates to organisms by conduction and reflected radiation. Because brightness influences the degree of heat absorption, substrates could affect the evolution of integumentary optical properties. Here, we show that vipers (Squamata:Viperidae) inhabiting hot, highly radiative and superficially conductive substrates have evolved bright ventra for efficient heat transfer. We analyzed the brightness of 4161 publicly available images from 126 species, and we found that substrate type, alongside latitude and body mass, strongly influences ventral brightness. Substrate type also significantly affects dorsal brightness, but this is associated with different selective forces: activity-pattern and altitude. Ancestral estimation analysis suggests that the ancestral ventral condition was likely moderately bright and, following divergence events, some species convergently increased their brightness. Vipers diversified during the Miocene and the enhancement of ventral brightness may have facilitated the exploitation of arid grounds. We provide evidence that integument brightness can impact the behavioral ecology of ectotherms.

Exposure to UV radiance predicts repeated evolution of concealed black skin in birds.

Plumage is among the most well-studied components of integumentary colouration. However, plumage conceals most skin in birds, and as a result the presence, evolution and function of skin colour remains unexplored. Here we show, using a database of 2259 species encompassing >99% of bird genera, that melanin-rich, black skin is found in a small but sizeable percentage (~5%) of birds, and that it evolved over 100 times. The spatial distribution of black skin follows Gloger's rule, which states that pigmentation of endothermic animals increases towards the equator. Furthermore, most black-skinned birds inhabit high irradiation regions, and tend to be bald and/or have white feathers. Thus, taken together, our results suggest that melanin-rich, black skin helps to protect birds against ultraviolet irradiation. More generally, our results illustrate that feathered skin colour varies taxonomically, ontogenetically and temporally, providing an additional dimension for avian colour research.

The golden age of arthropods: ancient mechanisms of colour production in body scales.

Insect colour is extremely diverse and produced by a large number of pigmentary and nanostructural mechanisms. Considerable research has been dedicated to these optical mechanisms, with most of it focused on chromatic colours, such as blues and greens, and less on achromatic colours like white and gold. Moreover, studies on the evolution of these colours are less common and largely limited to inferences from extant organisms, in part because of the limited amount and types of available fossil material. Here, we directly compare nanostructure and colour of extant and amber-preserved (approx. 15 and 99 Myr old, respectively) gold-coloured representatives of micromoths (Lepidoptera: Micropterigidae) and springtails (Collembola: Tomoceridae). Using electron microscopy, microspectrophotometry and finite domain time difference optical modelling, we show that golden coloration in the extant micromoth is produced by nanometre-scale crossribs that function as zero-order diffraction gratings and in the springtail by a diffraction grating without crossribs. Surprisingly, nanostructure and thus predicted colour of the amber-preserved specimens were nearly identical to those of their extant counterparts. Removal of amber enabled direct colour measurement of the fossil micromoth and further revealed that its colour matched both that of the extant specimen and the predicted colour, providing further support for our optical models. Our data thus clearly show an early origin and striking conservation of scale nanostructures and golden coloration, suggesting strong selection pressure either on the colour itself or on the mechanisms that produce the colour. Furthermore, we show the thus-far untapped potential for the use of amber-preserved specimens in studies on the evolution of organismal coloration.