Elucidating the mechanisms of colour production in organisms is important for understanding how selection acts upon a variety of behaviours. Spiders provide many spectacular examples of colours used in courtship, predation, defence and thermoregulation, but are thought to lack many types of pigments common in other animals. Ommochromes, bilins and eumelanin have been identified in spiders, but not carotenoids or melanosomes. Here, we combined optical microscopy, refractive index matching, confocal Raman microspectroscopy and electron microscopy to investigate the basis of several types of colourful patches in spiders. We obtained four major results. First, we show that spiders use carotenoids to produce yellow, suggesting that such colours may be used for condition-dependent courtship signalling. Second, we established the Raman signature spectrum for ommochromes, facilitating the identification of ommochromes in a variety of organisms in the future. Third, we describe a potential new pigmentary–structural colour interaction that is unusual because of the use of long wavelength structural colour in combination with a slightly shorter wavelength pigment in the production of red. Finally, we present the first evidence for the presence of melanosomes in arthropods, using both scanning and transmission electron microscopy, overturning the assumption that melanosomes are a synapomorphy of vertebrates. Our research shows that spiders have a much richer colour production palette than previously thought, and this has implications for colour diversification and function in spiders and other arthropods.

In a hybrid zone between two tropical lekking birds, yellow male plumage of one species has introgressed asymmetrically replacing white plumage of another via sexual selection. Here, we present a detailed analysis of the plumage trait to uncover its physical and genetic bases and trace its evolutionary history. We determine that the carotenoid lutein underlies the yellow phenotype and describe microstructural feather features likely to enhance color appearance. These same features reduce predicted water shedding capacity of feathers, a potential liability in the tropics. Through genome-scale DNA sequencing of hybrids and each species in the genus, we identify BCO2 as the major gene responsible for the color polymorphism. The BCO2 gene tree and genome-wide allele frequency patterns suggest that carotenoid-pigmented collars initially arose in a third species and reached the hybrid zone through historical gene flow. Complex interplay between sexual selection and hybridization has thus shaped phenotypes of these species, where conspicuous sexual traits are key to male reproductive success.

Most studies of animal coloration focus on spectral colours, which are colours evoked by single peaks within the wavelengths of visible light. It is poorly understood how non-spectral colours (those produced by a combination of reflectance peaks) are produced, despite their potential significance to both animal communication and biomimicry. Moreover, although both pigmentary and structural colour production mechanisms have been well characterized in feathers independently, their interactions have received considerably less attention, despite their potential to broaden the available colour spectrum. Here, we investigate the colour production mechanisms of the purple feathers of the purple-breasted cotinga (Cotinga cotinga). The purple feather colour results from both the coherent scattering of light by a sphere-type nanomatrix of β-keratin and air (spongy layer) in the barbs, which produces a blue–green colour, and the selective absorption of light in the centre of the bird-visible spectrum by the methoxy-carotenoid, cotingin. This unusual combination of carotenoid and nanostructure with a central air vacuole, in the absence of melanin, is a blueprint of a synergistic way to produce a non-spectral colour that would be difficult to achieve with only a single colour production mechanism.

Unlike humans, some animals have evolved a physiological ability to deposit porphyrins, which are pigments produced during heme synthesis in cells, in the skin and associated integument such as hair. Given the inert nature and easiness of collection of hair, animals that present porphyrin-based pigmentation constitute unique models for porphyrin analysis in biological samples. Here we present the development of a simple, rapid, and efficient analytical method for four natural porphyrins (uroporphyrin I, coproporphyrin I, coproporphyrin III and protoporphyrin IX) in the Southern flying squirrel Glaucomys volans, a mammal with hair that fluoresces and that we suspected has porphyrin-based pigmentation. The method is based on capillary liquid chromatography-mass spectrometry (CLC-MS), after an extraction procedure with formic acid and acetonitrile. The resulting limits of detection (LOD) and quantification (LOQ) were 0.006–0.199 and 0.021–0.665 µg mL⁻¹, respectively. This approach enabled us to quantify porphyrins in flying squirrel hairs at concentrations of 3.6–353.2 µg g⁻¹ with 86.4–98.6% extraction yields. This method provides higher simplicity, precision, selectivity, and sensitivity than other methods used to date, presenting the potential to become the standard technique for porphyrin analysis.

While the specific mechanisms of colour production in biological systems are diverse, the mechanics of colour production are straightforward and universal. Colour is produced through the selective absorption of light by pigments, the scattering of light by nanostructures or a combination of both. WhenTigriopus californicuscopepods were fed a carotenoid-limited diet of yeast, their orange-red body coloration became faint, but their eyespots remained unexpectedly bright red. Raman spectroscopy indicated a clear signature of the red carotenoid pigment astaxanthin in eyespots; however, refractive index matching experiments showed that eyespot colour disappeared when placed in ethyl cinnamate, suggesting a structural origin for the red coloration. We used transmission electron microscopy to identify consecutive nanolayers of spherical air pockets that, when modelled as a single thin film layer, possess the correct periodicity to coherently scatter red light. We then performed microspectrophotometry to quantify eyespot coloration and confirmed a distinct colour difference between the eyespot and the body. The observed spectral reflectance from the eyespot matched the reflectance predicted from our models when considering the additional absorption by astaxanthin. Together, this evidence suggests the persistence of red eyespots in copepods is the result of a combination of structural and pigmentary coloration.