Painting the Bunting: Carotenoids and structural elements combine to produce the feather coloration of the male Painted Bunting

ABSTRACT Male Painted Buntings (Passerina ciris) display at least 6 distinct plumage colors that encapsulate much of the visible light spectrum, yet the specific mechanisms responsible for generating this diversity of color have not been identified. Here, we show that metabolically derived carotenoids and nanostructures capable of producing structural color were ubiquitous across feather patches. We used digital photography, light microscopy, spectrophotometry, carotenoid extraction, and high-performance liquid chromatography to show that the resulting color of each feather patch depended on the concentration of carotenoids, melanins, and underlying feather nanostructures. For example, we found that the blue-violet head feathers contained low concentrations of ketolated carotenoids, which is not typical of blue-violet structurally colored feathers. Additionally, the red breast and orange belly feathers contained a green-tuned structural color visible after carotenoid extraction, which is not typical of feathers that contain ketolated carotenoids. Although, none of these abnormal combinations of carotenoids and structural coloration appeared to significantly impact feather color. Conversely, we found the purple rump, dark green greater coverts, and bright yellow-green mantle feather coloration resulted from the combination of high concentrations of carotenoids and the presence of structural color. For the first time, we identify the combination of red ketolated carotenoids and blue structural color as a mechanism to produce purple feather coloration. Identifying the specific mechanisms that give rise to the diversity of colors within this species will facilitate the study of the—to date—unknown signaling functions of colors produced through the combination of carotenoids and nanostructures in Painted Buntings and other songbirds. LAY SUMMARY Some of the most spectacular feather colors result from 2 distinct processes: light interacting with specific pigments or from the reflection of light by microscopic feather components. Red Northern Cardinal feathers are colored by only red pigments called carotenoids, whereas only feather nanostructures create the blue color of Blue Jay feathers. We demonstrate that all the different colored feather patches on male Painted Bunting contain both carotenoid pigments and structures capable of producing color. For the first time, we also detail how red carotenoid pigments and blue structural color are combined to create a purple-colored rump feather in the male Painted Bunting. These findings will inform future studies on the physiology, ecology, and evolution of bird coloration and provide insight into which genes underly color variation among individuals and species. RESUMEN Los machos de Passerina ciris muestran al menos seis colores de plumaje distintos que encapsulan gran parte del espectro de luz visible, pero no se han identificado los mecanismos específicos responsables de generar esta diversidad de colores. Aquí, mostramos que los carotenoides derivados metabólicamente y las nano-estructuras capaces de producir color estructural fueron ubicuos en los parches de plumas. Utilizamos fotografía digital, microscopía óptica, espectrofotometría, extracción de carotenoides y cromatografía líquida de alta resolución para demostrar que el color resultante de cada parche de plumas dependía de la concentración de carotenoides, melaninas y nano-estructuras de plumas subyacentes. Por ejemplo, encontramos que las plumas de la cabeza de color azul violeta contenían bajas concentraciones de carotenoides cetolados, lo que no es típico de las plumas de color estructural azul violeta. Adicionalmente, las plumas rojas del pecho y naranja del vientre contenían un color estructural afinado verde visible después de la extracción de carotenoides, que no es típico de las plumas que contienen carotenoides cetolados. Sin embargo, ninguna de estas combinaciones anormales de carotenoides y de coloración estructural pareció afectar significativamente el color de las plumas. Por el contrario, encontramos que la rabadilla morada, las coberteras mayores de color verde oscuro y la coloración de las plumas del manto de color amarillo verdoso brillante resultaron de la combinación de altas concentraciones de carotenoides y la presencia de color estructural. Por primera vez, identificamos la combinación de carotenoides cetolados rojos y color estructural azul como un mecanismo para producir la coloración púrpura de las plumas. Identificar los mecanismos específicos que dan lugar a la diversidad de colores dentro de esta especie facilitará el estudio de las funciones de señalización desconocidas hasta la fecha de los colores producidos a través de la combinación de carotenoides y nano-estructuras en P. ciris y otras aves canoras.