1272 INTRODUCTION Over evolutionary time, metazoans, and birds in particular, have occupied large portions of available color gamut, in part because of the diversity of color production mechanisms available to them (Stoddard and Prum, 2011). Coloration can result from selective light absorption by pigments deposited in feathers. Although more than five classes of pigments have been found in bird feathers, those most commonly present are melanins and carotenoids (McGraw, 2006). Melanins, found within membrane-bound organelles (melanosomes), can produce colors ranging from black to reddish browns and pale oranges (McGraw, 2006). Carotenoid pigments are acquired by birds through their diet (Goodwin, 1984) and are responsible for most of the bright red, orange and yellow colors (Brush, 1978). A second mechanism of color production is caused by the interaction between incident light and nano-scale reflective tissues (structural coloration) of feather barbs and barbules. Colors produced in this manner include the blue, violet, ultraviolet (UV) and iridescent parts of plumage (Auber, 1957; Dyck, 1976). It has long been recognized that structural and pigment-based colors are not mutually exclusive and can interact with one another to attain colors not possible by either mechanism alone (Dyck, 1971a; Prum et al., 1999a). For example, the combination of structural blue colors with yellow colors caused by pigments is thought to give rise to most green plumage colors (Auber, 1957; Fox, 1976) (but see Prum et al., 1998). Surprisingly, however, with the exception of a few studies (Dyck, 1971a; Shawkey and Hill, 2005; Shawkey and Hill, 2006), the interaction between pigment and structure has not been examined in detail. Understanding these interactions is crucial because different mechanisms of plumage coloration vary in, for example, their developmental cost and thus may convey different information to conspecifics. Furthermore, they may provide inspiration for new materials with novel optical properties. As a result of over 150 years of captive breeding, the color diversity of budgerigars [Aves, Psittacidae, Melopsittacus undulatus (Shaw 1805); Fig.1A] now far exceeds that of their green wild ancestors (Taylor and Warner, 1986). Color morphs include achromatic whites and greys as well as chromatic colors ranging from purple to yellow (World Budgerigar Organisation, www.world-budgerigar.org/). These latter colors are thought to be produced through various combinations of both nanostructures and pigment (Simon, 1971; Parker, 2002), but as far as we are aware, no data exist to support this hypothesis. Because of their diversity in color and (potentially) color production mechanisms, as well as rich genetic data from breeders, budgerigars may serve as a model system for understanding both physical and genetic bases of color evolution. Here, we use multiple techniques to identify the physical bases of color production in seven morphs of the budgerigar to determine the relative contribution of pigments and structure to plumage color. The Journal of Experimental Biology 215, 1272-1277 © 2012. Published by The Company of Biologists Ltd doi:10.1242/jeb.064907 RESEARCH ARTICLE Relative contributions of pigments and biophotonic nanostructures to natural color production: a case study in budgerigar (Melopsittacus undulatus) feathers Liliana DʼAlba*, Leah Kieffer and Matthew D. Shawkey Department of Biology and Integrated Bioscience Program, University of Akron, Akron, OH 44325-3908, USA *Author for correspondence (liliana@uakron.edu) Accepted 13 December 2011 SUMMARY Understanding the mechanistic bases of natural color diversity can provide insight into its evolution and inspiration for biomimetic optical structures. Metazoans can be colored by absorption of light from pigments or by scattering of light from biophotonic nanostructures, and these mechanisms have largely been treated as distinct. However, the interactions between them have rarely been examined. Captive breeding of budgerigars (Aves, Psittacidae, Melopsittacus undulatus) has produced a wide variety of color morphs spanning the majority of the spectrum visible to birds, including the ultraviolet, and thus they have been used as examples of hypothesized structure–pigment interactions. However, empirical data testing these interactions in this excellent model system are lacking. Here we used ultraviolet–visible spectrometry, light and electron microscopy, pigment extraction experiments and optical modeling to examine the physical bases of color production in seven budgerigar morphs, including grey and chromatic (purple to yellow) colors. Feathers from all morphs contained quasi-ordered air–keratin ʻspongy layerʼ matrices, but these were highly reduced and irregular in grey and yellow feathers. Similarly, all feathers but yellow and grey had a layer of melanin-containing melanosomes basal to the spongy layer. The presence of melanosomes likely increases color saturation produced by spongy layers whereas their absence may allow increased expression of yellow colors. Finally, extraction of yellow pigments caused some degree of color change in all feathers except purple and grey, suggesting that their presence and contribution to color production is more widespread than previously thought. These data illustrate how interactions between structures and pigments can increase the range of colors attainable in birds and potentially in synthetic systems. Supplementary material available online at http://jeb.biologists.org/cgi/content/full/215/8/1272/DC1 Key words: structural color, psittacofulvin. THE฀JOURNAL฀OF฀EXPERIMENTAL฀BIOLOGY