History and mechanisms of carotenoid plumage evolution in the New World orioles (Icterus) Nicholas R. Friedman a,c, ⁎, Kevin J. McGraw b , Kevin E. Omland a a Department of Biological Sciences, University of Maryland, Baltimore County, Baltimore, MD 21250, USA b School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA c Department of Zoology & Laboratory of Ornithology, Faculty of Science, Univerzita Palackého v Olomouci, Olomouc 779 00, Czech Republic abstract article info Article history: Received 10 December 2013 Received in revised form 18 March 2014 Accepted 27 March 2014 Available online 3 April 2014 Keywords: Bird coloration Ancestral state reconstruction Carotenoids HPLC While many recent studies focus on the functions of carotenoids in visual signaling, they seldom address the phylogenetic origins of plumage coloration and its mechanisms. Here, we used the New World orioles (Icterus) as a model clade to study the history of orange carotenoid-based coloration and pigmentation, sampling 47 muse- um specimens from 12 species. We examined the identity and concentration of carotenoids in oriole feathers using high-performance liquid chromatography, and used phylogenetic comparative methods to compare these obser- vations to reflectance measurements of plumage. Each of the seven yellow oriole species we sampled used only lutein to color their feathers. Ancestral state reconstruction of this trait suggests that the oriole common ancestor had yellow feathers pigmented with lutein. We found keto-carotenoids in small concentrations in the plumage of each of the five species scored as orange. This correlation suggests that discrete gains and losses of keto- carotenoids are behind independent gains of orange coloration in orioles. In contrast, total carotenoid concentra- tion was not associated with hue, and total concentration of keto-carotenoids poorly explained variation in hue among species where they were present. These findings suggest that orioles most likely evolved orange plumage coloration at least twice, each time by gaining the ability to metabolize dietary carotenoids by C4-oxygenation. Given that red coloration is generated by this same oxygenation process in a wide range of bird species, it raises the question of why, if orioles possess this metabolic capability, no red oriole species exist. © 2014 Elsevier Inc. All rights reserved. 1. Introduction For many decades, coloration has served as a convenient model for understanding general evolutionary processes. There is now abundant evidence explaining how colors may evolve to function in camouflage, social signaling, or mimicry (see Hill and McGraw, 2006). However, fewer investigations have sought to track the evolutionary history of the mechanisms behind color production. Such an approach is a synthe- sis of two of Tinbergen's four questions, phylogeny and mechanism, and is now feasible due to modern advances in biochemical and phylogenetic methods, and has been used successfully to study the evolution of color- ation in mice, irises, and butterflies (Tinbergen, 1963; Steiner et al., 2009; Reed et al., 2011; Smith and Rausher, 2011). With widespread interest in the functions of bird coloration, studies are needed that address the history and mechanisms of this system as well. The use of carotenoid pigments for coloration is widespread among animals, particularly in songbirds (McGraw, 2006). Carotenoids are diet- derived molecules that confer much of the yellow, orange, or red plumage colors. For example, Yellow Warblers (Setophaga petechia) appear yellow due to the presence of lutein, a common carotenoid that the species ingests from insects (McGraw et al., 2003). However, many types of carot- enoids found in plumage are not directly acquired from food, but are instead metabolic derivatives of ingested carotenoids (Brush, 1967; Fox et al., 1969). For example, House Finches (Haemorhous mexicanus; Inouye et al., 2001; McGraw et al., 2006) produce their red coloration through oxygenation of the C4 site on a carotenoid end-ring. This meta- bolic change results in a longer conjugated system, thus allowing the ca- rotenoid to absorb longer wavelengths of light (Britton, 1995). Such modified red compounds are commonly responsible for the red plumage coloration exhibited by other bird species as well (see McGraw, 2006), al- though diet-derived keto-carotenoids may occasionally be incorporated in the growth of anomalously red feathers (typically due to a diet supple- mented with exotic food items; Hudon and Brush, 1989; Mulvihill et al., 1992; Hudon et al., 2013). Indeed, many evolutionary transitions from yellow to red coloration in songbirds are likely the result of gains of keto- carotenoids via this mechanism of C4-oxygenation (Andersson et al., 2007; Prager and Andersson, 2009; Friedman et al., 2013). Orange carotenoid-based coloration is similarly produced by the deposition of keto-carotenoids into feathers (Hudon, 1991), but is a Comparative Biochemistry and Physiology, Part B 172–173 (2014) 1–8 ⁎ Corresponding author at: Department of Zoology & Laboratory of Ornithology, Faculty of Science, Univerzita Palackého v Olomouci, Tř. 17. Listopadu 50, 779 00 Olomouc, Czech Republic. E-mail address: friedmn1@umbc.edu (N.R. Friedman). http://dx.doi.org/10.1016/j.cbpb.2014.03.004 1096-4959/© 2014 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part B journal homepage: www.elsevier.com/locate/cbpb