Comparative Biochemistry and Physiology, Part B 172–173 (2014) 1–8

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Comparative Biochemistry and Physiology, Part B

j ourna l homepage: www.e lsev ie r .com/ locate /cbpb

History and mechanisms of carotenoid plumage evolution in the NewWorld 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, USAb School of Life Sciences, Arizona State University, Tempe, AZ 85287, USAc Department of Zoology & Laboratory of Ornithology, Faculty of Science, Univerzita Palackého v Olomouci, Olomouc 779 00, Czech Republic

⁎ Corresponding author at: Department of Zoology & Laof Science, Univerzita Palackého v Olomouci, Tř. 17. ListopRepublic.

E-mail address: friedmn1@umbc.edu (N.R. Friedman)

http://dx.doi.org/10.1016/j.cbpb.2014.03.0041096-4959/© 2014 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 10 December 2013Received in revised form 18 March 2014Accepted 27 March 2014Available online 3 April 2014

Keywords:Bird colorationAncestral state reconstructionCarotenoidsHPLC

While many recent studies focus on the functions of carotenoids in visual signaling, they seldom address thephylogenetic origins of plumage coloration and its mechanisms. Here, we used the New World orioles (Icterus)as amodel clade to study the history of orange carotenoid-based coloration and pigmentation, sampling 47muse-um specimens from12 species.We examined the identity and concentration of carotenoids in oriole feathers usinghigh-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 onlylutein to color their feathers. Ancestral state reconstruction of this trait suggests that the oriole common ancestorhad yellow feathers pigmented with lutein. We found keto-carotenoids in small concentrations in the plumageof 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 hueamong species where they were present. These findings suggest that orioles most likely evolved orange plumagecoloration 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 raisesthe 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 forunderstanding general evolutionary processes. There is now abundantevidence 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 ofthemechanisms behind color production. Such an approach is a synthe-sis of two of Tinbergen's four questions, phylogeny andmechanism, andis now feasible due tomodern advances in biochemical and phylogeneticmethods, and has been used successfully to study the evolution of color-ation inmice, irises, and butterflies (Tinbergen, 1963; Steiner et al., 2009;Reed et al., 2011; Smith and Rausher, 2011). With widespread interestin the functions of bird coloration, studies are needed that address thehistory and mechanisms of this system as well.

The use of carotenoid pigments for coloration is widespread amonganimals, particularly in songbirds (McGraw, 2006). Carotenoids are diet-

boratory of Ornithology, Facultyadu 50, 779 00 Olomouc, Czech

.

derivedmolecules that confermuch of the yellow, orange, or red plumagecolors. For example, YellowWarblers (Setophaga petechia) appear yellowdue to the presence of lutein, a common carotenoid that the speciesingests from insects (McGrawet al., 2003). However,many types of carot-enoids found in plumage are not directly acquired from food, but areinstead metabolic derivatives of ingested carotenoids (Brush, 1967; Foxet al., 1969). For example, House Finches (Haemorhous mexicanus;Inouye et al., 2001; McGraw et al., 2006) produce their red colorationthrough 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). Suchmodified red compounds are commonly responsible for the red plumagecoloration exhibited by other bird species aswell (seeMcGraw, 2006), al-though diet-derived keto-carotenoids may occasionally be incorporatedin 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 fromyellow 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 thedeposition of keto-carotenoids into feathers (Hudon, 1991), but is a

2 N.R. Friedman et al. / Comparative Biochemistry and Physiology, Part B 172–173 (2014) 1–8

less common phenotype among passerines than yellow or red (N.Friedman and S. Lutrell unpublished data). Perhaps due to this rarity,few studies have focused on songbird species with orange plumage(McGraw et al., 2004; Hofmann et al., 2007; Reudink et al., 2009).Fewer still have examined the proximate mechanisms by whichsome birds produce orange coloration using carotenoids (Prum et al.,2012). However, the NewWorld orioles (Icterus; family Icteridae) offera promising opportunity to study the evolution of orange plumage be-cause they are a speciose genus with plumage that varies continuouslyfrom yellow to orange (Jaramillo and Burke, 1999; Hofmann et al.,2006). Orioles have been studied extensively as a model clade, dueto their diverse color patterns, migratory behaviors, and life histories(e.g., Omland and Lanyon, 2000; Price et al., 2007; Friedman et al.,2009). The phylogenetic relationships of Icterus species have beeninferred using multiple independent loci (Omland et al., 1999; AllenandOmland, 2003; Jacobsen et al., 2010), and their phylogeny continuesto be resolved as both sequencing and phylogenetic inference methodsadvance (Jacobsen and Omland, 2011). Studies in other blackbird cladeshave described discrete variation in carotenoid coloration and pigmen-tation between yellow and red species (Kiere et al., 2009; Friedmanet al., 2013). The continuous variation of oriole coloration from yellowto orange (Hofmann et al., 2006) raises a question that can be uniquelyaddressed in orioles: which biochemical changes explain pigment-based variation in plumage coloration between yellow and orangeplumage?

Themolecular and genetic mechanisms of carotenoid color variationamong bird species are still unknown, though hypothetical models ofthese mechanisms have been proposed (see McGraw, 2006; Pragerand Andersson, 2009; Prum et al., 2012). Because the enzyme responsi-ble for keto-carotenoid production and corresponding orange and redplumage color is currently unidentified, an important first step in un-derstanding the evolution of carotenoid coloration is to examine howpigments vary among related species as a function of color variation.This approach has beenusedbyprevious studies examining evolutionarytransitions from yellow to red coloration in birds (Prager and Andersson,2010; Friedman et al., 2013).

Several studies have examined variation in and control mechanismsunderlying carotenoid-based coloration within species (reviewed inDale, 2006; McGraw, 2006). Much of the work on House Finches hassuggested that variation in keto-carotenoid concentration explains var-iation in hue among individuals (Hill et al., 1994; McGraw et al., 2006).Does oriole plumage coloration vary among species in a similar fashion?Specifically, does continuous variation in carotenoid concentrationexplain variation in color from yellow to orange? Alternatively, theremight be discrete pigment differences involved in transitions from yel-low to orange, as seen in transitions from yellow to red (Prager andAndersson, 2009; Friedman et al., 2013). In addition, we ask whetherdifferent oriole species have evolved orange coloration independentlyusing similar carotenoid pigments, or different ones (see Friedmanet al., 2013). To address these questions, we had three primary goals:(1) use high performance liquid chromatograpy (HPLC) biochemistrytechniques to extract, identify, and quantify the carotenoids present inthe breast feathers of yellow and orange orioles; (2) reconstruct theevolutionary history of gains and losses of carotenoid pigments on theoriole phylogeny; and (3) use phylogenetic comparative methods totest which types of evolutionary transitions in pigmentation are respon-sible for the evolution of orange coloration in orioles.

2. Materials and methods

2.1. Sampling

Vouchered museum specimens are an ideal means to obtain rich,repeatable data for comparative studies (Barlow and Flood, 1983).Consequently, we sampled 3–5 mg of feather material (barbs cut fromcontour feathers) from the breast patches of 47 specimens from 12

Icterus species at the Academy of Natural Sciences in Philadelphia andthe Delaware Museum of Natural History (Table 1; Clements, 2007).We sampled adult males collected during the breeding season, particu-larly skins that appearedwell preserved (i.e., lacking obvious staining ordamage). Females inmany, but not all, oriole species have bright yellowor orange plumage that is indistinguishable from males' (Jaramillo andBurke, 1999). Previous studies have thoroughly described interspecificvariation in sexual dichromatism in orioles (Hofmann et al., 2007;Hofmann et al., 2008a; Hofmann et al., 2008b), and so to limit thisstudy's destructive sampling of museum specimens, we did not samplefemale specimens.

2.2. Color scoring

Prior to feather removal, we collected reflectance spectra fromthe focal carotenoid-based plumage of each museum specimen. Weused an Ocean Optics USB2000 reflectance spectrometer with a PX-2 pulsed xenon light source (Ocean Optics, Dunedin, FL), calibratedwith a white Spectralon standard (Labsphere, North Sutton, NH).Orioles' carotenoid-based plumage varies among species primarilyin hue (Hofmann et al., 2006), and so to isolate variation in hue wecalculated reflectance midpoint values (λR50; Montgomerie, 2006;hereafter spectral location) for each reflectance spectrum. Thesevalues describe the hue of carotenoid-colored plumage, and havebeen used for this purpose in similar studies (e.g., Hofmann et al.,2006; Andersson et al., 2007). To score carotenoid hue as a discretecharacter, we followed the character states delineated in Friedmanet al. (2011), which show that there is a bimodal distribution of spec-tral location values across the New World blackbirds (Icteridae).Species with coloration that appears yellow or orange to humanshad spectral location values from 500 to 560 nm, whereas thosespecies that appear red to humans had spectral location valuesfrom 580 to 605 nm (Friedman et al., 2011; their Fig. 1A). Amongthe blackbirds, only species in the oriole genus Icterus had spectrallocation values from 540 to 560 nm (Friedman et al., 2011; theirFig. 1B). Consequently, in that study we assigned character statesbased on spectral location values: “yellow” for 500–539 nm, “orange”for 540–560 nm, and “red” for 580–605 nm (Friedman et al., 2011). Inthis study, we scored discrete character states for color based on thosesame criteria.

2.3. HPLC biochemistry

We usedmethods described in Friedman et al. (2013) to extract andidentify carotenoid pigments in oriole feathers. FollowingMcGraw et al.(2005), we extracted carotenoid pigments from 3 to 5 mg of trimmedfeather barbs using heated acidified pyridine. Carotenoids were trans-ferred to an organic layer bymixingwithwater and a solution of hexaneand t-butyl methyl ether (1:1, v/v) and then centrifugation at 3000 rpmfor 5min. After this process, we visually inspected feathers for the pres-ence of phaeomelanin (i.e., remaining brown pigmentation), but did notobserve any. Prior to analysis with HPLC, we evaporated the organiclayer under nitrogen for overnight storage at −80 °C. We then resus-pended the carotenoids in the HPLC mobile phase (methanol:acetoni-trile:dichloromethane, 42:42:16, v/v/v). Samples were analyzed usinga Waters 2695 HPLC instrument and a Waters YMC Carotenoid column(5 μm, 4.6 mm × 250 mm; Waters Corp., Milford, MA) at 30 °C. Absor-bance data (from 260 nm to 600 nm) was collected for each sampleusing a Waters 2996 photodiode array detector. As in Toomey andMcGraw (2007), we used a three-part gradient HPLC protocol that iscapable of separating and detecting both carotenes and xanthophylls.We identified and quantified carotenoids and their concentrationsusing external standards (following Rowe and McGraw, 2009) whileblind to each sample's taxonomic identity.

Table 1Mean concentrations of carotenoid pigments extracted from oriole breast feathers, and color characters scored from reflectance measurements taken from specimens prior to featherremoval. All concentrations below (indicated by brackets) are in μg/g; "-" refers to pigments that were absent, and "+" refers to pigments that were present in an unquantifiableconcentration. Taxa follow Clements (2007). Discrete hue scored as in Friedman et al. (2011). Column “n” describes the number of individuals sampled from each species.

Keto-carotenoids Dietary yellow Modified yellow Total concentrations Color characters

Taxon n [3HE] [ECH] [CXN] [AXN] [ADX] [ADR] [LUT] [ZXN] [CXA] [CXB] Total[keto-carotenoid]

Total[carotenoid]

Spectral location(nm)

Discretehue

I. bullockii 5 – 0.7 5.1 – – – 83.4 – 21.2 11.3 5.8 121.7 552 OrangeI. cucullatus 6 – – – – – – 99.6 – – – – 99.6 534 YellowI.dominicensis 3 – – – – – – 68.6 – – – – 68.6 514 YellowI. galbula 5 – 1.7 8.8 – – – 56.8 – 57.1 32.8 10.5 157.2 551 OrangeI.graduacauda 6 – – – – – – 115.4 – – – – 115.4 514 YellowI. gularis 4 1.4 – 0.4 – – – 78.6 7.7 45.2 24.2 1.9 157.5 551 OrangeI. croconotus 3 + – – – – – 76.7 – – – + 76.7 549 OrangeI. mesomelas 3 – – – – – – 60.4 – – – – 60.4 519 YellowI. nigrogularis 3 – – – – – – 121.8 – – – – 121.8 527 YellowI. pectoralis 3 – – – – – – 271.3 – – – – 271.3 526 YellowI. pustulatus 3 4.4 – 1.0 – – – 71.6 24.6 60.7 34.0 5.4 196.3 557 OrangeI.prosthemelas 3 – – – – – – 140.0 – – – – 140.0 517 Yellow

3HE = 3-hydroxy-echinenone, ECH = echinenone, AXN = astaxanthin, ADX = α-doradexanthin, ADR = adonirubin, LUT = lutein, ZXN = zeaxanthin, CXA = canary xanthophyll A,CXB = canary xanthophyll B.

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2.4. Phylogenetic comparative methods

To examine the evolution of carotenoid coloration and pigmentationin a phylogenetic context, we used the phylogenetic relationshipsinferred by Jacobsen et al. (2010) based on six Z-linked nuclear introns.For our analyses, we used the maximum posterior probability treefrom that study, which was produced in MrBayes 3.1 (Ronquist andHuelsenbeck, 2003). This tree exhibits minor differences in topologycompared to the mitochrondrial tree (Omland et al., 1999), particularlyin clade C. As the topology of amore recentmulti-locus species tree alsodiffers in clade C (Jacobsen and Omland, 2011), we compared ancestral

β-carotene

β-cryptoxant

zeaxanthin(ZXN)

3-dehydroluteincanary xanthophyll B(CXB)

canary xanthophyll A(CXA)

lutein(LUT)

Dietary YeCaroteno

Carotenoids

Legend

C3-oxygenation

C4-oxygenation

change observedin this study

Modified Yellow

Fig. 1. Carotenoid structures and hypothesizedmetabolic pathways. Abbreviated diagrams of cafor their metabolism adapted from Andersson et al. (2007). Central polyene groups are consobserved in this study have abbreviations below that are used throughout. Arrows pointing to(C3-oxygenation), while arrows pointing to the right indicate the addition of a ketone group aresponsible for gains of orange plumage coloration in orioles are shown as open arrows.

state reconstructions in clade C across these three published phyloge-nies. For use in likelihood methods (which cannot accept taxa withmissing data), we included only species for whichwe collected pigmentdata.

We reconstructed ancestral states for color and pigment charac-ters (Table 1) using both parsimony and likelihood-based inferencemethods inMesquite 2.74 (Maddison andMaddison, 2010). Ancestralstate reconstruction requires the estimation of many parameters,and may be vulnerable to violations of its assumptions (e.g., thatthe rate model used is accurate; Omland, 1999; Wiens et al., 2007).To address this, we compared ancestral states across 1-parameter

echinenone(ECH)

canthaxanthin(CXN)

hin 3-hydroxy-echinenone(3HE)

adonirubin

β-doradexanthin astaxanthin

α-doradexanthin

llowids (Keto-carotenoids)

Modified Red Carotenoids

rotenoid pigments commonly observed in bird and the hypothesized reactions responsibletant across the compounds shown, and are here replaced by a dashed line. Compoundsthe left indicate the addition of a ketone group at the C3 position on the carotenoid ringt the C4 position on the carotenoid ring (C4-oxygenation). Reactions hypothesized to be

0 2 4 6 8 10

520

540

[Keto-carotenoids] (µg/g)

Spe

ctra

l Loc

atio

n (n

m) 55

053

0

ECH CXN,ECHCXN,3HE

Lutein only

Keto- presentdogrprme

peni

cu

crgu

pu

buga

Fig. 2. Ketocarotenoid concentration versus carotenoidhue. Comparison of breast plumagecoloration and the total concentration of keto-carotenoids in feathers sampled from thatsame plumage across oriole species. Points filled dark gray denote species in which keto-carotenoids were observed as present; points filled light gray denote species in whichketo-carotenoids were observed as absent. Labels indicate which keto-carotenoids wereobserved in each species (see Table 1 for key to abbreviations). Note that echinenone(ECH) was observed in an unquantifiable concentration in I. croconotus. The dotted lineat 540 nm refers to the arbitrary cutoff for the discrete character states yellow and orangedescribed in Friedman et al. (2011; see text).

4 N.R. Friedman et al. / Comparative Biochemistry and Physiology, Part B 172–173 (2014) 1–8

(Lewis, 2001) and 2-parameter (Pagel, 1994) rate models estimatedfrom data in this study and estimated from amore thorough taxonomicsampling by Friedman et al. (2011).

We used phylogenetic comparative methods to study the relation-ships between pigment and color characters. Using the ouch package(version 2.8; King and Butler, 2009) in R (version 3.0.1; R Core Team,2013), we tested whether the Ornstein–Uhlenbeck model (Butler andKing, 2004) was a significantly better fit to our data and tree thanthe Brownian Motion (BM) model; it was not. Consequently, we usedPhylogenetic Generalized Least Squares (PGLS; Grafen, 1989) under aBM model in the ape package (version 3.0; Paradis et al., 2004) to testrelationships among continuous and discrete characters. To examinerelationships solely among discrete characters, we used Pagel's Discretetest (Pagel, 1994) as implemented in Mesquite 2.74. Together, thesemethods correct for the phylogenetic non-independence of compara-tive data. By combining multiple scoring and comparative methods,we test for correlated evolution of traits in a way that is robust to thelimitations of a single method (Freckleton, 2009).

3. Results

3.1. Carotenoid compounds observed

We detected the presence of ten distinct carotenoid compounds inoriole plumage, all of which were xanthophylls (Table 1). Based onprior descriptions of avian diets (see McGraw, 2006), we suspect thattwo of these pigments are dietary in origin: lutein and zeaxanthin.Lutein was themajor extraction product in the plumage of each speciesexamined in this study (concentration: mean 103.67 ± 37.35 μg/g).We detected zeaxanthin in low to intermediate concentrations inthe Altamira Oriole (Icterus gularis) and the Streak-backed Oriole(I. pustulatus; Table 1).

We observed keto-carotenoids at low total concentrations (mean4.7 ± 5.02 μg/g) in the plumage of five species examined in thisstudy, each of which exhibited plumage scored as “orange”. Amongclade C orioles, we observed four of six species with keto-carotenoid-containing plumage. The Baltimore Oriole (I. galbula) and Bullock'sOriole (I. bullockii) had plumage containing canthaxanthin andechinenone, while I. gularis and I. pustulatus had plumage containingcanthaxanthin and 3-hydroxy-echinenone (Table 1). In the plumageof the Orange-backed Troupial (I. croconotus), examination of chroma-tography data suggested the presence of echinenone in a low, unquan-tifiable concentration (i.e., at the detection limit of the instrument).

3.2. Color and keto-carotenoid concentration

Reflectance spectrometric analyses revealed five species ashaving “orange” coloration (Table 1). We found keto-carotenoids in theplumage of each of these species. While these keto-carotenoids showedconsiderable variation in their concentration, keto-carotenoid concentra-tion did not appear to explain color variation among species with “or-ange” coloration (Fig. 2). Furthermore, the types of keto-carotenoidcompounds present in each species' plumage did not appear to explaincolor variation among species with “orange” coloration (Fig. 2).

Phylogenetic comparative methods revealed that carotenoid hue(scored discretely as yellow or orange) and the presence or absence ofketo-carotenoids were perfectly correlated among species withoutphylogenetic correction (Fig. 3; Table 2). Keto-carotenoidswere presentin each “orange” species, and absent in every “yellow” species. UsingPagel's (1994) discrete method, we found a significant correlationbetween these two characters (df = 8, p b 0.05). With PGLS, we alsofound significant correlations between total keto-carotenoid concentra-tion and both spectral location (p b 0.05) and discrete hue (p = 0.01;Table 2). Furthermore, we found a strong correlation between the pres-ence or absence of keto-carotenoids and spectral location (p b 0.001).However, there was little evidence for any relationship between total

carotenoid concentration and either discrete or continuous scoring ofhue (Table 2).

3.3. Ancestral state reconstruction

Each reconstruction method used in this study supported thatlutein was present in the oriole common ancestor. We found two in-dependent gains and one loss of “orange” coloration using unorderedparsimony (Fig. 3). However, several likelihood reconstructionmethods conflicted with this result: ancestral state reconstructionusing a 2-parameter likelihood model showed orange coloration asancestral to clade C with two subsequent losses, and reconstructionusing a 1-parameter likelihood model was uninformative due to aflat likelihood surface. The other yellow xanthophylls found in thisstudy, zeaxanthin and the canary xanthophylls A and B, were recon-structed as gained in the clade C orioles, but not in clade B orioles.Canary xanthophylls A and B were most likely gained concurrentlywith orange coloration in clade C, as inferred by both parsimonyand likelihood. Zeaxanthin was most likely either gained twice inde-pendently in I. gularis and I. pustulatus, or gained once in their com-mon ancestor with a subsequent loss in I. nigrogularis.

Of the keto-carotenoids found in oriole feathers, ancestral statereconstruction showed canthaxanthin as gained concurrently with“orange” coloration in the clade C orioles (Fig. 3), but not in clade Borioles. Ancestral states for echinenone and 3-hydroxy-echinenonewere reconstructed with considerable uncertainty individually. Un-ordered parsimony was uninformative for both characters withinclade C. Likelihood methods showed repeated terminal gains ofboth characters in clade C orioles. However, the composite characterbased on the presence or absence of keto-carotenoids (see Table 1;reconstructed in Fig. 3) showed the most resolved reconstructionof keto-carotenoids across the New World orioles, with a transitionfrom producing echinenone to producing 3-hydroxy-echinenoneoccurring in clade C.

4. Discussion

4.1. Carotenoid pigments in oriole feathers

We found that gains and losses of keto-carotenoids best explainedvariation in carotenoid-based breast coloration among orioles, a

I. graduacauda

I. bullockii

I. galbula

I. pustulatus

I. gularis

I. nigrogularis

I. croconotus

I. mesomelas

I. pectoralis

I. cucullatus

I. prosthemelas

I. dominicensis

+ CXN, CXA, CXB, ECH

+ 3HE, ZXN- ECH

- ALL KETO, ZXN

+ ECH

LUT Present in MRCA

Clade C

Clade B

Clade A

Fig. 3. Ancestral state reconstruction of color and carotenoid pigmentation. Parsimony reconstruction of ancestral color and pigment character states on the Jacobsen et al. (2010) Icterusphylogeny, with un-sampled taxa pruned, and using a composite of keto-carotenoid characters. The shade of each branch and node represents the color of its inferred ancestral state; darkgray represents orange plumage coloration, and light gray represents yellow. Gains of carotenoid pigments are indicated under each branch in blackwith a “+”, while losses are indicatedin red with a “–”. The most recent common ancestor (MRCA) of the New World orioles was reconstructed with yellow plumage and the presence of lutein. Parsimony reconstructionsof independent keto-carotenoid characters show equivocal reconstructions for ECH and 3HE, while likelihood reconstructions of these characters show them as repeatedly gained. Allketo-carotenoids were reconstructed as lost in I. nigrogularis lineage, as denoted on the tree by “– all keto”.

5N.R. Friedman et al. / Comparative Biochemistry and Physiology, Part B 172–173 (2014) 1–8

conclusion strongly supported by significant correlations between ca-rotenoid presence and hue, as inferred by multiple methods of scoringand analysis (Table 2). Each gain of “orange” coloration saw a concur-rent gain of keto-carotenoids (Fig. 3). Continuous measures of keto-carotenoid concentration were also correlated with coloration (PGLS,p b 0.05). However, variation in keto-carotenoid concentration did notappear to explain variation in hue among the subset of taxa that are“orange” (Fig. 2). Furthermore, keto-carotenoids were absent or unde-tectable in all taxa with an observed spectral location value less than540 nm, including those described as “orange–yellow” in the literature(I. cucullatus, I. pectoralis; Jaramillo and Burke, 1999). This finding is

Table 2Results of comparative phylogenetic analyses using phylogenetic generalized leastsquares (PGLS) and Pagel's discrete method (Pagel, 1994). Explanatory variables arelisted on the left and response variables on the right. [Total] = Total carotenoidconcentration. [Keto] = Total keto-carotenoid concentration. Unstandardized coefficients(b) are reported with their standard error (SE).

Variables Test b (SE) P Relationship

Discrete keto vs. discrete color Pagel 94 N/A b0.05 +Discrete keto vs. spectral location PGLS 32.17 (3.24) b0.01 +[Keto] vs. spectral location PGLS 2.75 (1.16) 0.04 +[Total] vs. spectral location PGLS 0.00 (0.00) 0.38 +[Keto] vs. discrete color PGLS 0.1 (0.03) 0.01 +[Total] vs. discrete color PGLS 0.00 (0.00) 0.23 +

similar to results from similar studies in blackbirds and in widowbirds(Prager and Andersson, 2009; Friedman et al., 2013) in that gains ofketo-carotenoids produced by C4-oxygenation are likely responsiblefor changes in carotenoid hue.

Previous studies have described color variation among oriole speciesas continuous and unimodal (Hofmann et al., 2006; Friedman et al.,2011). Consequently, we had expected that continuous variation ofketo-carotenoid concentration would explain color variation amongorioles. Variation in pigment concentration has been reported to controlintraspecific variation in hue in several species, including BaltimoreOrioles (I. galbula; McGraw and Gregory, 2004; McGraw et al., 2006;Hudon et al., 2013). Furthermore, Andersson et al. (2007) reported ashift in carotenoid hue towards longer wavelengths in widowbirds asthe result of a substantial increase in carotenoid concentration.

However, our findings suggest that orioles instead gained orangeplumage coloration by discrete changes in the pigments used. Specifi-cally, we found repeated gains of C4-oxygenation from an ancestorthat lacked this trait. This result has important implications for themolecular mechanisms of color variation. The primary role of gainsand losses of C4-ketocarotenoids in oriole color variation suggests thatdiscrete changes in the presence or absence of a C4-oxygenase gene'sactivity, as opposed to continuous changes in its expression, havebeen responsible for the evolution of carotenoid hue variation in orioles.As keto-carotenoids are (and have long been) present within oildroplets in the avian retina (Toomey and McGraw, 2009), the neo-

Taxon Specimen # Museum Locality

Icterus bullockii 44399 DMNH USA, Kansas, Morton Co.Icterus bullockii 44405 DMNH USA, Idaho, Latah Co.,

MoscowIcterus bullockii 44723 ANSP USA, California, San DiegoIcterus bullockii 182969 ANSP USA, Texas, TomGreen Co.Icterus bullockii 182970 ANSP USA, Texas, TomGreen Co.Icterus croconotus strictifrons 111815 ANSP Brazil, DescalvadosIcterus croconotus strictifrons 111818 ANSP Brazil, DescalvadosIcterus croconotus strictifrons 119411 ANSP Bolivia, ChataronaIcterus cucullatus cucullatus 40817 ANSP USA, Texas, BrownsvilleIcterus cucullatus cucullatus 40818 ANSP USA, Texas, BrownsvilleIcterus cucullatus cucullatus 40824 ANSP USA, Texas, BrownsvilleIcterus cucullatus nelsoni 8306 DMNH USA, Arizona, Fort LowellIcterus cucullatus nelsoni 44217 DMNH USA, Arizona, Pima Co.,

TusconIcterus cucullatus nelsoni 45130 DMNH USA, Arizona, Pima Co.,

TusconIcterus dominicensis 3458 ANSP Dominican Republic,

San DomingoIcterus dominicensis 25105 ANSP Dominican Republic,

San DomingoIcterus dominicensis 35479 ANSP Dominican Republic,

San DomingoIcterus galbula 28095 ANSP USA, Iowa, Winnebago Co.Icterus galbula 42697 DMNH USA,Maine, Kennebec Co.

6 N.R. Friedman et al. / Comparative Biochemistry and Physiology, Part B 172–173 (2014) 1–8

localization of retinal C4-oxygenase expression to developing featherfollicles is a compelling possible pathway to explain the evolution oforange or red coloration.

4.2. Why no red orioles?

Orange plumage in orioles is caused by the deposition of smallconcentrations of keto-carotenoid compounds into feathers, as in theGuianan Cock-of-the-rock (Rupicola rupicola, Prum et al., 2012). Thesesame compounds, when found in slightly larger concentrations in thefeathers of other Icterid species, cause red coloration (Friedman et al.,2013). This finding raises a perplexing question: why have no NewWorld oriole species evolved red coloration? We propose two hy-potheses below that explore the evolutionary causes of this aspectof color evolution in orioles: one based on selection, and one based onconstraint.

First, selectionmay favor orange coloration over red for one of manyreasons. Sensory bias towards orange objects mightmake orange plum-age particularly stimulating to potential mates or rivals (Endler andBasolo, 1998; Murphy et al., 2009). Alternatively, a genetic correlationbetween trait and preference couldmaintain orange coloration as an ar-bitrary ornament in orioles, as with yellow coloration in Spinus tristis(Hill and McGraw, 2004; Prum, 2010). A good genes model ofintersexual selection appears to be a poor fit to our observations in Icter-us: if red coloration is the optimal means for honestly signaling condi-tion using carotenoids (Hill, 1996), its conspicuous absence in theoriole genus requires explanation. Second, biochemical mechanismsmay constrain color evolution in this group, either by environmentalor physiological limitation of keto-carotenoid production. Environmen-tal limitation seems unlikely in this case however, as orioles' successfuluptake of xanthophyll precursor compounds is evidenced by the depo-sition of high concentrations of these compounds in feathers. However,it is possible that such efficient deposition of xanthophylls could com-pete with their use as precursors for keto-carotenoid production, andthus provide a physiological limit to the redness of oriole plumage.

4.3. History of carotenoid pigmentation in orioles

Our ancestral state reconstruction results generally show repeatedgains of C4-oxygenation from an oriole common ancestor that lackedthis ability (Fig. 3). Keto-carotenoid pigment types differed amongspecies in clade C, and between clades B and C. These independentgains of keto-carotenoids in twodifferent oriole cladesmay be the resultof either parallel or convergent evolution (sensu strictu; Smith andRausher, 2011; Friedman et al., 2013). If the same enzyme performsC4-oxygenation ofβ-carotene,β-cryptoxanthin, and echinenone to pro-duce all the keto-carotenoids observed in this study, then our resultswould support a conclusion of parallel evolution of orange coloration.Within clade C, our parsimony reconstruction of the composite caroten-oid character suggests that there has been one gain of orange coloration,with a switch from echinenone to 3-hydroxy-echinenone in the com-mon ancestor of I. gularis and I. pustulatus.

Ancestral state reconstruction methods are varied, parameter-intensive, and potentially misleading when their assumptions are vio-lated (Wiens et al., 2007). These assumptions include using the correctphylogeny, the correct model of evolution, and the correct scoringof characters as composite or independent (McLennan and Brooks,1993; Omland, 1999). While our results appear to be sensitive to rea-sonable perturbations in several of these parameters, they are robustin their agreement on concurrent gains of orange coloration and keto-carotenoid pigmentation: they show one gain of keto-carotenoidsin an ancestor of clade C, and one gain of keto-carotenoids in clade B.Indeed, we found that multiple topologies, multiple reconstructionmethods, and multiple comparative methods all indicate that gainsand losses of orange coloration in orioles are the result of discretegains and losses of keto-carotenoids.

5. Conclusions

Together with our results in a similar study of the close relatives oforioles, caciques and meadowlarks (Friedman et al., 2013), we haveidentified the proximate mechanisms responsible for five independentshifts from yellow towards orange or red plumage coloration. Oriolesappear to have evolved keto-carotenoid production through metabo-lism of β-carotene and β-cryptoxanthin, but not lutein and zeaxanthinas in meadowlarks and caciques (Andersson et al., 2007; Friedmanet al., 2013). Thus, we have described the evolution of several pig-mentation mechanisms that are convergent in their use of differentpigments, butmay still be parallel in their use of C4-oxygenation to pro-duce keto-carotenoids. Future studies using novelmolecular techniquesmay be able to address this distinction between parallel and convergentgains at the genetic level (Pointer et al., 2012;Walsh et al., 2012). How-ever, this study and those that precede it (Friedman et al., 2011;Friedman et al., 2013) have used newmethods of analysis and inferenceto describe a detailed history of carotenoid coloration in the Icteridae. Byusing the present to infer the past, phylogenetic comparative methodsand ancestral state reconstruction are a more useful tool for under-standing past visual signals and behaviors than fossils may ever be(Cunningham et al., 1998), especially for forms of color like carotenoidpigmentation that do not preserve as well as other types (i.e., melanin).Here, we have used such methods to produce a detailed phylogeneticand mechanistic description of carotenoid color evolution, thus provid-ing the phylogenetic framework necessary for future studies to examinethe functional causes underlying the repeated evolution of carotenoid-based coloration.

Acknowledgments

The authors would like to thank Jean Woods and the DelawareMuseum of Natural History, and Nate Rice and the Academy of NaturalSciences in Philadelphia for their support in this project. The authorswould also like to thank M. Toomey and M. Rowe for their assistancewith HPLC analysis. NRF was supported by grants from Sigma Xi andthe Maryland Ornithological Society, and project no. CZ.1.07/2.3.00/30.0041. KEO was supported by a National Science Foundation CAREERgrant DEB-0347083 and DEB-1119506.

Appendix A

(continued)

Taxon Specimen # Museum Locality

Icterus galbula 42706 DMNH USA, Minnesota,Fillmore Co.

Icterus galbula 65963 ANSP USA, Delaware, RehobothIcterus galbula 187398 ANSP USA, New Jersey,

Salem Co.Icterus graduacauda audobonii 40795 ANSP USA, Texas, BellvilleIcterus graduacauda audobonii 65695 ANSP USA, Texas, BellvilleIcterus graduacauda audobonii 34844 DMNH Mexico, GuerreroIcterus graduacauda audobonii 44410 DMNH USA, Texas, Webb Co.,

LaredoIcterus graduacauda audobonii 44412 DMNH Mexico, Nuevo Leon,

MonterreyIcterus graduacauda graduacauda 35486 ANSP UnknownIcterus gularis gularis 129106 ANSP Mexico, Oaxaca, ChivelaIcterus gularis tamaulipensis 44609 DMNH Mexico, TamaulipasIcterus gularis tamaulipensis 44614 DMNH Mexico, TamaulipasIcterus gularis tamaulipensis 44615 DMNH Mexico, VeracruzIcterus mesomelas taczanowski 108092 ANSP Peru, ChagúalIcterus mesomelas taczanowski 116521 ANSP Peru, La LajaIcterus mesomelas taczanowski 116523 ANSP Peru, PalambaIcterus nigrogularis 54428 ANSP UnknownIcterus nigrogularis 58655 ANSP Venezuela, Buette TristeIcterus nigrogularis 63340 ANSP Colombia, Santa MartaIcterus pectoralis 30821 DMNH Mexico, Oaxaca, San Ga-

brielIcterus pectoralis 30831 DMNH Mexico, Guerrero, Tres

PalosIcterus pectoralis 30839 DMNH Mexico, Guerrero,

JoluchucaIcterus prosthemelas 76837 ANSP Nicaragua, PrinzapolkeIcterus prosthemelas 85971 ANSP Panama, ChanguinolaIcterus prosthemelas 91033 ANSP Honduras, LancetillaIcterus pustulatus pustulatus 17246 DMNH Mexico, Colima, ColimaIcterus pustulatus pustulatus 27410 DMNH Mexico, GuerreroIcterus pustulatus pustulatus 27411 DMNH Mexico, Guerrero

Appendix A (continued)

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