RESEARCH ARTICLE

Impairment of mixed melanin-based pigmentation in parrotsAna Carolina de Oliveira Neves1, Ismael Galván2,* and Dirk Van den Abeele3

ABSTRACTParrots and allies (Order Psittaciformes) have evolved an exclusivecapacity to synthesize polyene pigments called psittacofulvins atfeather follicles, which allows them to produce a striking diversity ofpigmentation phenotypes. Melanins are polymers constituting themost abundant pigments in animals, and the sulphurated form(pheomelanin) produces colors that are similar to those produced bypsittacofulvins. However, the differential contribution of these pigmentsto psittaciform phenotypic diversity has not been investigated. Giventhe color redundancy, and physiological limitations associated withpheomelanin synthesis, we hypothesized that the latter would beavoided by psittaciform birds. Here, we tested this using Ramanspectroscopy to identify pigments in feathers exhibiting colorssuspected of being produced by pheomelanin (i.e. dull red, yellow,greyish-brown and greenish-brown) in 26 species from the three mainlineages of Psittaciformes. We detected the non-sulphurated melaninform (eumelanin) in black, grey and brown plumage patches, andpsittacofulvins in red, yellow and green patches, but there was noevidence of pheomelanin. As natural melanins are assumed to becomposed of eumelanin and pheomelanin in varying ratios, our resultsrepresent the first report of impairment of mixed melanin-basedpigmentation in animals. Given that psittaciforms also avoid the uptakeof circulating carotenoid pigments, these birds seem to have evolved acapacity to avoid functional redundancy between pigments, likely byregulating follicular gene expression. Our study provides the firstvibrational characterization of different psittacofulvin-based colors andthus helps to determine the relative polyene chain length in thesepigments, which is related to their antireductant protection activity.

KEYWORDS: Pheomelanin, Color redundancy, Plumage coloration,Polyenes, Psittacofulvin, Raman spectroscopy

INTRODUCTIONVirtually all organisms have evolved pigmentation based onmelanins, mainly owing to the benefits derived from theirbroadband absorbance properties and capacity to protect cellsagainst the damaging effects of solar ultraviolet (UV) radiation(Brenner and Hearing, 2008). Animal melanins occur in two primaryforms: eumelanin, polymers of indole units, and pheomelanin,oligomers of sulfur-containing heterocycles, containing sulfhydrylgroups from the amino acid cysteine (Ito andWakamatsu, 2008). Thischemical heterogeneity is responsible for the optical properties ofmelanins, which provide animals with a wide diversity of colorsranging from black, brown and grey hues generated by eumelanin to

reddish, orange and yellowish hues generated by pheomelanin(Galván and Wakamatsu, 2016). The biosynthesis of melanins isconsidered a mixed process that leads to the formation of botheumelanin and pheomelanin in varying ratios (Ito and Wakamatsu,2008). Indeed, despite the existence of pheomelanin synthesis infishes being unclear (Ito and Wakamatsu, 2003; Kottler et al.,2015), eumelanin and pheomelanin are known to co-occur atdifferent ratios in the integument of molluscs (Speiser et al., 2014),insects (Galván et al., 2015) and all vertebrates including humans(Ito, 2003; d’Ischia et al., 2015; Del Bino et al., 2015).

The apparent wide distribution of both melanin forms in animalssuggests that mixed melanogenesis had an early evolutionary origin.This has probably been favored by the kinetics of the synthesisprocess, which consists of a ‘default’ pathway (i.e. in the absence ofsulfhydryls) that leads to the production of eumelanin from theoxidation of the amino acid tyrosine and subsequent polymerizationof intermediate compounds. However, sulfhydryl groups arealways incorporated into this pathway, leading to the formation ofpheomelanin, as long as cysteine is present in the cells (melanocytes invertebrates) above a certain threshold concentration (Ito andWakamatsu, 2008). Cysteine and its metabolites play a role inseveral essential processes, ranging from energy supplementation toantioxidant protection; thus, cysteine is prevalent in cells (Wu et al.,2004; Lambert et al., 2015; Bender and Martinou, 2016). Therefore,the kinetics of melanin synthesis seems to easily favor mixedmelanogenesis in cells, and it is not likely that pheomelanin hasexperiencedmany evolutionary losses, if any. In fact, the presence of aunique form of melanin has not been reported in the pigmentation ofany vertebrate class. Mixed melanogenesis seems to be prevalent inanimals, most notably in vertebrates. However, nothing is knownabout possible evolutionary losses of the mixed pigmentation processwithin animal classes.

The plumage coloration of birds (Class Aves) is one of the mostdiverse phenotypes in nature, and melanins are the most abundantpigments that contribute to it (Galván and Solano, 2016). However,some orders or families of birds have evolved a biochemical abilityto synthesize unique pigments, such as the porphyrins turacin andturacoverdin in turacos (Order Musophagiformes) (Church, 1892),spheniscins in penguins (Order Sphenisciformes) (Thomas et al.,2013), vitamin A in tropical starlings (Family Sturnidae) (Galvánet al., 2019) and psittacofulvins in parrots and allies (OrderPsittaciformes) (Stradi et al., 2001). This exclusivity of pigmentsallowed the evolution of conspicuous color phenotypes in thesebirds, which in some groups such as parrots is associated with astrikingly high color diversity (Martin, 2002; Berg andBennett, 2010). Some of the colors resulting from these exclusivepigments recall those resulting from melanins (Toral et al., 2008),and as pigment synthesis entails the use of limiting resources andphysiological costs (Galván and Solano, 2015), herewe hypothesizethat the evolution of novel metabolic pathways to pigmentation mayhave favored the loss of mixed melanin-based pigmentation owingto the benefits of reducing metabolic costs and the absence ofbenefits of the functional redundancy of pigments.Received 31 March 2020; Accepted 5 May 2020

1Institute of Chemistry, Federal University of Rio Grande do Norte, 59072-970 Natal,Brazil. 2Department of Evolutionary Ecology, Don ̃ana Biological Station, CSIC,41092 Sevilla, Spain. 3Ornitho-Genetics VZW, 9260 Wichelen, Belgium.

*Author for correspondence (galvan@ebd.csic.es)

A.C. de O.N., 0000-0001-7741-8454; I.G., 0000-0002-6523-8592

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© 2020. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2020) 223, jeb225912. doi:10.1242/jeb.225912

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In particular, we predict that the exclusive evolution inpsittaciform birds of polyene pigments called psittacofulvins,which create yellow, orange and red plumage coloration (McGrawand Nogare, 2005), may have promoted the evolutionary loss ofpheomelanin, given that this pigment also creates (albeit duller)yellow, orange and reddish plumage coloration (Toral et al., 2008;Galván and Wakamatsu, 2016) and that its synthesis implies thereduction of the availability of the precursor of the main intracellularantioxidant (glutathione, GSH) in melanocytes (i.e. cysteine;Galván, 2018). Indeed, psittacofulvin evolution has promoted theloss of plumage coloration based on other polyenic but dietaryacquired pigments also able to create similar plumage coloration(i.e. carotenoids) despite being present at high circulating levels inparrots (McGraw and Nogare, 2004). An absence of pheomelanin in

the feathers of parrots would represent the first report of impairedmixed melanogenesis in animals. Here, we tested this hypothesisusing Raman spectroscopy to identify the pigments responible forplumage coloration in 26 species belonging to the three mainlineages of Psittaciformes (Psittacoidea, Cacatuoidea andStrigopoidea) and displaying color hues suspected of beingproduced by pheomelanin, i.e. dull yellow, orange, reddish andbrown coloration (Galván and Wakamatsu, 2016).

MATERIALS AND METHODSSpecies selection and feather samplingPsittaciform species were selected on the basis of plumage patchesof colors suspected of being produced by pheomelanin. Althoughpheomelanin produces yellow, orange and red hues similar to thoseproduced by psittacofulvins, the colors produced by pheomelaninare not bright, but dull (Galván and Wakamatsu, 2016). Thus, weselected 26 psittaciform species whose plumage included a patchdisplaying dull yellow, orange, reddish or brown coloration, whoselow level of brightness makes them likely candidates to be producedby pheomelanin (Fig. 1, Table 1). We also included some greyish-brown as well as green plumage patches to explore a possiblepresence of brown barbules in feathers (Table 1). The selectedspecies covered a high phylogenetic diversity, thus being asignificant representation of the Order Psittaciformes (Fig. 2). Forsimplicity, the plumage patch colors are here described as red,yellow, green, grey and brown (Table 1).

The selection of species was made by examining bookillustrations (Juniper and Parr, 2001) and with the advice of parrotcaptive breeders in Belgium and The Netherlands, who alsoprovided feather samples. We included two mutations that appear incaptivity in two species, the whiteface mutation of the cockatielNymphicus hollandicus and the opaline mutation of Bourke’sparrot, Neopsephotus bourkii, as mutated birds exhibit changes inplumage pigmentation potentially owing to the synthesis ofpheomelanin (Martin, 2002) (Fig. 1). The whiteface mutation isinherited as an autosomal recessive mutation, while the opalinemutation is inherited as a sex-linked recessive mutation (Van denAbeele, 2016; van der Zwan et al., 2019).

One or two feathers from the studied plumage patches werecollected from an adult bird of each species and analyzed for

Fig. 1. Images of psittaciform species that were sampled for feathersduring the study. The species belong to the families Cacatuidae [1:Calyptorhynchus banksia; 2:Nymphicus hollandicus (wild type); 3:Nymphicushollandicus (whiteface mutation)], Psittacidae (4: Amazona leucocephala;5: Ara severus; 6: Aratinga weddelli; 7: Aratinga auricapillus; 8: Deroptyusaccipitrinus; 9: Enicognathus leptorhynchus; 10: Forpus coelestis; 11: Pionuschalcopterus; 12: Primolius auricollis; 13: Primolius maracana; 14: Pyrrhuracruentata; 15: Pyrrhura egregia), Psittaculidae [16: Eclectus roratus;17: Agapornis nigrigenis; 18: Chalcopsitta duivenbodei; 19: Chalcopsittascintillate; 20: Neopsephotus bourkii (wild type); 21: Neopsephotus bourkii(opaline mutation); 22: Psephotus haematonotus; 23: Pseudeos fuscata;24: Psittacula cyanocephala; 25: Psittacula eupatria], Psittrichasiidae(26: Coracopsis nigra) and Nestoridae (27: Nestor notabilis; 28: Nestormeridionalis). The color plumage patches that were studied in each species aredescribed in Table 1. Photo credits: Dirk Van den Abeele (images 2, 3, 10 and17), Danny Roels (with permission; images 1, 8, 9, 20-22 and 24) and PhilippeRocher (with permission; images 4, 6, 7, 11, 14, 16 and 19). The other imagesare under CC BY-SA license [image 26 (Hedwig Storch): https://creativecommons.org/licenses/by-sa/1.0; images 5 (Eric Savage),12 (Bernard Dupont) and 18 (Thomas Quine): https://creativecommons.org/licenses/by-sa/2.0; image 23 (Doug Janson): https://creativecommons.org/licenses/by-sa/3.0; images 13 (Etemenanki3), 15 (Gazelle74), 25 (RajuKasambe) and 28 (Maree McLeod): https://creativecommons.org/licenses/by-sa/4.0]. Image 28 is in the public domain.

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pigment identification using Raman spectroscopy (see below). Theuse of one or two samples from adults per species has been provensufficient to characterize the pigmentation phenotype of bird species(Galván et al., 2018a). All feather samples were collected from birdsbred and kept in captivity. The breeders had all necessary permits tohold the birds following guidelines from Belgian and Dutchauthorities.

Raman spectroscopyWe used a Thermo Fisher DXR confocal dispersive Ramanmicroscope (Thermo Fisher Scientific, Madison, WI, USA)operating at the National Museum of Natural Sciences (MNCN,CSIC) in Madrid, Spain, to identify the pigments responsible for thecolor of the studied feathers. Dispersive Raman spectroscopy candetect pigment molecules in solid samples at concentrations as lowas 0.05–0.1% (w/w) (Bouffard et al., 1994; Massonnet et al., 2012).The system has a point-and-shoot Raman capability of 1 µm spatialresolution. We used an excitation laser source at 780 nm and a slitaperture of 25 µm to analyze the wavenumber range of 300–2500 cm−1. When the samples contained psittacofulvins, we used a50× confocal objective lens, a laser power of 7 mW, and anintegration time of 3 s and 12 accumulations. When the samplescontained melanins, to avoid the burning of samples owing to theirdarker color and to optimize results, we used a 100× confocalobjective lens, a laser power of 1 mW, and an integration time of 3 sand 30 accumulations. The systemwas operated with Thermo FisherOMNIC 8.1 software. We analyzed one to two feathers from onebird of each species. One barb and one barbule chosen at randomwere analyzed in each feather. The average Raman spectracorresponding to each pigment were calculated for each species.

Computational analysis including importing and pre-processing(baseline correction and normalization) of data was performed withMATLAB®R2014b (MathWorks Inc., Natick, MA, USA) using thePLS Tollbox version 7.9.3 (Eigenvector Research Inc., USA). Theidentification of pigments was made on the basis of diagnosticvibrational bands. The Raman spectrum of eumelanin showsdefined Raman bands at 1380 and 1580 cm−1 resembling the D andG bands characteristic of disordered graphite, in addition to aweakerband at 500 cm−1 (Huang et al., 2004; Galván et al., 2013a, 2018b).In contrast, the Raman spectrum of pheomelanin shows wideRaman bands at approximately 500, 1490 and 2000 cm−1

(Wang et al., 2016; Polidori et al., 2017; Megía-Palma et al.,2018), and like eumelanin, it can be detected by direct examinationof the surface of feathers by Raman spectroscopy (Galván et al.,2013a,b, 2018b; Galván and Rodríguez-Martínez, 2018).In contrast, the Raman spectrum of psittacofulvins shows twostrong bands at approximately 1130 and 1530 cm−1, and an absenceof the band at approximately 1000 cm−1 owing to the vibration ofthe methyl group; however, this is present in the Raman spectra ofother polyenic pigments such as carotenoids (Maia et al., 2014;Fernandes et al., 2015).

RESULTSWe found Raman signals of eumelanin in nine of the species ormutations included in the study (Fig. 3). These corresponded tothree species with grey plumage patches and two species with brownplumage patches (Table 1, Fig. 1). Additionally, eumelanin wasfound spatially segregated in the red or brown plumage patchesof other three species (the Cuban amazon, Amazonaleucocephala, the chestnut-fronted macaw, Ara severus, and

Table 1. Description of colors and plumage patches whose pigments were investigated in the psittaciform species included in the study

Species Color analyzed Description of plumage patch Pigment

Calyptorhynchus banksii Grey Grey stripes in the breast of females EumelaninNymphicus hollandicus Grey Mantle (grey in wild-type,

grey-brownish in whiteface mutation)Eumelanin (wild-type and whiteface mutation)

Amazona leucocephala Red Red belly Psittacofulvin (red barbs), eumelanin (black barbules)Ara severus Red Red forehead Psittacofulvin (red barbs), eumelanin (black barbules)Aratinga weddelli Grey Head EumelaninAratinga auricapillus Red, green Belly PsittacofulvinDeroptyus accipitrinus Red Red neck PsittacofulvinEnicognathus leptorhynchus Red, green Belly PsittacofulvinForpus coelestis Green Wing PsittacofulvinPionus chalcopterus Red Orange-reddish covert wing feathers PsittacofulvinPrimolius auricollis Red Tail PsittacofulvinPrimolius maracana Red Belly PsittacofulvinPyrrhura cruentata Red Tail PsittacofulvinPyrrhura egregia Red Tail PsittacofulvinEclectus roratus Red Red body feathers in females PsittacofulvinAgapornis nigrigenis Brown Head Psittacofulvin (red barbs), eumelanin (grey barbules)Chalcopsitta duivenbodei Brown Brownish-greenish mantle PsittacofulvinChalcopsitta scintillata Red Forehead PsittacofulvinNeopsephotus bourkii Brown, red Brown mantle in wild-type, red breast in

opaline mutationPsittacofulvin (opaline mutation), eumelanin(wild-type)

Psephotus haematonotus Red Red rump in males PsittacofulvinPseudeos fuscata Red Breast PsittacofulvinPsittacula cyanocephala Red Red shoulder in males PsittacofulvinPsittacula eupatria Red Shoulder PsittacofulvinCoracopsis nigra Brown Breast EumelaninNestor notabilis Yellow, red Under wing primary covert feathers PsittacofulvinNestor meridionalis Yellow Yellowish breast feathers Psittacofulvin

The main pigment identified in the studied color patches by means of Raman spectroscopy is indicated. When not specified, the indicated pigment was detectedin both barbs and barbules of feathers. In the Cuban amazon (Amazona leucocephala), the chestnut-fronted macaw (Ara severus) and the black-cheeked lovebird(Agapornis nigrigenis), the color plumage patches are generated by a spatial segregation of psittacofulvin and eumelanin between barbs and barbules in feathers.

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the black-cheeked lovebird, Agapornis nigrigenis) (Fig. 1), inwhich eumelanin was found in black or grey barbules whilepsittacofulvin was found in red barbs (Table 1). In contrast, noRaman signal of pheomelanin was found in any of the plumagepatches analyzed. This indicates that pheomelanin is not presentin the feathers of psittaciforms, or that it is present in non-significant amounts.Raman signals of psittacofulvin were detected in all red plumage

patches analyzed, corresponding to 18 species (Fig. 4, Table 1).Psittacofulvins were also found to cause dull yellow plumagecoloration in two species (Fig. 5A) and green coloration in sixspecies (Fig. 5B). Psittacofulvin was also found to be the pigmentcausing dark brown coloration in two species (Fig. 5D), the brownlory (Chalcopsitta duivenbodei) and the black-cheeked lovebird(Fig. 1), though in the latter species the brown plumage patch isproduced by a combination of red psittacofulvin-containing barbsand grey eumelanin-containing barbules (Table 1). However, theRaman spectra of yellow, green and brown plumage patches alsoincluded eumelanin signal (Fig. 5), indicating that, whilepsittacofulvin is the main pigment responsible for yellow, greenand brown colors in the studied species, eumelanin is mixed withpsittacofulvin, contributing to the resulting plumage coloration.This is particularly notable in green feathers (Fig. 5B). Thesubtraction of eumelanin signal from the Raman spectra ofpsittacofulvin led to a clear prevalence of the two Raman bandsof psittacofulvin, the shape of the resulting spectra thus beingsimilar to that of red psittacofulvin (Figs 4 and 5).

The Raman spectra of red, yellow, green and brown plumagepatches were remarkably similar, all showing the two diagnosticbands of psittacofulvin. Variation between color groups in thefrequency of the first band (∼1130 cm−1) was notably low, as it waslocated at 1131 cm−1 in the spectra of red feathers, 1134 cm−1 in thespectra of yellow feathers, 1136 cm−1 in the spectra of greenfeathers and 1133 cm−1 in the spectra of brown feathers (Fig. 6).However, variation was more marked in the frequency of the secondband (∼1530 cm−1), being located at 1525 cm−1 in the spectra ofred feathers, 1531 cm−1 in the spectra of yellow feathers, 1536 cm−1

in the spectra of green feathers and 1528 cm−1 in the spectra ofbrown feathers (Fig. 6).

DISCUSSIONOur results show that pheomelanin does not contribute to theplumage pigmentation of psittaciform birds, despite assumptionsmade in some previous studies lacking analytical evidence(Tinbergen et al., 2013; Delhey and Peters, 2017). As we includedin our analyses a significant representation of psittaciform specieswith plumage colors suspected of being produced by pheomelanin(Galván and Wakamatsu, 2016) and from a high phylogeneticdiversity that included the three main lineages of Psittaciformes, theseresults provide evidence that psittaciform birds synthesize eumelaninto pigment feathers black, grey and brown, but do not synthesizepheomelanin or synthesize it at negligible amounts. As naturalmelanins are considered mixed pigments in which eumelanin andpheomelanin are present in varying ratios (Ito andWakamatsu, 2008),

Pseudeos fuscata

Chalcopsitta duivenbodei

Agapornis nigrigenis

Psephotus haematonotus

Neopsephotus bourkii

Psittacula eupatria

Psittacula cyanocephala

Coracopsis nigra

Enicognathus leptorhynchus

Pyrrhura cruentata

Pyrrhura egregia

Primolius maracana

Primolius auricollis

Ara severus

Aratinga auricapillus

Deroptyus accipitrinus

Forpus coelestis

Pionus chalcopterus

Amazona leucocephala

Calyptorhynchus banksii

Nymphicus hollandicus

Nestor notabilis

Nestor meridionalis

Fig. 2. Phylogenetic relationships between the 26 species ofpsittaciform bird species included in the study.Branch lengths areproportional to nucleotide substitutions. The tree is the least-squaresconsensus phylogenetic tree obtained, using the R package phytools(Revell, 2012), from the mean patristic distance matrix of a set of 100probable phylogenies obtained using the phylogeny subsets tool inwww.birdtree.org (Jetz et al., 2012).

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parrots represent the first animals in which an impaired mixedmelanin-based pigmentation system is reported. The presence ofpheomelanin (and eumelanin) in organisms such as molluscs andinsects (Speiser et al., 2014; Galván et al., 2015) and in all vertebrates(d’Ischia et al., 2015; Kottler et al., 2015) suggests that the absence of

pheomelanin in psittaciform birds is an exclusive evolutionary loss inthis order within Aves. Indeed, no other birds are known to lackpheomelanin in their feathers (Galván and Solano, 2016).

Psittaciform birds have evolved an exclusive pigmentationsystem by expressing the MuPKS gene, which codes for a

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Fig. 3. Raw Raman spectra of eumelanin in the feathers of ninepsittaciform species andmutations included in the study. The inset imageshows a mantle body feather from one of the species included, a cockatielNymphicus hollandicus with whiteface mutation.

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Fig. 4. Raw Raman spectra of psittacofulvin in feathers of 18 psittaciformspecies with red plumage coloration included in the species. The insetimage shows a tail feather from one of the species included, an ochre-markedparakeet, Pyrrhura cruentata.

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Fig. 5. Raw Raman spectra (upper) of psittacofulvin and the corresponding normalized spectra after subtracting eumelanin signal (lower) inpsittaciform species with different plumage coloration. (A) Yellow (two species), (B) green (six species) and (C) brown (two species). Inset images showexamples of feathers from these species displaying yellow (under wing primary covert of a kea, Nestor notabilis, also displaying red psittacofulvin; see Fig. 4),green (secondary wing feather of a Pacific parrotlet, Forpus coelestis) and brown (mantle feather of a brown lory, Chalcopsitta duivenbodei) coloration.

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polyketide synthase (Cooke et al., 2017), in feather follicles. Thisleads these birds to synthesize polyene pigments calledpsittacofulvins that produce colors that may be similar in hue tothose produced by pheomelanin (Toral et al., 2008; Galván andWakamatsu, 2016). Given this functional redundancy and thatpheomelanin synthesis may be physiologically limiting underenvironmental stress because it reduces GSH availability inmelanocytes (Galván, 2018), and that psittacofulvin synthesisdoes not seem to imply any comparable physiological limitation, wehypothesized an impairment of mixed melanin-based pigmentationin psittaciforms. Indeed, psittaciforms do not deposit carotenoids(dietary polyene pigments very commonly causing plumagecoloration in other birds) in feather follicles, despite circulatingthem through the blood at high levels (McGraw and Nogare, 2004).The psittacofulvin-based pigmentation system of psittaciforms thusseems to have blocked the physiological activity of otherpigmentation systems.

Interestingly, our results show that psittaciforms do notsynthesize pheomelanin, but synthesize eumelanin, whichproduces dark colors (black, grey and dark brown) not resemblingthose produced by psittacofulvins (Toral et al., 2008; Galván andWakamatsu, 2016). This makes it likely that the avoidance offunctional redundancy between pigments is the evolutionary causethat has favored the impairment of the mixed melanin-basedpigmentation system in psittaciforms. This impairment might beexerted through a regulation of the expression of genes controllingpheomelanin synthesis in melanocytes at the dermal papillae offeather follicles (Lin et al., 2013). Candidate genes in this regulationare those coding for the antagonist peptides of the melanocortin 1receptor in the membrane of melanocytes, namely agouti-signalling(ASIP) and agouti-related (AGRP) proteins (Nadeau et al., 2008),and also genes that control the availability of cysteine inmelanocytes such as that encoding the cystine/glutamateantiporter xCT (Slc7a11; Chintala et al., 2005) and that encoding

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Fig. 6. Comparison of Raman spectra of psittacofulvin from all plumage color groups included in the study. Red, yellow, green and brown spectracorrespond to the respective groups. Bottom panels show a magnification of the spectral regions around the two main Raman bands of psittacofulvinowing to C–C stretching (∼1130 cm−1) and C=C stretching (∼1530 cm−1) of the polyene chain.

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cystinosin (CTNS; Town et al., 1998). The activation of theMuPKSgene in feather follicles, which is observed in psittaciforms but notin other birds (Cooke et al., 2017), has thus probably led to a co-regulation of other genes, impairing the synthesis of pheomelaninand the uptake of circulating carotenoids (McGraw and Nogare,2004), and promoting a selectivity for the psittacofulvin-basedpigmentation system. Interestingly, this likely gene regulationmechanism in feather follicles would parallel the loss of geneduplicates that is observed when they exhibit functional redundancy(Lynch and Conery, 2000; Cooke et al., 2017). Future studies shouldinvestigate this gene regulation mechanism leading to a prevailingpigmentation system.Our study provides the first vibrational characterization of

psittacofulvins giving rise to different plumage colors.Psittacofulvins giving rise to yellow plumage are composed ofpolyene chains of 14, 16 and 18 carbon atoms in the form ofconjugated fatty acids, whereas psittacofulvins giving rise to redplumage are probably synthesized by reducing yellow ones to formfully conjugated aldehydes of 14, 16, 18 and 20 carbon atom chains(Stradi et al., 2001; Cooke et al., 2017). Psittacofulvins giving rise togreen plumage are composed of yellow psittacofulvins, combinedwith a structural effect of feather morphology (Cooke et al., 2017).Our results show that psittacofulvins can also give rise to brownplumage coloration when combined with eumelanin. We foundgreat similarity in the vibrational spectra of psittacofulvins of thesecolor groups, which suggests that these colors are produced bysimilar mixtures of polyenes of different chain length, as alreadyshown for yellow and red psittacofulvins (see above), but thatcertain chain length values must prevail in each color group to giverise to the observed differences in the absorbance properties, i.e.colors. This is supported by a close examination of the frequency ofthe two Raman bands of psittacofulvins. Although the band atapproximately 1130 cm−1, which is due to C–C stretching in thepolyene chains, is remarkably constant between species andplumage color groups, the band at approximately 1530 cm−1,which is due to C=C stretching (Maia et al., 2014), is more variablebetween groups (see Fig. 6). C=C stretching depends on the lengthof the polyene chain (Maia et al., 2014), and accordingly, a cleardifferentiation can be observed in the frequency of C=C stretchingof red psittacofulvins and that of yellow and green psittacofulvins(Fig. 6), which lack C20 chains (Cooke et al., 2017). Thisobservation is physiologically relevant, because psittacofulvinsare good electron acceptors (Martínez, 2009), and this antireductantactivity seems to increase with chain length in at least polyenals(Tagliazucchi et al., 2006). This suggests that psittacofulvins givingrise to red plumage may exert a lower cellular protection activity thanthose giving rise to yellow or green plumage, or that psittacofulvinsgiving rise to a given color (e.g. yellow) but composed of polyenesof different chain lengths may differ in cellular protection capacity.Our results show that Raman spectroscopy may represent a usefulnon-invasive tool to determine the relative chain length ofpsittacofulvins and thus investigate their physiological potential asantirreductant compounds.In conclusion, psittaciform birds have evolved an impairment of

the mixed melanin-based pigmentation system by blocking,probably by a regulation of genes involved in melanogenesis infollicular melanocytes, the synthesis of pheomelanin. We proposethat this impairment, which adds to the blocking of the uptake ofcirculating carotenoids by feather follicles in these birds, has likelybeen favored by a functional redundancy between pigments. Giventhe apparent solution to this redundancy shown here, it could bepossible that birds may modulate the activity of feather follicles to

maximize the diversity of color phenotypes while minimizing theuse of pigment resources. Future studies should provide furtherdetails about the physiology of psittacofulvin synthesis to determinewhether a comparison of physiological implications betweenpigments can explain the exclusive prevalence of the psittacofulvin-based pigmentation system in Psittaciformes.

AcknowledgementsWe thank Daniel Nuijten, Bert Van Gils, Nico Rosseel, Jef Kenis, Eric Gennissen,Heinz Schnitker and Glenn Ooms for their help in selecting psittaciform species forthe study and for feather sampling. Philippe Rocher and Danny Roels kindly allowedus to use their photographs shown in Fig. 1.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: A.C.d.O.N., I.G., D.V.d.A.; Methodology: A.C.d.O.N., I.G.;Formal analysis: A.C.d.O.N., I.G.; Investigation: A.C.d.O.N., I.G.; Resources:D.V.d.A.; Writing - original draft: A.C.d.O.N., I.G.; Visualization: A.C.d.O.N.;Supervision: I.G.; Funding acquisition: D.V.d.A.

FundingA.C.d.O.N. thanks the Brazilian entity Coordenaça ̃o de Aperfeiçoamento dePessoal de Nıv́el Superior for financial support (PNPD/CAPES). I.G. is a recipient ofa Ramón y Cajal Fellowship (RYC-2012-10237) from the Ministerio de Economıá yCompetitividad (MINECO) of the Spanish Government. D.V.d.A. was funded byOrnitho-Genetics VZW.

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