Impairment of Mixed Melanin-Based Pigmentation in Parrots Ana Carolina De Oliveira Neves1, Ismael Galván2,* and Dirk Van Den Abeele3

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

ABSTRACT reddish, orange and yellowish hues generated by pheomelanin Parrots and allies (Order Psittaciformes) have evolved an exclusive (Galván and Wakamatsu, 2016). The biosynthesis of melanins is capacity to synthesize polyene pigments called psittacofulvins at considered a mixed process that leads to the formation of both feather follicles, which allows them to produce a striking diversity of eumelanin and pheomelanin in varying ratios (Ito and Wakamatsu, pigmentation phenotypes. Melanins are polymers constituting the 2008). Indeed, despite the existence of pheomelanin synthesis in most abundant pigments in animals, and the sulphurated form fishes being unclear (Ito and Wakamatsu, 2003; Kottler et al., (pheomelanin) produces colors that are similar to those produced by 2015), eumelanin and pheomelanin are known to co-occur at psittacofulvins. However, the differential contribution of these pigments different ratios in the integument of molluscs (Speiser et al., 2014), to psittaciform phenotypic diversity has not been investigated. Given insects (Galván et al., 2015) and all vertebrates including humans ’ the color redundancy, and physiological limitations associated with (Ito, 2003; d Ischia et al., 2015; Del Bino et al., 2015). pheomelanin synthesis, we hypothesized that the latter would be The apparent wide distribution of both melanin forms in animals avoided by psittaciform birds. Here, we tested this using Raman suggests that mixed melanogenesis had an early evolutionary origin. spectroscopy to identify pigments in feathers exhibiting colors This has probably been favored by the kinetics of the synthesis ‘ ’ suspected of being produced by pheomelanin (i.e. dull red, yellow, process, which consists of a default pathway (i.e. in the absence of greyish-brown and greenish-brown) in 26 species from the three main sulfhydryls) that leads to the production of eumelanin from the lineages of Psittaciformes. We detected the non-sulphurated melanin oxidation of the amino acid tyrosine and subsequent polymerization form (eumelanin) in black, grey and brown plumage patches, and of intermediate compounds. However, sulfhydryl groups are psittacofulvins in red, yellow and green patches, but there was no always incorporated into this pathway, leading to the formation of evidence of pheomelanin. As natural melanins are assumed to be pheomelanin, as long as cysteine is present in the cells (melanocytes in composed of eumelanin and pheomelanin in varying ratios, our results vertebrates) above a certain threshold concentration (Ito and represent the first report of impairment of mixed melanin-based Wakamatsu, 2008). Cysteine and its metabolites play a role in pigmentation in animals. Given that psittaciforms also avoid the uptake several essential processes, ranging from energy supplementation to of circulating carotenoid pigments, these birds seem to have evolved a antioxidant protection; thus, cysteine is prevalent in cells (Wu et al., capacity to avoid functional redundancy between pigments, likely by 2004; Lambert et al., 2015; Bender and Martinou, 2016). Therefore, regulating follicular gene expression. Our study provides the first the kinetics of melanin synthesis seems to easily favor mixed vibrational characterization of different psittacofulvin-based colors and melanogenesis in cells, and it is not likely that pheomelanin has thus helps to determine the relative polyene chain length in these experienced many evolutionary losses, if any. In fact, the presence of a pigments, which is related to their antireductant protection activity. unique form of melanin has not been reported in the pigmentation of any vertebrate class. Mixed melanogenesis seems to be prevalent in KEY WORDS: Pheomelanin, Color redundancy, Plumage coloration, animals, most notably in vertebrates. However, nothing is known Polyenes, Psittacofulvin, Raman spectroscopy about possible evolutionary losses of the mixed pigmentation process within animal classes. INTRODUCTION The plumage coloration of birds (Class Aves) is one of the most Virtually all organisms have evolved pigmentation based on diverse phenotypes in nature, and melanins are the most abundant melanins, mainly owing to the benefits derived from their pigments that contribute to it (Galván and Solano, 2016). However, broadband absorbance properties and capacity to protect cells some orders or families of birds have evolved a biochemical ability against the damaging effects of solar ultraviolet (UV) radiation to synthesize unique pigments, such as the porphyrins turacin and (Brenner and Hearing, 2008). Animal melanins occur in two primary turacoverdin in turacos (Order Musophagiformes) (Church, 1892), forms: eumelanin, polymers of indole units, and pheomelanin, spheniscins in penguins (Order Sphenisciformes) (Thomas et al., oligomers of sulfur-containing heterocycles, containing sulfhydryl 2013), vitamin A in tropical starlings (Family Sturnidae) (Galván groups from the amino acid cysteine (Ito and Wakamatsu, 2008). This et al., 2019) and psittacofulvins in parrots and allies (Order chemical heterogeneity is responsible for the optical properties of Psittaciformes) (Stradi et al., 2001). This exclusivity of pigments melanins, which provide animals with a wide diversity of colors allowed the evolution of conspicuous color phenotypes in these ranging from black, brown and grey hues generated by eumelanin to birds, which in some groups such as parrots is associated with a strikingly high color diversity (Martin, 2002; Berg and Bennett, 2010). Some of the colors resulting from these exclusive 1Institute of Chemistry, Federal University of Rio Grande do Norte, 59072-970 Natal, pigments recall those resulting from melanins (Toral et al., 2008), Brazil. 2Department of Evolutionary Ecology, Doñana Biological Station, CSIC, 41092 Sevilla, Spain. 3Ornitho-Genetics VZW, 9260 Wichelen, Belgium. and as pigment synthesis entails the use of limiting resources and physiological costs (Galván and Solano, 2015), here we hypothesize *Author for correspondence ([email protected]) that the evolution of novel metabolic pathways to pigmentation may A.C. de O.N., 0000-0001-7741-8454; I.G., 0000-0002-6523-8592 have favored the loss of mixed melanin-based pigmentation owing to the benefits of reducing metabolic costs and the absence of

Received 31 March 2020; Accepted 5 May 2020 benefits of the functional redundancy of pigments. Journal of Experimental Biology

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Fig. 1. Images of psittaciform species that were sampled for feathers during the study. The species belong to the families Cacatuidae [1: Calyptorhynchus banksia;2:Nymphicus hollandicus (wild type); 3: Nymphicus hollandicus (whiteface mutation)], Psittacidae (4: Amazona leucocephala; 5: Ara severus;6:Aratinga weddelli;7:Aratinga auricapillus;8:Deroptyus accipitrinus;9:Enicognathus leptorhynchus; 10: Forpus coelestis; 11: Pionus chalcopterus; 12: Primolius auricollis; 13: Primolius maracana; 14: Pyrrhura cruentata; 15: Pyrrhura egregia), Psittaculidae [16: Eclectus roratus; 17: Agapornis nigrigenis; 18: Chalcopsitta duivenbodei; 19: Chalcopsitta scintillate; 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: Nestor meridionalis). The color plumage patches that were studied in each species are described in Table 1. Photo credits: Dirk Van den Abeele (images 2, 3, 10 and 17), Danny Roels (with permission; images 1, 8, 9, 20-22 and 24) and Philippe Rocher (with permission; images 4, 6, 7, 11, 14, 16 and 19). The other images are 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 (Raju Kasambe) and 28 (Maree McLeod): https://creativecommons.org/licenses/ by-sa/4.0]. Image 28 is in the public domain.

the feathers of parrots would represent the first report of impaired mixed melanogenesis in animals. Here, we tested this hypothesis using Raman spectroscopy to identify the pigments responible for plumage coloration in 26 species belonging to the three main lineages of Psittaciformes (Psittacoidea, Cacatuoidea and Strigopoidea) and displaying color hues suspected of being produced by pheomelanin, i.e. dull yellow, orange, reddish and brown coloration (Galván and Wakamatsu, 2016).

MATERIALS AND METHODS Species selection and feather sampling Psittaciform species were selected on the basis of plumage patches of colors suspected of being produced by pheomelanin. Although pheomelanin produces yellow, orange and red hues similar to those produced by psittacofulvins, the colors produced by pheomelanin are not bright, but dull (Galván and Wakamatsu, 2016). Thus, we selected 26 psittaciform species whose plumage included a patch displaying dull yellow, orange, reddish or brown coloration, whose low level of brightness makes them likely candidates to be produced by pheomelanin (Fig. 1, Table 1). We also included some greyish- brown as well as green plumage patches to explore a possible presence of brown barbules in feathers (Table 1). The selected species covered a high phylogenetic diversity, thus being a significant representation of the Order Psittaciformes (Fig. 2). For simplicity, the plumage patch colors are here described as red, yellow, green, grey and brown (Table 1). In particular, we predict that the exclusive evolution in The selection of species was made by examining book psittaciform birds of polyene pigments called psittacofulvins, illustrations (Juniper and Parr, 2001) and with the advice of parrot which create yellow, orange and red plumage coloration (McGraw captive breeders in Belgium and The Netherlands, who also and Nogare, 2005), may have promoted the evolutionary loss of provided feather samples. We included two mutations that appear in pheomelanin, given that this pigment also creates (albeit duller) captivity in two species, the whiteface mutation of the cockatiel yellow, orange and reddish plumage coloration (Toral et al., 2008; Nymphicus hollandicus and the opaline mutation of Bourke’s Galván and Wakamatsu, 2016) and that its synthesis implies the parrot, Neopsephotus bourkii, as mutated birds exhibit changes in reduction of the availability of the precursor of the main intracellular plumage pigmentation potentially owing to the synthesis of antioxidant (glutathione, GSH) in melanocytes (i.e. cysteine; pheomelanin (Martin, 2002) (Fig. 1). The whiteface mutation is Galván, 2018). Indeed, psittacofulvin evolution has promoted the inherited as an autosomal recessive mutation, while the opaline loss of plumage coloration based on other polyenic but dietary mutation is inherited as a sex-linked recessive mutation (Van den acquired pigments also able to create similar plumage coloration Abeele, 2016; van der Zwan et al., 2019). (i.e. carotenoids) despite being present at high circulating levels in One or two feathers from the studied plumage patches were parrots (McGraw and Nogare, 2004). An absence of pheomelanin in collected from an adult bird of each species and analyzed for Journal of Experimental Biology

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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 Eumelanin Nymphicus hollandicus Grey Mantle (grey in wild-type, Eumelanin (wild-type and whiteface mutation) grey-brownish in 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 Eumelanin Aratinga auricapillus Red, green Belly Psittacofulvin Deroptyus accipitrinus Red Red neck Psittacofulvin Enicognathus leptorhynchus Red, green Belly Psittacofulvin Forpus coelestis Green Wing Psittacofulvin Pionus chalcopterus Red Orange-reddish covert wing feathers Psittacofulvin Primolius auricollis Red Tail Psittacofulvin Primolius maracana Red Belly Psittacofulvin Pyrrhura cruentata Red Tail Psittacofulvin Pyrrhura egregia Red Tail Psittacofulvin Eclectus roratus Red Red body feathers in females Psittacofulvin Agapornis nigrigenis Brown Head Psittacofulvin (red barbs), eumelanin (grey barbules) Chalcopsitta duivenbodei Brown Brownish-greenish mantle Psittacofulvin Chalcopsitta scintillata Red Forehead Psittacofulvin Neopsephotus bourkii Brown, red Brown mantle in wild-type, red breast in Psittacofulvin (opaline mutation), eumelanin opaline mutation (wild-type) Psephotus haematonotus Red Red rump in males Psittacofulvin Pseudeos fuscata Red Breast Psittacofulvin Psittacula cyanocephala Red Red shoulder in males Psittacofulvin Psittacula eupatria Red Shoulder Psittacofulvin Coracopsis nigra Brown Breast Eumelanin Nestor notabilis Yellow, red Under wing primary covert feathers Psittacofulvin Nestor 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 detected in 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. pigment identification using Raman spectroscopy (see below). The Computational analysis including importing and pre-processing use of one or two samples from adults per species has been proven (baseline correction and normalization) of data was performed with sufficient to characterize the pigmentation phenotype of bird species MATLAB® R2014b (MathWorks Inc., Natick, MA, USA) using the (Galván et al., 2018a). All feather samples were collected from birds PLS Tollbox version 7.9.3 (Eigenvector Research Inc., USA). The bred and kept in captivity. The breeders had all necessary permits to identification of pigments was made on the basis of diagnostic hold the birds following guidelines from Belgian and Dutch vibrational bands. The Raman spectrum of eumelanin shows authorities. defined Raman bands at 1380 and 1580 cm−1 resembling the D and G bands characteristic of disordered graphite, in addition to a weaker Raman spectroscopy band at 500 cm−1 (Huang et al., 2004; Galván et al., 2013a, 2018b). We used a Thermo Fisher DXR confocal dispersive Raman In contrast, the Raman spectrum of pheomelanin shows wide microscope (Thermo Fisher Scientific, Madison, WI, USA) Raman bands at approximately 500, 1490 and 2000 cm−1 operating at the National Museum of Natural Sciences (MNCN, (Wang et al., 2016; Polidori et al., 2017; Megía-Palma et al., CSIC) in Madrid, Spain, to identify the pigments responsible for the 2018), and like eumelanin, it can be detected by direct examination color of the studied feathers. Dispersive Raman spectroscopy can of the surface of feathers by Raman spectroscopy (Galván et al., detect pigment molecules in solid samples at concentrations as low 2013a,b, 2018b; Galván and Rodríguez-Martínez, 2018). as 0.05–0.1% (w/w) (Bouffard et al., 1994; Massonnet et al., 2012). In contrast, the Raman spectrum of psittacofulvins shows two The system has a point-and-shoot Raman capability of 1 µm spatial strong bands at approximately 1130 and 1530 cm−1, and an absence resolution. We used an excitation laser source at 780 nm and a slit of the band at approximately 1000 cm−1 owing to the vibration of aperture of 25 µm to analyze the wavenumber range of 300– the methyl group; however, this is present in the Raman spectra of 2500 cm−1. When the samples contained psittacofulvins, we used a other polyenic pigments such as carotenoids (Maia et al., 2014; 50× confocal objective lens, a laser power of 7 mW, and an Fernandes et al., 2015). integration time of 3 s and 12 accumulations. When the samples contained melanins, to avoid the burning of samples owing to their RESULTS darker color and to optimize results, we used a 100× confocal We found Raman signals of eumelanin in nine of the species or objective lens, a laser power of 1 mW, and an integration time of 3 s mutations included in the study (Fig. 3). These corresponded to and 30 accumulations. The system was operated with Thermo Fisher three species with grey plumage patches and two species with brown OMNIC 8.1 software. We analyzed one to two feathers from one plumage patches (Table 1, Fig. 1). Additionally, eumelanin was bird of each species. One barb and one barbule chosen at random found spatially segregated in the red or brown plumage patches were analyzed in each feather. The average Raman spectra of other three species (the Cuban amazon, Amazona corresponding to each pigment were calculated for each species. leucocephala, the chestnut-fronted macaw, Ara severus,and Journal of Experimental Biology

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Nymphicus hollandicus Fig. 2. Phylogenetic relationships between the 26 species of psittaciform bird species included in the study. Branch lengths are Calyptorhynchus banksii proportional to nucleotide substitutions. The tree is the least-squares consensus phylogenetic tree obtained, using the R package phytools Amazona leucocephala (Revell, 2012), from the mean patristic distance matrix of a set of 100 Pionus chalcopterus probable phylogenies obtained using the phylogeny subsets tool in www.birdtree.org (Jetz et al., 2012). Forpus coelestis

Deroptyus accipitrinus

Aratinga auricapillus

Ara severus

Primolius auricollis

Primolius maracana

Enicognathus leptorhynchus

Pyrrhura egregia

Pyrrhura cruentata

Coracopsis nigra

Psittacula cyanocephala

Psittacula eupatria

Neopsephotus bourkii

Psephotus haematonotus

Agapornis nigrigenis

Chalcopsitta duivenbodei

Pseudeos fuscata

Nestor meridionalis

Nestor notabilis the black-cheeked lovebird, Agapornis nigrigenis) (Fig. 1), in The Raman spectra of red, yellow, green and brown plumage which eumelanin was found in black or grey barbules while patches were remarkably similar, all showing the two diagnostic psittacofulvin was found in red barbs (Table 1). In contrast, no bands of psittacofulvin. Variation between color groups in the Raman signal of pheomelanin was found in any of the plumage frequency of the first band (∼1130 cm−1) was notably low, as it was patches analyzed. This indicates that pheomelanin is not present located at 1131 cm−1 in the spectra of red feathers, 1134 cm−1 in the in the feathers of psittaciforms, or that it is present in non- spectra of yellow feathers, 1136 cm−1 in the spectra of green significant amounts. feathers and 1133 cm−1 in the spectra of brown feathers (Fig. 6). Raman signals of psittacofulvin were detected in all red plumage However, variation was more marked in the frequency of the second patches analyzed, corresponding to 18 species (Fig. 4, Table 1). band (∼1530 cm−1), being located at 1525 cm−1 in the spectra of Psittacofulvins were also found to cause dull yellow plumage red feathers, 1531 cm−1 in the spectra of yellow feathers, 1536 cm−1 coloration in two species (Fig. 5A) and green coloration in six in the spectra of green feathers and 1528 cm−1 in the spectra of species (Fig. 5B). Psittacofulvin was also found to be the pigment brown feathers (Fig. 6). causing dark brown coloration in two species (Fig. 5D), the brown lory (Chalcopsitta duivenbodei) and the black-cheeked lovebird DISCUSSION (Fig. 1), though in the latter species the brown plumage patch is Our results show that pheomelanin does not contribute to the produced by a combination of red psittacofulvin-containing barbs plumage pigmentation of psittaciform birds, despite assumptions and grey eumelanin-containing barbules (Table 1). However, the made in some previous studies lacking analytical evidence Raman spectra of yellow, green and brown plumage patches also (Tinbergen et al., 2013; Delhey and Peters, 2017). As we included included eumelanin signal (Fig. 5), indicating that, while in our analyses a significant representation of psittaciform species psittacofulvin is the main pigment responsible for yellow, green with plumage colors suspected of being produced by pheomelanin and brown colors in the studied species, eumelanin is mixed with (Galván and Wakamatsu, 2016) and from a high phylogenetic psittacofulvin, contributing to the resulting plumage coloration. diversity that included the three main lineages of Psittaciformes, these This is particularly notable in green feathers (Fig. 5B). The results provide evidence that psittaciform birds synthesize eumelanin subtraction of eumelanin signal from the Raman spectra of to pigment feathers black, grey and brown, but do not synthesize psittacofulvin led to a clear prevalence of the two Raman bands pheomelanin or synthesize it at negligible amounts. As natural of psittacofulvin, the shape of the resulting spectra thus being melanins are considered mixed pigments in which eumelanin and similar to that of red psittacofulvin (Figs 4 and 5). pheomelanin are present in varying ratios (Ito and Wakamatsu, 2008), Journal of Experimental Biology

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Fig. 3. Raw Raman spectra of eumelanin in the feathers of nine Fig. 4. Raw Raman spectra of psittacofulvin in feathers of 18 psittaciform psittaciform species and mutations included in the study. The inset image species with red plumage coloration included in the species. The inset shows a mantle body feather from one of the species included, a cockatiel image shows a tail feather from one of the species included, an ochre-marked Nymphicus hollandicus with whiteface mutation. parakeet, Pyrrhura cruentata. parrots represent the first animals in which an impaired mixed pheomelanin in psittaciform birds is an exclusive evolutionary loss in melanin-based pigmentation system is reported. The presence of this order within Aves. Indeed, no other birds are known to lack pheomelanin (and eumelanin) in organisms such as molluscs and pheomelanin in their feathers (Galván and Solano, 2016). insects (Speiser et al., 2014; Galván et al., 2015) and in all vertebrates Psittaciform birds have evolved an exclusive pigmentation (d’Ischia et al., 2015; Kottler et al., 2015) suggests that the absence of system by expressing the MuPKS gene, which codes for a

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Fig. 5. Raw Raman spectra (upper) of psittacofulvin and the corresponding normalized spectra after subtracting eumelanin signal (lower) in psittaciform species with different plumage coloration. (A) Yellow (two species), (B) green (six species) and (C) brown (two species). Inset images show examples 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. Journal of Experimental Biology

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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 spectra correspond to the respective groups. Bottom panels show a magnification of the spectral regions around the two main Raman bands of psittacofulvin owing to C–C stretching (∼1130 cm−1) and C=C stretching (∼1530 cm−1) of the polyene chain. polyketide synthase (Cooke et al., 2017), in feather follicles. This Interestingly, our results show that psittaciforms do not leads these birds to synthesize polyene pigments called synthesize pheomelanin, but synthesize eumelanin, which psittacofulvins that produce colors that may be similar in hue to produces dark colors (black, grey and dark brown) not resembling those produced by pheomelanin (Toral et al., 2008; Galván and those produced by psittacofulvins (Toral et al., 2008; Galván and Wakamatsu, 2016). Given this functional redundancy and that Wakamatsu, 2016). This makes it likely that the avoidance of pheomelanin synthesis may be physiologically limiting under functional redundancy between pigments is the evolutionary cause environmental stress because it reduces GSH availability in that has favored the impairment of the mixed melanin-based melanocytes (Galván, 2018), and that psittacofulvin synthesis pigmentation system in psittaciforms. This impairment might be does not seem to imply any comparable physiological limitation, we exerted through a regulation of the expression of genes controlling hypothesized an impairment of mixed melanin-based pigmentation pheomelanin synthesis in melanocytes at the dermal papillae of in psittaciforms. Indeed, psittaciforms do not deposit carotenoids feather follicles (Lin et al., 2013). Candidate genes in this regulation (dietary polyene pigments very commonly causing plumage are those coding for the antagonist peptides of the melanocortin 1 coloration in other birds) in feather follicles, despite circulating receptor in the membrane of melanocytes, namely agouti-signalling them through the blood at high levels (McGraw and Nogare, 2004). (ASIP) and agouti-related (AGRP) proteins (Nadeau et al., 2008), The psittacofulvin-based pigmentation system of psittaciforms thus and also genes that control the availability of cysteine in seems to have blocked the physiological activity of other melanocytes such as that encoding the cystine/glutamate pigmentation systems. antiporter xCT (Slc7a11; Chintala et al., 2005) and that encoding Journal of Experimental Biology

6 RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb225912. doi:10.1242/jeb.225912 cystinosin (CTNS; Town et al., 1998). The activation of the MuPKS maximize the diversity of color phenotypes while minimizing the gene in feather follicles, which is observed in psittaciforms but not use of pigment resources. Future studies should provide further in other birds (Cooke et al., 2017), has thus probably led to a co- details about the physiology of psittacofulvin synthesis to determine regulation of other genes, impairing the synthesis of pheomelanin whether a comparison of physiological implications between and the uptake of circulating carotenoids (McGraw and Nogare, pigments can explain the exclusive prevalence of the psittacofulvin- 2004), and promoting a selectivity for the psittacofulvin-based based pigmentation system in Psittaciformes. pigmentation system. Interestingly, this likely gene regulation mechanism in feather follicles would parallel the loss of gene Acknowledgements duplicates that is observed when they exhibit functional redundancy We thank Daniel Nuijten, Bert Van Gils, Nico Rosseel, Jef Kenis, Eric Gennissen, Heinz Schnitker and Glenn Ooms for their help in selecting psittaciform species for (Lynch and Conery, 2000; Cooke et al., 2017). Future studies should the study and for feather sampling. Philippe Rocher and Danny Roels kindly allowed investigate this gene regulation mechanism leading to a prevailing us to use their photographs shown in Fig. 1. pigmentation system. Our study provides the first vibrational characterization of Competing interests psittacofulvins giving rise to different plumage colors. The authors declare no competing or financial interests. Psittacofulvins giving rise to yellow plumage are composed of Author contributions polyene chains of 14, 16 and 18 carbon atoms in the form of Conceptualization: A.C.d.O.N., I.G., D.V.d.A.; Methodology: A.C.d.O.N., I.G.; conjugated fatty acids, whereas psittacofulvins giving rise to red Formal analysis: A.C.d.O.N., I.G.; Investigation: A.C.d.O.N., I.G.; Resources: plumage are probably synthesized by reducing yellow ones to form D.V.d.A.; Writing - original draft: A.C.d.O.N., I.G.; Visualization: A.C.d.O.N.; fully conjugated aldehydes of 14, 16, 18 and 20 carbon atom chains Supervision: I.G.; Funding acquisition: D.V.d.A. (Stradi et al., 2001; Cooke et al., 2017). Psittacofulvins giving rise to Funding green plumage are composed of yellow psittacofulvins, combined A.C.d.O.N. thanks the Brazilian entity Coordenação de Aperfeiçoamento de with a structural effect of feather morphology (Cooke et al., 2017). Pessoal de Nıveĺ Superior for financial support (PNPD/CAPES). I.G. is a recipient of Our results show that psittacofulvins can also give rise to brown a Ramón y Cajal Fellowship (RYC-2012-10237) from the Ministerio de Economıaý plumage coloration when combined with eumelanin. We found Competitividad (MINECO) of the Spanish Government. D.V.d.A. was funded by great similarity in the vibrational spectra of psittacofulvins of these Ornitho-Genetics VZW. color groups, which suggests that these colors are produced by References similar mixtures of polyenes of different chain length, as already Bender, T. and Martinou, J.-C. (2016). The mitochondrial pyruvate carrier in health shown for yellow and red psittacofulvins (see above), but that and disease: to carry or not to carry? Biochim. Biophys. Acta Mol. Cell Res. 1863, certain chain length values must prevail in each color group to give 2436-2442. doi:10.1016/j.bbamcr.2016.01.017 Berg, M. L. and Bennett, A. T. D. (2010). 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