© 2020. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2020) 223, jeb225912. doi:10.1242/jeb.225912 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 1 RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb225912. doi:10.1242/jeb.225912 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