1 1 Unique evolution of vitamin A as an external pigment in 2 tropical starlings 3 4 Ismael Galván1*, Khaled Murtada2,3, Alberto Jorge4, Ángel Ríos2,3, and 5 Mohammed Zougagh2,5 6 7 1Department of Evolutionary Ecology, Doñana Biological Station, Consejo Superior de 8 Investigaciones Científicas (CSIC), 41092 Sevilla, Spain 9 2Regional Institute for Applied Scientific Research (IRICA), University of Castilla-La 10 Mancha, 13071 Ciudad Real, Spain 11 3Department of Analytical Chemistry and Food Technology, Faculty of Chemical Science 12 and Technology, University of Castilla-La Mancha, 13071 Ciudad Real, Spain 13 4Laboratory of Non-Invasive Analytical Techniques, National Museum of Natural Sciences, 14 Consejo Superior de Investigaciones Científicas (CSIC), 28006 Madrid, Spain 15 5Department of Analytical Chemistry and Food Technology, Faculty of Pharmacy, 16 University of Castilla-La Mancha, 02071 Albacete, Spain 17 18 *Author for correspondence ([email protected]) 19 20 Running title: Evolution of retinol-based pigmentation 21 22 KEY WORDS: Biological pigments, Birds, Carotenoids, Color evolution, Retinol 23 24 25 2 26 Summary statement 27 We have discovered a new class of external biological pigments, after showing that an 28 Afrotropical starling has physiological capacity to deposit retinol in its integument and 29 create bright yellow coloration. 30 31 ABSTRACT 32 Pigments are largely responsible for the appearance of organisms. Most biological 33 pigments derive from the metabolism of shikimic acid (melanins), mevalonic acid 34 (carotenoids) or levulinic acid (porphyrins), which thus generate the observed diversity of 35 external phenotypes. Starlings are generally dark birds despite iridescence in feathers, but 36 10 % of species have evolved plumage pigmentation comprising bright colors that are 37 known to be produced only by carotenoids. However, using micro-Raman spectroscopy, 38 we have discovered that the bright yellow plumage coloration of one of these species, the 39 Afrotropical golden-breasted starling Cosmopsarus regius, is not produced by carotenoids, 40 but by vitamin A (all-trans-retinol). This is the first organism reported to deposit significant 41 amounts of vitamin A in its integument and use it as a body pigment. Phylogenetic 42 reconstructions reveal that the retinol-based pigmentation of the golden-breasted starling 43 has independently appeared in the starling family from dark ancestors. Our study thus 44 unveils a unique evolution of a new class of external pigments comprised by retinoids. 45 46 47 48 49 50 3 51 INTRODUCTION 52 The evolution of organisms is mediated in a significant part by their appearance. External 53 coloration greatly determines the capacity to adapt to the environment (Manceau et al., 54 2011), and reinforces the differentiation of incipient species (Sætre et al., 1997; 55 Seehausen et al., 2008). Highly diversified animal clades are indeed often associated to 56 the evolution of conspicuous color traits, because sexual selection, which plays an 57 important role in the generation of isolating mechanisms by adaptive radiation (Price 58 1998), favors conspicuous phenotypes (Maan and Seehausen, 2011). 59 Many of the bright colors shown by photosynthetic organisms are produced by 60 biosynthesizing tetraterpene compounds termed carotenoids (Alvarez et al., 2013). With 61 the exception of few invertebrates able to synthesize these pigments (Moran and Jarvik, 62 2010), animals accumulate and modify carotenoids taken with the plant products of their 63 diet (Toews et al., 2017). Consequently, carotenoids constitute a main class of pigments 64 responsible for the color of organisms (Alvarez et al., 2013). Another large class of 65 biological pigments is represented by melanins, polymers derived from the oxidation of 66 tyrosine or phenolic compounds that virtually all organisms synthesize (d'Ischia et al., 67 2015). The third most common class of pigments is represented by porphyrins, aromatic 68 rings produced in virtually all cells as intermediates during the synthesis of heme, but that 69 only certain groups of birds use as external pigments (Galván et al., 2018). Carotenoids, 70 melanins and porphyrins derive from the metabolism of three chemical precursors: 71 mevalonic, shikimic and levulinic acids, respectively (Gudin, 2003). Other exclusive 72 classes of pigments are only found in particular groups of animals, such as psittacofulvins 73 in psittaciform birds (parrots and allies; Martínez, 2009), spheniscins in penguins (Thomas 74 et al., 2013) and ommochromes in spiders (Hsiung et al., 2017). 75 Therefore, the synthesis, accumulation or modification of carotenoids, melanins and 76 porphyrins, together with their interaction with specialized morphological structures (Maia 4 77 et al., 2013), are main responsible for the observed diversity of biological colors. Here, we 78 report the evolution of a novel class of pigments responsible for bright body coloration in a 79 tropical species of the family Sturnidae (starlings), which comprises passerine birds widely 80 distributed in the Old World (Fig. 1). 81 82 MATERIALS AND METHODS 83 Reconstruction of ancestral pigmentation patterns 84 We investigated the pigmentation patterns of 107 species of starlings, representing the 85 entire family Sturnidae (Lovette and Rubenstein, 2007). The two-three species of dark 86 pigmented Philippine creepers (Rhabdornis spp.) were excluded, as their position within 87 Sturnidae still requires additional support (Zuccon et al., 2006). By examining starling 88 illustrations (Feare and Craig, 1998), we categorized each species as bright, if they 89 exhibited brilliant yellow or red/pink plumage patches regardless their size or number, or 90 dark, if they did not exhibit any plumage patch with such colors. These colors are known to 91 be produced by the deposition of carotenoid pigments in feathers (McGraw, 2006). We did 92 not consider iridescence in feathers nor color of skin. 93 We obtained 1000 probable phylogenies for the 107 starling species with branch 94 lengths expressed as proportions of nucleotide substitutions using the phylogeny subsets 95 tool in www.birdtree.org (Jetz et al., 2012). We then obtained the least-squares consensus 96 phylogenetic tree (Fig. 2) from the mean patristic distance matrix of the set of 1000 97 phylogenies using the package phytools (Revell, 2012) in R environment (R Core Team, 98 2019). The reconstruction of ancestral states was made using an Mk model as 99 implemented in phytools, assuming that transitions among states follow a continuous-time 100 Markov chain process (Pagel, 1994; Lewis, 2001). Using the set of 1000 phylogenies, we 101 calculated the empirical Bayesian posterior probabilities and represented them as pie 102 charts on the nodes of the consensus tree (Fig. 2). 5 103 104 Animals 105 We collected six breast yellow feathers from a golden-breasted starling Cosmopsarus 106 regius and six head yellow feathers from a golden-crested myna Ampeliceps coronatus 107 (Fig. 1). The feathers were obtained from adult birds kept in captivity at Attica Zoological 108 Park, Athens, Greece. Additionally, we collected pink breast feathers from an adult rosy 109 starling Sturnus roseus and undertail yellow feathers from an adult yellow-faced myna 110 Mino dumontii (Fig. 1), using skins deposited at the Museum of Vertebrate Zoology of the 111 University of California at Berkeley, USA (specimens MVZ 70159 and MVZ 93404). 112 Feather samples were stored in the dark until analyses. The plumage colors of these four 113 species thus cover the entire color diversity shown by starling species with bright plumage, 114 and are therefore significant representatives of the Sturnidae family (Fig. 2). 115 116 Raman spectroscopy 117 To obtain a molecular characterization of pigments responsible for the bright colors 118 observed in starlings (which we hypothesized to be carotenoids), we analyzed pigmented 119 feathers from the four species described above (golden-breasted starling, golden-crested 120 myna, yellow-faced myna and rosy starling) by non-destructive micro-Raman 121 spectroscopy. Raman spectra were obtained with an inVia Renishaw Microscope 122 spectrometer (Renishaw, Gloucestershire, UK). A 532 nm laser beam was used as an 123 excitation source, which was focused on the samples using a 100x objective. Acquisition 124 time was 10 s, and spatial resolution was 1 µm. Laser power was fixed to a range of 1-5 125 mW/µm2, avoiding heating damage to samples. Thirty-forty spectra were recorded for each 126 feather, and an average spectrum was then calculated for each species. The control of the 127 Raman system and recording of data were made using the WiRE interface (Renishaw, 128 v4.4). The spectra were baseline corrected and normalized prior to analyses. 6 129 Vibrational signatures recorded from Raman spectra allowed the molecular 130 identification of pigments. The main Raman bands of carotenoids are usually termed 131 �! − �! and arise from stretching vibrations of C=C double bonds (�!), from stretching 132 vibrations of C-C single bonds coupled with C-H in-plane bending vibration (�!), and from 133 in-plane rocking vibrations of methyl groups (�!) (Arteni et al., 2005; Kish et al., 2015). 134 135 RESULTS AND DISCUSSION 136 Most starling species exhibit metallic hues due to iridescence in their plumage that is 137 produced by interaction between specially ordered nanostructures and melanin granules in 138 feathers (Maia et al., 2013). Despite this, the general appearance