The Case of Deirocheline Turtles

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1 Body coloration and mechanisms of colour production in Archelosauria:

2 The case of deirocheline turtles

3 Jindřich Brejcha1,2*†, José Vicente Bataller3, Zuzana Bosáková4, Jan Geryk5, 4 Martina Havlíková4, Karel Kleisner1, Petr Maršík6, Enrique Font7

5 1 Department of Philosophy and History of Science, Faculty of Science, Charles University, Viničná 7, Prague 6 2, 128 00, Czech Republic 7 2 Department of Zoology, Natural History Museum, National Museum, Václavské nám. 68, Prague 1, 110 00, 8 Czech Republic 9 3 Centro de Conservación de Especies Dulceacuícolas de la Comunidad Valenciana. VAERSA-Generalitat 10 Valenciana, El Palmar, València, 46012, Spain. 11 4 Department of Analytical Chemistry, Faculty of Science, Charles University, Hlavova 8, Prague 2, 128 43, 12 Czech Republic 13 5 Department of Biology and Medical Genetics, 2nd Faculty of Medicine, Charles University and University 14 Hospital Motol, V Úvalu 84, 150 06 Prague, Czech Republic 15 6 Department of Food Science, Faculty of Agrobiology, Food, and Natural Resources, Czech University of Life 16 Sciences, Kamýcká 129, Prague 6, 165 00, Czech Republic 17 7 Ethology Lab, Cavanilles Institute of Biodiversity and Evolutionary Biology, University of Valencia, C/ 18 Catedrátic José Beltrán Martinez 2, Paterna, València, 46980, Spain

19 Keywords: Chelonia, Trachemys scripta, Pseudemys concinna, nanostructure, pigments, chromatophores 20

21 Abstract

22 Animal body coloration is a complex trait resulting from the interplay of multiple colour-producing mechanisms. 23 Increasing knowledge of the functional role of animal coloration stresses the need to study the proximate causes of 24 colour production. Here we present a description of colour and colour producing mechanisms in two non-avian 25 archelosaurs, the freshwater turtles Trachemys scripta and Pseudemys concinna. We compare reflectance spectra; 26 cellular, ultra-, and nanostructure of colour-producing elements; and carotenoid/pteridine derivatives contents in 27 the two species. In addition to xanthophores and melanocytes, we found abundant iridophores which may play a 28 role in integumental colour production. We also found abundant dermal collagen fibres that may serve as 29 thermoprotection but possibly also play role in colour production. The colour of yellow-red skin patches results from 30 an interplay between carotenoids and pteridine derivatives. The two species differ in the distribution of pigment cell 31 types along the dorsoventral head axis, as well as in the diversity of pigments involved in colour production, which 32 may be related to visual signalling. Our results indicate that archelosaurs share some colour production mechanisms 33 with amphibians and lepidosaurs, but also employ novel mechanisms based on the nano-organization of the 34 extracellular protein matrix that they share with mammals. 35 *Jindřich Brejcha ([email protected] ). †Present address: Department of Philosophy and History of Science, Faculty of Science, Charles U., Viničná 7, Prague, 128 00, Czech Republic

38 39 The evolutionary origins and the selective processes responsible for the maintenance of complex 40 morphological traits remains a persistent challenge in biology. Complex traits may in some cases arise by exaptation 41 of already existing structures into new functions [1]. Animal coloration is one of such complex morphological traits 42 in which composing subunits evolve for various roles and only later are co-opted for signalling or for other functions 43 [2]. Colour-producing elements interact to enhance or reduce each other’s contribution to observable colour [3]. 44 However, colour-producing elements can also develop and evolve independently of each other [4]. Variation in body 45 coloration during evolution is thus the result of two interacting processes: functional integration of colour 46 ornaments and morphological modularity of colour-producing elements [5]. These intriguing relationships between 47 colour-producing subunits and their functional role stress the need to study the proximate causes of colour 48 production [6]. Even though there has been progress in our understanding of colour production and its biological 49 functions [7,8], our knowledge of colour production mechanisms in most groups of animals is still scarce. 50 Animal colours are produced by ultrastructural elements of the integument interacting with incident light, 51 by light-absorbing pigments, or by a combination of both. In vertebrates, the majority of compounds responsible for 52 the coloration of the integument are found in pigment cells which are derived from the neural crest [9–11]. Pigment 53 cells are classified into five categories: Reflecting dermal pigment cells (iridophores, leucophores), absorbing dermal 54 pigment cells (xanthophores, erythrophores, cyanophores), melanocytes with non-motile organelles (dermal 55 melanocytes), melanocytes with motile organelles (dermal melanophores), and organelle transferring melanocytes 56 (interfollicular and follicular epidermal melanocytes) [12]. Iridophores contain reflecting platelets of guanine, while 57 absorbing dermal pigment cells contain pteridine derivatives in pterinosomes, or carotenoids dissolved in lipids in 58 carotenoid vesicles. Melanocytes and melanophores contain melanins in melanosomes. Single cells with 59 characteristics of two or more of pigment cell types, e.g. xanthophore and iridophore, are occasionally found and 60 are known as mosaic chromatophores [13,14]. Often, pigment cells of the same type are arranged in layers that are 61 roughly parallel to the skin surface. The vertical layering and relative thickness of the different pigment cells, 62 thickness, orientation, number, and geometry of reflecting elements, as well as characteristics of the pigments 63 within the cells determine the final colour of the skin [8,15,16]. Cell types of another embryonic origin, ectodermal 64 keratinocytes of the epidermis and mesenchymal fibroblasts of the dermis, also participate in colour production via 65 reflection of light on the intra/extracellular protein matrix these cells produce [17–21]. 66 Turtles are an early-diverging clade of Archelosauria, the evolutionary lineage of tetrapods leading to 67 crocodiles and birds [22]. Although many turtles have a uniform dull colour, conspicuous striped and spotted 68 patterns are common in all major lineages of turtles (for a comprehensive collection of photographs see [23–26]). 69 These conspicuous colour patterns may be present in the hard-horny skin of shells, and/or in the soft skin of the 70 head, limbs or tail. The dark areas of the skin of turtles may have a threefold origin consisting either of dermal, 71 epidermal, or both epidermal and dermal melanocytes. Colourful bright regions are thought to be the result of the 72 presence of xanthophores in the dermis [27] and their interplay with dermal melanophores [28]. Iridophores have 73 never been shown to play role in coloration of turtles [27,29]. 74 Pigment-bearing xanthophores were first described in the dermis of the Chinese softshell turtle (Pelodiscus 75 sinensis) [29]. Xanthophores have also been found sporadically in the dermis of the spiny softshell turtle Apalone 76 spinifera, the Murray river turtle (Emydura macquarii) and in the painted turtle (Chrysemys picta) [27]. Such scarcity 2

bioRxiv preprint doi: https://doi.org/10.1101/556670; this version posted February 21, 2019. The copyright holder for this preprint (which 77 wasof notcarotenoid/pteri certified by peer dreview)ine derivatives is the author/funder,-containing who cells has granted is in contrast bioRxiv a withlicense chemical to display analysesthe preprint of in theperpetuity. yellow It andis made red available under aCC-BY-NC-ND 4.0 International license. 78 regions of the integument of the red-eared slider (Trachemys scripta elegans) and C. picta [30,31]. Two major 79 classes of carotenoids have been described in the integument of these turtles: short wavelength absorbing 80 apocarotenoids and longer wavelength-absorbing ketocarotenoids [30]. In addition to carotenoids, Steffen et al. 81 [30] speculated that small quantities of some pteridine derivatives could play a minor role in colour production in 82 the integument of turtles. In support of this hypothesis, Alibardi [27] observed pterinosomes with characteristic 83 concentric lamellae in the xanthophores of turtles. However, to this date there is no direct evidence of pteridine 84 derivatives being present in colourful regions of the skin of turtles, or in the skin of any other archelosaur. 85 Deirochelyinae are freshwater turtles of the family Emydidae, all of which, except one species (the 86 diamond-back terrapin, Malaclemys terrapin), display a pattern of conspicuous stripes on the skin of the head and 87 legs [32,33]. This striped pattern usually consists of alternating yellow and black regions, but red regions are also 88 found in some species. The coloration of deirocheline turtles is thought to play a role in mate choice [30,34]. Some 89 species are sexually dichromatic, with males often displaying the brightest, most conspicuous colours (reviewed in 90 [19]); however sexual dichromatism is limited to particular regions of the body, whereas other regions show no 91 sexual differences [35,36]. Colour ornaments are designed to maximize their conspicuousness only in certain, 92 ecologically relevant contexts [37], e.g. when exposed to rivals during contests or to mates during courtship [38,39]. 93 Therefore, it has been suggested that some of the colourful regions found particularly in males may potentially be 94 involved in intersexual communication and mate choice [35,40]. In freshwater turtles courtship takes place 95 underwater and males adopt characteristic positions relative to the female [41]. Deirocheline turtles are 96 characterized by elaborated courtship behaviour including a complex forelimb display known as titillation [41]. 97 Males in most species of deirocheline turtles perform the titillation display facing the female while swimming 98 backwards in front of her, mostly near the surface; however, males of the genus Pseudemys titillate swimming 99 above and parallel to the female, facing the same direction as her [41–46]. Thus, depending on the position adopted 100 during courtship (face-to-face vs swim above) males of different species expose different body areas to females. By 101 comparing different deirocheline species, it may be possible to examine whether there is a relationship between 102 colour and colour-producing mechanisms and courtship position in these turtles. 103 In this study we compare the structure and pigment contents of striped skin in two species of deirocheline 104 freshwater turtles, the pond slider (Trachemys scripta) and the river cooter (Pseudemys concinna), with contrasting 105 strategies in courtship behaviour. Specifically, we predict that the brightest, most conspicuous skin regions of males 106 will be located precisely in those areas that are most exposed to females during courtship, which will differ 107 depending on the species-specific male position. Males of T. scripta court females in a face-to-face position while 108 males of P. concinna court females in a swim-above position. In T. scripta two of three subspecies (the yellow- 109 bellied slider T. s. scripta, and the Cumberland slider T.s. troosti) possess only yellow stripes on the head, while the 110 other subspecies (T.s. elegans) also has red markings in the postorbital region. All Pseudemys turtles show only 111 yellow stripes on the head. We particularly focus on a comprehensive description of the cellular and ultrastructural 112 composition of those body regions that likely play role during courtship behaviour (head, limbs) and on the chemical 113 composition of pigments involved in colour production. We combine multiple approaches to study the mechanisms 114 of colour production: 1) reflectance spectrophotometry to objectively determine the colour of the turtles’ body 115 surfaces, 2) transmission electron microscopy and image analyses to study the composition of the skin and the 116 ultrastructure of colour-producing elements, and 3) liquid chromatography coupled with mass spectrometry to 117 determine the contribution of carotenoid/pteridine derivatives to the turtles’ coloration. We discuss our results

122 123 2.1 Animals and handling

124 All turtles examined in this study were collected for purposes of control of invasive freshwater turtles as a 125 continuation of project ‘LIFE-Trachemys’ (LIFE09 NAT/ES000529) to the Servicio de Vida Silvestre, D.G. Medi Natural 126 (Generalitat Valenciana). Turtles (Pseudemys concinna: 4 males, 8 females; Trachemys scripta elegans: 30 males, 39 127 females; Trachemys scripta scripta: 1 male, 2 females) were captured during 2014-2017 with floating or hoop traps 128 in coastal marshes or artificial ponds at several localities in the Comunidad Valenciana (Spain). Turtles were 129 euthanized with an overdose of sodium pentobarbital (Eutanax, FATRO IBERICA, S.L) by authorized personnel. 130 131 2.2 Spectral reflectance measurements

132 To quantify colour, we measured the spectral reflectance of skin between 300 and 700 nm using a 133 spectrophotometer. Reflectance spectra were taken before euthanasia with an Ocean Optics Jaz System (QR400-7- 134 SR-BX: 400 µm reflection probe attached to JAZ-UV-VIS: Deuterium-Tungsten light source module) and a SONY Vaio 135 computer running SpectraSuite software (Ocean Optics, Inc.). The internal trigger was set to 30, integration time to 136 30, scans average to 30, and boxcar width to 10. The system was calibrated using a Spectralon WS-1 diffuse 137 reflectance standard, also from Ocean Optics. Measurements were taken in a darkened room at a distance of 5 mm 138 and perpendicular to the skin surface (i.e. coincident normal measuring geometry [47]). Turtles were kept alive in 139 clean water inside black plastic tanks and the skin surface was dried with a piece of cloth just before taking 140 reflectance measurements. The main median chin yellow stripe (CBC), dorsal head background coloration (DHC), 141 main bright/yellow forelimb stripe (FLBS) and the postorbital marking on the left side (PM) were measured in all 142 specimens of P. concinna and T. s. elegans (Fig. 1). Unfortunately, as T. s. scripta is rarely found in the Valencia 143 region, reflectance spectra could be obtained from only one specimen (male). The yellow zygomatic patch (YP) 144 instead of PM was measured in T. s. scripta (see Fig. 1B). During postnatal ontogenesis adult males of T. s. elegans 145 become melanistic as they get older [48]; therefore, we have not included reflectance spectra of any of the males 146 determined as melanistic in our analyses. 147 148 2.3 Processing and statistical analyses of reflectance spectra

149 Reflectance spectra binned by 0.37 nm were restricted to the range between 300 and 700 nm. Calculations were 150 performed in R 3.3.2 [49] using the package PAVO [50]. Spectra were processed by smoothing (span = 0.3) and 151 negative values were fixed to be zero. Mean processed reflectance spectra for each region of each individual of each 152 taxon were summarized by the following variables: luminance or brightness (B1: sum of the relative reflectance over 153 the entire spectral range), hue (H1: wavelength of maximum reflectance), and chroma (relative contribution of a 154 spectral range to total brightness). We calculated five measures of chroma based on the wavelength ranges 155 normally associated with specific colour hues: 300-400 nm (S1.UV), 400-510 nm (S1.blue), 510-605 nm (S1.green), 156 550-625 nm (S1.yellow), 605-700 nm (S1.red) [50–52]. Reflectance spectra of T. s. scripta were not included in the 4

bioRxiv preprint doi: https://doi.org/10.1101/556670; this version posted February 21, 2019. The copyright holder for this preprint (which 157 wasstatistical not certified analyses by peer review) due to is thethe author/funder, small sample who sizehas grantedavailable bioRxiv for a this license lineage to display. Redundancy the preprint in anaperpetuity.lysis (RDA) It is made was available under aCC-BY-NC-ND 4.0 International license. 158 performed on summary variables scaled to zero mean and unit variance using the package vegan 2.0-10 in R [53]. 159 Species identity was the constraining variable to compare P. concinna and T. s. elegans. Significance of results was 160 assessed by ANOVA like permutation tests (9,999 permutations) for redundancy analysis (anova.cca) using vegan 161 2.0-10 package (see script at [Reference to be added]). 162 163 2.4 Microscopy

164 To investigate the cellular composition of the turtles’ integument we took samples from CBC, FLBS, and PM 165 regions of P. concinna (females = 2) and CBC, DHC, FLBS, and PM regions of T. s. elegans (males = 5, females = 3). We 166 also took samples of the CBC, PM, and YP regions of T. s. scripta (female = 2). Pieces of integument measuring ca. 1 167 cm2 from target regions were excised immediately after sacrifice and placed in Karnovsky’s fixative (2% 168 paraformaldehyde, 2.5% glutaraldehyde in PB buffer), left overnight at room temperature, and stored in the 169 refrigerator at 4°C for seven days. Smaller pieces (ca. 2 mm3) were cut from the original pieces of tissue. These were 170 washed with 0.1M PB, postfixed with 2% osmium tetra oxide (in 0.1M PB solution), dehydrated in an increasing 171 ethanol series, washed with 2% uranyl acetate in 70% ethanol, washed in propylenoxide, after which they were 172 transferred to resin (Durcupan, Sigma). Polymerized resin blocks were cut on a Leica UCT Ultracut ultramicrotome. 173 Semi-thin sections were stained with toluidine blue and observed under bright field and phase contrast in a Nikon 174 Eclipse E800 photomicroscope. Ultra-thin sections were stained with lead citrate and observed and photographed 175 using a FEI Tecnai Spirit G2 TEM equipped with a digital camera (Soft Image System, Morada) and image capture 176 software (TIA 4.7 SP3, FEI Tecnai). Magnification ranged from 1250x to 43000x depending on the structures 177 observed; intensity of the electron beam was adjusted to be in the optimal range for different magnifications. 178 179 2.5 Reflecting platelets of iridophores

180 To infer role of iridophores in colour production we analysed the geometric properties of intact reflecting 181 platelets as well as of the empty holes that remain after the reflecting platelets dissolve due to the embedding 182 process. For analyses we used multiple electromicrographs of 4500-9900x magnification: P. concinna: PM (N = 8), 183 CBC (N = 8), FLBS (N = 8); T. s. elegans: PM (N = 19), CBC (N = 15), FLBS (N = 9). The scale of each image in nm/pixel 184 was set based on the number of pixels in the scale bar of the original electromicrographs. The length of the minor 185 axis (height of reflecting platelet) and of the major axis (width of reflecting platelet) of intact reflecting platelets was 186 measured directly from electromicrographs using the digital callipers utility in ImageJ (1.52a; [54]). Empty holes 187 were analysed automatically by greyscale thresholding and subsequent measurement tools in ImageJ similarly to 188 [16,55]. Briefly, electromicrographs were optimized for contrast and ellipses were fitted inside the platelets’ bodies. 189 Geometric parameters such as length of major axis of ellipse, length of minor axis, and angle between major axis of 190 the ellipse and x axis of electromicrographs were calculated (see electromicrographs of reflecting platelets at 191 [Reference to be added]). 192 Based on the length of the minor axis of both intact platelets and empty holes we calculated putative 193 reflective wavelengths of reflecting platelets following equation (2) in Morrison [56] as applied by Haisten et al. [57] 194 with refractive index of reflecting platelets 1.83 [58]. However, this model of colour production applies only to 195 reflecting platelets that produce colour through thin-film interference [59], which is characterized by reflecting 196 platelets organized in planes perpendicular to the direction of propagation of incoming light (i.e. parallel to the skin 197 surface). Disorganized reflecting platelets on the other hand act as broadband reflectors producing incoherent

199 region [16]. Following Saenko et al. [16] we calculated the A/y0 ratio to determine the relative amount of reflecting 200 platelets parallel to the skin surface from the Gaussian curve fitted to the distribution of the density of different 201 angles of the ellipses. A is the amplitude of the Gaussian curve above the background of randomly oriented

202 platelets, and y0 is the background level of randomly oriented platelets, i. e. intersection of Gaussian curve with y

203 axis. The lower the A/y0 ratio the lower the portion of platelets parallel to the skin surface, with 0 corresponding to

204 either no platelets parallel to the skin surface, or completely disorganized platelets [16]. High A and low y0 indicate 205 predominant orientation of platelets parallel to the surface and low density of disordered platelets. Full width of 206 half maximum (FWHM) of the peak of Gaussian curve was computed to characterize the spread of the Gaussian 207 curve fitted to the data, i. e. width of angular distribution, as in Saenko et al. [16]. 208 209 2.6 Fourier analysis of spatial distribution of dermal collagen arrays

210 To determine the role of the abundant extracellular collagen fibres in colour production we performed two 211 dimensional discrete Fourier analyses of the spatial distribution of dermal collagen arrays in the superficial layers of 212 the dermis using a Fourier tool [60] in MATLAB R2018a [61] kindly provided by Dr. Richard Prum 213 (https://prumlab.yale.edu/research/fourier-tool-analysis-coherent-scattering-biological-nanostructures). Because 214 the tool was originally developed for earlier versions of MATLAB, script syntax had to be updated to secure 215 compatibility with current version of MATLAB (see description of changes at [Reference to be added]). The analyses 216 followed published procedures [18,60]. Briefly, from each micrograph (9900x magnification) of cross-sections of the 217 collagen fiber arrays we selected a standardized square portion of the array (800 pixels2), and optimized and 218 standardized contrast in Adobe Photoshop (CS3, [62]). The scale of the image in nm/pixel was set based on the 219 number of pixels in the scale bar of the original electromicrographs. The darker pixels of collagen fibres and lighter 220 pixels of mucopolysaccharide were assigned representative refractive indices (1.42 and 1.35 respectively,[63]). We 221 used the Fourier tool with the 2-D fast Fourier transform (FFT2) algorithm [64], resulting in a 2-D Fourier power 222 spectrum expressing the amount of periodicity in the spatial frequencies of the original data. Cumulative analyses 223 were performed using multiple images per each examined region (P. concinna: PM, N = 8; CBC, N = 8; FLBS, N = 8; T. 224 s. elegans: PM, N = 19; CBC, N = 9; FLBS, N = 9; T. s. scripta: CBC, N = 6; YP, N = 11) (see electromicrographs of 225 collagen fibres at [Reference to be added]). For results we pooled images of the CBC region of both T. scripta 226 subspecies to increase the sample size for this skin region. Predicted reflectivity in visible spectra of the regions 227 were calculated for 51 concentric bins of the 2-D Fourier power spectra (corresponding to 51 10 nm wide 228 wavelength intervals from 300 to 800 nm) for each region as described in Prum & Torres [60]. To visualize the 229 composite predicted shape of reflectivity from multiple measurements of collagen fibre arrays per region we fitted a 230 smoothed curve (span= 0.3) to the data using the local fitting loess function in R. 231 232 2.7 Pigment content analyses

233 Integument from P. concinna (N = 3), T.s. elegans (N = 2) and T.s. scripta (N = 2) was used for carotenoid 234 analyses. Sampled regions were CBC, FLBS, PM, and YP. Small pieces of integument were removed using micro- 235 scissors and tweezers, cleaned mechanically, washed briefly with distilled water to get rid of potential 236 contamination from muscles or body fluids, and frozen at -20°C. Samples were then transported on ice to Prague 237 (Czech Republic) by plane, where they were stored frozen at -20°C until analyses, but never longer than one month.

bioRxiv preprint doi: https://doi.org/10.1101/556670; this version posted February 21, 2019. The copyright holder for this preprint (which 238 wasSamples not certified were by extracted peer review) with is the 0.5 author/funder, mL ethyl acetate. who has The granted vials bioRxiv containing a license the to extracts display thewere preprint stored in perpetuity. for 3 days It isat made room available under aCC-BY-NC-ND 4.0 International license. 239 temperature in complete darkness. The extracts were then evaporated to dryness by stream of nitrogen at 27°C and 240 stored at -18°C. Immediately prior to the analyses samples were diluted in 200µl of ethyl acetate (EtOAc). 241 Standards of astaxanthin, canthaxanthin, lutein, and zeaxanthin were purchased from Sigma Aldrich 242 (Munich, Germany). Stock solutions of the external standards were prepared at a concentration of 0.1 mg/mL by 243 dissolving in EtOAc. The working solution of the mixture of all the studied carotenoids in EtOAc was prepared at 244 concentrations of 50 ng/mL, 100 ng/mL, 200 ng/ml, 500ng/mL and 1000 ng/mL from the stock solutions. 245 Carotenoids were determined using UPLC system Dionex Ultimate 3000 (Thermo Fischer, USA) coupled 246 with photodiode array (PDA) detector followed by ultra-high resolution accurate mass (HRAM) Q-TOF mass 247 spectrometer (IMPACT II, Bruker Daltonik, Germany). Carotenoid separation was performed on Kinetex C18 RP 248 column ( 2.6 m, 150 x 2.1 mm; Phenomenex, USA) maintained at 35°C using acetonitrile (A), methanol/water 1:1 249 v/v (B) and a mixture of tert-Butyl methyl ether/acetonitrile/methanol - 86:86:8 v/v/v (C) as mobile phases for 250 gradient elution (supplementary Table S1) with constant flow rate 0.2 mL/min. Chromatograms were monitored at 251 445 and 472 nm. The identity of the particular carotenoids was confirmed by HRAM mass spectrometry. Ions were 252 detected in positive mode with electrospray (ESI) as well as atmospheric pressure chemical (APCI) ionization (see 253 details in supplementary Table S2). A chromatogram of the all carotenoid standards mixture in EtOAc at a 254 concentration of 1000 ng/mL is shown as supplementary Fig. S1a, Chromatograms of the individual carotenoid 255 standards with their absorbance spectra are shown in supplementary Fig. S1 c – f. 256 For analyses of pteridine derivatives we used integument from one individual of each of the examined taxa. 257 Sampled regions were CBC, FLBS, PM, and YP. Samples of integument were removed, treated and transported as for 258 carotenoid analyses. Samples were extracted with dimethyl sulfoxide (DMSO) following a previously published 259 procedure [65].

260 Standards of 6-biopterin, D-neopterin, leucopterin, pterin, and pterin-6-carboxylic acid (pterin-6-COOH) 261 were purchased from Sigma Aldrich (Munich, Germany). Isoxanthopterin and xanthopterin were obtained from

262 Fluka (Buchs, Switzerland). L-sepiapterin was purchased from the Cayman Chemical Company (Ann Arbor, MI, USA). 263 Erythropterin was kindly provided by Dr. Ron Rutowski. Stock solutions of the external standards were prepared at a 264 concentration of 0.1 mg/mL by dissolving in DMSO. The working solution of the mixture of all the studied pteridine 265 derivatives in DMSO was prepared at a concentration of 0.01 mg/ml from the stock solutions. 266 All chromatographic measurements were carried out in a HPLC system Agilent series 1290 coupled with a 267 Triple Quad 6460 tandem mass spectrometer (Agilent Technologies, Waldbronn, Germany). For data acquisition, the 268 Mass Hunter Workstation software was used. A ZIC®-HILIC (4.6 mm × 150 mm, 3.5 μm) column, based on 269 zwitterionic sulfobetaine groups, was chosen (Merck, Darmstadt, Germany). The chromatographic conditions were 270 adapted from Kozlík et al. [66]. The isocratic elution at a flow rate of 0.5 ml/min with the mobile phase consisted of 271 acetonitrile/5mM ammonium acetate, pH 6.80 at a volume ratio of 85:15 (v/v) was used for the separation. Tandem 272 mass spectrometric (MS/MS) measurements were performed in the selected reaction monitoring mode (SRM) with 273 positive ionization. SRM conditions used for MS/MS determination are listed in supplementary Table (S3). Under

274 these conditions pteridine derivatives eluted with the following retention times (min): L-sepiapterin t = 7.00, pterin t 275 = 9.20, isoxanthopterin t = 12.5, 6-biopterin t = 12.6, xantopterin t = 16.4, leukopterin t = 18.4, erythropterin t =

276 25.0, pterin-6-carboxylic acid t = 28.0 and D-neopterin t = 28.8. A chromatogram of the mixture of all pteridine 277 derivative standards in DMSO at a concentration of 0.01 mg/mL, measured in the TIC (total ion current) mode is 278 shown as supplementary Fig. S2a, SRM chromatograms of the individual derivatives are shown as supplementary 279 Fig. S2b.

282

283 3.1 Spectral reflectance

284 Average standardized reflectance spectra of Pseudemys concinna and Trachemys scripta are shown in Fig. 2 285 (see reflectance data of each individual at [Reference to be added]). The dorsal head background coloration (DHC) 286 of both T. scripta and P. concinna has a flat reflectance spectrum and low overall reflectance. Moreover, the 287 reflectance of DHC is further reduced beyond 550 nm. The red postorbital marking (PM) of T. s. elegans also has a 288 relatively flat reflectance spectrum, with low overall reflectance but, in contrast to DHC, the red PM of T. s. elegans 289 shows increased reflectance at wavelengths longer than 550 nm giving rise to a plateau between 600-700 nm. All 290 reflectance spectra from yellow regions (i. e. main median chin stripe – CBC, and main forelimb stripe – FLBS, of 291 both species, and PM of P. concinna as well as zygomatic patch – YP of T. s. scripta) are similar in that they show two 292 peaks, one minor peak in the UV part of the spectrum between 300 and 400 nm and a larger, primary peak between 293 500 and 600 nm. The width, shape and height of the primary peak differ slightly among the yellow regions. 294 Means of summary variables derived from reflectance spectra and their standard deviations for each region 295 of each taxon are shown in Table 1 (see summary variables of each individual at [Reference to be added]). Summary 296 variables derived from reflectance spectra of one individual T. s. scripta overlapped with summary variables (mean 297 +/- standard deviation) of T. s. elegans but fall outside the values (mean +/- standard deviation) of the summary 298 variables for P. concinna. 299 The ordination plot resulting from redundancy analyses (RDA) based on summary variables of both T. s 300 elegans and P. concinna is shown in Fig. 3 a. There are significant differences in coloration between P. concinna and 301 T. s. elegans (ANOVA-like permutation test, F = 16.39, p < 0.001). Constrained axis (RDA1) explains 17% of the total 302 variance in the data. The first residual axis (PC1) explains 18% and the second residual axis (PC2) explains 17% of the 303 total variance. Even though our primary goal was not to investigate sexual dichromatism, the results suggest sexual 304 differences in the reflectance of the red and yellow skin patches in T. scripta and, to a lesser extent, P. concinna (Fig. 305 2, See the supplementary materials for details). 306 Variables pertaining to background colour (DHC) are associated with PC1 rather than with RDA1 (compare 307 values of RDA1 scores of the DHC to scores of summary variables of other regions – Fig. 3B) and contribute very 308 little to colour differences between both species. Interpretation of the RDA1 axis, and therefore of differences 309 between both species, seems to be region specific. Pseudemys concinna shows increased brightness (B1) along the 310 RDA1 axis in CBC and PM, but decreased brightness in FLBS compared to T. s. elegans. The yellow CBC and FLBS of P. 311 concinna show higher chroma (S1) in the blue and UV segments of reflectance spectra, but the yellow CBC and FLBS 312 T. s. elegans have higher chroma (S1) in the yellow, red and green segments. The yellow PM of P. concinna shows 313 higher chroma in the green, yellow and blue segments of reflectance spectra, while the red PM of T. s. elegans 314 shows higher chroma in the red segment, but also in the UV segment of reflectance spectra (Fig. 3 b). The increased 315 chroma in the UV segment of the PM in T. s. elegans is a consequence of the overall low reflectance of this region 316 rather than exceptionally high reflectance in the UV. Reported patterns of interspecific differences did not change 317 when the PM region was not included in the analyses (data not shown here but see R script together with input data 318 at [Reference to be added]).

bioRxiv preprint doi: https://doi.org/10.1101/556670; this version posted February 21, 2019. The copyright holder for this preprint (which 319 was not certifiedIn short, by peer the review) major is differences the author/funder, in colour who ofhas examined granted bioRxiv regions a license between to display both thespecies preprint examined in perpetuity. are: It1) is made available under aCC-BY-NC-ND 4.0 International license. 320 shorter wavelengths contribute more to yellow colour of the CBC and FLBS of P. concinna compared to T. s. elegans; 321 2) CBC and PM are brighter in P. concinna, but the FLBS is brighter in T. s. elegans. 322 323 3.2 Microscopy

324 The epidermis is separated from the dermis by a conspicuous basal lamina (solid arrows in Figs. 4, 5). The 325 epidermis differs in thickness between regions and contains multiple layers of keratinocytes. The corneous layer of 326 the epidermis is relatively thin in all regions except in FLBS where it represents about one fifth of the thickness of 327 the entire epidermis (of all the regions examined only the FLBS is covered by scales). Arrays of collagen fibres are 328 highly abundant in the dermis of every region examined. Four basic pigment cells types are present in the 329 integument of all studied turtles: epidermal melanocytes, and dermal iridophores, melanocytes, and xanthophores 330 (Figs. 4; 5; 6). We have not observed finger-like projections of dermal melanocytes, that are characteristic for 331 dermal melanophores with motile organelles [12]. Mosaic pigment cells were also found in the dermis of T. s. 332 elegans (Fig. 6 d, g, h). The pigment cell types present in each of the studied regions are summarized in Table 2. 333 Epidermal melanocytes are present in the epidermis in the PM and DHC of T. scripta (Figs. 7 e; 8 a,b,c), and 334 DHC of P. conncina. Epidermal melanocytes are located just above the basal lamina and are surrounded by 335 keratinocytes. In samples with epidermal melanocytes the surrounding keratinocytes contain melanosomes. In 336 some samples, however, the epidermal melanocytes are contracted and contain only a few melanosomes; those 337 samples lack melanosomes deposited in keratinocytes. The PM of T. s. elegans contains mostly contracted 338 epidermal melanocytes and almost no transferred melanosomes (Fig. 8 a). In contrast, the PM of T. s. scripta 339 contains enlarged epidermal melanocytes and melanosomes distributed amongst many keratinocytes (Fig. 8 b, c). 340 Pseudemys concinna does not have epidermal melanocytes in the PM (Fig. 5, Fig. 8 e, f). 341 Iridophores are present especially in the dermis of the CBC, FLBS, and PM of P. concinna, and in the CBC, 342 FLBS of T. scripta, but are missing in the PM and YP of T. scripta. Iridophores are present also in the DHC, but these 343 are very rare, small and seem haphazardly distributed (Fig 7 e). The iridophores of P. concinna do not form a 344 continuous layer. In the CBC and FLBS of P. concinna iridophores are rather scarce (Fig. 7 b, d), but in the PM of P. 345 concinna they are more abundant (Fig. 8 e). In the CBC and FLBS of T. scripta iridophores are abundant and form an 346 almost continuous layer several cells thick (Fig. 7 a, c). 347 Dermal melanocytes are abundant in the DHC (Fig. 7 e, f) and in the narrow black lines outlining the yellow- 348 red regions (Fig. 6). In the DHC melanocytes are dispersed throughout the upper parts of dermis but do not 349 generally form a continuous layer. In contrast, the dermal melanocytes in the narrow black lines form a continuous 350 layer. Dermal melanocytes were present also in the PM of a melanistic male of T. s. elegans (Fig. 8 b). 351 Xanthophores are present in the dermis of all regions examined just beneath the basal lamina and just 352 above iridophores when those are present. In the CBC, FLBS (Fig. 7 b,d) and PM (Fig.8 e, f) of P. concinna the 353 xanthophores are sparse and do not form a continuous layer. The xanthophores form a thick continuous layer in the 354 PM of T. s. elegans (Fig. 8 a) and in the YP of T. s. scripta (Fig. 8 d). In the CBC and FLBS of T. s. elegans (Fig. 7 a, c) 355 and PM of T. s. scripta (Fig. 8 c) the xantophore layer is also continuous but its thickness is reduced compared to the 356 PM of T. s. elegans or YP of T. s. scripta. Xanthophores are also found in the DHC where they form aggregates of 357 interconnected cells, but not a continuous layer (Fig 7 e). Xanthophores contain three types of pigment bearing 358 organelles: two types of carotenoid vesicles with different electron density but without internal structure, and 359 pterinosomes (Fig. 9). Unlike carotenoid vesicles, mature pterinosomes are characterized by the presence of

378 The reflecting platelets present in the iridophores of all regions examined are roughly rectangular (Fig. 11). 379 Unfortunately, measurements of iridophores from the FLBS of P. concinna are not included in the results because 380 the platelets were not well preserved. Characteristics and counts of intact reflecting platelets as well as empty holes 381 are summarized in Table 2. Only in the FLBS of T. s. elegans and the CBC of P. concinna reflecting platelets are 382 mostly parallel to the skin surface (predominant angles of orientation are -1.98° and 2.49°, respectively), while the 383 major axes of reflecting platelets in the CBC of T. s. elegans and in the PM of P. concinna have average angles 384 differing from zero (30.86° and 68.3°, respectively). Moreover, the reflecting platelets of the FLBS of T. s. elegans are

385 more aligned with the skin surface and regularly organized (A/y0 = 607.28, FWHM = 59.19°) than other regions

386 examined (CBC of P. concinna - A/y0 = 10.85, FWHM = 95.3°; CBC of T. s. elegans - A/y0 = 6.31, FWHM = 106.24°; PM

387 of P. concinna - A/y0 = 4.91, FWHM = 112.42°) (see supplementary Fig. S3 a, b, c, d for distributions of angles of 388 reflecting platelets for different regions; see measurements of reflecting platelets at [Reference to be added]). 389 Only the FLBS of T. s. elegans meets the conditions of the thin layer interference model. Figure 12 a, b 390 shows the predicted reflectivity of iridophores of the FLBS of T. s. elegans. The peak of the highest density at the 391 predicted reflected wavelength differs slightly depending on whether intact reflecting platelets or empty holes are 392 measured (see supplementary Fig. S3 e, f, g, h for distributions of thickness of reflecting platelets for different 393 regions based of the two methods of measurement), being 618 nm based on measurement of intact platelets and 394 637 nm based on measurement of empty holes. 395 Due to the organization of their platelets, the iridophores in the CBC of P. concinna probably function as 396 broadband reflectors. The same probably applies to the iridophores in the CBC of T. s. elegans and the PM of P. 397 concinna, which in addition to disorganized reflecting platelets also possess platelets of variable size (Fig. 12 c). Only 398 the iridophores of the FLBS of T. s. elegans seem capable of intense narrowband colour production. 399 400 3.4 Fourier analysis of spatial distribution of dermal collagen arrays 10

bioRxiv preprint doi: https://doi.org/10.1101/556670; this version posted February 21, 2019. The copyright holder for this preprint (which 401 wasThe not 2certified-D Fourier by peer power review) spectra is the author/funder, show that who none has of granted the single bioRxiv collagen a license fibreto display arrays the preprint examined in perpetuity. (see examples It is made of available under aCC-BY-NC-ND 4.0 International license. 402 electromicrographs analyzed in Fig. 13; see output files of Fourier analyses for each elctromicrograph analysed at 403 [Reference to be added]) are organized randomly with respect to wavelengths of light, i. e. the distances between 404 collagen fibres are of the same order of magnitude as wavelengths of light [60]. However, the collagen arrays of T. s. 405 elegans show variation in the spatial frequencies of the 2-D Fourier power spectra within regions (supplementary 406 Fig. S4 a, b, c). In contrast, the power spectra of collagen arrays of P. concinna show reduced variation in 407 organization within regions (supplementary Fig. S4 d, e, f). Collagen arrays of the PM of P. concinna are 408 unambiguously organized in a way which results in small disc pattern of 2-D Fourier power spectra at low spatial 409 frequencies across all micrographs analysed. Collagen arrays of the FLBS of P. concinna show two-sided symmetric 410 pattern of 2-D Fourier power spectra at intermediate spatial frequencies rather than a disc pattern, suggesting 411 predominant periodicity in one direction perpendicular to the fibres. In the CBC of P. concinna power spectra show a 412 ring pattern at intermediate spatial frequencies, pointing to equivalent nanostructure in all directions perpendicular 413 to the fibres. 414 Normalized average radial means of the Fourier power spectra for each region of P. concinna and T. s. 415 elegans are shown in Fig 14. Radial means of the Fourier power spectra of all regions of T. s. elegans and the YP of T. 416 s. scripta show increased power values in the lowest spatial frequencies (< 0.0034 nm-1) compared to spatial 417 frequencies relevant to coherent scattering in the visible light (from 0.0034 to 0.0092 nm-1) (Fig. 14 a, b, c; 418 supplementary Fig. S5 a). Radial means of the Fourier power spectra of all regions of P. concinna also show 419 increased power values at the lowest spatial frequencies (Fig. 14 d, e, f). These values of spatial frequencies indicate 420 that the largest portion of light reflected by collagen fibres arrays in these regions belongs to the infra-red part of 421 the light spectrum. However, for the CBC of P. concinna this peak of low spatial frequencies lies immediately next to 422 the segment of spatial frequencies relevant to coherent scattering in visible light (Fig. 14 e). Radial means of spatial 423 frequencies of collagen fibres of CBC of P. concinna relevant to coherent scattering in visible light are in comparison 424 with radial means of all regions of T. scripta and the PM of P. concinna (Fig. 14 d) relatively elevated. In the FLBS of 425 P. concinna collagen fibres are organized at appropriate spatial frequencies (between 0.0034 and 0.0092 nm-1) to 426 produce coherent scattering in the visible spectrum (Fig. 14 f). 427 Predicted normalized reflectivity of collagen fibre arrays of P. concinna and T. s. elegans between 300 and 428 700 nm based on multiple radial means of the Fourier power spectra are shown in Fig. 15 together with normalized 429 measured reflectance spectra. All predicted normalized reflectivity curves have three distinct peaks (360–400 nm, 430 450–500 nm, and 560–570 nm) except for the YP of T. s. scripta which has four peaks (370, 450, 520, and 570 nm; 431 supplementary Fig. S5 b). All predicted normalized reflectivity curves increase beyond 600 nm, but in the CBC of 432 both T. s. elegans (Fig. 15 b) and P. concinna (Fig. 15 e) there is an additional peak/plateau at 660–680 nm. The 433 overall shapes of the predicted normalized reflectivity of collagen fibre arrays are more similar among regions than 434 among species. The PMs of both species (Fig. 15 a, d) have few distinct predicted peaks with a pronounced peak at 435 560 nm in the PM of P. concinna. The CBC of both species (Fig. 15 b, e) have four significant predicted peaks with 436 the highest at 490–500 nm. The FLBSs (Fig. 15 c, f) were characterized by predicted major peaks in the UV spectrum 437 at 360–380 nm. 438 Peaks of the predicted normalized reflectivity of collagen fibre arrays correspond roughly to peaks of 439 normalized measured reflectance spectra. However, there are differences in the overall shapes of the predicted and 440 measured curves. Differences between measured and predicted spectra probably arise due to interactions between 441 colour-producing pigment cells and the intercellular matrix of collagen fibre arrays. Even though the radial means of 442 the Fourier power spectra of the FLBS of P. concinna suggest appropriate spatial frequencies to produce colour by

452 Results of pigment content analyses for carotenoids and pteridine derivatives are summarized in Table 4. 453 Examples of UPLC chromatograms resulting from carotenoid content analyses can be found in the supplementary 454 materials (Fig. S1 b, g, h, i, j). The red PM of T. s. elegans contains a rich mixture of various carotenoids which, 455 according to UV/VIS absorbance spectra, are predominantly ketocarotenoids. The two most pronounced retention

+ 456 peaks were determined as astaxanthin (RT = 11.8 min, λmax = 471nm, [M+H] = 597.3944) and canthaxanthin (RT =

+ 457 13.7 min, λmax = 466, [M] = 565.4046). Other regions (CBC, FLBS of both species, PM of P. concinna, and PM and YP 458 of T. s. scripta) had no ketocarotenoids, but contained a relatively complex mixture of hydroxylated xanthophylls, of

•+ •+ 459 which lutein (RT = 12.9 min, λmax = 444, [M] = 568.4254) and zeaxanthin (RT = 13.1 min. , λmax = 449, [M] = 460 568.4253) were by far the most abundant. 461 Examples of SRM chromatograms of pteridine analyses are given in the supplementary material (Fig. S6). Out of nine

462 pteridine derivatives tested, seven were found in the integument of the turtles analysed. L-sepiapterin and 463 erythropterin were not detected in any sample. All other pteridine derivatives, i. e. 6-biopterin, pterin-6-COOH,

464 isoxanthopterin, leucopterin, D-neopterin, pterin, and xanthopterin, were found together only in the PM of T. s. 465 elegans. The yellow CBC and FLBS of both lineages of T. scripta and the YP of T. s. scripta contained all types of 466 pteridine derivatives except pterin. Only four types of pteridine derivatives, 6-biopterin, pterin-6-COOH, leucopterin,

467 and D-neopterin, were detected in the CBC, FLBS, and PM of P. concinna. 468 The red PM of T. s. elegans is unique among the examined region because it contains ketocarotenoid and seven 469 pteridine derivatives, including pterin, which was found in no other region. The ventral (CBC, FLBS) and dorsal 470 yellow regions (YP) do not differ in pigment contents in T. scripta. All yellow regions of T. scripta (CBC, FLBS, YP) and 471 P. concinna (CBC, FLBS) have the same carotenoid contents. On the other hand, pteridine derivatives are more 472 diverse in the yellow regions of T. scripta than in those of P. concinna. Two additional pteridine derivatives found in 473 the integument of T. scripta (isoxanthopterin and xanthopterin) are known to produce yellow colour. The 474 integument of P. concinna contains the same pigment types in every region examined (PM, CBC, FLBS). 475

476 4. Discussion

477 478 Although they are not known for their vivid, conspicuous body coloration, many turtles are in fact as colourful as 479 other vertebrates [69]. However very few studies have addressed the mechanisms of turtle colour production 480 [27,29,30]. In fact, there is more information on colour production in some fossil extinct taxa than in extant turtles 481 [70–76]. Research on the functional role of turtle chromatic signals is also limited [41], although it is known that at 482 least some species change colour during the reproductive season [19]. Given the revised phylogenetic position of

bioRxiv preprint doi: https://doi.org/10.1101/556670; this version posted February 21, 2019. The copyright holder for this preprint (which 483 wasturtles not certified as part by ofpeer archelosauria review) is the author/funder,[22,77–79], studyingwho has granted turtle bioRxivcoloration a license and tocolour display-producing the preprint mechanisms in perpetuity. Itcrucial is made to available under aCC-BY-NC-ND 4.0 International license. 484 understanding the evolution of colour and colour-producing mechanisms in vertebrates in general. 485 In this study we analysed the pigment cell organization of the integument, the ultrastructure of colour- 486 producing elements, and the chemical nature of pigments that produce skin colour in two freshwater turtles, 487 Pseudemys concinna and Trachemys scripta, with contrasting courtship behaviour. We found striking interspecific 488 differences in the organization of the chromatophores found in the dorsal and ventral head surfaces. Trachemys 489 scripta shows a different chromatophore composition in the ventral and dorsal sides of the head, whereas P. 490 concinna shows similar chromatophore composition on both sides. Also, there are more pigment types present in 491 the colour patches of T. scripta than in those of P. concinna. 492 Turtles employ colour producing mechanisms well known in amphibians and lepidosaurs, but not in other 493 extant archelosaurs (crocodiles and birds). In addition to xanthophores and melanophores previously reported in 494 turtles [27], and crocodiles [80], we found abundant iridophores containing rectangular reflecting platelets in yellow 495 skin of both species. To our knowledge, this is the first report of iridophores playing a role in integumental colour 496 production in any archelosaurian species [80,81]. Carotenoids are involved in colour production in the feathers of 497 birds [82], while pteridines are responsible for the colour of the iris of some birds [83] and perhaps also the feathers 498 of penguins [84]. However, our pigment analyses suggest that both pteridine derivatives and carotenoids are 499 involved in the production of the yellow-red skin colours of turtles, which had not been clearly documented in any 500 archelosaur to this date. 501 We also show that abundant collagen fibre arrays in the skin may serve to prevent infra-red light from 502 penetrating into the body of turtles, but in some cases, they may be capable of reflecting visible wavelengths of light 503 as well. Taken together, these results suggest that turtle coloration and colour-producing mechanisms are more 504 complex than previously thought. 505 506 507 4.1 The coloration of deirocheline turtles

508 Our results show that the conspicuous yellow stripes in the head and limbs of deirocheline turtles exhibit, in 509 addition to a primary reflectance peak responsible for the human-perceived yellow hue, a small secondary peak of 510 reflectance in the UV range of the spectrum. This confirms the results of several previous studies [30,85]. However, 511 we did not find support for the spectral shapes reported by Wang et al. [86]; in fact, we contend that the 372 nm 512 peak that these authors found in all their reflectance spectra (see their Figure 1 [86]) is an artefact possibly caused 513 by the illumination source. As turtles are capable of seeing into the UV range [87,88], the small peak of UV 514 reflectance may account for chromatic variation among different individuals and contribute to the conspicuousness 515 of the yellow stripes, at least during close-range interactions with conspecifics [30]. It is also possible that the 516 pattern of alternating dark and light stripes may provide camouflage through background matching (e.g. when 517 viewed against underwater vegetation) or as disruptive coloration creating false edges and visually breaking the 518 outline of the body, thus hindering detection of turtles by predators [89]. 519 Our results reveal interspecific differences in colour-producing mechanisms at multiple levels: differences 520 can be found in the distribution of chromatophores along the dorsoventral body axis, in the ultrastructure of 521 pigment-bearing vesicles of xanthophores, and in the chemical nature of pigments involved in colour production. As 522 for the distribution of chromatophores, Trachemys scripta has both xanthophores and iridophores in the yellow-red 523 regions of the ventral side of the head, but only xanthophores on the dorsal side. Such dorso-ventral patterning is 524 lacking in P. concinna which has xanthophores and iridophores on both sides of the head (Fig. 10). As iridophores 13

bioRxiv preprint doi: https://doi.org/10.1101/556670; this version posted February 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 525 increase the overall reflectance of a patch of skin [3,20,90], their presence on the ventral side of the head may 526 provide some sort of countershading to enhance crypsis ([91–93]), or may be related to signalling (see below). The 527 fact that both species of turtles also have a light plastron, but a dark coloured carapace is consistent with the 528 hypothesis that the increased reflectance caused by the presence of iridophores on the ventral side of the head 529 might serve a camouflage function. The background head coloration (DHC), which has been previously reported to 530 change in response to substrate colour [36,94–96], is produced in both T. scripta and P. concinna by epidermal 531 melanosomes and a combination of all three dermal pigment cell types (Fig. 7 e, f). However, we found epidermal 532 melanocytes in the PM of both lineages of T. scripta (Fig. 5, Fig. 8 e, f), but not in the PM of P. concinna (Fig. 4, Fig. 8 533 a, b, c) or in other yellow regions of the examined turtles (Fig. 7 b, d). 534 The red and yellow regions of turtle skin contain at least three morphologically distinct types of vesicles in 535 xanthophores. Two of these correspond to what have been previously [68,97,98] identified as carotenoid vesicles, 536 and differ in electron density, shape and size (Fig. 9 a, b, d, e). The third type of xanthophore vesicles are 537 pterinosomes, likely containing pteridine derivatives (Fig 9 c, f). These are recognizable by their prominent 538 concentric lamellae [67,68]. Xanthophores containing oval carotenoid vesicles with high electron density (Fig. 9 a, d) 539 are abundant only in the red PM of T. scripta elegans, while xanthophores containing round carotenoid vesicles with 540 lower electron density (Fig. 9 b, e) and/or pterinosomes (Fig. 9 c, f) are found in yellow regions of both species. We 541 assume that differences in the ultrastructure of carotenoid vesicles reflect differences in their pigment content, as in 542 the case of red and yellow retinal oil droplets [99] known to differ in carotenoid content [31,100,101]. However, 543 similar structures have been previously described as vesicles in different stages of maturation [102]. Further study 544 using appropriate methods is necessary to unambiguously determine the types of pigments contained in vesicles of 545 different ultrastructure. 546 Continuous layers of iridophores are found only in T. scripta. Moreover, the reflecting platelets of 547 iridophores from the CBC of T. scripta are highly organized with predicted reflected wavelengths between 618 and 548 637 nm (Fig. 12 a, b). Other regions containing iridophores, i. e. yellow FLBS of T. scripta and all yellow regions of P. 549 concinna, do not have organized platelets and therefore function as broadband reflectors which should enhance the 550 overall brightness/luminance of the corresponding skin patch. Previous reports have described iridophores with 551 randomly organized platelets in red and white skin of Phelsuma grandis where they produce broadband reflection 552 [16]. In contrast, reflecting platelets in orange and yellow skin regions of Uta stansburiana [57], Sceloporus 553 undulatus, and S. magister [56] are highly organized to reflect orange and yellow wavelengths. Even though 554 broadband reflecting iridophores do not influence perceived colour, they may increase the brightness of colour 555 regions and their overall conspicuousness to observers [3,103]. 556 Unlike all yellow regions of both species, which contain xanthophylls lutein and zeaxanthin, the red PM of 557 T. s. elegans contains the ketocarotenoids astaxanthin and canthaxanthin (Table 4, Fig. S1). Such difference is in 558 partial agreement with previous analyses of carotenoid contents in yellow and red regions of T. s. elegans and 559 Chrysemys picta [30]. Two major classes of carotenoids have been described in the integument of these turtles: 560 short wavelength absorbing apocarotenoids and longer wavelength absorbing ketocarotenoids [30]. In the yellow 561 chin of C. picta only apocarotenoids are present, whereas the orange neck and leg contain apocarotenoids and 562 ketocarotenoids. Previous reports indicated that apocarotenoids are abundant in tissue from yellow chin and yellow 563 neck stripes of T. scripta, its “orange” postorbital region containing only ketocarotenoids [30]. Our results, however, 564 suggest a major role of xanthophylls, rather than apocarotenoids, in yellow colour production in T. scripta and P. 565 concinna. Xanthophylls have been shown to produce yellow coloration in various vertebrates [82,104,105]. In C.

bioRxiv preprint doi: https://doi.org/10.1101/556670; this version posted February 21, 2019. The copyright holder for this preprint (which 566 waspicta not certifiedincreased by peeringestion review) of is the the author/funder,xanthophyll carotenoidwho has granted lutein bioRxiv results a license in an increaseto display inthe the preprint yellow in perpetuity. and red chromaIt is made of available under aCC-BY-NC-ND 4.0 International license. 567 yellow and red skin patches [28]. In contrast, it is unclear whether apocarotenoids participate in colour production 568 in animals. Apocarotenoids are cleavage products of carotenoids [106–108]. Transparent and clear cone oil droplets 569 in the retina of birds contain the apocarotenoid galloxanthin, but yellow oil droplets contain the xanthophyll 570 zeaxanthin and red oil droplets contain the ketocarotenoid astaxanthin [109]. The unique yellow colour of the 571 macula lutea in primates is due to the accumulation of xanthophylls in the cells of the retina via down-regulation of 572 their cleavage pathway to apocarotenoids [110]. Various carotenoid precursors give rise to a large diversity of 573 apocarotenoids [106], but specific types of apocarotenoids have not been reported in previous studies of turtle 574 coloration [30]. The role of apocarotenoids in turtle coloration will thus remain unclear until the relationship 575 between precursor carotenoids in the skin and apocarotenoids is elucidated. 576 Steffen et al. [30] suggested that the yellow-red skin of C. picta and the yellow skin (but not the red skin) of 577 T. scripta contain small amounts of pteridine derivatives. Our results show that the opposite is true (Table 4). The 578 red PM shows more types of pteridine derivatives than the yellow skin in T. scripta. Yellow skin in P. concinna 579 contains only a limited number of pteridine derivatives in comparison with all regions of T. scripta. Our results thus 580 show that the red PM of T.s. elegans is more elaborate in terms of its pteridine derivative contents than all the 581 yellow regions studied herein which contrasts with the previous study of Steffen et al. [30]. The yellow and red 582 colours of the turtles examined here result from an interplay between carotenoids and pteridine derivatives. 583 Whether the differences in colour and colour-producing mechanisms among deirocheline turtles are of 584 functional significance has yet to be determined. However, it is already well established that turtles are capable of 585 distinguishing different yellow and red colours [34,87,88] and it has been suggested that body coloration plays a 586 role in mate choice in emydid turtles [30,34,35]. Furthermore, there is some evidence that the red and yellow 587 regions of T. scripta could play a role in signalling individual quality based on the correlation of colour and induced 588 immune response [111]. Moreover, the spectral properties of colour patches of C. picta is influenced by the amount 589 of ingested carotenoids [28] which is one of the basic assumption of individual quality signalling [112–114]. 590 Is there a relationship between chromatophore distribution and male-female position during courtship? 591 Trachemys scripta and P. concinna differ in the distribution of chromatophore and pigment types along the 592 dorsoventral body axis (Fig. 10). The presence of xanthophores together with iridophores in the ventral CBC, but not 593 in the dorsal PM, as well as the presence of epidermal melanocytes in the PM, may enhance the cryptic appearance 594 of T. scripta in aquatic environments (see above). It is not clear, however, why P. concinna does not possess a similar 595 distribution of pigment cell types. Instead, P. concinna has no epidermal melanosomes in the PM, and xanthophores 596 and iridophores are distributed dorsally (PM) and ventrally (CBC). This should make P. concinna rather conspicuous 597 when viewed from above. Many animals solve the trade-off between making themselves conspicuous to 598 conspecifics and avoiding detection by predators by restricting bright colours to body surfaces that are exposed to 599 conspecifics but remain hidden to predators (called signal partitioning; e.g. [115,116]). This is often the case with 600 sexual signals which are made visible to mates during courtship [38,39]. Female mate choice is presumably 601 widespread among emydid turtles [117]. Males of T. scripta court females in a face-to-face position, so that both the 602 dorsal and ventral sides of the head as well as the limbs of the male are simultaneously exposed to the female. On 603 the other hand, males of P. concinna swim above the female during the final stages of courtship [44]. In this position 604 it may be more difficult for the female to perceive simultaneously the dorsal and ventral sides of the male’s head. 605 Thus, the location of the potentially brighter surfaces on the head of the males does not match predictions based on 606 the relative positions of males and females during courtship, at least in the two species examined here.

632 Results of Fourier analyses show that turtle collagen fibres reflect in the infrared part of the spectrum in all regions 633 examined (Fig. 14). Long wavelengths of light that pass through the xanthophores and iridophores may be absorbed 634 by the melanophores, or they may be reflected back to the surface by the underlying collagenous connective tissue 635 when melanophores are absent or scarce [125]. In many fish and amphibian larvae, the collagen fibres form 636 subepidermal collagenous lamella overlying the dermal chromatophores [20,126,127]. In lizards and snakes 637 collagenous connective tissue is found in the superficial fascia between the integument and the underlying muscle 638 [125], but in other animals such as frogs [128], mammals [17], or birds [18] collagen fibres are part of the dermis. 639 Here we show highly abundant dermal collagen fibre arrays located below the chromatophore layers in turtles (Figs. 640 4, 5). Unlike lizards [16,57,97] turtles lack a layer of dermal melanophores in the red-yellow skin regions. Turtles 641 bask to thermoregulate and overheating may represent an immediate risk for them [129], but basking could also 642 have a social function [130] for which it may be advantageous to accumulate heat at a slower rate. Therefore, the 643 function of dermal collagen fibres may be to prevent long infrared wavelengths from reaching the body core to 644 avoid overheating. A thermoprotective function of colour-producing structures has been recently suggested for the 645 deep iridophores found in chameleons [55] or plumage of birds [131]. The role of melanin in thermoregulation is 646 widespread across a broad range of organisms [132]. Thus, it seems that colour-producing elements are commonly

bioRxiv preprint doi: https://doi.org/10.1101/556670; this version posted February 21, 2019. The copyright holder for this preprint (which 647 wasacquired not certified to by thermoregulate peer review) is the and, author/funder, conversely, who skin has granted structures bioRxiv that a license play to role display in thermoregulationthe preprint in perpetuity. may It inis made some available under aCC-BY-NC-ND 4.0 International license. 648 instances be co-opted to produce visible colour. 649 Dermal collagen may appear achromatic [128], but colour production by coherent scattering on 650 nanostructured collagen fibre arrays has been previously reported in mammals [17] and birds [15,18]. In mammals 651 the colour produced by scattering on collagen fibres is limited to the blue part of the light spectrum [17], but in birds 652 other colours, such as orange in Trogopan coboti, are produced by coherent scattering on collagen fibre arrays [18]. 653 The yellow FLBS of P. concinna shows sufficient nanostructural organization to produce yellow colour by coherent 654 scattering (Fig. 14 f). In most of the examined regions of T. scripta and P. concinna there is also small peak in the UV 655 part of the spectrum which is predicted by Fourier analyses. Our results predict colour production by collagen fibres 656 in all regions (Fig. 15). However, in most regions the collagen fibers do not conform to the conditions of coherent 657 scattering or the predictions do not match the measured reflectance spectra very well. Even though the reflected 658 light by collagen fibre arrays would be achromatic their presence could still affect the reflectance spectra. It has 659 been shown theoretically that xanthophores have the highest influence and iridophore the lowest influence on 660 perceived colour when the melanophore layer is absent and reflected light off the collagen fibres passes back to the 661 surface [3]. As in the CBC and FLBS of P. concinna chromatophores are scarce, it is possible that collagen fibres play 662 a major role in colour production in this species. 663 664 665 4.3 Significance for vertebrate skin colour evolution

666 Most of vertebrates do produce various colours by superposition of different dermal pigment cells, however 667 homeothermic tetrapods, the birds and the mammals, acquire their coloration mainly due to the physiological 668 actions of epidermal melanocytes [133]. The archelosaurian clade, comprising turtles, crocodiles, and birds [22], has 669 undergone dramatic evolutionary changes affecting colour production mechanisms [134]. Some mechanisms of 670 colour production, such as melanogenesis, were modified perhaps with the acquisition of feathers [134], while 671 others, such as the use of ketocarotenoids for red coloration, may have been acquired early during the evolution of 672 the archelosaurian clade [31]. Iridophore and xanthophore differentiation or colour production by pteridine 673 derivatives have been lost or reduced during the evolution of the archelosaurian clade [84,133,135–138]. However, 674 cells with lipid vesicles suspected to contain pigments similar to xanthophores have been reported in both the 675 epidermis and the dermis of non-feathered bare skin of some bird taxa [18]. Crocodiles develop iridophores and 676 xanthophores that participate together with dermal melanophores/melanocytes in the production of brown-yellow 677 background colour, but the iridophores of crocodiles do not poses differentiated reflecting platelets [80]. Previous 678 results suggest that crocodiles may have gone through a nocturnal bottleneck after their divergence from the 679 lineage leading to birds while reducing their colour discrimination capacity [139], and thus may not represent an 680 ideal group to study colour evolution in the archelosaurian clade. Here we show that turtles possess all three types 681 of dermal pigment cells (xanthophores, iridophores, and melanocytes), shared with fish [140,141], amphibians 682 [142,143], and lepidosaurs [144–146]. Analyses of the pigments involved in the production of yellow-red colours in 683 turtles suggest that pteridine derivatives contribute, together with carotenoids, to these colours, which was not 684 previously documented in non-avian archelosaurs, but has been reported in fish [112], amphibians [147], and lizards 685 [104,148]. We show that dermal collagen fibres participate in colour production in turtles which has been reported 686 in mammals [17] and birds [18] and adult amphibians, but not in fish [20,126], amphibian larvae [127], and lizards 687 [125]. Turtles have also been reported to produce colour by hyperproliferation of keratin in epidermal keratinocytes 688 [19]. The keratin matrix is known to play a vital role in the coloration of birds’ feathers [149,150]. The evidence thus 17

698 More research is needed to further examine if the evolution of coloration in turtles is constrained by the position 699 during courtship and to test the idea of modular evolution of colour ornaments in turtles. Future studies should 700 consider a broader range of species of turtles and apply comparative cophylogenetic methods to map ancestral 701 states of coloration, its mechanisms of production, and relevant functional traits such as courtship behaviour. 702 In this study we have not addressed the ontogeny of colour-producing mechanisms, as our knowledge of 703 the early development of chromatophores in turtles is still scarce [123]. Also, the postnatal ontogeny of colour in 704 turtles is studied insufficiently and long-standing questions remain unresolved, e. g. melanophores in T. scripta are 705 found mostly in the dermis while older “melanistic” males also have elaborate epidermal melanocytes [48], which 706 raises the question how melanophores make the transition from one skin layer to another. 707 Our results also suggest that early archelosaurs share colour-producing mechanisms with amphibians and 708 lepidosaurs but also used novel ways to produce skin colour such as scattering on dermal collagen fibres. Studies of 709 colour-producing mechanisms on a broad phylogenetic scale are critical to clarify the evolution of colour-producing 710 mechanisms across vertebrates and should be encouraged in the future. 711 Turtles are a critically endangered group with 61% of species threatened or already extinct [154], which 712 may seriously limit the opportunities to collect biological samples. On the other hand, T. scripta is considered one of 713 the most widespread invasive alien species [155] and intensive management measures are taken to control its 714 introduced populations. Pertinent culling of individuals from introduced species offers an excellent opportunity to 715 collect rare biological samples which should not be missed. 716 717 718 Acknowledgments 719 We are thankful to Piscífatoria del Palmar for the exceptional hospitality. This study would not be possible without Vicente 720 Sancho and Ignacio Lacomba who pioneered turtle conservation in the Comunidat Valencia and are responsible for JB and EF 721 getting to know each other. This study would have not been possible without Alice Exnerová and Prof. Pavel Štys who put JB and 722 ZB in contact. We are thankful to Centro de recuperación de fauna, Granja El Saler, for the veterinary expertise. We are thankful 723 to José Manuel Garcia-Verdugo, his lab personnel (Patricia García-Tárraga, Arantxa Cebrian-Silla, Mariana Fill), and Mario Soriano 724 for support in electronic microscopy techniques. We are thankful to Jan Krajíček, who conducted the initial analyses of pteridine 725 derivatives. JB is thankful to Radek Šanda for his patience and support as Head of the Department of Zoology in the National 726 museum. We are thankful to Jan Raška and Gerardo Antonio Cordero for comments on the manuscript. JB dedicates this study to 727 his (growing) family. 728 729 730 Ethical Statement

bioRxiv preprint doi: https://doi.org/10.1101/556670; this version posted February 21, 2019. The copyright holder for this preprint (which 731 wasNo not human certified tissues by peer were review) used isin the this author/funder, study. All the who turt hasles grantedused in bioRxiv this study a license were tocaptured display thein thepreprint wild inin perpetuity. accordance It iswith made the available under aCC-BY-NC-ND 4.0 International license. 732 Regional Decrees 14/2013 and 213/2009 on control measures on invasive alien species in the Valencian Community. All 733 procedures performed were in accordance with the ethical standards of Direcció General de Medi Natural i d' Avaluació 734 Ambiental, specially the 4/1994 Regional Act on Pets Protection. All applicable international, national, and/or institutional 735 guidelines for the care and use of animals were followed. 736 737 Funding Statement 738 JB was financially supported by the Charles University (SVV 260434/2018) by the Ministry of Culture of the Czech Republic 739 (DKRVO 2019-2023/ 6.VII.a, 00023272). MH was financially supported by the Charles University, project GA UK No 760216 and 740 the project of the Specific University Research (SVV260440). 741 742 Data Accessibility 743 Reflectance spectra data, reflectance spectra summary variables, R script of spectral analyses, reflecting platelets 744 electromicrographs, reflecting platelets measurements and Fourier analyses output files: Dryad deposition underway, the 745 authors will contact the journal with an updated manuscript shortly 746 747 Competing Interests 748 We have no competing interests. 749 750 Authors' Contributions 751 JB conceived the study, designed the study, coordinated the study, measured reflectance spectra, collected sample tissues, 752 carried out the histological lab work, carried out microscopical observation, carried out the statistical analyses, carried out 753 reflective platelets measurements and analyses, carried out Fourier analyses, participated in carotenoid analyses, drafted 754 manuscript; JVB carried out the field work, communicated with local authorities; ZB supervised and carried out the pteridine 755 derivatives analyses, helped to draft the manuscript; JG participated in Fourier analyses; MH carried out pteridine derivatives 756 analyses; KK supervised and financially supported JB, supervised statistical analyses, helped to draft the manuscript; PM 757 conceived the study, carried out the carotenoid analyses, helped to draft the manuscript; EF conceived the study, designed the 758 study, financially supported the study, supervised histological and microscopical techniques, coordinated the study, drafted 759 manuscript. All authors gave final approval for publication. 760

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1066

Table 1 - Summary color variables for each taxon

Taxon (number of individuals) Region Variable (SD) B1 S1.UV S1.blue S1.green S1.yellow S1.red H1

P. concinna (N=12) CBC 15584(2670) 0.102(0.034) 0.272(0.019) 0.34(0.023) 0.26(0.019) 0.294(0.025) 515.25(10.244) DHC 1904.4282(713.37) 0.357(0.046) 0.348(0.013) 0.272(0.037) 0.141(0.024) 0.03(0.035) 365.167(85.58) FLBS 10572.43(3027.39) 0.14(0.037) 0.256(0.03) 0.338(0.029) 0.257(0.026) 0.274(0.031) 540.167(12.511) PM 7775.415(2765.69) 0.147(0.03) 0.271(0.028) 0.347(0.029) 0.248(0.022) 0.242(0.037) 529.75(11.787)

T. s. elegans (N=68) CBC 11923.4(4287.2) 0.095(0.036) 0.221(0.027) 0.382(0.036) 0.291(0.027) 0.31(0.029) 547.882(27.173) DHC 1792.31(1070) 0.353(0.042) 0.354(0.027) 0.28(0.029) 0.134(0.031) 0.02(0.053) 432.088(119.64) FLBS 12026.9(3134.57) 0.086(0.025) 0.186(0.027) 0.406(0.024) 0.318(0.023) 0.33(0.029) 552.912(10.215) PM 5991.65(2426.26) 0.223(0.037) 0.231(0.045) 0.237(0.029) 0.207(0.032) 0.316(0.089) 622.382(123.418)

T. s. scripta (N=1) CBC 11467.67 0.094 0.229 0.375 0.28 0.309 518 DHC 801.896 0.29 0.37 0.346 0.159 0 549 FLBS 10980.87 0.078 0.192 0.405 0.309 0.334 541 1067 YP 12102.164 0.172 0.252 0.323 0.239 0.261 518 1068

Taxon Region Pigment cell type epidermal dermal melanocytes iridophores melanocytes xantophores

P. concinna CBC ✖ ✔ ✖ ✔ DHC ✔ ✔ ✔ ✔ FLBS ✖ ✔ ✖ ✔ PM ✖ ✔ ✖ ✔

T. s. elegans CBC ✖ ✔ ✖ ✔ DHC ✔ ✔ ✔ ✔ FLBS ✖ ✔ ✖ ✔ PM ✔ ✖ ✖ ✔

T. s. scripta CBC ✖ ✔ ✖ ✔ DHC ✔ ✔ ✔ ✔ FLBS ✖ ✔ ✖ ✔ YP ✖ ✖ ✖ ✔ 1069 1070

Table 3 - Reflective platelets characteristics

Variable P. concinna T. s. elegans CBC PM CBC FLBS Number of platelets measured - intact 65 100 59 74 Number of platelets measured - empty holes 456 625 2777 2366 Mean minor axis lenght - intact +/- s.d. (nm) 143.44(64.52) 91.5(44.57) 86.34(30.65) 85.85(33.49) Mean minor axis lenght - empty holes +/- s.d. (nm) 124.63(47.55) 100.95(47.55) 104.43(51.20) 93.95(42.65) Mean major axis lenght - intact +/- s.d. (nm) 457.51(221.97) 414.93(222.31) 370.4(145.88) 395.52(179.60) Mean major axis lenght - empty holes +/- s.d. (nm) 420.30(223.63) 333.70(208.98) 364.25(240.22) 347.40(221.04)

A/y0 (A;y0) 10.85(0.0090; 4.91(0.0069; 6.31(0.0076; 607.28(0.0158; 0.0008) 0.0014) 0.0012) 0.000026) FWHM (°) 95.3 112.42 106.24 59.19 1072 Predominant orientation (°) 2.49 68.3 30.86 -1.98

Table 4 - Pigment contents for particular regions of skin of examined taxa

Taxon Region Pteridine Derivatives Predominant Carotenoids 6-biopterin pterin-6-COOH Erythropterin Isoxanthopterin Leucopterin L-sepiapterin D-Neopterin Pterin Xanthopterin Astaxanthin Canthaxanthin Lutein Zeaxanthin P. concinna CBC ✔ ✔ ✖ ✖ ✔ ✖ ✔ ✖ ✖ ✖ ✖ ✔ ✔ FLBS ✔ ✔ ✖ ✖ ✔ ✖ ✔ ✖ ✖ ✖ ✖ ✔ ✔ PM ✔ ✔ ✖ ✖ ✔ ✖ ✔ ✖ ✖ ✖ ✖ ✔ ✔

T. s. elegans CBC ✔ ✔ ✖ ✔ ✔ ✖ ✔ ✖ ✔ ✖ ✖ ✔ ✔ FLBS ✔ ✔ ✖ ✔ ✔ ✖ ✔ ✖ ✔ ✖ ✖ ✔ ✔ PM ✔ ✔ ✖ ✔ ✔ ✖ ✔ ✔ ✔ ✔ ✔ ✖ ✖

T. s. scripta CBC ✔ ✔ ✖ ✔ ✔ ✖ ✔ ✖ ✔ ✖ ✖ ✔ ✔ FLBS ✔ ✔ ✖ ✔ ✔ ✖ ✔ ✖ ✔ ✖ ✖ ✔ ✔ 1073 YP ✔ ✔ ✖ ✔ ✔ ✖ ✔ ✖ ✔ ✖ ✖ ✔ ✔

1075 1076 Figure 1 1077

1078 1079

bioRxiv preprint doi: https://doi.org/10.1101/556670; this version posted February 21, 2019. The copyright holder for this preprint (which 1080 wasFigure not certified 2 by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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1126 1127 Table captions

1128 Table 1 Summary colour variables for each taxon (Pseudemys concinna, Trachemys scripta elegans, Trachemys scripta 1129 scripta) involved in this study. B1 – sum of the relative reflectance over the entire spectral range; H1 – wavelength of 1130 maximum reflectance; relative contributions of a spectral ranges to the total brightness: 300-400nm (S1.UV), 400- 1131 510nm (S1.blue), 510nm-605nm (S1.green), 550nm-625nm (S1.yellow), 605-700nm (S1.red). CBC: main median chin 1132 yellow stripe; DHC: dorsal head background coloration; FLBS: main bright stripe of the fore limb; PM: postorbital 1133 marking; YP: yellow zygomatic patch. Note that for T. s. scripta the values of summary variables resulted from 1134 measurements of only one specimen. 1135 1136 Table 2 Contribution (✔ present/ ✖ absent) of different pigment cell types in particular regions of integument in 1137 studied taxa (Pseudemys concinna, Trachemys scripta elegans, Trachemys scripta scripta). CBC: main median chin yellow 1138 stripe; DHC: dorsal head background coloration; FLBS: main bright stripe of the fore limb; PM: postorbital marking; YP: 1139 yellow zygomatic patch. 1140 1141 Table 3 Characteristics of reflective platelets of iridophores in CBC (main median chin yellow stripe), FLBS (main bright 1142 stripe of the fore limb) regions of Pseudemys concinna, and CBC, PM (postorbital marking) of Trachemys scripta elegans. 1143 A/y0 ratio determine relative number of reflecting platelets parallel to the skin surface. A: amplitude of Gaussian curve 1144 above the background of randomly oriented platelets; y0: background level of randomly oriented platelets; FWHM: full 1145 width of half maximum of the peak of Gaussian curve fitted to the data, i. e. width of angular distribution of reflecting 1146 platelets. 1147 1148 Table 4 Contribution (✔ present/ ✖ absent) of different types of carotenoid and pteridine derivatives in regions of 1149 integument in studied taxa (Pseudemys concinna, Trachemys scripta elegans, Trachemys scripta scripta). CBC: main 1150 median chin yellow stripe; FLBS: main bright stripe of the fore limb; PM: postorbital marking; YP: yellow zygomatic 1151 patch. 1152

1153 Figure captions

1154 Figure 1 Photographs of skin regions examined in this study. (a) lateral view of the head of Trachemys scripta elegans; 1155 (b) lateral view of the head of female of Trachemys scripta scripta; (c) lateral view of head of female Pseudemys 1156 concinna; (d) ventral side of head of female of T. scripta; (e) front limb of T. scripta; (f) ventral side of P. concinna; (g) 1157 front limb of P. concinna. CBC: main median chin yellow stripe; DHC: dorsal head background coloration; FLBS: main 1158 bright stripe of the fore limb; PM: postorbital marking; YP: yellow zygomatic patch. 1159 1160 Figure 2 Mean (+/- SEM) reflectance spectra of focal regions of (a) females of Pseudemys concinna (N=8), (b) males of P. 1161 concinna (N=4), (c) females of Trachemys scripta elegans (N=39), and (b) males of T. s. scripta elegans (N=30). CBC: main

bioRxiv preprint doi: https://doi.org/10.1101/556670; this version posted February 21, 2019. The copyright holder for this preprint (which 1162 wasmedian not certified chin by yellow peer review) stripe; is DHC:the author/funder, dorsal head who background has granted bioRxivcoloration a license; FLBS: to display main the bright preprint stripe in perpetuity. of the fore It is made limb; PM: available under aCC-BY-NC-ND 4.0 International license. 1163 postorbital marking. The yellow zygomatic patch (YP) of T. s. scripta (N=1) is shown in (d). 1164 1165 Figure 3 Overall differences in colour between the two studied species. (a) Biplot representing results of RDA of 1166 summary variables derived from reflectance spectra of Pseudemys concinna and Trachemys scripta elegans. First axis 1167 (RDA1) is constrained by species (explains 17% of total variance). The first residual axis (PC1) explains 18% of total 1168 variance. B1: sum of the relative reflectance over the entire spectral range; H1: wavelength of maximum reflectance; 1169 relative contributions of a spectral ranges to the total brightness: 300-400 nm (S1.UV), 400-510 nm (S1.blue), 510-605 1170 nm (S1.green), 550-625 nm (S1.yellow), 605-700 nm (S1.red). CBC: main median chin yellow stripe; DHC: dorsal head 1171 background coloration; FLBS: main bright stripe of the fore limb; PM: postorbital marking; YP: yellow zygomatic patch. 1172 (b) Schema of the differences between species (distribution along RDA1 axis) in chroma (S1) and brightness (B1) of 1173 examined regions. Note that the DHC region aligned rather more with the first residual axis (PC1). (c) Table of scores of 1174 summary variables of all colour regions of both species on RDA1 axis. Thick line denotes zero. 1175 1176 Figure 4 Red postorbital region (PM) of Trachemys scripta elegans. (a) micrograph of semi-thin section stained with 1177 toluidine blue viewed under light microscope; (b) area denoted by rectangle in (a) viewed at higher magnification; (c) 1178 electromicrograph showing xanthophores residing in dermis under basal lamina (solid arrows); (d) closer view of 1179 xanthophore and its carotenoids vesicles; (e) detail of melanosomes of epidermal melanocyte. D: dermis; E: epidermis; 1180 c: collagen fibres; k: keratinocyte; m: melanocyte; x: xanthophore; arrows: basal lamina. 1181 1182 Figure 5 Yellow postorbital region (PM) of Pseudemys concinna. (a) semi-thin section stained by toluidine blue viewed 1183 under light microscope; (b) area denoted by rectangle in (a) viewed at higher magnification; (c) Electromicrograph 1184 showing xanthophores and iridophores in the dermis; (d) closer view of iridophore; (e) closer look on xanthophore with 1185 detail of pterinosome in the inset. D: dermis; E: epidermis; c: collagen fibres; k: keratinocyte; solid arrows: basal lamina; 1186 clear arrows: xanthophore; arrowheads: iridophores. 1187 1188 Figure 6 Boundary between the red postorbital region (PM) and its surrounding black line of Trachemys scripta elegans; 1189 (a) photography of the lateral view of the head, region of interest highlighted by white rectangle. (b) edge between two 1190 different regions viewed under binocular microscope; (c) electromicrograph of section of red postorbital region, orange 1191 line: xanthophores; white line: basal lamina; (d) edge of black region, turquoise line: iridophore; white line: basal 1192 lamina; black arrowhead: epidermal melanocyte; white arrow: dermal melanocytes; asterisk: mosaic chromatophore; 1193 (e) detail of vesicles of xanthophores of the red postorbital region – carotenoid vesicles and pterinosomes; (f) detail of 1194 vesicles of dermal melanocytes of black region, melanosomes; (g) detailed view of mosaic chromatophore in the dermis. 1195 White line: basal lamina; asterisk: mosaic chromatophore; (h) high magnification of vesicles of mosaic chromatophore, 1196 showing co-localization of carotenoid vesicles and melanosomes. 1197 1198 Figure 7 Electromicrographs of different regions of turtles’ integument. (a) yellow chin (CBC) region of Trachemys 1199 scripta; (b) CBC region of Pseudemys concinna; (c) yellow front limb (FLBS) region of T. scripta; (d) FLBS region of P. 1200 concinna; (e) Dorsal dark head (DHC) region of T. s. elegans; (f) DHC region of T. s. scripta. White line: basal lamina; 1201 orange line: xanthophores; turquoise line: iridophores; black arrowheads: epidermal melanocytes; white arrows: dermal 1202 melanocytes. Scale bar represents 10µm. Note that magnification differs.

bioRxiv preprint doi: https://doi.org/10.1101/556670; this version posted February 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1203 1204 Figure 8 Electromicrographs of different regions of turtles’ integument. (a) red postorbital (PM) region of Trachemys 1205 scripta elegans, note small epidermal melanocytes and no melanosomes in keratinocytes; (b) red PM region of 1206 melanistic male of T. s. elegans, note enlarged epidermal melanocytes, but few melanosomes in keratinocytes; (c) dark 1207 PM region of T.s. scripta, note enlarged epidermal melanocytes and abundant melanosomes in keratinocytes; (d) yellow 1208 zygomatic patch (YP) region of T.s. scripta, note that there are no epidermal melanocytes; (e) yellow PM region of P. 1209 concinna; (f) yellow PM region of different specimen of P. concinna than in e), see the difference in abundance of 1210 xanthophores and iridophores, here xanthophores form almost continuous layer, it is possible that these differences are 1211 due to the different localization within the PM stripe on head, or there these differences may be due to intraspecific 1212 variability. White line: basal lamina; orange line: xanthophores; turquoise line: iridophores; black arrowheads: 1213 epidermal melanocytes; white arrows: dermal melanocytes. Scale bar represents 10µm. Note that magnification differs. 1214 1215 Figure 9 Examples of three different types of vesicles present in the xanthophores of examined turtles. (a) electron 1216 dense vesicles present predominantly in the PM of Trachemys scripta elegans; (b) vesicles with lower electron density 1217 present predominantly in the YP of Trachemys scripta scripta; (c) pterinosomes; (d) detail of electron dense vesicles 1218 from a), these vesicles lack any internal structure and possibly contain ketocarotenoids; (e) detail of vesicles with lower 1219 electron density shown in (b), there is no pronounced structure visible inside these vesicles. In context of chemical 1220 analysis, it seems these vesicles contain predominantly yellow carotenoids; (f) detail of pterinosomes shown in (c), 1221 typical by concentric lamellae. Scalebar 2µm (a, b, c) and 500nm (d, e, f). Note that magnification of (a) differ. 1222 1223 Figure 10 Schematic diagram of cellular composition of colourful region of Emydinae. Ventral chin stripe (CBC), and 1224 dorsal postorbital marking (PM) and zygomatic patch (YP) of examined turtles compose of epidermal melanocytes and 1225 dermal xanthophores and iridophores. Both ventral and dorsal regions of Pseudemys concinna (e) consist of 1226 xanthophores and iridophores (a). However, integument of Trachemys scripta elegans (f) and T. s. scripta (g) contain 1227 xanthophores together with iridophores on ventral side. On dorsal side there are only xanthophores in the dermis (b, c) 1228 present in both subspecies of T. scripta. There are no epidermal melanocytes in neither PM or CBC of P. concinna (a), 1229 but there are epidermal melanocytes in dorsal integument of T. scripta. These epidermal melanocytes are small, 1230 without apparent melanosome transferring activity (b) in red PM of T. s. elegans and yellow zygomatic region (YP) of T. 1231 s. scripta. In dark PM region of T. s. scripta (c) epidermal melanosomes are enlarged and epidermal keratinocytes 1232 contain transferred melanosomes (grey dots). The dermis contains abundant collagen fibres. 1233 1234 Figure 11 Electromicrograph of iridophores in the turtle integument. E: epidermis; D: dermis; x: xanthophore; 1235 arrowheads: iridophore. In the inset is detailed view of reflecting platelet. 1236 1237 Figure 12 Distribution of predicted reflectivity of reflecting platelets of iridophores. (a) based on size of intact reflecting 1238 platelets of FLBS - yellow front limb region of Trachemys scripta. (b) based on size of empty holes of FLBS region of T. 1239 scripta left after staining of EM sample. Dashed line in (a) and (b) represent shape of reflectance spectra predicted from 1240 reflecting platelets properties based on thin layer interference model. (c) distribution of predicted reflectivity of 1241 reflecting platelets of iridophores in regions where assumption of thin layer interference model does not seem to be 1242 appropriate.

bioRxiv preprint doi: https://doi.org/10.1101/556670; this version posted February 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made 1243 available under aCC-BY-NC-ND 4.0 International license. 1244 Figure 13 Electromicrographs of details of collagen fibre arrays. (a) Postorbital marking (PM) of the Trachemys scripta. 1245 (b) Main median chin yellow stripe (CBC) of T. scripta. (c) Main bright stripe forelimb stripe of T. scripta. (d) PM region 1246 of Pseudemys concinna. (e) CBC region of P. concinna. (f) FLBS region of P. concinna. Scalebar represent 200 nm. 1247 1248 Figure 14 Radial means of the Fourier power spectra of electromicrographs of collagen fibre arrays. (a) Postorbital 1249 marking (PM) of the Trachemys scripta. (b) Main median chin yellow stripe (CBC) of T. scripta. (c) Main bright stripe 1250 forelimb stripe of T. scripta. (d) PM region of Pseudemys concinna. (e) CBC region of P. concinna. (f) FLBS region of P. 1251 concinna. Grey rectangle denotes spatial frequencies relevant to coherent scattering in visible light, on the other hand 1252 lower frequencies (to the left from the grey rectangle) are relevant to scattering in the infra-red part of light spectrum. 1253 1254 Figure 15 Measured reflectance spectra of the skin regions (solid line) and reflectance spectra predicted by Fourier 1255 analyses of dermal collagen fibres (dashed line). (a) Postorbital marking (PM) of the Trachemys scripta. (b) Main median 1256 chin yellow stripe (CBC) of T. scripta. (c) Main bright stripe forelimb stripe of T. scripta. (d) PM region of Pseudemys 1257 concinna. (e) CBC region of P. concinna. (f) FLBS region of P. concinna. Grey rectangle denotes spatial frequencies 1258 relevant to coherent scattering in visible light, on the other hand lowever frequencies (to the left from the grey 1259 rectangle) are relevant to scattering in the infra-red part of light spectrum. Note that both measured reflectance and 1260 Fourier power are both normalised to have minimum zero and maximum one. 1261 1262 Figure 15 Measured reflectance spectra of the skin regions (solid line) and reflectance spectra predicted by Fourier 1263 analyses of dermal collagen fibres (dashed line). (a) Postorbital marking (PM) of the Trachemys scripta. (b) Main median 1264 chin yellow stripe (CBC) of T. scripta. (c) Main bright stripe forelimb stripe of T. scripta. (d) PM region of Pseudemys 1265 concinna. (e) CBC region of P. concinna. (f) FLBS region of P. concinna. Grey rectangle denotes spatial frequencies 1266 relevant to coherent scattering in visible light, on the other hand lowever frequencies (to the left from the grey 1267 rectangle) are relevant to scattering in the infra-red part of light spectrum. Note that both measured reflectance and 1268 Fourier power are both normalised to have minimum zero and maximum one. 1269 1270 Figure 16 Phylogeny of extant tetrapods [156] illustrating distribution of composition of colour-producing elements in 1271 the skin of tetrapods. All of the tetrapods may pose epidermal melanocytes in the epidermis. (a) composition of colour- 1272 producing elements of amphibian larvae (on the left) is similar to that of fish. Basal lamina is underlined by layer of 1273 subepidermal collagen fibres beneath which are xanthophores, iridophores and melanophores/melanocytes located in 1274 the dermis. Adults of amphibians dermal xanthophores, iridophore and melanophores that may form dermal 1275 chromatophore units. (b) lepidosaurs pose similar organization of colour-producing elements to that of adults of 1276 amphibians. (c) birds do not pose any additional pigment cell type to the epidermal melanocytes. Birds are known to 1277 produce colour by organized collagen fibre arrays. Means of colour production of plumage are not depicted. (d) 1278 crocodiles pose dermal pigment cells: xanthophores, iridophores, melanocytes, that have not been reported to form 1279 continuous layers. Iridophores of crocodiles have been reported to not contain rectangular reflecting platelets. (e) 1280 turtles do pose in dermis continuous layers of single xanthophores in some regions, other regions may be characterized 1281 by presence of continuous double layer of xanthophores together with iridophore in the dermis, which have not been 1282 found underlined by melanophores/melanocytes in neither of the skin regions. Single continuous layer of dermal 1283 melanocytes has been found in turtles. Dermal collagen fibres participate in colour production of turtles (f) mammals do