SUPPLEMENTARY INFORMATION

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1 SUPPLEMENTARY INFORMATION doi: /nature Supplementary Methods a) Extant sampling and analyses Samples of extant amniote integument attempted to capture the diversity of melanosomes associated with melanin-based colors found in each clade. Melanin-based variation in hair and skin color is limited to black, brown and grey 1. Iridescent colors in mammals are very rare, thus far only documented in golden moles 2 (Family Chrysochloridae). Golden mole iridescent color is produced by interference from multiple layers of keratin in the hair cortex. While melanin is present in the medulla of these hairs, it does not serve a direct role in interference 2. Thus, because golden mole color has been documented to not be melanin-based, they were not a priority for sampling. Iridescent skin colors in squamates are also not produced by melanin, but rather by specialized pigment cells called iridiphores 1. By contrast, the thin films, hexagonally packed crystals and other nanostructures that produce iridescent colors in feathers are composed of melanosomes 3. Even the simplest iridescent colors in feathers require an organized layer of melanosomes beneath a keratin cortex, and this layer actively scatters light 4. Melanosomes from iridescent feathers were also key to include as they are morphologically distinct 5. A further class of morphologically distinct melanosomes divergent from all other birds has also been identified and is so far only known from a single extant avian clade (Spheniscidae 6 ). These Penguin-type melanosomes are included to fully represent avian melanosome diversity. Because our focus was specifically melanin (thus far the only amniote pigment known to be preserved in the fossil record), we did not sample any integument that had colors reported to be or otherwise consistent with carotenoidor other pigment-based coloration. Extant hairs were pulled from study specimens of mammals in the collections of the University of Akron and the San Diego Museum of Natural History, and small (~5mm 2 ) sections of skin were cut from melanized portions near the forelimb of formalin-preserved reptiles in the collection of the Smithsonian Museum of Natural History. Grey pelage was more rare than black and brown hair and, when found, was typically found to be caused by mixtures of separate black and white hairs, or single hairs with both black and white areas. Thus, sample sizes for grey hair were much smaller (n=7) than for black and brown hair (n=18 and 26, respectively). Sampled species are listed in Supplementary Table 2. We obtained data on melanosome morphology from these samples as previously described 6. First, skin or hair was embedded in Epon by dehydration using 100% ethanol (20 min.) twice, and infiltration with 15, 50, 70 and 100% Epon (24 h each step). Infiltrated samples were then placed in block molds and polymerized at 60º C for 16 hours. We then cut thick (5 µm) longitudinal sections of blocks with a glass knife on a Leica UC-6 ultramicrotome, mounted them on stubs with carbon tape, sputter-coated them with silver and viewed them on a JEOL JSM7401F SEM. From the resulting SEM images we used the image-processing program ImageJ (available for download at rsbweb.nih.gov/ij/) to measure melanosome morphology as described previously 6. Melanosomes from hairs were sparsely distributed and difficult to locate; therefore, sample sizes of hair melanosomes were 1

2 RESEARCH SUPPLEMENTARY INFORMATION smaller than those from skin and feathers. We measured maximum linear short and long axis length of melanosomes that were oriented perpendicular to line of sight, and from these data calculated aspect ratio (long:short axis). These ratios are an index of shape, and values close to 1 indicate sphericity while values further from 1 indicate cylindricity. The distribution of melanosome morphology within feathers was frequently skewed towards one type of morphology, therefore we also calculated the skewness of long and short axis. Using the R environment, we compared these values between extant colored integument using ANOVA (aov function) and Tukey s posthoc test (TukeyHSD function). Results are summarized in Figure 1 and Table S1. We used a quadratic canonical discriminant analysis to determine if color could be predicted from melanososome morphology in hair and skin. This standard method identifies the combination of variables that best separates two or more classes of objects (in this case, melanosomes from the different categories of hair or skin) 7. Using data from training points, the classifying variables are loaded onto factors that are then used to estimate categories, and the probability of correct classification, of unknowns. We used a quadratic rather than linear discriminant analysis because some variables showed evidence of collinearity, but use of linear discriminant analysis did not significantly alter results. We used a forward stepwise method to choose variables that contributed significantly (p<0.05) to the analysis and to eliminate those that did not. For hairs, only one variable, length (p<0.001), was retained for use in the analysis. No other variable significantly contributed to the analysis (all p>0.15). The discriminant function as a whole was highly significant (Pillai's trace, F 2,48 =31.66, p<0.001). Canonical axis 1 (length) had an Eigenvalue of 0.74, and canonical correlation of Only 59% of hair colors were correctly predicted using this analysis. Predictive power improved when grey hairs were removed from the analysis. Length was again the only significant variable in the analysis (p<0.001), and the discriminant function was highly significant (Pillai s trace, F 2,48 =17.77, p<0.001). Canonical axis 1 (length) had an Eigenvalue of 0.75 and canonical correlation of % of hairs were correctly predicted as either black or brown. Increased sample size may improve discriminant ability, but examination of macroscale patterns may be needed for detection of grey colors in hairs. In any case, we were at least able to distinguish between black and brown hairs, indicating a bimodal distribution of size (long eumelanosomes and short pheomelanosomes ) that is associated with color. By contrast, we detected no association between melanosome shape and color in reptilian skin. No variable significantly contributed to the analysis (all p>0.28), so the analysis could not be performed. Melanosomes in reptilian skin were all uniformly small and low aspect ratio (Fig. 1, Extended Data Figure 9). The causes of these differences in melanosome diversity and associations between shape and color are currently unknown, but are likely related proximately to genes involved in melanin and melanosome production, including MC1R, ASIP 8, and Pmel 9. b) Fossil sampling and taphonomy We sampled fossils with preserved integument spanning the range of known forms, including skin, protofeathers, pycnofibers and pinnate feathers. We collected morphological data from SEM images of fossil integument from the Jehol biota in the same manner as the extant samples. Small (~1 mm 2 ) samples were taken from preserved integument of fossils as before 6. Sampled specimens are shown in section I. 2

3 SUPPLEMENTARY INFORMATION RESEARCH Internal organs like liver contain melanosomes 10, so we restricted our samples to areas that were far from them, e.g. skin on the tail of Yabeinosaurus and Psittacosaurus or on the limbs of the unidentified turtle (See sampling maps in Extended Data). Each specimen was sampled as thoroughly as possible to maximize the potential diversity of melanosomes while maintaining the integrity of the fossil. The samples were coated with silver (30 seconds) and studied with either a ZEISS SUPRA-55 VP field emission scanning electron microscope (situated at China University of Geosciences, Beijing) or the JEOL environmental scanning electron microscope used for extant samples. Morphological measurements from melanosomes preserved as imprints and in three dimensions were assembled in the same manner as the modern samples. There was no taxonomic variation in the frequency of distinct preservational styles. These data are summarized in Figure 3-4 and Table S2. Our data and those of others strongly suggest that these microstructures are melanosomes and not bacteria. Every paper examining fossilized microstructures in feathers, eyes and ink sacs since 11,12 has confirmed their identity as melanosomes. These papers have used chemical and/or distributional 11,14,18,19 data. None of these papers have supported a bacterial identity for these microstructures. Similarly, here we show that microstructures are a) preserved in nearly an identical fashion to previously identified melanosomes, b) are present within the organic matrix, c) have a much more narrow distribution of shapes and sizes than bacterial films and d) as stated in the manuscript, these sizes and shapes of melanosomes from extant and fossil skin overlap nearly exactly (slightly less so if we assume they shrank 20%, see below). The most parsimonious conclusion is that these microstructures are melanosomes. Recent experiments 20 have shown that melanosomes subjected to extremely high temperatures and pressures shrink in both length and diameter by, on average, ~20%. In no cases did melanosomes increase in size, in variability, or assume high aspect ratio forms. Short axis shrinkage (20±7.9%) was statistically indistinguishable from long axis shrinkage (18±9.2%). These temperature and pressure conditions modeled potential burial at depth of deposits containing soft-tissue. We modeled potential alteration (shrinkage) of sampled fossil melanosomes by 20% based on these estimated values. Scaling up the length and diameter of measured fossil melanosomes by this amount does not affect the recovered pattern of variation across taxa: greater variation and higher aspect ratio forms are still found in birds and Maniraptora (see Extended Data Figure 10). It should also be noted that another more recent publication did not recover support for changes in shape during diagenesis 18. Sampled taxa also show the same pattern in melanosome variation regardless of the deposits from which they were recovered; e.g., turtles and maniraptorans from the Tiaojishan Formation show the same pattern in variation in shape and size as recovered in lizards and maniraptorans from the Yixian Formation. Only if fossil melanosomes from the same deposits altered shape and size in radically different ways, and these trajectories differed systematically by taxonomic groups to match extant taxa could our conclusions be affected. This improbable scenario would imply that melanosomes in both fossil skin and feathers were larger in life than extant melanosomes, but shrank and changed shape during fossilization to overlap the extant range nearly exactly. The more parsimonious scenario is that extant and fossil melanosomes of each integument are of similar size and shape and have potentially 3

4 RESEARCH SUPPLEMENTARY INFORMATION undergone less alteration due to burial conditions than recently modeled or retained their original size due to early taphonomic processes (e.g., during emplacement, compaction, and early burial) that remain to be fully modeled. Melanosomes are found not only in integument but also in internal organs such as the liver. We infer that all samples taken are from fossil integument. However, because some of our samples were located relatively near the potential sites of internal organs we tested the effect of removal of these samples from our database (Extended Data Figure 10). There was no effect on the range of melanosome morphologies for each taxonomic group and no effect on the conclusions we present from the complete dataset (Extended Data Figure 10). We performed a CDA using the extant feather data from 5 to predict the average color of Caudipteryx, Beipiaosaurus and the two pterosaur specimens. Both the tail and body feathers of Caudipteryx were predicted as black with moderate confidence (probability=82 and 83%, respectively). By contrast, Beipiaosaurus and the two pterosaurs were all predicted as brown with varying levels of confidence (0.94, 1.00 and 0.56, respectively). All fossil and extant specimens are in recognized public repositories. Specimen numbers are indicated in Supplemental Table 3. Samples taken for SEM are parts of these specimens and available through data requests to the respective institutions. 2. Supplementary Tables Hair Feathers Skin F Ratio 2.1±0.2 a 3.5±0.1 b 1.8±0.2 a 34.4 Long axis (nm) ± 48.4 a ± 26.7 a ± b 19.8 Short axis (nm) ± 17.4 a ± 9.6 b 324.2±20.7 b 24.5 Supplementary Table 1: Means, ANOVA and Tukey HSD of melanosome morphology in integumentary types of extant samples. All ANOVAs are significant (p<0.001). Values in columns not connected by the same letter are significantly different from one another. 4

5 SUPPLEMENTARY INFORMATION RESEARCH Species Common name Color Integument Length CV Diameter CV Ratio CV Anchiornis huxleyi BMNHC PH828 N/A N/A Fossil feather Archaeopteryx lithographica MB.Av.100 N/A N/A Fossil feather Caudipteryx zoui PMOL AD00020 N/A N/A Fossil feather Confuciusornis sanctus CUGB G N/A N/A Fossil feather Inkayacu paracasensis MUSM 1444 N/A N/A Fossil feather undescribed ornithurine CUGB G N/A N/A Fossil feather Microraptor BMNHC PH881 N/A N/A Fossil feather Undescribed enantiornithine CUGB P1201 N/A N/A Fossil feather Undescribed enantiornithine CUGB G N/A N/A Fossil feather Beipiaosaurus BMNHC PH N/A N/A Protofeather Sinosauropteryx IVPP V14202 N/A N/A Protofeather Undescribed pterosaur c.f. Jianchangnathus robustus N/A N/A BMNHC PH Pycnofiber Undescribed pterosaur PMOL AP00022 N/A N/A Pycnofiber Psittacosaurus lujiatunensis PKUP V1050 N/A N/A Fossil skin Psittacosaurus lujiatunensis PKUP V1051 N/A N/A Fossil skin Undescribed turtle PKUP V1070 N/A N/A Fossil skin Xianglong zhaoi PMOL N/A N/A Fossil skin Yabeinosaurus sp. PKUP V1059 N/A N/A Fossil skin Mephitis mephitis Striped Skunk Black Extant hair Urocyon cinereoargenteus Grey Fox Black Extant hair Mustela erminea Stoat (tail) Black Extant hair Tamias minimus Least Chipmunk Black Extant hair Martes americana American Marten Black Extant hair Ammospermophilus leucurus White-tailed Antelope Squirrel Black Extant hair Procyon lotor Northern Raccoon Grey Extant hair Lemmus trimucronatus Brown Lemming Black Extant hair Tamias striatus Eastern Chipmunk Brown Extant hair Crocidura bicolor Tiny Musk Shrew Brown Extant hair Urocyon cinereoargenteus Grey Fox Brown Extant hair Geomys pinetis Southeastern Pocket Gopher Grey Extant hair Lemniscomys griselda Griselda's Striped Grass Mouse Black Extant hair Herpestes urva Crab-eating Mongoose Black Extant hair Sciurus carolinensis Grey Squirrel Black Extant hair Pelomys fallax Creek Groove-toothed Swamp Rat Black Extant hair Napaeozapus insignis Woodland Jumping Mouse Brown Extant hair Peromyscus maniculatus Deer Mouse Brown Extant hair Neovison vison American Mink Brown Extant hair

6 RESEARCH SUPPLEMENTARY INFORMATION Crocidura cyanea Reddish-gray Musk Shrew Grey Extant hair Scalopus aquaticus Common Mole Grey Extant hair Parascalops breweri Hairy-tailed Mole Brown Extant hair Scalopus aquaticus Eastern Mole Brown Extant hair Dipodomys merriami Merriam's Kangaroo Rat Brown Extant hair Blarina brevicauda Northern Short-tailed Shrew Grey Extant hair Eutamias sonomae alleni Sonoma Chipmunk Brown Extant hair Eutamias amoenus affinis Yellow-pine Chipmunk Black Extant hair Notiosorex crawfordi Desert Shrew Grey Extant hair Sciurus chiricahua Fox Squirrel Brown Extant hair Sciurus griseus nigripes Grey Squirrel Black Extant hair Sciurus griseus anthonyi Grey Squirrel Black Extant hair Marmota monax rufescens Groundhog Brown Extant hair Peromyscus eremicus Cactus Mouse Brown Extant hair Scapanus oriarius orarius Coast Mole Brown Extant hair Sorex fumeus fumeus Smoky Shrew Grey Extant hair Felis catus Domestic Cat Black Extant hair Plecotus austriacus Grey Long-eared bat Brown Extant hair Nyctalus noctula Common Noctule Brown Extant hair Plecotus austriacus Grey Long-eared Bat Black Extant hair Diphylla ecaudata Hairy-legged Vampire Bat Brown Extant hair Diphylla ecaudata Hairy-legged Vampire Bat Brown Extant hair Desmodus rotundus Common Vampire Bat Brown Extant hair Desmodus Common Vampire Bat Brown Extant hair Plecotus auritus Brown Long-eared Bat Brown Extant hair Plecotus auritus Brown Long-eared Bat Black Extant hair Sturnira illium Little Yellow-shouldered Bat Brown Extant hair Molossus rufus nigricans Black Mastiff Bat Black Extant hair Phyllostomus discolor Pale Spear-nosed Bat Brown Extant hair Phyllostomus discolor Pale Spear-nosed Bat Brown Extant hair Myotis myotis Mouse-eared Bat Brown Extant hair Myotis myotis Mouse-eared Bat Brown Extant hair Ctenosaura pectinata Mexican Spiny-tailed Iguana Grey Extant skin Sauromalus ater Common Chuckwalla Black Extant skin Crotalus pricei Twin-spotted Rattlesnake Grey Extant skin Leiocephalus carinatus Northern Curly-tailed Lizard Brown Extant skin Agkistrodon contortrix Copperhead Brown Extant skin Agkistrodon piscivorus Water Moccasin Grey Extant skin Lialis burtonis Burton s Legless Lizard Grey Extant skin Acanthodactylus erythrurus Red-tailed Spiny-footed Lizard Black Extant skin Eumeces fasciatus Five-lined Skink Black Extant skin Varanus salvator Water Monitor Grey Extant skin Tropidurus oreadicus Calango Grey Extant skin Elgaria multicarinata California Alligator Lizard Black Extant skin Ameiva ameiva Giant Ameiva Grey Extant skin Mabuya unimarginata Central American Mabuya Brown Extant skin Mabuya rudis Rough Mabuya Brown Extant skin Aspidoscelis costata Western Mexico Whiptail Black Extant skin Mabuya macularia Bronze Mabuya Brown Extant skin Xantusia henshawi Granite Night Lizard Black Extant skin Sphenodon punctatus Tuatara Brown Extant skin Cordylus giganteus Giant Girdled lizard Black Extant skin Hemidactylus leschenaultia Leschenault s Leaf-toed Gecko Black Extant skin Xantusia vigilis Desert Night Lizard Grey Extant skin Amphisbaena fuliginosa Black and White Worm Lizard Black Extant skin Leposternon microcephalum Smallhead Worm Lizard Brown Extant skin

7 SUPPLEMENTARY INFORMATION RESEARCH Ramphotyphlops braminus Brahminy Blind Snake Brown Extant skin Crotalus aquilus Queretaro Dusky Rattlesnake Black Extant skin Hemidactylus frenatus Common House Gecko Black Extant skin Melanosuchus niger Black Caiman Black Extant skin Leptotyphlops sp. Slender Blind Snake Brown Extant skin Microlophus albemarlensis Galapagos Lava Lizard Grey Extant skin Lepidophyma flavimaculatum Yellow-spotted Tropical Night lizard Black Extant skin Uroplatus fimbriatus Leaf-tailed Gecko Brown Extant skin Chrysemys picta Painted Turtle Black Extant skin Uroplatus fimbriatus Leaf-tailed Gecko Brown Extant skin Gekko vittatus Lined Gecko Black Extant skin Urosaurus ornatus Tree Lizard Grey Extant skin Supplementary Table 2: Means (in nm) and coefficients of variation for morphological measurements of melanosomes sampled from fossil feathers, extant hairs, fossil skin, and extant skin in this study. 7

8 RESEARCH SUPPLEMENTARY INFORMATION Taxonomy Taxon Catalog number Horizon Locality Sampled in Testudines Unnamed PKUP V1070 Tiaojishan Formation Linglongta locality, Jianchang County, Liaoning This study Lepidosauria Yabeinosaurus sp. PKUP V1059 Yixian Formation Jinganshan locality, Yixian County, Liaoning This study Lepidosauria Xianglong zhaoi PMOL Yixian Formation Zhuangchengzi locality, Yixian County, Liaoning This study Pterosauria Unnamed PMOL AP00022 Jiufotang Formation Sihedang Locality, Lingyuan City, Liaoning This study Pterosauria c.f.jianchangnathus robustus BMNHC PH Tiaojishan Formation Linglongta locality, Jianchang County, Liaoning This study Ornithischia Psittacosaurus PKUP V1050 Yixian Formation Sihetun locality, Beipiao County, Liaoning This study lujiatunensis Ornithischia Psittacosaurus lujiatunensis PKUP V1051 Yixian Formation Sihetun locality, Beipiao County, Liaoning This study Theropoda Sinosauropteryx IVPP Yixian Formation 21 Dawangzhangzi locality, Lingyuan City, Liaoning Theropoda Beipiaosaurus BMNHC Yixian Formation Sihetun locality, Beipiao County, Liaoning This study inexpectus PH Theropoda Caudipteryx zoui PMOL AD00020 Yixian Formation Beipiao County, Liaoning This study Theropoda Microraptor BMNHC PH881 Yixian Formation 5 Lamadong locality, Jianchang County, Liaoning Theropoda Anchiornis huxleyi BMNHC PH828 Tiaojishan Daxishan locality, Jianchang County, Liaoning 22 Formation Theropoda Archaeopteryx MB.Av.100 Solnhofen Solnhofen, Germany 23 Theropoda Confuciornis sanctus CUGB Yixian Formation Beipiao County, Liaoning This study G Theropoda Enantiornithes Undescribed CUGB G Yixian Formation Fengning County, Hebei This study Theropoda Enantiornithes Undescribed CUGB P1201 Jiufotang Formation Lamadong locality, Jianchang County, Liaoning This study Theropoda Ornithurae undescribed CUGB Jiufotang Formation Jianchang County, Liaoning This study G Theropoda Aves: Sphenisciformes Inkayacu paracasensis MUSM 1444 Otuma Formation Paracas Reserve, Peru 6 Supplementary Table 3: Fossil specimens from which measurements of melanosomes were taken in this study. 8

9 SUPPLEMENTARY INFORMATION RESEARCH 3. Supplemental references 1 Fox, D. L. Animal Biochromes and Structural Colours. Berkeley: University of California Press. (1976). 2 Snyder, H. K., et al., Iridescent colour production in hairs of blind golden moles (Chrysochloridae). Biol. Lett. 8, (2012). 3 R. O. Prum, in Bird Coloration, Vol.1, Mechanisms and Measurements, G. E. Hill, K. J. McGraw, Eds. (Harvard Univ. Press, Boston, 2006), pp M. D. Shawkey, M. E. Hauber, L. K. Estep, G. E. Hill, J. Roy. Soc. Interface 3, 777 (2006). 5 Li, Q., et al. Reconstruction of Microraptor and the evolution of iridescent plumage. Science 335, (2012) 6 Clarke, J. A. et al., Fossil evidence for evolution of the shape and color of penguin feathers. Science 330, (2010). 7 T. Hastie, R. Tibishirani, and J. H. Friedman, The Elements of Statistical Learning. (Springer, New York, 2001). 8 Hubbard, J. K., Uy, J. A. C., Hauber, M. E., Hoekstra, H. E. & Safran, R. J. Vertebrate pigmentation: from underlying genes to adaptive function. Trends Genet. 26, (2010). 9 Hellström, A.R. et al. Inactivation of Pmel alters melanosome shape but has only a subtle effect on visible pigmentation. PLoS Genet. e (2011). 10 S. Sciuto, R. Chillemi, A. Patti, G. Sichel and M. Saclia, Melanosomes from liver and skin of Rana esculenta L. A comparative chemical study, Comp. Biochem. Phys. B: Comp. Biochem. 90, (1988). 11 J. Vinther, D. E. G. Briggs, R. O. Prum, V. Saranathan, Biol. Lett. 4, 522 (2008). 12 J. Vinther, D. E. G Briggs, J. Clarke, G. Mayr, R. O. Prum, Biol. Lett. 6, 128 (2010). 13 Barden, H.E., Wogelius, R.A., Li, D., Manning, P.L., Edwards, N.P., and van Dongen, B.E. Morphological and Geochemical Evidence of Eumelanin Preservation in the Feathers of the Early Cretaceous Bird, Gansus yumenensis. PLoS ONE 6(10): e25494 (2011). 14 Carney R.M., Vinther J., Shawkey M.D., D Alba, L., Ackermann J. New evidence on the colour, ultrastructure, and nature of the isolated Archaeopteryx feather. Nature Communications. 24:633:637. (2012). 9

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