LETTER doi:10.1038/nature12973 Melanosome evolution indicates a key physiological shift within feathered dinosaurs Quanguo Li 1 , Julia A. Clarke 2 , Ke-Qin Gao 3 , Chang-Fu Zhou 4 , Qingjin Meng 5 , Daliang Li 6 , Liliana D’Alba 7 & Matthew D. Shawkey 7 Inference of colour patterning in extinct dinosaurs 1–3 has been based on the relationship between the morphology of melanin-containing organelles (melanosomes) and colour in extant bird feathers. When this relationship evolved relative to the origin of feathers and other novel integumentary structures, such as hair and filamentous body covering in extinct archosaurs, has not been evaluated. Here we sample melanosomes from the integument of 181 extant amniote taxa and 13 lizard, turtle, dinosaur and pterosaur fossils from the Upper-Jurassic and Lower-Cretaceous of China. We find that in the lineage leading to birds, the observed increase in the diversity of melanosome morphologies appears abruptly, near the origin of pin- nate feathers in maniraptoran dinosaurs. Similarly, mammals show an increased diversity of melanosome form compared to all ecto- thermic amniotes. In these two clades, mammals and maniraptoran dinosaurs including birds, melanosome form and colour are linked and colour reconstruction may be possible. By contrast, melanosomes in lizard, turtle and crocodilian skin, as well as the archosaurian filamentous body coverings (dinosaur ‘protofeathers’ and pterosaur ‘pycnofibres’), show a limited diversity of form that is uncorrelated with colour in extant taxa. These patterns may be explained by con- vergent changes in the key melanocortin system of mammals and birds, which is known to affect pleiotropically both melanin-based colou- ration and energetic processes such as metabolic rate in vertebrates 4 , and may therefore support a significant physiological shift in man- iraptoran dinosaurs. Melanin-based colour is a ubiquitous feature of amniote integument, found in feathers of every major bird clade 5,6 , as well as in skin and hair (the scales covering skin in reptiles are typically transparent 7 ). We examined the relationship between integumentary structure and mela- nosome morphology in a phylogenetic context. Individual melanosomes were measured from scanning electron microscope (SEM) images of the hairs of 44 species of extant mammals (n 5 51 samples) and the skin of 36 extant species (n 5 36 samples), sampling across Lepidosauria, Testudines and Crocodylia (Supplementary Tables 1–3). These mea- surements were compared with our previously published data from 101 extant avian species (n 5 168 feather samples 3 ). Samples targeting the full range of melanin-based colours for each integument type included blacks, browns and greys for feathers, hair and skin. As feathers uniquely show a broad range of melanin-based iridescent colours, and these are known to be associated with distinctive melanosome morphologies 3 , they were also included. Although iridescent colours are found in rep- tile skin 8 and (rarely) in hair 9 , they are produced through light scatter- ing from iridiphores and multilayer keratin films, respectively. As these colours are not melanin-based, the integuments in which they are pro- duced were not sampled. To capture melanosome diversity fully also demanded inclusion of morphotypes associated with black colour that are so far known only from extant penguins 10 . Preserved integument from 13 fossil amniotes from the Upper Jurassic and Lower Cretaceous of northeast China were sampled using described protocols 2,3,10 (Supplementary Methods). Skin was sampled from two fossil lepidosaurs, a turtle, and two specimens of the ornithischian dinosaur Psittacosaurus (Extended Data Figs 1–3 and Supplementary Tables 2 and 3). Although extant amniote epidermal appendages are limited to scales, hairs and feathers 11 , additional structures are observed in the fossil record. These include basally bunched or single hollow fila- ments in theropod dinosaurs proposed to be homologous with modern feathers 12–14 , bristles and filaments in ornithischian dinosaurs 15,16 , and pterosaur pycnofibres, the identity and homology of which are more controversial 17 . Filamentous structures were sampled from the theropod dinosaur Beipiaosaurus and two pterosaurs (Extended Data Figs 4, 5 and Supplementary Tables 2, 3). Feathers were sampled from Caudipteryx, Confuciusornis, one ornithurine and two enantiornithine birds (Extended Data Figs 2, 6–8 and Supplementary Tables 2 and 3). Published SEM images and data were assessed for filamentous structures in the non- maniraptoran coelurosaurian dinosaur Sinosauropteryx 1 , feathers from the basal paravians Anchiornis 2 , Microraptor 3 and Archaeopteryx 18 and 1 State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Beijing 100083, China. 2 Department of Geological Sciences, University of Texas at Austin, 1 University Station C1100, Austin, Texas 78712, USA. 3 School of Earth and Space Sciences, Peking University, Beijing 100871, China. 4 Institute of Paleontology, Shenyang Normal University, Shenyang 110034, China. 5 Beijing Museum of Natural History, 126 Tianqiao South Street, Beijing 100050, China. 6 Museum of China University of Geosciences (Beijing), 29 Xueyuan Road, 100083, China. 7 Department of Biology and Integrated Bioscience Program, University of Akron, Akron, Ohio 44325-3908, USA. 500 1,000 1,500 2,000 Length (nm) vwx z v vw wx vw yz xyz yz yz yz Integument colour Extant feathers Mammalian hair Lepidosaur, testudine and archosaur skin Figure 1 | Melanosome length observed in extant feathers, lepidosaur, testudine and archosaur skin, and mammalian hair. Boxplot colours correspond with integument colour: black, brown and grey. For feathers, ‘penguin-like’ is shown in blue, and iridescent is shown in purple. Lines are median values, boxes are quartiles, lines are range. Boxplots sharing the same letter (v, w, x, y, z) are not significantly different (two-sided Tukey HSD; P , 0.05) from one another; melanosome shape correlates with distinct colours in feathers and hair but not in skin. Extant feathers, n 5 168; lepidosaur, testudine and archosaur skin, n 5 36; mammalian hair, n 5 51. 350 | NATURE | VOL 507 | 20 MARCH 2014 Macmillan Publishers Limited. All rights reserved ©2014