Molecular evidence of keratin and melanosomes in feathers of the Early Cretaceous bird Eoconfuciusornis Yanhong Pana,1, Wenxia Zhengb, Alison E. Moyerb,2, Jingmai K. O’Connorc, Min Wangc, Xiaoting Zhengd,e, Xiaoli Wangd, Elena R. Schroeterb, Zhonghe Zhouc,1, and Mary H. Schweitzerb,f,1 aKey Laboratory of Economic Stratigraphy and Palaeogeography of the Chinese Academy of Sciences, Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences Nanjing 210008, China; bDepartment of Biological Science, North Carolina State University, Raleigh, NC 27695; cKey Laboratory of Vertebrate Evolution and Human Origins of the Chinese Academy of Sciences, Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences, Beijing 100044, China; dInstitute of Geology and Paleontology, Linyi University, Linyi City, Shandong 276005, China; eShandong Tianyu Museum of Nature, Pingyi, Shandong 273300, China; and fNorth Carolina Museum of Natural Sciences, Raleigh, NC 27601 Contributed by Zhonghe Zhou, October 20, 2016 (sent for review August 10, 2016; reviewed by Dominique G. Homberger and Roger H. Sawyer) Microbodies associated with feathers of both nonavian dinosaurs reported microbodies were embedded. Whether keratinous and early birds were first identified as bacteria but have been proteins were preserved in these feathers, and the potential ex- reinterpreted as melanosomes. Whereas melanosomes in modern tent of this preservation, has not been explored. feathers are always surrounded by and embedded in keratin, Indeed, if both microbodies and matrix are preserved, tests can melanosomes embedded in keratin in fossils has not been be conducted to chemically characterize the composition of each. If demonstrated. Here we provide multiple independent molecular these bodies are melanosomes, they should be contained within a analyses of both microbodies and the associated matrix recovered keratinous matrix; if they are microbial in origin, this matrix should from feathers of a new specimen of the basal bird Eoconfuciusornis consist of exopolymeric substances secreted by the microbes and from the Early Cretaceous Jehol Biota of China. Our work represents subsequently mineralized. To distinguish between these alternative the oldest ultrastructural and immunological recognition of avian hypotheses, we applied multiple methods, well-established for beta-keratin from an Early Cretaceous (∼130-Ma) bird. We apply molecular and chemical characterization of modern materials, to immunogold to identify protein epitopes at high resolution, by chicken (Gallus gallus) feathers and to preserved feathers of a new – localizing antibody antigen complexes to specific fossil ultrastructures. specimen of the bird Eoconfuciusornis [Shandong Tianyu Museum Retention of original keratinous proteins in the matrix surrounding of Nature (STM) 7-144] (Pygostylia: Confuciusornithiformes) (Fig. electron-opaque microbodies supports their assignment as mela- 1A) from the 130-Ma Protopteryx horizon of the Huajiying Forma- nosomes and adds to the criteria employable to distinguish melano- tion in Fengning, northern Hebei, China. Our results are consistent somes from microbial bodies. Our work sheds new light on molecular with the retention of original organic components derived from preservation within normally labile tissues preserved in fossils. both keratin and melanin, thus supporting a melanosome origin for these ancient microstructures. keratinous protein | immunogold | ChemiSTEM | melanosome | Early Cretaceous Results Fossil Feather Histology. Scanning electron microscopy (SEM) was eathers and feather-like epidermal structures are now well- used to demonstrate that samples 201 to 205 were preserved as Fdocumented in several groups of nonavian dinosaurs and three-dimensional, thick carbon sheets. SEM images of the fossil basal birds, with the most abundant evidence coming from the Middle feathers (Fig. 1 F–J) were compared with those of G. gallus – Jurassic-to-Early Cretaceous deposits in northeastern China (1 3). remiges (Fig. 1 B–E). At higher magnifications, much of the Round-to-elongated microbodies associated with these feathers and fossil is composed of 3D microbodies, with some regions feather-like structures were first interpreted as microbes (4, 5), leading to the hypothesis that microbial activity played a key role in Significance the preservation of these normally labile remains (5). This hypothesis was based on and supported by experiments and observations of bacterial decomposition of feather keratins in modern birds (6, 7). We report fossil evidence of feather structural protein (beta- More recently, these bodies were reinterpreted as remnant keratin) from a 130-My-old basal bird (Eoconfuciusornis)from melanosomes (pigment-containing intracellular organelles) (8–11) the famous Early Cretaceous Jehol Biota, which has produced and, subsequently, hypotheses of dinosaurian color (8–13), be- many feathered dinosaurs, early birds, and mammals. Multiple havior (10), habitat (14), and physiology (15) were proposed based independent molecular analyses of both microbodies and asso- upon this reinterpretation. However, melanosomes and microbes ciated matrix recovered from the fossil feathers confirm that overlap completely in size and shape (16–18), and thus these hy- these microbodies are indeed melanosomes. We use trans- potheses are equally plausible. Furthermore, the majority of the mission electron microscopy and immunogold to show localized data presented to support a melanosome origin are based not on binding of antibodies raised against feather protein to matrix the morphology of the bodies themselves but rather on their im- filaments within these ancient feathers. Our work sheds new pressions within a structural matrix (9–15, 19) that is presumed, light on molecular constituents of tissues preserved in fossils. but not demonstrated, to be keratin. Author contributions: Y.P., W.Z., Z.Z., and M.H.S. designed research; Y.P. and W.Z. per- The most rigorous test for the origin of these microbodies is formed research; A.E.M. contributed new reagents/analytic tools; X.Z. and X.W. provided the chemical characterization of both the microbodies and the materials for analysis; Y.P., W.Z., J.K.O., M.W., X.Z., X.W., and E.R.S. analyzed data; and Y.P., matrix in which they were embedded. However, where chemical Z.Z., and M.H.S. wrote the paper. data were presented previously, with few exceptions (20, 21) the Reviewers: D.G.H., Louisiana State University; and R.H.S., University of South Carolina. data were not sufficiently specific to support or eliminate either The authors declare no conflict of interest. – hypothesis (22 26). Whether the melanin-related chemical sig- 1To whom correspondence may be addressed. Email: [email protected], zhouzhonghe@ nals originated from the microbodies or from the surrounding ivpp.ac.cn, or [email protected]. matrix (22–26) could not be determined, with the exception of 2Present address: Department of Biology, Drexel University, Philadelphia, PA 19104. one study by Lindgren et al. (21). No detailed morphological or This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. chemical studies have been conducted on the matrix in which the 1073/pnas.1617168113/-/DCSupplemental. E7900–E7907 | PNAS | Published online November 21, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1617168113 Downloaded by guest on September 23, 2021 PNAS PLUS Fig. 1. New specimen of Eoconfuciusornis (STM 7-144) collected from the Early Cretaceous lake deposits in Hebei, northern China, and SEM images of as- sociated feather material, compared with SEM images of a black feather from the extant chicken G. gallus.(A) Photograph of the new fossil specimen, indicating sample locations (boxes; not to scale). (B–E) SEM images of a black feather from G. gallus.(B) Low-magnification image. (C–E) High-magnification images of the boxed areas in B.(F–J) SEM images of the feather samples 111 (crural feathers) and 202 (interpreted as the tertials of the left wing) from Eoconfuciusornis.(F) Low-magnification image. (G) High-magnification image of the boxed area in F.(H) Melanosomes are shown located immediately below the structured thin layer, which is indicated with yellow arrowheads. (I and J) High-magnification images of the boxed areas in H. These bodies are embedded within the feathers and not observed on the surface of the feathers. (Scales are as indicated.) retaining a very thin carbon layer (Fig. 1 F–I) that covers the matrix (Fig. 4 G–I), but were sparsely distributed relative to microbodies (Fig. 1J). This thin layer displays fibrous bundles in binding in modern controls (Fig. 4 D and F–L). However, other arrays that form regular angles (Fig. 1 G and I), a feature pre- structures, including the electron-dense microbodies and needle- viously reported in the epicortex of G. gallus feathers (27) and shaped structures probably of diagenetic origin, were completely also observed in Fig. 1 C and D. unlabeled (Figs. 4E and 5A). No gold particles were visible in the Transmission electron microscopy (TEM) confirms micro- negative controls (omitting the primary antiserum) (Fig. 4 A–C), structural similarity between Eoconfuciusornis and extant similar to results from immunofluorescence. The higher-resolution G. gallus feathers (Fig. 2). The microbodies preserved in Eocon- immunoelectron localization of gold particles generally confirms fuciusornis STM 7-144 are distinct, relatively uniform, elongated, the binding of the anti-feather