Significance During the dinosaur–bird transition, feathers of bird ancestors must have been molecularly modified to become biomechanically suitable for flight. We report molecular moieties in fossil feathers that shed light on that transition. Pennaceous feathers attached to the right forelimb of the Jurassic dinosaur Anchiornis were composed of both feather β-keratins and α-keratins, but were dominated by α-keratins, unlike mature feathers of extant birds, which are dominated by β-keratins. Data suggest that the pennaceous feathers of Anchiornis had some, but not all, of the ultrastructural and molecular characteristics of extant feathers, and may not yet have attained molecular modifications required for powered flight.

Abstract Dinosaur fossils possessing integumentary appendages of various morphologies, interpreted as feathers, have greatly enhanced our understanding of the evolutionary link between birds and dinosaurs, as well as the origins of feathers and avian flight. In extant birds, the unique expression and amino acid composition of proteins in mature feathers have been shown to determine their biomechanical properties, such as hardness, resilience, and plasticity. Here, we provide molecular and ultrastructural evidence that the pennaceous feathers of the Jurassic nonavian dinosaur Anchiornis were composed of both feather β-keratins and α-keratins. This is significant, because mature feathers in extant birds are dominated by β-keratins, particularly in the barbs and barbules forming the vane. We confirm here that feathers were modified at both molecular and morphological levels to obtain the biomechanical properties for flight during the dinosaur–bird transition, and we show that the patterns and timing of adaptive change at the molecular level can be directly addressed in exceptionally preserved fossils in deep time.

Feathers are a key avian feature, used to identify and diagnose birds in the fossil record for centuries. The appearance of feathers has been closely tied to the origin of flight in birds. Although feathers have been used as a taxonomic character of birds, discoveries of fossils from Middle-Late Jurassic to Early Cretaceous sediments in western Liaoning, northern Hebei, and Inner Mongolia of China have revealed a remarkable diversity of nonavian dinosaur fossils displaying a wide range of integumentary appendages interpreted as feathers or feather-like structures (1⇓⇓–4). Some of these structures are present as simple filamentous structures without aerodynamic function, and these are widespread in more basal and flightless dinosaurs, including some ornithischians not on the bird lineage (4⇓–6). This distribution supports the hypothesis that feathers may have originated before the capacity for powered flight and, thus, were first employed for other purposes.

Complex pennaceous feathers with rachises and branching barbs and barbules have been described on the tail and limbs of Middle-Late Jurassic paravian dinosaurs, the best known among them being Anchiornis (3). Anchiornis represents a taxon that is significantly older (∼160 Ma) (7) than the first recognized bird, Archaeopteryx (∼150–155 Ma) (8), but strongly resembles it. The skeletal and feather anatomy, small body size, and long forelimbs (3, 9, 10) of Anchiornis suggest volant abilities, which is supported by anatomy-based computer models and wind-tunnel studies (11). However, although the pennaceous structure is confirmed for feathers in Anchiornis, barbules that interlock to form feather vanes critical for flight have not been identified yet (12).

In addition to morphological features, the ultrastructure and molecular composition are critical in determining whether the mechanical properties of feathers are suitable for flight (13, 14). Mature feathers of extant birds are primarily composed of β-keratins, a family of proteins found only in birds and reptiles (15). However, α-keratins, as a more basal protein family found in all vertebrates (16, 17), are coexpressed with derived β-keratins in the embryonic feathers of extant birds (15, 17) and in all other sauropsid keratinous tissues.

In mammals, α-keratins can be divided into “hard” (hair, nails, hooves) and “soft” (skin) keratins based on the number of intra- and intermolecular cross-links (14). Sauropsid-specific β-keratins are universally harder, because they incorporate a greater number of sulfur-bearing amino acids, which form stabilizing cross-links and form β-sheets as opposed to helices, as in α-keratins (14). The β-keratins in extant birds are further divided into subfamilies (e.g., basal claw and scale β-keratin subfamilies) (14, 18), and the more derived feather β-keratins. The latter are distinguished by a peptide deletion, resulting in the loss of the glycine-rich tail in the C-terminal region, a shorter amino acid sequence and, as a consequence, lower molecular weight, than other avian β-keratins (14, 18). This deletion creates a structural protein that is biomechanically more flexible, imparting to avian feathers the unique properties required for flight (16, 17, 19⇓⇓⇓–23).

The ultrastructure and molecular composition of the pennaceous feathers in Anchiornis may, therefore, shed light on the controversial issue (24) of its volant behavior. We argue that unless the pennaceous feathers of Anchiornis exhibit a molecular composition dominated by specific feather β-keratins, they were unlikely to support powered flight.

Molecular clock studies have suggested that feather β-keratins began to diverge from other β-keratins by ∼143 Ma (95% SD ∼ 176–110 Ma) (23). These studies supported the hypothesis that pennaceous feathers preceded flight, and led to the prediction that Anchiornis (∼160 Ma) expressed only more basal β-keratins, but not the derived feather β-keratins identified in extant birds.

To test this hypothesis and to determine the distribution of keratins across the dinosaur–bird transition, we employed multiple high-resolution analytical methods and strict controls (including various extant tissues) to elucidate the endogenous preservation and molecular expression of keratins in fossilized feathers. We compared pennaceous feathers attached to the forelimb of Anchiornis with fossil feathers from taxa that are phylogenetically more derived than Anchiornis (e.g., pennaceous feathers possibly from the left forelimb of an Early Cretaceous Dromaeosauridae indet., wing feathers of the right forelimb of Eoconfuciusornis, tail feathers near the distal end of the left pubis of Yanornis, and an isolated Oligocene flight feather). As controls, we included short fibers around the perimeter of the bones of the Late Cretaceous Shuvuuia deserti (a nonavian dinosaur) and claw sheath material from the Late Cretaceous Citipati (a nonavian dinosaur), as well as modern comparable tissues, including flight feathers of the chicken, goose, duck, white leghorn chicken, and emu; rhamphothecas of the chicken and emu; claws of the chicken, emu, and ostrich; and scales of the chicken and ostrich (detailed in SI Appendix and SI Appendix, Figs. S1–S4 and Table S1).

In this study, we demonstrate specific expressions of feather β-keratins and differentiate feather β-keratins from other β-keratins in numerous fossil taxa. These data shed light on the evolution of the proteinaceous components of feathers during the dinosaur–bird transition and provide a tool for assessing potential flight abilities in extinct feathered dinosaurs.

Discussion Keratins have a higher preservation potential than most other proteins, largely because of their molecular structure (14, 29, 30). All keratins, but particularly β-keratins, incorporate many hydrophobic residues that exclude water and a high concentration of sulfur-bearing amino acids that facilitate intra- and intermolecular disulfide bond formation, which in turn confers and enhances stability (30). Hence, keratins resist degradation by most proteolytic enzymes (29) and are somewhat protected against hydrolysis, thereby enhancing their preservation potential over other nonbiomineralized tissues. We have used multiple high-resolution analytical methods (SEM, TEM, ChemiSTEM), as well as IF and IG labeling, to investigate the protein composition of the feathers of Anchiornis by comparing them with materials from several related fossils and tissues from extant birds. Our study not only supports the presence of endogenous keratins in the fossil materials but also shows that specific antibodies, when combined with ultrastructural data, can be used to differentiate cornified tissues in feathers of extant and extinct birds and dinosaurs or other integumentary structures. We demonstrate that keratins within preserved integumentary structures can be identified in nonavian dinosaurs and birds, revealing potential biomechanical properties. Our data support the hypothesis that feather β-keratins are coexpressed and preserved with more basal α-keratins in the Anchiornis fossil integumentary materials, but that α-keratins take the predominant position, which is clearly different from comparable flight feathers in extant birds. These molecular data are supported by ultrastructural data showing the presence of thick filaments resembling α-keratins in the Anchiornis feathers. Because mature feathers of extant birds are dominated by feather β-keratins, the coexpression of α-keratins and feather β-keratins, in combination with the ultrastructural patterns shown here, suggests that feathers of Anchiornis may represent an evolutionary transition between more ancestral integumentary appendages and extant bird feathers. Subsequent predominance of feather β-keratins in mature feathers of extant birds have been shown to greatly affect the mechanical properties, increasing resilience and plasticity (13, 14). Thus, these modifications may have evolved in tandem with the evolution of powered flight. Several genomic studies indicate that feather β-keratin genes were probably present in the genome and expressed in the epidermis of scaled archosaurians and lepidosaurs before the emergence of feathers (31⇓⇓–34). However, the formation of feather placodes and the origin of the axial rachis and hierarchical branching of barbs and barbules, as well as different feather types with different functions, required increasing specialization of the feather β-keratin genes (15). This is reflected by both a reduction in the number of α-keratin genes and a significant expansion of β-keratin genes in the bird genomes relative to those in mammals and reptiles (19, 35), thus indicating that β-keratin gene duplications and mutations may be linked to feather evolution and adaptations of birds to different ecological niches (19, 20, 36, 37). Thus, at the molecular and ultrastructural levels, the pennaceous feathers of Anchiornis may represent an intermediate stage in feather evolution. In Anchiornis, thin filaments of feather β-keratins are not yet widely distributed, nor have the thick filaments of α-keratin been superseded by successive β-keratin expression in this ancient paravian. The molecular composition and ultrastructure of Anchiornis feathers suggest that their pennaceous feathers may have lacked the biomechanical properties suitable for flight, unlike mature feathers of extant birds. When these data are considered within the well-established phylogenetic framework of Aves and their closest relatives (38) (Fig. 5), we conclude that specific feather β-keratins most likely evolved outside the clade Aves, being expressed in the feathers of paravians (e.g., Anchiornis); basal α-keratin coexpression is retained in at least some paravian feathers; and although the feathers of the Early Cretaceous nonavian dinosaurs (e.g., Dromaeosauridae indet.) and basal birds (e.g., Eoconfuciuornis) also coexpressed feather β-keratins and α-keratins, they were more similar at the ultrastructural levels to the feathers of more derived birds (e.g., Yanornis), including neornithines, which demonstrate an absence of observable thick filaments composed of α-keratins. Therefore, we hypothesize that feathers continued to evolve throughout the Cretaceous until they reached the condition in Neornithes in which feather β-keratins predominated, thereby producing flexible and resilient feathers suitable for powered flight. Our data also suggest a mosaic pattern in the evolution of protein expression in dinosaurs that are closely related to birds. For example, Shuvuuia fibers show reactivity to the general β-keratin antiserum, but do not show evidence of the deletion event specific to feathers. However, the Shuvuuia fibers lost the coexpression of α-keratin, whereas Anchiornis feathers expressed the feather β-keratins together with α-keratins, as is observed in other cornified sauropsid tissues. Fig. 5. Time-scaled evolution of molecular composition and ultrastructure of feathers within a simplified Mesozoic avian and nonavian phylogeny (38), suggesting that the Anchiornis feather were composed of both feather β-keratins and α-keratins, but dominated by α-keratins, unlike feathers from younger fossils and mature feathers of extant birds, which are dominated by β-keratins. Filled stars showing the distribution of tested fossil feathers and related integumentary tissues used in this study: (1) Anchiornis (STM 0–214), (2) Dromaeosauridae indet. (STM5-12), (3) Eoconfuciusornis (STM7-144), (4) Yanornis (STM9-5), (5) Isolated flight feather (DY 1502006), (6) Shuvuuia deserti (IGM 100/977), and (7) Citipati (MPC-D). β+, positive reaction to the general β-keratin antiserum; Fβ+, positive reaction to the antiserum specific feather β-keratins; α+, positive reaction to the anti-pan cytokeratin antiserum; “Fβ+” in bold, thin β-keratin filaments is dominant in ultrastructure; “α+” in bold, thick α-keratin filaments is dominant in ultrastructure. This study not only confirms the preservation of endogenous keratins in various Mesozoic and Cenozoic fossil materials but also demonstrates the possibility of conducting rigorous molecular studies to address the relative timing of molecular events in cornified tissues. It also highlights the importance of integrating morphological, developmental, and molecular data (including those directly from fossils) in proposing and testing evolutionary hypotheses. However, to achieve a statistical significance of such studies, greater access to fossil materials and, particularly, to those with better preservation is needed.

Materials and Methods Experimental procedures for sampling, electron microscope observation, ChemiSTEM elemental mapping, in situ immunohistochemistry, and strict controls are described in SI Appendix, Materials and Methods.

Acknowledgments We thank Chuanzhao Wang of the State Key Laboratory of Paleaobiology and Stratigraphy of the Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences; Yang Liu of the Analytical Instrumentation Facility of North Carolina State University; Yuru Yang of the Key Laboratory of Unconventional Oil and Gas Geology, China Geological Survey; and Dr. Lulu Cong of the Analytical Center of Nanjing Institute of Geography & Limnology, Chinese Academy of Science, for technical assistance. The research was financially supported by the second Tibetan Plateau Scientific Expedition Program Grant XDA20070300, NSFC Grant 41688103, Strategic Priority Research Program of Chinese Academy of Sciences Grant XDB26000000, Open-Lab Grants of the State Key Laboratory of Paleaobiology and Stratigraphy, National Science Foundation (INSPIRE program EAR-1344198), and funding from Franklin Orr and Susan Packard Orr and Vance and Gail Mullis. F.W. is funded by the Strategic Priority Research of Chinese Academy of Sciences Grant XDA20070203. Y.P. is also supported by the Youth Innovation Promotion Association, Chinese Academy of Sciences, and NSFC Grant 41872016. This work was performed in part at the Analytical Instrumentation Facility at North Carolina State University, which is supported by the State of North Carolina, Key Laboratory of Unconventional Oil and Gas Geology, Analytical Center of Nanjing Institute of Geography & Limnology, Chinese Academy of Sciences, and the Center of Electron Microscopy, Zhejiang University. Antibody experiments were conducted in the labs of M.H.S., where tools, equipment, and reagents were provided.

Footnotes Author contributions: Y.P., Z.Z., and M.H.S. designed research; Y.P. and W.Z. performed research; R.H.S., M.W.P., and F.W. contributed new reagents/analytic tools; Y.P., W.Z., R.H.S., X.Z., X.W., M.W., L.H., J.O., T.Z., Z.L., E.R.S., and X.X. analyzed data; and Y.P., Z.Z., and M.H.S. wrote the paper.

Reviewers: D.G.H., Louisiana State University; and C.J., National Center for Protein Sciences-Beijing.

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1815703116/-/DCSupplemental.