Molecular evidence of keratin and melanosomes infeathers of the Early Cretaceous bird EoconfuciusornisYanhong 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 Laboratoryof Vertebrate Evolution and Human Origins of the Chinese Academy of Sciences, Institute of Vertebrate Paleontology and Paleoanthropology, ChineseAcademy of Sciences, Beijing 100044, China; dInstitute of Geology and Paleontology, Linyi University, Linyi City, Shandong 276005, China; eShandongTianyu 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 dinosaursand early birds were first identified as bacteria but have beenreinterpreted as melanosomes. Whereas melanosomes in modernfeathers are always surrounded by and embedded in keratin,melanosomes embedded in keratin in fossils has not beendemonstrated. Here we provide multiple independent molecularanalyses of both microbodies and the associated matrix recoveredfrom feathers of a new specimen of the basal bird Eoconfuciusornisfrom the Early Cretaceous Jehol Biota of China. Our work representsthe oldest ultrastructural and immunological recognition of avianbeta-keratin from an Early Cretaceous (∼130-Ma) bird. We applyimmunogold to identify protein epitopes at high resolution, bylocalizing antibody–antigen complexes to specific fossil ultrastructures.Retention of original keratinous proteins in the matrix surroundingelectron-opaque microbodies supports their assignment as mela-nosomes and adds to the criteria employable to distinguish melano-somes frommicrobial bodies. Our work sheds new light onmolecularpreservation within normally labile tissues preserved in fossils.

keratinous protein | immunogold | ChemiSTEM | melanosome |Early Cretaceous

Feathers and feather-like epidermal structures are now well-documented in several groups of nonavian dinosaurs and

basal birds, with the most abundant evidence coming from the MiddleJurassic-to-Early Cretaceous deposits in northeastern China (1–3).Round-to-elongated microbodies associated with these feathers andfeather-like structures were first interpreted as microbes (4, 5),leading to the hypothesis that microbial activity played a key role inthe preservation of these normally labile remains (5). This hypothesiswas based on and supported by experiments and observations ofbacterial decomposition of feather keratins in modern birds (6, 7).More recently, these bodies were reinterpreted as remnant

melanosomes (pigment-containing intracellular organelles) (8–11)and, subsequently, hypotheses of dinosaurian color (8–13), be-havior (10), habitat (14), and physiology (15) were proposed basedupon this reinterpretation. However, melanosomes and microbesoverlap completely in size and shape (16–18), and thus these hy-potheses are equally plausible. Furthermore, the majority of thedata presented to support a melanosome origin are based not onthe morphology of the bodies themselves but rather on their im-pressions within a structural matrix (9–15, 19) that is presumed,but not demonstrated, to be keratin.The most rigorous test for the origin of these microbodies is

the chemical characterization of both the microbodies and thematrix in which they were embedded. However, where chemicaldata were presented previously, with few exceptions (20, 21) thedata were not sufficiently specific to support or eliminate eitherhypothesis (22–26). Whether the melanin-related chemical sig-nals originated from the microbodies or from the surroundingmatrix (22–26) could not be determined, with the exception ofone study by Lindgren et al. (21). No detailed morphological orchemical studies have been conducted on the matrix in which the

reported microbodies were embedded. Whether keratinousproteins were preserved in these feathers, and the potential ex-tent of this preservation, has not been explored.Indeed, if both microbodies and matrix are preserved, tests can

be conducted to chemically characterize the composition of each. Ifthese bodies are melanosomes, they should be contained within akeratinous matrix; if they are microbial in origin, this matrix shouldconsist of exopolymeric substances secreted by the microbes andsubsequently mineralized. To distinguish between these alternativehypotheses, we applied multiple methods, well-established formolecular and chemical characterization of modern materials, tochicken (Gallus gallus) feathers and to preserved feathers of a newspecimen of the bird Eoconfuciusornis [Shandong Tianyu Museumof Nature (STM) 7-144] (Pygostylia: Confuciusornithiformes) (Fig.1A) from the 130-Ma Protopteryx horizon of the Huajiying Forma-tion in Fengning, northern Hebei, China. Our results are consistentwith the retention of original organic components derived fromboth keratin and melanin, thus supporting a melanosome origin forthese ancient microstructures.

ResultsFossil Feather Histology. Scanning electron microscopy (SEM) wasused to demonstrate that samples 201 to 205 were preserved asthree-dimensional, thick carbon sheets. SEM images of the fossilfeathers (Fig. 1 F–J) were compared with those of G. gallusremiges (Fig. 1 B–E). At higher magnifications, much of thefossil is composed of 3D microbodies, with some regions

Significance

We report fossil evidence of feather structural protein (beta-keratin) from a 130-My-old basal bird (Eoconfuciusornis) fromthe famous Early Cretaceous Jehol Biota, which has producedmany feathered dinosaurs, early birds, and mammals. Multipleindependent molecular analyses of both microbodies and asso-ciated matrix recovered from the fossil feathers confirm thatthese microbodies are indeed melanosomes. We use trans-mission electron microscopy and immunogold to show localizedbinding of antibodies raised against feather protein to matrixfilaments within these ancient feathers. Our work sheds newlight on molecular constituents of tissues preserved in fossils.

Author contributions: Y.P., W.Z., Z.Z., and M.H.S. designed research; Y.P. and W.Z. per-formed research; A.E.M. contributed new reagents/analytic tools; X.Z. and X.W. providedmaterials for analysis; Y.P., W.Z., J.K.O., M.W., X.Z., X.W., and E.R.S. analyzed data; and Y.P.,Z.Z., and M.H.S. wrote the paper.

Reviewers: D.G.H., Louisiana State University; and R.H.S., University of South Carolina.

The authors declare no conflict of interest.1To whom correspondence may be addressed. Email: yhpan@nigpas.ac.cn, zhouzhonghe@ivpp.ac.cn, or mhschwei@ncsu.edu.

2Present address: Department of Biology, Drexel University, Philadelphia, PA 19104.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1617168113/-/DCSupplemental.

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retaining a very thin carbon layer (Fig. 1 F–I) that covers themicrobodies (Fig. 1J). This thin layer displays fibrous bundles inarrays that form regular angles (Fig. 1 G and I), a feature pre-viously reported in the epicortex of G. gallus feathers (27) andalso observed in Fig. 1 C and D.Transmission electron microscopy (TEM) confirms micro-

structural similarity between Eoconfuciusornis and extantG. gallus feathers (Fig. 2). The microbodies preserved in Eocon-fuciusornis STM 7-144 are distinct, relatively uniform, elongated,electron-dense, and embedded in a less electron-dense filamen-tous matrix (Fig. 2 A and B), patterns also observed in extantfeathers (Fig. 2 C and D). Both filaments and microbodies aremore densely packed in the fossils, suggesting some diageneticalteration perhaps involving compression.

Immunohistochemistry. A polyclonal antiserum raised against ex-tracts of mature modern white chicken feathers (28) was used totest the hypothesis that the exceptional microstructural preserva-tion in these fossil feathers extended to the molecular level. Bothimmunofluorescence and immunogold labeling support the pres-ence of epitopes in fossil tissues consistent with beta-keratin, afamily of proteins not produced by either mammals or microbesbut that comprise ∼90% of proteins in extant feathers (28) and arecoexpressed with alpha-keratin in the skin, beak, and claw tissuesof reptiles and birds (29). These are also referred to as corneousproteins (30).Specific immunoreactivity to components within the fossil

feathers of Eoconfuciusornis (Fig. 3) was revealed by tissue-spe-cific, although weaker, localization of fluorescence signal (Fig. 3 Cand D) in a pattern comparable to extant black chicken feathersused as a positive control (Fig. 3 G and H). No fluorescence waspresent in any controls, where the primary antiserum was omitted,but all other steps were kept identical (Fig. 3 A, B, E, and F).Furthermore, antibodies to hemoglobin (a protein found in ver-tebrate red blood cells, used as an irrelevant control) and pepti-doglycan (a glycosaminoglycan produced exclusively by bacteria)did not react with the fossil feather sample (Fig. S1), supportingboth the persistence of keratinous components and the specificityof the anti-feather antibody.We applied the same anti-feather antiserum (28) to ultrathin

sections (∼90-nm) of fossil and modern black feathers. Bindingwas detected using a secondary antibody conjugated to 12-nm goldparticles. The gold particles were seen associated with fibril bun-dles measuring ∼3 nm, consistent with filaments of beta-keratin

matrix (Fig. 4 G–I), but were sparsely distributed relative tobinding in modern controls (Fig. 4 D and F–L). However, otherstructures, including the electron-dense microbodies and needle-shaped structures probably of diagenetic origin, were completelyunlabeled (Figs. 4E and 5A). No gold particles were visible in thenegative controls (omitting the primary antiserum) (Fig. 4 A–C),similar to results from immunofluorescence. The higher-resolutionimmunoelectron localization of gold particles generally confirmsthe binding of the anti-feather antiserum visualized in fluores-cence microscopy, and verifies that these fossil feathers partiallypreserved the molecular structure of the feather–keratin epitopes,which remains immunoreactive.

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-magnificationimages 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) fromEoconfuciusornis. (F) Low-magnification image. (G) High-magnification image of the boxed area in F. (H) Melanosomes are shown located immediately belowthe structured thin layer, which is indicated with yellow arrowheads. (I and J) High-magnification images of the boxed areas in H. These bodies are embeddedwithin the feathers and not observed on the surface of the feathers. (Scales are as indicated.)

Fig. 2. TEM images of the crural feathers sampled in Eoconfuciusornis STM7-144 (A and B, sample 111) compared with that of a black feather fromG. gallus (C andD). (A) Low-magnification image. (B) High-magnification imageof the boxed area in A. (C) Low-magnification image. (D) High-magnificationimage of the boxed area in C. Both melanosomes and beta-keratin filamentsare more densely distributed in the fossil sample. (Scales are as indicated.)

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ChemiSTEM. We obtained high-resolution elemental maps of ultra-thin sections of fossil feathers using ChemiSTEM technology [FEITitan G2 series of Cs-corrected STEM (scanning/transmission elec-tron microscopy)]. This technology reveals the elemental distributionof nanogold particles bound to antibodies (Fig. 5 B–D and I–K) andidentifies, at high spatial resolution, other elements (e.g., N) thatwould be expected to be present in organic remains. This technologyalso identifies and localizes potential diagenetic compounds (e.g.,calciummineral morphs). Antibodies tagged with gold localize to andoverlap with relatively high concentrations of sulfur in both ancient

(Fig. 5 A–C) and modern (Fig. 5 E, I, and J) feathers. Keratin mol-ecules incorporate the S-bearing amino acid residue cysteine in rel-atively high concentrations (31, 32), supporting the hypothesis thatthese antibodies are most likely binding keratin.We applied the same technology to modern feathers to show that

extant melanosomes contain higher concentrations of copper, sulfur,and calcium (Fig. 6 G–J) than are seen in the keratin matrix. Simi-larly, the elements Cu, S, and Ca localize to electron-dense micro-bodies visualized in the fossil feathers (Fig. 6 A–D). We also notethat the keratinous matrix surrounding melanosomes in modern

Fig. 3. Immunofluorescence results of fossil feathers (sample 205, propatagium, which is covered dorsally and ventrally by feathers) compared with feathers of G. gallus.(A, C, E, and G) Overlay images showing where the antibodies bind to tissues (green) superimposed on transmitted light images. (B, D, F, and H) Fluorescence imagesshowing only antibody binding, as represented by green fluorescence of the FITC label. (A and B) Negative control corresponding to C andD, where the primary antibody isomitted. (C and D) Eoconfuciusornis feathers exposed to anti-feather antibody demonstrates specific and localized antibody binding, as reflected by green fluorescencesignal. (E and F) Negative control, with the primary antibody omitted. (G and H) In situ fluorescence signal corresponding to bound antibodies on the feather tissue of G.gallus. Binding patterns are similar between samples, although the ancient feathers show reduced binding as indicated by less intense fluorescence signal. All data werecollected under identical conditions and all images were processed identically. (Scales are as indicated.)

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feathers (Fig. 6 K and L) contains greater concentrations of carbonand nitrogen than are seen in fossil feathers (Fig. 6 E and F), in-dicating a possible diagenetic effect.The fossil feathers also show needle-shaped structures (Fig. 5A)

that are high in Ca (Fig. 5F). We propose that these structures aremost likely of diagenetic origin (Fig. 5F) and possibly microbiallymediated, because they are not found in modern feathers andtheir shape is consistent with early diagenetic morphs of calcium-containing minerals. However, phosphorus is barely measurable, anddoes not map to the Ca signal, so a CaPO4 mineral morph can beexcluded. The elemental maps obtained from lower-magnificationimages using environmental scanning electron microscope with en-ergy dispersive spectroscopy (ESEM-EDS) (Fig. S2) indicate ahigher concentration of Ca in the fossil feather material, which mapsto both the microbodies and the needle-like mineral structures.

DiscussionThe smooth and dense outer cortex of the feathers of Eocon-fuciusornis was imaged using SEM and compared with that ofextant chicken feathers at the same magnification. In both sam-

ples, immediately below this dense cortex, 3D microbodies werepresent. Melanosomes are suspended in a beta-keratin matrix (33), apattern and distribution similar to what is seen in Eoconfuciusornis.SEM analyses of modern feathers rarely showed melanosomes, asthese are obscured by the dense outer feather cortex, and melano-somes herein were not visualized unless this cortex was removed(27). However, this dense cortex is rarely observed in fossil feathers,exposing microbodies for easier observation. It is likely that thecortex disintegrated early in diagenesis, as seen in taphonomic ex-periments of feather degradation.TEM of 90-nm sections taken from Eoconfuciusornis feathers

was comparable to images of modern feathers in that the matrixof both samples contained filaments consistent with fibrillarkeratin and electron-dense bodies consistent with melanosomesin size, orientation, and location.However, although suggestive, imaging alone is not conclusive.

Therefore, we hypothesized that the exceptional ultrastructuralpreservation may extend to the molecular level. Capitalizing onthe specificity and sensitivity of the vertebrate immune response,which has been used successfully to characterize other originally

Fig. 4. Immunogold labeling of fossil feathers (sample 201) compared with feathers of G. gallus. (A–C) Negative control of D–F, when the primary antiserumis omitted. (B) High-magnification image of the boxed area in A. (C) High-magnification image of the boxed area in B. (D–I) In situ nanogold immunohis-tochemistry of Eoconfuciusornis feathers exposed to anti-feather antibody. (E) Melanosomes from the same section of D and F; no nanogold particles wereseen to specifically bind. (F) High-magnification image of the boxed area in D; arrowheads show binding of the nanogold beads. (G–I) High-angle annulardark-field (HADDF) images show binding of the nanogold beads from the same section of D–F. (H) High-magnification image of the boxed area in G. (I) High-magnification image of H; arrowheads show the filaments at the bead-binding regions. (J–L) In situ nanogold immunohistochemistry of G. gallus feathersexposed to anti-feather antibody. Nanogold particles specifically bind the keratinous tissues but do not bind the melanosomes, visualized in cross-section.(K) High-magnification image of the boxed area in J. (L) High-magnification image of the boxed area in K. (Scales are as indicated.)

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keratinous fossil material (34, 35), we show that antibodies raisedagainst feather proteins (28) react specifically with ancientfeathers in in situ experiments. Binding, as visualized by greenfluorescence signal, is reduced in intensity but comparable inpattern and distribution to that of extant feathers. The decreasedintensity of antibody binding in the fossil feather may be due toseveral factors: (i) greater degradation resulting in a reducednumber of recognizable epitopes; (ii) some epitopes present inmodern feathers (against which the antibody was raised) may nothave been present at this early stage in avian evolution (29, 36);or (iii) epitopes may be blocked or masked by infiltration withminerals or other factors resulting from diagenetic processes.To confirm the results we observed by immunofluorescence

labeling, we applied nanogold (12-nm)-labeled antibodies to90-nm sections of fossil feathers and observed localized bindingunder TEM, which allows much higher resolution. We show that

the anti-feather antibodies localize to filaments within the ma-trix, supporting the hypothesis that the matrix is composed ofmaterial consistent with filamentous beta-keratin proteins. Nogold labeling was detected in or associated with the microbodiesembedded in this matrix in either fossil or extant feathers (Fig.4), supporting the specificity of antibody binding to keratinousmatrix but not other components of the feathers. These experi-ments produced complementary data that confirm the presenceof molecules consistent with feather keratins in the fossil tissues.High-resolution elemental mapping (ChemiSTEM) adds addi-

tional information. This technique showed that regions bindingantibodies complexed to gold also demonstrate a relatively highconcentration of both sulfur and nitrogen, which would be expectedin keratinous material. The keratin protein family incorporates highconcentrations of amino acids rich in sulfur (31, 32), whereas carbonand nitrogen are common to all amino acids and proteins. However,

Fig. 5. ChemiSTEM data derived from the region immunoreactively binding nanogold particles and needle-shaped structures of the Eoconfuciusornisfeathers (sample 205), and compared with feathers of G. gallus. (A) HADDF image of an immunogold-labeled section of the Eoconfuciusornis feather, in-dicating the region selected for elemental mapping. (B–D) Various element distributions for the immunogold-labeled region (arrowhead). (B) Gold (Au)distribution map indicating the localization of nanoparticles. (C) Sulfur map showing the distribution of this element of overlap with antibody-bound Aubeads. (D) Nitrogen map suggesting the entire tissue is dominated by N, consistent with a proteinaceous source. (F–H) Element distributions for the needle-shaped structures observed in some regions of the fossil feather. (F) Calcium distribution outlines the shape of the structure, indicating it contains a relativelyhigh Ca level. (G and H) Nitrogen and phosphorus distribution maps, respectively, which are not specifically localized within the needle-shaped structures.(E) HADDF image of an immunogold-labeled section of G. gallus feather, indicating the region selected for elemental mapping. (I–K) Various elementdistributions for the immunogold-labeled region. (I) Au distribution map. (J) S map. (K) N map. (Scales are as indicated.)

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the possibility that the sulfur signals might be enlarged by the an-tibodies cannot be fully excluded based on the present experiments.Recently, it has been demonstrated that certain trace ele-

ments, such as copper, sulfur, and calcium, are present in bothfossil and modern melanosome-bearing tissues, and it has beensuggested that these elements may be used as biomarkers formelanin-derived compounds in fossils (23–25). This is problem-atic, however, in that all Cu, S, and Ca are also derived frombacterially induced decay as well as many diagenetic processes(37, 38). One of the ways to differentiate between melanosomesand morphologically similar bacteria is to directly correlate, athigh resolution, these trace elements to the microstructures butnot the surrounding matrix. However, the resolution of eitherESEM-EDS or synchrotron rapid scanning X-ray fluorescence(SRS-XRF) maps is not sufficient to attribute the elementsspecifically to any organelles (25). The high-resolution elementalmaps achieved by ChemiSTEM technology display high levels ofcopper and sulfur in both the modern melanosomes and ancient

microbodies. It thus confirms that both Cu and S colocalize toproposed melanin-bearing organelles. Although S is also observedin both the fossil feather matrix and the microbodies, Cu is foundonly within the melanosomes, indicating little diagenetic movementand supporting the original distribution of this element. Hence, weconclude that the microbodies examined herein are indeed thefossilized remains of melanin-bearing organelles and that theycontain high concentrations of the original trace elements of theliving organisms preserved through geological time.Another notable trace metal is calcium, which was once thought to

be chelated by and affect the chemical properties of melanin (39).Because of this association, it was proposed that Ca distribution couldbe used as a proxy for melanin distribution in fossil feathers (23). Inprevious studies, the SRS-XRF maps of Ca in the neck region of aspecimen of Confuciusornis sanctus showed a strong correlation withCu (23). Our ESEM-EDS maps (Fig. S2) also suggest that the dis-tribution of Ca strongly correlates with diagenetically altered featherstructures. However, the high-resolution elemental maps show that

Fig. 6. ChemiSTEM data derived from the microbodies of feather samples from Eoconfuciusornis (A–F), compared with melanosomes of a black feather fromG. gallus (G–L). (A) HADDF image showing the selected region for elemental mapping. (B–F) Various element distributions for the selected region. (B) Cu map.(C) S map. (D) Ca map. (E) C map. (F) N map. (G) HADDF image showing the region on the G. gallus feather selected for elemental mapping. (H–L) Variouselement distributions for the selected region. (H) Cu map. (I) S map. (J) Ca map. (K) C map. (L) N map. (Scales are as indicated.)

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Ca signal is much weaker in melanosomes of extant featherscompared with that in the microbodies of fossil feathers and, al-though Cu is localized within proposed melanosomes in the fossil,Ca maps to both microbodies and the needle-shaped structureswithin the feather–keratin matrix. These Ca-concentrated needle-shaped structures are absent in extant feathers. Thus, we hy-pothesize that precipitation of calcium, possibly mediated bymicrobes during the fossilization process, facilitated ultrastructuraland molecular preservation of the feathers by stabilizing them atthe molecular level before they could completely degrade (5, 40).It has long been known that the association with mineral sub-strates greatly enhances the preservation potential of biomolecules(35). Furthermore, it has been proposed that calcium may in-corporate into molecular fragments, conferring stability, and thatthis process may be microbially mediated (35). Because the sur-rounding sediments of this Eoconfuciusornis specimen consistmainly of Al silicates, with little or no calcium detected, the sourceof the calcium observed in these feathers remains unknown.Molecular dating techniques suggest that feather beta-keratin

diverged from “feather-like” beta-keratins about 143 Ma (29). Ifthese molecular dates are correct, feathers of Archaeopteryx andAnchiornis were constitutively distinct from those of extant birdfeathers, and thus feather structure preceded modern featherproteins. Molecular data show that only in the Early Cretaceousdid feather beta-keratins begin to diversify to their modern forms.These molecular changes conferred greater elasticity to feathersthan is seen in materials composed of other types of beta-keratin(29), thus optimizing the material properties of feathers for flight(41). The diversification of feather beta-keratin genes coincidedwith and perhaps facilitated the radiation of modern birds.Another factor that may contribute to the unique biomaterial

properties of feathers is the epidermal differentiation cysteine-richprotein (42). This molecule has the same gene structure as beta-keratins, and is coexpressed with them at certain stages of develop-ment in most keratinized tissues of living birds. However, it is only infeathers that this protein continues to be detected throughout life(42). The greatly elevated cysteine levels in this protein facilitateintramolecular cross-linking, and contribute to the stability and re-sistance of feathers to degradation. Further molecular recovery ofancient feather materials may allow direct testing of these hypotheses.

ConclusionThe multiple independent analyses used in this study providestrong evidence for the retention of original and phylogeneticallysignificant protein components in Eoconfuciusornis STM 7-144,and are consistent with the identification of melanosomes as well askeratin filaments in these ancient (∼130-Ma) tissues. This is theoldest report of ultrastructure consistent with beta-keratin from anEarly Cretaceous bird feather. Identifying keratin is key to rulingout a microbial source for microbodies identified in fossils. Boththe depositional context and mode of preservation of this bird aresimilar to those of numerous other Early Cretaceous Jehol birds(and dinosaurs) and Jurassic Yanliao dinosaurs in China (1–3, 10,11, 13, 15). This similarity suggests that other fossils preserved atthese localities could provide additional sources of molecular in-formation. More importantly, our study incorporating specific an-tibody reactivity in this Early Cretaceous specimen demonstratesthe benefit of testing other fossil tissue samples with antiseragenerated against specific protein epitopes. Such tests have thepotential not only to distinguish between keratinous feathers andskin-derived collagen fibers (43, 44) but also to characterize themolecular composition of early feathers and elucidate steps leadingto the evolution, at the molecular level, of modern bird feathers.

Materials and MethodsEight samples (S102, S111, S112, S201, S202, S203, S204, and S205) removedfrom STM 7-144 were used in this study. Samples S102, S111, and S112 weredirectly observed by SEM without coating. Samples S201, S202, S203, S204,

and S205, whichwere selected formolecular analysis, were washed successivelyin 75% (vol/vol) ethanol and E-pure water and ultrasonically cleaned to avoidpotential contaminants. The samples were then dried under a biological hood,and the surface structure was documented using SEM. Subsequently, thesamples were put into 50% HF for 3 to 5 d to dissolve the silicon minerals ofthe matrix and washed with E-pure water three times. Approximately 1- to2-mm3 samples were selected to be dehydrated in two changes of 70% ethanolfor 30 min each, followed by one change of a mixture of LR White (medium-grade) and 70% ethanol (2:1) for 1 h, followed by another two changes of100% LR White for 1 h each. The process was carried out at room temperature(RT). Then the samples were moved to gelatin capsules, filled with 100% LRWhite, covered to exclude oxygen, and allowed to polymerize for 24 h at60 °C. A Leica EM UC6 ultramicrotome with a DiATOME 45° knife was used tocut 200-nm sections for immunohistochemistry analysis and 90-nm sections forTEM observations, immunogold tests, and ChemiSTEM analysis. Extant feath-ers were treated in the same way but sectioned with a different diamondknife. Neither fossils nor extant feathers were fixed, and only the extantfeathers were stained with uranyl acetate (5 min) and Reynold’s lead citrate(8 min) before the TEM morphological observation. All experiments wererepeated multiple times to validate results.

SEM. The dried samples were mounted on stubs with double-sided carbon tape(S111 and S112), and one sample from an extant chicken feather was coated with∼10 nm of gold/palladium for 30 s. The SEM-EDS data were collected from un-coated S102, S201, S203, S204, and S205. Samples were imagedwith a Leo 1530 VPanalytical scanning electron microscope controlled by JEOL InTouchScope version1.05A software. Two detectors were used for taking SEM images: (i) a normalsecondary electron detector, used at high vacuum and 1 to 5 keV, and (ii) a var-iable-pressure secondary electron detector, used at low vacuum and 10 to 20 keV.

TEM. The 90-nm sections were mounted on carbon-coated copper grids.Samples were stained with 5% methanolic uranyl acetate (5 min), washed inE-pure water, stained with Reynold’s lead citrate (8 min), and washed inE-pure water. Sections were imaged using a JEM-2100 transmission electronmicroscope (AATC) from Nanjing Forestry University and a JEM-2100Plustransmission electron microscope from the State Key Laboratory of LakeScience and Environment, Nanjing Institute of Geography and Limnology,Chinese Academy of Sciences, at 80 to 100 keV.

Immunohistochemistry. For the production of anti-feather antibody, seeMoyer et al. (28).

The autofluorescence immune-labeling protocol was modified fromSchweitzer et al. (45). The 200-nm sections were mounted on six-well Teflon-coated slides, with about 10 sections in each well, and dried overnight at 42 °C.Sections were etched with 25 μg/mL proteinase K in 1× PBS at 37 °C for 15 min,followed by 0.5 M EDTA (pH 8.0) (30 min) and last with NaBH4 (twice for 10 mineach). Incubations were interrupted by two 5-min washes in PBS. Following thesesteps to accomplish epitope retrieval and quenching of autofluorescence, 4%normal goat serum in PBS was applied to occupy nonspecific binding sites andprevent spurious binding. Sections were incubated in primary antibody (poly-clonal rabbit anti-feather, 1:200; polyclonal rabbit anti-hemoglobin, 1:200;monoclonal mouse anti-peptidoglycan, 1:200) in primary dilution buffer over-night at 4 °C. All sections were then incubated with secondary antibody [bio-tinylated goat anti-rabbit IgG (H+L) (antibody recognizes both heavy and lightchains)] (Vector Laboratories; BA-1000) diluted 1:500 for rabbit primary anti-bodies, and biotinylated goat anti-mouse IgG (H+L) (Vector Laboratories;BA-9200) diluted 1:500 for monoclonal mouse anti-peptidoglycan, for 2 h at RT.Sections were then incubatedwith fluorescein avidin D [fluorescein isothiocyanate(FITC); Vector Laboratories; A-2001] for 1 h at RT. All incubations were interruptedby sequential washes (twice for 10 min each) in PBS/Tween 20 followed by two10-min rinses in PBS. Finally, sections were mounted with Vectashield mountingmedia (Vector Laboratories; H-1000) and coverslips were applied. Sections wereexamined with a Zeiss Axioskop 2 Plus biological microscope and captured usingan AxioCam MRc 5 (Zeiss) with 10× ocular magnification on the Axioskop 2 Plusin the AxioVision software package (version 4.7.0.0).

The postembedding TEM immunogold-labeling protocol wasmodified from themethods shared in the protocol database from IHCWORLD Life Science Products &Services (www.ihcworld.com/_protocols/em/post_immunoem_embed812.htm). The90-nm sections were collected on carbon-coated nickel grids (EMS; CFT200-NI). Thegrids were incubated in PBS/Tween 20 for 10 min. Four percent normal donkeyserum in PBS was applied to occupy nonspecific binding sites and prevent spuriousbinding for 1 h at RT. The grids were incubated in primary antibody (polyclonalrabbit anti-feather, 1:20) in primary dilution buffer for 3 h at RT and then washedwith PBS/Tween 20, 10 times for 2 min each. All grids were then incubated withsecondary antibody [12-nm Colloidal Gold AffiniPure Donkey Anti-Rabbit

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IgG (H+L), 1:20; Jackson ImmunoResearch; 711-205-152] for 1 h. The grids wererinsed with PBS/Tween 20, 10 times for 2 min each and in E-pure water threetimes for 30 s each, and dried with filter paper. The sections were observedusing the FEI Titan G2 80-300 electron microscope at the Analytical In-strumentation Facility (AIF) of North Carolina State University.

ChemiSTEM. Sections (90-nm) were collected on carbon-coated nickel grids(EMS; CFT200-NI) and analyzed with an FEI Titan G2 80-300 electron micro-scope at the AIF of North Carolina State University.

The FEI Titan G2 series of Cs-corrected scanning/transmission electron micro-scopes with the revolutionary ChemiSTEM technology is applied in this study. Thisnew technology greatly enhances EDX (energy dispersive X-ray spectroscopy) de-tection sensitivity due to a number of innovations in the system architecture, in-cluding the X-FEG (a high-brightness Schottky field emission gun source), Super-XEDXdetector system(fourwindowless silicondriftdetectorswith shutters integrateddeeply into the objective lens), and high-speed electronics capable of 100,000spectra readouts. This new system architecture provides many performance ben-efits, such as improved light element detection, better sample tilt response, fastermapping, and atomic chemical mapping capabilities of crystalline structures.

ACKNOWLEDGMENTS. We thank technician Ms. Chunzhao Wang of theState Key Laboratory of Palaeobiology and Stratigraphy (LPS) of Nanjing In-stitute of Geology and Palaeontology, Chinese Academy of Sciences, ResearchScholars Chuanzhen Zhou and Yang Liu of the Analytical InstrumentationFacility (AIF) of North Carolina State University, Dr. Lulu Cong of the Analyt-ical Center of Nanjing Institute of Geography & Limnology, Chinese Academyof Sciences, and Dr. Lingfei Ding and Dr. Yang Jing of the Advanced AnalysisTest Center of Nanjing Forestry University for technical assistance. We alsobenefited from discussions with Dr. Johan Lindgren and Prof. Franz Fürsich.This work was performed in part at the AIF at North Carolina State University,which is supported by the State of North Carolina and the National ScienceFoundation (Award ECCS-1542015). The AIF is a member of the North Caro-lina Research Triangle Nanotechnology Network, a site in the National Nano-technology Coordinated Infrastructure. The research was supported by theStrategic Priority Research Program of the Chinese Academy of Sciences(XDB18030501), National Natural Science Foundation of China (41472009and 91514302), Open-Lab Grants of the LPS and National Science Founda-tion USA (INSPIRE Program EAR-1344198 to M.H.S., W.Z., and E.R.S., andDGE01252376 to A.E.M.), as well as funds from the David and Lucile PackardFoundation (to M.H.S.).

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