Fig 1.
Light micrographs (LM) of feathers used in this study.
A-C are control feathers, kept at room temperature (RT) for the 10-year duration of the experiment, and additional six years until present analyses. A) overview, showing original color distributions of feathers from Perdix perdix. B) red, and C) white regions of the feather. D-F show the condition 1 feather, kept at 60°C with intermittent watering for 3 years, then buried at RT until analyses. Colored regions are still distinct, but barbs show fraying and loss of integrity. Panels G-L show fragmented remains of feathers buried and maintained at 350°C for ten years, then at RT until analyses. No evidence of original color remains; barbules are not in evidence (but see J, arrow). G) shows a region of hollow rachis, with pith (p) internal, and a dark carbonized external cortex (c). H) Shows a region of the rachis (identified by diameter comparisons with A-C) preserved in three dimensions. Although no original color remains, the offset barb ridges (arrows) allow us to determine this shaft is from a remige (flight feather). I) is a longitudinal section through the shaft. Barb ridges can be seen on one side of this structure (arrows) and a lighter colored pith is visible filling the rachis. J) shows a tiny barbule with presumed hooklets arising from it (arrow) associated with a small fragment of a feather rachis. K) and L) represent other rachises (or parts) preserving the offset barbs intact and in three dimensions (arrows). M-O represent the remnants of a silicified coot feathers, collected from Yellowstone National Park. We interpret M) to be a degraded feather rachis, displaying fibrous surface. N) A small region interpreted to represent overlapping barbs, forming a vane. Additional fibrous remnants still embedded in silicified coated region can be seen in panel O (arrows).
Fig 2.
Scanning electron micrographs (SEM) of experimental (A-L), and Yellowstone (M-P) feathers.
A-D) barbs arising from a rachis at low (A) and higher (B-D) magnifications. Feather structure is virtually unaltered, and both barbs (b) and barbules (bu) can be seen. In Fig 2B, hooklets are seen arising from distal bars (arrows). C) Internal regions of a barb, with cortex (c) and pith (p) clearly discernible. D) Highly fibrous region of what is interpreted to be the distal rachis. E-H represent the condition 1 feathers. Loss of integrity is more obvious than in LM. E) Fraying of the rachis (arrows) reveals fibrous structure. F) Twisted and compressed barbules (arrows) and debris on the rachis and barbs (**). G) Higher magnification of rachis and barbs, with debris (**) that may be from the burial sands, or from degrading keratin flakes. H) Bent and twisted barbules (arrows) with presumed keratinous flakes (f) on the surface of feather structures. Panels I-L show the microstructural integrity of the condition 2 (350°C) feather. I) Rachis, with smooth external cortex (c) and internal pith (p). Barbs are seen arising from the surface (arrows). J) Higher magnification image showing pith (p) and cortex (c), but the cortex demonstrates thin cracks in the surface. A curved barb is still attached (arrow). K) Compressed barbules (arrows) arising from flattened barbs.; debris can be seen across the surface of these feather structures. L) Highly fibrous region of the condition 2 feather, very similar in structure to that seen in the control (D). Panels M-P show the three dimensional, coated structure of the silicified coot feather. M) Fibrous structure and overlapping barbs in low magnification, with evidence of fungal hyphae (fu) interspersed throughout. N) Region of overlapping barbs, with thin mineral coating (**). O) Thin mineral coating on the barbs in higher magnification (**); silicified fungal hyphae can also be seen (fu). P) Feather at higher magnification, revealing a fibrous outer cortex (c), and altered pith (p) interior to the cortex. Scale bars: A, E, I, M are 100 μm; K, N, P = 20 μm; O = 3 μm; all others = 10 μm.
Fig 3.
In situ immunofluorescence on feather tissues.
A, C, E, and G are overlay images; B, D, F, and H are fluorescence images, showing localized binding of antiserum raised against modern feathers to these experimental feathers. A, B) show in situ binding of the serum to feather rachis and barbs in control feathers. Antibody-antigen (ab-ag) complexes are demonstrated by localized green signal under fluorescent light. C, D) Virtually undiminished binding of antibodies to the condition 1 feather barbs. No spurious binding is seen on the embedding polymer, and ab-ag complexes are specific to feather structures. E, F) Cross section of a feather barb from condition 2. A thin cortex can be seen, with very thin rami of pith in E). F) Weak, but highly localized binding of antiserum to feather structures, with no binding observed outside of the tissues. G, H) Localization of ab-ag complexes to the surface of tissues seen in the Yellowstone feather. Binding is restricted to feather structure, as can be seen in G, but is intermittent and, although structurally preserved, not all feather material binds this antiserum.
Fig 4.
Transmission electron micrographs (TEM) and immunogold labeling of experimental and fossil tissues with antiserum to feather keratin.
Ab-ag complexes are demonstrated by electron-opaque gold beads attached to the secondary antibody. A-C) Localization of ab-ag complexes to keratinous tissues in the control feather. Melanosomes can be seen in A, B), but virtually no gold beads localize to these structures, and remain localized only to the filamentous matrix, supporting antibody specificity. C) gold beads are specifically associated with electron-lucent filaments against a darker background. D-F show the same immunoassay results on the condition 1 feathers. Melanosomes can be seen, but they are less electron dense than in A), and most exhibit hollow cores, possibly indicating initial degradation. Again, gold beads, reflecting the location of ab-ag complexes, are localized to the keratinous matrix interspersed between melanosomes, although these are reduced in density from the control feathers. F) Edge of a melanosome (arrow); no binding of the small gold beads is observed on the melanosome, but is localized to the matrix surrounding the melanosome. G-I) Immunolabelling on a small region of the condition 2 feather; although no melanosomes are seen in feathers from this condition, the keratinous matrix remains. Binding of antibodies is sparse, but is specific and highly localized to remnants of electron-lucent filaments (H, I). J-L) Localization of gold beads to Yellowstone coot feathers under low (J) and higher (K, L) magnification. No melanosomes are visible in TEM, but gold beads are strictly localized to regions of small electron lucent filaments, as in the other conditions presented here.
Fig 5.
Positive ion ToF-SIMS data of the control (RT) and condition 2 (350 °C) feathers, keratin reference sample and tape support.
A) Ion images show the signal intensity distributions of C4H8N+ (green), representing protein, C5H9+ (red), representing hydrocarbons, and Ca+ +CaOH+ (blue). Scale bar 100 μm. B) Mass spectra generated from the protein-rich areas of the control and 350°C feathers, a keratin reference, and the tape substrate onto which the feather samples were attached (see text). Strong peaks at m/z 70, 30 and 44 in the keratin spectrum and the relatively strong signal of these peaks also in the feather spectra are consistent with proteinaceous material in both feather samples.”w” indicates hydrocarbon ions, and”p” indicates nitrogen-containing ions that show strong intensity for proteins. Optical micrographs of the feather samples are provided in S6 Fig.