Chemical, experimental, and morphological evidence for diagenetically altered melanin in exceptionally preserved fossils

Abstract

In living organisms, color patterns, behavior, and ecology are closely linked. Thus, detection of fossil pigments may permit inferences about important aspects of ancient animal ecology and evolution. Melanin-bearing melanosomes were suggested to preserve as organic residues in exceptionally preserved fossils, retaining distinct morphology that is associated with aspects of original color patterns. Nevertheless, these oblong and spherical structures have also been identified as fossilized bacteria. To date, chemical studies have not directly considered the effects of diagenesis on melanin preservation, and how this may influence its identification. Here we use time-of-flight secondary ion mass spectrometry to identify and chemically characterize melanin in a diverse sample of previously unstudied extant and fossil taxa, including fossils with notably different diagenetic histories and geologic ages. We document signatures consistent with melanin preservation in fossils ranging from feathers, to mammals, to amphibians. Using principal component analyses, we characterize putative mixtures of eumelanin and phaeomelanin in both fossil and extant samples. Surprisingly, both extant and fossil amphibians generally exhibit melanosomes with a mixed eumelanin/phaeomelanin composition rather than pure eumelanin, as assumed previously. We argue that experimental maturation of modern melanin samples replicates diagenetic chemical alteration of melanin observed in fossils. This refutes the hypothesis that such fossil microbodies could be bacteria, and demonstrates that melanin is widely responsible for the organic soft tissue outlines in vertebrates found at exceptional fossil localities, thus allowing for the reconstruction of certain aspects of original pigment patterns.

Keywords: diagenesis; mass spectrometry; melanosome; paleocolor; pigmentation.

Fig. 1.

Range of fossils analyzed with TOF-SIMS imaged using photography and under SEM. (A and B) Fossil complete bird with associated feathers, Messelornis, Messel, Eocene, Germany. (C and D) Articulated bird associated with feathers, undescribed, Fur Formation, Eocene, Denmark. (E and F) Isolated feather previously described as iridescent (2), Messel, Eocene, Germany. (G and H) Complete cyclostome fish preserving the eye, detailed, Mazon Creek, Carboniferous. (I and J) The bat Hassianycteris, Messel, Eocene, Germany. (K and L) The bat Palaeochiropteryx, Messel, Eocene, Germany. (M and N) Fossil ink sac, Lyme Regis, Lower Jurassic, England. (O and P) Fossil octopus (?Keuppia) associated with ink sac, Cretaceous, Hakel, Lebanon; UV image courtesy of Jonathan Jackson. (Q and R) Fossil frog preserving eyes, Paleobatrachus, Messel, Eocene, Germany. (S and T) Same frog as in Q, detailing skin preserved on the hind leg, Messel, Eocene, Germany. (U and V) Fossil frog, Pipidae, Mush Valley, Miocene, Ethiopia. (W and X) Fossil tadpole, Pelobates, Oligocene, Enspel, Germany. Specimens were photographed under normal light, except in O, which is imaged under UV light as well as scanning electron microscopy images. See SI Appendix, Table S1, for details. (Scale bars, 1 µm.)

Fig. 2.

TOF-SIMS intensity maps of selected positive and negative secondary ions from an Eocene iridescent feather, SMF-ME 3850 from Messel, Germany. (A) Entire feather imaged under normal light. (B) Microscopic image of sample analyzed under TOF-SIMS. TOF-SIMS intensity distributions are labeled by the predicted secondary ion. Note the correlation of secondary ions, previously noted in melanins (7) in the feather barb relative to the matrix, whereas SiO₃−, Al+, and CHO₂− are more intense in the matrix, which can be attributed to the presence of aluminosilicates, whereas the presence of CHO₂− could be due to other organic-rich sources, such as algae or pollen. Sodium (Na+) is localized to the feather, whereas the presence of copper (Cu+) shows no particular correlation to the feather imprint. Bacterial biomarkers for hopanoids were also mapped: m/z = 149.113+, m/z = 177.164+, and m/z = 191.18+. These did not exhibit any particular localization, or any significant quantity in the spectra obtained.

Fig. 3.

PCA of fossil melanin TOF-SIMS spectra and comparative samples. (A) Principal component plot of TOF-SIMS spectra with the area defined by each sample category (fresh, fossil, matured 200 °C/250 bar, 250 °C/250 bar) indicated with distinct color. (B) Schematic overview of distribution of samples according to inferred/observed color/taxonomy. For loadings, see SI Appendix, Fig. S4.

Fig. 4.

Scanning electron images of experimentally matured melanosomes. (A) Gallus gallus feather (red/brown). (B) Troglodytes aedon feather (brown). (C) Junco hyemalis feather (gray). (D) Corvus corone feather (black). (E) Meleagris gallopavo feather (iridescent). (Scale bar, 1 µm.)