Sediment-encased P/T maturation shows macroscopic staining in fossil feathers is driven by melanin
These experiments show that sediment-matured samples are highly comparable to carbonaceous compression fossils from various lagerstätten in terms of appearance and chemistry. Such macroscopic carbonaceous stains inform pigmentation and patterning in fossil taxa (e.g. stripes, mottling, bars, bandit masks, countershading)5, 46, 47.
All feathers in these sediment-based experiments, irrespective of initial colour, leave behind brown stains on the bentonite matrix after P/T maturation. The staining appears most intense in the ~190–225°C treatments and completely disappears at ~300°C, leaving behind mainly impressions. The lower temperatures yield samples that resemble fossil feathers from the Mesozoic Altmühltal Formation48, Crato Formation5, 49, Jehol Group6, 46, 47 and Koonwarra fossil beds50 , Cenozoic Green River Formation51 and Fur Formation52 and many other konservat lagerstätten. However, sediment-matured iridescent feathers leave brown stains on the matrix and do not resemble the highly iridescent fossil feather (SMF ME 3850)53 from the Eocene of Messel Formation, although this type of original iridescence retention is admittedly rare in fossils53. Simultaneous unidirectional compaction and P/T maturation would likely be required to preserve original alignment of melanosome layers in order to preserve iridescence in stains.
A significant body of prior evidence aligns against preservation of endogenous proteins in carbonaceous compression fossils. Saitta, Kaye 28 showed that sediment-matured samples lose protein components (evidenced by volume loss of feathers/carcasses) and retain exposed melanosomes on the sediment matrix. This pattern is also noted in fossils5, 6, for example, where only darkly stained striped sections of feathers preserve melanosome films, whereas unpigmented sections show only sediment matrix without any proteinaceous ultrastructural features. Therefore, if proteins were to survive (relatively intact or cross-linked with sugars or lipids) organic mass in the feather tissue would not be significantly lost and melanosomes would be obscured by these polymers54. Total ion pyrochromatograms by both Saitta, Rogers 29 and Cincotta, Nguyen Tu 55 showed a lack of enriched peaks resulting from the pyrolysis of modern feather protein in both P/T-matured and fossil samples. ToF-SIMS spectra of experimental samples and fossils are quite different from
α/β-keratin controls from Schweitzer, Zheng 56 (Supplementary Fig. S3).
An alternative view57 suggests that brown stains in fossils can be readily produced by remnant proteinaceous components oxidatively condensing with sugar/lipid moieties to yield N-linked melanoidin-like heterocyclic polymers (i.e., advanced glycation end products [AGE] and advanced lipooxidation end products [ALE]). Wiemann, Fabbri 58 conducted their experiments at low temperatures (45–120°C) for short durations (10 mins–1hr) and did not elevate pressure. Vapourisation and removal of water from hotplate-heated samples of Wiemann, Fabbri 58 likely promoted condensation reactions between biomolecules59, enhancing AGE and ALE formation. Drying of tissues during maturation experiments are not realistically representative of typical burial environments preserving keratinous integumentary structures60 (i.e., extensively waterlogged, reducing lacustrine or marine settings). In contrast, the use of a pressurised setup in this study inhibits water from boiling off, instead directing proteins along hydrolytic reaction pathways.
PCA further supports this position. The sulphur content of extracted melanin originates from benzothiazole moieties of phaeomelanin19 whereas that of sediment-matured samples could derive from phaeomelanin, unleached keratin breakdown products, or from melanoidin-like polymers58. PC3 explains 9.86 % of the variation in the dataset (Supplementary Fig. S4 a, b, c). S-bearing fragments have a net positive loading on PC3 of either ~2.47 or ~2.22, depending on the ambiguous identity of the negatively loading fragment with m/z = ~134. The only S-bearing fragment(s) with negative loading on PC3 is/are large (containing six carbons and a nitrogen), possibly indicating incorporation within a larger polymer through thermal alteration, while the S-bearing fragments with positive loading on PC3 are typically smaller, possibly indicating that they are susceptible to volatilisation and loss through thermal alteration. Similar PC3 scores are observed across samples, but with slightly higher values in the sediment-matured feathers relative to the unmatured and capsule-matured melanin extracts as well as the fossils. There may also be a decrease in PC3 values as temperature increases in sediment maturation, consistent with the loss of volatile S-moieties, but sample sizes are small. The enrichment of presumably more volatile S-bearing moieties in the sediment-matured feathers could be indicative of unleached keratin breakdown products28 (e.g., hydrolysed peptide fragments or free/degraded amino acids) due to (a) a lack of a decay treatment to reduce protein concentrations prior to maturation or (b) maturation in dry sediment, limiting dissolution of protein breakdown products. S-bearing fragments in sediment-matured feathers could also come from melanoidin-like polymers formed through maturation58, again due to high protein and lipid concentrations when no decay has occurred prior to maturation or dry sediment that might favour condensation reactions. Regardless, this minor discrepancy in PC3 scores is not solely influenced by S-bearing fragments, used here as a proxy (albeit imperfect due to phaeomelanin confounding) for keratin-derived molecules, and would at most explain <10% of the variation in the data. Therefore, we can confidently say that any protein products or melanoidin-like polymers in sediment-matured samples are in very low quantities (consistent with observed volume loss in the impressions) and that PCA mostly describes variation in melanin chemistry. Similar PC3 scores between fossils and melanin extracts further highlight that protein loss is natural during fossilisation.
Sediment-encased P/T maturation elucidates melanin diagenesis
Animal integumentary structures (e.g., skin, scales, feathers, and hair) are composed of organic mixtures63 of proteins, lipids (e.g., waxes/oils), and pigments. When integumentary structures undergo fossilisation, complex organics proceed through multiple steps. Comparison of experimental results with fossils in terms of appearance and chemistry suggests that experimental maturation of feathers results in thermobaric decomposition of organics. We hypothesise that, like natural diagenesis, P/T-maturation favours hydrolytic loss of integumentary proteins – evidenced by significant volume loss leading to voids/impressions as noted in prior work28, 30.
Lipids, such as waxes and triglycerides, are predicted to undergo thermally mediated hydrolytic cleavage of ester bonds (e.g., between hydrophobic fatty acids and hydrophilic glycerol/glycerol phosphate groups64). After hydrolysis, severed hydrocarbon chains can undergo in situ polymerisation to be retained within fossils as insoluble aliphatic hydrocarbons (i.e. kerogen)25, 65. PCA distinguishes fossils from modern and experimental (capsule- and sediment-matured) samples. Lower PC2 values in modern/experimental samples compared to fossils are driven largely by enrichment in hydrocarbons (CxHy–), possibly originating from residual fatty acids and labile lipids (e.g., epidermal oils/waxes or melanosome lipid bilayers) in the modern/experimental samples that are depleted in fossils (Supplementary Fig. S5 a, b). Labile lipid hydrocarbons could have been lost from fossils during post-mortem peroxidation (i.e., early stage decay) and/or through prolonged late stage oxidative weathering of kerogen.
Melanin pigments are thought to have linear heteropolymeric, stacked oligomeric structures, or combination of both66, and have been shown to be resistant to hydrolysis except under harsh alkaline or acidic conditions67, 68. Multiple lines of evidence suggest that increasing PC1 scores correlate with increasing P/T alteration consistent with diagenesis: (1) At fixed pressure, PC1 scores increase with increasing maturation temperature; (2) open systems are shifted to higher PC1 scores; (3) non-hydrocarbon, especially N-bearing, fragments heavily load PC1 (e.g., CxSy–, CxHyS–, CxN–, CxHyN–, CxNS–, CxNO–, CxNSO–) (Fig. 3a), where certain smaller fragments (i.e., volatiles) have negative loading while large fragments (i.e., components of polymers) have positive loading.
When fragment masses were plotted against PC1 loading (Fig. 3b), large fragments had strong positive loading whereas certain small fragments had strong negative loadings, suggesting that these small volatiles are progressively lost during maturation whereas larger moieties are enriched at higher temperatures through polymerisation. Unmatured melanin extract with the lowest PC1 scores are the least altered and enriched in only certain small fragments. Capsule-matured melanin extracts have lower PC1 scores compared to sediment-matured feathers at the same temperature, most likely because they are enriched in those small fragments (i.e., the closed system traps volatiles/labile products otherwise lost in open-system sediment). These observations agree with previously published trends13, 31 and suggest that loss of certain volatile/labile moieties concurrent with polymerisation/crosslinking occurs during melanin diagenesis, especially with respect to heteroatomic compounds such as N/S-bearing compounds.
We hypothesise that melanin undergoes thermal decomposition in several overlapping steps (i.e. volatile loss and polymerisation are overlapping at certain temperatures, but different processes are dominant at different temperatures). First, certain volatile/labile N/S/O-bearing compounds are lost as evidenced by smaller to larger secondary ion mass with both increased maturation temperature and increased system openness. Next, thermal crosslinking of eumelanin monomers (i.e., dihydroxyindoles and dihydroxyindole carboxylic acids) and oligomerisation of phaeomelanin benzothiazole units are expected occur54, 68, 69. We infer this in our experiments from the dark stains left on the bentonite matrix (~190–225°C).
Finally, significant thermal decomposition of melanin (i.e., chemical /alteration of the polymer) occurs at higher temperatures leading to carbonisation/charring followed by decarbonisation/oxidation around 300°C for eumelanin and maybe even lower (e.g., closer to 250°C) for phaeomelanin. In sediment-matured feathers, phaeomelanin-dominated samples (i.e., reddish-brown and grey) start to show signs of stain fading at ~250°C, whereas eumelanin-dominated samples (i.e., black and iridescent) do so by ~300°C. PCA shows very high thermal alteration and enrichment of polymers at ~250°C followed by reversal towards the PC1 origin at ~300°C, consistent with increased carbonisation/charring followed by decarbonisation/oxidation, leading first to carbon-rich then organic-depleted samples (Fig. 2 a, b).
We posit that black and iridescent feather stains can survive harsher temperature regimes compared to brown and grey feathers. The observations of (a) stain retention at higher temperatures, (b) possible trends in PC1 scores according to melanin colour within each treatment/sample category, and (c) apparent reversal of this pattern at ~300°C suggest that that eumelanin has higher diagenetic stability than phaeomelanin. Seemingly intermediate positions occupied by mixed/organ melanin and grey colours along PC1 would suggest an intermediate composition and maturation stability, consistent with other studies70, 71.
Future work
The ultimate goal of maturation setups is to simulate natural diagenesis in the laboratory. The expected result would produce matured tissues that are structurally and chemically comparable to carbonaceous compression fossils. While our current setup closely mimics the macroscopic staining and key aspects of diagenetic chemical signatures, compaction and P/T-maturation are temporally decoupled. The original orientation of melanosome layers in specimens (e.g. in structurally iridescent arrays) can be altered through the current setup28. To minimise this, future setups should compact and mature specimens concurrently.
Additionally, this study compares previously published capsule-maturation of enzymatically extracted melanin to sediment-encased maturation of whole feathers with pigments, proteins, lipids, etc., so the effect of greater chemical complexity in the whole tissues of fossils and sediment-matured samples should be further controlled in future work. Although, as noted above, there is good evidence that these ToF-SIMS signatures are driven primarily by melanin rather than keratin protein (for comparisons of raw spectra see Supplementary Fig. S2). Chemical variation between modern/experimental samples and fossils is most possibly due to lipid loss during early decay or late oxidative weathering. This can be potentially accounted for in future experimental designs through the addition of pre-maturation decay treatments or by subjecting samples to oxidation after maturation using warm, moist, oxygenated air.
Lastly, temperature can fluctuate by ~2–5 °C inside the sample chamber of the current maturation rig, albeit of minimal concern when examining samples across a 300°C temperature range. Temperature gradients are more noticeable with scaling up to larger sample chambers. Future improvements in chamber design should help to minimise temperature fluctuations and produce samples that are even more similar to natural fossils.