Chemical Traces of Melanin Preserved in the Fur of a Three-Million-Year-Old Apodemus atavus

Millions of years after an animal dies, the original color of its fur is usually lost, and the resulting fossil no longer retains the brown, red, or black hues it displayed in life. Yet the disappearance of visible color does not necessarily erase every chemical trace of pigmentation. Some pigments form chemical complexes with trace metals that can survive prolonged fossilization, giving paleontologists a way to reconstruct aspects of an animal's original pigmentation from the chemical residues preserved in its tissues.

Two Pliocene mammal fossils recovered from Willershausen, Germany, belong to the extinct field mouse Apodemus atavus and date to approximately 3 million years ago. Both specimens preserve remarkably complete body outlines. In addition to the skeleton, remnants of soft tissues, including skin and fur, remain around the body.

Holotype specimen of Apodemus atavus (GZG.W.20027b). (b) False-color synchrotron rapid scanning X-ray fluorescence (SRS-XRF) image showing phosphorus (blue), zinc (green), and organic sulfur (red). Areas where zinc and organic sulfur overlap appear yellow（Image source：Manning PL et al. (2019)， CC BY 4.0 ）

Two major forms of melanin contribute to mammalian fur coloration: eumelanin and pheomelanin. Eumelanin produces black to dark brown coloration, whereas pheomelanin is associated with lighter reddish, yellowish-brown, and reddish-brown hues. The two pigments commonly occur together, meaning that a single hair may contain both types of melanin in different proportions.

One approach traditionally used to reconstruct coloration in fossils is to identify melanosomes, the intracellular organelles that contain melanin. In some fossils, these can be preserved as oval or spheroidal structures only a few micrometers in size. Morphology alone, however, is not always a reliable diagnostic criterion because microbial remains or mineralized structures can have similar shapes. Even when the structures can confidently be identified as melanosomes, observations from a minute area of a fossil cannot necessarily be extrapolated to the coloration of the entire animal.

Pheomelanin has a distinctive chemical feature: its synthesis requires cysteine, resulting in sulfur-containing structures such as benzothiazole and benzothiazine units. Some of this sulfur can bind directly to zinc. Eumelanin lacks these sulfur-bearing structures. Previous analyses of modern feathers had shown that in pheomelanin-rich tissues, zinc and pheomelanin-associated organic sulfur are concentrated in the same regions and display a strong spatial correlation. If the zinc and organic sulfur in the fur of a 3-million-year-old mouse retained a similar relationship, their distribution could provide evidence for the former presence of pheomelanin.

The two A. atavus fossils were therefore examined using Synchrotron Rapid Scanning X-ray Fluorescence imaging. This nondestructive method can map the distribution of different elements across a specimen without requiring pieces of fossil tissue to be removed. The scans revealed that both zinc and reduced organic sulfur were concentrated within remnants of the skin and fur, and their distributions closely overlapped. At higher spatial resolution, zinc followed elongated, hair-like structures, indicating that these chemical signals remained closely associated with the original fur.

Comparison of synchrotron rapid scanning X-ray fluorescence images of two Apodemus atavus fossil specimens. (a–c) Laterally compressed specimen GZG.W.20027b; (d–g) dorsoventrally compressed specimen GZG.W.17393a. (a, d) Distribution of total sulfur; (b, e) distribution of reduced organic sulfur, revealing remnants of soft tissues such as fur and skin; (c, f) distribution of inorganic sulfate obtained after subtraction of the organic sulfur signal; (g) false-color elemental image of specimen GZG.W.17393a showing phosphorus (blue), zinc (green), and organic sulfur (red). Areas where zinc and organic sulfur overlap appear yellow（Image source：Manning PL et al. (2019)， CC BY 4.0 ）

The surrounding sediment itself contains abundant sulfate minerals, so the researchers also used X-ray absorption spectroscopy to determine the chemical states in which these elements were preserved. Sulfur analysis detected a benzothiazole-type organic sulfur signal within the fossil soft tissues. Keratin, however, is itself rich in disulfide bonds. After millions of years of oxidation and degradation, sulfur compounds derived from keratin create a complex chemical background that can progressively obscure the much weaker signal originally contributed by pheomelanin. Sulfur alone is therefore unlikely to provide an unambiguous indicator of pheomelanin in ancient fossils.

X-ray absorption spectroscopy of zinc in Apodemus atavus fossils and samples from the extant wood mouse Apodemus sylvaticus. (a) Zinc K-edge XANES spectra comparing the A. atavus fossils, A. sylvaticus fur, human hair, and several zinc-bearing standards. The two vertical dashed lines indicate the positions corresponding to Zn–S, in which the first coordination shell consists entirely of sulfur, and Zn–O, in which zinc is coordinated by oxygen. (b) Comparison of the first derivatives of the absorption spectra. The zinc sulfide (ZnS) signal is shifted toward lower energy, whereas the Zn–O signal associated with eumelanin occurs at higher energy. Red and blonde human hair containing pheomelanin fall between these two positions, indicating a mixed coordination environment in which zinc is surrounded by both oxygen and sulfur. (c) Fourier-transformed EXAFS spectra comparing the relative distances of different atoms surrounding zinc; the thick dashed line marks the position of the Zn–S coordination shell. The distances shown have not been corrected for phase shift and are therefore shorter than the actual bond lengths, although the relative positions of the coordinating atoms are unaffected. (d) Two zinc coordination environments identified in A. sylvaticus fur and fossilized A. atavus fur. In one, zinc is primarily surrounded by oxygen or nitrogen; in the other, zinc is coordinated by three oxygen atoms and one sulfur atom. The abundance of organosulfur-coordinated zinc in the skin and fur remnants of A. atavus supports the presence of substantial pheomelanin pigmentation in life. Atom colors: zinc, dark gray; carbon, light gray; oxygen, red; sulfur, yellow; nitrogen, blue; hydrogen, white（Image source：Manning PL et al. (2019)， CC BY 4.0 ）

Wood mouse (Apodemus sylvaticus)（Image source：Hans Hillewaert， CC BY-SA 3.0 ）

The researchers next examined the atoms directly coordinated to zinc. In the fossil fur, zinc was surrounded by both oxygen and sulfur, and the Zn–S bond distances fell within the range expected for zinc coordinated to organosulfur compounds. This coordination environment closely resembled that found in modern red and blonde human hair and in the reddish fur of living Apodemus sylvaticus.

Copper within the fossils also showed a degree of spatial correlation with zinc. Organically bound copper can serve as a chemical indicator of eumelanin, while the mixed coordination environment surrounding zinc points to the presence of pheomelanin-derived compounds as well. Together, these observations support the interpretation that the original fur contained both eumelanin and pheomelanin. The body of A. atavus was therefore probably rich in both pigments and may have carried brown to reddish-brown fur.

The finer details of its color pattern remain less certain. Both fossils show relatively little organosulfur-bound zinc in the feet and tail, which could indicate that these regions were less heavily pigmented in life. At the same time, soft-tissue preservation is poorer in the tail, so some of the pigment signal may simply have been lost along with the fur itself. The chemical maps support a melanin-rich body containing both eumelanin and pheomelanin, but they are not sufficient to reconstruct the precise coloration of every part of the animal.

The true appearance of a 3-million-year-old Apodemus atavus can no longer be observed directly, yet the molecules that once produced its fur color have not vanished completely from the fossil record. Its pigmentation has survived in another form: a chemical map of elements and molecular bonds preserved within the remnants of its fur.

Author: Shui-Ye You

Reference:

Manning PL et al. (2019). Pheomelanin pigment remnants mapped in fossils of an extinct mammal. Nature Communications.