Soft tissues preserve in oxidative settings

Twenty-four specimens (Supplementary Tables 1–5, Supplementary Figs. 1–3) of biomineralized vertebrate tissues ranging in age from modern to Late Triassic (ca. 205 mya), and representing environments from oxidative to reducing, were decalcified to release any soft tissues present, and their mineralogy and organic content were analyzed (Supplementary Table 1–5). All specimens exhibited dark brown to gray-black colors (Supplementary Figs. 1–2). Decalcification did not affect key molecular features and their potential transformation products in soft tissues (Supplementary Fig. 4).

Soft tissues were present in all modern samples, but only in those fossils from oxidative settings. The Jurassic (Oxfordian) paleonisciform scales preserve a fully articulated three-dimensional vascular system together with a dense meshwork of unbranched tubular nerve projections resembling the dental tubuli in modern vertebrate teeth (Fig. 1a, Supplementary Fig. 1), the first such features discovered in a fossil. The blood vessels are stained brown. The tubular nerve projections are hollow and appear translucent with walls that are beige in color. The soft tissues are brittle and cracked, and account for about 70% by volume of the ganoid scale prior to decalcification (compare Fig. 1a, Supplementary Fig. 1).

Fig. 1 Decalcified vertebrate hard tissues (representing a total of 7 specimens). a Paleonisciform ganoid scale (Oxfordian (Jurassic), Xinjiang, China) showing articulated blood vessels (abv) of the dentine and organic matrix with peripheral aligned and ordered (otpn), or unordered (utnp), tubular nerve projections. The left scale bar equals 500 μm, the right one 250 μm. b Blood vessel (bv) fragments from a diplodocid sauropod (Jurassic, Wyoming, US). The basal lamina (bslm) shows original stratification even though the vascular wall is thickened compared to modern archosaur blood vessels. Osteocytes (oc) with dense filipodia (f) are embedded in the originally collagenous extracellular matrix (ecm). Osteocytes adjacent to the blood vessel preserve cellular detail, but elsewhere they have degraded leaving the osteocyte lacunae (ocl) in the organic matrix. The left scale bar equals 100 μm, the right one 25 μm. c Extracellular matrix from an Allosaurus fragilis vertebra (Late Jurassic, Wyoming, US). The originally collagenous matrix fibers are preserved, and osteocytes with filipodia are dark and infilled. The scale bar equals 25 μm. d Extracellular matrix from an Apatosaurus sp. bone (Late Jurassic). Osteocyte lacunae are preserved. Decalcified avian and non-avian dinosaur eggshells. The scale bar equals 25 μm. e (Left) Rhea (modern, in captivity, Montana, US), artificially matured spongy layer. The maturation gradient increases to the right of the image. The original spongy layer is green due to pigmentation, while the matured part is brown. The scale bar equals 500 μm. (Center) Psammornis rothschildi (Holocene, Algeria), degraded spongy layer fragments. The scale bar equals 25 μm. (Right) Oviraptorid Heyuannia huangi (Late Cretaceous, Jiangxi, China) spongy layer. The scale bar equals 25 μm Full size image

The organic remains in the bone samples are also brittle. The Jurassic diplodocid bone yielded large (up to 1500 µm) extracellular matrix fragments which preserve anastomosing vascular canals and interconnected (max. 25 µm length) osteocytes with extensive filipodia in some areas, but only osteocyte lacunae elsewhere (Fig. 1b, compare Fig. 1d). The extracellular matrix harboring the osteocytes is the most intensively stained, and occurs as homogenous patches without any identifiable fibers. The Jurassic Allosaurus fragilis preserves an extracellular matrix made up of fibers resembling collagen (Fig. 1c). Osteocytes (max. 25 µm length) are evident as three-dimensional opaque ellipsoidal structures with associated filipodia. The Jurassic Apatosaurus sp. preserves only small patches of extracellular matrix (up to 150 µm in dimension) comprising a homogenous organic scaffold (Fig. 1d). Neither osteocytes nor blood vessels are preserved, but ellipsoidal osteocyte lacunae are evident as impressions in the organic matrix.

Experimentally matured (see Methods Experimental Maturation) modern Rhea americana (Fig. 1e) eggshell yielded a spongy layer with an intense brown color which disintegrated into small, sheath-like fragments (<30 µm). The eggshell of Holocene Psammornis rothschildi preserves similar fragments (up to 10 µm) stained blackish-brown (Fig. 1e). That of Cretaceous Heyuannia huangi preserves fragments up to 40 µm in length (Fig. 1e). The organic material in the fossil eggshells has lost its elasticity.

Experimental maturation (oxidative crosslinking) of modern soft tissues released by decalcification of enamel scales of Lepisosteus osseus, bones of Gallus domesticus, and eggshells of Rhea americana, resulted in discoloration from translucent white to progressively darker brown as temperature (45–120 °C) and duration of air exposure (10–60 min) were increased, yielding colors similar to those in the fossil soft tissues (compare Fig. 1, Fig. 2). Samples from reducing depositional environments did not release any soft tissue structures (Supplementary Fig. 9).

Fig. 2 Aligned Raman spectra of decalcified modern, matured, and fossil organic material. All spectra were obtained in aqueous solution, 500–1800 cm−1, 20 s exposure time, 4 accumulations. These spectra are based on a total of 12 specimens with soft tissue preservation used for high-resolution point measurements, while Raman spectra for an additional 12 specimens without soft tissue preservation can be found in Supplementary Fig. 9; a total of additional 29 specimens was used for experimental maturation. a Teeth and enamel scales. b Bones. c Eggshells. Differences in the color of the sampled material are represented in the icons on the right. Brown spectral band: advanced glycoxidation and lipoxidation end products (AGEs), oxidative crosslinks; yellow spectral bands: peptide amides. AGE bands increase in intensity relative to amide I bands (dotted lines) with age and artificial maturation. Protein degradation and deamidation over time are represented by a decrease in the band intensities identified as amide III and amide I. Details on experimentally matured reference tissues at different temperatures (autoxidation) on the right of the spectra show that oxidative, brown discoloration already occurs at low temperatures. The color scale associated with temperatures and incubation times for the maturation experiments range from red (relatively high temperatures) to blue (low temperatures). The scale bar equals 500 μm Full size image

Fossil soft tissues consist mainly of non-proteinaceous N-heterocyclic polymers

Raman Spectroscopy (Fig. 2) of extracted soft tissues yielded amide signals representing peptide bonds (Supplementary Table 3): amide III between 1230 and 1250 cm−1 and amide I between 1650 and 1690 cm−1 (Supplementary Table 6). AGE/ALE bands between 1550 and 1600 cm−1 (Supplementary Table 6) generally increase in intensity with the age of the fossil, whether enamel scale, tooth, bone, or eggshell (Fig. 2). This, the most prominent band in the spectra, is assigned to the C=C stretching of a (transition metal chelating) imidazole ring (vibrations at 1434–1440 cm−1 and 1340–1352 cm−1 confirm this assignment, Supplementary Table 6). In the modern samples, this band is far less prominent, and corresponds to the N-heterocycle of the amino acid tryptophane, and the few age-related AGEs/ALEs present (Gallus versus Alligator bone in Fig. 2). The spectra also show a N-heterocycle pyridine-like ring stretch component, minor carbonyl stretching at a higher wavenumber, and N-H deformation at lower wavenumbers, all characteristic of AGEs and ALEs36,38 (Fig. 2, Supplementary Table 6). The intensity of the AGE/ALE bands shows a reciprocal relationship to that of the amide bands36,39 (Supplementary Fig. 5).

Oxidative crosslinking transforms proteins during fossilization

The ratio of AGE/ALE band intensity to amide I band intensity represents the proportion of oxidative crosslinks relative to unaltered protein/peptide mass36,38,39, and is reflected in the discoloration of extracted soft tissues (Fig. 2, Supplementary Table 4): An increased amount of AGEs/ALEs relative to unaltered peptides yields a more intense brown stain (Fig. 2, Supplementary Table 4). The AGE/ALE to amide I band intensity ratio of the experimentally matured modern soft tissue samples, and the intensity of their brown stain, fall between those of the unaltered modern samples and the fossil samples (Fig. 2, Supplementary Fig. 5). The Raman shift of the fossil soft tissue amide III and amide I bands is indicative of the structural organization of preserved crosslinked peptides, and indicates the presence of (unordered) secondary structures in most samples (Supplementary Table 6). Disulfide-bridges indicating the presence of tertiary structures are generally absent in fossil materials, showing that potentially preserved oligopeptides are of very short chain length (Fig. 2); some fossil samples appear to be composed of fully transformed, non-proteinaceous AGEs/ALEs (Fig. 2). This is corroborated in our Raman maps (Fig. 3): N-rich heterocycles produce a spatial signal restricted to soft tissue surfaces where they appear to be the major constituents (Fig. 3a, b). Pentosidine, a classic AGE marker, corresponds closely in its spatial distribution to the N-rich heterocyclic polymers (Fig. 3c), while the amide I signal (indicative of peptide bonds) is most prominent in areas with low N-rich heterocycle signal (Fig. 3d). High-resolution point spectra also revealed a range of minor compounds identified as degradation products of lipids (Fig. 2, Supplementary Table 6, Supplementary Fig. 6). In situ Raman spectra of vertebrate hard tissues from reducing environments showed no evidence for amide bands representing peptide bonds (Supplementary Fig. 9).

Fig. 3 High-resolution Raman maps (3 s exposure time, 3 accumulations) of remains of extracellular matrix extracted from Allosaurus fragilis bone in aqueous hydrochloric acid (pH = 3). The dotted rectangles mark the area mapped, and the different shades of red represent the signal intensity of the protein fossilization product (PFP). The colored spheres framing the compound structures represent a N-heterocycle polymer context, and the red shading labels the functional unit that gives rise to each signal mapped out. The scale bar equals 300 μm. a Dark residue of soft tissue. b Map of N-rich heterocycles (1550–1610 cm−1), which cover most of the sample surface. c Pentosidine (980 cm−1), the AGE marker, which corresponds to the spatial distribution of N-rich heterocycles. d Amide I generated by peptide bonds, which is a less precise match for the N-rich heterocycles (1690 cm−1). e–g The organic functional units representing the selected Raman shifts are shaded (red) below each map Full size image

A Principal Component Analysis (PCA) based on spectral intensities at seventeen representative Raman shifts (compositionally informative on peptide bonds and N-heterocycles) showed for the complete set of modern, matured, fossil and control samples (including in situ spectra of samples from reducing depositional environments, and various potential contaminants) a clear separation between two clusters formed by control samples and soft tissues (Fig. 4a). An additional PCA (Fig. 4b) focusing on the signal within the spectra of fresh, matured, and fossil soft tissues revealed a cluster of fresh soft tissues which is distinct from the cluster of fossil soft tissues, and a third cluster of experimentally matured soft tissues that bridged the other two sample clusters. Comparing the average composition (n = 4) of fresh soft tissue with that of fossil soft tissue (n = 7) (Fig. 4c), reveals a net enrichment during fossilization that corresponds to the N-heterocyclic crosslink pentosidine. Pentosidine is a classic and reliable marker for AGEs and ALEs30.