Nicholas P. Edwards

Trace Metals as Biomarkers for Eumelanin Pigment in the Fossil Record

X-ray maps of fossil feather pigments reveal color patterning in extinct bird species. Well-preserved fossils of pivotal early bird and nonavian theropod species have provided unequivocal evidence for feathers and/or downlike integuments. Recent studies have reconstructed color on the basis of melanosome structure; however, the chemistry of these proposed melanosomes has remained unknown. We applied synchrotron x-ray techniques to several fossil and extant organisms, including Confuciusornis sanctus, in order to map and characterize possible chemical residues of melanin pigments. Results show that trace metals, such as copper, are present in fossils as organometallic compounds most likely derived from original eumelanin. The distribution of these compounds provides a long-lived biomarker of melanin presence and density within a range of fossilized organisms. Metal zoning patterns may be preserved long after melanosome structures have been destroyed.

Infrared mapping resolves soft tissue preservation in 50 million year-old reptile skin

Non-destructive Fourier Transform InfraRed (FTIR) mapping of Eocene aged fossil reptile skin shows that biological control on the distribution of endogenous organic components within fossilized soft tissue can be resolved. Mapped organic functional units within this approximately 50 Myr old specimen from the Green River Formation (USA) include amide and sulphur compounds. These compounds are most probably derived from the original beta keratin present in the skin because fossil leaf- and other non-skin-derived organic matter from the same geological formation do not show intense amide or thiol absorption bands. Maps and spectra from the fossil are directly comparable to extant reptile skin. Furthermore, infrared results are corroborated by several additional quantitative methods including Synchrotron Rapid Scanning X-Ray Fluorescence (SRS-XRF) and Pyrolysis-Gas Chromatography/Mass Spectrometry (Py-GC/MS). All results combine to clearly show that the organic compound inventory of the fossil skin is different from the embedding sedimentary matrix and fossil plant material. A new taphonomic model involving ternary complexation between keratin-derived organic molecules, divalent trace metals and silicate surfaces is presented to explain the survival of the observed compounds. X-ray diffraction shows that suitable minerals for complex formation are present. Previously, this study would only have been possible with major destructive sampling. Non-destructive FTIR imaging methods are thus shown to be a valuable tool for understanding the taphonomy of high-fidelity preservation, and furthermore, may provide insight into the biochemistry of extinct organisms.

Synchrotron-based chemical imaging reveals plumage patterns in a 150 million year old early bird

Charles Darwin acknowledged the importance of colour in the natural selection of bird plumage. Colour can indicate age, sex, and diet, as well as play roles in camouflage, mating and establishing territories. Feather and integument colour depend on both chemical and structural characteristics and so melanosome structure and trace metal biomarkers can be used to infer colour and pigment patterns in a range of extant and fossil organisms. In this study, three key specimens of Archaeopteryx were subjected to non-destructive chemical analysis in order to investigate the potential preservation of original pigmentation in early fossil feathers. Synchrotron Rapid Scanning X-ray Fluorescence (SRS-XRF) maps are combined with sulphur X-ray Absorption Near Edge Structure (XANES) spectroscopy to provide the first map of organic sulphur distribution within whole fossils, and demonstrate that organically derived endogenous compounds are present. The distribution of trace-metals and organic sulphur in Archaeopteryx strongly suggests that remnants of endogenous eumelanin pigment have been preserved in the feathers of this iconic fossil. These distributions are used here to predict the complete feather pigment pattern and show that the distal tips and outer vanes of feathers were more heavily pigmented than inner vanes, contrary to recent studies. This pigment adaptation might have impacted upon the structural and mechanical properties of early feathers, steering plumage evolution in Archaeopteryx and other feathered theropod dinosaurs.

Morphological and geochemical evidence of eumelanin preservation in the feathers of the Early Cretaceous bird, Gansus yumenensis

Recent studies have shown evidence for the preservation of colour in fossilized soft tissues by imaging melanosomes, melanin pigment containing organelles. This study combines geochemical analyses with morphological observations to investigate the preservation of melanosomes and melanin within feathers of the Early Cretaceous bird, Gansus yumenensis. Scanning electron microscopy reveals structures concordant with those previously identified as eumelanosomes within visually dark areas of the feathers but not in lighter areas or sedimentary matrices. Fourier transform infrared analyses show different spectra for the feathers and their matrices; melanic functional groups appear in the feather including carboxylic acid and ketone groups that are not seen in the matrix. When mapped, the carboxylic acid group absorption faithfully replicates the visually dark areas of the feathers. Electron Paramagnetic Resonance spectroscopy of one specimen demonstrates the presence of organic signals but proved too insensitive to resolve melanin. Pyrolysis gas chromatography mass spectrometry shows a similar distribution of aliphatic material within both feathers that are different from those of their respective matrices. In combination, these techniques strongly suggest that not only do the feathers contain endogenous organic material, but that both geochemical and morphological evidence supports the preservation of original eumelanic pigment residue.

Synchrotron imaging reveals bone healing and remodelling strategies in extinct and extant vertebrates

Current understanding of bone healing and remodelling strategies in vertebrates has traditionally relied on morphological observations through the histological analysis of thin sections. However, chemical analysis may also be used in such interpretations, as different elements are known to be absorbed and used by bone for different physiological purposes such as growth and healing. These chemical signatures are beyond the detection limit of most laboratory-based analytical techniques (e.g. scanning electron microscopy). However, synchrotron rapid scanning–X-ray fluorescence (SRS–XRF) is an elemental mapping technique that uniquely combines high sensitivity (ppm), excellent sample resolution (20–100 µm) and the ability to scan large specimens (decimetre scale) approximately 3000 times faster than other mapping techniques. Here, we use SRS–XRF combined with microfocus elemental mapping (2–20 µm) to determine the distribution and concentration of trace elements within pathological and normal bone of both extant and extinct archosaurs (Cathartes aura and Allosaurus fragilis). Results reveal discrete chemical inventories within different bone tissue types and preservation modes. Chemical inventories also revealed detail of histological features not observable in thin section, including fine structures within the interface between pathological and normal bone as well as woven texture within pathological tissue.

Leaf metallome preserved over 50 million years

Large-scale Synchrotron Rapid Scanning X-ray Fluorescence (SRS-XRF) elemental mapping and X-ray absorption spectroscopy are applied here to fossil leaf material from the 50 Mya Green River Formation (USA) in order to improve our understanding of the chemistry of fossilized plant remains. SRS-XRF of fossilized animals has previously shown that bioaccumulated trace metals and sulfur compounds may be preserved in their original distributions and these elements can also act as biomarkers for specific biosynthetic pathways. Similar spatially resolved chemical data for fossilized plants is sparsely represented in the literature despite the multitude of other chemical studies performed. Here, synchrotron data from multiple specimens consistently show that fossil leaves possess chemical inventories consisting of organometallic and organosulfur compounds that: (1) map discretely within the fossils, (2) resolve fine scale biological structures, and (3) are distinct from embedding sedimentary matrices. Additionally, the chemical distributions in fossil leaves are directly comparable to those of extant leaves. This evidence strongly suggests that a significant fraction of the chemical inventory of the examined fossil leaf material is derived from the living organisms and that original bioaccumulated elements have been preserved in situ for 50 million years. Chemical information of this kind has so far been unknown for fossilized plants and could for the first time allow the metallome of extinct flora to be studied.

The mapping and differentiation of biological and environmental elemental signatures in the fossil remains of a 50 million year old bird

The preservation of fossils reflects the interplay of inorganic and organic chemical processes, which should be clearly differentiated to make interpretations about the biology of extinct organisms. A new coliiformes bird (mouse bird) from the � 50 million year old Green River Formation (Wyoming, USA) has here been analysed using synchrotron X-ray fluorescence and environmental scanning electron microscopy with an attached X-ray energy dispersive system (ESEM-EDS). The concentration and distribution of 16 elements (Si, P, S, Cl, K, Ca, Ti, Mg, Fe, Ni, Cu, Zn, As, Br, Ba, Hg) has been mapped for individual points on the sample. S, Cu and Zn map distinctly within visibly preserved feathers and X-ray Absorption Spectroscopy (XAS) shows that S and Cu within the feathers are organically bound in a similar manner to modern feathers. The morphological preservation of the feathers, on both macro- and microscopic scales, is variable throughout the fossil and the differences in the lateral microfacies have resulted in a morphological preservation gradient. This study clearly differentiates endogenous organic remains from those representing exogenous overprinted geochemical precipitates and illustrates the chemical complexity of the overall taphonomic process.

Bacteria or melanosomes? A geochemical analysis of micro-bodies on a tadpole from the Oligocene Enspel Formation of Germany

Many exceptionally preserved fossils have long been thought the product of preservation by bacterial autolithification, based largely upon the presence of, micron-sized, spherical or elongate bodies on their surface. This has recently been challenged by studies of similar fossils which cite morphological and geochemical evidence that these structures could be fossilized melanosomes, melanin-containing organelles. We geochemically analysed a tadpole from the Oligocene Enspel Formation, Germany, which displays such spherical bodies on its surface. Pyrolysis gas chromatography mass spectroscopy (Py-GCMS) and Fourier transform infrared spectrometry (FTIR) indicate that the organic remains of the tadpole are original and are not the result of external contamination, shown by the different chemical compositions of the fossil and its enclosing matrix. Py-GCMS also demonstrates the presence of bacterial and plant biomarkers in the matrix but not the tadpole, suggesting that the spherical bodies are unlikely to be bacterial, and also that such fossils do not develop their dark colour from incorporating plant material, as has been suggested. X-ray absorption spectroscopy (XAS) shows high levels of organically bound Zn(II) in the fossilized soft tissue, a metal known to chelate both eu- and pheomelanin. The zinc in the tadpole shows greater similarity to that bound in pheomelanized extant samples than to that in eumelanized ones. Though further geochemical analysis of both pure pheomelanin and bacterial samples is required to completely exclude a bacterial origin, these results are in line with a pheomelanic origin for the spherical bodies on the tadpole.

Elemental characterisation of melanin in feathers via synchrotron X-ray imaging and absorption spectroscopy

Melanin is a critical component of biological systems, but the exact chemistry of melanin is still imprecisely known. This is partly due to melanin’s complex heterogeneous nature and partly because many studies use synthetic analogues and/or pigments extracted from their natural biological setting, which may display important differences from endogenous pigments. Here we demonstrate how synchrotron X-ray analyses can non-destructively characterise the elements associated with melanin pigment in situ within extant feathers. Elemental imaging shows that the distributions of Ca, Cu and Zn are almost exclusively controlled by melanin pigment distribution. X-ray absorption spectroscopy demonstrates that the atomic coordination of zinc and sulfur is different within eumelanised regions compared to pheomelanised regions. This not only impacts our fundamental understanding of pigmentation in extant organisms but also provides a significant contribution to the evidence-based colour palette available for reconstructing the appearance of fossil organisms.

Identification of Shell Colour Pigments in Marine Snails Clanculus pharaonius and C. margaritarius (Trochoidea; Gastropoda)

Colour and pattern are key traits with important roles in camouflage, warning and attraction. Ideally, in order to begin to understand the evolution and ecology of colour in nature, it is important to identify and, where possible, fully characterise pigments using biochemical methods. The phylum Mollusca includes some of the most beautiful exemplars of biological pigmentation, with the vivid colours of sea shells particularly prized by collectors and scientists alike. Biochemical studies of molluscan shell colour were fairly common in the last century, but few of these studies have been confirmed using modern methods and very few shell pigments have been fully characterised. Here, we use modern chemical and multi-modal spectroscopic techniques to identify two porphyrin pigments and eumelanin in the shell of marine snails Clanculus pharaonius and C margaritarius. The same porphyrins were also identified in coloured foot tissue of both species. We use high performance liquid chromatography (HPLC) to show definitively that these porphyrins are uroporphyrin I and uroporphyrin III. Evidence from confocal microscopy analyses shows that the distribution of porphyrin pigments corresponds to the striking pink-red of C. pharaonius shells, as well as pink-red dots and lines on the early whorls of C. margaritarius and yellow-brown colour of later whorls. Additional HPLC results suggest that eumelanin is likely responsible for black spots. We refer to the two differently coloured porphyrin pigments as trochopuniceus (pink-red) and trochoxouthos (yellow-brown) in order to distinguish between them. Trochopuniceus and trochoxouthos were not found in the shell of a third species of the same superfamily, Calliostoma zizyphinum, despite its superficially similar colouration, suggesting that this species has different shell pigments. These findings have important implications for the study of colour and pattern in molluscs specifically, but in other taxa more generally, since this study shows that homology of visible colour cannot be assumed without identification of pigments.