Holly E. Barden

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.

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.

Evidence for sexual dimorphism in the stegosaurian dinosaur Kentrosaurus aethiopicus from the Upper Jurassic of Tanzania

ABSTRACT Sexual dimorphism, the condition whereby males and females differ from one another physically, is one of the most fundamental aspects of the biology of any animal. However, sexually dimorphic characters can be subtle and are mainly related to soft tissue anatomy. They are, therefore, difficult to identify reliably in the fossil record particularly when dealing with small sample sizes and osteology alone. The first geometric morphometric analysis of dimorphism in a thyreophoran (armored) dinosaur shows that the femora of the stegosaurian dinosaur Kentrosaurus aethiopicus (Upper Jurassic, Tanzania) bear a statistically significant shape difference of the proximal end, which is independent of size and is therefore proposed to be a sexual difference. Although the disarticulated nature of the material means that intraspecific variation in other skeletal elements, such as the enigmatic dermal armor, cannot be identified as sexual dimorphism at this time, this study provides a methodology for further work on articulated stegosaurian specimens and has the potential to reveal additional information regarding the paleobiology and population dynamics of this poorly understood clade.

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.

Identification of the earliest collagen- and plant-based coatings from Neolithic artefacts (Nahal Hemar cave, Israel)

Mortuary practices in human evolution record cognitive, social changes and technological innovations. The Neolithic Revolution in the Levant was a watershed in this domain that has long fascinated the archaeological community. Plaster modelled skulls are well known at Jericho and several other Neolithic sites, and in Nahal Hemar cave (Israel, ca. 8200 −7300 cal. BC) excavations yielded six unique human skulls covered with a black organic coating applied in a net pattern evoking a headdress. This small cave was used as storage for paraphernalia in the semi-arid area of the Judean desert and the dry conditions preserved other artefacts such as baskets coated with a similar dark substance. While previous analysis had revealed the presence of amino acids consistent with a collagen signature, in the present report, specific biomarkers were characterised using combined proteomic and lipid approaches. Basket samples yielded collagen and blood proteins of bovine origin (Bos genus) and a large sequence coverage of a plant protein charybdin (Charybdis genus). The skull residue samples were dominated by benzoate and cinnamate derivatives and triterpenes consistent with a styrax-type resin (Styrax officinalis), thus providing the earliest known evidence of an odoriferous plant resin used in combination with an animal product.

Geochemical Evidence of the Seasonality, Affinity and Pigmenation of Solenopora jurassica

Solenopora jurassica is a fossil calcareous alga that functioned as an important reef-building organism during the Palaeozoic. It is of significant palaeobiological interest due to its distinctive but poorly understood pink and white banding. Though widely accepted as an alga there is still debate over its taxonomic affinity, with recent work arguing that it should be reclassified as a chaetetid sponge. The banding is thought to be seasonal, but there is no conclusive evidence for this. Other recent work has, however demonstrated the presence of a unique organic boron-containing pink/red pigment in the pink bands of S. jurassica. We present new geochemical evidence concerning the seasonality and pigmentation of S. jurassica. Seasonal growth cycles are demonstrated by X-ray radiography, which shows differences in calcite density, and by varying δ13C composition of the bands. Temperature variation in the bands is difficult to constrain accurately due to conflicting patterns arising from Mg/Ca molar ratios and δ18O data. Fluctuating chlorine levels indicate increased salinity in the white bands, when combined with the isotope data this suggests more suggestive of marine conditions during formation of the white band and a greater freshwater component (lower chlorinity) during pink band precipitation (δ18O). Increased photosynthesis is inferred within the pink bands in comparison to the white, based on δ13C. Pyrolysis Gas Chromatography Mass Spectrometry (Py-GCMS) and Fourier Transform Infrared Spectroscopy (FTIR) show the presence of tetramethyl pyrrole, protein moieties and carboxylic acid groups, suggestive of the presence of the red algal pigment phycoerythrin. This is consistent with the pink colour of S. jurassica. As phycoerythrin is only known to occur in algae and cyanobacteria, and no biomarker evidence of bacteria or sponges was detected we conclude S. jurassica is most likely an alga. Pigment analysis may be a reliable classification method for fossil algae.