Diamond Annual Review 2019/20

76 77 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 1 9 / 2 0 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 1 9 / 2 0 Spectroscopy Group Beamline I18 Adding colour to the past: investigating the pigmentation of a three-million- year-oldmouse Related publication: Manning P. L., Edwards N. P., Bergmann U., Anné J., SellersW. I., vanVeelen A., Sokaras D., EgertonV. M., Alonso-Mori R., Ignatyev K., van Dongen B. E.,Wakamatsu K., Ito S., Knoll F. &Wogelius R. A. Pheomelanin pigment remnants mapped in fossils of an extinct mammal. Nat. Commun. 10 , 2250 (2019). DOI: 10.1038/s41467-019-10087-2 Publication keywords: Pheomelanin; Pigment; Mouse; Synchrotron; Fossil C olour has played a vital role in evolution for hundreds of millions of years. Until very recently, techniques used to study fossils could not explorethepigmentationofancientanimals.ResearchersusingtheMicrofocusSpectroscopybeamline(I18) haveachievedabreakthrough in the ability to resolve fossilised colour pigments. They mapped key elements associated with the pigment melanin in an extinct, three million-year-old species ofmouse. Different forms ofmelanin can give rise to a black or dark brown colour, or a reddish or yellow colour. Bybathing fossils in intenseX-rays, the research teamwereable todiscern the tracemetals in thepigments and to reconstruct the reddish colour in themouse’s fur. They were able to determine that the fossilisedmouse fur incorporated tracemetals in the same way that tracemetals bond to pigments in animals with red fur. Their work provides a new chemical method for resolving melanin pigments in both recent and extinct tissue (e.g. hair, skin, feather) samples. Alongtheway,theylearnedmuchmoreaboutthechemistryofpigmentationthroughouttheanimalkingdom.Theteamhopesthattheirresults will allowus to reconstruct extinct animals withmore confidence, adding another dimension to the study of the evolution of life on Earth. Colour plays a vital role in the selective processes that have steered the evolution of life for hundreds of millions of years. The many techniques used to study fossils, until recently, were not capable of exploring the pigmentation of ancient animals, a character pivotal to reconstructing what an organism might have looked like. A breakthrough in synchrotron-based imaging techniques now gives insight into the chemistry in long-extinct species by mapping key elements associated with soft tissue structures (Fig. 1), but also with the pigment melanin, the dominant group of compounds that colour life (both in vertebrates and invertebrates). Eumelanin pigment gives a black or dark brown colour, but in the formofpheomelanin,itproducesareddishoryellowcolour.Untilrecently,research has focused on the traces of elements known to be associated with eumelanin 1 . Previous experiments revealed dark and light pigment patterns in feathers of the Jurassic bird Archaeopteryx 2 and the Cretaceous Confuciusornis sanctus (Fig. 1). However, even though melanin pigments are a critical component of biological systems,theexactchemistryofmelaninisstillimpreciselyknown.Thecombination ofnewsynchrotron-based imagingtechniqueswithuniquefossilshasenabledthe chemistry of this illusivemolecule to be further refined. A suite of new imaging techniques, including elemental mapping techniques used at synchrotron light sources, provides one with the ability to peer deep into the chemical history of a fossil organism and also unpick the processes that preserved itstissues(Fig.1) 1,2 .Whereoncewesimplysawminerals,nowweaimto resurrect‘biochemicalghosts’oflongextinctspecies,mappingthewholeorganism and embeddingmatrix but without causing any damage to often fragile fossils. A key goal from an earlier study undertaken by the team was to differentiate between eumelanin and pheomelanin pigment chemistry in modern bird feathers 3 . The feathers analysed in this earlier study possessed a distinct chemical signature for zinc and sulfur, with a significant portion of the zinc inventory bonded to sulfur, almost certainly through the sulfur contained within the pheomelanin molecule (not the sulfur bound within the surrounding keratinous feather matrix) 3 . This was a tipping point for the understanding of pigments and being able to identify chemical signatures that could help resolve pigmentation in ancient animals, especially those that exhibit exceptionally preserved ‘soft’ tissue (feathers, hair and even skin). A strong foundation had to be built using the modern animal tissue (feathers) before the technique could be applied to more ancient samples 4 . Through using synchrotron-based imaging techniques, this work provided a chemical‘fingerprint’for our most recent study of a three-million- year-old mouse, Apodemus atuvas (Fig. 2) 4 . Two well-preserved fossil specimens of Apodemus atavus from Willershausen (Germany) were selected, the holotype GZG. W.20027b (Fig. 2) and a second specimen GZG. W.17393a. This Pliocene species of field mouse is closely related to the extant (living) related species Apodemus sylvaticus and Apodemus flavicollis . Extant members of this genus are reddish coloured and thus the close relationship of these extant species would imply that the related extinct species would also have had significant amounts of pheomelanin pigment in their fur. The spatial mapping of the mouse fossil (Fig. 2), at the Stanford Synchrotron Radiation Lightsource (USA), provided a beautiful chemical image of the fossil using Synchrotron Rapid Scanning X-ray Fluorescence (SRS-XRF). However, the chemistry of the melanin pigment required careful X-ray Absorption Spectroscopy (XAS)tobeundertakenatDiamondLightSourcetoconstraintheprecisegeometry and electronic structure of any compounds present.The study used a large number of chemical standards (Fig. 3) to provide comparative XAS spectra. The SRS-XRF showed that the distributions of zinc and organic sulfur correlatedwithin the fossil Apodemus fur,justasinpheomelanin-richmodernintegument(feathers)observed bytheearlierstudy 3 .Furthermore,thezinccoordinationchemistrywithinthisfossil furwascloselycomparabletothatdeterminedfrompheomelanin-richfurandhair standards that were also analysed using X-ray Absorption Near Edge Structure (XANES) spectroscopy at Diamond (Fig. 3b). The XANES showed that the zinc was directly bonded to the associated organic sulfur compounds, which is diagnostic of pheomelanin. However, given that the fossil soft tissue displayed enrichment in organic sulfur compounds, and that both the structural protein (keratin within fur) and red pigment (pheomelanin) in extant mouse fur contain sulfur species, it was critical that detailed sulfur XANES studies were undertaken in order to accurately characterise the oxidation state and origin of the sulfur inventory. Fig. 3a presents the results from fossil and extant material along with several sulfur standard compounds used to resolve each species. All spectra are quite different fromeachother,reflectingthestrong impactthatchangingtheoxidationstateand coordination environment has on XANES spectra for sulfur 4 . Melanins (e.g. pheomelanin and eumelanin) are complex molecules and their chemistry is hard to resolve, even in modern samples. However, the detailed spectroscopy undertaken at synchrotron light sources has shed new light on the structure of these molecules and identified the key elements, such as zinc, copper and sulfur, that are at the very heart of the pigment 1,2,3 . Probing the preserved chemical environment with X-rays allowed the team to reconstruct a reddish colour in the 3-million-year-old pelt. The study on the three-million-year-old mouse concludes that the zinc-organosulfur compounds present within the fossil were the residue of pheomelanin and indicated that Apodemus atavus was pigmented in a similar way to its extant related species 4 . The results also helped explain why the detection of pheomelanin pigment residue is challenging, as the high original quantities of sulfur ubiquitous with keratinous integument (e.g. feather, hair, skin) produce degradation products rich in oxidised sulfur, which obscure the organic sulfur residue derived frompheomelanin pigment. In order to resolve the pheomelanin signal embeddedwithin the complex mixture of organic degradation products, the bonding environment for organically complexed zinc was the most sensitive and stable chemical indicator.The study clearly shows that the resolution of pheomelanin pigment residue is possible, using a combination of chemical imaging and X-ray spectroscopy at synchrotron light sources, at least for specimens with an age equal to or less than threemillion years. References: 1. Wogelius R. A. et al. Tracemetals as biomarkers for eumelanin pigment in the fossil record. Science 333 , 1622 (2011). DOI: 10.1126/science.1205748 2. Manning P. L. et al. Synchrotron-based chemical imaging reveals plumage patterns in a 150million year old bird. J. Anal. At. Spectrom. 28 , 1024 (2013). DOI: 10.1039/C3JA50077B 3. Edwards N. P. et al. Elemental characterisation of melanin in feathers via synchrotron X-ray imaging and absorption spectroscopy. Sci. Rep. 6 , 34002 (2016). DOI: 10.1038/srep34002 4. Manning P. L. et al. Pheomelanin pigment remnants mapped in 3million year oldmammal fossils. Nat. Commun. 10 , 2250 (2019). DOI: 10.1038/s41467-019-10087-2 Funding acknowledgement: The University of Gottingen graciously loaned us the fossil A. atuvus specimens. Fundingwas provided by a UK Natural Environment Research Council grant NE/ J023426/1. Portions of this researchwere also carried out at Diamond Light Source, beamline I18 (UK, allocations SP12948, SP11865, SP9488, SP8597, and SP7749). Portions of this researchwere carried out at the Stanford Synchrotron Radiation Lightsource (CA, USA), a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences.We thank support staff at DLS and SSRL. PLM thanks the Science and Technology Facilities Council for their support (ST/M001814/1).We are grateful for access to the DRIAMAnalytical Service, Dalton Research Institute, Manchester Metropolitan University. Corresponding author: Prof PhillipManning, University of Manchester, phil.manning@manchester.ac.uk Figure 2: Optical and X-ray images of Apodemus atavus. (a) Optical image of A. atavus (GZG.W.20027b). (b) False-colour SRS-XRF image reveals exceptional preservation of integument as well as bone. This image is a combination of three maps, two standard single-element maps (blue= phosphorus, green = zinc), plus a third map that has been produced to especially emphasise the distribution of a specific oxidation state of organic sulfur (red= organic S (thiol)) in order to highlight the clear correlation between the distribution of zinc and organic sulfur which together appear as bright yellow (reproduced fromManning et al., 2019). Figure 3: (a) Sulfur K-edge XANES standard, extant/fossil specimens. A benzothiazole, key component of pheomelanin, B Zn-cysteine (terminal S organic functional group), C oxidised glutathione (disulfide) comparator for sulfur in keratin, D methionine sulfoxide, E Zn sulfate. Two extant mice spectra with linear combination fits (dashed-lines) computed as a binary benzothiazole/disulfide system. Red circles highlight resonance in the benzothiazole standard resolvable in red fur. Dashed vertical line indicates benzothiazole peak discernible in red fur, not albino fur. Normalised spectra from fossils presented with Linear Combination Fitting (LCF) fits calculated using standards. Fits shown as dashed lines for soft tissues analysed. (b) Fossil/extant mouse zinc K-edge XANES, human hair, Zn-bonded eumelanin, Zn-acetate heptahydrate, and ZnS. Vertical lines indicate absorption spectrummaxima (pure first shell Zn-S and pure first shell Zn-O species). Figure 1: Optical (a) and Synchrotron Rapid Scanning X-ray Fluorescence (SRS-XRF) map (b) of Confuciusornis sanctus (MGSF315). SRS-XRF false-colour image of MGSF315 main slab (red, Cu; blue, Ca; green, Zn). Additional spectroscopy indicated that the copper was residue of eumelanin pigment preserved within the feathers of C. sanctus, (reproduced fromWogelius et al., 2011). A B