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    Supporting Online Material for

    Trace Metals as Biomarkers for Eumelanin Pigment in the Fossil Record

    R. A. Wogelius,* P. L. Manning, H. E. Barden, N. P. Edwards, S. M. Webb, W. I. Sellers, K. G. Taylor, P. L. Larson, P. Dodson, H. You, L. Da-qing, U. Bergmann

    *To whom correspondence should be addressed. E-mail:

    Published 30 June 2011 on Science Express DOI: 10.1126/science.1205748

    This PDF file includes:

    Materials and Methods SOM Text Figs. S1 to S15 Tables S1 to S6 References and Notes

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    Materials and Methods Materials 1. Confuciusornis sanctus (MGSF315) is on loan to the University of Manchester from the Chinese Institute for Vertebrate Paleontology and Paleoanthropology (IVPP). See Figure 1 for photograph. (MGSF accession prefixes refer to samples held in the collection of the University of Manchester School of Earth, Atmospheric, and Environmental Sciences). 2. The second Confuciusornis sanctus (LL12418) is on loan to the Manchester Museum from the IVPP; photograph below (scale bar 10 cm total, markings 1 cm each). (LL and BB accession prefixes refer to the Manchester Museum, a public museum affiliated with the University.) 3. BHI-6358 is a small exceptionally preserved feather (unidentified) approximately 2 cm in length (for photograph see Figure 3A). Accession numbers with a BHI prefix refer to the Black Hills Institute Museum, part of the Black Hills Institute (Hill City, South Dakota). 4. BHI-6319 is a single slab from the Green River Formation (26) consists of a small slab with a preserved fish (Gosiutichthys parvus, ~2.5 cm in length) and feather (unidentified, ~2cm in length). For photograph see Figure 3B. 5. Gansus yumenensis (MGSF317) is a single feather collected by one of the authors (H. You) from the Lower Cretaceous (~115 to 105 Mya) Xiagou Formation near Changma, Gansu Province northwestern China (3). Phylogenetic analysis places Gansus within the Ornithurae, making it the oldest known member of the clade (3). The anatomy of Gansus, like that of other

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    non-neornithean (nonmodern) ornithuran birds, indicates specialization for an amphibious life- style (3). 6. A single black feather identified tentatively as from Haliaeetus leucocephalus (Figure 3D) was generously provided from a captive bird at a bird sanctuary. 7. Blue Jay feather (Figure 3E; Cyanocitta cristata) was provided by the Manchester Museum (specimen BB.9013.1). 8. Fossil squid Rachiteuthis tent. (BHI-2243B) is from Hakel, Lebanon (Late Cretaceous, ~90 Mya). See Figure 3F for photograph. 9. Extant squid (Figure 3G; Sepia officinalis) was purchased from a fisherman’s market. 10. Green River Feathered Wing (unidentified; HMNS 2010.185.02) is curated by the Houston Museum of Natural History who graciously allowed us access. See photograph below for details (note scale bar at upper right).

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    11. Green River Feather BHI-6403 (unidentified) is pictured below. Note scale bar at bottom left. 12. Archaeopteryx lithographica holotype (MB.Av.100) pictured below was graciously loaned by the Museum für Naturkunde, Humboldt University, Berlin. Feather is approximately 7 cm long and 1.5 cm wide.

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    13. Natural melanin (Sepia officinalis) was obtained commercially from Sigma-Aldrich. FTIR was completed on an as received portion. In order to produce Cu-melanin complexes for EXAFS and XANES standards, a part of this natural melanin was prepared and reacted with a copper solution as summarized below. This protocol is based on previously reported methods (21, 23). I. Remove the metals already bound to the melanin:

    1) Mix 50 g melanin with EDTA (10ml) 2) Centrifuge mixture at 2200 rpm and retain the supernatant 3) Add nanopure (DI) water (10ml) to the pellet and mix 4) Centrifuge and remove the water 5) Repeat this wash 3 more times with nanopure water

    II. Saturate the melanin with copper: 6) Add 50 ml of Cu2SO4.5H2O 0.1M and leave overnight 7) Centrifuge mixture and keep the supernatant 8) Add approximately 10ml of HCl pH 4, stir, centrifuge and remove the supernatant 9) Repeat the HCl wash once more 10) Repeat the procedure twice more with nanopure (DI) water 11) Freeze dry

    Methods 1. SRS-XRF Imaging was performed at wiggler beam lines 6-2 at SSRL. The beam line was operated in its standard configuration with a collimating mirror upstream of a Si (111) monochromator, and a pair of total reflection focusing mirrors downstream. For light element XRF images (Cl, S, P, and K) the excitation energy was chosen at 3.15 keV, for the heavier element images (Cu, Ca, Zn, Fe, Mn, Se, Ba, and Pb) an excitation energy of 12.0 or 13.5 keV was chosen. Flux at the sample surface varied between approximately 1010 and 1011 photons s-1 depending on the specific analytical conditions. At low incident energy light elements such as phosphorous and sulfur were easily resolved. Increasing the incident energy of the beam allowed analysis of heavier elements, especially the transition metals. The beam was focused onto a 100 µm thick tantalum pinhole of 50, 80, or 100 µm diameter and placed at a distance varying for the high energy measurements between approximately 1 and 8 mm from the fossil surfaces. Due to the very long focal depth of the setup, the beam striking the sample was only insignificantly larger than the pinhole diameter. Samples were mounted at either 45o (high incident energies) or 67o (low incident energy) relative to the incident beam. A photon counting single element silicon drift detector (Vortex, SII NanoTechnology USA Inc.) combined with Gaussian shaping amplifiers (Canberra) employing 0.125 µsec shaping times plus single channel analyzers was used to detect the XRF signals. For each element, the electronic windows were set to capture the fluorescent photons from the Kα or Lα emission lines. The width of the electronic windows corresponded to typically 200 – 350 eV per fluorescence line.

    For scans at high incident beam energy the detector was placed at a 90o angle to the beam in order to minimize the unwanted scattering signal. For scans at 3.15 keV where the scattering was much smaller the detector was placed normal to the sample

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    surface. All fossil and extant samples were held within a purpose built sample chamber which was carefully mounted on a computerized x-y translational stage and rapid scans were performed by continuously translating the sample horizontally across the beam. At the end of each horizontal line a vertical step (50, 80, or 100 µm/step, depending on pinhole) was performed, and the horizontal scan direction was reversed. Data were recorded on the fly in both horizontal directions at a rate corresponding to a travel distance of 50, 80, or 100 µm (to match pinhole size) per ~3 ms readout. The beam intensity I0 was monitored with a He filled ion chamber upstream of the pinhole. During scanning, small drifts of the beam with respect to the pinhole were corrected periodically. For the light element XRF imaging an X-ray transparent window was placed on the sample chamber and the chamber was purged of air with helium. Window material was an ~30 µm thick polyethylene film that touched the detector and was located ~2 cm from the pinhole. Sample surface to detector distance was varied from 200 to 14.5 mm, depending on the major element fluorescence intensity. At high energy, in order to reduce the very large Ca signal we furthermore placed a 50.8 µm thick Al foil in front of the detector. Both the air absorption and the Al foil reduced the Ca Kα fluorescence by a factor of 500 whereas the 3d transition metal signals were only reduced by between 20 and 80%.

    Supplementary scans of BHI-6358 and BHI-6319 were completed at 11.5 keV incident beam energy on beam line 10-2a using a 100 µm pinhole and similar scanning geometry. In this case the detector was placed at 10 cm distance from the point of analysis and the fluorescence signal was recorded “on the fly” at 5 ms time intervals corresponding to a travel distance of 100 µm to match the pinhole spot size. Detector output was collected using digital xMAP x-ray spectrometers from XIA in buffered mapping mode. In order to synchronize the readout with the continuous stage motion, the signal was gated electronically using TTL output from the stage motion to advance each pixel. SRS-XRF images presented in the main text and below represent raw data that have not been processed except for maximum intensity being clipped at between 90 and 99% of the maximum value in order to best show contrast relative to the background sedimentary matrix. For actual elemental concentration values, see point analysis information below. 2. Micro-XRF images were collected at the Stanford Synchrotron Radiation Lightsource (SSRL) using beam line 2–3. The incident x-ray energy was set to 10 keV using a Si (111) double crystal monochromator. The fluorescence lines of the elements of interest, as well as the intensity of the total scattered X-rays, were also monitored using a silicon drift Vortex detector. In addition to these regions of interest, the entire fluorescence spectrum was also collected at each data point. The microfocused beam of 2 x 2 µm was provided by a Rh-coated Kirkpatrick-Baez mirror pair (Xradia Inc.) The incident and transmitted x-ray intensities were measured with nitrogen-filled ion chambers. Samples we