Widespread preservation of fossilized biomolecules in many fossil animals has recently been reported in six studies, based on Raman microspectroscopy. Here, we show that the putative Raman signatures of organic compounds in these fossils are actually instrumental artefacts resulting from intense background luminescence. Raman spectroscopy is based on the detection of photons scattered inelastically by matter upon its interaction with a laser beam. For many natural materials, this interaction also generates a luminescence signal that is often orders of magnitude more intense than the light produced by Raman scattering. Such luminescence, coupled with the transmission properties of the spectrometer, induced quasi-periodic ripples in the measured spectra that have been incorrectly interpreted as Raman signatures of organic molecules. Although several analytical strategies have been developed to overcome this common issue, Raman microspectroscopy as used in the studies questioned here cannot be used to identify fossil biomolecules.

Origin of the spectra used in this study

In Figure 1 of our manuscript, we reused already published baseline-subtracted spectra provided along the original publications by Wiemann et al. (2018a) and McCoy et al. (2020). Specifically, we used the spectrum collected from the eggshell of the extant flightless bird Rhea americana (Wiemann et al., 2018a), and the spectrum collected from the fossil crustacean Acanthotelson stimpsoni specimen YPM52348 from the Carboniferous (ca. 307 Ma) Mazon Creek Lagerstätte, USA (McCoy et al., 2020).

The transmission spectrum of the 532 nm RazorEdge^® ultrasteep long-pass edge filter (Semrock) analyzed in Figure 2 is available online on the manufacturer’s website. URL: https://www.semrock.com/FilterDetails.aspx?id=LP03-532RE-25.

Raman microspectroscoy data shown in Figures 3 and 4 are original data collected at the University of Lausanne (see next paragraph for details on the method and data collection and processing). Fossil specimens shown in Figure 3a and 3b come from the Carboniferous (ca. 307 Ma) Mazon Creek Lagerstätte, USA, and the Upper Cretaceous (ca. 95 Ma) Jbel oum Tkout Lagerstätte from southeastern Morocco, and are housed at the Musée cantonal de géologie (MCG; Lausanne, Switzerland), and the Muséum national d’Histoire naturelle (MNHN; Paris, France), respectively. Note, however, that the latter material belongs to the Musée d’Histoire naturelle de Marrakech (MHNM; Marrakech, Morocco) and is currently only housed at the MNHN for study, within an agreement between both museums. The dried specimen of the modern shrimp Neocaridina davidii studied in Figure 3c come from the aquarium lab of the University of Lausanne. An adult N. davidii specimen was used in this study. Live specimens were purchased commercially through aquarium suppliers in Nyon, Switzerland. The shrimp were cultured in freshwater aquaria at the University of Lausanne, Switzerland. The specimen used was euthanized via anaesthesia by overdose of clove oil solution for 10 minutes, and then was washed and rinsed in de-ionised water continuously until all oily residue was removed, and finally left to dry.

Raman microspectrometry

Raman data in Figures 3 and 4 were collected in analytical conditions similar to those described by Wiemann et al. (2020), using a Horiba Jobin Yvon LabRAM 800 HR spectrometer (UNIL, Lausanne, Switzerland) in a confocal configuration, equipped with an Ar⁺ laser (532 nm) excitation source and an electron multiplying charge-coupled device (EMCCD). Measurements were performed at constant room temperature, directly on the sample surface, by focusing the laser beam with a 300 µm confocal hole using a long working distance ×50 objective (NA = 0.70). This configuration provided a ≈2 µm spot size for a laser power delivered at the sample surface below 1 mW. Light was dispersed using a 1800 gr/mm diffraction grating.

Wavelet analyses

Continuous wavelet transform and wavelet multiresolution analyses were performed in R using the dplR and waveslim packages, respectively. In this study, the Morlet wavelet (a Gaussian-modulated sinewave) was chosen for the continuous wavelet transform, and we used a 95% level for the significance test. The hatched area delineating the edges of the spectrum (so-called "cone of influence") marks parts of the spectrum where energy bands are likely to appear less powerful than they actually are because of the increasing importance of edge effects.

Baseline subtraction

Baseline subtraction (Figure 3d, dotted line) was performed using the SpectraGryph 1.2 spectroscopic software (adaptive baseline, 15%, no offset, minimally smoothed through rectangular averaging over an interval of 4 points), following protocols in (Wiemann et al., 2018a, 2018b, 2020; Fabbri et al., 2020; McCoy et al., 2020; Norell et al., 2020). Depending on the composition of the sample, the shape and quality of the baseline fit, and therefore the subtracted spectrum (Figure 3e) varies when using the same snuggling curved baseline percentage for all spectra instead of adapting the parameter to each spectrum.

All spectra used in this work have been gathered in text file "Alleon_etal_data.txt" or, alternatively, Excel file "Alleon_etal_data.xlsx". The R script "R_script_Alleon_etal.r" allows one to process the spectra and generate the plots used in our figures.

References mentionned above:

Fabbri, M., Wiemann, J., Manucci, F., Briggs, D.E.G. (2020) Three‐dimensional soft tissue preservation revealed in the skin of a non‐avian dinosaur. Palaeontology 63, 185-193.

McCoy, V.E., Wiemann, J., Lamsdell, J.C., Whalen, C.D., Lidgard, S., Mayer, P., Petermann, H., Briggs, D.E.G. (2020) Chemical signatures of soft tissues distinguish between vertebrates and invertebrates from the Carboniferous Mazon Creek Lagerstätte of Illinois. Geobiology.

Norell, M.A., Wiemann, J., Fabbri, M., Yu, C., Marsicano, C.A., Moore-Nall, A., Varricchio, D.J., Pol, D., Zelenitsky, D.K. (2020) The first dinosaur egg was soft. Nature 583, 406–410.

Wiemann, J., Yang, T.-R., Norell, M.A. (2018a) Dinosaur egg colour had a single evolutionary origin. Nature 563, 555-558.

Wiemann, J., Fabbri, M., Yang, T.-R., Stein, K., Sander, P.M., Norell, M.A., Briggs, D.E.G. (2018b) Fossilization transforms vertebrate hard tissue proteins into N-heterocyclic polymers. Nature Communications 9, 1-9.

Wiemann, J., Crawford, J.M., Briggs, D.E.G. (2020) Phylogenetic and physiological signals in metazoan fossil biomolecules. Science Advances 6, eaba6883.

Follow the R script "R_script_Alleon_etal.r" to process the spectra and generate the plots used in our figures. This script opens data from the text file "Alleon_etal_data.txt". It includes at the beginning all needed libraires/packages needed to process the spectra, which need to be installed prior to running the script. Comments throughout the script should make its use straightforward.