Identification of the polysaccharide-containing remains

The fossilized material consists of brownish anastomosing fibers (diameter ~100 μm) in an arrangement typical of Vauxia (Fig. 1). The dimension of the fibers is characteristic of poriferans. There are no reports of fungi or filamentous bacteria containing any fibers of similar diameter (see for comparison4). In our preparation and analytical protocol, which is described in the following, we tried to avoid any possible problems that may arise from modern contaminants. A 14C analysis only yielded a low fraction of modern 14C of 0.0057, where “modern” is defined as 95% of the radiocarbon concentration (in AD 1950) of NBS Oxalic Acid I (SRM 4990B) normalized to δ13C VPDB = −19 per mil. This confirms that the analysed organic material must contain ancient carbon (see Supplementary Information, text). Raman spectroscopy of the organic material suggests that all investigated samples had the same origin with respect to thermal low-grade metamorphism (Supplementary Information, Fig. S2). The results from the DNA identification study (Supplementary Information, Fig. S21) revealed the absence of DNA in the material isolated from fossilized V. gracilenta. This is a good indicator that there are no modern bacteria or fungi contaminates in the fossils, or in the instrumentation used to investigate them.

Figure 1 Optical micrographs from the studied sample. Burgess Shale sample with the fossil demosponge V. gracilenta (a) with detail of the exceptionally preserved skeleton (b). Full size image

Initially, three samples (Supplementary Information, Fig. S1) were examined under a fluorescence microscope to highlight any polysaccharide-based organic matter using the specific Calcofluor White (CFW) staining. Calcofluor White is a fluorescent marker capable of making hydrogen bonds with β-(1.4)- and β-(1.3)-linked polysaccharides and shows a high affinity for chitin7,8,9. Recently, it was confirmed experimentally that CFW specifically binds to carbohydrate residues and not to the protein matrix, even when staining glycoproteins21. This method has previously been used successfully at a microscopic scale to identify chitin-containing organisms attached to the surfaces, or embedded within different kind of materials22. If a chitin-containing material is present, the chitin will bind the dye and, upon exposure to light, the chitin-containing material may be visualized and can then be removed for further investigations. We used material from the third fossil sample (Supplementary Information, Fig. S1), which is preserved in remarkable morphological detail (Fig. 1). We selected areas on the surface of sample (Fig. 1b) where the fibrous morphology of the Vauxia skeleton is clearly visible in a binocular microscope and then stained them with CFW. The skeleton was stained with variable intensity, which is clearly visible using fluorescence microscopy (Fig. 2 a, b). We found several fragments (Supplementary Information, Fig. S4a) that were highly stained by CFW and precisely resembled the fibres observed by electron microscopy in size and shape. These preliminary results indicate the presence of polysaccharide material localized within well-preserved fossilized fibers of the sponge skeleton.

Figure 2 Preliminary identification of chitin. Calcofluor white staining of the cleaned surface of V. gracilenta with fluorescence, indicating the presence of polysaccharide-based compounds (a, b). Isolated and HF-demineralized fibers were identified as chitin using NEXAFS spectroscopy (c). Full size image

Selected fibers showing the presence of polysaccharides were carefully broken from the host rock using a very sharp steel needle under a stereomicroscope. Some of the fragments obtained in this way and showing a well-preserved fibrous structure (Fig. 3) were investigated as removed using light, fluorescence and scanning electron microscopy (SEM). The majority of the fragments were transferred to plastic vessels with 48% HF for 24 h at room temperature to remove aluminosilicates (Supplementary Information, Fig. S4b). Following this, the samples were centrifuged and the insoluble residue was washed five times using deionized water.

Figure 3 Structural features of the fossil sponge skeleton. A selectively isolated fragment (b) of the aspiculate fibrous skeleton of V. gracilenta (a) shows autofluorescence (c) that is characteristic for chitin. The non-homogenous fluorescence can be explained by the presence of a residual mineral phase observed using SEM (d, e). Full size image

The residual material was placed onto glass slides that had been cleaned in acetone. Micro-fibers or micro-particles were excluded from the slide using light and fluorescence microscopy. The 25 slides with residual material were observed using light and fluorescence microscopy and we isolated fragments possessing fibrillar microstructure. All of these show strong autofluorescence in the region of 470–510 nm, consistent with that of chitin (Supplementary Information, Fig. S5).

Identification of chitin

Our criteria for the positive identification of chitin are based on comparative investigations between a chitin standard and selected samples using the highly sensitive analytical techniques shown below; as well as the detection of D-glucosamine and the use of the chitinase test. Thus, selected samples of isolated material were analysed by near-edge X-ray absorption fine structure (NEXAFS) spectroscopy, Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM). Chitin, which may be considered a polymer of 2-acetamido-2-deoxy-CX-D-glucopyranose (N-acetyl-D-glucosamine), yields D-glucosamine on acid hydrolysis. Detection of D-glucosamine is required as the final step in supporting the survival of chitin in the fossil record23. Other samples were therefore hydrolysed to test for the presence of D-glucosamine (DGlcN) using High Performance Liquid Chromatography (HPLC), High Performance Size Exclusion Chromatography (HPSEC), High Performance Capillary Electrophoresis (HPCE) and Electrospray Ionization Mass Spectrometry (ESI-MS). One fragment was also used for experimental chitinase digestion. In all these experiments the samples from the surrounding rock of the analysed fibers were tested as a negative control. Thus, those modern contaminations capable of surviving the purification techniques we used would be present in both the analysed fibers and the surrounding rock.

NEXAFS spectroscopy was used to explore site-specific electronic properties of isolated fibres (measured on 500 μm × 500 μm areas). The carbon K-edge spectrum of investigated material showed all the typical absorption features of chitin (~288.5 eV)24 (Fig. 2c). We also show that the characteristic C = O absorption peak of chitin is clearly distinguishable from a strong cellulose peak reported at 289.5 eV24.

The results of the structural and spectroscopic analyses performed using NEXAFS (Fig. 2c), FTIR and CFW staining (Supplementary Information, Fig. S6) and electron diffraction (Supplementary Information, Fig. S7) agreed that the demineralized fibrous material isolated from fossilized V. gracilenta consists in part of α-chitin. The results of our analyses for the investigated fractions of the fibers were fully consistent with those of previous reports on the physicochemical identification of chitin in other organisms7,8,9.

Chitinase digestion experiments (Supplementary Information, Fig. S8) again confirmed the chitinous nature of the isolated V. gracilenta fibers. Additionally, results obtained using HPLC, HPSEC, HPCE and ESI-MS (Fig. 4) clearly indicate that the sample contains a species that is highly similar in its properties to DGlcN. The presence of D-glucosamine in the exceptionally preserved fibrous matter which is isolated from the rock, but absent in the surrounding rock (Supplementary Information, Fig. S22 and S23), clearly shows its fossil origin.