Research ArticlePALEONTOLOGY

Molecular identification of fungi microfossils in a Neoproterozoic shale rock

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Science Advances  22 Jan 2020:
Vol. 6, no. 4, eaax7599
DOI: 10.1126/sciadv.aax7599
  • Fig. 1 Textures and structures observed via light or scanning electron microscopy of a thin section from the fossiliferous dolomitic shale rock from BIIc8 (MMS).

    (A) Composite image of light microscopy (LM) views of dark, nontranslucide, interconnected filaments forming part of a large mycelium-like structure covering ~0.2 mm2 detailed in fig. S1 (as well as other mycelium-like structures). Note that the area shown in (B) was exposed to WGA-FITC in Fig. 2. (C) Scanning electron microscopy micrograph illustrating the presence of cells (~20 to 25 μm in length) regularly spaced by putative septa or pseudosepta (white arrows) and showing putative anastomoses (red arrow).(D) LM image of a mycelium network of dark-colored filaments with Y- and T-branching (red arrows); black arrow marks the position of an FIB foil #4984 cut across a filament in the thin section. Inset shows a portion of mycelial network composed of dark, nontranslucide filaments branching at a right angle. (E) Histogram showing the width frequency distribution of filaments, with the solid line depicting the median at 5.5 μm (the dashed line as first and third quartiles at 4.5 and 6.7 μm). (F) Encrusted aspect of these fungal networks and the absence of tunneling patterns suggesting the syngenicity of these remains.

  • Fig. 2 Confocal laser scanning fluorescence microscopy using WGA-FITC of mycelium-like structure illustrated in Fig. 1.

    Overview image in (A) shows the absence of large natural autofluorescence (i.e., without WGA-FITC) of the thin section. In contrast, with WGA-FITC labeling, the same area exhibits filamentous, mycelium-like structure visible in (B) (top section). (C to F) High-resolution confocal fluorescence views of the mycelium-like structures. The WGA-FITC binds specifically on the cell wall of the fossil filaments, which appears cylindrical (as evidenced by a rounded cross section of filaments). Several septa (s) are also stained. Note in (C) that the arrows highlight septa present in putative anastomosing filaments (a) illustrated in Fig. 1A (arrows; images are mirrored as confocal microscope is inverted). Insets in (F) illustrate septa (ps) with perforation or “bulged,” which resembles pseudosepta (39). Scale bars, 10 μm (C to F).

  • Fig. 3 Confocal raman spectroscopy of the filamentous network illustrated in Fig. 1A-B.

    (A) Reflected LM of a filamentous network in Fig. 1A. (B) G band intensity map of the filament shown in (A). a.u., arbitrary units. (C) Representative spectra showing the D and G bands analyzed (marked “D” at ~1350 cm−1 and “G” at ~1600 cm−1 on the spectra) from the areas marked by crosses in (B). The coloration of the spectra corresponds to the color of the crosses seen in (B). (D) G band peak center map from the same area (i.e., B) that shows significant variations in the G band peak center across the filamentous network. (E) Representative Raman spectrum illustrating the deconvolution in D1, D3, D4, D5, and G bands used to calculate diagenetic peak temperature and the determination of the parameter I(1350)/I(1600). The thermal maturation of the fossilized filament was derived according to the FWHM-D1 geothermometer relationship in (26).

  • Fig. 4 Synchrotron-μFTIR (average over 31 10 by 10–μm areas) of fragments of fossilized filaments illustrated in Fig. 1E.

    Assignments of absorption bands and vibration modes (δ = deformation; ν = stretching; s = symmetric; as = asymmetric) are indicated in parentheses (see table S1 for full details). Note the strong contributions of amide functional groups (at 1650, 1540, and 3286 cm−1) and aliphatic CH2 and CH3 moieties in the 2800 to 3000 cm−1 region, which is also evidenced by the presence of 727 cm−1 peak indicating long (CH2)n chains with n ≥ 4. (Inset) Detail of the 2800 to 3000 cm−1 spectral region used to calculate the branching index (i.e., R3/2—the relative ratio of CH3 to CH2 carbon molecules) based on the intensity of their respective asymmetric stretching.

  • Fig. 5 Combined FTIR and Raman biosignatures of fungi microfossils.

    (A) Plots of the μFTIR parameter R3/2 versus Raman parameter I(1350)/I(1600) of fossilized filament from MMS (red) and comparison to eukaryotic (green) and prokaryotic (blue) fossils from previous studies (2931). Within the Rhynie chert plant fossils, several regions were measured including cell wall, epiderm, protoplasm, and extracellular carbon. Our fossilized filaments fall close to the protoplasm of plants. The colored areas provide possible discrimination between eukaryote and prokaryote fossils based on their respective organic matter R3/2 and I(1350)/I(1600). The average values with SDs are plotted. (B) Box-and-whisker plot of the R3/2 ratio of a range of modern organisms spanning across the three domains of life [data for eukaryotes, prokaryotes, and Archaea are from (46, 47)]. R3/2 ratio quantifies the extent of branching and length of aliphatic moieties of the precursor membrane and can be used as a domain-specific proxy. As fossilization proceeds, it seems that the R3/2 values tend to decline (47), which also seem to be the case here for fungi. *Note that “Extant fungi” values were calculated from the FTIR spectra (4851) of Rhizopus sp., Motierella alpina, Mucor circinelloides, Paxillus involutus, Aspergillus nidulans, Neurospora sp., Penicillium glabrum, and Umbelopsis isabellina.

  • Fig. 6 X-ray absorption near edge spectroscopy at the C and N K-edge performed on FIB foil 4984.

    (A) Compilation of C-XANES spectra for a range of modern fungi (Paxillus involutus and Aspergillus fumigatus), fungal hypha in lichen (Parmelia saxatalis), cyanobacteria (Gloeobacter violaceus) (36), α-chitin, and fossil filament (FIB foil #4984; fig. S7 for detailed views). The colored bands denote the energy range for which various C functional groups are expected (see table S2). For the fossilized filament, the main peaks are centered at 285.1 eV (aromatic C═C), 286.7 (ketone and phenol C), and a shoulder in amide and carboxylic energy bands (288 to 288.7 eV). A complete deconvolution of the C-edge for fossilized filament was performed (fig. S7). Note that for P. involutus, the XANES was acquired on the FIB section at the interface between fungi and biotite; the double peak between 297 and 300 eV are, thus, absorption peaks for potassium. (B) N-XANES spectra of fossilized filament, fungal hypha in Parmelia saxatalis, and α-chitin (32). The fossilized filament exhibits spectral features at 398.7 (imine), at 399.8 eV related to pyridine (35), and a shoulder at 401.3 eV, which corresponds well with the main spectral feature of the α-chitin (N 1 s-3p/σ* transition in amide). Note that all those features are visible in the spectra of P. saxatalis.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/4/eaax7599/DC1

    Paleoenvironment and petrography of the BIIc subgroup

    Fig. S1. High-resolution SEM micrographs of the mycelial networks.

    Fig. S2. Geological map of the SMMLL Basin and synthetic stratigraphic and chemostratigraphic column of the MMS.

    Fig. S3. Optical micrographs of petrographic thin sections cut at the depth or in close vicinity of the fossiliferous sediment in BIIc8 (15) (BK 457b, 118.2 m) showing subaerial exposure feature and little to no compaction of fossiliferous dolomitic shale.

    Fig. S4. Fluorescence micrographs of thin sections in the BIIc6 shale bed (controls of WGA-FITC staining specificity).

    Fig. S5. Comparison of μFTIR spectra of fossil filaments, cyanobacteria, extant fungi, fossil sponge V. gracilenta, and α-chitin.

    Fig. S6. SEM, TEM (HAADF) micrographs, and elemental (STEM-EDS) and mineralogical analyses (selected-area electron diffraction) on FIB foil #4984 sampled collected fossil filament and associated paragenetic mineral phases.

    Fig. S7. Deconvolution of the C-XANES edge for fossil filament FIB foil #4984.

    Table S1. μFTIR assignments based on the following references.

    Table S2. Peaks detected in XANES at the C and N 1s edges and used for the deconvolution in fig. S7.

    References (5258)

  • Supplementary Materials

    This PDF file includes:

    • Paleoenvironment and petrography of the BIIc subgroup
    • Fig. S1. High-resolution SEM micrographs of the mycelial networks.
    • Fig. S2. Geological map of the SMMLL Basin and synthetic stratigraphic and chemostratigraphic column of the MMS.
    • Fig. S3. Optical micrographs of petrographic thin sections cut at the depth or in close vicinity of the fossiliferous sediment in BIIc8 (15) (BK 457b, 118.2 m) showing subaerial exposure feature and little to no compaction of fossiliferous dolomitic shale.
    • Fig. S4. Fluorescence micrographs of thin sections in the BIIc6 shale bed (controls of WGA-FITC staining specificity).
    • Fig. S5. Comparison of μFTIR spectra of fossil filaments, cyanobacteria, extant fungi, fossil sponge V. gracilenta, and α-chitin.
    • Fig. S6. SEM, TEM (HAADF) micrographs, and elemental (STEM-EDS) and mineralogical analyses (selected-area electron diffraction) on FIB foil #4984 sampled collected fossil filament and associated paragenetic mineral phases.
    • Fig. S7. Deconvolution of the C-XANES edge for fossil filament FIB foil #4984.
    • Table S1. μFTIR assignments based on the following references.
    • Table S2. Peaks detected in XANES at the C and N 1s edges and used for the deconvolution in fig. S7.
    • References (5258)

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