Diamond Annual Review 2019/20

52 53 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 Imaging andMicroscopy Group Beamline I08 and B22 Identifying the earliest known fungi Related publication: Bonneville S., Delpomdor F., Préat A., Chevalier C., Araki T., Kazemian M., Steele A., Schreiber A., Wirth R., & Benning L. G. Molecular identification of fungi microfossils in a Neoproterozoic shale rock. Sci. Adv. 6 , eaax7599 (2020). DOI: 10.1126/sciadv.aax7599 Publication keywords: Fungi fossils; Neoproterozoic; Chitin F ossils of fungi are scarceandhard to tell apart fromothermicroorganisms, andonly twopercent of species in thekingdomFungi havebeen identified. Until now, the oldest confirmedmushroomfossil was 460million years old. With nomethod to distinguish fungi fossils from the remains of other organisms, identification had to be based onmorphological criteria - the size and shape of the fossil. This has hindered our understanding of the early evolution of fungi and the beginnings of more complex life forms. Researchers used synchrotron techniques to investigate fossilised filamentous networks in rocks formed between 715 and 810 million years ago. They were looking for traces of chitin, a constituent of fungal cell walls. Using X-ray absorption near-edge spectroscopy (XANES) on the Scanning X-ray Microscopy beamline (I08), they investigated ultrathin sections of the fossilised filaments. They combined these results with complementary µFTIR spectroscopy on theMultimode InfraRed Imaging AndMicrospectroscopy (MIRIAM) beamline (B22). Their results clearly show that traces of chitin are present in the filamentous fossils, meaning that they have chemically identified the oldest known fossil fungi.Theseearly fungiwerealreadypresent onEarthbetween715and810millionyears ago, almost 300millionyears earlier than previously thought. In that era, fungi would have been a crucial partner for the first plant to grow on land. The fungi fossils examined were preserved in rocks deposited in the transition zone between land and sea. As such, those fungi couldhave participated in the establishment of thefirst landplants. Fungi form vast microscopic filamentous networks in symbiosis with plant roots that facilitate nutrient uptake from soil particles 1 . Because of these unique capabilities, ancient fungi are thought to have been crucial partners of early plants involved in terrestrial invasion 2 . However, the timing of this major evolutionary transition is largely unknown because of the notorious scarcity and ambiguous nature of the Precambrian fossil record for fungi. A number of Precambrian remains (e.g., Hurionospora , Shuiyousphaeridium, Tappania) , filaments and spores from 0.9-1 Ga in Arctic Canada, in Siberia (Lakhanda microbiota) or even older structures in 2.4-billion-year-old basalt were inferred to be of fungal nature, yet their conclusive attribution to Fungi remains problematic 3,4 . In this study, we report the discovery of fungi fossils in a dolomitic shale from the Mbuji-Mayi Supergroup (MMS) from the Sankuru- Mbuji-Mayi-Lomami-Lovoy Basin (SMMLL), south-central of the Democratic Republic of Congo (DRC). This shale (BIIc8 unit) was deposited in a coastal, perennial lacustrine pond between 715 and 810 million-years ago. The remains are dark, cylindrical filaments typically between 3.5 µm to 11.5 µm in width, extending over hundreds of microns in length and sometimes forming a dense network (Fig. 1). The size of the fossils fits well with fungal hypha dimensions; however, size alone cannot be a criterion as prokaryotes also form filaments and networks of similar dimensions. In order to determine whether those filamentous networks represent Neoproterozoic fungal or cyanobacterial remains, an in-depth chemical characterisation is required. Chitin is a biopolymer of N-acetyl-glucosamine which is abundant in fungal cell walls and more generally in animal taxa (i.e., ciliates, arthropods, chrysophytes, diatoms) yet absent in prokaryotic organisms. The chitin- producing organisms listed above are morphologically distinct from our fossils, thus finding chitin in the filament would be a strong argument for a fungal affinity. We detected chitin using Wheat Germ Agglutinin conjugated with Fluorescein IsothyoCyanate (WGA-FITC), a highly specific dye of N-acetyl glucosamine trimers. We observed that the WGA-FITC binds to extensive portions of the mycelial structure (Fig. 1) whereas staining was negative when WGA-FITC was exposed to inclusions of “non-fungal” organic matter. Considering the fungus-like morphology, the binding of the WGA-FITC to the cell wall, we assert that this positive staining can be used as a clear indicator of chitinous remnants and thus that those filamentous fossils are of fungal origin. Synchrotron radiation Fourier Transformed InfraRed spectroscopy (SR- µFTIR) performed on B22 revealed important information about the functional groups in the fossil filaments (Fig. 2). First, the kerogenization is evidenced by (i) the aromatic bands (1597 cm -1 , 3070 cm -1 and 1270 cm -1 ) and (ii) the strong aliphatic peaks (~2800~-3000 cm -1 ) and at lower wavenumber 1458 cm -1 , 1408 cm -1 and 727 cm -1 . As kerogen matures, aromaticity tends to increase relative to the aliphatic character, with fully aromatic graphite being the ultimate end member. Here, this process is far from complete as the aliphatic vibrations are still preeminent. Those long aliphatic chains result from the alteration of the protein-chitin complex of the fungal cell wall which produce free aliphatic groups and unsaturated carbon which then polymerise into long polymethylenic chains 5 . Here, even though the aliphatic signal is strong, the degradation of the protein-chitin complex appears incomplete as we found sharp vibrations characteristic of amide at 1651 and 1540 cm -1 assigned to amide I (v C=O), amide II (δ N-H). As for carbohydrates (950-1200 cm -1 ), although depleted, we could still detect a pyranose peak at 1170 cm -1 .The fact that, in addition to the aromatic and aliphatic vibrations, amide and pyranose peaks are present is a testimony of the exceptional preservation of those fossils and is also consistent with the presence of vestigial‘chitin’. Ultrathin sections were sampled across filaments by Focus Ion Beam (FIB) and analysed by X-ray Absorption Near Edge spectroscopy (XANES) at the C and N K-edge on I08 (Fig. 3). We detected two characteristic peaks of chitin at 285.1 eV (C=C 1s-π* transition in aromatic carbon) and at 288.6 eV (1s-π* transition of amide carbonyl and C-N bonds). Compared to pure chitin, the strengthening of the aromatic carbon peak (285.1 eV) and the occurrence of two additional features in the filament spectra (286.7 eV C=O/C=N bonds and 287.5 eV for aliphatic carbon) are consistent with the SR- µFTIR results and illustrate the incomplete degradation of the fossil. Our N-XANES data confirmed the presence of amydil N (401.3 eV), which corresponds to the main spectral feature of chitin, while the 398.7 and 399.7 eV peaks are associated with pyridine and its derivatives, which are known alteration-products of chitinous tissues. When compared to modern fungi, our XANES data exhibit the same trend as when it is compared to chitin, i.e., more intense aromatic/ olefinic and imine/ketone/phenolic peaks (at 285.1/398.8-399.9 eV and 286-287.2 eV) and reduced amide/carboxylic peaks (at 288.6 eV/401.3 eV). Equivalent C- and N-XANES trends were observed in a recent study simulating burial-induced maturation of various modern microorganisms 6 .This difference in C-XANES is particularly marked for ( Aspergillus fumigatus, not shown here), but also visible C and N-XANES for Parmelia saxatalis although to a lesser extent (Fig. 3) . The latter fungal species exhibit significant aromatic/olefinic peak and generally have C-XANES spectra close to the one of our fossil filaments. Although not directly diagnostic, the fossil filaments exhibit C- and N-XANES spectral features consistent with the presence of vestigial chitin, and also share some similarities with modern fungal species. Using an array of microscopic (SEM and TEM - and confocal laser scanning fluorescence microscopy) and spectroscopic techniques (Raman, FTIR, XANES), we demonstrated the presence of vestigial chitin in these fossil filaments. Based on the combined evidences of presence of vestigial chitin, syngenicity, size and morphology, those fossil filaments and mycelium-like structures are identified as remnants of fungal networks and represent the oldest, molecularly identified remains of Fungi. As such, this discovery pushes well into the Neoproterozoic the possibility that fungi helped to colonise land surface, almost 300 million years prior to the first evidence of land plants. References: 1. Bonneville B. et al. Tree-mycorrhiza symbiosis accelerate mineral weathering: evidences from nanometer-scale elemental fluxes at the hypha–mineral interface. Geochim. Cosmochim. Acta 75 , 6988 (2011). DOI: 10.1016/j.gca.2011.08.041 2. Selosse M. -A et al. Origins of the terrestrial flora: a symbiosis with fungi? BIO Web of Conferences 4 , 00009 (2015). DOI: 10.1051/bioconf/20150400009 3. Butterfield N. J. Probable Proterozoic fungi. Paleobiology 31 , 165 (2005). DOI: 10.1666 /0094-8373(2005)031< 0165:PPF >2.0.CO; 2 4. Cody G. D. et al. Molecular signature of chitin-protein complex in Paleozoic arthropods. Geology 39 , 255 (2011). DOI: 10.1130/G31648.1 5. Alleon J. et al. Organic molecular heterogeneities can withstand diagenesis. Sci. Rep. 7 , 1508 (2017). DOI: 10.1038/s41598-017-01612-8 Funding acknowledgement: This research was supported by the“Van Buren”funds from ULB and the Fonds National de la Recherche Scientifique (FRS-FNRS PDR T.1012.14). We thank Diamond Light Source and their beamline scientists for access to beamline B22 (SM15050) and I08 (SP14953) that contributed to the results presented here. Corresponding author: Dr Steeve Bonneville, Biogeochimie et Modelisation du Systeme Terre, steeve.bonneville@ulb.ac.be Figure 1: Textures and structures observed via (a) scanning electron and (b) light microscopy of a thin section from the fossiliferous dolomitic shale rock from BIIc8 (Mbuji-Mayi Supergroup). (c) Confocal laser scanning fluorescence microscopy using WGA-FITC of mycelium-like structure. Figure 2: Synchrotron-µFTIR (average over 31 10 by 10 µm areas) of fragments of fossilised filaments. Assignments of absorption bands and vibration modes (δ=deformation; ν=stretching; s=symmetric; as=asymmetric) are indicated in parentheses. Figure 3: (a) C-XANES spectra for fungal hypha in lichen (Parmelia saxatalis), chitin and fossil filament. For the fossil, the main peaks are centered at 285.1 eV (aromatic C=C), 286.7 eV (ketone and phenol C) and a shoulder in amide/carboxylic acids energy bands (288–288.7 eV). (b) N-XANES spectra of the above-mentioned samples. The fossilised filament exhibits spectral features at 398.7 eV (imine), at 399.8 eV related to pyridine and a shoulder at 401.3 eV which corresponds well with the main spectral feature of the chitin (N 1s-3p/σ* transition in amide). Note that all those features are visible in the spectra of P. saxatalis. Normalised absorption (a.u.) Normalised absorption (a.u.)