Abstract The Paleoproterozoic Era witnessed crucial steps in the evolution of Earth's surface environments following the first appreciable rise of free atmospheric oxygen concentrations ∼2.3 to 2.1 Ga ago, and concomitant shallow ocean oxygenation. While most sedimentary successions deposited during this time interval have experienced thermal overprinting from burial diagenesis and metamorphism, the ca. 2.1 Ga black shales of the Francevillian B Formation (FB2) cropping out in southeastern Gabon have not. The Francevillian Formation contains centimeter-sized structures interpreted as organized and spatially discrete populations of colonial organisms living in an oxygenated marine ecosystem. Here, new material from the FB2 black shales is presented and analyzed to further explore its biogenicity and taphonomy. Our extended record comprises variably sized, shaped, and structured pyritized macrofossils of lobate, elongated, and rod-shaped morphologies as well as abundant non-pyritized disk-shaped macrofossils and organic-walled acritarchs. Combined microtomography, geochemistry, and sedimentary analysis suggest a biota fossilized during early diagenesis. The emergence of this biota follows a rise in atmospheric oxygen, which is consistent with the idea that surface oxygenation allowed the evolution and ecological expansion of complex megascopic life.

Citation: El Albani A, Bengtson S, Canfield DE, Riboulleau A, Rollion Bard C, Macchiarelli R, et al. (2014) The 2.1 Ga Old Francevillian Biota: Biogenicity, Taphonomy and Biodiversity. PLoS ONE 9(6): e99438. https://doi.org/10.1371/journal.pone.0099438 Editor: Lorenzo Rook, University of Florence, Italy Received: February 27, 2014; Accepted: May 14, 2014; Published: June 25, 2014 Copyright: © 2014 El Albani et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding of this study was provided by University of Poitiers, Centre National pour la Recherche Scientifique and Fond Européen pour le Développement Régional. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Reports of Paleoproterozoic macrofossils tend to be controversial, and considerable uncertainty persists about the nature of such remains. The report of centimeter-sized pyritized fossils in the ca. 2.1 Ga Francevillian black shales of Gabon [1] portended a new window on the history of macroscopic multicellular life. Their putative biological origin was investigated with the help of non-invasive structural investigations in combination with Δ34S analysis elucidating the process of pyritization. The study concluded that the objects meet commonly accepted criteria of biogenicity [2] and thus are likely to be fossils. The fossils were interpreted to represent organized and spatially discrete populations of colonial organisms, exemplifying a possible pathway toward the emergence of multicellular macroorganisms. Multicellularity has arisen a multitude of times in prokaryotes and eukaryotes [3], [4]. It has a long geological history [5] and most likely occurred also in numerous lineages not represented in today's biota. The concept comprises a wide spread of phenomena, from cooperation between cells in a colony to highly organized and genetically regulated cell and tissue differentiation within complex bodies. The evolutionary pathways from simple coloniality to complex multicellularity are probably diverse, and involve various issues of cell–cell recognition, competition, co-operation, and adhesion. A fundamental distinction is between divisional (cells staying together after division) and aggreagational (individual cells coming together during part of their life cycle) types of multicellularity; they have different evolutionary origins [6], [7], and the aggregational type seems confined to terrestrial environments [3]. Multicellularity does not necessarily imply large body size, but because of the limitations of diffusion rates and streaming in cytoplasm, non-microscopic body size requires multicellularity (or syncytiality, multiple nuclei within a connected cytoplasm, which is functionally equivalent to multicellularity and sometimes interchangeable with it) [8]. The purported significance of the Gabon fossils is thus not multicellularity as such but the evidence they provide for a first appearance in the fossil record of macroscopic individuality [1]. In extensive investigation of the FB2 Subunit outcropping near Franceville, Gabon, we have now identified at least forty-five fossiliferous black-shale levels and collected more than 400 specimens, including diverse types not represented in the originally described assemblage. We are aware of the potentially confounding effects of some sedimentary (e.g., the binding of sediments by microbial mats) and diagenetic (e.g., the growth of concretions) processes that may produce macroscopic structures. The need to test mode of formation and origin is, therefore, critical. Accordingly, to evaluate further the spatial and chemical relationships of the structures to the surrounding rock, to elucidate their taphonomic history, and to assess their outer and inner structural variation and related biological diversity, we have submitted the newly available assemblage to detailed geochemical (δ34S) and morphological–structural (including microtomography) investigations.

Geological, geochronological and sedimentary setting The Francevillian basin consists of 35,000 km2 of unmetamorphosed sedimentary rocks, with no indication of hydrothermal influence [9]–[11], deposited during the Paleoproterozoic Eon in an epicontinental setting in what is now the Republic of Gabon, western equatorial Africa (Figure 1A). The sediment package is between 1000 and 2500 m thick and is subdivided into four lithostratigraphic units, FA to FD, which rest unconformably on Archean basement rocks [12] (Figure 1B, C). The FA unit consists of mainly fluviatile and deltaic sandstones. At the top, it contains uranium enrichments and hosts the well-known Oklo nuclear reactors [13]. The FB unit consists of marine sediments deposited mainly below storm wave base. Because of its diverse lithological composition, the FB unit is further divided into the FB1 (a, b, and c) and FB2 (a and b) subunits. The FB1a and FB1b subunits consist of interlayered shales, sandstones, and conglomerates, fining upwards to predominantly shales at the top. The FB1c subunit mainly consists of shales, but it also contains a thin iron formation overlain by black shales and a thick interval rich in manganese (Mn). The FB2a subunit consists of sandstone beds deposited in channels near the fair-weather wave base. These are sharply overlain by the FB2b subunit including finely-laminated black shales interbedded with thin siltstone layers deposited by waning storm surges. The previously reported large colonial organisms [1], and the new specimens presented here were collected from the FB2b black shales. In the fossiliferous quarry, the FB2b black shales are 5 m thick. The overlying FC Unit is dominated by dolomites and stromatolitic cherts, indicating shallow-water conditions. Stromatolites are found on topographic highs at the base of the FC unit [14]. The FD unit corresponds to black shales deposited during a transgressive phase. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Geological map of the Francevillian basin and lithostratigraphy of the Paleoproterozoic Francevillian Series. (A) The location of the fossiliferous quarry is indicated by a star. (B) The Francevillian Series consists of four formations (FA to FD). The star indicates the FB2 Subunit. (C) Detailed lithology of the FB2 Subunit in the fossiliferous quarry. https://doi.org/10.1371/journal.pone.0099438.g001 Diagenetic illites from the top of the FB1b subunit yielded a Sm-Nd age of 2099±115 Ma [15], while a more precise U-Pb zircon age of 2083±6 Ma was reported from a welded tuff near the top of the FD unit [16]. In addition, a Rb-Sr whole-rock age of 2143±143 Ma has been reported for coarse-grained syenites of the N'Goutou alkaline complex, the emplacement of which is believed to be contemporaneous with the deposition of the base of the FB1 subunit [17]. Overall, the large portion of the Francevillian Series was deposited during the ca. 2.22-2.1 Ga Lomagundi carbon isotope excursion [18]. The preservation of randomly ordered smectite-rich illite/smectite mixed layer minerals (R0-type) demonstrates unusually slow mineral transformation and only a moderate degree of diagenesis, which is remarkable, considering the Paleoproterozoic age of the sedimentary succession [11]. The Francevillian fossiliferous black shales were deposited from an oxygenated water column in a quiet, low-energy marine environment [1], [18]. They are interbedded with thin sandstone layers lacking macrofossils. Lithofacies analysis indicates the absence of bedding-parallel microbial mats throughout the entire fossiliferous sequence. The macrofossils are distributed without significant overlap on the black-shale bedding planes. Fine laminae, prevailing in the enclosing shales, surround the specimens, indicating that the structures were in place before burial compaction [1]. Most specimens are completely to partially pyritized, yet some are only represented by impressions. Others are coated with iron oxides from pyrite oxidation. Both moulds and impressions are commonly preserved [1].

Methods High-resolution micro-computed X-ray tomography (micro-CT) of the macrofossils was run on a X8050-16 Viscom AG. Reconstructions were done using DigiXCT v.3.0 (Digisens) 64-bit version running on a Carry Systems workstation with 2 processors Intel Xeon 6 core 2.66 GHz, with 24 GB of DDR3 1333Mhz and 3 NVIDIA graphic cards (Quadro 6000 and 2 Tesla C2070). Virtual sections and 3D rendering were performed with Avizo Fire v.7.0 (VSG, Visualization Sciences Group). Sulfur isotopes (34S to 32S ratios) were measured by Secondary Ion Mass Spectrometry (SIMS) using Cameca IMS1270 and 1280 instruments at CRPG (Nancy, France) and at the NordSIM facility (Stockholm, Sweden). The SIMS method for analysis of sulfur isotopes was described in detail in [19] and [20]. The sulfur isotopic compositions were measured using a 20 µm Cs+ primary beam of ≈2–5 nA. Sulfur isotopes were measured in multicollector mode using two off-axis Faraday cups (L2 and H1). The gains of the Faraday cups were intercalibrated at the beginning of the analytical session and the offsets were determined before each analysis during the pre-sputtering (300 s). Typical ion intensities of 3×109 counts per second (cps) were obtained on 32S−, so that an internal error better than ±0.1‰ could be reached. Instrumental mass fractionation and external reproducibility were determined by multiple measurements of the in-house reference material Pyr3B (δ34S = +1.41‰) at CRPG, and Ruttan pyrite (δ34S = +1.2‰) and Balmat pyrite (δ34S = +15.1‰) at NordSIM. The external reproducibility ranged between 0.05‰ and 0.40‰ (1 σ) depending on the analytical session. For palynology, about 25 g of each sample (n = 24) were macerated with HF/HCl followed by settling and decanting. Part of the residue was mounted on microscope slides. Single microfossils were handpicked under an inverted microscope by using a micropipette, then deposited uncoated on an aluminum stub and imaged by backscattered electron microscopy with a Jeol Environmental Scanning Electron Microscope (ESEM). Further analyses (FTIR, Raman, FIB, TEM, STXM, and Ultramicrotomy) of single palynomorphs are described below. Scanning transmission electron microscopy (STEM) observations were performed using the high-angle annular dark field mode (HAADF) and a probe size of 1 nm. In this mode, brighter areas correspond to regions with higher atomic numbers. Energy dispersive x-ray spectrometry (EDXS) mapping was performed using the STEM mode. For Focused Ion beam (FIB) sectioning, an ultrathin electron-transparent foil was prepared by focused ion beam (FIB) milling using a FEI strata Dual beam at Lille University. FIB foils were lifted up and welded on one side onto a copper TEM grid in situ before final polishing. The FIB foils was studied at IMPMC (Paris, France) by transmission electron microscopy (TEM) using a JEOL 2100F (FEG) operated at 200 kV and equipped with a field emission gun, a high-resolution UHR pole piece, and a Gatan energy filter GIF 200. Scanning transmission x-ray microscopy (STXM) and x-ray absorption near-edge structure spectroscopy (XANES) analyses were carried out on the FIB ultrathin section at the carbon K-edge (C K-edge) at the Advanced Light Source (Lawrence Berkeley National Laboratory, Berkeley, USA) on the Polymer STXM 5.3.2.2 beamline, providing theoretical energy resolution better than 0.1 eV and a spatial resolution better than 25 nm. Raman spectra were collected on isolated microfossils. Spectra were recorded from nearly twelve different points in each sample to ensure the representative nature of the spectra, by means of a Renishaw inVia Reflex Raman Microprobe using a Peltier-cooled charge. FTIR Spectra were recorded on single microfossils using an IR source and Cesium Iodine (CsI) beamsplitter from a Nicolet 6700 FT-IR spectrometer coupled with a Thermo Scientific Nicolet Continuum FT-IR microscope equipped with a Mercury Cadmium Telluride (MCT) detector cooled with liquid nitrogen. Analyses were performed in transmission mode in the Middle Infrared (MIR) domain between 4000 and 400 cm−1. For ultramicrotomy preparation, single microfossils were embedded in agar and dehydrated in ethanol solution. Samples were polymerized at 60°C for 12 h. Ultra-thin sections (50–60 nm-thick) were cut from the resin blocks with a diamond knife. Illustrated specimens are deposited at the University of Poitiers and the Swedish Museum of Natural History, Stockholm (SMNH numbers). Here below the references: (G-FB2-s-615), (G-FB2-s-614), (G-FB2-s-608), (G-FB2-s-606), (G-FB2-s-605), (G-FB2-s-604), (G-FB2-s-601), (G-FB2-s-600), (G-FB2-s-593), (G-FB2-s-589), (G-FB2-s-586), (G-FB2-s-82), (G-FB2-s-259), (G-FB2-s-71), (G-FB2-s-576), (G-FB2-s-575), (G-FB2-s-573), (G-FB2-s-423) (G-FB2-s-123), (G-FB2-s-49a), (G-FB2-s-118), (G-FB2-s-148), (G-FB2-s-160). Details of permits: the permits are provided by the Centre National Pour la Recherche Scientifique et technique du Gabon (CENAREST). Permit number: GA/488.

Discussion The crucial issue with regard to the interpretation of the Francevillian specimens is not biogenicity as such, because microbial processes are almost always involved in sedimentary pyrite formation during early diagenesis. Instead, the question is whether the morphology observed in the specimens reflects the shape of fossilized macroscopic organisms or colonies, or whether it was formed by taphonomic/diagenetic processes. We have earlier argued [1], on grounds of crystallography and isotopic composition, that fossilization of the Francevillian organisms was a prolonged process, where the sheet fabric was original, preserved through microbially induced pyritization in an open environment, whereas the central lumps of pyrite, the cores, were formed in a close environment after burial as pyrite concretions. The new data confirm this model, but also add evidence for intermediate pyritization processes in some specimens (Fig. 11), where secondary growth of spongy pyrite modified the original structure but without forming a distinct core. The biogenic morphology of the earlier reported sheet-like specimens is ostensibly challenged by their similarity with flat pyritic concretions such as pyrite “suns” and “flowers” [1]. Undulate or lobate outer edges, seen in such concretions as well as in the Francevillian sheets, can be created inorganically by fingering-driven Saffman-Taylor instability in a mixture of fluids with different viscosities [45]. However, the required pressure should then affect all comparable structures on the same bedding plane [45], which is not observed in the Francevillian material (Figure 3A, B). The textural and isotopic analysis of new specimens showing very different morphologies and the comparison with the two Phanerozoic pyrite concretions clearly demonstrate that the Francevillian specimens differ from the Phanerozoic pyrite concretions in both texture and isotopic composition and thus the latter cannot be used as a model for the formation of the former. We have earlier proposed that the flexible sheets with radial fabric represent colonial organisms showing incipient multicellular organization [1]. The presence of lobate, elongated-to-rod-shaped, and discoidal structures, as well as the circular aggregation, expands the morphological diversity of the Francevillian organisms. The specimens showing a string connected to a sheet-shaped macrofossil (Figures 4C–F, 5, 6, Figure S1 in File S1) suggest that these two structures represent the same organism. A combination of elongated and flattened stages of life opens up the possibility that the organism had an organization similar to that of cellular slime mould, Dictyostelia. These organisms go through a “slug” phase in which amoeboid cells congregate into multicellular “slugs” that move along a mucus tube to a place where a sedentary fruiting body is formed [46], [3]. Dictyostelia are understood to branch from a deep position in eukaryote phylogeny [47]. The aggregational style of dictyostelid multicellularity, however, seems to confine them to the terrestrial environment [3], [48]. Given the strong evidence for a marine setting of the FB2 unit, Dictyostelia are therefore unsuitable even as an analogue of the behavior implied for the Francevillian organisms. The occasional evidence for radial fabric in a narrow portion joined to a sheet-like morphology (Figures 6A–B; 4E–F) suggests that the rod-shaped portion does not represent a mucus tube, but an organic fabric similar to that which makes up the associated sheets. The difference with the rods lacking radial fabric may reflect preservation, but an alternative explanation is that the strings adjacent to a sheet in some cases represent a portion where the cells came to rest while still in the “slug” configuration. The second most abundant member of the Francevillian biota is represented by the small non-pyritized to lightly pyritized disks (Figures 7A–F; 8A–B). With their positive relief and somewhat concentric and centrally radial texture, they are reminiscent of the small sand-volcano-like structures in the Cambrian King Square Formation in Canada [49]. Such an interpretation has the appeal of easily explaining why most of these disks are non-pyritized or weakly pyritized, while the other fossils from the Francevillian Series are pyritized. However, the Francevillian disks occur in organic matter-rich shales and are not directly associated with sandy or silty layers. Moreover, the Francevillian disks are generally smaller than the sand-volcano-like structures, and their profile is highly repetitive, while sand-volcano-like structures show variable morphologies [49]. The Francevillian disks are therefore likely not related to fluid escape. The size and morphology of the Francevillian disks also resemble some Ediacaran disks [50], however, they differ from the latter as they appear in positive epirelief, while most Ediacaran disks are preserved in positive hyporelief. Moreover, the morphology of the Francevillian disks does not exactly correspond to any of the described Ediacaran disks [50]. Finally, the Francevillian disks are somewhat similar to the giant Proterozoic acritarch Chuaria [51], but differ in their larger size and radially striated centre. Partial pyritization of some of the Francevillian disks gives support to their original organic composition, but the scarcity of this pyritization also suggests that their nature was somewhat different from that of the pyritized specimens. In its organization, the circular aggregate shown in Figures 4F–G and 5F–G is reminiscent of the tight assemblage of circular structures of Nemiana and Beltanelloides [52]. Nemiana, however, appears in high positive hyporelief, while the Francevillian aggregate is preserved in low positive epirelief. Moreover, although Nemiana and Beltanelloides subunits tend to be packed in a similar way, they do not form discrete rounded aggregates. A superficial resemblance also exists to the trace fossil Paleodictyon nodosum, an enigmatic structure observed at the surface of deep-sea sediments [53], [54]. Though Paleodictyon is mostly described from Tertiary flysch sequences, it has a larger stratigraphic distribution, and its oldest occurrences are in Cambrian sediments [54]–[56]. Paleodictyon generally corresponds to a strictly hexagonal network of tunnels or tubes delineating isodiametric hexagons seen in positive hyporelief. Compared to Paleodictyon nodosum, the Francevillian fossil consists of rounded bodies of different sizes, the packing of which only occasionally produces a hexagonal pattern. Overall, the Francevillian biota represents an exceptional Paleoproterozoic oxygenated ecosystem [1], [18] comprising several types of macroscopic organisms, including the pyritized fossils, non-pyritized or lightly pyritized disks and circular aggregates, as well as carbonaceous microorganisms. As illustrated by the sulfur isotope record, early pyritization in a quiet depositional setting [21] is likely the most critical factor responsible for the exceptional preservation of these fossils. The emergence of this biota follows a rise in atmospheric oxygen, which is consistent with the idea that surface oxygenation allowed the evolution and ecological expansion of complex megascopic life [57], [58], [39], [18]. The disappearance of the Francevillian macrofossils in the upper part of the FB2b black shales is apparently related to increased energy in the environment. The macrofossils are not observed in the overlying Francevillian black shales of the FD Formation that were deposited under an anoxic and sulfidic (euxinic) water column [18]. Their absence from the later fossil record might ultimately be related to the fall in the atmospheric oxygen level that followed the ca. 2.22-2.1 Ga Lomagundi carbon isotope excursion [18], [59]–[63], followed by a long-lived and extensive marine anoxia that forms the hallmark for the most of the Proterozoic Era after ca. 2.1 Ga [59], [18]. Oxygen content in surface environments is not universally accepted as a major driver for the evolution and complexification of multicellular life [64], [65]. However, the emergence and later disappearance of megascopic life in association with oxygen overshoot and fall in the early Paleoproterozoic Eon [18], [59]–[63] is consistent with oxygen availability as a driver of evolutionary adaptation [39], [66], including aspects of body size [67]. The Francevillian deposits represent exceptionally well-preserved Paleoproterozoic sedimentary rocks deposited in shallow-marine, oxygenated environment [11], [12], [17]. This, coupled with pyritization during early diagenesis, provides a unique window on the early Paleoproterozoic biosphere during one of the most critical time periods in Earth's history. The Francevillian biota formed a diverse ecosystem. It appears to represent a first experiment in megascopic multicellularity.

Acknowledgments Ministry of Education, Research and Culture; Centre National Pour la Recherche Scientifique et technique du Gabon (CENAREST); Ministry of Mines, Oil, Energy and Hydraulic Resources; General Direction of Mines and Geology of Gabon; Sylvia Bongo Foundation; Agence Nationale des Parcs Nationaux of Gabon; University of Masuku; COMILOG-Company; French Embassy at Libreville; and Institut Français du Gabon, French Ministry for Foreign Affairs, The Danish National Research Foundation (grant DNRF53); ERC, “Oxygen”; Intervie-INSU, ERC PaleoNanoLife, are acknowledged for collaboration and support. Permit was provided by CENAREST (GA/488). For information and scientific discussion, we thank N. Butterfield, P. Garcia, F. Idiata, R. Laffont, F. Mayaga-Mikolo, P. Mouguiama, F. Pambo, P. Strother, and F. Weber. For assistance, we acknowledge Y. Batonneau, E. Bere, P. Devautour, Ch. Franzén, N. Guignard, C. Laforest, E. Marcon, Ph. Recourt, A. Texier, K. White and Studio LUDO. For continues support, we acknowledge the French CNRS-INSU, FEDER, the University of Poitiers, and the Région Poitou-Charentes. Data used in this study are available at the University of Poitiers.

Author Contributions Conceived and designed the experiments: AEA S. Bengtson DEC AR A. Meunier EH RM. Performed the experiments: AEA DEC S. Bengtson AR CRB LNP EH MW PB MC ACPW OR A. Mazurier IMM CC KB S. Bernard CF. Analyzed the data: AEA S. Bengtson DEC AR CRB EH RM AB KB S. Bernard FGL MV. Contributed reagents/materials/analysis tools: AEA S. Bengtson DEC AR CRB JMGR ECF LNP EH AB PB MC ACPW OR MW A. Meunier GV AT IMM ECF LW. Wrote the paper: AEA S. Bengtson DEC AR EH RM AB.