Tubular microfossils from 2.8 to 2.7 Ga-old lacustrine deposits of South Africa : a sign for early origin of eukaryotes?

Supported by the National Science Centre, Poland (grant 
2011/01/B/ST10/06479 to J. K) and by the Program of Scientific 
and Technological Co-operation between the Governments of the 
Republic of Poland and the Republic of South Africa (to J. K. and 
W. A.). We appreciate the Europlanet TransNational Access Programme 
for funding access to NanoSIMS facilities at the Milton 
Keynes Open University (UK). Cyprian Kulicki and Krzysztof 
Owocki (Warsaw) helped with SEM-EDS and Cameca microprobe 
analysis. Katarzyna Janiszewska (Warsaw) kindly prepared the 
micro-tomograph movie and 2-D slices. Comments and suggestions 
by the Editor and Reviewer are greatly appreciated. We thank 
the late Edwin Jackson, owner of the Omdraaivlei Farm, for his support, 
hospitality and interest in stromatolites and early life. The 
research was partially supported by the European Union within 
the European Regional Development Fund, through the Innovative 
Economy Operational Programme POIG.02.02.00-00-025/09/ supported 
by NanoFun POIG.02.02.00-00-025/09.


Geographic and geological location
The 2.8-2.7 Ga Sodium Group, from which the tubular microfossils described herein were derived, is a volcano-sedimentary succession correlated with the Neoarchean Ventersdorp Supergroup (Kaapvaal Craton, South Africa; Fig. 1A) (Grobler et al., 1989;Buck, 1980;Altermann and Lenhardt, 2012). Coccoidal, bacteria-like microfossils and stromatolites were previously described from the Sodium Group and the Ventersdorp Supergroup (Buck, 1980;van der Westhuizen et al., 1991;Altermann and Lenhardt, 2012). The lacustrine deposits in which the eukaryote-like microfossils were found belong to the 2.74-2.71 Ga Omdraaivlei Formation (Altermann and Lenhardt, 2012), where nine carbonate intervals occur in tuffaceous shale (Fig. 1B), as beds and lenses of stratiform and pseudocolumnar stromatolites (stratiform to laterally linked, hemispheroidal, narrow columns) ( Fig. 2A). The stromatolitic carbonate beds (lithostromes) are commonly topped by symmetrical or asymmetrical ripples ( Fig. 2B), preserved probably due to microbial mat overgrowth that stabilized the fine volcaniclastic sediment (Altermann and Lenhardt, 2012). In some stromatolitic beds, laterally linked columns are uniformly inclined and, together with the rippled stromatolite tops, indicate current action. Thinner beds of domical and stratiform stromatolites formed in a similar but less agitated, low energy shallow lacustrine environment (Buck, 1980;Altermann, 2007;Altermann and Lenhardt, 2012). Above the uppermost stromatolitic bed, sedimentation continued with shale and tuffacoeus sandstone intercalations, covered by lava flows. The carbonate beds are strongly silicified and recrystallization of chert and carbonate is common.
Partial early diagenetic silicification is expressed by diagenetic silica, visible as minute quartz crystallites mixed with fine carbonaceous flakes (kerogen) within the laminae.
Subsequently, SiO 2 has recrystallized to coarser, clean chert, with carbonaceous material squeezed between the chert crystallites of c. 10 µm diameter. Nevertheless, organosedimentary structures like laminae and tufts are very well preserved (Fig. 2C).

The tubular microfossils: distribution, morphology, composition
The tubular microfossils discussed herein occur with varying frequency in all stromatolitic intervals of the studied section. They are particularly abundant in the main stromatolitic unit (bed 11) indicated on Fig. 1B with a red arrow head. This c. 90 cm thick microbialitic bed is composed of strongly silicified narrow columns inclined in places towards the current, and of wavy laminites, both intercalating with each other and exhibiting rippled surfaces as described above.
The best-preserved microfossil specimens occur as dichotomously and trichotomously branched, mineralized tubes, up to several hundred micrometers long and 20 to 70 µm in diameter ( Fig. 2D-F). Ten well branched specimens were found in petrographic thin-sections  4). The external surfaces of the tubes are irregular and rough whereas their internal surfaces are smooth. The lumen of the tubes is in most specimens filled with a kerogenous-mineralic substance of flaky to microgranular appearance, which in transparent longitudinal sections is visible as continuous brownish strand (or band). These strands range from thick, filling almost entirely the tube space (Figs. 2E, F), to thinner, occupying, often discontinuously, no more than half of the tube (Fig. 4B). When the strands are absent, the tubes are filled with translucent chert (Fig. 4A). The jagged ends of the tube fragments indicate that they are parts of mechanically broken, longer tubular structures (Figs. 2F, 5A).
Chemical and spectral analyses of the tubular structures by EPMA, NanoSIMS and SEM/EDS  revealed the presence of two kinds of aluminosilicates: (i) Al-K-Fe-Mg silicate forming the walls of the tubes, and (ii) Al-Fe-Mg silicate comprising the continuous brownish bands in the tube interiors. Bulk X-ray diffractometry of the cherty sediment enclosing the tubular microfossils (Fig. 8) confronted with the EPMA and SEM/EDS elemental composition of the tubes (Figs. 5-7) indicate chamosite forming the mineral walls and muscovite-2M1 the internal strands. The element totals determined by EPMA are always below 100% for these samples, suggesting the presence of light elements in the tubular structures: about 2% in the tube walls and almost 10% in the brownish internal strands.
NanoSIMS and micro-Raman analyses show the presence of particulate organic carbon in both cases (Fig. 5). In addition to C, NanoSIMS analyses confirmed the presence of S and N, both in the tube walls and in the brownish internal strands, indicating their biogenicity.
The tubes and their carbon-rich internal bands lack cross walls, constrictions or other signs of septation or compartmentalization. After brief etching of the polished longitudinal sections with 40% HF, the tube walls exposed numerous small spheroid and rod-shaped, bacteria-like bodies in SEM images (Fig. 9A, B). Elemental electron dispersive spectra (SEM-EDS) of these bodies showed enrichment in Si and Ti, in contrast to the Ti-free Al-K-Mg-Fe silicate material building the bulk of the tube mineral walls (Fig. 9C, D). Micro-Raman spectra suggest a mixture of anatase and rutile as mineral components of the Ti-enriched bodies (Fig.   9E).

Arguments for siphonalean microalgae as analogs
The continuous carbon-rich strands (bands) inside most specimens examined can be interpreted as remains of variously shrunken cell content. They suggest similarity of these Neoarchean microfossils to coenocytic life forms known in diverse and unrelated groups of algae (green, yellow-green, red) and fungi. In the simplest modern case, a coenocytic construct is a multinucleate single giant tubular cell (called siphon) lacking cross walls (Kaplan and Hagemann, 1991;Niklas, 2000;Graham et al., 2009). Hence the adjective 'siphonous' used for such thallus organization. More advanced 'siphonous' forms (some red algae and fungal mycelia) are differently as the continuously open Omdraaivlei tubes characterized by multicellular thalli composed of coenocytic cells. In typical siphonous algae, in which nuclei are not organized in regularly spaced cytoplasm units, the cytoplasm exhibits streaming enabling transportation of organelles and nutrients across the thallus. The densely packed epiparietal chloroplasts are discoid or united in a reticulum. Both non-calcifying and calcifying representatives are known in modern siphonous algae, ranging in size between tens of micrometers to several meters in length (Vroom and Smith, 2003).
In most siphonous species the tubular central (axial) stipe is branched and the arrangement of the branches is used to describe their anatomical complexity and as taxonomic traits. The mode of branching of the Omdraaivlei tubular microfossils, with terminally located distinct branches of first and second order make them similar to modern representatives of siphonous green algae (Ulvophyta) classified to the order Bryopsidales (Figs. 3L, M; 10G, 12A) and to members of yellow-green algae (Xanthophyta) of the order Vaucheriales. The latter are known for their common extremely heavy calcification of thalli (Freytet and Verrecchia, 1998;Golubic et al., 2008;Gradziński, 2010). Vaucheriacean algae are typically inhabitants of hard water lakes and fluvial waters, although brackish and marine forms are also known (Graham et al., 2009). Since all the tubular Omdraaivlei fossils are fragmented, we can only assume from their morphological similarity to these modern siphonous microalgae that they occurred as tiny brush-or bush-like turf adhered to the substratum by a system of rhizoids.
The sparse dichotomous and trichotomous branching of the Omdraaivlei microalgae (Figs. 3A-E; 12B, C) makes them particularly similar to the branching mode of thalli characteristic of modern species of the order Bryopsidales such as Chlorodesmis (Ducker, 1967), Rhipilia (Verbruggen and Schils, 2012) and particularly Pseudochlorodesmis and Bryopsis (Børgesen, 1925) (Fig. 3L, M). The diminutive siphon of Pseudochlorodesmis, if branched at all, does so only a few times (Fig. 3L). With the exception of some constrictions occurring only close the ramified rhizoids, the 70 µm thick cylindrical thalli of Pseudochlorodesmis are without constrictions through their whole length. Such simple sparsely branching siphons are regarded as ancestral thallus anatomy in siphonous algae (Verbruggen et al., 2009). Although the Omdraaivlei Neoarchean microfossils support such claims, their preservation is too fragmentary for incontrovertible conclusions.
As shown above, many morphological and mineralogical features of the tubular structures found in the Omdraaivlei stromatolites are akin to modern taxa of siphonous algae. A petrographic thin section however, only exhibits a 30 to 100 µm thick section of a microfossil and the different planes of focus are often insufficient to judge the three-dimensional morphology of structures (Schopf and Kudryavtsev, 2012). Therefore, beside microtomographic sectioning ( Fig. 3F-K, Suppl. movie) for three dimensional examination of the tubular morphology and other anatomical details of the Omdraaivlei microfossils, small cubes were cut from the same rock samples as the thin sections and macerated with 40% hydrofluoric acid (HF). The residua from the dissolved rock samples yielded numerous very brittle fragments of 3D-preserved translucent mineralic-carbonaceous tubes (Figs. 10,11) corresponding in size, shape and elemental composition with the tubular mineral objects visible in thin-sections. Most of them showed only simple non-septated, tube-like morphologies, but a few displayed traits permitting indeed a direct comparison with thalli of modern green algae of the order Bryopsidales (class Ulvophyta). These common in modern, particularly tropical seas microalgae are regarded as a core group of green thallophytes (Lam and Zechman, 2006). They have biphasic heteromorphic life-history -a gametophytic generation alternating with a sporophytic generation (Morabito et al., 2003). As shown in Fig.   10, some of the traits observed in the best preserved macerated specimens are characteristic for modern gametophytic generation like terminal branches, gametangia-like structures, or propagation buds. An example of modern analog of the Omdraaivlei gametangium-like structure shown in Fig. 10F is presented in Fig. 10G, whereas thinner, thread-like thalli with characteristic bulging (Fig. 10H, I) can be interpreted as analogs of thalli of modern sporophytic generation (Neumann, 1969;Burr and West, 1970;Chang et al., 2003;Ye et al., 2010). Sparsely branching siphonous thalli and free-living microthalli, and elongate, simple thread-like tubules that could have developed from germinated spores (protonema) (Neumann, 1969;Morabito et al., 2003) were also recognized in the macerated material ( Fig.   10J, K). Due to the strong fragmentation of the thalli recovered by maceration, it is unfortunately not possible to apprise whether they belong to one algal taxon or represent a mixture of fragments of several siphonous taxa.
Weak C-signals obtained from SEM-EDS spectra of HF-macerated thalli confirm the presence of carbonaceous material as also documented by NanoSIMS analyses. Traces of Si, K, Al, Ca, Fe, Cl visible in the SEM-EDS spectra may represent remains of the thalli permineralizing silicate phases with possible trace admixture of calcium carbonate (Fig. 11).
Siphonous algae are well documented in the fossil record. Their hitherto oldest organically preserved representatives (Proterocladus), ascribed to siphonocladaleans, have been found in Middle and Late Proterozoic marine strata of Spitsbergen (Butterfield et al., 1994;Butterfield, 2004Butterfield, , 2015. Since at least the Neoproterozoic, siphonous algae were common components of shallow-marine biota, their calcified members even achieved a rock-building status in many Phanerozoic geological formations (Riding, 1991). Today, they are represented by a great variety of forms, many with complex thalli, and belong to the most common and ecologically dominant groups of organisms known from tropical marine habitats (Vroom et al., 2006).

Arguments for exclusion of filamentous cyanobacteria as analogs
As mentioned above, the brownish, non-segmented, C-rich, Fe-Mg aluminosilicate strands observable in thin-sections in the interiors of many Omdraaivlei microfossils (Figs. 2-4, 12B, C) which most probably represent permineralized kerogenous residues of the cell content

Discussion
Accepting the siphonous microalgae affinity of the Omdraaivlei tubular microfossils, the thick mineral envelopes can be explained as probably largely the product of an association between algae and bacteria (Lachnit et al., 2000). Such alliances are known in living siphonous algae and may be beneficial or detrimental for both parties. The bacteria reside either on the surface or within the algal cells (Hollants et al., 2011). Most algae, including siphonous varieties, excrete extracellular polymeric substances on their cell surface (Percival and McDowell, 1981;Ciancia et al., 2012). These excreta are densely settled and utilized by a great variety of heterotrophic bacteria (Lachnit et al., 2000;Goecke et al., 2010;Hollants et al., 2010Hollants et al., , 2012. Depending on the hydrochemistry of the environment (particularly pH and the load of metals), the concerted metabolic activity of the algae and of their bacterial settlers changing pH and alkalinity in their proximity, may cause precipitation of various mineral phases. In siphonous algae, calcium carbonate is usually precipitated in such a way on or within the polysaccharide-rich extracellular sheaths, as in many ulvacean and vaucheriacean taxa thriving in alkaline and calcium carbonate oversaturated marine, lacustrine and fluvial environments (Freytet and Verrecchia, 1998;Golubic et al., 2008;Gradziński, 2010). In the case of the purported Omdraaivlei siphonous microalgae, the lacustrine habitat located in a volcanic setting (Altermann and Lehnhardt, 2012) was probably characterized by circumneutral pH and less alkaline hydrochemistry (Altermann et al., 2009;Bristow and Milliken, 2011). It seems that such an environment was apparently prone for in vivo or early diagenetic precipitation of such aluminosilicate mineral phase as chamosite on, or in the polysaccharide sheaths covering the algae, and of muscovite in the decaying cell content (Figs. 7, 8).
Chamosite and muscovite may occur in natural environments as authigenic aluminosilicates.
They have been observed in close association with bacteria and microalgae over a range of chemical conditions. Experimental studies have shown (Ferris et al., 1986;Konhauser et al., 1993;Schultze-Lam et al., 1995;Fortin and Beveridge, 1997;Fortin et al., 1997;Konhauser and Urrutia, 1999) that biologically formed chamosite and chamositic clays start to precipitate as amorphous or poorly crystalline phases which, with time, converse continuously to more crystalline forms. The highly variable morphology of the chamosite forming the mineral coatings on the alleged Omdraaivlei microalgae, from microgranules to almost idiomorphic crystals (Figs.2-4) confirms these observations. The in vivo or very early post mortem precipitation of the aluminosilicate coatings on the Omdraaivlei algae was most probably the critical factor responsible for their unique 3-D preservation by making the cell wall resistant to lytic processes and the thalli stiff to compaction. Both chamosite and muscovite are mineral phases preferring lower oxygen fugacity and circum-neutral pH in the ambience, during formation (Harder, 1978;Merino et al., 1989). Their association with the Omdraaivlei microalgae-like organisms may therefore be an indirect evidence for their existence in dysoxic or at least not highly oxygenated environment. Generally, precipitation of metal silicates and oxides by various strains of free-living heterotrophic bacteria, cyanobacteria and diminutive green algae is a well-known phenomenon, particularly in metal-loaded, weakly acidic to circum-neutral environments (e.g., West et al., 1980;Steinberg and Klee, 1984;Köhler et al., 1994;Konhauser et al., 1994;Urrutia and Beveridge, 1994;Tazaki, 1997;Bischoff, 1997;Konhauser and Urrutia, 1999).
Interestingly, the high Ti signals obtained from SEM-EDS spectra and micro-Raman of the rods and granules exposed by HF-etching of the mineral coatings of Omdraaivlei microfossils ( Fig. 9C-E) may indicate an uptake of Ti either post mortem by particles of degraded algal mucus sheaths or in vivo by some of the mucus-associated microbes and formation of titanium biominerals (here identified by micro-Raman as mixture of anatase and rutile). Titanium is known to be sub-toxic or toxic for many microorganisms and can be neutralized through complexation by their extracellular polymeric substances (Glamoclija et al., 2009).
NanoSIMS generated elemental maps of the Omdraaivlei tubular microfossils exhibit clear relationships among carbon, nitrogen and sulfur, similar to those shown in organically preserved filamentous and spheroidal microfossils from the 0.85 Ga Bitter Springs Formation of Australia (Oehler et al., 2010). The sizes and shapes of the patchily distributed "biogenic" elements in the NanoSIMS maps of the tubular microfossils correspond also to the SEM images of the Ti-rich minute objects and may be an argument for their biogenicity (Oehler et al., 2006;Schopf et al., 2007).
The exceptional preservation of the described microalgae-like microfossils may have been the result of in vivo mineralization and fast burial in the lacustrine-volcanic depositional environment (Altermann and Lenhardt, 2012). Because of lack of (bio-)degradation of resistant components in cell walls, the fossilization potential of non-mineralized thalli of siphonalean algae is very low. Fossil remains of organic components of thalli are mostly known from carbonaceous impressions on bedding surfaces of shales. Extremely rare, 3D findings of whole organic thalli of calcareous siphonaleans preserved, like some of the Omdraaivlei thalli, due to very early silicification of the calcareous envelops of thalli, were described from marine Ordovician limestone by Kozłowski and Kaźmierczak (1968a, b). The high fragmentation of the studied microfossils and their co-occurrence with the fine volcaniclastic sediment incorporated in the stromatolitic laminae suggest that the alleged microalgae were probably not directly associated with the stromatolite-forming microbial mats during life-time, but episodically delivered by stormy winds or floods, as debris from adjacent areas to the stromatolite-sustaining lacustrine environment.

Conclusions
The mineralized tubular microfossils from the Neoarchean Omdraaivlei Formation seem to represent the earliest well-preserved microfossils with body organization characteristic for eukaryotic siphonalean organisms. Morphological and mineralogical features, along with the presence and mode of distribution of carbonaceous material in the walls and interiors of the tubular structures, indicate unmistakably their biogenicity. The anatomical organization of these microfossils is well documented and comparable to siphonous thalli of geologically much younger and modern ulvacean (Chlorophyta) or vaucheriacean (Xanthophyta) microalgae. Their thick mineral coatings indicate an early origin for the bio-induced and/or bio-mediated mineralization processes. Since all siphonous algae are oxygenic organisms and obligate aerobes (Graham et al., 2009), our findings support results of recent research advocating early oxygenation of the atmosphere or at least the existence of "oxygen oases" in the Neoarchean or even earlier environment (Buick, 1992;Crowe et al., 2013;Lyons et al., 2014;Mukhopadhyay et al., 2014;Planavsky et al., 2014;Riding et al., 2014;Satkoski et al., 2015). Our studies also create basis for recalibration of the proposed relaxed molecular clock models for siphonous green algae, currently estimated to Neoproterozoic (Verbruggen et al., 2008). The presence of siphonous microalgae-like fossils in the Neoarchean biosphere corroborates with morphological (Niklas, 2000) and conceptual (Egel, 2012) models assuming siphonous/coenocytic body plan as one of the earliest cellular systems in the evolution of life. The described microalgae-like fossils are over 1.5 byr older than the oldest known lacustrine eukaryotic fossils (Strother et al., 2011) and demonstrate the importance of non-marine environments in the evolution of early life. The evolutionary advanced character of the Omdraaivlei siphonous body plan is underscored by the presence of such anatomical traits as terminal branches, lateral appendages and reproductive structures (gametangia) characterizing most modern bryopsidalean algae. It is, however, possible that their reproduction could had also proceeded by fragmentation of thalli, which is a common reproductive strategy in some extant siphonous algae (Vroom and Smith, 2003).