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| 24 May 2023
The Volyn biota (Ukraine) – indications of 1.5 Gyr old eukaryotes in 3D preservation, a spotlight on the “boring billion”
The Volyn biota, fossilized organisms with a minimum age of 1.5 Ga, werefound in cavities in granitic pegmatites from the Korosten Pluton, NWUkrainian shield. Fossilization was due to an influx of hydrothermalfluorine-rich waters, which silicified the outermost part of the organisms,thus preserving the 3D morphology. Details of the morphology (investigatedby scanning electron microscopy) show that the majority of the specimens arefilamentous, of a large variety with diameters ranging from∼ 10 to∼ 200 µm, thin filaments with typicalbranching and thick filaments with ball-shaped outgrowths and dented surface.Filaments can be straight or conical, curvilinear, or strongly curved, up tomillimeters in length, some with a central channel. Some filaments show indicationsof segmentation and are grown as sessile organisms onto substrate; others showboth intact ends, indicating a non-sessile, free-living lifestyle. Objectswith flaky morphology and agglutinating filaments are interpreted as fossilbiofilms. Other objects are hollow and show a large variety of forms;spherical objects are scarce. Infrared spectroscopy indicates the presenceof chitosan in one filament type, electron microprobe analysis of nanometer-sizedinclusions in filaments identified the presence of Bi(Te,S) minerals, andboth observations are compatible with the interpretation as fungi-likeorganisms. Stable C- and N-isotope data of bulk samples are in the range of−31 ‰ to−47 ‰δ13C and of+3 ‰ to+10 ‰δ15N, indicating possible methanogens aspart of the subsurface microecosystem. The Volyn biota indicate that at 1.5 Ga complex forms of life existed in the continental deep biosphere, wellabove the microscopic level, including fungi-like organisms resemblingeukaryotes.
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Precambrian fossils are generally not well preserved because of the absenceof skeletal parts. In addition, most Precambrian fossil record is fromsedimentary rocks with strong diagenetic or even metamorphic overprint,which destroyed much of the original morphology and in extreme cases of veryold organisms left only an isotopic signature (e.g., Alleon et al., 2018;Berbee et al., 2020). Therefore, their biogenicity is often disputedespecially when the organic matter (OM) is completely replaced, often bysilica or pyrite. A preservation of 3D morphology is very rare and requiresspecial fossilization conditions, which include first prevention of rapiddecay of the OM and then preservation of the space around the fossil inorder to preserve its original morphology. These conditions were fulfilledin pegmatites of the Volyn pegmatite field, Ukraine, associated with theKorosten Pluton. These so-called “chamber pegmatites” contain largemiarolitic cavities in which OM named (oxy)-kerite was found and in previousinvestigations interpreted as an example of abiogenic formation (Ginzburget al., 1987; Luk'yanova et al., 1992), later re-interpreted as fossilcyanobacteria (Gorlenko et al., 2000; Zhmur, 2003) from a geyser typedeposit. Ginzburg et al. (1987) give a composition of 60 wt %–76 wt % C, 5 wt %–7 wt % H, 9 wt %–23 wt % O, 8 wt %–9 wt % N, and 2 wt %–3 wt % S and an empiricalformula of C491H386O87(S)N. Gorlenko et al. (2000) and Zhmur (2003) mention masses of up to 3 kg of kerite in one of the cavities with anirregular distribution within the pegmatite.
The organisms lived in these cavities and provide an example of thePrecambrian deep biosphere. Their fossilization conditions included suddeninflux of hot hydrothermal waters in the geyser system, where magmaticfluids rich in SiF4 mixed with meteoric waters (Franz et al., 2022a),infiltration of Si–Al into the outermost layer of the fossils, and formationof dominantly clay mineral encrustations. The 1.76 Ga intrusion age of thepegmatites (U–Pb zircon; Shumlyanskyy et al., 2021) provides a maximum ageof the fossils; the minimum age of 1.5 Ga is provided by the age offormation of a breccia, which contains degraded OM, brown opal with OM,buddingtonite whose NH4 content was provided by the degraded OM, andmuscovite (40Ar–39Ar laser ablation data; Franz et al., 2022b). Anadditional argument for this age comes from the brown opal, which cementsthe breccia and contains organic matter (Franz et al., 2017, and referencestherein). Therefore, we assume that breccia formation must have occurredwhen organic matter was already present in the “chambers”. Although some ofthe miarolitic chambers collapsed, producing the muscovite–opal-bearingbreccia, other chambers are still intact and have been mined since the 1930sfor piezo quartz and until now for pegmatite minerals such as beryl andtopaz (Ivanovich and Alekseevich, 2007; Lyckberg et al., 2009, 2019).
We report here details about the morphology and the internal structure ofthe fossils, investigated by scanning electron microscopy (SEM) and electronmicroprobe analysis (EMPA), and provide stable C–N isotope and infraredspectroscopy (FTIR) data, which allow speculating about the types oforganisms. An important point is that these “micro”-fossils in many casesreach a size well above the microscopic level, with filaments of several millimetresin length. The age of the fossils is 1.5 Ga in the middle of the “boringbillion” and gives insight into the organisms of the deep continentalbiosphere.
Figure 1(a) Location of the Volyn pegmatite field in the Ukrainian shield,which hosts the Volyn biota, and sketch map of the Korosten Pluton.(b) Conceptual model for the fossilization inthe miarolitic cavities (“chambers”) in the pegmatite. Current exposure isfrom the erosion level to a depth of 600 m. Kerite is attached to the walls,often to feldspar, but also(c) on topaz and beryl crystals.(d)Fossilization produces a thin rim of Si–Al enrichment and encrustations ofclay minerals.(e) In collapsed chambers, a breccia formed with pseudomorphof buddingtonite + muscovite + opal after beryl. Decaying kerite producedNH4 for formation of buddingtonite. The 1.5 Ga Ar–Ar age of muscovite isconsidered the minimum age for the fossils.(f) View into a chamber, arrowspoint to black kerite (all images reproduced from Franz et al., 2022a).
The locality in the Ukrainian Precambrian shield is associated with theKorosten anorthosite–mangerite–charnockite–granite plutonic complex(Shumlyanksyy et al., 2012) (Fig. 1). The samples were recovered fromunderground in shaft 3 of the mine from a depth of approximately 100 m. Onesample was obtained from the mineralogical museum of the Academy ofSciences, Kyiv, and one beryl sample with kerite on beryl was collected fromthe mine tailings (Table 1). Two additional samples of topaz from the museumin Kyiv with kerite (Fig. 2) were not investigated in detail. The samplesfrom underground could be simply picked up with no need for separation fromrock matrix and were stored in plastic sample bags. Kerite has not beenfound outside the cavities, which are in the core of the pegmatite. Itexists as fine fiber masses between fragments of the wall of the cavitiesand as larger masses hanging freely on the walls, attached to feldspar andoften around topaz (Fig. 2b, c) and to beryl (Fig. 2d–j). The bottom of thecavities is covered with soft clay.
Figure 2(a) Photograph of sample no. 0, illustrating the pieces of broken,solitary kerite filaments of millimeter length.(b) Photograph of kerite filamentson topaz (white crystals with iron oxide hydroxide staining; inset shows topazcrystal with 5 cm large matchbox for scale, image diameter approximately 2 mm).(c) Filaments of different diameter on topaz (arrows; image diameterapproximately 3 mm).(d) SEM image (with combined back scattered mode) ofberyl prism surface with characteristic etch pits. Rectangle indicatesposition of(e), which shows filamentous kerite together with kerite inirregular shape (dark contrast indicates organic matter).(f) SEM image,arrows point to kerite with irregular shape.(h) Kerite filaments withbranching (arrows) in dissolution feature of beryl.(i) Kerite filament andspherical kerite (arrow) in an etch pit of beryl; rectangle indicatesposition of(j), illustrating the irregular diameter of the filament.
Sample no. 0 consists of broken filaments of several millimeters in length (Fig. 2a),and it is likely that the original length was much larger on the centimeter scale.On beryl it wasfound attached to dissolution features on the surface of the crystals, butnot only in the common filamentous form, but also in irregular shape (Fig. 2d–j) and rarely in spherical shape (Fig. 2i). Although the previous reportsmention mostly filaments with smooth surface, our new observations revealeda large variety of different types of filaments, described below.
The samples were investigated by SEM and EMPA. SEM images were obtained witha Hitachi SU8030 instrument, equipped with an EDAX EDS system with a 30 mm2 silicon drift detector (SDD) fitted with a silicon nitride window.Samples were coated with an approximately 5 nm thick Ir layer allowing forhigh-resolution imaging of the filaments' surfaces without the structure ofcommonly applied Au coating. The kerite samples without further cleaning orpreparation were mounted on Al stubs stickered with conductive carbon tabs.The beryl crystals with kerite filaments were dust-cleaned with compressedair and coated with C.
The JEOL JXA-8530F field emission electron microprobe at TU Berlin was usedto investigate mounts embedded in epoxy, but with C-coating, forquantitative results and less absorbance (compared to Ir). EMPA data forelement distribution maps of cross sections or of parts of the rim of thefilaments and flaky kerite were acquired in the wavelength dispersive modeusing an 8 kV, 20 nA beam with a probe diameter of 64 nm. Back-scatteredelectron (BSE) images were taken to select appropriate sites. Mappings weredone in stage scan modus with pixel resolution between 277 and 360 × 180 and265, with a pixel size of mostly 80 nm, and a dwell time per pixel of 200 ms. Total scan areas varied between 70 × 36 and 33.2 × 31.8 µm.
Stable isotope analysis and concentration measurements of nitrogen andcarbon were performed simultaneously with a Thermo Finnigan MAT V isotoperatio mass spectrometer, coupled to a Thermo Flash EA 1112 elementalanalyzer via a Thermo Finnigan Conflo IV interface in the stable isotopelaboratory of the Museum für Naturkunde, Berlin. Isotope ratios areexpressed in the conventional delta notation (δ13C δ15N) relative to atmospheric N (Mariotti, 1983) and VPDB (ViennaPeeDee Belemnite standard). Standard deviation of repeated measurements oflab standard material (peptone) is generally better than 0.15 ‰ for both N and C. Standard deviations ofconcentration measurements of replicates of our lab standard are< 3 % of the concentration analyzed.
FTIR absorption spectra of several small, 40–60 µm wide, translucentdark-brown fragments of kerite (sample no. 0, which showed the leastmineralization crust) were measured in the spectral range 7000–700 cm−1 at room temperature using a Bruker IFS 66 spectrometer equippedwith an IR microscope. The kerite fragments were selected under a binocularmicroscope and placed on an IR-transparent KBr plate. Spectra were taken inthe transmittance mode at a spectral resolution of 4 cm−1 with ameasuring spot diameter of 40 µm. The reference spectra were measuredthrough the same KBr plate. The time-averaged signal was collected over 200scans in both reference and sample spectra. For comparison, absorptionspectra of chitin (poly-(1,4)-β-N-acetyl-D-glucosamine) and> 75 % deacetylated chitin, or chitosan (2-amino-2-deoxy-(1,4)-β-D-glucopyranan), both produced by Sigma-Aldrich Chemie GmbH(C7170-100G, C3646-10G) from shrimp shells, were measured in several singleflattened, 30–50 µm thick transparent flakes of these materials under thesame conditions. Band assignments are based on literature comparison (Table S1).
4.1 Morphology
4.1.1 Filaments
Filaments are curvilinear with smooth surfaces and circular cross section(Fig. 3) with different types of ends (Fig. 4). Other types have astructured surface, some are conical, others strongly curved (Figs. 5, 6).Branching is typical for filaments with a smooth surface and was observed asY, T, and double-T branching (Fig. 3b, h), as multiple branching (Fig. 3c), and combined Y–T branching (Fig. 3d). Clear indications ofanastomosing filaments were not found. Multiple branching represents thebeginning of growth of filaments (Fig. 3e). In others, globular outgrowthspossibly mark the beginning of new branches (Fig. 3g). Whereas the diameterof the individual filaments can be homogeneous between approximately 10and 20 µm (sample no. 0), others (e.g., sample no. 3; Fig. 3f) show different diameters, between a few micrometers and several tens ofmicrometers. Ball-shaped outgrowths at the end of a filament occur togetherwith a conical thinning-out filament (sample no. 1; Fig. 3i). Conical,thinning-out filaments originate in Y branching from a thicker filament withconstant diameter (Fig. 3m). One object was identified with multiple conicalfilaments, with claw-like curved ends (sample no. 6; Fig. 3j, k, l). Thebottom part can be interpreted as the beginning of growth of the filaments on asubstrate, i.e., the clay mineral assemblage in the miarolitic cavities.
Most filaments are broken pieces of larger filaments, and preserved lengthis on the order of millimeters, and it can be assumed that the original length was upto centimeters. Complete filaments were observed, with one end ball-shaped, the otherend thinning out (Fig. 6i, o). Whereas the beginning of a filament is rarelyobserved, ends are frequently preserved (Fig. 4) and can be either simplyround (Fig. 4a), ball-shaped (Fig. 4b–f), rarely with oval shape (Fig. 4e),or conical thinning-out (Fig. 4g, l, m).
Figure 3SEM images of curvilinear filaments with smooth surfaces and circularcross section.(a) Overview of sample no. 0, illustrating the amount ofmaterial with homogeneous diameter of approximately 10 µm, length ofmore than 1 cm, round ends.(b) Branching with Y, T, anddouble-T junctions.(c) Multiple branching and(d) combined Y andT branching.(e) Possible multiple branching representing the beginning ofthe filaments.(f) Overview (sample no. 3) with filaments of variablediameter and(g) multiple branching (upper left) and small outgrowths(arrows).(h) Sample no. 4 with Y branching.(i) Sample no. 1 showing 3filaments, one thinning out (upper left), one with constant diameter withball-shaped outgrowths on end (below), and a slightly conical one (above).(j, k, l) Image of multiple, conical filaments with claw-like ends, growingfrom a common center; view of the same object (sample no. 6) in differentperspectives. In(k) and(l) numbers 1 and 2 identify the same beginning andend of a filament; arrows point to a fluorite crystal.(m) Y branching of athinning-out filament (arrow) starting from a filament with constantthickness. The star-like shape in the center is not branching; it showsdifferent filaments in different heights.
Ball-shaped outgrowths (Fig. 4h) and multiple ball-shaped ends (Fig. 4i)possibly mark the beginning of new branches, and balls can be situatedasymmetrically at the end of a filament (Fig. 4j). The structured surface ofthis ball-shaped end is caused by the fossilization process, as indicated bythe round pores in the surface, together with mineral incrustation (Fig. 4k). This is also seen on the surface of a 300 µm long conicalfilament fragment (Fig. 4m, n), which has a micrometer-wide rim of mineralincrustation with a homogeneous interior part (Fig. 4o).
The structured surface is only partly a result of the fossilization process.Figure 5a–f show a filament with approximately 4 mm preserved length andoval cross section (120×80 µm thick on one end), which has a dentedsurface and bulbous outgrowths (Fig. 4d). Another example of a stronglycurved filament (Fig. 4g–l) with bulbous surface, several millimeters in length andclose to 200 µm diameter, shows irregular segmentation in distancesbetween 35 and 70 µm. On the surface of the filament,relicts of a sheath are visible; partly the sheath is intact. The transitionbetween the intact sheath and the remnants exhibits a polygonal structureand circular 1–2 µm wide holes, probably caused bydecay/fossilization. Segmentation is also seen in a branched filament withapproximately 3–5 µm wide ridges (Fig. 4m, n, o). This filament has amineralized outer part of clay minerals with irregular ridges; however,where branching starts, the surface is intact. We interpret these irregularridges as irregular segmentation of the filament, accentuated and emphasizedby fossilization.
Some samples have joint occurrence of filaments with smooth, slightly, andstrongly bulbous surfaces (Fig. 6a, b) and joint occurrence of straight,slightly, and strongly curved filaments with irregular segmentation (Fig. 6c, d). The strongly bulbous filaments are transitional to outgrowths (Fig. 6d). Segmentation is indicated (Fig. 6e) and the surface can be stronglysculptured. The filaments have variable diameters from 75 µm (Fig. 6e) to approximately 250 µm (Fig. 6d, f). Some thin filaments showclear indication of segmentation (Fig. 6g, h). The strongly sculpturedsurface consists of small ball-shaped outgrowths. Joint occurrence offilaments with strongly sculptured surface and smooth surface and withslight striation perpendicular to filament length and filaments with strongsculptured surface (Fig. 6k, l, m, n) indicates that these are probablydifferent types of organisms, not different stages of fossilization.
Figure 4SEM images of ends of filaments with a smooth surface.(a) Simple roundend (sample no. 0).(b) Ball-shaped end of straight and curved filament(sample no. 3).(c) Ball-shaped end of conical filament (sample no. 1).(d)Ball-shaped end of straight filament (sample no. 5).(e) Oval-shapedoutgrowths near end of filament (sample no. 7).(f) Ball-shaped end (sampleno. 1).(g) Complete filament with one end thinning out, one with a roundend (sample no. 1).(h) Ball-shaped outgrowths and ends (sample no. 3).(i)Double ball at end of filament (sample no. 1)(j) Ball-shaped end; rectangleindicates position of(k), surface of the ball with mineral incrustationand porosity, interpreted as result of decay/fossilization (sample no. 6).(l) Thinning-out of a filament (sample no. 5).(m, n) Cone-shaped filamentin different perspective, approximately 300 µm preserved length(sample no. 6); white triangle indicates position of(o) detail of the 1–2 µm wide rim with mineral incrustation.
Figure 5Filaments with structured, rough surface.(a) Conical filament ofapproximately 4 mm preserved length, upper oval diameter (b) 440 µm × 320 µm,(c) thin end 70 µm (sample no. 5); triangles point todetails shown in(d), bulbous outgrowths, and(e, f) dented surface.(g) Strongly curved filament with bulbous surface, several millimeters in length and closeto 200 µm diameter (sample no. 5). Rectangle shows position of(h),bulbous surface with irregular segmentation in distances between 35 and 70 µm; rectangle indicates position of(i), white triangleposition of(j).(i) In the upper part of the filament, relicts of a sheathare visible (single arrow), in the lower part the sheath is intact(triangles point to the contact).(j) The transition between the intactsheath and the remnants in the lower part of the filament exhibits apolygonal structure and(k, l) circular 1–2 µm wide holes, probablycaused by decay/fossilization.(m) Branched filament with approximately 3–5 µm wide ridges (sample no. 2). Note the intact surface where branchingstarts (arrow).(n) Detail of central part of(m). Platy objects are clayminerals.(o) Similar feature of filament surface (sample no. 4) withirregular ridges, indicating irregular segmentation.
Figure 6SEM images of filaments with structured, rough surface 2.(a) Overview illustrating joint occurrence of smooth, slightly, and stronglybulbous surfaces (sample no. 5).(b) Joint occurrence of straight, slightlyand strongly curved filaments; rectangle indicates detail in(c) withirregular segmentation of the slightly curved filament. The straightfilament also shows a slight structure on the surface (lower right).(d) Joint occurrence of slightly bulbous (right) and strongly bulbous filaments,transitional to outgrowths.(e) Filament with indication of segmentation(right) and filament with strongly sculptured surface; note the small diameter(75 µm) compared to the large filament in (d).(f) Thick filamentwith bulbous outgrowths, next to thin agglutinated filaments.(g, h) Thinfilaments with indication of segmentation (white triangles).(i) Completefilament of approximately 1 mm length with strongly sculptured surface andoutgrowths.(j) Part of a filament with strongly sculptured surface.(k) Joint occurrence of filaments with strongly sculptured surface and smoothsurface, together with an irregularly shaped object (center).(l) Detail ofstrongly sculptured surface, which consists of small ball-shaped outgrowths.Note the fluorite crystal in upper right, below label(m), which shows jointoccurrence of thick filament (top) with slight striation perpendicular tofilament length, and filament with strong sculptured surface, detail shownin (n).(o) Almost 2 mm long complete filament: one thin end, one withoutgrowths.
4.1.2 Hollow objects
Some objects appear hollow (Fig. 7); one object (Fig. 7a, b) has a hollowlower part transitional into a more solid upper, strongly bulbous part. Thehollow rather irregular objects (Fig. 7c) occur together with filaments.Filaments can also be hollow (Fig. 7d–h), and the thickness of the outer rimis approximately 2 µm (Fig. 7h). This is the width of the fossilizedouter part of filaments, which we documented in the previous study (Franz etal., 2022a), and therefore we interpret the hollow objects as organisms inwhich the interior part was completely decayed during and after thefossilization process. Some of the hollow objects are bowl-shaped (Fig. 7i–n). One such object (Fig. 8) is> 1 mm large, and from the viewin different perspectives is can be seen that it is grown onto mineralsubstrate; in addition to the clay minerals fluorite is a characteristic mineraland indicates a high fluorine activity in the fossilizing fluid (Franz etal., 2022a). The base of mineral substrate is followed by an approximately10 µm thick solid rim with bulbous outgrowths.
Figure 7SEM images of hollow objects. (a) Irregular-bulbous base of astrongly sculptured object, with(b) detail of the transition (center ina); sample no. 5).(c) Irregular hollow object below filaments (sampleno. 6).(d) Hollow filament, approximately 1 mm preserved length; positionof enlarged parts in(e)–(h) is indicated (sample no. 5). The mineralized rimis 1–2 µm wide, diameter near 20 µm.(f) Bulbous outgrowthsare also hollow.(i) Filament with an attached hollow form, similar tooutgrowths, but much larger (sample no. 6).(j, k) Same object as in(i),enlarged in two different perspectives; white triangle indicates identicalpoint.(l) Hollow filament next to a filament with a central channel (sampleno. 6).(m, n) Isolated hollow bowl-shaped object in two differentperspectives; white triangle indicates identical point (sample no. 6).(o) Irregular object, partly hollow (sample no. 6).
Figure 8SEM images of> 1 mm large bowl-shaped object (sampleno. 5)(a) seen from below, grown onto mineral substrate; euhedral crystalis fluorite; white triangle indicates position of(b), enlarged part of therim. Rectangle indicates position of(c) illustrating the base of mineralsubstrate (right) followed by an approximately 10 µm thick solid rimwith bulbous outgrowths.(d) Detail of the solid rim with several fluoritecrystals.
4.1.3 Spherical objects
Most spherical objects (Fig. 9) appear as rather complete, with only someparts broken off. One object with a double-ball shape (Fig. 9a, b) is clearlygrown onto the substrate (Fig. 9c). The double-ball with remnants of asheath points to cell separation. Note the different size of the objectsfrom< 10 µm (Fig. 9m) to> 1 mm (Fig. 9g). Twosmall objects identified on the etched beryl surface appear like seeds orspores (Fig. 9l, m).
Figure 9SEM images of spherical objects.(a, b, c) Same object in differentperspective and magnification; arrows in(a) point to a sheath; the euhedralcrystal in(c) is fluorite. The object grew from a flat mineral surfaceinto a double-ball with dented surface.(d, e) Same object in differentorientation; white triangle indicates identical position; bud – buddingtonite.(f) The thickness measured at one point is approximately 6 µm.(g, h, i) Approximately 0.5 mm large object in differentperspective with mineral incrustation.(j, k) Irregular, partly hollowobject in different perspective.(l) Perfectly round object, sitting on afilament, on etched surface of beryl (compare Fig. 2d); the circular roundstructure on its top is beam damage.(m, n, o) Oval object on etched surfaceon beryl (compare Fig. 2i). The lower contrast (dark) in the central partindicates less-dense (partly hollow) material.
4.1.4 Irregular objects
Irregular, flaky objects are abundant, especially on the surface of theberyl crystal (Fig. 2e, f), but also in many samples (e.g., Figs. 6k, 7a, c,o, 8, 9j, k). They show the same fossilization features as the filamentswith a thin rim enriched in Si, Al, Ca, and P, loss of N, and oxygenation(Franz et al., 2022a). In some samples (Fig. 6f) filaments appearagglutinated by OM and we interpret these as well as the irregular objectson the beryl crystals as fossilized biofilm.
4.2 Internal structure
For investigation of the internal structure we used SEM images of brokenfilaments and other objects, as well as polished sections embedded in epoxy,investigated by BSE images including mapping of element distribution. Dataof open-pyrolysis and TEM data (Franz et al., 2022a) had shown that the OMis highly mature, amorphous oxy-kerite. Indications of an outer cell wallare absent, because the outer rim of the fossils is silicified, partly withformation of mineral incrustation.
Segmentation of filaments, which might be a characteristic phenomenon forcertain organisms and is observed in the filaments' morphology (Figs. 5g, h,6b, c, e, h), is not obvious in the cross section, but one section shows internalcracks, separating the filament in∼ 50 to 100 µm wide segments (Fig. 10a, b). A section of a bulbous fossil showscracks, which separate the individual bulbs from each other (Fig. 10g, h).
The outer rim of the filament shows the typical enrichment of Si and Al(Fig. 10b), and the inner, homogenous and not silicified part showsabundant nanometer-sized mineral inclusions (Fig. 10c). They are located in thecentral part and thus not related to the fossilization process, irregularlydistributed or in linear array of several crystals (Fig. 10e, h). Theminerals were analyzed with the EDS system, and due to their small size onthe order of a few nanometersmuch smaller than the excitation volume of the electron beam, only mixedanalyses with the organic material could be obtained (Table 2).Recalculation of the analyses without the organic compounds C, O, and Nyielded an atomic ratio of Bi : (S,Te) near1:1, indicating minerals such asingodite Bi(S,Te) or joseite Bi4(S,Te)3. The example of the bulbousfilament (Fig. 10g) with inclusions also shows a Bi(S,Te) mineral, locatedin the central part. The heterogenous BSE contrast is caused by differenttrace compounds of Fe and Cu. Element distribution of N and O (Fig. 10j, k)in a bulbous fossil, indicated by different BSE contrast (Fig. 10i), shows aninternal structure, possibly indicating a primary separation into differentcells, whereas S (Fig. 10l) shows a systematic decrease towards the rims ofthe object, as a result of decay and/or fossilization.
Figure 10BSE images of filamentous(a–f) and bulbous fossils(g, h, i),embedded in epoxy, polished thin section, and element distribution(j, k, l).(a) Part of curved filament; orientation of section is shown in rectangle(dashed lines), position of enlargement(b) in rectangle (solid lines). Opencracks (black contrast, with impurities from polishing material) indicateapproximately 50 to 100 µm wide segments.(b) Silicifiedouter rim (white contrast, irregular) and a narrow, up to 10 µm wideinner rim are interpreted as the effect of fossilization. The homogeneousappearing central part shows in the enlarged image(c) irregularlydistributed inclusions, tens of nanometers in size, of Bi–S–Te minerals.(d)Filament with two centrally oriented Bi–S–Te mineral inclusions,approximately 50 µm in length and 1–2 µm wide, enlargement shownin (e) and (f). Arrows in(e) point to straight aligned inclusions, and(f)shows irregular contrast, possibly caused by heterogeneous distribution ofFe and Cu in the Bi–S–Te minerals.(g) Bulbous fossil, with silicified rimand encrustations of chlorite and fluorite. Cracks, partly filled withepoxy, separate individual bulbs from each other.(h) Enlarged part showingirregularly distributed and aligned nanometer-sized Bi–S–Te mineral inclusions, andepoxy-filled crack.(i) Bulbous fossil with element distribution of N(j), O(k), and S(l), indicating an interior structure with possible former cellwalls. The color code goes from cold to warm: blue denotes low concentration, whilered denotes high concentration.
Table 2EDS analyses of bismuth sulfide telluride inclusions.
a Fig. 10h.b Fig. 10f inclusion in channel.c Average of 18analyses, inclusions in matrix, Fig. 10b, c.d Normalized; n.d. – notdetected.
Figure 11SEM images of broken filamentous fossils, illustrating the centralchannel.(a, b, c) Six-sided channel in filament with(a) smooth outersurface,(b) dented surface, and(c) strongly mineralized surface.(d, e, f, g) Rectangular channel;(e) is enlarged part of(d).(h) Round, slightlyirregular channel;(i) 4 µm × 6 µm wide channel on filamentwith dented surface.(j) Round channel, enlarged from(k), approximately 12 µm wide in a filament of nearly 70 µm diameter.(l) Slightlyconical end of a filament with large, round channel.(m) Two filaments,with small micrometer-wide channels.(n)Channel in a filament with sheath-like structure.(o) Two filaments withsix-sided channels.
A very characteristic feature of the filaments is a central channel (Fig. 11), observed in many but not all of the filaments. The cross section of thechannel can be six-sided (Fig. 11a–c, m), rectangular (Fig. 11d–f), or round(Fig. 11h–l). The channel diameter is variable and ranges from approximately0.5 to 25 µm in filaments with an outer diameter betweenapproximately 5 and 100 µm; examples in Fig. 11 show 5 µm with a channel of 260 nm × 550 nm(a), 50 µm with a channelof approximately 20 µm(b), 10 µm with a channel of 2.5 µm × 4 µm(c), 100 µm with a channel of 400 nm × 560 nm(d, e), and 41 µm with a channel of 14 µm(i).
4.3 Stable isotopes and C N variation
Stable isotopes of C and N were obtained from all bulk samples (Table 1); itwas not possible to determine individual fossilized objects. In addition, wedetermined OM in black opal and OM adherent to topaz (see sample list inFranz et al., 2022a).
Results ofδ13C andδ15N determination and themolar C N show a large variation (Fig. 12). Allδ13C values arenegative, and for kerite fossils vary between−47 ‰ (sample 2) and−31 ‰ (sample 1);δ15N values vary between∼ 3–4 ‰ (samples kerite 0, 4) and∼ 10 ‰ (samples 1, 3). OM associated withopal and topaz (considered “secondary”) and buddingtonite, which obtainedits N from decayed OM, is less negative and homogeneous inδ13Cwith values between−25 ‰ and−27 ‰. The C values shouldbe considered maximum values, since alteration either by deep-seatedCO2 from the mafic magmas or from meteoric waters would have increasedδ13C. The close group ofδ13C andδ15N values for secondary OM indicates that during maturation and decaythey all have reached a similar value. The variation of the N isotopes isnot correlated with the C isotopes, and there is also no correlation withC N.
Table 3Results ofδ15N,δ13C, and molar C N ofbulk kerite samples.
Figure 12(a) Results of determination ofδ13C andδ15N of Volyn biota and degraded kerite. Symbols: blue diamonds –dominantly filamentous kerite, with small amounts of flaky and spherical OM;yellow triangle – black opal with OM; blue triangle – OM adherent to topaz;green dots – buddingtonite from breccia (from Franz et al., 2017). Fields ofmodern fungi from Mayor et al. (2009) and methanogens are summarized inStruck (2012).(b) Molar C N ratio of kerite fossils and degraded OM. Rangeof C N of modern fungi from Mayor et al. (2009).
4.4 FTIR investigation
All measured FTIR spectra of morphologically different kerite fragments insample no. 0 are very similar (Fig. 13a) and resemble closely thechitosan spectrum (Fig. 13b); both spectra are dominated by two main groupsof absorption bands located in the regions of 3500–2500 cm−1 and 1800–900 cm−1. The first group consist of overlappingbroad bands due to O–H and N–H stretching vibrations, with a group ofcharacteristic narrow peaks of C–H stretching vibrations on theirlong-wavelength wing in the region of 2960–2870 cm−1 (Fig. 13; fordetailed band assignments and for spectra of chitin see Table S1). The peakin vicinity of 1650 cm−1 is diagnostic of C = O group (Wanjun et al., 2005; Coates, 2011;Loron et al., 2019); the band at 1560 cm−1 (broad shoulder near 1570 cm−1 in kerite spectra) was assigned to N–Hbending vibrations in amide group. The relatively weak band near 1420 cm−1 (1450 cm−1 in kerite) was attributed to C–H bend (Loron etal., 2019), and the sharp peak at 1380 cm−1, which was reported incellulose, chitosan, and chitin spectra, was assigned to superposition of theO–H bend (pyranose ring; Li et al., 2009) and symmetrical bend of CH3group. A band centered near 1315 cm−1 in chitin and chitosan spectradue to C–N stretching vibrations in amide group (Vasilev et al., 2019;Wanjun et al., 2005) is not observed in kerite.
Figure 13FTIR spectra of filamentous fossil compared to standard materialschitin and chitosan.(a) Complete spectra of three pieces of sample keriteno. 0, the sample with less mineralization, showing two main regions ofabsorption: 3500 to 2800 cm−1 and 1850 to 900 cm−1.(b) Standard material chitosan. Compared to chitosan the majorabsorption bands in kerite spectra are broader; the weak shoulder near 3100 cm−1 in chitosan spectrum is not present in kerite. The narrow tripletnear 2950 cm−1 is observed as doublet in chitosan, shifted to lowerwavenumbers. In the part from 1800 to 700 cm−1, kerite showsonly broad absorption, shifted towards higher wavenumbers compared tochitosan, with three superimposed distinct weak peaks at 1450,1380 and 1038 cm−1; the first is not present in chitosan, whichhas a number of distinct peaks in this region.
A broad, weak band at around 2100 cm−1 is present in spectra ofkerite and chitosan (Fig. 13), and the same type of weak bands is shown inpublished chitosan spectra (see Table S1), but not mentioned and assigned.It can probably be attributed to overtone or combination bands of pyranosering vibrations. At lower wavenumbers, in all measured spectra there is aseries of strong (1150, 1180, 1030 cm−1) and several weak bands causedby different types of C–O vibrations in polysaccharides (Nakamoto, 1997;Wanjun et al., 2005; Li et al., 2009; Coates, 2011; Loron et al., 2019;Vasilev et al., 2019).
A general observation is that in kerite spectra, compared to chitosan, allcharacteristic absorption bands of the amide group and the pyranose ringbecome broader and weaker, in agreement with earlier studies ofspectroscopic changes during chitin/chitosan degradation (Wanjun et al.,2005; Zawadzki and Kaczmarek; 2010; Vasilev et al., 2019). Nevertheless, themain absorption features caused by an amide group, diagnostic of chitosan, arestill present in kerite spectra.
5.1 Interpretation of morphological and internal characteristics
The Volyn biota show an astonishingly large variation of different types offilaments and other forms, pointing to the interpretation that differentorganisms were involved. We have already interpreted the flaky objects of OMon the surface of beryl crystals (Fig. 2e, f) as biofilms (Franz et al.,2022a). Agglutinated filaments (Fig. 6f) and the hollow object agglutinatedto a filament (Fig. 7i) can similarly be interpreted as fossilized biofilms.The sheath structure (obvious, e.g., in Fig. 5i, j) is also an indication ofthe presence of a biofilm or extracellular polymeric substance (EPS).
Some objects have a base onto which they grew (Figs. 3j–l, 8, 9a–c), and oneobject shows a hollow lower part, from which bulbous outgrowths originate(Fig. 7a, b), pointing to sessile organisms. Filaments are generallyfragmented, but a few filaments have been found with two intact ends (Figs. 4c, g, 6i, o), and we interpret these as non-sessile, free-living organisms.
Thickness of the filaments varies from≤ 10 to> 200 µm. In filaments with diameters up to approximately 30 µm,branching with thinning-out of the branch clearly shows that these arewithin-species variations (irregular diameters of filaments, Fig. 2i, j, areinterpreted as collapse structures during fossilization). However, verythick filaments with diameters in the range of≥ 200 µm with astructured, bulbous surface (e.g., Fig. 6), or conical objects (Fig. 4m) areinterpreted as different species. The length of both types of filamentsreaches the millimeter range and, since they are fragments, possibly up to centimeter length.
Branching as an indication of growth of the organisms is typical in the thinfilaments, with Y, T, double-T, and multiple branching (Fig. 3), but anastomosing was notobserved. In thick filaments with diameters near 200 µm, branching wasnot found. The ends of filaments also hint to the type of growth. Simpleround ends are rare; more typical are ball-shaped ends (Fig. 4). Ball-shapedoutgrowths along filaments are interpreted as the beginning of a branching (Fig. 4h). In the complete filaments (Fig. 4c, g) with one end thinning out, onewith a ball-shaped end, the thinning-out end is possibly the origin, theball-shaped protrusions the growing end, because ball-shaped ends are rathercontinuous in shape, from a small protrusion (Fig. 4b) to a more completeball (Fig. 4f, i). Similar protrusions were found at the end of recent,large bacterial filaments (Volland et al., 2022). However, branched,thinning-out ends of the filaments (Fig. 3j–l, m) indicate ends similar toSpitzenkörper, what in modern fungi is described as a continuous andindefinite process of cell extension (Fischer et al., 2008).
Segmentation in thin filaments (Figs. 5m, 6g, h) with distances of a fewmicrometers up to tens of micrometers is accentuated by mineralization (Fig. 5n), with irregular ridges caused by mineralization. Thick filaments do notshow a clear segmentation; the morphology is more irregular and showsrounded, polygonal structures on the surface with dimensions ofapproximately 20–30 µm (parallel to filament axis) × 35–70 µm(perpendicular to filament axis) (Figs. 5g, h, i, 6b, c). Between thepolygonal structures on the surface, remnants of a sheath are visible. Inthe cross section (Fig. 10) segmentation is clearly visible by cracks with adistance of approximately 50–100 µm.
Bulbous forms (Figs. 7a, b, 8) mark the beginning of growth of some objects,and bulbous outgrowths are very typical for thick filaments (Fig. 6, d, f),which extend into approximately 20 µm large objects, which consist ofsmaller bulbs (Fig. 6l, n). In thin filaments with typical branching, theoutgrowths are rare and more regularly ball-shaped (Figs. 3f, g, 4h),indicating one species with prominent growth by branching of thin filaments,and another species with growth by outgrowths along thick filaments.
Among the spherical objects, only the small ones with a size of a fewmicrometers (Fig. 9l–o) resemble spores or other types of seeds/fruit bodies.The irregular, large objects several hundred micrometers in size (Fig. 9d–k)do not fit into any scheme of known organisms. Similarly, there is noobvious interpretation for the large bowl-shaped and irregular hollowobjects (Fig. 8). The small double-object with a partly preserved sheath(Fig. 9a–c) grown on a substrate has some similarities with cell division.
The function of the conspicuous central channel (Fig. 11) in many but notall filaments with different shape in cross section is speculative, likelyproviding pathways for transport of components for cell extension along thefilament axis. In one example we observed a type of filling in the channel(Fig. 11g), so in the original organisms it might have been filled with aneasily degradable substance. It is not clear whether a hollow form (Fig. 7e, l)is a different phenomenon or due to special preservation conditions. Thewidth of the preserved rim is on the same order of magnitude as thesilicified rim (1–2 µm), and therefore it might just be a remnant of afilament, in which the central part was completely degraded.
Another special feature of the internal structure is the nanometer-sizedmineral inclusions of Bi–S–Te minerals (Fig. 7). The organisms were able toconcentrate these elements, either irregularly distributed (Fig. 7c) orrod-like aligned (in a bulbous object; Fig. 7h) or within the channel (Fig. 7e). It is unclear whether the relatively large Bi–S mineral with some Cu and Fecontents in the center of a thick filament in the central channel is theoriginal position of the Bi–S concentration or an effect of fossilization.Modern fungi are able to concentrate Te (and Se) as nanometer-sized crystals (Lianget al., 2020) and could be used in technology for soil mycoremediation(Liang et al., 2019). In black shales, the organophilic element Bi mightbehave similarly to Se (Budyak and Brukhanova, 2012). Biogeochemistry of Te isprobably analogous to Se (Missen et al., 2020), but little is known aboutthe link of Bi to S and Te in OM (such as in coal; e.g., Finkelman et al.,2019). The concentration of Bi–S–Te in the organisms of the Volyn biota isanother indication of fungi-like organisms, although other organisms suchas bacteria are also able to concentrate Te (Missen et al., 2020).
Remnants of cell membranes, separating individual cells, could not beidentified, and the answer to the question of whether some of the organisms weremulticellular is speculative. However, the large size of many objects of theVolyn biota already indicates that possibly they were not single-celled butmulticellular, notwithstanding that single-cell bacteria (Thiomargarita magnifica; Volland et al.,2022) can reach the size of centimeters. These macroscopic single-cell bacteria showa very simple straight filament, whereas the large objects from the Volynbiota show a much more complicated form; the surface of large filamentsshows a bulbous structure with sizes on the order of tens of micrometers(Figs. 5g–i, 6c, f, 9a, b), well visible with a polygonal network (Fig. 5j).In the internal structure we also see phenomena that could be explained asseparate cells, such as the gaps in a filament (Fig. 10a) or in a bulbousobject (Fig. 10g). The interior structure visible in the elementdistribution of N (Fig. 7j) might indicate the original distribution informer interior cell walls, in which chitin-like substance was concentrated.Finally, the small spherical object shown in Fig. 9a and b might be taken astwo cells, with an envelope of a sheath.
5.2 Stable isotopes
Modern fungi show a very wide variation ofδ15N from−5 ‰ to+25 ‰, with the main clusterbetween−5 ‰ and+12 ‰, andδ13C is restricted to−19 ‰ to−29 ‰δ13C, with the main cluster at−22 ‰ to−28 ‰δ13C (Mayoret al., 2009; Fig. 12a). Whereas the N-isotopic signature of kerite isconsistent with the interpretation as fossil fungi, the C-isotopic signatureis much lower than that of modern fungi. However, fungi live fromconsumption of organic matter, and the C-isotopic signature is thentransferred to the fungi without a strong isotopic effect (Peterson and Fry,1987). That is, during incorporation of carbon from modern plants to fungi, theδ13C signature of−27 ‰ to−30 ‰ in plants changes to−25 ‰ to−27.5 ‰δ13C in fungi (e.g., Högberg etal., 1999). Assuming that the isotope fractionation in the Volyn biota wassimilar, the consumed organism had a C-isotopic signature of ca.−35 ‰ to−50 ‰δ13C. Thesevery low values are consistent with the interpretation that the primaryorganisms were methanogens. Another factor, which must be considered, isintracellular heterogeneity as observed in bacteria (Lepot et al., 2013).The membrane (lipids) can have a signature of 10 ‰δ13C lower than the bulk cell, and degradation duringfossilization of the proteins and polysaccharides can lower the nowdetermined C signature. It is also possible that the fungi consumed biofilm.Fossil biofilms of the 2.75 Ga Hardey Formation (Australia), probablycoexisting with methanogens, methanotrophs, and sulfur-metabolizing bacteria,haveδ13C of−55 ‰ to−43 ‰ (Rasmussen et al., 2009), well in the range ofδ13C values observed here. The biofilms, described by Rasmussen et al. (2009), lived in synsedimentary cavities similar to stromatolites, pointingto the importance of cavities for the preservation of organic matter,similarly to the biofilms at Volyn in the deep biosphere.
Maturation clearly affects the C- and N-isotope ratios, which we see indegraded OM preserved in black opal, in OM adherent to topaz, andbuddingtonite which obtained its NH4 from OM. These samples have muchmore positiveδ13C values around−26 ‰ andmore homogeneousδ15N values near+1.5 ‰ to+3 ‰ (Fig. 12a). In contrast, the large variation ofδ15N between 3 ‰ and 10 ‰ in the kerite samples (Fig. 12a) and C N between 10and> 50 (Fig. 12b) possibly indicates a variation of thespecies. These values were less influenced by maturation, as there is nocorrelation betweenδ13C and C N in all samples (fossils anddegraded OM). Alleon et al. (2018) in their description of the 3.4 Gyr oldStrelley Pool microfossils (Western Australia) argued that though thefossils experienced heating up to 300 ∘C, the C N did not changesignificantly. Also, for anthracite coal it has been shown that the originalC N did not vary with coalification (Gosh et al., 2020).
Loron et al. (2019) reported fossil fungi from the 1 Ga Grassy Bay Formation,Canada, and provided proof via chitin remnants (FTIR) and showing thecharacteristic bilayered fungal cell walls (TEM data). However, the few SEMimages for the Grassy Bay biota do not allow a comparison with the Volynbiota. Following their discussion, the FTIR investigation of the filamentousVolyn sample shows good indications for preserved chitosan as part of theOM. Degradation studies of chitosan (Wanjun et al., 2005; Zawadzki andKaczmarek; 2010; Vasilev et al., 2019) showed that the spectra of kerite havethe same characteristic bands as chitosan at approximately 250 ∘C; at lower as well as at higher temperatures these bands disappear.Completely independent temperature estimates for the fossilization based onphase equilibria of Be minerals yielded the same temperature range (Franz etal., 2017).
5.3 Taxonomy and comparison with Precambrian biota
Film-like microfossils were described from the 3.4 Gyr old Strelley Pool(Western Australia; Alleon et al., 2018), the 3.3–3.5 Gyr old OnverwachtGroup (Australia; Westall et al., 2001), and from the 2.75 Gyr old HardeyFormation (Australia; Rasmussen et al., 2009). There is little doubt thatbiofilms existed for a long time in the Earth's history and are an integralcomponent of the ancient life cycle (Hall-Stoodley et al., 2004). It seemssafe to assume that the irregular (Fig. 2f, and images in Franz et al.,2022a) and sheath-like structures (Figs. 5i,j, 6f, 9a) of the Volyn biotawere biofilms.
We have already pointed out that some of the organisms show analogies tofungi. Based on the molecular clock technique, Wang et al. (1999) estimatedthe divergence between the three-way split of the animal–plant–fungikingdoms at1.58±9 Ma, much earlier than the “Precambrian explosion”.This age is in the same range as the minimum age of the Volyn biota. Othermolecular clock estimates indicate that the first zygomycetous fungioccurred on Earth during the Precambrian, approximately 1.2–1.4 Gyr ago(review in Krings et al., 2013). Diversification of fungi and transition toland was dated at ca. 720 Ma (Lutzoni et al., 2018), and they estimate theorigin of fungi at ca. 1240 Ma, similarly to Berbee et al. (2020), whoplaced the origin of fungi at ca. 1300 Ma. If indeed the Volyn biota containfungi-like organisms, their origin as well as colonization of land occurredearlier than ca. 1500 Ma.
Bengtson et al. (2017) reported fungus-like organisms in the 2.4 Ga OngelukFormation (South Africa) from the deep biosphere, which are however notterrestrial but marine. The important fact is that these fossils were foundalso in open cavities, though of a completely different size of millimeter amygdalesin low-grade metamorphic basalt, in contrast to the huge cavities of tens ofmeters size in the pegmatites from Volyn. The filaments from the Ongelukbiota with a diameter of ca. 2 to 12 µm are generallythinner than the Volyn biota and show anastomosis, but also Y andT branching, and sometimes bulbous protrusions, 5–10 µm in diameter.A special feature is what Bengtson et al. (2017) call “broom structure”,diverging filaments growing from a substrate of clay minerals (chlorite),and the filaments consist also of the same type of chlorite. Thesestructures (shown in 2D in thin sections) could be similar to the objectfrom the Volyn biota (Fig. 3j, k, l) and what we called “multiplebranching” (Fig. 3c, e, g). A significant difference between the two biotais the fossilization process, which resulted in the Ongeluk biota incomplete replacement of the filaments by clay minerals, whereas at Volynfossilization is restricted to the outermost rim and most of the C ispreserved (Franz et al., 2022a).
Good evidence of fungi-like organisms was reported from the early-Ediacaran Doushantuo biota, at approximately 635 Ma (Gan et al., 2021).These fossils are pyritized, but with remnants of organic matter, andconsist of branching filaments (Y, T branching, but also with A and H typeand anastomosis) and associated hollow spheres. Compared to the Volyn biota,the filaments are thinner (two types, one with average 6.8 µm, onewith average 2.7 µm), whereas the observable length in thin sectionwith hundreds of micrometers is possibly in the same range as in the Volynbiota. The spheres of the Doushantuo biota are hollow and coaxially alignedbut also similar to what we described as ball-shaped outgrowths; their sizevaries from an average of 16 to 20 µm in small ones and largespheres with 36 to 102 µm, similarly to the Volyn biota(Fig. 4h, i for the small spheres, Fig. 4j for large spheres). The fact thatthe spheres of the Doushantuo Formation are hollow is possibly due to thefact that they are mostly pyritized; i.e., most of the organic matter wasdecomposed. The small spheres were interpreted (Gan et al., 2021) aspossible spores; the larger ones were possibly symbiontic organisms livingtogether with the fungi.
Myxomycetes (slime molds) are other possible eukaryotes, which might haveexisted in the Proterozoic, although Stephenson et al. (2008) considered 50 Myr as the oldest fossil record. Their diverse morphology during thedifferent stages of their life cycle including amoeboid forms leaves muchroom for speculation. Filamentous millimeter-long sporocaps, such as shown in Fig. 3a in Rikkinen et al. (2019), are similar to what we see in Fig. 4b. Thestructured surfaces shown in Fig. 6 are somehow similar to what Dagamac etal. (2017) showed in their Figs. 7–9 from recentArcyria complex, though on the micrometer scale, whereas those from the Volyn biota are much larger. The image ofmultiple, conical filaments with claw-like ends, growing from a commoncenter (Fig. 3j, k, l), is similar toCopromyxa protea shown by Schnittler et al. (2012) intheir Fig. 4-2. Hollow objects (Fig. 7, i–k, m, n) resemble open sporocapsofLicea sp. (Schnittler et al., 2012, in their Fig. 5-12). Finally, largeobjects such as the open, bowl-shaped one with bulbous outgrowths (Fig. 8)could be interpreted as the plasmodium of a myxomycete with beginningdevelopment of fruiting bodies (e.g., Fig. 2, life cycle of myxomycetes,transition from stage H1 to A; Stephenson and Schnittler, 2016).
Other possible organisms described from the Precambrian are all different from the Volyn biota and are excluded as possible analogs. Palynomorphs, which are among the earliest clear records of terrestrial life (Wellman and Strother, 2015), the 1.67 Gyr old eukaryotic Changcheng biota (Miao et al., 2019), or vase-shaped metazoan microfossils, considered as the oldest evidence for heterotrophic protists (e.g., Urucum Formation, Brazil; Morais et al., 2017), have a very different morphology.
Most of the Precambrian biota listed in the literature are consideredphotosynthetic organisms, probably not a likely analog for the Volyn biota.For example, the 770 Ma (Cryogenian) Chichkan Formation in Maly Karatau, Kazakhstan(Sergeev and Schopf, 2010), contains biota in fine-grained black chert, whichwere deposited in a mid-shelf and a near-shore environment withstromatolites. Most of the biota listed by Sergeev and Schopf (2010) arecyanobacteria, rather small mostly up to the 10 µm range and thus donot serve as analogues for the Volyn biota. They also list a number oflarger protista (incertae sedis) in the 100 µm range, however with littlemorphological similarity to the Volyn biota. No similarity was found toeukaryotes (acritarchs) from 1.1 Gyr old Taoudeni Basin, Mauretania (Beghinet al., 2017). Red algae (Rhodophyta) from the 1.05 Ga Hunting Formation,considered among the oldest eukaryotes (Butterfield, 2000; Gibson et al.,2018), are photosynthetic organisms and can also be excluded.
5.4 Model for a Precambrian deep biosphere ecosystem
The Volyn occurrence is a well-preserved example of a fossil ecosystem ofthe deep continental biosphere. We exclude an abiotic origin as previouslypostulated (Ginzburg et al., 1987; Luk'yanova et al., 1992) because of theextremely lowδ13C values and the large variation inmorphology. Abiotic pseudofossils have been produced experimentally, e.g.,by Nims et al. (2021) and references therein, when sulfide is oxidized inthe presence of organics. These “organic biomorphs” show a large variety ofmorphologies, mostly filamentous, but also globular. In a siliceousenvironment (for many cases chert) such organic biomorphs can be replaced bysilica, and their morphology can be well preserved. However, for the Volynbiota such a sulfide-rich environment did not exist. Additionally, we takethe presence of chitosan as another indication of a true fossil. McMahon (2019) provided another example of pseudo-fossils, which is howeverrestricted to an iron-rich environment; these pseudo-fossils consist ofhematite or iron oxides–hydroxides, conditions not realized in the highlydifferentiated pegmatites, which are very poor in Fe. Rouillard et al. (2018) produced another type of pseudofossils with an amazing large varietyof morphologies, which might occur in hydrothermal, silica-rich rocks, butrequire a high activity of Ba, for which there is no indication in theVolyn pegmatites.
In combination with textural arguments, the age determination of muscovite,formed in pseudomorphs after beryl, points to a minimum age of 1.5 Ga (Franzet al., 2022b); the maximum age is restricted by the intrusion of theigneous rocks at 1.760 Ga (Shumlyanksyy et al., 2021).
The geological context argues for a continental, terrestrial environment,because the KPC intruded into continental crust most likely in awithin-plate tectonic setting (Shumlyanskyy et al., 2012, 2017). Afterintrusion uplift to the erosion level occurred, documented by anunconformity, sedimentation started with sandstones and shales atapproximately 1.4 Ga (Zbranki Formation; Gorokov et al., 1981), later thanor coeval with the pseudomorph formation and the minimum age of themicrofossils. The depth, where the organisms lived, is an open question, butthe occurrence in the underground mines indicates a depth of up to at least150 m. The age of 1.5 Ga is much later than the Great Oxidation Event of theEarth's atmosphere, allowing for the evolution of complex species andecosystems on the land (sub)surface. The supply of organic matter to theunderground for the production of the high amounts of kerite is speculative.In a geyser system, which we invoke for the whole geological situation,intense growth of organisms at the surface is a common observation. In suchsystems continuous exchange between surface and depth is evident. This alsoexcludes the very deep (more than several hundreds of meters) biosphere. Thebiota were more likely located near to the surface. Unfortunately, noinformation is available right now on which of the many pegmatites from theVolyn pegmatite field contain kerite and which – in what depth – aredevoid of kerite. This remains to be investigated in the future.
Drake et al. (2017) reported partly mineralized fungi from the deepcontinental (granitic) biosphere (up to 740 m). The fossilization processalso included maturation of the OM and final mineralization by clayminerals. The source of carbohydrates was living or dead bacterial biofilms,similar to what we speculate about the Volyn biota.
The large size of the filaments up to a centimeter in length is atypical for bacteriaand archaea. Although Volland et al. (2022) described recent centimeter-longbacteria, these are still the exception, and it is more likely that some ofthe Volyn biota were multi-cellular eukaryotes. Their suggested age of 1.5 Ga is the age range given for the first appearance of eukaryotes (see reviewin Butterfield, 2015). Putative centimeter-sized Precambrian fossils (different fromthe Volyn biota) were reported from the 2.1 Gyr old Francevillian biota (ElAlbani et al., 2014); however, they are completely pyritized and occur indiagenetically overprinted black shales, which makes the interpretationdifficult.
The Volyn biota must have been highly radiation resistant, because aU–Th–K-rich granitic–pegmatitic system has a high radiation level. There area number of different organisms, such as bacteria (e.g.,Deinococcus radiodurans), archaea(Thermococcus gammatolerans) or microscopic fungi (e.g.,Cladosporium sphaerospermum), which fulfill this requirement; see reviewin Matusyak (2016). During the mining operations in Soviet times, a high Rncontent was measured inside cavities, when they were broken into. Thegeneral radiation levels, 3000 times higher than the allowed limit at thattime, were even higher 1.5 billion years ago. Deeply black-colored quartzcrystals in the pegmatites are of the “morion” type and also indicate highradiation. Recent observations at the Chernobyl power plant have led tospeculation about radiotrophic fungi (e.g., Matusiak, 2019; Prothmann andZauner, 2014), which produce melanin as a protection against radiation andenhancement of fungal growth via capture of ionizing radiation for energyconversion (Dadachova et al., 2007; Tugay et al., 2017). Mycoremediation isat least a well-documented mechanism for a very effective method of radionuclide pollutant removal considering the versatility of fungi in terms oftheir ecology, nutritional modes, adaptability, morphology, physiology, andmetabolism (Shourie and Vijayalakshmi, 2022). Fungi are known asextremophilic organisms (e.g., Blachowicz et al., 2019), and we can expectthat in the Proterozoic or possibly already earlier in the Earth's history similarorganisms were active and resistant to a high radiation level, in an epochwhen the ozone layer was not yet fully developed.
The exceptional 3D preservation of the 1.5 Ga Volyn biota is due to thefossilization conditions in open cavities, with SiF4-rich fluids as thedriving agent. There are a number of indications that fungi-like organismswere likely an important part of the microecosystem – filaments with branching(though not anastomosing), growth in thinning-out ends, and also in bulbousextrusion, both at the end of filaments and along the filaments. Sheath-likestructures are clearly visible, and there are good indications of a formerbiofilm and extracellular proteinic substance. The large size and internalstructure of the organisms and the segmentation visible on thick filamentspoints to multicellular organisms, and the nano-sized inclusions of Bi(S,Te)crystals have an astonishingly good analog in recent fungi. Other organisms,which might have been present in this subsurface microecosystem, aremyxomycetes or myxomycete-like. The stable N- and C-isotopic signature is inaccordance with such an interpretation.
The fungi-like organisms possibly lived from lithotrophic methanogens;alternatively or additionally bacteria such as cyanobacteria weretransported from the surface downwards into the cavities. The geyser systemof the Korosten Pluton provided an ideal framework for growth of bacterialor algal organisms at the surface. In the deep biosphere, attached as wellas free-living forms of organisms are observed.
The Volyn biota show that fungi-like organisms developed before 1 Ga (Loronet al., 2019), and support the speculation that the fossils from the 2.4 GaOngeluk Formation were fungi-like organisms (Bengtson et al., 2017).Molecular clock data, especially the three-way split of the kingdomsanimals–plants–fungi at1.58±9 Ma (Wang et al., 1999), are stilluncertain, but our data indicate that it must have occurred early in theProterozoic.
The Volyn biota also prove that a deep continental biosphere was alreadypresent in the early Mesoproterozoic/late Paleoproterozoic. It is known thatin the subseafloor environment microbial life existed in the Archean(Cavalazzi et al., 2021), as described from the 3.4 Gyr old Onverwacht Groupof the Barberton greenstone belt, but from the continental environment thishas not yet been reported.
All data are as tables in the text and in the Supplement.
The supplement related to this article is available online at: https://doi.org/10.5194/bg-20-1901-2023-supplement.
Concept, writing, interpretation, EMPA and SEM data acquisition: GF; IR spectra, writing: VK; sampling and geological information: VC, PL; stableisotopes: US; SEM – UG; EMPA – JN.
The contact author has declared that none of the authors has any competing interests.
Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
We thank three anonymous reviewers for helpful and constructive suggestions,Tina Treude for editorial handling, Martin Schnittler for discussion andliterature about myxomycetes, Frederik Börner for his advice about Te biogeochemistry, and Felix Haesler for discussion about fungi.
Alexander von Humboldt foundation provided support for Vladimir Khomenko during his stay inBerlin in 2022–2023.
This paper was edited by Tina Treude and reviewed by three anonymous referees.
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