Evidence for microbes in early Neoproterozoic stromatolites

Journal Pre-proof Evidence for microbes in early Neoproterozoic stromatolites

Zhongwu Lan, Shujing Zhang, Maurice Tucker, Zhensheng Li, Zhuoya Zhao PII:

S0037-0738(20)30001-4

DOI:

https://doi.org/10.1016/j.sedgeo.2020.105589

Reference:

SEDGEO 105589

To appear in:

Sedimentary Geology

Received date:

29 October 2019

Revised date:

1 January 2020

Accepted date:

4 January 2020

Please cite this article as: Z. Lan, S. Zhang, M. Tucker, et al., Evidence for microbes in early Neoproterozoic stromatolites, Sedimentary Geology(2020), https://doi.org/10.1016/ j.sedgeo.2020.105589

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© 2020 Published by Elsevier.

Journal Pre-proof

Evidence for microbes in early Neoproterozoic stromatolites

Zhongwu Lan*1,2,3, Shujing Zhang1, Maurice Tucker4, Zhensheng Li5, Zhuoya Zhao5

1

State Key Laboratory of Lithospheric Evolution, Institute of Geology and Geophysics, Chinese

State Key Laboratory of Palaeobiology and Stratigraphy, Nanjing Institute of Geology and

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2

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Academy of Sciences, Beijing 100029, China.

State Key Laboratory of Geological Processes and Mineral Resources, China University of

Geosciences, Wuhan 430074, Hubei, China.

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3

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Palaeontology, Chinese Academy of Science, Nanjing 210008, Jiangsu, China.

School of Earth Sciences, University of Bristol, BS8 1RJ, UK.

5

School of Resources and Environmental Engineering, Hefei University of Technology, Anhui

ABSTRACT

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230009, China.

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The biogenesis of stromatolites has long been discussed since many lack convincing evidence of their biological origin. This has particularly been the case with older Precambrian examples where widespread diagenetic and metamorphic processes have commonly destroyed microbial relics; in some cases however, evidence has been preserved through very early mineralisation. Precambrian stromatolites are relatively abundant but it is extremely rare that they contain calcified or dolomitized cyanobacteria and biofilms. This study provides evidence from early Neoproterozoic 1

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columnar stromatolites in the Jiuliqiao Formation from the southeastern margin of the North China Platform for fossilized extracellular polymeric substances (EPS) and cyanobacteria, along with nanospheres which may represent permineralised viruses or viral-like particles. A similar chemical composition and colour along with their embedded and cross-cutting relationships with the matrix suggest they are syn-depositional products, and not modern artefacts or the result of sample

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preparation. The interweaving of cyanobacterial filaments with detrital grains such as quartz and

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intraclasts indicates trapping, baffling and binding processes. The presence of virus-like particles

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and mineralized EPS with anhedral, fine-grained calcite suggests they may have provided initial

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nucleation sites for subsequent carbonate precipitation. This study demonstrates the use of a

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high-definition scanning electron microscope to detect fossilized (calcified) filamentous and

stromatolites;

Neoproterozoic;

EPS;

cyanobacteria;

nanospheres;

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virus/coccoid bacteria

Early

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Keywords:

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spheroidal bacteriomorphs within ancient stromatolites in order to confirm their biogenesis.

INTRODUCTION

Stromatolites are typically carbonate rocks that consist of alternating fine laminae formed through the presence of a microbial mat on the sediment surface; they are developing today in a variety of modern shallow-water depositional environments (Vasconcelos et al., 2014). They are formed by means of baffling, trapping and binding of sediment by a microbial biofilm, some dominated by filamentous 2

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cyanobacteria, along with calcification of microbes and precipitation of carbonate cement under favorable physical and chemical conditions (Riding, 2000; Allwood et al., 2009; Tang et al., 2013a; Perri et al., 2018). In the exploration of the origin and evolution of early life, stromatolites are commonly interpreted as fossil microbial mats and growth forms that represent early morphological evidence suggestive of the

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presence of life on Earth (Riding, 2000; Allwood et al., 2006; Knoll, 2012). There are

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cases of exceptional preservation, e.g., where mineralization by silicification has

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occurred prior to microbe decay, and then the stromatolites do indeed preserve

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et al., 2009; Tang et al., 2013a).

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organosedimentary fabrics regarded as relics of ancient microbial mats (e.g., Allwood

Widespread deformation and metamorphic recrystallization, particularly of

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Precambrian sedimentary rocks, however, have commonly destroyed the direct

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biological evidence within organosedimentary fabrics, such that proving the former

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presence of microbial life is challenging (Grotzinger and Knoll, 1999; McLoughlin et al., 2008; Allwood et al., 2009). Even when putative organic materials have been identified, it remains uncertain whether they actively participated in the formation of the stromatolitic laminae or were only passively present, or they represent purely abiotic carbon derived from the mantle via hydrothermal processes (Brasier et al., 2002). In the last case, the laminae in stromatolites are hard to differentiate from abiotic carbonate precipitates generated by purely physicochemical processes without the involvement of microbial activities (e.g., Pope et al., 2000; McLoughlin et al., 2008). 3

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Fortunately, stromatolites do host evidence of microbial biomineralization throughout the entire geological record, although evidence of fossilized primary microbes is usually lacking (Riding, 2000). In this regard, microscopic identification of mineralized microbial life-forms within stromatolite laminae has provided confirmation of how stromatolites form and demonstrated the participation of benthic

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microbial communities. In view of the morphological similarity, syndepositional

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planar, filamentous and spheroidal structures documented from ancient and modern

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carbonates are frequently interpreted as mineralized extracellular polymeric

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substances (EPS), and filamentous and coccoidal bacteria, respectively (Reid et al.,

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2000; Mastandrea et al., 2006; Perri and Tucker, 2007; Dupraz et al., 2009; Perri et al., 2012; Tang et al., 2013a; Sánchez-Román et al., 2008, 2014; Mettraux et al., 2014,

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2015). Given that the complex interaction between microbial metabolisms, organic

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substances and environmental factors facilitates the precipitation and nucleation of

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carbonate minerals (Pace et al., 2016), such planar, filamentous and spheroidal structures can be regarded as index signatures reflecting microbial activities in stromatolite laminae (e.g., Perri et al., 2018). Recently,

we discovered

abundant

microscopic

syndepositional planar,

filamentous and spheroidal structures within columnar stromatolites from the early Neoproterozoic Jiuliqiao Formation of the southeastern part of the North China Platform. These structures are morphologically similar to EPS, filamentous and coccoid bacteria, and viral-like particles (VLP), which thus add further biological evidence to demonstrate the biogenicity of Precambrian stromatolites and will shed 4

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light on the search for more evidence of microbial life in ancient stromatolites.

GEOLOGICAL SETTING

Sedimentary successions of presumed Neoproterozoic age are exposed in the

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Huainan region and were deposited on the southeastern margin of the North China

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Platform (Figure 1). In ascending order, the Huainan and Feishui groups overlie the

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Palaeo-Mesoproterozoic Fengyang Group and underlie the Fengtai Formation of

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purportedly Ediacaran age (Sun et al., 1986) (Figure 1). The Huainan Group is mainly

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composed of siliciclastic rocks, and is divisible into the Caodian, Bagongshan and Liulaobei formations in ascending order. In contrast, the Feishui Group is dominated

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by carbonates, and can be further divided into the Shouxian, Jiuliqiao and Sidingshan

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formations in ascending order.

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The Shouxian Formation is composed of a 35-90 m thick cross-stratified sandstone indicating a shoreface environment. The Jiuliqiao Formation consists of a 26-119 m thick micritic/stromatolitic limestone interbedded with thin siltstone layers, with local small-scale cross-bedding. It was largely deposited within a peritidal environment (Xiao et al., 2014). Microscopic and macroscopic fossils with variable morphologies have been documented from the Jiuliqiao Formation (Zang and Walter, 1992; Hong et al., 2004; Dong et al., 2008), as has been documented from other worldwide pre-Ediacaran successions (Butterfield, 2009). The overlying Sidingshan Formation is composed of ca. 300 m thick intertidal stromatolitic dolomite 5

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interbedded with chert and nodules (Cao et al., 1985). The Fengtai Formation disconformably overlies

the

Sidingshan Formation

and

mainly comprises

poorly-stratified diamictite with outsized clasts in a calcareous mudstone and dolomite matrix, suggestive of glaciogenic deposits (Wang et al., 1984). The Fengtai diamictite has been inferred to be the stratigraphic equivalent of late Ediacaran glacial

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diamictites (Xiao et al., 2004; Shen et al., 2010). The lower Cambrian Houjiashan

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Formation disconformably overlies the Fengtai diamictite, and is composed of a basal

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phosphatic conglomerate succeeded by carbonate rocks.

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No reliable radiometric ages have been obtained from the Huainan region;

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imprecise Rb–Sr and K–Ar ages (900–750 Ma) from the Huainan and Feishui groups suggest an early Neoproterozoic age (Dong et al., 2008, and citations therein). A

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Cryogenian–Ediacaran age was proposed by Xing (1989), but this has been

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challenged by recent biostratigraphic and chemostratigraphic studies (Tang et al.,

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2013b; Xiao et al., 2014). For instance, the occurrence of Sinosabellidites, Pararenicola, Protoarenicola in the Liulaobei and Jiuliqiao formations suggests a Tonian age because of similar fossil assemblages present in early Neoproterozoic rocks in Russia and India, and this is also supported by comparable Tonian δ13Ccarb patterns with Laurentia (Xiao et al., 2014).

METHODS

Fifteen stromatolite samples were collected from the early Neoproterozoic 6

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Jiuliqiao Formation within the Anhui Province, on the southeast margin of the North China Platform. Polished slabs were prepared by cutting samples perpendicular to the bedding plane, so as to detect typical internal microstructures. The nano- to micro-scale fossilized microbial bodies can only be observed under the scanning electron microscope (SEM). Before SEM imaging, samples were pre-treated to

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remove impurities and other pollutants from the sample surfaces. Acid etching and

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gold coating were not utilized in this study as etching using weak HCl (1-5%) tends to

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produce bacterial-like artefacts with the increase in strength of acid and the duration

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of etching (Kirkland et al., 1999), and gold can produce bacteria-like artefacts if the

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coating time is not appropriately controlled (Folk and Lynch, 1997). As such, ultrasonic cleaning with alcohol followed by carbon-coating was applied to freshly

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broken surfaces of stromatolite prior to SEM observations utilizing a Zeiss 1555

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VP-FESEM. The SEM was specifically manipulated to get an optimal resolution at

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50,000–200,000X magnification. It was tuned to an optimal working distance of 7–15 mm and a voltage of 10–20 kV. Semi-quantitative analyses were performed with an energy dispersive spectrometer (EDS) attached to the SEM to detect the presence of major elements in the submicron to nanometer-sized structures. The operating conditions for spot analyses were 15 kV, 120 μm and 16 mm distance with a beam diameter of 30 nm to 1 μm. Clay minerals were determined by means of X-ray diffraction (XRD) on oriented mounts of clay-sized particles. Clay minerals are minor constituents in our sample, so their signals in the XRD spectrum are undetectable for the whole rock powder analyses. Thus, acid was used to dissolve the carbonate and 7

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concentrate clay minerals. As hydrochloric acid can not only dissolve carbonate but also clay minerals (Li et al., 2008), acetic acid was used to remove the carbonate given their negligible influence on clay minerals (Yokoo et al., 2004). A 10% H2O2 solution was utilized to remove organic matter within samples. The pipette method was applied to prepare the oriented specimens on glass slides at room temperature. A

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PANalytical X'Pert PRO X-ray diffractometer with CuKα radiation was used with

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operating conditions of 40 kV and 40 mA. Scanning parameters were 3 to 28°2θ with

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a step size of 0.0167°2θ and a scan speed of 0.0711°/s. A range of 2-28°2θ was

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determined with a step size of 0.004°2θ and a scan speed of 0.006°/s. Clay minerals

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were identified mainly on the basis of correlation of position of (001) series of basal

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RESULTS

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reflections on the XRD diagrams (Moore and Reynolds, 1989; Peng et al., 2014).

Field observations show that the Jiuliqiao stromatolite unit examined is composed of two major pale brown compacted-pressure dissolved sediment horizons sandwiching grey columns with no evidence of subaerial exposure (Figure 2). The upper horizon has a thickness in the range of 5-10 cm (Figure 2A), and thicken upwards from 0.4 to 5 cm, extending laterally continuously in a near-sinuous way (Figure 2B). In the upper part, microbial laminites occur between the columns and are downwardly connected to other microbial laminites (Figure 2B). Photographs from polished slabs show that the microbial laminites commonly show crinkly/folded 8

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surfaces (Figure 2C). In some places, laminites are composed of compacted-pressure dissolved sediments containing abundant detrital quartz and carbonate intraclasts in a size range of 30-250 μm, with their long axes commonly aligned parallel to the lamina surface (Figure 2C). Between the two horizons of compacted-pressure dissolved sediments, there occur abundant columns that have heights of 25-30 cm and widths of

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2-6 cm that remain nearly constant upwards (Figure 2A, 3A). The columns are

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composed of numerous alternating thin dark and bright laminae (commonly 1-2 mm)

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curving downwards. Several columns, growing in different directions, seem to have

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branched from other columns (Figures 2B; 3B). The top of a typical stromatolite unit

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consists of small irregular columns and carbonate clasts of 2-6 cm high, aligned in varying directions and entangled with microbial laminites of 0.3-2.5 cm thick (Figure

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3C). Some of the columns show internal laminae, whereas others have no laminae.

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These irregular columns and carbonate clasts are contained in the compacted-pressure

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dissolved sediments forming “eyeball structures”. Pores and vugs of irregular morphology occur between columns where there is no sediment or cement (right parts of Figure 2A; left parts of Figure 3A, B). The laminae are dominantly composed of alternating layers of dark micrite and bright coarse-grained calcite crystals of 10-40 μm diameter. Scanning electron microscope observations show the presence of multiple thin (5-100 μm thick), commonly isopachous, curved, sinuous or folded sheets, protruding from the sample surface (Figure 4A-C). Some sheets appear to be porous structures defining a polygonal morphology (Figure 4A). Cavities are visible between individual 9

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microbial sheets, and between microbial sheets and host rocks. Stromatolites are dominated by a calcite mineralogy (Figure 4D). The sheets show an identical dark grey colour to the host rock, and have a dominant calcite composition with Ca and O contents of 81% and 12.3%, respectively (Figure 5A). In some places, the sheets show the presence of multiple elements such as O, Mg, Fe, Al, Si, K and Ca (Figure 5B). In

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contrast, the host rocks have Ca and O concentrations of 88.5% and 5.8%,

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respectively (Figure 5C). Other elements in subsidiary amounts include Yb, Fe, Si, K

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and Al, accounting for less than 7% in total. Since samples are carbon coated, the

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content of carbon could not be determined (cf, Gong et al., 2008), but signals of

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carbon are still visible in the EDX spectrum (Figure 5). The host rock, 300-500 μm distant from the microbial laminites, is dominated by dark grey, closely packed

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anhedral to subhedral crystals in the size range of 8-40 μm (Figure 4D).

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Within the microbial laminites, abundant filamentous structures of 15-135 μm

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long and 4-5 μm wide are preserved in intercrystalline pore spaces. Most of them are twisted together and aligned in varying directions (Figure 6A, F). The width of individual filaments commonly remains constant along their length. All these filaments have their ends embedded into, rather than sitting upon, the matrix (Figure 6A-F). Also present are spheroidal structures that are commonly 0.7-5 μm in diameter (Figure 7A, B, D), and nanospheres of 100-500 nm (Figure 7C, E, F). The light-coloured euhedral to subeuhedral calcite (5-30 μm) layers (Figure 7G), alternating with dark layers, contain these special structures. By means of combined SEM imaging observation and EDX composition analyses, the filamentous, 10

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spheroidal structures and nanospheres are shown to be dominated by Ca and O with concentrations of 88% and 6.8%, respectively, and they show an identical dark grey colour to the host rock and laminite structures (Figure 5D). Other minor elements such as Yb, Si, Fe and Al account for less than 6%. For samples without acid-digestion, the XRD spectra show a dominant peak of

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calcite and a secondary peak of quartz (Figure 8A). For samples after acetic acid

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digestion, the XRD spectra show a dominant peak of quartz and a secondary peak of

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calcite (Figure 8B). Also, a peak of illite is present, although less distinct (c.f. Peng et

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al., 2014).

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DISCUSSION

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The Jiuliqiao Formation is dominated by stromatolitic and micritic limestones

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which show abundant evidence of microscopic and macroscopic microbial activity (Xiao et al., 2014). The stromatolitic limestone has δ13Ccarb and δ18Ocarb values in the range of 0 to -1 ‰ and -4 to -8 ‰, respectively (Xiao et al., 2014). The δ13Ccarb values are close to early Neoproterozoic biogenic carbonate of marine origin (Derry et al., 1992; Jacobsen and Kaufman, 1999; Shields-Zhou et al., 2012), but the δ18Ocarb values are quite negative. The low negative values could reflect seawater, but the more negative ones indicate some diagenetic alteration (Shields-Zhou et al., 2012). The alternations of dark micritic layers representing mineralized microbial mats with light-coloured carbonate grain-bound layers are typical of ancient and modern 11

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stromatolites that are formed by means of microbial trapping and binding of particles (Reid et al., 2000; Riding, 2000; Perri and Tucker, 2007; Tang et al., 2013a). The filamentous structures are very similar to modern and fossilized cyanobacteria from microbial mats and stromatolites in size and morphology (e.g., Reid et al., 2000, their figure 2; Tang et al., 2013a, their figure 7C; Xiao et al., 2014,

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their figure 4C; Mettraux et al., 2015, their figures 8, 9, 11; Perri et al., 2018, their

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figure 4C). All the filaments show the same colour with the host rock with their ends

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typically embedded into calcite matrix, suggesting a syndepositional origin rather than

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a later diagenetic or a modern artefact, or inappropriate sample preparation. The

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filaments have the same calcite-dominated composition as the host micrite crystals which suggest very early calcite precipitation and exclude a later diagenetic origin,

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which would commonly result in much larger calcite crystals that would easily mask

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such a fine detailed texture. The filamentous structures are present within continuous

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and isopachous micrite laminae with no evidence of subaerial exposure, which could potentially bring syndepositional contamination. The sinuous morphology with an apparent original flexible nature supports a biogenic fossilization process rather than chemical precipitation of an inorganic mineral form, which would be expected to display sharp terminations with rigid regular crystal structures. Based on this circumstantial evidence, the filamentous structures can be interpreted as fossilized remains of syn-depositional bacterial forms (cf., Perri and Tucker, 2007; Lan and Chen, 2012a; Tang et al., 2013a; Mettraux et al., 2014, 2015). It is well known that a complex community of microbes including cyanobacteria and other forms of bacteria 12

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are present in present-day calcifying mats which induce carbonate precipitation (Riding, 2000). Carbonate precipitation inhibited the early decay of microbes, which requires the preferential entombment of filamentous bacteria by calcium carbonate upon their final death. Preservation of these bacterial bodies is ascribed to preferential calcification of the original organic microbial material by calcite.

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The planar sheet-like structures can be compared with EPS within modern and

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fossilized stromatolites in terms of size and morphology (e.g., Reid et al., 2000, their

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figure 2; Perri and Tucker, 2007, their figure 4A, B; Sánchez-Román et al., 2008, their

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figure 2C; Perri et al., 2012, their figure 7B; Tang et al., 2013a, their figure 7D, E;

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Mettraux et al., 2014, their figures 18A-E; Mettraux et al., 2015, their figure 9F; Perri et al., 2018, their figure 4A). The planar sheet-like structures are embedded in the

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matrix, cross-cut calcite crystals and have the same colour and calcite-dominated

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chemical composition as the host rock, indicating a syndepositional origin rather than

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being later diagenetic or a modern artefact. The lack of a rigid, regular crystalline morphology coupled with the folded flexible morphology point to a biogenic origin. Studies have shown that amorphous Ca-Mg-Si-Al-O-C can be initially precipitated in mats by replacing EPS, followed by their transformation into palygorskite and calcite upon diagenesis (Perri et al., 2018). As such, the presence of Mg, Al, Si, O, C, Ca and K signals in the EDX spectrum (Figure 5B) could be an indication of signals from clay mineral and calcite, as evidenced by the mineral assemblage (Figure 4E, F; Figure 7F). Yb is a kind of heavy rare earth element (HREE) which may be related to clay minerals. As such, the presence of Yb suggests the presence of clay minerals 13

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although it is a negligible amount (Figure 5; Figure 8). The presence of other elements such as Si, K and Al, coupled with the XRD spectrum, suggests the clay mineral is illite. This was probably present in the original stromatolite matrix, as commonly trapped by means of EPS-mucilage (e.g. Pace et al., 2016). The illite shows a boxwork-like structure similar to that documented previously (Gualtieri et al., 2008).

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The larger, micron-sized spheroidal structures resemble fossilized coccoid

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cyanobacteria, whereas the nanospheres, 100-500 nm in diameter are closer to the size

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of viruses or VLP, documented from modern and ancient stromatolites in terms of

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size and morphology (Perri and Tucker, 2007, their figure 3A, B; Perri et al., 2012,

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their figure 3C, D; Tang et al., 2013a, their figure 7F; Perri et al., 2018, their figures 5-7). These microorganisms are frequently regarded as important agents in carbonate

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precipitation where they could play an important role as centres of nucleation for

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subsequent crystal growth (Folk, 1999; Dupraz et al., 2004; Perri and Tucker, 2007).

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Viruses have the ability to implant their viral material into host cells where abundant new viruses are produced, causing the death of the host cell and the release of new viruses. Spheroidal viruses commonly display an icosahedral morphology with flat surfaces, as is the case of the nanospheres of the Jiuliqiao stromatolite (Figure 7C, E, F). The size of these nanospheres and their intimate spatial relationships with likely coccoid and filamentous bacterial cells make them similar to fossilized viruses (Pacton et al., 2014; Perri et al., 2018). Cyanobacterial filaments and coccoidal bacteria in microbial mats are responsible for baffling and binding sediment, organic detritus and calcium carbonate precipitates 14

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in the water column. The cohesive and sticky EPS-coated surface of cyanobacteria and coccoidal bacteria in the biofilm contributes to trapping detrital grains which then become enveloped in filaments to form the microbial laminae (Figures 2, 3). Earlier microbial laminae provide a template for the growth of the next biofilm of microbes and accommodate clasts and pellets, thus forming upward-directed growth of multiple

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laminae (Gerdes, 2007; Vasconcelos et al., 2014). Upon trapping by the

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compacted-pressure dissolved sediments, detrital grains would be adjusted by any

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current energy and gravity to stable positions with their long axes aligned parallel to

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the bedding plane, forming wavy to lenticular laminae and lamina-specific grain

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arrangements. The “eyeball structures” (Figures 2, 3) are commonly an indication of microbial response to disturbance by settling of clasts or minerals (cf., Gerdes, 2000).

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Such an active response contributes to binding the detrital particles together within the

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matrix that would finally entomb them. Hydrodynamic conditions directly influence

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the baffling and trapping activities of filamentous bacteria; this means that laminites formed within highly turbulent hydrodynamic settings tend to be thin and incorporate more detrital grains, whereas those formed within low-energy quiet settings would entrap fewer detrital grains (Gerdes, 2000). In this regard, the alternating columns with laminites in the Jiuliqiao stromatolites would point to changing hydrodynamic conditions during peritidal deposition. Although detection of organic carbon itself would be regarded as further evidence of the biogenesis of these stromatolites, in general the most common process for organic matter preservation is very early silicification (Allwood et al., 2006, 2009; 15

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Tang et al., 2013a). In the absence of an earlier silicification event, precipitation of carbonate minerals in the form of amorphous nanoparticles and nanospheres on to a cell surface (calcification) can replace the organic matter, including bacterial cells and EPS (e.g., Dupraz et al., 2004; Sánchez-Román et al., 2007; Lan and Chen, 2012b; Pan et al., 2019). The assemblage of nano- and micro- particles/spheres has been

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frequently reported as biogenic, from a wide range of modern and ancient microbial

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carbonates (Perri et al., 2018, and literature cited therein), fabrics of which strikingly

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differ from those of abiogenic origin (Hofmann et al., 2008). Also, morphological

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correlation has been taken as direct evidence of biogenesis for these filamentous and

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spheroidal structures from terrestrial and even extraterrestrial samples (Buseck et al., 2001; Brasier et al., 2002; Schopf et al., 2002). Thus, integrated petrographic,

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biological and geochemical characteristics suggest a biogenic carbonate origin for the

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stromatolitic limestone of the Jiuliqiao Formation.

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The early Neoproterozoic witnessed the flourishing of stromatolites which theoretically should all contain abundant calcified cyanobacterial microfossils but in fact they are very rare (Riding, 2000; Hoffman and Jackson, 2007). This inconsistency is unlikely to be caused by a reduction in calcification potential at this time, as previously suggested (Grotzinger, 1990), but may be because of inadequate microscopic observations, particularly using high-resolution SEM image analysis, given that calcified filamentous microbial bodies have been increasingly discovered from modern and ancient biogenic carbonates (Reid et al., 2000; Perri and Tucker, 2007; Tang et al., 2013a; Mettraux et al., 2014, 2015; Perri et al., 2018). It is predicted 16

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that with more high-definition SEM image analyses, more calcified cyanobacterial microfossils, as well as permineralised viruses or VLP, will be discovered which would reconcile the stromatolite-microfossil inconsistencies. Meanwhile, the roles of submicron to nanometer-scale spheroidal bacteria and viruses should not be underestimated, in terms of their ability to act as initial nucleation sites for further

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carbonate mineral precipitation.

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CONCLUSIONS

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Early Neoproterozoic columnar stromatolites are documented from the Jiuliqiao Formation at the southeastern margin of the North China Platform. They are

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morphologically similar to modern and ancient columnar stromatolites that typically

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consist of alternating dark micrite and light calcite-bound laminae. Abundant planar,

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filamentous and spheroidal structures have been discovered within micrite laminae that are reminiscent of fossilized extracellular polymeric substances, cyanobacteria and viruses in terms of size and morphology. The likely biogenesis of these structures is also supported by their chemical composition (Mg, Si, O, C and Ca) that suggest transformation of amorphous Ca-Mg-Si-Al-O-C into calcite and clay mineral upon diagenesis. Ductile deformation features, coupled with fabric relationships within the host rock, suggest a syndepositional origin rather than post-depositional effects or the presence of modern artefacts. New evidence of likely microbial relics assists in reconciling the inconsistencies in the occurrence of microbial fossils within 17

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stromatolites in the Proterozoic sedimentary record. The roles that viruses played in providing initial nucleation sites should be explored in comparison to filamentous bacteria. Meanwhile, high-definition scanning electron image analyses are encouraged to search for direct evidence of fossilized microorganisms in carbonate rocks.

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ACKNOWLEDGEMENTS

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Thanks are directed to Xin Yan and Jiangyan Yuan for their assistance with

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FESEM observation. The study was supported by National Natural Science

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Foundation of China (41673016), State Key Laboratory of Palaeobiology and Stratigraphy, Nanjing Institute of Geology and Palaeontology, Chinese Academy of

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Sciences (No. 193112) and State Key Laboratory of Geological Processes and

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Mineral Resources, China University of Geosciences (No. GPMR201902).

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Figure captions

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Figure 1. (A) Sketch map showing the study area within the southeastern margin of the North

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China Block. (B) Road map showing location of studied section. (C) Stratigraphic column around the study area showing the contact relationships between the Jiuliqiao and other formations.

Figure 2. Occurrences of Jiuliqiao Formation stromatolites. (A) Cross-section view of thick compacted-pressure dissolved sediments in the middle and upper parts bracketing a near-parallel layer of columnar stromatolites. (B) Cross-section view of the basal stromatolites showing the presence of thick compacted-pressure dissolved sediments within stromatolite domes. (C) Cross-section

view

of

polished

slab

showing

stromatolite

columns

entrapped

in

compacted-pressure dissolved sediments. The column is composed of alternating dark and light 26

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grey laminae. The light brown coloured materials between the columns are mixed Fe-rich carbonate and clay minerals. Pen cap in Figure A is 5 cm in length. Pen for scale in Figure B is ca. 15 cm in length. Coin in Figure C is 2 cm in diameter. Red arrowhead in all figures indicates microbial laminites.

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Figure 3. Occurrences of Jiuliqiao Formation stromatolites. (A) Cross-section view of the middle

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and upper parts of columnar stromatolites showing the presence of alternating thick and thin

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microbial laminites intercalated with carbonate laminae. Most of the columns show near-parallel

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growth. (B) Cross-section view of the top of stromatolites showing the presence of thick microbial

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laminites entangling short columnar stromatolites. Some vugs/cavities occur, possibly formed through later stage carbonate dissolution. (C) Cross-section view showing a series of near-parallel

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columnar stromatolites sitting on compacted-pressure dissolved sediments. Pen cap in Figure A is

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5 cm in length and also apply to Figure C. Hammer in Figure B is about 35 cm in length. Red

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arrowhead in all figures indicates microbial laminites.

Figure 4. SEM images showing the microscopic texture of stromatolites. (A) Cross-section view showing the presence of a porous polygonal structure which could possibly be a mineralised relic of extracellular polymeric substances (EPS), contained within a micritic calcite matrix. (B)-(C) Cross-section views showing the presence of folded-planar calcite sheets which could be mineralized relics of extracellular polymeric substances (EPS), protruding from a freshly broken sample surface. Red arrowhead indicates possible fossilized EPS. (D) Anhedral to euhedral calcite forming carbonate laminae of the columnar stromatolites. (E)-(F) Boxwork-like illite clay 27

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embedded in calcite matrix. The cross-cutting nature of the possible EPS structures (A-C) and boxwork-like illite (E-F) with the calcite matrix exclude the likelihood of them being modern artefacts.

Figure 5. EDX spectra showing the mixed clay and calcite composition of the compacted-pressure

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and filamentous, coccoidal and nanospheroidal structures (D).

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dissolved carbonate-clay sediments (A, B), and dominant calcite composition of the host rock (C)

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Figure 6. SEM images showing the microscopic texture of stromatolites. Calcified filamentous

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bacteria (red arrows) and nanospheroids (green arrows) within microbial laminites of a columnar stromatolite. The embedded nature of the filamentous and nanospheroidal structures within the

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matrix excludes the possibility of them being modern artefacts.

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Figure 7. SEM images showing the microscopic texture of stromatolites. (A), (B), (D) and (E) Calcified coccoidal bacteria. (C) SEM image showing the presence of abundant nanospheroids which could possibly be calcified viruses (or VLP), which also occur in (E) and (F), indicated by red arrowheads. The boxwork-like mineral in (F) is illite. (G) Micron-sized euhedral to subhedral calcite crystals possibly indicating continued growth after bacteria/virus mineralisation.

Figure 8. XRD patterns for the stromatolites of the Jiuliqiao Formation without acid digestion (A) and with acetic acid digestion (B).

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This manuscript contains no research interest conflict with other researches. The data does not cover experiments with secret rocks/minerals or other related materials. Once published, it can be available to all researchers.

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