Can NanoSIMS probe quantitatively the geochemical composition of ancient organic-walled microfossils? A case study from the early Neoproterozoic Liulaobei Formation

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Can NanoSIMS probe quantitatively the geochemical composition of ancient organic-walled microfossils? A

case study from the early Neoproterozoic Liulaobei Formation

Frédéric Delarue, François Robert, Romain Tartese, Kenichiro Sugitani, Qing Tang, Rémi Duhamel, Sylvain Pont, Shuhai Xiao

To cite this version:

Frédéric Delarue, François Robert, Romain Tartese, Kenichiro Sugitani, Qing Tang, et al.. Can NanoSIMS probe quantitatively the geochemical composition of ancient organic-walled microfossils?

A case study from the early Neoproterozoic Liulaobei Formation. Precambrian Research, Elsevier,

2018, 311, pp.65 - 73. �10.1016/j.precamres.2018.03.003�. �hal-01829638�

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Contents lists available atScienceDirect

Precambrian Research

journal homepage:www.elsevier.com/locate/precamres

Can NanoSIMS probe quantitatively the geochemical composition of ancient organic-walled microfossils? A case study from the early Neoproterozoic Liulaobei Formation

Frédéric Delarue

^a,⁎

, François Robert

^a

, Romain Tartèse

^b

, Kenichiro Sugitani

^c

, Qing Tang

^d

, Rémi Duhamel

^a

, Sylvain Pont

^a

, Shuhai Xiao

^d

aMuséum National d'Histoire Naturelle, Sorbonne Université, UMR CNRS 7590, IRD, Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie, IMPMC, 75005 Paris, France

bSchool of Earth and Environmental Sciences, University of Manchester, Manchester M13 9PL, UK

cDepartment of Environmental Engineering and Architecture, Graduate School of Environmental Studies, Nagoya University, Nagoya, Japan

dDepartment of Geosciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, United States

A R T I C L E I N F O

Keywords:

Acritarchs Early life Hydrogen Microfossils NanoSIMS Nitrogen

A B S T R A C T

Assessing the biogenicity of Precambrian putative remnants of life requires solid criteria. Among possible criteria, searching for evidence of pristine biological signatures and identifying various biological organic matter (OM) precursors in close association with microfossil morphology are of interest. Nano-scale Secondary Ion Mass Spectrometry (NanoSIMS) can provide a quantitative geochemical proxy at the scale of the individual microfossil but its use has remained limited because of potential analytical biases related to matrix effects and microtopography that may result in inaccurate NanoSIMS-derived measurement. No study so far has assessed whether these potential analytical biases were strong enough to preclude any identification of pristine OM degradation products and of organic precursors in ancient sediments. In this study, we characterized the geochemical composition of organic-walled microfossils from the early Neoproterozoic Liulaobei Formation in North China using NanoSIMS. The¹²CH⁻/¹²C₂⁻ionic ratio allows us to distinguishfilament from spheroid acritarchs, re- vealing the co-occurrence of two distinct pristine OM signatures that differ by their H and/or aliphatic contents.

In addition, NanoSIMS data show that morphological degradation was tightly linked to a loss of H and/or hydrogenated organic compounds in spheroid acritarchs. In contrast,in situN/C atomic ratios are homogeneous across all organic-walled microfossils studied. Although highly coherent with Proterozoic N/C atomic ratios from the literature, such homogeneity may alternatively reflect (i) a similar N content for different organic precursors or (ii) an extensive homogenization related to early degradation. Overall, these data obtained on microfossils from the Proterozoic Liulaobei Formation are the first to demonstrate that the quantitative capability of NanoSIMS can be used to track ancient OM precursors and to probe the effects of degradation on pristine OM.

Theseﬁndings open up tremendous perspectives and put forward new criteria for assessing the biogenicity of the putative early traces of life found in Archean metasediments.

1. Introduction

The emergence and widespread development of life during the Precambrian are unanimously recognized, since microfossils have been identiﬁed in numerous Precambrian geological formations throughout the world (Oehler et al., 1977; Green et al., 1988; Awramik, 1992;

Javaux et al., 2001; Altermann and Kazmierczak, 2003; Brasier et al., 2006; Javaux and Marshall, 2006; Knoll et al., 2006; Schopf et al., 2007; Sugitani et al., 2007; Javaux et al., 2010; Knoll, 2015; Sugitani

et al., 2015; Kazmierczak et al., 2016). However, proving their bio- genecity can sometimes be a contentious issue since microfossil biological morphological traits tend to vanish in response to early diagenesis and associated thermal alteration, making it diﬃcult to distinguish between biological remnants and mineral/carbonaceous biomorphs (Garcia-Ruiz et al., 2003; Brasier et al., 2006; Cosmidis and Templeton, 2016; Wacey et al., 2016). Along with the loss of morphological traits, early diagenesis and thermal alteration also cause geochemical changes recorded at the elemental scale in organic matter (OM). Indeed, during

https://doi.org/10.1016/j.precamres.2018.03.003

Received 13 July 2017; Received in revised form 14 February 2018; Accepted 4 March 2018

⁎Corresponding author at: Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie, Muséum National d’Histoire Naturelle, Sorbonne Universités, CNRS, UPMC & IRD, 75005 Paris, France.

E-mail address:fdelarue@mnhn.fr(F. Delarue).

Available online 06 March 2018

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burial, biological signatures of OM precursor(s) evolve through biode- gradation and thermal aromatization – i.e. loss of heteroatoms and aliphatic units–smoothing and, eventually, erasing pristine biological signatures when high degrees of thermal alteration are reached. Even if this geochemical pattern has been addressed at the scale of bulk OM (seeVandenbroucke and Largeau, 2007and references therein), it re- mains poorly documented at the scale of individual microfossils. In- deed, only a few studies have dealt with the characterization of the elemental and isotopic compositions of individual Precambrian microfossils (House et al., 2000; Kaufman and Xiao, 2003; Oehler et al., 2009, 2010; Alleon et al., 2016; Delarue et al., 2017), despite the potential crucial linking of geochemical with morphological preservation states to assess the biogenicity of microfossils through the distinction of various biological OM precursors, and of degradation patterns of pristine biological signature(s) (Alleon et al., 2017).

During the last decade, analytical tools, such as Nano-scale Secondary Ion Mass Spectrometry (NanoSIMS), have been developed to characterize elemental compositions at the sub-micrometer scale. Apart from stable isotope analysis, NanoSIMS has so far been mainly used to investigate the distribution of“organic elements”such as C or N across Precambrian organic-walled microfossils (Oehler et al., 2006; Wacey et al., 2010; McLoughlin et al., 2011; Peng et al., 2016). In parallel, it

has been demonstrated that the emission of secondary ionic species used to monitor elements such as C or N can also be quantitatively associated with the geochemical elemental composition of both bulk OM (Thomen et al., 2014; Alleon et al., 2016) and individual organic- walled microfossils (Oehler et al., 2009, 2010; Delarue et al., 2017). For instance, the¹²C¹⁴N⁻/¹²C2−

secondary molecular ion ratio allows the determination of the N/C atomic ratio (Thomen et al., 2014), which is often used as a proxy for OM degradation during early diagenesis (Watanabe et al., 1997; Beaumont and Robert, 1999). However, the quantitative capabilities of NanoSIMS have been little used since some analytical biases may signiﬁcantly alter¹²C¹⁴N⁻and¹²C2−emissions.

Among them, sample surface microtopography can cause variations of the emission intensity of ¹²C¹⁴N⁻ and¹²C2−, which can lead to in- accuracy of measured¹²C¹⁴N⁻/¹²C2−molecular ion ratios of up to ca.

20% in non-ﬂat objects (Thomen et al., 2014). Recently,Delarue et al.

(2017)have demonstrated that microtopography features below 10 µm do not signiﬁcantly modify the slope (used as anin situN/C proxy) of the correlation line relating the emissions of the¹²C¹⁴N⁻and¹²C2−

secondary molecular ion species. Comparison between OM samples with diﬀerent organic structure (for instance, the proportion of aliphatic and aromatic moieties) can also be problematical since the structure of OM can bias the emissions of secondary molecular ion Fig. 1.Geological map and stratigraphic column of the Proterozoic sequence in Huainan region, North China (modiﬁed fromXiao et al., 2014). Stars denote the geographic location and stratigraphic horizon from which the sample of organic-walled microfossils was collected (GPS coordinates: 32°37′51.52″N, 116°46′0.19″E).

Biostratigraphic and geochronological data are given byDong et al., 2008; Tang et al., 2013; Xiao et al., 2014; andTang et al., 2017. Pal-Mes.: Paleoproterozoic- Mesoproterozoic; Ton.: Tonian; Edi.: Ediacaran; Cam.: Cambrian; Fm.: Formation; Gp.: Group.

F. Delarue et al. Precambrian Research 311 (2018) 65–73

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species, and in turn molecular ion ratios, through the so-called“matrix effects”(Sangely et al., 2005, 2007; Lepot et al., 2013). However, there has been no systematic study investigating whether biases introduced by microtopography and matrix effects preclude distinction of the geochemical composition of organic-walled microfossil population(s) or not. In other words, if quantitative NanoSIMS-derived data are biased, it should not be possible to distinguish both precursor and degradation effects within a microfossil population using NanoSIMS quantitative parameters. On the other hand, linking geochemical proxies determined in situ with morphological preservation criteria would provide new tools to assess the biogenicity of ancient organic-walled microfossils, allowing us to differentiate various biological OM precursors and to provide evidence for degradation of pristine biological signature(s).

Here, we aim to test new criteria for biogenicity based on (i) the geochemical distinction of various biological precursors and (ii) the close association between geochemical and morphological degradations in a Precambrian microfossil assemblage. Using NanoSIMS, we investigated the geochemical composition of individual organic-walled microfossils from the early Neoproterozoic Liulaobei Formation in North China. The taxonomy and morphologies of these organic-walled microfossils have been characterized using light and electron microscopy, and both precursor (biological) and degradational features have been documented (Tang et al., 2013). Therefore, the microfossil assemblage of the Liulaobei Formation is ideal for trying to determine precursor and degradation geochemical signatures at the scale of microfossil morphological types by NanoSIMS.

2. Geological setting

The geological setting of the Liulaobei Formation and of its microfossil assemblage has been extensively described in the literature (Zang and Walter, 1992; Yin and Sun, 1994; Steiner and Reitner 2001; Tang et al., 2013). Brieﬂy, the Liulaobei Formation is located in the Huainan region in the southeastern margin of North China. In this region, the Liulaobei Formation of the lower Huainan Group conformably overlies the Bagongshan Formation and is subdivided into three members. All microfossils studied originated from shale of the upper Liulaobei For- mation (Fig. 1; see sample 11-LLB-A inTang et al., 2013), which also contains sandstone, calcareous siltstone and argillaceous limestone. The age of the Liulaobei Formation is constrained between the Mesopro- terozoic and Ediacaran, and it was probably deposited between ca. 900 and 750 Ma based on Rb-Sr and K-Ar ages (Dong et al., 2008). Organic- walled microfossils are deposited at the Virginia Polytechnic Institute Geosciences Museum (VPIGM, Blacksburg, Virginia, USA).

3. Material and methods

3.1. Microfossil isolation and Scanning electron microscopy imaging

Organic-walled microfossils were isolated from mineral matrix using HF maceration techniques (Butterfield et al., 1994). Microfossils were then pipetted andfiltered on a polycarbonatefilter (10 µm pore size), which was directly gold coated (20 nm thick) for further analyses.

Organic-walled microfossils were identiﬁed and characterized using a TESCAN VEGA II Scanning Electron Microscope (SEM) operating with an accelerating voltage of 15 kV at the Museum National d’Histoire Naturelle (MNHN) facility.

3.2. NanoSIMS analyses

Organic-walled microfossils from the Liulaobei Formation were analyzed together with reference materials (type III kerogen and resin;

Table 1) using the CAMECA NanoSIMS 50 at the MNHN during the same analytical session. Before measurements, selected microfossils were pre-sputtered using a 400 pA Cs⁺primary beam rastered over 45 × 45 µm² areas in order to remove surﬁcial contamination and

attain constant secondary ion count rates after reaching saturation ﬂuence (Thomen et al., 2014). Analyses were then carried out using a 1 pA primary current (150 µm aperture diaphragm) on smaller areas to avoid pre-sputtering edge artifacts. An electron gun was used for elec- tronic charge compensation. The secondary molecular ion species

12CH⁻,¹²C2−and¹²C¹⁴N⁻were collected simultaneously in electron multipliers. NanoSIMS raw data were corrected for a 44 ns dead time on each electron multiplier and were processed using the Limage software (developed by L. Nittler, Carnegie Institution, Washington DC, USA).

For each organic-walled microfossil analyzed, a grid of regions of interest (ROI) was drawn over the studied microfossil. Note that ROI were only drawn on areas where N was present since the polycarbonate ﬁlter does not contain N. In addition, grid cells avoided microfossil edges to exclude any microtopographic features beyond 10 µm (Delarue et al., 2017). In each cell of the grid, ¹²C2−, ¹²C¹⁴N⁻ and ¹²CH⁻ emissions were then extracted in order to study the spatial relationship between¹²C2−and¹²C¹⁴N⁻or between¹²C2−and¹²CH⁻molecular ion emissions at the microfossil scale. Using ¹²C¹⁴N⁻/¹²C2− and

12CH⁻/¹²C2−molecular ionic ratio as proxy for thein situN/C and H/C atomic ratios assumes (i) a linear spatial relationship between¹²C2−

and¹²C¹⁴N⁻or between¹²C2−and¹²CH⁻molecular ion emissions and (ii) a zero intercept. In the presence of signiﬁcant levels of N, the spatial relationship between¹²C¹⁴N⁻and¹²C2−is indeed linear but it presents a non-zero intercept, which can be probably related to the sample surface microtopography (Delarue et al., 2017). This spatial relationship between¹²C¹⁴N⁻and¹²C2−is characterized by a slope“α”, whose value is a function of the N/C atomic ratio (Delarue et al., 2017). Here, this procedure was also applied to ¹²C2− and¹²CH⁻molecular ion emissions. In the following,“αN”corresponds to the slope of the linear regression relating the emissions of¹²C¹⁴N⁻and¹²C2−and“αH”is the slope of the linear regression relating¹²CH⁻and¹²C2−. The external reproducibility (1σrep) was calculated through the determination of the standard deviation of the meanαHandαNvalues obtained for the resin standard across the whole analytical session (n = 7;Table 1). Oneσrep

was determined on the resin standard, which isﬂat and geochemically homogeneous, thus errors related to microtopography and geochemical heterogeneity are not counted twice (in the standard and in the studied organic-walled microfossils).

For each microfossil analysis, correlations between the¹²C2−and both the¹²C¹⁴N⁻and¹²CH⁻molecular ion emissions were tested using a Spearman's rank correlation (Table 2). In the presence of a signiﬁcant correlation between the emissions of secondary ions (p-values < 0.05), linear regressions were then performed to calculate the values and the standard errors of the slopes αHandαN (1σreg). An absence of sig- niﬁcant correlation between the emissions of secondary ions (p-values > 0.05) is related to large emission heterogeneities induced by geochemical heterogeneity and/or analytical biases (i.e. microtopographic variations), making it impossible to determine a representative slope at the scale of the microfossil studied. For each microfossil, the total uncertainty (1σtot) onαHandαNwas determined as follows:

Table 1

NanoSIMS-derived parameters determined on the type III kerogen and resin standards, for which corresponding bulk N/C atomic ratios and related analytical error (1σBulk) were analyzed. For each standard (“n”refers to the number of analyses), and¹²C₂⁻vs.¹²C¹⁴N⁻and¹²C₂⁻vs.¹²CH⁻correlations were tested using Spearman’s rank correlation. Systematically,p-values were sig- niﬁcant (below 0.05). Slopes of¹²C2−vs.¹²C¹⁴N⁻(αN) and¹²C2−vs.¹²CH⁻ (αH) linear regressions were calculated following the procedure deﬁned in the material and methods section. Oneσrepcorresponds to the reproducibility error.

Bulk N/C atomic ratio

1σBulk n αN 1σrep αH 1σrep

Type III kerogen

0.015 0.00012 3 0.15 0.004 0.093 0.011

Resin 0.053 0.00024 7 0.52 0.036 0.082 0.014

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= +

σ σ σ

1 _tot 1 _rep² 1_reg² (1)

The determination of the N/C atomic ratio at the scale of the individual microfossil was calibrated using theαNslopes and the bulk N/

C atomic ratios measured on the type III kerogen and the resin standards (Table 1). The observed relationship – αN= 9.8051 × N/

C + 0.0029 –is equivalent to those previously determined by Nano- SIMS onﬂattened OM (Thomen et al., 2014; Alleon et al., 2016). This is consistent with a wealth of literature data showing that the determination of the N/C atomic ratioin situis not biased by matrix eﬀects (Van Zuilen et al., 2007; Thomen et al., 2014; Alleon et al., 2016; Delarue et al., 2017). In contrast to the¹²C¹⁴N⁻molecular ionic species, the

12CH⁻one undergoes matrix eﬀects. Thus, calibration ofαHagainst the bulk H/C atomic ratio was not warranted since it would have required the use of standards sharing organic structures and H contents similar to those of the microfossils studied. αH was only used as a proxy to compare H contents and/or OM structure changes across the microfossil assemblage of the Liulaobei Formation.

3.3. Variations of N/C atomic ratio across the Precambrian

Variations of N/C atomic ratio determined in whole rock samples up to ca. 3.8 billion years old were assessed using a simple linear regression (XLSTAT; AddinSoft software). N/C atomic ratios determined in Precambrian (meta)sedimentary rocks (Schopf, 1983; Beaumont and

Robert, 1999; Jia and Kerrich, 2004; Van Zuilen et al., 2007) were considered as dependent variables whereas their ages were used as quantitative explanatory variables. About 50% of the variability observed for Precambrian N/C atomic ratios can be explained by their age (p< 0.001). The linear model followed the relationship:

= − − ×

log N C( / ) 1.28 0.38 age in( Ga) (2)

4. Results and discussion

4.1. Taphonomic and precursor geochemical signatures as revealed by NanoSIMS

The microfossil assemblage of the Liulaobei Formation is dominated byﬁlms (Fig. 2A-B), ﬁlamentous microfossils (Fig. 2C-D) such asSi- phonophycusspp. (Fig. 2A-B) and by sphaeromorph acritarchs (mainly Leiosphaeridiaspp. andSynsphaeridiumsp.;Fig. 2E–L), consistent with previous observations fromTang et al. (2013). Among sphaeromorph acritarchs, a wide range of morphological degradation was observed.

Taphonomic degradation was dominated by compression and disrup- tion, which distinguishes degraded and well-preserved sphaeromorph acritarchs occurring alone or as a colony (Fig. 2L). On the simple basis of morphological degradation and microfossil types, four microfossil populations were defined: organicfilms (labelledfilm),filaments (la- belled Fil), thin and disrupted sphaeromorphs (labeled DS) and well- Table 2

Quantitative NanoSIMS-derived parameters determined on organic-walled microfossils from the early Neoproterozoic Liulaobei Formation (Film: organicﬁlms, Fil:

filaments; DS: degraded spheroids; WPS: Well-preserved Spheroids; UOP: undefined organic particles). Note thatFig. 1X refer to organic-walled microfossils presented inFig. 1. For each microfossil,¹²C2−vs.¹²C¹⁴N⁻and¹²C2−vs.¹²CH⁻correlations were tested using Spearman’s rank correlation.p-values are considered significant if below 0.05, whereas“n.s.”corresponds top-values above 0.05. In the case of significant correlations, slopes¹²C₂⁻vs.¹²C¹⁴N⁻(αN) and¹²C₂⁻vs.

12CH⁻(αH) linear regressions and associated errors (1σregand 1σtot) were calculated following procedures described in the material and methods section.In situN/C ratios and associated error (1σN/C) were then calculated according to the linear calibration derived from standards presented inTable 1(see text for details).

12C2−vs¹²C¹⁴N⁻ ¹²C2−vs¹²CH⁻

Microfossil type (Fig. 1X) Spearmanp-value αN 1σreg 1σtot in situN/C 1σN/C Spearman p-value αH 1σreg 1σtot

Film (Fig. 1A) 0.003 0.18 0.04 0.05 0.018 0.005 < 0.0001 0.14 0.03 0.03

Film (Fig. 1B) 0.047 0.53 0.15 0.15 0.054 0.015 < 0.0001 0.09 0.02 0.03

Film 0.001 0.43 0.13 0.13 0.043 0.013 n.s. – – –

Fil (Fig. 1C) n.s. – – – – – 0.004 0.15 0.02 0.02

Fil (Fig. 1D) n.s. – – – – – < 0.0001 0.12 0.02 0.02

Fil < 0.0001 0.94 0.10 0.11 0.096 0.011 < 0.0001 0.13 0.01 0.02

DS1 (Fig. 1E) n.s. – – – – – < 0.0001 0.09 0.01 0.01

DS2 (Fig. 1F) n.s. – – – – – 0.004 0.03 0.01 0.02

DS3 (Fig. 1G) < 0.0001 0.27 0.07 0.08 0.027 0.007 < 0.0001 0.07 0.01 0.02

DS4 (Fig. 1H) < 0.0001 0.37 0.05 0.06 0.038 0.006 < 0.0001 0.03 0.00 0.01

DS5 < 0.0001 0.81 0.07 0.08 0.082 0.008 n.s. – – –

DS6 ((Fig. 1L) < 0.0001 0.26 0.05 0.06 0.027 0.006 0.007 0.08 0.03 0.03

WPS1 (Fig. 1I) < 0.0001 1.04 0.05 0.06 0.106 0.006 < 0.0001 0.04 0.00 0.01

WPS2 (Fig. 1J) n.s. – – – – – n.s. – – –

WPS3 (Fig. 1K) < 0.0001 0.63 0.10 0.10 0.064 0.010 n.s. – – –

WPS4 (Fig. 1L) n.s. – – – – – 0.005 0.10 0.02 0.02

WPS5 (Fig. 1L) 0.001 0.25 0.07 0.08 0.026 0.008 0.001 0.08 0.02 0.03

WPS6 (Fig. 1L) < 0.0001 0.36 0.04 0.06 0.036 0.006 < 0.0001 0.13 0.02 0.02

WPS7 (Fig. 1L) < 0.0001 0.44 0.06 0.07 0.045 0.007 n.s. – – –

UOP1 (Fig. 1M) < < 0.0001 1.01 0.35 0.35 0.103 0.035 < 0.0001 0.07 0.00 0.01

UOP2 (Fig. 1N) 0.007 0.92 0.13 0.13 0.093 0.013 < 0.0001 0.15 0.01 0.02

UOP3 (Fig. 1O) < 0.0001 0.48 0.04 0.05 0.049 0.005 < 0.0001 0.07 0.01 0.01

UOP4 (Fig. 1P) < 0.0001 3.22 0.46 0.46 0.328 0.047 < 0.0001 0.08 0.00 0.01

UOP5 n.s. – – – – – < 0.0001 0.06 0.00 0.01

UOP6 n.s. – – – – – 0.027 0.05 0.02 0.03

UOP7 n.s. – – – – – < 0.0001 0.06 0.00 0.01

UOP8 n.s. – – – – – < 0.0001 0.10 0.01 0.01

UOP9 n.s. – – – – – < 0.0001 0.06 0.01 0.01

Microfossil type n αN S.E. n αH S.E.

Film 3 0.38 0.10 2 0.12 0.03

Fil 1 0.94 n.d. 3 0.14 0.01

DS 4 0.43 0.13 5 0.06 0.01

WPS 5 0.55 0.14 4 0.09 0.02

UOP 4 1.41 0.61 9 0.08 0.01

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preserved sphaeromorphs (labeled WPS). In addition, some amorphous particles with no distinct morphological features, except a lack of compression, were also investigated (Fig. 2M-P; labeled UOP).

All these microfossils are carbonaceous structures as conﬁrmed by NanoSIMS images (Fig. 3). The elemental composition of each microfossil was then assessed usingαNandαHas proxies of the preservation of N and H contents, respectively, or of OM structure. Seventeen out of 28 organic-walled microfossils are characterized by a signiﬁcant spatial correlation between the¹²C2−and¹²C¹⁴N⁻(Table 2;Fig. 4). Thus, only these 17 organic-walled microfossils are considered in the following.

Excluding one outlier αN value of 321.5 × 10⁻², the remaining αN

values range from 18.2 × 10⁻² to 104.4 × 10⁻² (Table 2). Twenty three out of 28 organic-walled microfossils are characterized by a

significant spatial correlation between¹²C2−and¹²CH⁻(Table 2). In these specimens,αHvalues range between 3 × 10⁻²and 14.9 × 10⁻². SignificantαNandαHvalues can be calculated simultaneously on 13 specimens (Table 2). CombiningαNandαHvalues does not allow clear distinction of any OM precursor, except forfilaments showing higher αHvalues, and degradation effects among the analyzed organic-walled microfossils (Table 2). However, the large range ofαNandαHvalues suggests that there is significant geochemical heterogeneity among these organic-walled microfossils.

αHandαNvalues were then used to probe the geochemical composition at the scale of the microfossil/carbonaceous particle populations deﬁned on the basis of microfossil morphological types. For that purpose, we considered the standard error relative to the dispersion of Fig. 2.Organic-walled microfossils isolated from the early Neoproterozoic Liulaobei Formation. (A–B) Organic Films (Film 1 and Film 2 specimens). (C-D) Filaments (Fil 1 and Fil 2 specimens). (E-H) Degraded spheroids (DS1 to DS4 specimens). (I-L) Well-preserved spheroids (WPS1 to WPS2 specimens). (M-P) Undeﬁned organic particles (UOP1 to UOP4 specimens). Scale bar, 20 µm.

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the studied variable (αN andαHvalues) at the scale of the different organic-walled microfossil populations. In other words, if the analytical procedure was not reproducible because of analytical biases such as microtopography and matrix effect(s), differences between microfossil populations would not arise.

At the scale of the various microfossil populations,αNvalues were of ca. 38.0 ± 10.3 × 10⁻², 94.5 × 10⁻², 42.9 ± 12.9 × 10⁻², 54.6 ± 13.9 × 10⁻², and 140.6 ± 61.4 × 10⁻²in the Film, Fil, DS, WPS, and UOP groups, respectively (Table 2;Fig. 5A). As for individual

microfossils,αNvalues did not allow the distinction of any precursor or degradation eﬀects on the geochemical composition determined at the scale of the diﬀerent microfossil-type populations. In contrast toαN

values,αHvalues are more contrasted at the scale of organic-walled microfossil populations. NanoSIMS analyses yielded an average αH

value of ca. 11.5 ± 3.0 × 10⁻²; 13.6 ± 0.7 × 10⁻², 5.8 ± 1.2 × 10⁻², 8.8 ± 1.7 × 10⁻² and 7.8 ± 1.0 × 10⁻² for Film, Fil, DS, WPS, and UOP groups, respectively (Table 2;Fig. 5B).

Hence, αH values allow the distinction of both precursor and Fig. 3.NanoSIMS molecular ion images (¹²C₂⁻,¹²C¹⁴N⁻and¹²CH⁻; scale bar, 2 µm) of the Film 2, Fil 1, DS3, WPS4, and UOP4 organic-walled microfossil specimens (scale bar, 20 µm).

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taphonomic degradation effects. First, theαHmean value is higher in filamentous microfossils than in spheroid acritarchs (Fig. 5B). Since derivingin situH/C atomic ratios from measuredαHvalues has not been calibrated because of matrix effects, it is not possible to argue that these different αH values for different microfossil populations are solely caused by variations of their H content. Nonetheless, H content is a function of aliphatic moieties contained in OM, leading to emission matrix effects (Sangely et al., 2005, 2007). Thus, the differences inαH

values determined forﬁlamentous microfossils and spheroid acritarchs may point to diﬀerences in their H and/or aliphatic contents. Although searching for distinct geochemical precursor signatures has been carried out in various Proterozoic geological formations (Arouri et al., 1999; Arouri et al., 2000; Javaux et al., 2004; Marshall et al., 2005;

Igisu et al., 2009), these results are theﬁrst distinguishing such signatures at the scale of various organic-walled microfossil types within Precambrian rocks. Indeed, studying morphologically-distinct

leiospheres from the Mesoproterozoic Roper Group (Javaux et al., 2004, Marshall et al., 2005) did notfind any difference in their geochemical composition using Micro-Fourier Transform Infrared spectroscopy. In the Liulaobei microfossil assemblage, morphological differences between microfossil types are more easily recognizable, which may have favored the distinction of various geochemical signatures. In future studies, combining Micro-Fourier Transform Infrared spectroscopy with NanoSIMS should yield better constraints on the strict significance of the variation ofαHvalues.

Destruction of microfossil morphology during early diagenesis has also been reported as a taphonomic feature, and this can be expressed within a single rock sample (Turner et al., 2000). In a single rock sample, studying taphonomic heterogeneities can be considered as a way to characterize the eﬀect of early diagenesis on OM degradation since all microfossils experienced the same external geological condi- tions after deposition. As detailed above, spheroid acritarchs present variable levels of morphological preservation within the early Neo- proterozoic Liulaobei Formation (Fig. 2E–L). Degraded spheroid acritarchs are characterized by a lowerαHvalue (hence lower H/C ratios and/or aliphatic content) than well-preserved ones (Fig. 5B). These NanoSIMS results show a close association between morphological and geochemical degradation, suggesting that NanoSIMS has the potential to detect pristine OM geochemical signature and degradational history Fig. 4.(A) Relationships between the emissions of¹²C2−and¹²CH⁻and (B)

between¹²C2−and¹²C¹⁴N⁻ions in an organic-walled microfossil (seeFig. 1I for corresponding SEM image).

Fig. 5.(A)αHvalues determined for the diﬀerent morphological groups. (B)αN

values determined for the diﬀerent morphological groups (Film, Fil, DS, WPS and UOP).

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at the scale of the individual organic-walled microfossils.

4.2. Towards a framework to evaluate the signiﬁcance of in situ N/C atomic ratios

The N/C atomic ratio has often been used as a proxy for bulk OM preservation status (Watanabe et al., 1997; Beaumont and Robert, 1999). Consequently, it has also been used as a proxy for the geochemical preservation at the scale of individual Precambrian microfossils (Oehler et al., 2009, 2010; Alleon et al., 2016). As stressed before, αN values did not allow any distinction of precursors and degradation eﬀects among the organic-walled microfossil populations investigated. αN values can further be used to calculate in situN/C atomic ratios and compared to bulk and in situ N/C atomic ratios measured in Precambrian geological formations up to ca. 3.8 billion years old. There is a signiﬁcant relationship between bulk N/C ratios taken from the literature (Schopf, 1983; Beaumont and Robert, 1999;

Jia and Kerrich, 2004; Van Zuilen et al., 2007) and the age of the formations in which the OM is preserved (Fig. 6). This progressive de- crease of bulk N/C atomic ratios with time suggests a strong link with the degree of thermal carbonization, since the latter increases with age in ancient metasediments (Delarue et al., 2016). This observed linear correlation (associated with its 95% conﬁdence interval; grey band in Fig. 6) provides a basis to discuss the signiﬁcance of N/C atomic ratios determinedin situon microfossils by illustratingin situN/C atomic ratio anomalies.

N/C atomic ratios determined by NanoSIMS on organic-walled microfossils from the 0.8 Gyr-old Bitter Springs, the 3.0 Gyr-old Farrel Quarzite and the 3.4 Gyr-old Strelley Pool Formations (Oehler et al., 2009, 2010) are coherent with bulk N/C atomic ratio measured on formations of similar ages (Fig. 6). On the other hand, most organic- walled microfossils from the 1.9 Gyr-old Gunﬂint Formation (Alleon et al., 2016) present anomalously highin situN/C atomic ratios compared to literature data (Fig. 6). Raman spectroscopy suggests that all these microfossils are characterized by an aromatic structure indicating

their syngenecity (Alleon et al., 2016). Therefore,Alleon et al. (2016) argued that their high N/C atomic ratios indicate exceptionally well- preserved OM. Alternatively, these very highin situN/C atomic ratios may not be representative of the whole microfossil population/bulk OM contained within these Gunﬂint siliciﬁed metasediments.

Thein situN/C atomic ratios determined on organic-walled microfossils from the Liulaobei Formation range between 1.8 ± 0.5 × 10⁻² and 10.6 ± 0.6 × 10⁻², except for one value reaching up to 32.8 ± 4.7 × 10⁻², measured on an amorphous OM particle, and this value is outside the 95% confidence envelope defined by bulk N/C atomic ratios (Fig. 6). The medianin situN/C ratio of ca. 4.95 × 10⁻² confirms that N/C atomic ratios determined in situon the Liulaobei microfossils are highly consistent with Proterozoic bulk N/C atomic ratios (Fig. 6). These N/C atomic ratios measuredin situare also fairly homogeneous across organic walled microfossils and amorphous carbonaceous particles, and they do not seem to relate to precursor type or degradation effects, in contrast toαHderived using NanoSIMS. This is consistent with the fact that N is known to be contained in organic compounds that are easily degraded during early diagenesis stages, in contrast to H-enriched compounds that partially persist during early degradation before being further degraded during later thermal alteration (Tissot and Welte, 1978; Vandenbroucke and Largeau, 2007).

In the specific case of the Liulaobei Formation, temperature-related alteration was not intense enough to completely erase precursor and degradation effects on the H content of organic-walled microfossil populations but early degradation appears to have been strong enough to smooth any geochemical distinction related to the occurrence of N compounds through selective degradation of OM (Meyers, 1994). Al- ternatively, the observed N/C ratio homogeneity may also reflect the homogeneity of the N abundance across pristine OM precursors.

5. Conclusion

In this study, we demonstrate the utility of NanoSIMS to quantitatively probe both precursor and degradation eﬀects on the geochemical composition of a microfossil assemblage from the early Neoproterozoic Liulaobei Formation in North China. This paves the way for further investigations dedicated to Archean organic-walled microfossils.

Indeed, these can contain significant amounts of H and N at the scale of the individual microfossil as a consequence of early silicification, which can counterbalance the effect of thermal alteration on their geochemical composition. Searching for the occurrence of various OM precursors and of degradation pathways in close association with microfossil morphology can then provide a solid criterion to assess the biogenicity of controversial and putative remnants of life in Archean rocks.

Acknowledgments

The authors are thankful to J.J. Pantel for his help in sample crushing. This research is supported by ERC Grant No. 290861 – PaleoNanoLife (PI F. Robert), the Japanese Society for the Promotion of Science (a grant-in-aid No. 24654162), and NASA Exobiology and Evolutionary Biology Program (NNX15AL27G). The authors warmly thank Roger Hewins for English editing and Xiaotong Peng and Guochun Zhao for their constructive comments.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, athttp://dx.doi.org/10.1016/j.precamres.2018.03.003.

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