Comparative Multi-Scale Analysis of Filamentous Microfossils from the ∼850 Ma Bitter Springs Group and Filaments from the ∼3

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Accepted Manuscript Journal of the Geological Society

Comparative multi-scale analysis of filamentous microfossils from the ∼850 Ma Bitter Springs Group and filaments from the 3460 Ma Apex chert

David~ Wacey, Kate Eiloart & Martin Saunders DOI: https://doi.org/10.1144/jgs2019-053

Received 27 March 2019 Revised 16 May 2019 Accepted 20 May 2019

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Comparative multi-scale analysis of filamentous microfossils from the ~850 Ma

Bitter Springs Group and filaments from the ~3460 Ma Apex chert

Abbreviated title: Multi-scale analysis of Precambrian microfossils

David Wacey1*, Kate Eiloart1, and Martin Saunders1,2

1Centre for Microscopy, Characterisation and Analysis, The University of Western

Australia, 35 Stirling Highway, Perth, WA 6009, Australia.

2School of Molecular Sciences, The University of Western Australia, 35 Stirling

Highway, Perth, WA 6009, Australia.

*Correspondence ([email protected])

MANUSCRIPT ABSTRACT: Filamentous microfossils belonging to Cephalophytarion from the 850

Ma Bitter Springs Group have previously been used as key analogues in support of a

biological interpretation for filamentous objects from the 3460 Ma Apex chert. Here

we provide a new perspective on this interpretation by combining Raman with

correlative electron microscopy data from both Cephalophytarion and Apex

specimens. We show that, when analysed at high spatial resolution, the Apex

filamentsACCEPTED bear no morphological resemblance to the younger Bitter Springs

microfossils. Cephalophytarion filaments are shown to be cylindrical, comprising

chains of box-like cells of approximately constant dimensions with lateral kerogenous

walls and transverse kerogenous septa. They exhibit taphonomic shrinkage and

folding, possess fine cylindrical sheaths and are permineralised by sub-micrometric Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

quartz grains. They fulfil all established biogenicity criteria for trichomic

microfossils. In contrast, Apex filaments do not possess lateral cell walls, are not

cylindrical in nature, and vary considerably in diameter along their length. Their

kerogenous carbon does not have a cell-like distribution and their chemistry is

consistent with an origin as exfoliated phyllosilicate grains. This work demonstrates

the importance of high resolution data when interpreting the microstructure, and

origins, of putative Precambrian microfossils.

Abstract end

Filamentous microstructures found within hydrothermal black chert veins intruding

the ~3460 Ma Apex Basalt of Western Australia have been at the centre of a long

running and high profile controversy surrounding Earth’s oldest cellular life. First described and interpreted as an assemblageMANUSCRIPT of at least 11 species of filamentous prokaryotes (Schopf & Packer 1987; Schopf 1993), doubts were subsequently raised

over the authenticity of these objects (Brasier et al. 2002, 2004, 2005, 2006, 2011) but

a biological interpretation was in turn vigorously defended (Schopf et al. 2002, 2007;

Schopf & Kudryavtsev 2009, 2012, 2013). More recently, evidence for an abiotic

origin was provided by two studies (Brasier et al. 2015; Wacey et al. 2016a) that used

electron microscopy to show that many Apex filaments comprise elongated stacks of angularACCEPTED aluminosilicate grains onto which carbon could have migrated. These authors suggested that the morphology and chemistry of the Apex filaments could be

explained by the hydration, heating and exfoliation of mica minerals, plus the

redistribution and adsorption of barium, iron and carbon within an active

hydrothermal system, a scenario consistent with the hydrothermal feeder vein Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

geological setting (Brasier et al. 2005, 2011) of the host rock. One putative Apex

species (Eoleptonema apex) has also convincingly been shown to be a carbon filled

intra-granular crack (Bower et al. 2016). Most recently, contrary to these data,

proponents of a cellular origin have inferred that multiple biological metabolisms are

represented within the assemblage of filamentous objects based on in situ carbon

isotope data (Schopf et al. 2018).

Here, we focus on the most fundamental line of evidence that must be provided to

assign a microfossil origin to an object, the possession of plausible biological

morphology. In so doing, we revisit previous claims that the morphology of Apex

filaments is comparable to younger definitive Precambrian permineralized

filamentous organisms (e.g., Schopf et al. 2010). In a series of papers (Schopf &

Kudryavtsev 2005, 2009; Schopf et al. 2007), filamentous organisms of the genus Cephalophytarion from the 850 Ma BitterMANUSCRIPT Springs Group were used as morphological analogues for Apex filaments, especially for Apex filaments assigned to the genus

Primaevifilum which are reported to be the most abundant in the assemblage (Schopf

1993) and from which most data have been presented (e.g., Schopf & Kudryavtsev

2012). This contribution provides correlative light microscopy, Raman micro-

spectroscopy, and higher spatial resolution electron microscopy data from

Cephalophytarion filaments from the Ross River locality (as first described by Schopf 1968)ACCEPTED of the Bitter Springs Formation. We then compare and contrast these data with data obtained in precisely the same manner from Primaevifilum filaments from the

‘Chinaman Creek microfossil locality’ of the Apex chert (Schopf 1993). We go on to

show that as one increases the spatial resolution of data obtained the Apex

Primaevifilum filaments are shown to be in no way morphologically comparable to Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

Cephalophytarion microfossils, possessing no distinctive cellular morphology, and

hence it is very difficult to envisage an origin as microfossils of filamentous

prokaryotes. This work provides an important correlative link between different

spatial scales of data collection, showing that higher resolution electron microscopy

data can be deconstructed to construct a landscape of lower resolution Raman-scale

mapping. In turn, this permits more refined abiotic models to be advanced for

complex microstructures that have previously been explained solely using biological

reasoning.

MATERIAL AND METHODS

Sample localities

The Bitter Springs material studied here (sample RH5; GR -23.58367, 134.49767)

was collected in 2017 from the Ross River area approximately 65 km east-northeast of Alice Springs. It was collected from a ridgeMANUSCRIPT 1.1 km north-northeast of the Ross River Resort (previously known as the Ross River Tourist Camp or Love’s Creek

Homestead), and comprises part of a carbonaceous black chert lense hosted within a

sedimentary carbonate succession, the same unit (to the best of our knowledge since

GPS coordinates are not present in the original report) from which the original Ross

River locality microfossils were described by Schopf (1968). Details of the geology of

the Ross River area can be found in Wells et al. (1970). ACCEPTED The Apex material studied here (sample CHIN-3) comes from the original Chinaman

Creek locality (Schopf & Packer 1987; Schopf 1993) and was collected in 2001. Data

come from a series of thin sections all prepared from the same CHIN-3 hand sample

as used in Brasier et al. (2015) and Wacey et al. (2016a,b). As detailed in Wacey et Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

al. (2016a), it is prohibited to perform any destructive or intrusive analyses on the

type specimens held by the Natural History Museum (NHM), London. Hence,

standard (~30 m thick) polished geological thin sections of CHIN-3 are as close to

the type specimens as we can feasibly analyse by modern techniques (cf. Schopf et al.

2018). Crucially, filamentous microstructures found in CHIN-3 are morphologically

comparable to those found in the type material held at NHM (which are thicker

sections, hence the filaments often appear darker; see also extensive details in Brasier

et al. 2015 and Wacey et al. 2016a, plus discussion herein) and in the non-type

material recently investigated by Schopf et al. (2018). Details of the geology of the

Chinaman Creek area of the Apex Basalt, including evidence for a hydrothermal

setting for the host black chert can be found in Brasier et al. (2005, 2011). This

hydrothermal geological setting for the host chert is now widely agreed upon,

including by proponents of a cellular microfossil origin for these structures (e.g., Schopf & Kudryavtsev 2012). MANUSCRIPT

Optical microscopy

The ~30 m thick, polished, uncovered thin sections were examined by optical

microscopy (transmitted and reflected light) to gain an understanding of the filament

distributions and morphologies, and to select the most appropriate samples for

detailed study. This was carried out using Leica DM2500M and Zeiss Axioskop

microscopes,ACCEPTED with 5x, 10x, 20x, 50x, and 100x objective lenses, located within the

Centre for Microscopy, Characterisation and Analysis (CMCA) at The University of

Western Australia (UWA). Images were captured using a digital camera and

Toupview imaging software.

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Confocal laser Raman microspectroscopy

Raman was performed on a WITec alpha 300RA+ instrument with a Toptica

Photonics Xtra II 785 nm laser source at the CMCA, UWA. Laser excitation intensity

at the sample surface was in the 1-5 mW range, below the intensity that may damage

carbonaceous material (e.g., Everall et al. 1991) and comparable to previous studies

of the Apex chert and Bitter Springs Formation (e.g., Olcott Marshall et al. 2012;

Sforna et al. 2014). The laser was focused through a 100x/0.9 objective to obtain a

spot size of smaller than 1 m. Spectral acquisitions were obtained with a 600 l/mm

grating and a peltier-cooled (-60 °C) 1024 x 128 pixel CCD detector. Laser centering

and spectral calibration were performed daily on a silicon chip with characteristic Si

Raman band of 520.4 cm-1. Spectra were collected in the 100-1800 rel. cm-1 region in

order that both 1st order mineral vibration modes and 1st order carbonaceous vibration

modes could be examined simultaneously. Raman maps were acquired with the spectral centre of the detector adjusted to 944MANUSCRIPT cm-1, with a motorised stage allowing XYZ displacement with precision of better than 1 m. 3D analysis was performed

with a 1 m z-spacing interval. Spectral decomposition and subsequent image

processing were performed using WITec Project FOUR software, with baseline

subtraction using a 3rd or 4th order polynomial. Carbon maps were created by

integrating over the ~1600 cm-1 ‘G’ Raman band. The ~1350 cm-1 carbon ‘D’ Raman

band was not used to construct maps because this may suffer from interference from theACCEPTED ~1320 cm-1 hematite Raman band in some Apex samples (cf. Marshall & Olcott Marshall 2013). All analyses were conducted on material embedded below the surface

of the thin section to avoid artefacts in the Raman spectra resulting from polishing

and/or surface contamination. Only filaments occurring entirely in the top ~12 m of Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

the thin sections were analysed because of loss of Raman signal and spatial resolution

at greater depth (Schopf et al. 2005; Marshall & Olcott Marshall 2013).

It is notable that two phases subsequently detected using electron microscopy (nano-

grains of iron oxyhydroxide, and sheet-like aluminosilicates) were not readily

detected using Raman. It is likely that the iron oxyhydroxide phase was not detected

due to it being nano-crystalline and having a weak signal compared to that of the

surrounding carbonaceous material. Aluminosilicate clays generally show only weak

Raman bands and their detection requires rather specialised analytical conditions,

such as low-fluence lasers, which we did not have at our disposal. This highlights

some of the potential limitations of Raman for studying very small complex objects

and may also explain why these phases have not been detected in the type material.

Focussed ion beam (FIB) preparation of TEMMANUSCRIPT samples TEM samples were prepared using a FEI Helios Nanolab G3 CX at the CMCA,

UWA. Electron beam imaging was used to identify microstructures of interest in the

polished thin sections coated with c. 10 nm of gold or platinum, allowing site-specific

TEM samples to be prepared. The TEM sections were prepared using a slightly

modified version of the protocol for microstructure extraction given in Wacey et al.

(2012), with milling and imaging parameters optimised to suit the specific type of sampleACCEPTED (i.e. carbonaceous and phyllosilicate-rich objects within a silica matrix). Briefly, regions of interest (ROI) were covered with a protective (c. 2 μm-thick)

platinum layer. Initial large trenches were milled either side of the ROI with a 21 nA

Ga+ ion beam, and the trench faces cleaned up using a 9.3 nA beam. Element

mapping within the FIB-SEM using energy-dispersive X-ray spectroscopy (EDS) was Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

performed on some cleaned trench faces to gain a preliminary understanding of the

chemistry of the filaments and their surrounding matrix. SEM-BSE imaging was also

performed on cleaned trench faces during the thinning process. On reaching a

thickness of c. 1.5-2 m the ROI was extracted using an in situ micromanipulator, and

attached to a Pelco copper TEM grid by welded platinum strips. This ‘welding’

protocol means that there is no carbon film underneath the wafer, simplifying

subsequent carbon elemental mapping in the TEM. Subsequent thinning of the ROI

was performed with decreasing ion beam currents (0.79 nA then 0.23 nA). Final

thinning was performed at lower voltage (16 kV and 1.3 nA current) followed by face

cleaning at 5 kV and 15 pA. Average final wafer thicknesses were in the range of 150-

200 nm.

TEM analysis of FIB-milled wafers TEM and STEM (scanning transmission electronMANUSCRIPT microscopy) data were obtained using an FEI Titan G2 80-200 TEM/STEM with ChemiSTEM technology operating at

200 kV equipped with a Gatan SC1000 camera located in the CMCA at UWA.

Crystal orientation and mass/density difference data were gained from high angle

annular dark-field (HAADF) and bright field (BF) STEM imaging. Energy-dispersive

X-ray spectroscopy (EDX) via the ChemiSTEM system provided elemental maps.

Lattice spacings of crystals were obtained via high resolution TEM (HRTEM). ACCEPTED Reconciling EM and Raman data

We applied a Gaussian blurring filter using ImageJ software to our electron

microscopy carbon elemental map data to mimic the effect that the lower resolution

Raman technique would have on our ability to interpret the microstructure. The width Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

of the Gaussian filter was progressively increased, starting with a 20 pixel full width

half maximum (FWHM) followed by increases in increments of 10 pixels until a 150

pixel FWHM was reached. This was done in order to test how decreases in spatial

resolution affect the imaged morphology of the structures of interest. Depending on

the image magnification, the application of a Gaussian blur filter with a FWHM of

between 100 and 150 pixels resulted in our electron microscopy data having a similar

appearance to our Raman data and that of previously reported Raman data (e.g.,

Schopf et al. 2018). This is consistent with the ratio of the pixel size of our EDS maps

and the resolution of the Raman technique.

RESULTS

Nanoscale characterisation of 850 Ma Bitter Springs microfossils Optical and Raman data show that CephalophytarionMANUSCRIPT filaments comprise distinct, almost cubic compartments outlined by carbonaceous walls with kerogen-like

composition (Fig. 1a-d), and these compartments are each infilled by quartz (Fig. 1d).

Higher spatial resolution electron microscopy data from longitudinal sections through

Cephalophytarion filaments reinforce the Raman data showing both distinct

carbonaceous lateral walls and carbonaceous transverse walls (septa) outlining

cellular compartments (Fig. 1f). Individual cells are ~4 m in maximum diameter, and 3-5ACCEPTED m in length, joined to form a cylindrical filament that is demonstrably circular to elliptical in latitudinal cross section (Fig. 1e). Although there is some minor variation

in cell morphology due to taphonomic effects such as cell wall shrinkage, the

carbonaceous septa in individual Cephalophytarion filaments are more or less

regularly spaced and each are of very similar thickness (Fig. 1f). For example, septa Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

maximum thicknesses for the filament shown in Fig. 1 are between 70 nm and 100

nm, and the spacings between septa in this organism show a relatively narrow range

of between 3.1 and 4.2 m (n=11).

Individual cellular compartments had evidently lost much of their cell contents prior

to silicification, although preservation in places is of such high quality that potential

remnants of organic cell contents remain, mostly found close to the inner margins of

the cell walls (Fig. 1e-f, 2a-b). This organic material frequently outlines sub-spherical

quartz nanograins that have permineralised the organism, suggesting only localised

modification of organic structure during mineralisation (Fig. 2a-b). The remainder of

the interior of the cellular compartments now comprises a mosaic of nanocrystalline

quartz (Fig. 2a-b, f).

Electron microscopy shows that the lateralMANUSCRIPT carbonaceous cell walls are continuous, gently curved and sometimes slightly folded or shrunken, consistent with minor

taphonomic decay of the specimen prior to silicification (Fig. 1f, 2a). The kerogen-

like material retains significant sulfur content (Fig. 2d, 3a-b) consistent with

biological material, and is also associated with some calcium (Fig. 2c, 3a-b). Calcium

carbonate grains are relatively common within the matrix in the vicinity of the

microfossil (Fig. 1d) but the calcium associated with the organic material of the microfossilACCEPTED does not correlate with oxygen so is not present as a carbonate or sulfate phase. The calcium and carbon EDX maps (Fig. 2b-c) show close correlation but in

some places calcium appears to extend up to a few tens of nanometers away from the

carbon. These data suggest that calcium had bound to the cyanobacterial organic

material and had not yet been released by heterotrophic decay of the organism at the Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

time of fossilisation (cf. Dupraz et al. 2009), though further work is needed to fully

understand this process in Cephalophytarion. A very fine, cylindrical, carbonaceous,

sheath-like structure is also revealed (Fig. 1e-f, 2a-b, 3a), which could not be detected

using light microscopy or Raman. This is consistent with the original description of

Cephalophytarion in which it was remarked “sheath, if present, indistinct” (Schopf

1968). Finally, nanopores possessing significant chlorine content (Fig. 2e, 3a, c) may

reflect incorporation of hypersaline ambient porewater (cf. Southgate 1986) during the

final stages of permineralisation.

Nanoscale characterisation of Apex microstructures

Our Raman analyses of Primaevifilum from the Apex chert show that some portions

of these filaments have a kerogen-like carbonaceous composition (Fig. 4a-d; Fig. 5a-

d). However, this carbon does not define lateral walls and corresponding transverse walls, except superficially on very rare occasionsMANUSCRIPT (e.g., Fig 4b-c arrows); such a carbon distribution is entirely consistent with previous Raman analyses of Apex

filaments (Schopf et al. 2002, 2007, 2018; Schopf & Kudryavtsev 2009, 2012; see for

example figs. 1-2 of Schopf et al. 2018). For the most part, structures that could be

interpreted as lateral cell walls are absent, with the interiors of filaments characterised

by clumps of carbon, that are significantly thicker than the septa observed in

Cephalophytarion, interspersed with non-carbonaceous regions of variable morphologyACCEPTED and chemical composition (Fig. 4b-d; Fig. 5c-d). Frequently, the pattern of carbon distribution expected for cells (i.e. narrow carbonaceous cell walls

interspersed with thicker compartments filled by quartz or other minerals) is reversed,

with apparent thick solid masses of carbon (but see electron microscopy data below

for true distribution of carbon) being separated by very narrow mineralic ‘walls’ (Fig. Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

5b-c arrows). Carbonaceous particles are also dispersed throughout the matrix in the

vicinity of the filaments (Fig. 4b-c; Fig. 5b-d). Anatase is relatively common in the

matrix (cf. Bower et al. 2016) and can also occur within the filaments where the

morphology of the grains may give a false impression of cellular

compartmentalisation (i.e. an anatase grain surrounded by carbon mimics the

morphology of a cell; Fig. 4d).

Correlative electron microscopy performed on the same Primaevifilum filaments

provides a clearer picture of their morphology. Lateral carbonaceous ‘walls’ outlining

the filaments are mostly absent (Fig. 4g; Fig. 5e-f; Fig. 6a-b). Instead, the carbon

dominantly forms narrow sub-vertical features; on rare occasions these may

superficially resemble the septa of filamentous organisms but throughout the majority

of the filaments they define angular structures, with variable orientations, variable lengths and thicknesses, and irregular spacingsMANUSCRIPT between one another (Fig. 4g; Fig. 5e- f; Fig. 6a-b). Compared to the septa of Cephalophytarion, these sub-vertical features

have significantly more variable thicknesses (< 10 nm to > 300 nm; n=40), and much

more variable spacings between one another (<30 nm to ~2 m; n=38) within a single

filament. More than 80% of measured spacings are <250 nm meaning that there are an

order of magnitude more vertical carbonaceous components in a given length of

Primaevifilum filament compared to the same length of Cephalophytarion filament, andACCEPTED the majority of ‘septa’ spacings in Primaevifilum are too small to feasibly equate to cellular compartments (Fig. 4g; Fig. 5e-f; Fig. 6a-b). In longitudinal cross section,

many Primaevifilum filaments change width quite dramatically along their length,

varying between <1 m and >4 m in width perpendicular to the plane of the

geological thin section (Fig. 4g; Fig. 6a). The filaments are not cylindrical, having Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

transverse cross sectional profiles of diverse, mostly angular morphologies (Fig. 4e-f).

Branching is also common (Wacey et al. 2016a).

Electron microscopy also shows that the Primaevifilum filaments possess a much

more complex chemistry than is observed using Raman alone. This chemistry was

first described by Brasier et al. (2015) and Wacey et al. (2016a) from specimens in a

separate CHIN thin section, and the new specimens examined herein provide some

additional data. The filaments comprise chains of angular sheet-like aluminosilicate

mineral grains. As well as ubiquitous Al, Si, and O, these contain minor K (~2-4%), a

patchy distribution of Ba (<1%) (Fig. 6-7) and unevenly distributed trace amounts of

Fe and Mg. Al and Si occur in an approximately equal amount. HRTEM data herein

(Fig. 8b) reinforce the electron diffraction data of Wacey et al. (2016a) showing the

dominant 1 nm spacing of the Al-Si-O sheets characteristic of 2:1 phyllosilicates, for example micas and some clay minerals suchMANUSCRIPT as illite. It is not possible to constrain their exact mineralogy such is the heterogeneity of the distribution of minor elements

(but see part three of the Discussion below for the possible origin of this phase).

At least three other phases, in addition to kerogen, are present within the filaments.

An iron-oxygen-rich phase occurs as small needles in between sheets of the

phyllosilicate and towards the outer tips of many of the silicate sheets (Fig. 6b). This phaseACCEPTED also contains minor As (~1%) (Fig. 6d, 7), and sometimes trace amounts of Ni and/or Cr. HRTEM reveals a crystal structure consistent with the iron oxyhydroxide

mineral goethite (Fig. 8c), potentially with minor distortions due to the small amounts

of As, Ni and/or Cr in the lattice. Titanium oxide (anatase) occurs infrequently as

nano- to micro-scale grains within and just exterior to the filaments (Fig. 4d). Finally, Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

quartz, which comprises the vast majority of the matrix of the host rock, is often

found inter-grown with these other phases within the filaments (Fig. 4d, 6a, f).

The plate-like aluminosilicates also frequently occur nearby to the Primaevifilum

filaments (Fig. 8d-f) and elsewhere in the CHIN thin sections, and here they may not

be organised into chains and thereby lack a filamentous appearance; some of these

occurrences are also associated with carbon and goethite. Hence, there is a

morphological continuum of aluminosilicate objects, only some of which resemble

filamentous microfossils. Within Primaevifilum filaments carbon does not appear to

preferentially occur associated with any specific mineral phase (Fig. 6b). Carbon is

also found elsewhere within the quartz matrix, where it can occur at quartz grain

boundaries or, most notably, in partially carbon-filled fractures in the quartz which

occur in close proximity to some Primaevifilum filamentous objects (Fig. 8e-i). MANUSCRIPT

DISCUSSION

Microfossil criteria

In any test of a microfossil origin for an ancient microstructure, the primary trait that

must be demonstrated is that of biological morphology (Schopf & Walter 1983; Buick

1990; Schopf et al. 2010). If an object does not demonstrate a plausible biological morphologyACCEPTED it cannot be considered a microfossil even if it possesses secondary characteristics that may be consistent with biology (e.g., kerogenous chemistry with a

13C of biological magnitude). This point cannot be overstated and it is this point that

separates a true cellular microfossil from other objects that may be remnants of life

but are composed of remobilised organic material. Indeed, proponents of a microfossil Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

origin for the Apex filaments have stated that resolution of the Apex microfossil

controversy hinges on ‘whether the Apex fossils are cellular and composed of

kerogenous carbon’ (Schopf & Kudryavtsev 2012).

Three of the critical morphological features of fossilised filamentous trichomes

highlighted in previous appraisals of putative microfossils are (after Schopf et al.

2010): 1) they should be cylindrical (or, if distorted during preservation, initially

cylindrical) with their shape defined by distinct carbonaceous lateral walls; 2) they

should have essentially uniform diameter throughout their lengths (again, with

allowances for distortion during preservation, and for tapering towards their apices);

3) they should be partitioned by septa into discrete cells (box like or spheroidal) of

more or less uniform size and shape that are predominantly hollow (later mineral

infilled). These morphological features are obviously specific to a discussion of filamentous trichomes (the interpretation putMANUSCRIPT forward by Schopf (1993) for these Primaevifilum objects) and different, less stringent, criteria may apply for potential

non-cellular cylindrical microfossils such as those derived from sheaths.

It should be noted at this point that we are not using Cephalophytarion as an exact

potential analogue for the Primaevifilum filaments since the geological settings, and

hence types of biota likely inhabiting the two localities, are different. We are using CephalophytarionACCEPTED for two reasons: 1, Cephalophytarion was one of the main organisms used by Schopf and colleagues in their initial comparative work with Apex

filaments (e.g., Schopf & Kudryavtsev 2009) so it must be investigated whether these

comparisons hold up under higher resolution scrutiny; 2, Cephalophytarion is a well

preserved example of a bona fide filamentous Precambrian organism, fossilised by Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

silica, that should contain the fundamental morphological features of a fossilised

trichome outlined above.

Our electron microscopy analysis of Cephalophytarion filaments show that they fully

satisfy the criteria for a filamentous microfossil listed above, being of essentially

uniform diameter, with cylindrical cross sections outlined by clear carbonaceous

lateral cell walls, and being partitioned into box-like segments by carbonaceous septa

of essentially uniform diameter and length (Figs. 1-2). Notably, several other silicified

Archean (Wacey et al. 2012) and Proterozoic (Wacey et al. 2012; Fadel et al. 2017;

Lekele Baghekema et al. 2017; Lepot et al. 2017) filamentous microfossils display

some, or all, of these biological features when examined using electron microscopy.

In contrast, none of the fundamental morphological features expected of a fossilised trichome are present in the Primaevifilum MANUSCRIPTfilaments when examined using electron microscopy (Figs. 4-8). Features that could be interpreted as lateral carbonaceous

walls are absent; transverse cross sections show angular filament morphologies with

very variable widths controlled by the location of the aluminosilicate minerals;

transverse carbonaceous features have morphologies inconsistent with biological

septa, and the spaces between these features are too small to be compatible with an

origin as cellular compartments. Nor is the morphology of the Primaevifilum filamentsACCEPTED comparable with other potential filamentous microfossils such as those derived from sheaths. The lack of fundamental cellular features cannot be explained

by simple physical distortion during preservation since there is no evidence for

folding, tearing or flattening of original cell walls.

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Could Apex filaments be heavily degraded microfossils?

The above data and discussion show that the Apex Primaevifilum specimens cannot

be well-preserved filamentous bacteria as previously claimed (e.g., Schopf 1993)

since they possess none of the morphological features of such organisms, particularly

when viewed at high resolution. However, we next need to consider whether

Primaevifilum filaments could be heavily degraded versions of ancient organisms.

That is, we must address whether it is plausible that biodegradation, thermal decay,

and diagenetic and metamorphic mineral growth/replacement could be responsible for

transforming an original trichome of box-like cells (or indeed any other biological

morphology) into the acellular morphology now observed.

It has previously been claimed that the Apex filaments may represent heavily

degraded sheathed colonies of coccoid cyanobacteria (Kazmierczak & Kremer 2002, 2009). This was based on superficial morphologicalMANUSCRIPT similarities (examined using only low resolution light microscopy) between some Apex filaments and thermally altered

remnants of colonial coccoid cyanobacteria from Silurian cherts of Poland. However,

3-D analysis of those Silurian colonies show that although they may have flattened,

roughly filamentous cross sections perpendicular to bedding, they are disc-shaped

parallel to bedding (Kazmierczak & Kremer 2009). 3-D analysis of Apex specimens

clearly shows that they are not disc-shaped in any orientation (Schopf et al. 2007; SchopfACCEPTED & Kudryavtsev 2009; Wacey et al . 2016a, and herein) so they are not analogous to the younger Silurian objects. Note also that the Apex filaments do not

occur in bedded cherts, as erroneously assumed by Kazmierczak & Kremer (2002,

2009), they occur in a black chert vein intruded at a high angle into basalt, a very

different environment to their Silurian specimens. Furthermore, based on recent Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

molecular clock estimates, such an interpretation seems implausible because sheathed

colonial coccoid cyanobacteria likely evolved some 600 million years or more after

the formation of the Apex chert (Sanchez-Baracaldo et al. 2017).

Where heavily degraded carbonaceous objects co-occur in assemblages with much

better preserved individuals that possess clear biological morphology (e.g., Knoll et

al., 1988) a case can be made for their interpretation as microfossils. In such cases this

can be a valid argument so long as one can show a logical progression from well

preserved to poorly preserved morphotypes. Such an argument cannot be made for the

Apex chert because there are no specimens that possess biological morphology.

Furthermore, the presence of additional identical aluminosilicate grains in the host

chert in the vicinity of the Primaevifilum filaments, often occurring in pairs or short

stubby chains but without the distinct microfossil-like filamentous organisation, emphasises that the Primaevifilum filamentsMANUSCRIPT are merely one end member of a morphological continuum of mineralic objects.

The mineralogy of the Apex filaments does little to support or refute a biogenic

origin. Microorganisms can occasionally be fossilised by diagenetic aluminosilicates,

and K-rich clays such as illite have been reported within ~ 1 billion year old cells

from northwest Scotland (Wacey et al. 2014). However, the small crystal size of clay mineACCEPTEDrals usually results in very high quality preservation of the organisms, with cell walls (and even some cell contents) remaining intact and only nano-scale modification

of the overall morphology of a given cell (Wacey et al. 2014). There is also usually

some spatial relationship between the chemistry of the clays and cellular components:

for example, Wacey et al. (2014) found that Fe- and Mg-rich clays preferentially Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

occurred in the vicinity of cell walls, and K-rich clays preferentially occurred in cell

interiors, suggesting some biological influence on the nano-scale distribution of clay

chemistry. While it is possible that increased thermal degradation and chemical

modification could have significantly overprinted original biochemical patterns in the

silicates, some zonation of silicate phases relative to biological components may still

be expected to be preserved even in highly metamorphosed rocks (Bernard et al.

2007, 2008). It must also be remembered that the oldest example of this style of

microfossil preservation is approximately 2.5 billion years younger than the Apex

chert and occurred in a freshwater lacustrine setting (Wacey et al. 2014) not in a

complex hydrothermal system.

There is some evidence that nano-domains between sheets of clays may be more

conducive to preservation of organic matter (e.g., Curry et al., 2007) than, for example, interfaces between clay mineralsMANUSCRIPT and quartz, which may partially account for the greater amount of organic material in the interior of the filaments than at the

outer border. However, if the Apex filaments are highly degraded filamentous

organisms it would require the lateral cell walls of a trichome (or sheath walls) to

have been almost completely destroyed by mineral growth, and a large portion of this

carbon to be redistributed along crystal faces perpendicular to the original walls in the

interior of the filament, a scenario which to the best of our knowledge is unproven in theACCEPTED geological record. Alternatively, it would require the lateral cell walls to have been destroyed but much of the cell contents to have been preserved along crystal

boundaries which is contrary to the known degradation pathways of organisms (e.g.,

Knoll & Barghoorn 1975).

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Plausibility of an abiogenic formation mechanism

If the Apex filaments are not heavily degraded microfossils then what alternative

mechanism may explain their formation? Here, additional data from the newly

examined specimens permit us to propose a more comprehensive abiotic formation

mechanism, building upon the work of Brasier et al. (2015) and Wacey et al.

(2016a,b).

Micas (e.g., biotite, muscovite) are common accessory minerals in several rock types

that originally formed the Pilbara Craton, in particular the massive granitoids that

comprise >50% of the East Pilbara Terrane (Van Kranendonk et al. 2007). Micas are

2:1 layered silicates where each layer comprises two (Si, Al)O4 tetrahedra and one

MO6 octahedron, where M is commonly Al, Mg, or Fe. The strong negative charge of

these layers is counterbalanced by interlayer cations such as K+, with these electrostatic forces holding the layers togetherMANUSCRIPT (Deer et al. 2013). The chemistry of the Apex 2:1 phyllosilicates is complex but is closest to muscovite in composition, with

an approximate 1:1 ratio of Al:Si, and K present as the next most abundant element.

Other micas such as the biotite subgroup, or K-rich 2:1 clay minerals such as illite,

would be expected to have much lower Al:Si ratios (Deer et al. 2013). However, the

Apex phyllosilicate is severely depleted in K compared to natural muscovite and has

elevated levels of Ba, plus trace amounts of Fe and Mg, which are all rather heterogeneouslyACCEPTED distributed. HRTEM shows that the 1 nm sheet spacing has been retained throughout much of the phyllosilicate structure but there are hundreds of

occasions where the layers have separated and are now filled with carbon, goethite or

void space.

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Muscovite layers are not as easily separated (exfoliated) as some other micas such as

biotite, but exfoliated muscovite has been observed in natural geological settings (e.g.,

Rutherford 1987) and created in the laboratory (e.g., Jia et al. 2017). Muscovite

exfoliation typically involves heating in the presence of water and organic solvents to

replace some of the interlayer K ions and weaken the interlayer electrostatic attraction

(Nicolosi et al. 2013; Tominaga et al. 2017). We propose that the Apex hydrothermal

dykes provided suitable conditions for the partial exfoliation of muscovite creating a

range of mineralic objects, some of which resemble filamentous microfossils. This is

consistent with partial loss of K and presence within the filaments of Ba, As, Ni, and

Cr, elements typically found within the Apex hydrothermal system (cf. Brasier et al.

2005). The Pilbara black chert dykes are carbon-rich (Lindsay et al. 2005) and this

organic material may have helped facilitate exfoliation of the muscovite, and would

also have readily migrated onto muscovite crystal faces and/or been trapped in the narrow gaps that opened up between the muscoviteMANUSCRIPT sheets (cf. Medeiros et al. 2009). This indigenous carbon was supplemented by non-syngenetic carbon ingress as

potentially evidenced by multiple partially carbon-filled fractures that occur close to

several Primaevifilum filaments (Fig. 8e-i). This is consistent with previous reports of

multiple generations of carbon within these samples, including a demonstrably later,

less thermally mature phase (Olcott Marshall et al. 2012). Whether some or all of this

carbon ultimately had a biological source is still open to debate but abiotic synthesis viaACCEPTED Fischer-Tropsch-Type reactions (Fu et al. 2007; McCollum 2013) remains, in our opinion, a logical scenario for carbon found in these hydrothermal systems.

The presence of goethite in the filaments indicates that there has been modern

weathering of these samples, consistent with the study material being obtained from Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

surface outcrop. Based on the presence of sporadic, partially weathered sulphide

grains elsewhere in the CHIN thin sections, and abundant sulphides elsewhere in

Apex chert dyke samples (e.g., Brasier et al. 2005), we infer that the goethite is a

weathering product of small grains of iron (plus As, Ni) sulphides initially formed

within the hydrothermal system. Goethite could also have resulted from the modern

weathering of Fe-rich micas such as biotite (cf. Banfield 1985) but we see no firm

evidence of the residual silicate component of that reaction, kaolinite.

In summary, the observed mineral phases, their distribution relative to one another,

their minor elemental composition, and the distribution of kerogen-like carbon in the

Apex filaments can be most parsimoniously explained by alteration of a micaceous

mineral within a carbonaceous hydrothermal environment, followed by a modern

weathering event. The variety of colours of Apex filaments observed in optical microscopy, from almost colourless, to orange,MANUSCRIPT to very dark brown (e.g., Schopf 1993; Brasier et al. 2005, 2015; Wacey et al. 2016a, and herein) can simply be attributed to

differences in the initial Fe content of the micas or abundance of Fe-sulfides,

differences in the amount of carbon accreted onto the phyllosilicates, and variations in

the thickness of the thin sections used for analysis.

Reconciling Raman and electron microscopy data PreviousACCEPTED data concerning the Apex filaments have mostly been obtained using Raman spectroscopy; this is especially true for the proponents of a microfossil origin who

have used Raman mapping of kerogen-like carbon to demonstrate the apparent

cellularity of the Apex filaments (e.g., Schopf et al. 2007, 2018; Schopf &

Kudryavtsev 2009, 2012). In contrast, higher spatial resolution electron microscopy Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

data herein (plus examples in Brasier et al. 2015 and Wacey et al. 2016a,b) show no

such evidence of cellularity. Hence, it is necessary to reconcile the differences in

carbon patterns observed using Raman with those observed using electron

microscopy. To investigate whether the spatial resolution of the respective imaging

techniques can explain the differences in these data we applied a Gaussian blurring

filter (see methods) to our electron microscopy data to mimic the lower spatial

resolution of the Raman method (Figs. 9-10).

This approach is imperfect as it cannot take into account the differences in z-depth

imaged by the different techniques. For example, our TEM samples are 150-200 nm

thick but the confocal Raman is collecting signal across a depth of more than 500 nm

in a single image, which means a greater volume of carbon has potentially been

sampled by Raman. Application of the Gaussian filter to the electron microscopy carbon data can only realistically account MANUSCRIPTfor the lower x and y spatial resolution of the Raman carbon data, and will likely lead to an underestimation of the carbon

feature size in the resultant blurred image as we are not accounting for the additional

carbon signal that may be present in the larger z depth of material sampled by the

Raman technique. In addition, Raman data was collected in the plane parallel to the

surface of the geological thin section, while the electron microscopy data was

collected in the plane perpendicular to the surface of the geological thin section. For theseACCEPTED reasons the blurred electron microscopy data will not be expected to perfectly match the Raman data.

Nonetheless, application of this Gaussian filter to the Apex electron microscopy data

shows how angular strips of carbon, often with diameters of only ~10 nm, are Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

transformed into more rounded features that amalgamate into larger semi-solid zones

of carbon, closely comparable to those observed in our correlative Raman maps (Fig.

9a-e; Fig. 10a-f). The application of the same filter to the Cephalophytarion data also

results in the electron microscopy data emulating the Raman data (Fig. 9i-l).

Moreover, this simple experiment provides a consistent and logical explanation of

previous Apex Raman data (e.g., Schopf et al. 2002, 2007, 2018) that show

predominantly solid carbonaceous filaments interspersed with rather random zones of

carbon-poor material (Fig. 9f-h). On rare occasions, fortuitous spacing of

carbonaceous features can give the impression of cellular compartments in Raman

maps (e.g., Fig. 4b-c, arrows; see also Schopf et al. 2018 fig. 1t-z) but here we

demonstrate that these can be explained as coincidental artefacts of the limited spatial

resolution of the Raman data.

A recent report, based on Raman plus a smallMANUSCRIPT 13C dataset from 11 carbonaceous Apex microstructures (averages of 13C = -31‰ to -39‰), inferred that multiple

metabolic pathways were present amongst Apex microfossils (Schopf et al. 2018).

None of the new objects analysed in that work have cellular organisation, with Raman

maps showing mostly solid carbonaceous filamentous objects, some with angular

facets, plus large quantities of carbon and potential iron staining in the surrounding

matrix (see Schopf et al. 2018 figures 1 and 2); hence, none of those newly described objectsACCEPTED pass accepted criteria for classification as microfossils (Schopf et al. 2010). Doubts also exist within the carbon isotope data presented by Schopf et al. (2018):

Attempts are made to claim that five different taxa are represented within their 13C

analyses (averages of -31‰ to -39‰), including both Archaea and Proteobacteria,

exhibiting up to three different metabolisms. Yet, only 11 filaments were analysed in Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

total and two supposed taxa only yielded a single 13C data point. Of the supposed

taxa that yielded more than one data point, the variability of 13C within a taxon is of

a similar magnitude as the variability of 13C between taxa, and there is even

significant 13C variability (in excess of 10‰) in measurements of the laboratory

standard (Schopf et al. 2018, table 1 and supporting information).

Regardless of the quality of the Schopf et al. (2018) isotopic data, our demonstration

here that Apex filaments lack a plausible biological morphology, can simply be

explained by alteration of flakes of mica, and likely contain a later generation of

carbon, renders the inference of multiple metabolic pathways in these objects

untenable. The 13C patterns observed by Schopf et al. (2018) are more likely a result

of the multiple sources and generations of carbon present within these

hydrothermally-influenced Apex samples (cf. Olcott Marshall et al. 2012; Sforna et al. 2014). As mentioned previously, it is ofMANUSCRIPT course possible that one or more of these carbon sources could be remobilised biological material, since life was very likely

established on Earth by this time (e.g., Noffke et al. 2013). However, abiotic sources

of carbon within the filaments, for example resulting from Fischer-Tropsch-type

synthesis of organic carbon (Fu et al. 2007; McCollum 2013) remain consistent with

the geological setting (cf. Holm & Charlou 2001), associated Ba-rich and As-rich

hydrothermal minerals, structure and bonding (cf. De Gregorio & Sharp 2006), and isotopicACCEPTED signature (cf. McCollom & Seewald 2006) of the Apex carbon.

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CONCLUSION

We have here examined filamentous microfossils belonging to Cephalophytarion

from the 850 Ma Bitter Springs Group plus filamentous objects (Primaevifilum) from

the 3460 Ma Apex chert at a range of spatial scales. We have shown that the

previously held view that these two types of filaments are morphologically

comparable (e.g., Schopf & Kudryavtsev 2005, 2009; Schopf et al. 2007) is

unsupported, particularly when analysed at high spatial resolution. Raman analyses

show superficial morphological similarities between Cephalophytarion and

Primaevifilum but there are subtle differences in the distribution of carbon in each

case. In the former, carbon clearly corresponds to cell walls outlining hollow, almost

cubic cellular compartments. In the latter, carbon occurs as more random clumps

without cellular distribution.

Higher spatial resolution electron microscopyMANUSCRIPT data highlight the complete lack of morphological comparison between Cephalophytarion and Primaevifilum filaments.

The former satisfy established biogenicity criteria for fossilised filamentous

microorganisms, having cylindrical cross sections defined by distinct carbonaceous

lateral walls, uniform diameter (excepting their apices), and being partitioned by septa

into discrete box-like cells of more or less uniform size and shape. The latter fulfil

none of these criteria: they lack lateral carbonaceous walls; are angular in transverse crossACCEPTED section; have rapidly changing diameters along their length; and are not partitioned into box-like cells.

Most previous work on the Apex filaments (e.g., Schopf et al. 2018 and references

therein) has tended to resort to biological explanations for the preserved Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

microstructures, perhaps due to a lack of abiological models to explain complex

mineral-organic morphology. By providing high resolution electron microscopy data,

and deconstructing these data to inform upon previous Raman-scale mapping, we

provide additional morphological and compositional details that were not available

using traditional light microscopy and Raman analyses. In so doing, this contribution

builds upon previous work (Brasier et al. 2015, Wacey et al. 2016) to continue to

refine abiological models that can act as alternative explanations in the assessment of

Archean microfossil-like objects.

In summary, when examined at an appropriate spatial scale, Apex Primaevifilum

filaments do not possess any of the cellular features found in bona fide Precambrian

fossilised filamentous organisms. Hence, they fail the most essential criterion

established for the evaluation of putative microfossils and can no longer be interpreted as some of Earth’s oldest cellular life. Rather,MANUSCRIPT these new data provide further support to a hypothesis that the Apex filaments are mineralic objects, formed as a result of the

alteration of mica, onto which organic carbon has migrated to create pseudo-fossils.

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Acknowledgements and Funding Information

We acknowledge the facilities, scientific and technical assistance of the Microscopy

Australia Research Facility at the Centre for Microscopy Characterisation and

Analysis, The University of Western Australia. This facility is funded by the University,ACCEPTED State and Commonwealth Governments. DW was funded by the Australian Research Council via a Future Fellowship grant (FT140100321). KE was

supported by an Australian Government Research Training Program Stipend and a

UWA Safety-Net Top-Up Scholarship. We acknowledge the late Prof. Martin Brasier

for numerous discussions during the formative stages of this work. We acknowledge Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

Martin van Kranendonk, Andrew McPherson, Alex Brasier, Owen Green, Cris

Stoakes, Nicola McLoughlin, The Geological Survey of Western Australia, and the

late John Lindsay for fieldwork assistance. We also acknowledge the staff of the Ross

River Resort and the Grollo family for enabling access to the Bitter Springs outcrops.

Paul Strother and an anonymous reviewer are thanked for their constructive

comments, and Phil Donoghue is acknowledged as the handling editor.

Figure Legends

Fig. 1. Correlative microscopy of a filamentous microfossil belonging to

Cephalophytarion from the 850 Ma Bitter Springs Formation. (a) Optical

photomicrograph showing the morphology of the microfossil and locations of detailed

analyses. Dashed black lines separate images taken at different focal depths in the thin

section. (b-c) Raman maps (from red boxedMANUSCRIPT area in (a)) of the ~1600 cm-1 carbon ‘G’

band from two different focal depths, showing the distribution of kerogen-like carbon.

(d) Three colour overlay Raman maps of the ~1600 cm-1 carbon ‘G’ band (red), 465

cm-1 quartz band (blue) and ~1090 cm-1 carbonate band (green) showing the infilling

of cellular compartments by quartz. (e) SEM-BSE image of a transverse section

through the microfossil (from green line in (a)) demonstrating its cylindrical nature,

presence of lateral cell wall (green arrow) and thin sheath (red arrow). (f) SEM-BSE

imageACCEPTED of a longitudinal section through the microfossil (from blue dashed line in (a))

showing a chain of box-like cellular compartments with both lateral cell walls and

septa that are sometimes taphonomically contracted or folded (e.g., green arrow). A

thin sheath is also visible in places (e.g., red arrow).

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Fig. 2. Elemental analysis of Cephalophytarion. (a) Bright field STEM image of part

of the microfossil shown in Fig. 1. (b-f) Corresponding EDX elemental maps created

using the K peaks for carbon (b), calcium (c), sulphur (d), chlorine (e) and silicon (f).

Note the thick lateral carbonaceous cell walls and septa, plus very fine outer

carbonaceous sheath (red arrows), plus preservation of some potential organic cell

contents around nanospherular quartz grains (e.g., yellow arrows).

Fig. 3. EDX spectra from Cephalophytarion. (a) High angle annular dark field

(HAADF)-STEM image of the same microfossil as Fig. 2. Boxed regions show

localities where EDX spectra were obtained. (b) EDX spectrum from an area

encompassing the organic microfossil walls showing significant C, Ca and S peaks.

significant Cl peak. In both spectra, Si and O come from the quartz matrix, and Cu and Ga are instrumental artefacts (from theMANUSCRIPT Cu TEM grid and Ga ion implantation respectively).

Fig. 4. Correlative microscopy of a filamentous object from the ~3460 Ma Apex

Basalt. (a) Optical photomicrograph of a filamentous object morphologically identical

to objects previously classified (Schopf, 1993) as Primaevifilum. (b-c) Raman maps

(from red boxed area in (a)) of the ~1600 cm-1 carbon ‘G’ band from two different focalACCEPTED depths, showing the distribution of kerogen-like carbon. Note presence of box- like to spheroidal carbon-poor compartments distributed rather randomly throughout

the filament (blue arrows). (d) Three colour overlay Raman maps (from white dashed

box in (c)) of the ~1600 cm-1 carbon ‘G’ band (red), 465 cm-1 quartz band (blue) and

~145 cm-1 anatase band (green). (e-f) SEM-BSE images of transverse sections Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

through the filament (from green dashed lines in (a)) demonstrating its non-cylindrical

nature, absence of lateral carbonaceous walls, and angular, plate-like mineralic

morphology. (g) SEM-BSE image of a longitudinal section through the filament (from

dashed blue line in (a)) showing absence of lateral carbonaceous walls and significant

changes in diameter along its length. The filament mostly comprises angular stacks of

plate-like aluminosilicate mineral crystals (light grey) interspersed with quartz grains

(mid grey; blue arrows), carbon and void space (black), plus iron oxyhydroxides

(white). Note significant differences between SEM-BSE images in Fig. 4e-g and those

shown in Fig. 1e-f.

Fig. 5. Raman and electron microscopy analysis of a second Apex filament. (a)

Optical photomicrograph of a filamentous object morphologically equivalent to

objects previously classified (Schopf, 1993) as Primaevifilum. (b-d) Raman maps of the ~1600 cm-1 carbon ‘G’ band from threeMANUSCRIPT different focal depths, showing the distribution of kerogen-like carbon. Note presence of mostly solid carbonaceous

features interspersed with narrow mineralic ‘walls’ (blue arrows). (e) HAADF-STEM

image of a longitudinal cross section through the same filament shown in (a-d). The

filament mostly comprises angular stacks of plate-like aluminosilicates (light grey)

interspersed with some quartz grains (mid grey; e.g., blue arrows), carbon and void

space (black), plus iron oxyhydroxides (bright grey/white) that are often As-rich. Note howACCEPTED the orientation of the plate-like aluminosilicates changes towards the left of the image. (f) TEM-EDS map of carbon obtained at x 40,000 magnification. Note the

very narrow, angular sub-vertical distribution of carbon, lack of any morphology that

could be interpreted as cellular, and stark contrast to the Raman maps in (b-d).

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Fig. 6. Detailed elemental analysis of an Apex filamentous object. (a) HAADF-STEM

image of part of the Apex filament shown in Fig. 4. (b) EDX elemental maps of

carbon (red), iron (green) and aluminium (blue) from the boxed region of (a). These

show that carbon mostly occurs as narrow linear features with a morphology

controlled by the shapes of the Fe-rich and Al-rich crystal faces. There is no evidence

for cellular morphology. The majority of the filament body comprises

aluminosilicates plus iron oxyhydroxides, as shown by the element maps in (c-f) and

EDX spectra in Fig. 7.

Fig. 7. EDX spectra from the aluminosilicate and oxyhydroxide mineral phases within

the Apex filaments. (a) HAADF-STEM image of a longitudinal cross section through

the same filament shown in Fig. 6 with boxed areas showing regions where EDX

spectra were obtained. (b) EDX spectrum from a Ba-poor region of the aluminosilicate. Note K, Al, Si, O peaks, plusMANUSCRIPT minor Fe and C. (c) EDX spectrum from a Ba-rich region of the aluminosilicate. (d) EDX spectrum from the

oxyhydroxide phase showing large Fe peaks, minor As, trace of Ni, plus moderate

levels of C. The K, Al and Si peaks in this spectrum come from the surrounding

aluminosilicate and quartz minerals. Cu and Ga peaks in all spectra come from

instrumental artefacts due to Cu TEM grids and Ga ion implantation respectively.

Fig.ACCEPTED 8. Insights into the mineralogy and carbon syngeneity of Apex filaments from electron microscopy. (a) Overview of part of a Primaevifilum filament showing the

location of the HRTEM images in (b) and (c). (b) HRTEM image of the phyllosilicate

phase showing the 1 nm spacing characteristic of 2:1 phyllosilicates such as

muscovite. (c) HRTEM image of the Fe-O-rich phase consistent with the <102> zone Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

axis of goethite. White lines denote the {211} planes. (d) Aluminosilicate grains

(yellow arrows), in close proximity to Primaevifilum filaments, which are not

arranged into chains and therefore lack a filamentous microfossil-like morphology;

carbon can sometimes be associated with such grains. (e) A partially carbon-filled

crack (white arrows) that occurs in close proximity to a branched Primaevifilum

filament. Note also the aluminosilicate grains associated with carbon but not part of

the main filament (yellow arrow). (f-g) A network of partially carbon-filled cracks

(white arrows) occurring close to one end of a Primaevifilum filament. Again note the

small aluminosilicate grain associated with carbon and iron oxyhydroxide some way

away from the main filament (yellow arrow). (h-i) A further carbon filled fracture

adjacent to a Primaevifilum filament.

Fig. 9. Reconciling electron microscopy and Raman data for Apex filaments and a Cephalophytarion microfossil. (a) TEM-EDXMANUSCRIPT elemental map of the distribution of carbon within part of the Apex filament shown in Fig. 4. (b-d) TEM-EDX map of

carbon with a 50 pixel (b), 100 pixel (c) and 150 pixel (d) Gaussian blur filter applied.

Note how narrow linear objects are transformed into more rounded thicker objects. (e)

Raman map of the ~1600 cm-1 carbon ‘G’ band from part of the same Apex filament.

Note that (a-d) are necessarily taken from a different orientation of the filament to (e)

but despite this the Raman map and TEM map with 150 pixel blur filter show similar patternsACCEPTED of carbon, including hollow (mineralic) compartments (arrows). (f-h) Raman maps of the ~1600 cm-1 carbon ‘G’ band from three paratype examples of

Primaevifilum previously illustrated by Schopf et al. (2002). Note similar features to

(e) including mostly solid-looking regions of kerogen-like material and occasional

hollow (mineralic) compartments of variable size. (i) TEM-EDX elemental map of the Downloaded from http://jgs.lyellcollection.org/ by guest on October 2, 2021

distribution of carbon within part of the Cephalophytarion filament shown in Fig. 1.

(j-k) TEM-EDX map of carbon with a 50 pixel (j) and 100 pixel (k) Gaussian blur

filter applied. (l) Raman map of the ~1600 cm-1 carbon ‘G’ band from part of the

same Cephalophytarion filament. Note that (i-k) are necessarily taken from a different

orientation of the filament to (l) but despite this the Raman map and TEM map with

100 pixel blur filter show similar morphological features. Parts (f-h) are reproduced

with permission from Schopf et al. (2002).

Fig. 10. A further example of reconciling electron microscopy and Raman data for an

Apex filament. (a) TEM-EDX elemental map of the distribution of carbon within an

Apex filament (same specimen as shown in Fig. 5). (b-e) TEM-EDX maps of carbon

with a 20 pixel (b), 50 pixel (c), 100 pixel (d), and 150 pixel (e) FWHM Gaussian blur

filter applied. (f) Raman map of the ~1600 cm-1 carbon ‘G’ band from the same Apex filament. Note that (b-e) are necessarily takenMANUSCRIPT from a different orientation of the filament to (f) but despite this the carbon ‘G’ Raman map and TEM carbon map with

150 pixel blur filter show similar morphological features, including narrow mineralic

‘walls’ (arrows) separating more solid-looking regions of carbon. Scale bars are 1 m.

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