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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
© 2019 The Author(s). This is an Open Access article distributed under the terms of the Creative
Commons Attribution 4.0 License (http://creativecommons.org/licenses/by/4.0/). Published by The
Geological Society of London. Publishing disclaimer: www.geolsoc.org.uk/pub_ethics
To cite this article, please follow the guidance at http://www.geolsoc.org.uk/onlinefirst#cit_journal
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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 (David.Wacey@uwa.edu.au)
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
filaments 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
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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 assemblage 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
angular 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
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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 Bitter 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) 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
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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 ridge 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).
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
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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).
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, 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 944 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
the ~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
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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 TEM 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
sample (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
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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 electron 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).
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
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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 Cephalophytarion 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
3-5 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
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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 lateral 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
microfossil 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
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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 occasions (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
morphology 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.
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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 spacings 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,
and 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
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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 such 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
phase 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,
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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).
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
morphology 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
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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 put 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
Cephalophytarion 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
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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 filaments 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
filaments 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 morphological 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;
Schopf & 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
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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 filaments 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
minerals 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
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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 minerals 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
the 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 together (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
heterogeneously 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 muscovite 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
via 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
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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, 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
Previous 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
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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 for 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
these 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
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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 small 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
objects 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
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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 of 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
isotopic 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 microscopy 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
cross 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
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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, 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, 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
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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 boxed 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
image 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.
(c) EDX spectrum from nano-porous quartz infilling the microfossil showing
significant Cl peak. In both spectra, Si and O come from the quartz matrix, and Cu
and Ga are instrumental artefacts (from the 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
focal 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
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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 three 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
how 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, plus 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. 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
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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-EDX 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
patterns 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
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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 taken 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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