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New perspectives on ancient microbes and microbialites: from isotopes to immunology

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New perspectives on ancient microbes and microbialites: from isotopes to immunology

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Content NEW PERSPECTIVES ON ANCIENT MICROBES AND MICROBIALITES: FROM
ISOTOPES TO IMMUNOLOGY
by
Jake Vincent Bailey
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(EARTH SCIENCES)
August 2008
Copyright 2008 Jake Vincent Bailey
ii
Acknowledgements
My decision to come to USC for graduate school was largely based on my interest
in working with my advisor, Frank Corsetti. In retrospect, it was the best decision I could
have made. Frank struck the perfect balance between giving me the freedom to find my
own paths, and providing the support, advice, and training I needed to succeed. His
mentoring and dedication to excellence in research and teaching will unquestionably
serve as a model for me to follow in my own academic career. Frank is thanked not only
for his excellence and professionalism as an advisor, but also for his friendship.
My spouse, Beverly Flood, has been my partner and best friend throughout my
five years at USC. Her passion for microbial ecology is infectious and our conversations
have served as a source of constant inspiration and reflection. We have both grown
together as people, and as scientists during our time at USC. It is with great excitement
and happiness that I look forward to our future together.
Chapters 2 through 6 form the core of manuscripts published (Chp. 3 & 4),
submitted for publication (Chp. 2), or in preparation for submission (Chp. 5 & 6), and
involved input from collaborators. Chapter 2, entitled “Isotopic signatures in n-alkanes
from Holocene lacustrine stromatolites: Implications for the detection of ancient layered
microbial communities”, was submitted to Organic Geochemistry with co-authors Jian
Peng and Frank Corsetti and is currently under revision for eventual resubmission. I
thank Arndt Schimmelman of Indiana University and Miguel Rincon of the University of
Southern California for help on the isotope calibration standards. I also thank Nate
Lorentz, and Alex Sessions for analytical support and discussions. Stan Awramik
(UCSB) initially pointing out the Walker Lake stromatolites localities. I am grateful for
iii
the support of Jian Peng and David Tsukada at the Southern California Coastal Water
Research Project for supporting my organic geochemical studies and providing me with
invaluable training. Chapter 3 was published in the journal Palaios under the title:
“Evidence of microbial communities preserved in siliciclastic sediments. Microbially-
Mediated Environmental Influences on Metazoan Colonization of Matground
Ecosystems: Evidence from the Lower Cambrian Harkless Formation”, with co-authors
Frank Corsetti, David Bottjer, and Katherine Marenco. Donn Gorsline assisted with x-
radiography. The bulk of the material in Chapter 4 was published as a letter to Nature,
under the title “Evidence of giant sulfur bacteria in Neoproterozoic phosphorites” and
involved Karen Kalanetra, Samantha Joye, Beverly Flood, and Frank Corsetti. Chapters 5
& 6 are in preparation to be submitted for publication. The work in Chapter 5 was made
possible by the generous contributions of by Crescentin antibodies and strains CB15, and
∆cres provided by Christine Jacobs-Wagner and Matthew Cabeen. Jeanne Poindexter
provided isolates of C. leidyia and Caulobacter sp. CM243. John Smit and John
Nomellini provided isolates of Maricaulis washingtonensis MSC6, C. halobacteroides
CM13a, Hyphomonas adhaerens MHS-1, Woodsholea maritime CM243, and Maricaulis
maris CM11. Chapter 6 involved David Caron, Frank Corsetti, Mike Moldowan, Fred
Fago, and Wei Shao. I am also very thankful for the generous support provided by Dr.
Alan Epstein of the Keck School of Medicine who was instrumental in production of the
monoclonal antibodies used in Chapter 6.
The Agouron Geobiology Course, MBL Microbial Diversity Course, and the
professors and students in USC Earth Sciences and Marine Environmental Biology were
essential to my development as an interdisciplinary scientist. I would especially like to
iv
thank my committee members, David Bottjer, Will Berelson, and David Caron for their
suggestions, mentoring, thesis editing, and support of my laboratory research. Al Fischer
is greatly thanked for his advice on my thesis proposals. This work was financially
supported by fellowships and grants from the University of Southern California, the
National Science Foundation, the Geological Society of America, the American
Association of Petroleum Geologists, and NASA.
v
Table of contents
Acknowledgements ii
List of figures vii
List of tables x
Abstract xi
Chapter I: Introduction
Geobiology 1
Sedimentary Structures 4
Microfossils 6
Biomarkers 9
Chapter II: Isotopic signatures in n-alkanes from Holocene lacustrine
stromatolites: Implications for the detection of ancient layered
microbial communities
Abstract 12
Introduction 13
Methods 17
Results 23
Discussion 28
Conclusions 42
Chapter III: Evidence of microbial communities preserved in
siliciclastic sediments. Microbially-mediated environmental
influences on metazoan colonization of matground ecosystems:
Evidence from the Lower Cambrian Harkless Formation
Abstract 45
Introduction 46
Geologic Context 49
Methods 51
Results 53
Discussion 58
Conclusions 72
Chapter IV: Evidence of giant sulfur bacteria in Neoproterozoic
phosphorites
Abstracts 73
Introduction 73
Methods 75
Colorless Sulfur Bacteria – Background 76
Morphology and Context of Doushantuo Microfossils 80
vi
Comparison of Thiomargarita with Doushantuo Microfossils 81
Other Associated Fossils and Structures 93
Sulfur Bacteria and Phosphogenesis 96
Conclusions 101
Chapter V: Is cell shape conserved over geologic timescales? Clues
from a cytoskeletal protein in the dimorphic prosthecate bacteria
Abstract 103
Introduction 104
Methods 107
Results 111
Discussion and Conclusions 118
Chapter VI: Assessing biomarker syngeneity: An in-situ approach using
monoclonal antibodies to squalane
Abstract 124
Introduction 125
Materials and Methods 128
Results 132
Discussion 143
Conclusions 154
Bibliography 156
Appendix A: Brief Communication Arising by Xiao et al. 186
Appendix B: Reply to Brief Communication Arising 192
vii
List of figures
2-1 Map of Walker Lake 18
2-2 Field photographs of Walker Lake stromatolites 18
2-3 Encrusted Walker Lake algae 19
2-4 Photomicrograph of Walker Lake stromatolites 19
2-5 Isotopic composition of n-alkanes from stromatolites and biomass 27
2-6 Isotope mixing model cartoon 38
2-7 Comparative sampling strategy illustration 41
3-1 Stratigraphic section of the White-Inyo succession 50
3-2 Field photograph of Lower Cambrian wrinkle structures 50
3-3 Field photographs of Harkless Formation sedimentary structures 52
3-4 Map of Cedar Flat locality 52
3-5 Detail of wrinkle structure sedimentary features 54
3-6 Fossils associated with wrinkle structures 54
3-7 Detail of agglutinated fossil, Volborthella 56
3-8 Matground ecosystem cartoon 69
4-1 Detail of Thiomargarita ultrastructure 77
4-2 Comparison of Thiomargarita with Doushantuo microfossils 78
4-3 Thiomargarita tetrad exhibiting distortion of the division planes 84
4-4 Thiomargarita tetrad with decussate geometry 84
4-5 Thiomargarita tetrad with tetrahedral geometry 84
4-6 Thiomargarita octad 85
4-7 Multi-cell Thiomargarita cells 86
viii
4-8 Internal inclusion aggregates in Thiomargarita 90
4-9 Thiomargarita mat from the Gulf of Mexico
4-10 Collapsed Thiomargarita cell 94
4-11 Other Doushantuo microfossils 94
4-12 Microbial filaments associated with Thiomargarita cells 95
4-13 Neoproterozoic spread of the colorless sulfur bacteria 100
5-1 Caulobacter wild type and creS mutant 106
5-2 Immunofluorescent detection of crescentin in prosthecate bacteria 112
5-2 16S rDNA dendrogram of examined strains 114
5-3 Growth curves for Caulobacter wild type and creS mutant 115
5-4 Caulobacter capillary tube motility assay results 116
5-5 Caulobacter warm agar motility results 117
6-1 ELISA results for squalane 133
6-2 ELISA results for tetracosane 133
6-3 ELISA results for alkane mixture 134
6-4 ELISA results for pristine and phytane 134
6-5 ELISA results for lycopane 134
6-6 ELISA results for stigmastane 135
6-7 ELISA results for cholesterol 135
6-8 Immunoreagent wells on rock section 141
6-9 Immunofluorescent detection of squalane in organic laminae 141
6-10 Immunofluorescence negative control wells 142
6-11 Immunofluorescence of squalane-coated silylated glass slides 142
ix
6-12 GC-MS chromatogram of Green River Formation extract 144
6-13 Mineral autofluorescence in imaged sections 144
6-14 Coccoid microfossils from the Green River Formation 151
6-15 Fish fossils from the Green River Formation 151
6-15 Biomarker distribution and syngeneity cartoon 151
x
List of tables
2-1 Walker Lake stromatolite locality and alkane data 20
2-2 δ
13
C values for n-alkanes: stromatolite-associated organic matter 25
2-3 δ
13
C values for n-alkanes: modern Walker Lake biomass 26
2-4 Isotopic composition of alkanes: hypothetical mat community A 34
2-5 Isotopic composition of alkanes: hypothetical mat community B 35
5-1 Bacterial strains and media 108
6-1 ELISA reactivities of squalane, controls, and various other purified 139
and mixed hydrocarbons to the anti-squalane monoclonal antibody
xi
Abstract
Morphologic data has always played a foundational role in our understanding of
ancient life. Visual comparisons often show remarkable similarities between fossils and
extant organisms, and yet the modern molecular and geochemical era has taught us that
appearances can deceive, and that the genetic and metabolic diversity of the microbial
world is greater than anything that could have been previously imagined. What can these
revelations in our understanding of the modern microbial biosphere tell us about ancient
life, particularly where morphology is conservative?
Sedimentary structures such as stromatolites and winkle structures are often
thought to result from the activities of metabolically-complex microbial mat
communities. A compound-specific stable isotope approach is used here to analyze n-
alkanes from Holocene lacustrine stromatolites. Stromatolite-associated n-alkanes are
isotopically similar to those is surrounding sediments – a finding that is perhaps
inconsistent with the assumption that metabolically-diverse microbial mat communities
were once associated with these structures. Metabolic diversity in microbial mat
communities may however, explain the correlation of a particular fossil assemblage with
wrinkle structures from the Lower Cambrian Harkless Formation described here.
Fossil cells also provide a window onto the evolution of microbial life. The shape
of fossil cells is thought to be an important indicator of their phylogenetic affinities.
However, an immunofluorescence and cell biology study of Caulobacter crescentus and
its prosthecate relatives suggests that shape-influencing genes may be labile over
geologic time scales. Convergent evolution may also result in morphological similarities
xii
between distantly-related organisms. Morphologies in modern giant sulfur bacteria are
presented that closely resemble abundant globular microfossils from the 600 Ma
Doushantuo Formation commonly thought to represent animal embryos. If correct, this
reassessment may also explain some lithological and geochemical features of the
Neoproterozoic record, such as sulfur isotope excursions and phosphorite proliferation.
Finally, molecular detection methods used to study extant life may also be
adapted for the study of ancient life. Monoclonal antibodies allow for the in situ detection
of squalane in Eocene-age rocks and suggests the potential to use immunological probes
to visualize the distribution of molecular fossils in microfossils, rock fabrics etc.
1
Chapter I: Introduction
In 1880, Sergei Winogradski discovered Beggiatoa, a bacterium that was capable
of fixing cell carbon from CO
2
while obtaining energy by oxidizing hydrogen sulfide
(Thornton, 1953). Around the same time, the Dutch microbiologist, Martinus Beijerinck,
pioneered the use of enrichment culturing and discovered bacteria that use sulfate as a
terminal electron acceptor in anaerobic respiration (Theunissen, 1996). Beijerinck also
discovered bacteria that fix atmospheric N
2
, converting it to a bioavailable form.
Winogradski and Beijerinck recognized that these processes have important implications
for the cycling of elements and transfer of energy in the environment. The century-old
discovery of microorganisms that derived energy and nutrients from the oxidation or
reduction of inorganic substrates paved the way for a modern interdisciplinary field,
known as geobiology. Geobiology, by the definition accepted here, is the study of the
interaction of the biosphere with the geosphere through time.
While the field of geobiology is not limited to the study of single-celled
organisms, they have received the most attention because their metabolisms are the most
diverse, and their activities have had the most profound effect on the geosphere over the
last four billion years (Newman and Banfield, 2002). For example, bacteria were the first
organisms to produce free oxygen – a process that transformed the Earth’s early
atmosphere and eventually its oceans and rocks (e.g., Holland, 1992). Bacteria and
archaea have transformed the geochemistry of the ocean and sediments through their
oxidation and reduction of various inorganic chemical species such as Fe, Mn, and S
among others (Ehrlich, 1995; Lovley and Philips, 1988; Myers and Nealson, 1988;
2
Nealson and Berelson, 2003) and through the precipitation and dissolution of numerous
minerals (Bell et al., 1987; Kotska and Nealson, 1995; Reith, 2006; Schulz and Schulz,
2005). The respiration of methane by bacteria and archaea may even influence global
climate (Boetius et al., 2000; Dickens, 2003; Ziebis and Haese, 2005).
One important aspect of the field of geobiology is the recognition that
geobiological processes have a history that is most assuredly relevant to our
understanding of the evolution of the biosphere and the Earth itself. Many geobiologists
have posited links between modern geobiological processes and lithological,
paleontological, or geochemical features in the rock record (Canfield and Raiswell, 1999;
Shen et al., 2001). Some purported connections between modern microbial processes and
ancient geologic features remain quite controversial or poorly understood. For example,
banded iron formations were originally thought to have resulted from oxygen produced
by cyanobacteria (Cloud, 1965; Holland, 1992). But other investigators suggest that
reduced iron in the oceans was oxidized directly by anoxygenic phototrophs (Kappler et
al., 2005). While our understanding of the ancient biosphere will never be complete, it is
only through the confluence of our study of modern processes and the rock record that we
will broaden our perspectives, improve our confidence in our interpretations, and
appreciate the magnitude of our ignorance. My fascination with the ancient record of
microbial biogeochemical processes drove my selection of the diverse topics investigated
in this dissertation. This dissertation represents a five-year effort to use specific
discoveries in the biological sciences to re-examine aspects of microbial paleobiology.
3
In 1670, the Italian paleontologist Agostino Scilla argued that the senses do not
deceive – fossils are the remains of ancient organisms similar to those living today. In the
era of Scilla, and later Linnaeus, and even through much of the 20
th
century,
morphological comparison was used as the basis for interpretation and classification, both
in biology and paleontology. But with the advent of the molecular age, much has been
revealed that was hidden behind the façade of morphology. A recent example from the
metazoan world was the discovery that the worm Buddenbrockia, long thought to be a
bilaterian, is actually a cnidarian (Jiménez-Guri et al. 2007)! Morphology in
microorganisms is often much more conservative than in metazoans and molecular
techniques have not only revolutionized our appreciation of microbial diversity but also
massively reorganized microbial phylogenies. The concomitant discovery of microbial
interactions with the geosphere, particularly its mineralogy and chemistry, have changed
our perceptions of modern microbial ecosystems, and must also be used to reevaluate our
view of ancient ecosystems and the fossil, sedimentary, and chemical biosignatures that
record them. The unifying theme of chapters 2-5 is that observations from modern cell
biology, genetics, biogeochemistry, and ecology of modern microbial ecosystems can
help to inform, or cast doubt upon, our understanding of ancient life that has historically
relied almost solely on morphological data. In Chapter 6, it is the tools of modern
molecular biology in the form of monoclonal antibodies that are used to help improve our
understanding of the history of microbial life.
Indicators of ancient life, or biosignatures, take a variety of forms. Microbes can
shape their physical environment by influencing sedimentary processes, as well as the
4
dissolution and precipitation of minerals. The influence of these processes can be seen in
the structure and composition of sedimentary structures, such as some stromatolites and
wrinkle structures (Awramik, 1977; Hagadorn and Bottjer, 1997; Riding, 1990). Under
certain circumstances, microbial cells themselves can be preserved, either as mineral-
entombed organic matter, or as permineralized structures (e.g., Schopf and Klein, 1992).
In other cases, microbial life can leave behind organic or inorganic chemical signatures
that are characteristic of their clade or metabolic activities (e.g., Brocks and Summons,
2003). In the investigations presented here, my collaborators and I have contributed to
our understanding of these indicators of ancient microbial life with new observations,
novel interpretations, and the development of original analytical techniques. The
following background material on the various modes of preservation and representation
in the rock record sets the context for the chapters that follow:
Sedimentary Structures
Certain sedimentary structures, such as stromatolites and “Kinneyia”, were
thought to represent fossilized plant or algal material as early as the late-19
th
century
(Matthew, 1890; Walcott, 1883; Walcott, 1914). These interpretations were highly
controversial in their era. It was only after modern microbial mats associated with recent
stromatolites were shown to trap and bind sedimentary grains that stromatolite
biogenicity became more widely-accepted (Logan et al., 1964; Schopf et al., 1971). More
recently, stromatolite lithification has also been postulated to have been influenced my
microbial processes (Chafetz and Buczynski, 1992; Visscher et al., 2000). The pendulum
5
has now swung the other way, and stromatolite biogenicity is often taken as a default
assumption (Schopf et al., 1974), despite considerable evidence that stromatolites can
form the in the absence of microbial activity (Grotzinger, 1999; Lowe, 1994, Mcloughlin
et al., 2008). New criteria or indicators are needed to evaluate the biogenicity of
stromatolites. In Chapter 2 my co-authors and I investigate the possibility that
differences in the isotopic composition of n-alkanes preserved in stromatolites vs. those
in surrounding sediments might be used as a means of evaluating whether or not such
structures formed in association with the type of metabolically-complex, layered
microbial communities commonly thought to be involved in stromatolite petrogenesis.
The approach of comparing geochemical signatures in putative microbialites with those
in surrounding sediments, may provide a fruitful avenue for distinguishing microbially-
mediated sedimentary structures from abiotic constructs.
Stromatolites are not the only sedimentary structures commonly thought to have
been mediated by microorganisms. Convolute patterns on bedding plane surfaces in
siliciclastic sediments (e.g., wrinkle structures, Kinneyia, elephant hide structures, etc.)
are thought to result from the trapping and binding of sediment grains by extra cellular
polymeric substances produced by microbes (Hagadorn and Bottjer, 1997, 1999; Gehling,
1986, Noffke et al., 2001), although some workers urge prudence in the application of
this interpretation to similar structures that can be produced abiotically (Porada and
Bouougri, 2007). Wrinkle structure-covered bedding plane surfaces from the Lower
Cambrian Harkless Formation co-occur with a distinctive metazoan fossil assemblage. If
the Harkless Formation wrinkle structures do in fact indicate the presence of microbial
6
mats, the geochemical conditions associated with such communities, as inferred from
conditions beneath modern mats, may well have influenced their inhabitation by early
metazoans. Evidence for such a relationship is presented in Chapter 3.
Microfossils
Although potential microfossils of purported Precambrian age were known more
than a century ago, it wasn’t until the discovery of fossilized cells in the 1.9 Ga Gunflint
Formation that the search for evidence of life in the Precambrian began in earnest
(Barghoorn and Tyler, 1965; Cloud and Hagen, 1965; Tyler and Barghoorn, 1954). The
Gunflint microbiota contained perhaps a dozen distinctive morphotypes, and was closely
associated with stromatolites and banded iron formations. These associations inspired
investigators such as Preston Cloud to consider a possible link between the evolution of
microbial life and the evolution of the Precambrian environment as recorded in certain
lithologic features (Cloud, 1973; Cloud, 1965). Other discoveries followed the discovery
of the Gunflint microbiota - discoveries that demonstrated a rich record of microbial life
dating back to the Archean (Barghoorn et al., 1965; Barghoorn and Schopf, 1966;
Schopf, 1968). For example, filamentous organic-walled structures interpreted as bacteria
preserved in 3.5 Ga cherts from the Warrawoona Group in Australia were described by
(Schopf and Packer, 1987). Although the biogenic interpretation of the Warawoona
Group structures has been challenged (Brasier et al., 2002; M.D. Brasier), the presence of
probable microbial filaments within carbonaceous laminae from the Buck Reef Chert (3.4
Ga) of South Africa support a ~3.5 Ga age for the earliest fossil microbes (Ticeand Lowe,
7
2004). Microfossils from the Paleoproterozoic Transvaal Group (Nagy, 1974), the
Mezoproterozoic Skillogalee Dolomite (Knoll et al., 1975) and Neoproterozoic Bitter
Springs Formation (Knoll and Golubic, 1978; Schopf, 1968; Schopf, 1972; Schopf and
Blacic, 1971) show remarkable morphological similarity to modern cyanobacteria – a
similarity that lead to a paradigm in which fossil structures are often thought to represent
organisms with physiologies identical to those organisms possessing similar
morphologies today (Schopf, 1968; Schopf, 1992).
While it is reasonable that selective pressures have maintained the morphology of
some bacteria for billion of years, the common rearrangement of genomes (Miller and
Day, 2004), and the ability for one gene to change an organism’s cell shape (Ausmees et
al., 2003), suggest that the morphology of most microorganisms is unlikely to remain
unchanged over geological time scales without considerable selective pressure. In
Chapter 4, I show that one such shape-changing gene is restricted to only two organisms
among a host of genetically and ecologically related α-proteobacteria. Furthermore, the
gene’s phenotype, a curved rather than straight cell shape, appears to offer little selective
advantage in laboratory tests and is not expressed the same way in both organisms.
These observations invite the question, “Just how reliable of a phylogenetic indicator is
bacterial shape over geologic time scales?” Perhaps future investigations will be able to
answer that question, but for now, it is noteworthy that we have justification to ask it.
The record of microbial life in not restricted to preservation of representatives
from the bacterial and archaeal domains. Some fossil microbiotas record important
intervals in the evolution of eukaryotic life. For example, the oldest fossils readily
8
assignable to a modern clade (the red algae) are preserved in the 1200 Ma Hunting
Formation of Canada (Butterfield et al., 1990), while the Neoproterozoic Wynniatt
Formation (800-900 Ma) is thought to preserve the oldest known fungi (Butterfield,
2005; Butterfield et al., 1990). The Tindir Group microbiota, which includes probable
bacteria, fungi, acritarchs, and microscopic metazoans (Allison and Awramik, 1989;
Allison and Hilgert, 1986), offers a glimpse into microbial and metazoan life preceding
the Cambrian explosion. While some workers have proposed criteria for differentiating
eukaryotic microfossils from those of bacteria and archaea (Javaux et al., 2003), the
phylogenetic affinities of fossilized cells can be difficult to determine because microbial
morphology can often be conservative (Young, 2006), taphonomy can produce artifacts
that can be mistaken for biological features (Knoll and Barghoorn, 1975), and convergent
evolution can produce seemingly distinctive features in phyogenetically-distant groups.
For example, one of the most famous Precambrian microfossil assemblages is that hosted
by the ~ 600 Ma Doushantuo Formation (Awramik et al., 1985; Zhang, 1984). The
Doushantuo Formation contains a variety of microfossils thought to represent
cyanobacteria and algae (Zhang, 1981), but is perhaps best known for a population of
phosphatized globular microfossils. Initially interpreted as algae, Xiao et al. (1998) re-
interpreted these structures as cleavage-stage animal embryos based on the observation
that cell clusters maintained relatively constant volume as the number of internal bodies
increased. They also noted that their large size, and distortions of the plane of cell
division were incompatible with an algal interpretation (Xiao, 2002; Xiao and Knoll,
1999). In Chapter 5, my collaborators and I offer an alternative interpretation of the
9
most common Doushantuo globular microfossils as giant sulfur bacteria similar to the
modern genus Thiomargarita. The potential importance of this re-interpretation lies not
only in the testing of the theory that these fossils represent the oldest animal life, but also
that these sulfur bacteria are important in the mediation of phosphorites and other
biogeochemical cycles, which they may have begun to influence during the
Neoproterozoic.
Biomarkers
Biomarkers are the geologically-preserved hydrocarbon derivatives of lipids or
pigments that are thought to serve as indicators of a particular clade or metabolic class of
organism based on the restricted occurrence of the precursor biomolecule in the modern
biosphere (Brocks and Summons, 2003a; Peters et al., 2005). Biomarkers have been used
to discover much about ancient microbial ecosystems. However, the potential for ancient
rocks to be contaminated by hydrocarbons – either through natural processes or through
anthropogenic contamination, during or after sampling, makes establishing syngeneity an
important aspect of biomarker analyses (Brocks et al., 2003).
One approach to establishing biomarker syngeneity is to recognize that the spatial
distribution of sygenetic compounds should, in most cases, differ from the distribution of
contaminants. In Chapter 6, I introduce a novel lipid antibody approach for detecting
biomarkers in situ within mineral-bound organic matter. One application of this in situ
detection capability is that it allows for biomarkers to by visually associated with rock
fabrics. If the associated rock fabrics are primary, such as organic-rich laminae or
10
microfossils, then chances are good that the biomarkers included within them are also
primary. Similarly, if the biomarkers are associated with secondary features, such as
fracture surfaces, then one might reasonably conclude that the biomarkers themselves
were introduced as part of some post-depositional process.
While the in situ immunodetection of hydrocarbons certainly holds
tremendous potential for establishing biomarker syngeneity in ancient rocks, this
antibody-based approach may transform other aspects of the geobiological sciences. This
technique may provide a new window onto the microfossils record because the
association of biomarkers with microfossils could potentially reveal the phylogenetic
affinities of many organic-walled microfossils of uncertain origins. Potentially, these
results could revolutionize our understanding of the evolution of many deeply-rooted
microbial clades. For example, acritarchs are large organic-walled microfossils of
unknown phylogenetic affinities. Most are regarded as the resting cysts of eukaryotes.
Acritarchs appear in the early Proterozoic and their morphologically-defined record
shows an extended period of low diversity through the Mezoproterozoic prior to several
intervals of diversification. While acritarchs remain phylogenetically unresolved, they
almost certainly record an important interval in early eukaryote evolution – an interval
that we still no little about. If biomarkers characteristic of certain eukaryotic clades could
be reliably associated with these organic-walled fossils, this cryptic record may be
revealed to us. The research presented in this dissertation illustrates that our knowledge
of the modern microbial biosphere, its interactions with the geosphere, and even some of
the modern methods used to study it, can serve to broaden our understanding of the
11
ancient record. The present may not always be the key to the past, but without a thorough
understanding of the present, our understanding of the history of life will undoubtedly
remain incomplete.
12
Chapter II: Isotopic signatures in n-alkanes from Holocene lacustrine stromatolites:
Implications for the detection of ancient layered microbial communities
Abstract
Layered microbial communities are commonly associated with modern
stromatolites and are often assumed to have been important in the formation of ancient
stromatolites. Modern layered microbial communities utilize diverse metabolic
pathways. Isotope effects associated with these various pathways produce recognizable
fractionations in resulting biomass. We predict that the metabolic diversity common in
layered mat communities should be reflected in the isotopic composition of endogenous
stromatolite-associated hydrocarbons, particularly in comparison with hydrocarbons
sourced from the surrounding depositional environment. Alkanes extracted from
Holocene stromatolites of Walker Lake, Nevada were analyzed by GC-IRMS and
compared with alkanes from the modern limnic biomass. The isotopic composition of
stromatolite-associated alkanes at all chain lengths was largely indistinguishable from the
composition of modern algae. These results suggest that layered microbial communities
were either not present during stromatolite petrogenesis, or evidence for their existence
was replaced with an ambient signal by diagenetic processes. Both possibilities have
important implications for the detection of layered microbial communities associated
with ancient stromatolites, and we suggest a simple sampling strategy for testing these
possibilities in future studies of stromatolite-associated organic matter.
13
Introduction
Stromatolites are laminated accretionary growth structures (Semikhatov et al.,
1979) generally considered to have microbial origins (e.g., Allwood et al., 2006; Buick,
1992; Kalkowski, 1908; Semikhatov, 1976; Walter, 1976). Morphogenesis of some
modern stromatolites is demonstrably influenced by the trapping and binding of sediment
grains by microbially-excreted extracellular polymeric substances (e.g., Black, 1933;
Logan, 1961; Riding and Awramik, 2000). Microbial metabolic processes are also
thought to mediate stromatolite lithification in some instances (Chafetz & Buczynski,
1992; Guo et al., 1996; Reid et al., 2000; Visscher et al., 1998, 2000). Gross
morphological similarities between modern stromatolites that co-occur with extant
microbial mat communities and stromatolites in ancient rocks has led to a general
presumption of stromatolite biogenicity (e.g., Gebelein, 1974; Schopf et al., 1971, Reid et
al., 2000). However, some stromatolites are also interpreted to have formed in the
absence of microbial mats (e.g., Walter, 1976; Lowe, 1994), and numerical models
suggest that many stromatolite morphologies can also be produced without the need to
invoke biologic processes (Grotzinger & Knoll, 1999; Grotzinger & Rothman, 1996).
These observations and analyses emphasize that assumptions about stromatolite
biogenicity should be regarded with caution in the absence of other indicators of
microbial-mediated petrogenesis.
Establishing robust criteria for substantiating microbial involvement in the
formation of stromatolites is of considerable paleobiological importance, given that
Precambrian stromatolites are widely thought to represent some of Earth’s oldest traces
14
of life (e.g., Schopf et al., 1971; Buick, 1992; Allwood et al., 2006). Despite the common
presumption of stromatolite biogenicity, geochemical and lithological indicators that
might conclusively demonstrate their biological origins, which are both unambiguously
diagnostic of microbial involvement and present in ancient stromatolites, remain elusive.
The presence of fossilized remnants of a mat community within a stromatolite might
seem to suggest its biogenecity. However, fossilized microbes are only observed in a very
small percentage of ancient stromatolites (Grotzinger & Knoll, 1999). Furthermore, the
mere presence of fossilized microbes does not establish that the organisms played a role
in the formation of the stromatolite (Hoffman, 1973; Grotzinger & Knoll, 1999).
Isotopic and molecular signatures preserved in stromatolites might also reveal
details of ancient stromatolite-associated microbial ecosystems as outlined by McKirdy
(1976), and a number of studies have examined the biomarker and isotopic composition
of microbial mats with the goal of predicting what types of geochemical biosignatures
might be found in ancient stromatolites (e.g., Winters et al., 1969; Boon et al., 1981;
Jahnke & Summons, 2006). Modern mat ecosystems are commonly composed of layered
microbial communities containing bacteria and archaea with diverse metabolic pathways
that are spatially segregated by the availability of various wavelengths of light, as well as
the presence or absence of various terminal electron acceptors (e.g., O
2
, NO
3
-
, Fe3
+
, Mn
4+
,
SO
4
2-
) (Canfield & Des Marais, 1993; Fourcans et al., 2004; Jørgensen et al., 1979;
Nealson & Berelson, 2003). Autotrophic bacteria and archaea that inhabit layered
microbial communities are known to use four different pathways to fix carbon: the Calvin
cycle (the reductive pentose phosphate cycle), the reductive acetyl-CoA pathway, the 3-
15
hydroxypropionate cycle, and the reverse tricarboxylic acid cycle (rTCA) (House et al.,
2003; Preuβ et al., 1989). These four pathways, which are discussed further in section
4.2. preferentially incorporate
12
C to varying degrees, resulting in distinct ranges of
isotopic compositions for the biomass resulting from each type of carbon fixation (e.g.,
Guy et al., 1993; House et al., 2003). In addition to the effects of these autotrophic
pathways, methane depleted in
13
C can be used as a carbon source to produce
isotopically-light biomass by methanotrophic bacteria (Gelwicks et al., 1994; Summons
et al., 1994). Other heterotrophic processes common in microbial mats can affect the
isotopic composition of evolved CO
2
(Blair et al., 1985), but the importance of these
processes of the isotopic composition of mat-associated organic matter are likely to be
relatively minor.
If a particular stromatolite underwent petrogenesis in the presence of a
metabolically-diverse layered community, then we predict that isotopic values of
endogenous organic matter derived from such a community, if preserved, would reflect
the influences of those diverse metabolisms. An example of such a localization of
isotopically-distinctive lipids in microbially-mediated sedimentary structures is illustrated
by carbonate chimneys from the Black Sea that enclose mats of microbial consortia that
anaerobically oxidize methane (Michaelis et al., 2002). Lipids derived from these
communities have been recovered from the chimneys, with δ
13
C values ranging from –89
to -95‰ (Michaelis et al., 2002). Similarly, if a stromatolite’s formation was mediated by
microbial activity in the presence of a metabolically-diverse biotic community, then
stromatolite-associated, mat-derived syngenetic organic matter should likely retain a
16
diverse lipid isotopic signature distinct from organic matter derived from ambient
organisms unrelated to the stromatolite. Conversely, stromatolites precipitating via
abiotic processes are less likely to record organic biomarkers or isotopic signatures that
differ from the surrounding sediments, as both are likely to incorporate similar
compounds derived from the same water column or benthic organic detritus.
In this study we present an isotopic analysis of n-alkanes extracted from Holocene
stromatolites exposed on the shoreline of Walker Lake, Nevada, and compare them with
n-alkanes extracted from modern Walker Lake algal biomass. A compound-specific
isotope analysis extends the possibility of capturing isotopic signals from more than one
biological source (Collister et al., 1994; Hayes et al., 1990) because alkanes of various
chain lengths can result from the degradation and alteration of a variety of biomolecular
precursors produced by metabolically-diverse organisms (Johns, 1986; Peters et al.,
2005). For example, long-chain alkanes and their precursors are more abundant in higher
plants, whereas middle and short chain length alkanes and precursors are more
characteristic of algae and bacteria (Peters et al., 2005.
Geological setting
Walker Lake, a remnant of Pleistocene Paleolake Lahontan, is a saline, alkaline
lake located in the Great Basin, Nevada on the eastern edge of the Sierra Nevada
Mountains (Fig. 2-1) (e.g., Russell, 1885; Koch et al., 1979). Its terminal basin hydrology
has resulted in lake level fluctuations in response to Holocene climatic change/headwater
diversion (Meyers and Benson, 1987; Benson et al., 1991; Yuan et al., 2004; Yuan et al.,
17
2006; Adams 2007), and more recent anthropogenic diversions of the Walker River
(Benson & Leach, 1979; Beutel et al., 2001). Carbonate-cemented sediments and
calcareous “tufa” deposits can be found on modern shoreline and paleo strand lines
(Newton & Grossman, 1988; Russell, 1885). Steady lake level retreat since 1882 has
exposed small stromatolites (<10 cm thick) encrusting granitic boulders (Osborne et al.,
1982; Awramik et al., 1992). Some of the boulder-encrusting stromatolites are currently
submerged in shallow water along the lake margin (Fig. 2-2a). Subaqueous stromatolites
are subjected to intense wave action (Fig. 2-2b) and inundation by rafts of decaying
organic detritus (Fig. 2-2c). In cross section, Walker Lake stromatolites often consist of a
distinct well-laminated lower portion, overlain by a poorly-laminated <2 cm-thick outer
rind often covered with organic debris, such as Cladophora filaments (Fig. 2-3). Within
the well-laminated portion, laminae are generally continuous and isopachous, with lamina
thicknesses on the order of ~100 µm. In thin section, Walker Lake stromatolites exhibit a
high porosity (blue-stained epoxy in Figure 2-4), both as void space between laminae
(Fig. 4-4a), and as the result of abundant internal fractures (Fig. 2-4b).
Methods
Sample collection
Hand samples of stromatolites were collected from the western shore of Walker
Lake (38
o
39’32.2”N, 118
o
45’34.8”W) (Fig. 2-2a and Table 2-1 contains sample numbers
and sampling locations relative to the modern shoreline). All carbonate samples were
packed and stored in pre-combusted aluminum foil in the field. Samples of algal biomass
18
from the water column, rafts of living Cladophora from surface waters, and desiccated
Figure 2-1. Map of stromatolite collection locality on the western shore of Walker Lake
near Hawthorne, Nevada.
Figure 2-2. Walker Lake stromatolites encrust shoreline boulders (a, b), and are exposed
to wave action (c) and rafts of algal and plant debris (d).
19
Figure 2-3. Filamentous algae, such as Cladophora glomerata, can be found on many
Walker Lake shoreline substrates, including stromatolites. The filaments trap carbonate
grains, and are themselves encrusted in a thin crust of calcium carbonate.
Figure 2-4. Walker Lake stromatolites exhibit considerable internal porosity (here filled
with blue epoxy), including fractures and voids between carbonate laminae. Additionally,
two distinct zones: a lower well-laminated portion, capped by a massive, poorly-
laminated layer, generally < 1 cm-thick.
20
algal mats from the external surfaces of the stromatolites were collected using a solvent-
washed spatula and forceps before being transferred to pre-cleaned glass jars with
Teflon
®
-lined lids. Sample containers were packed in dry ice in the field and during
transport, and then stored at –30
o
C until extraction and analysis.
Table 2-1
Location and n-alkane composition of Walker Lake stromatolite-associated organic matter.
Sample # and Type Distance
From
Shore (m)
Most
Abundant
Chain Length
Carbon
Preference
Index
Average
Chain
Length
NT4 – Stromatolite Submerged C
26
0.97 23.5
BD2 – Stromatolite 2 C
24
0.96 23.5
BD3 – Stromatolite 3 C
26
1.14 23.3
NT5 – Stromatolite 20 C
26
0.98 23.4
NT6 – Stromatolite 40 C
26
0.96 24.0
NT7 – Stromatolite 60 C
26
0.92 23.7
Distance reported is the horizontal landward distance of host stromatolite from shoreline (m).
Carbon Preference Index=0.5*Σ (X
23
–X
29
)/(X
22
–X
28
) + 0.5*Σ (X
23
–X
29
)/(X
24
–X
30
), X is abundance.
Average chain length = Σ(i*X
i
)/ ΣX
i
)
21
Organic matter extraction and n-alkane isolation
Stromatolite hand samples were broken with a hammer to expose internal
surfaces. A Dremel

tool with drill bits washed in triplicate with 6:1 DCM/MeOH was
used to produce sample powders from internal surfaces. This method was used to prevent
the incorporation of desiccated modern biomass on external carbonate surfaces and
biomass in internal sediment-filled voids and tubes that would have accompanied
crushing of bulk samples. As discussed below, the stromatolites are composed of a lower
well-laminated portion and an upper non-laminated portion: our samples were drilled
from the lower laminated portion. ~10 g of drilled powder from each sample was placed
into Teflon
®
vessels. The powders were then extracted with DCM/MeOH (9:1) using a
microwave-accelerated reaction system (MARS). Lipids were extracted from biological
samples using a modified Bligh-Dyer extraction (Bligh & Dyer, 1959; Kates, 1986).
After solvent exchange into hexane, activated copper was added to sample extract and
was allowed to react overnight to remove elemental sulfur. Lipid extracts volumes were
reduced to near dryness using a Rotovap
®
before being separated using silica gel column
chromatography. The saturated hydrocarbon fraction (F1) was then subject to urea
adduction following the method by Bull et al. (1999). Briefly, samples were dissolved in
hexane/acetone (2:1) and vortex-mixed while a solution of urea-saturated methanol was
added dropwise, causing a crystalline urea precipitate to form. The solvent was
evaporated under N
2
, and the urea crystals were then re-suspended with DCM and
centrifuged. The DCM containing the non-adduct was removed with a pipette and passed
22
through a glass wool filter to ensure purification of the non-adduct. The adduction and re-
suspension/washing procedures were repeated twice before the urea crystals were
dissolved in double-distilled water, and the adducted hydrocarbons were extracted via
liquid-liquid extraction with DCM in a separatory funnel. The urea-saturated aqueous
phase was removed. The addition of water, mixing, and removal was repeated to ensure
complete removal of the urea. The organic phase containing the adduct was passed
through a column containing sodium-sulfate to remove residual water. Powdered granite
served as a procedural blank and underwent identical extraction and processing steps.
GC-IRMS and GC-FID analyses
Carbon isotope ratios of individual urea-adducted n-alkanes were determined
using a Trace GC interfaced with a ThermoFinnigan Delta
Plus
XP isotope ratio mass
spectrometer at the Southern California Coastal Water Research Project.. The GC
temperature program ramped from 80
o
C (held for 1 min.) to 130
o
C at 20
o
C/min., then
ramped to 320
o
C at 5
o
C/min. (held for 5 min.). Samples were injected in splitless mode,
and were co-injected with perdeuterated alkane reference standards (C
16
D
34
, C
24
D
50
,
C
30
D
62
) of known δ
13
C values (from the Biogeochemical Laboratories at Indiana
University) for peak identification and isotope ratio calibration. CO
2
reference gas was
introduced as multiple pulses during each run as a calibration standard. Reference alkanes
of known isotopic composition, ranging from C
16
-C
30
, were analyzed separately to assess
machine precision. Delta notation is used to report the isotopic composition of n-alkanes,
relative to Vienna Pee Dee Belemnite (VPDB) according to the standard equation:
23
δ
13
C(‰) = [(
13
C/
12
C)
sample
/(
13
C/
12
C)
VPDB
– 1] x 10
3
(1)
All samples were run in triplicate with standard deviations < 1‰. N-alkane abundance
was quantified using a flame ionization detector (FID) interfaced with a Trace GC.
Radiocarbon dating
Powdered calcium carbonate samples were dissolved using 6M HCl. Residues
were washed with Milli-Q water before being dried overnight at 80
o
C. Organic residues
and untreated calcium carbonate powders of stromatolite samples NT4 and NT5 were
14
C-dated at the UC-Irvine Keck Carbon Cycle AMS facility (Southon et al., 2004) and
reported using the convention of Stuiver & Polach (1977).
Results
Geochronology
Some subaqueous stromatolites are covered with accumulations of algae and were
initially assumed to be currently accreting (Osborne et al., 1982). However, bulk calcium
carbonate powders drilled from the laminated portion of stromatolite NT4 produced a
14
C
age of 2140±20 yrs BP and stromatolite NT5 produced a
14
C age of 1935±20 yrs BP.
These dates are similar to the laminated Walker Lake “tufa” determined by Newton and
Grossman (1988) to have an apparent radiocarbon age of 2105±90 yrs BP. Carbonate
from the outermost rind of another stromatolite from this locality was
14
C dated at ~700
24
yrs BP, suggesting that Walker Lake stromatolites are not currently accreting. This would
also be true if a ~300 year correction for a reservoir effect, which was applied by Yuan et
al. (2004) to dating of Walker Lake sediment core samples (Broecker and Walton (1959)
Yuan et al. (2004), was also applied to these stromatolites.
Organic matter from stromatolite NT4 returned a date of 1525 ±15 YBP, while
organic matter from stromatolite sample NT5 was dated at 1085 ±20 YBP. Because of
the low TOC content, it was necessary to drill several spots to produce enough powder to
date the organic fraction, so some amount of time averaging is inevitable, and offsets
between the organic and inorganic ages are not unexpected, despite the fact that splits of
the homogenized sample were used for both organic and inorganic analyses.
Gas chromatography of saturated hydrocarbons in stromatolites
Extraction of hydrocarbons from stromatolite-associated organic matter resulted
in a saturated fraction dominated by n-alkanes. Isoprenoid compounds were also
detected, but in concentrations too low for reliable isotopic analyses. Walker Lake
stromatolites have average TOC values of 0.56 ±0.1%. Clade-specific biomarkers
produced by mat-inhabiting photoautotrophs, such as methylhopanoids or mid-chain
branched alkanes have been used to demonstrate the presence of cyanobacteria (Shea et
al., 1990; Summons et al., 1999). However, these compounds were not detectable in
extracts from Walker Lake stromatolites.
25
Stable isotope analyses
Figure 2-5 shows carbon isotope values of n-alkanes from stromatolites (filled
symbols) compared with those from extant Walker Lake water column algae and algae-
dominated mats (open symbols). The reported δ
13
C values represent the mean of three
replicate measurements. The δ
13
C values of alkanes extracted from stromatolites range
from –24.8‰ to –33.7‰. The mean for stromatolite-associated alkanes of all chain
lengths was –29.2‰ ±1.1‰ (1σ). Stromatolite-associated long-chain-length alkanes
Table 2-2. δ
13
C values for n-alkanes extracted from stromatolite-associated organic
matter.
NT4 NT5 NT6 NT7 BD2 BD3
C
16
-28.6 ± 0.0 -27.4 ± 0.2 ** -29.1 ± 0.5 -29.6 ± 0.2 -29.2 ± 0.3
C
17
-30.5 ± 0.1 -28.1 ± 0.4 ** -28.9 ± 0.6 -29.5 ± 0.4 -29.2 ± 0.2
C
18
-28.6 ± 0.1 -27.2 ± 0.2 -27.9 ± 0.9 -28.1 ± 0.5 -28.1 ± 0.6 -28.6 ± 0.0
C
19
** **
** -28.4 ± 0.4 -28.4 ± 0.6 -28.3 ± 0.5
C
20
** **
** -28.2 ± 0.5 -29.0 ± 0.9 -28.1 ± 0.5
C
21
** **
** -28.7 ± 0.3 -28.5 ± 0.2 -28.3 ± 0.6
C
22
** **
** -29.7 ± 0.6 -29.5 ± 0.4 -29.4 ± 0.1
C
23
-28.1 ± 0.9 **
** -30.2 ± 0.0 -30.1 ± 0.2 -30.9 ± 0.2
C
24
-30.1 ± 0.6 -28.5 ± 0.8 -28.1 ± 0.0 -29.3 ± 0.2 -29.9 ± 0.5 -29.2 ± 0.6
C
25
-30.8 ± 0.4 -30.7 ± 0.1 -30.7 ± 0.4 -30.8 ± 0.0 -30.5 ± 0.5 -30.6 ± 0.2
C
26
-29.2 ± 0.3 -29.2 ± 0.1 -29.6 ± 0.0 -30.1 ±0.2 -30.8 ± 0.1 -30.1 ± 0.5
C
27
-33.4 ± 0.8 -31.5 ± 0.3 -30.5 ± 0.2 -31.0 ± 0.0 -31.6 ± 0.5 -30.5 ± 0.4
C
28
-31.3 ± 0.3 -29.3 ± 0.1 -29.2 ± 0.0 -30.5 ± 0.2 -30.3 ± 0.2 -29.9 ± 0.4
C
29
-29.9 ± 0.2 -30.2 ± 0.3 -30.7 ± 0.7 -31.5 ± 0.4 -31.0 ± 0.6 -30.4 ± 0.6
C
30
-30.4 ± 0.3 -29.7 ± 0.1 -30.3 ± 0.2 -30.3 ± 0.8 -30.1 ± 0.2 -29.9 ± 0.4
Indicated uncertainties are standard deviations of triplicate analyses
** Indicates that abundances were non-existent or too low for isotopic analysis
26
Table 2-3. δ
13
C values for n-alkanes extracted from modern Walker Lake organic matter.
DESC CLAD MIX
C
16 -26.9 ± 0.6 -28.4 ± 0.1 -28.1 ± 0.1
C
17 -28.8 ± 0.4 -28.5 ± 0.0 -28.5 ± 0.2
C
18 -28.0 ± 0.3 -27.9 ± 0.1 -27.9 ± 0.1
C
19 -27.6 ± 0.3 -27.7 ± 0.2 -27.7 ± 0.2
C
20 -27.6 ± 0.5 -27.6 ± 0.3 -27.5 ± 0.4
C
21 -30.3 ± 0.5 -27.8 ± 0.0 -26.7 ± 0.2
C
22 -28.4 ± 0.2 -29.4 ± 0.1 -28.6 ± 0.3
C
23 -30.8 ± 0.1 -30.4 ± 0.3 -27.9 ± 0.5
C
24 -29.1 ± 0.2 -28.2 ± 0.5 -27.7 ± 0.4
C
25 -30.2 ± 0.2 -29.2 ± 0.1 -30.5 ± 0.2
C
26 -29.2 ± 0.4 -29.0 ± 0.4 -29.6 ± 0.5
C
27 -30.1 ± 0.7 -31.5 ± 0.3 -29.9 ± 0.3
C
28 -29.6 ± 0.6 -30.5 ± 0.6 -30.3 ± 0.7
C
29 -29.9 ± 0.5 -30.7 ± 0. 5 -30.7 ± 0.2
C
30 -29.2 ± 0.6 -30.2 ± 0.3 -29.2 ± 0.0
(e.g., C
25
-C
29
), on average, are depleted in
13
C (-30.3‰) relative to short chain (C
17
-C
21
)
alkanes (-28.1‰). The slight depletion in
13
C observed in the longer chain alkanes was
also observed in the modern biomass samples that range from –26.7‰ to –31.5‰. This
could be the result of two different biomoleculular precursors present in the same alga
(e.g., fatty acids vs. algaenans). Another, perhaps more likely possibility is that the
sampled algal mats that appeared to be dominated by Cladophora, actually include a
consortium of organisms that contributed the shorter-chain-length alkanes enriched in
δ
13
C, and Cladophoran algae are known to host both eukaryotic and eubacterial epibionts
(Carballeira et al., 1997). Five of six stromatolite-associated alkane assemblages have
isotopic values within 1‰ of algae-derived alkanes at any given chain length.
27
Figure 2-5. Isotopic compositions of n-alkanes extracted from the ambient algae-
dominated modern lake biomass (open symbols) exhibit very similar δ
13
C values to
alkanes extracted from Walker Lake stromatolites (filled symbols).
28
Comparisons between stromatolite and biomass-associated alkane assemblages did not
show a statistically-significant isotopic difference as shown by a paired t-test; all two-
tailed P values >>0.05. Alkanes from a sixth stromatolite sample (NT4) exhibit isotopic
values more depleted in
13
C at C
17
, C
27
, and C
28
than all algal samples, but have similar
values to the algal samples at all other chain lengths (paired t-test P=0.09).
Discussion
Origin of organic matter in Walker Lake Stromatolites
As shown in Figure 2-5, at all chain lengths, alkanes from multiple stromatolite
samples closely track the isotopic composition of extant Walker Lake water column algae
and algae-dominated benthic mats. These data also suggest that the organic matter
associated with Walker Lake stromatolites was derived from organisms of low metabolic
diversity, primarily utilizing Calvin cycle carbon fixation, rather than the metabolically-
diverse organisms that typify layered microbial mat communities. The absence of an
even-over-odd chain-length preference in Walker Lake stromatolite-associated alkanes
suggests an algal or bacterial, rather than land plant, source. Leaf waxes are known to
show an increasing depletion in
13
C with increasing chain length (Lockheart et al., 1997),
however, the same pattern is shown in alkanes extracted from Walker Lake Cladophora
mats in this study and in long chain fatty acids from the freshwater chlorophyte
Scenedesmus communis (Schouten et al., 1998). Walker Lake shorelines are seasonally
inundated by algal mats dominated by the filamentous, branching alga Cladophora
glomerata. Large rafts of algae and organic debris are common, and stromatolites are
29
occasionally exposed to a brown soup of humic material resulting from the shoreline
decay of plants and algae (Fig. 2-2d). Desiccated epilithic Cladophora mats are often
encrusted by a thin film of recently-precipitated carbonate (Fig. 2-3). Although
Cladophora is the most conspicuous mat-forming organisms, other green algae such as
Ulothrix aequalis, and cyanobacteria such as Schizothrix sp., Amphithrix janthina, and
Lyngbya sp. are also known to grow on the exterior surfaces of Walker Lake
stromatolites (Osborne et al., 1982). Cyanobacteria such as Nodularia spumigena and
Anabaena sp. can also, at times, dominate phytoplankton biomass in Walker Lake
(Beutel et al., 2001). These other organisms likely contributed to the biomass of the
Cladophora-dominated mats analyzed here.
Hydrocarbons extracted from Walker Lake stromatolites appear to be derived
from a limnic, rather than terrestrial source. Carbon preference indices (CPI) in five of
the six samples (Table 2-1) show even chain length predominance (CPI<1.0), which is
consistent with a primarily algal and bacterial, rather than land plant origin for Walker
Lake stromatolite hydrocarbons (Peters et al., 2005). Microalgae, macrophytes like
Cladophora, and bacteria are all possible sources of long-chain alkanes lacking an odd-
over-even predominance (Nishimoto, 1974; Volkman et al., 1998; Wakeham et al.,
1991).
Alkanes associated with Walker Lake stromatolites also resemble n-alkane
compositions found in Precambrian stromatolites that lack an odd-over-even
predominance and contain alkanes ranging from C
16
to C
36
(e.g., McKirdy, 1976; Smith,
1970). Like the Walker Lake stromatolites, the chain length patterns in alkanes associated
30
with Precambrian stromatolites may have resulted from diagenetic alteration or
contamination.
The close match between both the shape and the absolute value of the two
compound-specific stable isotope datasets in Figure 2-5 suggest that those values did not
result from biological sources with substantially different fractionation factors acting on
DIC that was isotopically different from modern values.
The low TOC values and the paucity of biomarker compounds other than alkanes
in Walker Lake stromatolites is likely a consequence of stromatolite exposure to meteoric
diagenesis and aerobic bacterial biodegradation (Peters et al., 2005). Stromatolite
morphogenesis and mineralogical preservation are also known to be affected by meteoric
diagenesis (Nehza & Woo, 2006), and labile organic matter is generally more susceptible
to degradation under subarial conditions (e.g., Britton, 1984). Many ancient stromatolites
are thought to have formed in, or been exposed to, supratidal to intertidal environments
and would have experienced similar subaerial early diagenetic conditions. Stromatolites
that lithify in subtidal depositional environments are perhaps less susceptible to early
diagenetic degradation of organic matter due to their potential for rapid burial (Playford
and Cockbain, 1976).
Layered microbial communities
Layered microbial communities (LMCs) are comprised of organisms that utilize
disparate metabolic pathways producing lipids and other biomolecules with a variety of
isotopic signatures. The isotopic signature of a geolipid fraction derived from LMCs
31
represents a source mixture of isotopic signatures produced by multiple metabolic
pathways. All photosynthetic eukaryotes and many phototrophic bacteria utilize the
Calvin cycle to fix CO
2
(e.g., Roeske and O’Leary, 1984). Freshwater cyanobacterial
lipids show a depletion of 22-30‰ relative to DIC (Sakata et al., 1997), which is similar
to the range of isotopic values observed in lipids produced by lacustrine eukaryotic algae.
Although cyanobacteria are often thought to be the most abundant organisms is modern
stromatolite-associated microbial communities, in Shark Bay, Australia they comprise
only ~5% of the genetic diversity of the microbial population (Papineau et al., 2005).
While there are biases inherent in translating sequence diversity into cell abundance, the
similarity of multiple samples analyzed by Papineau et al. (2005) suggests that 5% is
likely a reasonable approximation of abundance. The relatively large size and rapid
turnover of cyanobacteria likely results in contributions to mat lipid content that exceeds
5%, though the preservation potential for their lipids may be poor, in light of their initial
exposure to heterotrophic processes at the surface, and eventual exposure to anaerobic
heterotrophs deeper in the mat.
Some common microbial mat-inhabiting photoautotrophs, such as photosynthetic
sulfur bacteria, utilize the reverse tricarboxylic acid cycle (Quandt et al., 1977; Sirevåg &
Ormerod, 1970). The reverse TCA cycle produces biomass enriched in
13
C relative to
biomass resulting from Calvin cycle CO
2
fixation (van der Meer et al., 1998). In addition
to some archaea, the phototrophic bacterium Chloroflexus, which lives in thermophilic
layered communities, utilizes a third carbon fixation pathway known as the 3-
hydroxypropionate cycle (Hügler et al., 2003; Strauss & Fuchs, 1993). Like the reverse-
32
TCA cycle, the 3-hydroxypropionate cycle produces biomass that is more enriched in
13
C
than that produced by the Calvin cycle (van der Meer et al., 2001). Chlorflexus is
associated with modern stromatolites forming in Yellowstone Hot Springs (Doemel &
Brock, 1974) and has been hypothesized as a candidate for the role of primary autotroph
in ancient stromatolite-associated microbial communities (Papineau et al., 2005).
Autotrophic sulfate reducing bacteria (SRB), acetogenic bacteria, and
methanogenic archaea use a fourth pathway, known as the acetyl Co-A pathway.
Representatives of these three major polyphyletic groups all commonly inhabit layered
communities (Javor & Castenholz, 1981; Nicholson, 1987). For example, sulfate-
reducing bacteria are found in layered communities associated with Bahamian
stromatolites (Visscher et al., 1998, 2000) and the sulfide produced from their
metabolism can fuel anoxygenic photosynthesis that fixes carbon via the reverse TCA
cycle. SRB are metabolically diverse, and grow both heterotrophically on organic
compounds, and autotrophically on inorganic CO
2
using the acetyl Co-A pathway
(Londry & Des Marais, 2003). Some SRB also use a modified citric acid cycle. In
addition to the more-diagnostic iso- and anteiso- alkanes and fatty acids, sulfate-reducing
bacteria produce n-alkanes in the C
15
to C
31
range (e.g., Han & Calvin, 1969). Alkanes
and their straight-chain lipid precursors are also found in methanogens, but constitute a
minor component of total methanogen lipid extracts that are dominated by phytanyl
glycerol ethers and isoprenoids (Tornabene et al., 1978). Sulfate concentrations are
sufficient to support microbial dissimilatory sulfate reduction in Walker Lake, and both
sulfate reduction and methanogenesis are known to occur in its sediments, and in the
33
hypolimnion during water column stratification (Domagalski et al., 1989, Beutel et al.,
2001).
These diverse mat inhabitants utilizing various metabolic pathways can
contribute a wide variety of molecules with a range of δ
13
C values, and compounds
specific to a certain organism might be enriched within particular horizons of the mat at
any given time, but the fate of the compounds through diagenetic space is likely far more
complex.
Mixing of organic compounds in layered communities
Layered communities are biogeochemically-stratified, as biogeochemical zones
migrate over time, so do the microbial populations inhabiting particular zones.
Stromatolite accretion slowly displaces the absolute position of the sediment/water
interface, but not the relative positions of redox interfaces. As a result, any given
subsurface horizon will be inhabited by microbes from each successive redox zone that
can contribute their own lipids and associated isotopic signatures to each superjacent
horizon. In addition, some microbes occupy microenvironments within zones that are
dominated by organisms that thrive on the most prevalent biogeochemical conditions
within a particular zone (e.g., Okabe et al., 1999). These processes are likely to result in a
mixing of compounds derived for multiple biological sources, even if the source
organisms were vertically segregated while alive. The importance of the relative
contributions of compounds from two or more sources with different isotopic values can
be calculating using a mixing model, such as:
34
δ
13
C
Sorg
= ƒ
X
(δ
13
C
X
) + ƒ
Y
(δ
13
C
Y
); ƒ
X
+

ƒ
Y
= 1

(2)
where the subscript
Sorg
represents stromatolite-associated organic matter, and the
subscripts
X
and
Y
represent different biological sources, while ƒ represents the fractional
contribution from each source that is preserved in the stromatolite. Figure 2-6 shows a
mixing model for a series of n-alkanes derived from two hypothetical layered microbial
communities. Purple boxes in Figure 2-6 represent a layered community that includes
photosynthetic sulfur bacteria utilizing the reverse TCA-cycle (Population Y), in addition
to phototrophs that fix carbon via the Calvin cycle (Population X). Hypothetical values
for the average isotopic composition for each population at that chain length, as well as
the fraction of alkanes contributed from organisms using that metabolic pathway, are
listed in table 2-3.
Table 2-4
Alkane
Carbon #
δ
13
C X Cont. X δ
13
C Y Cont. Y
16 -28.0 0.8 -12.0 0.2
17 -28.2 0.8 -15.0 0.2
18 -28.4 0.8 -10.0 0.2
19 -28.8 0.8 -13.0 0.2
20 -29.0 0.9 -13.0 0.1
21 -29.2 0.85 -13.0 0.15
22 -29.5 0.9 -13.0 0.1
23 -29.7 0.9 -13.0 0.1
24 -29.9 0.9 -14.0 0.1
25 -30.0 0.95 -14.0 0.05
26 -30.1 0.95 -14.0 0.05
27 -30.2 0.95 -15.0 0.05
28 -30.4 0.95 -15.0 0.05
29 -30.5 0.95 -15.0 0.05
30 -30.7 0.95 -16.0 0.05
Yellow boxes in Figure 2-6 represent a layered community containing organisms that in
addition to the Calvin cycle carbon fixing organisms (Population X) utilize the acetyl
35
CoA pathway (Population Y) as outlined in Table 2-4. Methanotrophs are also
represented (Population Z).
Table 2-5
Alkane
Carbon #
δ
13
C X Cont. X δ
13
C Y Cont. Y δ
13
C Z Cont. Z
16 -28.0 0.85 -34.0 0.1 -80.0 0.05
17 -28.2 0.85 -34.0 0.1 -80.0 0.05
18 -28.4 0.85 -34.0 0.1 -80.0 0.05
19 -28.8 0.85 -34.0 0.1 -80.0 0.05
20 -29.0 0.85 -34.0 0.1 -80.0 0.05
21 -29.2 0.95 -34.0 0.1 -80.0 0.05
22 -29.5 0.95 -34.0 0.1 -80.0 0.05
23 -29.7 0.95 -34.0 0.1 -80.0 0.05
24 -29.9 0.95 -34.0 0.1 -80.0 0.05
25 -30.0 0.95 -34.0 0.05 -80.0 0.001
26 -30.1 0.95 -34.0 0.05 -80.0 0.001
27 -30.2 0.95 -34.0 0.05 -80.0 0.001
28 -30.4 0.95 -34.0 0.05 -80.0 0.001
29 -30.5 0.95 -34.0 0.05 -80.0 0.001
30 -30.7 0.95 -34.0 0.05 -80.0 0.001
Hypothetical Community A includes cyanobacteria, sulfate-reducing bacteria and
methanogens. Hypothetical Community B is influenced by sulfide-oxidizing phototrophs
that utilize the reverse TCA pathway. The colored regions indicate observed isotopic
compositions of lipids, as well as fractionation factors subtracted from DIC values of
zero. However, the isotopic composition of lipids can also be affected by environmental
conditions such as temperature, salinity and substrate availability (Laws et al., 1998).
Because of these various complicating variables, the cartoons in Figure 2-6 do not
adequately show the range of isotopic compositions possible in natural systems and
therefore are not intended as guides for predicting a particular isotopic composition for
ancient stromatolite-associated organic matter. Hypothetical isotopic values (shown as
36
solid boxes) of alkane assemblages from two types of layered community as compared to
the ambient biomass (hatched boxes), are intended to convey our prediction that
metabolically-diverse layered microbial communities are unlikely to produce isotopic
signatures patterns that are identical to those produced by the ambient organic matter,
which is dominated by the Calvin cycle used by cyanobacteria, plants, and eukaryotic
algae. The effects of these alternate metabolic pathways specifically on stromatolite-
associated alkanes are most likely to be observed in the low and mid-chain length alkanes
(see patterns in Figure 2-6b) because microbes that utilize these metabolic pathways
primarily produce alkanes and fatty acids of relatively short chain lengths – though the
preservation potential of these compounds within a microbial mat is largely unknown. It
is possible that in certain layered communities, lipids depleted in
13
C produced by acetyl-
CoA-utilizing organisms could offset the isotopic contributions of lipids enriched in
13
C
produced by organisms using the reverse TCA cycle, though it is unlikely that such an
offset would occur equally at each chain length. It is also possible that ambient n-alkane
input deposited in both the stromatolite and coeval sediments could be much greater than
the contribution from the mat community – which could potentially dilute the mat-
derived signal to the point where its contribution is undetectable. Because Walker Lake is
a relatively eutrophic water body, algal contributions that exceed those contributed by
mat biomass are feasible, but only if those contributions contributed significantly at each
alkane chain length.
37
Implications of Walker Lake stromatolite-associated alkanes
The close relationship between the isotopic signatures of Walker Lake
stromatolite-associated organic matter and the endemic aquatic algae suggests that
Walker Lake stromatolites do not preserve evidence that they formed in the presence of a
metabolically-diverse community. We propose the following possible explanations for
these results: 1) Walker Lake stromatolites formed in the presence of low-diversity
benthic algal mats; 2) Walker Lake stromatolites formed by abiotic processes; or 3)
syngenetic organic matter that would have indicated the presence of a complex
community was destroyed during diagenesis, and the currently-hosted organic matter was
either diagenetically-emplaced, or perhaps was more recalcitrant than the hydrocarbons
contributed by bacteria and archaea, a possibility suggested by the work of Sinninghe
Damsté & Schouten (1997).
We do not favor the algal mat hypothesis for the finely laminated portion of the
stromatolite; the large size of Cladophora would preclude the formation of such fine
laminae. In fact, most modern marine stromatolites are much coarser grained and crudely
laminated (if at all), because of the presence of larger eukaryotes such as diatoms
(Awramik & Riding, 1988). The uptake of CO
2
by algae may induce carbonate
precipitation in marine waters of near neutral pH, but the alkaline nature of Walker Lake
would negate the metabolic effects on pH (cf., Arp et al). The currently available
evidence may not be sufficient to distinguish between the remaining hypotheses, or if
aspects of each may have contributed to the observed results. On the one hand, the
isopachous lamination that characterize the lower portions of the stromatolites are
38
Figure 2-6. Carbon isotopic compositions of n-alkanes derived from ambient sources
(hatched boxes) in many water bodies will be dominated by Calvin cycle CO
2
fixation by
phytoplankton and benthic photoautotrophs. Stromatolite-associated organic matter (solid
boxes) derived from these sources (6a) should show similar isotopic compositions at
various chain lengths to the ambient signal. Conversely, stromatolite-associated organic
matter produced by a metabolically-diverse inhabitants of layered microbial communities
(6b) should have isotopic values distinct from those of the ambient aquatic biota of the
stromatolite-hosting water body. For example, a layered community containing
photosynthetic sulfur bacteria (Purple boxes) that utilize the reverse-TCA cycle would
lead to short and mid-chain length alkanes enriched in
13
C, while the strong influence of
sulfate-reducing bacteria, methanogens, and methanotrophs would tend to result in
alkanes more depleted in
13
C (represented by yellow boxes) relative to the ambient signal.
39
suggestive of in situ precipitation that is commonly, though not exclusively, associated
with abiotic processes (Grotzinger and Knoll, 1999), whereas the non-laminated upper
portion of the stromatolites is more characteristic of stromatolites that form in the
presence of modern microbial communities. On the other, the hypothesis of Sinninghe
Damste and Schouten (1997), which suggests bacterial and archaeal biomass generally
contributes little to preserved Phanerozoic sedimentary organic matter, is not incongruent
with our data.
Despite the recent formation of these structures, their exhumation and exposure to
meteoric weathering is likely responsible for the biodegradation of associated lipids –
destroying much or all of the primary signal.
14
C dating provides a slightly younger age
for stromatolite-associated organic matter than the carbonates that host them. This
discrepancy might constitute evidence for the diagenetic replacement of syngenetic
organic matter with younger material derived from ambient lacustrine biomass. The high
internal porosity and common exposure of the stromatolites to wave action that carries
copious amounts of organic detritus provides ample opportunity for natural post-
depositional organic contamination. The slightly younger ages could have result from the
mixture of minor amounts of recent diagenetic contamination mixed with a
predominantly syngenetic signal. Core samples from Walker Lake show a similar
discrepancy between the apparent radiocarbon ages of the carbonate fraction vs. the
organic fraction (Bradbury et al., 1988).
Although the data from Walker Lake stromatolites closely tracks the isotopic
composition of the ambient biomass, the C
17
, C
27
, and C
28
alkanes from the subaqueous
40
stromatolite (NT4) showed δ
13
C values that were slightly more negative than the extant
biomass. Though the isotopic differences between the NT4 assemblage and algal alkane
assemblages may not be statistically-significant (e.g., t-test P=0.09), lighter values are all
restricted to the one sample that is currently submerged, which may indicate practical
significance. These negative values may result from the contribution of
13
C-depleted
lipids from one or more organisms not present in the analyzed modern biota, and perhaps
this contribution is more diagenetically labile than ambient algae-derived compounds.
Alkanes from the other stromatolites that are all exposed along the shoreline do not
exhibit noticeably different isotopic values at any chain length than the ambient modern
biomass.
A sampling strategy for detecting layered communities in ancient stromatolites
These results suggest the potential importance of comparing the isotopic
composition of biomarkers from stromatolite laminae with that from organic matter
trapped in the surrounding non-stromatolitic rock matrix (Fig. 2-7), which presumably is
exposed to ambient organic matter deposition. The difference between
13
C values of
geolipids inside the stromatolite versus those preserved in sediments surrounding the
stromatolite (Δ
13
C
in-out
) can be used as a metric for relatedness of the biological sources
between a potential community associated with the stromatolite, and the ambient
biomass. A large Δ
13
C
in-out
would suggest the possible presence of a layered community,
with potential implications for understanding stromatolite petrogenesis and paleoecology.
For example, sulfate reducing bacteria are thought to mediate carbonate precipitation in
41
Figure 2-7. A significant difference between the isotopic composition of biomarkers
from within the stromatolite laminae vs. from surrounding sediments (Δ
13
C
in-out
) is
suggestive of the former presence of a layered community, whereas little or no difference
is indicative of ambient biomass incorporation. Such a determination might be made by
the comparative sampling strategy illustrated with this Cambrian stromatolite. Scale bar =
0.5 cm.
42
some modern stromatolites (Visscher et al., 1998, 2000). If such a process were
important for the petrogenesis of ancient stromatolites, then we would predict a large
Δ
13
C
in-out
value for those stromatolites. Conversely, if a particular ancient stromatolite
formed largely by abiotic processes, and organic matter was incorporated from the
trapping of ambient detritus, a very small Δ
13
C
in-out
would be expected. Likewise, the
diagenetic degradation of syngenetic organic matter, and subsequent incorporation of
allochthonous organic debris is likely to result in a Δ
13
C
in-out
near zero. Unfortunately, it
may be difficult to distinguish between these two latter states.
Finally, the possibility exists that ancient stromatolites formed in the presence of
microbial mats of low metabolic diversity, although metabolic conditions, such as CO
2
and metabolite concentrations would likely differ between mat communities and
surrounding sediments, even in stromatolites associated with low-diversity mat
ecosystems. Such environmental conditions are known to influence isotopic values of
autotroph lipids, therefore stromatolites formed in the presence of such mats would still
likely result in a non-zero Δ
13
C
in-out
.
Conclusions
Sediments and sedimentary rocks serve as the primary archives of ancient
molecules and organic matter. However the organic biomarker record preserved in
sediments is not unlike a palimpsest of the molecular contribution of biocoenoses –as
diagenesis partially or completely destroys the original record before overwriting it with
the biomolecules of later sediment inhabitants. Stromatolites are not true body fossils,
43
but rather sedimentary structures that are often assumed to have formed in the presence of
microbial life that is somehow distinct from the microbiota of the surrounding
environment. Mounting evidence suggests that stromatolites originate from a variety of
processes than run the gamut of biological involvement and microbial habitation, from
the purely abiotic to extensive mediation by microbes. Organisms with diverse metabolic
pathways are commonly present in layered microbial communities such as those
postulated to have been associated with ancient stromatolites. Therefore we predict that
stromatolites that formed in the presence of layered-microbial communities will exhibit a
marked isotopic difference from organic matter not associated with stromatolites from the
same locality. Holocene stromatolites from Walker Lake fail this test, as stromatolite-
associated n-alkanes are isotopically-indistinguishable from those in the modern local
ambient biota. Perhaps these stromatolites formed in part via abiotic processes? Or,
perhaps diagenetic degradation and contamination may have played a role in replacing
layered community isotopic compositions with those of recent ambient lake biota?
Likewise, the possibility that ambient n-alkane contributions to the preserved organic
matter diluted mat-derived contributions to the point of making them undetectable, must
be considered. At this point, the isotopic composition of n-alkanes from Walker Lake
stromatolites cannot be used to support the former presence of a layered microbial
community during their formation. Perhaps the analysis of other biomarkers present at
only trace levels will eventually resolve these issues. Regardless, the approach used in
this study, which compares stromatolite-associated organic matter with ambient organic
matter, offers a sampling strategy that might answer the question of whether or not
44
layered microbial communities contributed to organic biosignatures preserved in ancient
stromatolites.
45
CHAPTER III: Microbially-Mediated Environmental Influences on Metazoan
Colonization of Matground Ecosystems: Evidence from the Lower Cambrian
Harkless Formation
Abstract
Prior to the advent of widespread bioturbation during Cambro-Ordovician times,
microbial mats may have covered large expanses of the continental shelf. Evidence of
matgrounds in shallow-marine settings is provided by abundant wrinkle structures in
Lower Cambrian strata of the Great Basin, United States. Wrinkle structures from the
Lower Cambrian Harkless Formation commonly co-occur with a distinctive assemblage
of invertebrate fossils, providing evidence for the possibility of selective metazoan
colonization of matground substrates. Molds of linguliform brachiopods are abundant on
many wrinkle surfaces. The agglutinated problematica, Volborthella tenuis, is also found
on wrinkle surfaces and in laminations beneath wrinkle-structure surfaces. Bedding-
parallel trace fossils, such as Planolites, Diplichnites, and Taphrhelminthopsis,
commonly cross-cut wrinkle structures, while vertically-oriented trace fossils are absent.
Microbial mats containing layered microbial communities would have
considerably compressed redox zones beneath the sediment water interface in marine
shelf settings. Sulfidic and anoxic conditions within and beneath microbial mats would
have precluded habitation by many metazoans, while those that adapted to such
conditions may have found matgrounds a unique, though temporally fleeting, ecological
46
niche. The distinctive low-diversity fossil assemblage found in association with the
wrinkle structures in the Great Basin suggests that some early animals may have been
adapted to hypoxic and sulfidic conditions found in matground substrates, while others
may have been physiologically excluded from these environments.
Introduction
Utilization of ecospace by benthic and infaunal marine animals is largely
regulated by trophic opportunities and the physiological limitations of organisms to
survive within a particular set of environmental conditions (e.g., Bambach, 1983; Orr,
2003). Fossil assemblages and paleoenvironmental data can provide indicators of the
evolution of ecospace utilization (Ausich and Bottjer, 1982). For example, soft-bodied
Neoproterozoic Ediacaran organisms were most likely restricted to a limited number of
passive trophic strategies in primarily benthic habitats (McMenamin, 1986; Seilacher,
1999; Clapham et al., 2003). Later, during the Proterozoic-Phanerozoic transition, marine
invertebrates, with a rapidly evolving range of physiologies, expanded into newly
inhabitable environments, while others found new ways to exploit old habitats (e.g.,
Bottjer et al., 2000). As metazoan evolution progressed through the Early Cambrian,
feeding strategies and environmental tolerances expanded, as evidenced in the fossil
record by the increasing numbers of filter-feeding and deposit-feeding organisms (Ausich
and Bottjer, 1982; Bottjer and Ausich, 1986). The diversification of the ichnological
record during the Early Cambrian also marks an explosion of infaunal ecospace
47
utilization (Seilacher and Pflüger, 1994; McIlroy and Logan, 1999; Jensen et al., 2000;
Droser et al., 2002), which expanded during the Paleozoic.
Explanations for rapid metazoan radiation during the Cambrian include intrinsic
and extrinsic factors. Biological triggers, such as the advent of biomineralization
(Lowenstam and Margulis, 1980; Lowenstam, 1981; Kirschvink and Hagadorn, 2000)
and the evolution of hox genes may have provided mechanisms for rapid physiological
change (Valentine and Campbell, 1975; Valentine, 1994; Peterson and Davidson, 2000).
Environmental influences such as Neoproterozoic glacial activity (e.g., Hoffman et al.,
1998) and the attainment of a critical atmospheric oxygen threshold (Berkner and
Marshall, 1965; Cloud, 1973; Knoll, 1996; Fedonkin, 2003) have also been cited as
possible triggers of rapid Cambrian diversification. Only recently have investigators
begun to consider the ways in which the Precambrian-Cambrian transition was influenced
by microbial processes, recognizing that the metazoan radiation occurred in the retreating
shadow of a world dominated by microorganisms (McIlroy and Logan, 1999; Seilacher,
1999; Bottjer et al., 2000; Dornbos and Bottjer, 2000; Buatois and Mángano, 2003;
Dornbos et al., 2004).
Neoproterozoic-Cambrian metazoans lived on and above substrates that were
extensively colonized by microbes. Work by Gehling (1986, 1996, 1999, 2000),
Schieber (1986), and Hagadorn and Bottjer (1997, 1999), suggests that large tracts of
Proterozoic and Lower Cambrian marine seafloors may have been covered by extensive
microbial mats as evidenced by microbially-induced sedimentary structures (Noffke et
al., 2001a) such as wrinkle structures, and by the exceptional preservation of soft-bodied
48
Ediacaran organisms in siliciclastic sediments. Additional evidence of microbe-bound
sediments in other ancient siliciclastic subtidal settings originates from Ordovician rocks
in France (Noffke, 2000), Neoproterozoic rocks in Namibia (Noffke et al., 2002), and
Mesoarchean rocks in South Africa (Noffke et al., 2003). Wrinkle structures and other
microbially-induced sedimentary structures from Precambrian-Cambrian siliciclastic
strata are also reported in publications that predate the proposal of their microbial origins
(Walcott, 1916; Martinsson, 1965; Moore, 1976; Cooper et al., 1982; Hiscott, 1982;
Ranger et al., 1984; Kopaska-Merkel and Grannis, 1990).
If large tracts of the continental shelf were covered by bacterial mats during much
of the Neoproterozoic and Early Cambrian, shallow sediment porewaters, and perhaps
surface sediments, would have been anoxic, and likely sulfidic, at much shallower depths
than in modern marine sediments (e.g., McIlroy and Logan, 1999). In matground
environments, metabolic activity of benthic and subsurface microbial communities would
have controlled porewater chemistry up to the sediment-water interface and perhaps even
influenced bottom waters. These conditions would likely have imposed significant
biogeochemical stresses for metazoans attempting to colonize such sediments. Mat
habitats may also have provided new trophic opportunities to those metazoans that could
modify or tolerate anoxic and sulfidic conditions.
Indicators of anoxia and dysoxia in the rock record are often accompanied by
low-diversity or distinctive fossil assemblages (Rhoads and Morse, 1971; Byers, 1977;
Savrda et al.,1984; Savrda and Bottjer, 1987; Gaines and Droser, 2003). Given the nature
of oxygen consumption and sulfide production in mat environments, strata containing
49
microbially-induced sedimentary structures should possess similar paleontological
characteristics. This chapter (1) presents petrographic data from Lower Cambrian wrinkle
structures in the Harkless Formation that suggest microbially-mediated
paleoenvironmental conditions, (2) examines the trace fossils and body fossils associated
with these microbial sediments, and (3) explores the possible significance of matgrounds
for metazoan ecology and evolution during the Early Cambrian.
Geologic Context
The Harkless Formation is a component of the Neoproterozoic-Cambrian White-
Inyo succession (Fig. 3-1) (Nelson, 1962; Stewart, 1970), a series of shallow marine
siliciclastic and carbonate strata deposited along a thermally subsiding passive margin
formed in the aftermath of late Neoproterozoic continental rifting (Stewart, 1970;
Christie-Blick and Levy, 1989; Mount and Signor, 1991; Mount and Bergk, 1998).
Wrinkle structures are found throughout the siliciclastic-dominated Harkless Formation,
but are best exposed where folding has revealed large bedding plane surfaces and dip
slopes (Fig. 3-2). Wrinkle-structure-bearing strata contain other sedimentary structures,
such as symmetrical ripple marks (Fig. 3-3A), trough cross-bedding (Fig. 3-3B),
hummocky cross-stratification, and wavy laminations, which indicate deposition in
shallow subtidal settings similar to those described from Lower Cambrian strata
throughout the White-Inyo mountains (Stewart, 1970; Moore and Fritsche, 1976; Robison
and Rowell, 1976; Cooper et al., 1982). Strata from the White-Inyo succession are
characterized by alternations between siliciclastic-dominated and carbonate-dominated
50
Figure 3-1. Partial stratigraphic section of the White-Inyo succession showing the
occurrences of features discussed in the report and similar features in subjacent rock units
(after Nelson, 1962; Stewart 1970 and Corsetti and Hagadorn, 2003).
Figure 3-2. Wrinkle structures on a bedding plane surface from the Harkless Formation
in the White-Inyo Mountains, California (Cedar Flat locality; see Fig. 3-4).
51
units commonly termed “grand cycles” (Mount and Signor, 1991). Carbonates are also
found as minor components of many of the siliciclastic-dominated sections. For example,
within the Harkless Formation strata exposed at Cedar Flat, an isolated ~30 cm thick bed
of oncoidal packstone is locally exposed within an otherwise locally continuous sequence
of siliciclastic strata.
Methods
Hand samples were taken from wrinkle-structure-covered bedding-plane surfaces
and cross-sectional exposures in the Lower Cambrian Harkless Formation that crop out in
the Cedar Flat area of the White-Inyo Mountains, Inyo County, CA (Fig. 3-4). Wrinkle
structures, body fossils, and ichnofossils were catalogued. The intensity of vertical
bioturbation was estimated using ichnofabric indices (Droser and Bottjer, 1986). Samples
were slabbed and photographed, and thin sections were made, parallel and perpendicular
to the wrinkle-structure surfaces. Thin sections were analyzed using a Zeiss Axioplan
polarizing microscope equipped with a Zeiss 12-bit digital imaging system. Thin slabs (1-
2 cm thick) containing wrinkle structures, were cut perpendicular to bedding from
selected samples for x-radiography. The sections were then x-rayed using a Penetrex
industrial x-ray unit and 25.4 X 30.5 cm Kodak Industrex M x-ray film. Lead numbers
and letters were used for labeling. Film was exposed at 96kVA/8mA for 9, 12, or 21
minutes depending on sample thickness.
52
Figure 3-3. Sedimentary structures that indicate wrinkle structure formation in subtidal
environments include ripple marks (A), and trough cross bedding (B), and the
conspicuous absence of intertidal or supratidal indicators.
Figure 3-4. Map showing Lower Cambrian outcrops in gray and the Cedar Flat locality
(star) in the Harkless Formation, White-Inyo Mountains, California.
53
Results
Wrinkle Structures
Bedding plane surfaces of quartz-rich, fine-grained sandstone from the Harkless
Formation are commonly covered with wrinkle structures (Hagadorn and Bottjer 1999).
Wrinkle structures primarily occur in patches <5 m in diameter. In hand sample, wrinkle
structures exhibit a variety of oversteepened crests arranged in quasi-polygonal, complex,
contorted patterns, as described in detail by Hagadorn and Bottjer (1997, 1999). Wrinkle
surfaces commonly overlie thinly laminated fine-grained meta-sediments that are
separated by horizons that are rich in pyrite and other heavy mineral grains (Fig. 3-5A).
Wrinkle structures are also commonly found beneath hummocky cross-stratified units
(storm beds).
In thin section, the sediment forming the wrinkle structures consists of fine-
grained quartz arenite matrix along with an assortment of accessory heavy mineral grains
such as zircons, opaques such as iron oxides and pyrite, and low-grade metamorphic
minerals including chlorite and muscovite. Photomicrographs of wrinkle structures in
cross section reveal crests composed almost entirely of quartz grains, whereas the wrinkle
troughs are replete with bedding-parallel chlorite and mica grains (Fig. 3-5B).
Brachiopods
Aggregates of linguliform brachiopod casts and molds are common on wrinkle-
structure-covered bedding surfaces in the Harkless Formation (Fig. 3-6, A-C). Casts and
molds are ovoid and span an average 11 mm in breadth, and ~8 mm from valve apex to
54
Figure 3-5. Wrinkle-structure features: (A) Sub-cm to mm-thick laminations beneath the
wrinkle surface contain concentrations of heavy mineral grains such as pyrite. Scale bar =
1.0 cm. (B) Photomicrograph of wrinkle trough exhibiting a concentration of oriented,
filamentous phyllosilicate grains. Scale bar = 0.5 mm.
Figure 3-6. Harkless Formation strata contain: (A) Molds of linguliform brachiopods on
a wrinkle-structure-covered bedding surface. (B) Bed-parallel burrows, such as
Planolites, and brachiopods are common on wrinkle-structure-covered surfaces. (C)
Casts of linguliform brachiopods (D) Rusophycus and Cruziana, which are ubiquitous
trace fossils in massively-bedded rocks that do not contain wrinkle structures.
55
anterior. Little taxonomic information is available from the cast-and-mold style of
preservation. Mold and cast margins are similar to the valve dimensions and ovoid shape
of many Early Cambrian brachiopods, including the linguliform brachiopods Obolella sp.
and Mickwitzia occidens, which are both known from the Bonnia-Olenellus zone in the
Great Basin (Mount, 1974; Rowell, 1977).
Volborthella
Cone-shaped agglutinated fossils, indistinguishable from descriptions of
Volborthella tenuis (Hagadorn and Waggoner, 2002), were observed on wrinkle surfaces
during this study, and were subsequently found to be very common on wrinkle-structure-
covered bedding surfaces (Fig. 3-7A). Despite extensive searching, Volborthella was not
observed in rocks that did not contain wrinkle structures, although specimens in have
been reported from both the Poleta and Harkless Formations (Hagadorn and Waggoner,
2002).
Petrographic examinations reveal that Volborthella specimens average ~5 mm in
length and are composed of heavy mineral grains, such as pyrite, ilmenite, and zircon.
The specimen in Figure 3-7B has been sectioned through its long axis and is shown here
under plain polarized light. Under higher magnification, Volborthella specimens exhibit
an arrangement of agglutinated grains arranged in concentric curved laminae (Fig. 3-7C).
Grain orientation within the laminae is convex toward the small aperture of the cone, as
reported by Hagadorn and Waggoner (2002). Lithic grains incorporated into
Volborthella include ellipsoidal, rod-shaped, and cubic grains, many of which are opaque
56
Figure 3-7. (A) The cone-shaped agglutinated fossil, Volborthella on a wrinkle-structure-
covered bedding plane. Scale bar is 5 mm. (B) Photomicrograph of Volborthella from a
wrinkle crest in plane-polarized light. Scale bar is 1 mm. (C) Volborthella is constructed
of concentric, curved laminae composed of quartz, zircons, and pyrite grains as shown in
this photomicrograph. Scale bar = 100 µm. (D) X-ray radiograph showing cross-sectional
views of two Volborthella, and perhaps a third viewed on end. The area marked “W” is
the bedding surface with wrinkle structures.
57
in polarized light. Petrographic identification of grain mineralogy in this study was
consistent with the quartz, zircon, and iron oxide pseudomorphs after pyrite as reported
from EDS spectra of Volborthella from the Wood Canyon Formation (Hagadorn and
Waggoner, 2002), an age-correlative unit that is exposed to the southwest (Stewart, 1970;
Corsetti and Hagadorn, 2000, 2003).
X-radiographs of the laminae beneath wrinkle-bedding surfaces also show
abundant x-ray silhouettes with shapes and dimensions consistent with Volborthella (Fig.
3-7D). X-radiographs of small slabs (x: 5 cm, y: 1cm, z: 0.5 cm) show up to twenty
Volborthella silhouettes from a single sedimentary horizon. Mm-thick laminae directly
subjacent to buried Volborthella specimens commonly contain heavy mineral grains that
appear as bright horizontal stringers on negative exposures of radiographs.
Trace Fossils
Wrinkle-structure surfaces contain abundant trace fossils, in particular, a variety
of horizontal forms such as Planolites (Fig. 3-6B), Diplichnites, and Taphrhelminthopsis
similar to those described by Hagadorn and Bottjer (1997). Trace fossils associated with
wrinkle structures are consistently bed-parallel forms. Laminated sediments beneath
wrinkle structures are not disrupted by vertical bioturbation (ichnofabric index [hereafter
abbreviated ii] of 1). In adjacent rocks from within the same stratigraphic sections,
vertically-oriented traces such as Bergaueria and Skolithos commonly occur on bedding
surfaces that lack wrinkle structures, and often top mottled, heavily-bioturbated beds (ii
58
4-6). Traces of arthropod activity, such as Rusophycus, Diplichnites, and Cruziana, are
very common in massively-bedded strata (Fig. 3-6D) adjacent to those that contain
wrinkle structures. However, these ichnofossils are not observed in association with
wrinkle structures.
Discussion
Rocks from the Harkless Formation that contain abundant wrinkle structures
possess fossil assemblages and other sedimentary characteristics that are distinct from
those rocks in which wrinkles are absent and bioturbation is more extensive. If wrinkle-
structure formation was induced by the presence of subtidal microbial mats, and if
biogeochemical conditions in ancient mat environments were similar to those shared by
microbial mats from numerous modern environments (e.g., Williams and Reimers, 1983;
Nealson and Berelson, 2003), then anoxic and sulfidic conditions would likely have
placed physiological stresses on organisms that colonized mat-bound sediments. Modern
anoxic and/or sulfidic environments are generally characterized by restricted to
nonexistent macrofaunal burrowing activity and low species diversity. Evidence of
matground habitats in the rock record, by inference, might then be characterized by a
dearth of vertically-oriented trace fossils, and low-diversity, perhaps matground-endemic,
fossil assemblages. These characteristics are observed in association with wrinkle
structures in rocks from the Harkless Formation.
Microbially-Stabilized Substrates
59
Wrinkle structures and sediment-stabilizing microbes: Morphological similarities
between wrinkle structures from Ediacaran-Cambrian siliciclastic strata and microbial
mat surfaces from Redfish Bay, Texas, led Hagadorn and Bottjer (1997, 1999) to
conclude that wrinkle structures in subtidal siliciclastic deposits from the Great Basin,
USA, and Newfoundland were likely formed by microbial trapping and binding of
sediments. Extensive studies of microbially-induced sedimentary structures in modern
marine sediments also provide support for microbial sediment-stabilization as the
biological process responsible for wrinkle structure petrogenesis (Noffke et al., 2001 a,b,
2003a). Adhesive extracellular polymeric substances, filamentous trichomes, and
phototactic migration through sediments make filamentous cyanobacteria the most
common biostabilization agents in modern substrates (Gerdes, 2000; Noffke 2003a).
Cyanobacteria are also commonly invoked as the microorganisms responsible for ancient
microbial-induced sedimentary structures such as wrinkle structures (Noffke et al., 2002,
2003b).
Although cyanobacteria are the organisms most commonly associated with the
formation of microbially-induced sedimentary structures, other filamentous microbes
could potentially have served as the trapping and binding agents responsible for wrinkle
structure formation. Eukaryotic green algae such as Enteromorpha sp. and Cladophora
sp., and prokaryotic filamentous sulfur-oxidizing bacteria, such as members of the genera
Beggiatoa, and Thioploca, also form thick mats that have the potential to trap and bind
sediment (Neumann et al., 1970; Ward et al., 1992; Fossing et al., 1995; Jørgensen and
Gallardo, 1999; Pihl et al., 1999). Microbial mats dominated by sulfur-oxidizing bacteria
60
inundate modern shelf sediments in upwelling areas, such as the coast of Namibia
(Gallardo et al., 1998), and in oxygen-poor sediments off the western coast of South
America (Gallardo, 1977), the Cariaco Basin, (Tuttle and Jannasch, 1973), and the Santa
Barbara Basin (Soutar and Crill, 1977). Morphological characteristics of wrinkle
structures do not provide sufficient information to determine the identity of the trapping
and binding microbes, though the composition of the surface microbial population may
have influenced matground paleoenvironmental conditions. Variability in physiology
within groups like cyanobacteria could have had significant ramifications for metazoan
matground inhabitants. For example, some cyanobacteria produce animal toxins (e.g., Li
et al., 2001), while others make use of anoxygenic photosynthesis when hydrogen sulfide
is present (Cohen et al., 1975).
Microbially-Mediated Redox Conditions: If oxygenic phototrophs were the
primary microorganisms responsible for wrinkle-structure formation, then mat surfaces
and the overlying water column may have been well-oxygenated during daylight hours.
However, in modern microbial mats, heterotrophic microbes remineralize organic matter
from the water column and from the mat biomass itself - consuming O
2
in the process.
Microbial respiration rapidly consumes oxygen from the water column and from
oxygenic phototrophs at the mat surface (Froelich et al., 1979; Reimers and Suess, 1983;
Bender and Heggie, 1984), creating anoxic conditions less than two millimeters beneath
the mat/water interface during the day (Jørgensen et al., 1992). During diel darkness, in
turbid waters, or in mats dominated by sulfur bacteria, anoxic conditions often extend to
the sediment-water interface (Fig. 3-8A). Anoxic conditions can reach the mat surface
61
even if the overlying water column is rich in oxygen (Jørgensen and Revsbech, 1985).
This occurs primarily in organic-rich sediments with a high oxygen demand and a thick
diffusive boundary layer, criteria that are met by microbial mat-covered substrates.
Analogous instabilities in oxygen concentration and sulfide production would almost
certainly have characterized Cambrian matground environments.
Although the identities of the filamentous microbes that comprised ancient mat
surfaces remain unclear, features that are common to modern mat-covered substrates can
be used to infer some of the likely subsurface geochemical characteristics of Cambrian
matgrounds. The presence of layered microbial communities would have been the
primary factor controlling redox conditions in sediments beneath mat surfaces. Stabilized
substrates, such as microbial-mat-covered sediments, foster the formation of layered
microbial communities (e.g., Nealson and Berelson, 2003). The high productivity of
benthic microbial mats would have made abundant organic carbon available for oxidation
by sulfate-reducing bacteria and other anaerobes within the subsurface microbial
community (Jørgensen et al., 1992; Deming and Baross, 1993). In the anaerobic
porewaters of modern layered communities, bacteria such as Desulfovibrio sp. use
geochemical substrates such as sulfate as the terminal electron acceptor for anaerobic
decomposition of organic matter (Berner, 1981). Sulfate reduction is carbon-limited in
modern marine environments. In Cambrian subtidal matground environments, sources of
lactate, pyruvate, and fatty acids are likely to have been abundant due to high primary
productivity at the mat surface. Interestingly, bacterial reduction of seawater sulfate is
thought to have been at its zenith during the Cambrian, as evidenced by the highest
62
fractional burial of reduced sulfur in the Phanerozoic, along with high S/C ratios and
abundant sedimentary pyrite (Raiswell and Berner, 1986; Berner and Canfield, 1989;
Strauss, 2004). Benthic microbial mats may have supplied the organic matter that drove
the increased efficiency of sulfate reduction that was responsible for these geochemical
data.
Because sulfate reduction generates toxic hydrogen sulfide, its production would
have had important implications for matground ecology, particularly with respect to
metazoan life. Once produced at depth in sediments, hydrogen sulfide can dissolve in
porewaters or diffuse upward where it reacts with dissolved iron and/or oxygen. Sulfide
can exist in oxygenated water for a period of a few hours (Morse et al., 1987) but will
eventually be oxidized to elemental sulfur. In reactive-iron-containing sediments,
hydrogen sulfide also induces the formation of iron sulfides, which eventually results in
the precipitation of pyrite (e.g., Canfield, 1989). If wrinkle structures from the Harkless
Formation represent microbial stabilization of sediments, then the presence of pyrite-rich
laminations at mm to cm depths below the wrinkle-surfaces may provide evidence for
ancient layered microbial communities that included sulfate-reducing bacteria.
Although layered microbial communities are present in modern shelf sediments,
the presence of a diffuse oxygenated mixed-layer, along with the ventilating influence of
active bioirrigation, often necessitates that the anoxic zones of layered communities be
located at depths ranging from tens of centimeters to several meters below the sediment-
water interface (Nealson and Berelson, 2003). During the Early Cambrian, shelf
sediment porewater biogeochemistry was likely in a state of flux between geochemical
63
profiles that were controlled by shallow, layered microbial communities, such as those
that characterized Proterozoic mat-stabilized sediments (Fig. 3-8A), and profiles wherein
bioturbation limited microbial controls on porewaters, such as those found in modern
bioturbated shelf sediments (Fig. 3-8B).
Metazoan Life In Matground Ecosystems
Oxygen depletion and hydrogen sulfide both pose significant challenges to animal
physiology. It is likely that these biogeochemical characteristics of matground habitats
would have had profound implications for the ecology and evolution of metazoans living
on and in these substrates. Several faunal associations with wrinkle structures provide
support for this conclusion, For example, wrinkle-structure-covered bedding planes in
Cambrian strata from the Great Basin contain only bedding-parallel trace fossils, which
suggests that burrowing organisms may have been restricted to the uppermost, well-
oxygenated sediments. Cambrian-age burrowing organisms may have had similar
environmental tolerances to those of many modern infaunal animals, which are limited or
otherwise influenced by subsurface redox conditions and burrowing activity that follows
redox boundaries (Wetzel, 2002). Sulfide concentrations ranging from 0 to >200 µM are
found in burrows of modern invertebrates (Völkel, 1995). These concentrations are
deadly to animals that do not have physiological or symbiotic mechanisms for dealing
with the effects of hydrogen sulfide. H
2
S is toxic to aerobic organisms because it
interferes with cytochrome c oxidase by reversibly binding to the iron-containing heme
group. Many modern infaunal organisms have evolved various means of reducing the
64
toxicity of sulfide (Grieshaber and Völkel, 1998). Some invertebrates rely on symbiotic
chemoautotrophic bacteria to oxidize hydrogen sulfide, while others use specialized
compounds found in their blood that provide a similar function. A number of
invertebrates, such as the lugworm Arenicola marina (Völkel and Gieshaber, 1993), the
priapulid Halicryptus spinulosus (Oeschger and Vetter, 1992), and the polychaete
Heteromastus filiformis (Oeschger and Vismann, 1994), actively oxidize sulfide to
thiosulfate in their mitochondria. The absence of vertical burrows in wrinkle-structure-
containing beds suggest that many Early Cambrian burrowing animals may not have
possessed the physiological adaptations necessary for extended infaunal activity in
sulfidic environments.
The observation that vertically-oriented trace fossils such as Skolithos and
Bergaueria can be found in massively-bedded, heavily-bioturbated rocks within tens of
meters of wrinkle-bearing beds, and yet are conspicuously absent from wrinkle surfaces
provides additional evidence that sulfidic conditions beneath microbial mats excluded or
limited infaunal activity. The possibility that euxinia beneath mat surfaces limited
infaunalization by early burrowing animals may impose certain restrictions on the nature
of the agronomic revolution (Seilacher and Pflüger, 1994). One possible scenario is that
grazers living above microbial mat surfaces eventually depleted the microbial biomass,
and thus reduced the organic carbon flux needed to sustain high rates of oxygen
consumption and sulfide production. Another possibility is that some early burrowing
organisms rapidly evolved adaptations to sulfidic conditions and thereafter destabilized
matgrounds so rapidly that a record of their co-existence was not preserved. A third
65
possibility is that matgrounds were patchily distributed, either because of locally
unfavorable conditions such as turbid water and nutrient limitations or transient
disturbances such as periodic erosion by storms, which is supported by the occurrence of
wrinkled bedding planes beneath hummocky cross-stratified units.
In areas where microbial activity was hampered by high levels of erosion, or in
very turbid or nutrient-poor waters, vertically-oriented bioturbators, such as those
responsible for Skolithos piperock may have been able to gain a foothold, gradually
oxygenating porewaters, destabilizing sediments, and beginning to exploit deeper
infaunal settings. The agronomic revolution may have been characterized by local
metazoan coups coexisting alongside patches of persisting matgrounds.
Linguliform Brachiopods and Wrinkle Structures
Linguliform brachiopod casts and molds are very abundant on wrinkle-structure-
covered bedding planes. If the casts and molds represent brachiopods in life position, or
nearly so if attached to a short pedicle and suspended above the mat, then their presence
may indicate a filter-feeding trophic strategy that took advantage of the high bacterial
biomass at the mat surface.
Although linguliform brachiopods can be found in rocks from a variety of marine
settings throughout the Phanerozoic, they are one of the few organisms commonly found
on bedding plane exposures of black shales that were likely formed under oxygen-poor
conditions (Moore, 1952; Thompson and Newton, 1987). The blood of many
brachiopods, along with members of the Priapulida and Sipunculida, contains a non-
heme respiratory pigment called hemerythrin (Manwell, 1960). Hemerythrin can both
66
store and transport oxygen, and, in the case of Lingula, has an increased Bohr effect,
which allows these brachiopods to survive under oxygen-deficient, and perhaps sulfidic,
conditions. Modern brachiopods have demonstrated the capacity to survive for periods of
up to several days in hypoxic settings (Brunton, 1982). If the physiological tolerance of
linguliform brachiopods to anoxic conditions extends back to the Cambrian, it would
likely make them one of the few invertebrate groups capable of tolerating fluctuating
environmental conditions on or above oxygen-poor matground substrates. Although
linguliform brachiopods may have used a pedicle to maintain a position above the
sulfidic and anoxic waters at the mat surface, physiological tolerance to changing
conditions would likely have been necessary for animals living just above mat substrates.
Volborthella
Volborthella, and the related fossil Salterella, are agglutinated cone-shaped fossils
found in Lower Cambrian strata from North America, Greenland, and Northern Europe
(Yochelson and Kisselev, 2003). Seilacher (1999) suggested that millimetric cone-
shaped fossils such as Cloudina and Volborthella lived as “mat stickers” that used the
adhesive properties of microbial mat substrates as a way of anchoring themselves in the
sediment. This study reports, for the first time, Volborthella specimens found in
association with wrinkle structures. Abundant Volborthella specimens found on, and
beneath, wrinkle surfaces support the hypothesis that the organism lived on, or in,
microbial-mat-dominated substrates. The hypothesis that Volborthella passively adhered
to the mat surface during transport is not supported, as other, more abundant bioclasts
found in superjacent strata are absent from the wrinkle beds (see further discussion,
below).
67
Although many authors have suggested a variety of organisms that could have
constructed Volborthella, there is still considerable debate surrounding the zoological
classification of these enigmatic fossils. Signor and Ryan (1993) proposed that
Volborthella represents a spine or sclerite of a large soft-bodied worm, although
Hagadorn and Waggoner (2002) and Yochelson and Kisselev (2003) criticized this
interpretation. Some investigators have suggested that Volborthella may be a member of
an extinct phylum, Agmata (Yochelson and Kisselev, 2003). Volborthella has also been
interpreted as the agglutinated tube of an annelid (Glaessner, 1976). The gross
morphological resemblance to the conical agglutinated tubes of the Pectinariidae and
many other polychaetes is consistent with this hypothesis. The organism responsible for
the construction of Volborthella appears to have been selective about the size and
composition of the agglutinated grains. Grain size and compositional selectivity is
commonly exhibited in test construction by agglutinated foraminifera (e.g., Heron-Allen,
1915) and in tube construction by polychaete worms. Yochelson (1977) proposed that the
organism responsible for Salterella (and presumably Volborthella) used tentacles to
collect food and construct the agglutinated tube. This interpretation is again consistent
with the physiology and life mode of the tube-building, tentacled polychaetes (e.g.,
Paralvinella sp.). The objection raised by Yochelson and Kisselev (2003), that such small
agglutinated tests could not have anchored themselves in even slightly agitated marine
substrates does not account for the possibility that Volborthella lived as a mat sticker
(Seilacher, 1999), or, inverted, as a mat-scratcher. This second possibility is more likely
given that Volborthella is found oriented parallel to bedding plane surfaces. If
68
Volborthella lived as a mat sticker, then it is likely that at least some specimens would
exhibit a bed-perpendicular orientation.
Interpretations of Volborthella as a mat-dwelling organism are strongly supported
by the association between Volborthella and wrinkle structures presented here. A
speculative cartoon schematic of matground life modes (Fig. 3-8A) depicts Volborthella
as a tube-dwelling tentacled polychaete that uses its tentacles to probe into the mat
substrate, a feeding mode similar to that of modern pectinarids. However, the evidence
presented in this study suggests only a matground habitat and possible life position for
Volborthella and does not directly further elucidate its physiology or taxonomic affinity.
Studies of modern mat ecosystems show that small metazoan grazers do not inhibit the
development of microbial mats (Farmer, 1992). If Volborthella was bactivorous, then its
feeding may not have had a destructive effect on microbial mats.
Significance of Associations and Matground Fossil Endemicity
The fossil assemblages found associated with wrinkle structures are distinct from
the fossil assemblages observed in rocks without wrinkle structures from the same
localities. This correlation could result from: 1) sampling bias, 2) a taphonomic effect
resulting from burial in mat-covered sediments that resulted in a preservational bias,
and/or 3) an endemic matground paleoecology.
Sampling bias cannot be fully excluded from possible interpretations of the
evidence presented here. Brachiopods are found elsewhere in siliciclastic rocks from the
Harkless Formation and the underlying Poleta Formation (Rowell, 1977). However,
69
Figure 3-8. A) Schematic of a hypothetical matground ecosystem that contains tentacled
polychaetes (Volborthella) and linguliform brachiopods. Porewater oxygen (dashed line)
and hydrogen sulfide (gray line) relative concentration profiles (0-100%) are depicted vs.
depth. Oxygen disappears within 1 mm of the surface and is inversely related to the
hydrogen sulfide concentration that decreases as it becomes oxidized near the surface (B)
Schematic showing heavily bioturbated sediments that are characterized by oxygenated
sediments and low concentrations of hydrogen sulfide. Trilobite image after Gon, 1999.
70
brachiopods are far less common in rocks that do not contain wrinkles than in those that
do contain wrinkles. In addition, brachiopod-bearing sediments without wrinkles often
appear to be unbioturbated (ii 1), suggesting that they may have also contained microbial
mats or low oxygen conditions, but did not preserve wrinkle structures. The preservation
of brachiopod molds and casts on wrinkle surfaces, rather than shell material, suggests
that perhaps matground substrates preserve linguliform brachiopods under conditions that
do not commonly preserve phosphatic shell material. Ediacaran organisms are thought to
have been preserved as impressions on mat surfaces (e.g., Gehling, 1999), and the
brachiopod molds and casts observed here may have resulted from a similar taphonomic
process. The physiological resiliency of brachiopods to anoxic (and perhaps sulfidic)
conditions offers another possible explanation for these associations, wherein linguliform
brachiopods were one of the few metazoans capable of colonizing Cambrian matgrounds.
Agglutinated fossils have also been reported previously from the Harkless
Formation (Signor and Ryan, 1993). Shell lags of Salterella, a cone-shaped fossil similar
to Volborthella, can be found higher in the Harkless Formation, although these fossils are
larger and differently-shaped from the Volborthella specimens reported here and are
preserved only in shell lag deposits (unpublished data). The x-ray and petrographic
evidence presented here shows that Volborthella specimens are abundant on and beneath
wrinkle surfaces. Volborthella specimens are not oriented in a particular direction on
bedding planes, do not form clast-supported aggregates, and do not appear broken or
abraded, whereas lag deposits of Salterella from elsewhere in the Harkless Formation
exhibit all of these features. The absence of lag deposit indicators suggests that
71
Volborthella specimens associated with wrinkle structures were not transported but rather
represent in-situ components of a matground fossil assemblage. The construction of
Volborthella from grains that include pyrite may indicate that the organisms responsible
for its construction lived in or just above euxinic porewaters where pyrite was actively
forming.
Finally, bioturbation in wrinkle structure-containing rocks is distinct from that
found in adjacent rocks where wrinkles structures are absent. Rocks from the Harkless
Formation preserve burrow structures in considerable detail. The excellent preservation
of ichnofossils in these rocks, along with the fact that laminated sediments beneath
wrinkle surfaces are not disrupted by bioturbation, strongly suggests that differences in
burrowing characteristics were due to paleoenvironmental conditions and not taphonomic
or sampling bias.
The possible significance of sampling bias and/or taphonomic bias for interpreting
the association of linguliform brachiopods and/or Volborthella cannot be discounted.
However, the interpretation of the observed fossil associations as evidence of a
physiologically-resilient, matground-endemic biota is physiologically realistic within the
context of the anoxic and sulfidic conditions that early metazoans would certainly have
encountered within matground substrates. Additional studies of associations between
fossil assemblages and microbially-induced sedimentary structures from other regions
will be required to determine whether or not the associations observed in the Harkless
Formation are representative of matground ecologies in other Early Cambrian shelf
settings.
72
Conclusions
If wrinkle structures were induced by the presence of subtidal microbial mats, and
if conditions beneath mats were similar to those observed in modern mat-containing
substrates, then anoxic and sulfidic pore-waters would most certainly have characterized
the substrates recorded by wrinkle-structure-bearing rocks. Anoxic and sulfidic
matground substrates would have been difficult to exploit for many infaunal, and
possibly epibenthic, metazoans. Linguliform brachiopods, grazing or shallowly-
burrowing animals, and possibly the organisms that constructed Volborthella were likely
part of the metazoan ecology of matground substrates.
Some matground inhabitants may have been adapted to the physical, rather than
geochemical, characteristics of matground habitats and perhaps went extinct in their
absence. The adaptations of other matground inhabitants to anoxic or sulfidic conditions
may have helped them to expand into other hypoxic infaunal habitats once matgrounds
were eventually supplanted by bioturbation. Euxinic conditions in microbial matgrounds
may have served as evolutionary proving grounds for those invertebrates that eventually
colonized microbe-dominated infaunal habitats and deep-water marine environments.
73
Chapter IV: Evidence of giant sulfur bacteria in Neoproterozoic phosphorites
Abstract
The recent discovery of in situ phosphatization

(Schulz and Schulz, 2005) and
reductive cell division (Kalanetra et al., 2005) within the vacuolate sulfur-oxidizing
bacteria leads us to interpret certain Neoproterozoic Doushantuo Formation (~600 m.y.
BP) microfossils, including structures previously interpreted as the oldest known
metazoan eggs and embryos (Chen, 2004; Chen et al., 2006; Chen et al., 2004; Chen et
al., 2002; Chen et al., 2000; Li et al., 1998; Xiao and Knoll, 2000; Xiao et al., 2000; Xiao
et al., 1998) as giant vacuolate sulfur bacteria. Sulfur bacteria of the genus Thiomargarita
exhibit similar sizes and morphologies to many Doushantuo microfossils, including
symmetric cell clusters that result from multiple stages of reductive division in three
planes. We also propose that Doushantuo phosphorite precipitation was mediated by
these bacteria, as demonstrated in modern Thiomargarita-associated phosphogenic sites,
thus providing the taphonomic conditions that preserved other early metazoan fossils
known from the Doushantuo Formation.
Introduction
Fossil cyanobacteria (Awramik et al., 1985), spherical organic-walled
microfossils known as acritarchs (Chuanming et al., 2001), algae (Zhang et al., 1998),
and putative microscopic metazoans (e.g., Chen et al., 2004; Chen et al., 2002; Chen et
al., 2000; Li et al., 1998)

have been described from the Neoproterozoic (~599 ± 4.2 Myr
74
BP (Barfod et al., 2002) Doushantuo Formation in South China, but the unit is perhaps
most noted for producing the world’s oldest animal eggs (Megasphaera) and animal
embryos (Parapandorina) (Chen et al., 2006; Xiao and Knoll, 2000; Xiao et al., 1998).
Initially regarded as algae (Xue et al., 1995), solitary spheroids were reinterpreted as
animal eggs because of their external envelope and large size, and multiple bodies
enclosed by envelopes were reinterpreted as embryos based on their size and apparent
evidence for reductive division by non-rigid cells (Chen et al., 2006; Xiao and Knoll,
2000; Xiao et al., 1998). These putative eggs and embryos, along with other Doushantuo
microfossils commonly regarded as metazoans (Chen et al., 2004; Chen et al., 2002;
Chen et al., 2000; Li et al., 1998), currently provide the earliest direct evidence of animal
life.
However, a number of questions remain regarding the origins and preservational
context (taphonomy) of this unprecedented accumulation of cellular microfossils.
Conspicuously absent are intermediate stages linking very abundant single bodies
interpreted as eggs (Megasphaera) and less common low-cell-number clusters interpreted
as early blastula stages (Parapandorina), with the very-rare, controversial structures
interpreted as gastrulae, larvae and adult forms (Chen et al., 2004; Chen et al., 2002;
Chen et al., 2000; Dornbos et al., 2005; Xiao and Knoll, 2000). Furthermore, recent x-ray
tomography investigations report no evidence of epithelialization via gastrulation in
putative Doushantuo embryos, a developmental process that is ubiquitous in modern
metazoan embryos

(Hagadorn et al., 2006). Taphonomic studies advocate that a bias
toward preservation of early developmental stages could result from the resistance
75
conferred by the embryonic envelope (Raff et al., 2006, Martin et al., 2005). However,
many specimens are missing all or part of the outer envelope, a condition that if present
prior to mineralization would result in degradation within hours (Raff et al., 2006).
Preservation of blastomeres within an intact fertilization envelope has been achieved in
laboratory experiments, but experimental conditions required unrealistically large
concentrations (100 millimolar) of β-mercaptoethanol as a hypothetical simulant of
extremely sulfidic conditions (Raff et al., 2006). Even if conditions were capable of
continuously preventing autolysis and microbial degradation of eggs and embryos,
evidence for a specific molecular mechanism of phosphatization in these cells is lacking
(Xiao and Knoll, 1999b). Here we reinterpret Megasphaera inornata and Parapandorina
as solitary and reductively-dividing giant vacuolate sulfur bacteria. This explanation is
consistent with the morphology, taphonomic robustness, and paleogeochemical
conditions required to explain many Doushantuo globular microfossils, while providing a
biochemical mechanism for phosphatization, which likely facilitated the preservation of
other Doushantuo fossils.
Methods
Thiomargarita sample collection: Gulf of Mexico sediment samples from a
variety of hydrocarbon seeps were collected by Dr. Mandy Joye (Univeristy of Georgia)
during a July 2002 research expedition (R/V Seward Johnson). Push cores were collected
using the Johnson Sea-Link II submersible
2
. Sediment samples were preserved (2.5%
glutaraldehyde) at 4°C until microscopic analysis. Microscopic images and cell diameter
76
measurements were made using phase contrast microscopy, with the exception of Figure
4-2e, which was stained with fluorescein isothiocyanate (FITC) and imaged using a Leica
TCS-SP Laser Scanning Confocal Microscope fitted with an Argon ion laser (488 nm).
Colorless sulfur bacteria – background
In modern organic-rich marine sediments, sulfur-oxidizing bacteria, such as
Beggiatoa, Thioploca, and Thiomargarita oxidize hydrogen sulfide generated by
bacterial sulfate-reduction

(Jørgensen and Nelson, 2004; Schulz, 2006; Schulz et al.,
1999; Schulz and Schulz, 2005). In order to meet their metabolic needs, these organisms
live at gradients between sulfide and microaerophilic levels of oxygen or nitrate.
Thiomargarita, the focus of our investigation, is currently the largest known bacterium,
with average cell diameters between 100-400 µm (some cells grow as large as 750 µm)
(Schulz, 2006; Schulz et al., 1999). Thiomargarita cells are generally spherical and
appear hollow, with the central vacuole occupying the majority of the cell volume
(Kalanetra et al., 2005; Schulz, 2006; Schulz et al., 1999) (Fig. 4-1a-b, Fig. 4-2a-e).
Thiomargarita stores nitrate in the vacuole, presumably used as the oxidant for sulfide
oxidation, at concentrations thousands of times that of seawater. A thin (0.5-2 µm-thick)
outer layer of cytoplasm surrounding the vacuole contains abundant spherical inclusions
(Schulz and Schulz, 2005). Thiomargarita from the Gulf of Mexico occur primarily as
solitary cells or clusters of cells surrounded by a mucus-filled sheath (Fig. 4-1a-b)
(Schulz, 2006; Schulz et al., 1999). Dense colliform clusters of smaller sulfur-globule-
containing cells (~20 µm) are also observed (Schulz, 2006). While Thiomargarita cells
77
Figure 4-1. Phase contrast images of Thiomargarita cellular structure. (a) A multi-
layered ultrastructure (white arrows) surrounding cytoplasm that hosts abundant spherical
inclusions (labeled “i” with black arrows) and (b) a mucous-filled sheath (white arrows)
that hosts abundant microbial filaments. Scale bar in a = 100 µm; scale bar for b = 60
µm.
78
Figure 4-2. Comparisons of light micrograph images of translucent unmineralized
modern Thiomargarita cells (left column) with SEM images of opaque mineralized
Doushantuo microfossils (right column). a, Solitary Thiomargarita cell from the Gulf
of Mexico (after Kalanetra et al., 2005). b, Two-cell cluster of Thiomargarita. c, Three-
cell Thiomargarita cluster, thought to result from the incomplete division of a two-cell
cluster. Greek letters identify each of the three cells. d, Tetragonal Thiomargarita tetrad
resulting from reductive division in two planes. e, Offset between opposing cells in
rhomboidal Thiomargarita tetrads resembles offset in some Doushantuo tetrads (Xiao
and Knoll, 2000) and cross-furrows in four-cell blastulas. Arrows indicate thin sheath
surrounding cell cluster. a', Megasphaera inornata, from the Doushantuo Formation
(Xiao and Knoll, 2000). b', Two appressed hemispherical bodies enclosed by an external
envelope (after Xiao and Knoll, 2000). c', Thiomargarita triplets occasionally result from
incomplete division, which results in two cells with a combined volume roughly equal to
the third undivided cell (Xiao and Knoll, 2000). This Doushantuo globular triplet exhibits
similar relative volumes. Modified after Chen, 2004. d’, Parapandorina tetrad resulting
from division in two planes. Modified after Chen, 2004. e’, Doushantuo rhomboidal
tetrad modified after (Xiao and Knoll, 2000). Scale bar for b' = 150 µm; scale bars for a-
e, a', c', d', e' = 100 µm.
79
80
from Namibia commonly divide in one plane, and more rarely in two planes

(Schulz,
2006), recently described Thiomargarita cells from the Gulf of Mexico undergo division
along three planes, resulting in clusters of 2, 4, and 8 cells

(Kalanetra et al., 2005) (Fig. 4-
2 b-e, Figs. 4-3 through 4-7). No statistically significant difference was observed in the
biovolumes of cell clusters, regardless of cell number, an observation consistent with
reductive cellular division (Kalanetra et al., 2005). Clusters of three cells occasionally
resulted from an incomplete second stage of reductive division (Fig. 4-2d)

(Kalanetra et
al., 2005).
Morphology and geologic occurrence of the Doushantuo Globular Microfossils
The Neoproterozoic Doushantuo Formation exposed in southwestern China hosts
a unique assemblage of phosphatized globular microfossils. The most famous fossil
locality, the Weng’an phosphorite, exposes two phosphatic intervals that are thought to
represent marine shallowing-upward sequences deposited above storm wave base (e.g.
Dornbos et al., 2006). The globular microfossils generally occur as phosphoclasts within
a dolomitic matrix. Although numerous shapes and sizes of globular microfossil are
known from the Weng’an assemblage, the following discussion focuses on the most
abundant and best-known forms: Solitary spherical bodies some 600 ±200 µm in
diameter that are surrounded by an envelope are given the genus name Megasphaera.
Some spherical bodies have a textured envelope (Megasphaera ornata), though the
majority of specimens possess a smooth envelope (Megasphaera inornata). Many
81
Megasphaera specimens possess no envelope at all, but are assumed to represent a
taphonomic variant of the enveloped specimens (Xiao and Knoll, 1999, 2000). Globular
microfossils that possess multiple, usually 2
n
(2 , 4, 8, 16, 32 etc.) appressed bodies are
known as Parapandorina raphospissa. While Parapandorina specimens are known to
occur with more than a hundred internal bodies, these are rare, and Parapandorina most
commonly contains 8 or fewer internal bodies. Parapandorina raphospissa range from
400 to 800 µm in diameter (Xiao and Knoll, 1998, 2000). In general, the volume of these
structures does not increase along with the number of internal bodies. Parapandorina is
often, but not always, surrounded by a thin envelope, generally 1-10 µm thick,
constructed of collophane. Most specimens exhibit a smooth envelope, although
Parapandorina clusters were discovered more recently that possess a textured envelope
(Xiao et al., 2007). Megaclonophycus onustus is a third form of globular microfossil that
consists of hundreds of internal bodies within an internal envelope, but these internal
bodies are very loosely compacted and do not possess the polyganol shapes characteristic
of the bodies enclosed by Parapandorina envelopes (Xiao and Knoll, 2000).
Comparison of Thiomargarita with Doushantuo Microfossils
Morphology and Geometry
Many of the single-celled globular Doushantuo Formation microfossils exhibit
sizes, morphologies, and cellular division geometries consistent with Thiomargarita. The
Doushantuo microfossil Megasphaera inornata, for example, is a large unornamented
spheroidal microfossil (~500 µm) that may or may not be surrounded by a thin (~10 µm)
phosphatic envelope (Xiao and Knoll, 2000) (Fig. 4-2a'), similar to large single cells of
82
Thiomargarita (Fig. 4-1a, b). In the absence of additional morphological differentiation,
such spherical bodies convey limited information, highlighting a perpetual issue in the
investigation of morphologically-conservative fossil structures.
A more striking comparison can be made between certain Thiomargarita clusters
and the Doushantuo microfossil known as Parapandorina (Xiao et al., 1998). Typically,
Parapandorina contains even-numbered (2
n
) internal bodies (2, 4, 8, 16, 32) and exhibits
little or no apparent change in diameter, regardless of the number of internal bodies
present, suggesting that the internal bodies resulted from reductive cellular division (Xiao
and Knoll, 2000, Xiao et al., 1998) (Fig. 4-2 b'-e'). The observation of reductive division
led to their interpretation as animal embryos, as this process was at that time unknown
outside the metazoa in cells of comparable size. The recent discovery of reductive
division in Thiomargarita now warrants a reevaluation of the Doushantuo structures.
Both Thiomargarita and Parapandorina consist of a number of polyhedral to spherical
bodies surrounded by a thin envelope, display reductive cell division, and are of similar
size (Figs. 4-2 through 4-7). Thiomargarita and Parapandorina cells both appear to be
distorted by adjacent cells, suggesting a non-rigid cell wall, a fact previously used to
discount the latter’s interpretation as an alga Xiao, 2002. Both Doushantuo and
Thiomargarita diads exhibit an undisturbed division plane (Fig. 4-2b, b’). Three-cell
clusters, thought to result from the incomplete division of a two-cell stage are present in
both Parapandorina and Thiomargarita (Fig. 4-2c, c’). Whether or not the undivided
larger cell of the Thiomargarita triads later undergoes reductive division to produce a
tetrad is presently unknown. Both Thiomargarita and Parapandorina tetrads exhibit a
83
variety of cell configurations. Four-radiate cross-junctions of division planes in tetragonal
Doushantuo tetrads are shown in Figure 4-2d’, a configuration common in extant non-
mammalian embryos and in Thiomargarita tetrads (Fig. 4-2d). Some Doushantuo octads
are also observed to exhibit radiate cross-junctions (Fig. 4-1c in Dornbos et al., 2005),
which undoubtedly resulted from division of tetragonal tetrads. Rhomboidal tetrads,
which are characterized by two opposite bodies in contact, and two opposite bodies
separated by a gap, are also observed in Parapandorina (Fig. 7.5 from Xiao and Knoll,
(2000)), reproduced in Figure 4-2e’), and in Thiomargarita tetrads (Fig. 4-2e).
Perhaps a more common topology observed in Doushantuo tetrads are decussate
or tetrahedral configurations, which result from deformation of preexisting cell-division
planes (Xiao, 2002). Such deformation is thought to require non-rigid cell
walls/membranes and produces Y-shaped triple junctions of division planes (Fig. 4-3).
Although bacterial cell walls were once thought to be rigid structures, they are now
known to be quite elastic (Doyle and Marquis, 1994). Deformation of division planes
resulting in Y-shaped triple junctions are observed in multi-planar Thiomargarita tetrads,
including partially-deformed tetragonal tetrads (Fig. 4-3a), decussate tetrads (Fig. 4-4),
and tetrahedral tetrads (Supplementary Fig. 4-5). Y-shaped triple junctions are also
observed in 8-cell Thiomargarita and multi-cell Parapandorina specimens (Figure 4-6,
4-7c, d). Although Thiomargarita from off the coast of Namibia are known to form
chains (Schulz, 1999), Thiomargarita from the Gulf of Mexico, the focus of this study,
have not been observed to form chains.
84
Figure 4-3. Thiomargarita tetrad exhibiting deformation of the preceding division planes
with Y-shaped triple junctions; (see arrows) as observed in a cartoon modified after
(Xiao, 2002) showing an intermediate stage in the deformation thought to result in many
Doushantuo Parapandorina tetrads. Scale bar = 100 µm.
Figure 4-4. Thiomargarita tetrad in an approximately decussate geometric configuration,
with two cell pairs approximately at right angles to one another, similar to the geometry
observed in some Parapandorina tetrads (cartoon modified after Xiao, 2002). The dark
material surrounding the cluster is composed primarily of small filamentous and spherical
cells, with sizes and morphologies similar to the filamentous and spherical structures
commonly found on surfaces of Doushantuo microfossils. Scale bar = 100 µm.
85
Figure 4-5. Thiomargarita tetrad exhibiting an approximately tetrahedral geometry akin
to another geometry commonly observed in microfossil tetrads from the Doushantuo
Formation. Scale bar = 100 µm.
Figure 4-6. Thiomargarita octads exhibit Y-shaped junctions (white arrows), similar to
those observed in multi-cell Parapandorina clusters (after Xiao and Knoll, 2000, Fig.
8.8). Scale bar =100 µm.
86
Figure 4-7. Thiomargarita cell clusters (a: 3-cell cluster, b: 4-cell cluster, c, d:
probable 8-cell clusters) show a variety of geometries that indicate deformation of the
previous division plane, as observed in Parapandorina clusters. All scale bars = 100 µm.
87
Four, and possible up to six reductive divisions (16-cell to 64-cell stage) are the
maximum number currently observed in Thiomargarita, although such division could
continue to produce clusters with more cells. Reductive division in Thiomargarita and
other bacteria is thought to be a survival response to starvation (Kalanetra et al., 2005).
We postulate that Thiomargarita cells, if entombed within precipitating hydroxyapatite
and cut off from their vital metabolic substrates, sulfide and nitrate, could continue to
divide, perhaps resulting in a morphology similar to the rare Parapandorina specimens
that contain more than eight cells. Of the 56 specimens examined by Hagadorn et al., 4-
cell Parapandorina specimens were the most abundant (28%) with 74% of specimens
containing 8 or fewer cells (Hagadorn et al., 2006). Specimens with >16 cells were less
abundant (18%). In the 207 specimens examined by Dornbos et al., Parapandorina with
four internal bodies were also the most abundant (48%) and those containing >16 cells
were absent (Dornbos et al., 2005). Parapandorina is much less common than
Megasphaera in Doushantuo phosphorites, which is consistent with the interpretation of
Megasphaera inornata as abundant solitary cells that reductively divide as a stress
response, producing rare Parapandorina.
Thiomargarita cell clusters have yet to be observed containing hundreds of
internal bodies, such as Megaclonophycus, another globular microfossil from the
Doushantuo Formation. However, these microfossils have never been demonstrated to be
part of a developmental continuum with Parapandorina (Xiao and Knoll, 2000).
Megaclonophycus are loosely-packed clusters that contain rounded, rather than
88
polyganol, internal bodies (Xiao and Knoll, 2000), and do not exhibit a blastocoel, as
would be expected in embryos with similar numbers of cells

(Hagadorn et al., 2006), thus
calling into question their metazoan affinities.
Internal bodies
Thiomargarita cells also include abundant inclusions and globules that can
contain sulfur, polyphosphate, or glycogen (labeled “i” in Fig. 4-1a)

(Schulz 2006) and
can form larger aggregates (Fig. 4-8). Subcellular structures in some Parapandorina
specimens (Hagadorn, 2006) may have resulted from diagenetic alteration of such
inclusions in ancient Thiomargarita. Such bodies are consistent with diagenetically-
altered inclusions of the type that are common in Thiomargarita, but as with other
hypotheses, their origin is ultimately ambiguous. A few Parapandorina specimens
(n=10) include larger spherical-to-reniform internal structures (Hagadorn et al., 2006).
Hagadorn et al. (2006) suggest that these bodies might be organelles, however they also
allow for the possibility that the larger internal bodies resulted from inorganic mineral
precipitation or shrunken cytoplasm as observed in other microfossils (Knoll and
Barghoorn, 1975), including algae from the Doushantuo Formation (Fig. 3H in Hagadorn
et al. (2006)). As a sheathed organism with vacuole contents that are chemically-distinct
from surrounding waters, Thiomargarita cells could also have undergone cytoplasmic
shrinkage or internal diagenetic mineral precipitation resulting in intracellular structures.
Inclusions in Thiomargarita also occasionally form aggregates (Fig. 4-8), likely as a
result of cytoplasmic degradation, that are similar in size and shape to the large
89
intracellular structures observed in a small number of Parapandorina specimens. These
aggregates often exhibit approximate symmetry across division planes (Fig. 4-8b), and
can sometimes be found as pairs in each cell of a multi-cell cluster, as is also observed in
at least one Doushantuo tetrad (Hagadorn et al., 2006), though these aggregates likely
result from degradational, rather than physiological, processes.
Abundance
The unusual abundance of globular microfossils in the Doushantuo Formation has
long been considered problematic for the animal embryo interpretation (Xue et al., 1999);
mass spawning and concentration via sedimentary processes have been proposed as
possible solutions Xiao and Knoll (1999). More parsimoniously, Thiomargarita cells of
similar sizes (ca. 500 mm) can be found in great abundances (up to 10
7
cells m
-2
) and
would not require unusual circumstances to explain large accumulations of their fossils
(Schulz, 1999). The unusual abundance of globular microfossils in the Doushantuo
Formation has long been considered problematic for the animal embryo interpretation
(Xue et al., 1999); concentration via sedimentary processes has been proposed as a
possible solution (Xiao and Knoll, 1999a). Such a circumstance does fall within the realm
of possibility. However, the globular microfossils generally occur as grains within larger
reworked clasts (Xiao and Knoll, 1999b; Xiao and Knoll, 2000). Literature on this topic
suggests that the fossils were phosphatized, size sorted by currents, cemented together,
ripped-up, and re-deposited elsewhere within the larger clasts

(Xiao and Knoll, 1999b).
Each stage of cementation followed by reworking and re-deposition loses the memory of
90
Figure 4-8. Aggregates of internal inclusions, presumably resulting from cytoplasmic
degradation, appear as large spheroidal to reniform intracellular bodies in a small number
of a) solitary Thiomargarita cells, and b, c) multi-cell clusters. All scale bars = 70 µm.
Figure 4-9. Hydrocarbon-seep sediments covered with very abundant Thiomargarita
cells. The width of the pH microelectrode tip at center is ~500 µM.
91
the former clast size. Such multiple reworking would more likely dilute, not concentrate,
the microfossils in the deposit. Thus, we do not find concentration by sedimentary
processes to be the most likely solution.
The interpretation of the microfossils as bacteria more easily explains their
abundance, as Thiomargarita is known to occur in great abundance (up to 200 g m
-2
),
which would translate to approximately 10
7
cells per m
-2
. In the Gulf of Mexico,
abundance is somewhat lower at ~ 10
5
cells per m
-2
(Fig. 4-9). In addition, stages of
reductive division in Thiomargarita are separated by months to years (Kalanetra et al.,
2005), allowing a longer window for the observed preferential preservation of 2, 4 and 8-
cell clusters, unlike invertebrate embryos that develop from fertilized egg, through the
blastula stages to a gastrula in a matter of a few hours (Costello et al., 1971).
Envelopes and ultrastructure
The Doushantuo Formation contains abundant large spherical microfossils that
exhibit a range of morphological features. Abundant indistinct smooth spheres of
uncertain affinities, generally categorized as sphaeromorphic acritarchs, are often thought
to represent algal resting cysts. Some Doushantuo spherical bodies are encased in an
envelope (Megasphaera). It has been recognized that some of these envelopes possess
external surface ornamentation (Megasphaera ornata), while others lack ornamentation
(Megasphaera inornata). This ornamentation has been central to the argument for a
metazoan egg interpretation of Megasphaera (Xiao and Knoll, 1998). Recently, it was
92
proposed that Megasphaera results from taphonomic alteration of the acanthomorphic
acritarch Tianzhushiana tuberifera (Yin et al., 2004), which is characterized by external
processes that penetrate a multi-lamellate outer wall (Yin and Li, 1978, Zhang et al.,
1998). Similarities between the middle wall of T. tuberifera and the outer wall of M.
ornata, and the finding of an additional outer wall on a few M. ornata specimens lead to
the hypothesis that they represent the same species. Based on the hypothesis that
unornamented globular fossils may have once had an ornamented outer wall that was not
preserved, the unornamented M. inornata was also proposed to be synonymous with
Tianzhushania (Yin et al., 2004). Given the great abundance of unornamented globular
microfossils in the Doushantuo phosphorites, we find this explanation insufficient to
explain most unornamented specimens. Therefore, we retain the use of M. inornata,
which we interpret as possible fossilized giant sulfur bacteria based on its abundance in
phosphorites. Deflated envelopes lacking internal bodies are also observed in the
Doushantuo microbiota (Fig. 4-10a’), and similar deflation is commonly exhibited in
Thiomargarita cells with ruptured internal vacuoles (Fig. 4-10a). However, modern
animal eggs and phosphatized Doushantuo acritarchs are also observed to exhibit
deflation and collapse. Ultimately, unornamented globular fossils are taxonomically
ambiguous and could represent more than one organism.
The recent discovery of ornamented envelopes surrounding a handful of multi-cell
globular microfossils (e.g., Parapandorina) lead Xiao et al. (2007) (Appendix A) to
question the bacterial interpretation presented here. However, this criticism doesn’t
account for the complex ultrastructures observed in some modern relatives of
93
Thiomargarita, nor does it acknowledge the possibility that the textured surface observed
around only a few of these structures may have been produced by diagenetic processes as
explained by Bailey et al. in Appendix B. Beyond those microfossils that have been
previously interpreted as animal embryos, other structures preserved in Doushantuo
phosphorites suggest the preservation of various morphotypes of colorless sulfur bacteria.
For example, Figure 4-11a shows a large smooth envelope surrounding multiple internal
spherical bodies that exhibits a similar appearance to Thiomargarita morphotypes that
contain more than a dozen isolated cells within a mucous sheath (Fig. 4-11b).
Other Associated Fossils and Structures
Filaments surrounding globular fossils
Doushantuo globular microfossils, but not acritarchs and algae, are commonly
associated with phosphatized filaments and spheroids previously interpreted to be
fossilized infesting bacteria (Fig. 3b from Xiao and Knoll, 1999). Figure 4-12a shows
Thiomargarita cells from Namibia covered by filamentous bacteria interpreted to be
symbiotic sulfate-reducing bacteria (Schulz, 2006). Thiomargarita cells from the Gulf of
Mexico also commonly co-occur with filamentous and spheroidal bacteria. Taphonomic
studies indicate that infesting bacteria rapidly destroy embryonic animal cells (Raff et al.,
2006), making the preferential co-preservation of embryos and the offending degradative
bacteria unlikely, though not unprecedented (Xiao and Knoll, 1999). Conversely, the
preservation of bacteria with their natural host Thiomargarita cells requires no such
fortuitous mineralization of transient putrefying remains.
94
Figure 4-10. Deflated Megasphaera (a’) (after Xiao and Knoll, 1998) may indicate an
initially hollow interior, such as the large vacuole in Thiomargarita, which is
occasionally observed to rupture resulting in a deflated cell (a). Scale bar = 100 µm.
Figure 4-11. Some Doushantuo microfossils closely resemble sulfur bacteria, but not
animal embryos. For example, the spherical envelope in b surrounding non-appressed
internal spherical cells resembles some less common Thiomargarita morphotypes (a),
and the large septate filament in d resembles Beggiatoa, Thiomargarita’s closest known
relative.
95
Figure 4-12. Filaments associated with Thiomargarita cells and Doushantuo
microfossils. a. Thiomargarita from Namibia (modified after Schulz, 2006) and the Gulf
of Mexico are often covered with filamentous bacteria that are likely sulfate-reducing
symbionts. b, Doushantuo globular microfossils, such as this Parapandorina specimen,
are commonly covered with filaments thought to be mineralized embryo-infesting
bacteria (after Xiao and Knoll, 2000). Scale bar = 50µm for a; 100 µm for b.
96
Very Large Septate Filamentous Fossils
In addition to globular microfossils, the Doushantou Formation also preserves
septate filaments approximately 50 µm wide and more than a millimeter in length (Fig. 4-
11d). These large filamentous microfossils resemble marine Beggiatoa morphotypes (4-
11c) that commonly inhabit similar environments as Thiomargarita. Marine Beggiatoa
are most-closely related to Thiomargarita, with which they share a common physiology -
included the propensity to accumulate internal polyphosphate.
Sulfur bacteria and phosphogenesis
Phosphorite deposition is relatively uncommon and poorly understood throughout
the geologic record. The Neoproterozoic and Cambrian have long been known as periods
of widespread marine phosphogenesis, with approximately 10% of world phosphate
reserves coming form rocks of this interval (Notholt and Sheldon, 1986) (Figure 4-12).
Archean and Mesoproterozoic phosphorites are largely associated with igneous and
metamorphic rocks. Minor phosphorites are known from Mesoproterozoic rocks, and
some are associated with stromatolites. However, the accumulation of massive marine
chemo-sedimentary phosphorites on multiple continents about the Precambrian/Cambrian
boundary is unprecedented and was likely driven by changes in ocean biogeochemistry.
Recently, sulfide-oxidizing bacteria have been demonstrated to mediate phosphorite
deposition in modern environments, (Schulz and Schulz, 2005). Thiomargarita cell
accumulations correlate with increased pore water phosphate and accumulations of P-
97
containing minerals, which amounted to >50 g kg
-1
of dry sediment (Schulz and Schulz,
2005). Laboratory experiments suggest that metabolically-driven phosphate release by
Thiomargarita controls phosphate mineral precipitation, thus providing a microbially-
mediated mechanism of phosphorite formation Schulz and Schulz, (2005). Dense
accumulations of Thiomargarita inhabit modern phosphogenic pore waters, leading
(Schulz and Schulz, 2005) to conclude that sulfur bacteria contribute significantly to the
formation of modern phosphorites. Schulz and Schulz (2005) also suggested that this
process was likely responsible for the formation of many ancient phosphorites.
The release of orthophosphate into discrete porewater horizons by these sulfide-
oxidizing bacteria, may be sufficient to induce the precipitation of hydoxyapatite or its
precursors. Additionally, the precipitation of phosphatic minerals may also be catalyzed
by a reduction in porewater pH resulting from the oxidation of H
2
S (Krajewski et al.,
1994). These biological processes lead to porewater conditions that are conducive to the
abiotic precipitation of phosphatic minerals, thus providing a biogeochemical explanation
for the remarkable preservation of Doushantuo Weng’an microfossils and perhaps the
formation of other abundant Ediacaran and Phanerozoic phosphorites.
Multiple geochemical studies suggest the initial spread of oxygen to the
sediment/water interface during the Late Ediacaran (e.g., (Canfield et al., 2007; Fike et
al., 2006; McFadden et al., 2008). This oxygen penetration would almost certainly have
been preceded or accompanied by the spread of nitrate to the benthic realm. Both oxygen
and nitrate at microaerophilic concentrations, act as terminal electron acceptors for
sulfur-oxidizing bacteria such as Thiomargarita. In modern low-oxygen marine benthic
98
settings, such as off the coast of Chilé and Peru, colorless sulfur bacteria comprise the
largest microbial-mat ecosystems in the world (e.g., Gallardo et al., 1977). Widespread
coastal shelf settings may have experienced widespread microaerophilic levels of oxygen
for the first time during the Neoproterozoic – perhaps leading to conditions ripe for the
evolutionary and/or biogeographic radiation of the colorless sulfur bacteria. The Late
Neoproterozoic spread of oxygen correlates with, and is often indicated by, a rise in
Δδ
34
S values above 45‰ – a value that is the maximum produced by sulfate-reduction
that does not involve recycling by sulfur oxidation. As initially suggested by Canfield
and Teske (1996), the colorless sulfur bacteria may have contributed greatly to the
nascent oxidative sulfur cycle and the formation and utilization of sulphur intermediates
suggested by Δδ
34
S values above 45‰. Molecular clock extrapolations also suggest the
divergence of the sulfur bacteria at 0.62 ± 0.2 Gyr. The correlation of sulfur isotope
values that suggest benthic oxidative processes, molecular clock data that suggest
colorless sulfur bacterial divergence, unprecedented phosphorite proliferation, and the
presence of microfossils that resemble giant sulfur bacteria strongly suggests that
colorless sulfur bacteria radiated during the Neoproterozoic in response to the spread of
oxygen and/or nitrate to benthic marine settings, and that these bacteria had a profound
effect on cycling of sulfur and phosphorous, and perhaps nitrogen and carbon as well
(Figure 4-12).
Paleoenvironmental Considerations
The geochemical conditions during the deposition of the Doushantuo Formation,
but not necessarily the paleoenvironment itself, may have been analogous to modern
99
Figure 4-13: The proliferation of phosphorites about the Precambrian/Cambrian
Boundary correlates closely with Δδ
34
S, fossil evidence, and molecular clock divergence
estimates that all suggest the co-existence, and perhaps a genetic link between colorless
sulfur bacteria and the unprecedented PC/C phosphogenic event. Includes data adapted
from Cook (1992), Canfield and Teske (1996) and Fike et al. (2007).
100
101
upwelling zones where Thiomargarita was discovered (Schulz et al., 1999). Multiple
lines of paleontological and lithological evidence point to enriched nutrient
concentrations in Doushantuo depositional environments (Zhou et al., 2001).
Additionally, indicators of sulfidic conditions, which would be a requirement for
Thiomargarita, are abundant in the Doushantuo Formation. Heavy sulfur and oxygen
isotope values of sulfate from Doushantuo Formation phosphorites provide evidence of
probable bacterial sulfate reduction, perhaps coupled to sulfide oxidation Goldberg et al.,
2005). Abundant pyrite in embryo-bearing phosphorites and in microfossils (Xiao and
Knoll, 1999) is also suggestive of sulfidic conditions. In an environment with nutrient-
rich, productive surface waters and euxinic bottom waters, the remains of planktonic
algae, and perhaps metazoans, from the overlying water column, along with
allochthonous benthic organic debris, would co-occur with abundant sulfide-oxidizing
bacteria as in the modern counterparts, resulting in the fossil assemblages rich in
phosphatic spheroidal and globular forms, such as those observed in the Doushantuo
Formation.
Conclusions
We do not suggest that the Doushantuo microbiota is composed entirely of sulfur
bacterial remains, as there are many structures in the Doushantuo that clearly do not
resemble sulfur bacteria. Furthermore, the hypothesis posed here does not invalidate the
possibility that some Doushantuo globular microfossils are indeed animal eggs and
embryos, since the two hypotheses are not mutually exclusive. Instead, our reassessment
102
provides a morphologically-plausible alternative interpretation of the most abundant
globular microfossils (Megasphaera inornata and Parapandorina) as giant vacuolate
sulfur bacteria, and a mechanism of preservation via microbially-mediated phosphate
release by microorganisms, as noted in modern habitats. Given the possibility that
Megasphaera inornata and Parapandorina represent giant bacteria, and the controversy
surrounding other Doushantuo putative metazoans (e.g., Xiao et al., 200, Bengston and
Budd, 2004), the interpretation of Doushantuo globular microfossils as metazoan
embryos deserves further scrutiny.
103
Chapter V: Is cell shape conserved over geologic timescales? Clues from a
cytoskeletal protein in dimorphic prosthecate bacteria.
Abstract
The bacterial intermediate-filament homolog protein, crescentin, plays a major
role in the cell shape of Caulobacter crescuntus. This study investigates the phylogenetic
distribution of this protein, and attempts to determine its selective advantage with the
goal of predicting its potential for conservation over geologic time scales. Isolates of
eight prosthechate α-proteobacteria were screened for the presence of the bacterial
intermediate-filament homolog, crescentin. The protein was not detectable by
immunofluorescence techniques in any of the screened isolates, with the exception of
Woodsholea maritime, where anti-crescentin antibody binding exhibited a diffuse
cytoplasmic occurrence in fully-extended prosthecate cells, a narrow mid-cell band
localization in swarmer cells, and an extended helical localization in intermediate-sized
prosthecate cells. These results suggest that crescentin is not universally utilized among
the prosthecate bacteria, despite its occurrence in the model dimorphic prosthecate
bacterium, Caulobacter crescentus. Although the marine bacterium W. maritime, appears
to contain crescentin, localization patterns and cell morphology hint at an alternative role
for the cytoskeletal protein. Additionally, previous results indicating that C. crescentus
mutants lacking crescentin suffered no detectable fitness loss are further supported here
by growth, motility and chemotaxis assays that show little difference between the
laboratory performance of the C. crescentus wild type and creS- deficient mutant.
104
Introduction
Much of our understanding of life in the Precambrian relies on interpretations of
morphologic features exhibited by fossilized cells. Certain morphological characteristics,
such as size, shape, ultrastructure, presence or absence of peripheral processes, etc. are
generally used to assign fossils to specific clades (e.g., Javaux et al., 2002). Some
morphological features are only thought to be diagnostic at the domain level, whereas
other characteristics, or groups of characteristics, are frequently used to assign
microfossils down to the genus level e.g., (Schopf, 1992). But how reliable are
characteristics such as cell shape? Can the genes that control cell shape be horizontally
transferred between distantly-related organisms? Do closely-related organisms share a
common morphology? Is cell shape conserved over hundreds of millions, or even
billions, of years? The molecular tools necessary to answer the questions are now
becoming available. Cytoskeletal proteins serve as an important mediator of cell shape.
Originally known only in eukaryotes, the recent discovery of cytoskeletal protein
homologs in bacteria and archaea has greatly increased our understanding of cell
structure and function in bacteria and archaea (Carballido-López, 2006; Jones et al.,
2001; Kruse et al., 2005). Bacterial cytoskeletal proteins are now known to play a pivotal
roll in cell shape, cell division, and cell polarity (Cabeen and Jacobs-Wagner, 2005; Klint
et al., 2007; Møller-Jensen and Löwe, 2005). A bacterial homolog to the intermediate
filaments, known as crescentin, was recently discovered when transposon insertions in
the genome of the prosthecate bacterium Caulobacter crecsentus (Ausmees et al., 2003)
105
resulted in mutants that formed straight rod-shaped cells (Fig. 5-1b), rather than the
vibroidal shape characteristic of the wild type (Fig. 5-1a). Structural and sequence
similarities suggest a homology between crescentin and eukaryotic intermediate
filaments. Preliminary unpublished results by Ausmees et al., (2003) indicated similar
laboratory growth by both the wild type and the mutant lacking crescentin, prompting
questions about the selective advantage of a protein that confers a vibroidal cell shape
(Young, 2006). The authors suggested that the selective advantage of crescentin might
only be present under natural conditions that have yet to be simulated in the laboratory.
Testing other physiological factors of the vibroid wild type and comparing them with the
rod-shaped mutant may reveal selective advantages associated with crescentin.
Additionally, understanding the distribution of crescentin in prosthecate bacteria isolated
from a range of environmental conditions might also provide clues about crescentin’s
selective advantage, if such an advantage does in fact exist. Here I report on the use of
immunofluorescence techniques to investigate the occurrence of crescentin among eight
prosthecate α-proteobacteria related to C. crescentus that inhabit a wide range of
environmental conditions, in the hopes that the occurrence of crescentin in other closely-
related organisms might offer clues about the reliability of using cell shape to infer
phylogeny. Additionally, I present the results of experiments that compare the growth,
motility, and chemotactic responses of the C. crescentus wild type with the CresC:Tn 5
mutant that lacks crescentin. The goal of this comparison is to determine what selective
benefit cell shape might confer on these organisms and whether or not this selective
pressure is sufficient to maintain such a trait over millions of years.
106
Figure 5-1. Caulobacter crescentus wild type (a) exhibits a vibroidal cell shape, while the
Crescentin-deficient mutant (b) appears as a straight-rod.
107
Methods
Bacterial strains and growth conditions
Each of the bacterial strains used in this study are listed in Table 5-1. Caulobacter
crescentus CB15N, CJW763, and C. leidyia were grown in PYE or minimal media at
30
o
C with shaking. All marine strains were grown on CPS complex media at 25
o
C with
shaking. Marine strains were grown on Caulobacter Complex Marine Media.
Hyphomicrobium sp. was isolated from Woods Hole, Massachusetts soil using methanol
as the carbon source under denitrifying conditions as described by (Sperl and Hoare,
1971). Biomass for immunofluorescence was collected during mid-exponential phase or
during stationary phase. Both conditions were examined to monitor crescentin expression
by different morphotypes.
Media compositions
1) PyE: 0.2% (w/v) Peptone, 0.1% Yeast Extract, 0.02% MgSO
4
• 7H
2
O.
2) PyCM: 0.25% peptone, 0.05% yeast extract, 1mM CaCl
2
, 1 mM MgSO
4
, 1% Bacto
agar, prepared in milli-Q water.
3) CPS Complex Medium: 0.05% casamino acids, 0.05 % Bacto peptone, 80%
seawater.
4) MG2 Minimal media: 20X M2 Salts (17.4 g Na2HPO4, 10.6 g KH2PO4, 10.0 g
NH
4
Cl) dissolved in 1L of Milli-Q water was autoclaved. 50 ml of salt solution,
was added to 938ml of sterile water, followed by the sequential addition of 0.5 ml
of 1M MgSO
4
, 10ml of 20% glucose, 1 ml of 10mM FeSO
4
/10mM EDTA, and
108
0.5 ml of CaCl
2
. Fe-deficient minimal media was made using the same recipe, but
adding only 1 mM FeSO
4
/1mM EDTA. Similarly, P-deficient minimal media
contained 1X phosphate salts.
Table 5-1
Bacterial strains and media used in this study:
Species/Strain Name Description Media
Caulobacter crescentus CB15N Wild type strain, positive control creS PYE
C. crescentus CJW763 CB15N creS::Tn5, negative control creS PYE
C. leidya CB37 Now assigned to the genus Asticcacaulis PYE
Hyphomicrobium sp. Unnamed denitrifying Hyphomicrobium
isolate from soil, Woods Hole, MA
HYP
C. halobacteroides CM13a Marine Caulobacter CPS
Hyphomonas adhaerens MHS-1 Uncharacterized isolate, mud slough CPS
Maricaulis washingtonensis
MSC6
Marine Maricaulis CPS
Woodsholea maritime CM243 Formerly Caulobacter halobacteroides
CM243
CPS
Maricaulis maris CM11 Isolated from sea water, Anacortes, WA,
USA
CPS
Caulobacter sp. CM261 Unnamed marine Caulobacter/Maricaulis CPS
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Immunofluorescence assays
Immunofluorescence microscopy using anti-crescentin polyclonal antibodies was
performed using methods similar to those described by Pogliano et al. (1995). Cells
grown under the growth conditions provided in Table 1 were fixed using 8%
paraformaldehyde. After 4x centrifuge washes with PBS at 11,000 rpm, cells were re-
suspended in 75 µl of GTE. 25 µl of 0.01 mg/ml lysozyme diluted in GTE was added to
the cell suspension and ~20 µl was spotted on poly-l-lysine-coated, 8-well glass slides.
After 10-minute of incubation, the lysozyme/cell solution was aspirated. 2% filtered BSA
in PBS was then used to block non-specific binding sites for 15 minutes in a humid
chamber. Anti-crescentin antibodies diluted 1:40 in 2% BSA were then added and
allowed to incubate for 1 hr. at 25
o
C. Following aspiration and 10X washing with
2%BSA in PBS, FITC-labeled goat anti-rabbit secondary antibodies (diluted 1:200) were
applied and allowed to incubate in the dark for 1 hr. at 25
o
C. Following 10X washes with
PBS, 5 µl of 2 µM FM 4-64 (Invitrogen) was added to Citfluor (Citifluor Ltd., London)
anti-fade mounting solution and applied to each well before adding a cover slip and
imaging. Images were acquired using a Hsm Axiocam attached to a Zeiss M1 Imager
microscope. Image processing was performed using Axiovision Rel 4.6 and Photoshop
CS2 software. All isolates were evaluated in triplicate. Each immunofluorescence 8-well
slide included C. crescentus as a positive control and the CresC:Tn 5 mutant as a negative
control.
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Motility assays
0.3% agar swarm plates were inoculated with 5 µl of saturated culture (either
CB15 or the creS::Tn5 mutant) normalized to the same optical density (OD
600
). The assay
employed here was similar to those used by (Pierrce et al., 2006). In cases where
cultures were maintained on peptone yeast extract, and later transferred to experiments on
minimal media, all cultures were transferred and grown overnight on minimal media
prior to motility plate inoculation. All plates were incubated and kept at room
temperature in sealed humid containers for 4.5 days before swarm halos were measured.
Chemotaxis assays
10 µm-diameter glass capillary tubes containing PyE media were suspended in the
center of culture plate wells. Each well contained 300 µl of freshwater base, and was
inoculated with 5 µl of exponential phase culture (either CB15 or the creS::Tn5 mutant).
Chemotactic response, as inferred by the number of cells present in the capillary tubes,
was compared 12 hours after inoculation by expelling the capillary fluid onto a Hausser
counting chamber slide and counting cells in nine randomly-selected 1 sq. mm. grids.
Nine grids were averaged per capillary tube, and 6 replicate experiments were performed
for both CB15 and the creS::Tn5 mutant
Phylogenetic tree construction
16S rDNA sequences for all organisms except for Hyphomicrobium sp. and
Caulobacter strain CM261were downloaded from GenBank and the Ribosomal Database
111
Project (RDP) and aligned using the alignment tools provided on the RDP II website
(Cole, 2007). Organisms selected to show the phylogenetic context of the organisms
studied within the tree were selected based in part on the groupings of prosthecate α-
Proteobacteria that resulted from the analyses of (Sly et al., 1999). A 16S rDNA
phylogenetic tree was constructed using the RDP Weighbor-weighted, neighbor-joining
tree building algorithm.
Results
Immunofluorescence microscopy
In this study, polyclonal antibodies against crescentin allowed for the screening of
isolates of prosthecate bacteria from the environment. In each immunofluorescence slide
imaged, the C. crescentus wild type was used as a positive control and the CresC:Tn 5
mutant served as a negative control. In previous investigations, a GFP-crescentin hybrid
protein was shown to localize to the inner curvature of the vibroidal C. crescentus wild
type, and was absent in the CresC:Tn 5 mutant (Ausmees et al., 2003). Localization of
the anti-cres antibodies to the concave portion of the cell was consistently observed in
positive control cells (Fig. 5-2 a-c). Negative controls exhibited no, or very low, levels of
background binding, and no localization of anti-crescentin antibodies (Fig. 5-2d).
Similarly, no consistent anti-crescentin antibody binding or localization was observed in
Caulobacter halobacteroides CM13a, an unnamed Hyphomicrobium sp., Caulobacter
leidya, Hyphomonas MHS-1, Maricaulis washingtonensis, Maricaulis maris CM11, and
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Figure 5-2. Caulobacter crescentus CB15 wild type showing localization of FITC-
labeled antibody binding to anti-crescentin antibodies (a-c). Binding is localized to the
concave side of the cell. The wild type served as the immunofluorescence positive
control. Red staining is the amphiphilic dye FM 4-64; d. creS::Tn5 mutant, lacks
crescentin and functions here as an immunofluorescence negative control; Several
prosthecate bacteria did not exhibit anti-crescentin antibody binding, such as e.
Maricaulis maris; f. Hyphomonas adhaerens MHS-1; g. Hyphomicrobium sp.; h.
Maricaulis washingtonensis; i-l. Woodholea maritime exhibits crescentin antibody
binding with localization dependent on cell morphotype. Scale bars in a and g = 5µm.
Scale bar in l = 10 µm. Scale bar in j = 20 µm.
113
marine Caulobacter strain CM261 (Fig. 5-2 e-h). Binding of anti-crescentin antibodies
was consistently observed in Woodsholea maritime at population coverages
indistinguishable from those in C. crescentus (e.g., >90% cells showed strong binding),
but with very different patterns of localization observed in different cell morphotypes
(Fig. 5-2 i-l). A diffuse pattern of antibody-binding was observed throughout the cell
(but not in the stalk) in Woodsholea cells with elongated prosthecate and bud formation
(Fig. 5-2j). In some elongated mother cells, antibody binding localized to a coiled
filament-like structure (Fig. 5-2k), while in swarmer cells, antibody-binding was largely
localized to one or two narrow bands running along the cell’s short axis (Fig. 5-2l). The
phylogenetic distribution of organisms screened for crescentin in this study, along with
the presence/absence results of the screening, are presented in a dendrogram of the 16S
rDNA sequences of the strains used in this study, as well as related prosthechate α-
proteobacteria (Fig. 5-3).
Growth studies
Similar growth rates for C. crescentus wild type and the CresC:Tn 5 mutant growing on
PYE media at 30
o
C are shown in Figure 5-4.
Chemotaxis assays
The chemotactic response for both the C. crescentus wild type and the CresC:Tn
5 mutant, as measured by the relative numbers of bacteria in each of six replicate
114
Figure 5-3. Dendrogram based on 16S rDNA sequences of selected prosthecate α-
proteobacteria. Isolates observed to contain crescentin in this study are outlined in green,
while those isolates that did not show anti-crescentin antibody binding are outlined in red.
Hyphomicrobial phylotypes are outlined in pink indicating the assumed, but uncertain,
phylogenetic affinity of the denitrifying Hyphomicrobium sp. examined in this study.
(averages of 9 grids each) for the C. crescentus wild type, while average cell counts for
CresC:Tn 5 were 12, 15, 18, 18 22, and 32 cells/mm
2
.
115
Figure 5-4. Semi-log plot of exponential phase growth in Caulobacter crescentus
CB15N (green symbols) and the mutant creS::Tn5 (red symbols) on PYE media at 30
o
C.
116
Figure 5-5. Caulobacter crescentus CB15N (green symbols) and the mutant creS::Tn5
(red symbols) show a similar chemotactic response to capillary tubes containing peptone-
yeast extract suspended in an inoculated freshwater base.
117
Figure 5-6. Caulobacter crescentus CB15N (green symbols) and the mutant creS:Tn5
(red symbols) showed similar motility on swarm agar plates with four different media
compositions.
118
capillary tubes after 12 hours, is presented in Figure 5-5. 1 sq. mm counting-grid squares
from each of the six replicate tubes contained 10, 14, 14, 23, 25, and 25 cells/mm
2
Motility assays
Swarm diameters for C. crescentus wildtype and the mutant are shown in Figure
5-6. On PyE swarm plates, C. crescentus wild type swarm halos averaged 1.3 ± 0.13 cm,
while the CresC:Tn 5 mutant produced swarm diameters of 1.28 ± 0.17 cm. C. crescentus
wild type swarm halos averaged 1.37 ± 0.08 cm, while the CresC:Tn 5 mutant produced
swarm diameters of 1.33 ± 0.08 cm on minimal media swarm plates. Minimal media
plates with reduced Fe resulted in C. crescentus wild type swarm halos that averaged 1.8
± 0.12 cm, while the CresC:Tn 5 mutant produced swarm diameters of 1.7 ± 0.09 cm.
While minimal media plates with reduced P contents resulted in C. crescentus wild type
swarm halos that averaged 1.0 ± 0.2 cm, while the CresC:Tn 5 mutant produced swarm
diameters of 1.0 ± 0.14 cm. Minimal media plates with reduced Fe concentrations
produced the largest diameter swarm halos, while those plates containing reduced-P
minimal media produced the smallest halos. Swarm halos on plates with reduced
phosphorous minimal media were also characterized by a concentric banding not
observed in the other plates.
Discussion and conclusions
The dimorphic prosthecate bacteria (DPB) are a group of a-proteobacteria in
which cell morphological features are intimately associated with ecophysiology. Cell
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division produces one small, flagellated motile cell, and one non-motile, prosthechate cell
- a reproductive strategy that provides for separation of sessile mother cells from motile
offspring under nutrient-limited conditions (Hallez et al., 1004; Poindexter, 2006).
Additionally, prosthecate - thin appendages that increase surface area available for
nutrient uptake, are morphological adaptations to nutrient limitation regulated on a
genetic level. Because morphology plays such an important role in the physiology of the
DPB, it would perhaps be unsurprising to find that the cytoskeletal protein crescentin,
which imparts a vibroidal cell shape on C. crescentus, provides a physiological benefit to
C. crescentus, or to other species within the DPB. However, given the similarities
between the growth rates, swarm motility, and chemotactic response of the C. crescentus
wild type vs. the creS mutant observed in this study and others, the functional role of
crescentin, and a vibroid cell shape in general amongst the DPB, remains unclear.
Immunofluorescence microscopy did not reveal the presence of crescentin in any
of the DPB screened in this study, with the exception of W. maritime. The absence of
crescentin within the Hyphomonads is further supported by the sequenced genome of H.
neptunium, which shares many genes with C. crescentus, but lacks the crescentin-
encoding gene, creS (Badger et al., 2006). H. neptunium and C. crescentus share many
more genes than are shared between H. neptunium and Silicibacter pomeroyi (the closest
relative to H. neptunium by 16S rDNA)(Badger et al., 2006), making crescentin’s
absence in the Hyphomonads all the more conspicuous.
Confirmation of the absence of crescentin, or homologous proteins, in the other
DPB will need to await additional genome sequencing. For now, the apparent paucity of
120
crescentin in dimorphic prosthecate bacteria suggests that any advantage it might confer
might be limited to particular ecological niches, and is not a requirement of a dimorphic
reproductive strategy or the development of prosthecae. Many of the prosthecate bacteria
that exhibited no crescentin antibody-binding in this study are marine organisms, whereas
C. crescentus is a representative of a monophyletic cluster of freshwater Caulobacters
(which now excludes members of the genus Asticcacaulis). Any selective advantage
conferred by crescentin might only be significant in terrestrial habitats or under specific
conditions that are not easily duplicated in the laboratory. C. leidyia is a freshwater
dimorphic prosthecate bacterium that apparently lacks crescentin, however 16S rDNA
analyses suggest that it is quite distantly related to the other freshwater Caulobacters, and
should be grouped within the genus Asticcacaulis in the 4α- subclass of the
Proteobacteria (Sly et al., 1999).
One caveat with regard to the possible differential role in terrestrial vs. marine
environments, is the occurrence of the protein in the marine prosthecate bacterium
Woodholea maritime, as interpreted from the antibody binding affinity and localization
observed in this study. W. maritime is most closely related to other marine Caulobacters
now placed within the genus Maricaulis, but is distinguished from these other organisms
by 16S rDNA and its distinctive lipids (Abraham et al., 2004). The diffuse occurrence of
crescentin in extended prosthecate W. maritime cells, and its distinct banding in swarm
cells, is reminiscent of the localization observed in the bacterial microtubule analog, ftsZ
(Bi and Lutkenhaus, 1991), and somewhat unlike crescentin asymmetric localization
along the concave ultrastructure observed in C. crescentus (Fig. 5-2a-c this study, and
121
(Ausmees et al., 2003). However, the apparent helical occurrence of crescentin in
intermediate-length W. maritime cells (Fig. 5-2l) resembles the coiled crescentin
localization observed in C. crescentus. The occurrence of crescentin as a narrow band in
W. maritime swarm cells may be the result of compression of the helical geometry in
more-compact cells similar to the differences in crescentin geometries observed in
elongated stationary-phase C. crescentus filaments vs. smaller swarm cells, as noted by
(Cabeen and Jacobs-Wagner, 2005). These results suggest that crescentin might be
expressed differently in W. maritime than in C. crescentus, but that the protein shares
basic structural characteristics in both organisms. Despite some underlying structural
similarities, W. maritime is not vibroidal in shape, and the possibility exists that
crescentin may play an entirely different role in this organism than it does in C.
crescentus.
The presence of crescentin in freshwater caulobacters and one marine strain, but
the apparent absence of crescentin in several other closely-related groups of dimorphic
prosthecate bacteria suggests a complex evolutionary history for the gene. One interesting
possibility is that the presence of crescentin in C. crescentus and/or W. maritime is the
result of horizontal gene transfer of creS. Deviations from the whole genome in GC
content, codon usage bias, and di/tri-nucleotide signature analysis might be employed to
determine if evidence exists for such a horizontal transfer of the crescentin gene
(Syvanen, 1994). Other comparisons between the nucleotide composition of the whole
genome and creS, such as silent site substitutions, might determine whether or not the
gene is under selective pressure. If creS is under positive selective pressure, this would
122
imply that crescentin, and the vibroidal morphology it confers, provide a selective
advantage that has yet to be demonstrated under laboratory conditions. In any case, the
sporadic presence of a sophisticated protein architecture that determines the shape of at
least one organism, and whose adaptive role has yet to be identified, demonstrates the
significant limitations in our understanding of the importance of cell shape in bacteria
and archaea.
Paleobiological Implications
From a paleobiological standpoint, the apparent absence of crescentin in
genetically and ecologically similar organisms provokes several questions. If only one
member of the clade possesses the CreS gene and the resulting curved cell phenotype,
then just how good of a phylogenetic indicator is shape? If we were to examine fossils of
the bacteria studies here, would we conclude that the vibroidal Caulobacter was closely
related to these other organisms? If the selective advantage conferred by a shape-
influencing protein is not sufficient for it to be transferred and retained by related
organisms, or even expressed as a vibroid morphology in the case of Woodsholensia,
what chance does this gene have for being conserved over millions of years? Perhaps
even more troubling from a paleobiologic standpoint, is the potential for shape-
influencing genes such as crescentin, to be transferred between distantly-related
organisms. Horizontal gene transfer in bacteria and archaea occurs at such a rate that, in
the absence of strong selective pressures, entire genomes can be rewritten over the course
of a billion years (Lawrence and Ochman, 1997, 1998). When faced with the discoveries
123
of the molecular era, can we really be confident that 2-billion year old septate filaments
really represent a representative of the genus Oscillatoria?
It is clear from the questions raised by these observations that recent discoveries
in microbial cell biology, molecular biology, and genetics have much to contribute to our
understanding of the microfossil record and that experimentation in these areas may cast
a new light on ancient life.
124
Chapter VI. Assessing biomarker syngeneity: An in-situ approach using monoclonal
antibodies to squalane
Abstract
Lipid biomarkers preserved in ancient rocks have the potential to reveal much
about ancient ecosystems. However, establishing that the compounds of interest are
syngenetic has proven to be an analytically challenging task. Traditional biomarker
analyses rely on extraction of large quantities of powdered rock, making the association
of molecules with sedimentary fabrics difficult, if not impossible. In modern biology,
antibodies are used as extremely sensitive tools for molecular labeling. When coupled
with fluorescent labels, antibodies allow for the visual localization of molecules. Here
we show that monoclonal antibodies that bind specifically to geolipid compounds can be
used as a tool for in situ detection and labeling of such compounds in etched mineral-
bound organic macerals. Monoclonal antibodies to squalene, produced for human health
studies, react with the geolipids squalane and lycopane, but not with other common
hydrocarbons found in sedimentary bitumens. An immunofluorescence labeling
methodological test case showed that squalane antibodies bind specifically to isolated
organic-rich lamina in squalane-containing rocks from the Eocene Green River
Formation. These results suggest that squalane is confined to discrete sub-millimetric
organo-sedimentary fabrics within those rocks, providing evidence for its syngeneity, and
suggests that an in situ antibody detection approach may be useful for establishing
syngeneity in older rocks.
125
Introduction
Lipid biomarkers are organic compounds with hydrocarbon skeletons that can be
related to biochemical precursors that are presently thought to be restricted to specific
taxonomic groups (e.g., Brocks and Summons, 2003a; Peters et al., 2005). Many lipid
biomarkers are stable over geologic time, and the identification of these compounds in
rocks and sediments has provided an alternative source of evidence for the appearance of
specific taxa in the rock record (Brocks and Pearson, 2005; Brocks and Summons,
2003a; Moldowan and Talyzina, 1998; Brocks et al., 1999). However, biomarker studies
are commonly complicated by the possibility of contamination. The mobilization of
petroleum can introduce non-indigenous hydrocarbons into sedimentary rocks along the
pathway of migration (Curiale and Bromley, 1996). Anthropogenic petroleum products
can also contaminate samples during collection and storage (Hoering, 1965; 1966). For
these reasons, establishing the syngeneity of geolipids is a critical prerequisite to their use
as palebiological indicators (Brocks et al., 2003; Meinschein, 1965; Nagy, 1970).
Brocks et al. (2003) presented a number of analytical approaches that were
thought to provide evidence of biomarker syngeneity in Archean rocks. Such approaches
include matching molecular indicators of thermal maturity with the age and lithological
maturity of the host rocks, comparing the stable isotope values of sample kerogen and
bitumen, assessing the geological context of the host rocks for potential contamination by
migrating hydrocarbons, and evaluating the spatial distribution of hydrocarbons within
the sample. However, these approaches are not always possible, nor are their results fully
conclusive.
126
One alternative approach would be to test the association of specific compounds
with primary or secondary organo-sedimentary fabrics, as indicators of endemicity or
contamination, respectively. In this paper, we show that the distribution of specific
compounds within rock fabrics can be visualized through a new in situ detection method
using monoclonal antibodies that bind to specific geolipids. As a test case, anti-squalene
antibodies were applied to squalane-containing mudstones from the Eocene Green River
Formation. We also evaluated the specificity of the antibodies by measuring their binding
affinity for a variety of purified hydrocarbons that represent many of the most common
hydrocarbon classes found in sedimentary bitumen extracts.
Antibodies
The immune systems of higher animals use antibodies as part of an adaptive
response to recognize and disable, or destroy, potentially harmful compounds and
pathogens (e.g., Abbas et al., 1991; Birch and Lennox, 1995; Roitt, 1991). Because
antibodies bind to epitopes on their target molecules with remarkable specificity and
sensitivity, they are commonly used as probes for the detection of specific molecules in
modern biology applications ranging from cancer treatment (Berinstein and Levy, 1987;
Biela et al., 2003; Scheinberg, 1991) to microbial ecology (Caron et al., 2003; Faude and
Hofle, 1997; Lin and Carpenter, 1996). Immunodetection approaches have even been
used in paleontological studies. For example, Muyzer and Westbroek (1989) and Muyzer
et al. (1984) raised antibodies against the organic matrix of recent bivalve shells. The
127
antibodies also reacted with biopolymers preserved in Pleistocene bivalve shells
suggesting a chemical similarity between the modern and ancient shell matrices.
In immunodetection approaches, once the primary antibody binds to the target
compound, a secondary antibody, which binds specifically to the primary antibody, is
commonly introduced to deliver one of several extraordinarily sensitive labels that allow
the bound antibody to be detected, either in situ or in an extract (Blanchette-Mackie et al.,
1989; Compère et al., 1995). Biological antigens are typically proteins and
polysaccharides that have epitopes derived from their complex functional groups and
three-dimensional stereochemistry (Li et al., 1998; Liddell and Cryer, 1991). Although
lipids, especially in their saturated hydrocarbon form, are chemically simple molecules
compared to other antigenic biomolecules, antibodies that exhibit specific binding to
lipids are well documented (Fogler et al., 1987; Maneta-Peyret et al., 1992; Swartz et al.,
1989). For example, human blood is known to contain antibodies to cholesterol, which is
thought to act as a mechanism for regulating low-density lipoprotein production (Alving
and Swartz, 1991; Alving et al., 1989; Swartz et al., 1989). Maule et al. (2004) produced
a polyclonal antibody serum that showed some reactivity to hopanes for use as a stand-
alone biomarker detection tool to be used as an alternative to standard GC-MS methods
for NASA life-detection applications. However, the heterogeneous nature of polyclonal
antibodies, which consist of a mixture of multiple antibodies derived from multiple cells,
fundamentally limits confidence in their capability to bind only to the target compound in
complex molecular assemblages of structurally-similar hydrocarbons, such as those found
in sedimentary bitumen and petroleum.
128
More promising for studies of natural organic matter, Matyas et al. (2000, 2002)
produced several monoclonal antibodies (antibodies of a single type derived from clones
of a single cell) to the lipid squalene in a study related to Gulf War syndrome. Some
isolated clones also produced antibodies that react with the saturated geolipid form,
squalane. While squalane is not a true biomarker, as it is found in representatives from
all three domains, it is common in ancient bitumen extracts and thought to be an indicator
of archaea in certain depositional environments (e.g., Brocks and Summons, 2000; Grice
et al., 1998). In this study, the antibodies produced by Matyas et al. (2000) are evaluated
for their molecular specificity by testing their potential to bind other common
sedimentary hydrocarbons and to function as molecular probes in sedimentary organic
matter.
Materials and Methods
Hybridoma Culturing and Antibodies
Hybridoma clone PTA6538-SQE14 was obtained from the American Type
Culture Collection and grown up in hybridoma growth medium (GIBCO; Formula
number 06-5012EL with 3% characterized fetal calf serum). Cells were grown initially
in T75 flasks. When confluent, the cells were further expanded to T250 flasks and then
added to 3L spin flasks. When cells reached a concentration of 1 x 10
6
cell/ml, the flasks
were aerated for an addition 3-5 days of growth. After culture, the cell suspension was
poured into 500 ml conical centrifuge tubes and spun at 2,400 rpm for 15 minutes at 4
o
C.
The supernatant was then filtered through 0.2 micron 1L filter tops into a 5 L sterile glass
129
bottle. IgM Antibody, CD75 Clone LN-1, which reacts with a proteinaceous antigen on
certain lymphocytes, was used as an irrelevant control antibody in all ELISA and
immunofluorescence experiments.
Assessing Antibody Binding Characteristics
The Enzyme-linked Immunosorbent Assay (ELISA) is a commonly used and
sensitive method of detecting and quantifying antibodies or antigens in a sample. Here
we used ELISA as a platform for screening the binding specificity of the squalene
antibodies produced by Matyas et al. (2000). Standard ELISA protocols were modified
by Matyas et al. (2002; 2000) for implementation on PVDF plates. This study used
ELISA protocols similar to those of Matyas et al., but with additional emphasis placed on
limiting hydrocarbon contamination from plastics. The ELISA protocol was as follows:
Hydrocarbon standards dissolved in 100 µl of hexane, were added via a 6x solvent-
washed glass syringe to polyvinyldiene-fluoride (PVDF) membranes on Millipore
Multiscreen IP 96-well plates. Positive controls of anti-mouse label with a murin positive
control (an irrelevant mouse-derived antibody), and controls with no antigen (solvent-
only), no primary antibody, and no secondary antibody were included with each plate
analyzed.
Hexane added to the well with a syringe was evaporated leaving the purified
hydrocarbon standard dried onto the surface of the hydrophobic PVDF membrane. After
further drying overnight at 4
o
C, membrane surfaces were blocked by adding 300 µl of
phosphate buffered saline (PBS) - 2% delipidized bovine serum albumin (BSA). The foil-
130
covered 96-well plate was allowed to incubate for 2 hours at 22
o
C. Block solution was
tapped out, and 100 µl of antibody-containing culture supernatant was added to each
well. The 96-well plate was covered with an aluminum plate-sealing film and was shaken
on an orbital plate shaker at 800 rpm for 1 hour. The supernatant was tapped out, and
each well washed four times using 300 µl of PBS-2% BSA. Following the initial wash
step, peroxidase-linked goat anti-mouse IgM antibodies was diluted 1 to 1000 in PBS-2%
BSA and 100 µl was added to each well. Again, the plate was covered with aluminum
sealing film and placed on an orbital shaker at 800 rpm for 1 hour. After tapping out the
antibody solution, each well was washed four times with 300 µl of PBS-2%BSA. 150 µl
of 2,2'-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid (ABTS) solution (Invitrogen) was
added to each well, the plate was covered with aluminum foil and shaken at 800 rpm for
30 minutes. Finally, 100 µl of ABTS solution was removed from each well and
transferred to a clear flat-bottom 96-well plate. The absorbance of the ABTS solution
was read at λ = 405 nm using a Molecular Devices Thermo Max spectrophotometric
microplate reader. Solvent washed PTFE pipette tips were initially used to eliminate a
possible source of hydrocarbon contamination, but were found to give similar results to
standard pipette tips. All standards were stored and diluted in glass vials with Teflon-
lined caps. All reagents were allowed to come to room temperature before application, as
reagents used immediately upon removal from refrigeration produced inconsistent results.
Sample Collection & Immunofluorescence Protocol
Unweathered hand samples of lacustrine kerogen-rich mudstones from the Green
131
River Formation outcropping in Fossil Basin were collected from a fossil quarry near
Kemmerer, Wyoming (Lat. 41.798; Long. -110.732) and wrapped in pre-combusted
aluminum foil for transport and storage. Rock samples were slabbed with a water-cooled
rock saw. One face was used for immunofluorescence and the other for conventional
GC/MS analysis. Press-on immunoreagent wells (1 cm diameter) were applied to the slab
surfaces to keep control reagents from mixing with experimental reagents (Figure 6-8).
Slab surfaces used for immunofluorescence were etched in 0.2% HCl/Milli-Q before
being rinsed 3x with pure Milli-Q water. Rock surfaces were blocked with PBS/2%BSA
and allowed to incubate in a humid chamber to prevent dehydration. Blocking solution
was aspirated before 60 µl of antibody-containing supernatant diluted 1:10 in
2%BSA/PBS was added to each well and allowed to incubate in a humid chamber for 1
hour at ~25
o
C. Upon aspiration, each well was washed 10x with 260 µl drops
2%BSA/PBS with aspiration between washes. 20 µl of secondary goat anti-mouse FITC
antibody-containing solution diluted in 2%BSA/PBS was then added to each well and
allowed to incubate in the dark for one hour. After 10x washing with 20 µl drops
2%BSA/PBS and aspiration between washes, 20 µl of VectaShield anti-fade mounting
media was added and a coverslip attached. FITC-conjugated antibodies were visualized
using a Zeiss Axioscope equipped with Zeiss Filter Set 09 (BP) 450–490, (BMS) 510,
(LP) 515, Zeiss, Jena, Germany). Image post processing involving uniform image level
adjustments was performed using Adobe Photoshop CS3.
Two types of controls, with two replicate wells for each control, were contained
on each immunofluorescence slide. First, all reagents were applied to un-etched portions
132
of each section. Second, etched portions of the slide received a control treatment identical
to the experimental treatment, but with an irrelevant primary antibody (CD75).
GC-MS
For this study, cuttings from the same hand sample used in the
immunofluorescence detection experiment were powdered and analyzed by GC-MS to
provide independent confirmation of squalane in the samples used for
immunofluorescence. Powdered rock was extracted using dichloromethane. A saturate
fraction was isolated using silica gel chromatography using hexane as a mobile phase. A
branched/cyclic fraction was obtained using silicalite (ZSM-5) chromatography with
isooctane as a mobile phase. GC-MS analysis was performed on a VG Autospec-Q mass
spectrometer coupled to an HP Series II gas chromatograph fitted with an Agilent DB-1
silica capillary column (60 meters long, 0.25mm ID, 0.25um phase). The temperature
program was as follows: 150
o
C for 1 minute, 150
o
to 320
o
C. at 2 deg/ min., hold at
320
o
C for 20 minutes.
Results
Evaluating antibody specificity
The common Enzyme Linked Immunosorbent Assay (ELISA) was used to
compare antibody binding to squalane with binding to other purified hydrocarbons and
mixtures. Figures 6-1 through 6-7 shows ELISA results for antibodies from Clone
SQE14 binding to 1 µg/well of squalane, lycopane, tetracosane, pristine, phytane, and a
133
Figure 6-1. ELISA results from PVDF plates show that SQE14 antibodies react strongly
to squalane.
Figure 6-2. ELISA results from PVDF plates show that SQE14 antibodies do not react
with individual alkanes such as tetracosane.
134
Figure 6-3. ELISA results from PVDF plates show that SQE14 antibodies also do not
react with more complex mixtures of n-alkanes, such as a mixture of C
16
through C
30
.
Figure 6-4. ELISA results from PVDF plates show that SQE14 antibodies do not react
with the isoprenoids pristine and phytane.
135
Figure 6-5 SQE14 antibodies do show reactivity with the tail-to-tail isoprenoid,
lycopane.
Figure 6-6. ELISA results from PVDF plates show that SQE14 antibodies do not react
with the sterane, stigmastane.
136
Figure 6-7. SQE14 antibodies also do not appear to react with sterols, such as
cholesterol.
137
hexane blank. The x-axis shows the reciprocal of the dilution for the antibody-containing
hybridoma supernatant, while the y-axis shows absorbance in units of optical density
obtained on a spectrophotometric plate reader. The results show a strong antibody-
concentration-dependent binding to squalane (Avg. OD = 1.526 ± 0.062 at 1:40
supernatant dilution), which is clearly distinguished from the minimally concentration-
dependent blank (Avg. OD = 0.335 at 1:40 supernatant dilution).
Normal alkanes are the most abundant compounds in natural mature oils and
bitumen (Brocks and Summons, 2003b), and are found in nearly all un-biodegraded
organic matter preserved in the rock record. SQE14 antibodies tested against alkanes,
Table 6-1 – ELISA reactivities of squalane, controls, and various other purified and
mixed hydrocarbons to the SQE14 monoclonal antibody measured as absorbance on
spectrophotometric plate reader at 405nm. Antibody supernatant dilution increases from
left to right.
SQE14-Squalane (Positive Control)
Ab Dil. 1:40 1:80 1:160 1:320 1:640 No Ab
R1 1.44 0.71 0.44 0.26 0.31 0.27
R2
1.54 0.98 0.53 0.30 0.30 0.23
R3 1.62 0.88 0.49 0.33 0.32 0.25
Mean 1.53 0.86 0.48 0.30 0.31 0.25
Std. Dev. 0.09 0.14 0.04 0.04 0.01 0.02
138
SQE14-Hexane Only (Negative Control)
Ab Dil. 1:40 1:80 1:160 1:320 1:640 No Ab
R1 0.24 0.29 0.26 0.23 0.20 0.19
R2 0.36 0.36 0.22 0.23 0.23 0.25
R3 0.40 0.37 0.26 0.18 0.22 0.14
Mean 0.34 0.34 0.24 0.21 0.22 0.19
Std. Dev. 0.08 0.05 0.02 0.03 0.01 0.06
Irrelevant Antibody CB75 with squalane (Negative Control)
Ab Dil. 1:40 1:80 1:160 1:320 1:640 No Ab
R1 0.17 0.18 0.23 0.21 0.22 0.11
R2 0.26 0.26 0.17 0.31 0.21 0.35
R3 0.45 0.27 0.21 0.24 0.24 0.35
Mean 0.29 0.23 0.20 0.25 0.22 0.27
Std. Dev. 0.14 0.05 0.03 0.05 0.02 0.14
Tetracosane
Ab Dil. 1:40 1:80 1:160 1:320 1:640 No Ab
R1 0.34 0.32 0.30 0.25 0.23 0.15
R2 0.41 0.30 0.24 0.21 0.27 0.17
R3 0.34 0.36 0.26 0.25 0.19 0.17
Mean 0.36 0.33 0.27 0.23 0.23 0.16
Std. Dev. 0.04 0.03 0.03 0.02 0.04 0.01
Pristane & Phytane Mixture
Ab Dil. 1:40 1:80 1:160 1:320 1:640 No Ab
R1 0.47 0.39 0.34 0.32 0.28 0.14
R2 0.38 0.39 0.34 0.30 0.25 0.14
R3 0.42 0.45 0.26 0.30 0.18 0.16
Mean 0.42 0.41 0.31 0.30 0.24 0.15
Std. Dev. 0.04 0.03 0.04 0.01 0.05 0.01
Alkane Mixture C
16
-C
30
Ab Dil. 1:40 1:80 1:160 1:320 1:640 No Ab
R1 0.56 0.39 0.33 0.20 0.24 0.12
R2 0.32 0.30 0.50 0.19 0.24 0.33
R3 0.34 0.58 0.27 0.24 0.14 0.21
Mean 0.41 0.43 0.37 0.21 0.20 0.22
Std. Dev. 0.13 0.14 0.12 0.03 0.06 0.11
139
Lycopane
Ab Dil. 1:40 1:80 1:160 1:320 1:640 No Ab
R1 0.91 0.50 0.36 0.20 0.24 0.14
R2 0.91 0.59 0.58 0.19 0.14 0.14
R3 0.98 0.58 0.43 0.24 0.24 0.16
Mean 0.93 0.56 0.45 0.21 0.20 0.15
Std. Dev. 0.04 0.05 0.11 0.03 0.06 0.01
Stigmastane
Ab Dil. 1:40 1:80 1:160 1:320 1:640 No Ab
R1 0.32 0.38 0.29 0.14 0.30 0.37
R2 0.30 0.26 0.29 0.25 0.27 0.20
R3 0.33 0.44 0.23 0.22 0.32 0.40
Mean 0.31 0.36 0.27 0.20 0.30 0.32
Std. Dev. 0.01 0.09 0.04 0.06 0.03 0.11
Cholesterol
Ab Dil. 1:40 1:80 1:160 1:320 1:640 No Ab
R1 0.40 0.24 0.38 0.36 0.25 0.18
R2 0.32 0.27 0.36 0.39 0.29 0.25
R3 0.32 0.27 0.30 0.37 0.23 0.26
Mean 0.35 0.26 0.34 0.37 0.25 0.23
Std. Dev. 0.04 0.02 0.04 0.02 0.03 0.05
such as heptadecane, octadecane, and tetracosane did not exhibit binding above that of
the background blank signal. The same was true for a mixture of C
16
-C
30
n-alkanes.
Anti-squalane antibodies did not react with common isoprenoids, such as pristine and
phytane. Nor did they exhibit reactivity to sterols, or their derivatives. Figure 6-3 shows
representative results for cholesterol and stigmastane. Anti-squalene antibodies did show
some reactivity with the tail-to-tail isoprenoid lycopane (Avg. O.D. = 0.908) (Fig. 6-5).
140
In situ detection in kerogen-rich marlstone using immunofluorescence
Rock sections from the Eocene Green River Formation were examined using
fluorescence microscopy after etching and immunoreagent incubations. The examined
rock sections consisted of kerogen-rich laminated mudstones characterized by alternating
calcite and kerogen laminae. As noted above, the presence of squalane was established by
GC-MS. The mineralogy of the examined rocks consists primarily of calcite, but with
minor amounts of dolomite, quartz, feldspar, and clay, as previously reported by
Buchheim (1994) and Buchheim and Eugster (1998). Kerogen occurs primarily as
continuous, wavy organic-rich laminae, ~10-50 µm thick. Many of the laminae in the
sampled rocks are discontinuous and evidence for soft-sediment deformation is present in
hand sample.
Of the 119 cm
2
examined
,
only three regions showed extensive antibody binding
along sections of individual laminae several hundred microns in length (Fig. 6-9 a-c).
These segments of antibody binding accumulation present as convolute undulations
within the organic lamina connecting small irregularly-shaped signal “patches” ~5 µm in
diameter (Fig. 6-9, Arrows). In total, antibody binding was observed in thirteen separate
regions – although in all but three cases, these occurred as small patches rather than
signal horizons that followed bedding. Antibody binding was not observed in control
wells that contained un-etched surfaces (Fig. 6-10a). Examination of the control wells in
which an irrelevant primary antibody was used also revealed no FITC signal (Fig. 6-10b).
Controls wells covered an area of ~30cm
2
. Silylated glass slides coated with squalane,
used to assess the approximate sensitivity of the immunofluorescence technique showed
141
Figure 6-8. Immunoreagent wells (1-cm diameter) were applied to laminated rock
sections from the Green River Formation.
Figure 6-9. Immunofluorescence imaging of FITC conjugated antibodies revealed
discrete signal accumulation (arrows) suggesting the presence of squalane associated with
individual laminae. Scale bar = 50 µm.
142
Figure 6-10. Negative controls of un-etched sections and etched sections with an
irrelevant primary antibody (CD75) did not exhibit an immunofluorescence signal.
Scale bar = 100 µm.
Figure 6-11. Squalane immobilized on aldehyde silylated glass slides exhibits an obvious
signal over the blank (a) at squalane concentrations of ~1 ng/cm
2
(b) and an even greater
signal at concentrations of ~10 ng/cm
2
.
143
obvious FITC signal at concentrations of 13 ng/cm (Fig. 6-11c) and at ~1ng/cm
2
(Fig. 6-
11b) with minimal background (Fig. 6-11a).
Some minerals within the examined rock sections exhibited substantial
autofluorescence. Autofluorescence manifested itself as a dull greenish-yellow glow in
the carbonate matrix, or as very bright greenish-yellow emanating from isolated mineral
grains (Figure 6-11). Kerogenous laminae and macerals such as microfossils were not
autofluorescent, though most microfossils exhibited a reddish hue characteristic of spores
in the sub-bituminous to high-volative coal maturity and thermal alteration index of 2.0 –
3.0.
GC-MS
GC-MS analysis was performed on sample cuttings removed from the same slabs
used for the immunofluorescence analysis. Figure 6-13, a partial GC-MS chromatogram,
indicates the presence of squalane in extracts from the cuttings at concentrations of ~100
ppb.
Discussion
Evaluating Antibody Binding Specificity
The potential for antibodies to be used as probes for specific biomarker
compounds (or groups of compounds) in ancient rocks is directly related to the binding
affinity of the antibody for the target compound, as compared to other hydrocarbons that
144
Figure 6-12. GC-MS chromatogram of extractable hydrocarbons indicating the presence
of squalane, but at relatively-low concentrations.
Figure 6-13. Mineral autofluorescence was present in almost all imaged sections. The
dull yellow background autofluorescence, and diffuse bright greenish-yellow
autofluorescence associated with certain mineral grains, is distinct from the sharply-
localized green fluorescence of the antibody-conjugated FITC fluorochrome (Fig. 3).
Scale bar = 50 µm.
145
are also common in extracts from ancient organic matter. Matyas et al. (2000) reported
that antibodies from some clones (e.g., PTA6538-SQE16) react only with squalene,
whereas antibodies from other clones (e.g., PTA6538-SQE 14) reacts with both squalene
and squalane on PVDF plates. SQE 14 antibodies were employed in this study. The
results of this study using SQE14 on squalane-coated PVDF wells were similar to those
of Matyas et al. (2000), who reported binding to squalene and squalane (Fig. 6-1). The
experiments reported on here were performed using similar ELISA protocols to Matyas et
al. (2000).
Anti-squalane antibodies showed no reactivity (indistinguishable from the
control) for individual alkanes, such as tetracosane (Fig. 6-2). Reactivity was also
indistinguishable from the negative control for the isoprenoids pristane and phytane (Fig.
6-4). The alkanes and isoprenoids tested are extremely common in the majority of
bitumens and oils (Brocks and Summons, 2003b), and are chemically-representative of
the alkanes and isoprenoids that comprise the bulk of bituminous saturated hydrocarbons.
The SQE14 antibodies showed moderate levels of binding to the isoprenoid
lycopane. (Fig. 6-5) Lycopane is an isoprenoid, as are phytane, pristine, and squalane.
However, pristine and phytane are both regular isoprenoids, whereas squalane and
lycopane are both tail-to-tail isoprenoids. The observation that the anti-squalane
antibodies react with lycopane, but not to other isoprenoids, suggests that their reactivity
may be specific to the tail-to-tail isoprenoids, rather than squalane alone. Lycopane is
structurally identical to squalane, but with one additional isoprenoid unit on either end of
146
the molecule. Testing was not performed on other tail-to-tail isoprenoids, such as
crocetane, because standards were unavailable.
The anti-squalane antibodies showed little or no reactivity with sterols, such as
cholesterol, and their derivatives, such as stigmastane (Fig. 6-6 and 6-7). The lack of
reactivity to sterols further supports the hypothesis that the epitope for the anti-squalane
antibodies is related to the tail-to-tail isoprenoid linkage, as sterols are chemically similar
to their squalene-derived precursors. It may seem surprising that a molecule as seemingly
simple as squalane could present sufficient structural uniqueness so as to provide a
unique antibody binding site. However, squalene is a flexible molecule that undergoes
folding in solution (Pogliani et al., 1994), and perhaps when immobilized on a membrane
or when adsorbed to kerogen and mineral surfaces. Indeed the enzymes responsible for
the biosynthesis of hopanoids and sterols, such as squalene-hopane cyclase, possesses
structural properties that allow for site-specific reactions with squalene. It is possible that
squalane and lycopane fold in a similar manner to squalene, creating a folded binding site
for the anti-squalene antibodies. Alternatively, some aspect of the tail-to-tail linkage itself
may serve as an antibody binding site.
Although squalane is not, strictly speaking, a biomarker because of its widespread
occurrence as a biosynthetic precursor, the capability of developing antibodies that react
with lipids and other hydrocarbons, such as squalane and cholesterol, hints at the
potential for developing antibodies to other compounds that could then be localized in
rocks, sediments, fossils, modern cell membranes etc. Based on the cross-reaction of anti-
squalene antibodies with lycopane, it remains to be seen whether anti-hydrocarbon
147
antibodies will recognize specific molecules, or whether they will only be specific to
groups of structurally-similar compounds.
In Situ Detection
As part of their efforts to evaluate biomarker syngeneity in Archean rocks, Brocks
et al. (2003) noted that the spatial distribution of contaminants in a rock sample should
differ from the distribution of syngenetic compounds. For example, some Hamersley and
Fortescue Group drill cores contain decreasing concentrations of low molecular weight
hydrocarbons from the center to the surface of the rock sample, while the concentration
of high-molecular-weight compounds increases with distance from the center of the core.
Brocks et al. attribute this heterogeneous spatial distribution to either surficial
contamination or migration of the low-molecular-weight compounds during a pressure
release brought about by drilling and core extraction. Certain gross heterogeneities may
indeed indicate contamination, however we suggest that given the proper detection tools,
the spatial distribution of molecules relative to sedimentary fabrics can provide more
detailed information that could be used for the purposes of testing syngeneity and
detecting contamination. For example, by analogy to Steno’s Principle of Inclusion,
molecular fossils included within primary sedimentary fabrics or macerals suggest that
the molecules themselves are syngenetic (Fig. 6-16 a-b), as it is unlikely that
contamination, natural or anthropogenic, would deposit molecules only within isolated
laminae or macerals. Similarly, associations between secondary features such as fractures
or drill core margins would seem to suggest that the molecules of interest were
148
secondarily emplaced (Fig. 6-16 c-d). The homogenous distribution of hydrocarbons
within a uniform rock matrix may present a case in which spatial analysis would be
unhelpful for establishing the origins of included hydrocarbons.
GC-MS analyses typically require large samples sizes (grams to tens of grams) to
produce a detectable signal, which makes the possibility of conducting spatial analysis by
selectively analyzing organic matter from discrete macerals or horizons by conventional
techniques difficult to impossible. As a result, syngeneity cannot generally be
demonstrated by establishing an association between molecules and organo-sedimentary
fabrics in the host rock. Time of flight secondary ion mass spectrometry (TOF-SIMS)
which allows for the analysis of molecules ablated from a sample offers one possible
solution – although TOF-SIMS analyses are commonly plagued by surface effects and
the absence of standardized spectra (Toporski and Steele, 2004). The immunodetection
approach described here offers another, perhaps more promising, possibility. The
detection of squalane within organic-rich laminae in rocks from the Green River
Formation (Fig. 6-9) demonstrates that a monoclonal antibody detection approach may be
quite effective as a means of visualizing the distribution of biomarkers or hydrocarbon
types within a rock sample. The low number or laminae that exhibited binding, as well as
the absence of signal from negative control wells, suggests that the antibodies are indeed
reacting specifically to squalane, or a chemically-similar compound, that is concentrated
within the partially mineral-bound organic matter. Antibody binding to many or all
laminae would most likely result from non-specific binding. The relatively low
concentrations of extractable squalane in these rocks, as detected by GC-MS, is
149
consistent with the immunofluorescence results that suggest squalane is restricted to only
a few discrete macerals. Given the apparent reactivity of these antibodies to lycopane,
they may also have reacted with other tail-to-tail isoprenoids, or similar polymer-bound
structures within the kerogen matrix. This possibility seems reasonable, given the low
concentration of extractable squalane and the small surface area of organic matter
exposed for binding on each etched slab face. The squalane antibodies applied to
squalane dried onto silylated glass slides exhibit an obvious signal over blank background
(Figure 6-10a) levels at concentration of 1ng/cm
3
, but detection limits may well be lower,
as a “bulls-eye” effect was seen in the signal strength observed on the glass slides, likely
resulting from the uneven adsorption of squalane onto the glass surface during drying. It
is also unclear how the levels of antibody sensitivity observed on the glass slides predicts
the sensitivity of the antibodies when used in kerogen and bitumen partially-encased in
the rock matrix.
Squalane source
If the antibodies are indeed binding to squalane or other similar structures derived
from squalene, there are several possible sources for such molecules. Squalene occurs in
organisms from all three domains of life, where it acts as a molecular precursor for the
biosynthesis of steroids, terpenoids, and carotenoids (Kannenberg and Poralla, 1999;
Peters et al., 2005) – however, these precursors are generally transient, and only a handful
of organisms bio-accumulate squalene. One possible source of concentrated squalene-
bearing biomass in the Green River Formation is fish debris. Green River mudstones are
150
well-known for their extremely common preservation of fish fossils (Fig. 6-15),
particularly Knightia sp., a small fish of the family Clupidae – the fish family which
contains sardines and herrings. Squalene can be a substantial constituent of fish oils, such
as herring oil (Gershbein and Singh, 1969), and Knightia tissues may well have contained
concentrated squalene. Knightia mass mortality beds occur within meters of the rocks
sampled in this study.
Squalene is also used as a primary lipid membrane constituent in some archaea,
including methanogens, halophiles and thermoacidophiles (Tornabene, 1978; Tornabene
et al., 1979; Tornabene et al., 1978). Halophilic archaea, such as Halobacterium,
Haloarcula, Halococcus, and Haloferax etc., are found in great abundance in many
hypersaline lakes and brines (Arahal et al., 1996; Oren, 1994), and squalane is common
in sediments and sedimentary rocks deposited in hypersaline settings where it has been
interpreted as a sedimentary hydrocarbon biomarker for halophilic archaea (Grice et al.,
1998; ten Haven et al., 1988). Some of the rock slabs examined via immunofluorescence
contain clusters of organic-walled coccoidal microfossils with cell diameters in the ~1
µm range (Fig. 6-14). The sizes and shapes of these microfossils are compatible with
many bacteria, microbial eukaryotes, and archaea. However, no antibody signal was
detected in association with these microfossils. Although the microfossils themselves did
not exhibit antibody reactivity, it is possible that some kerogen-rich laminae themselves
represent benthic mat material as postulated by Schieber (2007). Discontinuous laminae
were the focus of Schieber’s mat interpretations, and while one lamina in this study
exhibited both reactivity and discontinuity, this sample showed obvious evidence of soft-
151
Figure 6-14. Coccoid organic-walled microfossils from the examined sections had a
reddish-appearance, unlike the organic matter that comprised the organic-rich laminae.
Microfossils did not exhibit antibody binding. Scale bar = 10 µm.
Figure 6-15 The Green River Formation is particularly well known for the preservation
of fossil fish such as Knightia eocaena. Squalane may have been derived from fish
debris.
152
sediment deformation disrupting continuous laminae at the hand sample scale. Therefore,
sedimentologic evidence does not necessarily support a microbial mat origin for these
particular laminae.
Autofluorescence
One factor that may potentially complicate immunofluorescence imaging of rock
samples is the difficulty of distinguishing fluorescent probe signal from autofluorescence.
Carbonate minerals within the marls tested here commonly exhibited dull background
autofluorescence with rare isolated grains showing bright yellow autofluorescence
(Figure 6-12). Organic macerals and kerogenous laminae from the analyzed samples were
not autofluorescent (e.g., Figure 6-10). Fluorescent FITC signal was distinguished from
autofluorescence because of its distinctive greenish color and sharp margins – however,
these color differences were only obvious when viewed through wide-pass filter sets.
Autofluorescence appeared greenish-yellow under the wide-pass filter and exhibited
diffuse margins. The FITC probe fluorophore also photobleached during observation,
whereas the autofluorescence of the mineral grains maintained a constant intensity. In
certain rock samples, the autofluorescent characteristics of minerals or organic matter
may render fluorophore-based detection systems unreliable. However, the availability of
non-fluorescent antibody reported systems, such as immunogold, may offer an alternative
reporter system under such conditions.
153
Figure 6-16. The localization of biomarkers within primary organo-sedimentary rock
fabrics and macerals, such as organic-rich laminae (A) and (micro)fossils (B) is
consistent with biomarker syngeneity. Conversely, contamination is suggested by the
association of biomarkers with secondary features such as fractures (C), or patterns which
are suggestive of diffusion, such as those in illustration D - a cartoon of a core sample
contaminated by drilling fluids.
154
Conclusions
Antibodies are powerful tools for molecular detection. Targeted antigens for
biological applications are typically complex macromolecules, such as proteins.
However, numerous studies document the production of antibodies to lipids and
membrane-rigidifying compounds such as cholesterol. This study demonstrates, for the
first time, that monoclonal antibodies can be used for the in situ detection and
visualization of geolipids within partially mineral-bound organic matter. ELISA tests
show that anti-squalene antibodies bind to squalane and lycopane, but not other common
geolipids. Immunofluorescence analysis of squalane-containing, organic-rich mudstones
from the Eocene Green River Formation demonstrated that squalane antibodies bind to
isolated portions of organic-rich laminae. These findings are significant for organic
geochemical studies in that they allow for the association of molecules with organo-
sedimentary fabrics providing critical evidence of syngeneity. Furthermore,
paleontological or sedimentological features of the host organic accumulation may
provide important clues about the biological source of certain biomarkers. In situ
detection could provide evidence of syngeneity, or even show an association between
organic-walled microfossils and biomarkers that could be used to establish their
phylogenetic affinities.
Because the potential exists for non-specific binding, immunodetection should
be used to augment, rather than replace, conventional GC-MS techniques. Additionally,
secondary reporting systems that can be easily distinguished from autofluorescence may
me an important consideration for future use in sedimentary rocks. Future development
155
of this technique could include the production of antibodies against a host of biomarkers
that could then be used to detect these compounds in sediments, rocks, fossils and
modern cells.
156
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Appendix A: Comment and Reply on Bailey et al. 2007
Following the publication of our manuscript, Xiao et al. published the following Brief
Communication Arising online in Nature:
Undressing and redressing Ediacaran embryos
Arising from: J. V. Bailey, S. B. Joye, K. M. Kalanetra, B. E. Flood & F. A. Corsetti
Nature 445, 198–201 (2006). doi:10.1038/nature05457.
Abstract
Bailey et al.1 propose that the Ediacaran microfossils Megasphaera and
Parapandorina, previously interpreted as animal resting eggs and blastula embryos2,
represent Thiomargarita-like sulfide oxidizing bacteria. They further contend that
the bacterial interpretation better explains their abundance and taphonomy.
However, the authors ignored important observations that significantly weaken the
bacterial interpretation.
Morphological Features. To avoid merging morphotaxa before their ontogeny and
taphonomy are completely understood, the initial report3 described eggs and embryos
from the Ediacaran Doushantuo Formation in several taxa—Megasphaera inornata (one
cell enclosed in smooth envelope), M. ornata (one cell in ornamented envelope),
Parapandorina raphospissa (multiple polyhedral cells in smooth envelope), and
Megaclonophycus onustus (large number of spheroidal cells in smooth envelope).
Subsequent studies4–6 have shown that M. ornata, Parapandorina, and a spiral
microfossil (interpreted as post-blastula embryo) are all surrounded by one or more
envelopes. The outermost envelope is typically ornamented with tubercular, polygonal,
cerebral, and fractal sculptures (Fig. 1A–I), and may also bear cylindrical processes7. The
187
sculptured envelope is distinct from botryoidal coating on cell surface or membrane of
Doushantuo microfossils (fig. 85E–F of ref.5), and finds no comparison in the simple
sheath of Thiomargarita or other bacteria. Instead, it is similar to modern animal egg
cases3 and implies a diapause stage—a physiological feature unknown in Thiomargarita.
While implicitly acknowledging that M. ornata and ornamented Parapandorina could
be animal eggs and embryos, Bailey et al.1 interpret M. inornata and smooth-walled
Parapandorina as Thiomargarita-like sulfur bacteria that underwent reductive cell
division when stressed. However, their similar size, cell configuration, developmental
sequence, and occurrence suggest that the smooth-walled and ornamented populations
have close phylogenetic relationships. Alternatively and more likely, the partial
preservation of ornamented envelope (Fig. 1A–G) at various cell division stages indicates
that the smooth-walled microfossils illustrated in Bailey et al.1 are poorly preserved
specimens, with their ornamented outer envelope taphonomically removed7. Hence,
Bailey et al.’s description of Megasphaera and Parapandorina is incomplete, their
comparison to Thiomargarita inconclusive, and their interpretation questionable.
A bacterial interpretation is also inconsistent with the organelle-like subcellular
structures8. These structures have consistent size, shape, occurrence, and location,
indicating biological rather than taphonomic origins8. They are different from irregularly
shaped cytoplasmic degradation structures in Thiomargarita1. Furthermore, its spatial
distribution of vacuoles and cell numbers (typically <8) also suggest that Thiomargarita
is a poor interpretive guide for Doushantuo microfossils9.
Taphonomic Features. Bailey et al.1 argue that a bacterial interpretation can explain
how Doushantuo microfossils were phosphatized, because Thiomargarita concentrates
phosphate in pore water. If Thiomargarita provides a complete answer to the mystery of
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phosphatization, why isn’t there phosphotized Thiomargarita in modern sediments?
Certainly, phosphate enrichment by some sort of bacteria may be important10, but it does
not necessarily mean that what was phosphatized are exclusively or preferentially
bacteria.
Bailey et al.1 further argue that a bacterial interpretation explains the preservation
of two-, four-, or eight-cell stages of Parapandorina, because “stages of reductive
division in Thiomargarita are separated by months to years, allowing a longer window
for the observed preferential preservation of two-cell, four-cell and eight-cell clusters”.
The preservability of a Parapandorina individual is determined by the competing
degradation and phosphatization processes, not by cell division rate, because
phosphatization occurs after death, not between cell divisions. The relative abundance of
different cell-division stages in the preserved population reflects the age structure of the
death assemblage, which may include individuals of any cell-division stages, regardless
whether they are separated by years or minutes.
Finally, Bailey et al.1 question earlier interpretation that Doushantuo microfossils
were concentrated by reworking and winnowing10,11, and argue that a bacterial
interpretation explains their abundance, citing the great abundance of Thiomargarita cells
in modern sediments. Megasphaera and Parapandorina occur abundantly in the grey
facies of Doushantuo phosphorites11. Abrasion, rounding, and inclusion in intraclasts
(Fig. 1J–K; fig. 3.7 of ref.12) clearly suggests intrabasinal reworking and winnowing,
which concentrated Doushantuo microfossils in the grey facies. Megasphaera and
Parapandorina also occur in the less reworked black facies of Doushantuo
phosphorites11, but in much less abundance. In fact, they are so few in the more pristine
black facies that a comparison with the abundant occurrence of Thiomargarita seems
inappropriate.
To summarize, the naked Doushantuo microfossils chosen to be illustrated in
189
Bailey et al.1 were likely undressed by taphonomic processes. When redressed and
considered with other Doushantuo fossils, their similarity to Thiomargarita is restricted
to reductive cell division—a convergent physiological response to limited external
nutrient supply. A bacterial interpretation does not adequately address the taphonomy and
abundance of Doushantuo microfossils.
Shuhai Xiao*, Chuanming Zhou†, Xunlai Yuan†
*Department of Geosciences, Virginia Polytechnic Institute and State University,
Blacksburg, Virginia 24060, USA e-mail: xiao@vt.edu
†State Key Laboratory of Paleobiology and Stratigraphy, Nanjing Institute of Geology
and Paleontology, Chinese Academy of Sciences, Nanjing 210008, China
1. Bailey, J. V., Joye, S. B., Kalanetra, K. M., Flood, B. E. & Corsetti, F. A.
Evidence of giant sulphur bacteria in Neoproterozoic phosphorites. Nature 445,
198–201 (2007).
2. Xiao, S., Zhang, Y. & Knoll, A. H. Three-dimensional preservation of algae and
animal embryos in a Neoproterozoic phosphorite. Nature 391, 553–558 (1998).
3. Xiao, S. & Knoll, A. H. Phosphatized animal embryos from the Neoproterozoic
Doushantuo Formation at Weng'an, Guizhou, South China. Journal of
Paleontology 74, 767–788 (2000).
4. Yuan, X. et al. Doushantuo Fossils: Life on the Eve of Animal Radiation (China
University of Science and Technology Press, Hefei, China, 2002).
5. Chen, J. The Dawn of Animal World (Jiangsu Science and Technology Press,
Nanjing, 2005).
6. Xiao, S., Hagadorn, J. W., Zhou, C. & Yuan, X. Rare helical spheroidal fossils
190
from the Doushantuo Lagerstätte: Ediacaran animal embryos come of age?
Geology 35, 115–118 (2007).
7. Yin, C., Bengtson, S. & Yue, Z. Silicified and phosphatized Tianzhushanian,
spheroidal microfossils of possible animal origin from the Neoproterozoic of
South China. Acta Palaeontologica Polonica 49, 1–12 (2004).
8. Hagadorn, J. W. et al. Cellular and subcellular structure of Neoproterozoic
embryos. Science 314, 291–294 (2006).
9. Donoghue, P. C. J. Embryonic identity crisis. Nature, 155–156 (2007).
10. Xiao, S. & Knoll, A. H. Fossil preservation in the Neoproterozoic Doushantuo
phosphorite Lagerstätte, South China. Lethaia 32, 219–240 (1999).
11. Dornbos, S. Q. et al. Environmental controls on the taphonomy of phosphatized
animals and animal embryos from the Neoproterozoic Doushantuo Formation,
southwest China. Palaios 21, 3–14 (2006).
12. Zhang, Y., Yin, L., Xiao, S. & Knoll, A. H. Permineralized fossils from the
terminal Proterozoic Doushantuo Formation, South China. The Paleontological
Society, Memoir 50, 1–52 (1998).
191
Figure 1. Doushantuo microfossils. a–i, SEM images showing partial preservation of
ornamented envelope in Megasphaera ornata (a), Parapandorina raphospissa
(b–g), and a spiral microfossil (h–i)6. Apparent uneven thickness of envelope (a)
is due to eccentric location of cell and unequal cement fills in space between cell
and envelope. j–k, Planar- (j) and cross-polarized (k) light microphotographs of
microfossils (circled) within two phosphatic intraclasts in grey facies. Micrite is
absent and intraclasts are cemented by dolospars. Scale bar in (a) applies to (a–c),
in (d) applies to (d–i), in (j) applies to (j–k).
192
Appendix B
The following reply to “Undressing and redressing Ediacaran embryos" was also
published online in Nature.
Xiao et al.
1
suggest that the presence of a textured capsule surrounding some
Doushantuo globular microfossils calls into question the alternative interpretation of
these structures as giant sulfur bacteria similar to modern Thiomargarita
2
. However, the
outer coatings figured by Xiao et al.
1
are morphologically similar to known bacterial
features, and the texture, location, and thickness change of the capsule is inconsistent
with a fertilization envelope. Thus, we are not convinced that the bacterial hypothesis
has been falsified.
Xiao et al.
1
provide new images of the Doushantuo globular structures that reveal
a thick, ornamented outer capsule on some specimens, and suggest that the patterns on
the capsule are too complex to be microbial in origin. Our original discussion centered on
Doushantuo specimens with smooth envelopes (Megasphaera inornata and
Parapandorina), as smooth and textured examples had been assigned different Linnean
names
3
with the implication being that they were different organisms. On the one hand,
we note that the embryo and bacterial hypotheses are not mutually exclusive: both could
have existed together in the same deposit, just as eukaryotic debris can be found with
Thiomargarita today. This possibility might explain the abundance of specimens that
possess smooth (or entirely lack) envelopes, the absence of Tianzhushania-like peripheral
processes in ubiquitous phosphatized specimens (but see discussion in
4
), and the multiple
193
geometric configurations observed in cell-clusters at the same stage of reductive division,
which are not easily explained by a single metazoan species. On the other hand, we
welcome the opportunity to discuss the textured examples in light of the bacterial
hypothesis, given the tacit assumption by Xiao et al. that all Doushantuo globular
structures may have at one time had such elaborate envelopes.
Gulf of Mexico Thiomargarita are surrounded by a multilayered ultrastructure
and display a mucous filled sheath (Fig. 2 in
2
), similar in thickness to the capsule noted
by Xiao et al., and if fossilized, would make an excellent textural match to the
Doushantuo envelopes. As for the complex pattern figured by Xiao et al., similar
complex patterns are well-documented from the microbial world
5-8
. Inclusion-bearing
sulfide-oxidizing bacteria in the same family as Thiomargarita (Fig. 1A) have strikingly
similar outer textures
8
to those figured by Xiao et al., demonstrating that such features are
not exclusive to the Eukarya. Thus, the presence of an ornamented capsule is entirely
consistent with our original hypothesis.
Note that the capsule surrounding Megasphaera ornata shown by Xiao et al.
1
exhibits an extreme variation in thickness across the structure (Figure 1B). A sculptured
capsule is also observed to enclose two internal bodies on their distal sides and to act as a
medial boundary between the same two bodies (Figure 1C). Such occurrences are
incompatible with the interpretation of the textured capsule as a fertilization envelope.
Additionally, the spiral grooves and pits on capsules shown by Xiao
1
exhibit a
remarkably-similar geometry to pyrite trails (e.g., Figure 124C in
10
) considered by Xiao
194
and Knoll
9
to be abiotic. Thus these features fail to provide a valid falsification of the
bacterial hypothesis.
Xiao et al. are also compelled by the presence of paired reniform structures noted
via computed tomography techniques
11
within a few Parapandorina specimens
11
, and
suggest that their presence falsifies the bacterial hypothesis. We are not so compelled.
Only 10 (out of 162, screened from thousands) contain large internal structures, and only
a few of those would be characterized as “regular” in appearance; we consider the
attention given to these structures overly selective. We offer that the reniform sulfur or
polyphosphate inclusion clusters observed in degrading Thiomargarita (Supplementary
Figure 6 in
2
) better explain the occurrence of compositionally-distinct regions that occur
in their specimens.
New discoveries consistently highlight the complexity of the microbial world.
Features once considered diagnostic for one group (e.g., size, cellular division patterns,
complex ultrastructural patterns) are now found in groups once considered “too simple”
to possess such features. Perhaps a more convincing test of the bacterial and animal
interpretations will arise with future research. For now, we are content to say the emperor
has no clothes with respect to undressing and redressing the Doushantuo globular
structures.
- Jake V. Bailey, Samantha B. Joye, Karen M. Kalanetra, Beverly E. Flood, & Frank A.
Corsetti
195
Figure 1. An exclusively metazoan interpretation of bodies enclosed in textured
envelopes is called into question by a, complex patterns on bacterial cells, such as
polygonal patterns on inclusion-bearing Achromatium, a close relative of Thiomargarita.
(after ref.
8
) and diagenetic features in Doushantuo microfossils such as b, Megasphaera
ornata specimen showing internal diagenetic processes and thickening of envelope (after
ref.
3
) and c, Parapandorina specimen showing ornamented envelope that passes between
internal bodies (after ref.
10
). Scale bars, 25 µm (a), 200 µm (c).
196
References:
1 Xiao, S., Zhou, C., and Yuan, X. Undressing and redressing Neoproterozoic
embryos. Nature (2007).
2 Bailey, J. V. et al. Evidence of giant sulphur bacteria in Neoproterozoic
phosphorites. Nature 445, 198-201 (2007).
3 Xiao, S. and Knoll, A. H. Phosphatized animal embryos from the Neoproterozoic
Doushantuo Formation at Weng'an, Guizhou, South China. Journal of
Paleontology 74, 767-788 (2000).
4 Yin, C., Bengston, S., and Yue, Z. Silicified and phosphatized Tianzhushania,
spheroidal microfossils of possible animal origin from the Neoproterozoic of
South China. Acta Palaeontologica Polonica 49, 1-12 (2004).
5 Strohl, W. R., Howard, K. S., and Larkin, J. M. Ultrastructure of Beggiatoa alba
Strain B15LD. Journal of General Microbiology 128, 73-84 (1982).
6 Thornley, M. J., Glauert, A. M., and Sleytr, U. B. Isolation of outer membranes
with an ordered array of surface subunits from Acinetobacter. Journal of
Bacteriology 114, 1294-1308 (1973).
7 Thar, R. and Kühl, M. Conspicuous veils formed by vibroid bacteria on sulfidic
marine sediment. Applied and Environmental Microbiology 68, 6310-6320
(2002).
8 Babenzien, H. D. and Sass, H., in Ergebnisse der Limnologie: Aquatic Microbial
Ecology, edited by M. Simon, H. Güde, and T. Weisse (E. Schweizerbart,
Stuttgart, 1996), Vol. 48, pp. 247-251.
197
9 Xiao, S. and Knoll, A. H. Fossil preservation in the Neoproterozoic Doushantuo
phosphorite Lagerstätte, South China. Lethaia 32, 219-240 (1999).
10 Chen, J.-Y. The Dawn of Animal World. (Publishing House of Jiangsu Science
and Technology, Nanjing, 2004).
11 Hagadorn, J. W. et al. Cellular and subcellular structure of Neoproterozoic animal
embryos. Science 314, 291-294 (2006).

Abstract (if available)

Abstract Morphologic data has always played a foundational role in our understanding of ancient life. Visual comparisons often show remarkable similarities between fossils and extant organisms, and yet the modern molecular and geochemical era has taught us that appearances can deceive, and that the genetic and metabolic diversity of the microbial world is greater than anything that could have been previously imagined. What can these revelations in our understanding of the modern microbial biosphere tell us about ancient life, particularly where morphology is conservative?

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Asset Metadata

Creator Bailey, Jake Vincent (author)

Core Title New perspectives on ancient microbes and microbialites: from isotopes to immunology

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Geological Sciences

Publication Date 07/11/2010

Defense Date 06/06/2008

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Caulobacter,Doushantuo,microfossils,OAI-PMH Harvest,squalane,stromatolites,Thiomargarita

Language English

Advisor Corsetti, Frank A. (committee chair), Berelson, William M. (committee member), Bottjer, David J. (committee member), Caron, David (committee member)

Creator Email jvbailey@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1339

Unique identifier UC1284579

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Dmrecord 86051

Document Type Dissertation

Rights Bailey, Jake Vincent

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

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