RECOGNIZING AND INTERPRETING THE FOSSILS OF

EARLY EUKARYOTES

EMMANUELLE J. JAVAUX1∗ , ANDREW H. KNOLL1 and MALCOLM WALTER2

1 Organismic and Evolutionary Biology Department, Botanical Museum, Harvard University,
Cambridge, MA, U.S.A.; 2 Australian Centre for Astrobiology, NSW 2109, Australia
(∗ author for correspondence)

(Received 27 July 2002; accepted in revised form 10 December 2002)

Abstract. Using molecular sequence data, biologists can generate hypotheses of protistan phylogeny
and divergence times. Fossils, however, provide our only direct constraints on the timing and envir-
onmental context of early eukaryotic diversification. For this reason, recognition of eukaryotic fossils
in Proterozoic rocks is key to the integration of geological and comparative biological perspectives
on protistan evolution. Microfossils preserved in shales of the ca. 1500 Ma Roper Group, northern
Australia, display characters that ally them to the Eucarya, but, at present, attribution to any particular
protistan clade is uncertain. Continuing research on wall ultrastructure and microchemistry promises
new insights into the nature and systematic relationships of early eukaryotic fossils.

Keywords: chemistry, early eukaryotes, evolution, molecular phylogeny, morphology, Proterozoic,

ultrastructure

1. Introduction

Fossils formed early in the evolution of a major group commonly defy paleobi-
ological interpretation. Ediacaran impressions, for example, are widely accepted
as the remains of early animals, but what kinds of animals remains contentious
and alternative interpretations range from seaweeds (Xiao et al., 2002) to giant
coenocytic protists, or even bacterial colonies (see Runnegar, 1995, for review).
Fossils interpreted with confidence as stem group members of extant phyla appear
more than thirty million years after the first Ediacarans, and unambiguous crown
group members of most phyla appear later yet.
Early eukaryotic fossils present similar interpretational challenges. Phanerozoic
(<543 Ma) rocks commonly brim with the fossils of eukaryotic organisms, both
macroscopic and microscopic, and most can be related to major clades present in
the modern biota. In contrast, Neoproterozoic (1000–543 Ma) fossil assemblages
mix fossils of known systematic origin with remains that are unambiguously euka-
ryotic, but impossible to relate to specific clades within the domain. And receding
backward still further, late Paleo- and Mesoproterozoic (ca. 1800 to 1000 Ma)
assemblages include fossils that are problematic even at the level of domain.

Paper presented at the Astrobiology Science Conference, NASA Ames Research Center, 7–11
April 2002.

Origins of Life and Evolution of the Biosphere 33: 75–94, 2003.

© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
76 E. J. JAVAUX ET AL.

Since the birth of modern research on Precambrian life, paleontologists have

endeavored to recognize and interpret fossils of early eukaryotes on the basis of
preserved morphology. Early on, size was suggested as a criterion (Schopf, 1977);
after all, many eukaryotic cells are larger than 10–20 µm in diameter, while most
bacteria are no more than 1–2 µm in maximum dimension. Unfortunately, however,
size fails us at the size classes most commonly found in Precambrian rock.
Eukaryotes can form 1–10 µm spheres, but so can cyanobacteria as well as
members of other bacterial and archaeal clades. Larger vesicles (up to a few hun-
dred µm) can be the preserved walls of algae or heterotrophic protists, but, in
principle, they can also be large sulfur bacteria (Schulz et al., 1996) or the preferen-
tially preserved envelopes of colonial cyanobacteria (Waterbury and Stanier, 1978).
Among the largest fossils known from mid-Proterozoic cherts are cylindrical forms
more than 100 µm long interpreted as akinetes of nostocalean blue greens (Golubic
et al., 1995).
Another early hope was that small blebs of organic matter found within mi-
crofossils might be the preserved remnants of nuclei or other organelles (Schopf,
1968). Again, however, confidence in any such interpretation was dashed by the
recognition that nuclei have an extremely low preservation potential, and both field
observations (Golubic and Barghoorn, 1977) and laboratory experiments (Knoll
and Barghoorn, 1975) showed that organic remnants within fossils are mostly col-
lapsed and partially decayed cytoplasm. Perhaps the strongest case for organellar
preservation in Proterozoic microfossils was advanced by Oehler (1977) on the
basis of TEM images of small unicells from the Neoproterozoic Bitter Springs
Formation. Within fossilized walls or envelopes, Oehler found tiny electron-dense
structures that she interpreted as preserved pyrenoids. Pyrenoids are proteinaceous
microstructures commonly associated with storage products in algal chloroplasts
– green algal cells sometimes accumulate starch bodies about pyrenoid structures.
Proteins have a low probability of preservation, but starch bodies are more likely
to survive in fossils; thus, one possibility is that the structures imaged by Oehler
are preserved starch grains within fossilized green algae (Knoll, 1981). At present,
however, we lack taphonomic studies of modern protists that might illuminate the
relative probabilities or distinguishing features of pyrenoid, starch, or collapsed
cytoplasm preservation.
To date, the most compelling interpretations of Proterozoic cells as eukaryotic
have been drawn on the basis of morphology – by the identification of structural
features known to be fashioned by one or more eukaryotic groups but unknown
among Bacteria or Archaea. Thus, vase-shaped microfossils 100 µm long, with
shapes and wall composition known today only within specific genera of lobose
and filose amoebae, provide unambiguous records not only of eukaryotic microor-
ganisms, but more specifically of testate amoeban clades (Porter and Knoll, 2000;
Porter et al., in press). Large (commonly >200 microns) spinose fossils found
globally in terminal Proterozoic rocks are likewise considered to be unambiguously
eukaryotic based on their pattern of ornamentation and microchemistry (e.g., Zang
 RECOGNIZING AND INTERPRETING THE FOSSILS OF EARLY EUKARYOTES 77

and Walter, 1992; Zhang et al., 1998; Arouri et al., 1999, 2000, see discussion
below), although more specific systematic attribution is currently impossible.
In this article, we explore the biological relationships of spheroidal acritarchs
from ca. 1500 Ma shales of the Roper Group, northern Australia. Roper fossils
epitomize the challenges inherent in the recognition and interpretation of early eu-
karyotes. They also illustrate how new approaches may improve our understanding
of mid-Proterozoic biology, among other things enhancing our ability to calibrate
molecular phylogenies and relate them to Earth’s physical history.

2. Eukaryotic fossils in the early Mesoproterozoic Roper Group, Australia

Biologists easily differentiate between prokaryotic and eukaryotic organisms using

myriad features of molecular and cell biology, but these characters rarely survive
fossilization and so are not generally available to the paleontologist. Phylogenetic-
ally informative lipid molecules may be preserved in carbonaceous sediments, and
these provide a geochemical record of Proterozoic eukaryotes (e.g., Summons and
Walter, 1990). Morphologically preserved fossils can be identified as eukaryotic
based on a number of features thought to be diagnostic of the domain (Javaux et al.,
2002a). These include: (1) wall structure and surface ornamentation, (2) processes
that extend from vesicle walls, (3) excystment structures (openings through which
cysts liberate their cellular contents), (4) wall ultrastructure and (5) wall chemistry.
Other early eukaryotes can be recognized on the basis of a regular and well-defined
macroscopic morphology (e.g., Walter et al., 1976; Walter and Du Rulin, 1990;
Grey and Williams, 1990).

2.1. G EOLOGICAL SETTING

The Roper Group comprises a thick ramp-like succession of siliciclastic rocks de-
posited in a rapidly subsiding intracratonic basin, located in the Northern Territory
of Australia, West of the Gulf of Carpentaria (Abbott and Sweet, 2000). The Roper
Group is well dated at its base by U-Pb SHRIMP analyses of zircons from an ash
bed within the Mainoru Formation that fix an age of 1492±3 Ma (Jackson and
Raiswell, 1991). A Rb-Sr age of 1429±31 Ma, obtained from illite in dolomitic
siltstones near the top of the succession is consistent with the zircon age, albeit
less reliable (Kralik, 1982).
Our study of 5 drill cores revealed abundant and exceptionally well preserved
microfossils distributed among four biofacies stacked repeatedly throughout the
group (Javaux et al., 2001). There is a clear onshore-offshore pattern of decreas-
ing abundance, declining diversity, and changing dominance among Roper micro-
fossils. Highly carbonaceous shales in basinal deposits of the Velkerri Formation
contain low abundances of steranes sourced from eukaryotic organisms (Summons
et al., 1988a). Recent geochemical research by Shen and Knoll (2002) shows
78 E. J. JAVAUX ET AL.

that a strong redoxcline existed within the basin, likely within the photic zone.
Shen and Knoll (2002) also identified a strong correlation between facies and the
isotopic abundances of sulfur in early diagenetic pyrites – likely only if sulfate
levels and, by implication, oxygen concentrations were much lower than in present
day oceans (Canfield, 1998; Kah et al., 2002; Shen et al., 2002). Thus, paleon-
tology and geochemistry concur in their recognition of a strong onshore-offshore
pattern in the Roper seaway. Similar microfossil assemblages occur in the slightly
younger Ruyang Group of China (Yin, 1998; Xiao et al., 1997); the ca. 1.3 Ga Totta
Formation, Siberia (Sergeev, 2002, pers. comm.), and the poorly dated but broadly
correlative Sanda Formation, Ganga Basin, India (Prasad and Asher, 2001).

2.2. M ORPHOLOGICAL EVIDENCE OF ROPER EUKARYOTES

2.2.1. Surface Ornamentation and Wall Structure

Resting cells and reproductive cysts of many protists display micron-scale patterns
of lineations, fields, spines or bosses not known among prokaryotic organisms.
This being the case, Neoproterozoic and Paleozoic acritarchs with similar orna-
mentation are ascribed to eukaryotes with confidence. Roper microfossils do not
display the strong ornamentation seen in some younger populations. Nonetheless,
the ornamentation and wall structure of some Roper taxa identify them as protists.
For example, Valeria lophostriata is a spherical acritarch easily distinguished by its
distinctive ornament of concentric striations (Figure 1: 1). SEM observation shows
that these striations consist of parallel ridges spaced 1 µm apart that traverse the
inner surface of the vesicle (Figure 1: 2). Dictyosphaera, as well, displays surface
ornamentation, in this case small interlocking polygons (Figure 1: 3).
Many Bacteria and almost all Archaea show a surface layer ornamentation of
oblique, square or hexagonal crystalline arrays of glycoprotein subunits (called the
S-layer; Sara and Sleytr, 2000). S-layers function as a molecular sieve, providing
a protection from bacterial parasites and viruses, a template for mineralization,
and adhesion sites for exoenzymes. The subunits are synthesized intracellularly,
then translocated through the cell wall and incorporated into the existing S-layer
lattice. S-layers subunits are easily isolated by chemical agents, and they retain the
ability to recrystallize into regular arrays in suspension or on surfaces (Sara and
Sleytr, 2000). This type of proteinic surface ornamentation occurs at a nanoscale
and would probably not be preserved since it is easily removed by chemicals and
easily lost in cultures.
Some prokaryotic cells are ornamented with concentric rings of juxtaposed fil-
aments, or fibrils, pili and fimbriae, but again these features occur at the nanoscale
(Boone and Castenholz, 2001), not the microscale seen in Roper acritarchs. Pili or
fimbriae are hair-like appendages that extend out from the bacterial cell surface.
Their composition and assembly are well studied because of their role in the col-
onization of host cells during bacterial infections (Soto and Hultgren, 1999). Pili
consist of a large variety of adhesive proteinic structures assembled through at least
 RECOGNIZING AND INTERPRETING THE FOSSILS OF EARLY EUKARYOTES 79

Figure 1. Eukaryotic microfossils from the Roper Group, Australia. 1–2: Valeria lophostriata,
partially enrolled half vesicle, likely resulting from a medial split, 2: SEM showing striated or-
namentation consisting of ridges spaced at 1 µm intervals on the internal surface of the vesicle;
3: Dictyosphaera, with polygonal ornamentation; 4–9: Tappania plana, 4: specimen with hetero-
morphic processes (including a branched process-see arrow) distributed asymmetrically about the
vesicle and budding (double arrow), 5: detail of branched process in 4, 6: SEM of branched process,
7: specimen with heteromorphic processes, 8: specimen with excystment structure (arrow), 9: SEM
showing structural continuity between vesicle wall and the base of a process; 10–11: Satka favosa,
10: specimen showing wall made of polygonal plates, 11: SEM showing detail of juxtaposed poly-
gonal plates. The scale bar in 4 is 30 µm for 1, 2 µm for 2, 15 µm for 3, 35 µm for 4 and 7, 10 µm
for 5, 5 µm for 6 and 11, 20 µm for 8, 3 µm for 9, and 40 µm for 10.
80 E. J. JAVAUX ET AL.

four distinct molecular pathways. These pathways are complex and range from
secretion and precipitation on the cell surface where a specialized molecule serves
for nucleation, to intracellular secretion and assembly followed by translocation
of the assembled pili across the outer membrane to the surface, or two different
chaperone mediated pathways where chaperone proteins form complexes with pilin
components, forming fibers that are then transported across the membrane (Soto
and Hultgren, 1999). Pili can reach sizes up to 4,000 nm long and width of 6–
7 nm, and some types may branch. Thus, even if these nanoscale proteinic bacterial
appendages could be preserved, they could not be confused with ornamentations of
Roper acritarchs. Neither would myxobacterial cells that can produce an extracel-
lular matrix of polysaccharide and proteinaceous fibrils with diameters of 10 nm
and lengths of 60 to 100 nm (Kim et al., 1999). Myxobacteria live mostly in soils,
dung, decaying plant material and bark, and their spores resistant to desiccation
can sometimes be found in seashore sediments but few myxobacterium are known
to be able to live in seawater (Reichenbach and Dworkin, 2001; Iizuka et al., 2002).
Most myxobacteria enclose their myxospores in unornamented sporangioles up to
30 to 50 µm in diameter, with a wall up to 1 µm thick. Sporangioles may occur
singly or in groups and may rest directly on or in the substratum or on simple
or branched stalks. Nothing is known about the chemical composition of stalks,
sporangioles and pigments composing myxobacterial fruiting bodies (Reichenbach
and Dworkin, 2001), so their preservation potential is unknown although the spores
are resistant to desiccation. Although vegetative cells of Actinobacteria can be rel-
atively large (a few microns) and form complex branching colonies, their spores are
0.5 to 2 or 3 µm in diameter and form chains (Holt, 1984; Boone and Castenholz,
2001; Miyadoh, 1997). Some of these actinomycete spores are ornamented by rod-
lets, spines, warts, cristae, or hair-like tufts, but again, the scale differs from Roper
and other Proterozoic fossils. In several species of Streptomyces, this ornamenta-
tion is formed by a fibrous protein sheath covering the peptidoglycan cortex of the
spores (Miyadoh et al., 1997; Claessen et al., 2002). It is not known if this protein
layer could be preserved in the fossil record, although the sheath can be dissociated
into submicroscopic rods or tubules upon sonic oscillation (Dworkin, 1985). The
soil actinobacteria Actinoplanes, can form sporangia of variable morphology and
size (up to 5–15 µm) that may be covered by short hairs, but again at a nanoscale
(Matsumoto et al., 2000).
Cyanobacterial cell walls are commonly covered by S-layers or by carbohydrate
structures forming slime or sheaths. These sheaths are preserved in the fossil re-
cord, in preference to the peptidoglycan-rich cell walls, as shown by taphonomic
experiments (Bartley, 1996). They are composed of polysaccharides such as cellu-
lose-like homoglucan fibrils cross-linked by minor monosaccharides (Phormidium,
Nostoc), pectin-like exopolysaccharides (Microcystis), or two different complex
polysaccharides (Anabaena), and pigments like scytonemin (Hoiczyk and Hansel,
2000). These extracellular fibrillar carbohydrates provide a protective coat for the
cells against UV radiation and desiccation as they maintain the cells in a highly
 RECOGNIZING AND INTERPRETING THE FOSSILS OF EARLY EUKARYOTES 81

hydrated gel-like matrix. However, these tube-like or vesicle-like sheaths are not
ornamented.
Thus it seems that while prokaryotic organisms can synthesize both cell wall
ornament and preservable structures, wall ornamentation rarely occurs on the size
scale observed in Proterozoic fossils, and ornamentation seldom is found on pre-
servable structures.
For these reasons, we interpret Roper Valeria and Dictyosphaera as the pre-
served walls of mid-Proterozoic eukaryotes. More specific systematic interpreta-
tion will require TEM and microchemical analysis (see below).
Complex wall structure can also be a sign of eukaryotic affinity. Satka favosa
wall is made of interlocking polygonal plates 10–15 µm in maximum dimension
(Figure 1: 10–11). No prokaryotic cells build comparable walls. Note that the form
genus Satka currently contains several species of potentially disparate biological
origins. Satka squamifera consist of thin envelopes distended by ellipsoidal cells
and could be prokaryotic. Only S. favosa is known to construct complex walls of
interlocking plates.

2.2.2. Processes
Tappania plana comprises 20 to 160 µm vesicles that bear 0 to 20 or more elong-
ated extensions or processes (Figure 1: 4–9). Those processes are heteromorphic,
are distributed asymmetrically about the vesicle surface and have variable length
(25 to 60 µm). Processes communicate freely with the vesicle interior, have dark
slightly expanded closed ends and may branch (Figure 1:4–6). Specimens may
also bear up to 3 bulbous protuberances and/or neck-like extensions. The irregular
morphology and asymmetric distribution of processes in Tappania stand in marked
contrast to the regular size and distribution of processes in Palaeozoic acritarchs
and suggest that Tappania might have been an actively growing cell or germinating
cyst rather than a metabolically inert spore (as acritarchs are generally assumed to
represent). The bulbous protrusions in some specimens further suggest vegetative
reproduction by budding (Figure 1: 4), although some neck-like extensions might
be excystment structures (Figure 1: 8, see below).
As far as we are aware, the complex morphology of Tappania does not occur in
prokaryotes. Some prosthecate bacteria have wart to tube-like appendages extend-
ing from the cell surface that are 0.5 to 2–3 µm in length and may bear bundles
of fimbriae (Schlesner, 1987). Caulobacter produces a single stalk extending from
its 1–2 µm cell, with a length usually 1 to 2 µm but ranging up to 20–30 µm in
phosphate-depleted waters; the stalk terminates in a holdfast (composed of com-
plex polysaccharide) that attaches to surfaces (Brun and Janakiraman, 2000). Be-
cause stalks increase the surface-to-volume ratio of the cells, they are thought to
increase nutrient absorption in oligotrophic habitats. Stalks might also help main-
tain the cell at the air-water interface and unidirectional growth in biofilms. The
cell surface layers are continuous with those of the cell body and include the
cytoplasmic membrane, the peptidoglycan layer, and the outer membrane (Brun
82 E. J. JAVAUX ET AL.

and Janakiraman, 2000). The stalk is traversed by peptidoglycan rings or cross-

bands synthesized at each cell cycle (Pondexter and Staley, 1996). Biogenesis of
polar structures in Bacteria is linked to asymmetric protein localization near the
cytoplasmic membrane (Lybarger and Maddock, 2001). In Caulobacter, localized
signaling proteins are used to coordinate the cell-cycle so that dissimilar progeny
is generated at each division: a swarmer cell with a polar flagellum and a stalked
cell (Shapiro et al., 2002). Temporally controlled localization of structural and
regulatory proteins to the cell pole and subsequent proteolysis and release of these
polar components in preparation for the next cell cycle maintain asymmetry in
Caulobacter (Shapiro et al., 2002). The biosynthesis of stalks is poorly understood
but occurs only at one pole (or two poles in the case of Asticcacaulis biprosthecum)
of the cell where the swarmer cell ejects its flagellum. One hypothesis is that FtsZ,
the tubulin-like protein that polymerizes into a ring structure associated with the
cytoplasmic membrane at the site of cell division, could also be involved in the
topological reorientation of peptidoglycan synthesis required for stalk synthesis
(Quardokus et al., 1996). It is unknown if the wall of these gram-negative bac-
teria would withstand fossilization and maceration in acids (technique used to
extract organic-walled microfossils from shales). Moreover these morphologies
differ from Tappania by their vesicle size; appendage diameter, number, distri-
bution and morphology (not branching, not heteromorphic); and the absence of
neck-like extensions (a morphology unknown in prokaryotes).
Some actinobacterial spores can produce up to three germ tubes, but these tubes
originate within the spore interior and perforate the spore wall as they proliferate.
Thus, their morphology bears little in common with Tappania; moreover, they are
much smaller (Holt, 1984). Streptomyces can have 100 µm protoplasts from which
hyphae emerge (Cavalier-Smith, 2002), but these consist of amorphous masses
with open expanded tubes and do not closely resemble the Tappania vesicles il-
lustrated clearly in a number of publications (Prasad and Asher, 2001; Xiao et
al., 1997; Yin, 1998), or other Mesoproterozoic spiny acritarchs, despite cavalier
claims to the contrary (Cavalier-Smith, 2002). Indeed, as Cavalier-Smith (2002,
p. 37) pointed out, ‘cysts with spines or reticulate surface sculpturing would prob-
ably have required both an endomembrane system and a cytoskeleton, the most
fundamental features of the eukaryotic cell, for their construction’. We agree, and
underscore the fact that such fossils appear in the fossil record only slightly later
than 1500 Ma.
Could large prokaryotes form large appendages? The answer to this question
is not known although no examples have been reported from nature, as far as
we know. Heterotrophic and chemotrophic bacteria absorb nutrients over their
external surface. Thus the surface-to-volume ratio of the cells is important and
might explain why most bacteria are small rods, filaments, or vibrios (Koch, 1996).
Another solution to optimize nutrient absorption is to branch and produce a colony
with hyphae branching out in all directions as the actinobacteria and myxobacteria
do. The largest bacteria live in special nutrient-rich environments such as fish gut
 RECOGNIZING AND INTERPRETING THE FOSSILS OF EARLY EUKARYOTES 83

(heterotrophic gut bacteria Epulopiscium, 80 × 600 µm) or sulfide-rich seafloor

(sulfur bacteria Thiomargarita namibiense, 750 µm; filamentous sulfur Bacteria
Beggiatoa, bundles of filaments in sheath, 160 × 50 µm), where limitation of dif-
fusion through a large cell would not be a problem (Schulz and Jorgensen, 2001).
As mentioned above, some cyanobacteria can produce large colonial envelopes
up to 30–50 µm in diameter (Waterbury and Stanier, 1978) or cylindrical akinetes
more than 100 µm long (Golubic et al., 1995), and myxobacteria can produce spor-
angioles up to 50 µm in diameter. However none of these large bacterial structures
are ornamented.

2.2.3. Excystment Structures

The cyst walls of Phanerozoic protists commonly contain well-defined openings
through which motile cells escape. These excystment structures range from simple
perforations that run the circumference of cyst walls (‘medial splits’) to the poly-
gonal archaeopyles of dinoflagellates. Several preserved features of Roper micro-
fossils may document excystment in mid-Proterozoic eukaryotes.
The simplest candidate excystment structures are, unsurprisingly, medial splits,
as found in Valeria lophostriata (Figure 1: 1). One must be careful to distin-
guish between accidental tearing of the vesicle and biologically programmed splits.
Observation of regular openings in a large population of vesicles would provide
convincing evidence of excystment, but Roper populations display only limited
numbers of splits. Thus, their interpretation remains conjectural. (Note that some
pleurocapsalean cyanobacteria cells rupture to liberate their baeocytes (Waterbury
and Stanier, 1978); these cells can reach sizes up to 30 µm, but ruptured walls do
not separate clearly into two halves and do not roll-up.)
As noted in the preceding section, Tappania plana exhibits what appears to
be a sophisticated excystment structure consisting of an opening at the end of a
neck-like extension (Figure 1: 8). No prokaryotes, as far as we know, exhibit a
comparable morphology.

2.2.4. Wall Ultrastructure

In conjunction with observations of living protists, TEM observations of wall ul-
trastructure may provide evidence for specific eukaryotic affiliation. For example,
the walls around phycomata of some prasinophyte green alga have a diagnostic
ultrastructure consisting of a homogeneous electron-dense wall punctuated by pore
canals (Wall, 1962; Jux, 1968). Talyzina and Moczydlowska (2000) have identified
such an ultrastructure in the Early Cambrian prasinophyte Tasmanites tenellus,
demonstrating that this feature can be preserved in ancient microfossils. To date,
few ultrastructural studies of Proterozoic acritarchs have been completed. Oehler
(1977) reported unilayered walls of small unicells from the Neoproterozoic Bitter
Springs Formation. Peat (1981) illustrated the homogeneous wall of three leio-
spherids from the McMinn Formation of the Roper Group. Arouri et al. (1999,
2000) studied the ultrastructure and chemistry of Neoproterozoic acritarchs from
84 E. J. JAVAUX ET AL.

Australia and suggested a dinoflagellate affinity for some species with a fibrillar
multilayered wall and a chlorophycean affinity for other species with possibly
preserved TLS (trilaminar sheath structure, characteristic of many green algae). Ta-
lyzina (2000) found a single-layered, electron-dense homogenous wall ultrastruc-
ture for the species Chuaria circularis from the Neoproterozoic Visingso Group,
Sweden. The same species from the Neoproterozoic Pendjari Formation, West
Africa, (Amard, 1992) showed a multilamellar ultrastructure with pore canals. We
are currently conducting ultrastructural research on the walls of Roper and other
Mesoproterozoic fossils. Preliminary results indicate that some Mesoproterozoic
acritarchs display comlex, multi-layered wall ultrastructure similar in general to
extant protists (Javaux et al., 2002b).

2.2.5. Wall Chemistry

Conventional extraction of lipid biomarker molecules can identify the presence of
eukaryotic organisms in an ancient ecosystem (Summons and Walter, 1990), but
correlation of biomarkers with specific microfossil taxa is fraught with difficulty.
Moldowan and Talyzina (1998) reported the strong correlation of specific ster-
anes such as dinosterane with particular acritarch populations in Lower Cambrian
shales. It is certainly possible that a single species made both the biomarkers and
the preserved cyst walls, but insofar as sterols (the parent molecules of geologically
stable steranes) are not wall constituents (but, rather, part of the lipid bilayer of
the cell membrane), it is hard to reject the alternative explanation that steranes
and cyst walls reflect two different organisms that lived in ecological association.
Paleobotanists, who commonly find isolated seeds and leaves on a single bedding
plane, will immediately recognize the problem.
Another major difficulty resides in the fact that very little is known about the
chemical composition of various potentially fossilizable structures (such as ve-
getative cells and cysts) of recent protists. The few biomarkers characteristic of
eukaryotes include a variety of C28−30 steranes with side-chains alkylation pat-
terns (Summons and Walter, 1990; Brocks, 2003) among which dinosteranes that
are derived from dinosterol produced by dinoflagellates (and one diatom species;
Volkman et al., 1993).
Recently developed geochemical techniques now permit microchemical ana-
lyses of individual microfossils (e.g., Arouri et al., 1999, 2000; Boyce et al., 2002;
House et al., 2000; Kempe et al., 2002; Kudryavtsev et al., 2001; Schopf et al.,
2002). In particular, laser micropyrolysis of single acritarchs may show biomark-
ers specific for eukaryotes (steranes) and even diagnostic of particular groups.
However, it is possible that steranes would adhere to the surface of cell walls
and not originate from the cell membrane that was once contained in that wall.
Conclusive evidence (that the biomarker comes from the analyzed microfossil)
would be obtained if various taxa from the same sample show different biomarker
signatures that also differ from the host sediment. In conjunction with ultrastruc-
tural study, chemical analyses of single Neoproterozoic acritarchs (Arouri et al.,
 RECOGNIZING AND INTERPRETING THE FOSSILS OF EARLY EUKARYOTES 85

Figure 2. Phylogenetic tree for eukaryotes (modified from combined-protein tree of Baldauf et al.,
2000) calibrated using the paleobiological record (shaded boxes: biomarkers ; white boxes: body
fossils – see text for details) and inferred minimum time of divergence.

1999, 2000) suggested possible affiliations with chlorophycean green algae (based
on the finding of highly aliphatic biomacromolecules resembling the algaenan
biomacromolecules of Chlorophyceae) and with dinoflagellates (among the two
biomarkers of dinoflagellates, dinosterane and 4α-methyl-24-ethylsterane, only the
latter rather not specific was found). Scanning transmission X-ray microscopy
and spectroscopy provides another means of obtaining micron-scale chemical ana-
lysis of organically preserved acritarch walls (Boyce et al., 2002). Microchemical
studies of Roper fossils are planned, in conjunction with chemical analysis of
acid-resistant structures of recent protists.

3. Discussion

The criteria for fossil interpretation outlined above will not eliminate all uncertain-
ties in Proterozoic paleontology, but they will help us to fill in the gap between the
origin of the eukaryotic kingdom and its Neoproterozoic morphological diversific-
ation. This, in turn, will permit more accurate and reliable geological calibration of
eukaryotic phylogenies constructed using molecular sequence data (e.g., Baldauf
et al., 2000). At present, the fossil record provides minimum ages for a number of
events in early eukaryotic evolution (Figure 2).
86 E. J. JAVAUX ET AL.

Biomarkers in 2.7 Ga kerogens of the Fortescue Group, Australia, indicate

that contemporaneous cells were able to synthesize sterols (Brocks et al., 1999,
2003a,b). Sterol biosynthesis is a characteristic of eukaryotic cells – sterols have
been found in bacteria, but known cases either result from the incorporation of
molecules made by eukaryotic organisms (Bloch, 1994) or synthesis directed by
genes transferred laterally from eukaryotic cells (Lamb et al., 1998; Gamieldien et
al., 2002). As far as we know, only one bacterium (Methylococcus capsulatus) has
been reported to produce methyl-sterols; these, however, differ structurally from
eukaryotic sterols (Bouvier et al., 1976). Thus, accepting SSU rRNA phylogenies,
Fortescue biomarkers would seem to set a minimum date for the divergence of
Archaea and Eucarya. The presence in Fortescue rocks of carbon isotopic sig-
natures thought to reflect methanogenic archaeans and methanotrophic bacteria
independently suggests the same thing. By themselves, steranes in 2.7 Ga rocks
do not necessarily imply the Archean evolution of eukaryotic cells with nuclei,
cytoskeletons and endomembranes – although it is certainly not ruled out by the
scanty evidence available in very old sediments.
Fossils thought to be eukaryotic occur in Paleo- and Mesoproterozoic rocks
around the world, but at present, few of these can be related to specific protistan
clades. Han and Runnegar (1992) interpreted millimetric coiled filaments found
in the 1.87 Ga Negaunee Iron Formation, Michigan (Schneider et al., 2002), as
eukaryotic, based on their broad resemblance to large helical fossils in 1.4 Ga
rocks from China (Geoyuzhuan Formation; Walter and Du Rulin, 1990), the west-
ern United States (Walter et al., 1976) and India (Kumar, 1995) that are more
clearly eukaryotic (based on morphological features such as coiling and transverse
septae). Recently, however the interpretation of the 1.87 Ga coiled structures has
been questioned, as there is no preserved microscopic detail (Samuelsson and But-
terfield, 2001). Large (up to 200 µm) sphaeromorphic acritarchs possibly in the
2.4–2.5 Ga Hutuo Formation of China but very poorly illustrated (Sun and Zhu,
1998) and in the 1.8 Ga Chuanliggou Formation of China (Zhang, 1986) are also
candidates for early eukaryotes, although none are known to exhibit the features of
wall ornamentation, processes or excystment structures observed in Roper fossils.
As discussed above, more convincing microfossil evidence of eukaryotic mi-
croorganisms appears around 1.5–1.4 Ga in the Roper Group of Australia (Javaux
et al., 2001) and other early Mesoproterozoic successions. Microfossils from the
Ruyang Group, northern China, include not only the taxa found in Roper rocks,
but also acritarchs with numerous regularly arranged cylindrical processes (Guan
et al., 1988; Xiao et al., 1997; Yin, 1998). Such fossils strongly indicate that
eukaryotic organisms of marked cytological and genetic complexity existed 1,500–
1,300 million years ago. Relative to earlier assemblages, early Mesoproterozoic
protists show higher diversity and more obvious ecological heterogeneity (Javaux
et al., 2001).
At present, however, we do not know how to relate early Mesoproterozoic
fossils to specific branches of the eukaryotic tree. More satisfying in this regard are
 RECOGNIZING AND INTERPRETING THE FOSSILS OF EARLY EUKARYOTES 87

ca. 1.2 Ga fossils from the Hunting Formation in arctic Canada (Butterfield et al.,
1990; Butterfield, 2000) that can be related with confidence to the bangiophyte red
algae, based on observation of a nearly complete ontogenetic sequence for the pre-
served haploid generation, diagnostic cell division patterns, and morphologically
distinct sexual dimorphs in a large and exceptionally well-preserved population.
This provides a firm calibration point for molecular phylogenies, implying that by
1,200 million years ago, major branches of the eukaryotic tree were already in
place.
At 1 Ga, Palaeovaucheria, a xanthophyte from the recently well-dated Lakhanda
Formation, Siberia (German, 1990; Rainbird et al., 1998; Woods et al., 1998),
indicates the appearance of stramenopiles (which include diatoms, xanthophytes,
and brown algae) and of secondary symbiosis (involving a red alga-like endosym-
biont). Populations of Paleovaucheria display morphological traits characteristic of
vaucherian xanthophytes such as branching at right angles, 2 sizes of filaments on
the same individual, and terminal pores and septae at filament ends (Woods et al.,
1998). Vaucherians recently discovered in ca. 700–800 Ma shales in Spitsbergen
display a more complete range of vaucherian morphologies (Butterfield, 2002).
Upper Mesoproterozoic/Lower Neoproterozoic rocks have also yielded bio-
markers of alveolates (which include dinoflagellates and ciliates, among other
groups). Dinosterane, derived from dinosterol produced by dinoflagellates, occurs
in the 1.1 Ga Nonesuch Formation, United States (Pratt et al., 1991); the 830 Ma
Bitter Springs Formation, Australia; and the 590–570 Ma Pertatataka Formation,
also Australia (Summons and Walter, 1990; Summons et al., 1992; Moldowan et
al., 1996). Gammacerane, derived from tetrahymenol produced by ciliates has been
found in ca. 750 rocks of the Chuar Group, Arizona (Summons et al., 1988b)
however it could also be derived from some bacteria (Kleeman et al., 1990). It
is unknown whether Proterozoic dinoflagellates were photosynthetic.
Proterocladus, a Cladophora-like alga from the ca. 750 Ma Svanbergfjellet
Formation of Spitsbergen (Butterfield et al., 1994), suggests that chlorophyte di-
versification was well advanced by the mid-Neoproterozoic. As noted above, filose
and lobose testate amoebae from >742±6 Ma rocks of the Chuar Group, Arizona,
provide a firm calibration point for the great clade that includes animals, fungi and
the amoebozoans (Baldauf et al., 2000; Bapteste et al., 2002), not to mention direct
evidence for heterotrophic eukaryotes and eukaryotic biomineralization (Porter and
Knoll, 2000; Porter et al., in press).
Finally the Doushantuo Formation of China, recently dated at ca. 598±2 Ma
(Barfod et al., 2002), hosts multicellular green, red and, possibly brown algae,
as well as animal embryos, possible stem group cnidarians, and putative sponges
(Xiao et al., 1998a, b, 2002; Li et al., 1998; Xiao and Knoll, 2000).
Molecular clocks estimate phylogenetic divergence times, whereas fossils re-
cord the evolution of recognizable body plans within clades that diverged earlier.
Thus, Proterozoic fossils furnish only minimum dates for eukaryotic clade diver-
gence. Nonetheless, the fossil record suggests that the Mesoproterozoic and early
88 E. J. JAVAUX ET AL.

Neoproterozoic eras were crucial times for eukaryotic diversification (Figure 2).
By the time that large animals appear in the latest Proterozoic, much diversification
among and within major eukaryotic clades had already taken place.
This view contrasts strongly with Cavalier-Smith’s (2002) inexplicable view
that eukaryotes evolved only 850 million years ago. (Actually, Cavalier-Smith’s
claim is inexplicable only from the standpoint of actual observations of Proterozoic
fossils. It finds ready explanation in his argument that most principal clades of
eukaryotes diverged rapidly from one another early in the history of the domain.
Accepting Ediacaran fossils as faithful indicators of animal – and, hence, euka-
ryotic divergence – Cavalier-Smith is forced to doubt all significantly older records
of nucleated organisms. There is, of course, a more reasonable reconciliation of
fossils and molecular phylogeny – tissue-grade metazoans doubtfully reflect accur-
ately (or even remotely) the timing of eukaryotic origins. Using Cavalier-Smith’s
own criteria for the recognition of eukaryotic fossils (2002, p. 37, quoted above),
we must conclude that protists played a role in marine ecosystems 1,500 Ma and,
conceivably, much earlier. Later Proterozoic diversification with eukaryotic clades
reflects environmental opportunity (e.g., Anbar and Knoll, 2002) and the poly-
phyletic rise of complex multicellularity, likely in combination (e.g., Knoll and
Carroll, 1999; Butterfield, 2000).

4. Conclusions

In combination with molecular biology and geology, detailed studies of organic-

walled microfossils focusing on wall morphology, structure, ultrastructure, and
chemistry will help us to understand better the early evolution of eukaryotic or-
ganisms. In particular, studies of Paleo- and Mesoproterozoic fossils, including our
research on the Roper Group, Australia, will help to fill the gap between the earliest
biogeochemical record of protists and their late Mesoproterozoic/Neoproterozoic
diversification. Future developments of this work must include the refinement of
TEM and microchemical techniques that hold out the promise of better systematic
characterization of morphologically simple or enigmatic microfossils.

Acknowledgements

This article is a contribution to the Australian Geological Survey Organisation’s

NABRE project. We thank Jim Jackson, Peter Southgate and other members of
the NABRE team for access to unpublished observations, helpful discussions, and
advice on sampling. Core library staff in Canberra (AGSO) and Darwin (North-
ern Territory Geological Survey) greatly facilitated sample collection. We thank
Nick Butterfield and an anonymous reviewer for helpful comments. We also thank
Jochen Brocks for information on biomarker chemistry and Richard Losick for
 RECOGNIZING AND INTERPRETING THE FOSSILS OF EARLY EUKARYOTES 89

discussion on bacterial morphology. Yin Leiming provided helpful information

on Chinese stratigraphy, and Volodya Sergeev on Russian paleontology. Research
supported in part by NASA Exobiology Grant NAG5-3645 and the NASA Astro-
biology Institute, the Australian Research Council and Macquarie University.

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