A new approach in deciphering early protist paleobiology and … · A new approach in deciphering early protist paleobiology and evolution: Combined microscopy and microchemistry

lynology 139 (2006) 1–15www.elsevier.com/locate/revpalbo

Review of Palaeobotany and Pa

A new approach in deciphering early protist paleobiology andevolution: Combined microscopy and microchemistry

of single Proterozoic acritarchs

E.J. Javaux a,⁎, C.P. Marshal b

a Department of Geology, Geophysics, Oceanography, University of Liège, 4000 Liège Sart-Tilman, Belgiumb Australian Centre for Astrobiology, Macquarie University, NSW 2109 Sydney, Australia

Available online 3 March 2006

Abstract

Beside a few cases, the biological affinities of Proterozoic and Paleozoic acritarchs remain, by definition, largely unknown.However, these fossils record crucial steps in the early evolution of microorganisms and diversification of complex ecosystems.

We present how combining microscopy (light microscopy, scanning and transmitted electron microscopy) with microchemicalanalyses of individual microfossils may offer further insights into the paleobiology and evolution of early microorganisms. We useour ongoing work on early Mesoproterozoic and Neoproterozoic assemblages, as well as other published work, as examples toillustrate how this approach may clarify the evolution of early microorganisms and we underline how useful this approach could befor palynologists working on younger material. Such a multidisciplinary approach offers new possibilities to investigate thebiological affinities of acritarchs and the record of early life on Earth and beyond.© 2006 Elsevier B.V. All rights reserved.

Keywords: acritarchs; biological affinities; microscopy; microchemistry; Proterozoic; Paleozoic

1. Introduction

Acritarchs are organic-walled microfossils of un-known biological affinities. They are conventionallyinterpreted as algal cysts but most probably include alarger range of organisms such as prokaryotic sheaths,heterotroph protists or even parts of multicellular beings(Van Waveren, 1992; Martin, 1993; Colbath andGrenfell, 1995; Butterfield, 2005). Biologists easilydifferentiate between prokaryotic and eukaryotic organ-isms using molecular and cell biology, but thesecharacters rarely survive fossilization and so are notgenerally available to the paleontologist. However,

⁎ Corresponding author. Fax: +32 4 366 2921.E-mail address: [email protected] (E.J. Javaux).

0034-6667/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.revpalbo.2006.01.005

acritarchs record crucial steps in the early evolutionand diversification of the biosphere. Identifying thebiological affinities of these microscopic organisms willclarify Proterozoic and Paleozoic microbial paleobiol-ogy and food webs (see Butterfield, 1997, 2000 fordiscussions on plankton food webs and ecologicaltiering in the late Proterozoic). It will also contributeto phylogenetic reconstructions and improve ourunderstanding of early evolutionary mechanisms andpatterns, and the early interactions between environmentand life.

Comparative biology is useful when organismsdisplay characteristic morphological attributes, but thisrarely occurs among acritarchs (for example earlydinoflagellates might not show tabulation). Most acri-tarchs have relatively simple morphologies; basically a

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2 E.J. Javaux, C.P. Marshal / Review of Palaeobotany and Palynology 139 (2006) 1–15

microscopic organic ball or tube (often flattenedwhen preserved in shales) with or without variousornaments, that make comparison with recent cladesdifficult. New approaches are necessary to elucidatetheir paleobiology.

Study of the wall ultrastructure can differentiatebetween prokaryotic and eukaryotic acid-resistantmicrofossils and, in some cases, even identify particularclades (review in Javaux et al., 2003, 2004a). Combin-ing microscopy (light microscopy, scanning and trans-mitted electron microscopy) with microchemicalanalyses of individual microfossils offers furtherinsights into the paleobiology and evolution of earlymicroorganisms. A combination of techniques applied

Table 1Summary of techniques that can be applied on single acritarchsextracted from shales by acid-maceration and possible paleobiologicalinformation

Techniques Data Paleobiological information

Transmittedlightmicroscopy

Morphology(ornamentation, color,branching, etc.)

Assemblage diversity

No. of specimens Characteristic morphologyNo. of species Cell division patterns

Reproduction modeWall thickness, flexibilityTaphonomyMulticellularityBiological affinities insome cases

Scanningelectronmicroscope

Detailed outer andinner wall features

Wall ornamentation andstructure sometimesindicative of eukaryotccomplexity

Wall thicknessTransmissionelectronmicroscopy

Wall ultrastructure Biological affinities atlevel of kingdom,class in some cases

Fluorescenceconfocalmicroscopy

Fluorescence ofwall polymers

Linked to wall chemistry,applicable on veryimmature material

EDEX Elementalcomposition

Details onpreservation mode,

FT-infraredspectrometry

Biopolymercomposition

Biologicalaffinities insome cases

FT-Ramanspectrometry

Aromaticity andthermal maturity oforganic matter

To use incombination withFTIR for furthercharacterizationof wall chemistry

PyrolysisGC/MS

Biomarkercomposition

Biological affinitiesin some cases

SIMS C isotopes Metabolism in somecases, in combinationwith morphology

to single or very few microfossils allows characteriza-tion of the fine ultrastructure and microchemistry of thematerial studied. In some cases, it is possible to relatethese with similar known features of recent organismsand clarify the paleobiology of the fossils. Ideally, thisapproach should be conducted within a good geologicalframework, in well-dated successions, with detailedsedimentological reconstruction of paleoenvironments.

This paper focus on techniques that can be applied toisolated acid-resistant organic-walled microfossilsextracted from shales, in order to characterize theirmorphology, wall ultrastructure and chemistry and toreconstruct their paleobiology (Table 1). Other techni-ques (briefly mentioned in the text) can be used forvisualizing microfossils still embedded in a mineralmatrix (and too fragile to withstand extraction) and forcharacterizing their elemental composition and theirdistribution in the rock, and the biological–mineralinteractions.

2. Materials

Most of the fossils used as examples here come fromcarbonaceous shales of the early Mesoproterozoic RoperGroup, northern Australia (Fig. 1). The sedimentaryarchitecture of the Roper Basin is well characterized(Abbott and Sweet, 2000). The shales preserve abundantand exquisite organic-walled microfossils distributed inassemblages showing an onshore–offshore pattern ofdecreasing abundance, declining diversity and changingdominance (Javaux et al., 2001). U–Pb SHRIMPanalyses of zircons from an ash bed in the MainoruFormation fix an age of 1492±3 Ma for early Roperdeposition (Page et al., 2000). A 1429±31 Ma Rb–Srage for illite in dolomitic siltstones near the top of thesuccession is consistent with the zircon age, even if lessreliable (Kralik, 1982). Highly carbonaceous shales inbasinal deposits of the Velkerri Formation, near the topof the Roper Group, also contain low abundances ofsteranes sourced by eukaryotic organisms (Summons etal., 1988). The Roper microfossils have been variouslyinterpreted as the oldest unambiguous evidence foreukaryotes (Javaux et al., 2001, 2003), as complexprokaryotes (Cavalier-Smith, 2002) and as fungi(Butterfield, 2005). Their exquisite preservation andtheir age makes them ideally suited for paleobiologicalinvestigations.

One highly ornamented fossil also treated here,Shuiyousphaeridium macroreticulatum, comes fromshales of the top of the Ruyang Group, northern China(Yin, 1998). Ruyang deposition is not well constrainedby radiometric dates, but appears to be at least broadly

Fig. 1. Location and generalized stratigraphy of the Roper Group,northern Australia, showing stratigraphic distribution of facies andmicrofossils. Modified from Javaux et al. (2001).

3E.J. Javaux, C.P. Marshal / Review of Palaeobotany and Palynology 139 (2006) 1–15

coeval with Roper sedimentation. A ca. 1000 Ma granite(U–Pb zircon date) intrudes the Ruyang succession,providing a minimum age for the group; moreover,abundant microdigitate precipitates and C-isotopicprofiles that vary little from 0‰ in thick, overlyingcarbonates suggest that Ruyang shales are older than ca.1250 Ma (Xiao et al., 1997). Ruyang shales shareseveral distinctive taxa (species of Tappania, Valeriaand Dictyosphaera) with the Roper Group.

The palaeoecological distribution, morphology, wallultrastructure and microchemistry of the acritarchstreated here have been described for Roper and Ruyangsamples by Javaux et al. (2001, 2003, 2004a,b) andMarshall et al. (2005), and for Neoproterozoic acritarchsfrom the Tanana Formation, Australia (ca. 590–565 Ma)by Arouri et al. (1999, 2000) and Grey (1998).

Microfossils were extracted from shales using amodified palynological method involving slow hydro-fluoric acid digestion with minimal agitation (Grey,1999). Single acritarchs were handpicked from kero-gen isolate under a stereomicroscope using amicropipette.

2.1. Characterization of acritarch morphology andultrastructure

2.1.1. Light microscopyLight microscopy is routinely used to examine the

morphology of the fossils mounted on glass slideswith transmitted light, as well as for determining thediversity of assemblages. In some cases, comparativemorphology with extant groups might reveal adivision or reproduction pattern or other morpholog-ical feature (for example, tabulation in dinoflagellates)unique to certain biological groups, therefore permit-ting the phylogenetic positioning of the specimens.Butterfield (2000) compared the division patterns ofcells in various groups of algae and bacteria andconcluded to a bangiophyte affiliation for a populationof 1.2–1 Ga (gigayear-old) fossil preserved in chertsof the Hunting Formation, arctic Canada. This carefulstudy identified the earliest eukaryote so far that canbe related to an extant lineage. This approach has alsobeen very successful in other cases, identifyingcyanobacteria genera ranging from the Proterozoic tonow (Knoll and Golubic, 1992). Examination of alarge population of well-preserved specimens from asingle bedding plane might also permit to unifyseemingly unicellular disparate forms into a multicel-lular organism (for example, a diverse Neoproterozoicassemblage has been shown to be the disarticulatedremains of a single vaucheriacean metaphyte, Butter-field, 2004).

We have outlined several criteria for differentiatingeukaryotic from prokaryotic fossil cells and to evaluatetheir degree of complexity: the presence of processesand/or other surface ornamentation, the wall structure,the presence of excystment structures and the wallultrastructure can all indicate a level of complexityunknown in prokaryotes (Javaux et al., 2003).

Features such as excystment structures or details ofornamentations are important for acritarch classificationand may demonstrate eukaryotic origin (Javaux et al.,2003). Resting cells and reproductive cysts of manyprotists display micron-scale patterns of lineations,fields, spines or bosses not known among prokaryoticorganisms. While prokaryotic organisms can synthesizeboth cell wall ornament and preservable structures, wallornamentation consists mostly on nanoscale proteina-ceous (not acid-resistant) spines or bosses, and preserv-able structures such as cyanobacterial envelopes are notornamented (see review in Javaux et al., 2003).Cyanobacterial sheaths are preserved in the fossilrecord, in preference to the peptidoglycan-rich cellwalls, as shown by taphonomic experiments (Bartley,


1996). It is unknown if the wall of other prokaryoteswould withstand fossilization in shale and maceration inacids (technique used to extract organic-walled micro-

fossils from shales); however, they can be easilypreserved by mineralization with silica (chert), calciumcarbonate (calcite, aragonite), calcium phosphate


(apatite), iron carbonate (siderite), iron oxide (hematite)and iron sulfur sulfide (pyrite) (Westall, 1999).

The cyst walls of Phanerozoic protists commonlycontain well-defined openings through which motilecells escape (e.g. Le Hérissé, 1984). These excystmentstructures range from simple perforations that run thecircumference of cyst walls (‘medial splits’) to thepolygonal archaeopyles of dinoflagellates. Excystmentstructures are common in the Neoproterozoic and mightalso occur in early Mesoproterozoic microfossils fromthe ∼1.5 Ga Roper Group, Australia in the form ofenrolled half vesicles resulting from medial split(Valeria) or opening at the end of a neck-like extension(Tappania) although this need to be confirmed byexamination of a larger population (Javaux et al., 2001,2003) (Fig. 2).

Among the ∼1.5 Ga Roper Group microfossilassemblage, population of Tappania plana consists onvesicles bearing 0 to 20 heteromorphic processes,irregularly distributed about the vesicle. The processesmay branch (Fig. 2: 2), they communicate with thevesicle interior and they have closed dark expanded end(Fig. 2: 5). Some specimens have a neck-like expansion,open in two specimens and suggesting possible excyst-ment structure (Fig. 2: 3–4). Others bear up to threerounded extensions, possibly suggesting reproductionby budding (Fig. 2: 2). The population of process-bearing species T. plana includes vegetative cells with acomplex and irregular morphology, possible excystingcysts and possible evidence for reproduction bybudding. Some of these features are characteristic ofmetabolically active cells with cytoskeletal sophistica-tion, suggesting an eukaryotic grade of organization(Javaux et al., 2001, 2003). In one specimen, theprocesses have a septum (Fig. 2: 1), suggestingmulticellularity, as proposed by Butterfield (2005).However, a larger number of specimens displayingthis character should be examined before confirmingthis degree of complexity in early Mesoproterozoiceukaryotes.

These few examples illustrate how careful examina-tion of microfossil assemblages with transmitted lightmicroscopy may yield important paleobiological infor-mation. However, in many cases, other additional

Fig. 2. Ornamentation and possible excystment structures in Late Palemicrofossils). All specimens illustrated are from the Roper Group of northern A5) Light photographs of Tappania plana, Roper Group. Arrows shows septumopening of neck-like expansion suggesting excystment structures in 3 and 4Group. (6–7) Light photographs, two halves possibly resulting from medialorganic plates. (9–11) Valeria lophostriata. (9, 11) Light photographs showinresult from medial split; (10) SEM image showing striations consisting on 1 μm25 μm for 2, 16 μm for 3, 20 μm for 4, 24 μm for 5 and 6, 10 μm for 7, 5

observational and analytical techniques are required ifthe work is not limited to descriptive or stratigraphicobjectives.

2.1.2. Scanning electron microscopy (SEM)Scanning electron microscopy (SEM) of microfossils

mounted on aluminium stubs and covered with a thinlayer of gold or gold/palladium permits us to studydetails of surface sculpture or ornaments (e.g. detailedstudy of excystment structures in Silurian acritarchs, LeHérissé, 1984) or even wall structure in fracturedspecimens.

For example, Valeria lophostriata, a species knownfrom the late Paleoproterozoic to the Neoproterozoic onfour continents, is easily distinguished by its concentricstriations observable by light microscopy (Fig. 2: 9–11)(Hoffman and Jackson, 1996; Xiao et al., 1997;Hoffman, 1999; Javaux et al., 2001, 2004a,c). SEMobservation of this species from the ∼1.5 Ga RoperGroup, Australia, shows these striations to consist ofparallel ridges uniformly spaced 1 μm apart on theinternal surface of the vesicle (Fig. 2: 10). At ourknowledge, this micron-scale pattern in an acid-resistantwall is unknown in prokaryotes and strongly suggests aneukaryotic affinity (Javaux et al., 2004a). SEM study ofanother species from the Roper assemblage, Satkafavosa, shows clearly that the wall consists of polygonalplates that form a tessellated pattern, a wall constructionunknown in prokaryotes at our knowledge (Fig. 2: 6–8)(Javaux et al., 2004a). The acanthomorphic acritarchShuiyousphaeridium macroreticulatum, from the∼1.3 Ga Ruyang Group, China, has a reticulated surfaceand numerous regularly spaced cylindrical processesthat flare outward (Fig. 3: 10–13) (Xiao et al., 1997;Javaux et al., 2001, 2004a). Close SEM examination ofits wall, and more precisely through cracks in the wall,revealed a wall structure consisting of closely packed,beveled hexagonal plates (Fig. 3: 11). This speciesdisplays clearly eukaryotic morphology: as Cavalier-Smith (2002, p. 37) has pointed out, “cysts with spinesor reticulate surface sculpturing would probably haverequired both an endomembrane system and a cytoskel-eton, the most fundamental features of the eukaryoticcell, for their construction”.

oproterozoic and Early Mesoproterozoic acritarchs (organic-walledustralia, except specimen in Fig. 1(11), from the McArthur Group. (1–in processes in 1, branching of processes and bud like extensions in 2,, and dark rounded ends of processes in 5. (6–8) Satka favosa, Ropersplit in 7; (8) SEM image showing wall made of imbricated polygonalg characteristic concentric ornamentation, rolled half vesicle in 9 mightspaced ridges on the inner part of the vesicle. Scale bar is 18 μm for 1,

μm for 8, 22 μm for 9 and 11, and 2 μm for 10.

Fig. 3. Morphology and wall ultrastructure of simple leiospheres from the Roper Group, northern Australia and complex acanthomorphic acritarchfrom the Ruyang Group of China (Mesoproterozoic) (see text and Javaux et al., 2004a,b,c,d for detailed explanations). (1–3) Leiosphaeridia jacutica;(4–6) L. crassa; (7–9) L. tenuissima; (1, 4, 7) light photographs; (2, 5, 8) SEM images; (3, 6, 9) TEM images showing different multilayered walls inthe three species. (10–13) Shuiyousphaeridium macroreticulatum. (10, 11) SEM images showing details of wall made of imbricated beveledpolygonal organic plates (10) and ornamentation with furcated processes (11), reticulate outer ornamentation; (12) TEM image illustratingmultilayered wall with organic plates; (13) light photograph showing numerous regularly distributed processes and reticulate ornamentation. Scalebar in a is 30 μm for 1, 7 and 8, 50 μm for 2 and 13, 1.7 μm for 3, 10 μm for 4 and 5, 0.3 μm for 6, 1.6 μm for 9, 7 μm for 11, 9 μm for 10, and 1.5 μmfor 12.


Scanning electron microscope with back-scatteredelectron imaging (SEM-BSE ) permits the visualizationof microfossils still embedded in their mineral matrix

when ultrastructural elements (such as the wall) aremineralized and their inorganic features differ from thatof the matrix. This technique is promising for studying


in situ the microorganism–mineral interface, especiallyfor endolithic or mat-forming microorganisms thatcannot withstand dissolution of the rock and determin-ing biogenecity of the observed structures. Samplepreparation includes fixation, staining and embedding inresin, followed by sectioning of the carbon-coatedsample (Ascaso and Wierzchos, 2002).

2.1.3. Transmission electron microscopy (TEM)Transmission electron microscopy (TEM) is rarely

used to elucidate the wall ultrastructure of singlespecimens. It is indeed a lengthy and delicate processto embed single properly oriented microfossils in resinsand to cut ultrathin sections for examination in the TEM.However, in conjunction with observations of livingmicroorganisms, this technique has proved very usefulin identifying simple microfossils at the level of domain(Javaux et al., 2004a) and even in some cases at the levelof class (Talyzina and Moczydlowska, 2000).

Examination of acritarch wall ultrastructure usingTEM was initiated in the late 1960s to 1970s mostly onPaleozoic microfossils (e.g. Jux, 1968, 1971, 1977;Kjellström, 1968a,b; Loeblich, 1970; Martin andKjellström, 1973; Oehler, 1977). More recently, affinityto green algae (including prasinophytes) has beenshown for a few Neoproterozoic acritarch species fromthe Australian Central Superbasin (Grey, 1998; Arouri etal., 1999, 2000). Peat (1981) illustrated the homoge-neous wall of 3 unidentified sphaeromorphs from theMcMinn Formation in the Mesoproterozoic RoperGroup of Australia. Recent work by Talyzina andMoczydlowska (2000) on Lower Cambrian acritarchsrevealed four structural types of vesicle wall in additionto their single- to multilayered composition and thevariable thickness of the wall. The leiospheres studiedshowed a multilayered and composite wall similar to thewall of chlorophycean algae, Order Chlorococcales(Talyzina and Moczydlowska, 2000). Some prasino-phyte green algae have a diagnostic ultrastructureconsisting of a homogeneous electron-dense wallpunctuated by pore canals (Wall, 1962; Jux, 1968) thathas been recognized in Cambrian Tasmanites tenellus(Talyzina and Moczydlowska, 2000).

Our research on microfossils from the ∼1.5–1.4 GaRoper Group from Australia and Ruyang Group ofChina lead to the discovery of eukaryotic ultrastructurespreserved in acid-resistant walls of eight species oforganic-walled microfossils (Javaux et al., 2004a). Inparticular, our discovery of complex eukaryotic ultra-structures in the walls of morphologically simple mid-Proterozoic microfossils (leiospheres) indicates thatTEM can provide an important tool in paleontological

efforts to decipher the earliest records of eukaryoticdiversification. When observed under transmitted lightand SEM, leiospheres could be interpreted as cyano-bacterial envelopes (Fig. 3: 1–2, 4–5, 7–8). However,these cyanobacterial envelopes differ from protistanwalls at the ultrastructural level, consisting of fibrouslayers (Waterbury and Stanier, 1978, Fig. 1; J. Water-bury, pers. comm., 2003) quite distinct from any of theultrastructures described in Javaux et al. (2004a) (Fig. 3:3, 6, 9). Observations with TEM revealed exquisitelypreserved wall ultrastructures of other Mesoproterozoicmicrofossils ranging from single, homogeneous, elec-tron-dense layers of variable thickness- and variablyornamented- to multilayered walls differentiated byelectron density and texture (Javaux et al., 2004a).Among the Roper microfossils, a single homogeneouswall ultrastructure occurs in ornamented acritarchs suchas Tappania plana, Satka favosa and Valeria lophos-triata that clearly exhibit eukaryotic features (wallornamentation). Simple wall ultrastructure does not initself indicate eukaryotic affinity since diagenesis couldtheoretically give this appearance to a fibrous prokary-otic sheath. Other Roper and Ruyang acritarchsincluding the simple leiospheres and the complexacanthomorphic Shuiyousphaeridium macroreticulatumdisplay multilayered walls. Thus, existing data indicatethat the structural complexity of eukaryotic cell wallscan be preserved in ancient microfossils and distin-guished from acetolysis-resistant structures formed bybacteria (Javaux et al., 2003). This, in turn, suggests thatultrastructural features can provide evidence for eu-karyotic affinities, even in older Proterozoic fossils,where simple morphology may be non-diagnostic.

Combined analysis of the fine structure with SEMand TEM permitted also to show that an acanthomorphacritarch from the Ruyang Group of China, Shuiyou-sphaeridium macroreticulatum, has a reticulate wallconsisting of imbricated beveled polygonal organicplates (Javaux et al., 2004a), and not of thickened,polygonal cells as could be suggested on the base oflight microscopy alone (Butterfield, 2005). This speciesmay also show medial split excystment structures,suggesting a cyst-like morphology, but whether it was ametabolically inert stage of a unicellular or multicellularorganism, or whether it had a phototrophic or hetero-trophic metabolism, is unknown, as underlined byButterfield (2005).

2.2. Microchemistry of acritarch wall

Colbath and Grenfell (1995, p. 24) suggested that“further advances in understanding the chemistry of


sporopollenin-like compounds hold considerable prom-ise for establishing biological affinities, when and if themethods can be applied to fossil material”. Indeed,recently developed geochemical techniques now permitmicrochemical analyses of individual microfossils (forexample, Lee et al., 1998; Arouri et al., 1999, 2000;Marshall et al., 1999, 2001; House et al., 2000;Kudryavtsev et al., 2001; Ascaso and Wierzchos,2002; Boyce et al., 2002; Foster et al., 2002; Kempeet al., 2002; Schopf et al., 2002). Micro-Fouriertransform infrared (FTIR) spectroscopy enables thedetermination of biopolymer composition. Micro-Raman spectroscopy determines the degree of aroma-ticity and the thermal maturity of the macromolecularstructure of the cell wall. Ion microprobe measures thecarbon isotopes. Significantly, all these techniques canbe applied on a single acritarch. By comparison withbulk biomarker analyses, micro-FTIR and micro-Ramanspectroscopy techniques have the great advantage to beapplicable on a very small sample (such as onemicrofossil), to provide data on the chemical composi-tion of the microfossil with previously describedmorphology and ultrastructure, and to avoid contami-nation problems. Thus, these techniques permit us torelate directly a morphology to a chemical composition,instead of dealing with bulk rock analyses or mixedmicrofossil assemblages. Micropyrolysis or flash pyrol-ysis permits us to determine the presence of biomarkermolecules on a few dozen specimens. Confocalmicroscopy can be used to detect autofluorescence ofthe wall biopolymer on recent dinoflagellates (dinos-porin autofluorescence differs from that of sporopollen-in) (Graham and Wilcox, 2000) and on Cambrianacanthomorph acritarchs (Talyzina et al., 2000), but didnot give any results on our older Mesoproterozoicmaterial (Javaux, personal observation). Other techni-ques seem promising but need further development(Cady et al., 2003).

2.2.1. Energy dispersive X-ray spectroscopy (EDX) inthe SEM

Energy dispersive X-ray spectroscopy (EDX) in theSEM permits a qualitative and quantitative analysis ofelements in microfossils such as C, O, P, S, Si and Fe.This data might explain in part the different textures ofmicrofossil walls seen with SEM and their mode ofpreservation (such as mineralization, carbonization orphosphatization). For example, detailed SEM study ofNeoproterozoic vase-shaped microfossils from theChuar Group, Arizona, combined with elementalanalysis, revealed different modes of preservation.These fossils, identified as testate amoebae, were

originally composed of non-resistant organic matter,and preserved as mineralized casts and molds coatedwith organic debris or iron minerals (Porter and Knoll,2000). Elemental analysis has also been used in somecases to infer biogenicity of putative fossil bacteria, incombination with other parameters (Ascaso and Wierz-chos, 2002). Elemental analysis can provide crucialinformation for the paleontologist, by revealing thatseemingly biogenic morphologies can be artifacts ofpreparation (Edwards et al., 2004).

Combining EDX facility with SEM-BSE permits toanalyze microfossils still embedded in the mineralmatrix and sometimes to identify biomobilization ofelements (Al, K, Fe) at the former place of cellularultrastructural zones such as pyrenoids, chloroplasts andcell walls (Ascaso and Wierzchos, 2002).

2.2.2. In situ ion microprobe analysis of carbonisotopes

Ion microprobe analyses of the carbon isotopes canbe carried out on individual organic-walled microfossils.House et al. (2000) measured δ13C values rangingbetween −21.3‰ and −45.4‰ in microfossils pre-served in stromatolitic cherts from the 850 Ma BitterSpring Fm and the 2100 Ma Gunflint Fm, and suggestedthese values to be consistent with a cyanobacterialaffinity based on morphological characteristics. Kauf-man and Xiao (2003) have analyzed microfossilsextracted from shales of the Mesoproterozoic RuyangGroup in North China and calculated δ13C values of−25‰ for specimens of Dictyosphaera delicata. Basedon these values and the morphological complexity of D.delicata, the authors suggested that the Calvin cycle wasin place at least 1400 Myr ago (Kaufman and Xiao,2003).

However, the selectively preserved acritarch wall isthe end product of complex biosynthetic pathway andtherefore is likely to have different values of isotopicfractionation than the primary photosynthetic metabolite(G. Versteegh, pers. comm.). Isotope fractionation mayalso vary with cell size and cell compartment (Popp etal., 1998; Schouten et al., 1998). The authors'assumption that this species is a photosynthetic algahas not been confirmed by our ultrastructural andmicrochemical investigations of another species fromthe same beds in the Ruyang Group, Shuiyousphaer-idium macroreticulatum, suggested to be a betterpreserved variant of D. delicata (Xiao et al., 1997).Shuiyousphaeridium macroreticulatum does not showevidence of a trilaminar structure (TLS) in its wall(Javaux et al., 2004a), nor the presence of the aliphaticbiopolymer algaenan (Marshall et al., 2005); two


characteristics of several extant chlorococcalean andeustigmatophycean green algae. However, some othergreen algae do not show these features, so their absencedoes not preclude a green algal affinity. Butterfield(2005) suggested a possible fungal affinity for S.macroreticulatum. Further isotopic analyses by A.J.Kaufman (Univ. Maryland) of these species and otheracritarchs we studied from the coeval Roper Group ofAustralia will aim to characterize the isotopic variationswithin and between species and facies, and mightanswer this question, along with other chemicalanalyses.

2.2.3. Micro-Fourier transform infrared spectroscopy(micro-FTIR spectroscopy)

FTIR spectroscopy is a sensitive and efficienttechnique commonly used for characterizing complexorganic macromolecules such as kerogen. We havedeveloped a micro-FTIR spectroscopic method tailoredto microscale characterization of acritarchs for elucidat-ing their cell-wall biopolymer composition and thusdelineate biological affinity (Javaux et al., 2004b,c;Marshall et al., 2005).

Micro-Fourier transform infrared (FTIR) spectrosco-py involves the measurement of the wavelength andintensity of the absorption of IR radiation by a sample.The wavelengths of IR adsorption bands are character-istic of specific types of chemical bonds, allowing theidentification of organic compounds. Measuring theintensity of aliphatic C–H stretch (2800–3000 cm−1),aromatic C–H stretch (3000–3100 cm−1) and carbonylgroups CfO (1715–1740 cm−1) provide information onkerogen type or biopolymer type possibly specific ofparticular clades.

Sample preparation includes soaking in dichloro-methane to remove extraneous surface contaminants,before placing the acritarch samples on potassiumbromide slides (details in Marshall et al., 2005).

Cell wall chemistry of organic-walled microfossilscan provide clues to the biological relationships ofProterozoic and Paleozoic fossils. However, a majordifficulty resides in the fact that very little is knownabout the chemical composition of potentially fossiliz-able structures (for algae, see review by Versteegh andBlokker, 2004). Acritarch wall is assumed to becomposed of sporopollenin, the component of theresistant outer layer of spore and pollen walls, becauseof its resistance to acids. Algaenans (Tegelaar et al.,1989) are highly aliphatic biopolymers present in thevegetative cell wall of several marine and fresh watermicroalgae in the classes of Chlorophyceae andEustigmatophyceae, and in one dinoflagellate species

(Derenne et al., 1992; Gelin et al., 1999). Dinosporin is ahighly resistant aromatic macromolecular substancecompletely different from that of algaenan and hasbeen isolated in the cyst of one marine dinoflagellateLingulodinium polyedrum (Kokinos, 1994; Kokinos etal.,1998). Versteegh et al. (2004a) reported a highlyaliphatic composition of fossil dinoflagellate casts, butthese were not cyst walls enclosing a cavity, but ratherthe secondary inside fillings of former vegetative cells,so their chemical composition is not comparable to abiopolymer. Prokaryotic biopolymers called cyanobac-teran or bacteran has been shown to be artifacts ofchemical isolation procedure (Allard et al., 1997, 1998).

Arouri et al. (1999, 2000) have attempted to elucidatethe biological affinities of single acritarchs using Fouriertransform infrared and Raman spectroscopy, with mixedsuccess due to technical problems. Micro-FTIR spec-troscopy of the microfossil Reduviasporonites by one ofus (C.M.) in Foster et al. (2002) demonstrated that thisfossil thought to be a fungal spore and occurring asspikes at the Permo–Triassic boundary in variouslocations was in fact algal in origin.

Our microchemical analyses of the wall of theNeoproterozoic acritarch Tanarium conoideum fromObservatory Hill, Australia, has revealed the presence ofa highly aliphatic hydrocarbon with a compositionconsistent with that of algaenan found in resistant wallsof some microgreen algae (Fig. 4) (Javaux et al., 2004b,c;Marshall et al., 2005). The Neoproterozoic Leiosphaer-idia sp. from Observatory Hill, Australia may comprise anew class of biopolymer containing significant aliphatic,branched aliphatic and saturated/olefinic carbon constitu-ents. Mesoproterozoic acritarch cell walls, includingShuiyousphaeridium macroreticulatum, contain a pre-dominantly aromatic biopolymer consisting of shortaliphatic chains that are highly branched, with a fewoxygenated functionalities (Fig. 4) (Javaux et al., 2004c;Marshall et al., 2005). Differences among species ofsimilar thermal history occur in the presence andabundance of aliphatic carbon and oxygen functionalities.Among early Mesoproterozoic species of the RoperGroup, Australia, three species of leiopheres showed verydifferent multilayered wall ultrastructure (Javaux et al.,2004a) but similar chemical composition (Marshall et al.,2005), underlying the importance of combining both themorphological and ultrastructural studies with the micro-chemical analyses for characterizing these earlyeukaryotes.

2.2.4. Micro-Raman spectroscopyLaser micro-Raman spectroscopy measures the

wavelength and intensity of inelastically scattered light

Fig. 4. Representative micro-FTIR spectra for Neoproterozoic Tanarium conoideum from the Tanana Fm, Australia (a) and for the MesoproterozoicShuiyousphaeridium macroreticulatum, Ruyang Group, China (b). See explanations in the text.


from molecules. Laser micro-Raman spectroscopy isused as a technique for the surface characterization ofcarbonaceous materials to elucidate carbon structure andits thermal maturity. Sample preparation includescleaning the acritarchs with dichloromethane, beforedeposition on clean aluminum microscope slides(Marshall et al., 2005).

In our experience, Raman spectroscopy does notprovide useful information about the molecular compo-sition of Proterozoic acritarchs, but rather elucidates thecarbon structure and thermal alteration of constituentorganic matter (Marshall et al., 2005). Neither it can beused by itself to determine biogenicity of Archeancarbonaceous material (Marshall et al., 2004).

However pigments of living microbial cells inAntarctic mats have been detected in situ using Ramanspectroscopy acquired at another laser excitation line(Wynn-Williams et al., 2002) that unfortunately cannotbe applied on fossil material.

2.2.5. Micropyrolysis gas chromatography/mass spec-trometry (GC/MS) and flash pyrolysis

Conventional extraction of lipid biomarker mole-cules can identify the presence of eukaryotic organismsin an ancient ecosystem (Summons and Walter, 1990),but correlation of biomarkers with specific microfossiltaxa is difficult. Moldowan and Talyzina (1998)

reported the strong correlation of specific steranessuch as dinosterane with particular acritarch populationsin Lower Cambrian shales. It is certainly possible that asingle species made both the biomarkers and thepreserved cyst walls, but insofar as sterols (the parentmolecules of geologically stable steranes) are not wallconstituents (but, rather, part of the lipid bilayer of thecell membrane), it is hard to reject the alternativeexplanation that steranes and cyst walls reflect twodifferent organisms that lived in ecological association(Javaux et al., 2003). Paleobotanists, who commonlyfind isolated seeds and leaves on a single bedding plane,will immediately recognize the problem.

Another major difficulty resides in the fact that verylittle is known about the chemical composition ofvarious potentially fossilizable structures (such asvegetative cells and cysts) of recent protists. The fewbiomarkers determined so far include lipids (derivedfrom the lipid bilayer of cell membranes) and othermolecules constituent of cell walls. They have a longfossil record and permit to characterize the diversity ofancient assemblages producing preservable and charac-teristic organic molecules but not necessarily preserv-able cellular structures. For example, some steranes arebiomarkers for eukaryotes and include a variety of C28–30 steranes with side-chains alkylation patterns (Sum-mons and Walter, 1990; Pearson et al., 2003).


Dinosterane are derived from dinosterol produced bydinoflagellates (and one diatom species; Volkman et al.,1993). 2-Methylhopanes are biomarkers for cyanobac-teria (Summons et al., 1999).

Laser micropyrolysis of single acritarchs may showbiomarkers specific for eukaryotes (steranes) and evendiagnostic of particular groups. However, it is possiblethat steranes would adhere to the surface of cell wallsand not originate from the cell membrane that was oncecontained in that wall. Conclusive evidence (that thebiomarker comes from the analyzed microfossil) wouldbe obtained if various taxa from the same sample showdifferent biomarker signatures that also differ from thehost sediment. Arouri et al. (1999, 2000) haveattempted to elucidate the biological affinities of singleacritarchs using micropyrolysis GC/MS but couldobtain only limited chemical data, possibly due totechnical problems (Marshall et al., 2005). Greenwoodet al. (2000) analyzed the Permian Tasmanites pickedfrom Tasmanite oil samples, suggesting a tricyclicterpenoid composition. Recently, Versteegh et al.(2004b) performed flash pyrolysis on a mixture oflate Cambrian–early Ordovician galeate acritarchspresenting morphological, paleoecological and paleo-geographical similarities with dinoflagellate cysts. Thistechnique seems promising, especially if applied tomonospecific samples.

3. Discussion

3.1. Paleobiology

As illustrated above, an approach combining micros-copy andmicrochemistry might clarify several importantsteps in the evolution of early biosphere paleobiologyand paleoecology, by identifying ancestors of extantclades or at least important steps in cellular, biochemicaland ecological evolution. We have applied this approachto Proterozoic microfossils exquisitely preserved at thestructural and ultrastructural level. Different wall ultra-structures characterize different taxa, implying diversebiological affinities and a level of diversity undetectedvia light microscopy. Moreover, the morphologicalcomplexity shown by taxa with processes and/or withwalls made of polygonal plates imply cytologicalcomplexity in early Mesoproterozoic protists—theevolution of a eukaryotic cell with nucleus, cytoskeletonand internal membranes involved in secretion and self-assembly of wall components (Javaux et al., 2001,2003). In particular, the complex and highly variablemorphology of Tappania plana shows that metabolicallyactive cells with cytoskeletal sophistication limited

entirely to a eukaryotic grade of organization werealready present in early Mesoproterozoic (1.5 Ga)ecosystems (Javaux et al., 2001, 2003). We have recentlydiscovered ornamented acritarchs in late Paleoproter-ozoic shales from China (1800–1625 Ma ChuanlinggouFormation) and Australia (1650 Ma Mallapunyah Fm,Fig. 1: 11) permitting us to extend significantly thestratigraphic range of fossil evidence for early eukar-yotes (Javaux et al., 2004d). This, in turn, implies anearlier evolution of the domain Eucarya, as suggested bythe presence of 2.7 Ga biomarkers in the FortescueGroup of Australia (Brocks et al., 1999, 2003). Fossilevidence and molecular phylogeny studies indicate anearly origin of photosynthetic eukaryotes in the latePaleoproterozoic (Su et al, 2004) or early Mesoproter-ozoic (Hedges et al., 2004) and diversification in theMesoproterozoic (Knoll, 1996; Anbar and Knoll, 2002;Douzery et al., 2004; see review in Javaux et al., 2003and in Porter, 2004). The presence of abundant and well-preserved multicellular red algae in ∼ 1.2 Ga chert fromArctic Canada (Butterfield, 2000) indicates the earlyevolution of multicellularity, sexual reproduction, pri-mary plastid endosymbiosis and consequently priorevolution of heterotrophy (when an eukaryotic hostengulfed a cyanobacterial ancestor of the chloroplast).Xanthophyte algae (Palaeovaucheria) from the 1 GaLakhanda Formation, Siberia (German, 1990; Woods etal., 1998) and the ca. 700–800 Ma shales in Spitsbergen(Butterfield, 2004) indicate the appearance of strameno-piles (which include diatoms, xanthophytes and brownalgae) and of secondary symbiosis (involving a red alga-like endosymbiont). Upper Paleoproterozoic throughLower Neoproterozoic rocks have also yielded biomar-kers of alveolates (which include dinoflagellates andciliates, among other groups) (Summons and Walter,1990; Pratt et al., 1991; Summons et al., 1992;Moldowan et al., 1996). It is unknown whetherProterozoic dinoflagellates were photosynthetic. Filoseand lobose testate amoebae from ∼750 Ma rocks of theChuar Group, Arizona (Porter et al., 2003) andmetazoans from the ∼600 Ma Doushantuo Formationof China (Xiao and Knoll, 1999, 2000; references inJavaux et al., 2003) provide a firm calibration point forthe great clade that includes animals, fungi and theamoebozoans (Baldauf, 2003; Nikolaev et al., 2004), notto mention direct evidence for heterotrophic eukaryotes,eukaryotic biomineralization and possibly predation.Finally, the recently discovered Neoproterozoic(∼850 Ma) Tappania record complex multicellularityof probably heterotrophic organisms, possibly micro-predator fungi, suggesting a high complexity in trophicinteractions in the early Neoproterozoic (Butterfield,


2005). These Neoproterozoic Tappania are suggested tobe related to the simpler Mesoproterozoic specimens(Butterfield, 2005), despite the presence of neck-likeextensions and the absence of hyphal fusion in the latter.In this context, it is interesting to note that theMesoproterozoic Tappania, bearing expansions sugges-tive of reproduction by budding, may resemble someyeast with bud scars however this feature occurs also inother eukaryotes. Very significantly, if further ultrastruc-tural and chemical studies confirm the fungal affinity ofthe Neoproterozoic Tappania, and its relationship toearly Mesoproterozoic Tappania, as suggested byButterfield (2005), it would provide the oldest taxonom-ically resolved eukaryote on record, the oldest knownexample of complex multicellularity, and a ∼1.4 Gacalibration point for the Opisthokonths (Butterfield,2005; Stokstad, 2005). A combination of microchemicaland ultrastructural analyses of the Neoproterozoic andMesoproterozoic Tappania fossils might give a moredefinitive answer.

In this paper, we have illustrated how in combinationwith molecular biology and geology, detailed studies oforganic walled microfossils focusing on wall morphol-ogy, structure, ultrastructure and chemistry will help usto understand better the early evolution of eukaryoticorganisms in the Proterozoic. This approach could alsoimprove our understanding of a more recent evolution oflife, in the Paleozoic. Paleozoic acritarchs are abundantin siliciclastic successions and surely played an impor-tant role in early food webs and ecosystems. Were theydinoflagellates without tabulation or something else?Were they photosynthetic or heterotrophic? Do theyrepresent many different biological groups? Thematerialis abundant but studied mostly for stratigraphy purposes,leaving these fundamental questions unanswered.

3.2. Exopaleobiology

The techniques described and illustrated in this shortreview have great potential for detecting and character-izing life–as we know it–beyond our planet, if it existsand is ever found. Instruments performing Ramanspectroscopy and mass spectrometry, and pyrolysisGC/MS are being miniaturized for use by robots on theplanet Mars (ESA Newsletter 4, 2004). Microscopicimager is already in use by the NASA Rovers Spirit andOpportunity for close examination of sedimentarystructures and rock textures, as well as other spectro-meters to characterize their mineralogy (Squyres et al.,2004). The Pasteur payload of the rover EXOMARSfrom ESA (ESA Pasteur Payload Newsletter, 2004)might include a drilling mole to study underground

samples protected from harsh surface conditions, higherresolution microscopes to permit detection of fossils andvarious instruments to measure carbon isotopic frac-tionation, and detect the presence of biosignatures suchas biomarkers, amino acids, genetic markers, pigmentsand biogenic minerals (Simoneit et al., 1998). Of course,meteorites and, in the future, returned samples fromMars can be examined in far greater details in Earthlaboratories equipped with instruments that cannot beminiaturized or manipulated by robots because ofsample preparation requirements (such as transmissionelectron microscopy).

4. Conclusions

Thus a combination of various techniques isavailable to determine the paleobiology of singleorganic-walled microfossils. We are now expandingour research to older Paleoproterozoic and Archeansiliciclastic and younger Paleozoic successions, as wellas characterizing chemical and morphological signa-tures of recent prokaryotes and protists that producefossilizable cells, to compare with the fossils. Indeed, agreat limitation in detecting the biological affinities ofmicrofossils is our currently limited knowledge of themorphology and chemical composition of decay-resistant cellular structures produced by various livingmicroorganisms. Our ongoing research includes thedetermination and characterization of resistant biopoly-mers in a range of living prokaryotes, protists and fungiby combined microscopy and microchemistry, thedetermination of the modifications of chemical compo-sition due to thermal alteration, and the comparison withcombined microscopy and microchemical analyses ofProterozoic and Archean microfossils. This will permitus to characterize biosignatures (morphology, biomar-kers, biopolymers and FTIR spectra) needed forpaleobiology and astrobiology. Understanding how lifeappeared and evolved on the only planet where it isknown so far is crucial if we are to look for lifeelsewhere. Such a multidisciplinary approach offers newpossibilities to investigate the record of early (and not soearly) life on Earth and beyond.

Acknowledgments

We thank the guest editor Thomas Servais forinviting our contribution in this volume issue followingthe 11th IPC meeting in Granada, Spain. We are verygrateful to the reviewers Nick Butterfield and GerardVersteegh for useful suggestions to improve ourmanuscript. We also thank Nick Butterfield for


discussion about Neoproterozoic Tappania. This re-search has benefited from discussions and/or collabora-tions in other papers with J. Brocks, K. Grey, A.H.Knoll, Y. Leiming, M.R. Walter and S. Xiao. Financialsupport came from Exobiology Grant NAG5-3645 andthe NASA Astrobiology Institute (granted to A.H.Knoll), the Australian Research Council and MacquarieUniversity, and the Belgian Science Federal PolicyOffice.

References

Abbott, S.T., Sweet, I.P., 2000. Tectonic control on third-ordersequences in a siliciclastic ramp-style basin: an example from theRoper Superbasin (Mesoproterozoic), northern Australia. Austra-lian Journal of Earth Sciences 47, 637–657.

Allard, B., Templier, J., Largeau, C., 1997. Artifactual origin ofmycobacterial bacteran: formation of melanoidin-like artifactualmacromolecular material during the usual isolation process.Organic Geochemistry 26, 691–703.

Allard, B., Templier, J., Largeau, C., 1998. An improved method forthe isolation of artifact-free algaenans from microalgae. OrganicGeochemistry 28, 543–548.

Anbar, A.D., Knoll, A.H., 2002. Proterozoic ocean chemistry andevolution: a bioinorganic bridge? Science 297, 1137–1142.

Arouri, K., Greenwood, P.F., Walter, M.R., 1999. A possiblechlorophycean affinity of some Neoproterozoic acritarchs. OrganicGeochemistry 30, 1323–1337.

Arouri, K., Greenwood, P.F., Walter, M.R., 2000. Biological affinitiesof Neoproterozoic acritarchs from Australia: microscopic andchemical characterisation. Organic Geochemistry 31, 75–89.

Ascaso, C., Wierzchos, J., 2002. New approaches to the study ofAntarctic lithobiontic microorganisms and their inorganic traces,and their application in the detection of life in Martian rocks.International Microbiology, 15 pp. doi:10.1007/s10123-002-0088-6.

Baldauf, S.L., 2003. The deep roots of eukaryotes. Science 300,1703–1706.

Bartley, J.K., 1996. Actualistic taphonomy of Cyanobacteria:implications for the Precambrian fossil record. Palaios 11,571–586.

Boyce, C.K., Cody, G.D., Feser, M., Knoll, A.H., Wirick, S., 2002.Organic chemical differentiation within fossil plant cell wallsdetected with X-ray spectromicroscopy. Geology 30 (11),1039–1042.

Brocks, J.J., Logan, G.A., Buick, R., Summons, R.E., 1999. Archeanmolecular fossils and the early rise of eukaryotes. Science 285,1033–1036.

Brocks, J.J., Buick, R., Summons, R.E., Logan, G.A., 2003. Areconstruction of Archean biological diversity based on molecularfossils from the 2.78–2.45 billion year old Mount BruceSupergroup, Hamersley Basin, western Australia. Geochim.Cosmochim. Acta 67 (22), 4321–4335.

Butterfield, N.J., 1997. Plankton ecology and the Proterozoic–Phanerozoic transition. Paleobiology 23, 247–262.

Butterfield, N.J., 2000. Bangiomorpha pubescens n. gen., n. sp.:implications for the evolution of sex, multicellularity and theMesoproterozoic–Neoproterozoic radiation of eukaryotes. Paleo-biology 26, 386–404.

Butterfield, N.J., 2004. A vaucheriacean alga from the middleNeoproterozoic of Spitsbergen: implications for the evolution ofProterozoic eukaryotes and the Cambrian explosion. Paleobiology30, 231–252.

Butterfield, N.J., 2005. Probable Proterozoic fungi. Paleobiology 31(1), 165–182.

Cady, S.L., Farmer, J.D., Grotzinger, J.P., Schpf, J.W., Steele, A.,2003. Morphological signatures and the search for life on Mars.Astrobiology 3, 351–368.

Cavalier-Smith, T., 2002. The neomuran origin of archaebacteria, thenegibacteria root of the universal tree and bacteria megaclassi-fication. International Journal of Systematic Microbiology 52,7–76.

Colbath, G.K., Grenfell, H.R., 1995. Review of biological affinities ofPaleozoic acid-resistant, organic walled eukaryotic algal micro-fossils (including acritarchs). Review of Palaeobotany andPalynology 86, 287–314.

Derenne, S., Largeau, C., Berkaloff, C., Rousseau, B., Wilhelm, C.,Hatcher, P.G., 1992. Non-hydrolysable macromolecular constitu-ents from outer walls of Chlorella fusca and Nanochlorumeucaryotum. Phytochemistry 31, 1923–1929.

Douzery, E.J.P., Snell, E.A., Bapteste, E., Delsuc, F., Philippe, H.,2004. The timing of eukaryotic evolution: does a relaxed molecularclock reconcile proteins and fossils? Proceedings of the NationalAcademy of Sciences 101 (43), 15386–15391.

Edwards, D., Axe, L., Parkes, R.J., 2004. Validating fossil bacteria:some cautionary tales from the mid-Palaeozoic. 48th Palaeonto-logical Association Annual Meeting, Lille 2004, Abstracts WithPrograms, p. 118.

ESA, 2004, Progress Letter Pasteur Instrument Payload for theExoMars Rover Mission. Number 4, August 2004.

Foster, C.B., Stephhenson, M.H., Marshall, C.P., Logan, G.A.,Greenwood, P.F., 2002. A revision of Reduviasporonites Wilson1962: description, illustration, comparison and biological affini-ties. Palynology 26, 35–58.

Gelin, F., Volkman, J.K., Largeau, C., Derenne, S., Sinninghe Damste,J.S., De Leeuw, J.W., 1999. Distribution of aliphatic, nonhydro-lyzable biopolymers in marine microalgae. Organic Geochemistry30, 147–159.

German, T.N., 1990. Organic World One Billion Years Ago. Nauka,Leningrad.

Graham, L.E., Wilcox, L.W., 2000. Algae. Prentice Hall Inc. 640 pp.Greenwood, P.F., Arouri, K.R., George, S.C., 2000. Tricyclic terpenoid

composition of Tasmanites kerogen as determined by pyrolysisGC-MS. Geochem. Cosmochim. Acta 64, 1249–1263.

Grey, K., 1998. Ediacarian acritarchs of Australia. Ph.D. Thesis,Macquarie University, Australia.

Grey, K., 1999. A modified palynological preparation technique forthe extraction of large Neoproterozoic acanthomorph acritarchsand other acid insoluble microfossils. Record-Geological Surveyof Western Australia 199/10, 1–23.

Hedges, S.B., Blair, J.E., Vebturi, M.L., Shoe, J.L., 2004. A moleculartimescale of eukaryote evolution and the rise of complexmulticellular life. BMC Evolutionary Biology 2004, 4, 2. http://www.biomedcentral.com/1471-2148/4/2.

Hoffman, H.J., 1999. Global distribution of the Proterozoic sphaer-omorph acritarch Valeria lophostriata (Jankauskas). Acta Micro-paleontologica Sinica 16, 215–224.

Hoffman, H.J., Jackson, G.D., 1996. Notes on the geology andpaleontology of the Thule Group, Ellesmere Islands, Canada andNorth-West Greenland. Bulletin-Geological Survey of Canada495, 1–26.

http://www.biomedcentral.com/1471-2148/4/2

http://www.biomedcentral.com/1471-2148/4/2

http://dx.doi.org/10.1007/s10123-002-0088-6


House, C.H., Schopf, J.W., McKeegan, K.D., Harrison, T.M., Stetter,K.O., 2000. On isotopic composition of individual Precambrianmicrofossils. Geology 28 (8), 707–710.

Javaux, E.J., Knoll, A.H., Walter, M.R., 2001. Morphological andecological complexity in early eukaryotic ecosystems. Nature 412,66–69.

Javaux, E.J., Knoll, A.H., Walter, M.R., 2003. Recognizing andinterpreting the fossils of early eukaryotes. Origins of Life andEvolution of the Biosphere 33, 75–94.

Javaux, E.J., Knoll, A.H., Walter, M.R., 2004a. TEM evidence foreukaryotic diversity in mid-Proterozoic oceans. Geobiology 2,121–132.

Javaux, E.J., Knoll, A.H., Marshall, C.P., Walter, M.R., 2004b.Recognizing early life on Earth and Mars. Special Paper-EuropeanSpace Agency 545, 127–130.

Javaux, E.J., Knoll, A.H., Marshall, C.P., Walter, M.R., 2004c.Protistan evolution in the Precambrian: a new multidisciplinaryapproach combining microscopy and microchemistry. GeologicalSociety of America annual meeting, Denver, CO, Abstracts withProgram, pp. 171–173.

Javaux, E.J., Knoll, A.H., Marshall, C.P., Xiao, S., Walter, M.R.,2004d. Early eukaryotes in Paleoproterozoic and Mesoproterozoicoceans. 48th Palaeontological Association Annual Meeting, Lille2004, Abstracts with programs.

Jux, U., 1968. Uber den Feinbau derWandung bei TasmanitesNewton.Palaeontographica Abteilung B Paläophytologie 124, 112–124.

Jux, U., 1971. Uber den Feinbau der Wandungen einiger paläozoischerBaltisphaeidiacean. Palaeontographica Abteilung B Paläophytolo-gie 136, 115–128.

Jux, U., 1977. Uber die wandstrukturen sphaeromorpher acritarchen:Tasmanites Newton, Tapajonites Sommer and Van Boekel,ChuariaWalcott. Palaeontographica Abteilung B Paläophytologie160, 1–16.

Kaufman, A.J., Xiao, S., 2003. High CO2 levels in the Proterozoicatmosphere estimated from analyses of individual microfossils.Nature 425, 279–282.

Kempe, A., Schopf, J.W., Altermann, W., Kudryavtsev, A.B., Heckl,W.M., 2002. Atomic force microscopy of Precambrian microscop-ic fossils. Proceedings of the National Academy of Sciences 99(14), 9117–9120.

Kjellström, G., 1968a. Remarks on the chemistry and ultrastructure ofthe cell wall of some Palaeozoic leiospheres. GeologiskaFöreningens I Stockholm Förhandlingar 90, 118–221.

Kjellström, G., 1968b. Remarks on the chemistry and ultrastructure ofthe cell wall of some Palaeozoic leiospheres. GeologiskaFöreningens I Stockholm Förhandlingar 90, 118–228.

Knoll, A.H., 1996. Archaean and Proterozoic palaeontology. In:Jansonius, J., McGregor, D.C. (Eds.), Palynology: Principlesand Applications. American Association of Stratigraphic Paly-nologists Foundation, vol. 1. Publishers Press, Salt Lake City,pp. 51–80.

Knoll, A.H., Golubic, S., 1992. Proterozoic and living cyanobacteria.In: Schidlowski, et al. (Ed.), Early Organic Evolution: Implicationsfor Mineral and Energy Resources. Springer-Verlag, Berlin,pp. 450–462.

Kokinos, J.P., 1994. Studies on the cell wall of dinoflagellate restingcysts: morphological development, ultrastructure, and chemicalcomposition. Ph.D. Thesis, Massachusetts Institute of Technology/Woods Hole Oceanographic Institution (Technical ReportWHOI-94-10).

Kokinos, J.P., Eglinton, T.I., Goñi, M.A., Boon, J.J., Martoglio, P.A.,Anderson, D.M., 1998. Characterization of a highly resistant

biomacromolecular material in the cell wall of a marinedinoflagellate resting cyst. Organic Geochemistry 28, 265–288.

Kralik, M., 1982. Rb–Sr age determinations on Precambrian carbonaterocks of the Carpentarian McArthur Basin, Northern Territory,Australia. Precambrian Research 18, 157–170.

Kudryavtsev, A.B., Schopf, J.W., Agresti, D.G., Wdowiak, T.J., 2001.In situ laser-Raman imagery of Precambrian microscopic fossils.Proceedings of the National Academy of Sciences of the UnitedStates of America, vol. 98, pp. 823–826.

Le Hérissé, A., 1984. Microplankton à paroi irganique du Silurien deGotland (Suède): observations au microsocpe électronique destructures de désenkystement. Review of Palaeobotany andPalynology 43, 217–236.

Lee, G.S.H., Mar, G.L., Rose, H.R., Marshall, C.P., Young, B.R.,Skilbeck, C.G., Wilson, M.A., 1998. X-ray photoelectronspectroscopy of conodonts. Organic Geochemistry 28, 759–765.

Loeblich, T.R., 1970. Morphology, ultrastructure and distribution ofPalaeozoic acritarchs. Proceedings of the North AmericanPalaeontological Convention, pp. 705–788.

Marshall, C.P., Rose, H.R., Lee, G.S.H., Mar, G.L., Wilson, M.A.,1999. Structure of organic matter in conodonts with differentcolour alteration indexes. Organic Geochemistry 30, 1339–1352.

Marshall, C.P., Mar, G.L., Nicoll, R.S., Wilson, M.A., 2001. Organicgeochemistry of artificially matured conodonts. Organic Geo-chemistry 32, 1055–1071.

Marshall, C.P, Allwood, A.C., Walter, M.R., Van Kranendonk, M.J.,Summons, R.E., 2004. Characterization of the carbonaceousmaterial in the 3.4 Ga Strelley Pool Chert, Pilbara Craton, westernAustralia. Geological Society of America annual meeting, Denver,CO, Abstracts with programs, vol. 36 (5), p. 453.

Marshall, C.P., Javaux, E.J., Knoll, A.H., Walter, M.R., 2005.Combined micro-Fourier transform infrared (FTIR) spectroscopyand micro-Raman spectroscopy of Proterozoic acritarchs: a newapproach to palaeobiology. Precambrian Research 138, 208–224.

Martin, F., 1993. Acritarchs: a review. Biological Review 68,475–538.

Martin, F., Kjellström, G., 1973. Ultrastructural study of someOrdovician acritarchs from Gotland, Sweden. Neues Jahrbuchfür Geologie und Paläontologie. Monatshefte 1, 44–54.

Moldowan, J.M., Talyzina, N.M., 1998. Biogeochemical evidences fordinoflagellate ancestors in the early Cambrian. Science 281,1168–1170.

Moldowan, J.M., Dahl, J., Jacobson, S.R., Huizinga, B.R., Fago, F.J.,Shetty, R., Watt, D.S., Peters, K.E., 1996. Chemostratigraphicreconstruction of biofacies: molecular evidence linking cyst-formingdinoflagellates with pre-Triassic ancestors. Geology 159–162.

Nikolaev, S.I., Berney, C., Farhni, J.F., Bolivar, I., Polet, S., Mylnikov,A.P., Aleshin, V.V., Petrov, N.B., Pawloski, J., 2004. The twilightof the Heliozoa and rise of Rhizaria, an emerging supergroup ofamoeboid eukaryotes. Proceedings of the National Academy ofSciences of the United States of America 101, 8066–8071.

Oehler, D.Z., 1977. Pyrenoid-like structures in late Precambrian algaefrom the Bitter Springs Formation of Australia. Journal ofPaleontology 51, 885–901.

Page, R.W., Jackson, M.J., Krasay, A.A., 2000. Constraining sequencestratigraphy in north Australian basins: SHRIMP U–Pb zirconchronology between Mt. Isa and McArthur River. AustralianJournal of Earth Sciences 47, 431–459.

Pearson, A., Budin, M., Brocks, J., 2003. Phylogenetic andbiochemical evidence for sterol synthesis in the bacteriumGemmata obscuriglobus. Proceedings of the National Academyof Sciences 100, 15352–15357.


Peat, C.J., 1981. Comparative light microscopy, scanning electronmicroscopy and transmission electron microscopy of selectedorganic walled microfossils. Journal of Microscopy 122, 287–294.

Popp, B.N., Laws, E.A., Bidigare, R.R., Dore, J.E., Hanson, K.L.,Wakeham, S.G., 1998. Effect of phytoplankton cell geometry oncarbon isotopic fractionation. Geochemica et Cosmochemica Acta62, 69–77.

Porter, S.M., 2004. Early eukaryotic diversification. In: Lipps, J.,Waggoner, B. (Eds.), Neoproterozoic–Cambrian Biological Revo-lutions. Paleontological Society Papers, vol. 10, pp. 35–50.

Porter, S.M., Knoll, A.H., 2000. Testate amoebae in the Neoproter-ozoic era: evidence from vase-shaped microfossils in the ChuarGroup, Grand Canyon. Paleobiology 26, 60–385.

Porter, S.M., Meisterfeld, R., Knoll, A.H., 2003. Vase-shapedmicrofossils from the Neoproterozoic Chuar Group, GrandCanyon: a classification guided by modern testate amoebae.Journal of Paleontology 77, 409–429.

Pratt, L.M., Summons, R.E., Hieshima, G.B., 1991. Sterane andtriterpane biomarkers in the Precambrian Nonesuch Formation,North American Midcontinent Rift. Geochimica et CosmochimicaActa 55, 911–916.

Schopf, J.W., Kudryayvtsev, A.B., Agresti, D.G., Wdowiak, T.J.,Czaja, A.D., 2002. Laser-Raman imagery of Earth's earliestfossils. Nature 416, 73–76.

Schouten, S., Breteler, W.C.M.K., Blokker, P., Schogt, N., Rupstra, W.I.C., Grice, K., Baas, M., Sinninghe Damsté, J.S., 1998. Biosyntheticeffects on the stable carbon isotopic compositions of algal lipids:implications for deciphering the carbon isotopic biomarker record.Geochimica et Cosmochimica Acta 62, 1397–1406.

Simoneit, B.R.T., Summons, R.E., Jahnke, L.L., 1998. Biomarkers astracers for life on early Earth and Mars. Origins of Life andEvolution of the Biosphere 28, 475–483.

Squyres S.W., and 49 authors, 2004. The Opportunity Rover's Athenascience investigation at Meridiani Planum, Mars. Science. 306,1698–1703.

Stokstad, E., 2005. Primordial fungus. Science 307, 204.Su, H., Hackett, Y.D., Ciniglia, C., Pinto, G., Bhattacharya, D., 2004.

A molecular timeline for the origin of photosynthetic eukaryotes.Molecular Biology and Evolution 21 (5), 809–818.

Summons, R.E., Walter, M.R., 1990. Molecular fossils and micro-fossils from Proterozoic sediments. American Journal of Science290-A, 212–244.

Summons, R.E., Powell, T.G., Boreham, C.J., 1988. Petroleumgeology and geochemistry of the Middle Proterozoic McArthurBasin, northern Australia: III. Composition of extractable hydro-carbons. Geochimica et Cosmochimica Acta 51, 3075–3082.

Summons, R.E., Thomas, J., Maxwell, J.R., Boreham, C.J., 1992.Secular and environmental constraints on the occurrence ofdinosterane in sediments. Geochimica et Cosmochimica Acta 56,2437–2444.

Summons, R.E., Jahnke, L.L., Hope, J.M., Logan, G.A., 1999. 2-Methylhopanoids as biomarkers for cyanobacterial oxygenicphotosynthesis. Nature 400, 554–557.

Talyzina, N.M., Moczydlowska, M., 2000. Morphological andultrastructural studies of some acritarchs from the Lower CambrianLukati Formation, Estonia. Review of Palaeobotany and Palynol-ogy 112, 1–21.

Talyzina, N.M., Moldowan, J.M., Johannisson, A., Fago, F.J., 2000.Affinities of early Cambrian acritarchs studied by using micros-copy, fluorescence flow cytometry and biomarkers. Review ofPalaeobotony and Palynology 108, 37–53.

Tegelaar, E.W., de Leeuw, J.W., Derenne, S., Largeau, C., 1989. Areappraisal of kerogen formation. Geochimica et CosmochimicaActa 53, 3103–3106.

Van Waveren, I., 1992. Morphology of probable planktonic crustaceaneggs from the Holocene of the Banda Sea (Indonesia). In: Head, M.J., Wrenn, J.H. (Eds.), Neogene and Quaternary DinoflagellateCysts and Acritarchs. American Association of StratigraphicPalynologists Foundation, Dallas, pp. 89–120.

Versteegh, J.M., Blokker, P., 2004. Resistant macromolecules of extantand fossil microalgae. Phycological Research 52, 325–339.

Versteegh, G.J.M., Blokker, P., Wood, G.D., Collinson, M.E.,Sinninghe Damste, J.S., de Leeuw, J.W., 2004a. An example ofoxidative polymerization of unsaturated fatty acids as a preserva-tion pathway for dinoflagellate organic matter. Organic Geochem-istry 35, 129–1139.

Versteegh, G.J.M., Raevskaya, E., Servais, T., 2004b. On thebiological affinities of galeate acritarchs: morphological andbiogeochemical data. Abstracts of XI Int. Palynol. Congress,Grenada, Spain. Polen, vol. 14, pp. 130–131.

Volkman, J.K., Barrett, S.M., Dunstan, G.A., Jeffrey, S.W., 1993.Geochemical significance of the occurrence of dinosterol and other4-methyl sterols in a marine diatom. Organic Geochemistry 20 (1),7–15.

Wall, D., 1962. Evidence from recent plankton regarding the biologicalaffinities of TasmanitesNewton 1875 and Leiosphaeridia Eisenack1958. Geological Magazine 4, 353–362.

Waterbury, J.B., Stanier, R.Y., 1978. Patterns of growth anddevelopment in Pleurocapsalean cyanobacteria. MicrobiologicalReview 42, 2–44.

Westall, F., 1999. The nature of fossil bacteria. Journal of GeophysicalResearch 104, 16437–16451.

Woods, K.N., Knoll, A.H., German, T.N., 1998. Xanthophyte algaefrom the Mesoproterozoic/Neoproterozoic transition: confirmationand evolutionary implications. Abstracts with Programs-Geolog-ical Society of America 30, A 232.

Wynn-Williams, D.D., Edwards, H.G.M., Newton, E.M., Holder, J.M.,2002. Pigmentation as a survival strategy for ancient and modernphotosynthetic microbes under high ultraviolet stress on planetarysurfaces. International Journal of Astrobiology 1, 39–49.

Xiao, S., Knoll, A.H., 1999. Fossil preservation in the NeoproterozoicDoushantuo phosphorite Lagerstätte, South China. Lethaia 32,219–240.

Xiao, S., Knoll, A.H., 2000. Phosphatized animal embryos from theNeoproterozoic Doushantuo Formation at Weng'an, Guizhou,South China. Journal of Paleontology 74, 767–788.

Xiao, S., Knoll, A.H., Kaufman, A.J., Yin, L., Zhang, Y., 1997.Neoproterozoic fossils in Mesoproterozoic rocks? Chemostrati-graphic resolution of a biostratigraphic conundrum from the NorthChina Platform. Precambrian Research 84, 197–220.

Yin, L., 1998. Acanthomorphic acritarchs from Meso-Neoproterozoicshales of the Ruyang Group, Shanxi, China. Review of Palaeo-botany and Palynology 98, 15–25.

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