A New Approach in Deciphering Early Protist Paleobiology and Evolution: Combined Microscopy and Microchemistry of Single Proterozoic Acritarchs ⁎ E.J

Review of Palaeobotany and Palynology 139 (2006) 1–15 www.elsevier.com/locate/revpalbo

A new approach in deciphering early protist paleobiology and evolution: 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, Belgium b 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 microchemical analyses of individual microfossils may offer further insights into the paleobiology and evolution of early microorganisms. We use our ongoing work on early Mesoproterozoic and Neoproterozoic assemblages, as well as other published work, as examples to illustrate how this approach may clarify the evolution of early microorganisms and we underline how useful this approach could be for palynologists working on younger material. Such a multidisciplinary approach offers new possibilities to investigate the biological 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 record crucial steps in the early evolution and diversification of the biosphere. Identifying the Acritarchs are organic-walled microfossils of un- biological affinities of these microscopic organisms will known biological affinities. They are conventionally clarify Proterozoic and Paleozoic microbial paleobiol- interpreted as algal cysts but most probably include a ogy and food webs (see Butterfield, 1997, 2000 for larger range of organisms such as prokaryotic sheaths, discussions on plankton food webs and ecological heterotroph protists or even parts of multicellular beings tiering in the late Proterozoic). It will also contribute (Van Waveren, 1992; Martin, 1993; Colbath and to phylogenetic reconstructions and improve our Grenfell, 1995; Butterfield, 2005). Biologists easily understanding of early evolutionary mechanisms and differentiate between prokaryotic and eukaryotic organ- patterns, and the early interactions between environment isms using molecular and cell biology, but these and life. characters rarely survive fossilization and so are not Comparative biology is useful when organisms generally available to the paleontologist. However, display characteristic morphological attributes, but this rarely occurs among acritarchs (for example early ⁎ Corresponding author. Fax: +32 4 366 2921. dinoflagellates might not show tabulation). Most acri- E-mail address: [email protected] (E.J. Javaux). tarchs have relatively simple morphologies; basically a

0034-6667/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.revpalbo.2006.01.005 2 E.J. Javaux, C.P. Marshal / Review of Palaeobotany and Palynology 139 (2006) 1–15 microscopic organic ball or tube (often flattened to single or very few microfossils allows characteriza- when preserved in shales) with or without various tion of the fine ultrastructure and microchemistry of the ornaments, that make comparison with recent clades material studied. In some cases, it is possible to relate difficult. New approaches are necessary to elucidate these with similar known features of recent organisms their paleobiology. and clarify the paleobiology of the fossils. Ideally, this Study of the wall ultrastructure can differentiate approach should be conducted within a good geological between prokaryotic and eukaryotic acid-resistant framework, in well-dated successions, with detailed microfossils and, in some cases, even identify particular sedimentological reconstruction of paleoenvironments. clades (review in Javaux et al., 2003, 2004a). Combin- This paper focus on techniques that can be applied to ing microscopy (light microscopy, scanning and trans- isolated acid-resistant organic-walled microfossils mitted electron microscopy) with microchemical extracted from shales, in order to characterize their analyses of individual microfossils offers further morphology, wall ultrastructure and chemistry and to insights into the paleobiology and evolution of early reconstruct their paleobiology (Table 1). Other techni- microorganisms. A combination of techniques applied ques (briefly mentioned in the text) can be used for visualizing microfossils still embedded in a mineral matrix (and too fragile to withstand extraction) and for Table 1 characterizing their elemental composition and their Summary of techniques that can be applied on single acritarchs distribution in the rock, and the biological–mineral extracted from shales by acid-maceration and possible paleobiological information interactions. Techniques Data Paleobiological information 2. Materials Transmitted Morphology Assemblage diversity light (ornamentation, color, microscopy branching, etc.) Most of the fossils used as examples here come from No. of specimens Characteristic morphology carbonaceous shales of the early Mesoproterozoic Roper No. of species Cell division patterns Group, northern Australia (Fig. 1). The sedimentary Reproduction mode architecture of the Roper Basin is well characterized Wall thickness, flexibility (Abbott and Sweet, 2000). The shales preserve abundant Taphonomy Multicellularity and exquisite organic-walled microfossils distributed in Biological affinities in assemblages showing an onshore–offshore pattern of some cases decreasing abundance, declining diversity and changing Scanning Detailed outer and Wall ornamentation and dominance (Javaux et al., 2001). U–Pb SHRIMP electron inner wall features structure sometimes analyses of zircons from an ash bed in the Mainoru microscope indicative of eukaryotc complexity Formation fix an age of 1492±3 Ma for early Roper Wall thickness deposition (Page et al., 2000). A 1429±31 Ma Rb–Sr Transmission Wall ultrastructure Biological affinities at age for illite in dolomitic siltstones near the top of the electron level of kingdom, succession is consistent with the zircon age, even if less microscopy class in some cases reliable (Kralik, 1982). Highly carbonaceous shales in Fluorescence Fluorescence of Linked to wall chemistry, confocal wall polymers applicable on very basinal deposits of the Velkerri Formation, near the top microscopy immature material of the Roper Group, also contain low abundances of EDEX Elemental Details on steranes sourced by eukaryotic organisms (Summons et composition preservation mode, al., 1988). The Roper microfossils have been variously FT-infrared Biopolymer Biological interpreted as the oldest unambiguous evidence for spectrometry composition affinities in some cases eukaryotes (Javaux et al., 2001, 2003), as complex FT-Raman Aromaticity and To use in prokaryotes (Cavalier-Smith, 2002) and as fungi spectrometry thermal maturity of combination with (Butterfield, 2005). Their exquisite preservation and organic matter FTIR for further their age makes them ideally suited for paleobiological characterization investigations. of wall chemistry Pyrolysis Biomarker Biological affinities One highly ornamented fossil also treated here, GC/MS composition in some cases Shuiyousphaeridium macroreticulatum, comes from SIMS C isotopes Metabolism in some shales of the top of the Ruyang Group, northern China cases, in combination (Yin, 1998). Ruyang deposition is not well constrained with morphology by radiometric dates, but appears to be at least broadly E.J. Javaux, C.P. Marshal / Review of Palaeobotany and Palynology 139 (2006) 1–15 3

2.1. Characterization of acritarch morphology and ultrastructure

2.1.1. Light microscopy Light microscopy is routinely used to examine the morphology of the fossils mounted on glass slides with transmitted light, as well as for determining the diversity of assemblages. In some cases, comparative morphology with extant groups might reveal a division or reproduction pattern or other morphological feature (for example, tabulation in dinoflagellates) unique to certain biological groups, therefore permitting the phylogenetic positioning of the specimens. Butterfield (2000) compared the division patterns of cells in various groups of algae and bacteria and concluded to a bangiophyte affiliation for a population of 1.2–1 Ga (gigayear-old) fossil preserved in cherts of the Hunting Formation, arctic Canada. This careful study identified the earliest eukaryote so far that can be related to an extant lineage. This approach has also been very successful in other cases, identifying cyanobacteria genera ranging from the Proterozoic to now (Knoll and Golubic, 1992). Examination of a large population of well-preserved specimens from a single bedding plane might also permit to unify seemingly unicellular disparate forms into a multicel- Fig. 1. Location and generalized stratigraphy of the Roper Group, northern Australia, showing stratigraphic distribution of facies and lular organism (for example, a diverse Neoproterozoic microfossils. Modified from Javaux et al. (2001). assemblage has been shown to be the disarticulated remains of a single vaucheriacean metaphyte, Butter- field, 2004). coeval with Roper sedimentation. A ca. 1000 Ma granite We have outlined several criteria for differentiating (U–Pb zircon date) intrudes the Ruyang succession, eukaryotic from prokaryotic fossil cells and to evaluate providing a minimum age for the group; moreover, their degree of complexity: the presence of processes abundant microdigitate precipitates and C-isotopic and/or other surface ornamentation, the wall structure, profiles that vary little from 0‰ in thick, overlying the presence of excystment structures and the wall carbonates suggest that Ruyang shales are older than ca. ultrastructure can all indicate a level of complexity 1250 Ma (Xiao et al., 1997). Ruyang shales share unknown in prokaryotes (Javaux et al., 2003). several distinctive taxa (species of Tappania, Valeria Features such as excystment structures or details of and Dictyosphaera) with the Roper Group. ornamentations are important for acritarch classification The palaeoecological distribution, morphology, wall and may demonstrate eukaryotic origin (Javaux et al., ultrastructure and microchemistry of the acritarchs 2003). Resting cells and reproductive cysts of many treated here have been described for Roper and Ruyang protists display micron-scale patterns of lineations, samples by Javaux et al. (2001, 2003, 2004a,b) and fields, spines or bosses not known among prokaryotic Marshall et al. (2005), and for Neoproterozoic acritarchs organisms. While prokaryotic organisms can synthesize from the Tanana Formation, Australia (ca. 590–565 Ma) both cell wall ornament and preservable structures, wall by Arouri et al. (1999, 2000) and Grey (1998). ornamentation consists mostly on nanoscale proteina- Microfossils were extracted from shales using a ceous (not acid-resistant) spines or bosses, and preserv- modified palynological method involving slow hydro- able structures such as cyanobacterial envelopes are not fluoric acid digestion with minimal agitation (Grey, ornamented (see review in Javaux et al., 2003). 1999). Single acritarchs were handpicked from kero- Cyanobacterial sheaths are preserved in the fossil gen isolate under a stereomicroscope using a record, in preference to the peptidoglycan-rich cell micropipette. walls, as shown by taphonomic experiments (Bartley, 4 E.J. Javaux, C.P. Marshal / Review of Palaeobotany and Palynology 139 (2006) 1–15

1996). It is unknown if the wall of other prokaryotes fossils from shales); however, they can be easily would withstand fossilization in shale and maceration in preserved by mineralization with silica (chert), calcium acids (technique used to extract organic-walled micro- carbonate (calcite, aragonite), calcium phosphate E.J. Javaux, C.P. Marshal / Review of Palaeobotany and Palynology 139 (2006) 1–15 5

(apatite), iron carbonate (siderite), iron oxide (hematite) observational and analytical techniques are required if and iron sulfur sulfide (pyrite) (Westall, 1999). the work is not limited to descriptive or stratigraphic The cyst walls of Phanerozoic protists commonly objectives. contain well-defined openings through which motile cells escape (e.g. Le Hérissé, 1984). These excystment 2.1.2. Scanning electron microscopy (SEM) structures range from simple perforations that run the Scanning electron microscopy (SEM) of microfossils circumference of cyst walls (‘medial splits’) to the mounted on aluminium stubs and covered with a thin polygonal archaeopyles of dinoflagellates. Excystment layer of gold or gold/palladium permits us to study structures are common in the Neoproterozoic and might details of surface sculpture or ornaments (e.g. detailed also occur in early Mesoproterozoic microfossils from study of excystment structures in Silurian acritarchs, Le the ∼1.5 Ga Roper Group, Australia in the form of Hérissé, 1984) or even wall structure in fractured enrolled half vesicles resulting from medial split specimens. (Valeria) or opening at the end of a neck-like extension For example, Valeria lophostriata, a species known (Tappania) although this need to be confirmed by from the late Paleoproterozoic to the Neoproterozoic on examination of a larger population (Javaux et al., 2001, four continents, is easily distinguished by its concentric 2003)(Fig. 2). striations observable by light microscopy (Fig. 2:9–11) Among the ∼1.5 Ga Roper Group microfossil (Hoffman and Jackson, 1996; Xiao et al., 1997; assemblage, population of Tappania plana consists on Hoffman, 1999; Javaux et al., 2001, 2004a,c). SEM vesicles bearing 0 to 20 heteromorphic processes, observation of this species from the ∼1.5 Ga Roper irregularly distributed about the vesicle. The processes Group, Australia, shows these striations to consist of may branch (Fig. 2: 2), they communicate with the parallel ridges uniformly spaced 1 μm apart on the vesicle interior and they have closed dark expanded end internal surface of the vesicle (Fig. 2: 10). At our (Fig. 2: 5). Some specimens have a neck-like expansion, knowledge, this micron-scale pattern in an acid-resistant open in two specimens and suggesting possible excyst- wall is unknown in prokaryotes and strongly suggests an ment structure (Fig. 2:3–4). Others bear up to three eukaryotic affinity (Javaux et al., 2004a). SEM study of rounded extensions, possibly suggesting reproduction another species from the Roper assemblage, Satka by budding (Fig. 2: 2). The population of process- favosa, shows clearly that the wall consists of polygonal bearing species T. plana includes vegetative cells with a plates that form a tessellated pattern, a wall construction complex and irregular morphology, possible excysting unknown in prokaryotes at our knowledge (Fig. 2:6–8) cysts and possible evidence for reproduction by (Javaux et al., 2004a). The acanthomorphic acritarch budding. Some of these features are characteristic of Shuiyousphaeridium macroreticulatum,fromthe metabolically active cells with cytoskeletal sophistica- ∼1.3 Ga Ruyang Group, China, has a reticulated surface tion, suggesting an eukaryotic grade of organization and numerous regularly spaced cylindrical processes (Javaux et al., 2001, 2003). In one specimen, the that flare outward (Fig. 3:10–13) (Xiao et al., 1997; processes have a septum (Fig. 2: 1), suggesting Javaux et al., 2001, 2004a). Close SEM examination of multicellularity, as proposed by Butterfield (2005). its wall, and more precisely through cracks in the wall, However, a larger number of specimens displaying revealed a wall structure consisting of closely packed, this character should be examined before confirming beveled hexagonal plates (Fig. 3: 11). This species this degree of complexity in early Mesoproterozoic displays clearly eukaryotic morphology: as Cavalier- eukaryotes. Smith (2002, p. 37) has pointed out, “cysts with spines These few examples illustrate how careful examina- or reticulate surface sculpturing would probably have tion of microfossil assemblages with transmitted light required both an endomembrane system and a cytoskel- microscopy may yield important paleobiological infor- eton, the most fundamental features of the eukaryotic mation. However, in many cases, other additional cell, for their construction”.

Fig. 2. Ornamentation and possible excystment structures in Late Paleoproterozoic and Early Mesoproterozoic acritarchs (organic-walled microfossils). All specimens illustrated are from the Roper Group of northern Australia, except specimen in Fig. 1(11), from the McArthur Group. (1– 5) Light photographs of Tappania plana, Roper Group. Arrows shows septum in processes in 1, branching of processes and bud like extensions in 2, opening of neck-like expansion suggesting excystment structures in 3 and 4, and dark rounded ends of processes in 5. (6–8) Satka favosa, Roper Group. (6–7) Light photographs, two halves possibly resulting from medial split in 7; (8) SEM image showing wall made of imbricated polygonal organic plates. (9–11) Valeria lophostriata. (9, 11) Light photographs showing characteristic concentric ornamentation, rolled half vesicle in 9 might result from medial split; (10) SEM image showing striations consisting on 1 μm spaced ridges on the inner part of the vesicle. Scale bar is 18 μm for 1, 25 μm for 2, 16 μm for 3, 20 μm for 4, 24 μm for 5 and 6, 10 μm for 7, 5 μm for 8, 22 μm for 9 and 11, and 2 μm for 10. 6 E.J. Javaux, C.P. Marshal / Review of Palaeobotany and Palynology 139 (2006) 1–15

Fig. 3. Morphology and wall ultrastructure of simple leiospheres from the Roper Group, northern Australia and complex acanthomorphic acritarch from 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 in the three species. (10–13) Shuiyousphaeridium macroreticulatum. (10, 11) SEM images showing details of wall made of imbricated beveled polygonal organic plates (10) and ornamentation with furcated processes (11), reticulate outer ornamentation; (12) TEM image illustrating multilayered wall with organic plates; (13) light photograph showing numerous regularly distributed processes and reticulate ornamentation. Scale bar 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 μm for 12.

Scanning electron microscope with back-scattered when ultrastructural elements (such as the wall) are electron imaging (SEM-BSE ) permits the visualization mineralized and their inorganic features differ from that of microfossils still embedded in their mineral matrix of the matrix. This technique is promising for studying E.J. Javaux, C.P. Marshal / Review of Palaeobotany and Palynology 139 (2006) 1–15 7 in situ the microorganism–mineral interface, especially efforts to decipher the earliest records of eukaryotic for endolithic or mat-forming microorganisms that diversification. When observed under transmitted light cannot withstand dissolution of the rock and determin- and SEM, leiospheres could be interpreted as cyano- ing biogenecity of the observed structures. Sample bacterial envelopes (Fig. 3:1–2, 4–5, 7–8). However, preparation includes fixation, staining and embedding in these cyanobacterial envelopes differ from protistan resin, followed by sectioning of the carbon-coated walls at the ultrastructural level, consisting of fibrous sample (Ascaso and Wierzchos, 2002). layers (Waterbury and Stanier, 1978, Fig. 1; J. Water- bury, pers. comm., 2003) quite distinct from any of the 2.1.3. Transmission electron microscopy (TEM) ultrastructures described in Javaux et al. (2004a) (Fig. 3: Transmission electron microscopy (TEM) is rarely 3, 6, 9). Observations with TEM revealed exquisitely used to elucidate the wall ultrastructure of single preserved wall ultrastructures of other Mesoproterozoic specimens. It is indeed a lengthy and delicate process microfossils ranging from single, homogeneous, elec- to embed single properly oriented microfossils in resins tron-dense layers of variable thickness- and variably and to cut ultrathin sections for examination in the TEM. ornamented- to multilayered walls differentiated by However, in conjunction with observations of living electron density and texture (Javaux et al., 2004a). microorganisms, this technique has proved very useful Among the Roper microfossils, a single homogeneous in identifying simple microfossils at the level of domain wall ultrastructure occurs in ornamented acritarchs such (Javaux et al., 2004a) and even in some cases at the level as Tappania plana, Satka favosa and Valeria lophos- of class (Talyzina and Moczydlowska, 2000). triata that clearly exhibit eukaryotic features (wall Examination of acritarch wall ultrastructure using ornamentation). Simple wall ultrastructure does not in TEM was initiated in the late 1960s to 1970s mostly on itself indicate eukaryotic affinity since diagenesis could Paleozoic microfossils (e.g. Jux, 1968, 1971, 1977; theoretically give this appearance to a fibrous prokary- Kjellström, 1968a,b; Loeblich, 1970; Martin and otic sheath. Other Roper and Ruyang acritarchs Kjellström, 1973; Oehler, 1977). More recently, affinity including the simple leiospheres and the complex to green algae (including prasinophytes) has been acanthomorphic Shuiyousphaeridium macroreticulatum shown for a few Neoproterozoic acritarch species from display multilayered walls. Thus, existing data indicate the Australian Central Superbasin (Grey, 1998; Arouri et that the structural complexity of eukaryotic cell walls al., 1999, 2000). Peat (1981) illustrated the homoge- can be preserved in ancient microfossils and distin- neous wall of 3 unidentified sphaeromorphs from the guished from acetolysis-resistant structures formed by McMinn Formation in the Mesoproterozoic Roper bacteria (Javaux et al., 2003). This, in turn, suggests that Group of Australia. Recent work by Talyzina and ultrastructural features can provide evidence for eu- Moczydlowska (2000) on Lower Cambrian acritarchs karyotic affinities, even in older Proterozoic fossils, revealed four structural types of vesicle wall in addition where simple morphology may be non-diagnostic. to their single- to multilayered composition and the Combined analysis of the fine structure with SEM variable thickness of the wall. The leiospheres studied and TEM permitted also to show that an acanthomorph showed a multilayered and composite wall similar to the acritarch from the Ruyang Group of China, Shuiyou- wall of chlorophycean algae, Order Chlorococcales sphaeridium macroreticulatum, has a reticulate wall (Talyzina and Moczydlowska, 2000). Some prasino- consisting of imbricated beveled polygonal organic phyte green algae have a diagnostic ultrastructure plates (Javaux et al., 2004a), and not of thickened, consisting of a homogeneous electron-dense wall polygonal cells as could be suggested on the base of punctuated by pore canals (Wall, 1962; Jux, 1968) that light microscopy alone (Butterfield, 2005). This species has been recognized in Cambrian Tasmanites tenellus may also show medial split excystment structures, (Talyzina and Moczydlowska, 2000). suggesting a cyst-like morphology, but whether it was a Our research on microfossils from the ∼1.5–1.4 Ga metabolically inert stage of a unicellular or multicellular Roper Group from Australia and Ruyang Group of organism, or whether it had a phototrophic or hetero- China lead to the discovery of eukaryotic ultrastructures trophic metabolism, is unknown, as underlined by preserved in acid-resistant walls of eight species of Butterfield (2005). organic-walled microfossils (Javaux et al., 2004a). In particular, our discovery of complex eukaryotic ultra- 2.2. Microchemistry of acritarch wall structures in the walls of morphologically simple mid- Proterozoic microfossils (leiospheres) indicates that Colbath and Grenfell (1995, p. 24) suggested that TEM can provide an important tool in paleontological “further advances in understanding the chemistry of 8 E.J. Javaux, C.P. Marshal / Review of Palaeobotany and Palynology 139 (2006) 1–15 sporopollenin-like compounds hold considerable prom- originally composed of non-resistant organic matter, ise for establishing biological affinities, when and if the and preserved as mineralized casts and molds coated methods can be applied to fossil material”. Indeed, with organic debris or iron minerals (Porter and Knoll, recently developed geochemical techniques now permit 2000). Elemental analysis has also been used in some microchemical analyses of individual microfossils (for cases to infer biogenicity of putative fossil bacteria, in example, Lee et al., 1998; Arouri et al., 1999, 2000; combination with other parameters (Ascaso and Wierz- Marshall et al., 1999, 2001; House et al., 2000; chos, 2002). Elemental analysis can provide crucial Kudryavtsev et al., 2001; Ascaso and Wierzchos, information for the paleontologist, by revealing that 2002; Boyce et al., 2002; Foster et al., 2002; Kempe seemingly biogenic morphologies can be artifacts of et al., 2002; Schopf et al., 2002). Micro-Fourier preparation (Edwards et al., 2004). transform infrared (FTIR) spectroscopy enables the Combining EDX facility with SEM-BSE permits to determination of biopolymer composition. Micro- analyze microfossils still embedded in the mineral Raman spectroscopy determines the degree of aroma- matrix and sometimes to identify biomobilization of ticity and the thermal maturity of the macromolecular elements (Al, K, Fe) at the former place of cellular structure of the cell wall. Ion microprobe measures the ultrastructural zones such as pyrenoids, chloroplasts and carbon isotopes. Significantly, all these techniques can cell walls (Ascaso and Wierzchos, 2002). be applied on a single acritarch. By comparison with bulk biomarker analyses, micro-FTIR and micro-Raman 2.2.2. In situ ion microprobe analysis of carbon spectroscopy techniques have the great advantage to be isotopes applicable on a very small sample (such as one Ion microprobe analyses of the carbon isotopes can microfossil), to provide data on the chemical composi- be carried out on individual organic-walled microfossils. tion of the microfossil with previously described House et al. (2000) measured δ13C values ranging morphology and ultrastructure, and to avoid contami- between −21.3‰ and −45.4‰ in microfossils pre- nation problems. Thus, these techniques permit us to served in stromatolitic cherts from the 850 Ma Bitter relate directly a morphology to a chemical composition, Spring Fm and the 2100 Ma Gunflint Fm, and suggested instead of dealing with bulk rock analyses or mixed these values to be consistent with a cyanobacterial microfossil assemblages. Micropyrolysis or flash pyrol- affinity based on morphological characteristics. Kauf- ysis permits us to determine the presence of biomarker man and Xiao (2003) have analyzed microfossils molecules on a few dozen specimens. Confocal extracted from shales of the Mesoproterozoic Ruyang microscopy can be used to detect autofluorescence of Group in North China and calculated δ13C values of the wall biopolymer on recent dinoflagellates (dinos- −25‰ for specimens of Dictyosphaera delicata. Based porin autofluorescence differs from that of sporopollen- on these values and the morphological complexity of D. in) (Graham and Wilcox, 2000) and on Cambrian delicata, the authors suggested that the Calvin cycle was acanthomorph acritarchs (Talyzina et al., 2000), but did in place at least 1400 Myr ago (Kaufman and Xiao, not give any results on our older Mesoproterozoic 2003). material (Javaux, personal observation). Other techni- However, the selectively preserved acritarch wall is ques seem promising but need further development the end product of complex biosynthetic pathway and (Cady et al., 2003). therefore is likely to have different values of isotopic fractionation than the primary photosynthetic metabolite 2.2.1. Energy dispersive X-ray spectroscopy (EDX) in (G. Versteegh, pers. comm.). Isotope fractionation may the SEM also vary with cell size and cell compartment (Popp et Energy dispersive X-ray spectroscopy (EDX) in the al., 1998; Schouten et al., 1998). The authors' SEM permits a qualitative and quantitative analysis of assumption that this species is a photosynthetic alga elements in microfossils such as C, O, P, S, Si and Fe. has not been confirmed by our ultrastructural and This data might explain in part the different textures of microchemical investigations of another species from microfossil walls seen with SEM and their mode of the same beds in the Ruyang Group, Shuiyousphaer- preservation (such as mineralization, carbonization or idium macroreticulatum, suggested to be a better phosphatization). For example, detailed SEM study of preserved variant of D. delicata (Xiao et al., 1997). Neoproterozoic vase-shaped microfossils from the Shuiyousphaeridium macroreticulatum does not show Chuar Group, Arizona, combined with elemental evidence of a trilaminar structure (TLS) in its wall analysis, revealed different modes of preservation. (Javaux et al., 2004a), nor the presence of the aliphatic These fossils, identified as testate amoebae, were biopolymer algaenan (Marshall et al., 2005); two E.J. Javaux, C.P. Marshal / Review of Palaeobotany and Palynology 139 (2006) 1–15 9 characteristics of several extant chlorococcalean and (Derenne et al., 1992; Gelin et al., 1999). Dinosporin is a eustigmatophycean green algae. However, some other highly resistant aromatic macromolecular substance green algae do not show these features, so their absence completely different from that of algaenan and has does not preclude a green algal affinity. Butterfield been isolated in the cyst of one marine dinoflagellate (2005) suggested a possible fungal affinity for S. Lingulodinium polyedrum (Kokinos, 1994; Kokinos et macroreticulatum. Further isotopic analyses by A.J. al.,1998). Versteegh et al. (2004a) reported a highly Kaufman (Univ. Maryland) of these species and other aliphatic composition of fossil dinoflagellate casts, but acritarchs we studied from the coeval Roper Group of these were not cyst walls enclosing a cavity, but rather Australia will aim to characterize the isotopic variations the secondary inside fillings of former vegetative cells, within and between species and facies, and might so their chemical composition is not comparable to a answer this question, along with other chemical biopolymer. Prokaryotic biopolymers called cyanobac- analyses. teran or bacteran has been shown to be artifacts of chemical isolation procedure (Allard et al., 1997, 1998). 2.2.3. Micro-Fourier transform infrared spectroscopy Arouri et al. (1999, 2000) have attempted to elucidate (micro-FTIR spectroscopy) the biological affinities of single acritarchs using Fourier FTIR spectroscopy is a sensitive and efficient transform infrared and Raman spectroscopy, with mixed technique commonly used for characterizing complex success due to technical problems. Micro-FTIR spec- organic macromolecules such as kerogen. We have troscopy of the microfossil Reduviasporonites by one of developed a micro-FTIR spectroscopic method tailored us (C.M.) in Foster et al. (2002) demonstrated that this to microscale characterization of acritarchs for elucidat- fossil thought to be a fungal spore and occurring as ing their cell-wall biopolymer composition and thus spikes at the Permo–Triassic boundary in various delineate biological affinity (Javaux et al., 2004b,c; locations was in fact algal in origin. Marshall et al., 2005). Our microchemical analyses of the wall of the Micro-Fourier transform infrared (FTIR) spectrosco- Neoproterozoic acritarch Tanarium conoideum from py involves the measurement of the wavelength and Observatory Hill, Australia, has revealed the presence of intensity of the absorption of IR radiation by a sample. a highly aliphatic hydrocarbon with a composition The wavelengths of IR adsorption bands are character- consistent with that of algaenan found in resistant walls istic of specific types of chemical bonds, allowing the of some microgreen algae (Fig. 4)(Javaux et al., 2004b,c; identification of organic compounds. Measuring the Marshall et al., 2005). The Neoproterozoic Leiosphaer- intensity of aliphatic C–H stretch (2800–3000 cm− 1), idia sp. from Observatory Hill, Australia may comprise a aromatic C–H stretch (3000–3100 cm− 1) and carbonyl new class of biopolymer containing significant aliphatic, groups CfO (1715–1740 cm− 1) provide information on branched aliphatic and saturated/olefinic carbon constitu- kerogen type or biopolymer type possibly specific of ents. Mesoproterozoic acritarch cell walls, including particular clades. Shuiyousphaeridium macroreticulatum,containapre- Sample preparation includes soaking in dichloro- dominantly aromatic biopolymer consisting of short methane to remove extraneous surface contaminants, aliphatic chains that are highly branched, with a few before placing the acritarch samples on potassium oxygenated functionalities (Fig. 4)(Javaux et al., 2004c; bromide slides (details in Marshall et al., 2005). Marshall et al., 2005). Differences among species of Cell wall chemistry of organic-walled microfossils similar thermal history occur in the presence and can provide clues to the biological relationships of abundance of aliphatic carbon and oxygen functionalities. Proterozoic and Paleozoic fossils. However, a major Among early Mesoproterozoic species of the Roper difficulty resides in the fact that very little is known Group, Australia, three species of leiopheres showed very about the chemical composition of potentially fossiliz- different multilayered wall ultrastructure (Javaux et al., able structures (for algae, see review by Versteegh and 2004a) but similar chemical composition (Marshall et al., Blokker, 2004). Acritarch wall is assumed to be 2005), underlying the importance of combining both the composed of sporopollenin, the component of the morphological and ultrastructural studies with the micro- resistant outer layer of spore and pollen walls, because chemical analyses for characterizing these early of its resistance to acids. Algaenans (Tegelaar et al., eukaryotes. 1989) are highly aliphatic biopolymers present in the vegetative cell wall of several marine and fresh water 2.2.4. Micro-Raman spectroscopy microalgae in the classes of Chlorophyceae and Laser micro-Raman spectroscopy measures the Eustigmatophyceae, and in one dinoflagellate species wavelength and intensity of inelastically scattered light 10 E.J. Javaux, C.P. Marshal / Review of Palaeobotany and Palynology 139 (2006) 1–15

Fig. 4. Representative micro-FTIR spectra for Neoproterozoic Tanarium conoideum from the Tanana Fm, Australia (a) and for the Mesoproterozoic Shuiyousphaeridium macroreticulatum, Ruyang Group, China (b). See explanations in the text. from molecules. Laser micro-Raman spectroscopy is reported the strong correlation of specific steranes used as a technique for the surface characterization of such as dinosterane with particular acritarch populations carbonaceous materials to elucidate carbon structure and in Lower Cambrian shales. It is certainly possible that a its thermal maturity. Sample preparation includes single species made both the biomarkers and the cleaning the acritarchs with dichloromethane, before preserved cyst walls, but insofar as sterols (the parent deposition on clean aluminum microscope slides molecules of geologically stable steranes) are not wall (Marshall et al., 2005). constituents (but, rather, part of the lipid bilayer of the In our experience, Raman spectroscopy does not cell membrane), it is hard to reject the alternative provide useful information about the molecular compo- explanation that steranes and cyst walls reflect two sition of Proterozoic acritarchs, but rather elucidates the different organisms that lived in ecological association carbon structure and thermal alteration of constituent (Javaux et al., 2003). Paleobotanists, who commonly organic matter (Marshall et al., 2005). Neither it can be find isolated seeds and leaves on a single bedding plane, used by itself to determine biogenicity of Archean will immediately recognize the problem. carbonaceous material (Marshall et al., 2004). Another major difficulty resides in the fact that very However pigments of living microbial cells in little is known about the chemical composition of Antarctic mats have been detected in situ using Raman various potentially fossilizable structures (such as spectroscopy acquired at another laser excitation line vegetative cells and cysts) of recent protists. The few (Wynn-Williams et al., 2002) that unfortunately cannot biomarkers determined so far include lipids (derived be applied on fossil material. from the lipid bilayer of cell membranes) and other molecules constituent of cell walls. They have a long 2.2.5. Micropyrolysis gas chromatography/mass spec- fossil record and permit to characterize the diversity of trometry (GC/MS) and flash pyrolysis ancient assemblages producing preservable and charac- Conventional extraction of lipid biomarker mole- teristic organic molecules but not necessarily preserv- cules can identify the presence of eukaryotic organisms able cellular structures. For example, some steranes are in an ancient ecosystem (Summons and Walter, 1990), biomarkers for eukaryotes and include a variety of C28– but correlation of biomarkers with specific microfossil 30 steranes with side-chains alkylation patterns (Sum- taxa is difficult. Moldowan and Talyzina (1998) mons and Walter, 1990; Pearson et al., 2003). E.J. Javaux, C.P. Marshal / Review of Palaeobotany and Palynology 139 (2006) 1–15 11

Dinosterane are derived from dinosterol produced by entirely to a eukaryotic grade of organization were dinoflagellates (and one diatom species; Volkman et al., already present in early Mesoproterozoic (1.5 Ga) 1993). 2-Methylhopanes are biomarkers for cyanobac- ecosystems (Javaux et al., 2001, 2003). We have recently teria (Summons et al., 1999). discovered ornamented acritarchs in late Paleoproter- Laser micropyrolysis of single acritarchs may show ozoic shales from China (1800–1625 Ma Chuanlinggou biomarkers specific for eukaryotes (steranes) and even Formation) and Australia (1650 Ma Mallapunyah Fm, diagnostic of particular groups. However, it is possible Fig. 1: 11) permitting us to extend significantly the that steranes would adhere to the surface of cell walls stratigraphic range of fossil evidence for early eukar- and not originate from the cell membrane that was once yotes (Javaux et al., 2004d). This, in turn, implies an contained in that wall. Conclusive evidence (that the earlier evolution of the domain Eucarya, as suggested by biomarker comes from the analyzed microfossil) would the presence of 2.7 Ga biomarkers in the Fortescue be obtained if various taxa from the same sample show Group of Australia (Brocks et al., 1999, 2003). Fossil different biomarker signatures that also differ from the evidence and molecular phylogeny studies indicate an host sediment. Arouri et al. (1999, 2000) have early origin of photosynthetic eukaryotes in the late attempted to elucidate the biological affinities of single Paleoproterozoic (Su et al, 2004) or early Mesoproter- acritarchs using micropyrolysis GC/MS but could ozoic (Hedges et al., 2004) and diversification in the obtain only limited chemical data, possibly due to Mesoproterozoic (Knoll, 1996; Anbar and Knoll, 2002; technical problems (Marshall et al., 2005). Greenwood Douzery et al., 2004; see review in Javaux et al., 2003 et al. (2000) analyzed the Permian Tasmanites picked and in Porter, 2004). The presence of abundant and well- from Tasmanite oil samples, suggesting a tricyclic preserved multicellular red algae in ∼ 1.2 Ga chert from terpenoid composition. Recently, Versteegh et al. Arctic Canada (Butterfield, 2000) indicates the early (2004b) performed flash pyrolysis on a mixture of evolution of multicellularity, sexual reproduction, pri- late Cambrian–early Ordovician galeate acritarchs mary plastid endosymbiosis and consequently prior presenting morphological, paleoecological and paleo- evolution of heterotrophy (when an eukaryotic host geographical similarities with dinoflagellate cysts. This engulfed a cyanobacterial ancestor of the chloroplast). technique seems promising, especially if applied to Xanthophyte algae (Palaeovaucheria) from the 1 Ga monospecific samples. Lakhanda Formation, Siberia (German, 1990; Woods et al., 1998) and the ca. 700–800 Ma shales in Spitsbergen 3. Discussion (Butterfield, 2004) indicate the appearance of strameno- piles (which include diatoms, xanthophytes and brown 3.1. Paleobiology algae) and of secondary symbiosis (involving a red alga- like endosymbiont). Upper Paleoproterozoic through As illustrated above, an approach combining micros- Lower Neoproterozoic rocks have also yielded biomar- copy and microchemistry might clarify several important kers of alveolates (which include dinoflagellates and steps in the evolution of early biosphere paleobiology ciliates, among other groups) (Summons and Walter, and paleoecology, by identifying ancestors of extant 1990; Pratt et al., 1991; Summons et al., 1992; clades or at least important steps in cellular, biochemical Moldowan et al., 1996). It is unknown whether and ecological evolution. We have applied this approach Proterozoic dinoflagellates were photosynthetic. Filose to Proterozoic microfossils exquisitely preserved at the and lobose testate amoebae from ∼750 Ma rocks of the structural and ultrastructural level. Different wall ultra- Chuar Group, Arizona (Porter et al., 2003)and structures characterize different taxa, implying diverse metazoans from the ∼600 Ma Doushantuo Formation biological affinities and a level of diversity undetected of China (Xiao and Knoll, 1999, 2000; references in via light microscopy. Moreover, the morphological Javaux et al., 2003) provide a firm calibration point for complexity shown by taxa with processes and/or with the great clade that includes animals, fungi and the walls made of polygonal plates imply cytological amoebozoans (Baldauf, 2003; Nikolaev et al., 2004), not complexity in early Mesoproterozoic protists—the to mention direct evidence for heterotrophic eukaryotes, evolution of a eukaryotic cell with nucleus, cytoskeleton eukaryotic biomineralization and possibly predation. and internal membranes involved in secretion and self- Finally, the recently discovered Neoproterozoic assembly of wall components (Javaux et al., 2001, (∼850 Ma) Tappania record complex multicellularity 2003). In particular, the complex and highly variable of probably heterotrophic organisms, possibly micro- morphology of Tappania plana shows that metabolically predator fungi, suggesting a high complexity in trophic active cells with cytoskeletal sophistication limited interactions in the early Neoproterozoic (Butterfield, 12 E.J. Javaux, C.P. Marshal / Review of Palaeobotany and Palynology 139 (2006) 1–15

2005). These Neoproterozoic Tappania are suggested to samples protected from harsh surface conditions, higher be related to the simpler Mesoproterozoic specimens resolution microscopes to permit detection of fossils and (Butterfield, 2005), despite the presence of neck-like various instruments to measure carbon isotopic frac- extensions and the absence of hyphal fusion in the latter. tionation, and detect the presence of biosignatures such In this context, it is interesting to note that the as biomarkers, amino acids, genetic markers, pigments Mesoproterozoic Tappania, bearing expansions sugges- and biogenic minerals (Simoneit et al., 1998). Of course, tive of reproduction by budding, may resemble some meteorites and, in the future, returned samples from yeast with bud scars however this feature occurs also in Mars can be examined in far greater details in Earth other eukaryotes. Very significantly, if further ultrastruc- laboratories equipped with instruments that cannot be tural and chemical studies confirm the fungal affinity of miniaturized or manipulated by robots because of the Neoproterozoic Tappania, and its relationship to sample preparation requirements (such as transmission early Mesoproterozoic Tappania, as suggested by electron microscopy). Butterfield (2005), it would provide the oldest taxonom- ically resolved eukaryote on record, the oldest known 4. Conclusions example of complex multicellularity, and a ∼1.4 Ga calibration point for the Opisthokonths (Butterfield, Thus a combination of various techniques is 2005; Stokstad, 2005). A combination of microchemical available to determine the paleobiology of single and ultrastructural analyses of the Neoproterozoic and organic-walled microfossils. We are now expanding Mesoproterozoic Tappania fossils might give a more our research to older Paleoproterozoic and Archean definitive answer. siliciclastic and younger Paleozoic successions, as well In this paper, we have illustrated how in combination as characterizing chemical and morphological signa- with molecular biology and geology, detailed studies of tures of recent prokaryotes and protists that produce organic walled microfossils focusing on wall morphol- fossilizable cells, to compare with the fossils. Indeed, a ogy, structure, ultrastructure and chemistry will help us great limitation in detecting the biological affinities of to understand better the early evolution of eukaryotic microfossils is our currently limited knowledge of the organisms in the Proterozoic. This approach could also morphology and chemical composition of decay- improve our understanding of a more recent evolution of resistant cellular structures produced by various living life, in the Paleozoic. Paleozoic acritarchs are abundant microorganisms. Our ongoing research includes the in siliciclastic successions and surely played an impor- determination and characterization of resistant biopoly- tant role in early food webs and ecosystems. Were they mers in a range of living prokaryotes, protists and fungi dinoflagellates without tabulation or something else? by combined microscopy and microchemistry, the Were they photosynthetic or heterotrophic? Do they determination of the modifications of chemical compo- represent many different biological groups? The material sition due to thermal alteration, and the comparison with is abundant but studied mostly for stratigraphy purposes, combined microscopy and microchemical analyses of leaving these fundamental questions unanswered. Proterozoic and Archean microfossils. This will permit us to characterize biosignatures (morphology, biomar- 3.2. Exopaleobiology kers, biopolymers and FTIR spectra) needed for paleobiology and astrobiology. Understanding how life The techniques described and illustrated in this short appeared and evolved on the only planet where it is review have great potential for detecting and character- known so far is crucial if we are to look for life izing life–as we know it–beyond our planet, if it exists elsewhere. Such a multidisciplinary approach offers new and is ever found. Instruments performing Raman possibilities to investigate the record of early (and not so spectroscopy and mass spectrometry, and pyrolysis early) life on Earth and beyond. GC/MS are being miniaturized for use by robots on the planet Mars (ESA Newsletter 4, 2004). Microscopic Acknowledgments imager is already in use by the NASA Rovers Spirit and Opportunity for close examination of sedimentary We thank the guest editor Thomas Servais for structures and rock textures, as well as other spectro- inviting our contribution in this volume issue following meters to characterize their mineralogy (Squyres et al., the 11th IPC meeting in Granada, Spain. We are very 2004). The Pasteur payload of the rover EXOMARS grateful to the reviewers Nick Butterfield and Gerard from ESA (ESA Pasteur Payload Newsletter, 2004) Versteegh for useful suggestions to improve our might include a drilling mole to study underground manuscript. We also thank Nick Butterfield for E.J. Javaux, C.P. Marshal / Review of Palaeobotany and Palynology 139 (2006) 1–15 13 discussion about Neoproterozoic Tappania.Thisre- Butterfield, N.J., 2004. A vaucheriacean alga from the middle search has benefited from discussions and/or collabora- Neoproterozoic of Spitsbergen: implications for the evolution of Proterozoic eukaryotes and the Cambrian explosion. Paleobiology tions in other papers with J. Brocks, K. Grey, A.H. 30, 231–252. Knoll, Y. Leiming, M.R. Walter and S. Xiao. Financial Butterfield, N.J., 2005. Probable Proterozoic fungi. Paleobiology 31 support came from Exobiology Grant NAG5-3645 and (1), 165–182. the NASA Astrobiology Institute (granted to A.H. Cady, S.L., Farmer, J.D., Grotzinger, J.P., Schpf, J.W., Steele, A., Knoll), the Australian Research Council and Macquarie 2003. Morphological signatures and the search for life on Mars. Astrobiology 3, 351–368. University, and the Belgian Science Federal Policy Cavalier-Smith, T., 2002. The neomuran origin of archaebacteria, the Office. negibacteria root of the universal tree and bacteria megaclassi- fication. International Journal of Systematic Microbiology 52, 7–76. References Colbath, G.K., Grenfell, H.R., 1995. Review of biological affinities of Paleozoic acid-resistant, organic walled eukaryotic algal micro- Abbott, S.T., Sweet, I.P., 2000. Tectonic control on third-order fossils (including acritarchs). Review of Palaeobotany and sequences in a siliciclastic ramp-style basin: an example from the Palynology 86, 287–314. Roper Superbasin (Mesoproterozoic), northern Australia. Austra- Derenne, S., Largeau, C., Berkaloff, C., Rousseau, B., Wilhelm, C., lian Journal of Earth Sciences 47, 637–657. Hatcher, P.G., 1992. Non-hydrolysable macromolecular constitu- Allard, B., Templier, J., Largeau, C., 1997. Artifactual origin of ents from outer walls of Chlorella fusca and Nanochlorum mycobacterial bacteran: formation of melanoidin-like artifactual eucaryotum. Phytochemistry 31, 1923–1929. macromolecular material during the usual isolation process. Douzery, E.J.P., Snell, E.A., Bapteste, E., Delsuc, F., Philippe, H., Organic Geochemistry 26, 691–703. 2004. The timing of eukaryotic evolution: does a relaxed molecular Allard, B., Templier, J., Largeau, C., 1998. An improved method for clock reconcile proteins and fossils? Proceedings of the National the isolation of artifact-free algaenans from microalgae. Organic Academy of Sciences 101 (43), 15386–15391. Geochemistry 28, 543–548. Edwards, D., Axe, L., Parkes, R.J., 2004. Validating fossil bacteria: Anbar, A.D., Knoll, A.H., 2002. Proterozoic ocean chemistry and some cautionary tales from the mid-Palaeozoic. 48th Palaeonto- evolution: a bioinorganic bridge? Science 297, 1137–1142. logical Association Annual Meeting, Lille 2004, Abstracts With Arouri, K., Greenwood, P.F., Walter, M.R., 1999. A possible Programs, p. 118. chlorophycean affinity of some Neoproterozoic acritarchs. Organic ESA, 2004, Progress Letter Pasteur Instrument Payload for the Geochemistry 30, 1323–1337. ExoMars Rover Mission. Number 4, August 2004. Arouri, K., Greenwood, P.F., Walter, M.R., 2000. Biological affinities Foster, C.B., Stephhenson, M.H., Marshall, C.P., Logan, G.A., of Neoproterozoic acritarchs from Australia: microscopic and Greenwood, P.F., 2002. A revision of Reduviasporonites Wilson chemical characterisation. Organic Geochemistry 31, 75–89. 1962: description, illustration, comparison and biological affini- Ascaso, C., Wierzchos, J., 2002. New approaches to the study of ties. Palynology 26, 35–58. Antarctic lithobiontic microorganisms and their inorganic traces, Gelin, F., Volkman, J.K., Largeau, C., Derenne, S., Sinninghe Damste, and their application in the detection of life in Martian rocks. J.S., De Leeuw, J.W., 1999. Distribution of aliphatic, nonhydro- International Microbiology, 15 pp. doi:10.1007/s10123-002- lyzable biopolymers in marine microalgae. Organic Geochemistry 0088-6. 30, 147–159. Baldauf, S.L., 2003. The deep roots of eukaryotes. Science 300, German, T.N., 1990. Organic World One Billion Years Ago. Nauka, 1703–1706. Leningrad. Bartley, J.K., 1996. Actualistic taphonomy of Cyanobacteria: Graham, L.E., Wilcox, L.W., 2000. Algae. Prentice Hall Inc. 640 pp. implications for the Precambrian fossil record. Palaios 11, Greenwood, P.F., Arouri, K.R., George, S.C., 2000. Tricyclic terpenoid 571–586. composition of Tasmanites kerogen as determined by pyrolysis Boyce, C.K., Cody, G.D., Feser, M., Knoll, A.H., Wirick, S., 2002. GC-MS. Geochem. Cosmochim. Acta 64, 1249–1263. Organic chemical differentiation within fossil plant cell walls Grey, K., 1998. Ediacarian acritarchs of Australia. Ph.D. Thesis, detected with X-ray spectromicroscopy. Geology 30 (11), Macquarie University, Australia. 1039–1042. Grey, K., 1999. A modified palynological preparation technique for Brocks, J.J., Logan, G.A., Buick, R., Summons, R.E., 1999. Archean the extraction of large Neoproterozoic acanthomorph acritarchs molecular fossils and the early rise of eukaryotes. Science 285, and other acid insoluble microfossils. Record-Geological Survey 1033–1036. of Western Australia 199/10, 1–23. Brocks, J.J., Buick, R., Summons, R.E., Logan, G.A., 2003. A Hedges, S.B., Blair, J.E., Vebturi, M.L., Shoe, J.L., 2004. A molecular reconstruction of Archean biological diversity based on molecular timescale of eukaryote evolution and the rise of complex fossils from the 2.78–2.45 billion year old Mount Bruce multicellular life. BMC Evolutionary Biology 2004, 4, 2. http:// Supergroup, Hamersley Basin, western Australia. Geochim. www.biomedcentral.com/1471-2148/4/2. Cosmochim. Acta 67 (22), 4321–4335. Hoffman, H.J., 1999. Global distribution of the Proterozoic sphaer- Butterfield, N.J., 1997. Plankton ecology and the Proterozoic– omorph acritarch Valeria lophostriata (Jankauskas). Acta Micro- Phanerozoic transition. Paleobiology 23, 247–262. paleontologica Sinica 16, 215–224. Butterfield, N.J., 2000. Bangiomorpha pubescens n. gen., n. sp.: Hoffman, H.J., Jackson, G.D., 1996. Notes on the geology and implications for the evolution of sex, multicellularity and the paleontology of the Thule Group, Ellesmere Islands, Canada and Mesoproterozoic–Neoproterozoic radiation of eukaryotes. Paleo- North-West Greenland. Bulletin-Geological Survey of Canada biology 26, 386–404. 495, 1–26. 14 E.J. Javaux, C.P. Marshal / Review of Palaeobotany and Palynology 139 (2006) 1–15

House, C.H., Schopf, J.W., McKeegan, K.D., Harrison, T.M., Stetter, biomacromolecular material in the cell wall of a marine K.O., 2000. On isotopic composition of individual Precambrian dinoflagellate resting cyst. Organic Geochemistry 28, 265–288. microfossils. Geology 28 (8), 707–710. Kralik, M., 1982. Rb–Sr age determinations on Precambrian carbonate Javaux, E.J., Knoll, A.H., Walter, M.R., 2001. Morphological and rocks of the Carpentarian McArthur Basin, Northern Territory, ecological complexity in early eukaryotic ecosystems. Nature 412, Australia. Precambrian Research 18, 157–170. 66–69. Kudryavtsev, A.B., Schopf, J.W., Agresti, D.G., Wdowiak, T.J., 2001. Javaux, E.J., Knoll, A.H., Walter, M.R., 2003. Recognizing and In situ laser-Raman imagery of Precambrian microscopic fossils. interpreting the fossils of early eukaryotes. Origins of Life and Proceedings of the National Academy of Sciences of the United Evolution of the Biosphere 33, 75–94. States of America, vol. 98, pp. 823–826. Javaux, E.J., Knoll, A.H., Walter, M.R., 2004a. TEM evidence for Le Hérissé, A., 1984. Microplankton à paroi irganique du Silurien de eukaryotic diversity in mid-Proterozoic oceans. Geobiology 2, Gotland (Suède): observations au microsocpe électronique de 121–132. structures de désenkystement. Review of Palaeobotany and Javaux, E.J., Knoll, A.H., Marshall, C.P., Walter, M.R., 2004b. Palynology 43, 217–236. Recognizing early life on Earth and Mars. Special Paper-European Lee, G.S.H., Mar, G.L., Rose, H.R., Marshall, C.P., Young, B.R., Space Agency 545, 127–130. Skilbeck, C.G., Wilson, M.A., 1998. X-ray photoelectron Javaux, E.J., Knoll, A.H., Marshall, C.P., Walter, M.R., 2004c. spectroscopy of conodonts. Organic Geochemistry 28, 759–765. Protistan evolution in the Precambrian: a new multidisciplinary Loeblich, T.R., 1970. Morphology, ultrastructure and distribution of approach combining microscopy and microchemistry. Geological Palaeozoic acritarchs. Proceedings of the North American Society of America annual meeting, Denver, CO, Abstracts with Palaeontological Convention, pp. 705–788. Program, pp. 171–173. Marshall, C.P., Rose, H.R., Lee, G.S.H., Mar, G.L., Wilson, M.A., Javaux, E.J., Knoll, A.H., Marshall, C.P., Xiao, S., Walter, M.R., 1999. Structure of organic matter in conodonts with different 2004d. Early eukaryotes in Paleoproterozoic and Mesoproterozoic colour alteration indexes. Organic Geochemistry 30, 1339–1352. oceans. 48th Palaeontological Association Annual Meeting, Lille Marshall, C.P., Mar, G.L., Nicoll, R.S., Wilson, M.A., 2001. Organic 2004, Abstracts with programs. geochemistry of artificially matured conodonts. Organic Geo- Jux, U., 1968. Uber den Feinbau der Wandung bei Tasmanites Newton. chemistry 32, 1055–1071. Palaeontographica Abteilung B Paläophytologie 124, 112–124. Marshall, C.P, Allwood, A.C., Walter, M.R., Van Kranendonk, M.J., Jux, U., 1971. Uber den Feinbau der Wandungen einiger paläozoischer Summons, R.E., 2004. Characterization of the carbonaceous Baltisphaeidiacean. Palaeontographica Abteilung B Paläophytolo- material in the 3.4 Ga Strelley Pool Chert, Pilbara Craton, western gie 136, 115–128. Australia. Geological Society of America annual meeting, Denver, Jux, U., 1977. Uber die wandstrukturen sphaeromorpher acritarchen: CO, Abstracts with programs, vol. 36 (5), p. 453. Tasmanites Newton, Tapajonites Sommer and Van Boekel, Marshall, C.P., Javaux, E.J., Knoll, A.H., Walter, M.R., 2005. Chuaria Walcott. Palaeontographica Abteilung B Paläophytologie Combined micro-Fourier transform infrared (FTIR) spectroscopy 160, 1–16. and micro-Raman spectroscopy of Proterozoic acritarchs: a new

Kaufman, A.J., Xiao, S., 2003. High CO2 levels in the Proterozoic approach to palaeobiology. Precambrian Research 138, 208–224. atmosphere estimated from analyses of individual microfossils. Martin, F., 1993. Acritarchs: a review. Biological Review 68, Nature 425, 279–282. 475–538. Kempe, A., Schopf, J.W., Altermann, W., Kudryavtsev, A.B., Heckl, Martin, F., Kjellström, G., 1973. Ultrastructural study of some W.M., 2002. Atomic force microscopy of Precambrian microscop- Ordovician acritarchs from Gotland, Sweden. Neues Jahrbuch ic fossils. Proceedings of the National Academy of Sciences 99 für Geologie und Paläontologie. Monatshefte 1, 44–54. (14), 9117–9120. Moldowan, J.M., Talyzina, N.M., 1998. Biogeochemical evidences for Kjellström, G., 1968a. Remarks on the chemistry and ultrastructure of dinoflagellate ancestors in the early Cambrian. Science 281, the cell wall of some Palaeozoic leiospheres. Geologiska 1168–1170. Föreningens I Stockholm Förhandlingar 90, 118–221. Moldowan, J.M., Dahl, J., Jacobson, S.R., Huizinga, B.R., Fago, F.J., Kjellström, G., 1968b. Remarks on the chemistry and ultrastructure of Shetty, R., Watt, D.S., Peters, K.E., 1996. Chemostratigraphic the cell wall of some Palaeozoic leiospheres. Geologiska reconstruction of biofacies: molecular evidence linking cyst-forming Föreningens I Stockholm Förhandlingar 90, 118–228. dinoflagellates with pre-Triassic ancestors. Geology 159–162. Knoll, A.H., 1996. Archaean and Proterozoic palaeontology. In: Nikolaev, S.I., Berney, C., Farhni, J.F., Bolivar, I., Polet, S., Mylnikov, Jansonius, J., McGregor, D.C. (Eds.), Palynology: Principles A.P., Aleshin, V.V., Petrov, N.B., Pawloski, J., 2004. The twilight and Applications. American Association of Stratigraphic Paly- of the Heliozoa and rise of Rhizaria, an emerging supergroup of nologists Foundation, vol. 1. Publishers Press, Salt Lake City, amoeboid eukaryotes. Proceedings of the National Academy of pp. 51–80. Sciences of the United States of America 101, 8066–8071. Knoll, A.H., Golubic, S., 1992. Proterozoic and living cyanobacteria. Oehler, D.Z., 1977. Pyrenoid-like structures in late Precambrian algae In: Schidlowski, et al. (Ed.), Early Organic Evolution: Implications from the Bitter Springs Formation of Australia. Journal of for Mineral and Energy Resources. Springer-Verlag, Berlin, Paleontology 51, 885–901. pp. 450–462. Page, R.W., Jackson, M.J., Krasay, A.A., 2000. Constraining sequence Kokinos, J.P., 1994. Studies on the cell wall of dinoflagellate resting stratigraphy in north Australian basins: SHRIMP U–Pb zircon cysts: morphological development, ultrastructure, and chemical chronology between Mt. Isa and McArthur River. Australian composition. Ph.D. Thesis, Massachusetts Institute of Technology/ Journal of Earth Sciences 47, 431–459. Woods Hole Oceanographic Institution (Technical Report Pearson, A., Budin, M., Brocks, J., 2003. Phylogenetic and WHOI-94-10). biochemical evidence for sterol synthesis in the bacterium Kokinos, J.P., Eglinton, T.I., Goñi, M.A., Boon, J.J., Martoglio, P.A., Gemmata obscuriglobus. Proceedings of the National Academy Anderson, D.M., 1998. Characterization of a highly resistant of Sciences 100, 15352–15357. E.J. Javaux, C.P. Marshal / Review of Palaeobotany and Palynology 139 (2006) 1–15 15

Peat, C.J., 1981. Comparative light microscopy, scanning electron Talyzina, N.M., Moldowan, J.M., Johannisson, A., Fago, F.J., 2000. microscopy and transmission electron microscopy of selected Affinities of early Cambrian acritarchs studied by using micros- organic walled microfossils. Journal of Microscopy 122, 287–294. copy, fluorescence flow cytometry and biomarkers. Review of Popp, B.N., Laws, E.A., Bidigare, R.R., Dore, J.E., Hanson, K.L., Palaeobotony and Palynology 108, 37–53. Wakeham, S.G., 1998. Effect of phytoplankton cell geometry on Tegelaar, E.W., de Leeuw, J.W., Derenne, S., Largeau, C., 1989. A carbon isotopic fractionation. Geochemica et Cosmochemica Acta reappraisal of kerogen formation. Geochimica et Cosmochimica 62, 69–77. Acta 53, 3103–3106. Porter, S.M., 2004. Early eukaryotic diversification. In: Lipps, J., Van Waveren, I., 1992. Morphology of probable planktonic crustacean Waggoner, B. (Eds.), Neoproterozoic–Cambrian Biological Revo- eggs from the Holocene of the Banda Sea (Indonesia). In: Head, M. lutions. Paleontological Society Papers, vol. 10, pp. 35–50. J., Wrenn, J.H. (Eds.), Neogene and Quaternary Dinoflagellate Porter, S.M., Knoll, A.H., 2000. Testate amoebae in the Neoproter- Cysts and Acritarchs. American Association of Stratigraphic ozoic era: evidence from vase-shaped microfossils in the Chuar Palynologists Foundation, Dallas, pp. 89–120. Group, Grand Canyon. Paleobiology 26, 60–385. Versteegh, J.M., Blokker, P., 2004. Resistant macromolecules of extant Porter, S.M., Meisterfeld, R., Knoll, A.H., 2003. Vase-shaped and fossil microalgae. Phycological Research 52, 325–339. microfossils from the Neoproterozoic Chuar Group, Grand Versteegh, G.J.M., Blokker, P., Wood, G.D., Collinson, M.E., Canyon: a classification guided by modern testate amoebae. Sinninghe Damste, J.S., de Leeuw, J.W., 2004a. An example of Journal of Paleontology 77, 409–429. oxidative polymerization of unsaturated fatty acids as a preserva- Pratt, L.M., Summons, R.E., Hieshima, G.B., 1991. Sterane and tion pathway for dinoflagellate organic matter. Organic Geochem- triterpane biomarkers in the Precambrian Nonesuch Formation, istry 35, 129–1139. North American Midcontinent Rift. Geochimica et Cosmochimica Versteegh, G.J.M., Raevskaya, E., Servais, T., 2004b. On the Acta 55, 911–916. biological affinities of galeate acritarchs: morphological and Schopf, J.W., Kudryayvtsev, A.B., Agresti, D.G., Wdowiak, T.J., biogeochemical data. Abstracts of XI Int. Palynol. Congress, Czaja, A.D., 2002. Laser-Raman imagery of Earth's earliest Grenada, Spain. Polen, vol. 14, pp. 130–131. fossils. Nature 416, 73–76. Volkman, J.K., Barrett, S.M., Dunstan, G.A., Jeffrey, S.W., 1993. Schouten, S., Breteler, W.C.M.K., Blokker, P., Schogt, N., Rupstra, W.I. Geochemical significance of the occurrence of dinosterol and other C., Grice, K., Baas, M., Sinninghe Damsté, J.S., 1998. Biosynthetic 4-methyl sterols in a marine diatom. Organic Geochemistry 20 (1), effects on the stable carbon isotopic compositions of algal lipids: 7–15. implications for deciphering the carbon isotopic biomarker record. Wall, D., 1962. Evidence from recent plankton regarding the biological Geochimica et Cosmochimica Acta 62, 1397–1406. affinities of Tasmanites Newton 1875 and Leiosphaeridia Eisenack Simoneit, B.R.T., Summons, R.E., Jahnke, L.L., 1998. Biomarkers as 1958. Geological Magazine 4, 353–362. tracers for life on early Earth and Mars. Origins of Life and Waterbury, J.B., Stanier, R.Y., 1978. Patterns of growth and Evolution of the Biosphere 28, 475–483. development in Pleurocapsalean cyanobacteria. Microbiological Squyres S.W., and 49 authors, 2004. The Opportunity Rover's Athena Review 42, 2–44. science investigation at Meridiani Planum, Mars. Science. 306, Westall, F., 1999. The nature of fossil bacteria. Journal of Geophysical 1698–1703. Research 104, 16437–16451. Stokstad, E., 2005. Primordial fungus. Science 307, 204. Woods, K.N., Knoll, A.H., German, T.N., 1998. Xanthophyte algae Su, H., Hackett, Y.D., Ciniglia, C., Pinto, G., Bhattacharya, D., 2004. from the Mesoproterozoic/Neoproterozoic transition: confirmation A molecular timeline for the origin of photosynthetic eukaryotes. and evolutionary implications. Abstracts with Programs-Geolog- Molecular Biology and Evolution 21 (5), 809–818. ical Society of America 30, A 232. Summons, R.E., Walter, M.R., 1990. Molecular fossils and micro- Wynn-Williams, D.D., Edwards, H.G.M., Newton, E.M., Holder, J.M., fossils from Proterozoic sediments. American Journal of Science 2002. Pigmentation as a survival strategy for ancient and modern 290-A, 212–244. photosynthetic microbes under high ultraviolet stress on planetary Summons, R.E., Powell, T.G., Boreham, C.J., 1988. Petroleum surfaces. International Journal of Astrobiology 1, 39–49. geology and geochemistry of the Middle Proterozoic McArthur Xiao, S., Knoll, A.H., 1999. Fossil preservation in the Neoproterozoic Basin, northern Australia: III. Composition of extractable hydro- Doushantuo phosphorite Lagerstätte, South China. Lethaia 32, carbons. Geochimica et Cosmochimica Acta 51, 3075–3082. 219–240. Summons, R.E., Thomas, J., Maxwell, J.R., Boreham, C.J., 1992. Xiao, S., Knoll, A.H., 2000. Phosphatized animal embryos from the Secular and environmental constraints on the occurrence of Neoproterozoic Doushantuo Formation at Weng'an, Guizhou, dinosterane in sediments. Geochimica et Cosmochimica Acta 56, South China. Journal of Paleontology 74, 767–788. 2437–2444. Xiao, S., Knoll, A.H., Kaufman, A.J., Yin, L., Zhang, Y., 1997. Summons, R.E., Jahnke, L.L., Hope, J.M., Logan, G.A., 1999. 2- Neoproterozoic fossils in Mesoproterozoic rocks? Chemostrati- Methylhopanoids as biomarkers for cyanobacterial oxygenic graphic resolution of a biostratigraphic conundrum from the North photosynthesis. Nature 400, 554–557. China Platform. Precambrian Research 84, 197–220. Talyzina, N.M., Moczydlowska, M., 2000. Morphological and Yin, L., 1998. Acanthomorphic acritarchs from Meso-Neoproterozoic ultrastructural studies of some acritarchs from the Lower Cambrian shales of the Ruyang Group, Shanxi, China. Review of Palaeo- Lukati Formation, Estonia. Review of Palaeobotany and Palynol- botany and Palynology 98, 15–25. ogy 112, 1–21.