Cell evolution and Earth history: stasis and revolution

(b) The contrast between negibacteria and posibacteria

The most fundamental distinction within eubacteria is between Posibacteria, invariably bounded by one membrane (e.g. Bacillus, Streptomyces), and Negibacteria, always having two distinct membranes (e.g. Escherichia, Salmonella, cyanobacteria, spirochaetes; figure 3). The negibacterial inner membrane is homologous to the single cytoplasmic membrane (CM) of posibacteria and archaebacteria (collectively called unibacteria; Cavalier-Smith 2002a), whereas their OM is unique and homologous to the OM of mitochondria and chloroplasts, which evolved from enslaved negibacteria (Cavalier-Smith 2002a, 2006b, in press). All OM lipids are made in the CM and exported to the OM, probably through specific contact sites, Bayer's patches. OM proteins are made by ribosomes on the CM cytosolic face and transported across the periplasmic space (that between the CM and OM) helped by specific chaperones. These lipid- and protein-export mechanisms may be homologous for all negibacteria, but are well studied only in proteobacteria. In negibacteria, the murein peptidoglycan wall, when present—as it was ancestrally (lost only within planctobacteria and by mitochondria and most chloroplasts)—is much thinner than in posibacteria and lies between the CM and OM, to which it is attached by lipoproteins.

Figure 3

Membrane evolution and the fundamental contrast between negibacteria and posibacteria. Earliest cells were negibacteria bounded by two lipoprotein membranes: an inner cytoplasmic membrane (CM) impermeable to small hydrophilic molecules (with alpha-helical membrane proteins: green) and an OM, more permeable because of large proteinaceous pores, made by porin proteins. Between the two negibacterial membranes is the murein sacculus or cell wall: a gigantic hollow bag-shaped peptidoglycan molecule covalently cross-linked in three dimensions, rigidly enough to resist high internal osmotic pressures of more than 20 atm. The simplest negibacterial envelope (right) is in Chlorobacteria, which lack Omp85 that inserts OM β-barrel proteins in glycobacteria (left) and Hadobacteria (not shown: intermediate in complexity between chlorobacteria and glycobacteria), in both of which all known OM proteins are β-barrels. In eobacteria, the CM and OM both consist of acyl ester phospholipids plus embedded proteins. Glycobacteria retained these, but also have hopanoids that make membranes more rigid, and replaced phospholipids in the OM outer leaflet by the immensely more complex lipopolysaccharide (LPS), which so dramatically reduces OM permeability that they simultaneously evolved novel protein export machinery (using the β-barrel TolC protein) and the TonB importer of small molecules (Cavalier-Smith 2006a). For clarity, lipoproteins and Bayer's patches are not shown for glycobacteria. Posibacteria lack an OM, but have a much thicker wall of murein peptidoglycan supplemented by other polymers (teichoic acids in Endobacteria; a disparate array in Actinobacteria). Murein walls can grow and divide only by cleavage of covalent bonds by murein hydrolases. The transition from negibacteria to posibacteria involved loss of the cell wall and the origin of novel families of sortase enzymes that recognize a C-terminal LPXT motif on numerous extracellular proteins which they cleave and use for attaching them covalently to the cell wall, preventing their escape as would otherwise be likely without an OM. Posibacterial wall-attached proteins include murein lipoproteins with hydrophobic lipid tails embedded in the outer leaflet of the cytoplasmic membrane, not in the OM inner leaflet as in negibacteria.

The lipid tails of posibacterial lipoproteins are in the CM outer leaflet; their protein part is covalently attached to the thick murein wall by sortase enzymes that recognize cell wall sorting signals near the C-terminus of its precursor, after its export to the periplasmic space by SRP (Comfort & Clubb 2004). Prior to this, sortases cleave the hydrophobic C-terminal sequence that temporarily anchors it to the CM. Hundreds of cell wall proteins, mostly not lipoproteins, are thus anchored to the thick murein of posibacteria before the N-terminal signal sequence is cleaved. Most posibacteria have sortases of 2–4 subfamilies with different substrate specificities (Comfort & Clubb 2004). Sortases are a synapomorphy for posibacteria (comprising subphyla Endobacteria and Actinobacteria), strongly supporting their monophyly (Cavalier-Smith 2006a). They probably evolved to attach the wall firmly to the CM and prevent soluble periplasmic proteins escaping when their cenancestor arose by losing the negibacterial OM.

Endobacteria include ancestrally endospore-forming Gram-positive bacteria, e.g. Bacillus, Clostridium, Staphylococcus, and the parasitic Mollicutes (mycoplasmas, spiroplasmas) that secondarily lost murein, lipoproteins and the sortase machinery, and miniaturized their genomes. Endobacteria also exclude the ancestrally endospore-producing Eurybacteria (e.g. Heliobacteria, Sporomusa), which group with them on most trees (Woese et al. 1985), and so are often also misleadingly called low-GC Gram-positives. Eurybacteria are neither Gram-positive nor posibacteria, but negibacteria with OM and endospores; as discussed below, they are probably ancestral to posibacteria (Cavalier-Smith 2006a). Thermotogales are also Eurybacteria, despite frequent, probably artefactual, grouping on sequence trees with neomura and/or Aquificales (Cavalier-Smith 2006a).

(c) Negibacteria preceded posibacteria

The transition between negibacteria and posibacteria occurred in cells possessing eubacterial flagella (Cavalier-Smith 2006a). Determining whether flagella originally evolved in posibacteria or in negibacteria polarizes the transition. Eubacterial flagella have three parts: a static motor embedded in the CM, typically covalently attached to the murein wall; the cylindrical basal body, rotated by a proton current through the associated motor; the helical shaft outside the CM passively transmits rotation to the environment. As evolutionary precursors of both basal body and motor exist only in negibacteria, never posibacteria, flagella evolved first in negibacteria and were transmitted vertically to the first posibacterium (figure 4). This unambiguously polarizes evolution from negibacteria to posibacteria, not the reverse, so the ancestral posibacterium lost the OM; the simplest mechanistic cause of this loss was sudden murein hypertrophy thickening the wall enough to break contacts between the CM and OM at the Bayer's patches, thus preventing transfer of lipids and LPS to the OM, causing its irreversible loss (Cavalier-Smith 1987b). OM loss had two coevolutionary consequences: loss of flagellar L-rings that in negibacteria except spirochaetes embed the basal body in the OM, and origin of the posibacterial sortase enzymes to attach periplasmic proteins to the thick murein wall, thereby preventing their loss to the environment when the OM disappeared.

Figure 4

Flagellar origin unambiguously polarizes evolution from negibacteria to posibacteria. The TonB importer and its force-providing proton channel ExbB are homologues of the motor proteins MotB and MotA of eubacterial flagella and probably their evolutionary precursors (all in green); they are found in all glycobacterial phyla, including the totally non-flagellate Cyanobacteria, but never in Posibacteria or Eobacteria. The other key flagellar precursor was probably the hollow cylindrical junctional pore complex of cyanobacteria that carries a slime secretion nozzle used for gliding motility, which arguably became the flagellar basal body (Cavalier-Smith 2006a). Eubacterial flagella probably originated by novel functional association between these previously unrelated precursors: proton inflow through the MotA channel now powers basal body rotation instead of molecular import across the OM like ExcB. Both TonB and MotB are fixed to the rigid murein layer. To enable the cell to swim and not merely rotate relative to its secreted slime, it evolved the hook and shaft, made from a protein family that arose by gene duplication from unknown precursors (slime nozzle constituent?). As motor and basal body precursors are both totally absent from posibacteria, and MotB betrays a chimaeric ancestry from TonB and OM protein OmpA, also never in posibacteria, flagella cannot have arisen in a cell lacking an OM. They must have arisen in a negibacterium and been inherited by posibacteria. Therefore, posibacteria are derived from negibacteria. Evolution occurred in the direction of the arrows not its reverse. The L-ring bushing that lodges the distal end of the basal body in the OM of typical negibacteria was probably lost after the OM, when its function disappeared in the first posibacterium (Cavalier-Smith 2006a). Archaebacterial flagella are unrelated (see text); eubacterial flagella were lost with murein during the neomuran revolution. The phylogeny on the right shows the three taxa with eubacterial flagella in black; Gracilicutes include Proteobacteria and three other phyla (see figure 5). Cyanobacteria are sisters to these flagellate taxa, not their direct ancestors; therefore, the junctional pore complex evolved prior to their common ancestor.

A second argument polarizing the transition from negibacteria to posibacteria is the mechanistic difficulty of adding an OM in one step to a flagellate posibacterium; nobody has explained how this could be done while plausibly allowing OM biogenesis. The difficulty of evolution is that hypothetical direction is compounded by the closest negibacterial relatives of posibacteria being Eurybacteria, which are glycobacteria with a much more complex OM than eobacteria, which are arguably more primitive (Cavalier-Smith 2006a). If evolution were in that direction, the OM must immediately have evolved hugely complex LPS and other complexities shared by glycobacteria, e.g. hopanoids, porins, TolC. If instead the first negibacteria were eobacteria, especially chlorobacteria with much the simplest OM, their origin is much simpler; I explained how the OM could have evolved simply and gradually to generate the first eobacterium (Cavalier-Smith 2001).

(a) The age of eukaryote taxa

The oldest unambiguously eukaryotic fossils are vase-shaped (e.g. Melanocyrillium), going back at least 760 Myr and probably slightly earlier (Porter & Knoll 2000; Porter et al. 2003). All are probably arcellinid lobose testate amoebae (phylum Amoebozoa), which originated relatively early within class Lobosea (Nikolaev et al. 2005). Those forms sometimes suggested to be euglyphid filose testate amoebae could also be agglutinated tests of arcellinids; I do not accept them as evidence for Cercozoa. Undoubted euglyphid fossils occur much later in amber (Foissner & Schiller 2001), but most Cercozoa fossilize poorly; exceptions are ebriids, first in the Palaeocene and throughout the Tertiary (Tappan 1980), and Phaeodaria, from ca 75 Myr (Takahashi 2004). Table 1 references the earliest fossils reasonably attributable to specific protist phyla (see also figure 7). Most groups apparently originate close to or a little before the Cambrian animal explosion or at the beginning of the Mesozoic or Caenozoic. Thus, protist phyla mainly radiated 570–500 Myr ago, soon after snowball earth unfroze, whereas many now important classes originated later during recovery from the end Permian and end Cretaceous mass extinctions (Mundil et al. 2004). Protist diversification broadly resembles the familiar pattern for animal phyla: sudden origins of many phyla near the Precambrian boundary, and novel classes and/or orders in the Early Mesozoic and Cenozoic to exploit niches or whole adaptive zones emptied by the greatest mass extinctions.

The marked discrepancy between these late morphological records of all fossils unambiguously attributable to specific eukaryotic phyla and the very early occurrence of steranes often interpreted as eukaryotic derivatives (Brocks et al. 1999) is explained if the 2.7 Gyr ago steranes actually came from eubacteria, at least four groups of which can make sterols (Cavalier-Smith 2002a; Pearson et al. 2003). Of these, Mycobacteria make cholesterol like eukaryotes, and belong to actinobacteria, the probable ancestors of eukaryotes. Sterol biosynthesis evolved polyphyletically by modifying universal eubacterial isoprenoid metabolism after atmospheric oxygenation made it mechanistically possible, long before eukaryotes. It was probably vertically inherited by the first eukaryote from an actinobacterium. Claims that mycobacterial sterol synthesis enzymes were laterally transferred from eukaryote DNA have not been substantiated by phylogenetic analysis (Cavalier-Smith & Chao 2003b), and reflect the false assumption that the ancestors of eukaryotes were archaebacteria, not modified derivatives of actinobacteria that generated the eukaryote/archaebacterial cenancestor. Fossil steranes are not sound evidence for eukaryotes (Cavalier-Smith 2002b); even were they specific for eukaryotes, the potential for downward mobility of petroleum fractions containing them is a perpetual worry for age authenticity—this problem is absent for body fossils, though their identification as eukaryotic or bacterial is also sometimes practically impossible, sometimes overoptimistic.

Some palaeontologists, rightly I think, do not accept Grypania (1.8 Gyr old; Han & Runnegar 1992; redated by Knoll et al. 2006) as eukaryotic rather than a giant cyanobacterial sheath. Identity of the largest, most complex fossils ca 1.5 Gyr ago often called eukaryotic (Javaux et al. 2001, 2003) is problematic. While some might be stem eukaryotes (Javaux et al. 2003), this is not compellingly demonstrated. I consider it more likely that most, probably all, are simply unusually large and complex prokaryotes. Unlike Javaux et al. (2001), I do not think their morphology demands the presence of an internal cytoskeleton or endomembranes; it is within the morphogenetic capabilities of bacteria. I agree with Butterfield (2005) that the morphologically most complex of these fossils is probably not an alga as originally assumed, but a sporangial entity broken from a branching trophic hyphal network, as are his beautiful 0.8–0.9 Gyr fossils (mostly Tappania). In 2001, I microscopically examined the Harvard Tappania plana specimen shown in fig. 4 of Javaux et al. (2003); I told Javaux that I thought it was not a single algal cell and that the structure labelled there by an arrow was the broken base of a branched hypha passing through the outer ‘wall’ towards the dense central mass. However, I disagree with Butterfield's suggestion that these fossils are probably fungi. They could instead be actinobacteria similar to modern Amycolatopsis decaplanina and Kibdelosporangium that generate large pseudosporangia from mycelial networks (Wink et al. 2004); their pseudosporangia are very variable in size, but usually somewhat smaller than in the fossils, but the thickness of their hyphae is indistinguishable. Confusion of actinomycetes and fungi was long-standing even for extant cultivated species; actinomycetes were treated as fungi in my undergraduate mycology textbook (Alexopoulos 1952); it is markedly more difficult to differentiate between them in fossils.

I also do not accept Bangiomorpha (Butterfield et al. 1990; Butterfield 2000) as a red alga or eukaryote; it could be a mixture of two cyanobacterial species, possibly stigonematalean; I was mistaken in earlier calling it Oscillatoria-like (Cavalier-Smith 2002a). It is certainly not like the red alga Bangia, as it lacks the characteristic elongated projections of cells in its holdfast that result from intrusive growth, which had they been present would have supported a eukaryote nature. As some filamentous red algae lack such intrusive cells and are effectively indistinguishable at low resolution from Bangiomorpha, we cannot be sure it is not a red alga. However, the holdfast and all other morphological features are no more complex than in some cyanobacteria. Unfortunately, it lacks characters that require presence of a eukaryotic cytoskeleton or endomembrane system that would firmly make it eukaryotic or that prove that it cannot simply be a complex cyanobacterium. Assuming Bangiomorpha to be correctly dated at 1.2 Gyr (currently unclear; perhaps younger), Bangiomorpha is 600 Myr older than any fossil reasonably confidently a red alga (Xiao & Knoll 1999) and ca 400 Myr older than unambiguously eukaryotic fossils.

Another Late Proterozoic fossil type sometimes called eukaryotic are broad filaments, e.g. Palaeovaucheria, conventionally assigned to the heterokont Xanthophyceae and dating to ca 1 Gyr ago (Butterfield 2004). These lack features unambiguously identifying them as xanthophytes, heterokonts, or even eukaryotes; they could be large filamentous cyanobacteria. Although we cannot rule out their being a stem eukaryotic alga unattributable to phylum or kingdom, their early date makes it virtually impossible that they are xanthophytes, given the well-established heterokont molecular trees and recency of all well-fossilized heterokont groups (diatoms, haptophytes, chrysomonads, silicoflagellates); none go even into the Palaeozoic (table 1), and trees show xanthophytes as no older (figure 7; Cavalier-Smith & Chao 2006).

There are many technically inadequate attempts to date eukaryotes assuming a universal molecular clock (Graur & Martin 2004), even though this assumption is false and grossly misleading, as evolutionary rates of all molecules can change idiosyncratically across a molecular tree, sometimes by many orders of magnitude (Cavalier-Smith 2002a). Yet some stability in evolutionary rates of nucleotide or amino acid substitution exists in local parts of the tree for a given molecule, and assuming a local ‘clock’ is sometimes useful for interpolating between known fossil dates. Extrapolation backwards earlier than fossil dates is considerably more hazardous; nonetheless, with a statistical model that allows rates to change across the tree, reasonably realistic models of substitution, and calibration of many points on the tree by known fossil dates (not one as in many studies), it is worth attempting to try to assess congruence between the fossil record and molecular phylogenetic trees, and to improve interpretation of both if they mismatch; Roger & Hug (2006) discuss some of the problems.

Only two such studies, using a Bayesian ‘relaxed clock’, attain the highest current standards. One based on 129 proteins, 36 eukaryotes and six Phanerozoic fossil calibration points based on macro-organisms (three animals, two land plants, one fungal) deduced 950–1259 Myr ago (mean 1085) for the divergence of Amoebozoa from other eukaryotes (Douzery et al. 2004); given the difficulty of resolving the branching order of Amoebozoa and bikonts on trees (Cavalier-Smith 2004b), this probably approximates to an estimate for the cenancestral eukaryote. Berney (2005) and Berney & Pawlowski (2006), using 18S rRNA (1465 nucleotides) from 83 eukaryotes and 26 Phanerozoic microfossil time constraints, similarly dated the cenancestral eukaryote at 948–1357 Myr ago (mean 1126). Using microfossils to constrain trees has the advantage that such organisms as diatoms, radiolaria and foraminifera are superabundant, with billions of clearly identifiable fossils, so a group's first appearance is more accurately dated than for sparsely recorded macro-organisms and unlikely to be underestimated. However, Bayesian algorithms are unlikely to model episodic evolution (e.g. dramatic short-term changes in evolutionary rate) well. For rRNA, the evolutionary rate probably massively increased in the stem eukaryote lineage (possibly by approx. four orders of magnitude; Cavalier-Smith 2002a) then subsequently greatly slowed again. If slowing finished before the initial radiations, the earlier fast rates should not distort Berney's conclusion (though probably fatal for similar studies of the whole tree). If higher rates lasted after the primary eukaryotic divergence but declined before fossil calibration dates, this gene tree would overestimate the age of the cenancestral eukaryote. As many proteins used by Douzery et al. (2004) were ribosomal (coevolving with rRNA), their dates also could be overestimates. Many other proteins used in their dataset were novel eukaryotic proteins, expected to have evolved faster early on when function was freshly established than later when stabilizing and purifying selection would predominate; thus, the protein dates may be overestimates. This is supported by reanalysis of these data by a technically superior method; calculating trees separately for each gene and then combining likelihoods (Roger & Hug 2006) gave a younger estimate of ca 900 Myr ago. Based on numerous proteins, this should be more reliable than the 200 Myr earlier date from 18S rRNA (Berney 2005; Berney & Pawlowski 2006), especially if hyper-fast rRNA evolution in the pre-eukaryote stem (Cavalier-Smith 2002a) persisted somewhat after the earliest divergences.

Berney used a clever method to test identifications of contentious Proterozoic fossils like Palaeovaucheria, Tappania, Pterocladus (claimed to be a cladophoran green alga) and Bangiomorpha. He assumed palaeontologists correctly identified them and used their dates as constraints for Bayesian calculations, deducing a hypothetical origin time for groups with easily identified fossils, e.g. diatoms, dinoflagellates. Invariably, this dated these groups with a continuous unambiguous fossil record grossly earlier than their actual fossil dates (typically overestimating ages of rhizosolenid diatoms, pennate diatoms and coccolithophorid haptophytes by 4–5 times). This strongly supports the view that these fossils were misidentified as crown eukaryotes (Cavalier-Smith 2002b). Although some might be stem eukaryotes, all are likely simply complex bacteria. Likewise, assuming that some vase-like fossils are euglyphids (Porter & Knoll 2000; Porter et al. 2003) overestimates these dates 4–8 times (because euglyphids nest relatively shallowly in the cercozoan tree; Cavalier-Smith & Chao 2003a); by contrast arcellinids nest relatively deeply in the amoebozoan tree (Nikolaev et al. 2005); their date of more than 760 Myr ago (Porter & Knoll 2000; Porter et al. 2003) is consistent within the error range with estimates of ca 950 Myr ago for Amoebozoa (Berney 2005; Berney & Pawlowski 2006) and ca 0.9 Gyr ago for eukaryotes (Roger & Hug 2006).

My objection to identifying these fossils as eukaryotes is primarily on morphological grounds; morphology inadequately supports it. Their conflict with the phylogenetic evidence for approximately equal ages for bikonts and unikonts, and thus with plants and chromists being not dramatically older than animals, made me examine them critically, but I should be sceptical even were they much younger. The relaxed Bayesian estimates for red algae (ca 730 Myr ago from rRNA; less than 928 Myr ago from proteins) and Bangiophyceae (ca 680 Myr ago from rRNA) are consistent with ca 570 Myr old fossils being genuine florideophytes (Xiao et al. 2004). These estimates should not be called ‘molecular’. They synthesize fossil and molecular data with a model for rate change across the tree, i.e. fossil dates modulated and integrated by extensive comparative molecular evidence. Molecular trees and substitution models help integrate the partial fossil evidence with more representative molecular data and apply dates to groups lacking fossils. Molecular sequences alone give no dates.

Inferred cenancestral dates for chromalveolate groups (haptophytes 560 Myr ago; heterokonts 580 Myr ago; alveolates 600 Myr ago) are consistent with the requirement that chomalveolates postdate red algae (having originated by one red algal enslavement), but the mean unseparated protein date for chromalveolates (919 Myr ago, Douzery et al. 2004) is only just consistent with the red algal protein date and markedly earlier than the rRNA red algal date, suggesting an overestimate. Estimates for the red/green algal divergence of ca 930 (rRNA) and 1010 Myr ago (proteins, unseparated; Douzery et al. 2004) should be slightly younger than for the origin of Plantae as glaucophytes diverge earlier. However, as rRNA gives 812 Myr ago for the primary animal radiation and proteins 695 Myr ago, early backwardly extrapolated dates of these studies may be 100–300 Myr too old, it being unlikely that animals have been missed in the fossil record (Peterson & Butterfield 2005; Conway Morris 2006)—their true age is probably ca 555 Myr ago (if Vendobiota are not animals) or ca 570 Myr ago if Vendobiota are animals.

Taking fossils and molecular evidence together, 900±100 Myr ago is the most likely origin date for eukaryotes. Dating the origin of chloroplasts and Plantae is harder, but most likely 570–850 Myr ago, whereas ca 570 Myr ago is reasonable for the origin of chromalveolates, opisthokonts, Rhizaria and excavates, when snowball earth melted.

(b) Archaebacteria are the youngest prokaryotes

Archaebacteria were named when all known archaebacteria were methanogens: Woese (1977)assumed that metabolism using H₂ to reduce CO₂ to methane was very ancient. His primary reason for supposing archaebacteria to be ancient was that their rRNA seemed as divergent from that of eukaryotes and eubacteria as they were from each other; he implicitly assumed that rRNA evolved at the same rate in all three groups and that all three were equally old (Balch et al. 1977; Woese & Fox 1977). Fossil dates (eukaryotes much younger than eubacteria) were ignored. Thus, the widespread belief that archaebacteria are ancient stems from dubious assumptions not from direct evidence. Stackebrandt & Woese (1981) explicitly stated there was no evidence that rRNA evolved at the same rate in the three groups, but that important caveat has been generally ignored; numerous interpretations have assumed that substitution rates are uniform for rRNA, especially bacterial, despite this being false and misleading. Even that caveat overlooked the likelihood that quantum evolution during transitions between the three groups mainly caused the differences, not long elapsed time (Cavalier-Smith 1981, 1987b, 1991a), as critical interpretation of the trees in the light of fossils effectively proves (Cavalier-Smith 2002a). We cannot use phyletic depth on rRNA trees to determine age independently of fossils. But we can combine tree data with fossil evidence for relatives to estimate dates for groups without fossils. As archaebacteria are sisters of eukaryotes, they cannot be dramatically older. Thus, they are probably only ca 0.9 Gyr old. Eubacteria are probably 2.2–2.6 Gyr older.

It is unlikely that neomura are much older than either eukaryotes or archaebacteria. Probably eukaryotes and archaebacteria each originated almost immediately after neomura (Cavalier-Smith 2002a). It is especially unlikely that neomura can be as old as eubacteria. If they were, bacteria with a neomuran glycoprotein envelope, DNA-handling enzymes and ribosomes, but lacking specific archaebacterial properties (e.g. isoprenoid ether lipids, archaeosine, flagella), must have existed for 2.5 Gyr before eukaryotes without leaving any descendants not converted to eukaryotes or archaebacteria; it is incredible that a major niche for such an intermediate bacterium persisted for more than 2 Gyr, but was suddenly wiped out coincidentally with the origin of eukaryotes.

There is no morphological fossil record for archaebacteria and scant prospect of ever finding any. One potentially important biomarker unique to archaebacteria is the double-length C40 isoprenoid lipids that span the entire CM of hyperthermophilic (not other) archaebacteria. As hyperthermophily is probably the ancestral state (Cavalier-Smith 2002b), these probably first evolved in the cenancestral archaebacterium and would mark their origin. Unfortunately, they are unknown earlier than the Mesozoic, suggesting relative instability (they ought to date from the Early Neoproterozoic (ca 0.9 Gyr), like eukaryotes) that underestimates the age of archaebacteria (Cavalier-Smith 2002a). Linear C20–C30 isoprenoids ca 1.6 Gyr are proposed as biomarkers for halophilic archaebacteria, which typically make membrane lipid isoprenoids of C20 and C25 (Summons et al. 1998). However, all eubacteria have long-chain isoprenols of C50 or C55 lengths as membrane carriers, and one wonders whether partial degradation products of these or other eubacterial isoprenoids might persist and be hard to distinguish from halobacterial isoprenoids. R. E. Summons (2005, personal communication) noted that polyunsaturation of these eubacterial isoprenols makes them highly susceptible to degradation by light and oxidation, unlike saturated archaebacterial lipids. Given that atmospheric oxygen was substantially sparser ca 1.6 Gyr ago (Holland 2006) and the deep ocean might have been anoxic, possibly anoxic burial of eubacteria occurred rapidly enough for degradation to yield the observed spectrum of chain lengths.

Organic carbon samples 2.8 Gyr ago (and somewhat later) are markedly lighter isotopically than achievable by one-step CO₂ fixation by Rubisco (Δ^{C −38‰). Previously,} assuming that archaebacterial methanogenesis was ancient, this was interpreted as the result of recycling of biogenic methane made by archaebacteria (archaebacterial methanogenesis has exceptionally low ^{C : C ratios: ΔC −30–50‰) by methanotrophic bacteria into organic carbon (}Hayes 1983, 1994). Hayes suggested that the rise of oxygen restricted methanogen habitats sufficiently to make their hypothetical contribution quantitatively undetectable less than 2.2 Gyr ago. As phylogenetic and morphological fossil evidence in combination strongly indicate that archaebacteria are much younger than 2.0–2.8 Gyr, explanations for these light carbon deposits cannot involve archaebacteria. Straus et al. (1992) suggested that chemosynthetic bacteria or anoxygenic photosynthesis might participate in two-step fractionation that could produce lighter organic carbon than Rubisco alone. A specific suggestion was a chemotroph (presumably using Rubisco) taking as substrate CO₂ already made light by photosynthetic fixation and released by respiration. The lightness of the final product would depend on local recycling efficiency compared with mixing in the atmospheric pool. Section 5d develops an analogous explanation involving greater phototroph diversity generated by the glycobacterial revolution 2.8 Gyr ago.

Ultralight reduced carbon is also made by autotrophic acetogenesis; some Clostridiales thus make acetate from CO₂ and hydrogen, with similar ^{C depletion to methanogenesis (ΔC −59‰;} Gelwicks et al. 1989). As they use the same input materials as methanogens but are undoubtedly much older, they are a potentially more plausible explanation for the isotopic data. As anaerobes, their relative contribution to fixed carbon compared with aerobic phototrophs would have become insignificant just when the ultralight signal went. However, Clostridiales are Endobacteria, possibly not present as early as 2.7 Gyr ago: §4d suggests they arose 2.3–1.5 Gyr ago, but does not firmly exclude that they evolved earlier and contributed to the ultralight signal.

Another potential explanation is abiotic sources of methane plus aerobic biological methanotrophy. Some abiotic methane is not ultralight. Iron- and nickel-rich alloys, common in some minerals, catalyse production from bicarbonate at 200–300 °C of abiotic methane indistinguishably ultralight (Δ^{C−35–50‰) from archaebacterial methane. However, as} §5d argues, biotic depletion is more likely. But, given the potential of three very different kinds of explanation not involving archaebacteria for the 2.8–2.1 Gyr ago carbon isotopic data, they are not specific evidence for Archaean or Palaeoproterozoic archaebacteria, especially as all other evidence strongly contradicts that. Hayes (1994) recognized that ‘the antiquity of methanogens… is more speculative than proven’. I consider it now disproved (Cavalier-Smith 2006a).

(d) Probable intermediate age of posibacteria

Despite the usual (not universal) failure of Actinobacteria/neomura to group with Endobacteria, Posibacteria are most probably monophyletic (§2b and Cavalier-Smith 2006a). Therefore, as Endobacteria nest within Eurybacteria in virtually all sequence trees, Posibacteria must be substantially younger. If Eurybacteria are ca 2.75 Gyr old, Posibacteria can hardly be older than the 2.3 Gyr ago oxygenation event or else they would not nest so reliably within Eurybacteria on trees. I think they cannot be younger than ca 1.5 Gyr old or they would nest even more securely and distinctly more shallowly within Eurybacteria. Therefore, Posibacteria probably evolved ca 2.0–1.5 Gyr ago. While Actinobacteria are predominantly aerobic, endobacteria exploit aerobic and anaerobic niches to the full and include early diverging sulphate reducers. The extra-thick walls of Posibacteria and preadapted resistant endospores made them dominant bacteria in soils that became increasingly developed as a favourable microbial habitat after an ozone layer developed (and geographically more extensive by continental accretion). Endospores and thick walls protected from drying during wind dispersal and drought.

Although Actinobacteria are related to Endobacteria, it is uncertain if they are sisters to Endobacteria, and roughly equally old (previously assumed; Cavalier-Smith 2002), or derived from them, and thus younger. Many actinobacteria use the thick posibacterial walls to become morphologically highly complex; actinomycetes can be macroscopic and visible to the naked eye, like some cyanobacteria that they rival and sometimes exceed in morphological complexity. Actinomycetes are throughout the world in soils and sediments, even in deepest oceanic trenches. Surprisingly, palaeontologists almost never consider them possible candidates for more complex fossils, especially those conventionally seen as candidate stem eukaryotes. 1.5 Gyr ago is when distinctly larger than previously fossil thick-walled cysts begin to occur at low frequency (Schopf & Klein 1992; Knoll et al. 2006). Butterfield (2005) broke with the tradition that most if not all of these are algal, for which there was never compelling evidence; in his words, micropalaeontologists adopted ‘a search image weighted excessively in favour of unicellular plant protists’. He reasonably suggests that Tappania and possibly also Germinosphaera, Foliomorpha, Trachyhystrichosphaera, Shuiyousphaeridium and Dictyosphaera are not single cells but multi-genome marine benthic mycelial, osmotrophic, heterotrophs. He thought they were fungi, but such as Tappania could be actinomycetes similar to Amycolatopsis (Wink et al. 2004) and Kibdelosporangium. If they are, as I suspect, actinomycetes are probably at least 1.5 Gyr old if any Roper fossils (Javaux et al. 2001, 2003; Knoll et al. 2006) are actinomycetes (I agree with Knoll et al. (2006) that Roper T. plana may differ from Butterfield's Neoproterozoic fossils).

Since actinomycetes are just one derived subclade within the actinobacterial tree, unless Tappania is an extinct derivative of an earlier branching clade now represented only by morphologically simpler species, then actinobacteria are probably distinctly older. I suggest that Actinobacteria are no younger than ca 1.8 Gyr. Thus, we have a rough estimate of the maximum age of Endobacteria as 2.3 Gyr (2 Gyr more likely) and a minimum age for Actinobacteria of ca 1.8 Gyr. These estimates are compatible with their being sisters and both 2–1.8 Gyr old or with Actinobacteria being younger than Endobacteria and derived from them. The closeness of these estimates implies that even if Actinobacteria evolved from Endobacteria, they should nest deeply within Endobacteria, less than or equal to 10% of the distance from their base with perfect phylogenetic reconstruction. In the real world of imperfect phylogenetic algorithms and pervasive systematic biases, e.g. from the exceptionally high-GC nucleotide composition of actinobacterial DNA and unusually low GC in Endobacteria, we should not expect Actinobacteria and Endobacteria to group together on most single-gene or multigene trees, even if Posibacteria are monophyletic. If they are sisters, mild systematic biases—or sampling error on single-gene trees—could falsely separate them.

Similar considerations and more accurately dating Actinobacteria are important for interpreting sequence trees in relation to neomuran origins. If eukaryotes and neomura are only 0.9 Gyr old and Actinobacteria are 1.8 Gyr old, perfect trees ought to nest neomura within Actinobacteria. Previously, as some eukaryote-like properties are phylogenetically restricted, I assumed that actinobacteria are paraphyletic ancestors of neomura, and thus older (Cavalier-Smith 2002a). If instead neomura and Actinobacteria are sisters (not disproved) and ca 1.5 Gyr old, as in some fossil interpretations, neomura should NOT nest within Actinobacteria on perfect trees but should be their sisters (as they are weakly on some trees, especially if the often closer, probably artefactual, position near neomura of Thermotoga/Aquifex is set aside). Systematic bias from accelerated evolution of most neomuran genes may be sufficient to exclude neomura from Actinobacteria artefactually on most sequence trees, even if they differ in age by 800 Myr; thus we cannot use their normal exclusion to argue that their ages are markedly closer than that, or for their being sisters.

(b) Methane and the Precambrian biosphere

After water vapour and CO₂, methane is the most important greenhouse gas; per molecule its warming effect is ca 21 times that of CO₂. Today methane made by free-living and symbiotic archaebacteria probably greatly exceeds abiotic methane. The Archaean sun was weaker; climate modellers argue that more greenhouse gas was thus necessary to prevent global oceanic freezing. It was once thought that substantially higher levels of CO₂ would suffice (Kasting & Pollack 1984), but palaeosol evidence suggested atmospheric CO₂ levels below those that on their own would prevent global freezing (Kasting 1987). Following Lovelock's (1988) suggestion of methane as an important Archaean greenhouse gas, the assumption of palaeoclimatic models was that biogenic methane could solve the faint Archaean sun problem (Pavlov et al. 2000; Kasting & Siefert 2002; Kasting & Ono 2006). However, as discussed above, archaebacteria probably did not exist in the Archaean or Early Proterozoic. Some geologists argue that CO₂ levels were much higher than palaeosol data would allow (Ohmoto 2004; Ohmoto et al. 2004), and thus potentially sufficient to solve the faint sun problem, but arguments against that scenario seem convincing (Kasting 2004). Thus, at least some role for inorganic methane seems inescapable unless net heating by water vapour and clouds was proportionally higher than currently assumed or a fourth factor is overlooked. Abiotic methane (Horita & Berndt 1999; Foustoukos & Seyfried 2004; Scott et al. 2004a) is found, e.g. in deep mines in Canada and South Africa and in mid-oceanic rifts. Was there enough to solve the faint sun problem, with presently assumed levels of water vapour and the maximum CO₂ allowable by palaeosol data? Before the 2.3 Gyr ago oxygenation and origin of biological methanotrophy, methane would have been destroyed by oxidation more slowly than today. According to fig. 4 of Kasting & Ono (2006), the suggested maximum level of abiogenic methane of 1000 p.p.m. would be amply sufficient to solve the problem in conjunction with higher CO₂ levels, below the palaeosol limit, as would 10–20 times lower methane levels. Therefore an early origin of archaebacteria need not be postulated to save the Archaean earth from perpetual freezing. Phylogenetic evidence for the absence of archaebacteria from the Archaean usefully narrows the range of allowable possibilities for the levels of CO₂, methane, and temperature.

Methanotrophy and biological methanogenesis are both done by a suite of 15–16 different enzymes related to those that mediate other C1-compound conversions, some using unusual cofactors made by enzymes encoded by ca 10 other genes (Chistoserdova et al. 2004). Belief that these 25–26 genes were restricted to methanogenic archaebacteria and proteobacteria of subphylum Rhodobacteria (purple bacteria plus non-photosynthetic descendants) fostered hypotheses of LGT from archaebacteria to proteobacteria (Chistoserdova et al. 1998; Boucher et al. 2003; Martin & Russell 2003) or vice versa (Cavalier-Smith 2002a). Lateral transfer from archaebacteria, the more popular, was temporally impossible because methanogens are probably about three times younger than Rhodobacteria (ca 2.7 Gyr old), and practically highly improbable because their C1-related genes are scattered all over the chromosome and could hardly all be cotransferred. Reverse transfer from Proteobacteria to Archaebacteria is mechanistically more plausible as many are closely clustered in operons in eubacteria and could theoretically be cotransferred. However, discovery of related genes in Planctobacteria, probably sisters of Proteobacteria, and in non-methanogenic archaebacteria and one even in the actinobacterium Streptomyces, plus recent phylogenetic analysis argues strongly that almost all these genes have simply been inherited vertically, being repeatedly lost by lineages without them (Chistoserdova et al. 2004). Lateral transfer need be invoked only for one Fae homologue of unknown function from a proteobacterium to the planctomycete Pirellula. Phylogeny of six genes with straightforward history (three C1 pathway and three cofactor genes) is entirely congruent, if rooted between Proteobacteria/Planctobacteria (collectively Exoflagellata; Cavalier-Smith 2002a) and Archaebacteria, with my rooted universal tree (figure 5), favouring vertical inheritance. Gene phylogenies contradict suggestion D of Chistoserdova et al. (2004) of lateral transfer from Planctobacteria to Proteobacteria and Archaebacteria.

The simplest interpretation of evolution of methylotrophy and methanogenesis is not scenario E of Chistoserdova et al. (2004), which makes the usual incorrect assumption that the tree's root is between neomura and eubacteria, but that these C1 enzymes first evolved in the common ancestor of Gracilicutes and Eurybacteria, were inherited vertically by Planctobacteria, Rhodobacteria and Archaebacteria, and lost by eurybacteria, eukaryotes, most Posibacteria, Sphingobacteria and Spirochaetes. In Planctobacteria they do not mediate methylotrophy or methanogenesis; they originally probably oxidized C1 compounds, e.g. formaldehyde. Aerobic methylotrophy and methanotrophy occur only in α- and γ-proteobacteria, probably originating after they diverged from planctobacteria. If vertically inherited, these enzymes must date approximately to the ancestral rhodobacterium (2.7 Gyr; see above); C1 enzymes generally must be as old. Thus, methylotrophy extends back to the 2.78 ultralight C-isotope spike discussed above. Methanotrophy need not be that old; adding only one enzyme, methane monooxygenase, more easily transferred by LGT than the whole pathway, would convert a methylotroph to a methanotroph. However, the closest relative of proteobacterial methane monooxygenase is ammonium monooxygenase of β-proteobacteria, somewhat more divergent from methane oxygenases of α- and γ-proteobacteria than they are from each other, suggesting that divergence in function of the two paralogues occurred in the ancestral rhodobacterium and methanooxygenase and methanotrophy also originated ca 2.7 Gyr ago, as suggested for methylotrophy. Some aerobic methanotrophs can scavenge methane even from the atmosphere (Henckel et al. 2000). Aerobic methanotrophy needs more than 20 p.p.m. methane; atmospheric abiotic methane levels needed to solve the faint sun problem would have provided enough. The initial limiting factor was oxygen (Hayes 1994).

It is reasonable that C1 oxidation originated at virtually the same time as oxygenic photosynthesis providing the oxygen (2.8–2.9 Gyr ago). The initial impetus could have been protection against harmful molecules like formaldehyde by converting them to CO₂. Abiotic methane destruction was probably markedly accelerated ca 2.7 Gyr ago by aerobic methanotrophy. Lovelock (1988) and Pavlov et al. (2000) suggested that the origin of oxygenic photosynthesis would have accelerated methane removal by oxygenating the atmosphere and could thus have caused the 2.45–2.25 Gyr ago global snowball Earth episodes (probably two). Section 5d discusses this further.

Methanogenesis is probably younger than archaebacteria, as methanogens nest within one archaebacterial subphylum, Euryarchaeota (Gribaldo & Brochier 2006). As archaebacterial sequence trees are substantially non-clock like (Gribaldo & Brochier 2006), estimating the age of methanogens is somewhat hazardous. In the absence of a Bayesian relaxed clock analysis using an arbitrary age for the archaebacterial cenancestor to estimate how much younger methanogens may be, a rough estimate can be made: inspection of the concatenated ribosomal protein tree (Gribaldo & Brochier 2006) and a crude clock suggest that the cenancestral methanogen was 13–32% younger than the cenancestral archaebacterium. A rounded middle 20% and date of 0.9 Gyr ago for archaebacteria (as for eukaryotes; see above) gives ca 720 Myr ago for the origin of archaebacterial methanogenesis. As this coincides with onset of Neoproterozoic near-global freezing, I suggest this atmospheric infusion of biogenic methane caused snowball Earth indirectly by the mechanism of Schrag et al. (2002). Eventually stabilization occurred as the biosphere adapted to the innovation. One important adaptation would have been the origin of anaerobic methanotrophy, done only by still-uncultivated archaebacteria (Orphan et al. 2002). Methanotrophic archaebacteria all nest within the methanogens (Gribaldo & Brochier 2006), so must be younger. They probably evolved from methanogens by reversing the metabolic pathway and adding methane oxidase; small innovations, given prior evolution of methanogenesis (Chistoserdova et al. 2005). Anaerobic methanotrophs are most related to Methanosarcinales, which from the same concatenated tree (Gribaldo & Brochier 2006) are 24–57% younger; if methanogens are 720 Myr old and Methanosarcinales ca 30% younger, Methanosarcinales would be ca 500 Myr old. If anaerobic methanogens are their sisters, they are similarly aged or a little older. I suggest they evolved less than 570 Gyr ago, when snowball Earth episodes ceased, and by depleting methane close to its source (their methanogenic congeners) they have helped prevent excessive global warming by biogenic methane ever since.

(d) The glycobacterial revolution, biotic and abiotic lags and the Palaeoproterozoic snowball

Oxygenic photosynthesis provided most atmospheric oxygen and oxidized the oceans and surface rocks (Holland 2006). The striking coincidence of this great oxidation with the first snowball Earth episodes suggests they are causally connected. I argued above that Archaean climatic stability depended on greenhouse effects of both CO₂ and abiotic methane. Oxygenic photosynthesis by using a more abundant hydrogen source (water, not H₂ or H₂S) allowed life to expand immensely. By increasing photosynthetic flux, it would reduce CO₂ levels directly. Rising oxygen would reduce methane levels by abiotic atmospheric oxidation and enabling evolution of aerobic methanotrophy. Thus, it would reduce both major greenhouse gases, eventually bringing on snowball Earth. Unlike others proposing methane removal by atmospheric oxidation as the prime cause of Palaeoproterozoic global freezing (Pavlov et al. 2000; Kopp et al. 2005; Kasting & Ono 2006), I think the methane was not biogenic; nor should the roles of CO₂ draw-down by expanded phototrophy and of proteobacterial methanotrophy be ignored. If there was no biogenic methane, the drop in methane would have been even faster than they assume.

Kopp et al. (2005) argue that cyanobacterial expansion and oxygen rise were so fast that cyanobacteria must have evolved only ca 2.4 Gyr ago. But if the 2.7 Gyr bitumen biomarkers are truly endogenous (uncertain) that cannot be true. Apart from possible past downward mobility, Kopp et al. (2005) give three reasons for discounting that 2.7 Gyr hopanoid evidence for cyanobacteria: (i) some methylotrophs (e.g. Methylobacterium) make low levels of 2-methyl-bacteriohopanepolyol; (ii) other hopanols are not restricted to aerobes but present in Geobacter; (iii) methyl-bacteriohopanepolyol might have been made by ancestral cyanobacteria before oxygenic photosynthesis arose. All are refuted by phylogenetic arguments: Methylobacterium and other aerobic methylotrophs, and Geobacter are all proteobacteria, thus part of the Gracilicute clade, whose common ancestor must have had two photosystems like cyanobacteria (Cavalier-Smith 2006a). They are also part of a larger ancestrally flagellate eubacterial clade that is sister to the non-flagellate cyanobacteria. They cannot therefore be older than cyanobacteria and are probably somewhat younger. If any of these bacteria were present 2.7 Gyr ago, cyanobacteria must have been also. Hopanols evolved no later than the common ancestor of cyanobacteria and proteobacteria; as cyanobacteria are holophyletic (Gupta et al. 2003), their common ancestor with Gracilicutes had both hopanols and two contrasting photosystems. Unless a reason other than the origin of oxygenic photosynthesis can be found for divergence of two photosystems in one cell, then this glycobacterial common ancestor already had at least primitive oxygenic photosynthesis. As there is no evidence for hopanoids or two photosystems in Eobacteria, they probably evolved in this common ancestor immediately before it diverged to cyanobacteria and gracilicutes/eurybacteria. Thus, hopanols in general, not just 2-methyl-bacteriohopanepolyol, are probably good proxies for the age of cyanobacteria and (insignificantly earlier) glycobacteria as a whole. If my argument (based on catalase evolution) that oxygenic photosynthesis evolved in the common ancestor of glycobacteria and Hadobacteria is correct (Cavalier-Smith 2006a), it probably even slightly preceded the origin of glycobacteria. Even though cyanobacteria probably do not make sterols (Summons et al. 2006), their biosynthesis requires oxygen, suggesting that biotic oxygen was made in microbial mats as early as 2.715 Gyr ago. It is unwise to seek escape from this refutation in an unknown anaerobic mechanism for adding oxygen (Kopp et al. 2005).

Hayes & Waldbauer (2006) point out that the reservoir of reduced iron and sulphur able to mop up early biogenic oxygen was so huge that one expects a substantial lag after oxygenic photosynthesis before atmospheric oxygen levels rose. If oxygen was not generated faster than these minerals could remove it, a 400 Myr lag can easily be explained by that reservoir size. Kopp et al. (2005) calculated biogenic oxygen fluxes compared with removal by Fe, assuming that cyanobacterial population expansion was limited only by total oceanic P or N nutrients, and concluded that atmospheric oxygen should rise enough to deplete methane within a few million years. But this has two problems: it is the available stock of reduced Fe and S that matters initially not its regeneration rate, which Kopp et al. assumed limited O₂ sequestration; secondly, it is unrealistic that cyanobacteria were limited only by nutrients. Before an ozone layer they would have been seriously restricted by harmful UV radiation (Cockell 2000; Cockell & Horneck 2001).

Consider the isotopic evidence for the state of the carbon cycle at the putative time (2.8 Gyr) of the glycobacterial revolution. Rothman et al. (2003) and Hayes & Waldbauer (2006) showed that traditional steady-state carbon cycle models of the past 30 years have been oversimplified in two respects, making predictions disagree with the data. First, it is unrealistic to treat all oceanic/atmospheric carbon as one pool; one must differentiate between dissolved organic carbon and CO₂ (Rothman et al. 2003). Secondly, the crust/oceans/atmosphere is not self-contained; as geothermal activity continually injects CO₂ from the mantle, one must also weigh export of oxidizing power to the mantle in the balance (Hayes & Waldbauer 2006). Hayes & Waldbauer (2006) also note that as crustal burial processes important for redox mass balance calculations operate on a much slower time-scale than biological processes that directly generate or consume oxygen, isotopic fractionation of buried carbon need not correlate with atmospheric changes; large negative excursions of Δ^{C in organic carbon are not mirrored by complementary changes in inorganic carbonate from 2.8 to 2.0 Gyr ago, so they argue that changes in burial rates were not a major factor—in marked contrast to Neoproterozoic perturbations to the isotope record affecting both organic and inorganic deposits.}

A further complication not considered previously is heterogeneity of the environment where primary production occurs. For Phanerozoic oceans dominated by plankton a homogeneous well-mixed model is reasonable. But in the Archaean and Early Proterozoic more than 2.3 Gyr ago with no ozone layer, UV irradiation probably largely excluded photosynthetic bacterioplankton from the upper photic zone. Phototrophs were probably concentrated in shallow water where iron-impregnated mineral grains or snow overlying thin ice offered enough protection from UV radiation. Global productivity was much less than today; life concentrated in stratified microbial mats (fossilizable as stromatolites) rather than as well-mixed phytoplankton freely communicating with the atmospheric CO₂ pool. Mat complexity increased greatly with the glycobacterial revolution (figure 9) offering increased opportunity for internally recycling CO₂. Figure 9b shows a simplified model with only two strata, which allows two-stage fractionation by Rubisco. First, by cyanobacteria in the upper layer. Then by anaerobic bacteria, e.g. chlorobacteria or purple bacteria (proteobacteria) such as Chromatium in the lower layer. Fermentative and respiring heterotrophs would have been in both layers using organics generated by the phototrophs. CO₂ generated by these heterotrophs or phototrophs at night would be already depleted of ^{C by Rubisco carbon fixation. Recycling it within the mat could deplete it again—and again, producing increasingly light organic carbon for burial. For a strictly two stage recycling (}figure 9b) the depletion in buried organic carbon would be: ΔC+f_orrΔC, where ΔC is the depletion by a single stage, r is the fraction of CO₂ fixed by the second stage (assumed for simplicity to be solely purple bacteria, but in practice it could be any Rubisco-using phototroph in the mat) and f_or is the fraction of buried carbon that comes from the purple bacteria after recycling (1−f_or would come from cyanobacteria).

Figure 9

Ecosystems and the carbon cycle before and after the glycobacterial revolution. (a) In the age of chlorobacteria, life was mainly restricted to low diversity microbial mats protected from UV radiation by mineral particles. (b) After cyanobacteria evolved, mats became more complex with upper aerobic and lower anaerobic layers increasing the scope for internal CO₂ recycling, with serial ^{C isotope depletion by Rubisco. Isotopic composition of buried organic carbon depends on the fraction,} r, of once depleted CO₂ used by the second stage and on the fraction f_or of total C buried coming from second-stage organisms. (c) After flagella and proteobacteria evolved, they displaced chlorobacteria from most habitats and began to form a facultatively aerobic photosynthetic plankton, whose quantitative contribution greatly expanded with the ozone layer as UV inhibition declined and cyanobacterial phytoplankton evolved, especially prochlorophytes adapted to higher light intensities. As the fraction of buried organic carbon coming from plankton, f_op, increased, that from recycling within the mat (f_or) became quantitatively insignificant from Mid-Proterozoic to Phanerozoic, thus abolishing the ultralight carbon signal of stage (b).

If cyanobacterial photosynthesis and CO₂ trapping were very efficient, the upper layer could absorb most unfractionated CO₂ diffusing from the atmosphere, leaving only a small proportion for the lower layer. Thus, values for r could probably easily exceed 0.5 and could be as high as 1. Assuming a value of 0.9 and a value of 0.5 for f_or gives a total fractionation 1.45 times that of one stage. Note that f_or is determined by the relative biomass of the two layers, not by relative rates of synthesis. This is important because if photosynthesis in the lower layer were CO₂-limited, it would occur at a lower rate. But if the lower layer were thicker it could have a higher biomass, even if turnover was slower. If it were three times as thick and as dense, f_or would be 0.75 and total fractionation 1.65 times that of a single stage. In fact, mats can have much greater anaerobic than aerobic biomass (Sorensen et al. 2005). If one Rubisco stage can achieve −30‰, two could yield −45‰ and three could yield Δ^{C −63‰. I propose that a sudden increase in biological complexity of microbial mats, favouring multistage recycling, caused the negative ΔC spike 2.77 Gyr ago and that this negative spike is the best way of dating the glycobacterial revolution. There is no need to invoke methanogenesis or acetogenesis.} Logan et al. (1999) found that in terminal Proterozoic samples those from microbial mats were more depleted in ^{C than planktonic samples, fitting the idea that mats can favour recycling already lighter CO}₂ more than possible in suspension and thus serial light-biased fractionations by Rubisco. Mats often have three layers, with green-sulphur bacteria at the bottom, but in some habitats these largely replace the purple bacterial layer of figure 9c (Sorensen et al. 2005).

This stratified mat model explains timing of the onset and end of this unusual depletion. The negative spike would disappear completely when the photic zone was fully colonized by glycobacterial plankton (mainly cyanobacteria and proteobacteria) after the ozone layer was formed. If it is correct, the isotope record suggests that this expansion into the plankton was complete by 2.1 Gyr in the later stages of the great oxidation event. However, the spike is greatly reduced by 2.55 Gyr, well before even the slight oxidation that ended mass-independent oxygen 2.45 Gyr ago and the likely onset of the ozone layer (2.3 Gyr). This early decline of the negative spike clearly contradicts the methanogenesis explanation, which suggested that termination was caused by atmospheric oxidation of methane, which could not have occurred till after 2.45 Gyr. It is no problem for the stratification theory if a partial shift into the plankton occurred prior to the ozone layer. A sound phylogenetic reason expects such a partial shift: the origin of flagella. Flagella arose after the glycobacterial revolution, but before the origin of proteobacteria (Cavalier-Smith 2006a). I suggest that the spike peak represents the brief period when cyanobacteria had evolved, but proteobacteria had not. At that time the only phototrophs were chlorobacteria and cyanobacteria, both with gliding motility and no flagella. If that is correct, the second anaerobic stage of CO₂ recycling (figure 9b) must then have been by Rubisco-using chlorobacteria, not proteobacteria. If flagella evolved ca 2.75 Gyr ago, they would have enabled facultatively aerobic photosynthetic purple bacteria to swim above the mat and photosynthesize as plankton, reducing the recycling fraction r. If they kept in the lowest photic zone region they would suffer little UV damage.

This scenario is evolutionarily sounder than the original methanogenesis/methylotrophy as there is direct evidence from biomarkers that cyanobacteria and purple bacteria had both evolved by that date. If the overlying water column were also stratified, impeding downward flux of CO₂ from the atmosphere, this would have favoured preferential reuse of respired CO₂ compared with that from the atmospheric pool, increasing fractionation. However, this scenario does not require two different types of photosynthesis. It could occur by repeatedly recycling previously fixed and respired CO₂ by one kind of photosynthesizer alone, e.g. purple bacteria in an entirely anaerobic mat or as suggested by Straus et al. (1992) by a chemotroph within the mat recycling previously phototrophically fixed CO₂. The key thing is restriction of free mixing with the atmospheric CO₂ pool, i.e. the physical conditions not the precise organisms involved. Thus, extra-light carbon cannot be used to infer the presence of a particular kind of bacterium; rather it tells us either that special physical conditions to allow local recycling of gaseous products without global mixing were present in the particular habitats where the ultralight carbon was laid down or that little-known abiotic processes contributed to such a signature. However, although §4b cited some able to do so, there is no obvious rationale why abiotic causes should have been globally effective just then, neither before nor later, so they are probably irrelevant. Stratified mats, composed of the same globally distributed bacteria could give a consistent global isotopic signal without mat CO₂ having to mix freely globally. As Hayes & Waldbauer (2006) stress, kinetic effects can dominate the short term, and the spike was definitely short term compared with rock burial cycles. Microbial mats are not the only situation where isotopic fractionation can be higher than the standard model predicts. Biomass stemming from proteobacterial chemotrophs densely packed within animals is markedly more depleted (Δ^{C −30–35‰; much more than in phytoplankton) than the 24‰ caused in one step by the corresponding Rubisco (}Scott et al. 2004b). This is partly because the symbiotic system recycles already depleted inorganic carbon from the local environment; but CO₂ recycling within the animal's chemotroph mass could also occur.

The above is oversimplified. Like earlier models (Hayes 1983, 1994; Hayes & Waldbauer 2006) it ignores the fact that Δ^{C by Rubisco varies evolutionarily, being typically lower in bacteria than eukaryotes, usually} ca −20‰ not −30‰ as in spinach (Guy et al. 1993). This is a problem for the classical assumption that pre-2.77 Gyr ago Archaean Δ^{C of 35‰ reflects a single-stage Rubisco fractionation. There was no spinach in the Archaean. I suggest that two-stage recycling in mats is also needed to explain the more than 2.77 Gyr ago data—unless they were produced purely abiotically and the 2.77 Gyr spike is a marker not for glycobacteria but for the origin of life and photosynthesis, which cannot be conclusively rejected, but I think is unlikely. The problem for the chlorobacterial Early Archaean is even greater, because most chlorobacteria, except} Oscillochloris that uses Rubisco with Δ^{C of} ca 20‰ (Ivanovsky et al. 1999), do not use Rubisco, but the 3-hydroxypropionate pathway, showing less favouritism for light carbon (e.g. Chloroflexus). We currently cannot infer which carbon fixation pathway was ancestral for chlorobacteria and whether both or only one were present in the Archaean. Assuming an equal mixture gives Δ^{C of only −16‰, so much recycling would be needed to produce the observed values; some is unavoidable even if they only used Rubisco like that of} Oscillochloris (Ivanovsky et al. 1999). However, there is a vast unexplored diversity of chlorobacteria, the most neglected bacterial phylum; other mechanisms may exist with higher fractionation. Immensely more research is needed on Chlorobacteria if we are to understand Archaean ecosytems.

Another complication is the unpublished evidence for a temporary pulse of oxygen sufficient to abolish mass-independent sulphur isotope fractionation around 2.9 Gyr ago (Kasting & Ono 2006). Was the glycobacterial revolution, therefore, earlier than suggested? Not necessarily. One possibility is that this pulse came from pre-cyanobacterial oxygenic photosynthesizers, which probably evolved immediately before Cyanobacteria and Hadobacteria diverged (Cavalier-Smith 2006a). This could be a signal from that node in the tree, and could have caused the glaciation 2.9 Gyr ago, as suggested above. If it were, why did oxygen not remain high enough to prevent mass-independent fractionation? One possibility is that aerobic respiration evolved and reduced oxygen concentrations at source sufficiently for abiotic hydrogen and methane to accumulate enough to scavenge atmospheric oxygen, keeping it below the critical level more than 2.45 Gyr ago. Thereafter continued oxygenic photosynthesis produced the ozone layer, allowing cyanobacterial populations to expand rapidly and oxidize the earth. Once oxygen reached a certain threshold and the ozone layer began, positive feedback through population expansion would make oxygen levels rise explosively. Snowball Earth itself would have favoured phototroph expansion into the plankton through UV protection from snow on sea ice (Cockell et al. 2002; Cockell & Cordoba-Jabonero 2004), which would probably not have been too thick for light penetration (McKay 2000) and thus synergistic with ozone rise. As cytochrome oxidase probably evolved from an oxygen-independent oxidase previously used in anaerobic respiration, and the earlier part of the respiratory chain was just taken over from anaerobic respiration, aerobic respiration could have evolved rapidly after oxygen levels in mats became appreciable. This is important, as it must have happened fast enough to prevent oxygen rising above the threshold for oxidizing minerals like uraninite (only 10 times that which abolishes mass-independent fractionation: Kasting & Ono 2006) which remained reduced until 2.4 Gyr ago.

Finally, why did global glaciations cease for 1.6 Gyr? This is unexplained. Had the sun perhaps warmed enough to avoid global freezing altogether? Perhaps biology helped by invasion of land by cyanobacteria that the ozone layer allowed after 2.25 Gyr. Vast desert areas and parts of the Arctic are now covered by a thin dark cryptogamic crust of cyanobacteria, lichens and fungi (Johansen 1993). Prominent therein is the black cyanobacterium Scytosiphon, but actinobacteria and proteobacteria also abound (Nagy et al. 2005). They reduce albedo and in the Arctic can warm surfaces by 8–12 °C and soil by 4–5 °C (Gold 1998). Cyanobacterial blackening of Early-Mid-Proterozoic continents, albeit initially perhaps only half their present extent, possibly saved the day, until quantum evolution again intervened, starting the Neoproterozoic snowball.

In discussing carbonate isotopic levels 2.0–2.3 Gyr ago, Hayes & Waldbauer (2006) wrote ‘In all likelihood, the diagenetic alternative has failed to win popularity because an alternative does not appear to be needed.’ Likewise the biogenic methane explanation for the organic carbon negative spike and the increased ^{C in 2.0–2.3 Gyr carbonates remains popular because the necessity for an alternative is widely unrecognized. If the present explanation is found wanting, another must be found, not invoking methanogens, which were almost certainly absent more than 1.5 Gyr ago, and probably also more than 0.75 Gyr.}