doi: 10.1186/1745-6150-1-19.
Rooting the tree of life by transition analyses
Affiliations
- PMID: 16834776
- PMCID: PMC1586193
- DOI: 10.1186/1745-6150-1-19
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Rooting the tree of life by transition analyses
Biol Direct.
.
Abstract
Background: Despite great advances in clarifying the family tree of life, it is still not agreed where its root is or what properties the most ancient cells possessed--the most difficult problems in phylogeny. Protein paralogue trees can theoretically place the root, but are contradictory because of tree-reconstruction artefacts or poor resolution; ribosome-related and DNA-handling enzymes suggested one between neomura (eukaryotes plus archaebacteria) and eubacteria, whereas metabolic enzymes often place it within eubacteria but in contradictory places. Palaeontology shows that eubacteria are much more ancient than eukaryotes, and, together with phylogenetic evidence that archaebacteria are sisters not ancestral to eukaryotes, implies that the root is not within the neomura. Transition analysis, involving comparative/developmental and selective arguments, can polarize major transitions and thereby systematically exclude the root from major clades possessing derived characters and thus locate it; previously the 20 shared neomuran characters were thus argued to be derived, but whether the root was within eubacteria or between them and archaebacteria remained controversial.
Results: I analyze 13 major transitions within eubacteria, showing how they can all be congruently polarized. I infer the first fully resolved prokaryote tree, with a basal stem comprising the new infrakingdom Glidobacteria (Chlorobacteria, Hadobacteria, Cyanobacteria), which is entirely non-flagellate and probably ancestrally had gliding motility, and two derived branches (Gracilicutes and Unibacteria/Eurybacteria) that diverged immediately following the origin of flagella. Proteasome evolution shows that the universal root is outside a clade comprising neomura and Actinomycetales (proteates), and thus lies within other eubacteria, contrary to a widespread assumption that it is between eubacteria and neomura. Cell wall and flagellar evolution independently locate the root outside Posibacteria (Actinobacteria and Endobacteria), and thus among negibacteria with two membranes. Posibacteria are derived from Eurybacteria and ancestral to neomura. RNA polymerase and other insertions strongly favour the monophyly of Gracilicutes (Proteobacteria, Planctobacteria, Sphingobacteria, Spirochaetes). Evolution of the negibacterial outer membrane places the root within Eobacteria (Hadobacteria and Chlorobacteria, both primitively without lipopolysaccharide): as all phyla possessing the outer membrane beta-barrel protein Omp85 are highly probably derived, the root lies between them and Chlorobacteria, the only negibacteria without Omp85, or possibly within Chlorobacteria.
Conclusion: Chlorobacteria are probably the oldest and Archaebacteria the youngest bacteria, with Posibacteria of intermediate age, requiring radical reassessment of dominant views of bacterial evolution. The last ancestor of all life was a eubacterium with acyl-ester membrane lipids, large genome, murein peptidoglycan walls, and fully developed eubacterial molecular biology and cell division. It was a non-flagellate negibacterium with two membranes, probably a photosynthetic green non-sulphur bacterium with relatively primitive secretory machinery, not a heterotrophic posibacterium with one membrane.
Figures
The logic and problems of paralogue rooting. In theory (A), two genes that arose from a single parent by duplication immediately prior to the common ancestor of the group under study should yield two identical trees joined together by a line (shown extra thick) between the roots (stars) of each tree. Letters are taxa. In practice (B), stochasticity and systematic biases in evolutionary modes and rates yield trees with partially incorrect topology and often-misplaced roots [1]. Misplaced branches (red) are shown as extra long, but in practice misplaced taxa often do not reveal themselves so neatly. In practice, root positions in paralogue subtrees may both be right (very rare: I recall no examples), both wrong but the same (implying strong systematic biases), both wrong but different (often reflecting stochasticity and poor resolution), or one right and one wrong. When such conflicts occur among different paralogue pairs (or triples, etc.), as is almost invariable, other means are required to decide between them.
Evolutionary relationships among the four major kinds of cell. The horizontal red arrow indicates the position of the universal root as inferred from the first protein paralogue trees, i.e. between neomura and eubacteria. To determine whether the root is really there or within eubacteria, as suggested instead by many paralogue trees for metabolic enzymes, we must correctly polarize the direction of the negibacteria/posibacteria transition that took place in bacteria that had already evolved flagella. As argued in detail in the text, flagellar evolution and wall/envelope evolution both strongly favour a transition from negibacteria to posibacteria (continuous black arrow), not from posibacteria to negibacteria (broken red arrow). This places the root within Negibacteria and shows that the ancestral cell had two bounding membranes, not just one as traditionally assumed. A negibacterial root also fits the fossil record, which shows that Negibacteria are more than twice as old as eukaryotes [1, 129]. As negibacteria are the only prokaryotes that use sunlight to fix carbon dioxide this is also the only position that would have allowed the first ecosystems to have been based on photosynthesis, without which extensive evolution might have been impossible. Posibacteria, archaebacteria and eukaryotes were probably all ancestrally heterotrophs, whereas negibacteria are likely to have been ancestrally photosynthetic and diversified by evolving all the known types of photosystem and major antenna pigments.
Key molecular cladistic characters that help root the tree of life. Green bars mark major evolutionary innovations. Those explained in detail in previous publications [1, 24, 26] are labelled in blue. Those introduced for the first time or discussed in more detail in the present paper are in red. The three most fundamental changes in cell structure (the origin of unibacteria by loss of the negibacterial outer membrane [1, 5]; the neomuran revolution involving novel chromatin and glycoprotein secretion and much coadaptive macromolecular evolution [1, 5, 29, 62]; and the origin of the eukaryote cell [5, 27, 62]) are marked by thicker bars. So also are the three major transitions, whose key importance and decisiveness for rooting the tree of life are explained here for the first time: the origins of the proteasome, of flagella, and of Omp85 for insertion of OM β-barrel proteins. The three major kinds of cell from the viewpoint of their having fundamentally distinct membrane topology (eukaryotes, unibacteria, negibacteria) [5, 29, 56, 62] are shown by thumbnail sketches (isoprenoid ether lipids in red, outer membranes in blue). Thumbnail sketches also illustrate the inferred times of origin of two key cylindrical macromolecular assemblies (the OM β-barrel protein Omp85 and HslVU/proteasome ATP-dependent regulated proteases) and the two-step increased complexity of the latter. Negibacterial taxa are shown in black, Posibacteria in orange, and neomuran taxa in brown. Gracilicutes comprise four negibacterial phyla with either a very thin peptidoglycan layer or no peptidoglycan at all in their cell envelope: Proteobacteria, Planctobacteria, Spirochaetae, Sphingobacteria (Table 1 explains the formal bacterial taxon names used here for precision and brevity). Evidence for the relatively late dating of the neomuran revolution was explained in detail previously [1]. Note that although Chlorobacteria and Endobacteria are shown as holophyletic, either or both might actually be paraphyletic; I suspect that Endobacteria may be paraphyletic as the most divergent actinobacterium has endospores, but think that Chlorobacteria are probably not. Conversely, it is uncertain whether actinobacteria are paraphyletic as shown or paraphyletic; see text – further work is needed to decide. For simplicity, five additional polarizations within Gracilicutes that are also discussed are not shown; see the more comprehensive Fig. 7 for them and additional characters mapped onto the tree. Note that the ~2.8 Gy date for the origin of cyanobacteria is based solely on hopanoid biomarkers; since no earlier organic deposits have been found that are sufficiently well preserved and with enough extractable hydrocarbons for such biomarker analysis, this is a minimum date (though its validity also depends on the assumption that such hydrocarbons have not migrated vertically in the rocks since being formed, which is hard to test).
Schematic longitudinal sections through the two-tier HslV and the four-tier bacterial 20S proteasome core particle. Red dots are proteolytic active centres. Thumbnail sketches on the left of the main figure are cross sections through the proteolytic chamber showing respectively their 6-fold and 7-fold symmetry. Evolution from the 12-mer HslV to the 28-mer proteasome by duplication to form α- and β-subunits forming heptameric rings is shown by the arrow; loss of proteolytic activity by the new α-subunit (black) coupled with a new ability to stack onto the β-subunits would have expanded the digestive cavity radially and longitudinally and kept potentially vulnerable external proteins further away from the proteolytic centres. Changed dimensions and shape of the α-subunit's ATPase binding surface probably favoured replacement of the HslU ATPase ring by a different one. Hypothetical evolution in the reverse direction by loss of the α-subunit's would have created a less efficient purely β-subunit 14-mer that might have lost any ability to bind an ATPase ring through adapting to α-subunit binding instead and with a broader digestive cavity and entry pore more likely to digest the wrong proteins. It is unlikely that it could have survived purifying selection long enough to reduce its symmetry to sixfold and find a new ATPase partner to bind and thus generate HslVU. No selective advantage for simplification of a proteasome to HslV is apparent. Subunit shapes simplified from [199].
Proteasome evolution showing step-wise increase in complexity, first to the HslV ring protease, then to the 20S proteasome, and lastly to the 26S proteasome; the two major transitions in proteasome structure important for polarizing the tree are marked by grey bars. Blue bars mark four other important evolutionary transitions that also congruently polarize the tree. HslV has 6-fold symmetry (a 2-tiered ring of 12 identical subunits) and arose from a monomeric NTN hydrolase, probably just before Hadobacteria diverged. HslV rings interact with an unrelated chaperone ATPase, HslU, also having 6-fold ring symmetry, like ClpX chaperone from which it arguably evolved and virtually all AAA+ ATPase proteins, which originated in a burst of gene duplications prior to the last common ancestor of all life [19]. The 4-tier proteolytic core of the 6-tiered 20S proteasome evolved in a common ancestor of neomura and Actinomycetales (jointly proteates) of the subphylum Actinobacteria by another gene duplication that generated its catalytic β- and non-catalytic α-subunits from HslV, with an associated symmetry change to 7-fold: all four rings forming the core of the proteasomal cylinder have 7 subunits, but the 6-fold-symmetric HslU was replaced by another hexameric ATPase ring from a different AAA+ family to make the proteasome 'base' (red in the two-colour sketch of the archaebacterial proteasome at the top left). Glycobacteria [1] comprise all the typical negibacteria with OM lipopolysaccharide, i.e. all negibacterial phyla listed in Table 2 except Hadobacteria and Chlorobacteria).
Contrasting cell envelope structure in posibacteria and negibacteria. OM phospholipids, and when present possibly also lipopolysaccharides (LPS), may pass from their site of synthesis in the cytoplasmic membrane to the OM at the Bayer's patch contact sites, but this is not proven and only one protein (Imp) needed for LPS export is yet known. During its biosynthesis murein is secreted across the cytoplasmic membrane by isoprenol carriers. Lipoprotein (LP) is cotranslationally synthesised in both groups. Conversion of a negibacterial wall to a posibacterial wall as shown would be very much simpler than the reverse, requiring only a mutation causing sudden murein hypertrophy that could have broken the OM away from the Bayer's patches, preventing further lipid transfer and OM regrowth, plus the origin of sortases with a novel recognition system for covalently attaching murein lipoproteins (MLP) to the wall. As the negibacteria most closely related to Posibacteria (Eurybacteria) are glycobacteria with much more complex OM, secretion, and import mechanisms than Chlorobacteria (which lack lipopolysaccharide, most porins, Omp85, type I, II, and III secretion machinery, and probably the LolDE lipoprotein release mechanism, of more advanced bacteria), evolution in the reverse direction of such a complex OM in one step from a posibacteria would be practically impossible (see text) and immensely more difficult than the stepwise increase in its complexity possible with a chlorobacterial root of the tree. As the transitional stage between negibacteria and posibacteria had flagella, adding an outer membrane to a posibacterium and evolving a lipid export mechanism in one step would be even more complicated and improbable, as flagellar biogenesis would have had to be conserved and modified at the same time (see Fig. 8). No satisfactory mechanistic explanation has ever been given of how it could possibly have occurred.
The rooted tree of life emphasizing key novelties and synapomorphies. Thumbnail sketches show major variants in cell morphology (microtubular skeleton red; peptidoglycan wall brown; outer membrane blue). The most likely root position is as shown; the possibility that it may lie within Chlorobacteria instead cannot yet be ruled out. Lowest level groups including or consisting entirely of photosynthetic organisms are in green or purple. The frequently misplaced hyperthermophilic eubacteria are in red; indel analysis confirms that Aquifex is a very divergent proteobacterium [79]. The new negibacterial infrakingdom Gracilicutes segregates four phyla from the other negibacteria. Planctobacteria probably lost or reduced murein twice, as free-living Verrucomicrobia have murein. Note that 12 synapomorphies support the earliest branching of Chlorobacteria. The fact that mitochondria were present in the cenancestral eukaryote and that their ancestors, α-proteobacteria are a relatively recently derived of the eubacterial phylum Proteobacteria, proves that eubacteria must be significantly older than eukaryotes and decisively refutes suggestions that eubacteria may be derived from eukaryotes. As α-proteobacteria are nowhere near the root of the tree (irrespective of whether it is rooted beside or within chlorobacteria or as some mistakenly think between neomura and eubacteria) eukaryotes are substantially younger. The age of ~900 My for eukaryotes is based on a recent Bayesian analysis of 143 proteins multiply calibrated from the fossil record [35] and my own critical interpretation of the direct fossil record [129]. This tree, though constructed from rare discrete cladistic characters, is remarkably similar to a 31-protein, 191 species universal sequence tree published while this paper was being reviewed [175]; see responses to comments by referee 3 for discussion of the few differences, all but one (the position of Aquifex) in regions poorly supported on the sequence tree.
Schematic comparison of the three different basal body structures of eubacterial flagella with the putative ancestral junctional pore complex and the related type III secretion injector. The exoflagella of Proteobacteria and Planctobacteria (Exoflagellata), Sphingobacteria, and Eurybacteria project through the outer membrane, with which they are associated by a lipoprotein L-ring (made of FlgH protein units). Spirochaetes have endoflagella within the periplasmic space that do not penetrate the outer membrane and thus need no L-ring. Exoflagella and spirochaete endoflagella both have a P-ring (made of FlgI protein units) thought to act as a bushing for free rotation within the thin peptidoglycan wall (sacculus). Both P-ring and L-ring are absent from the exoflagella of Posibacteria (Actinobacteria and Endobacteria). Posibacterial flagella would automatically have become external when the ancestral outer membrane was lost. The more complex multiprotein shaft of spirochaetes, clearly a derived character (see text) is shown by its greater thickness. If junctional pore complexes also use a basal type III secretion apparatus, flagella and type III injectors probably evolved from them independently. If junctional pore complexes lack type III secretion homologues, it is likely that they evolved during the origin of flagella only and that type III injectors evolved later in the ancestral exoflagellate by simplification of flagella (dashed arrow); see text for discussion. The diagram assumes that ExbB/TonB/OmpA only associated with the basal body of the flagella and evolved into the flagellar stator MotAB during the origin of flagella.
Hypothetical phylogeny for photosynthetic reaction centres. Prior to the last common ancestor of all extant life the primitive reaction centre, a homodimer with two bound quinones, each donating electrons to a primitive cytochrome cc complex, evolved into the heterodimeric type found in green non-sulphur bacteria (Chlorobacteria). This was duplicated prior to divergence of cyanobacteria and gracilicutes to generate a modified homodimeric type of cytochrome bc1 complex with iron-sulphur clusters (FF); for a mechanistic explanation of this duplication see [126]. Cyanobacteria converted the two versions into photosystems I and II. Proteobacteria replaced chlorosomes in the original heterodimeric type by an H subunit with purple carotenoid, but did not retain the new duplicate with FeS clusters. By contrast, this was the only version retained by green sulphur bacteria (Sphingobacteria) and Heliobacteria, both losing the earlier heterodimeric type. This scenario is simplified from ref. 1 and congruent with the cladistic tree in Fig. 7 and the concatenated rRNA tree [80] and is compatible with photosynthetic protein trees, if properly rooted (see text).
Simplified summary of the 10 major cellular transitions in the history of life. Those discussed and polarized previously [1, 27, 33] are shown by grey bars, while those discussed in detail here for the first time have blue bars; the five shown by thick bars plus the origin of 20S proteasomes (within actinobacteria, so no bar) are especially decisively polarized; evolution in the reverse direction would have been highly improbable, whereas for the four shown by narrow blue bars evolution in the reverse direction would have been mechanistically possible but unparsimonious and have required numerous losses. Evolutionary loss has, however, sometimes played a crucial role, as in the origins of posibacteria and of neomura by the loss of the OM and murein wall respectively. The bacterial groups shown in green are either all photosynthetic (Cyanobacteria) or have a mixture of phototrophs and heterotrophs (the others); the entirely non-photosynthetic bacterial groups are in black. The fundamental changes involving murein peptidoglycan are shown in brown. NPC = nuclear pore complexes, several proteins of which are structurally related to those of coated vesicles, all probably arising in a single burst of gene duplication during the origin of eukaryotes [199]. The dotted line from Gracilicutes to mitochondrion signifies the intracellular enslavement of a probably photosynthetic purple bacterium by a protoeukaryote to make the chimaeric eukaryote cell [33, 129], which must have taken place long after the origin of proteobacteria, even longer after the origin of Gracilicutes and eubacteria, but probably only shortly after the neomuran revolution and bifurcation into archaebacteria (the youngest of all bacterial phyla) and the preeukaryote.
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