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International Journal of Systematic and Evolutionary Microbiology(2002), 52, 776 Printed in Great Britain

The neomuran origin of archaebacteria, thenegibacterial root of the universal tree andbacterial megaclassification

Department of Zoology,

University of Oxford,South Parks Road, OxfordOX1 3PS, UK

T. Cavalier-Smith

Tel: j44 1865 281065. Fax: j44 1865 281310. e-mail: tom.cavalier-smith!zoo.ox.ac.uk

Prokaryotes constitute a single kingdom, Bacteria, here divided into two new

subkingdoms: Negibacteria, with a cell envelope of two distinct genetic

membranes, and Unibacteria, comprising the new phyla Archaebacteria and

Posibacteria, with only one. Other new bacterial taxa are established in a

revised higher-level classification that recognizes only eight phyla and 29

classes. Morphological, palaeontological and molecular data are integrated into

a unified picture of large-scale bacterial cell evolution despite occasional lateral

gene transfers. Archaebacteria and eukaryotes comprise the clade neomura,

with many common characters, notably obligately co-translational secretion of

N-linked glycoproteins, signal recognition particle with 7S RNA and translation-

arrest domain, protein-spliced tRNA introns, eight-subunit chaperonin,

prefoldin, core histones, small nucleolar ribonucleoproteins (snoRNPs),

exosomes and similar replication, repair, transcription and translation

machinery. Eubacteria (posibacteria and negibacteria) are paraphyletic,

neomura having arisen from Posibacteria within the new subphylum

Actinobacteria (possibly from the new class Arabobacteria, from which

eukaryotic cholesterol biosynthesis probably came). Replacement of

eubacterial peptidoglycan by glycoproteins and adaptation to thermophily are

the keys to neomuran origins. All 19 common neomuran character suites

probably arose essentially simultaneously during the radical modification of

an actinobacterium. At least 11 were arguably adaptations to thermophily.

Most unique archaebacterial characters (prenyl ether lipids; flagellar shaft ofglycoprotein, not flagellin; DNA-binding protein 10b; specially modified tRNA;

absence of Hsp90) were subsequent secondary adaptations to

hyperthermophily and/or hyperacidity. The insertional origin of protein-spliced

tRNA introns and an insertion in proton-pumping ATPase also support the

origin of neomura from eubacteria. Molecular co-evolution between histones

and DNA-handling proteins, and in novel protein initiation and secretion

machineries, caused quantum evolutionary shifts in their properties in stem

neomura. Proteasomes probably arose in the immediate common ancestor of

neomura and Actinobacteria. Major gene losses (e.g. peptidoglycan synthesis,

hsp90, secA) and genomic reduction were central to the origin of

archaebacteria. Ancestral archaebacteria were probably heterotrophic,

anaerobic, sulphur-dependent hyperthermoacidophiles; methanogenesis and

halophily are secondarily derived. Multiple lateral gene transfers fromeubacteria helped secondary archaebacterial adaptations to mesophily and

genome re-expansion. The origin from a drastically altered actinobacterium of

neomura, and the immediately subsequent simultaneous origins of

archaebacteria and eukaryotes, are the most extreme and important cases of

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This paper is an elaboration of part of an invited presentation to the XIIIth meeting of the International Society for Evolutionary Protistology in CB eske!

Bude)jovice, Czech Republic, 31 July4 August 2000.

Abbreviations: ER, endoplasmic reticulum; GlcNac, N-acetylglucosamine; RuBisCO, ribulose-1,5-bisphosphate carboxylase/oxygenase; snoRNP, small

nucleolar ribonucleoprotein; TCA, tricarboxylic acid.

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T. Cavalier-Smith

quantum evolution since cells began. All three strikingly exemplify De Beers

principle of mosaic evolution: the fact that, during major evolutionary

transformations, some organismal characters are highly innovative and change

remarkably swiftly, whereas others are largely static, remaining conservatively

ancestral in nature. This phenotypic mosaicism creates character distributions

among taxa that are puzzling to those mistakenly expecting uniform

evolutionary rates among characters and lineages. The mixture of novel

(neomuran or archaebacterial) and ancestral eubacteria-like characters in

archaebacteria primarily reflects such vertical mosaic evolution, not chimaeric

evolution by lateral gene transfer. No symbiogenesis occurred. Quantum

evolution of the basic neomuran characters, and between sister paralogues in

gene duplication trees, makes many sequence trees exaggerate greatly the

apparent age of archaebacteria. Fossil evidence is compelling for the extreme

antiquity of eubacteria [over 3500 million years (My)] but, like their eukaryote

sisters, archaebacteria probably arose only 850 My ago. Negibacteria are the

most ancient, radiating rapidly into six phyla. Evidence from molecular

sequences, ultrastructure, evolution of photosynthesis, envelope structure and

chemistry and motility mechanisms fits the view that the cenancestral cell was

a photosynthetic negibacterium, specifically an anaerobic green non-sulphur

bacterium, and that the universal tree is rooted at the divergence between

sulphur and non-sulphur green bacteria. The negibacterial outer membranewas lost once only in the history of life, when Posibacteria arose about

2800 My ago after their ancestors diverged from Cyanobacteria.

Keywords: Unibacteria, Actinobacteria, thermophily and molecular co-evolution ofDNA-handling enzymes, origin of N-linked glycoprotein secretion,microbial fossils and evolution

Introduction and overview

Recent genome sequencing has fostered a simplisticview of organisms as essentially aggregates of genes.However, organisms are not simply a sum of theirgenes nor, as some biochemists were once wont to say,mere bags of enzymes. Genes and enzymes are bothfundamental, but play their vital roles as parts ofhighly organized growing and dividing cells. Their lifedepends on a mutualistic symbiosis of genes, catalysts,membranes and cell skeleton (Cavalier-Smith, 1987a,1991a, b, 2001). Co-adaptation between co-operatingnot selfish molecules is the key to understanding livingorganisms. The degree to which different cellularmacromolecules are co-adapted varies greatly ; formany metabolic enzymes, direct co-adaptation in

structure is low, integration being mediated throughnon-informational intermediary metabolites, but formany informational and structural molecules it is high.Genetic information is made manifest through physi-cal structure. DNA is physically inert genes do notmake organisms; they grow by physico-chemical inter-actions between effector macromolecules whose struc-ture and physico-chemical properties are geneticallydetermined. Membranes of lipids with embeddedproteins are centrally important: chromosomes, ribo-somes and the cytoskeleton physically attach to them;

the cells structural integrity and its character as agrowing and reproducing organism depend on these

direct physical interconnections. The ability of mem-branes to sequester food, grow and divide underliescell growth and reproduction. Like chromosomes, butunlike ribosomes and the skeleton, membranes showdirect genetic continuity: all are descended by growthand division from those bounding the first cell(Cavalier-Smith, 1991a, b). Membranes have a her-editary role as well as structural and physiologicalroles (Cavalier-Smith, 2000a, 2001). The unity of lifestems from the common origin and fundamentalsimilarity of these processes in all organisms.Organismal structural diversity, on the other hand,arises through variations in membrane topology andphysico-chemical properties as well as in the shapesformed by the cell skeleton, for both of which thegenically specified catalysts create the building blocks.This means that we cannot understand the evolutionof life without elucidating the evolution of cellorganization and reproduction as well as that of theindividual molecules that mediate them.

The most profound difference within the living worldlies between bacteria and eukaryotes (Stanier & VanNiel, 1962 ; Stanier, 1970; Cavalier-Smith, 1987b,1991a, b, 1998). Bacteria in this paper is used in the

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Eubacterial origins of life and of Archaebacteria

proper traditional sense to embrace all prokaryotes(Cavalier-Smith, 1992b, 1998; Mayr, 1998), never asa fashionable but highly confusing synonym foreubacteria only (Woese et al., 1990). Bacteria andeukaryotes differ fundamentally in the topologicalrelationships between membranes, genomes and ribo-somes and in their skeletons. In all bacteria, chromo-somal DNA and ribosomes making membrane

proteins are attached directly to the cytoplasmicmembrane, which grows by the direct insertion ofproteins and lipids. In eukaryotes, the chromosomesand ribosomes making membrane proteins are at-tached instead to the endoplasmic reticulum (ER)\nuclear envelope, which is topologically within, andunconnectedto,theplasmamembrane,whichgrowsbyfusion of vesicles budded from endomembranes; theER grows, like the bacterial cytoplasmic membrane,by the direct insertion of individual lipid moleculessynthesized by proteins embedded within the samemembrane. All eukaryotes have a complex endo-skeleton (the cytoskeleton) of microtubules and actinfilaments that use attached molecular motors to

mediate chromosome segregation and cell division,respectively. By contrast, bacteria have an exoskeleton(cell wall) important for DNA segregation and celldivision. There has been much discussion of how theseand other profound differences between bacteria andeukaryotes have arisen (Margulis, 1970 ; Cavalier-Smith, 1975, 1980, 1981, 1987b, 1990, 1991a, b, c,1992c, 1993, 2000b; de Duve, 1996; Faguy & Doolittle,1998), updated in a following paper (Cavalier-Smith,2002). The primary purpose of this paper is to discussthe origins of the less profound, but highly importantdifferences between the three major types of bacteria:the structurally simple archaebacteria (Woese &Fox, 1977) and posibacteria (Cavalier-Smith, 1987b)

and the topologically more complex negibacteria(Cavalier-Smith, 1987b).

Archaebacteria and posibacteria are bounded by asingle membrane only and are thus referred to col-lectively as unibacteria (Cavalier-Smith, 1998). Negi-bacteria, in sharp contrast, are bounded by twotopologically distinct membranes; the cytoplasmicmembrane, into which lipids and proteins are inserteddirectly, and the relatively porous outer membranethat grows more indirectly by their subsequent transferacross specific adhesion sites between the two. Thebiogenesis of the negibacterial envelope is more com-plex and requires extra chaperones. As the cytoplasmic

membrane of posibacteria and negibacteria is com-posed of acyl ester lipids, like eukaryotic membranes,they are grouped together as eubacteria, so as tocontrast them with archaebacteria, which are uniquein the living world in having prenyl ether lipids instead.

Two fundamentally different views have been pro-posed of the significance of this and other strikingdifferences between archaebacteria and eubacteria.One influential school of thought regards them asancient differences that reflect an early divergence soonafter the origin of life, before many cell characters had

become stabilized (Woese & Fox, 1977; Woese, 1998,2000; Graham et al., 2000). The second view is thatarchaebacteria are not an ancient group at all (Horiet al., 1982) but arose secondarily from eubacteriarelatively recently as an adaptation to hyperther-mophily (Cavalier-Smith, 1987a, b, 1991a, b, 1998;Forterre, 1996); although not all archaebacteria arethermophiles, it is argued that their last common

ancestor was a hyperthermophile and that it arosefrom a eubacterial ancestor by lipid replacement andother adaptations. Here, I review recent evidence andarguments that, in my view, support compellingly thesecondarily derived nature of archaebacteria. It is nowwell established that archaebacteria are either ancestralto (Van Valen & Maiorana, 1980) or, more likely(Cavalier-Smith, 1987b), sisters of eukaryotes, withwhich they share many important characters. Whenfirst proposing that archaebacteria and eukaryoteswere sister taxa, I called the clade that comprised themneomura (new walls), because I considered that theirshared N-linked glycoproteins were derived comparedwith the ancestral peptidoglycans of eubacteria, ar-

guing that the fossil record implied that neomura wereless than half the age of eubacteria (Cavalier-Smith,1987b). I also asserted that neomura evolved fromposibacteria by the replacement of peptidoglycan byN-linked glycoproteins and tentatively suggested thatneomura are more closely related to high-GjC Gram-positive bacteria (the subphylum Actinobacteria) thanto low-GjC Gram-positives (here collectivelygrouped with mycoplasmas and their heliobacterialand thermotogalean allies as a new subphylum, Endo-bacteria).

This paper reviews recent evidence that very stronglysupports such an actinobacterial origin for theneomura and develops the secondary hyperthermo-phily hypothesis of the origin of archaebacteria(Cavalier-Smith, 1987a, b) in more detail. A critical re-evaluation of the fossil record in the present paperindicates that eukaryotes are much younger than oftenthought (Cavalier-Smith, 1990) probably only about850 million years (My) old. The bacterial fossil recordclearly indicates that eubacteria are far more ancient,at least 3500 My old. Dating archaebacterial origins ismore problematic, but I shall argue that, likeeukaryotes, they are probably at least four timesyounger than eubacteria. The present paper alsoseverely criticizes arguments and assumptions thathave been used to suggest that archaebacteria and\or

eukaryotes may be more ancient than or as old aseubacteria. The somewhat revised classification of thekingdom Bacteria adopted here is summarized in Table1; my reasons for treating all prokaryotes as a singlekingdom Bacteria, and why eubacteria are not a cladeand are preferably not treated as a taxon, wereexplained previously (Cavalier-Smith, 1998).

My arguments that neomuran and archaebacterialcharacteristics are all relatively recently derivedcharacters in no way trivializes the importance of thenumerous differences between archaebacteria and

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Table 2. Major archaebacterial properties not found in eubacteria

(a) Neomuran properties (i.e. those shared with eukaryotes)

1. Signal recognition particle (SRP) with 7S RNA with a helix 6 that binds SRP19 protein; protein secretion generally

co-translational; SecA absent

2. Co-translational glycosylation of surface glycoproteins by transfer of GlcNAc and mannose-containing

oligosaccharides from a dolichol isoprenoid carrier to N-asparagine; homologous oligosaccharyl transferases; murein

absent3. Ribosomal rRNA pseudouridylated by C\D-box snoRNAs

4. Core histones with histone fold [secondarily lost in some archaebacteria (e.g. Thermoplasma) and some eukaryotes

(dinoflagellates)]

5. Replicative DNA polymerases B type ; inhibited by aphidicolin; replicative sliding clamp is PCNA-type, not part of a

type C DNA polymerase holoenzyme; novel replication factor complex

6. Flap endonuclease and RAD2 DNA-repair enzymes

7. Seven or more RNA polymerase holoenzyme subunits (not four as in eubacteria)

8. Many similarities of ribosomal RNA and proteins; a more substantial projecting bill on the small ribosomal subunit ;

ribosomes insensitive to chloramphenicol; anisomycin inhibits peptidyl transferase by binding to 23S\28S rRNA

9. CCT-type group II chaperonins with eightfold symmetry, not sevenfold symmetry as in their distant eubacterial Hsp60

relatives; with built-in cap; co-chaperonin Hsp10 absent ; prefoldin (GimC) channels nascent proteins to the chaperonin

lumen

10. Some similar tRNA modification

11. Exosomes; complex of 1116 proteins involved in exonucleolytic digestion of RNA ; exonucleases, helicases and RNA-binding proteins (Koonin et al., 2001)

12. More similar protein synthesis elongation factors (e.g. sensitive to ADP ribosylation by diphtheria toxin)

13. Co-translational selenocysteine insertion requires a SECIS-binding protein in addition to a selenocysteine-specific

elongation factor

14. CCA 3h terminus of tRNA added post-translationally, not encoded by the gene

15. Protein synthesis initiated by methionine not N-formyl methionine; several extra initiation factors (eIF-2, 2A, 2B and

5A)

16. 5h-OH\3h-phosphate protein-spliced tRNA introns with homologous endonucleases

17. Novel type II DNA topoisomerase VI\meiotic protein

18. Insertion in catalytic subunit of the vacuolar-type proton-pumping ATPase

19. Hexameric replicative DNA helicase Mcm instead of eubacterial DnaB (Poplawski et al., 2001)

(b) Unique archaebacterial properties

1. Prenyl ether instead of acyl ester lipids

2. Flagellar shaft of acid-insoluble glycoproteins related to pilin, not acid-soluble flagellin

3. DNA-binding protein 10b

4. Unique tRNA modifications, including archaeosine in -loop and absence of queuine

5. A tiny large subunit ribosomal protein, LX

6. Absence of Hsp90 chaperone

7. RNA polymerase A split into two proteins

8. Glutamate synthetase split into three separate proteins

eubacteria. Although the differences in organization of

the replication, transcription and translation machin-ery of archaebacteria are well known (Doolittle, 1998;Graham et al., 2000), the full extent of other majordifferences between archaebacteria and eubacteria incell organization is still insufficiently widely appreci-ated, some having only become apparent recently.Table 2 lists the key differences between archaebacteriaand eubacteria. The scale of these is so great that thispaper, which attempts to explain them all, is necessarilylong and detailed. To help the reader see the wood forthe trees, let me outline its basic structure. I shall argue

that all 19 features listed in Table 2(a) arose in the

common ancestor of eukaryotes and archaebacteria inassociation with the loss of eubacterial peptidoglycanand its functional replacement by neomuran N-linkedglycoproteins. This part of the neomuran theory isidentical to the original, except that the number ofuniquely shared neomuran character suites hasdoubled since the theory was originally proposed(Cavalier-Smith, 1987b), placing the relationship be-tween eukaryotes and archaebacteria beyond question.To save space, I refer readers to the original paper formore details of the basic rationale of the neomuran



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theory, including the sister relationship of archae-bacteria and eukaryotes (rather than an ancestordescendant one, as suggested by Van Valen &Maiorana, 1980; Rivera & Lake, 1992; Baldaufet al.,1996), the much more ancient ancestral character ofeubacteria and the changeover from peptidoglycan toglycoproteins, as well as for diagrams summarizing thecellular transformations (Cavalier-Smith, 1987b).

I concentrate here on six things. First are the keyinnovations of the present paper: the arguments thatthe majority of the novel neomuran characters arose asadaptations of the neomuran ancestor to thermophilyand that nearly all neomuran characters can be used topolarize unambiguously the direction of evolutionfrom posibacteria to neomura, not the reverse. Secondis the argument that, after the neomuran commonancestor adapted thus to thermophily, the archae-bacterial ancestor alone underwent a more extremeadaptation to hyperthermophily and hyperacidity thatproduced almost all the uniquely archaebacterialcharacters listed in Table 2(b). About a third of the

paper discusses the origin of each of these neomuranand archaebacterial characters. Having provided ex-tensive evidence from comparative biology that neo-mura are derived compared with eubacteria, I thendiscuss the fossil record for all three domains of life,which shows exactly the same thing and indicatesthat neomura are about four times younger thaneubacteria. Central to my re-evaluation of the fossilrecord is recent evidence that some actinobacteria, theprobable ancestors of eukaryotes, make sterols (Lambet al., 1998), which invalidates earlier palaeontologicalinterpretations of fossil steranes as eukaryoticmarkers ; this and other recent discoveries of mor-phological fossils make my earlier estimate of 850 My

for the origin of eukaryotes (Cavalier-Smith, 1980)more accurate than more recent ones giving an olderdate (Cavalier-Smith, 1987a, 1990). My fourth topic isthe application of the ideas of quantum and mosaicevolution to the interpretation of molecular sequencetrees. These principles explain many of the puzzlingconflicts between different trees. Still more import-antly, in conjunction with my discussion of theevidence for temporarily accelerated evolutionaffecting all the characters of Table 2 at the time oforigin of neomura, but not other more ancestralcharacters, they tell us that reciprocally rooted proteinparalogue trees and single-gene trees (e.g. for rRNA)based on them are so dimensionally distorted as to be

highly misleading about the temporal history of life;this has caused the misrooting of the universal tree oflife. Once we understand these distortions, we can seethat there is no genuine conflict between any moleculartrees and the fossil evidence that neomura are veryrecent. My fifth topic is to use this new understandingof the strengths and weaknesses of different moleculartrees to integrate their evidence with the fossil recordand cell-biological considerations so as to pinpoint theroot of the tree as accurately as is currently possible. Ishall argue that recent evidence concerning the evol-

ution of photosynthesis strongly supports earlierarguments that the root of the tree of life lies withinthe negibacteria (Cavalier-Smith, 1987a, b, 1991a, b,1992b). Although the precise position of the rootremains uncertain, it very likely lies within or im-mediately adjacent to the green bacteria, as suggestedpreviously (Cavalier-Smith, 1985a, 1987a). I point outthat many current interpretations of cell and molecular

evolution are fundamentally flawed by the seriousmisrooting of molecular trees and the misplacing ofsome long branches. My sixth concern is to show that,although lateral gene transfer is more frequent andconfusing in bacteria than in eukaryotes, we can stillconstruct sensible organismal phylogenies for bacteria,provided we emphasize organismal features thatdepend on strong co-adaptation between macro-molecules and do not overemphasize the evidencefrom any single molecule.

I emphasize that, for most of the history of life,immensely long periods of relative stasis have followedtwo explosive radiations or biological big bangs,each stimulated by revolutionary innovations in cellbiology: (i) the origin about 3700 My ago of the firsteubacterial cell with peptidoglycan walls and photo-synthesis (Cavalier-Smith, 2001) and (ii) the originabout 850 My ago of the ancestral neomuran cell,when N-linked glycoproteins replaced peptidoglycanand the pre-eukaryote neomurans evolved phago-trophy, internal skeletons and the endomembranesystem. The neomuran theory of the origin ofeukaryotes is further developed in another paper,published separately because of space constraints(Cavalier-Smith, 2002); however, the two papers needto be read together fully to appreciate and evaluate thisrevised neomuran theory of the simultaneous actino-

bacterial origins of archaebacteria and eukaryotes. Athird paper, on the origin of the negibacterial cell andthe genetic code (Cavalier-Smith, 2001), is com-plementary to both, since it shows that it is much easierto understand the origin of life if we root the treeamong photosynthetic negibacteria, rather than be-tween archaebacteria and eubacteria as suggested bymost reciprocally rooted protein paralogue trees.

I also discuss the early diversification of negibacteriathat constituted the first big bang, integrating bothfossil and recent evidence and arguing that thedifferences between the six phyla arose primarily asdivergent adaptations within the microlayers of earlymicrobial mats. In addition to these phylogenetic andevolutionary questions, I discuss briefly the higherclassification of bacteria and how it may be improved.

Secondary hyperthermophily and acidophily

and the origin of archaebacteria

It has long been argued that the prenyl ether lipids ofarchaebacteria evolved as replacements for the acylester lipids of eubacteria (Cavalier-Smith, 1987a, b) asa secondary adaptation to hot, acid environments



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Fig. 1. The origin and diversification ofArchaebacteria. Archaebacteria originatedby two successive revolutions in cell biology:a neomuran phase shared with their eu-karyote sisters followed shortly by a uniquelyarchaebacterial one. The first, neomuranphase was an adaptation to thermophily andinvolved a really major transformation of 19key characters, including replacement of thecell wall peptidoglycan murein by N-linkedglycoprotein and a great upheaval in thecells protein-secretion and DNA-handlingmachinery. The second, relatively minorphase of specifically archaebacterial inno-vations, notably replacement of acyl estermembrane by isoprenoid tetraether lipidsand of eubacterial flagellin by glycoproteins,

involvedfurtheradaptations to hyperthermo-phily and hyperacidity, respectively. Substan-tially later, several lineages independentlyreadapted secondarily to mesophily. Lateraltransfer of genes from the immensely olderand far more diverse eubacteria often playeda role in these secondary returns to mesophilyand may also have done in the origins ofarchaebacterial hyperthermophily, sulphatereduction by Archaeoglobus and methano-genesis. This phylogenetic interpretation isbased on a synthesis of discrete organismaland molecular characters treated cladistic-ally, sequence trees and palaeontology, asdiscussed in the text.

(Reysenbach & Cady, 2001). The presence of sulphur-dependent hyperthermophiles among both eury-archaeotes (Thermococcales and Methanothermus)and crenarchaeotes strongly suggests that the ancestralarchaebacterium was also a sulphur-dependenthyperthermophile (Woese, 1987; Barns et al., 1996).It is very unlikely, however, that the ancestral eu-bacterium or first cell was a thermophile or hyper-thermophile, as is sometimes suggested (Achenbach-Richter et al., 1987; Pace, 1991); the low thermalstability of essential organic molecules such as RNA

makes it far more likely that the first cell was amesophile (Levy & Miller, 1998) or even psychrophile(Cavalier-Smith, 2001). Hyperthermophilic environ-ments were probably the last to be colonized; thechimaeric origin of reverse gyrase implies that hyper-thermophiles evolved last of all (Forterre, 1996) andmaximum-likelihood reconstruction of the cenan-cestral base composition favours a mesophile (Galtieret al., 1999). The distribution of reverse gyrase withinarchaebacteria indicates that it was present in theircommon ancestor but was lost by Halobacteria and

Methanosarcinales (here grouped together as Halome-bacteria; Table 1) and by Thermoplasma (Lo! pez-Garc!a, 1999) and replaced by eubacterial DNA gyraseby lateral gene transfer. The fact that mesophilicmethanogens and halobacteria share a split RNApolymerase gene (RpoB protein exists as two distinctsubunits) uniquely with Archaeoglobales (Klenk et al.,1997) implies strongly that this clade (which I callNeobacteria; Table 1), and thus the mesophily ofHalomebacteria, is derived within the Archaebacteria(Fig. 1) and that reverse gyrase was replaced by DNA

gyrase independently in the thermophile Thermo-plasma, which has the ancestral unsplit RNA poly-merase gene. This secondary mesophily of Halome-bacteria was associated with the replacement of tetra-ether prenyl lipids, which form thermostable mono-layers, by biether prenyl lipids giving more fluidbilayers. I argued previously (Cavalier-Smith, 1987a)that there would probably be no selective advantagefor a secondary mesophile in replacing these lipids byeubacterial\eukaryotic acyl ester lipids, whereas re-placement of acyl esters by the more heat-stable and



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acid-stable prenyl ethers (initially tetraethers in thearchaebacterial ancestor), which are much more im-permeable to protons at higher temperature and clearlyadaptive to hot acid (Albers et al., 2000), wouldundoubtedly be selectively advantageous to a hyper-thermophile that evolved secondarily from a meso-philic ancestor. Thus, both the unique lipids andreverse gyrase indicate strongly that the direction of

evolution was from mesophilic eubacteria to hyper-thermophilic archaebacteria. Because of their novellipids, thermophilic archaebacteria can control theirpH and use proton gradients as energy sources, unlikeeubacterial thermophiles (Albers et al., 2000).

I now argue that secondary acidophily gives a simpleadaptive explanation to the otherwise puzzling factthat archaebacterial flagellar shafts lack classicalflagellins but are built of unrelated proteins (Faguy etal., 1994). Eubacterial flagellin filaments disassembleto monomers under very acid conditions, and atsomewhat acid pH eubacterial flagella undergo aremarkable phase transition to an abnormal, lessefficient, curly form (Kamiya et al., 1982). Replacing

an ancestral flagellin polymer by recruiting an acid-stable glycoprotein from pili, which the shaft resembles(Faguy et al., 1994), would have enabled archae-bacterial flagella to function in highly acid conditions,while retaining the same basal rotary motor. Asarchaebacterial flagella operate well in neutral con-ditions, there would be no selective advantage inreplacing them by flagellin in secondary mesophiles.

Paucity of unique features of archaebacteria

Apart from the special lipids and flagellar shafts, onlyfour other unique features have been so far identifiedas generally present in archaebacteria (Table 2b). Best

known are the unique post-transcriptional modifi-cations of their tRNAs. These are also almost certainlysecondary adaptations to thermophily. Kowalak et al.(1994) have shown that modifications greatly increasethe thermal stability of archaebacterial tRNAs and aremore extensive at higher temperatures. Unlike anyother organisms, archaebacteria replace a guanine atposition 15 in the -loop with archaeosine. Unlikeeubacteria and eukaryotes, they never use queuine inthe wobble position of the anticodon. Graham et al.(2000) assert that the transglycosylase protein thatinserts archaeosine is unique to archaebacteria. This isonly half true. The enzyme is clearly homologousin sequence to the queuine-inserting one of other

organisms. Rather than being a novel archaebacterialenzyme, a pre-existing one simply switched itsspecificity; I suggest that the switch was from queuineinsertion to archaeosine insertion. A more convinc-ingly unique archaebacterial protein is the small,10 kDa DNA-binding protein, 10b; in Sulfolobus, itsbinding produces passive negative supercoils at veryhigh temperatures, but not at mesic ones (Xue et al.,2000). I suggest that it evolved as a secondaryadaptation to hyperthermophily in the ancestralarchaebacterium, as a substitute for active negative

supercoiling by DNA gyrase, which was lost in theneomuran cenancestor (i.e. their last common an-cestor; Fitch & Markowitz, 1970). Too little is knownabout the other unique archaebacterial protein, thetiny, 77-amino-acid protein LX of the large ribosomalsubunit, to know whether it was also an adaptationto hyperthermophily or hyperacidity or evolved foranother reason. There is no reason to think that the

splitting of the RNA polymerase gene A to make twoseparate proteins or of glutamate synthetase into threewere adaptations; both were possibly neutral changesthat became incidentally fixed in the archaebacterialcenancestor.

Graham et al. (2000) identified 36 conserved hypo-thetical proteins found in all five of the archaebacterialgenomes then sequenced. When their functions areknown, it will be interesting to see how many are alsoadaptations to hyperthermophily and how few arereally unique to archaebacteria. It is likely that mostare evolutionarily related to eubacterial proteins, butdiverged so drastically during archaebacterial originsthat a relationship is not obvious from the sequences.Adaptation to hyperthermophily increases the chargedresidues in proteins (Cambillau & Claverie, 2000); insome proteins, such adaptation may have led to muchmore extensive changes. Four proteins claimed to beunique to archaebacteria (Graham et al., 2000) clearlyare not. Both A and B subunits of DNA topoisomeraseVI, though stated to be uniquely archaebacterial[Forterre & Philippe, 1999 ; Graham et al., 2000(mislabelled as topoisomerase IV in their table)], arestrongly related to those of a meiosis-specific proteinof eukaryotes. As no eubacterial relatives are certainlyknown, Table 2 shows them as neomuran, not simplyarchaebacterial characters; however, I shall argue thatthis neomuran topoisomerase evolved from DNAgyrase and is not a novel protein. The Holliday

junction cleavage resolvase is not unique to archae-bacteria (Graham et al., 2000) but is structurallyrelated to other nucleases widespread in eubacteria(Aravind et al., 2000); it even has distant primarystructure similarity to RuvC, the eubacterial Holliday

junction resolvase, despite the latters different fold.The transcription termination\inhibition factor isclearly related to the functionally equivalent NusGprotein of eubacteria and to elongation factor Spt5 ofeukaryotes.

To speak of an archaebacterial genomic signature(Graham et al., 2000) is misleading. The genome

organization of archaebacteria is fundamentally thesame as that of eubacteria. What is unique are therather small number of genes mentioned above, andeven their uniqueness is probably exaggerated byaccelerated sequence evolution. Graham et al. (2000)also exaggerate the uniqueness of archaebacteria byreferring to features present in only some archae-bacteria as archaebacterial signatures . However,such features as methanogenesis are not properties ofarchaebacteria as a whole; it is as misleading to callthem archaebacterial signatures as it would be to call



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feathers or hair vertebrate signatures, rather thanbird or mammal signatures. Many hundreds ofproteins they call archaebacterial signatures are ac-tually euryarchaeote, crenarchaeote or methanogensignatures, for example, and are irrelevant to theunderstanding of the origin of archaebacteria, mymain focus. However, some features found only inarchaebacteria, but not in all, will have been present in

their cenancestor but lost by a few lineages. The mostobvious of these are the two flagellar shaft proteinsand a flagellar accessory protein, which Table 2 doestreat as general archaebacterial proteins. Phylogeneticanalysis will eventually reveal other non-universalarchaebacterial proteins that were actually alsocenancestral.

Thus, all functionally understood unique and generalfeatures of archaebacteria are apparently adaptationsto hyperthermophily or hyperacidity. There is noreason to think that any are ancient or relics of earlyevolution. Archaebacteria are genomically and cyto-logically fundamentally the same as posibacterial

eubacteria. Their uniqueness among bacteria restsmuch less on the small number of unique archae-bacterial characters (Table 2b) than on the very largenumber of characters shared with eukaryotes (Table2a). These are especially important, as many are notsingle-gene characters but depend on numerous genes.Thus, it is exceedingly misleading to refer to archae-bacteria as a third form of life. Except for theirmembrane lipids, flagellar shafts, tRNA modificationsand the small proteins 10b and LX, they share virtuallyevery understood character with other bacteria or withtheir eukaryote sisters. Because the origin of neomurancharacters (Table 2a) is important for understandingthe origin of both archaebacteria and eukaryotes

(Cavalier-Smith, 1987b, 2002), I discuss them first.

Novel cell walls, thermophily and the origin

of neomura: rooting the tree in Eubacteria

At first sight, the changes listed in Table 2(a) seem anarbitrary set of molecular properties from thethousands that characterize bacteria. The neomurantheory argues, however, that virtually all are explicableas co-ordinated changes in the cell envelope andinteractions of ribosomes with it or with the adaptationof chromatin to thermophily. None involves changesin intermediary metabolism, to which the majority

of eubacterial genes are devoted. It is the intercon-nections between so many of these changes that areevolutionarily important. The replacement of peptido-glycan by glycoprotein involved novel protein-secretion mechanisms; these involved changes in theribosomes and in the chaperone machinery. Changesin DNA-binding proteins affected the replication,repair and transcription machinery. Though I shalldiscuss them one by one, the key point of the theorylies in the concerted evolution that radically trans-formed hundreds of genes and their interacting gene

products during one short evolutionary episode, butleft thousands more little changed.

The first 11 neomuran characters (Table 2a) can beinterpreted as adaptations by the ancestral neomuranto thermophily; as they have generally not beenreversed either in secondarily mesophilic eury-archaeotes or in eukaryotes, which probably becamemesophiles during eukaryogenesis (Cavalier-Smith,2002), this indicates that the reverse change from theneomuran to the eubacterial state would be unlikely tobe positively selected. Thus, these 11 characters plusthe first four unique archaebacterial characters, whichhave also not been reversed in secondary mesophiles,together provide 15 evolutionary valves that we canuse with high confidence to polarize the direction ofevolution from eubacteria to neomura rather than thereverse. Elsewhere (Cavalier-Smith, 2001), I arguedthat characters 1214 are so much more complex thanthose of eubacteria that each must be regarded asderived not primitive, while tRNA introns must bederived (Cavalier-Smith, 1991c). The insertion in the

catalytic subunit of the vacuolar proton-pumpingATPase, though an apparently trivial character un-connected with the others, is important because itsabsence in the paralogous non-catalytic subunitstrongly indicates that the eubacterial condition isancestral and the neomuran one was derived by aninsertion in the common ancestor of eukaryotes andarchaebacteria (Gogarten & Kibak, 1992). The selec-tive advantage of this universally conserved changeprobably lies in the increased complexity of the linkerproteins that join the ATPase to the membrane-spanning proteolipid that also underwent great changein the neomuran cenancestor (Hilario & Gogarten,1998). Overall, therefore, including reverse gyrase,

there are 20 different characters, most rather complex,that independently polarize the direction of evolutionfrom eubacteria to neomura. Not one supports thereverse. Fig. 2 summarizes this view of the tree of life.

First, consider the switch in protein-secretion mech-anism between eubacteria and neomura.

Derived neomuran protein-secretion and-glycosylation mechanisms

In eubacteria, proteins bearing a signal sequencefollow two distinct pathways. Membrane proteins withan uncleaved signal sequence are inserted co-trans-

lationally directly into the cytoplasmic membrane bythe interaction of their signal sequence with the signalrecognition particle (SRP); this causes ribosomes todock onto a ribosome receptor (the SecYEG proteincomplex) embedded in the membrane. Secretory pro-teins with cleavable signal sequences, by contrast, areoften released from the ribosome into the cytosol andare recognized by SecA protein, which directs thempost-translationally to the SecYEG channel for trans-location across the membrane into the periplasmicspace. In proteobacteria like Escherichia coli, most, if



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.....................................................................................................

Fig. 2. The rooted tree of life, showingkey innovations. The ancestral eubacterialdomain is about four times older thanthe archaebacteria and eukaryotes, which

jointly form a recent clade, designatedneomura (Cavalier-Smith, 1987b) becausethe ancestral eubacterial peptidoglycan wasreplaced by N-linked glycoprotein duringtheir common origin about 850 My ago.

Both the fossil record and the 20 charactersuites (Table 2) that polarize the tree fromeubacteria to neomura prove that eubac-teria are ancestral and paraphyletic the onlyUr-domain. The double envelope of negi-bacterial cells probably evolved well beforethe cenancestor by the fusion of obcells, asdescribed elsewhere (Cavalier-Smith, 2001) ;it was retained as the double envelope ofmitochondria and chloroplasts when theyoriginated from proteobacteria and cyano-bacteria. The negibacterial outer membranewas lost only once in the history of life, inthe ancestral posibacterium. This unimem-branous character of posibacteria was apre-adaptation for the much later originof neomura from a thermophilic actino-

bacterium similar to a mycobacterium. Afterthe origin of the 19 shared character suites(Table 2a), the neomuran ancestor divergedsharply into two contrasting lineages; oneformed a glycoprotein wall and becamehyperthermophilic, evolving prenyl etherlipids and losing many eubacterial genes,e.g. for H1 histones, to form the archae-bacteria, the other became much moreradically changed by using its glycoproteinsas a flexible surface coat, evolving phago-trophy, an endomembrane system, endo-skeleton and nucleus (N) and enslaving an-proteobacterium as a protomitochondrion(M) to become the first eukaryote, as ex-plained in detail elsewhere (Cavalier-Smith,2002).

not all, secretory proteins follow this post-translationalpathway. In Bacillus subtilis (and, very likely, otherposibacteria), however, only a minority of secretoryenzymes use the SecA post-translational mechanism;the great majority are probably secreted co-trans-lationally by the SRP mechanism (Tjalsma et al.,2000).

Neomura, however, do not have SecA and secreteessentially all proteins with a cleavable signal sequenceco-translationally. This is achieved through the pres-

ence of an additional translation-arrest domain on theSRP RNA and an extra 19 kDa SRP protein. Thetranslation-arrest domain delays the extension of thepolypeptide chain sufficiently for the signal sequenceto bind to the membrane receptor and for translationacross the membrane to be initiated prior to thecleavage of the signal peptide by the membrane-associated signal peptidase, which has its active site onthe periplasmic surface of the cytoplasmic membrane(Mason et al., 2000; Walter etal., 2000). This direct co-translational threading of the nascent polypeptide

through the cytoplasmic membrane to the outside,where it can fold immediately into its native con-figuration, would be especially advantageous for athermophile. With the eubacterial post-translationalSecA-based system, there is a much greater risk thatthe protein could become irreversibly denatured in thecytosol and lose its translocation competence or bedegraded by cytosolic proteases that recognize un-folded proteins. Eubacteria possess two other purelypost-translational translocases, TAT and YidC (Stuart& Neupert, 2000; Samuelson et al., 2000). These lattersystems would also be more prone to disruption byheat, which might denature proteins irreversibly beforethey ever reached the membrane, than would anobligately co-translational one.

The ancestral bacterium is unlikely to have done muchprotein secretion compared with the more complexmodern ones, and the smallest, simplest SRP of negi-bacteria is likely to be the ancestral type that evolvedinitially just for the insertion of membrane proteins,essential even for the simplest, most primitive cell



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Table 3. Neomuran characters shared by some or all actinobacteria but not other eubacteria.................................................................................................................................................................................................................................................................................................................

The ability to produce N-penicillin and cephalosporins is shared, as far as is known, only by fungi, which may therefore haveacquired them by lateral gene transfer. The listed, more generally distributed eukaryotic characters are more likely to have beeninherited vertically.

General neomuran characters

1. Proteasomes

2. 3h-Terminal CCA of tRNAs mostly (actinobacteria) or entirely (neomura) added post-transcriptionally

Characters shared by eukaryotes generally but not archaebacteria

1. Sterols

2. Chitin

3. Numerous serine\threonine phosphotransferases and protein kinases related to cyclin-dependent kinases (Av-Gay &

Everett, 2000)

4. Tyrosine kinases

5. Long H1 linker histone homologues related to eukaryotes ones throughout

6. Calmodulin-like proteins (Swan et al., 1987)*

7. Phosphatidylinositol (in all actinobacteria)

8. Three-dimensional structure of serine proteases

9. Primary structure of alpha amylases10. Fatty acid synthetase a complex assembly

11. Desiccation-resistant exospores

12. Double-stranded DNA repair Ku protein with C-terminal HEH domain (Aravind & Koonin, 2001)

* Xi et al. (2000) report a protein with calmodulin-like motifs, but its sequence is much less similar to calmodulin than those ofStreptomycetes and Arabobacteria, which are remarkably like those of sarcoplasmic reticulum.

(Cavalier-Smith, 2001). As mesophilic eubacteria be-came more complex and started to secrete proteins,they added the SecA mechanism to facilitate this. Oneparticular phylum alone, the Proteobacteria, made thefurther addition of the SecB chaperone to reduce the

problem of denaturation and degradation of proteinsprior to secretion. I suggest that this problem becameparticularly acute in actinobacteria, which are oftenthermophiles (never hyperthermophiles) that secretean unusually large number of proteins or peptides;pronounced protein secretion is a basic characteristicof posibacteria Bacillus subtilis secretes over 300(Tjalsma et al., 2000); its more-complex SRP hashelices 14 like neomura. These two features of theirlifestyle may explain why the ancestral actino-bacterium evolved proteasomes for the degradation ofmisfolded or denatured proteins. Proteasomes areconstitutively synthesized, cylindrical macromolecularassemblies in which protein digestion takes place

within the cylinder and which are found only inActinobacteria and neomura (Maupin-Furlow et al.,2001). This is one of a dozen important reasons (Table3; discussed briefly later in this paper and in moredetail by Cavalier-Smith, 2002) why Actinobacteriaare the most likely ancestors of neomura. It used to bethought that Thermoplasma, in which archaebacterialproteasomes were first characterized, also hadubiquitin, like eukaryotes, but the genome sequencecontradicts this (Ruepp et al., 2000). Since ubiquitinhas not been convincingly demonstrated in any archae-

bacterium, but is universal in eukaryotes, I suggestthat proteasome evolution occurred in two temporallydistinct phases: first, the origin of the basic 20Sproteasome in the common ancestor of actinobacteriaand neomura, then, very much later, in the pre-

eukaryotic lineage alone, the evolution of ubiquitinand the polyubiquitin system for tagging proteins fordegradation that the more complex eukaryotic 26Sproteasome uses. I argue elsewhere (Cavalier-Smith,2002) that the eukaryotic complexification of theproteasome was connected with the evolution ofnovel eukaryotic cell-cycle controls. The basic 20Sproteasome, though a prerequisite for these laterelaborations, had quite other origins in an earlyactinobacterium. The fact that inhibition of pro-teasome action in Thermoplasma has much moresevere effects during heat shock than in normalgrowth (Ruepp et al., 1998) supports my thesis thatproteasomes were initially an adaptation to thermo-

phily.

The narrow openings of the proteasome nano-compartment (Maupin-Furlow et al., 2001) wouldallow denatured proteins to enter, but not native ones,and probably not those complexed with SecA. Allother eubacteria lack proteasomes but have an HslUVenergy-dependent protease instead, which is inducibleby heat shock. It is reasonable to regard HslUV, whichmediates the heat-shock response of most eubacteria,as an adaptation by a mesophile to temporarily hot



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conditions. I suggest that this was replaced by theconstitutive proteasome in a thermophilic commonancestor of Actinobacteria (the GjC-rich posi-bacteria, the base composition of which is equallyreasonably interpretable as a secondary adaptation tothermophily). Having a constitutive proteasomewould, however, increase the risk of post-trans-lationally secreted proteins becoming denatured and

degraded before they could be secreted, especially ifprotection by being bound to SecA was only partial.Evolution of the SRP translation-arrest domain wouldsolve this problem and also make SecA no longeruseful, and thus lost rapidly in the ancestral neomuran.

Archaebacterial N-linked glycoproteins are madeco-translationally by the transfer of complex oligo-saccharides by a membrane-bound oligosaccharyltransferase from a dolichol phosphate carrier to thenascent protein associated with membrane-boundribosomes (Lechner et al., 1986), a process absent ineubacteria. In both eubacteria and archaebacteria,ribosomes are attached to the cytoplasmic membrane

by an SRP (Walter et al., 2000), in which Ffh proteinrecognizes the signal peptide and binds to the mem-brane SRP receptor FtsY, after which the protein isinserted by a trimeric SecYEG protein translocase.Eubacteria differ from archaebacteria and eukaryotes,however, in having a smaller SRP with 4n5S RNA not7S RNA, which is associated with their translocationbeing more often post-translational: many eubacterialproteins are brought to the SecYEG translocase post-translationally with the help of SecA protein andsoluble SecB or other chaperones. However, in theancestor of archaebacteria and eukaryotes, theeubacterial 4n5S SRP RNA (Walter et al., 2000)became extended to the neomuran 7S SRP by the

addition of an elongation-arrest domain (Mason et al.,2000); this prevents the secretory or membrane proteinemerging until the ribosome binds to the membrane,thereby protecting it more simply than would acomplex chaperone system from premature de-naturation. Elongation arrest presumably allowedarchaebacteria to rely less on the eubacterial post-translational secretion mechanism and to dispensewith Hsp90 chaperone and SecA and the TAT andYidC translocases: thermal streamlining. But, ifarchaebacterial 7S SRP had been the ancestral type, Icannot see why it should have been reduced to the 4n5SSRP, which would reduce the efficacy of a perfectlygood co-translational system. A key factor in the

greater emphasis on co-translational transfer may havebeen the origin of the co-translational N-linkedglycosylation of novel wall proteins, a key pre-adaptation for the origin of eukaryotes (Cavalier-Smith, 1987b). Note that the relatively much greaterrole for the co-translational mechanism in Posibacteriacompared with Proteobacteria means that they arepartially pre-adapted for the evolution of the neo-muran system. The relative importance of these twomechanisms in other eubacterial phyla is unknown,but ought to be studied.

Analogous arguments may account for the absence ofthe characteristically eubacterial Clp A proteaseactivity from the neomuran cytosol (secondarilyreacquired by eukaryotes in mitochondria and chloro-plasts and modified into an ATPase in archaebacteria).

The changeover from partially post-translational toessentially exclusively co-translational protein secre-tion discussed above was a key pre-adaptation for

the evolution of the rough endoplasmic reticulum(RER) and, therefore, the entire eukaryotic endo-membrane system, as discussed in detail separately(Cavalier-Smith, 2002).

The second fundamental neomuran innovation wasthe evolution of N-linked glycoproteins. I considerthat the biosynthesis of N-linked glycoproteins is toocomplex to have been present in the first cell. Acomplex, mannose-rich oligosaccharide core attachedto an isoprenoid carrier (dolichol phosphate) bytwo residues of N-acetylglucosamine (GlcNAc) issynthesized by a suite of different enzymes and movedto the non-cytosolic face of the membrane (RER in

eukaryotes ; cytoplasmic membrane in archaebacteria;Zhu & Laine, 1996) by the highly hydrophobicdolichol. Core oligosaccharides are cleaved from thedolichol and ligated to asparagine residues of proteinspartly translocated across the membrane by the SRP-associated machinery discussed above. Although suchglycoproteins might, in principle, have evolved ineubacteria for some proteins, there is no evidence thatthey did so prior to the loss of SecA and the exclusivereliance on co-translational secretion. As general co-translational insertion is probably a direct adaptationto thermophily, the origins of N-linked glycoproteinsmay be regarded as an indirect consequence of ther-mophily. The immediate selective force, however, may

have been the resistance it gave the early neomuran tothe -lactam and other antibiotics that inhibit mureinbiosynthesis or enzymes (lysozyme) that digest mureinand which were secreted by its actinobacterial relatives.Thus, this innovation makes adaptive sense if itoccurred in an environment such as soil and rottingorganic matter, rich in posibacterial synthesizers ofantibiotics. Glycoprotein is, in effect, an antibiotic-resistant replacement for murein peptidoglycan; asmuramopeptides are the target of-lactams, muramicacid (one of the two aminosugars constitutingeubacterial peptidoglycan) was lost but the otheraminosugar, GlcNAc, was retained as part of the coreoligosaccharide ofN-linked glycoproteins.

The above arguments make it easy to understand thechangeover from eubacterial murein and partiallypost-translational protein secretion to neomuran gly-coprotein and co-translational protein secretion. I cansee no adaptive reason why either change should havegone in the reverse direction. The streamlined neo-muran protein secretion is just as good for mesophilesas thermophiles. Neither mesophily nor the absence of-lactam antibiotics in an environment would favourthe replacement of glycoprotein by murein. Thus,these two changes, like the three discussed earlier,



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unequivocally polarize evolution from eubacteria toarchaebacteria, not the reverse. Most rRNA andseveral protein trees indicate that methanogenesisevolved after the divergence between euryarchaeotesand crenarchaeotes. Interestingly, some methanogenicarchaebacteria (Methanobacteriales) have secondarilyevolved pseudomurein as a replacement for glyco-protein (ability to make other glycoproteins is

retained). Far from being a sign of antiquity(Stackebrandt & Woese, 1981), the novel pseudo-murein is probably a late adaptation. GlcNAc is foundin pseudomurein, as in glycoproteins, but, as theancestral neomuran had entirely lost the capacity tomake muramopeptides, no murein is present and thesugar structure is novel.

Thermophily and the origins of H1, core histones andDNA topoisomerase VI

I also interpret the origin of core histones as anadaptation to thermophily, to induce negative super-coiling passively by wrapping the DNA round proto-

nucleosomes, with less exorbitant energy costs thanusing DNA gyrase. In itself, this does not tell us thedirection of evolution, since core histones can be lost,as they have been within eukaryotes in peridineandinoflagellates, where only H1 homologues remain(Kasinsky et al., 2001), and in euryarchaeotes, wherehistones have been lost in Thermoplasma (Ruepp etal.,2000). Histones have notbeen found in crenarchaeotes,which have eubacterial-type DNA-binding proteins.Although it is possible that core histones evolved onlyin the ancestral euryarchaeote and were never presentin crenarchaeotes, that could be so only if eukaryotesevolved directly from euryarchaeotes (Sandman &Reeve, 1998). If, as most evidence and evolutionary

arguments suggest, eukaryotes are sisters to archae-bacteria as a whole and not direct euryarchaeotedescendants (Cavalier-Smith, 2002), core histonesmust have evolved in the ancestral neomuran and havebeen lost in stem crenarchaeotes (unless they movedbetween eukaryotes and euryarchaeotes by lateral genetransfer, a possibility that I acknowledge but stronglydiscount). Hyperthermophilic crenarchaeotes are evenmore extreme hyperthermophiles than euryarchaeotehyperthermophiles. Since the psychrophilic or meso-philic crenarchaeotes (Cenarchaeales) are phylo-genetically derived (DeLong et al., 1998), the ancestralcrenarchaeote probably adapted to a hotter habitatthan did any previous bacteria; I suggest that this

caused the loss of histones and replacement of theirfunction by other proteins, for example the 66 aminoacid Sac7d DNA-binding protein ofSulfolobus, whichis exceedingly heat- and acid-stable and sharply kinksand stabilizes DNA by intercalation (Robinson et al.,1998).

Although core histones probably evolved in a stemneomuran, H1 linker histones did so much earlier, inthe eubacterial ancestors of neomura (Kasinsky et al.,2001). The fact that the H1 homologue of the actino-mycete Streptomyces is more similar in length and

sequence to that of eukaryotes than any yet knownfrom non-actinobacterial eubacteria (Kasinsky et al.,2001) is another piece of evidence for the actino-bacterial ancestry of neomura. The ancestral eukaryoteretained both the actinobacterial H1 histone (manyactinobacteria are thermophiles) and the novel neo-muran core histones. By contrast, their sisters, theancestral archaebacteria, must have lost H1 and

retained the novel core histone.The ancestral neomuran also lost eubacterial DNAgyrase activity and evolved a novel type II DNAtopoisomerase (topoisomerase VI), not found ineubacteria; in eukaryotes, the homologous enzyme isrestricted to meiosis and makes the double-strandedbreaks needed for crossing over, which probablyevolved as their ancestor was readopting mesophily.The B subunit of topoisomerase VI is very distantlyrelated to the B subunit of DNA gyrase, but the Asubunit (Spo11 in eukaryotes) is much shorter thanthat of DNA gyrase. DNA gyrase can be convertedartificially to a conventional type II topoisomerase bydeleting the C-terminal region of the A subunitresponsible for the active wrapping of DNA(Kampranis & Maxwell, 1996). I suggest that thishappened naturally in the ancestral neomuran as adirect consequence of the evolution for the first time ofpassive negative supercoiling by histones; this madeactive negative supercoiling by DNA gyrase redun-dant, so mutational truncation of the GyrA subunitwas no longer disadvantageous and the former gyraseevolved rapidly into the ancestral topoisomerase VI.Pre-eukaryotes and pre-archaebacteria then diverged.Once the originally thermophilic sister pre-eukaryotebegan to evolve phagotrophy and perfect the cyto-skeleton and endomembrane system, it reverted rap-

idly to mesophily, since such environments provideimmensely more food and the moderate temperatureswould be more compatible with the relatively fluid cellsurface that phagocytosis entails (Cavalier-Smith,2002). In eukaryotes, which ancestrally had four corehistones and H1, supercoiling is negative. Archae-bacteria probably never had more than two corehistones (Reeve et al., 1997). The archaebacterialcenancestor took the extra step into hyperthermophily,further modifying its chromatin.

Hyperthermophily and the late origins of reversegyrase and DNA-binding protein 10b

I suggest that, as one thermophilic stem neomuranlineage adapted to hyperthermophily, thereby becom-ing the archaebacterial cenancestor, it lost H1 andevolved DNA-binding protein 10b, which makesnegative supercoils in Sulfolobus only at very hightemperatures (Xue et al., 2000), when adapting tohyperthermophily. At the same time, reverse gyrase,found in hyperthermophiles, evolved to reduce the riskof denaturation of DNA at high temperature bysupercoiling it positively. Another unrelated proteinmay be involved in positive supercoiling in Sulfolobus



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(Napoli et al., 2001). The argument that reverse gyraseis an evolutionary chimaera of a eubacterial DNAhelicase and a eubacterial type of DNA topoisomeraseI is further proof of the eubacterial ancestry andrelatively later origin of archaebacteria (Forterre,1996). The various lines of evidence assembled here forthe secondary origin of archaebacteria leave littledoubt that hyperthermophilic environments were the

last major habitat colonized by free-living bacteria.I suggest that archaebacterial core histones, topo-isomerase VI and reverse gyrase are mutually co-adapted to function efficiently. In keeping with this,Thermoplasma, a thermophile but not a hyper-thermophile, has secondarily lost all three and replacedthem, apparently by lateral gene transfer from aeubacterium, by a two-subunit eubacterial DNAgyrase (Ruepp et al., 2000). However, they retained theDNA-binding protein 10b. The presence of bothprotein 10b and reverse gyrase in crenarchaeotespossibly allowed them to dispense with histones.

Molecular co-evolution and the origins of neomuranreplication, DNA repair and transcription machinery

It has long been baffling to molecular evolutioniststhat the replication and transcription machinery ofarchaebacteria and eukaryotes is so similar, despitetheir vastly different cell organization, yet so differentfrom that in eubacteria, which have a fundamentallysimilar cell organization to that of archaebacteria(despite repeated vociferous denials of this basic fact ofcell biology and bacteriology by a few influentialbiochemists). Attributing this striking differencemerely to early divergence (Woese & Fox, 1977;Woese, 1982, 2000) has always been contradicted

by fossil data and reasonable interpretations ofcell evolution (Cavalier-Smith, 1981, 1987b, 1990,1991a, b); the antiquity or progenote hypothesis thatmaintains, contrary to such evidence, that all threedomains are of equal age fails entirely to explain whyneomura have one system and bacteria another. Forall three reasons, the progenote hypothesis was a non-starter as a basic explanation, yet has been repeatedwidely, largely for want of a more convincing alterna-tive: my arguments, that there must have been arelatively radical but rapid changeover in the trans-criptional and translational machinery in stem neo-murans (Cavalier-Smith, 1987b), fell largely on deafears. The dangerous question, why are com-

ponents of the Central dogma a package deal(Belfort & Weiner, 1997), is best answered in terms ofmolecular co-adaptation between the variousmolecules that interact strongly with DNA and itsassociated proteins. I originally attributed the change-over to a genomic destabilization caused by the loss ofthe eubacterial murein cell wall, which is important forDNA segregation, and the sudden release of manyharmful transposable elements, which I also invokedin the origin of neomuran introns (Cavalier-Smith,1987b, 1991c, 1993).

Although such considerations might be importantcontributors to the origin of histones and DNAfolding, they never provided a very satisfactory ex-planation for the radical changes in DNA replicationand transcription machinery. The replacement of theeubacterial replicative polymerases by a novel type Bpolymerase has been particularly puzzling (Edgell &Doolittle, 1997). The simplest interpretation is that the

eubacterial type B repair DNA polymerase, which canalready interact with processivity factors, took overthe replication function of the eubacterial PolC poly-merase and underwent a gene duplication in theneomuran ancestor (Edgell et al., 1998). But whyshould such a changeover have occurred? To get togrips with the temporally concerted changes in somany (not all) of the DNA replication and tran-scription enzymes, we need a fundamental evolution-ary explanation. This is especially true now that it hasbecome apparent that the same dichotomy is found forDNA repair and recombination enzymes ; those ofarchaebacteria are much more like those of eukaryotesthan eubacteria. Thus, all four types of protein, which

I shall collectively refer to as the DNA-handlingmachinery, underwent drastic evolutionary change atthe eubacterial\neomuran transition in whicheverdirection it occurred.

I now suggest that that the origin of core histones inthe ancestral neomurans, yielding a primitive form ofchromatin, so changed the properties of the DNAperceived by many DNA-handling proteins that theyalso had to undergo co-adaptive changes in order tomaintain high-efficiency transcription and replication.This is because DNA-strand separation is an essentialpart of both processes that would have been impededandcomplicatedby the tight wrapping of DNA aroundnucleosome core particles. Both initiation of nucleicacid synthesis and chain elongation would have beenstrongly affected. DNA repair also involves transitionsbetween single- and double-stranded DNA or inter-actions between them that would have been pro-foundly modified by the origin of histones. Thedemonstration that core histones are widespread inarchaebacteria, and the deduction that they veryprobably evolved in the ancestral neomuran, whichwas not known when the neomuran theory was firstpresented (Cavalier-Smith, 1987b), therefore now pro-vide a much more convincing rationale for the change-over in DNA-handling machinery than was previouslypossible.

Transcription. The switch from eubacterial sigma factorsto the much more complex neomuran system with sixinteracting transcription factors was, I suggest, causeddirectly by the adoption of passive supercoiling ofDNA by densely attached histones in place of activesupercoiling by sparsely attached DNA gyrase. Thetight coiling of the DNA around the histones probablynecessitated a more active mechanism to initiatetranscription by the TATA-box-binding protein and



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associated transcription factors that mediate the bind-ing of RNA polymerase. TATA boxes themselvesprobably evolved from eubacterial Pribnow boxes inthe ancestral neomuran. Later, after archaebacteriaand pre-eukaryotes diverged, the RNA polymerasegenes of the latter underwent triplication to make threedistinct polymerases and TATA boxes were lost fromthe genes transcribed by RNA polymerases I and III,

but retained for the majority, which use polymerase II.The multiplication of the number of RNA polymeraseholoenzyme subunits from four in eubacteria to sevenor more in neomura was, I argue, in turn driven by co-adaptation to the novel neomuran transcription com-plex and core histones. Because of the much greatercomplexity of the neomuran transcription machinery,it is easier to understand the origin of transcription ifneomura evolved from eubacteria, as all the evidencesuggests, rather than the reverse. The splitting of thesecond largest (A) subunit into two parts that charac-terizes all archaebacteria must have occurred in a stemarchaebacterium after it diverged from the pre-eukaryote lineage ; this splitting of RNA polymerase A

is one of several reasons why eukaryotes are probablysisters of, rather than derived from, archaebacteria(Cavalier-Smith, 2002). A splitting of the largest (B)subunit occurred later in the common ancestorof Neobacteria alone (Fig. 1). The presence ofboth splits in methanogen but not eukaryote RNApolymerases doubly refutes both recent theoriesof a hydrogen-using methanogen as an ancestor toeukaryotes (Martin & Mu$ller, 1998; Moreira & Lo! pez-Garc!a, 1998).

Replication. It is well known that eukaryote replication

forks move about 50 times more slowly than those ineubacteria, which I have attributed to the greaterdifficulty of strand separation (Cavalier-Smith, 1985b).However, in archaebacteria, the speed is more like thatin eubacteria, probably because they have only twocore histones and lack H1. It is sometimes suggested,because of low conservation of DNA polymerasesequences (Doolittle & Edgell, 1997), that the DNA-replication machinery evolved independently ineubacteria and neomura (Leipe et al., 1999). Given theoverwhelming evidence presented here for a relativelyrecent transition from eubacteria to neomura, thisis simply not credible. The pattern of replication,bi-directional from a single origin in a circular

chromosome, is identical in archaebacteria andeubacteria (Myllykallio et al., 2000). Cann et al. (1999)have shown that the machinery for DNA replication isfundamentally the same in all three domains. Itconsists of a catalytic DNA polymerase and a slidingclamp that ensures its processivity by moving alongthe DNA with it. In neomura, the clamp is PCNA(proliferating nuclear antigen) and its archaebacterialhomologue, a torus-shaped molecule consisting ofthree identical subunits. In eubacteria, the beta subunitof the replicative polymerase, which has little sequence

similarity to PCNA, forms the sliding clamp. As bothclamps have an almost identical three-dimensionalstructure (Kong et al., 1992; Krishna et al., 1994), itseems virtually certain that one evolved from theother; naturally, I suggest the eubacterial version wasancestral.

It appears that the ancestral neomuran replaced theaphidicolin-resistant type C replicative DNA poly-

merase (pol III) alpha subunit by an aphidicolin-sensitive type B polymerase. Such aphidicolin-sensitivepolymerases are not only found in several bacterialviruses, but have a scattered distribution in bacteria: inEscherichia coli, as a less processive repair polymerase(pol II), in some cyanobacteria and in the thermo-philic posibacterium Bacillus caldotenax (Burrows &Goward, 1992). As they are fairly widespread asbacterial repair enzymes, there is no need to invoke aviral origin. A bacterial repair polymerase could simplyhave replaced the normal replicative polymerase. Therepair polymerase might have proved even better thanthe old replicator for handling DNA wound roundhistones and been positively selected for that reason. Ipostulate that the origin of histones stimulated markedchanges to the sliding clamp to allow it to continue tofunction properly. Such changes possibly reduced itsinteraction with the original pol III alpha subunit andcaused it to interact more efficiently with the type Brepair polymerase instead. Such direct interactionsbetween the PCNA sliding clamp and B-type poly-merases have been demonstrated (Bruck & ODonnell,2001). This histone-triggered co-evolution not onlyexplains why the changeover occurred, but allows anintermediate stage in which both DNA polymerasesmay have been able to interact to some degree,suggesting that a smooth functional transition wouldhave been possible without death of the cell.

The neomuran replication factor C is a hetero-pentameric complex responsible for loading the PCNAsliding clamp onto primed DNA. As homologues arenot known in eubacteria, I suggest that this factor alsoevolved radically in co-adaptation to PCNA becauseof the origin of histones.

Repair. There are several repair enzymes unique toneomura, e.g. flap endonuclease I (FEN-1), Rad2,RadA(archaebacteria)\Rad51 and Dmc (eukaryotes).As FEN-1 shares an octapeptide involved in binding tothe interdomain region of PCNA with neomuran PolBDNA polymerases and several other eukaryotic pro-

teins, it is clear that it is co-adapted specifically tofunction with PCNA. I suggest that all the novelneomuran repair enzymes and topoisomerases arosedirectly or indirectly as a result of the evolutionaryorigin of histones and the co-adaptive changes in otherDNA-handling proteins such as PCNA.

There is no necessity to argue that the unique proper-ties of the neomuran proteins reflect an early di-vergence from eubacteria. The weight of evidencecompels us to accept that it was a secondary change-over, not a primary divergence; the fossil evidence



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reviewed below suggests that the changeover wasremarkably recent.

Although I argue that histones were originally anadaptation to thermophily, they can also work per-fectly well in mesophiles and therefore need not be lostduring secondary reversion to mesophily; in fact, theyhave not been lost in secondarily mesophilic eury-archaeotes (Halomebacteria and others), which, as

argued above, are undoubtedly derived. The novelneomuran replicative and transcription machinery,though needed for DNA with histones, also need notundergo reversion to the eubacterial type, and wouldbe incapable of such a reversal except by highlyimprobable massive simultaneous lateral gene transfer.Thus, having evolved such machinery, neomura werestuck with it, even in lineages that later lost histones;this is known to be true for the archaebacteria[Thermoplasma (Ruepp et al., 2000) and crenar-chaeotes], but awaits explicit testing in dinoflagellates.On this molecular co-adaptive interpretation, thedifference between the eubacterial and neomurantranscription and replication machinery itself polarizesthe direction of evolution from eubacteria to neomura ;the comparative evidence indicates that histone losswas not accompanied by a major change in thismachinery, whereas histone gain was accompanied bysuch a change; the biophysical argument that strandseparation is more difficult when histones are presentexplains mechanistically why this is so. It is importantto stress that this co-adaptive explanation for theradical evolutionary transformation of the DNA-handling machinery is independent of the correctnessor otherwise of my hypothesis that histones originatedas an adaptation to thermophily. The two subtheoriesare logically independent and can stand or fall alone. Ihave presented them together because I think both areprobably true; if both are, then the origin of theneomuran transcription\replication novelties was, in-directly, partially caused by ancestral neomuran ther-mophily.

Not every detail of this machinery need have beendirectly adaptive. On a reasonable view of molecularco-adaptation, it need not be. It is likely that, some-times, one molecule becomes co-adapted to a mol-ecular feature of another that became fixed in the firstplace by drift or mutation pressure; the evolution ofdifferential intron splicing is a probable example ofselection for intermolecular interactions on molecularfeatures that originally spread by transposition press-

ure. The phenomenon of genetic hitch-hiking (Barton,2000), which is easily demonstrated experimentally inbacteria and probably occurs in the wild (Tenaillon etal., 1999), means that a selective sweep in response toone strong selective pressure may easily cause a neutralor even mildly deleterious mutation to become fixedindirectly; if both mutations are in the same gene (e.g.RNA polymerase), the hitch-hiking effect is particu-larly strong even in a population with active recom-bination. Compensatory base changes maintainingpairing in RNA are probably mostly examples of the

phenotypic correction of mildly deleterious mutationsthat may have spread initially by drift or hitch-hiking.There is no reason to think that the evolution ofproteins is immune to such inevitable basic evol-utionary forces. If early neomura were largely clonal,hitch-hiking could allow neutral mutations to spread;subsequent co-evolution of interacting moleculesfavoured by selection could stabilize originally neutral

mutations that had spread on the backs of thoseselected directly. I have stressed this because, althoughI have sought to find selective explanations for theorigins of all major neomuran novelties, I do not wishto argue that every molecular detail was selecteddirectly. Many may have arisen through a complexinterplay of mutational, selective, neutral, hitch-hikingand selfish principles acting on macromolecular com-plexes where direct physical interactions mean thatthey cannot evolve independently, subject to only oneevolutionary force at a time, as assumed in the moresimplistic models.

Co-adaptation of the DNA-handling proteins to theorigin of histones in response to selection for ther-

mophily simply solves the central conundrum of cellevolution, as Edgell & Doolittle (1997) dubbed it: thefact (surprising to them) that the largest quantum shiftin DNA-handling machinery in the history of lifeoccurred not in the ancestral eukaryote but in theancestral neomuran. Contrary to the preconceptionsof many molecular biologists, this major suite ofmacromolecular changes was not selected to allow theevolution of the more complex eukaryotic cell; selec-tion has no such foresight. Instead, it was selected tomake a prokaryote a more efficient thermophile. As Ihave long argued, the origin of eukaryote complexitywas not caused by innovations in the gene-expressionmachinery, but by the origin of the cytoskeleton and ofcytosis (membrane budding and fusion; Cavalier-Smith, 1975, 1987b, 2002); an obsession with geneexpression has prevented molecular biologists fromunderstanding cell evolution, for which novel proper-ties of gene products are fundamentally more im-portant.

Small nucleolar (sno) RNAs another neomuranadaptation to thermophily

Another novelty probably related to thermophily wasthe origin of extensive rRNA and some tRNAmethylation by C\D-box snoRNAs in archaebacteria(absent from eubacteria) ; such methylation appears to

be more extensive in hyperthermophiles (Omer et al.,2000). Whether there is also eukaryotic-like extensivepseudouridylation by H\ACA-box small nucleolarribonucleoproteins (snoRNPs) in archaebacteria is notknown, but the evidence that their pseudouridinesynthetases are more like those of eukaryotes that douse such snoRNPs (Watanabe & Gray, 2000) suggeststhat they may turn out to have them. If they do, suchextra pseudouridylation might also initially have beenan adaptation to thermophily, since pseudouridylationsignificantly rigidifies RNA (Charette & Gray, 2000),



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which might have been particularly beneficial tohyperthermophiles. Notethat thesemarkers of pseudo-uridylation sites are not ribozymes, they are simplybase-pairers, a very simple property of RNA. Contraryto the assumption of Poole et al. (1999), they couldeasily have evolved at any stage in evolution and neednot have done so early.

Most RNA complexity and ribozymes are derivedIt is often assumed and sometimes explicitly asserted(Poole et al., 1999) that all ribozymal functions arerelics of a hypothetical RNA world (Gilbert, 1986).This is illogical because, if RNA has an inherent abilityto evolve RNA catalysis, there is no a priori reasonwhy it could not have done so polyphyletically, inwhich case some examples may be phylogeneticallyearly and others late. Arbitrarily defining the presenceof genomic characters more prevalent in eukaryotes asancestral makes it a logical necessity that eukaryotesare ancestral. It is thus circular reasoning to assertthat the greater prevalence of ribozymes in eukaryotes

requires us to root the tree of life on them or,alternatively, on hypothetical organisms having theirgenomic but none of their cellular properties. Thelatter, purely imaginary, organisms would not beeukaryotic and it is nomenclaturally confusing andphylogenetically tendentious to call them so (Poole etal., 1999). It is not an independent deduction from thefacts, but a simple restatement of the phylogeneticallyquestionable, and I argue false, assumption thatall ribozymal activity must be monophyleticallydescended from a pre-protein world. The RNA worldis a purely speculative phylogenetic hypothesis; wecannot use such an uncorroborated hypothesis, plus alogically untenable assumption, to root the tree ob-

jectively! We do not know if an RNA world everexisted (Cavalier-Smith, 2001); some chemists think itlikely that RNA replaced an earlier polynucleotide(XNA) shortly after that had invented protein syn-thesis and protein catalysis provided the first ribo-nucleotides (Orgel, 1998; Nelson et al., 2000). Thesequence XNA world, XNAprotein world, RNAprotein world, DNARNAprotein world is as plaus-ible as the RNA world hypothesis at present.

To root the tree, we must use all the molecular, cell-biological and palaeontological evidence. Poole et al.(1999) castigate fragmented approaches and advo-cate a single continuous theory. But their analysis isitself fragmented, as it ignores all cell-biological and

palaeontological evidence and also all molecular evi-dence except that relating to ribozymes. Doing that isalmost certain to give the wrong answer. Theiradvocacy of a single continuous theory is a rhetoricaldevice, making it appear that ribozyme monophyly(single continuous theory) must be true and polyphyly(fragmented approach) false. To distinguish these apriori equally reasonable possibilities, we need actualphylogenetic evidence about the origins of every kindof ribozyme to establish whether it is ancient orderived, related to others or not. The structure of the

three best-known ribozymes does not support a com-mon origin (Herschlag, 1998). As they are associatedwith a virus, a viroid and a mobile type of intron, Isuggest that all three evolved after the origin of proteinsynthesis and originated not by cellular selection butindependently in different selfish genetic parasites. ThetRNA-cutting function of RNase P, the only trulycellular ribozyme, suggests strongly that it evolved

after protein synthesis had started and, in becomingperfected, required more precisely trimmed tRNAsthan earlier; this may have facilitated the basicdifferentiation between chromosomes and functionalRNA molecules even before cells arose (Cavalier-Smith, 1987a, 2001).

Poole et al. (1999) are also fragmentary in one-sidedlyciting the literature on the origins of spliceosomalintrons. They cite only those, like Gilbert (1986), whoonce believed they were all early, and none of thosewho have since demonstrated that they are phylo-genetically late (Logsdon, 1998 ; Stoltzfuss et al., 1997)and almost certainly evolved from group II self-splicing introns, which have now been shown to beretrotransposable mobile elements (Cousineau et al.,2000), during or after the origins of mitochondria andnuclei (Cavalier-Smith, 1985c, 1991c; Roger et al.,1994). This means that the RNA catalytic ancestors ofthe spliceosome are present in bacteria, not absent asPoole etal. (1999) assert; thus, on the eukaryotes lateview, group II introns are not a late invention but maybe very ancient, dating back at least to the commonancestor of Proteobacteria and Posibacteria, whichboth have them (unless they underwent more recentlateral transfers). Thus, the origin of spliceosomalintrons involved the recruitment of extra proteins to apre-existing posibacterial ribozyme; if the startingassumption of Poole et al. (1999) was correct, thiswould strongly favour eukaryotes late, the oppositeof what they assert.

Poole et al. (1999) ignore the selfish RNADNAevolutionary considerations that tell us that genomescan readily acquire vast numbers of transposableelements and therefore become much more complexthan their simpler ancestors. They puzzle over why allthose scores of snoRNAs should have been acquiredfor nucleolar processing of rRNA since bacteria get bywithout them. Perhaps this puzzlement can be removedon the eukaryotes late view in the same simple way asit was for introns. Conceivably, they were initiallyselfish mobile RNA (Cavalier-Smith, 2002); but

whether their spread was partially selfish or purelyorganismally selected, snoRNPs are fully consistentwith eukaryotes late. The persistence of some intronsand many snoRNPs in the cryptomonad nucleomorph(Douglas etal., 2001; Maier et al., 2000), which is morestrongly streamlined than any bacterial genome(Zauner et al., 2000; Maier et al., 2000; Douglas et al.,2001), refutes the assumption that both could bereadily lost if bacteria had evolved from eukaryotes(Poole et al., 1999), as does the discovery of anextensive methylating snoRNA system in archae-



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bacteria (Omer et al., 2000). Most snoRNAs are notribozymal, but are markers of sites for methylation orpseudouridylation (Smith & Steitz, 1997); which sitesthey mark depends simply on base pairing, so could bereadily acquired independently. Cleavage snoRNAsmay be ribozymes, I suggest, but the possibility thatthe protein part of their RNPs is the catalyst has notbeen excluded. If their RNA is ribozymal, it might

have evolved from RNA of RNase P, which cleavestRNA; even if it evolved de novo in the first eukaryote(or an ancestral neomuran if archaebacteria eventuallyprove to have them), RNA cleavage is not a novelfunction for a ribozyme, and this would be theslenderest of possible grounds for the cell-biologicallyabsurd view that bacteria evolved from eukaryotes.Poole et al. (1999) also misconstrue the significance ofeukaryotic telomerase. Contrary to their assertions, itis not a ribozyme; the catalysis is by a protein (Counteret al., 1997) and the RNA is simply a template. As anyR

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