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Theoretical Population Biology 61, 409–422 (2002) doi:10.1006/tpbi.2002.1592 Evolution of the Archaea Patrick Forterre Institut de Ge!ne!tique et Microbiologie, UMR 8621 CNRS, Bat 409, Universite! Paris-Sud, 91405 Orsay Cedex, France E-mail: forterre@igmors:u psud:fr and Celine Brochier and Herve! Philippe Phyloge!nie, Bioinformatique et Ge!nome, UMR 7622 CNRS, Universite! Pierre et Marie Curie, 9 quai St Bernard Ba#t. C 75005 Paris, France Received February 27, 2002 Archaea, members of the third domain of life, are bacterial-looking prokaryotes that harbour many unique genotypic and phenotypic properties, testifying for their peculiar evolutionary status. The archaeal ancestor was probably a hyperthermophilic anaerobe. Two archaeal phyla are presently recognized, the Euryarchaeota and the Crenarchaeota. Methanogenesis was the main invention that occurred in the euryarchaeal phylum and is now shared by several archaeal groups. Adaptation to aerobic conditions occurred several times independently in both Euryarchaeota and Crenarchaeota. Recently, many new groups of Archaea that have not yet been cultured have been detected by PCR amplification of 16S ribosomal RNA from environmental samples. The phenotypic and genotypic characterization of these new groups is now a top priority for further studies on archaeal evolution. & 2002 Elsevier Science (USA) Eubacteria). In the following decades, this discovery prompted a flood of new hypotheses about the nature of the Last Universal Cellular Ancestor (LUCA) and the phylogenetic relationships between the three domains of life (Archaea, Bacteria and Eukarya) (for recent reviews and alternative viewpoints, see Gupta, 1998; Forterre and Philippe, 1999; Lopez-Garcia and Moreira, 1999; Penny and Poole, 1999; Glansdorff, 2000; Woese, 2000; Cavalier-Smith, 2002; Gribaldo and Philippe, 2002, this issue). The evolution of Archaea per se is an important field of evolutionary biology. Since Archaea exhibit a wide variety of phenotypic and genotypic characters, it would be interesting to have a clear idea of the history of the archaeal domain, in order to understand the origin and evolution of these characters. In this review, we will try to sum up the state of the art regarding archaeal evolution. This is timely, as data from comparative 1. INTRODUCTION The discovery, by Woese and colleagues in the 1970s, that three ribosome forms are present in living organisms initiated a revolution in evolutionary biology (Woese and Fox, 1977; Woese, 1987; Woese et al., 1990). This tripartite division of the translation apparatus had been previously obscured by early observation of ribosome structure that fitted well with the binary classification of organisms into eukaryotes (80S ribosomes) and prokaryotes (70S ribosomes). However, when comparative sequence analysis of the ribosome components became available, it turned out that ribosomal RNAs (rRNA) and ribosomal proteins of some prokaryotes (the Archaea, formerly Archaebacteria) were drastically different from those of both eukaryotes and those of ‘‘classical’’ bacteria (the Bacteria, formerly 409 0040-5809/02 $35.00 # 2002 Elsevier Science (USA) All rights reserved. 410 Forterre, Brochier and Philippe genomics can now be combined with more traditional phylogenetic and taxonomic approaches. At the time of writing this review, 12 genomes of Archaea have been completely sequenced and are available in public databases (http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/a g.html). Furthermore, an up-to-date description of all identified archaeal species has been recently published in Volume I of the new Bergey’s Manual edition (Boone and Castenholz, 2001). Finally, archaeal rRNA probes have been widely used by molecular ecologists to investigate the worldwide distribution of the organisms of this domain, as well as its phylogenetic depth (for recent papers see Massana et al., 2000; LopezGarcia et al., 2001; Takai et al., 2001a, b). 2. BRIEF DESCRIPTION OF THE DOMAIN ARCHAEA The Archaea are prokaryotes (cell without nucleus) that cannot be easily distinguished from Bacteria by size or shape. However, although most Archaea look like typical Bacteria, some have morphologies that are not found in Bacteria, such as polygonal in halophilic Archaea or very irregular cocci in particular hyperthermophiles (Fig. 1). This could reflect the absence of a rigid cell wall in most Archaea (see below) and/or novel (still unknown) mechanisms for morphogenesis. Archaea exhibit a wide diversity of phenotypes, as is the case for Bacteria. The first three phenotypes to be recognized were the methanogens (strict anaerobes and methane producers), the halophiles (strict aerobes living in high-salt environments) and the thermoacidophiles (aerobes living in hot and acidic environments) (Woese and Fox, 1977). Organisms with such disparate phenotypes were first unified based on the similarities of their rRNA sequences, and later on also by the unique structure of their membrane phospholipids (see below). Many additional phenotypes were discovered among Archaea in the following decades, such as hyperthermophilic or psychrophilic methanogens, halophilic and/or alkaliphilic methanogens, anaerobic, alkaliphilic and neutrophilic hyperthermophiles (Mathrani et al., 1988; Franzmann et al., 1997; Ollivier et al., 1998; Huber et al., 2000). Finally, the explosion of environmental studies based on PCR amplification of 16S rRNA has revealed the widespread occurrence in water and soils of mesophilic Archaea with otherwise unknown phenotypes (Fig. 2). For example, a recent study has estimated that mesophilic Archaea of the phylum FIG. 1. (A) Electron micrograph of the euryarchaeon Haloarcula (formerly ‘‘square bacteria’’), from Dr. Rodriguez-Valera, (B) electron micrograph of the crenarchaeon Pyrodictium abyssi (a hyperthermophile that can grow up to 1108C), the cells are inserted into a reticulated network whose function is still unknown and (C) transmission electron micrograph of P. abyssi. EM pictures of P. abyssi are from Reinhard Rachel and Karl Stetter. Crenarchaeota account for nearly 20% of the total marine picoplankton oceanic biomass worldwide (Karner et al., 2001). Many of these not yet cultivated Archaea probably exhibit novel phenotypes. For instance, previously unsuspected archaeal methanotrophs were predicted recently from geomicrobiological data (Orphan et al., 2001). Archaea are often viewed as Evolution of the Archaea 411 FIG. 2. Phylogeny of the archaeal domain based on 16S rRNA of both cultured and uncultured species. A neighbour-joining phylogenetic tree was constructed from 342 archaeal nearly complete sequences of SSU rRNA (1235 positions). The distances were calculated with the Tamura and Nei method (Tamura and Nei, 1993) and the a-parameter was computed with PAUP 4b8. Scale bars correspond to 10 substitutions per 100 positions for a unit branch length. The tree was arbitrarily rooted. The size of the triangles is proportional to the number of sequences analysed. Grey triangles include SSU rRNA from both cultured and uncultured species, whereas the white triangle includes only environmental sequences (uncultured species). predominant over Bacteria in all extreme environments. This is indeed true for high-temperature environments, since only Archaea can thrive at temperatures above 958C (and up to 1138C) (Huber et al., 2000). However, in all other situations (high salt, low or high pH, low temperature, high pressure), Bacteria are found together with Archaea and Eukarya (Rothschild and Mancinelli, 2001). 412 The metabolic diversity of Archaea is also reminiscent of Bacteria. Apart from methanogenesis (presently unknown in Bacteria) all metabolic pathways discovered in Archaea also exist among Bacteria. They can be either heterotrophs or autotrophs (chemio- or photo-lithoautotroph) and use a large variety of electron donors and acceptors (Huber et al., 2000). Photosynthesis based on chlorophyll has not been found in Archaea, whereas photosynthesis based on bacteriorhodopsin, once believed unique to halophilic Archaea, has been recently detected in planktonic Bacteria as well (Beja et al., 2001). Spore formers or organisms with complex cell cycle and multicellular stages are unknown among Archaea, but this could be simply due to our incomplete sampling of this domain. The only way to discriminate between Archaea and Bacteria is thus by looking at the molecular level. Beside their specific rRNA, Archaea can be distinguished from Bacteria by the nature of their membrane glycerolipids that are ethers of glycerol and isoprenol, whereas bacterial and eukaryal lipids are esters of glycerol and fatty acids (Kates, 1993). Archaeal glycerolipids are also ‘‘reverse lipids’’, since the enantiomeric configuration of their glycerophosphate backbone is the mirror image of the configuration found in bacteria and eukaryal lipids. Another difference between Archaea and Bacteria is the absence of murein in Archaea, whereas this compound is present in the cell wall of most Bacteria. Archaea exhibit a great diversity of cell envelopes (Kandler and Konig, 1998), most Archaea have a simple S-layer of glycoproteins covering the cytoplasmic membrane, whereas a few of them (Thermoplasmatales) only have a cytoplasmic membrane containing glycoproteins. Some Archaea have a rigid cell wall based either on heteropolysaccharides (Halococcus) or pseudomurein (Methanobacteriales), the latter being Gram positive. The difference between Archaea and Bacteria at the molecular level is exemplified by the resistance of Archaea to most antibiotics active on Bacteria. Early studies on the molecular biology of Archaea have shown that this resistance was due indeed to critical differences in the antibiotic targets (e.g., RNA polymerase or ribosomal proteins) (for an early review on archaeal molecular biology, see Zillig, 1991). The coherence of Archaea at the molecular level is now also well documented by comparative genomics (Olsen and Woese, 1997; Forterre, 1997; Forterre and Philippe, 1999; Fitz-Gibbon and House, 1999; Makarova et al., 1999; Snel et al., 1999; Tekaia et al., 1999; Wolf et al., 2001). In all archaeal genomes sequenced so far, most encoded proteins give first hits Forterre, Brochier and Philippe with other archaeal proteins when their homologues are searched in public databases. This is especially true for informational proteins (those involved in DNA replication, transcription and translation) that are usually present in most archaeal genomes and are nearly always more similar between one archaeon and another than between one archaeon and any bacterium or eukaryote. These archaeal informational proteins are also usually much more similar to those of Eukarya than to those of Bacteria. In addition, archaeal genomes encode many informational proteins that have homologues in Eukarya but not in Bacteria. This is especially striking in the case of DNA replication, since archaeal genomes encode homologues of nearly all eukaryal DNA replication proteins but only one homologue of a bacterial DNA replication protein (Edgell and Doolittle, 1997; Forterre, 1999; Leipe et al., 1999). The eukaryotic-like putative replication proteins identified in archaeal genomes are most likely involved in actual archaeal DNA replication, as indicated by recent observation such as direct interaction in vivo between the chromosomal origin (oriC) and the eukaryotic-like initiator protein Cdc6 (Matsunaga et al., 2001) and the similar size of Okazaki fragments in Archaea and Eukarya (Matsunaga and Myllykallio, pers. comm.). It is often considered that informational proteins constitute the ‘‘core’’ of the genome of any organism, because they are less subject to lateral gene transfer (LGT), and are therefore more representative of the actual history of the organisms (Jain et al., 1999). Indeed, except for well-documented LGT between Archaea and Bacteria in the case of amino-acyl tRNA synthetases (Wolf et al., 1999; Woese et al., 2000), few LGTs of informational proteins have been identified between the two prokaryotic domains. For example, an exhaustive study has shown that no LGT had occurred between Archaea and Bacteria in the case of ribosomal proteins (Brochier et al., 2002) even if LGTs were discovered within Bacteria (Brochier et al., 2000, 2002) and Archaea (Matte-Tailliez et al., 2002). The close similarity between eukaryal and archaeal informational proteins can be explained by two hypotheses: either Archaea and Eukarya are sister groups, and the ‘‘eukaryotic’’ characters of Archaea are synapomorphies, or these characters are primitive (already present in the LUCA, thus symplesiomorphies) and divergent in Bacteria. We will not discuss this problem here (for a review see Forterre and Philippe, 1999; Poole et al., 1999; Woese, 2000; Cavalier-Smith, 2002; Gribaldo and Philippe, this issue) since we will focus on the evolution of the archaeal domain per se. Evolution of the Archaea Interestingly, in contrast to informational proteins, most operational proteins of Archaea are ‘‘bacteriallike’’. This category includes enzymes of the primary and secondary metabolisms, membrane receptors, transporters and so on, but also some cell division proteins (Koonin et al., 1997; Jain et al., 1999). This is often interpreted as testifying for extensive LGT of operational proteins between the two prokaryotic domains (Faguy and Doolittle, 1999; Makarova et al., 1999). However, although LGTs between hyperthermophilic Bacteria and Archaea have been well documented by phylogenetic and/or genome context analyses (Gribaldo et al., 1999; Forterre et al., 2000; Nesbo et al., 2001), a supertree inferred only on operational proteins is very similar to the one inferred from informational proteins (Daubin and Gouy, 2001), suggesting that LGTs are not so frequent for operational genes. More analyses are thus required to test the hypothesis that the similarity between Archaea and Bacteria for operational genes is only due to LGTs. Alternative hypotheses that have been proposed to explain this similarity are the fusion of ancient bacterial and eukaryotic lineages to produce extant Archaea (Koonin et al., 1997) or the monophyly of Archaea and Bacteria, if the universal tree of life is rooted in the eukaryotic branch (Forterre and Philippe, 1999). 3. GENOME EVOLUTION IN ARCHAEA Comparative genomics suggests that the mechanisms of genome evolution in Archaea are quite similar to those identified in Bacteria. This can be explained by the resemblance between the genome organizations in the two domains. For instance, despite eukaryotic-like replication proteins, hyperthermophilic Archaea of the genus Pyrococcus replicate their chromosome at high speed, bi-directionally and using a single replication origin, as in Bacteria (Lopez et al., 1999; Myllykallio et al., 2000; Matsunaga et al., 2001). Most highly transcribed genes are transcribed co-directionally with DNA replication, as in Bacteria, probably to avoid frequent head-to-head collision between the replication and transcription apparatuses (Zivanovic et al., 2002). Shine–Dalgarno sequences are found in some Archaea as in Bacteria, and archaeal genes are often organized in operon. Finally, mechanisms of gene regulation in Archaea involve bacterial-like regulator proteins (Aravind and Koonin, 1999; Bell and Jackson, 2001). Except for closely related organisms, archaeal and 413 bacterial genomes exhibit no synteny and very little conservation of gene order, in agreement with rapid genomic evolution by recombination (Huynen and Bork, 1998). Comparison of the two closely related Archaea Pyrococcus abyssi and Pyrococcus horikoshi has shown that the terminus of DNA replication is a hot spot of recombination, as in the case of Bacteria (Myllykallio et al., 2000). Furthermore, the main rearrangement between the two Pyrococcus genomes has occurred symmetrically to the axis formed by the origin and the terminus of replication (Makino and Suzuki, 2001; Zivanovic et al., 2002). This characteristic feature has been also observed in Bacteria, suggesting that major recombination events occurred between bidirectional replication forks (Tillier and Collins, 2000). These similarities in the mechanisms of genome evolution of the two prokaryotic domains could reflect a common structural organization of the replication apparatus (for example, association of the two replication forks into a single replication factory) and of the chromosome segregation machinery (in relation with the presence in Archaea of homologues of the bacterial proteins Xer C/D), which are involved in the resolution of recombinant chromosomes at the end of replication, and of the bacterial proteins MinD and FtsZ, which are involved in the formation of septum for cell division. However, it should be stressed that study of the mechanisms of chromosome structure and evolution in Archaea is still in its infancy, and that the diversity of Archaea in this respect remains to be explored. For instance, two possible (instead of one) replication origins have been predicted in silico in Halobacterium (Kennedy et al., 2001) and the bacterial cell division proteins FtsZ and MinD are lacking in all Crenarchaeota. In addition to chromosomal rearrangements linked to DNA replication, recombination events have been associated to IS elements in several archaeal genomes (Kennedy et al., 2001; Bru. gger et al., 2002). IS elements are especially abundant in S. solfataricus and Halobacterium NRC1. Comparison of the three completely sequenced Pyrococcus genomes suggested that a family of IS elements, only present in P. furiosus, has been involved in most chromosomal rearrangements observed in the latter species (Maeder et al., 1999; Lecompte et al., 2001). In Pyrococcus and Sulfolobus, chromosomal rearrangements linked to IS elements occur preferentially in the limit of one replichore (the half chromosome comprised between the origin and terminus of replication), suggesting a principle of genome organization that is not yet understood (Zivanovic et al., 2001, 2002; Bru. gger et al., 2002). 414 An important difference of the genome organization between the two prokaryotic domains is that all Bacteria contain a gyrase and a negatively supercoiled genome, whereas most Archaea have no gyrase and relaxed or positively supercoiled genome (Charbonnier and Forterre, 1994). Some Euryarchaeota, including the hyperthermophile Archaeoglobus fulgidus, have acquired bacterial gyrases by LGT and have negatively supercoiled genomes (Lopez-Garcia and Forterre, 1999). It is not known presently if differences in genome topology have an effect on the mechanisms of genome evolution. LGTs between different archaeal species, and also between Bacteria and Archaea, have been involved as a major mechanism of genome evolution in Archaea. LGTs appear to be predominant between organisms living in similar environment. This has been shown both by genome wide comparison (Aravind et al., 1998; Nelson et al., 1999; Ruepp et al., 2000) and in the case of some ribosomal proteins inside the archaeal domain (Matte-Tailliez et al., 2002). For example, many LGTs have been detected between the thermoacidophilic Archaea Thermoplasma and Sulfolobus that are otherwise phylogenetically very distant (Ruepp et al., 2000). IS elements are likely often involved in these LGTs. For instance, a 16 kb DNA fragment flanked by two IS has been transferred between Thermococcus littoralis and Pyrococcus furiosus (Diruggiero et al., 2000). IS elements could have also played a critical role in LGT between Bacteria and Archaea since many of the IS detected in Archaea belong to families previously detected in Bacteria (Bru. gger et al., 2002). The precise mechanism of LGT between the two prokaryotic domains remains mysterious. In particular, transformation by naked DNA appears highly unlikely in the case of hyperthermophiles considering the high instability of DNA fragments at high temperature (Marguet and Forterre, 1994). LGT could possibly involve transfection by viruses, especially if one considers that operational proteins similar between Bacteria and Archaea include some outer membrane proteins (e.g., sugar transporters) known to be potential viral receptors. One can thus imagine that bacteriophages sometimes interact with archaeal homologues of their usual bacterial receptors, and then inject by mistake their DNA into archaeal cells (and vice versa). Such rare events could readily lead to LGT if the injected DNA fragment contains mobile IS elements that can function in both prokaryotic domains. Viruses and plasmids seem to be as common in Archaea as in Bacteria (Benbouzid-Rollet et al., 1997; Forterre, Brochier and Philippe Prangishvili et al., 2001). It has been shown that IS elements present in Sulfolobus species have indeed triggered active recombination between chromosomes, plasmids and viruses (for review, see Bru. gger et al., 2002), in agreement with the above hypothesis on the role of viruses in LGT. The recent analysis of the complete genome of the halobacteriophage HF2 is especially relevant to the idea that viruses have been a major vector for LGT between domains, since it contains a mixture of archaeal, bacterial and bacteriophage-like genes (Tang et al., 2002). In particular, its DNA polymerase seems more closely related to bacteriophage T4 DNA polymerase than to archaeal DNA polymerases (Fil!ee et al., in press). Accordingly, LGTs are probably not limited by environmental barriers since mechanisms for genetic exchanges (e.g., virus or transposable elements) should have evolved and adapted themselves to various environments together with their hosts. 4. ARCHAEAL PHYLOGENY, THE TWO MAJOR PHYLA Phylogenetic trees of the archaeal domain based on 16S rRNA have led to split this domain into two phyla (according to Bergey’s Manual definition), the Euryarchaeota and the Crenarchaeota (Woese et al., 1990). The Euryarchaeota have been named from the Greek word euruB; meaning wide, because they encompass the greatest phenotypic diversity among known cultivable species, with the halophiles, the methanogens, some thermoacidophiles and some hyperthermophiles. In contrast, the phenotypic diversity of cultivable Crenarchaeota is much more limited, with only hyperthermophilic species. This phylum has thus been named from the Greek word krZnZ meaning spring, from the popular hypothesis that hyperthermophiles are at the origin of all present-day organisms. This name has been conserved, despite the detection by PCR in various environments of many groups of mesophilic and probably psychrophilic Crenoarchaeota that have not yet been cultivated (Fig. 2). Some data of comparative genomics support the division of Archaea into two distinct phyla, since many proteins present in all euryarchaeal genomes are missing in Crenarchaeota and vice versa. In particular, several striking differences have been noticed between the two phyla for some key proteins involved in DNA replication, chromosome structure and cell division, such as the Evolution of the Archaea absence of eukaryal-like histones, eukaryal RPA-like proteins and bacterial cell division proteins in all Crenarchaea (Bernander, 2000; Myllykallio and Forterre, 2000). However, it remains to be known if these differences are specific to cultivable hyperthermophilic Crenarchaeota, or if they are a general characteristic of this phylum. Furthermore, some global genomic analyses have failed to recover the division of Archaea into two phyla. For example, using five different approaches (e.g., gene content, gene order, concatenated ribosomal proteins) on ten archaeal genomes, Wolf et al. (2001) failed to recover the monophyly of the two phyla. Nevertheless, their phylogeny based on gene content is very likely biased by frequent LGTs between Thermoplasmatales (Euryarchaeota) and Sulfolobales (Crenarchaeota) (Ruepp et al., 2000). If Thermoplasmatales are not taken into account, the two phyla are monophyletic, in agreement with a previous study based on gene content (Huynen et al., 1999). Similarly, the use of a much more refined phylogenetic method (through maximum likelihood inference of individual genes and supertree reconstruction), performed on 459 genes from seven Archaea, also recovered the monophyly of the two phyla (Daubin and Gouy, 2001; see also Brown et al., 2001). The lack of monophyly in the phylogeny based on ribosomal proteins obtained by Wolf and coworkers is more surprising (Wolf et al., 2001). We recently performed a detailed analysis of 53 ribosomal proteins present in most of 14 archaeal species (Matte-Tailliez et al., 2002). We found that eight genes have likely 415 undergone LGT events during archaeal evolution, with a bias for LGTs between Thermoplasmatales and Crenarchaeota, and were thus not suitable for inferring species phylogeny. The phylogeny based on the remaining 45 genes was very similar to the rRNA phylogeny and strongly supported the partitions between Crenarchaeota and Euryarchaeota. We have now carried out the same analysis, but including the genomes of Sulfolobus tokodai and of Thermoplasma volcanium that were published after our initial work was completed, and we obtained similar results (Fig. 3). It should be noted that the Halobacteriales and the Thermoplasmatales display very long branches. Therefore, when a very distant outgroup is used to root the archaeal phylogeny (i.e., the Bacteria), the long-branch attraction artefact (Felsenstein, 1978) is a major problem (Philippe and Laurent, 1998). Accordingly, the long branches of Halobacteriales and Thermoplasmatales likely emerge early in ribosomal proteins’ phylogeny because they are attracted by the bacterial branch, as previously suggested (Wolf et al., 2001). In conclusion, the division of Archaea into Crenarchaeota and Euryarchaeota can be viewed as supported by phylogenetic analyses, at least for cultivable species (Fig. 2). Indeed, the analysis of archaeal sequences from the environment has somehow complicated the picture of the archaeal domain from the 16S rRNA viewpoint. Although many of the new archaeal lineages that have been detected by PCR analysis of environmental samples branch among Crenarchaeota and Euryarchaeota, some of them branch in between. A tentative FIG. 3. Phylogeny of the archaeal domain based on concatenated ribosomal protein amino acid sequences. Ribosomal protein sequences have been retrieved from completely sequenced genomes (with the exception of Haloarcula marismortui). Ribosomal proteins for which LGTs were suspected were removed from the analysis, with our recently developed method (Brochier et al., 2002). The only modification was that all the likelihood was computed with a G-law model. A maximum likelihood phylogenetic tree was constructed from the concatenated 47 ribosomal proteins sequence (6220 positions). The calculations of the best tree and the branch lengths were conducted using the program PUZZLE with a G-law correction. Numbers close to nodes are ML bootstrap supports computed with the RELL method upon 2000 top-ranking trees using the MOLPHY program without correction for among-site variation. Scale bars correspond to 10 substitutions per 100 positions for a unit branch length. The tree was arbitrarily rooted between Crenarchaeota and Euryarchaeota. 416 third phylum, the Korarchaeota (from the Greek word koroB; meaning young man), was suggested (Barns et al., 1996). Sequences that branch even deeper than Korarchaeota in the archaeal 16S rRNA tree were recently reported (Takai et al., 2001a, b). We thus constructed an archaeal phylogeny using all the Archaea for which rRNA sequences are almost complete (Fig. 2). This tree identifies a huge group of environmental sequences that cannot be attributed to either Euryarchaeota or Crenarchaeota. The Korarchaeota and other uncultured lineages form a clade with cultured Crenarchaeota, but with low bootstrap support (below 50%). It is thus not clear if the dichotomy of the archaeal domain will survive future studies. More phyla will very likely have to be defined in Archaea. This could eventually produce an archaeal classification more similar to the bacterial one (23 phyla are presently recognized in the Bacterial domain, Boone and Castenholz, 2001). Nevertheless, one has also to be careful in interpreting the diversity of environmental sequences, since divergent copies of rRNA can exist within a single organism (Mylvaganam and Dennis, 1992; Amann et al., 2000). For a long time, some authors have advocated the paraphyly of Archaea, suggesting that Crenarchaeota (called eocytes by Lake and colleagues) form a clade with Eukarya (Lake, 1988), based for example on an insertion in elongation factor 1a (Rivera and Lake, 1992). A phylogenetic analysis of rRNA taking amongsite rate variability into account supports this claim (Tourasse and Gouy, 1999). It is reasonable to assume that the monophyly of Archaea generally observed in rRNA trees (Woese et al., 1990) is due to a long-branch attraction artefact, because Bacteria and Eukarya display much longer branches than Archaea for rRNA. The use of methods less sensitive to this artefact (Tourasse and Gouy, 1999) provided a good evidence in favour of the paraphyly of Archaea. However, phylogenies based on genome content (Fitz-Gibbon and House, 1999; Huynen et al., 1999) as well as on multiple markers (Brown et al., 2001; Daubin and Gouy, 2001) recovered the monophyly of Archaea. In addition, recent analyses of two crenarchaeal genomes (Aeropyrum pernix and Sulfolobus solfataricus) failed to identify specific eukaryal-crenarchaeal proteins that could support the hypothesis of paraphyly (Faguy and Doolittle, 2000; Natale et al., 2000). On the contrary, the Crenarchaea lack several eukaryal proteins of the informational apparatus that are present in Euryarchaea (such as histones or some DNA replication proteins, Bernader, 2000; Myllykallio and Forterre, 2000). The completion of several genomes from Crenarchaeota, Korarchaeota and their relatives at the base of the 16S Forterre, Brochier and Philippe rRNA tree (Fig. 2), together with their analysis with the refined method, will be critical to confirm or invalidate the monophyly of Archaea. 5. STRUCTURE AND EVOLUTION OF THE TWO ARCHAEAL PHYLA The Euryarchaeota have been divided into nine orders, five orders for the methanogens (Methanobacteriales, Methanomicrobiales, Methanococcales, Methanosarcinales and Methanopyrales), one for the halophiles (Halobacteriales), one for the thermoacidophiles (Thermoplasmatales) and two for the hyperthermophiles (Thermococcales and Archaeoglobales) (Fig. 2). Phylogenetic trees based on 16S rRNA place the Methanopyrales at the base of the euryarchaeal tree, followed by Thermococcales, but with very weak statistical support (the bootstrap supports for the four basal nodes are between 4% and 8% in Fig. 2). Since Methanopyrales are hyperthermophiles (the only species of this order, Methanopyrus kandleri, can grow up to 1108C), this has suggested that ancestral Euryarchaeota were both methanogens and hyperthermophiles. The idea that methanogenesis (formation of methane from H2 and CO2 ) is an ancestral phenotype was first advocated to support the name ‘‘Archaebacteria’’ itself (Woese and Fox, 1977). The discovery of a subterranean community of methanogens thriving on H2 has recently prompted speculation about the possibility that methanogens are directly connected to a hot origin of life on Earth and possibly elsewhere (Chapelle et al., 2002). The exact position of Methanopyrales in the euryarchaeal tree is thus of critical importance. The recent sequencing of the M. kandleri genome is thus an important step in our understanding of archaeal evolution (Slezarev et al., 2002). Phylogenetic analysis of concatenated ribosomal proteins suggests that Methanopyrales do not branch first in the euryarchaeal tree but branch with other thermophilic and hyperthermophilic methanogens (Slesarev et al., 2002). Furthermore, our phylogenetic analysis of ribosomal proteins limited to the archaeal domain (thus using more positions and limiting the impact of long-branch attraction artefact) locates Thermococcales firmly at the base of the euryarchaeal tree (bootstrap support of 86%, MatteTailliez et al., 2002). This was confirmed by our new analysis with two additional species (bootstrap support of 89%, Fig. 3). The position of Thermococcales at the 417 Evolution of the Archaea base of the euryarchaeal tree and the grouping of M. kandleri with other methanogens thus suggest that methanogenesis (a very complex metabolic pathway) has originated in the euryarchaeal domain only after the divergence between Thermococcales and other Euryarchaea. This would be in agreement with the absence of relics of methanogenesis in the genomes of Thermococcales, whereas such relics have been found in Archaeoglobus (Klenk et al., 1997; Slesarev et al., 2002). Both rRNA and ribosomal protein based phylogenies have suggested that Halobacteriales are a sister group to Methanomicrobiales (Fig. 3). This grouping makes sense since Methanomicrobiales are the only order of methanogens that does not include thermophilic species, and contains halophilic species. This suggests that Halobacteriales originated from halophilic and mesophilic methanomicrobiales that have lost their ability to produce methane and have acquired both oxygen tolerance and respiratory enzyme complexes. Analysis of the genes involved in aerobic respiration that can be detected in the genome of Halobacterium sp. NRCl has indicated that these genes have been borrowed from aerobic Bacteria by LGT (Kennedy et al., 2001). Indeed, in one of these clusters, the genes for aerobic respiration present in Halobacterium NRCl have no archaeal counterpart, whereas in the others, they are more closely related to their bacterial than to their archaeal homologues. Both rRNA and ribosomal protein based phylogenies also suggest the existence of a large clade grouping Methanobacteriales, Thermoplasmatales, Archaeoglobales, Methanomicrobiales and Halobacteriales. However, phylogenies based on other genes or on gene content failed to retrieve it (Brown et al., 2001; Daubin and Gouy, 2001; Wolf et al., 2001). Furthermore, this group is not recovered when sequences from uncultured species are included in the 16s RNA tree (Fig. 2). The analysis of additional genomes will thus be required to sort out the phylogenetic relationships among these different lineages. The cultured Crenoarchaeota have been divided into three orders according to 16S rRNA: the Thermoproteales, the Desulfurococcales and the Sulfolobales (Fig. 2). The first two orders include only anaerobic hyperthermophiles that live at neutral pH and include the most hyperthermophilic organisms known today (with maximal growth temperature up to 1138C), whereas Sulfolobales, which live at low pH (2–3), can be either aerobes or anaerobes and have lower temperature maxima (up to 858C). Thermoproteales are a sister group to Sulfolobales in 16S rRNA trees. Genomic data cannot yet be used to test this phylogeny since genomes from Desulfurococcales have not yet been sequenced. As in the case of Euryarchaeota, oxygen respiration is most likely a secondary adaptation in Crenoarchaeota. However, we should be aware that hyperthermophilic Crenoarchaeota that have been discovered in the last two decades represent only a small fraction of this phylum. From examination of Fig. 2, it is striking that most of the archaeal groups in both Euryarchaeota and Crenarchaeota are in fact presently uncultured. There is therefore much research to be done both in classical microbiology and environmental genomics before a complete picture of the archaeal domain structure and evolution can be obtained. A recent development has been the discovery of parasitic archaea of reduced size (Nanoarchaeum equitans) and genome (less than 0:5 Mb) that live in parasitic association with hyperthermophilic Crenoarchaeota of the genus Ignicoccus and branch very deeply in the crenoarchaeotal 16S rRNA tree (Huber et al., 2002). 6. NATURE OF THE ARCHAEAL ANCESTOR Phylogenetic and genomic analyses of cultivable Archaea suggest that the ancestor of all present-day Archaea was an anaerobe that probably lived when oxygen was still absent from the terrestrial atmosphere. Complete adaptation to an aerobic lifestyle is a secondary adaptation in the archaeal domain that occurred several times independently: at least once in Crenarchaeota (Sulfolobales) and twice in Euryarchaeota (Halobacteriales and Thermoplasmatales). However, several Crenarchaeota are also microaerobes (Aeropyrum pernix and Pyrobaculum aerophilum), so we cannot exclude that the archaeal ancestor itself had some oxygen tolerance. As previously noticed, this ancestor was probably not a methanogen, at least if we consider indeed that the rooting of the archaeal tree between Thermococcales and Crenarchaeota (Fig. 3) is correct. It is also often considered that the archaeal ancestor was a chemiolithoautotroph, since chemiolithoautotrophic thermophiles are present in both archaeal phyla. For several authors, this is consistent with the theory of an autotrophic origin of life (Wachtershauser, 1990). However, since heterotrophic hyperthermophiles are present in both archaeal phyla (e.g., Thermococcales in Euryarchaeota and Pyrobaculum, Aeropyrum and Desulfurococcus in Crenarchaeota), one cannot exclude 418 that the archaeal ancestor was a heterotroph. In fact, considering the extent of LGTs in prokaryotes, it is difficult to raise firm conclusions about the origin and evolution of such metabolic pathways. Moreover, the presence of many uncultivated lineages at strategic points of the phylogeny renders inference of the ancestral metabolism very hypothetical until their metabolism will be characterized. A fascinating aspect of the archaeal domain is its connection with hyperthermophily. We have already mentioned that only Archaea have apparently been able to colonize biotopes with temperatures above 958C: Possibly, this can be explained by the unique structure of archaeal lipids that maintain the impermeability of the cytoplasmic membrane to ions at such high temperatures. When the membrane lipid layer becomes unable to maintain an efficient barrier to passive ion diffusion, the cell cannot build up the ion gradients that are required to produce ATP. The membrane of hyperthermophilic Archaea contains tetraether lipids that can form a monolayer highly resistant to passive ion transfer. This has been experimentally demonstrated in vitro by the analysis of ion transport in reconstituted vesicles formed with archaeal lipids. Hyperthermophilic Bacteria also have unusual lipids that ‘‘try’’ to resemble archaeal ones (tetraester or diether), but they have been apparently unable to produce membrane that can prevent ion leakage at temperatures above 958C (for a recent review on the importance of lipids and membrane permeability in thermoadaptation, see Albers et al., 2001). Interestingly, mesophilic and even some psychrophilic Archaea also have tetraether lipids (Schouten et al., 2000). Indeed, these unique lipids have the ability to maintain a correct membrane fluidity in a wide temperature range. The appearance of such lipids might have thus predated (and anticipated) the appearance of hyperthermophiles in the archaeal domain. Alternatively, these lipids might have appeared in a hyperthermophilic archaeal ancestor and their presence today in mesophilic and psychrophilic Archaea can be viewed as a heritage from their hyperthermophilic ancestor. The later hypothesis is supported by the presence of many hyperthermophic lineages that emerge at the base of both the euryarchaeal and the crenarchaeal phyla (at least for the cultivable species, the picture being much more complex with uncultured species). Furthermore, in both rRNA trees and ribosomal protein trees (Figs. 1 and 2), the hyperthermophilic Archaea have shorter branches than their mesophilic counterparts, suggesting that hyperthermophiles might have retained more ancestral characters, since their macromolecules have Forterre, Brochier and Philippe evolved more slowly (Kollman and Doolittle, 2000; Matte-Tailliez et al., 2002). The phylogeny of reverse gyrase, a unique DNA topoisomerase that introduces positive superturns in DNA, also argues for a hyperthermophilic archaeal ancestor, since it is coherent with the rRNA phylogeny (e.g., the separation of Euryarchaeota and Crenarchaeota) (Forterre et al., 2000). Reverse gyrase is present in all hyperthermophiles (both Archaea and Bacteria) and absent in all genomes from mesophiles or moderate thermophiles. From comparative genomics analysis performed using the ‘‘Phylogenetic Patterns Search Program’’ of the COG database (Tatusov et al., 2001), extended to the genome of Sulfolobus solfataricus (She et al., 2001), it even turned out that reverse gyrase is the only protein that exhibits this striking phylogenetic distribution (Forterre, 2002). Phylogenetic and genome context analyses have indicated that reverse gyrase has been transferred from Archaea to hyperthermophilic Bacteria by LGT (at least twice independently), suggesting that the common ancestor of all Bacteria was not a hyperthermophile and that the hyperthermophilic phenotype originated in Archaea (Forterre et al., 2000). This is consistent with the idea that LUCA was not a hyperthermophile, a viewpoint that is also supported by recent analysis suggesting that the GC content of LUCA rRNA was too low to be compatible with a hyperthermophilic lifestyle (Galtier et al., 1999). 7. CONCLUSION AND PERSPECTIVE The study of Archaea has confirmed the two initial predictions by Woese and Fox (1977), i.e., that Archaea will exhibit a phenotypic diversity at least comparable to that of Bacteria and that Archaea will be characterized by unique features at the molecular level. In addition, Archaea turned out to exhibit a mosaic of features from the two other domains that continue to stimulate discussions among evolutionists. In the course of their history, Archaea have in particular retained (or acquired) in their informational apparatus many characters that they only share now with Eukarya. Despite their differences in this respect, Archaea and Bacteria have been engaged in a continuous ‘‘dialogue’’ via LGTs, due to their similar prokaryotic lifestyles. Whereas some Bacteria learned from Archaea how to bypass the hyperthermophilic barrier (borrowing reverse gyrase), some Archaea learned from Bacteria Evolution of the Archaea how to utilize oxygen to get energy surplus. We have now, with some confidence, the idea of a hyperthermophilic and anaerobic archaeal ancestor, suggesting that the invention of reverse gyrase, the acquisition of tetraether lipids and other features of thermoadaptation have been critical for the emergence of Archaea. However, this view, as all others concerning the origin and evolution of phenotypic traits in Archaea, should still be taken with caution, considering the limited number of archaeal lineages for which cultivable species are available for phenotypic and phylogenomic analyses. In particular, it should be extremely important now to get genotypic and phenotypic information about all the archaeal lineages that are presently only known from their phylotypes, especially those branching in between Euryarchaeota and Crenarchaeota. ACKNOWLEDGMENTS We thank Allan Campbell and Samuel Karlin for providing us with the opportunity to write this review. Preliminary sequence data of Ferroplasma and Methanosarcina were obtained from the DOE Joint Genome Institute (JGI) at http://www.jgi.doe.gov/JGI microbial/ html/index.html. We are grateful to Karl O Stetter, Francisco Rodriguez-Valera and Purification Lopez-Garcia for the EM pictures. 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