The evidence that the tree of life is not rooted within the Archaea is unreliable: a reply to Skophammer et al

Skophammer et al. [Skophammer, RG, Herbold, CW, Rivera, MC, Servin, JA, Lake, JA. 2006. Evidence that the root of the tree of life is not within the Archaea. Mol Biol Evol, 23, 1648-1651] report evidence suggesting that the tree of life cannot be rooted within the Archaea domain. I have observed that an alignment used in their analysis is not reliable and that, therefore, their conclusions are unjustified.     

Evidence excluding the root of the tree of life from the actinobacteria

The Actinobacteria are found in aquatic and terrestrial habitats throughout the world and are among the most morphologically varied prokaryotes. They manufacture unusual compounds, utilize novel metabolic pathways, and contain unique genes. This diversity may suggest that the root of the tree of life could be within the Actinobacteria, although there is little or no convincing evidence for such a root. Here, using gene insertions and deletions found in the DNA gyrase, GyrA, and in the paralogous DNA topoisomerase, ParC, we present evidence that the root of life is outside the Actinobacteria.     

The effects of heavy meteorite bombardment on the early evolution — The emergence of the three Domains of life

A characteristic of many molecular phylogenies is that the three domains of life (Bacteria, Archaea, Eucarya) are clearly separated from each other. The analyses of ancient duplicated genes suggest that the last common ancestor of all presently known life forms already had been a sophisticated cellular prokaryote. These findings are in conflict with theories that have been proposed to explain the absence of deep branching lineages. In this paper we propose an alternative scenario, namely, a large meteorite impact that wiped out almost all life forms present on the early Earth. Following this nearly complete frustation of life on Earth, two surviving extreme thermophilic species gave rise to the now existing major groups of living organisms, the Bacteria and Archaea. [The latter also contributed the major portion to the nucleo-cytoplasmic component of the Eucarya]. An exact calibration of the molecular record with regard to time is not yet possible. The emergence of Eucarya in fossil and molecular records suggests that the proposed late impact should have occurred before 2100 million years before present (BP). If the 3500 million year old microfossils [Schopf, J. W. 1993: Science 260: 640–646] are interpreted as representatives of present day existing groups of bacteria (i.e., as cyanobacteria), then the impact is dated to around 3700 million years BP.The analysis of molecular sequences suggests that the separation between the Eucarya and the two prokaryotic domains is less deep then the separation between Bacteria and Archaea. The fundamental cell biological differences between Archaea and Eucarya were obtained over a comparatively short evolutionary distance (as measured in number of substitution events in biological macromolecules).Our interpretation of the molecular record suggests that life emerged early in Earth's history even before the time of the heavy bombardment was over. Early life forms already had colonized extreme habitats which allowed at least two prokaryotic species to survive a late nearly ocean boiling impact. The distribution of ecotypes on the rooted universal tree of life should not be interpreted as evidence that life originated in extremely hot environments.     

Microbial genescapes: phyletic and functional patterns of ORF distribution among prokaryotes

We have implemented a statistically based approach to comparative genomics that allows us to define and characterize distributional patterns of conceptually translated open reading frames (ORFs) at different confidence levels based on pairwise FASTA matches. In this report, we apply this methodology to nine microbial genomes, focusing particularly on phyletic and functional patterns of ORF distribution within and between the two prokaryotic domains of life, Bacteria and Archaea. We examine patterns of presence and absence of matches, determine the universal ORF set, analyze features of genome specialization between closely related organisms, and present genomic evidence for the monophyly of Archaea. These analyses illustrate how a quantitative approach to comparative genomics can illuminate questions of fundamental biological significance.     

Identification of genes involved in the biosynthesis and attachment of Methanococcus voltae N-linked glycans: insight into N-linked glycosylation pathways in Archaea

N-linked glycosylation is recognized as an important post-translational modification across all three domains of life. However, the understanding of the genetic pathways for the assembly and attachment of N-linked glycans in eukaryotic and bacterial systems far outweighs the knowledge of comparable processes in Archaea. The recent characterization of a novel trisaccharide [beta-ManpNAcA6Thr-(1-4)-beta-GlcpNAc3NAcA-(1-3)-beta-GlcpNAc]N-linked to asparagine residues in Methanococcus voltae flagellin and S-layer proteins affords new opportunities to investigate N-linked glycosylation pathways in Archaea. In this contribution, the insertional inactivation of several candidate genes within the M. voltae genome and their resulting effects on flagellin and S-layer glycosylation are reported. Two of the candidate genes were shown to have effects on flagellin and S-layer protein molecular mass and N-linked glycan structure. Further examination revealed inactivation of either of these two genes also had effects on flagella assembly. These genes, designated agl (archaeal glycosylation) genes, include a glycosyl transferase (aglA) involved in the attachment of the terminal sugar to the glycan and an STT3 oligosaccharyl transferase homologue (aglB) involved in the transfer of the complete glycan to the flagellin and S-layer proteins. These findings document the first experimental evidence for genes involved in any glycosylation process within the domain Archaea.     

Ancient gene duplications and the root(s) of the tree of life

Summary. Tracing organismal histories on the timescale of the tree of life remains one of the challenging tasks in evolutionary biology. The hotly debated questions include the evolutionary relationship between the three domains of life (e.g., which of the three domains are sister domains, are the domains para-, poly-, or monophyletic) and the location of the root within the universal tree of life. For the latter, many different points of view have been considered but so far no consensus has been reached. The only widely accepted rationale to root the universal tree of life is to use anciently duplicated paralogous genes that are present in all three domains of life. To date only few anciently duplicated gene families useful for phylogenetic reconstruction have been identified. Here we present results from a systematic search for ancient gene duplications using twelve representative, completely sequenced, archaeal and bacterial genomes. Phylogenetic analyses of identified cases show that the majority of datasets support a root between Archaea and Bacteria; however, some datasets support alternative hypotheses, and all of them suffer from a lack of strong phylogenetic signal. The results are discussed with respect to the impact of horizontal gene transfer on the ability to reconstruct organismal evolution. The exchange of genetic information between divergent organisms gives rise to mosaic genomes, where different genes in a genome have different histories. Simulations show that even low rates of horizontal gene transfer dramatically complicate the reconstruction of organismal evolution, and that the different most recent common molecular ancestors likely existed at different times and in different lineages. Correspondence and reprints: Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269-3125, U.S.A. Present address: Genome Atlantic, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada.     

Recovery and phylogenetic analysis of novel archaeal rRNA sequences from a deep-sea deposit feeder.

In 1992, two independent reports based on small-subunit rRNA gene (SSU rDNA) cloning revealed the presence of novel Archaea among marine bacterioplankton. Here, we report the presence of further novel Archaea SSU rDNA sequences recovered from the midgut contents of a deep-sea marine holothurian. Phylogenetic analyses show that these abyssal Archaea are a paraphyletic component of a highly divergent clade that also includes some planktonic sequences. Our data confirm that this clade is a deep-branching lineage in the tree of life.     

Archaea and the prokaryote-to-eukaryote transition.

Since the late 1970s, determining the phylogenetic relationships among the contemporary domains of life, the Archaea (archaebacteria), Bacteria (eubacteria), and Eucarya (eukaryotes), has been central to the study of early cellular evolution. The two salient issues surrounding the universal tree of life are whether all three domains are monophyletic (i.e., all equivalent in taxanomic rank) and where the root of the universal tree lies. Evaluation of the status of the Archaea has become key to answering these questions. This review considers our cumulative knowledge about the Archaea in relationship to the Bacteria and Eucarya. Particular attention is paid to the recent use of molecular phylogenetic approaches to reconstructing the tree of life. In this regard, the phylogenetic analyses of more than 60 proteins are reviewed and presented in the context of their participation in major biochemical pathways. Although many gene trees are incongruent, the majority do suggest a sisterhood between Archaea and Eucarya. Altering this general pattern of gene evolution are two kinds of potential interdomain gene transferrals. One horizontal gene exchange might have involved the gram-positive Bacteria and the Archaea, while the other might have occurred between proteobacteria and eukaryotes and might have been mediated by endosymbiosis.     

Phylogenomics of type II DNA topoisomerases

Type II DNA topoisomerases (Topo II) are essential enzymes implicated in key nuclear processes. The recent discovery of a novel kind of Topo II (DNA topoisomerase VI) in Archaea led to a division of these enzymes into two non-homologous families, (Topo IIA and Topo IIB) and to the identification of the eukaryotic protein that initiates meiotic recombination, Spo11. In the present report, we have updated the distribution of all Topo II in the three domains of life by a phylogenomic approach. Both families exhibit an atypical distribution by comparison with other informational proteins, with predominance of Topo IIA in Bacteria, Eukarya and viruses, and Topo IIB in Archaea. However, plants and some Archaea contain Topo II from both families. We confront this atypical distribution with current hypotheses on the evolution of the three domains of life and origin of DNA genomes.     

Microbial diversity in natural asphalts of the Rancho La Brea Tar Pits

Bacteria commonly inhabit subsurface oil reservoirs, but almost nothing is known yet about microorganisms that live in naturally occurring terrestrial oil seeps and natural asphalts that are comprised of highly recalcitrant petroleum hydrocarbons. Here we report the first survey of microbial diversity in ca. 28,000-year-old samples of natural asphalts from the Rancho La Brea Tar Pits in Los Angeles, CA. Microbiological studies included analyses of 16S rRNA gene sequences and DNA encoding aromatic ring-hydroxylating dioxygenases from two tar pits differing in chemical composition. Our results revealed a wide range of phylogenetic groups within the Archaea and Bacteria domains, in which individual taxonomic clusters were comprised of sets of closely related species within novel genera and families. Fluorescent staining of asphalt-soil particles using phylogenetic probes for Archaea, Bacteria, and Pseudomonas showed coexistence of mixed microbial communities at high cell densities. Genes encoding dioxygenases included three novel clusters of enzymes. The discovery of life in the tar pits provides an avenue for further studies of the evolution of enzymes and catabolic pathways for bacteria that have been exposed to complex hydrocarbons for millennia. These bacteria also should have application for industrial microbiology and bioremediation.     

Phylogenetic analysis of Group I marine archaeal rRNA sequences emphasizes the hidden diversity within the primary group Archaea

Archaea form one of the three primary groups of extant life and are commonly associated with the extreme environments which many of their members inhabit. Currently, the Archaea are classified into two kingdoms, Crenarchaeota and Euryarchaeota, based on phylogenetic analysis of ribosomal RNA (rRNA) sequences. Molecular techniques allowing the retrieval and analysis of rRNA sequences from diverse environments are increasing our knowledge of archaeal diversity. This report describes the presence of marine Archaea in north-east Atlantic waters. Quantitative estimates indicated that the marine Archaea constitute 8 per cent of the total prokaryotic rRNA in Irish coastal waters. Phylogenetic analysis of the archaeal rRNA gene sequences revealed sufficient genetic diversity within Archaea to indicate that the current two-kingdom classification of Crenarchaeota and Euryarchaeota is restrictive.     

N-glycosylation in Archaea—New roles for an ancient posttranslational modification

Genome analysis points to N-glycosylation as being an almost universal posttranslational modification in Archaea. Although such predictions have been confirmed in only a limited number of species, such studies are making it increasingly clear that the N-linked glycans which decorate archaeal glycoproteins present diversity in terms of both glycan composition and architecture far beyond what is seen in the other two domains of life. In addition to continuing to decipher pathways of N-glycosylation, recent efforts have revealed how Archaea exploit this variability in novel roles. As well as encouraging glycoprotein synthesis, folding and assembly into properly functioning higher ordered complexes, N-glycosylation also provides Archaea with a strategy to cope with changing environments. Archaea can, moreover, exploit the apparent species-specific nature of N-glycosylation for selectivity in mating, and hence, to maintain species boundaries, and in other events where cell-selective interactions are required. At the same time, addressing components of N-glycosylation pathways across archaeal phylogeny offers support for the concept of an archaeal origin for eukaryotes. In this MicroReview, these and other recent discoveries related to N-glycosylation in Archaea are considered.     

The euryarchaeotes, a subdomain of Archaea, survive on a single DNA polymerase: fact or farce?

Archaea is now recognized as the third domain of life. Since their discovery, much effort has been directed towards understanding the molecular biology and biochemistry of Archaea. The objective is to comprehend the complete structure and the depth of the phylogenetic tree of life. DNA replication is one of the most important events in living organisms and DNA polymerase is the key enzyme in the molecular machinery which drives the process. All archaeal DNA polymerases were thought to belong to family B. This was because all of the products of pol genes that had been cloned showed amino acid sequence similarities to those of this family, which includes three eukaryal DNA replicases and Escherichia coli DNA polymerase II. Recently, we found a new heterodimeric DNA polymerase from the hyperthermophilic archaeon, Pyrococcus furiosus. The genes coding for the subunits of this DNA polymerase are conserved in the euryarchaeotes whose genomes have been completely sequenced. The biochemical characteristics of the novel DNA polymerase family suggest that its members play an important role in DNA replication within euryarchaeal cells. We review here our current knowledge on DNA polymerases in Archaea with emphasis on the novel DNA polymerase discovered in Euryarchaeota.     

Evidence for a gram-positive, eubacterial root of the tree of life

Directed indels, insertions, and deletions within paralogous genes, have the potential to root the tree of life. Here we apply a newly developed rooting algorithm, top-down rooting, to indels found in informational and operational gene sets, introduce new computational tools for indel analyses, and present evidence (P < .01) that the root of the tree of life is not present in its traditional location, between the Eubacteria and the Archaebacteria. Using indels contained in the dihydroorotate dehydrogenase/uroporphyrinogen decarboxylase gene pair and in the ribosomal protein S12/beta prime subunit of the RNA polymerase gene pair, we exclude the root from within the clade consisting of the Firmicutes plus the Archaebacteria and their most recent common ancestor. These results, plus previous directed indel studies excluding the root from the eukaryotes, restrict the root to just four possible sites. One potential root is on the branch leading to the double-membrane prokaryotes, another is on the branch leading to the Actinobacteria, another is within the Actinobacteria, and the fourth is on the branch leading to the Firmicutes-Archaea clade. These results imply (1) that the cenancestral population was not hyperthermophilic, but moderate thermophily cannot be excluded for the root on the branch leading to the Firmicutes-Archaea clade, (2) that the cenancestral population was surrounded by ester lipids and a peptidoglycan layer, and (3) that parts of the mevalonate synthesis pathway were present in the population ancestral to the Bacilli and the Archaebacteria, including geranylgeranylglyceryl phosphate synthase, an enzyme thought to be partially responsible for the unique sn-1 stereochemistry of the archaeal glycerol phosphate backbone.     

New substitution models for rooting phylogenetic trees

The root of a phylogenetic tree is fundamental to its biological interpretation, but standard substitution models do not provide any information on its position. Here, we describe two recently developed models that relax the usual assumptions of stationarity and reversibility, thereby facilitating root inference without the need for an outgroup. We compare the performance of these models on a classic test case for phylogenetic methods, before considering two highly topical questions in evolutionary biology: the deep structure of the tree of life and the root of the archaeal radiation. We show that all three alignments contain meaningful rooting information that can be harnessed by these new models, thus complementing and extending previous work based on outgroup rooting. In particular, our analyses exclude the root of the tree of life from the eukaryotes or Archaea, placing it on the bacterial stem or within the Bacteria. They also exclude the root of the archaeal radiation from several major clades, consistent with analyses using other rooting methods. Overall, our results demonstrate the utility of non-reversible and non-stationary models for rooting phylogenetic trees, and identify areas where further progress can be made.     

Comparative analysis of ribosomal proteins in complete genomes: an example of reductive evolution at the domain scale

A comprehensive investigation of ribosomal genes in complete genomes from 66 different species allows us to address the distribution of r-proteins between and within the three primary domains. Thirty-four r-protein families are represented in all domains but 33 families are specific to Archaea and Eucarya, providing evidence for specialisation at an early stage of evolution between the bacterial lineage and the lineage leading to Archaea and Eukaryotes. With only one specific r-protein, the archaeal ribosome appears to be a small-scale model of the eukaryotic one in terms of protein composition. However, the mechanism of evolution of the protein component of the ribosome appears dramatically different in Archaea. In Bacteria and Eucarya, a restricted number of ribosomal genes can be lost with a bias toward losses in intracellular pathogens. In Archaea, losses implicate 15% of the ribosomal genes revealing an unexpected plasticity of the translation apparatus and the pattern of gene losses indicates a progressive elimination of ribosomal genes in the course of archaeal evolution. This first documented case of reductive evolution at the domain scale provides a new framework for discussing the shape of the universal tree of life and the selective forces directing the evolution of prokaryotes.     

Isolation of haloarchaea that grow at low salinities

Summary Archaea, the third domain of life, were long thought to be limited to environmental extremes. However, the discovery of archaeal 16S rRNA gene sequences in water, sediment and soil samples has called into question the notion of Archaea as obligate extremophiles. Until now, none of these novel Archaea has been brought into culture, a critical step for discovering their ecological roles. We have cultivated three novel halophilic Archaea (haloarchaea) genotypes from sediments in which the pore-water salinity was close to that of sea water. All previously reported haloarchaeal isolates are obligate extreme halophiles requiring at least 9% (w/v) NaCl for growth and are typically the dominant heterotrophic organisms in salt and soda lakes, salt deposits and salterns. Two of these three newly isolated genotypes have lower requirements for salt than previously cultured haloarchaea and are capable of slow growth at sea-water salinity (2.5% w/v NaCl). Our data reveal the existence of Archaea that can grow in non-extreme conditions and of a diverse community of haloarchaea existing in coastal salt marsh sediments. Our findings suggest that the ecological range of these physiologically versatile prokaryotes is much wider than previously supposed.     

Genetic technologies for Archaea

Members of the third domain of life, the Archaea, possess structural, physiological, biochemical and genetic features distinct from Bacteria and Eukarya and, therefore, have drawn considerable scientific interest. Physiological, biochemical and molecular analyses have revealed many novel biological processes in these important prokaryotes. However, assessment of the function of genes in vivo through genetic analysis has lagged behind because suitable systems for the creation of mutants in most Archaea were established only in the past decade. Among the Archaea, sufficiently sophisticated genetic systems now exist for some thermophilic sulfur-metabolizing Archaea, halophilic Archaea and methanogenic Archaea. Recently, there have been developments in genetic analysis of thermophilic and methanogenic Archaea and in the use of genetics to study the physiology, metabolism and regulatory mechanisms that direct gene expression in response to changes of environmental conditions in these important microorganisms.