Origin and early evolution of eukaryotes inferred from the amino acid sequences of translation elongation factors 1α/Tu and 2/G

Phylogenetic placements of archaebacteria and protozoa are important in understanding the origin and early evolution of eukaryotes. These problems have been analyzed mainly by comparisons of small subunit ribosomal RNA (SrRNA) sequences. However, the SrRNA phylogeny may sometimes be unreliable, especially when base compositions are biased among species. Because it is difficult to take full account of the bias in inferring the SrRNA tree, alternative examinations using protein sequence data have been very much desired. We analyzed the phylogenetic relationship among eukaryotes, archaebacteria, and eubacteria by the ML method of protein phylogeny using amino acid sequence data of EF-1α/Tu and 2/G. The unrooted tree analyses of both the EF-1α/Tu and 2/G consistently demonstrated that the ‘eocyte’ tree, in which archaebacteria are not monophyletic but eocytes are closer to eukaryotes than to other archaebacteria, is very likely. Further analysis using a composite tree of EF-1α/Tu and 2/G suggested that archaebacteria are closer to eukaryotes than to eubacteria but are not monophyletic. These results clearly support the hypothesis that eukaryotes have evolved from the eocyte-like organism. We also analyzed a protozoan phylogeny including mitochondrion-lacking species by the ML method using EF-1α and EF-2 data sets, and demonstrated (a) that two mitochondrion-lacking species, G. plecoglossi (Microsporidians) and G. lamblia (Diplomonads) probably represent the first and the second earliest offshoots of eukaryotes, respectively; (b) that Trypanosoma is not likely to have diverged next to Giardia as suggested by the SrRNA tree, but shows high affinity with higher eukaryotes; and (c) that protein phylogeny would give a robust estimation because amino acid compositions of conservative proteins do not differ significantly among species.     

A congruent phylogenomic signal places eukaryotes within the Archaea

Determining the relationships among the major groups of cellular life is important for understanding the evolution of biological diversity, but is difficult given the enormous time spans involved. In the textbook ‘three domains’ tree based on informational genes, eukaryotes and Archaea share a common ancestor to the exclusion of Bacteria. However, some phylogenetic analyses of the same data have placed eukaryotes within the Archaea, as the nearest relatives of different archaeal lineages. We compared the support for these competing hypotheses using sophisticated phylogenetic methods and an improved sampling of archaeal biodiversity. We also employed both new and existing tests of phylogenetic congruence to explore the level of uncertainty and conflict in the data. Our analyses suggested that much of the observed incongruence is weakly supported or associated with poorly fitting evolutionary models. All of our phylogenetic analyses, whether on small subunit and large subunit ribosomal RNA or concatenated protein-coding genes, recovered a monophyletic group containing eukaryotes and the TACK archaeal superphylum comprising the Thaumarchaeota, Aigarchaeota, Crenarchaeota and Korarchaeota. Hence, while our results provide no support for the iconic three-domain tree of life, they are consistent with an extended eocyte hypothesis whereby vital components of the eukaryotic nuclear lineage originated from within the archaeal radiation.     

Protein-based phylogenies support a chimeric origin for the eukaryotic genome

The phylogenetic position of the archaebacteria and the place of eukaryotes in the history of life remain a question of debate. Recent studies based on some protein-sequence data have obtained unusual phylogenies for these organisms. We therefore collected the protein sequences that were available with representatives from each of the major forms of life: the gram-negative bacteria, gram-positive bacteria, archaebacteria, and eukaryotes. Monophyletic, unrooted phylogenies based on these sequence data show that seven of 24 proteins yield a significant gram-positive-archaebacteria clade/gram-negative- eukaryotic clade. The phylogenies for these seven proteins cannot be explained by the traditional three-way split of the eukaryotes, archaebacteria, and eubacteria. Nine of the 24 proteins yield the traditional gram-positive-gram-negative clade/archaebacteria-eukaryotic clade. The remaining eight proteins give phylogenies that cannot be statistically distinguished. These results support the hypothesis of a chimeric origin for the eukaryotic cell nucleus formed from the fusion of an archaebacteria and a gram-negative bacteria.      

Accounting for evolutionary rate variation among sequence sites consistently changes universal phylogenies deduced from rRNA and protein-coding genes.

Phylogenetic analyses of gene and protein sequences have led to two major competing views of the universal phylogeny, the evolutionary tree relating the three kinds of living organisms, Bacteria, Archaea, and Eukarya. In the first scheme, called "the archaebacterial tree, " organisms of the same type are clustered together. In the second scenario, called "the eocyte tree," the archaeal phylum of Crenarchaeota is more closely related to eukaryotes than are other Archaea. A major property of the evolution of functional ribosomal and protein-encoding genes is that the rate of nucleotide and amino acid substitution varies across sequence sites. Here, using distance-based and maximum-likelihood methods, we show that universal phylogenies of ribosomal RNAs and RNA polymerases built by ignoring this variation are biased toward the archaebacterial tree because of attraction between long branches. In contrast, taking among-site rate variability into account gives support for the eocyte tree.     

Turning the crown upside down: gene tree parsimony roots the eukaryotic tree of life

The first analyses of gene sequence data indicated that the eukaryotic tree of life consisted of a long stem of microbial groups "topped" by a crown-containing plants, animals, and fungi and their microbial relatives. Although more recent multigene concatenated analyses have refined the relationships among the many branches of eukaryotes, the root of the eukaryotic tree of life has remained elusive. Inferring the root of extant eukaryotes is challenging because of the age of the group (～1.7-2.1 billion years old), tremendous heterogeneity in rates of evolution among lineages, and lack of obvious outgroups for many genes. Here, we reconstruct a rooted phylogeny of extant eukaryotes based on minimizing the number of duplications and losses among a collection of gene trees. This approach does not require outgroup sequences or assumptions of orthology among sequences. We also explore the impact of taxon and gene sampling and assess support for alternative hypotheses for the root. Using 20 gene trees from 84 diverse eukaryotic lineages, this approach recovers robust eukaryotic clades and reveals evidence for a eukaryotic root that lies between the Opisthokonta (animals, fungi and their microbial relatives) and all remaining eukaryotes.     

Phylogenetic Signal Dissection Identifies the Root of Starfishes

Relationships within the class Asteroidea have remained controversial for almost 100 years and, despite many attempts to resolve this problem using molecular data, no consensus has yet emerged. Using two nuclear genes and a taxon sampling covering the major asteroid clades we show that non-phylogenetic signal created by three factors - Long Branch Attraction, compositional heterogeneity and the use of poorly fitting models of evolution – have confounded accurate estimation of phylogenetic relationships. To overcome the effect of this non-phylogenetic signal we analyse the data using non-homogeneous models, site stripping and the creation of subpartitions aimed to reduce or amplify the systematic error, and calculate Bayes Factor support for a selection of previously suggested topological arrangements of asteroid orders. We show that most of the previous alternative hypotheses are not supported in the most reliable data partitions, including the previously suggested placement of either Forcipulatida or Paxillosida as sister group to the other major branches. The best-supported solution places Velatida as the sister group to other asteroids, and the implications of this finding for the morphological evolution of asteroids are presented.     

Phylogenetic analysis of 70 kD heat shock protein sequences suggests a chimeric origin for the eukaryotic cell nucleus

Background: The evolutionary relationships between archaebacteria, eubacteria and eukaryotic cells are of central importance in biology. The current view is that each of these three groups of organisms constitutes a monophyletic domain, and that eukaryotic cells have evolved from an archaebacterial ancestor. Recent studies on a number of highly conserved protein sequences do not, however, support this view and raise important questions concerning the evolutionary relationships between all extant organisms, particularly regarding the origin of eukaryotic cells.Results We have used sequences of 70 kD heat shock protein (hsp70) — the most conserved protein found to date in all species — to examine the evolutionary relationship between various species. We have obtained two new archaebacterial hsp70 sequences from the species, Thermoplasma acidophilum and Halobacterium cutirubrum. A global comparison of hsp70 sequences, including our two new sequences, shows that all known archaebacterial homologs share a number of sequence signatures with the Gram-positive group of bacteria that are not found in any other prokaryotic or eukaryotic species. In contrast, the eukaryotic homologs are shown to share a number of unique sequence features with the Gram-negative bacteria that are not present in any archaebacteria. Detailed phylogenetic analyses of hsp70 sequences strongly support a specific evolutionary relationship between archaebacteria and Gram-positive bacteria on the one hand, and Gram-negative bacteria and eukaryotes on the other. The phylogenetic analyses also indicate a polyphyletic branching of archaebacteria within the Gram-positive species. The possibility that the observed relationships are due to horizontal gene transfers can be excluded on the basis of sequence characteristics of different groups of homologs.Conclusion Our results do not support the view that archaebacteria constitute a monophyletic domain, but instead suggest a close evolutionary linkage between archaebacteria and Gram-positive bacteria. Furthermore, in contrast to the presently accepted view, eukaryotic hsp70s show a close and specific relationship to those from Gram-negative species. To explain the phylogenies based on different gene sequences, a chimeric model for the origin of the eukaryotic cell nucleus involving fusion between an archaebacterium and a Gram-negative eubacterium is proposed. Several predictions from the chimeric model are discussed.     

Comparisons of large subunit rRNAs reveal some eukaryote-specific elements of secondary structure

All large rRNAs possess a common core of secondary structure. However, large variations in the size of the molecule have arisen during evolution, which are accommodated over a dozen rapidly evolving domains. Most of the enlargement of the eukaryotic molecules (as compared to prokaryotes) is in fact restricted over only two of these divergent domains, which are dramatically expanded in vertebrates. We have derived secondary structure models for these two domains through a systematic comparison of all the pro- and eukaryotic sequences published so far. Within each of these domains, a subset of secondary structure elements which are specific to eukaryotes is detected. Archaebacterial-specific secondary structures can also be identified which appear to be maintained through a strong selective constraint. The relative preservation of such group-specific structures raises the issue of their potential involvement in some diversification of ribosomal functions among the three fundamental phylogenetic groups, eubacteria, archaebacteria and eukaryotes. We also show that eukaryotic ribosomal RNAs are subjected, over their entire length, to a unique type of compositional constraint which may largely differ among the major eukaryotic taxa.     

Informational gene phylogenies do not support a fourth domain of life for nucleocytoplasmic large DNA viruses

Mimivirus is a nucleocytoplasmic large DNA virus (NCLDV) with a genome size (1.2 Mb) and coding capacity ( 1000 genes) comparable to that of some cellular organisms. Unlike other viruses, Mimivirus and its NCLDV relatives encode homologs of broadly conserved informational genes found in Bacteria, Archaea, and Eukaryotes, raising the possibility that they could be placed on the tree of life. A recent phylogenetic analysis of these genes showed the NCLDVs emerging as a monophyletic group branching between Eukaryotes and Archaea. These trees were interpreted as evidence for an independent "fourth domain" of life that may have contributed DNA processing genes to the ancestral eukaryote. However, the analysis of ancient evolutionary events is challenging, and tree reconstruction is susceptible to bias resulting from non-phylogenetic signals in the data. These include compositional heterogeneity and homoplasy, which can lead to the spurious grouping of compositionally-similar or fast-evolving sequences. Here, we show that these informational gene alignments contain both significant compositional heterogeneity and homoplasy, which were not adequately modelled in the original analysis. When we use more realistic evolutionary models that better fit the data, the resulting trees are unable to reject a simple null hypothesis in which these informational genes, like many other NCLDV genes, were acquired by horizontal transfer from eukaryotic hosts. Our results suggest that a fourth domain is not required to explain the available sequence data.     

Cladistic analysis of 5S rRNA and 16S rRNA secondary and primary structure—The evolution of eukaryotes and their relation to archaebacteria

Summary The secondary structure of 5S rRNA has been elucidated by a cladistic analysis resulting in minimal models for eukaryotes, eubacteria, and halophilic-methanogenic archaebacteria, as well as for an ur-5S rRNA. This ancestor of all present-day 5S rRNA molecules is compared with an ur-tRNA and can be fitted into a tRNA-like structure allowing tertiary-structure interactions at the equivalent positions. A phylogenetic analysis of eukaryotic 5SrRNA and 16S rRNA sequences confirms particular monophyletic taxa: rhodophytes (red algae), chlorobionts (green algae and plants), metazoans (multicellular animals), euglenozoans (euglenids and trypanosomatids), a group of zygomycetes (excluding Kickxellales), a group of ascomycetes (excluding Protomycetales), two distinct groups of basidiomycetes, and a group consisting of phaeophyceans (brown algae) and oomycetes (water molds). The Euglenozoa show a distinct relation to the Eumycota (true fungi) and Metazoa. An analysis of archaebacterial sequences substantiates the paraphyletic nature of this third urkingdom defining the eubacteria as a sister group of the halophile-methanogens and defining the eukaryotes as a sister group of a particular lineage of the eocytes/sulfur-dependents. The latter fact implies that even the eocytes/sulfur-dependent archaebacteria are paraphyletic.Presented at the FEBS Symposium on Genome Organization and Evolution, held in Crete, Greece, September 1–5, 1986Dedicated to the memory of Erik Huysmans who died on July 8, 1986, at the age of 29.     

Evolution of glutamate dehydrogenase genes: Evidence for two paralogous protein families and unusual branching patterns of the archaebacteria in the universal tree of life

Summary The existence of two families of genes coding for hexameric glutamate dehydrogenases has been deduced from the alignment of 21 primary sequences and the determination of the percentages of similarity between each pair of proteins. Each family could also be characterized by specific motifs. One family (Family 1) was composed of gdh genes from six eubacteria and six lower eukaryotes (the primitive protozoan Giardia lamblia, the green alga Chlorella sorokiniana, and several fungi and yeasts). The other one (Family 11) was composed of gdh genes from two eubacteria, two archaebacteria, and five higher eukaryotes (vertebrates). Reconstruction of phylogenetic trees using several parsimony and distance methods confirmed the existence of these two families. Therefore, these results reinforced our previously proposed hypothesis that two close but already different gdh genes were present in the last common ancestor to the three Ur-kingdoms (eubacteria, archaebacteria, and eukaryotes). The branching order of the different species of Family I was found to be the same whatever the method of tree reconstruction although it varied slightly according the region analyzed. Similarly, the topological positions of eubacteria and eukaryotes of Family II were independent of the method used. However, the branching of the two archaebacteria in Family II appeared to be unexpected: (1) the thermoacidophilic Sulfolobus solfataricus was found clustered with the two eubacteria of this family both in parsimony and distance trees, a situation not predicted by either one of the contradictory trees recently proposed; and (2) the branching of the halophilic Halobacterium salinarium varied according to the method of tree construction: it was closer to the eubacteria in the maximum parsimony tree and to eukaryotesin distance trees. Therefore, whatever the actual position of the halophilic species, archaebacteria did not appear to be monophyletic in these gdh gene trees. This result questions the firmness of the presently accepted interpretation of previous protein trees which were supposed to root unambiguously the universal tree of life and place the archaebacteria in this tree. Offprint requests to: B. Labedan     

Molecular phylogenies based on ribosomal protein L11, L1, L10, and L12 sequences

Summary Available sequences that correspond to the E. coli ribosomal proteins L11, L1, L10, and L12 from eubacteria, archaebacteria, and eukaryotes have been aligned. The alignments were analyzed qualitatively for shared structural features and for conservation of deletions or insertions. The alignments were further subjected to quantitative phylogenetic analysis, and the amino acid identity between selected pairs of sequences was calculated. In general, eubacteria, archaebacteria, and eukaryotes each form coherent and well-resolved nonoverlapping phylogenetic domains. The degree of diversity of the four proteins between the three groups is not uniform. For L11, the eubacterial and archaebacterial proteins are very similar whereas the eukaryotic L11 is clearly less similar. In contrast, in the case of the L12 proteins and to a lesser extent the L10 proteins, the archaebacterial and eukaryotic proteins are similar whereas the eubacterial proteins are different. The eukaryotic L1 equivalent protein has yet to be identified. If the root of the universal tree is near or within the eubacterial domain, our ribosomal protein-based phylogenies indicate that archaebacteria are monophyletic. The eukaryotic lineage appears to originate either near or within the archaebacterial domain. Correspondence to: P. Dennis     

Early evolution and the origin of eukaryotes

Our understanding of evolutionary relationships in the eukaryotic world has been revolutionized by molecular systematics. Phylogenies based upon comparisons of rRNAs define five major eukaryotic assemblages plus a series of paraphyletic protist lineages. Comparison of conserved genes that were duplicated prior to the divergence of eubacteria, archaebacteria, and eukaryotes, positions the root of the universal tree within the eubacterial line of descent. In this review a novel model is presented which uses the rRNA and protein based phylogenies to describe the evolutionary origins of eukaryotes.     

Evolution of RNA polymerases and branching patterns of the three major groups of archaebacteria

Summary The amino acid sequences of the largest subunits of the RNA polymerases I, II, and III from eukaryotes were compared with those of archaebacterial and eubacterial homologs, and their evolutionary relationships were analyzed in detail by a recently developed tree-making method, the likelihood method of protein phylogeny, as well as by the neighbor-joining method and the parsimony method, together with bootstrap analyses. It was shown that the best tree topologies predicted by the first two methods are identical, whereas the last one predicts a distinct tree. The maximum likelihood tree revealed that, after the separation from archaebacteria, the three eukaryotic RNA polymerases diverged from an ancestral precursor in the eukaryotic lineage. This result is contrasted with the published result showing multiple origins for the three eukaryotic polymerases. It was shown that eukaryotic RNA polymerase I evolved much more rapidly than RNA polymerases II and III: The N-terminal half of RNA polymerase I shows an extraordinarily high evolutionary rate, possibly due to relaxed functional constraints. In contrast the evolutionary rate of archaebacterial RNA polymerase is remarkably limited. In addition, including the second largest subunit of the RNA polymerase, a detailed analysis for the branching pattern of the three major groups of archaebacteria was carried out by the maximum likelihood method. It was shown that the three major groups of archaebacteria are likely to form a single cluster; that is, archaebacteria are likely to be monophyletic as originally proposed by Woese and his colleagues.     

Base Compositional Bias and Phylogenetic Analyses: A Test of the “Flying DNA” Hypothesis

Phylogenetic methods can produce biased estimates of phylogeny when base composition varies along different lineages. Pettigrew (1994,Curr. Biol.4:277–280) has suggested that base composition bias is responsible for the apparent support for the monophyly of bats (Chiroptera: megabats and microbats) from several different nuclear and mitochondrial genes. Pettigrew's “flying DNA” hypothesis makes several predictions: (1) that metabolic constraints associated with flying result in elevated levels of adenine and thymine throughout the genome of both megabats and microbats, (2) that the resulting base compositional bias in bats is sufficient to mislead phylogenetic methods and account for the support for bat monophyly from several nuclear and mitochondrial genes, and (3) that phylogenetic analysis using pairwise distances corrected for compositional bias should eliminate the support for bat monophyly. We tested these predictions by analyzing DNA sequences from two nuclear and three mitochondrial genes. The predicted base compositional bias does not appear to exist in some of the genes, and in other genes the differences in AT content are very small. Analyses under a wide diversity of criteria and models of evolution, including analyses that take base composition into account (using log-determinant distances), all strongly support bat monophyly. Moreover, simulation analyses indicate that even extreme bias toward AT-base composition in bats would be insufficient to explain the observed levels of support for bat monophyly. These analyses provide no support for the “flying DNA” hypothesis, whereas the monophyly of bats appears to be well supported by the DNA sequence data.     

What are archaebacteria: life's third domain or monoderm prokaryotes related to Gram-positive bacteria? A new proposal for the classification of prokaryotic organisms

The evolutionary relationship within prokaryotes is examined based on signature sequences (defined as conserved inserts or deletions shared by specific taxa) and phylogenies derived from different proteins. Archaebacteria are indicated as being monophyletic by a number of proteins related to the information transfer processes. In contrast, for several other highly conserved proteins, common signature sequences are present in archaebacteria and Gram-positive bacteria, whereas Gram-negative bacteria are indicated as being distinct. For these proteins, archaebacteria do not form a phylogenetically distinct clade but show polyphyletic branching within Gram-positive bacteria. A closer relationship of archaebacteria to Gram-positive bacteria in comparison with Gram-negative bacteria is generally seen for the majority of the available gene/protein sequences. To account for these results and the fact that both archaebacteria and Gram-positive bacteria are prokaryotes surrounded by a single cell membrane, I propose that the primary division within prokaryotes is between monoderm prokaryotes (surrounded by a single membrane) and diderm prokaryotes (i.e. all true Gram-negative bacteria containing both an inner cytoplasmic membrane and an outer membrane). This proposal is consistent with both cell morphology and signature sequences in different proteins. The monophyletic nature of archaebacteria for some genes, and their polyphyletic branching within Gram-positive bacteria as suggested by others, is critically examined, and several explanations, including derivation of archaebacteria from Gram-positive bacteria in response to antibiotic selection pressure, are proposed. Signature sequences in proteins also indicate that the low-G + C Gram-positive bacteria are phylogenetically distinct from the high-G + C Gram-positive group and that the diderm prokaryotes (i.e. Gram-negative bacteria) appear to have evolved from the latter group. Protein phylogenies and signature sequences also show that all eukaryotic cells have received significant gene contributions from both an archaebacterium and a Gram-negative eubacterium. Thus, the hypothesis that archaebacteria and eukaryotes shared a common ancestor exclusive of eubacteria is not supported. These observations provide evidence for an alternate view of the evolutionary relationship among living organisms that is different from the currently popular three-domain proposal.     

Phylogenetic relationships between Sophophora and Lordiphosa, with proposition of a hypothesis on the vicariant divergences of tropical lineages between the Old and New Worlds in the family Drosophilidae

Despite many studies on the phylogeny of the subgenus Sophophora, its monophyly has not been established, especially in relation to its putative relative, the genus Lordiphosa. We analyzed their phylogenetic relationships using DNA sequence data of two mitochondrial genes (ND2 and COII) and two nuclear genes (Adh and 28SrRNA). In constructing phylogenetic trees, we accounted for the problem of among-taxa nucleotide compositional heterogeneity, and took a sequence-partitioning approach to allow multiple substitution models for nucleotide sequences that have evolved under different evolutionary processes, particularly developing a novel, sequence-partitioning procedure for Neighbor Joining (NJ) tree construction. Trees constructed by different methods showed an almost identical and strongly supported topology in which Sophophora was paraphyletic: Lordiphosa was placed as the sister to the Neotropical Sophophora consisting of the saltans and willistoni groups, and Sophophora was divided into the clade of Lordiphosa+Neotropical Sophophora and the clade of the obscura+melanogaster groups. Based on the estimated time, 45.9 Mya, of divergence between the Old World Lordiphosa and the Neotropical Sophophora and evidence from paleontology, paleo-geography and -climatology, we propose a hypothesis that this vicariant divergence should have occurred when the North Atlantic Land Bridge between Europe and North America broke in the middle Eocene Epoch.     

Evidence for the early divergence of tryptophanyl- and tyrosyl-tRNA synthetases

Each amino acid is attached to its cognate tRNA by a distinct aminoacyl-tRNA synthetase (aaRS). The conventional evolutionary view is that the modern complement of synthetases existed prior to the divergence of eubacteria and eukaryotes. Thus comparisons of prokaryotic and eukaryotic aminoacyl-tRNA synthetases of the same type (charging specificity) should show greater sequence similarities than comparisons between synthetases of different types—and this is almost always so. However, a recent study [Ribas de Pouplana L, Furgier M, Quinn CL, Schimmel P (1996) Proc Natl Acad Sci USA 93:166–170] suggested that tryptophanyl- (TrpRS) and tyrosyl-tRNA (TyrRS) synthetases of the Eucarya (eukaryotes) are more similar to each other than either is to counterparts in the Bacteria (eubacteria). Here, we reexamine the evolutionary relationships of TyrRS and TrpRS using a broader range of taxa, including new sequence data from the Archaea (archaebacteria) as well as species of Eucarya and Bacteria. Our results differ from those of Ribas de Pouplana et al.: All phylogenetic methods support the separate monophyly of TrpRS and TyrRS. We attribute this result to the inclusion of the archaeal data which might serve to reduce long branch effects possibly associated with eukaryotic TrpRS and TyrRS sequences. Furthermore, reciprocally rooted phylogenies of TrpRS and TyrRS sequences confirm the closer evolutionary relationship of Archaea to eukaryotes by placing the root of the universal tree in the Bacteria. Received: 7 December 1996 / Accepted: 11 February 1997