The Presence of a Haloarchaeal Type Tyrosyl-tRNA Synthetase Marks the Opisthokonts as Monophyletic
http://www.100md.com
分子生物学进展 2005年第11期
* Department of Biology, East Carolina University; NASA Astrobiology Institute at the Marine Biological Laboratory, Woods Hole, Massachusetts; Department of Molecular and Cell Biology, University of Connecticut; and Department of Biochemistry and Molecular Biology, University of Georgia
E-mail: huangj@mail.ecu.edu.
Abstract
Lateral gene transfer plays an important role in the evolution of life. Events of ancient gene transfer can transmit genetic novelties to descendent lineages and subsequently shape their genetic systems. We here present the analyses of the gene encoding tyrosyl-tRNA synthetase (tyrRS), which reveal two eukaryotic tyrRS lineages, one including the opisthokonts and the other the remaining eukaryotes. The different origins of tyrRS lineages between the opisthokonts and the remaining eukaryotes indicate a likely case of ancient lateral gene transfer of tyrRS from an archaeon to the opisthokonts, which lends further support for the monophyly of the latter group. Ancient paralogy followed by differential gene loss is an alternative, albeit less parsimonious explanation for the distribution of the two eukaryotic tyrRS types. In either case, the presence of a haloarchaeal tyrRS type in the opisthokonts marks this group as monophyletic. This finding also points to the potential utility of ancient gene transfer events as molecular markers for major organismal lineages.
Key Words: ancient gene transfer ? opisthokonts ? tyrosyl-tRNA synthetase ? eukaryotic evolution
Lateral gene transfer across species boundaries brings new enzymes, pathways, and other novelties to host organisms and plays an important role in the evolution of life (Ochman, Lawrence, and Groisman 2000; Gogarten, Doolittle, and Lawrence 2002; Woese 2002). Events of ancient gene transfer occurring during early evolutionary time can transmit novel genetic materials to descendent lineages. Considering the possibility that the novelties introduced by these ancient events are retained among descendent lineages and subsequently shape their genetic systems, ancient gene transfer theoretically may play a far greater role in evolution than those occurring within specific lineages or in more recent evolutionary time. Nevertheless, because the phylogenetic signal of anciently transferred genes is often obscured over the course of evolution, ancient gene transfer events are often difficult to identify. Here, we present analyses of the gene encoding tyrosyl-tRNA synthetase (tyrRS), which suggests a likely case of ancient gene transfer from an archaeon to the opisthokont ancestor. This ancient gene transfer event supports monophyly for the opisthokonts and points to the potential utility of ancient gene transfer events as molecular markers for major organismal lineages.
The group opisthokonts, including animals and fungi, was proposed by Cavalier-Smith (1987) to reflect their characteristics in flagellate cells and mitochondria. Many molecular phylogenetic analyses support the opisthokonts as a natural group (Baldauf and Palmer 1993; Wainright et al. 1993; Kumar and Rzhetsky 1996; Baldauf et al. 2000; Van de Peer et al. 2000; Lang et al. 2002; Philippe et al. 2004). In particular, this group shares an insertion of 12 aa in the otherwise conserved region of the elongation factor 1 (EF-1) (Baldauf and Palmer 1993). Nevertheless, several other recent studies did not recover the monophyly of the opisthokonts; these included data from protein domain content (Wolf, Rogozin, and Koonin 2004; Yang, Doolittle, and Bourne 2005) and concatenations of protein sequences selected from completely sequenced genomes (Stiller 2004; Philip, Creevey, and McInerney 2005), which indicate that plants and animals may actually be sister groups. In addition, a recent study on mRNA capping also showed that animals and plants share a capping apparatus distinct from that found in fungi, apicomplexans, kinetoplastids, diplomonads, and red algae (Hausmann et al. 2005). This shared mRNA capping apparatus between plants and animals suggests either a common origin or lateral gene transfer between these two groups (Hausmann et al. 2005). Resolving the bifurcations leading to plants, animals, and fungi based on molecular sequences is difficult because these events occurred a long time ago and because poor taxon sampling and biases generated through long branch attraction can lead to systematic artifacts. For these reasons, the question "do the opisthokonts constitute a natural group?" is still under debate.
Aminoacyl-tRNA synthetases (aaRSs) are responsible for attaching tRNAs to cognate amino acids during translation. Genes encoding aaRSs are prone to lateral transfers due to the loose interaction of their protein products with other components of the cellular network (Wolf et al. 1999; Woese et al. 2000). The eukaryotic tyrRS was once hypothesized to have shared a recent common origin with eukaryotic tryptophanyl-tRNA synthetase (trpRS) (Ribas de Pouplana et al. 1996), but this hypothesis was not supported by later studies (Brown et al. 1997; Diaz-Lazcoz et al. 1998; Moreira and Lopez-Garcia 2005). The later studies suggested independent ancient origins for both tyrRS and trpRS.
Consistent with these more recent studies of the tyrRS evolution, our sequence comparisons show an overall tyrRS protein sequence similarity for all living organisms. Many eukaryotes possess two copies of the tyrRS gene, one of which is closely related to the bacterial lineage. Given the frequent intracellular gene transfer from mitochondria (or chloroplasts) to the nuclear genome, these tyrRSs in eukaryotes may actually be of bacterial ancestry in essence. The other tyrRS is far more similar to the archaeal lineages of tyrRS than either is to the bacterial counterparts (see also Moreira and Lopez-Garcia 2005). This finding is consistent with the common belief that eukaryotes and archaea are more closely related, especially with respect to components of the information-processing machinery (Gogarten et al. 1989; Iwabe et al. 1989). Interestingly, the true eukaryotic tyrRSs apparently fall into two major groups. The tyrRS protein sequence identities between opisthokonts and the remaining eukaryotes are only 24%–31%, in sharp contrast to their overall sequence identities within each group (40%–64% and 41%–77%, respectively). The opisthokontal tyrRS sequences also are more similar to their homologs from euryarchaeotes (27%–41% identities). In particular, opisthokontal tyrRS shares several unique amino acid residues with that of Haloarcula maristmortui (fig. 1), a halophilic archaeon living in extremely saline environments. The tyrRS sequences from the remaining eukaryotes, including parabasalids (Trichomonas), diplomonads (Giardia), amoebozoa (Entamoeba), alveolates (Cryptosporidium, Plasmodium), kinetoplastids (Leishmania, Trypanosoma), green plants (Arabidopsis), and red algae (Cyanidioschyzon), group together as distinct from the opisthokontal tyrRS lineage (fig. 2). The former group of eukaryotes is traditionally considered basal in eukaryotic phylogeny and not a monophyletic, but rather a paraphyletic, group. However, their tyrRS protein sequences share many unique residues (fig. 1). The tyrRS from the nonopisthokonts probably represents the ancestral states of eukaryotic lineage.
FIG. 1.— Multiple protein sequence alignment for tyrRS. Boxed columns show shared amino acid residues within each lineage of tyrRS.
FIG. 2.— Phylogenetic analyses of tyrRS protein sequences. Numbers above the branch show posterior probability for Bayesian analyses, bootstrap values for maximum likelihood and distance analyses, respectively. Asterisks indicate values lower than 50%. Solid arrow indicates where the bacterial tyrRS lineage branches off, as inferred from analyses including a bacterial out-group (see Additional_Trees.pdf in Supplementary Material online). Dashed arrow shows where the bacterial tyrRSs branch off in the analysis from Moreira and Lopez-Garcia (2005).
As expected from the aligned sequences, phylogenetic analyses using multiple methods supported the closer relationship between archaeal and eukaryotic tyrRSs and the independent origins of the two eukaryotic tyrRS lineages (fig. 2). As many halophilic genomes are characterized by compositional bias, which could potentially affect phylogenetic reconstruction from sequence data, we removed sequences (Cryptosporidium, Plasmodium, and Cyanidioschyzon) from the analyses that failed the statistic test for compositional homogeneity. Still, analyses showed different ancestries of tyrRS for the opisthokonts and all other eukaryotes (see file Additional_Trees.pdf in Supplementary Material online). This indicates that the two eukaryotic tyrRS lineages indeed arose independently.
The phylogeny of the archaeal tyrRSs is similar to the archaeal phylogeny based on rRNA sequences (Woese 1987) and concatenated gene sets (Brochier, Forterre, and Gribaldo 2005): the Haloarchaea group with the methanogens, most of the crenarchaeal sequences group together, and among the euryarchaeotes the thermococci and thermoplasmata group close to the Crenarchaea. A single molecule should not be expected to provide perfect phylogenetic resolution. In particular, the determination of the archaeal sister group to the eukaryotes usually is a difficult problem. Apart from the Haloarcula tyrRS, which is distinctly related to the opisthokontal homologs, there appear to be four other subtypes of tyrRS existing in archaea (fig. 2). The tyrRS sequences in methanogens and Halobacterium (MH) are more related to those of Haloarcula and opisthokonts (HO), whereas the other three archaeal subtypes, including crenarchaeotes (CA), thermoplasmata (TP), and Nanoarchaeum, euryarchaeotes, and crenarchaeotes (NEC), appear to be more related to tyrRSs from nonopisthokontal eukaryotes (EU), though their relationships are less certain due to weak support. Although it is possible that these multiple subtypes of archaeal tyrRS might represent paralogs arising from an ancient gene duplication, this scenario requires several independent losses among groups of archaea. It is more plausible that the erosion of phylogenetic signal over long evolutionary time, combined with other factors such as lateral gene transfer and periods of accelerated evolution (Cavalier-Smith 2002) gave rise to the observed distribution and weak grouping of these archaeal subtypes. Supporting evidence for ancient duplication and differential gene loss is weak: all sampled archaea in our analyses have single-copied tyrRS except for the crenarchaeote Pyrobaculum, where one copy groups as expected with the crenarchaeal homologs and the second copy groups with homologs from Nanoarchaeum and Pyrococcus. Given the frequency of lateral gene transfer in prokaryotes (Ochman, Lawrence, and Groisman 2000; Gogarten, Doolittle, and Lawrence 2002), the second copy in Pyrobaculum was likely acquired from another species, presumably a euryarchaeote.
It is probable that the independent origins of tyrRSs in opisthokonts and in the remaining eukaryotes (fig. 2) represent a case of ancient gene transfer from an archaeon to eukaryotes. Because nonopisthokontal eukaryotes are often considered paraphyletic and emerged relatively early in eukaryotic evolution, this ancient lateral gene transfer likely occurred from an archaeon, presumably similar to Haloarcula, to the opisthokont ancestor. Gene duplication followed by differential loss can always be invoked as an alternative to horizontal gene transfer detected based on phylogenetic signal (Gogarten and Townsend 2005). Applied to the presence of the haloarchaeal tyrRS type in opisthokonts, this scenario requires many independent complementary gene loss events. It therefore appears less parsimonious and less likely to account for the presence of the haloarchaeal type tyrRS in the opisthokonts than the assumption of an ancient transfer event. However, even if the distribution of the two eukaryotic tyrRS types were due to an ancient gene duplication followed by differential gene loss, the presence of a distinct tyrRS type nevertheless would support the opisthokonts as a monophyletic group.
The group opisthokonts was not recovered in some recently published analyses (e.g., domain contents and concatenation of genome sequences). These results may be due to intrinsic limitations of the data. For example, the analyses of genome sequences (Philip, Creevey, and McInerney 2005) were based on few taxa only and included genes with conflicting evolutionary histories. One of the five universally distributed genes in the selected genomes apparently supports the group opisthokonts whereas the other four do not. Concatenation of these sequences of different evolutionary histories could result in a misleading topology. If the opisthokontal tyrRS was indeed acquired from archaea, an argument against monophyly of the opisthokonts would entail multiple, independent transfers of the tyrRS gene to that group. Although gene transfer is a dynamic process constantly occurring over the course of organismal evolution, multiple, independent transfers of the same gene to related organismal groups resulting twice in successful orthologous replacement appear to be a very unlikely scenario. Therefore, the ancient gene transfer of tyrRS supports the monophyly of the opisthokonts.
The tyrRS gene might have been directly transferred from a haloarchaeon to the opisthokont ancestor, or other organisms might have been involved as intermediate carriers. The former would suggest a high-salt environment for the opisthokont ancestor. However, given the few surviving lineages from ancient times and the still poor sampling of archaeal tyrRS, a direct transfer seems unlikely. The finding that an anciently transferred tyrRS unites the opisthokonts also points to the potential utility of ancient gene transfer as a molecular marker for major organismal lineages. This might be particularly true for genes encoding essential functions because they are likely ubiquitous in life. In fact, efforts have recently been made to propose a close relationship between Giardia and Trichomonas based on a case of ancient gene transfer (Andersson, Sarchfield, and Roger 2005). In addition, gene transfer between divergent organisms also allows correlating evolutionary events in different parts of the tree of life (Gogarten, Murphey, and Olendzenski 1999; Cavalier-Smith 2002). The donor had to live at the same time or before the recipient. If our interpretation of the tyrRS phylogeny is correct, this implies that the Haloarchaea, usually considered a derived group of archaea (Woese 1987), already existed at the time of the opisthokont ancestor. Given the dynamic occurrences of lateral gene transfer during the long course of organismal evolution, we expect that ancient transfer events will provide more valuable witness to the evolutionary history of life.
Materials and Methods
Data Sources
Nucleotide and amino acid sequences for Cyanidioschyzon merolae were from Cyanidioschyzon merolae genome project database (http://merolae.biol.s.u-tokyo.ac.jp). The tyrRS protein sequence of Trichomonas vaginalis was annotated based on a nucleotide sequence obtained from the Institute of Genomic Research (http://www.tigr.org). All other sequences were obtained from the National Center for Biotechnology Information GenBank database. A file (genbank_ids.pdf) containing tyrRS accession and gene identification numbers is available in the Supplementary Material online.
Phylogenetic Analyses
Sequences were selected for major groups within each domain of life. Multiple protein sequence alignment was performed using MUSCLE (Edgar 2004) and ClustalX (Thompson et al. 1997), followed by cross-comparisons and manual refinement. Two data sets were used for the analyses, one with samples from all three major domains of life, whereas the other with bacterial sequences excluded. Only unambiguously aligned sequence portions were used for phylogenetic analyses, resulting in 188 and 205 aa positions per sequence for data sets with and without bacterial sequences, respectively. The alignments are available as Supplementary Material online (tyrRS_alignment.faa, tyrRS_analyzed_no_Bact.faa, tyrRS_analyzed.faa) or from the authors upon request. Phylogenetic analyses were performed with Bayesian inference method using MrBayes 3.1 (Ronquist and Huelsenbeck 2003), a maximum likelihood method using PHYML (Guindon and Gascuel 2003) and distance matrix analyses using the program Neighbor of PHYLIP version 3.6a (Felsenstein 2004) with maximum likelihood distances. Posterior probabilities were estimated using two independent analyses with 200,000 generations each for Bayesian analyses; bootstrap support was estimated using 1,000 replicates for both maximum likelihood and distance analyses. For Bayesian analyses, the Jones-Taylor-Thornton (JTT) substitution matrix, four gamma-distributed rate classes, the autocorrelated gamma model, and the covarion model were used. All other maximum likelihood calculations were based on the JTT substitution matrix and a mixed model of four gamma-distributed rate classes plus invariable sites. Maximum likelihood distances for bootstrap analysis were calculated using Tree-Puzzle (Schmidt et al. 2002) and PUZZLEBOOT v1.03 (by Michael E. Holder and Andrew J. Roger, available from http://www.tree-puzzle.de). Chi-square tests on amino acid sequence composition were performed using Tree-Puzzle with the default maximum likelihood model.
Supplementary Material
The following supplementary files are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
Additional_Trees.pdf: Phylogenetic analyses and maximum likelihood analyses of tyrRS.
FIG. 1S—Phylogenetic analyses of tyrRS protein sequences including bacterial sequences.
FIG. 2S—Maximum likelihood analyses for tyrRS from archaea and eukaryotes. Sequences that failed the chi-square test on sequence composition were removed.
genbank_ids.pdf: Table containing tyrRS accession numbers.
tyrRS_alignment.faa: Alignment of tyrRS sequences, including bacterial homologs in Fasta format. The file can be imported directly into ClustalX.
tyrRS_analyzed_no_Bact.faa: Aligned positions used for analyses reported in figure 1. The file can be imported directly into ClustalX.
tyrRS_analyzed.faa: Aligned positions used for analyses reported in figure 1S. The file can be imported directly into ClustalX.
Acknowledgements
We thank Greg Fournier for discussions and two anonymous reviewers for constructive comments and suggestions. This work was performed while J.H. held a National Research Council Associateship Award at the NASA Astrobiology Institute at the Marine Biological Laboratory in Woods Hole, Mass. (NCC2-1054). Y.X.'s work was supported in part by the U.S. Department of Energy's Genomes to Life Program under project "Carbon Sequestration in Synechococcus sp.: from Molecular Machines to Hierarchical Modeling" (http://www.genomes-to-life.org) and by the National Science Foundation (#NSF/DBI-0354771, #NSF/ITR-IIS-0407204, and #NSF/MCB-0237197).
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E-mail: huangj@mail.ecu.edu.
Abstract
Lateral gene transfer plays an important role in the evolution of life. Events of ancient gene transfer can transmit genetic novelties to descendent lineages and subsequently shape their genetic systems. We here present the analyses of the gene encoding tyrosyl-tRNA synthetase (tyrRS), which reveal two eukaryotic tyrRS lineages, one including the opisthokonts and the other the remaining eukaryotes. The different origins of tyrRS lineages between the opisthokonts and the remaining eukaryotes indicate a likely case of ancient lateral gene transfer of tyrRS from an archaeon to the opisthokonts, which lends further support for the monophyly of the latter group. Ancient paralogy followed by differential gene loss is an alternative, albeit less parsimonious explanation for the distribution of the two eukaryotic tyrRS types. In either case, the presence of a haloarchaeal tyrRS type in the opisthokonts marks this group as monophyletic. This finding also points to the potential utility of ancient gene transfer events as molecular markers for major organismal lineages.
Key Words: ancient gene transfer ? opisthokonts ? tyrosyl-tRNA synthetase ? eukaryotic evolution
Lateral gene transfer across species boundaries brings new enzymes, pathways, and other novelties to host organisms and plays an important role in the evolution of life (Ochman, Lawrence, and Groisman 2000; Gogarten, Doolittle, and Lawrence 2002; Woese 2002). Events of ancient gene transfer occurring during early evolutionary time can transmit novel genetic materials to descendent lineages. Considering the possibility that the novelties introduced by these ancient events are retained among descendent lineages and subsequently shape their genetic systems, ancient gene transfer theoretically may play a far greater role in evolution than those occurring within specific lineages or in more recent evolutionary time. Nevertheless, because the phylogenetic signal of anciently transferred genes is often obscured over the course of evolution, ancient gene transfer events are often difficult to identify. Here, we present analyses of the gene encoding tyrosyl-tRNA synthetase (tyrRS), which suggests a likely case of ancient gene transfer from an archaeon to the opisthokont ancestor. This ancient gene transfer event supports monophyly for the opisthokonts and points to the potential utility of ancient gene transfer events as molecular markers for major organismal lineages.
The group opisthokonts, including animals and fungi, was proposed by Cavalier-Smith (1987) to reflect their characteristics in flagellate cells and mitochondria. Many molecular phylogenetic analyses support the opisthokonts as a natural group (Baldauf and Palmer 1993; Wainright et al. 1993; Kumar and Rzhetsky 1996; Baldauf et al. 2000; Van de Peer et al. 2000; Lang et al. 2002; Philippe et al. 2004). In particular, this group shares an insertion of 12 aa in the otherwise conserved region of the elongation factor 1 (EF-1) (Baldauf and Palmer 1993). Nevertheless, several other recent studies did not recover the monophyly of the opisthokonts; these included data from protein domain content (Wolf, Rogozin, and Koonin 2004; Yang, Doolittle, and Bourne 2005) and concatenations of protein sequences selected from completely sequenced genomes (Stiller 2004; Philip, Creevey, and McInerney 2005), which indicate that plants and animals may actually be sister groups. In addition, a recent study on mRNA capping also showed that animals and plants share a capping apparatus distinct from that found in fungi, apicomplexans, kinetoplastids, diplomonads, and red algae (Hausmann et al. 2005). This shared mRNA capping apparatus between plants and animals suggests either a common origin or lateral gene transfer between these two groups (Hausmann et al. 2005). Resolving the bifurcations leading to plants, animals, and fungi based on molecular sequences is difficult because these events occurred a long time ago and because poor taxon sampling and biases generated through long branch attraction can lead to systematic artifacts. For these reasons, the question "do the opisthokonts constitute a natural group?" is still under debate.
Aminoacyl-tRNA synthetases (aaRSs) are responsible for attaching tRNAs to cognate amino acids during translation. Genes encoding aaRSs are prone to lateral transfers due to the loose interaction of their protein products with other components of the cellular network (Wolf et al. 1999; Woese et al. 2000). The eukaryotic tyrRS was once hypothesized to have shared a recent common origin with eukaryotic tryptophanyl-tRNA synthetase (trpRS) (Ribas de Pouplana et al. 1996), but this hypothesis was not supported by later studies (Brown et al. 1997; Diaz-Lazcoz et al. 1998; Moreira and Lopez-Garcia 2005). The later studies suggested independent ancient origins for both tyrRS and trpRS.
Consistent with these more recent studies of the tyrRS evolution, our sequence comparisons show an overall tyrRS protein sequence similarity for all living organisms. Many eukaryotes possess two copies of the tyrRS gene, one of which is closely related to the bacterial lineage. Given the frequent intracellular gene transfer from mitochondria (or chloroplasts) to the nuclear genome, these tyrRSs in eukaryotes may actually be of bacterial ancestry in essence. The other tyrRS is far more similar to the archaeal lineages of tyrRS than either is to the bacterial counterparts (see also Moreira and Lopez-Garcia 2005). This finding is consistent with the common belief that eukaryotes and archaea are more closely related, especially with respect to components of the information-processing machinery (Gogarten et al. 1989; Iwabe et al. 1989). Interestingly, the true eukaryotic tyrRSs apparently fall into two major groups. The tyrRS protein sequence identities between opisthokonts and the remaining eukaryotes are only 24%–31%, in sharp contrast to their overall sequence identities within each group (40%–64% and 41%–77%, respectively). The opisthokontal tyrRS sequences also are more similar to their homologs from euryarchaeotes (27%–41% identities). In particular, opisthokontal tyrRS shares several unique amino acid residues with that of Haloarcula maristmortui (fig. 1), a halophilic archaeon living in extremely saline environments. The tyrRS sequences from the remaining eukaryotes, including parabasalids (Trichomonas), diplomonads (Giardia), amoebozoa (Entamoeba), alveolates (Cryptosporidium, Plasmodium), kinetoplastids (Leishmania, Trypanosoma), green plants (Arabidopsis), and red algae (Cyanidioschyzon), group together as distinct from the opisthokontal tyrRS lineage (fig. 2). The former group of eukaryotes is traditionally considered basal in eukaryotic phylogeny and not a monophyletic, but rather a paraphyletic, group. However, their tyrRS protein sequences share many unique residues (fig. 1). The tyrRS from the nonopisthokonts probably represents the ancestral states of eukaryotic lineage.
FIG. 1.— Multiple protein sequence alignment for tyrRS. Boxed columns show shared amino acid residues within each lineage of tyrRS.
FIG. 2.— Phylogenetic analyses of tyrRS protein sequences. Numbers above the branch show posterior probability for Bayesian analyses, bootstrap values for maximum likelihood and distance analyses, respectively. Asterisks indicate values lower than 50%. Solid arrow indicates where the bacterial tyrRS lineage branches off, as inferred from analyses including a bacterial out-group (see Additional_Trees.pdf in Supplementary Material online). Dashed arrow shows where the bacterial tyrRSs branch off in the analysis from Moreira and Lopez-Garcia (2005).
As expected from the aligned sequences, phylogenetic analyses using multiple methods supported the closer relationship between archaeal and eukaryotic tyrRSs and the independent origins of the two eukaryotic tyrRS lineages (fig. 2). As many halophilic genomes are characterized by compositional bias, which could potentially affect phylogenetic reconstruction from sequence data, we removed sequences (Cryptosporidium, Plasmodium, and Cyanidioschyzon) from the analyses that failed the statistic test for compositional homogeneity. Still, analyses showed different ancestries of tyrRS for the opisthokonts and all other eukaryotes (see file Additional_Trees.pdf in Supplementary Material online). This indicates that the two eukaryotic tyrRS lineages indeed arose independently.
The phylogeny of the archaeal tyrRSs is similar to the archaeal phylogeny based on rRNA sequences (Woese 1987) and concatenated gene sets (Brochier, Forterre, and Gribaldo 2005): the Haloarchaea group with the methanogens, most of the crenarchaeal sequences group together, and among the euryarchaeotes the thermococci and thermoplasmata group close to the Crenarchaea. A single molecule should not be expected to provide perfect phylogenetic resolution. In particular, the determination of the archaeal sister group to the eukaryotes usually is a difficult problem. Apart from the Haloarcula tyrRS, which is distinctly related to the opisthokontal homologs, there appear to be four other subtypes of tyrRS existing in archaea (fig. 2). The tyrRS sequences in methanogens and Halobacterium (MH) are more related to those of Haloarcula and opisthokonts (HO), whereas the other three archaeal subtypes, including crenarchaeotes (CA), thermoplasmata (TP), and Nanoarchaeum, euryarchaeotes, and crenarchaeotes (NEC), appear to be more related to tyrRSs from nonopisthokontal eukaryotes (EU), though their relationships are less certain due to weak support. Although it is possible that these multiple subtypes of archaeal tyrRS might represent paralogs arising from an ancient gene duplication, this scenario requires several independent losses among groups of archaea. It is more plausible that the erosion of phylogenetic signal over long evolutionary time, combined with other factors such as lateral gene transfer and periods of accelerated evolution (Cavalier-Smith 2002) gave rise to the observed distribution and weak grouping of these archaeal subtypes. Supporting evidence for ancient duplication and differential gene loss is weak: all sampled archaea in our analyses have single-copied tyrRS except for the crenarchaeote Pyrobaculum, where one copy groups as expected with the crenarchaeal homologs and the second copy groups with homologs from Nanoarchaeum and Pyrococcus. Given the frequency of lateral gene transfer in prokaryotes (Ochman, Lawrence, and Groisman 2000; Gogarten, Doolittle, and Lawrence 2002), the second copy in Pyrobaculum was likely acquired from another species, presumably a euryarchaeote.
It is probable that the independent origins of tyrRSs in opisthokonts and in the remaining eukaryotes (fig. 2) represent a case of ancient gene transfer from an archaeon to eukaryotes. Because nonopisthokontal eukaryotes are often considered paraphyletic and emerged relatively early in eukaryotic evolution, this ancient lateral gene transfer likely occurred from an archaeon, presumably similar to Haloarcula, to the opisthokont ancestor. Gene duplication followed by differential loss can always be invoked as an alternative to horizontal gene transfer detected based on phylogenetic signal (Gogarten and Townsend 2005). Applied to the presence of the haloarchaeal tyrRS type in opisthokonts, this scenario requires many independent complementary gene loss events. It therefore appears less parsimonious and less likely to account for the presence of the haloarchaeal type tyrRS in the opisthokonts than the assumption of an ancient transfer event. However, even if the distribution of the two eukaryotic tyrRS types were due to an ancient gene duplication followed by differential gene loss, the presence of a distinct tyrRS type nevertheless would support the opisthokonts as a monophyletic group.
The group opisthokonts was not recovered in some recently published analyses (e.g., domain contents and concatenation of genome sequences). These results may be due to intrinsic limitations of the data. For example, the analyses of genome sequences (Philip, Creevey, and McInerney 2005) were based on few taxa only and included genes with conflicting evolutionary histories. One of the five universally distributed genes in the selected genomes apparently supports the group opisthokonts whereas the other four do not. Concatenation of these sequences of different evolutionary histories could result in a misleading topology. If the opisthokontal tyrRS was indeed acquired from archaea, an argument against monophyly of the opisthokonts would entail multiple, independent transfers of the tyrRS gene to that group. Although gene transfer is a dynamic process constantly occurring over the course of organismal evolution, multiple, independent transfers of the same gene to related organismal groups resulting twice in successful orthologous replacement appear to be a very unlikely scenario. Therefore, the ancient gene transfer of tyrRS supports the monophyly of the opisthokonts.
The tyrRS gene might have been directly transferred from a haloarchaeon to the opisthokont ancestor, or other organisms might have been involved as intermediate carriers. The former would suggest a high-salt environment for the opisthokont ancestor. However, given the few surviving lineages from ancient times and the still poor sampling of archaeal tyrRS, a direct transfer seems unlikely. The finding that an anciently transferred tyrRS unites the opisthokonts also points to the potential utility of ancient gene transfer as a molecular marker for major organismal lineages. This might be particularly true for genes encoding essential functions because they are likely ubiquitous in life. In fact, efforts have recently been made to propose a close relationship between Giardia and Trichomonas based on a case of ancient gene transfer (Andersson, Sarchfield, and Roger 2005). In addition, gene transfer between divergent organisms also allows correlating evolutionary events in different parts of the tree of life (Gogarten, Murphey, and Olendzenski 1999; Cavalier-Smith 2002). The donor had to live at the same time or before the recipient. If our interpretation of the tyrRS phylogeny is correct, this implies that the Haloarchaea, usually considered a derived group of archaea (Woese 1987), already existed at the time of the opisthokont ancestor. Given the dynamic occurrences of lateral gene transfer during the long course of organismal evolution, we expect that ancient transfer events will provide more valuable witness to the evolutionary history of life.
Materials and Methods
Data Sources
Nucleotide and amino acid sequences for Cyanidioschyzon merolae were from Cyanidioschyzon merolae genome project database (http://merolae.biol.s.u-tokyo.ac.jp). The tyrRS protein sequence of Trichomonas vaginalis was annotated based on a nucleotide sequence obtained from the Institute of Genomic Research (http://www.tigr.org). All other sequences were obtained from the National Center for Biotechnology Information GenBank database. A file (genbank_ids.pdf) containing tyrRS accession and gene identification numbers is available in the Supplementary Material online.
Phylogenetic Analyses
Sequences were selected for major groups within each domain of life. Multiple protein sequence alignment was performed using MUSCLE (Edgar 2004) and ClustalX (Thompson et al. 1997), followed by cross-comparisons and manual refinement. Two data sets were used for the analyses, one with samples from all three major domains of life, whereas the other with bacterial sequences excluded. Only unambiguously aligned sequence portions were used for phylogenetic analyses, resulting in 188 and 205 aa positions per sequence for data sets with and without bacterial sequences, respectively. The alignments are available as Supplementary Material online (tyrRS_alignment.faa, tyrRS_analyzed_no_Bact.faa, tyrRS_analyzed.faa) or from the authors upon request. Phylogenetic analyses were performed with Bayesian inference method using MrBayes 3.1 (Ronquist and Huelsenbeck 2003), a maximum likelihood method using PHYML (Guindon and Gascuel 2003) and distance matrix analyses using the program Neighbor of PHYLIP version 3.6a (Felsenstein 2004) with maximum likelihood distances. Posterior probabilities were estimated using two independent analyses with 200,000 generations each for Bayesian analyses; bootstrap support was estimated using 1,000 replicates for both maximum likelihood and distance analyses. For Bayesian analyses, the Jones-Taylor-Thornton (JTT) substitution matrix, four gamma-distributed rate classes, the autocorrelated gamma model, and the covarion model were used. All other maximum likelihood calculations were based on the JTT substitution matrix and a mixed model of four gamma-distributed rate classes plus invariable sites. Maximum likelihood distances for bootstrap analysis were calculated using Tree-Puzzle (Schmidt et al. 2002) and PUZZLEBOOT v1.03 (by Michael E. Holder and Andrew J. Roger, available from http://www.tree-puzzle.de). Chi-square tests on amino acid sequence composition were performed using Tree-Puzzle with the default maximum likelihood model.
Supplementary Material
The following supplementary files are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
Additional_Trees.pdf: Phylogenetic analyses and maximum likelihood analyses of tyrRS.
FIG. 1S—Phylogenetic analyses of tyrRS protein sequences including bacterial sequences.
FIG. 2S—Maximum likelihood analyses for tyrRS from archaea and eukaryotes. Sequences that failed the chi-square test on sequence composition were removed.
genbank_ids.pdf: Table containing tyrRS accession numbers.
tyrRS_alignment.faa: Alignment of tyrRS sequences, including bacterial homologs in Fasta format. The file can be imported directly into ClustalX.
tyrRS_analyzed_no_Bact.faa: Aligned positions used for analyses reported in figure 1. The file can be imported directly into ClustalX.
tyrRS_analyzed.faa: Aligned positions used for analyses reported in figure 1S. The file can be imported directly into ClustalX.
Acknowledgements
We thank Greg Fournier for discussions and two anonymous reviewers for constructive comments and suggestions. This work was performed while J.H. held a National Research Council Associateship Award at the NASA Astrobiology Institute at the Marine Biological Laboratory in Woods Hole, Mass. (NCC2-1054). Y.X.'s work was supported in part by the U.S. Department of Energy's Genomes to Life Program under project "Carbon Sequestration in Synechococcus sp.: from Molecular Machines to Hierarchical Modeling" (http://www.genomes-to-life.org) and by the National Science Foundation (#NSF/DBI-0354771, #NSF/ITR-IIS-0407204, and #NSF/MCB-0237197).
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