Phylogenetic Analyses of Diplomonad Genes Reveal Frequent Lateral Gene Transfers Affecting Eukaryotes

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Provided by Elsevier - Publisher Connector Current Biology, Vol. 13, 94–104, January 21, 2003, 2003 Elsevier Science Ltd. All rights reserved. PII S0960-9822(03)00003-4 Phylogenetic Analyses of Diplomonad Genes Reveal Frequent Lateral Gene Transfers Affecting Eukaryotes

Jan O. Andersson,1,*A˚ sa M. Sjo¨ gren,1 tween the two prokaryotic domains [1]. The situation in Lesley A.M. Davis,1 T. Martin Embley,2 eukaryotes is less clear due to a lack of genome se- and Andrew J. Roger1 quence data and systematic analyses. However, the 1The Canadian Institute for Advanced Research number of convincing reports of LGT from prokaryotes Program in Evolutionary Biology to unicellular eukaryotes (protists) is steadily growing Department of Biochemistry and Molecular Biology [2–9], indicating that LGT may be an important evolution- Dalhousie University ary force in protists as well as in prokaryotes. This should Halifax, Nova Scotia B3H 4H7 not be surprising, since protists tend to live in environ- Canada ments that are rich in prokaryotes and often use them 2 Department of Zoology as food or harbor them as endosymbionts. Indeed, a The Natural History Museum gene transfer ratchet mechanism has been proposed Cromwell Road for protists by which ancestral eukaryotic genes may London SW7 5BD gradually be replaced with prokaryotic genes from food United Kingdom or endosymbionts over time [10]. We selected diplomonads for a study of the role of LGT in eukaryotic evolution. Diplomonads are a group of anaerobic and mostly parasitic protists that are very Summary interesting from an evolutionary point of view. They have been proposed to represent one of the earliest branching Background: Lateral gene transfer (LGT) is an important eukaryotes, which never had mitochondria [11]. Indeed, evolutionary mechanism among prokaryotes. The situa- the metabolism of the intestinal parasite Giardia lamblia, tion in eukaryotes is less clear; the human genome se- the most studied diplomonad species, has been de- quence failed to give strong support for any recent trans- scribed to be more similar to the metabolism of anaero- fers from prokaryotes to vertebrates, yet a number of bic prokaryotes than to the metabolism of mitochondrial LGTs from prokaryotes to protists (unicellular eukary- eukaryotes and was argued to be an ancestral “primi- otes) have been documented. Here, we perform a sys- tive” feature of these protists [12]. However, this attrac- tematic analysis to investigate the impact of LGT on the tive view of the evolutionary history of diplomonads has evolution of diplomonads, a group of anaerobic protists. been challenged during the last few years; genetic data Results: Phylogenetic analyses of 15 genes present in strongly suggest that diplomonads secondarily lost mi- the genome of the Atlantic Salmon parasite Spiro- tochondria [13–16], and phylogenetic reconstructions nucleus barkhanus and/or the intestinal parasite Giardia indicate that the similarities to anaerobic prokaryotes lamblia show that most of these genes originated via might be due to transfer of individual genes from anaero- LGT. Half of the genes are putatively involved in pro- bic prokaryotes to diplomonads [5, 8, 9], rather than cesses related to an anaerobic lifestyle, and this finding being an ancestral feature of the group and of eukary- suggests that a common ancestor, which most probably otes as a whole. Furthermore, the early branching phylo- was aerobic, of Spironucleus and Giardia adapted to an genetic position of diplomonads is controversial and anaerobic environment in part by acquiring genes via may well be an artifact resulting from inadequate phylo- LGT from prokaryotes. The sources of the transferred genetic methods [14, 17–19]. The aim of this study is to diplomonad genes are found among all three domains explore the role of LGT in the evolution of diplomonads of life, including other eukaryotes. Many of the phyloge- in particular and to act as a case study for microbial netic reconstructions show eukaryotes emerging in sev- eukaryotes in general. eral distinct regions of the tree, strongly suggesting that We are currently engaged in a sequence survey proj- LGT not only involved diplomonads, but also involved ect of the Atlantic Salmon parasite Spironucleus barkha- other eukaryotic groups. nus, and there is an ongoing whole-genome sequence Conclusions: Our study shows that LGT is a significant project on G. lamblia [20]. We used the sequence data evolutionary mechanism among diplomonads in par- from these two projects to identify and then fully charac- ticular and protists in general. These findings provide terize genes that may have originated via LGT. Many insights into the evolution of biochemical pathways in of the identified genes were acquired from anaerobic early eukaryote evolution and have important implica- prokaryotes, but we also found cases in which genes tions for studies of eukaryotic genome evolution and had been exchanged with other eukaryotes. Interest- organismal relationships. Furthermore, “fusion” hypoth- ingly, most of the phylogenetic trees indicated multiple eses for the origin of eukaryotes need to be rigorously LGT events involving many different eukaryote lineages, reexamined in the light of these results. suggesting that this mechanism has been, and still is, a significant force in the evolution of eukaryotic genomes.

Introduction Results

Comparative studies of prokaryotic genome sequences The Majority of the Putative LGTs Are Present have shown that LGT occurs frequently within and be- in Both Diplomonad Species Similarity searches followed by preliminary phylogenetic *Correspondence: [email protected] analyses identified 15 genes among the sequences re- Lateral Gene Transfers Affecting Eukaryotes 95

Table 1. Protein Functions and Number of Eukaryotic Clusters for the 15 Genes Number of Number of Gene Protein Functional Assignment Figure Euksa Clustersb proS Prolyl tRNA synthetase translation 1A 14 3 alaS Alanyl tRNA synthetase translation 1B 14 3 ilvE Branched chain amino acid transferase amino acid metabolism 2A 28 3 ilvA Threonine dehydratase amino acid metabolism 2B 20 4c pyrG CTP synthetase nucleotide metabolism S1 18 2c deoD Purine nucleoside phosphorylase nucleotide metabolism S2 8 2c deoC Deoxyribose-phosphate aldolase carbohydrate metabolism 3 9 2c nagB Glucosamine-6-phosphate isomerase aminosugar metabolism S3 11 2 accS Acetyl-CoA synthetase fermentation S4A 2 1 adhE Alcohol dehydrogenas E fermentation S4B 3 2 adh Class III alcohol dehydrogenase fermentation (?) 4 12 6 priS Hybrid-cluster protein anaerobic nitrate and/or nitrite respiration (?) 5A 3 1c fprA A-type flavoprotein oxidative stress response (?) 5B 3 2 hmpA Flavohemoglobin nitric oxide detoxification 5C 6 5c pfl Conserved hypothetical protein unknown 5D 1 1 a The number of eukaryotic sequences in the phylogenetic reconstruction. b The number of eukaryotic clusters separated either by a bootstrap support value Ͼ90% or statistically separated (p Ͻ 0.05) in pairwise AU tests [28]. See the Experimental Procedures for details. c AU tests were performed.

leased from the genome project of the diplomonad G. impossible to infer when these transfers happened; the lamblia [20] that potentially originated via gene transfer genes could have been lost in the Spironucleus lineage, from prokaryotic sources (Table 1). These genes were or the genes might be present in the S. barkhanus ge- amplified from genomic DNA with exact match primers, nome but not conserved enough to be amplified with and the PCR products were fully sequenced to confirm degenerate PCR. their sequences. The proteins are functionally very diverse, ranging from proteins involved in translation and Aminoacyl-tRNA Synthetases metabolism of small molecules to proteins involved in Two of the aminoacyl-tRNA synthetases, the dual-speci- functions useful for an anaerobic lifestyle (Table 1). We ficity prolyl-cysteinyl- and the alanyl-tRNA synthetase, identified 12 of these genes in another diplomonad, S. of G. lamblia have been shown to have a close relation- barkhanus, by scanning our ongoing in-house genome ship to archaeal homologs [22, 23]. We only included sequencing projects and performing degenerate PCR. one of the two distantly related versions of the enzyme All of the S. barkhanus genes showed high sequence in our analysis of the prolyl-tRNA synthetase, since it is similarity to the Giardia genes. Homologous sequences only possible to align a small fraction of the sites in the for the 15 genes were retrieved from the public data- molecule between the two versions. The phylogenetic bases and ongoing genome projects, which produced analysis shows three strongly supported groups: a eu- data sets ranging from 20 to 74 taxa suitable for phyloge- bacterial group with one plant sequence, an exclusively netic analysis, with 179–827 unambiguously aligned eukaryotic group, and an archaeal group with two diplo- amino acid positions (Table S1). Protein maximum likeli- monad sequences (Figure 1A). The presence of these hood phylogenies were inferred by using a mixed four- three groups indicates that this version of the enzyme category discrete ⌫-model of among-site rate variation was present in the last common ancestor of the three plus invariable sites (Figures 1–5 and S1–S4). Analyses domains. The Arabidopsis thaliana homolog that is using a model with uniform rates among sites yielded found within the eubacterial group possibly reflects a trees with quite similar topologies (data not shown), transfer event from a eubacterium; an endosymbiotic indicating that our findings are not due to an overcom- gene transfer from the plastid seems slightly less likely pensation for multiple substitutions by the ⌫ among-site since the known cyanobacteria sequences belong to the rate variation model. The two diplomonads formed a large group of distantly related prolyl-tRNA synthetases cluster in all phylogenetic reconstructions, except for excluded from our analysis [22]. pyrG, indicating that at least 11 of the 15 genes were The position of the two diplomonad sequences as an present in the last common ancestor of Giardia and immediate outgroup to the archaeal cluster indicates Spironucleus (see below and Figures 1–4, 5A, 5B, S3, that the dual-specificity aminoacyl-tRNA synthetase, and S4). These genes most likely were ancestral to diplo- which is only found in diplomonads and Archaea [22], monads, since S. barkhanus and G. lamblia represent most likely was introduced into the diplomonad lineage the two most distantly related clades of diplomonads from an archaeon (Figure 1A). An alternative intrepreta- [21]. Analyses of the codon usage and amino acid com- tion of the phylogenetic tree, which also involves an positions of the pyrG genes and the three genes in which interdomain LGT event, would be that the root of the only the Giardia homolog is known (see below and Fig- tree lies between diplomonads and archaea and that the ures 5C, 5D, S1, and S2) failed to indicate any significant gene was transferred from a eukaryote to a eubacterium. deviation from the other genes that would be indicative This would place the diplomonads at the root of the of very recent introductions of these genes into diplomo- eukaryotic domain (Figure 1A). However, we favor the nad genomes (data not shown). Therefore, it is currently archaea-to-diplomonads gene transfer scenario for two Current Biology 96

Figure 1. Maximum Likelihood Tree of Two Aminoacyl-tRNA Synthetase Protein Sequences (A and B) Maximum likelihood tree of (A) prolyl-tRNA synthetase and (B) alanyl-tRNA synthetase protein sequences. Only unambiguously aligned amino acid positions were used in the phylogenetic reconstructions, and the presented trees are based only on taxa that passed the amino acid composition test. Protein maximum likelihood phylogenies were inferred by using a mixed four-category discrete ⌫-model of among-site rate variation plus invariable sites. Protein maximum likelihood distance bootstrap values Ͼ50% for bipartitions are shown. The approximate position of the diplomonad sequences that failed the amino acid composition test is based on a separate analysis and is indicated with dotted branches. The bootstrap support values from this analysis are shown in parentheses. See the Experimental Procedures for further details. Eukaryotes are labeled red, Archaea are labeled blue, and Eubacteria are labeled black. The trees are arbitrary rooted. Only the genus names are shown. The numbers after the genus names refer to different homologs found in the same species. reasons; it is consistent with a single origin of the dual chaeotes, indicative of a transfer from a euryarchaeote specificity of the prolyl-cysteinyl-tRNA synthetase en- to an ancestral diplomonad. zyme with no subsequent losses, and it does not require The alanyl-tRNA synthetase phylogeny shows one that diplomonads are deeply branching eukaryotes, archaeal/diplomonad group, one eukaryotic group, and which indeed is questionable [14, 17–19]. Unfortunately, polyphyletic Eubacteria (Figure 1B). Unfortunately, a the source of the transfer is not easily identified since large number of sequences, including both diplomonad the diplomonads form an immediate outgroup to the sequences, were excluded from the initial data set due archaeal cluster. However, this could be an artifact of to significantly deviant amino acid compositions (Table phylogenetic reconstruction; the diplomonads have the S1). To get an indication of their phylogenetic affinities, longest branches in the archaeal/diplomonad part of the diplomonad sequences were included in a separate the tree and could be artifactually attracted to the long analysis (Figure 1B). Four eukaryotic sequences are internal branch leading to the archaeal/diplomonad found outside the major eukaryotic group; one of the cluster (Figure 1A) [24]. As a result, the archaeal/diplo- two A. thaliana sequences groups with the two cyano- monad subtree would then be incorrectly rooted, the bacteria, likely representing an endosymbiotic gene true root instead lying on the branch leading to the transfer from the plastid to the nucleus, and the two crenarchaeotes in the tree. This position of the root diplomonad and the E. histolytica sequences are found would then place the diplomonads within the euryar- as an immediate outgroup to the Archaea (Figure 1B). Lateral Gene Transfers Affecting Eukaryotes 97

The diplomonad alanyl-tRNA synthetase may represent quences [27]. Hence, additional events of gene transfer the ancestral eukaryotic version, while the other eukary- between eukaryotes probably occurred after the first otes have a eubacterial version acquired after the split prokaryote-to-eukaryote LGT event. between diplomonads and the rest of the eukaryotes, In the phylogenetic reconstruction of threonine dehy- as previously suggested [23]. However, that requires dratase, another enzyme involved in amino acid metabo- that the diplomonads are a deeply branching eukaryote lism, the eukaryotes are found in six clusters, four of group, which is a questionable assumption [14, 17–19]. which are separated with a bootstrap support of 66% In addition, both diplomonad sequences and the E. his- or higher and statistically separated in pairwise “approx- tolytica sequence failed the amino acid composition test imately unbiased” tests (AU tests) [28] (Figure 2B and and are long branches, which makes the precise place- Table 1). A eukaryotic cluster, consisting of two meta- ment of these three eukaryotes in the tree unreliable. zoan, two fungal, and one plant sequence, is nested Indeed, the maximum likelihood distance analysis used within a cluster of ␣-proteobacteria, and this arrange- to obtain statistical support values for the bipartitions ment is indicative of a transfer from the endosymbiont placed the group within the euryarchaeotes with moder- that gave rise to mitochondria. Interestingly, Drosophila ate (72%) bootstrap support (data not shown). Thus, it melanogaster and two of the three C. elegans ilvA homo- seems plausible that the diplomonads acquired their logs form a sister group to a Caulobacter crescentus alanyl-tRNA synthetase via gene transfer from an sequence and are clearly separated from the metazoan/ archaeon. fungal/plant cluster in pairwise AU tests (p Ͻ 0.005), Given current accounts of eukaryote phylogeny, the indicative of an LGT from ␣-proteobacteria to metazoa grouping of the E. histolytica sequence within diplomo- (Figure 2B). Surprisingly, the two diplomonad se- nads in the alanyl-tRNA synthetase tree is surprising; quences form a strongly supported group with the diplomonads and Entamoeba should not be related to Dictyostelium discoideum sequence to the exclusion of the exclusion of the other eukaryotes (Figure 1B). Rather, Entamoeba (Figure 2B). Dictyostelium and Entamoeba the Entamoeba sequence is expected to be related to are more closely related to each other than to diplomo- the Dictyostelium sequence [16, 25, 26]. Although the nads [25, 26], suggesting that the Dictyostelium/diplo- specific relationship between the E. histolytica and the monad group originated via a single gene transfer from S. barkhanus sequences should be viewed cautiously— a prokaryote, followed by a eukaryote-to-eukaryote LGT both represent long branches and have deviant amino event between the two lineages. Finally, two eukaryotic acid composition—the specific relationship between the clusters, one fungal and one plant, that are not clearly two diplomonad sequences and E. histolytica appear separated are found as outgroups to a diverse eubacter- solid and are suggestive of a eukaryote-to-eukaryote ial cluster, and this is indicative of at least one interdo- LGT between the two lineages. main transfer event involving eukaryotes.

Branched Chain Amino Acid Transferase Deoxyribose-Phosphate Aldolase and Threonine Dehydratase Deoxyribose-phosphate aldolase catalyzes the revers- Only small parts of the phylogenetic tree of branched ible aldol reaction of acetaldehyde and D-glyceralde- chain amino acid transferase, the last enzyme in the hyde-3-phosphate to form 2-deoxyribose-5-phosphate, metabolic pathways leading to isoleucine, valine, and a reaction in the pentose phosphate pathway. Again, leucine, show relationships that are consistent with ex- the eukaryotes are divided into multiple groups in phylo- pected organismal phylogenies, indicating numerous genetic trees (Figure 3 and Table 1). The metazoan se- transfers of the gene between lineages (Figure 2A). The quences are found in a paraphyletic cluster with ␣- and largest eukaryotic group, which includes fungal, myce- ␤-proteobacterial sequences nested within, indicative tozoan, metazoan, and euglenozoan sequences, is a of a eukaryote-to-prokaryote LGT event. However, the sister group to the Neisseria meningitidis sequence with identification of the donor lineage is not straightforward, moderate bootstrap support (75%), indicating that this due to the uncertainty of the phylogenetic relationship group most probably originated via LGT from a eubac- between Drosophila, Caenorhabditis, and humans. The terial lineage, possibly from a ␤-proteobacterium. All metazoa/proteobacteria group forms a strongly sup- plant sequences form a cluster with the single green ported sister group to Streptomyces coelicolor. The for- algal sequence as an immediate outgroup, and this find- mation of this cluster indicates that the metazoa/proteo- ing is in agreement with a single origin that is probably bacteria group originated via a transfer from an ancestor the result of gene transfer from a eubacterium, followed of S. coelicolor to the ancestor of the metazoan lineages by vertical inheritance (Figure 2A). The third eukaryotic (Figure 3). As in the cases of alaS and ilvE (Figures 1B cluster includes the two diplomonad sequences to- and 2A), the diplomonad sequences are found in an gether with a red algal sequence and two fungal se- unexpected eukaryotic grouping in the deoC tree; the quences. The position of this group within the eubacter- two sequences form a cluster with a fungal sequence ial group suggests an origin by LGT, although the source and a Trypanosoma sequence (Figure 3). The group is unclear. Strikingly, the five eukaryotic sequences in is nested within eubacteria, which is suggestive of a the group represent three distantly related eukaryotic prokaryote-to-eukaryote LGT event, followed by trans- groups; the fungal sequences are expected to branch fers within the eukaryotes. However, since it fails to with the other fungal sequences, and the red algal se- group specifically with any prokaryotic lineage, we can- quences should be more closely related to the plant not exclude the possibility that the group represents the sequences than to the diplomonad and fungal se- ancestral eukaryotic version. Current Biology 98

Figure 2. Maximum Likelihood Tree of Two Amino Acid Metabolism Protein Sequences (A and B) Maximum likelihood tree of (A) branch chain amino acid transferase and (B) threonine dehydratase protein sequences. Methods and labeling are as in Figure 1.

Class III Alcohol Dehydrogenase crude lysate of Giardia trophozoites [29], indicating that It has been shown that E. histolytica and G. lamblia the enzymes might have a different function or that the have acquired several of their fermentation enzymes via genes are expressed in different stages of the life cycle independent transfer events from anaerobic prokary- in Giardia. Our phylogenetic analysis of the class III otes [5, 8]. We present updated phylogenetic analyses alcohol dehydrogenase shows sequences from eubac- including homologs from S. barkhanus for three of these terial, archaeal, and eukaryotes scrambled with respect enzymes, acetyl-CoA synthetase, alcohol dehydroge- to organismal phylogeny, and this suggests that the nase E, and class III alcohol dehydrogenase (Figures 4 gene has been transferred very frequently. Amazingly, and S4). the 12 eukaryotic sequences are found in six clusters We found two previously undescribed genes coding that all group with different prokaryotic groups with for class III alcohol dehydrogenases in the G. lamblia strong support (Figure 4). One of the Giardia homologs genome as well as a homolog from S. barkhanus, and forms a strongly supported group with a Fibrobacter this finding suggests that these enzymes are functionally sequence, while the other three diplomonad sequences important for these organisms. However, no class III form an immediate outgroup to a group of two low G ϩ C alcohol dehydrogenase activity could be detected in the Gram-positive bacteria and the archaeon Thermococ- Lateral Gene Transfers Affecting Eukaryotes 99

Figure 3. Maximum Likelihood Tree of Deoxyribose-Phosphate Al- dolase Protein Sequences Methods and labeling are as in Figure 1. cus hydrothermalis (Figure 4). Only one eukaryotic cluster shows an affinity indicative of an endosymbiotic gene transfer. However, the strongly supported grouping of the fungal and Rhodospirillum sequences is more likely due to a prokaryote-to-eukaryote gene transfer; ␣-proteobacterial sequences are found in six other positions in the tree, and this version of the adh gene is only Figure 4. Maximum Likelihood Tree of Class III Alcohol Dehydroge- found in a limited diversity of eukaryotes. Therefore, all nase Protein Sequences eukaryotic clusters most likely originated via LGT from Methods and labeling are as in Figure 1. prokaryotes (Figure 4). found in obligate and facultative anaerobic prokaryotes Hybrid-Cluster Protein, Flavoprotein, Flavohemoglobin, [30]. Here, we show that the gene for the enzyme is also and a Conserved Hypothetical Protein present in the genomes of the anaerobic protists G. The hybrid-cluster protein (“prismane protein”), which lamblia, S. barkhanus, and E. histolytica. There is little has been suggested to have a functional role in anaero- agreement with expected organismal phylogeny in the bic nitrate and/or nitrite respiration, was previously only phylogenetic analysis of the hybrid-cluster protein; the Current Biology 100

Figure 5. Maximum Likelihood Tree of Four Protein Sequences with Diverse Functions (A–D) Maximum likelihood tree of (A) hybrid-cluster protein, (B) A-type flavoprotein, (C) flavohemoglobin, and a (D) conserved hypothetical protein protein sequences. Methods and labeling are as in Figure 1. four archaea are found in three well-supported groups, previously only been found in Archaea and Eubacteria and the three eukaryotes are split into two clusters, and have been suggested to have distinct functions due although the separation is not strongly supported (Fig- to different interactions with specific redox partners [31]. ure 5A and Table 1). Even though the eukaryotic se- At any rate, a role in oxidative stress response has been quences fail to group with specific prokaryotic se- suggested for the homolog from Moorella thermoace- quences with strong statistical support, an origin via tica [32]. The phylogenetic tree suggests multiple inter- vertical descent from the last common eukaryotic an- domain gene transfers between Archaea and Eubac- cestor is unlikely, since that implies several independent teria, as well as multiple transfers within Eubacteria losses of the gene among eukaryotes. Thus, the eukary- (Figure 5B). The two diplomonad sequences form a otic sequences were likely transferred from prokaryotes, strongly supported cluster, which is an outgroup to a possibly in two separate events. diverse cluster of prokaryotes, while the Entamoeba Flavoproteins bind one or both of the flavin cofactors sequence forms an outgroup to a cluster of mostly low FMN and FAD, which enable them to catalyze a variety G ϩ C Gram-positive bacteria. Neither of these se- of electron transfer reactions. A-type flavoproteins have quences is likely to represent the ancestral eukaryotic Lateral Gene Transfers Affecting Eukaryotes 101

enzyme, since that would imply a rooting of the prokary- eukaryotes, and the eukaryotic sequences are nested otic tree in improbable positions as well as multiple within prokaryotic sequences. This should not be sur- independent gene losses in the eukaryotic lineages. prising since the mitochondrion, which is likely ancestral Thus, the eukaryotic sequences were probably intro- to all studied eukaryotes [34, 35], shows a specific rela- duced from prokaryotes into the Entamoeba and diplo- tionship with a subgroup of the ␣-proteobacteria [36]; monad lineages after each of these diverged from the this relationship indicates that the prokaryotic diversity rest of the eukaryotes (Figure 5B). was large at the time of the last common eukaryotic It was only recently established that the flavohemo- ancestor. Anaerobic metabolism, for example, was most globins, a class of hemoglobins, function in response likely already present in diverse lineages of prokaryotes to nitric oxide and nitrosative stress under both aerobic when the lineage leading to diplomonads adapted to an and anaerobic conditions [33]. Strikingly, the six eukary- anaerobic lifestyle (see the Discussion below). There- otic sequences are divided into five groups in the phylo- fore, even in the absence of phylogenetic analyses, it genetic tree, all of which are separated by strong boot- is plausible to assume that genes were transferred in strap support or pairwise AU tests (Figure 5C and Table the direction to eukaryotes. Our phylogenetic analyses 1). Without a doubt, flavohemoglobins have been trans- of anaerobic proteins do not contradict this interpreta- ferred between prokaryotes and eukaryotes multiple tion (Figures 4, 5, and S4), and most analyses of the times and have possibly been transferred three times other proteins indicate the same direction of transfer; between prokaryotes and different lineages of fungi. The the diplomonads branch outside the main eukaryotic G. lamblia sequence is nested within ␥-proteobacteria, groups and likely acquired the genes from prokaryotes strongly suggesting a transfer event from a ␥-proteo- after they diverged from the other eukaryotes (Figures bacterium to an ancestor of G. lamblia. 1, 2A, S1, and S2). However, the interpretation of the Our search identified a Giardia gene with high similar- direction should be taken cautiously since the taxo- ity to a set of mostly archaeal genes, some of which nomic sampling represents prokaryotic diversity much are annotated as coding for putative pyruvate formate- better than eukaryotic diversity in most trees. Transfers lyase-activating enzymes. However, these genes only from eukaryotes to prokaryotes are certainly possible; show moderate sequence similarity to the functionally the deoxyribose-phosphate aldolase gene was most categorized enzymes, suggesting that the functional an- likely transferred to a proteobacterium from a metazoan notation should be treated as very preliminary. At any (Figure 3). rate, LGT seems to have distributed the enzyme across all domains of life, and G. lamblia, the only eukaryote, Frequent Transfers of Diplomonad Genes is nested within mostly anaerobic prokaryotes, suggesting Although the direction of the transfer and the source a gene transfer to the Giardia lineage (Figure 5D). cannot be determined in every single case, we have shown that more than a dozen diplomonad genes have been involved in LGT (Figures 1–5, S1, S2, and S4). In Discussion addition, several genes have previously been shown to have prokaryotic origin via LGT in diplomonads; the first The Source and Direction of the Transfers two enzymes and the fourth enzyme in the glycolytic Are Difficult to Identify pathway, glucokinase, glucosephosphate isomerase, To convincingly identify the source of a gene transfer and fructose-1, 6-bisphosphate aldolase, respectively, with phylogenetic methods, it is best if the recipient have origins distinct from other eukaryotes in diplomo- lineage is nested within a cluster of sequences that nads [4, 6, 37], the phosphoenolpyruvate carboxykinase otherwise matches the expected organismal phylogeny. gene has an archaeal origin [3], and the gene encoding If this pattern is obtained, a gene transfer most likely 3-hydroxy-3-methylglutaryl coenzyme A reductase has occurred from an ancestor of an organism within the a eubacterial origin [2]. Thus, both inter- and intradomain identified organismal group to an ancestor of the recipi- LGTs are significant evolutionary mechanisms in diplo- ent lineage. Unfortunately, we were unable to identify monads. However, the relative importance of these pro- the sources of most of the diplomonad genes by using cesses in diplomonad genome evolution compared to this rationale, partly due to the lack of statistical support other mechanisms is not yet well understood. We se- for the position of the diplomonad sequences within the lected for interdomain LGTs in our initial search, but we phylogenetic tree. However, a more serious problem for certainly did not detect all transfers (see the Experimen- the detection of the source is that most of the phyloge- tal Procedures for details). netic trees indicate extensive transfer within and between the three domains of life. Even if the diplomonad gene(s) forms a strongly supported group with a subset Independent Adaptation to the Anaerobic Lifestyle of prokaryotes, the source of the transfer is difficult, if in the Diplomonad and the Entamoeba Lineages not impossible, to identify if the prokaryotic group fails The presence of genes in the genome of diplomonads to match a subset of the expected prokaryotic phylog- with phylogenetic affinity to genes with mitochondrial eny. At any rate, our analyses indicate that the diplomo- origins in other eukaryotes [13, 15, 16], as well as the nads have acquired genes from a variety of sources, presence of an organelle with a probable mitochondrial and there are no indications that all, or even most, of origin in Carpediemonas, a closely related protist [14], these genes were acquired from the same single source. indicates that diplomonads are secondarily amitochon- Most of the interdomain LGTs in our analysis appear drial. This suggests that the lineage leading to diplomo- to have occurred in the direction from prokaryotes to nads likely made the transition from an aerobic to an Current Biology 102

anaerobic lifestyle. From that perspective, it is interest- are unlikely the result of frequent gene transfers from ing that many of the genes we have examined seem to mitochondria and chloroplasts. Only rarely do the eukary- have functions related to an anaerobic lifestyle (Table otic groups show any specific affinity to ␣-proteobac- 1). Acetyl-CoA synthetase and alcohol dehydrogenase teria or cyanobacteria, the ancestors of mitochondria and E are fermentation enzymes in Giardia [29, 38], and the cyanobacteria, respectively. Furthermore, endosymbiotic class III alcohol dehydrogenase might also be involved gene transfer could only explain two clades of eukary- in anaerobic metabolism [29]. In addition, the hybrid- otes arising within eubacteria, one with a mitochondrial cluster protein is expressed under anaerobic conditions origin and one with a chloroplast origin, in addition to [30], and the flavoprotein has been suggested to be an ancestral eukaryotic “nucleocytoplasmic” lineage. involved in oxidative stress response [32]. Both of these Another plausible explanation for the polyphyly of eu- enzymes, the flavohemoglobin and the hypothetical prokaryotes is that their last common ancestor possessed tein, are mostly found in anaerobic organisms, indicating multiple copies of the gene that was differentially lost that they likely encode functions for an anaerobic life- in the various lineages. However, in the absence of LGT, style in anaerobic protists (Figure 5). Most of these puta- such an “ancient paralogy” scenario is expected to re- tively anaerobic genes are present in both diplomonads, sult in “mirror” organismal phylogenetic trees for the whose sequences group together in phylogenetic analy- different copies, which we did not observe. Instead, ses, suggesting that the common ancestor of S. barkha- the eukaryotic sequences often show affinity to specific nus and G. lamblia had already adapted to an anaerobic prokaryote sequences. In such cases, “ancient paral- lifestyle. ogy” scenarios imply that multiple copies of present- In agreement with earlier studies of fermentation en- day single-copy genes have been retained in genomes zymes [5, 8], adaptations to anaerobiosis seem to have over long evolutionary times and have recently indepen- occurred independently in the lineages leading to diplo- dently been lost in multiple lineages. Therefore, “ancient monads and Entamoeba (Figures 4, 5, and S4), another paralogy” scenarios are unlikely to explain the unex- secondarily amitochondrial anaerobic protist [39]. Dur- pected topologies in our analyses. ing the transition process from an aerobic to an anaero- If a gene is transferred frequently amongst prokary- bic lifestyle, the two lineages appear to have acquired otes and rarely between prokaryotes and eukaryotes, the same genes via LGT, but from different prokaryotic the prokaryotic sequences will remain more similar to sources. These observations have implications for each other and will tend to group together in phyloge- hypotheses about the origin of anaerobic eukaryotes. netic trees. This is best explained by the following exam- Firstly, our data do not support the hypothesis that the ple. Imagine two ancient interdomain gene transfer anaerobic metabolism is an ancestral “primitive” feature events from closely related proteobacteria to eukary- in diplomonads [12]. Similarly, our data on fermentation otes, followed by prokaryote-to-prokaryote transfers in enzymes are inconsistent with the “hydrogen hypothe- which the genes in the proteobacterial donor lineages sis” for eukaryogenesis in its original form, which pre- are replaced by homologs from more distantly related dicted the enzymes for both aerobic and anaerobic lineages. In such a case, the proteobacterial “donor eukaryotic energy metabolism to be of a single eubac- lineages” will be nested within a “prokaryotic” cluster in terial source, the ␣-proteobacterial progenitor of mito- the phylogenetic reconstruction, and the two eukaryotic chondria and hydrogenosomes [40]. However, our data sequences will group together, despite the fact that two are inconclusive regarding a later version of the hypothe- ancient interdomain transfers have occurred. Thus, fre- sis, which only predicts that proteins involved in energy quent LGT amongst the prokaryotes, as in most of our metabolism in hydrogenosomes should share a single cases, does not invalidate the interpretation of transfers origin from the mitochondrial endosymbiont, while lat- between prokaryotes and eukaryotes in any way. eral gene transfer of other genes may have facilitated Rather, it is likely to bias the interpretations of gene colonization of anaerobic environment by present-day transfer events toward false negatives. eukaryotes [41]. Eukaryote-to-Eukaryote LGTs Several of our phylogenetic analyses showed unex- Eukaryotes Emerging in Several Distinct Regions pected groupings of eukaryotes with strong support of the Phylogenetic Tree Indicate Frequent LGT from bootstrapping that conflicted with known organis- Interestingly, eukaryote homologs are found in multiple mal phylogeny of eukaryotes (Figures 1B and 2). In the clusters in many of the phylogenetic trees of the gene absence of any obvious phylogenetic artifacts, a plausi- families that we have examined (Table 1), and this finding ble explanation for these groupings is that the genes is indicative of frequent transfer events between pro- have been exchanged between the eukaryotic lineages. karyotes and eukaryotes in addition to the events we It was recently argued that a part of the enolase gene have discussed from prokaryotes to diplomonads. The may have been transferred between two distantly re- approach we used explicitly selected for proteins that lated eukaryotic lineages [42]. However, to our knowl- were transferred from prokaryotes to diplomonads, but edge, these are the first well-documented cases of it did not select for proteins for which multiple transfer transfer of entire protein coding genes between eukary- events affecting eukaryotes had occurred. Therefore, it otes independent of secondary endosymbiotic events is remarkable that the Entamoeba genes seem to have [43]. If it turns out that transfers between eukaryotes are originated via LGT in eight of the nine cases (Figures common, shared prokaryote-to-eukaryote LGT events 2B, 3, 4, 5A, 5B, S3, and S4B) in which a homolog could should be treated cautiously as indicators of organismal be found. The polyphyly of the eukaryotes in many trees phylogeny among eukaryotes. Lateral Gene Transfers Affecting Eukaryotes 103

Implications for Eukaryote Origins NS, Canada), and John M. Logsdon, Jr. (Emory University, Atlanta, Our study shows that LGT is an important mechanism GA, USA). We thank Patricia L. Dyal (Natural History Museum, Lon- of genome evolution in microbial eukaryotes, and that don, UK) for technical support and sequencing and Alastair G.B. Simpson (Dalhousie University, Halifax, NS, Canada) for sequencing the latter have acquired genes from a variety of prokary- of the alaS EST clone. We utilized preliminary data generated by otic lineages, as well as from other eukaryotes. Thus, the Josephine Bay Paul Center for Comparative Molecular Biology the failure of the human genome sequence to give strong and Evolution, the Institute for Genomic Research, the Utah Genome support for any recent transfers from prokaryotes to Center, the DOE Joint Genome Institute, the Wellcome Trust Sanger vertebrates [44] is more likely due to the multicellularity Institute, the University of Oklahoma’s Advanced Center for Genome of vertebrates and their sequestered germline rather Technology, and the Genome Sequencing Centre Jena. J.O.A. was supported by a Post Doctoral Fellowship from the Wenner-Gren than to their eukaryotic nature per se [45]. The occur- Foundations. A.J.R. is supported by the CIAR Program in Evolution- rence of LGT among eukaryotes implies that the origin ary Biology. This work was supported by Natural Sciences and of a specific eukaryotic gene family will not necessarily Engineering Research Council (NSERC) Genomics Project Grant be the same for different eukaryotic lineages and there- 228263-99, awarded to A.J.R. fore cannot be determined by investigating the origin of the genes in one genome alone. Likewise, all genes in Received: August 2, 2002 any given eukaryotic genome cannot easily be divided Revised: September 16, 2002 into a few large groups that have different origins. Accepted: October 9, 2002 Published: January 21, 2003 These findings have important implications for “fusion” or “chimaeric” theories for the origins of eukaryotes. A common theme of such theories is a fusion event References between two cellular lineages that, in addition to the 1. Doolittle, W.F. (1999). Phylogenetic classification and the uni- mitochondrial and chloroplast endosymbioses, contrib- versal tree. 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