Gene Transfers from Nanoarchaeota to an Ancestor of Diplomonads
and Parabasalids
Jan O. Andersson,* Stewart W. Sarchfield,* and Andrew J. Roger*
*The Canadian Institute for Advanced Research, Program in Evolutionary Biology, Department of Biochemistry and
Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada; and Institute of Cell and Molecular Biology,
Uppsala University, Biomedical Center, Uppsala, Sweden
Rare evolutionary events, such as lateral gene transfers and gene fusions, may be useful to pinpoint, and correlate the
timing of, key branches across the tree of life. For example, the shared possession of a transferred gene indicates
a phylogenetic relationship among organismal lineages by virtue of their shared common ancestral recipient. Here, we
present phylogenetic analyses of prolyl-tRNA and alanyl-tRNA synthetase genes that indicate lateral gene transfer events
to an ancestor of the diplomonads and parabasalids from lineages more closely related to the newly discovered archaeal
hyperthermophile Nanoarchaeum equitans (Nanoarchaeota) than to Crenarchaeota or Euryarchaeota. The support for this
scenario is strong from all applied phylogenetic methods for the alanyl-tRNA sequences, whereas the phylogenetic
analyses of the prolyl-tRNA sequences show some disagreements between methods, indicating that the donor lineage
cannot be identified with a high degree of certainty. However, in both trees, the diplomonads and parabasalids branch
together within the Archaea, strongly suggesting that these two groups of unicellular eukaryotes, often regarded as the
two earliest independent offshoots of the eukaryotic lineage, share a common ancestor to the exclusion of the eukaryotic
root. Unfortunately, the phylogenetic analyses of these two aminoacyl-tRNA synthetase genes are inconclusive regarding
the position of the diplomonad/parabasalid group within the eukaryotes. Our results also show that the lineage leading to
Nanoarchaeota branched off from Euryarchaeota and Crenarchaeota before the divergence of diplomonads and
parabasalids, that this unexplored archaeal diversity, currently only represented by the hyperthermophilic organism
Nanoarchaeum equitans, may include members living in close proximity to mesophilic eukaryotes, and that the presence
of split genes in the Nanoarchaeum genome is a derived feature.
Introduction
Nanoarchaeum equitans is an enigmatic symbiont
that attaches to the surface of the crenarchaeon Ignicoccus
in submarine hot vents and represents the only species of
the newly discovered phylum Nanoarchaeota to be cultivated to date (Huber et al. 2002; Waters et al. 2003).
However, a large unexplored archaeal diversity may exist;
phylogenetic analyses show N. equitans as an early branching archaeon (Waters et al. 2003), and nanoarchaeotalike ribosomal RNA samples have been amplified from hot
environments around the world (Hohn, Hedlund, and
Huber 2002). It is unknown whether this potential diversity also includes representatives in cooler environments or
whether hyperthermophily is an ancient and exclusive trait
of this phylum.
Like the Archaea, an improved understanding of the
deep phylogeny of Eukaryota has been also achieved by the
identification of novel unicellular lineages in combination
with improved phylogenetic methods. It now seems clear
that the eukaryotic diversity falls into at least half a dozen
eukaryotic ‘‘supergroups’’ (superkingdoms) (CavalierSmith 2002; Simpson and Roger 2002; Baldauf 2003).
One of these, Excavata (Cavalier-Smith 2003; Simpson
2003), contains two important human pathogens: Giardia
lamblia, a diplomonad commonly causing diarrhea in
humans, and Trichomonas vaginalis, a parabasalid that
causes trichomoniasis. For the past 2 decades, it has been
widely thought that these organisms were the most
‘‘primitive’’ eukaryotes, branching as the first two lineages
of eukaryotes in rDNA and other molecular phylogenies
(Sogin 1991). However, the deep-branching emergence of
diplomonads and parabasalids in phylogenetic trees of
eukaryotes has recently been called into question; this
phylogenetic position may result from artifacts of the
methods employed, rather than reflect their true evolutionary positions (Embley and Hirt 1998; Philippe et al. 2000;
Simpson et al. 2002; Cavalier-Smith 2003; Simpson 2003).
Here, we address the phylogenetic relationships of diplomonads and parabasalids by using lateral-gene-transfer
events as discrete evolutionary markers, by extending the
eukaryotic taxonomic sampling of the prolyl-tRNA and
alanyl-tRNA synthetase genes (proS and alaS), which
were previously shown to have been transferred from the
Archaea to diplomonads (Andersson et al. 2003). Our
phylogenetic analyses of the updated data sets indicate that
diplomonads and parabasalids shared a most recent
common anaerobic protist ancestor that was the recipient
of transfers of homologs of both of these genes from an
archaeon of the Nanoarchaeota lineage. These data
indicate that diplomonads and parabasalids shared a common ancestor exclusive of the eukaryotic root, as well as
provide insights into the evolution and molecular biology
of the archaeal phylum Nanoarchaeota. Thus, gene-transfer
events may provide a range of biological information unrelated to the encoded functions of the transferred genes.
Material and Methods
PCR Amplifications
Key words: Nanoarchaeum equitans, Trichomonas vaginalis,
Giardia lamblia, lateral gene transfer, phylogeny.
E-mail: [email protected].
Mol. Biol. Evol. 22(1):85–90. 2005
doi:10.1093/molbev/msh254
Advance Access publication September 8, 2004
Using degenerate primers against conserved parts of
the proS and alaS amino acid alignments, with genomic
DNA in PCR reactions, we amplified proS and alaS
gene sequences from diverse eukaryotes. The proS gene
was amplified with PCR from Entamoeba invadens,
Molecular Biology and Evolution vol. 22 no. 1 Ó Society for Molecular Biology and Evolution 2005; all rights reserved.
86 Andersson et al.
E. moshkovskii, E. terrapinae (cell lysates were gifts from
C. G. Clark, London School of Hygiene and Tropical
Medicine), and the heterolobosean Naegleria gruberi
(genomic DNA and cDNA were gifts from Å. Sjögren,
Dalhousie University) using the forward primer PROSeuf2
(AAYTTYCCNGARTGGTA) and the reverse primers
PROSeur2 (TCGCCYTCRCACCANGGNGC) and PROSeur1 (GCNGTRTGNCCYTCYTGCCA) for the Entameoba species and Naegleria, respectively. The Cterminal part of N. gruberi proS were amplified with PCR
from cDNA using a specific internal primer and a primer
complimentary to the 39 end of the cDNA. alaS gene
sequences from E. invadens and E. moshkovskii were
amplified using the forward primer alaSswsF1 (GGAATCTAYTGYTWYCANCC) and the reverse primer
alaSswsR4 (TGCGTNCCRCARCANGCYTC), the N.
gruberi alaS was amplified using the forward primers
alaSswsBTf1 (CAYCAYACNTTYTTYGARATG) and
the reverse primer alaSswsBTr2 (TGCGTRCCNCCRCANAAYTC), and the pelobiont Mastigamoeba balamuthi
alaS was amplified using a specific reverse primer based on
the partial cDNA clone released to GenBank (gi: 18055573)
and the degenerate forward primer alaSBTf1 (genomic
DNA was a gift from E. Gill, Dalhousie University). T.
vaginalis proS and alaS gene sequences were obtained from
cDNA clones (gifts from P. Tang, Chang Gung University,
Taiwan). The PCR products and the cDNA clones were
sequenced using standard methods.
Assembly of the Data Sets
Unpublished proS and alaS sequences were retrieved
from various genome projects. Phytophthora sojae
(oomycete) and Thalassiosira pseudonana (diatom) sequences were retrieved from the DOE-Joint Genome
Institute (http://www.jgi.doe.gov/), sequences from Tetrahymena thermophila (ciliate) sequences were retrieved
from Genoscope (http://www.genoscope.cns.fr/), a proS
Dictyostelium discoideum (mycetozoa) sequence was
retrieved from the Dictyostelium discoideum Genome
Project (http://www.uni-koeln.de/dictyostelium/), and sequences from the ciliate Paramecium tetraurelia, Entamoeba histolytica, the kinetoplastid Trypanosoma brucei,
and the apicomplexan Cryptosporidium parvum, were
retrieved from The Institute for Genomic Research (http://
www.tigr.org/). Subsequently, 305 and 365 unambiguously aligned amino acid positions of prolyl-tRNA
synthetase and alanyl-tRNA synthetase data sets, respectively, were identified. The v2 tests for deviation of amino
acid frequencies implemented in Tree-Puzzle version 5.1
(Strimmer and von Haeseler 1996) were applied. Because
none of the commonly used phylogenetic methods are able
to deal with compositional heterogeneity in the data (Foster
and Hickey 1999), sequences that failed the test were
excluded from further analyses, except that the N. equitans
and the G. lamblia proS sequences and the N. equitans alaS
sequence were retained although they failed the test. In
principle, the recently published method for calculating
LogDet pairwise distances between amino acid sequences
(Thollesson 2004) could overcome the problem with amino
acid composition heterogeneity within the data sets, which
would make the exclusion of sequences that fail the test
unnecessary. However, the relative performance of the
LogDet method compared with maximum-likelihood (ML)
methods has not yet been rigorously tested for single-gene–
size data sets.
Phylogenetic Analyses
We chose to use two protein ML methods in our
analyses because they have different advantages. The
PROML method is an established method that employs
a fairly extensive heuristic tree-searching algorithm and is
widely used (Felsenstein 1989). By contrast, the PHYML
method is a much newer method with an ultrarapid (but less
extensive) heuristic tree-searching algorithm that has not yet
been applied to a wide array of data sets. It is much faster
than PROML, thereby allowing a larger number of bootstrap
replicates to be evaluated (Guindon and Gascuel 2003). ML
trees were inferred using PROML within the PHYLIP
version 3.6a3 program (Felsenstein 1989), using the JonesTaylor-Thornton (JTT) substitution model, a mixed fourcategory discrete-gamma model of among-site rate variation
plus invariable sites (JTT 1 ÿ 1 Inv) and 10 jumbles with
global rearrangements. The ÿ shape parameter, a, and the
fraction of invariable sites, Pinv, were estimated by using
Tree-Puzzle version 5.1 (Strimmer and von Haeseler 1996).
Protein ML bootstrap values based on 500 resampled data
sets generated using SEQBOOT within the PHYLIP
package (Felsenstein 1989) were calculated using PHYML
version 2.1b1 (Guindon and Gascuel 2003), with JTT 1 ÿ
1 Inv models. Bootstrap support values using other
methods and models were calculated for comparison.
Protein ML bootstrap values were calculated with PROML
with JTT 1 ÿ 1 Inv models based on 100 resampled data
sets. All other analyses were based on 500 resampled data
sets. Protein ML bootstrap values assuming uniform site
rates were calculated using PHYML (Guindon and Gascuel
2003), and protein ML distance bootstrap values with JTT
1 ÿ 1 Inv models were calculated using PUZZLEBOOT
(http://hades.biochem.dal.ca/Rogerlab/). LogDet amino
acid distance bootstrap support values were calculated with
the LDDist Perl module using the companion Perl script
PLD.pl (Thollesson 2004) with a model of invariable sites
and one category of variable site rates. The fractions of
invariable sites were calculated using Tree-Puzzle version
5.1 (Strimmer and von Haeseler 1996). The distance
matrices were analyzed using FITCH within the PHYLIP
package with global rearrangements and one jumble.
LogDet analyses with more site rate categories were not
performed, because the sequence lengths in our data sets
were judged too small to give valid results when divided into
several rate classes (Thollesson 2004; M. Thollesson,
personal communication). Finally, maximum-parsimony
bootstrap support values were calculated using PROTPARS
(Felsenstein 1989).
Results and Discussion
Interdomain Gene Transfers of proS and alaS
Previously we showed that the genes encoding prolyltRNA synthetase and alanyl-tRNA synthetase, respectively,
A Common Ancestor of Diplomonads and Parabasalids 87
A
B
Treponema
Spirochaetales
Leptospira
Thermus/Deinococcus group
Deinococcus
Mycetozoa
Dictyostelium discoideum 2
Cyanobacteria
Nostoc
90
100
Streptophyta
Arabidopsis thaliana 1
Cyanobacteria
Gloeobacter
Oomycete
Phytophthora sojae 2
Low
G+C
Gram
positives
Desulfitobacterium
Thermotogales
Thermotoga
100
Thermobifida
High G+C Gram positive
Bifidobacterium
Green nonsulfur bacteria
Chloroflexus
α-Proteobacteria
Sinorhizobium
80
γ-Proteobacteria
Escherichia
β-Proteobacteria
Nitrosomonas
δ-Proteobacteria
Geobacter
Low G+C Gram positives
Heliobacillus
ε-Proteobacteria
Campylobacter
100
Thermoanaerobacter
Low G+C Gram positives
Clostridium
Planctomycetes
Pirellula
100
Staphylococcus
Low G+C Gram positives
Bacillus
Oomycete
Phytophthora sojae 1
93
Diatom
Thalassiosira pseudonana 1
Streptophyta
Arabidopsis thaliana 2
Diatom
Thalassiosira pseudonana 2
Mycetozoa
Dictyostelium discoideum 1
Euglenozoa
Trypanosoma cruzi
96
Caenorhabditis elegans 2
Metazoa
Homo sapiens
76
Heterolobosea
Naegleria gruberi
C. elegans 1 Metazoa
83
100
Neurospora crassa
Fungi
Saccharomyces cerevisiae
Microsporidia
Encephalitozoon cuniculi
Chlamydiales
Chlamydophila
92
Bacteroides
Bacteroidetes/Chlorobi group
Chlorobium
Aquificales
Aquifex
100
100
Magnetospirillum
α-Proteobacteria
Sphingomonas
Pirellula
Planctomycetes
Bacteroides
58
Bacteroidetes/Chlorobi
group
Chlorobium
Chloroflexus
Green nonsulfur bacteria
Mycobacterium
53
High G+C Gram positive
56
Deinococcus
Thermus/Deinococcus group
Thermus
71
100 Oryza sativa
Streptophyta
Arabidopsis thaliana 1
89
Borrelia
Spirochaetales
50
98
Bacillus
Low G+C Gram positives
Clostridium
Halobacterium
Euryarchaeota
Entamoeba invadens
76
Entamoeba terrapinae
Entamoebidae
100
Entamoeba moshkovskii
98
65
57
100
ML+Γ: 100
ML+Γ*: 100
ML-Γ: 100
MLdist: 98
LogDet:100
Pars: 100
ML+Γ: 100
ML+Γ*: 98
ML-Γ: 100
MLdist: 97
LogDet: 98
Pars: 96
Entamoeba histolytica
Streptophyta
Arabidopsis thaliana 2
Caenorhabditis elegans
Metazoa
Homo sapiens
Neurospora crassa
Fungi
Saccharomyces cerevisiae
Trypanosoma brucei
Euglenozoa
Cryptosporidium parvum
Apicomplexa
Tetrahymena thermophila
Ciliata
Plasmodium falciparum
Apicomplexa
Paramecium tetraurelia
Ciliata
Diatom
Thalassiosira pseudonana
Oomycete
Phytophthora sojae
Mycetozoa
Dictyostelium discoideum
Microsporidia
Encephalitozoon cuniculi
Heterolobosea
Naegleria gruberi
Methanopyrus
Methanothermobacter
Methanocaldococcus
Euryarchaeota
Methanosarcina
79
54
Archaeoglobus
100
Aeropyrum
83
Sulfolobus
Crenarchaeota
Pyrobaculum
100
Ferroplasma
97
Thermoplasma
Euryarchaeota
Pyrococcus
Giardia lamblia
Spironucleus barkhanus Diplomonads
Trichomonas vaginalis
Parabasalid
Nanoarchaeum equitans Nanoarchaeota
ML+Γ: 73
ML+Γ*: 74
ML-Γ: 76
MLdist: 42
LogDet: 25
Pars: 72
ML+Γ: 100
ML+Γ*: 100
ML-Γ: 100
MLdist: 99
LogDet: 98
Pars: 100
ML+Γ: 70
ML+Γ*: 62
ML-Γ: 73
MLdist: 86
LogDet: 94
Pars: 39
10
91
ML+Γ: 81
ML+Γ*: 64
ML-Γ: 73
MLdist: 21
LogDet: 99
Pars: 62
57
76
55
100
100
ML+Γ: 100
ML+Γ*: 100
ML-Γ: 100
MLdist: 97
LogDet:100
Pars: 100
ML+Γ: 100
ML+Γ*: 99
ML-Γ: 100
MLdist: 98
LogDet: 98
Pars: 98
ML+Γ: 100
ML+Γ*: 100
ML-Γ: 100
MLdist: 98
LogDet: 99
Pars: 100
Methanothermobacter
Methanocaldococcus
Archaeoglobus
Euryarchaeota
Methanosarcina
Ferroplasma
Thermoplasma
Sulfolobus
Crenarchaeota
Aeropyrum
Pyrobaculum
76 Entamoeba moshkovskii
100 Entamoeba histolytica
Entamoebidae
51
Entamoeba invadens
92
Paramecium tetraurelia
Ciliata
Tetrahymena thermophila
Trichomonas vaginalis Parabasalid
Spironucleus barkhanus
Diplomonads
Giardia lamblia
Nanoarchaeota
Nanoarchaeum equitans
ML+Γ: 95
ML+Γ*: 81
ML-Γ: 85
MLdist: 91
LogDet: 8
Pars: 91
ML+Γ: 99
ML+Γ*: 100
ML-Γ: 94
MLdist: 74
LogDet:100
Pars: 93
10
FIG. 1.—Phylogenies of two aminoacyl-tRNA synthetase protein sequence data sets. ML trees of (A) prolyl-tRNA synthetase and (B) alanyl-tRNA
synthetase. Protein ML bootstrap values greater than 50% calculated using PHYML are shown (ML1ÿ). Bootstrap support for critical bipartitions from
additional analyses are shown within boxes for comparison; ML bootstrap values calculated using PROML (ML1ÿ*), protein ML assuming uniform
site rates (ML–ÿ), protein ML distance (ML–dist), LogDet distance (LogDet), and maximum parsimony (Pars). Eubacteria are labeled black, Archaea
are labeled blue, and Eukaryotes are labeled according to their classification into ‘‘supergroups’’ (Cavalier-Smith 2002; Simpson and Roger 2002;
Baldauf 2003): opisthokonts (orange), amoebozoa (purple), plantae (green), chromalveolates (red), and excavates (brown). The eukaryotic backbone is
labeled gray.
were most likely laterally transferred from Archaea to
diplomonads (Andersson et al. 2003). To explore these
interdomain transfers further, we have increased the
eukaryotic taxonomic sampling for these genes to include
sequences from three additional Entamoeba species, the
heterolobosean Naegleria, the pelobiont Mastigamoeba, the
parabasalid Trichomonas, the diatom Thalassiosira, the
oomycete Phytophthora, and the ciliates Paramecium and
Tetrahymena. On the whole, the phylogenetic analyses on
the updated data sets of the two proteins agree fairly well
with expected organismal phylogeny, with only a few easily
identified exceptions (fig. 1). Only two of these unexpected
branching patterns with high bootstrap support are observed
among the prokaryotes, both in the proS tree; the Pirellula
sequence shows close relationship with a-proteobacteria
and the Halobacterium sequence is found in a distinct
position from the other euryarchaeota sequences (fig. 1A).
Surprisingly, the eukaryotes show up in a handful unexpected positions. The D. discoideum and P. sojae alaS
sequences are found outside the main eukaryotic clade (fig.
1B). Unfortunately, the statistical support for the separation
is weak and, therefore, the origins of these sequences are
88 Andersson et al.
uncertain. Both proS and alaS plant sequences are found
nested within the Eubacteria with strong support (fig. 1),
most likely indicating two interdomain gene transfer
events—the alaS sequence almost certainly via the
chloroplast. Finally, a subset of the eukaryotes is found
nested within the Archaea in both trees (fig. 1), strongly
suggesting transfer of the genes between Archaea and
eukaryotes. The seemingly similar frequency of observed
gene transfer events affecting eukaryotes compared with
prokaryotes for these two aminoacyl-tRNA genes may be
somewhat surprising (fig. 1). However, we previously
reported that gene transfer appears to have affected
prokaryotes and microbial eukaryotes to a similar extent in
the glutamate dehydrogenase gene families (Andersson and
Roger 2003), indicating that lateral gene transfer may indeed
be a widespread evolutionary mechanism in microbial
eukaryotes (Andersson et al. 2003; Archibald et al. 2003;
Gogarten 2003).
A Single Archaeal Origin of the Diplomonad and
Parabasalid proS
The topology with a eukaryotic clade including
parabasalid and diplomonad sequences nested with the
Archaea to the exclusion of other eukaryotes is strongly
supported in the proS tree by all phylogenetic methods,
likely indicating an interdomain gene-transfer event from
the Archaea to a common ancestor of these eukaryotic
groups. The specific archaeal origin of the proS gene in
diplomonads and parabasalids is more difficult to identify
because of large disagreements between the results from
the various phylogenetic methods used. A specific relationship between the N. equitans and the eukaryotic
sequences is supported by a bootstrap value of 73% in the
ML analysis, and the relationship is recovered by the other
ML methods, as well as with parsimony (fig. 1A).
However, the LogDet distance analysis disagrees. The
Nanoarchaeum relationship is recovered in only 25% of
the bootstrap replicates (fig 1A), whereas 52% of the
replicates place the eukaryotic sequences basal to the
archaea (data not shown)—a position that is only found in
4% of the replicates in the ML bootstrap analyses that
incorporate the gamma model of rate heterogeneity (data
not shown). Because both the N. equitans and the G.
lamblia sequences failed the test for amino acid compositional heterogeneity applied to the data sets, and the
applied phylogenetic methods assume a uniform amino
acid composition within the data set (with the exception of
LogDet analysis), the specific relationship between the
Nanoarchaeota, diplomonad, and parabasalid sequences
could be the result of an artifactual attraction caused by the
amino acid compositional heterogeneity. On the other
hand, a model that incorporates rate heterogeneity within
the data set could not be applied to the LogDet analysis,
because that would require a larger data set (Thollesson
2004; M. Thollesson, personal communication), and the
attraction between the diplomonad and parabasalid
sequences—which represent the longest branches within
the archaea/diplomonad/parabasalid subtree (fig. 1A)—and
the long internal branch in this analysis could be an artifact
caused by the absence of a rate heterogeneity model.
Obviously, in the absence of an efficient method that
simultaneously can incorporate models of rate and amino
acid heterogeneities, the relationship within the archaea/
diplomonad/parabasalid subtree cannot be resolved with
a high degree of confidence for proS.
Gene-Transfer Events Have Distributed Archaeal alaS
in Diverse Microbial Eukaryotes
The eukaryotic group showing the largest diversity
within the alaS tree is found within eubacteria, and
a eukaryotic cluster including diplomonads, parabasalid,
Entamoeba, and ciliate sequences is found as a sister group
to the N. equitans sequence with high bootstrap support
(98% for all methods [fig. 1B]). Given current accounts of
eukaryotic phylogeny (Baldauf et al. 2000; Bapteste et al.
2002; Cavalier-Smith 2002; Simpson and Roger 2002;
Baldauf 2003), this topology is most easily explained by
a gene transfer to a common ancestor of diplomonads
and parabasalids, followed by two eukaryote-to-eukaryote
gene transfers that replaced the ancient eukaryotic version
in the ciliate and Entamoeba lineages. Specifically, the
well-supported relationship in ML and parsimony analyses
between the Trichomonas sequence and the Entamoeba
and ciliate sequences to the exclusion of diplomonads in
the alaS tree suggests that a parabasalid was the donor
eukaryotic lineage for the first of the two eukaryoteto-eukaryote gene transfers. Interestingly, the LogDet
analysis places the Entamoeba and ciliate sequences as
a sister clade to the diplomonads with a high bootstrap
support (92%). Because all sequences in this cluster passed
the test for amino acid compositional heterogeneity, the
inconsistency between ML and LogDet analyses probably
is explained by the absence of a model incorporating rate
heterogeneity in the latter—the Trichomonas sequence
represents the longest branch in the cluster and, indeed, is
attracted to the root of the cluster in the LogDet analysis.
Although only a few lateral gene transfers between
eukaryotes have been described (Andersson et al. 2003;
Archibald et al. 2003; Bergthorsson et al. 2003), the
inferred intradomain transfers should not be surprising,
because both Entamoeba and ciliates are phagotrophic
lineages that may ingest both microbial eukaryotes and
prokaryotes. The alternative hypothesis—that both alaS
versions were present in the last common eukaryotic
ancestor and subsequently differentially lost—is much less
likely for several reasons. Ciliates, Entamoeba, and
parabasalids/diplomonads are specifically related to the
apicomplexa, pelobionts, and heterolobosea, respectively
(Baldauf et al. 2000; Bapteste et al. 2002; Cavalier-Smith
2002; Simpson and Roger 2002; Baldauf 2003), which all
have the ‘‘bacterial’’ version (fig. 1B). Plasmodium
falciparum (apicomplexa) and M. balamuthi (pelobiont)
sequences were excluded from the phylogenetic analyses
because of strong amino acid heterogeneity and short
length, respectively (data not shown). Thus, many parallel
independent losses have to be posited if the ‘‘archaeal’’
version were ancestral to all eukaryotes. Also, all extant
eukaryotes have either the ‘‘bacterial’’ or the ‘‘archaeal’’
version of alaS—none has been found to encode both—
which argues against retention of both versions in a single
A Common Ancestor of Diplomonads and Parabasalids 89
genome over a long evolutionary timescale, as required by
an ‘‘ancient paralogy and differential loss’’ scenario.
Transfer of Two Unsplit Nanoarchaeota Genes
in a Single Event?
A gene-transfer ratchet mechanism could explain the
presence of the two archaeal genes in the eukaryotic
genomes (Doolittle 1998); the food ingested by the common ancestor of diplomonads and parabasalids may have
been rich in members of the Nanoarchaeota or close
relatives to the phylum, or a symbiotic relationship between
such an organism and the eukaryote may have existed.
Noticeably, the only described species from Nanoarchaeota
lives as a symbiont with a crenarchaeon (Huber et al. 2002;
Waters et al. 2003). However, the organization of the genes
in Nanoarchaeum hints that the transfer of the two tRNA
synthetase genes may have occurred in a single rather than
in multiple events. The N. equitans alaS gene is one of
several in that genome shown to be ‘‘split’’ into two
noncontiguous pieces (Waters et al. 2003). Curiously, the
gene corresponding to the C-terminus of alaS shows a close
genetic linkage to the proS gene; they are separated by only
a single gene encoding a hypothetical protein in the
Nanoarchaeum genome. If the ancestral unsplit alaS gene
in the nanoarchaeal lineage was located in the current position of the C-terminal gene, the transfer of a single DNA
fragment would be sufficient to transfer of both alaS and
proS in a single event to a common ancestor of diplomonads and parabasalids. The fact that Nanoarchaeum is
the only archaeal genome (among 18 full-genome
sequences available from this domain) that shows such
a close linkage of these two genes circumstantially supports
this scenario.
A Common Ancestor of Diplomonads and Parabasalids
to the Exclusion of the Root
These findings have several additional important
implications. Both diplomonads (Chihade et al. 2000)
and parabasalids (Keeling and Palmer 2000) have each
been suggested, individually, to represent the deepest eukaryotic branch, and rDNA phylogenies have long
depicted them as the two earliest emerging groups (Sogin
1991). Our data indicate that none of these proposals is
correct—the presence of two aminoacyl-tRNA synthetase
genes of archaeal ancestry are shared derived features that
distinguish them from the other eukaryotes included in the
study, indicating that they share a common ancestor. Thus,
the root of eukaryotes can neither lie on the branch leading
to diplomonads nor lie on the branch leading to parabasalids. Although this has been proposed previously
(Embley and Hirt 1998; Cavalier-Smith 2002; Simpson
and Roger 2002; Baldauf 2003; Cavalier-Smith 2003;
Simpson 2003), the support from phylogenetic analyses
has been relatively weak (Henze et al. 2001; Simpson et al.
2002; Cavalier-Smith 2003; Simpson 2003). Our data do
not directly bear on the phylogenetic position of the
diplomonad/parabasalid group within the eukaryotes.
Nevertheless, the confirmation of the specific relationship
between diplomonads and parabasalids will deepen the un-
derstanding of the evolution of two important human pathogens, G. lamblia and T. vaginalis, the genomes of which
will both be completely sequenced shortly. For instance,
this sister group relationship suggests that hydrogenosomes, mitochondrial-derived, hydrogen-evolving energygenerating organelles in T. vaginalis and the recently
discovered mitochondrial remnant organelles (mitosomes)
in G. lamblia (Tovar et al. 2003) may have common anaerobic ancestry. Indeed, because most extant archaeal
lineages, including N. equitans, exist in oxygen-poor
environments like those inhabited by free-living diplomonads and parabasalids, the transfers probably occurred in
an anaerobic ancestor of these two protist lineages that
could have already begun to lose canonical aerobic
mitochondrial functions.
Ancient Gene Transfers Provide Insights into
Nanoarchaeota
The phylogenies of the two transferred genes also
indicate that the lineage leading to Nanoarchaeota diverged
from Crenarchaeota and Euryarchaeota before the divergence between diplomonads and parabasalids. Moreover, the transfer of a continuous nanoarchaeon alaS gene
to a eukaryote indicates that the presence of split noncontiguous genes—one of which is alaS—on the genome of
N. equitans likely is a derived feature, rather than
a reflection of the ancestral state of genes in early microbial
evolution. Thus, the split N. equitans genes are probably not
indicators that the lineage represents ‘‘a living microbial
fossil’’ (Thomson et al. 2004). Unfortunately, it remains
unclear whether the divergent nature of the N. equitans
sequences is a consequence of the symbiotic lifestyle of the
lineage (Boucher and Doolittle 2002) or indicates a truly
ancient origin within the Archaea; further phylogenomic
studies are needed to confirm the phylogenetic position of
Nanoarchaeota within Archaea. In any case, our results
suggest that there have been mesophilic archaea that are
closer relatives to Nanoarchaeota than to Crenarchaeota or
Euryarchaeota because the common ancestor of diplomonads and parabasalids most likely was a mesophile
(Cavalier-Smith 2002) and physical proximity of the
organisms is likely an important factor that vastly increases
the probability of successful gene-transfer events. However, a transfer from a hyperthermophile to a mesophile
living close to a hyperthermophilic environment cannot be
excluded, and further studies are needed to clarify whether
mesophilic organisms related to Nanoarchaeota still exist
and wehether they live as symbionts with eukaryotes.
Hopefully, further examples of interdomain lateral gene
transfer discovered from genomic sequences will continue
to resolve the phylogeny within prokaryotes and eukaryotes
and allow us to determine the relative timing of major
evolutionary events in disparate regions of the tree of life.
Supplementary Material
The sequences obtained in this study were deposited
under GenBank accession numbers AY568067 to
AY568076.
90 Andersson et al.
Acknowledgments
We thank P. Tang, E. Gill, and C. G. Clark for generous
gifts of cDNA clones, genomic DNA, and cell lysates,
respectively. We also thank Å. Sjögren for experimental
assistance and critical reading of the manuscript. A.J.R. is
supported by the Canadian Institute for Advanced Research,
Program in Evolutionary Biology. This work was supported
by a Canadian Institutes of Health Research (CIHR) Grant
(MOP-62809) awarded to A.J.R and a Swedish Research
Council (VR) Grant awarded to J.O.A.
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Martin Embley, Associate Editor
Accepted September 1, 2004