*Department of Microbiology, Immunology, and Molecular Genetics, University of California at Los Angeles;
Institute of Geophysics and Planetary Physics, University of California at Los Angeles;
Canadian Institute for Advanced Research, Program in Evolutionary Biology, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax;
Department of Parasitology, Faculty of Science, Charles University, Prague
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Abstract |
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Introduction |
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In recent years, the archezoa hypothesis has fallen into disfavor primarily because of the discovery of genes of apparent mitochondrial origin within the nuclei of many putative Archezoa. The presence and phylogenetic affinities of these genes strongly suggest that the organisms bearing them had once had mitochondria but had subsequently lost or modified them. For example, mitochondrial isoformrelated chaperonin 60 (cpn60) or heat shock protein 70 (hsp70) genes (or both) have now been found in diplomonads, parabasalids, microsporidia, and Entamoeba (see Roger 1999
). Additionally, parabasalids and Entamoeba still harbor apparently mitochondrion-derived organelles, hydrogenosomes, and mitosomes, respectively (Mai et al. 1999
; Tovar, Fischer, and Clark 1999
; Dyall and Johnson 2000
; Rotte et al. 2000
). Organelles resembling hydrogenosomes-mitosomes have now also been reported in several pelobionts (Andresen, Chapman-Andresen, and Nillson 1968
; Chavez, Balamuth, and Gong 1986
; Seravin and Goodkov 1987
; Walker et al. 2001
), and the most recent molecular phylogenetic analyses seem to place pelobionts as the sister to entamoebae (Silberman et al. 1999
; Milyutina et al. 2001
), indicating that they too are secondarily amitochondriate. Recently, the first molecular phylogenies placing oxymonads in a robust position (ssu rRNA) indicate a close ancestry with Trimastix (Dacks et al. 2001
). Although no obvious double-membranebounded organelles have been described in oxymonads, the flagellated protist Trimastix does possess organelles that resemble mitochondria (Brugerolle and Patterson 1997
). This Trimastix-oxymonad clade emerges nowhere near the base of the eukaryotic tree, which is also consistent with a mitochondriate ancestry.
The only original archezoan whose mitochondrial status remains unchallenged by molecular data is the retortamonad. Retortamonads are a small group of flagellates comprising two subtaxa (genera), Chilomastix and Retortamonas. Several ultrastructural synapomorphies (most notably the arched fiber form of the B fiber and the presence of the dorsal lapel structure) support the monophyly of the group (Simpson and Patterson 1999
). Most retortamonads are obligate symbionts, commensals, or parasites inhabiting animal guts (Kulda and Nohynková 1978
), with one free-living species also known, Chilomastix cuspidata (Bernard, Simpson, and Patterson 1997
). In addition to lacking mitochondria, retortamonads also seem to lack Golgi, dictyosomes, and peroxisomes (Brugerolle and Müller 2000
).
Retortamonads have often been considered together with the similarly cytologically depauperate diplomonads and oxymonads, collectively referred to by many as the metamonads (Cavalier-Smith 1987
; Corliss 1994
; Brugerolle and Müller 2000
). However, there has never been strong morphological evidence uniting all three taxa (Brugerolle and Müller 2000
), or even any two of them, prompting some workers to avoid the use this grouping (Patterson 1994, 1999
). In fact, recent phylogenies based on molecular data do not group oxymonads and diplomonads (Moriya, Ohkuma, and Kudo 1998
; Dacks and Roger 1999
; Dacks et al. 2001
; Moriya et al. 2001
). More recently, ultrastructural data have emerged that link retortamonads to several mitochondrion-bearing flagellates, especially core jakobids (e.g. Reclinomonas and Jakoba) and Malawimonas (O'Kelly 1993, 1997
; O'Kelly and Nerad 1999
). The close structural similarities between retortamonads, core jakobids, Malawimonas, Trimastix, Carpediemonas, diplomonads, and Heterolobosea form the basis of the excavate hypothesis (Simpson and Patterson 1999
) which argues that these taxa have descended from a common ancestor. Regardless of the validity of the excavate hypothesis, it remains unclear whether the common ancestor of these organisms had mitochondria or whether excavate organisms are a grade from which most other living eukaryotes have descended, possibly straddling the mitochondrial acquisition event (O'Kelly 1993
; O'Kelly and Nerad 1999
; Simpson and Patterson 1999
). The latter possibility would allow retortamonads to be primitively amitochondriate, even if diplomonads were not. The relationships amongst excavate taxa remain poorly understood (O'Kelly and Nerad 1999
; Simpson, Bernard, and Patterson 2000
; Simpson and Patterson 2001
), and there is no convincing structural evidence placing retortamonads or diplomonads any closer to each other than to any of the other excavate taxa.
Here we report ssu rRNA gene sequences from several Retortamonas isolates. We demonstrate a strongly supported phylogenetic affinity between retortamonads and diplomonads. Because current evidence indicates that the ancestors of diplomonads once had mitochondria, a retortamonad + diplomonad clade suggests that retortamonads also descend from mitochondrion-bearing ancestors.
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Materials and Methods |
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Because a mixed protist culture of Retortamonas and Blastocystis was recovered from the guinea pig (C. G. Clark, London School of Hygiene and Tropical Medicine, London, England), internal primers (based on the ssu rDNA sequences of diplomonads and Retortamonas sp. ATCC 50375) Dip-F (5'-GGGACAGGTGAAAYAGGATGATCC-3') and Dip-R (5'- GGATCATCCTRTTTCACCTGTCCC-3') were used in conjunction with eukaryotic primers B and A, respectively, to specifically amplify the retortamonad ssu rRNA gene in two pieces. Each half of the ssu rRNA gene was T-A cloned and sequenced as described above. This Retortamonas sequence, labeled as Retortamonas caviae, has been previously used as an outgroup taxon in another study (Amaral Zettler et al. 2000
). All sequence data have been deposited with GenBank with accession numbers AF439344AF439351, and sequence alignments are available upon request (jsilber@ucla.edu).
Phylogenetic Analyses
Two data sets were constructed to ascertain the phylogenetic relationship of retortamonads among eukaryotes and to each other. A comprehensive data set, including ssu rDNA sequences from most major eukaryotic lineages plus an archaeal outgroup consisted of 40 taxa and 1,096 unambiguously aligned positions. Fine-scale relationships among the retortamonads were examined in a restricted data set that included all available retortamonad and diplomonad ssu rDNA sequences along with outgroup sequences from two separate excavate taxa (Heterolobosea and Trimastix) and the dictyostelid slime mold Dictyostelium discoideum (23 taxa, 1,177 positions).
Phylogenetic analyses were performed with PAUP* 4.0b8 (Swofford 2000
), using maximum likelihood (ML) distance (minimum evolution), and parsimony methods under a variety of evolutionary models of DNA substitution. The best available model, as determined by hierarchical nested likelihood ratio tests implemented in Modeltest version 3.06 (Posada and Crandall 1998
), was a general time-reversible model of substitution, incorporating a gamma distribution for among-site rate variation (four discrete rate categories) plus an estimate of invariable sites (GTR +
+ I). This model was employed in maximum likelihood and maximum likelihood distance tree reconstructions. Heuristic tree searches were conducted for each analysis with 20 and 100 random additions of taxa for maximum likelihood and parsimony analysis, respectively, each followed by tree bisectionreconnection topological rearrangements. Support for topological elements was assessed by tree reconstructions of bootstrap-resampled data sets with 200 (smaller-scale analysis) or 357 (large-scale analysis) replicates for maximum likelihood analyses and 1,000 replicates under distance and parsimony criteria. Because the ssu rRNA genes in some diplomonad species are highly biased toward G+C (up to 75% G+C in Giardia intestinalis), LogDet distance analyses incorporating an estimate of invariable sites (Gu and Li 1996
) were also performed. No significant differences in branching topology were found between LogDet distance and trees estimated from the other methods or models employed. Shimodaira-Hasegawa and Kishino-Hasegawa tests (SH and KH tests, respectively, Kishino and Hasegawa 1989
; Shimodaira and Hasegawa 1999
) were used to test whether the difference between the log-likelihood (lnL) scores of the optimal ML trees (where retortamonads fall within the diplomonads) and the lnL of the ML trees in which the retortamonads were constrained not to branch within a monophyletic diplomonad clade (
lnLobserved) was statistically significant.
For the smaller data set, we employed two additional tests: the parametric bootstrapping likelihood ratio test (described as the SOWH test by Goldman, Anderson, and Rodrigo 2000
) and the expected likelihood weights method (ELW), recently described by Strimmer and Rambaut (2002)
. For the SOWH test, 1,000 data sets were simulated with Seq-Gen, Version 1.2.4 (Rambaut and Grassly 1997
) using the optimal constrained monophyletic diplomonad tree and substitution model parameters (GTR +
+ I) and branch lengths optimized for this topology, given the data. For each simulated data set, substitution model parameters were reestimated, followed by a heuristic ML tree search (one random stepwise addition replicate) to derive the optimal tree. The differences in lnL between this maximum likelihood topology and the monophyletic diplomonad tree used to simulate the data were then calculated (
lnL) to form the null distribution against which the
lnLobserved was compared. A more detailed exposition of the SOWH test can be found in Goldman, Anderson, and Rodrigo (2000)
. To calculate confidence intervals for the optimal tree by the ELW method, two PERL scripts were created (elw.pl and calcwts.pl, available upon request from A.J.R., aroger@is.dal.ca) to automate the procedure using SEQBOOT (PHYLIP 3.57, Felsenstein 2000
) and PAUP* 4.0b8. The best trees found in the 200 maximum likelihood bootstrap replicates (described previously) comprised a set of 66 unique trees. These included the maximum likelihood tree for the observed data as well as the monophyletic diplomonad topology. The 95% confidence interval, given this set of 66 likely trees, was then calculated with the ELW method using 1,000 bootstrap replications, with substitution model parameters reestimated for each replicate over a Jukes-Cantor distancecorrected Neighbor-Joining topology.
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Results and Discussion |
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The extreme similarity among the ssu rRNA gene sequences from all the mammalian isolates is consistent with our inability to identify noteworthy morphological differences by light microscopy (J. D. Silberman, unpublished data). In fact, we are hard-pressed to differentiate these new isolates from the human commensal Retortamonas intestinalis based on morphological criteria. Considering morphological similarity along with ssu rDNA sequence conservation, the isolates from sheep, elk, goat, guinea pig, and human might provisionally be considered a single species. If conspecificity is accepted for all these mammalian isolates, the name R. intestinalis (Wenyon and O'Connor, 1917) Wenrich, 1932 has priority. On the other hand, even though the retortamonads isolated from the two amphibians are likewise morphologically indistinguishable from each other, the sequence dissimilarity between their ssu rRNA genes is somewhat greater than that found among the mammalian isolates, and it is unclear if they represent different species.
The Placement of Retortamonads in the Eukaryote Tree
All methods used here group retortamonads and diplomonads to the exclusion of all other eukaryotes (figs. 1 and 2
). In our broad-scale phylogenetic analyses (fig. 1
), this relationship is highly supported by bootstrap analyses using maximum likelihood, parsimony, and LogDet distance methods (maximum likelihood, 100%; parsimony, 89%; LogDet, 99%) and receives moderate support from maximum likelihood distance bootstrap analysis (66%). These analyses robustly recover many major eukaryotic clades, such as opisthokonts (Metazoa + Fungi + relatives), Viridiplantae, stramenopiles, alveolates, etc. and now Retortamonas + diplomonads. As with other rigorous analyses of ssu rDNA sequences, the relationships among major clades are often poorly resolved using models allowing among-site rate variation (Kumar and Rzhetsky 1996
; Silberman et al. 1999
).
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We further examined the relationships within the retortamonad + diplomonad clade with a second, more restricted analysis that included more aligned sites. This analysis included all the available retortamonad and diplomonad sequences, together with three eukaryotic outgroups. As with the broad-scale analysis, the retortamonads branch within the diplomonads (fig. 2 ). Support for the sisterhood of retortamonads and Giardia is actually slightly higher than in the broad-scale analyses.
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To our knowledge, a placement of retortamonads cladistically within diplomonads has not been seriously entertained before, other than in the now disfavored hypotheses in which diplomonads are the stem group for almost all extant eukaryotes (Cavalier-Smith 1992
). We are suspicious of this placement in our trees for three reasons: (1) the particularly unusual nature of Giardia ssu rDNA sequences (i.e., in terms of base composition), (2) the low bootstrap support for the Giardia + retortamonad clade, and (3) the failure of SH, KH, and ELW tests to reject the intuitive hypothesis that retortamonads and diplomonads are each monophlyetic. However, it is interesting to note that Retortamonas and Giardia ssu rRNA gene sequences share a single guanosine nucleotide insertion that is not present in any other sequenced eukaryote ssu rRNA gene (position 544, using the guinea pig Retortamonas sequence as reference).
Retortamonads have a conventional monomonad cell structure rather than the distinctive doubled-cell organization of well-known diplomonads (fig. 3
). A placement of retortamonads specifically with Giardia would therefore imply either a reversal to the monomonad condition or multiple origins of the doubled-cell phenotype. Both these alternatives seem unlikely. However, a collection of diplomonads called the enteromonads are usually observed in a monomonad state (Brugerolle and Müller 2000
). The placement of enteromonads with respect to other diplomonads is unclear (they lack the morphological synapomorphies for retortamonads but may or may not be basal to other diplomonads), and it is therefore possible that they too have reverted to a monomonad state. The only published analysis that includes both retortamonads and a wide diversity of diplomonads is the parsimony analysis of morphological data by Siddall, Hong, and Desser (1992)
. This analysis actually employs retortamonads as the outgroup to diplomonads. Nonetheless, there is no way of rerooting their maximum parsimony tree to recover a clade of retortamonads plus Giardia to the exclusion of Hexamita, Spironucleus, and Trepomonas; therefore, it is incompatible with our trees. However, their tree is also not reconcilable with the contemporary molecular-phylogenetic understanding of diplomonad phylogeny (Cavalier-Smith and Chao 1996
; Rozario et al. 1996
; Keeling and Doolittle 1997b
) and is not recovered in parsimony analyses of an updated morphological data set (A. G. B. Simpson, unpublished data). Thus, although we cannot nominate any morphological evidence to support a placement of retortamonads close to Giardia, we cannot exclude it as a possibility. Examination with more taxa and additional phylogenetic markers will be required to resolve the phylogeny within the diplomonad + retortamonad clade. It has been noted that Giardia proteins are translated with the universal genetic code, whereas proteins are translated with an alternative code in Hexamita, Trepomonas, and Spironucleus (TAA and TAG encode glutamine rather than termination; Rozario et al. 1996
; Keeling and Doolittle 1997b;
Horner, Hirt, and Embley 1999). Characterization of protein-encoding genes from retortamonads, in conjunction with phylogenic reconstruction, may prove especially informative for interpreting both retortamonad and diplomonad evolutionary history.
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Two ancillary observations from the broad-scale phylogenetic analysis deserve further comment: (1) Sawyeria marylandensis, an anaerobic-microaerophilic heteroloboseid branches with 100% bootstrap support with the other anaerobic amoeboflagellate, Psalteriomonas lanterna, thus forming an apparent clade of anaerobes embedded within a predominantly aerobic lineage; (2) As noted in previous analyses of Entamoeba ssu rRNA gene sequences (Silberman et al. 1999
), Endolimax nana tends to branch with the pelobiont Mastigamoeba balamuthi in phylogenies based on evolutionary distances. The present analysis also groups Endolimax with the pelobiont Mastigamoeba balamuthi to the exclusion of the other Entamoeba. In addition to a phylogenetic affinity, these organisms possess ssu rRNA genes that are similar in size to one another and significantly larger than those reported for Entamoeba (Silberman et al. 1999
). These observations suggest that Endolimax may really be more closely related to pelobionts than to Entamoeba or any other taxon.
Retortamonads and the Archezoa Hypothesis
Regardless of the internal phylogeny of the diplomonad-retortamonad clade, it now seems reasonable to consider diplomonads and retortamonads as one group with respect to mitochondrial history. The status of mitochondrial genes in diplomonads has been somewhat controversial in recent literature and is worth restating briefly here. Five genes, chaperonin 60 (cpn60), heat shock protein 70 (hsp70), valyl tRNA synthetase (Val-tRS), triose phosphate isomerase (TPI), and, most recently, pyridoxal-5'-phosphatedependent cysteine desulfurase (IscS) have been claimed as potential mitochondrial relics from the diplomonad Giardia (Keeling and Doolittle 1997a;
Hashimoto et al. 1998
; Roger et al. 1998
; Morrison et al. 2001
; Tachezy, Sánchez, and Müller 2001
). A mitochondrial relict cpn60 has also recently been reported from a second diplomonad, Spironucleus (Horner and Embley 2001
). In each case, the claims are based on phylogenetic analyses which place the diplomonad gene(s) with mitochondrial forms from other eukaryotes (cpn60, hsp70, and IscS) or with sequences from proteobacteria (or both), the bacterial clade from which the mitochondrial symbiont arose (Val-tRS, TPI). Two of these five cases now appear problematic: TPI and Val-tRS. For TPI, additional
-proteobacterial sequences have been obtained, and they do not clearly form a clade with the eukaryote sequences (B. Canbäck, S. G. E. Andersson, and C. G. Kurland, personal communication), casting doubt on earlier claims (Keeling and Doolittle 1997a
). Similarly, Val-tRS genes are now available from
-proteobacteria that appear to lack the
-proteobacterial + mitochondrial insertion, questioning the endosymbiotic origin of all eukaryote homologs (T. Hashimoto, personal communication). Furthermore, in hsp70 phylogenies, the close proximity of the highly divergent Giardia sequence to mitochondrial and
-proteobacterial forms is indicative of a mitochondrial origin, but the overall topology is poorly supported (Morrison et al. 2001
). On the other hand, the cpn60 and IscS phylogenies consistently and robustly place the Giardia sequence with, and often within, mitochondrial forms to the exclusion of all sequences from bacteria (Roger et al. 1998
; Horner and Embley 2001
; Tachezy, Sánchez, and Müller 2001
). Although additional
-proteobacterial IscS gene sequence data are desirable to confirm that they share a common ancestry with mitochondrial eukaryotic homologs, the case for cpn60 is most convincing. Although it is important to establish the presence of genes of mitochondrial origin in retortamonads and other diplomonads, the most straightforward interpretation of available evidence is that ancestors of both diplomonads and retortamonads once had mitochondria.
With the strong confirmation that retortamonads and diplomonads do form a clade, all of the taxa suggested as primitively amitochondriate under the archezoa hypothesis appear to have physical or genetic (or both) mitochondrial relics or form a strongly supported clade with a group bearing such a relic. The perpetuation of the original archezoa hypothesis requires the refuting of all the mitochondrial relics from one of the amitochondriate taxa, demolition of one of the clades, or the discovery of new amitochondriate taxa.
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Acknowledgements |
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Footnotes |
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Keywords: Retortamonas
Archezoa
ssu rRNA
evolution
mitochondria
Address for correspondence and reprints: J. D. Silberman, Department of Microbiology, Immunology, and Molecular Genetics, University of California at Los Angeles, 405 Hilgrad Avenue, 1602 Molecular Sciences Building, Los Angeles, California 90095. jsilber{at}ucla.edu.4
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