Evolutionary History of "Early-Diverging" Eukaryotes: The Excavate Taxon Carpediemonas is a Close Relative of Giardia1

Alastair G. B. Simpson*, Andrew J. Roger*, Jeffrey D. Silberman{dagger}, Detlef D. Leipe{ddagger}, Virginia P. Edgcomb§, Lars S. Jermiin||, David J. Patterson|| and Mitchell L. Sogin§

*Department of Biochemistry, Dalhousie University, Halifax, Canada;
{dagger}UCLA Institute of Geophysics & Planetary Physics and Department of Microbiology & Immunology;
{ddagger}National Center for Biotechnology Information, National Library of Medicine, Bethesda;
§Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole;
||School of Biological Sciences, University of Sydney


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Diplomonads, such as Giardia, and their close relatives retortamonads have been proposed as early-branching eukaryotes that diverged before the acquisition-retention of mitochondria, and they have become key organisms in attempts to understand the evolution of eukaryotic cells. In this phylogenetic study we focus on a series of eukaryotes suggested to be relatives of diplomonads on morphological grounds, the "excavate taxa". Phylogenies of small subunit ribosomal RNA (SSU rRNA) genes, {alpha}-tubulin, ß-tubulin, and combined {alpha}- + ß-tubulin all scatter the various excavate taxa across the diversity of eukaryotes. But all phylogenies place the excavate taxon Carpediemonas as the closest relative of diplomonads (and, where data are available, retortamonads). This novel relationship is recovered across phylogenetic methods and across various taxon-deletion experiments. Statistical support is strongest under maximum-likelihood (ML) (when among-site rate variation is modeled) and when the most divergent diplomonad sequences are excluded, suggesting a true relationship rather than an artifact of long-branch attraction. When all diplomonads are excluded, our ML SSU rRNA tree actually places retortamonads and Carpediemonas away from the base of the eukaryotes. The branches separating excavate taxa are mostly not well supported (especially in analyses of SSU rRNA data). Statistical tests of the SSU rRNA data, including an "expected likelihood weights" approach, do not reject trees where excavate taxa are constrained to be a clade (with or without parabasalids and Euglenozoa). Although diplomonads and retortamonads lack any mitochondria-like organelle, Carpediemonas contains double membrane-bounded structures physically resembling hydrogenosomes. The phylogenetic position of Carpediemonas suggests that it will be valuable in interpreting the evolutionary significance of many molecular and cellular peculiarities of diplomonads.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
From an evolutionary perspective diplomonads are one of the most interesting and enigmatic groups of eukaryotes. Diplomonads (and their close relatives, retortamonads; Silberman et al. 2002Citation ) are small, flagellated unicells that lack mitochondria and tend to have poorly developed endomembrane systems (Brugerolle 1991Citation ). Most are parasites, including the best known, the human pathogen Giardia lamblia. Early phylogenies of small subunit ribosomal RNA (SSU rRNA) genes and elongation factor proteins placed Giardia as one of the earliest diverging lineages in the eukaryotic evolutionary tree (Sogin et al. 1989Citation ; Hashimoto et al. 1994Citation ; Hashimoto et al. 1995Citation ). These results (and later studies incorporating other genes and taxa) supported the idea that diplomonads had diverged before the acquisition of mitochondria (Cavalier-Smith 1983Citation ). The possibility that diplomonads are early-diverging, "primitive" eukaryotes fuelled an explosion of interest in their molecular and cellular biology, culminating in the Giardia genome project (McArthur et al. 2000Citation ).

In recent years, controversy has surrounded the evolutionary significance of diplomonads. Several genes from Giardia (chaperonin 60, cpn60; pyridoxal-5'-phosphate-dependent cysteine desulfurase, IscS; heat shock protein 70, hsp70; triose phosphate isomerase, TPI; and valyl-tRNA synthetase) and another diplomonad, Spironucleus (cpn60), are candidates for having mitochondrial origins, based on their phylogenetic affinity with nuclear-encoded mitochondrial genes from other eukaryotes (Keeling and Doolittle 1997Citation ; Hashimoto et al. 1998Citation ; Roger et al. 1998Citation ; Horner and Embley 2001Citation ; Morrison et al. 2001Citation ; Tachezy, Sánchez, and Müller 2001Citation ). At face value these data suggest that mitochondria were present in ancestors of diplomonads. But the robustness of these cases varies. Eukaryotic TPI and valyl-tRNA synthetase lack a clear phylogenetic affinity with orthologs from {alpha}-proteobacteria, the closest relatives of the mitochondrial symbiont. Perhaps these genes entered early eukaryotic cells by independent lateral transfers from other prokaryotes. Mitochondrial hsp70 from most eukaryotes has a close phylogenetic affinity to hsp70 from {alpha}-proteobacteria, but the orthologous gene from Giardia branches separately from these mitochondrial forms in rigorous analyses and could represent a separate lateral transfer to diplomonads (Morrison et al. 2001Citation ). Only in the cases of cpn60 and probably IscS do the genes from diplomonads form strong clades with well-founded mitochondrial forms from other eukaryotes (Roger et al. 1998Citation ; Horner and Embley 2001Citation ; Tachezy, Sánchez, and Müller 2001Citation ). Given the lack of cytological relics of mitochondria in diplomonads, alternative models have been proposed whereby even the most promising putative mitochondrial genes from diplomonads were also acquired through other lateral gene transfers or were transferred from a mitochondrial symbiont before its stable incorporation as an eukaryotic organelle (Sogin 1997Citation ; Doolittle 1998Citation ; Chihade et al. 2000Citation ; Margulis, Dolan, and Guerrero 2000Citation ). These scenarios retain a "premitochondrial" status for diplomonads based on the assumption that they are basal eukaryotes. Further complicating the issue are indications that the basal portions of many eukaryotic gene phylogenies (including SSU rRNA and elongation factor proteins) may be artificial collections of phylogenetically misleading sequences (Philippe and Adoutte 1998Citation ; Hirt et al. 1999Citation ; Stiller and Hall 1999Citation ). This suggests that the evidence for diplomonads being basal eukaryotes is unreliable but fails to demonstrate that diplomonads are not early diverging.

A critical impediment to resolving the controversy is our poor understanding of the immediate relationships of diplomonads and retortamonads to other eukaryotes. A close relationship with the amitochondriate, hydrogenosome-bearing parabasalids is most commonly entertained, based on some molecular phylogenies (Embley and Hirt 1998Citation ; Baldauf et al. 2000Citation ; Cavalier-Smith 2000Citation ). But cytoskeletal morphology studies suggest that diplomonads and retortamonads might be closely related to some or all of a different array of protists; "core" jakobids, Malawimonas, Heterolobosea, Trimastix and Carpediemonas (O'Kelly 1993Citation ; O'Kelly, Farmer, and Nerad 1999Citation ; Simpson and Patterson 1999Citation ; Simpson, Bernard, and Patterson 2000Citation ; Simpson and Patterson 2001Citation ). Collectively these groups are termed the "excavate taxa", named for the shared possession of a distinctive form of feeding groove (Simpson and Patterson 1999Citation ). All excavate taxa other than diplomonads and retortamonads have mitochondria, albeit sometimes with unusually bacterial-like (primitive) mitochondrial genomes (Lang et al. 1997Citation ; Gray et al. 1998Citation ), or they possess double membrane-bounded organelles that have been proposed to be mitochondrial homologs (Brugerolle and Patterson 1997Citation ; O'Kelly, Farmer, and Nerad 1999Citation ; Simpson and Patterson 1999Citation ; Simpson, Bernard, and Patterson 2000Citation ). Excavate taxa have not been systematically examined by molecular phylogenetic methods, with just three or four of the seven known groups represented in any one analysis (Dacks et al. 2001Citation ; Edgcomb et al. 2001Citation ; Archibald, O'Kelly, and Doolittle 2002Citation ; Cavalier-Smith 2002Citation ; Silberman et al. 2002Citation ).

The small excavate flagellate Carpediemonas lives in oxygen-poor sediments and contains double membrane-bounded organelles of unknown function (Simpson and Patterson 1999Citation ; Bernard, Simpson, and Patterson 2000Citation ). Here we report the first gene sequences from Carpediemonas: SSU rRNA, {alpha}-tubulin, and ß-tubulin. We also report SSU rRNA sequences for several core jakobids, and for Malawimonas, the organism that, aside from having true mitochondria, displays perhaps the greatest morphological similarity to Carpediemonas (O'Kelly, Farmer, and Nerad 1999Citation ; Simpson and Patterson 1999Citation ). Our SSU rRNA alignment is the first molecular data set to include all seven known excavate taxa. Our phylogenetic analyses suggest that Carpediemonas is the sister group to the diplomonads + retortamonads clade.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Gene Amplification and Sequencing
Carpediemonas membranifera and Jakoba incarcerata were grown as described previously (Simpson and Patterson 1999Citation ; Bernard, Simpson, and Patterson 2000Citation ; Simpson and Patterson 2001Citation ), and genomic DNA were extracted using a Puregene kit (Gentra Systems). Reclinomonas americana (ATCC 50283) genomic DNA was isolated from a cell pellet obtained from the American Type Culture Collection (Rockville, MD) by standard SDS lysis, proteinase K digestion, and organic extractions. DNA from Jakoba libera and Malawimonas jakobiformis was provided kindly by Franz Lang (Université de Montréal). Sequences were amplified by PCR (see below). Reclinomonas americana SSU rRNA genes were cloned into M13 mp18 and mp19. All other amplification products reported were cloned in pGEMt or pGEMt easy (Promega Biotech). Bidirectional sequencing was performed using Li-Cor 4000 or 4200 automated sequencers, confirming earlier manual sequencing in the case of R. americana.

For C. membranifera, SSU rRNA genes were amplified as two overlapping fragments using the 5'-end primer "A" (Medlin et al. 1988Citation ) with the internal primer CCGTCAAATYCTTTAAGTTTC, and the 3'-end primer "B" (Medlin et al. 1988Citation ) with the internal primer "Dip-F" (Silberman et al. 2002Citation ). Annealing temperatures were 45–50°C. Pools of 13 and 11 clones of the respective fragments were sequenced, with heterogeneity detected at six sites. For J. incarcerata, J. libera, M. jakobiformis, and R. americana, SSU rRNA genes were amplified using primers A and B (annealing temperature 37°C). For J. incarcerata, eight clones were pooled for sequencing, with seven heterogeneous sites being found. Heterogeneities were recorded as ambiguities. Ten clones were pooled for sequencing for J. libera and M. jakobiformis. Five and eight M13 mp18 and mp19 R. americana clones, respectively, were pooled for sequencing. No heterogeneity was detected for the latter three taxa.

Near-complete (~90%) {alpha}- and ß-tubulin genes from C. membranifera were amplified using universal primers (Edgcomb et al. 2001Citation ) in the presence of acetamide (5% w/v) at annealing temperatures of 45–50°C. Two and six near-identical clones of {alpha}- and ß-tubulin, respectively, were examined; one of each was completely sequenced.

Phylogenetic Analysis—SSU rRNA
The new SSU rRNA gene sequences were aligned by eye with sequences from a diversity of other eukaryotes, and two prokaryotic outgroups (45 taxa in total). Within well-accepted groups, taxa that contribute short terminal branches were selected wherever possible. Some groups, notably microsporidia, were omitted altogether. Initial phylogenetic analyses used 717 sites. A second alignment of 1,018 sites excluded prokaryote, diplomonad, and Entamoeba sequences. Alignments are available at http://hades.biochem.dal.ca/Rogerlab. All analyses were performed using PAUP* 4.0b4 (Swofford 2000Citation ), as described below.

For the maximum-likelihood (ML) analysis, the Tajima-Nei substitution model was selected, with a "four-category discrete {Gamma} distribution plus invariable sites" allowance for among-site rate heterogeneity, after likelihood ratio tests in MODELTEST v3.0 (Posada and Crandall 1998Citation ). The parameters were estimated in PAUP* from a maximum-parsimony tree. Rate heterogeneity was estimated to be relatively strong ({alpha} = 0.631; proportion of invariable sites = 0.194). Treespace was explored with 20 random taxon additions, with tree bisection-reconnection (TBR) rearrangements. A bootstrap analysis with 108 replicates was performed (neighbor-joining starting trees, with TBR).

Three minimum-evolution distance analyses were performed. First, ML estimates of pairwise distances (MLdist) were calculated using the same model as for the full ML analysis but with eight categories of variable sites. Second, the LogDet method was used, after removal of constant sites. Third, the simple Kimura two parameter (K2P) model was used. In each case treespace was searched using 100 random taxon additions with TBR, and a 1,000 replicate bootstrap analysis was performed (five random additions and TBR).

A parsimony (Pars) analysis with a 1,000 replicate bootstrap was performed using the same search strategies as in the distance analyses.

This series of analyses was repeated with three restricted data sets. In the first, Giardia and Entamoeba sequences were removed. In the second, all diplomonads and Entamoeba were removed. In the third, prokaryotes, diplomonads, and Entamoeba were all excluded (retaining 37 eukaryote sequences), but the 1,018 site alignment was examined. All parameter estimations and searches were performed as mentioned above (ML bootstraps included 100–109 replicates).

One "long branch" that was excluded from consideration in the analyses described above was the oxymonad Pyrsonympha sp. This sequence branched strongly with Trimastix in a previous study (Dacks et al. 2001Citation ). To examine whether this Trimastix-oxymonad relationship was robust to the addition of more excavate taxa, we added the Pyrsonympha sequence to our 37 taxa, 1,018 sites data set. The inclusion of Pyrsonympha mandated the exclusion of another eight sites. Using this data set, we performed the same series of analyses as described above, excepting ML.

We used the 37 taxa, 1,018 sites data set to assess the evidence for polyphyly of the excavate taxa using two methods for examining the plausibility of alternative tree topologies, Shimodaira-Hasegawa (SH) tests (Shimodaira and Hasegawa 1999Citation ) and the "expected likelihood weights" method of Strimmer and Rambaut (Strimmer and Rambaut 2002Citation ). Preliminary to these tests, using PAUP* 4.0b8, we performed a heuristic search (one random addition sequence, with TBR) using the same ML model as before, and saved the 5,000 best trees found. The ML tree found was the same as in our original analysis. We also searched for the ML trees under two topological constraints. In the first, the excavate taxa were constrained to form a clade. In the second, excavate taxa plus Euglenozoa and parabasalids were constrained to form a clade (previous data suggests that Euglenozoa and parabasalids are related to Heterolobosea and diplomonads, respectively (Baldauf et al. 2000Citation ) and could fall "within" a clade of excavate taxa).

For the SH approach, the test implementation in PAUP* 4.0b8 was used, using the "RELL" method with 1,000 bootstrap replicates. First, the two ML-constrained trees were compared with the overall ML tree (i.e., three trees in total). Then, these three ML trees were compared together with the next 100 best trees found in the unconstrained search and then with the next 500 best trees. For contrast, the three ML trees only were also compared using one-tailed Kishino-Hasegawa tests.

There is concern about the utility of SH tests because they appear conservative (i.e., unlikely to reject) relative to other tests and can give very different results depending on which trees are considered (Andersson and Roger 2002Citation ; Silberman et al. 2002Citation ). But the most well-known alternative, parametric bootstrapping, may be overly prone to reject in practice (Strimmer and Rambaut 2002Citation ) and is also very expensive computationally. Another approach is the expected likelihood weights method (Strimmer and Rambaut 2002Citation ). This test distributes a "likelihood weight" across a set of test trees, averaged over a number of bootstrap replicates, considering the minimal collection of trees that contribute a given fraction of the total likelihood weight as the confidence set. In principle, the test requires the consideration of all reasonable trees, a condition often intractable in practice. For this study we approximated this condition, while keeping within sensible computational bounds by using the best trees from the ML bootstrap analysis to provide a broad sample from the landscape of "reasonable trees". For the test we therefore included the three ML trees (see above) together with the best trees from 100 bootstrap replicates. We considered using a greater number of MLdist bootstrap trees but found that they conferred substantially lower likelihood on the original data than did the bulk of the ML bootstrap trees, and thus could not be well sampling the best trees under the full ML criterion. For the test, 1,000 bootstrap replicates were generated using SEQBOOT (Felsenstein 2000Citation ), and the likelihoods for all 103 considered trees were calculated for each replicate using PAUP*, using the same model as described previously but with the parameter values for each replicate optimized on a Jukes and Cantor distance neighbor-joining tree. The PAUP file was created using a Perl script, elw.pl. Expected likelihood weights for each tree were then calculated using calcwts.pl (elw.pl and calcwts.pl are available on request from A.J.R.; aroger@is.dal.ca).

Phylogenetic Analysis—Tubulins
The {alpha}- and ß-tubulin genes from C. membranifera were converted to inferred amino acids that were aligned by eye with homologs from other eukaryotes. Taxa were selected to sample diversity broadly, while excluding highly divergent sequences. Notable omissions include microsporidia and (other) long-branching fungi, leaving chytrids to represent fungi sensu lato. All excavate taxa except retortamonads and Trimastix were represented. A combined {alpha}- + ß-tubulin data set was similarly constructed from organisms where both genes are known. To improve taxon representation, four "composite taxa" were also included: "parabasalid" (Monotrichomonas sp. {alpha}-tubulin and Trichomonas vaginalis ß-tubulin); "stramenopile" ({alpha}-tubulin from Pelvetia fastigiata, and ß- tubulin from Pythium ultimum); "Cercomonas" (Cercomonas sp. ATCC 50319 {alpha}-tubulin and Cercomonas sp. ATCC 50316 ß-tubulin) and "Leishmania" (Leishmania donovani {alpha}-tubulin and L. mexicana ß-tubulin).

All three data sets were analyzed by ML using PROML 3.6a (Felsenstein 2000Citation ), with a model incorporating four site-rate categories. The rates approximated a {Gamma} distribution estimated by TREE-PUZZLE 4.0.2 (Strimmer and von Haeseler 1996Citation ). The PAM substitution matrix was used (the only matrix available with PROML 3.6a). Treespace was explored by 20 random taxon additions with "global rearrangements". Bootstrap analyses with 250 replicates were also performed (random taxon addition, with rearrangement).

For the distance analyses (MLdist), ML estimates of pairwise distances were calculated by TREE-PUZZLE using the JTT substitution matrix, with eight categories of site-rates approximating a {Gamma} distribution. FITCH (Felsenstein 2000Citation ) was used to explore treespace (Fitch-Margoliash criterion; 100 random additions, with rearrangement). Bootstrap analyses (1,000 replicates) were performed using PUZZLEBOOT 1.03 (http://www.tree-puzzle.de/puzzleboot.sh) and FITCH (five additions, with rearrangement).

Parsimony analyses (with 1,000 replicate bootstraps) were completed in PAUP* using identical search strategies to the SSU rRNA analyses.

All analyses of tubulin data were repeated with sequences from "hexamitid" diplomonads (Hexamita and Spironucleus) excluded.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Small Subunit Ribosomal RNA
As with previous studies, the trees from the 45 taxon SSU rRNA data set can be viewed as having two regions, an "apical region" of mostly short branches (widely referred to as the "eukaryotic crown") and a "base" of long branches (fig. 1A ). The apical region includes opisthokonts (represented by an animal, a fungus, and a choanoflagellate), Viridiplantae, alveolates, stramenopiles, haptophytes, cryptomonads, the so-called "Cercozoa" (represented by a testate amoeba and Thaumatomonas sp.), rhodophytes, Acanthamoeba, Apusomonas, Trimastix and, in the ML analysis only, the pelobiont Mastigamoeba balamuthi. The base includes Mycetozoa (represented by myxogastrid and dictyostelid slime molds), Entamoeba, parabasalids, heteroloboseids, diplomonads, and retortamonads. Parabasalids are usually the most basal eukaryotic branch (51%–86% bootstrap support [BS]), although the ML tree itself does not have this topology. Opisthokonts and Cercozoa always form a weak clade (<55% BS). Diplomonads and retortamonads always form a strong clade (>90% BS). Otherwise, relationships among the "major groups" are poorly resolved.



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Fig. 1.—Small subunit ribosomal RNA phylogenies of eukaryotes. New sequences are denoted by large type. Excavate taxa are shaded. ML BS values are given on internal branches. Unmarked branches have <50% BS. Estimated-rate scale bar applies for both phylogenies. A. ML topology for the "full" 45 taxon, 717 site analysis (-ln L = 9872.93). The apical region (= eukaryotic crown) of the tree is denoted (M. balamuthi falls in the basal portion with other methods). Bootstrap support values for the Carpediemonas + retortamonads + diplomonads clade with all methods are displayed in the box. B. ML topology for the 717 site analysis with all diplomonads (and Entamoeba) excluded (-ln L = 8127.35). Bootstrap support values for the Carpediemonas + retortamonads clade with all methods are boxed

 
Of the new sequences, those from Reclinomonas, J. libera, J. incarcerata and Malawimonas all fall within the apical region. Reclinomonas and J. libera form a strongly supported clade with all methods (94%–100% BS). Malawimonas forms a very weak clade with Trimastix with ML and Pars (<25% BS). Neither clade clusters with the other or with J. incarcerata nor do they show any particular affinity for any other group of eukaryotes.

In contrast, Carpediemonas falls within the base, grouping specifically with diplomonads and retortamonads with all methods. This clade is weakly supported in Pars, K2P, and MLdist analyses (48%–58% BS), but support is quite strong with LogDet (71%) and likelihood (92%; see table 1 ).


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Table 1 Bootstrap Support for the Carpediemonas + Diplomonads + Retortamonads Clade in the SSU rRNA Analyses

 
Two factors are focused upon as potential sources of error in SSU rRNA analyses; base composition heterogeneity and highly divergent sequences. Both might be relevant to the placement of Carpediemonas in our analysis because sequences from Giardia are G+C rich, and sequences from diplomonads all contribute very long branches. We therefore performed two subsidiary "717 site" analyses. First, sequences from Giardia were excluded (along with the G+C poor Entamoeba). Second, all other diplomonads were also excluded, leaving the shorter branching retortamonads to represent the diplomonads + retortamonads clade. In both analyses the optimal trees are very similar to the first analysis, but the BS for Carpediemonas as sister to diplomonads-retortamonads increases with all methods, climbing to over 70% in all cases except one Pars run (table 1 ). Results are very similar when Entamoeba is retained (data not shown). Notably, when all diplomonads are excluded the ML tree loses the classical "crown plus base" structure, with stramenopiles appearing in the most basal position (fig. 1B ). Most of the long branches form a cluster, but Carpediemonas + retortamonads instead form a very weakly supported clade with the short-branching excavate taxa Trimastix and Malawimonas.

In the final analyses prokaryotic outgroups were excluded (in addition to diplomonads and Entamoeba), permitting more sites to be included but making it impossible to root the tree (figure not shown). Resolution of the relationships amongst most major groups remains poor, except that the support for a clade of opisthokonts and Cercozoa increases (55%–83% BS), and alveolates are recovered as a strong clade with all methods (70%+ BS). Nonetheless, the Carpediemonas + retortamonads grouping is always strongly supported (90%+ BS; table 1 ), although it still does not fall with other excavate taxa. The BS for the J. libera + Reclinomonas clade is 100% with all methods, but J. incarcerata still branches separately. Malawimonas and Trimastix form a very weak clade with ML and LogDet only. When the oxymonad Pyrsonympha is included this sequence forms a strong clade with Trimastix to the exclusion of other excavate taxa and other eukaryotes (67% BS with Pars, 81%–95% BS with other methods). The branching order and support values amongst eukaryotes are otherwise very similar to those from other analyses.

Using the SH test, we further examined evidence for the polyphyly of excavate taxa by comparing (1) the overall ML tree (-ln L 11922.75), (2) the best tree in which excavate taxa alone form a clade (-ln L 11948.03), and (3) the best tree in which excavate taxa, parabasalids, and Euglenozoa collectively form a clade (-ln L 11940.44). This test does not reject either alternative tree (2 and 3; P = 0.165 and 0.266, respectively). When more "good" trees are considered the margin from rejection increases further (with 100 next best trees added, P = 0.291 and 0.451, respectively; with 500 next best trees, P = 0.394 and 0.578, respectively). It seems likely that SH tests would fail to reject the alternative trees irrespective of which other trees were included in the test. More familiar (but technically invalid for this question—Goldman, Anderson, and Rodrigo 2000Citation ) individual Kishino-Hasegawa tests also fail to reject the alternative trees (P = 0.155 and 0.221, respectively).

With the expected likelihood weights test, the overall ML tree receives the highest score of the 103 trees included. Alternative tree 3 is included in the 46% confidence set, whereas alternative tree 2 is included in the 82% confidence set. As such, the test does not reject either alternative tree, although given our crude sampling of "reasonable treespace" the result for tree 2 at least should be considered tentative.

Tubulins
The basic topology of all the tubulin trees can be viewed as comprising two regions, an "animals, chytrids (fungi), diplomonads and parabasalids" cluster and a "plants plus other well-recognized groups of protists" cluster (figs. 2 and 3 ). Previously sequenced excavate taxa aside from diplomonads (i.e., Heterolobosea, core jakobids and Malawimonas) mostly fall as separate lineages within the "plant-protist" cluster. But the J. incarcerata {alpha}-tubulin sequence instead falls either as the sister to parabasalids (ML) or to diplomonads + Carpediemonas (MLdist and Pars—see below), always with <50% BS. In the ß-tubulin and combined tubulin analyses Reclinomonas and J. libera form a fairly strong clade (>90% BS with ß-tubulin, 65%–89% with combined tubulin) but fall separately to J. incarcerata. Reclinomonas and J. libera branch separately in {alpha}-tubulin analyses.



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Fig. 2.—Tubulin phylogenies. Excavate taxa are shaded. ML BS values are shown. Unmarked branches have <50% BS. Support values for the Carpediemonas + diplomonads clade with all methods are displayed in the boxes. Estimated-rate scale bar applies for both phylogenies. The phylogenies are unrooted. A. {alpha}-tubulin, ML topology (-ln L = 5011.23). B. ß-tubulin, ML topology (-ln L = 4854.69)

 


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Fig. 3.—Combined {alpha}- + ß-tubulin gene phylogeny. Excavate taxa are shaded. Unrooted ML topology depicted (-ln L = 9652.59). ML BS values are shown. Unmarked branches have <50% BS. Support values for the Carpediemonas + diplomonads clade with all methods are boxed

 
Carpediemonas is placed as the sister group to diplomonads with all data sets and methods examined. This position is very strongly supported (>90% BS) in all {alpha}-tubulin and combined analyses. Support is weaker in the ß-tubulin analyses (51%–71% BS; table 2 ). This diplomonads + Carpediemonas clade clusters with the parabasalids in the ß-tubulin analyses, and, weakly, in the ML analysis of the combined data set (table 2 ). Although not present in the best trees, this clustering also attracts some BS in the MLdist and Pars analyses of the combined data set (table 2 ).


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Table 2 Bootstrap Support for the Carpediemonas + Diplomonads Clade (Diplo, Carpe) and the Parabasalids + Carpediemonas + Diplomonads Clade (Para, Diplo, Carpe) in the Tubulin Analyses

 
The Carpediemonas branches are fairly long but are markedly shorter than those of the hexamitid diplomonads Hexamita and Spironucleus. When the hexamitids are excluded, leaving the less divergent Giardia sequences to represent diplomonads, all methods return very similar best trees. In the {alpha}-tubulin and combined analyses the bootstrap values for the diplomonads + Carpediemonas clade remain very high with all methods (90%+). For ß-tubulin, bootstrap values decline with Pars but improve with MLdist and, particularly, with likelihood (table 2 ). By contrast, BS for a sister group relationship between parabasalids and the diplomonads + Carpediemonas clade declines with all methods or remains negligible in the case of {alpha}-tubulin (table 2 ).


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The Positions of the Excavate Taxa
This study expands the sampling of SSU rRNA genes from excavate taxa such that all recognized major groups are now represented. This is the first molecular data set with such coverage. Our addition of sequences from Carpediemonas gives good (but not comprehensive) coverage of excavate taxa for the {alpha}- and ß-tubulin data sets.

Broadly speaking, our results are congruent with recent molecular phylogenetic studies. The close relationship between retortamonads and diplomonads and between J. libera and R. americana in SSU rRNA gene trees confirms recent examinations with more limited taxon sampling (and unpublished sequences in the latter case) (Cavalier-Smith 2000Citation ; Silberman et al. 2002Citation ). The remoteness of Malawimonas and J. incarcerata from the J. libera + Reclinomonas clade in both SSU rRNA and tubulin trees agrees with recent tubulin phylogenies (Edgcomb et al. 2001Citation ). The marked nonmonophyly of the excavate taxa (forming at least four separated clades) mirrors all previous molecular phylogenetic studies that include several excavate taxa, with one recent, unconfirmed exception (Cavalier-Smith 2002Citation ).

The molecular evidence for polyphyly of the excavate taxa should be viewed cautiously. The branches separating excavate taxa usually receive only weak BS and are often not consistent across different data sets and methods. For example, considering ML analyses, the only branches separating excavate taxa that receive greater than 50% BS are branches in tubulin trees linking Carpediemonas, diplomonads, and either Malawimonas or J. incarcerata (not both) to opisthokonts. There is no measurable support for such relationships with our SSU rRNA data (where opisthokonts instead form a moderately well-supported clade with Cercozoa). When used, statistical tests of alternative tree topologies did not reject trees in which excavate taxa form a clade (with or without Euglenozoa and parabasalids). It is possible that the apparent polyphyly of the excavate taxa is due to analysis artifacts caused by the wide variation in inferred evolutionary rate of the sequences examined. It is also possible that the divergences between some excavate taxa were extremely ancient or rapid, beyond the effective resolving power of our data sets. The disparity between molecular trees indicating polyphyly and morphological arguments that excavate taxa are descended from a common excavate ancestor (Simpson and Patterson 1999Citation ; Simpson, Bernard, and Patterson 2000Citation ) requires further exploration.

The Position of Carpediemonas
Our analyses strongly suggest that Carpediemonas is the closest known relative of diplomonads and retortamonads. The relationship is recovered with a variety of molecular markers, analysis methods, taxon sets, and alignments. For all data sets studied, diplomonads, retortamonads, and Carpediemonas constitute "long branches"; however, several considerations suggest their clustering is not a long-branch attraction artifact. First, other similarly long branches fail to attract Carpediemonas to the exclusion of diplomonads and retortamonads (in some cases where diplomonads are excluded, the retortamonads + Carpediemonas cluster actually falls separately from other long branches—fig. 1B ). Second, in most analyses, support for the position of Carpediemonas is higher with methods expected to be more resilient to long-branch artifacts (e.g., ML with models of among-site rate variation), compared with more susceptible methods (e.g., Pars, K2P distances). If anything, the opposite result might be expected if long-branch attraction was primarily responsible for the position of Carpediemonas. Third, removal of the most divergent diplomonad sequences almost always increases support for the position of Carpediemonas or maintains it at very high levels. Again, it might be expected that support would decrease if the association were due to long-branch attraction.

Before this study, parabasalids were seen by many as the best candidate for a sister group to diplomonads and retortamonads. This proposal was based on several protein phylogenies, none of which included any other excavate taxa except Heterolobosea (Embley and Hirt 1998Citation ; Roger et al. 1998Citation ; Roger 1999Citation ; Baldauf et al. 2000Citation ). With the data sets examined here, parabasalids, at closest, are the sister group to the diplomonads-retortamonads + Carpediemonas clade. Even this position receives support only from the ß-tubulin and combined tubulin data sets. Furthermore, exclusion of the long-branch Spironucleus sequences from these data sets markedly reduces the support for this position for parabasalids, hinting at an influence of long-branch attraction. It would not be surprising if evidence emerged placing other excavate taxa inside the minimal clade containing diplomonads and parabasalids.

The Mitochondrial Status of Diplomonads
Carpediemonas has double membrane-bounded organelles that structurally resemble hydrogenosomes-mitochondria (Simpson and Patterson 1999Citation ). We can envisage two simple scenarios for the symbiotic origin of these organelles. First, they could be descended from the same symbiosis as mitochondria. Second, they could be degenerate descendants of a different prokaryotic symbiont. At present we lack the genetic and biochemical data to directly distinguish between these two possibilities. But similar enigmatic organelles in other amitochondriate eukaryotes, such as the hydrogenosomes of parabasalids, some ciliates and some chytrids, and the mitosome of Entamoeba, have all turned out to be mitochondrial homologs when examined in detail (Roger 1999Citation ; Dyall and Johnson 2000Citation ; van der Giezen et al. 2002Citation ). It would seem most likely that this may also be the case for Carpediemonas. As such, there is now a candidate for a cytological mitochondrial relic in the diplomonads + retortamonads + Carpediemonas clade in addition to the possible genetic relics (e.g., cpn60, IscS) identified previously (Roger et al. 1998Citation ; Horner and Embley 2001Citation ; Tachezy, Sánchez, and Müller 2001Citation ). Provided there was a single origin for the mitochondrial organelle in eukaryotes (Gray, Burger, and Lang 1999Citation ), strong evidence that the Carpediemonas organelle is descended from the same symbiosis would make premitochondrial scenarios for diplomonads (including "transient symbiont" models such as the one suggested by Chihade et al. 2000Citation ) very difficult to sustain.

The Deep-Branching Status of Diplomonads
The molecular phylogenetic argument that diplomonads are "early-diverged" eukaryotes originated with SSU rRNA trees in which Giardia was one of the earliest emerging eukaryote lineages. It has been argued that this position may be an artifact of long-branch attraction (Philippe and Adoutte 1998Citation ). But no previous phylogenetic analysis of "all alignable sites" that we know of has been able to "overcome" the inferred artifact and place diplomonads topologically remote from prokaryote outgroups, even when BS for this basal position, and for the whole tree backbone, is low (as in our analyses; fig. 1A ). This has helped sustain the argument that the diplomonads really are very deep-branching eukaryotes.

Retortamonads and Carpediemonas have markedly less divergent SSU rRNA genes than diplomonads, inviting their use as surrogates for diplomonads in phylogenetic analyses. When diplomonads are excluded, our ML analysis places retortamonads + Carpediemonas deeply embedded within the short-branching eukaryotes, topologically remote from the prokaryotes, although the tree backbone remains poorly supported (fig. 1B ). The consistently deep placement for diplomonads in SSU rRNA trees thus does not seem to be a universal property of the entire diplomonads + retortamonads + Carpediemonas clade, illustrating the weakness of the "diplomonads early" case with this marker.

A Window into the Evolution of Diplomonads
By virtue of its inferred phylogenetic position, Carpediemonas could be an important organism in understanding the evolution of the molecular and cellular attributes of diplomonads. The little that is known about Carpediemonas hints at a much more "typical" eukaryote than are diplomonads or retortamonads. Carpediemonas is free-living, whereas most diplomonads and retortamonads are parasites or endocommensals, and it is possible that the entire clade is ancestrally endobiotic (Siddall, Hong, and Desser 1992Citation ). Carpediemonas has a well-developed endomembrane system, including a normal-looking Golgi apparatus (Simpson and Patterson 1999Citation ), a component that is cryptic in diplomonads and retortamonads (Brugerolle 1991Citation ; Gillin, Reiner, and McCaffery 1996Citation ). Most interestingly, although Carpediemonas lacks classical mitochondria it does possess double membrane-bounded organelles. No similar organelle of any kind has been detected in diplomonads or retortamonads. An intriguing possibility consistent with our phylogenies is that Carpediemonas approaches an intermediate stage in a process of mitochondrion loss by the diplomonad lineage. The limited data available make this suggestion speculative at present. But given the potential for insight into the process by which mitochondria may have been discarded by the canonical amitochondriate eukaryotes, greater attention to the evolutionary position and biology of Carpediemonas is well warranted.

Supplementary Information
The sequences reported here have been deposited with GenBank (accession nos. AY117416–AY117422).


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Thanks to M. Holder for computational assistance and to J. Andersson and J. Dacks for critical comments. This work was supported by a NAI Astrobiology grant NCC2-1054 and a NASA Exobiology Grant NG5-4895 to M.L.S., an Australia Research Council grant to D.J.P., and a NSERC (Canada) operating grant 227085-00 to A.J.R. A.G.B.S. is the recipient of a Canadian Institutes of Health Research postdoctoral fellowship. J.D.S. is supported by the NASA Astrobiology Institute.


    Footnotes
 
Geoffrey McFadden, Reviewing Editor

1 Research conducted at: The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, and at the Department of Biochemistry, Dalhousie University. Back

Abbreviations: BS, bootstrap support; K2P, Kimura two parameter model; ML, maximum-likelihood; MLdist, maximum-likelihood distance; Pars, parsimony; SSU rRNA, small subunit ribosomal RNA; TBR, tree bisection-reconnection. Back

Keywords: Archezoa diplomonads jakobids eukaryote evolution mitochondria protists Back

Address for correspondence and reprints: Alastair G. B. Simpson, Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7. simpson{at}hades.biochem.dal.ca Back


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Accepted for publication June 11, 2002.