Toward resolving the eukaryotic tree: the phylogenetic positions of jakobids and cercozoans

Resolving the global phylogeny of eukaryotes has proven to be challenging. Among the eukaryotic groups of uncertain phylogenetic position are jakobids, a group of bacterivorous flagellates that possess the most bacteria-like mitochondrial genomes known. Jakobids share several ultrastructural features with malawimonads and an assemblage of anaerobic protists (e.g., diplomonads and oxymonads). These lineages together with Euglenozoa and Heterolobosea have collectively been designated "excavates". However, published molecular phylogenies based on the sequences of nuclear rRNAs and up to six nucleus-encoded proteins do not provide convincing support for the monophyly of excavates, nor do they uncover their relationship to other major eukaryotic groups. Here, we report the first large-scale eukaryotic phylogeny, inferred from 143 nucleus-encoded proteins comprising 31,604 amino acid positions, that includes jakobids, malawimonads and cercozoans. We obtain compelling support for the monophyly of jakobids, Euglenozoa plus Heterolobosea (JEH group), and for the association of cercozoans with stramenopiles plus alveolates. Furthermore, we observe a sister-group relationship between the JEH group and malawimonads after removing fast-evolving species from the dataset. We discuss the implications of these results for the concept of "excavates" and for the elucidation of eukaryotic phylogeny in general.     

Evolutionary relationships among "jakobid" flagellates as indicated by alpha- and beta-tubulin phylogenies

Jakobids are free-living, heterotrophic flagellates that might represent early-diverging mitochondrial protists. They share ultrastructural similarities with eukaryotes that occupy basal positions in molecular phylogenies, and their mitochondrial genome architecture is eubacterial-like, suggesting a close affinity with the ancestral alpha-proteobacterial symbiont that gave rise to mitochondria and hydrogenosomes. To elucidate relationships among jakobids and other early-diverging eukaryotic lineages, we characterized alpha- and beta-tubulin genes from four jakobids: Jakoba libera, Jakoba incarcerata, Reclinomonas americana (the "core jakobids"), and Malawimonas jakobiformis. These are the first reports of nuclear genes from these organisms. Phylogenies based on alpha-, beta-, and combined alpha- plus beta-tubulin protein data sets do not support the monophyly of the jakobids. While beta-tubulin and combined alpha- plus beta-tubulin phylogenies showed a sister group relationship between J. libera and R. americana, the two other jakobids, M. jakobiformis and J. incarcerata, had unclear affinities. In all three analyses, J. libera, R. americana, and M. jakobiformis emerged from within a well-supported large "plant-protist" clade that included plants, green algae, cryptophytes, stramenopiles, alveolates, Euglenozoa, Heterolobosea, and several other protist groups, but not animals, fungi, microsporidia, parabasalids, or diplomonads. A preferred branching order within the plant-protist clade was not identified, but there was a tendency for the J. libera-R. americana lineage to group with a clade made up of the heteroloboseid amoeboflagellates and euglenozoan protists. Jakoba incarcerata branched within the plant-protist clade in the beta- and the combined alpha- plus beta-tubulin phylogenies. In alpha- tubulin trees, J. incarcerata occupied an unresolved position, weakly grouping with the animal/fungal/microsporidian group or with amitochondriate parabasalid and diplomonad lineages, depending on the phylogenetic method employed. Tubulin gene phylogenies were in general agreement with mitochondrial gene phylogenies and ultrastructural data in indicating that the "jakobids" may be polyphyletic. Relationships with the putatively deep-branching amitochondriate diplomonads remain uncertain.     

The chaperonin genes of jakobid and jakobid-like flagellates: implications for eukaryotic evolution

The jakobids are free-living mitochondriate protists that share ultrastructural features with certain amitochondriate groups and possess the most bacterial-like mitochondrial genomes described thus far. Jakobids belong to a diverse group of mitochondriate and amitochondriate eukaryotes, the excavate taxa. The relationships among the various excavate taxa and their relationships to other putative deep-branching protist groups are largely unknown. With the hope of clarifying these issues, we have isolated the cytosolic chaperonin CCTalpha gene from the jakobid Reclinomonas americana (strains 50394 and 50283), the jakobid-like malawimonad Malawimonas jakobiformis, two heteroloboseans (Acrasis rosea and Naegleria gruberi), a euglenozoan (Trypanosoma brucei), and a parabasalid (Monocercomonas sp.). We also amplified the CCTdelta gene from M. jakobiformis. The Reclinomonas and Malawimonas sequences presented here are among the first nuclear protein-coding genes to be described from these organisms. Unlike other putative early diverging protist lineages, a high density of spliceosomal introns was found in the jakobid and malawimonad CCTs-similar to that observed in vertebrate protein-coding genes. An analysis of intron positions in CCT genes from protists, plants, animals, and fungi suggests that many of the intron-sparse or intron-lacking protist lineages may not be primitively so but have lost spliceosomal introns during their evolutionary history. In phylogenetic trees constructed from CCTalpha protein sequences, R. americana (but not M. jakobiformis) shows a weak but consistent affinity for the Heterolobosea and Euglenozoa.     

Lateral transfer of the gene for a widely used marker, alpha-tubulin, indicated by a multi-protein study of the phylogenetic position of Andalucia (Excavata)

alpha-Tubulin is one of the most widely used markers for estimating deep-level phylogenetic relationships amongst eukaryotes. We sequenced 6-7 nuclear protein-coding genes, including alpha-tubulin, from the two described species of the enigmatic jakobid(-like) excavate protist Andalucia. Concatenated protein phylogenies place Andalucia in a clade with other jakobids, Euglenozoa and Heterolobosea. Individual gene trees, except that of alpha-tubulin, do not conflict strongly with this position. In alpha-tubulin trees, Andalucia instead falls in a strongly supported clade with diplomonads, parabasalids and opisthokonts (including animals and fungi), and branches with diplomonads. This clade is robust to changes in taxon sampling, and is unlikely to represent long-branch attraction, compositional heterogeneity artefact, or segmental gene conversion. Phylogenies estimated without alpha-tubulin strongly support the original position for Andalucia, and also reinforce recent studies in placing diplomonads and parabasalids with Preaxostyla, not opisthokonts. alpha-Tubulin seems to have experienced two or more eukaryote-to-eukaryote lateral gene transfer (LGT) events, one perhaps from an ancestral opisthokont to an ancestor of diplomonads and parabasalids, or vice versa, and one probably from the diplomonad lineage to Andalucia. Like EF-1alpha/EFL, alpha-tubulin has a complex history that needs to be taken into account when using this marker for deep-level phylogenetic inference.     

Evolutionary history of "early-diverging" eukaryotes: the excavate taxon Carpediemonas is a close relative of Giardia

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, beta-tubulin, and combined alpha- + beta-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.     

Inference of the phylogenetic position of oxymonads based on nine genes: support for metamonada and excavata

Circumscribing major eukaryote groups and resolving higher order relationships between them are among the most challenging tasks facing molecular evolutionists. Recently, evidence suggesting a new supergroup (the Excavata) comprising a wide array of flagellates has been collected. This group consists of diplomonads, retortamonads, Carpediemonas, heteroloboseans, Trimastix, jakobids, and Malawimonas, all of which possess a particular type of ventral feeding groove that is proposed to be homologous. Euglenozoans, parabasalids, and oxymonads have also been associated with Excavata as their relationships to one or more core excavate taxa were demonstrated. However, the main barrier to the general acceptance of Excavata is that its existence is founded primarily on cytoskeletal similarities, without consistent support from molecular phylogenetics. In gene trees, Excavata are typically not recovered together. In this paper, we present an analysis of the phylogenetic position of oxymonads (genus Monocercomonoides) based on concatenation of eight protein sequences (alpha-tubulin, beta-tubulin, gamma-tubulin, EF-1alpha, EF-2, cytosolic (cyt) HSP70, HSP90, and ubiquitin) and 18S rRNA. We demonstrate that the genes are in conflict regarding the position of oxymonads. Concatenation of alpha- and beta-tubulin placed oxymonads in the plant-chromist part of the tree, while the concatenation of other genes recovered a well-supported group of Metamonada (oxymonads, diplomonads, and parabasalids) that branched weakly with euglenozoans--connecting all four excavates included in the analyses and thus providing conditional support for the existence of Excavata.     

Centrohelida is still searching for a phylogenetic home: analyses of seven Raphidiophrys contractilis genes

By recent advance in evolutionary biology, the majority of eukaryotes are classified into six eukaryotic assemblages called as "supergroups". However, several eukaryotic groups show no clear evolutionary affinity to any of the six supergroups. Centrohelida, one of major heliozoan groups, are such an unresolved lineage. In this study, we newly determined the genes encoding translation elongation factor 2 (EF2), cytosolic heat shock protein 70 (HSP70), and cytosolic heat shock protein 90 (HSP90) from the centroheliozoan Raphidiophrys contractilis. The three Raphidiophrys genes were then combined with previously determined actin, alpha-tubulin, beta-tubulin, and SSU rRNA sequences to phylogenetically analyze the position of Centrohelida in global eukaryotic phylogeny. Although the multi-gene data sets examined in this study are the largest ones including the centroheliozoan sequences, the relationships between Centrohelida and the eukaryotic groups considered were unresolved. Our careful investigation revealed that the phylogenetic estimates were highly sensitive to genes included in the multi-gene alignment. The signal of SSU rRNA and that of alpha-tubulin appeared to conflict with one another: the former strongly prefers a monophyly of Diplomonadida (e.g., Giardia), Parabasalia (e.g., Trichomonas), Heterolobosea (e.g., Naegleria), and Euglenozoa (e.g., Trypanosoma), while the latter unites Diplomonadida, Parabasalia, Metazoa, and Fungi. In addition, EF2 robustly unites Rhodophyta and Viridiplantae, while the remaining genes considered in this study do not positively support the particular relationship. Thus, it is difficult to identify the phylogenetic relatives of Centrohelida in the present study, since strong (and some are conflicting) gene-specific "signals" are predominant in the current multi-gene data. We concluded that larger scale multi-gene phylogenies are necessary to elucidate the origin and evolution of Centrohelida.     

On Core Jakobids and Excavate Taxa: The Ultrastructure of Jakoba incarcerata

The cellular organisation of the 'excavate' flagellate Jakoba incarcerata Bernard, Simpson and Patterson 2000 is described. Cells have one nucleus and dictyosome. The putative mitochondria lack cristae. Two flagella (anterior and posterior) insert anterior to the feeding groove. The posterior flagellum bears a dorsal vane. An 'anterior' microtubular root arises against the anterior basal body. Two main microtubular roots, left and right, and a singlet 'root' arise around the posterior basal body and support the groove. Non-microtubular fibres termed 'A', 'B', 'I', and 'composite' associate with the right root. A multilaminar 'C' fibre associates with the left root. The cytoskeleton of J. incarcerata indicates a common ancestry with other excavate taxa (i.e. diplomonads, retortamonads, heteroloboseids, 'core jakobids', Malawimonas, Carpediemonas, and Trimastix). Overall, J. incarcerata is most similar to (other) core jakobids, namely Jakoba libera, Reclinomonas, and Histiona. We regard J. incarcerata as a core jakobid and identify the group by the synapomorphy 'vanes restricted to dorsal side of the posterior flagellum'. The anterior root and position of the B fibre (and presence of dense inclusions in the cartwheels and a conscpicuous singlet root-associated fibre) in J. incarcerata are novel for core jakobids and argue for close relationships with Trimastix and/or Heterolobosea. The C fibre is similar in substructure to the costal fibre of parabasalids and it is possible that the structures are homologous.     

Mitochondrial-type hsp70 genes of the amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and two microsporidians

Genes encoding putative mitochondrial-type heat shock protein 70 (mit-hsp70) were isolated and sequenced from amitochondriate protists, Giardia intestinalis, Entamoeba histolytica, and two microsporidians, Encephalitozoon hellem and Glugea plecoglossi. The deduced mit-hsp70 sequences were analyzed by sequence alignments and phylogenetic reconstructions. The mit-hsp70 sequence of these four amitochondriate protists were divergent from other mit-hsp70 sequences of mitochondriate eukaryotes. However, all of these sequences were clearly located within a eukaryotic mitochondrial clade in the tree including various type hsp70 sequences, supporting the emerging notion that none of these amitochondriate lineages are primitively amitochodrial, but lost their mitochondria secondarily in their evolutionary past.     

Oxymonads are closely related to the excavate taxon Trimastix

Despite intensive study in recent years, large-scale eukaryote phylogeny remains poorly resolved. This is particularly problematic among the groups considered to be potential early branches. In many recent systematic schemes for early eukaryotic evolution, the amitochondriate protists oxymonads and Trimastix have figured prominently, having been suggested as members of many of the putative deep-branching higher taxa. However, they have never before been proposed as close relatives of each other. We amplified, cloned, and sequenced small-subunit ribosomal RNA genes from the oxymonad Pyrsonympha and from several Trimastix isolates. Rigorous phylogenetic analyses indicate that these two protist groups are sister taxa and are not clearly related to any currently established eukaryotic lineages. This surprising result has important implications for our understanding of cellular evolution and high-level eukaryotic phylogeny. Given that Trimastix contains small, electron-dense bodies strongly suspected to be derived mitochondria, this study constitutes the best evidence to date that oxymonads are not primitively amitochondriate. Instead, Trimastix and oxymonads may be useful organisms for investigations into the evolution of the secondary amitochondriate condition. All higher taxa involving either oxymonads or Trimastix may require modification or abandonment. Affected groups include four contemporary taxa given the rank of phylum (Metamonada, Loukozoa, Trichozoa, Percolozoa), and the informal excavate taxa. A new "phylum-level" taxon may be warranted for oxymonads and Trimastix.     

Primary Structure and Phylogenetic Relationships of a Malate Dehydrogenase Gene from Giardia lamblia

The lactate and malate dehydrogenases comprise a complex protein superfamily with multiple enzyme homologues found in eubacteria, archaebacteria, and eukaryotes. In this study we describe the sequence and phylogenetic relationships of a malate dehydrogenase (MDH) gene from the amitochondriate diplomonad protist, Giardia lamblia. Parsimony, distance, and maximum-likelihood analyses of the MDH protein family solidly position G. lamblia MDH within a eukaryote cytosolic MDH clade, to the exclusion of chloroplast, mitochondrial, and peroxisomal homologues. Furthermore, G. lamblia MDH is specifically related to a homologue from Trichomonas vaginalis. This MDH topology, together with published phylogenetic analyses of β-tubulin, chaperonin 60, valyl-tRNA synthetase, and EF-1α, suggests a sister-group relationship between diplomonads and parabasalids. Since these amitochondriate lineages contain genes encoding proteins which are characteristic of mitochondria and α-proteobacteria, their shared ancestry suggests that mitochondrial properties were lost in the common ancestor of both groups. Received: 14 September 1998 / Accepted: 29 December 1998     

Ultrastructure and molecular phylogeny of Stephanopogon minuta: an enigmatic microeukaryote from marine interstitial environments

Although Stephanopogon was described as a putative ciliate more than a century ago, its phylogenetic position within eukaryotes has remained unclear because of an unusual combination of morphological characteristics (e.g. a highly multiflagellated cell with discoidal mitochondrial cristae). Attempts to classify Stephanopogon have included placement with the Ciliophora, the Euglenozoa, the Heterolobosea and the Rhizaria. Most systematists have chosen, instead, to conservatively classify Stephanopogon as incertae sedis within eukaryotes. Despite the obvious utility of molecular phylogenetic data in resolving this issue, DNA sequences from Stephanopogon have yet to be published. Accordingly, we characterized the molecular phylogeny and ultrastructure of Stephanopogon minuta, a species we isolated from marine sediments in southern British Columbia, Canada. Our results showed that S. minuta shares several features with heteroloboseans, such as discoidal mitochondrial cristae, a heterolobosean-specific (17_1 helix) insertion in the small subunit ribosomal RNA gene (SSU rDNA) and the lack of canonical Golgi bodies. Molecular phylogenetic analyses of SSU rDNA demonstrated that S. minuta branches strongly within the Heterolobosea and specifically between two different tetraflagellated lineages, both named 'Percolomonas cosmopolitus.' Several ultrastructural features shared by S. minuta and P. cosmopolitus reinforced the molecular phylogenetic data and confirmed that Stephanopogon is a highly divergent multiflagellated heterolobosean that represents an outstanding example of convergent evolution with benthic eukaryovorous ciliates (Alveolata).     

Molecular phylogenies of Parabasalia inferred from four protein genes and comparison with rRNA trees

The molecular phylogeny of parabasalids has mainly been inferred from small subunit (SSU) rRNA sequences and has conflicted substantially with systematics based on morphological and ultrastructural characters. This raises the important question, how congruent are protein and SSU rRNA trees? New sequences from seven diverse parabasalids (six trichomonads and one hypermastigid) were added to data sets of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase, alpha-tubulin and beta-tubulin and used to construct phylogenetic trees. The GAPDH tree was well resolved and identical in topology to the SSU rRNA tree. This both validates the rRNA tree and suggests that GAPDH should be a valuable tool in further phylogenetic studies of parabasalids. In particular, the GAPDH tree confirmed the polyphyly of Monocercomonadidae and Trichomonadidae and the basal position of Trichonympha agilis among parabasalids. Moreover, GAPDH strengthened the hypothesis of secondary loss of cytoskeletal structures in Monocercomonadidae such as Monocercomonas and Hypotrichomonas. In contrast to GAPDH, the enolase and both tubulin trees are poorly resolved and rather uninformative about parabasalian phylogeny, although two of these trees also identify T. agilis as representing the basal-most lineage of parabasalids. Although all four protein genes show multiple gene duplications (for 3-6 of the seven taxa examined), most duplications appear to be relatively recent (i.e., species-specific) and not a problem for phylogeny reconstruction. Only for enolase are there more ancient duplications that may confound phylogenetic interpretation.     

Cryptic diversity of free-living parabasalids, Pseudotrichomonas keilini and Lacusteria cypriaca n. g., n. sp., as inferred from small subunit rDNA sequences

Ultrastructural and molecular phylogenetic evidence indicate that the Parabasalia consists of seven main subgroups: the Trichomonadida, Honigbergiellida, Hypotrichomonadida, Tritrichomonadida, Cristamonadida, Spirotrichonymphida, and Trichonymphida. Only five species of free-living parabasalids are known: Monotrichomonas carabina, Ditrichomonas honigbergii, Honigbergiella sp., Tetratrichomonas undula, and Pseudotrichomonas keilini. Phylogenetic analyses show that free-living species do not form a clade and instead branch in several different positions within the context of their parasitic relatives. Because the diversity of free-living parabasalids is poorly understood, the systematics of these lineages is in a significant state of disarray. In order to better understand the phylogenetic distribution of free-living parabasalids, we sequenced the small subunit rDNA from three different strains reminiscent of P. keilini; the strains were isolated from different geographical locations: (1) mangrove sediments in Japan and (2) sediments in Cyprus. These data demonstrated that the free-living parabasalids P. keilini and Lacusteria cypriaca n. g., n. sp., form a paraphyletic assemblage near the origin of a clade consisting mostly of parasitic trichomonadids (e.g. Trichomonas vaginalis). This paraphyletic distribution of similar morphotypes indicates that free-living trichomonadids represent a compelling example of morphostasis that provides insight into the suite of features present in the most recent free-living ancestor of their parasitic relatives.     

Gene transfers from nanoarchaeota to an ancestor of diplomonads and parabasalids

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.     

Phylogenetic positions of several amitochondriate protozoa

Several groups of parasitic protozoa, as represented by Giardia, Trichomonas, Entamoeba and Microsporida, were once widely considered to be the most primitive extant eukaryotic group―Archezoa. The main evidence for this is their ‘lacking mitochondria’ and possessing some other primitive features between prokaryotes and eukaryotes, and being basal to all eukaryotes with mitochondria in phylogenies inferred from many molecules. Some authors even proposed that these organisms diverged before the endosymbiotic origin of mitochondria within eukaryotes. This view was once considered to be very significant to the study of origin and evolution of eukaryotic cells (eukaryotes). However, in recent years this has been challenged by accumulating evidence from new studies. Here the sequences of DNA topoisomerase II in G. lamblia, T. vaginalis and E. histolytica were identified first by PCR and sequencing, then combining with the sequence data of the microsporidia Encephalitozoon cunicul and other eukaryotic groups of different evolutionary positions from GenBank, phylogenetic trees were constructed by various methods to investigate the evolutionary positions of these amitochondriate protozoa. Our results showed that since the characteristics of DNA topoisomerase II make it avoid the defect of ‘long-branch attraction’ appearing in the previous phylogenetic analyses, our trees can not only reflect effectively the relationship of different major eukaryotic groups, which is widely accepted, but also reveal phylogenetic positions for these amitochondriate protozoa, which is different from the previous phylogenetic trees. They are not the earliest-branching eukaryotes, but diverged after some mitochondriate organisms such as kinetoplastids and mycetozoan; they are not a united group but occupy different phylogenetic positions. Combining with the recent cytological findings of mitochondria-like organelles in them, we think that though some of them (e.g. diplo-monads, as represented by Giardia) may occupy a very low evolutionary position, generally these organisms are not as extremely primitive as was thought before; they should be poly-phyletic groups diverging after the endosymbiotic origin of mitochondrion to adapt themselves to anaerobic parasitic life.     

Phylogenetic positions of several amitochondriate protozoa-Evidence from phylogenetic analysis of DNA topoisomerase II

Several groups of parasitic protozoa, as represented by Giardia, Trichomonas, Entamoeba and Microsporida, were once widely considered to be the most primitive extant eukaryotic group―Archezoa. The main evidence for this is their 'lacking mitochondria' and possessing some other primitive features between prokaryotes and eukaryotes, and being basal to all eukaryotes with mitochondria in phylogenies inferred from many molecules. Some authors even proposed that these organisms diverged before the endosymbiotic origin of mitochondria within eukaryotes. This view was once considered to be very significant to the study of origin and evolution of eukaryotic cells (eukaryotes). However, in recent years this has been challenged by accumulating evidence from new studies. Here the sequences of DNA topoisomerase II in G. lamblia, T. vaginalis and E. histolytica were identified first by PCR and sequencing, then combining with the sequence data of the microsporidia Encephalitozoon cunicul and other eukaryotic groups of different evolutionary positions from GenBank, phylogenetic trees were constructed by various methods to investigate the evolutionary positions of these amitochondriate protozoa. Our results showed that since the characteristics of DNA topoisomerase II make it avoid the defect of 'long-branch attraction' appearing in the previous phylogenetic analyses, our trees can not only reflect effectively the relationship of different major eukaryotic groups, which is widely accepted, but also reveal phylogenetic positions for these amitochondriate protozoa, which is different from the previous phylogenetic trees. They are not the earliest-branching eukaryotes, but diverged after some mitochondriate organisms such as kinetoplastids and mycetozoan; they are not a united group but occupy different phylogenetic positions. Combining with the recent cytological findings of mitochondria-like organelles in them, we think that though some of them (e.g. diplomonads, as represented by Giardia) may occupy a very low evolutionary position, generally these organisms are not as extremely primitive as was thought before; they should be polyphyletic groups diverging after the endosymbiotic origin of mitochondrion to adapt themselves to anaerobic parasitic life.     

Euglena gracilis and Trypanosomatids Possess Common Patterns in Predicted Mitochondrial Targeting Presequences

Euglena gracilis possessing chloroplasts of secondary green algal origin and parasitic trypanosomatids Trypanosoma brucei, Trypanosoma cruzi and Leishmania major belong to the protist phylum Euglenozoa. Euglenozoa might be among the earliest eukaryotic branches bearing ancestral traits reminiscent of the last eukaryotic common ancestor (LECA) or missing features present in other eukaryotes. LECA most likely possessed mitochondria of endosymbiotic ??-proteobacterial origin. In this study, we searched for the presence of homologs of mitochondria-targeted proteins from other organisms in the currently available EST dataset of E. gracilis. The common motifs in predicted N-terminal presequences and corresponding homologs from T. brucei, T. cruzi and L. major (if found) were analyzed. Other trypanosomatid mitochondrial protein precursor (e.g., those involved in RNA editing) were also included in the analysis. Mitochondrial presequences of E. gracilis and these trypanosomatids seem to be highly variable in sequence length (5?C118 aa), but apparently share statistically significant similarities. In most cases, the common (M/L)RR motif is present at the N-terminus and it is probably responsible for recognition via import apparatus of mitochondrial outer membrane. Interestingly, this motif is present inside the predicted presequence region in some cases. In most presequences, this motif is followed by a hydrophobic region rich in alanine, leucine, and valine. In conclusion, either RR motif or arginine-rich region within hydrophobic aa-s present at the N-terminus of a preprotein can be sufficient signals for mitochondrial import irrespective of presequence length in Euglenozoa.     

A high density of ancient spliceosomal introns in oxymonad excavates

Background  

Certain eukaryotic genomes, such as those of the amitochondriate parasites Giardia and Trichomonas, have very low intron densities, so low that canonical spliceosomal introns have only recently been discovered through genome sequencing. These organisms were formerly thought to be ancient eukaryotes that diverged before introns originated, or at least became common. Now however, they are thought to be members of a supergroup known as excavates, whose members generally appear to have low densities of canonical introns. Here we have used environmental expressed sequence tag (EST) sequencing to identify 17 genes from the uncultivable oxymonad Streblomastix strix, to survey intron densities in this most poorly studied excavate group.