Phylogenetic positions of several amitochondriate protozoa

Several groups of parasitic protozoa, as represented by Giardia, Trichomonas, En-tamoeba 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.     

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.     

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 Analysis of the Complete Genome Sequence of <Emphasis Type="Italic">Encephalitozoon cuniculi</Emphasis> Supports the Fungal Origin of Microsporidia and Reveals a High Frequency of Fast-Evolving Genes

Microsporidia are unicellular eukaryotes living as obligate intracellular parasites. Lacking mitochondria, they were initially considered as having diverged before the endosymbiosis at the origin of mitochondria. That microsporidia were primitively amitochondriate was first questioned by the discovery of microsporidial sequences homologous to genes encoding mitochondrial proteins and then refuted by the identification of remnants of mitochondria in their cytoplasm. Various molecular phylogenies also cast doubt on the early divergence of microsporidia, these organisms forming a monophyletic group with or within the fungi. The 2001 proteins putatively encoded by the complete genome of Encephalitozoon cuniculi provided powerful data to test this hypothesis. Phylogenetic analysis of 99 proteins selected as adequate phylogenetic markers indicated that the E. cuniculi sequences having the lowest evolutionary rates preferentially clustered with fungal sequences or, more rarely, with both animal and fungal sequences. Because sequences with low evolutionary rates are less sensitive to the long-branch attraction artifact, we concluded that microsporidia are evolutionarily related to fungi. This analysis also allowed comparing the accuracy of several phylogenetic algorithms for a fast-evolving lineage with real rather than simulated sequences.This article contains online supplementary material.Reviewing Editor: Dr. Wen-Hsiung LiSupplementary material is available at     

Early branching eukaryotes?

Recent phylogenetic analyses suggest that Giardia, Trichomonas and Microsporidia contain genes of mitochondrial origin and are thus unlikely to be primitively amitochondriate as previously thought. Furthermore, phylogenetic analyses of multiple data sets suggest that Microsporidia are related to Fungi rather than being deep branching as depicted in trees based upon SSUrRNA analyses. There is also room for doubt, on the basis of a lack of consistent support from analyses of other genes, whether Giardia or Trichomonas branch before other eukaryotes. So, at present, we cannot be sure which eukaryotes are descendants of the earliest-branching organisms in the eukaryote tree. Future resolution of the order of emergence of eukaryotes will depend upon a more critical phylogenetic analysis of new and existing data than hitherto. Hypotheses of branching order should preferably be based upon congruence between independent data sets, rather than on single gene trees.     

Evaluating hypotheses for the origin of eukaryotes

Numerous scenarios explain the origin of the eukaryote cell by fusion or endosymbiosis between an archaeon and a bacterium (and sometimes a third partner). We evaluate these hypotheses using the following three criteria. Can the data be explained by the null hypothesis that new features arise sequentially along a stem lineage? Second, hypotheses involving an archaeon and a bacterium should undergo standard phylogenetic tests of gene distribution. Third, accounting for past events by processes observed in modern cells is preferable to postulating unknown processes that have never been observed. For example, there are many eukaryote examples of bacteria as endosymbionts or endoparasites, but none known in archaea. Strictly post‐hoc hypotheses that ignore this third criterion should be avoided. Applying these three criteria significantly narrows the number of plausible hypotheses. Given current knowledge, our conclusion is that the eukaryote lineage must have diverged from an ancestor of archaea well prior to the origin of the mitochondrion. Significantly, the absence of ancestrally amitochondriate eukaryotes (archezoa) among extant eukaryotes is neither evidence for an archaeal host for the ancestor of mitochondria, nor evidence against a eukaryotic host. BioEssays 29: 74–84, 2007. © 2006 Wiley Periodicals, Inc.     

Retortamonad flagellates are closely related to diplomonads--implications for the history of mitochondrial function in eukaryote evolution

We present the first molecular phylogenetic examination of the evolutionary position of retortamonads, a group of mitochondrion-lacking flagellates usually found as commensals of the intestinal tracts of vertebrates. Our phylogenies include small subunit ribosomal gene sequences from six retortamonad isolates-four from mammals and two from amphibians. All six sequences were highly similar (95%-99%), with those from mammals being almost identical to each other. All phylogenetic methods utilized unequivocally placed retortamonads with another amitochondriate group, the diplomonads. Surprisingly, all methods weakly supported a position for retortamonads cladistically within diplomonads, as the sister group to Giardia. This position would conflict with a single origin and uniform retention of the doubled-cell organization displayed by most diplomonads, but not by retortamonads. Diplomonad monophyly was not rejected by Shimodaira-Hasegawa, Kishino-Hasegawa, and expected likelihood weights methods but was marginally rejected by parametric bootstrapping. Analyses with additional phylogenetic markers are needed to test this controversial branching order within the retortamonad + diplomonad clade. Nevertheless, the robust phylogenetic association between diplomonads and retortamonads suggests that they share an amitochondriate ancestor. Because strong evidence indicates that diplomonads have secondarily lost their mitochondria (rather than being ancestrally amitochondriate), our results imply that retortamonads are also secondarily amitochondriate. Of the various groups of eukaryotes originally suggested to be primitively amitochondriate under the archezoa hypothesis, all have now been found to have physical or genetic mitochondrial relics (or both) or form a robust clade with an organism with such a relic.     

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     

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.     

Mitochondrial type iron-sulfur cluster assembly in the amitochondriate eukaryotes Trichomonas vaginalis and Giardia intestinalis, as indicated by the phylogeny of IscS

Pyridoxal-5'-phosphate-dependent cysteine desulfurase (IscS) is an essential enzyme in the assembly of FeS clusters in bacteria as well as in the mitochondria of eukaryotes. Although FeS proteins are particularly important for the energy metabolism of amitochondrial anaerobic eukaryotes, there is no information about FeS cluster formation in these organisms. We identified and sequenced two IscS homologs of Trichomonas vaginalis (TviscS-1 and TviscS-2) and one of Giardia intestinalis (GiiscS). TviscS-1, TviscS-2, and GiiscS possess the typical conserved regions implicated in cysteine desulfurase activity. N-termini of TviscS-1 and TviscS-2 possess eight amino acid extensions, which resemble the N-terminal presequences that target proteins to hydrogenosomes in trichomonads. No presequence was evident in GiiscS from Giardia, an organism that apparently lacks hydrogenosmes or mitochondria. Phylogenetic analysis showed a close relationship among all eukaryotic IscS genes including those of amitochondriates. IscS of proteobacteria formed a sister group to the eukaryotic clade, suggesting that isc-related genes were present in the proteobacterial endosymbiotic ancestor of mitochondria and hydrogenosomes. NifS genes of nitrogen-fixing bacteria, which are IscS homologs required for specific formation of FeS clusters in nitrogenase, formed a more distant group. The phylogeny indicates the presence of a common mechanism for FeS cluster formation in mitochondriates as well as in amitochondriate eukaryotes. Furthermore, the analyses support a common origin of Trichomonas hydrogenosomes and mitochondria, as well as secondary loss of mitochondrion/hydrogenosome-like organelles in Giardia.     

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.     

Comparative Analysis of the Ribosomal Componentsof the Hydrogenosome-Containing Protist, Trichomonas vaginalis

The ribosomes of the amitochondriate but hydrogenosome-containing protist lineage, the trichomonads, have previously been reported to be prokaryotic or primitive eukaryotic, based on evidence that they have a 70S sedimentation coefficient and a small number of proteins, similar to prokaryotic ribosomes. In order to determine whether the components of the trichomonad ribosome indeed differ from those of typical eukaryotic ribosomes, the ribosome of a representative trichomonad, Trichomonas vaginalis, was characterized. The sedimentation coefficient of the T. vaginalis ribosome was smaller than that of Saccharomyces cerevisiae and larger than that of Escherichia coli. Based on two-dimensional PAGE analysis, the number of different ribosomal proteins was estimated to be approximately 80. This number is the same as those obtained for typical eukaryotes (approximately 80) but larger than that of E. coli (approximately 55). N-Terminal amino acid sequencing of 18 protein spots and the complete sequences of 4 ribosomal proteins as deduced from their genes revealed these sequences to display typical eukaryotic features. Phylogenetic analyses of the five ribosomal proteins currently available also clearly confirmed that the T. vaginalis sequences are positioned within a eukaryotic clade. Comparison of deduced secondary structure models of the small and large subunit rRNAs of T. vaginalis with those of other eukaryotes revealed that all helices commonly found in typical eukaryotes are present and conserved in T. vaginalis, while variable regions are shortened or lost. These lines of evidence demonstrate that the T. vaginalis ribosome has no prokaryotic or primitive eukaryotic features but is clearly a typical eukaryotic type.     

Biogenesis of iron-sulphur clusters in amitochondriate and apicomplexan protists

During the last 4 years there has been an enormous interest in the question how iron-sulphur ([Fe-S]) clusters, which are essential building blocks for life, are synthesised and assembled into apo-proteins, both in prokaryotes and in eukaryotes. The emerging picture is that the basic mechanism of this pathway has been well conserved during evolution. In yeast and probably all other eukaryotes the mitochondrion is the place where [Fe-S] clusters are synthesised, even for extramitochondrial [Fe-S] cluster-containing proteins, and a number of proteins have been functionally characterised to a certain extent within this pathway. However, almost nothing is known about this aspect in parasitic protists, although recent studies of amitochondriate protists and on the plastid-like organelle of apicomplexan parasites, the apicoplast, have started to change this. In this article I will summarise the current view of [Fe-S] cluster biogenesis in eukaryotes and discuss its implications for amitochondriate protists and for the plastid-like organelle of apicomplexan parasites.     

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.     

Microsporidia: a journey through radical taxonomical revisions

Microsporidia are obligate intracellular parasites of medical and commercial importance, characterized by a severe reduction, or even absence, of cellular components typical of eukaryotes such as mitochondria, Golgi apparatus and flagella. This simplistic cellular organization has made it difficult to infer the evolutionary relationship of Microsporidia to other eukaryotes, because they lack many characters historically used to make such comparisons. Eventually, it was suggested that this simplicity might be due to Microsporidia representing a very early eukaryotic lineage that evolved prior to the origin of many typically eukaryotic features, in particular the mitochondrion. This hypothesis was supported by the first biochemical and molecular studies of the group. In the last decade, however, contrasting evidence has emerged, mostly from molecular sequences, that show Microsporidia are related to fungi, and it is now widely acknowledged that features previously recognized as primitive are instead highly derived adaptations to their obligate parasitic lifestyle. The various sharply differing views on microsporidian evolution resulted in several radical reappraisals of their taxonomy. Here we will chronologically review the causes and consequences for these taxonomic revisions, with a special emphasis on why the unique cellular and genomic features of Microsporidia lured scientists towards the wrong direction for so long.     

微孢子虫系统进化研究的变迁与展望

微孢子虫(Microsporidia)是一类专性细胞内寄生的单细胞真核生物,在科研、医疗、农业、商业等领域具有重要影响。由于其不具有某些典型的真核生物细胞结构,如线粒体、过氧化物酶体、高尔基体、鞭毛,曾将其归属于古真核生物谱系,认为其进化历程先于这些细胞器的起源,该假说也得到了一些生物化学和分子生物学研究证据的支持。然而,在最近十年里,通过更深入的研究,尤其是基于分子序列的系统进化分析,表明微孢子虫和真菌具有一定亲缘关系,并认为其结构的简约性恰好体现了微孢子虫营寄生生活的高度退化现象。目前对微孢子虫的系统进化仍存在各种不同意见,对其进化研究历史进行探讨有着重要意义。本文将按照时间顺序回顾微孢子虫进化分类研究过程中的各种研究成果,并讨论为什么微孢子虫独特的细胞和基因组特性会导致众多的学者在其进化分类问题上争执这么久。     

A single eubacterial origin of eukaryotic pyruvate: ferredoxin oxidoreductase genes: implications for the evolution of anaerobic eukaryotes.

The iron sulfur protein pyruvate: ferredoxin oxidoreductase (PFO) is central to energy metabolism in amitochondriate eukaryotes, including those with hydrogenosomes. Thus, revealing the evolutionary history of PFO is critical to understanding the origin(s) of eukaryote anaerobic energy metabolism. We determined a complete PFO sequence for Spironucleus barkhanus, a large fragment of a PFO sequence from Clostridium pasteurianum, and a fragment of a new PFO from Giardia lamblia. Phylogenetic analyses of eubacterial and eukaryotic PFO genes suggest a complex history for PFO, including possible gene duplications and horizontal transfers among eubacteria. Our analyses favor a common origin for eukaryotic cytosolic and hydrogenosomal PFOs from a single eubacterial source, rather than from separate horizontal transfers as previously suggested. However, with the present sampling of genes and species, we were unable to infer a specific eubacterial sister group for eukaryotic PFO. Thus, we find no direct support for the published hypothesis that the donor of eukaryote PFO was the common alpha-proteobacterial ancestor of mitochondria and hydrogenosomes. We also report that several fungi and protists encode proteins with PFO domains that are likely monophyletic with PFOs from anaerobic protists. In Saccharomyces cerevisiae, PFO domains combine with fragments of other redox proteins to form fusion proteins which participate in methionine biosynthesis. Our results are consistent with the view that PFO, an enzyme previously considered to be specific to energy metabolism in amitochondriate protists, was present in the common ancestor of contemporary eukaryotes and was retained, wholly or in part, during the evolution of oxygen-dependent and mitochondrion-bearing lineages.