First multigene analysis of Archamoebae (Amoebozoa: Conosa) robustly reveals its phylogeny and shows that Entamoebidae represents a deep lineage of the group.

Archamoebae is an understudied group of anaerobic free-living or endobiotic protists that constitutes the major anaerobic lineage of the supergroup Amoebozoa. Hitherto, the phylogeny of Archamoebae was based solely on SSU rRNA and actin genes, which did not resolve relationships among the main lineages of the group. Because of this uncertainty, several different scenarios had been proposed for the phylogeny of the Archamoebae. In this study, we present the first multigene phylogenetic analysis that includes members of Pelomyxidae, and Rhizomastixidae. The analysis clearly shows that Mastigamoebidae, Pelomyxidae and Rhizomastixidae form a clade of mostly free-living, amoeboid flagellates, here called Pelobiontida. The predominantly endobiotic and aflagellated Entamoebidae represents a separate, deep-branching lineage, Entamoebida. Therefore, two unique evolutionary events, horizontal transfer of the nitrogen fixation system from bacteria and transfer of the sulfate activation pathway to mitochondrial derivatives, predate the radiation of recent lineages of Archamoebae. The endobiotic lifestyle has arisen at least three times independently during the evolution of the group. We also present new ultrastructural data that clarifies the primary divergence among the family Mastigamoebidae which had previously been inferred from phylogenetic analyses based on SSU rDNA.

Morphological and Molecular Diversity of the Neglected Genus Rhizomastix Alexeieff, 1911 (Amoebozoa: Archamoebae) with Description of Five New Species.

The genus Rhizomastix is a poorly known group of amoeboid heterotrophic flagellates living as intestinal commensals of insects, amphibians or reptiles, and as inhabitants of organic freshwater sediments. Eleven Rhizomastix species have been described so far, but DNA sequences from only a single species have been published. Recently, phylogenetic analyses confirmed a previous hypothesis that the genus belongs to the Archamoebae; however, its exact position therein remains unclear. In this study we cultured nine strains of Rhizomastix, both endobiotic and free-living. According to their light-microscopic morphology and SSU rRNA and actin gene analyses, the strains represent five species, of which four are newly described here: R. bicoronata sp. nov., R. elongata sp. nov., R. vacuolata sp. nov. and R. varia sp. nov. In addition, R. tipulae sp. nov., living in the intestine of crane flies, is separated from the type species, R. gracilis. We also examined the ultrastructure of R. elongata sp. nov., which revealed that it is more complicated than the previously described R. libera. Our data show that either the endobiotic lifestyle of some Rhizomastix species has arisen independently from other endobiotic archamoebae, or the free-living members of this genus represent a secondary switch from the endobiotic lifestyle.

Next-Generation Sequencing Assessment of Eukaryotic Diversity in Oil Sands Tailings Ponds Sediments and Surface Water.

Tailings ponds in the Athabasca oil sands (Canada) contain fluid wastes, generated by the extraction of bitumen from oil sands ores. Although the autochthonous prokaryotic communities have been relatively well characterized, almost nothing is known about microbial eukaryotes living in the anoxic soft sediments of tailings ponds or in the thin oxic layer of water that covers them. We carried out the first next-generation sequencing study of microbial eukaryotic diversity in oil sands tailings ponds. In metagenomes prepared from tailings sediment and surface water, we detected very low numbers of sequences encoding eukaryotic small subunit ribosomal RNA representing seven major taxonomic groups of protists. We also produced and analysed three amplicon-based 18S rRNA libraries prepared from sediment samples. These revealed a more diverse set of taxa, 169 different OTUs encompassing up to eleven higher order groups of eukaryotes, according to detailed classification using homology searching and phylogenetic methods. The 10 most abundant OTUs accounted for > 90% of the total of reads, vs. large numbers of rare OTUs (< 1% abundance). Despite the anoxic and hydrocarbon-enriched nature of the environment, the tailings ponds harbour complex communities of microbial eukaryotes indicating that these organisms should be taken into account when studying the microbiology of the oil sands.

Evidence of intraflagellar transport and apical complex formation in a free-living relative of the apicomplexa.

Since its first description, Chromera velia has attracted keen interest as the closest free-living relative of parasitic Apicomplexa. The life cycle of this unicellular alga is complex and involves a motile biflagellate form. Flagella are thought to be formed in the cytoplasm, a rare phenomenon shared with Plasmodium in which the canonical mode of flagellar assembly, intraflagellar transport, is dispensed with. Here we demonstrate the expression of intraflagellar transport components in C. velia, answering the question of whether this organism has the potential to assemble flagella via the canonical route. We have developed and characterized a culturing protocol that favors the generation of flagellate forms. From this, we have determined a marked shift in the mode of daughter cell production from two to four daughter cells per division as a function of time after passage. We conduct an ultrastructural examination of the C. velia flagellate form by using serial TEM and show that flagellar biogenesis in C. velia occurs prior to cytokinesis. We demonstrate a close association of the flagellar apparatus with a complex system of apical structures, including a micropore, a conoid, and a complex endomembrane system reminiscent of the apical complex of parasitic apicomplexans. Recent work has begun to elucidate the possible flagellar origins of the apical complex, and we show that in C. velia these structures are contemporaneous within a single cell and share multiple connections. We propose that C. velia therefore represents a vital piece in the puzzle of the origins of the apical complex.

Multivesicular bodies in the enigmatic amoeboflagellate Breviata anathema and the evolution of ESCRT 0.

Endosomal sorting complexes required for transport (ESCRTs) are heteromeric protein complexes required for multivesicular body (MVB) morphogenesis. ESCRTs I, II, III and III-associated are ubiquitous in eukaryotes and presumably ancient in origin. ESCRT 0 recruits cargo to the MVB and appears to be opisthokont-specific, bringing into question aspects of the current model of ESCRT mechanism. One caveat to the restricted distribution of ESCRT 0 was the previous limited availability of amoebozoan genomes, the supergroup closest to opisthokonts. Here, we significantly expand the sampling of ESCRTs in Amoebozoa. Our electron micrographic and bioinformatics evidence confirm the presence of MVBs in the amoeboflagellate Breviata anathema. Searches of genomic databases of amoebozoans confirm the ubiquitous nature of ESCRTs I-III-associated and the restriction of ESCRT 0 to opisthokonts. Recently, an alternate ESCRT 0 complex, centering on Tom1 proteins, has been proposed. We determine the distribution of Tom1 family proteins across eukaryotes and show that the Tom1, Tom1L1 and Tom1L2 proteins are a vertebrate-specific expansion of the single Tom1 family ancestor, which has indeed been identified in at least one member of each of the major eukaryotic supergroups. This implies a more widely conserved and ancient role for the Tom1 family in endocytosis than previously suspected.

A family of diatom-like silicon transporters in the siliceous loricate choanoflagellates.

Biosilicification is widespread across the eukaryotes and requires concentration of silicon in intracellular vesicles. Knowledge of the molecular mechanisms underlying this process remains limited, with unrelated silicon-transporting proteins found in the eukaryotic clades previously studied. Here, we report the identification of silicon transporter (SIT)-type genes from the siliceous loricate choanoflagellates Stephanoeca diplocostata and Diaphanoeca grandis. Until now, the SIT gene family has been identified only in diatoms and other siliceous stramenopiles, which are distantly related to choanoflagellates among the eukaryotes. This is the first evidence of similarity between SITs from different eukaryotic supergroups. Phylogenetic analysis indicates that choanoflagellate and stramenopile SITs form distinct monophyletic groups. The absence of putative SIT genes in any other eukaryotic groups, including non-siliceous choanoflagellates, leads us to propose that SIT genes underwent a lateral gene transfer event between stramenopiles and loricate choanoflagellates. We suggest that the incorporation of a foreign SIT gene into the stramenopile or choanoflagellate genome resulted in a major metabolic change: the acquisition of biomineralized silica structures. This hypothesis implies that biosilicification has evolved multiple times independently in the eukaryotes, and paves the way for a better understanding of the biochemical basis of silicon transport through identification of conserved sequence motifs.

Eukaryotic systematics: a user's guide for cell biologists and parasitologists.

Single-celled parasites like Entamoeba, Trypanosoma, Phytophthora and Plasmodium wreak untold havoc on human habitat and health. Understanding the position of the various protistan pathogens in the larger context of eukaryotic diversity informs our study of how these parasites operate on a cellular level, as well as how they have evolved. Here, we review the literature that has brought our understanding of eukaryotic relationships from an idea of parasites as primitive cells to a crystallized view of diversity that encompasses 6 major divisions, or supergroups, of eukaryotes. We provide an updated taxonomic scheme (for 2011), based on extensive genomic, ultrastructural and phylogenetic evidence, with three differing levels of taxonomic detail for ease of referencing and accessibility (see supplementary material at Cambridge Journals On-line). Two of the most pressing issues in cellular evolution, the root of the eukaryotic tree and the evolution of photosynthesis in complex algae, are also discussed along with ideas about what the new generation of genome sequencing technologies may contribute to the field of eukaryotic systematics. We hope that, armed with this user's guide, cell biologists and parasitologists will be encouraged about taking an increasingly evolutionary point of view in the battle against parasites representing real dangers to our livelihoods and lives.

Clarifying the taxonomic identity of a phylogenetically important group of eukaryotes: Planomonas is a junior synonym of Ancyromonas.

Ancyromonas was first described in 1882 by Saville Kent, with the modern concept of the genus dating from 1979 with the work of Hänel. Since then, organisms assigned to Ancyromonas have been found to be common in diverse ecosystems, and the group's isolated phylogenetic placement renders it of considerable evolutionary interest. However, in 2008 Cavalier-Smith et al. concluded that all modern accounts of Ancyromonas were of a different organism from that described by Saville Kent, and erected the new genus Planomonas to encompass modern observations of Ancyromonas, and several new species. We critique the rationale for creating this new genus, reexamining the original sources and making additional observations using light and electron microscopy. We find that almost all the differences between the genera are mistaken or insubstantial. In particular, (1) Cavalier-Smith et al. characterized Ancyromonas sensu Saville Kent as anchoring and Planomonas as gliding, while we find that each type of organism actually does both, and (2) it was claimed that Planomonas is flattened while Ancyromonas sensu Saville Kent is not, but this argument is inconsistent. We treat Planomonas as a junior synonym of Ancyromonas, and Planomonas mylnikovi as a junior synonym of Ancyromonas sigmoides. We transfer Planomonas cephalopora, Planomonas micra, Planomonas howeae and Planomonas limna to Ancyromonas. The genus Ancyromonas therefore includes: A. sigmoides, Ancyromonas cephalopora n. comb., Ancyromonas melba, Ancyromonas sinistra, Ancyromonas micra n. comb., Ancyromonas howeae n. comb., and Ancyromonas limna n. comb.

Implications of the new eukaryotic systematics for parasitologists.

An accurate understanding of evolutionary relationships is central in biology. For parasitologists, understanding the relationships among eukaryotic organisms allows the prediction of virulence mechanisms, reconstruction of metabolic pathways, identification of potential drug targets, elucidation of parasite-specific cellular processes and understanding of interactions with the host or vector. Here we consider the impact of major recent revisions of eukaryotic systematics and taxonomy on parasitology. The previous, ladder-like model placed some protists as early diverging, with the remaining eukaryotes "progressing" towards a "crown radiation" of animals, plants, Fungi and some additional protistan lineages. This model has been robustly disproven. The new model is based on vastly increased amounts of molecular sequence data, integration with morphological information and the rigorous application of phylogenetic methods to those data. It now divides eukaryotes into six major supergroups; the relationships between those groups and the order of branching remain unknown. This new eukaryotic phylogeny emphasizes that organisms including Giardia, Trypanosoma and Trichomonas are not primitive, but instead highly evolved and specialised for their specific environments. The wealth of newly available comparative genomic data has also allowed the reconstruction of ancient suites of characteristics and mapping of character evolution in diverse parasites. For example, the last common eukaryotic ancestor was apparently complex, suggesting that lineage-specific adaptations and secondary losses have been important in the evolution of protistan parasites. Referring to the best evidence-based models for eukaryotic evolution will allow parasitologists to make more accurate and reliable inferences about pathogens that cause significant morbidity and mortality.

Morphological and molecular evidence support a close relationship between the free-living archamoebae Mastigella and Pelomyxa.

Members of the archamoebae comprise free-living and endobiotic amoeboid flagellates and amoebae that live in anoxic/microoxic habitats. Recently, the group has been divided into four separate families, Mastigamoebidae, Entamoebidae, Pelomyxidae, and Rhizomastixidae, whose interrelationships have not been completely resolved. There still are several key members of the archamoebae, notably the genus Mastigella, from which sequence data are missing. We established 12 strains of 5 species of Mastigella and Pelomyxa in culture, examined their morphology and determined their actin gene sequences. In addition, we examined the ultrastructure of three strains and determined and analyzed SSU rDNA sequences of two strains. Our data strongly suggest that Mastigella is specifically related to Pelomyxa, and it is transferred into the family Pelomyxidae. Surprisingly, Mastigella is likely paraphyletic with Pelomyxa forming its internal branch. The two genera share several morphological features that point to their common evolutionary history. Three new species of Mastigella are described: M. erinacea sp. nov., M. rubiformis sp. nov. and M. ineffigiata sp. nov.

A duplex PCR-based assay for measuring the amount of bacterial contamination in a nucleic acid extract from a culture of free-living protists.

BACKGROUND: Cultures of heterotrophic protists often require co-culturing with bacteria to act as a source of nutrition. Such cultures will contain varying levels of intrinsic bacterial contamination that can interfere with molecular research and cause problems with the collection of sufficient material for sequencing. Measuring the levels of bacterial contamination for the purposes of molecular biology research is non-trivial, and can be complicated by the presence of a diverse bacterial flora, or by differences in the relative nucleic acid yield per bacterial or eukaryotic cell. PRINCIPAL FINDINGS: Here we describe a duplex PCR-based assay that can be used to measure the levels of contamination from marine bacteria in a culture of loricate choanoflagellates. By comparison to a standard culture of known target sequence content, the assay can be used to quantify the relative proportions of bacterial and choanoflagellate material in DNA or RNA samples extracted from a culture. We apply the assay to compare methods of purifying choanoflagellate cultures prior to DNA extraction, to determine their effectiveness in reducing bacterial contamination. Together with measurements of the total nucleic acid concentration, the assay can then be used as the basis for determining the absolute amounts of choanoflagellate DNA or RNA present in a sample. CONCLUSIONS: The assay protocol we describe here is a simple and relatively inexpensive method of measuring contamination levels in nucleic acid samples. This provides a new way to establish quantification and purification protocols for molecular biology and genomics in novel heterotrophic protist species. Guidelines are provided to develop a similar protocol for use with any protistan culture. This assay method is recommended where qPCR equipment is unavailable, where qPCR is not viable because of the nature of the bacterial contamination or starting material, or where prior sequence information is insufficient to develop qPCR protocols.

The microtubular cytoskeleton of the apusomonad Thecamonas, a sister lineage to the opisthokonts.

Apusomonads are of evolutionary interest because they are close relatives to the supergroup Opisthokonta, which contains both animals and fungi. There are no detailed morphological studies of 'amastigomonad' type apusomonads, such as Thecamonas trahens, despite this species having a sequenced genome. We use serial-section transmission electron microscopy and 3D reconstruction to examine the cell architecture and complete microtubular cytoskeleton of Thecamonas. Thecamonas has two flagella and an anteriorly projecting 'tusk'. The anterior basal body associates with one microtubular root, which travels leftward, and a non-root 'ribbon' of six microtubules that travels down the right side of the cell. The posterior basal body associates with three roots: an eight-membered right root, a doublet left root, and an intermediate singlet root. These rearrange into two bands, both on the left side of the cell. One comprises the left and singlet roots plus one right root microtubule. The other comprises the remaining right root microtubules. A splitting right root and supernumerary singlet root are also present in breviates, ancyromonads, and 'typical excavates', suggesting that these characters are ancestral for much of eukaryote diversity. If so, opisthokonts, and most or all living eukaryotes, probably arose from cells with complex microtubular cytoskeletons.

The flagellar apparatus of Breviata anathema, a eukaryote without a clear supergroup affinity.

Breviata anathema is an anaerobic amoeboid flagellate that does not branch within any established 'supergroup'. Molecular phylogenies suggest affinities to Amoebozoa, Opisthokonta, or apusomonads. Here we describe its flagellar apparatus ultrastructure. Breviata has two basal bodies. The flagellated anterior basal body (AB) is associated with a fan of ∼18 microtubules and a short singlet microtubular root. Three microtubular roots associate with the posterior basal body. One, the right root (RR), is initially a triplet that splits into two parts. The other two are singlets: the left root (LR), and the middle root (MR), which arises on the posterior side of the basal body. The MR, LR and smaller part of RR support the left ventral side of the cell, while the larger part of RR runs down the right. Outer dynein arms were not observed on the flagellar axoneme. The mitochondrion-like organelle sometimes contains some tubular cristae. The posterior flagellar apparatus resembles that of several eukaryotic lineages, particularly apusomonads, ancyromonads, excavates, and myxogastrid amoebozoans. This comparison suggests that the complex flagellar apparatus of myxogastrids is actually plesiomorphic within Amoebozoa. The widely distributed splitting right root and posterior singlet (MR in Breviata) may be plesiomorphies in many eukaryotic lineages, and thus could be features of the last eukaryotic common ancestor.

Evolution of Archamoebae: morphological and molecular evidence for pelobionts including Rhizomastix, Entamoeba, Iodamoeba, and Endolimax.

The archamoebae form a small clade of anaerobic/microaerophilic flagellates or amoebae, comprising the pelobionts (mastigamoebids and pelomyxids) and the entamoebae. It is a member of the eukaryotic supergroup Amoebozoa. We examined 22 strains of 13 species of Mastigamoeba, Pelomyxa and Rhizomastix by light-microscopy and determined their SSU rRNA gene sequences. The SSU rRNA gene sequences of Pelomyxa palustris and Mastigella commutans in GenBank are shown to belong to P. stagnalis and Mastigamoeba punctachora, respectively. Five new species of free-living archamoebae are described: Mastigamoeba abducta, M. errans, M. guttula, M. lenta, and Rhizomastix libera spp. nov. A species of Mastigamoeba possibly living endosymbiotically in Pelomyxa was identified. Rhizomastix libera, the first known free-living member of that genus, is shown to be an archamoeba. R. libera possesses an ultrastructure unique within archamoebae: a rhizostyle formed from a modified microtubular cone and a flagellum with vanes. While many nominal species of pelobionts are extremely hard to distinguish by light microscopy, transient pseudopodial characters are worthy of further investigation as taxonomic markers.

A genome-wide analysis of annexins from parasitic organisms and their vectors.

In this study, we conduct an in-depth analysis of annexin proteins from a diverse range of invertebrate taxa, including the major groups that contain the parasites and vector organisms that are harmful to humans and domestic animals. Using structure-based amino acid sequence alignments and phylogenetic analyses, we present a classification for this protein group and assign names to sequences with ambiguous annotations in public databases. Our analyses reveal six distinct annexin clades, and the mapping of genes encoding annexins to the genome of the human blood fluke Schistosoma mansoni supports the hypothesis of gene duplication as a major evolutionary event in annexin genesis. This study illuminates annexin diversity from a novel perspective using contemporary phylogenetic hypotheses of eukaryote evolution, and will aid the consolidation of annexin protein identities in public databases and provide a foundation for future functional analysis and characterisation of these proteins in parasites of socioeconomic importance.

Nitrile hydratase genes are present in multiple eukaryotic supergroups.

BACKGROUND: Nitrile hydratases are enzymes involved in the conversion of nitrile-containing compounds into ammonia and organic acids. Although they are widespread in prokaryotes, nitrile hydratases have only been reported in two eukaryotes: the choanoflagellate Monosiga brevicollis and the stramenopile Aureococcus anophagefferens. The nitrile hydratase gene in M. brevicollis was believed to have arisen by lateral gene transfer from a prokaryote, and is a fusion of beta and alpha nitrile hydratase subunits. Only the alpha subunit has been reported in A. anophagefferens. METHODOLOGY/PRINCIPAL FINDINGS: Here we report the detection of nitrile hydratase genes in five eukaryotic supergroups: opisthokonts, amoebozoa, archaeplastids, CCTH and SAR. Beta-alpha subunit fusion genes are found in the choanoflagellates, ichthyosporeans, apusozoans, haptophytes, rhizarians and stramenopiles, and potentially also in the amoebozoans. An individual alpha subunit is found in a dinoflagellate and an individual beta subunit is found in a haptophyte. Phylogenetic analyses recover a clade of eukaryotic-type nitrile hydratases in the Opisthokonta, Amoebozoa, SAR and CCTH; this is supported by analyses of introns and gene architecture. Two nitrile hydratase sequences from an animal and a plant resolve in the prokaryotic nitrile hydratase clade. CONCLUSIONS/SIGNIFICANCE: The evidence presented here demonstrates that nitrile hydratase genes are present in multiple eukaryotic supergroups, suggesting that a subunit fusion gene was present in the last common ancestor of all eukaryotes. The absence of nitrile hydratase from several sequenced species indicates that subunits were lost in multiple eukaryotic taxa. The presence of nitrile hydratases in many other eukaryotic groups is unresolved due to insufficient data and taxon sampling. The retention and expression of the gene in distantly related eukaryotic species suggests that it plays an important metabolic role. The novel family of eukaryotic nitrile hydratases presented in this paper represents a promising candidate for research into their molecular biology and possible biotechnological applications.

Comparative analysis of Tritrichomonas foetus (Riedmüller, 1928) cat genotype, T. foetus (Riedmüller, 1928) cattle genotype and Tritrichomonas suis (Davaine, 1875) at 10 DNA loci.

The parasitic protists in the genus Tritrichomonas cause significant disease in domestic cattle and cats. To assess the genetic diversity of feline and bovine isolates of Tritrichomonas foetus (Riedmüller, 1928) Wenrich and Emmerson, 1933, we used 10 different genetic regions, namely the protein coding genes of cysteine proteases 1, 2 and 4-9 (CP1, 2, 4-9) involved in the pathogenesis of the disease caused by the parasite. The cytosolic malate dehydrogenase 1 (MDH1) and internal transcribed spacer region 2 of the rDNA unit (ITS2) were included as additional markers. The gene sequences were compared with those of Tritrichomonas suis (Davaine, 1875) Morgan and Hawkins, 1948 and Tritrichomonas mobilensisCulberson et al., 1986. The study revealed 100% identity for all 10 genes among all feline isolates (=T. foetus cat genotype), 100% identity among all bovine isolates (=T. foetus cattle genotype) and a genetic distinctness of 1% between the cat and cattle genotypes of T. foetus. The cattle genotype of T. foetus was 100% identical to T. suis at nine loci (CP1, 2, 4-8, ITS2, MDH1). At CP9, three out of four T. suis isolates were identical to the T. foetus cattle genotype, while the T. suis isolate SUI-H3B sequence contained a single unique nucleotide substitution. Tritrichomonas mobilensis was 0.4% and 0.7% distinct from the cat and cattle genotypes of T. foetus, respectively. The genetic differences resulted in amino acid changes in the CP genes, most pronouncedly in CP2, potentially providing a platform for elucidation of genotype-specific host-pathogen interactions of T. foetus. On the basis of this data we judge T. suis and T. foetus to be subjective synonyms. For the first time, on objective nomenclatural grounds, the authority of T. suis is given to Davaine, 1875, rather than the commonly cited Gruby and Delafond, 1843. To maintain prevailing usage of T. foetus, we are suppressing the senior synomym T. suisDavaine, 1875 according to Article 23.9, because it has never been used as a valid name after 1899 and T. foetus is widely discussed as the cause of bovine trichomonosis. Thus bovine, feline and porcine isolates should all be given the name T. foetus. This promotes the stability of T. foetus for the veterinary and economically significant venereal parasite causing bovine trichomonosis.

The ultrastructure of Ancyromonas, a eukaryote without supergroup affinities.

The small heterotrophic flagellate Ancyromonas (=Planomonas) lacks close relatives in most molecular phylogenies, and it is suspected that it does not belong to any of the recognized eukaryote 'supergroups', making it an organism of great evolutionary interest. Proposed relatives include apusomonads and excavates, but limited understanding of the ancyromonad cytoskeleton has precluded identification of candidate structural homologies. We present a detailed analysis of the ultrastructure of Ancyromonas through computer-based reconstruction of serial sections. We confirm or extend previous observations of its major organelles (mitochondria, Golgi body, extrusomes, etc.) and pellicle, and distinguish a system of stacked endomembranes that may be developmentally connected to the glycocalyx. Ancyromonas has two basal bodies, each with its own flagellar pocket. The anterior basal body associates with two microtubular elements: a doublet root that runs from between the basal bodies to support the cell's rostrum, and a short singlet root. The posterior basal body is associated with two multi-microtubular structures and a singlet root. One multi-microtubular structure, L1, is a conventional microtubular root. The other structure appears as a curved ribbon of ∼8 microtubules near the basal body, but then flares out into two multi-microtubular elements, L2 and L3, plus two single microtubules. The posterior singlet root originates independently near this second complex. L1, the singlet, L2, and L3 all support the posterior flagellar pocket and channel. We also identified several groups of peripheral microtubules. Possible homologies with the flagellar apparatus of both apusomonads and excavates include a splitting root on the right side of the posterior basal body and a singlet root, both supporting a longitudinal channel or groove associated with the posterior flagellum. The anterior flagellar apparatus in each includes a root supporting structures to the left of the anterior flagellum. Given the probable deep divergences of Ancyromonas, apusomonads and excavates within eukaryotes, it is possible that the eukaryotic cenancestor also possessed these features.

Ultrastructural description of Breviata anathema, n. gen., n. sp., the organism previously studied as "Mastigamoeba invertens".

An understanding of large-scale eukaryotic evolution is beginning to crystallise, as molecular and morphological data demonstrate that eukaryotes fall into six major groups. However, there are several taxa of which the affinities are yet to be resolved, and for which there are only either molecular or morphological data. One of these is the amoeboid flagellate Mastigamoeba invertens. This organism was originally misidentified and studied as a pelobiont using molecular data. We present its first light microscopical and ultrastructural characterisation. We demonstrate that it does not show affinities to the amoebozoan pelobionts, because unlike the pelobionts, it has a double basal body and two flagellar roots, a classical Golgi stack, and a large branching double membrane-bound organelle. Phylogenetic analyses of small subunit ribosomal RNA suggest an affinity with the apusomonads, when a covariotide correction for rate heterogeneity is used. We suggest that previous molecular results have been subject to artefacts from an insufficient correction for rate heterogeneity. We propose a new name for the taxon, Breviata anathema; and the unranked, apomorphy-based name "Breviates" for Breviata and its close relatives.