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Unicellular eukaryotes, or protists, are among the most ancient organisms on Earth. Protists belong to multiple taxonomic groups; they are widely distributed geographically and in all environments. Their ability to discriminate among con- and heterospecifics has been documented during the past decade. Here we discuss exemplar cases of taxa-, clone-, and possible kin-discrimination in five major lineages: Mycetozoa (Dictyostelium, Polysphondylium), Dikarya (Saccharomyces), Ciliophora (Tetrahymena), Apicomplexa (Plasmodium) and Archamoebae (Entamoeba). We summarize the proposed genetic mechanisms involved in discrimination-mediated aggregation (self versus different), including the csA, FLO and trg (formerly lag) genes, and the Proliferation Activation Factors (PAFs), which facilitate clustering in some protistan taxa. We caution about the experimental challenges intrinsic to studying recognition in protists, and highlight the opportunities for exploring the ecology and evolution of complex forms of cell-cell communication, including social behavior, in a polyphyletic, still superficially understood group of organisms. Because unicellular eukaryotes are the evolutionary precursors of multicellular life, we infer that their mechanisms of taxa-, clone-, and possible kin-discrimination gave origin to the complex diversification and sophistication of traits associated with species and kin recognition in plants, fungi, invertebrates and vertebrates.

The taxonomic position of the uniciliate, unicentriolar zooflagellate Phalansterium is problematic; its distinctive ultrastructure with a pericentriolar microtubular cone placed it in its own order and suggested phenotypic closeness to the eukaryote cenancestor. We sequenced the 18S rRNA of a unicellular Phalansterium. Phylogenetic analysis shows that it belongs to Amoebozoa, decisively rejecting a postulated relationship with the cercozoan Spongomonas; Phalansterium groups with Varipodida ord. nov. (Gephyramoeba/Filamoeba) or occasionally Centramoebida emend. (Acanthamoebidae/Balamuthiidae fam. nov.), centrosomes of the latter suggesting flagellate ancestors. We also studied Phalansterium solitarium cyst ultrastructure; unlike previously studied P. solitarium, this strain has pentagonally symmetric walls like P. consociatum. We also sequenced 18S rRNA genes of further isolates of Hyperamoeba, an aerobic unicentriolar amoeboflagellate with conical microtubular skeleton; both group strongly with myxogastrid Mycetozoa. However, the four Hyperamoeba strains do not group together, suggesting that Hyperamoeba are polyphyletic derivatives of myxogastrids that lost fruiting bodies independently. We revise amoebozoan higher-level classification into seven classes, establishing Stelamoebea cl. nov. for Protosteliida emend. plus Dictyosteliida (biciliate former ‘protostelids’ comprise Parastelida ord. nov. within Myxogastrea), and new subphylum Protamoebae to embrace Variosea cl. nov. (Centramoebida, Phalansteriida, Varipodida), Lobosea emend., Breviatea cl. nov. for ‘Mastigamoeba invertens’ and relatives, and Discosea cl. nov. comprising Glycostylida ord. nov. (vannellids, vexilliferids, paramoebids, Multicilia), Dermamoebida ord. nov. (Thecamoebidae) and Himatismenida. We argue that the ancestral amoebozoan was probably unikont and that the cenancestral eukaryote may have been also.

Lobose amoebae are abundant free-living protists and important pathogenic agents, yet their evolutionary history and position in the universal tree of life are poorly known. Molecular data for lobose amoebae are limited to a few species, and all phylogenetic studies published so far lacked representatives of many of their taxonomic groups. Here we analyse actin and small-subunit ribosomal RNA (SSU rRNA) gene sequences of a broad taxon sampling of naked, lobose amoebae. Our results support the existence of a monophyletic Amoebozoa clade, which comprises all lobose amoebae examined so far, as well as the amitochondriate pelobionts and entamoebids, and the slime molds. Both actin and SSU rRNA phylogenies distinguish two well-defined clades of amoebae, the “Gymnamoebia sensu stricto” and the Archamoebae (pelobionts+entamoebids), and one weakly supported and ill-resolved group comprising some naked, lobose amoebae and the Mycetozoa.

The ultrastructural appearances of Mastigamoeba punctachora, Mastigamoeba simplex and Mastigella commutansare described. All three species have electron-dense membrane-bounded bodies, suggestive of mitochondrial homologues. All species have a single basal body giving rise to conical arrays of microtubules and to a single ribbon-like microtubular root. The proximal portion of the root is associated with a sheet of dense material. All species have ‘9+2’ flagellar axonemes, with basal bodies composed of triplets of microtubules, but axonemal outer dynein arms appear to be absent. All have a cylinder at the base of the transition zone. The transition zone of Mastigamoeba punctachora is elongate and also contains a column of electron-dense material. Ultrastructural data are compiled and analysed to assess two alternative views that the pelobionts are a primitive group of eukaryotes and the source of the other eukaryotes, or that they are related to eumycetozoan slime moulds.

We present a testable model for the origin of the nucleus, the membrane-bounded organelle that defines eukaryotes. A chimeric cell evolved via symbiogenesis by syntrophic merger between an archaebacterium and a eubacterium. The archaebacterium, a thermoacidophil resembling extant Thermoplasma , generated hydrogen sulfide to protect the eubacterium, a heterotrophic swimmer comparable to Spirochaeta or Hollandina that oxidized sulfide to sulfur. Selection pressure for speed swimming and oxygen avoidance led to an ancient analogue of the extant cosmopolitan bacterial consortium “Thiodendron latens.” By eubacterial-archaebacterial genetic integration, the chimera, an amitochondriate heterotroph, evolved. This “earliest branching protist” that formed by permanent DNA recombination generated the nucleus as a component of the karyomastigont, an intracellular complex that assured genetic continuity of the former symbionts. The karyomastigont organellar system, common in extant amitochondriate protists as well as in presumed mitochondriate ancestors, minimally consists of a single nucleus, a single kinetosome and their protein connector. As predecessor of standard mitosis, the karyomastigont preceded free (unattached) nuclei. The nucleus evolved in karyomastigont ancestors by detachment at least five times (archamoebae, calonymphids, chlorophyte green algae, ciliates, foraminifera). This specific model of syntrophic chimeric fusion can be proved by sequence comparison of functional domains of motility proteins isolated from candidate taxa.

Summary The taxon Archezoa was proposed to unite a group of very odd eukaryotes that lack many of the characteristics classically associated with nucleated cells, in particular the mitochondrion. The hypothesis was that these cells diverged from other eukaryotes before these characters ever evolved, and therefore they represent ancient and primitive eukaryotic lineages. The kingdom comprised four groups: Metamonada, Microsporidia, Parabasalia, and Archamoebae. Until recently, molecular work supported their primitive status, as they consistently branched deeply in eukaryotic phylogenetic trees. However, evidence has now emerged that many Archezoa contain genes derived from the mitochondrial symbiont, revealing that they actually evolved after the mitochondrial symbiosis. In addition, some Archezoa have now been shown to have evolved more recently than previously believed, especially the Microsporidia for which considerable evidence now indicates a relationship with fungi. In summary, the mitochondrial symbiosis now appears to predate all Archezoa and perhaps all presently known eukaryotes. BioEssays 20:87‐95, 1998. r 1998 John Wiley & Sons, Inc.

Summary Recent molecular and ultrastructural discoveries necessitate major changes in the higher level classification of the kingdom Protozoa. A new class Anaeromonadea and subphylum Anaeromonada are created for the anaerobic tetrakont flagellate Trimastix , which is grouped with Parabasala, ranked as a subphylum, to form the new protozoan phylum Trichozoa. To simplify the supraphyletic classification of Protozoa I dispense with the category parvkingdom by increasing Neozoa in rank from infrakingdom to subkingdom and creating a new subkingdom Eozoa for the eokaryote phyla Trichozoa, Percolozoa and Euglenozoa. The subkingdom Neozoa, containing all neokaryote Protozoa, is divided into four infrakingdoms: Sarcodina infrak. nov.; Alveolata Cavalier-Smith 1991; Actinopoda Calkins 1902, stat. nov.; and Neomonada infrak. nov. Sarcodina are subdivided into three superphyla: Eosarcodina superph. nov. (phyla Reticulosa and Mycetozoa); Neosarcodina (phyla Amoebozoa and Rhizopoda); and Haplosporidia (phylum Haplosporidia). Paramyxia are treated as a superclass within infraphylum Sporozoa of the Apicomplexa, not as a separate phylum. Major changes are made to the tubulicristate phylum Opalozoa: I transfer the partially pseudopodial opalozoan groups into Rhizopoda; I move Proteromonadida into the subphylum Opalinata, and group the remaining non-opalinate opalozoans with the Choanozoa as a new zooflagellate phylum Neomonada, leaving only the revised Opalinata in the Opalozoa; these residual Opalozoa are left as protists incertae sedis, as it is unclear whether they belong in the kingdom Protozoa or Chromista. Neomonada are divided into four subphyla: Choanozoa stat. nov. (classes Choanoflagellea, Coral lochytrea); Hemimastigophora; Apusozoa subphyl. nov. (classes Thecomonadea, Anisomonadea, Jacobea cl. nov., Ebridea) and Isomita subphyl. nov. (classes Telonemea and Cyathobodonea). Commation is transferred from Heterokonta to Apusozoa. Three apusozoan orders (Discocelida; Caecitellida; and Commatiida) are created. Within the revised Rhizopoda I group Sarcomonadea (revised by excluding proteomyxids and Jakoba ) and Filosea together as a new subphylum Monadofilosa, and group Chlorarachnea with the formerly opalozoan class Proteomyxidea as a second new rhizopod subphylum, Reticulofilosa; Phytomyxea are removed from Opalozoa and treated as a third new rhizopod subphylum, Phytomyxa. Lobose amoebae are excluded from Rhizopoda and placed in the separate sarcodine phylum Amoebozoa as a subphylum (Lobosa) comprising the classes Amoebaea and Testacealobosea. A new amoebozoan subphylum (Holomastiga) and class (Holomastigea) are created for Multicilia , and Archamoebae (classes Pelobiontea and Entamoebea) are treated as a third subphylum of Amoebozoa. A central role for amoeboflagellates in eukaryote cell evolution is proposed. I suggest that ancestral eukaryotes were non-amoeboid zooflagellates and that the amoeboflagellate condition originated only once in a percolozoan that evolved eruptive pseudopodia to create the Heterolobosea. Later, by evolving graded spatial fine-tuning of cortical properties, these evolved into the non-eruptive pseudopodia of Sarcodina. The non-amoeboid flagellate groups Euglenozoa, Neomonada, and Alveolata may have evolved secondarily by the independent suppression of the amoeboid phase of amoeboflagellates, just as the non-ciliated sarcodine groups evolved polyphyletically from amoeboflagellates by several independent losses of cilia. It is argued that Actinopoda arose from neosarcodine amoeboflagellates by evolving axopodia. The four higher kingdoms did not evolve directly from amoeboflagellates (Animalia and Fungi arose independently from the neomonad Choanozoa, and Plantae and Chromista probably from early alveolates); but, as the neomonads and alveolates themselves evolved from amoeboflagellates, the ancestors of all four higher kingdoms were indirectly descended from amoeboflagellates, and their descendants retained the potential to re-evolve a controlled amoeboid motility. The evolutionary significance of pseudophytoplankton for neokaryote evolution is discussed. An evolutionary explanation for the rare changes in the form of mitochondrial cristae during eukaryote cell evolution is proposed: cristal morphology coevolves with plasma membrane properties through the pleiotropic effects of genes affecting membrane composition. Selection acts primarily on plasma membrane properties, and cristal form is a basically neutral response accounted for by correlations of growth. Origin of non-eruptive pseudopods in the ancestral sarcodine could have caused the changeover from discoid to tubular cristae in the ancestral sarcodine, and converse changes in cell cortical properties may account for the polyphyletic flattening of cristae in Plantae, Cryptista, and Opisthokonta.

Summary A new species of pelobiont, Mastigamoeba schizophrenia n. sp., is described using light and electron microscopy. Three forms were observed: flagellates, aflagellated amoebae and cysts. This species is distinguished by having adhering paired nuclei. Most cells have a single pair of nuclei but large amoebae may have several pairs. The flagellar apparatus consists of a single basal body, connected to the most anterior nucleus by a cone of microtubules. The basal body is composed of doublets of microtubules. A single ribbonlike microtubular root originates alongside the basal body. Like other pelobionts, the cell has a comparatively simple endomembrane system which includes endoplasmic reticulum but lacks mitochondria, dictyosomes, contractile vacuole and other elements found in many eukaryotic cells. The taxonomy of the pelobionts is discussed, the genus Phreatamoeba is synonymised with Mastigamoeba, and arguments for a deep-branching position for the pelobionts among eukaryotes are discussed.

A new amoeba species pathogenic for Senegalese sole is described based on ultrastructural analysis and SSU rDNA phylogenetic inference. The parasite presents round to ovoid trophozoites (, This work was funded by the Spanish Ministry of Science and Innovation and by aquaculture companies through CSIC Internal Research Programmes (Intramural projects). Additional funding was granted by the regional Government (Generalitat Valenciana PROMETEO 2010/006 and ISIC 2012/003). Constenla was supported by a Ph.D. student grant of the Universitat Autònoma de Barcelona (‘P.I.F. Programme’).

Actin is an essential multifunctional protein encoded by two distinct ancient classes of genes in animals (cytoplasmic and muscle) and plants (vegetative and reproductive). The prevailing view is that each class of actin variants is functionally distinct. However, we propose that the vegetative plant and cytoplasmic animal variants have conserved functional competence for spatial development inherited from an ancestral protist actin sequence. To test this idea, we ectopically expressed animal and protist actins in Arabidopsis thaliana double vegetative actin mutants that are dramatically altered in cell and organ morphologies. We found that expression of cytoplasmic actins from humans and even a highly divergent invertebrate Ciona intestinalis qualitatively and quantitatively suppressed the root cell polarity and organ defects of act8 act7 mutants and moderately suppressed the root-hairless phenotype of act2 act8 mutants. By contrast, human muscle actins were unable to support prominently any aspect of plant development. Furthermore, actins from three protists representing Choanozoa, Archamoeba, and green algae efficiently suppressed all the phenotypes of both the plant mutants. Remarkably, these data imply that actin’s competence to carry out a complex suite of processes essential for multicellular development was already fully developed in single-celled protists and evolved nonprogressively from protists to plants and animals.

The morphology of Mastigamoeba aspera, a type species of the genus Mastigamoeba Schulze, 1875, has been investigated at the light- and electron-microscopical level. Motile individuals are oval or peach-shaped. Motile flagella is situated at the anterior end of uninucleate cells. During locomotion, the surface of mastigamoebes forms many conical or finger-shaped hyaline pseudopodia, wereas bulbous uroid is often formed at the posterior end of the cell. Micropopulations of M. aspera consist of uninucleate flagellate forms as well as multinucleate aflagellate ones. There is a thick layer ofglycocalix on the cell surface where many rod-shaped bacterial ectobionts live. The nucleus is vesicular with spherical central nucleolus. The flagellar apparatus of M. aspera is connected with nucleus to form so called kariomastigont. A single kinetosome is associated with many radial microtubules and a lateral root. A distinct microtubule organization centre (MTOC) is situated at the basal part of the kinetosome. Microtubules of the nuclear cone are connected with the MTOC. This microtubules take part in the formation of kariomastigont. The axoneme has a standart set of microtubules 9(2)+2. Digestive vacuoles are the main component of the cytoplasm of M. aspera. Beside, many light-difracted granules and glycogen bodies were found in the cells. Mitochondria, dictyosomes of the Golgi apparatus and microbodies were not revealed in the cytoplasm of M. aspera.

Here is reported the draft genome sequence of Methanobacterium formicicum DSM 3637, which was isolated from the methane-producing amoeba Pelomyxa palustris . This bacterium was determined to be an endosymbiont living in the cytoplasm of P. palustris and the source of methane; however, the global characteristics of its genome suggest a free-living lifestyle rather than an endosymbiotic one.

Iodamoeba is the last genus of obligately parasitic human protist whose phylogenetic position is unknown. Iodamoeba small subunit ribosomal DNA sequences were obtained using samples from three host species, and phylogenetic analyses convincingly placed Iodamoeba as a sister taxon to Endolimax. This clade in turn branches among free-living amoeboflagellates of the genus Mastigamoeba. Two Iodamoeba ribosomal lineages (RL1 and RL2) were detected whose sequences differ by 31%, each of which is found in both human and nonhuman hosts.

Using light and electron microscopy, the morphology of a new species of pelobionts Pelomyxa flava was studied. The coverings of P. flava are represented by plasma membrane bearing the thick layer of weakly structured glycocalyx on its outer surface. Numerous flagella are often located on the tops of short conical pseudopodia. Kinetosomes of flagella reach a length of 0.9 microm and are hollow with a pronounced central filament. Rootlet system is represented by three groups of microtubules: the radial, basal and microtubules of lateral root. The transition zone is short and does not exceed the level of cell surface; the axoneme is characterized by an unstable set of microtubules. Trophic stages of P. flava life cycle are presented by binuclear cells; plasmotomy is performed at the tetranuclear stage. Nuclei have a granular structure. Fibrillar nuclear bodies were revealed in karyoplasm. The nuclei shell has a complex organization. On its surface, the outer membrane has a layer of electron-dense material which contacts with short microtubules, located in a row at the surface of the nuclear envelope. The bubbles and cisterns of endoplasmic reticulum, which are the derivatives of the nuclear envelope, are located outward from the microtubules. The presence of structural and digestive vacuoles and grains of glycogen was noticed in P. flava endoplasm. Three types of prokaryotic cytobionts were revealed. Large multi-membranous organelles reaching 5 pm in diameter were described for the first time. We discuss morphology and biology features of P. flava in comparison with the previously studied Pelomyxa species.

The structure of a new pelomyxa species was investigated with the use of light- and electron-microscope technique. Motile individuals reach 800 microm in length. There is a thin layer of amorphous glycocalix on the cell surface. Many non-motile flagellae are found mainly in the uroidal zone. The axoneme has a non-stable set of microtubules. There are no any special elements in the transition zone. A short kinetosome is about 150 nm long. A bundle of 15-20 microtubules starts from the one side of kinetosome and pass below the cell surface. Structure vacuoles are one of the main cytoplasm components of Pelomyxa stagnalis. Glycogen bodies are surrounded with flattened reticulum cisterns often containing electron-dense material. Two morphological distinct species if prokaryote endobionts were found in the cells of P. stagnalis. The number of nuclei in the cells of P. stagnalis reaches 50 or more. The nuclei are rounded by a two-layer envelope including a multilaminar layer and outer layer, which is formed by small vesicles often containing electron-dense material. One nucleolus is situated at the center of a nucleus. In the nuclei, often in connection with the nucleolus, there are bodies formed by electron-dense threads.

Archamoebae is a small group of anaerobic protists belonging to the eukaryotic supergroup Amoebozoa. Historically, they were regarded as primitively amitochondriate. However, a mitochondrial remnant has been found in some archamoebae. Phylogenetic analyses showed that Archamoebae are closely related to the aerobic slime moulds (Mycetozoa). Trophozoites of archamoebae are amoeboflagellates or aflagellated amoebae. The group includes both parasitic (Entamoeba, Endolimax and, possibly, Endamoeba and Iodamoeba) and free-living (Mastigamoeba, Mastigella, Pelomyxa) genera. The genus Mastigina comprises both endozoic and free-living representatives. Flagellated genera Mastigina, Mastigamoeba, Mastigella and Pelomyxa possess a single basal body associated with a microtubular cone which may or may not be associated with nucleus. The cone is a common feature for Archamoebae and mycetozoan slime moulds. The phylogeny of Archamoebae has not been fully elucidated yet and the taxonomy of free-living representatives is confusing. In the present study, we obtained 42 stable isolates of free-living Archamoebae. We sequenced and analyzed SSU rDNA of 15 of them. The Archamoebae split into five lineages. Based on TEM, we were able to recognize genera Mastigamoeba and Mastigella. The isolate IND8 probably represents a new...

The archezoan phylum Archamoebae Cavalier-Smith, 1983 is here modified by adding a new order Phreatamoebida (presently containing only Phreatamoeba) and removing the family Entamoebidae. Entamoebidae are instead tentatively placed as a class Entamoebea together with the classes Heterolobosea, Percolomonadea and Pseudociliatea in the new protozoan phylum Percolozoa Cavalier-Smith, 1991. Thus emended the phylum Archamoebae is more homogeneous; it is distinguished from the other two phyla of the primitively amitochondrial kingdom and superkingdom Archezoa (i.e. Metamonada and Microsporidia) by having kinetids with only a single flagellum and basal body and a flagellar root consisting of a cone of evenly spaced microtubules. This unikont character of the archamoebae suggests that they may be ancestral to the tetrakont Metamonada, from which the non-flagellate Microsporidia possibly evolved. Higher eukaryotes (superkingdom Metakaryota) probably evolved from a tetrakont metamonad by the symbiotic origin of mitochondria and peroxisomes. If so, the Archamoebae are the most primitive extant phylum of eukaryotes; if molecular phylogenetic studies confirm this idea, Archamoebae will deserve intensive study, which could reveal much about the origin of the eukaryote condition and also establish what is truly universal among eukaryotes. Archamoebae, like other Archezoa, lack mitochondria and peroxisomes and have no obvious Golgi dictyosomes. Their evolutionary significance is discussed and a detailed classification is presented in which the two earlier classes are merged into a single one: Pelobiontea Page, 1976 stat. nov., containing two orders Mastigamoebida Frenzel, 1892 (Syn. Rhizo-Flagellata Kent, 1880 non Rhizomastigida auct.) (including Mastigamoeba, Mastigina, Mastigella, Pelomyxa and probably a few other genera, which have one or more flagella or cilia (motile or immotile, 9 + 2 or otherwise) in the amoeboid trophic phase), and Phreatamoebida ord. nov. (including only Phreatamoeba in the new family Phreatomoebidae, which has alternating phases of non-flagellate amoebae and uniflagellate cells). Mastigamoebida are divided into three families: Mastigamoebidae Goldschmidt, 1907; Mastigellidae fam. nov.; Pelomyxidae Schulze, 1877. Archamoebae may be uni- or multi-nucleate and either gut parasites or (more usually) free-living in soil, freshwater, or marine habitats. Some can form cysts that would probably fossilize; the earliest (1450 My old) smooth-walled fossil cells large enough to be probable eukaryotes might therefore be archamoebal cysts.

Mycetozoa, characterized by spore-bearing fruiting bodies, are the most diverse Amoebozoa. They traditionally comprise three taxa: Myxogastria, Dictyostelia and Protostelia. Myxogastria and Dictyostelia typically have multispored fruiting bodies, but controversy exists whether they are related or arose independently from different unicellular ancestors. Protostelid slime moulds, with single-spored fruiting bodies, are possible evolutionary intermediates between them and typical amoebae, but have received almost no molecular study. Protostelid morphology is so varied that they might not be monophyletic. We therefore provide 38 new 18S rRNA and/or EF-1alpha gene sequences from Mycetozoa and related species, including four protostelids and the enigmatic Ceratiomyxa fruticulosa. Phylogenetic analyses support the monophyly of Dictyostelia, Myxogastria, and Ceratiomyxa (here collectively called "macromycetozoa") and show that protostelids are Amoebozoa, mostly related to non-fruiting amoebae of the class Variosea, but may not be monophyletic; some phylogenetic relationships remain poorly resolved. Ceratiomyxa fruticulosa, originally regarded as a myxogastrid, but in recent decades included in Protostelia, is a deeply diverging sister to Myxogastria. The protostelids studied here plus varipodid amoebae and the flagellates Phalansterium and Multicilia together probably form the outgroup to macromycetozoa plus Archamoebae. Thus protostelids and Variosea are especially significant for understanding the evolutionary transition from solitary amoebae to macromycetozoa.

We isolated and described three new freshwater amoebozoan species that could be unambiguously assigned to the genus Flamella Schaeffer, 1926 by light microscopy. The phylogenetic position of the genus Flamella within the Amoebozoa was unknown, and gene sequence data were lacking. We sequenced the SSU rRNA gene of five Flamella spp., including a previously described F. aegyptia Michel et Smirnov, 1999. The phylogenetic trees inferred from these data showed, that Flamella is monophyletic and robustly branches within Amoebozoa. It belongs to a clade comprising Filamoeba spp., “Arachnula” sp., some protostelids and several SSU rRNA sequences of unidentified or uncultured eukaryotes. This clade consistently branched close to Archamoebae, Mycetozoa, Acramoeba dendroida and Multicilia marina; in contrast to the previous hypotheses, Flamella spp. did not show any relatedness either to Leptomyxida, or to Flabellinea. The ultrastructure of trophic amoebae and especially cysts of the species studied showed considerable similarity to Comandonia operculata Pernin et Pussard, 1979. We therefore suggest that Comandonia may be a junior synonym of Flamella, although more ultrastructural data about Comandonia operculata are necessary to test this hypothesis.

We develop a symbiogenetic concept of the origin of eukaryotic intracellular motility systems from anaerobic but aerotolerant spirochetes in sulfide-rich environments. The last eukaryotic common ancestors (LECAs) have extant archaeprotist descendants: motile nucleated cells with Embden-Meyerhof glycolysis and substrate-level phosphorylation that lack the α-proteobacterial symbiont that became the mitochondrion. Swimming and regulated O 2 -tolerance via sulfide oxidation already had been acquired by sulfidogenic wall-less archaebacteria (thermoplasmas) after aerotolerant cytoplasmic-tubule-containing spirochetes (eubacteria) attached to them. Increasing stability of sulfide-oxidizing/sulfur-reducing consortia analogous to extant sulfur syntrophies ( Thiodendron ) led to fusion. The eubacteria–archaebacteria symbiosis became permanent as the nucleus evolved by prokaryotic recombination with membrane hypertrophy, analogous to Gemmata obscuriglobus and other δ-proteobacteria with membrane-bounded nucleoids. Histone-coated DNA, protein-synthetic RNAs, amino-acylating, and other enzymes were contributed by the sulfidogen whereas most intracellular motility derives from the spirochete. From this redox syntrophy in anoxic and microoxic Proterozoic habitats LECA evolved. The nucleus originated by recombination of eu- and archaebacterial DNA that remained attached to eubacterial motility structures and became the microtubular cytoskeleton, including the mitotic apparatus. Direct LECA descendants include free-living archaeprotists in anoxic environments: archamoebae, metamonads, parabasalids, and some mammalian symbionts with mitosomes. LECA later acquired the fully aerobic Krebs cycle-oxidative phosphorylation-mitochondrial metabolism by integration of the protomitochondrion, a third α-proteobacterial symbiont from which the ancestors to most protoctists, all fungi, plants, and animals evolved. Secondarily anaerobic eukaryotes descended from LECA after integration of this oxygen-respiring eubacterium. Explanatory power and experimental predictions for molecular biology of the LECA concept are stated.

The amitochondrial flagellates comprise the Archamoebae, the Metamonada (orders: Retortamonadida, Diplomonadida, Oxymonadida) and the Parabasala. Molecular rDNA sequence comparison has shown that the diplomonads are very ancient and placed at the base of the tree, but the position of the parabasalids before or after the Euglenozoa and other mitochondriate protists is not definitively determined and such molecular data are required to place the other groups. Common cytological features such as one basal body and a basal body-nucleus connector show that the mastigamoebids or Archamoebae are an evolutionary lineage. The metamonad grouping is heterogenous; the three orders have in common two pairs ofbasal bodies, no Golgi and could be poly- or paraphyletic

In order to ascertain a phylogenetic position of the freshwater amitochondriate amoeboflagellate Pelomyxa palustris its small subunit (SSU) rRNA gene was amplified and sequenced. It was shown to be 3502 bp long. The predicted secondary structure of its rRNA includes at least 16 separate expansion zones located in all the variable regions (V1–V9), as well as in some conservative gene regions. Most insertions are represented by sequences of low complexity that have presumably arisen by a slippage mechanism. Relatively conservative, uniformly positioned motifs contained in regions V4 and V7, as well as in some others, made it possible to perform folding. In maximum likelihood, maximum parsimony, and neighbor-joining trees, P. palustris tends to cluster with amitochondriate and secondary lost mitochondria amoebae and amoeboflagellates Entamoeba , Endolimax nana , and Phreatamoeba balamuthi , comprising together with them and aerobic lobose amoebae Vannella , Acanthamoeba , Balamuthia , and Hartmannella a monophyletic cluster. Another pelobiont, Mastigamoeba invertens , does not belong to this cluster. No specific similarity was discovered between the SSU rRNA of P. palustris and amitochondriate taxa of ‘Archezoa’: Diplomonada, Parabasalia, Microsporidia. Pelomyxa palustris SSU rRNA does not occupy a basal position in the phylogenetic trees and could be ascribed to the so-called eukaryotic ‘crown’ group if the composition of the latter were not so sensitive to the methods of tree building. Thus, molecular and morphological data suggest that P. palustris represents a secondarily modified eukaryotic lineage.

Evolutionary relationships within Amoebozoa have been the subject of controversy for two reasons: 1) paucity of morphological characters in traditional surveys and 2) haphazard taxonomic sampling in modern molecular reconstructions. These along with other factors have prevented the erection of a definitive system that resolves confidently both higher and lower-level relationships. Additionally, the recent recognition that many protosteloid amoebae are in fact scattered throughout the Amoebozoa suggests that phylogenetic reconstructions have been excluding an extensive and integral group of organisms. Here we provide a comprehensive phylogenetic reconstruction based on 139 taxa using molecular information from both SSU-rDNA and actin genes. We provide molecular data for 13 of those taxa, 12 of which had not been previously characterized. We explored the dataset extensively by generating 18 alternative reconstructions that assess the effect of missing data, long-branched taxa, unstable taxa, fast evolving sites and inclusion of environmental sequences. We compared reconstructions with each other as well as against previously published phylogenies. Our analyses show that many of the morphologically established lower-level relationships (defined here as relationships roughly equivalent to Order level or below) are congruent with molecular data. However, the data are insufficient to corroborate or reject the large majority of proposed higher-level relationships (above the Order-level), with the exception of Tubulinea, Archamoebae and Myxogastrea, which are consistently recovered. Moreover, contrary to previous expectations, the inclusion of available environmental sequences does not significantly improve the Amoebozoa reconstruction. This is probably because key amoebozoan taxa are not easily amplified by environmental sequencing methodology due to high rates of molecular evolution and regular occurrence of large indels and introns. Finally, in an effort to facilitate future sampling of key amoebozoan taxa, we provide a novel methodology for genome amplification and cDNA extraction from single or a few cells, a method that is culture-independent and allows both photodocumentation and extraction of multiple genes from natural samples.

The origin of cells was crucial to the evolution of life: by creating the first discrete organisms it vastly strengthened organismic selection, at the expense of genie and population (group) selection which would have dominated early evolution. There are four major sorts of cell, two bacterial (kingdoms Eubacteria and Archaebacteria) and two eukaryotic (superkingdoms Archezoa and Meta- karyota): their origins are the most significant examples of quantum evolution in the history of life. Ultrastructural, molecular and fossil evidence suggest that the first cells were eubacteria, and that archaebacteria and the primitively amito- chondrial archezoa both evolved from a wall-less mutant eubacterium. Subsequently a tetraciliated archezoan metamonad phagocytosed purple non-sulphur and Gram-positive bacteria (both eubacteria), converting them into mitochondria and peroxisomes respectively, creating the first metakaryote. Metakaryota comprise five kingdoms: Protozoa, Chromista, Fungi, Animalia, Plantae. Chloroplasts evolved from symbiotic cyanobacteria, probably once only, either in the ancestral metakaryote or in the common ancestor of dinoflagellates, green and red algae. A separate symbiosis involving a proteromonad–like protozoan host and an aberrantly pigmented dinoflagellate symbiont created the kingdom Chromista, with chloroplasts located inside the rough endoplasmic reticulum. Euglenoids also may have obtained their chloroplasts secondarily from a eukaryotic symbiont, but a direct inheritance from the ancestral metakaryote cannot yet be ruled out. There is no evidence for a symbiotic origin of cilia or eukaryotes; the most primitive eukaryotes may be the monociliated archezoan Archamoebae. The first eubacteria were probably Gram-negative photoheterotrophs with an outer membrane outside their plasma membrane: this outer membrane probably evolved into the outer membrane of mitochondria and chloroplasts, but was lost in the ancestor (s) of Posibacteria and Thermotoga. The first cell perhaps evolved by “gastrulation” of an inside-out-cell bearing ribosomes and chromosomes on its outer surface. Archaebacteria and eukaryotes probably evolved from a Thermotoga-like eubacterium after it lost the murein cell wall: the archaebacterial ancestor evolved a new wall and isoprenoidal ether lipids, whilst the eukaryote ancestor instead evolved an actin/microtubulule endoskeleton allowing phagotrophy, endomembranes, nuclei, and sex (i.e., syngamy and meiosis) to evolve. The mechanisms and significance of these megaevolutionary events are discussed.

The hypothesis that protists without mitochondria, the so-called Archezoa of Cavalier-Smith, are primitive has received some support from rRNA sequence studies on Microsporidia and Diplomonadida. In spite of the lack of mitochondria the archezoan groups of protists show considerable differences in their organization: mastigont and cytoskeletal system, mitosis, Golgi apparatus, hydrogenosomes. This paper examines the characters of the flagellar apparatus and its associated cytoskeleton to obtain clues used for phylogenetic consideration on the three cited groups of flagellates. Archamoebae of the Pelobiontida order comprising families such as Pelomyxidae and Mastigamoebidae share common features: a rudimentary mastigont system composed of only one basal body giving rise to a poorly motile flagellum and a basal body associated microtubular cone capping the nucleus. No Golgi apparatus has been detected.

A new amoeba, isolated from well water in Gambia, West Africa, is described and named Phreatamoeba balamuthi n. g., n. sp. Requiring anaerobic conditions for growth, it is easily cultured monoxenically with Escherichia coli or axenically in complex, undefined organic media. Three phenotypes have been observed in the life cycle: an amoeba, a flagellate, and a cyst. The amoeba moves by monopodia, is predominantly multinucleate, and varies from 11 to 160 microns in length. The flagellate has a single flagellum and is from 6 to 50 microns long. The cyst is surrounded by a resistant wall that lacks pores and ranges from 9 to 18 microns in diameter. The transformation from amoeba to flagellate can be induced nutritionally, the exact inducing factor(s) being unknown. Sexual reproduction has not been observed.

Specimens of Pelomyxa palustris from five collecting sites had numerous nonmotile flagella. The structures are called flagella because of morphological similarities to flagella and because P. palustris has affinities with amoeboid flagellates. Flagella were photographed on living cells and studied by transmission and scanning electron microscopy. From 64 to 742 flagella per cell were estimated from scanning electron microscopy of ten cells 204 to 1269 micron in length. The nonmotile flagella arise from basal granules which were, in one strain, surrounded by radiating electron-dense microtubules. This strain also had excess axonemal microtubules. Abundant cytoplasmic microtubules were arranged in several different patterns. In about half of the P. palustris cells in which nuclei were studied, microtubules were either apposed to the nuclear membrane in a parallel alignment (with some also radiating) or radiating from the nuclear membrane (with none parallel). Bacteria associated with nuclei were of three characteristic types: Gram-negative rods, Gram-positive rods, and large rods. All nuclei within a given trophozoite had similar perinuclear features. Recent proposals for separation of Pelomyxa to its own phylum (based on its proposed primitive, unique nature) can not be justified. Pelomyxa is a complex, highly specialized organism adapted to live in a specific fresh-water environment. Mastigamoebid amoeboid flagellates of the genera Mastigamoeba, Mastigella, Mastigina, and possibly Dinamoeba are placed with Pelomyxa within the order Pelobiontida Page, 1976, emend., containing two families. Pelomyxidae Schulze, 1877, and Mastigamoebidae Goldschmidt, 1907.