187-gene phylogeny of protozoan phylum Amoebozoa reveals a new class (Cutosea) of deep-branching, ultrastructurally unique, enveloped marine Lobosa and clarifies amoeba evolution.

Monophyly of protozoan phylum Amoebozoa, and subdivision into subphyla Conosa and Lobosa each with different cytoskeletons, are well established. However early diversification of non-ciliate lobose amoebae (Lobosa) is poorly understood. To clarify it we used recently available transcriptomes to construct a 187-gene amoebozoan tree for 30 species, the most comprehensive yet. This robustly places new genus Atrichosa (formerly lumped with Trichosphaerium) within lobosan class Tubulinea, not Discosea as previously supposed. We identified an earliest diverging lobosan clade comprising marine amoebae armoured by porose scaliform cell-envelopes, here made a novel class Cutosea with two pseudopodially distinct new families. Cutosea comprise Sapocribrum, ATCC PRA-29 misidentified as 'Pessonella', plus from other evidence Squamamoeba. We confirm that Acanthamoeba and ATCC 50982 misidentified as Stereomyxa ramosa are closely related. Discosea have a strongly supported major subclade comprising Thecamoebida plus Glycostylida (suborders Dactylopodina, Stygamoebina; Vannellina) phylogenetically distinct from Centramoebida. Stygamoeba is sister to Dactylopodina. Himatismenida are either sister to Centramoebida or deeper branching. Discosea usually appear holophyletic (rarely paraphyletic). Paramoeba transcriptomes include prokinetoplastid Perkinsela-like endosymbiont sequences. Cunea, misidentified as Mayorella, is closer to Paramoeba than Vexillifera within holophyletic Dactylopodina. Taxon-rich site-heterogeneous rDNA trees confirm cutosan distinctiveness, allow improved conosan taxonomy, and reveal previous dictyostelid tree misrooting.

Multiple origins of Heliozoa from flagellate ancestors: New cryptist subphylum Corbihelia, superclass Corbistoma, and monophyly of Haptista, Cryptista, Hacrobia and Chromista.

Heliozoan protists have radiating cell projections (axopodia) supported by microtubular axonemes nucleated by the centrosome and bearing granule-like extrusomes for catching prey. To clarify previously confused heliozoan phylogeny we sequenced partial transcriptomes of two tiny naked heliozoa, the endohelean Microheliella maris and centrohelid Oxnerella marina, and the cercozoan pseudoheliozoan Minimassisteria diva. Phylogenetic analysis of 187 genes confirms that all are chromists; but centrohelids (microtubules arranged as hexagons and triangles) are not sisters to Endohelea having axonemes in transnuclear cytoplasmic channels (triangular or square microtubular arrays). Centrohelids are strongly sister to haptophytes (together phylum Haptista); we explain the common origins of their axopodia and haptonema. Microheliella is sister to new superclass Corbistoma (zooflagellate Telonemea and Picomonadea, with asymmetric microfilamentous pharyngeal basket), showing that these axopodial protists evolved independently from zooflagellate ancestors. We group Corbistoma and Endohelea as new cryptist subphylum Corbihelia with dense fibrillar interorganellar connections; endohelean axopodia and Telonema cortex are ultrastructurally related. Differently sampled trees clarify why corticate multigene eukaryote phylogeny is problematic: long-branch artefacts probably distort deep multigene phylogeny of corticates (Plantae, Chromista); basal radiations may be contradictorily reconstructed because of their extreme closeness and the Bayesian star-tree paradox. Haptista and Hacrobia are holophyletic, and Chromista probably are.

Multigene eukaryote phylogeny reveals the likely protozoan ancestors of opisthokonts (animals, fungi, choanozoans) and Amoebozoa.

Animals and fungi independently evolved from the protozoan phylum Choanozoa, these three groups constituting a major branch of the eukaryotic evolutionary tree known as opisthokonts. Opisthokonts and the protozoan phylum Amoebozoa (amoebae plus slime moulds) were previously argued to have evolved independently from the little-studied, largely flagellate, protozoan phylum, Sulcozoa. Sulcozoa are a likely evolutionary link between opisthokonts and the more primitive excavate flagellates that have ventral feeding grooves and the most primitive known mitochondria. To extend earlier sparse evidence for the ancestral (paraphyletic) nature of Sulcozoa, we sequenced transcriptomes from six gliding flagellates (two apusomonads; three planomonads; Mantamonas). Phylogenetic analyses of 173-192 genes and 73-122 eukaryote-wide taxa show Sulcozoa as deeply paraphyletic, confirming that opisthokonts and Amoebozoa independently evolved from sulcozoans by losing their ancestral ventral groove and dorsal pellicle: Apusozoa (apusomonads plus anaerobic breviate amoebae) are robustly sisters to opisthokonts and probably paraphyletic, breviates diverging before apusomonads; Varisulca (planomonads, Mantamonas, and non-gliding flagellate Collodictyon) are sisters to opisthokonts plus Apusozoa and Amoebozoa, and possibly holophyletic; Glissodiscea (planomonads, Mantamonas) may be holophyletic, but Mantamonas sometimes groups with Collodictyon instead. Taxon and gene sampling slightly affects tree topology; for the closest branches in Sulcozoa and opisthokonts, proportionally reducing missing data eliminates conflicts between homogeneous-model maximum-likelihood trees and evolutionarily more realistic site-heterogeneous trees. Sulcozoa, opisthokonts, and Amoebozoa constitute an often-pseudopodial 'podiate' clade, one of only three eukaryotic 'supergroups'. Our trees indicate that evolution of sulcozoan dorsal pellicle, ventral pseudopodia, and ciliary gliding (probably simultaneously) generated podiate eukaryotes from Malawimonas-like excavate flagellates.

Invalidation of Hyperamoeba by transferring its species to other genera of Myxogastria.

The genus Hyperamoeba Alexeieff, 1923 was established to accommodate an aerobic amoeba exhibiting three life stages-amoeba, flagellate, and cyst. As more species/strains were isolated, it became increasingly evident from small subunit (SSU) gene phylogenies and ultrastructure that Hyperamoeba is polyphyletic and its species occupy different positions within the class Myxogastria. To pinpoint Hyperamoeba strains within other myxogastrid genera we aligned numerous myxogastrid sequences: whole small subunit ribosomal (SSU or 18S rRNA) gene for 50 dark-spored (i.e. Stemonitida and Physarida) Myxogastria (including a new "Hyperamoeba"/Didymium sequence) and a approximately 400-bp SSU fragment for 147 isolates assigned to 10 genera of the order Physarida. Phylogenetic analyses show unambiguously that the type species Hyperamoeba flagellata is a Physarum (Physarum flagellatum comb. nov.) as it nests among other Physarum species as robust sister to Physarum didermoides. Our trees also allow the following allocations: five Hyperamoeba strains to the genus Stemonitis; Hyperamoeba dachnaya, Pseudodidymium cryptomastigophorum, and three other Hyperamoeba strains to the genus Didymium; and two further Hyperamoeba strains to the family Physaridae. We therefore abandon the polyphyletic and redundant genus Hyperamoeba. We discuss the implications for the ecology and evolution of Myxogastria, whose amoeboflagellates are more widespread than previous inventories supposed, being now found in freshwater and even marine environments.

Multidomain ribosomal protein trees and the planctobacterial origin of neomura (eukaryotes, archaebacteria).

Palaeontologically, eubacteria are > 3× older than neomura (eukaryotes, archaebacteria). Cell biology contrasts ancestral eubacterial murein peptidoglycan walls and derived neomuran N-linked glycoprotein coats/walls. Misinterpreting long stems connecting clade neomura to eubacteria on ribosomal sequence trees (plus misinterpreted protein paralogue trees) obscured this historical pattern. Universal multiprotein ribosomal protein (RP) trees, more accurate than rRNA trees, are taxonomically undersampled. To reduce contradictions with genically richer eukaryote trees and improve eubacterial phylogeny, we constructed site-heterogeneous and maximum-likelihood universal three-domain, two-domain, and single-domain trees for 143 eukaryotes (branching now congruent with 187-protein trees), 60 archaebacteria, and 151 taxonomically representative eubacteria, using 51 and 26 RPs. Site-heterogeneous trees greatly improve eubacterial phylogeny and higher classification, e.g. showing gracilicute monophyly, that many 'rDNA-phyla' belong in Proteobacteria, and reveal robust new phyla Synthermota and Aquithermota. Monoderm Posibacteria and Mollicutes (two separate wall losses) are both polyphyletic: multiple outer membrane losses in Endobacteria occurred separately from Actinobacteria; neither phylum is related to Chloroflexi, the most divergent prokaryotes, which originated photosynthesis (new model proposed). RP trees support an eozoan root for eukaryotes and are consistent with archaebacteria being their sisters and rooted between Filarchaeota (=Proteoarchaeota, including 'Asgardia') and Euryarchaeota sensu-lato (including ultrasimplified 'DPANN' whose long branches often distort trees). Two-domain trees group eukaryotes within Planctobacteria, and archaebacteria with Planctobacteria/Sphingobacteria. Integrated molecular/palaeontological evidence favours negibacterial ancestors for neomura and all life. Unique presence of key pre-neomuran characters favours Planctobacteria only as ancestral to neomura, which apparently arose by coevolutionary repercussions (explained here in detail, including RP replacement) of simultaneous outer membrane and murein loss. Planctobacterial C-1 methanotrophic enzymes are likely ancestral to archaebacterial methanogenesis and ß-propeller-α-solenoid proteins to eukaryotic vesicle coats, nuclear-pore-complexes, and intraciliary transport. Planctobacterial chaperone-independent 4/5-protofilament microtubules and MamK actin-ancestors prepared for eukaryote intracellular motility, mitosis, cytokinesis, and phagocytosis. We refute numerous wrong ideas about the universal tree.

Phylogeny of novel naked Filose and Reticulose Cercozoa: Granofilosea cl. n. and Proteomyxidea revised.

Naked filose and reticulose protozoa were long lumped as proteomyxids or left outside higher groups. We cultivated eight naked filose or reticulose strains, did light microscopy, 18S rDNA sequencing and phylogeny (showing all are Cercozoa), and sequenced 80 environmental 18S-types. Filose species belong in subphylum Filosa and reticulose ones in subphylum Endomyxa, making proteomyxids polyphyletic. We therefore transfer the classically mainly reticulose Proteomyxidea to Endomyxa, removing evident filosans as new class Granofilosea (including Desmothoracida, Acinetactis and new heliomonad family Heliomorphidae (new genus Heliomorpha (=Dimorpha)). Five new species of Limnofila gen. n. (L. mylnikovi; L. anglica; L. longa; L. oxoniensis; L. borokensis, previously misidentified as Biomyxa (=Gymnophrys) cometa) form a large freshwater clade (new order Limnofilida). Mesofila limnetica gen., sp. n. and Nanofila marina gen., sp. n. group separately in Granofilosea (Cryptofilida ord. n.). In Endomyxa, a new genus of reticulose proteomyxids (Filoreta marina, F. japonica, F. turcica spp. n., F. (=Corallomyxa) tenera comb. n.) forms a clade (Reticulosida) related to Gromiidea/Ascetosporea. Platyreta germanica gen., sp. n. and Arachnula impatiens are related vampyrellids (Aconchulinida) within a large clade beside Phytomyxea. Biomyxidae and Rhizoplasmidae fam. n. remain incertae sedis within Proteomyxidea. Gymnophrydium and Borkovia are revised. The reticulose Corallomyxa are unlike Filoreta and possibly Amoebozoa, not Cercozoa.

Planomonadida ord. nov. (Apusozoa): ultrastructural affinity with Micronuclearia podoventralis and deep divergences within Planomonas gen. nov.

Gliding zooflagellates previously misidentified as Ancyromonas sigmoides, Metopion or Heteromita constitute a new genus Planomonas. Three new Planomonas species (marine P. micra and P. mylnikovi: freshwater P. limna) have extremely divergent 18S rRNA and subtly but consistently different light microscopic morphology, distinguishable from P. (=Ancyromonas) melba comb. nov. and P. (=Bodo) cephalopora comb. nov. Ultrastructurally, P. micra and P. mylnikovi have a sub-plasma membrane dense pellicular layer (except in the ventral feeding pocket whose rim is supported by microtubules), kinetocysts, and flat mitochondrial cristae. Centrioles, connected at approximately 80 degrees by short fibres, have a dense amorphous distal plate below a double axosome and four microtubular roots. Microbody, mitochondrion, and dictyosomes associate with the nucleus. Longitudinal cytokinesis is slow and peculiar; ciliary transformation is from anterior to posterior as in other bikonts. Planomonads, like the non-flagellate Micronuclearia (here grouped with planomonads as Hilomonadea cl. nov.), have an indistinguishable single dense pellicular layer, not a double layer like apusomonads (comprising emended class Thecomonadea, phylum Apusozoa). We also sequenced 18S rDNA for Planomonas howeae sp. nov. and Micronuclearia podoventralis, plus actin genes of P. micra, Micronuclearia, Amastigmonas marina. All were analysed phylogenetically; the Planomonas clade is ancient, diverse and robust: it sometimes groups weakly as sister to Micronuclearia.

Morphology and phylogeny of Sainouron acronematica sp. n. and the ultrastructural unity of Cercozoa.

Sainouron are soil zooflagellates of obscure taxonomy. We studied the ultrastructure of S. acronematica sp. n. and sequenced its extremely divergent 18S rDNA and that of Cholamonas cyrtodiopsidis (here grouped as new family Sainouridae) to clarify their phylogeny. Ultrastructurally similar, they weakly group together, deeply within Monadofilosa. Sainouron has three cytoplasmic microtubules; all organelles specifically link to them or the nucleus. Mature centrioles have fibrous rhizoplasts. The posterior centriole bearing the motile cilium (with cortical filaments) has a transitional hub-lattice; a dense spiral fibre links its thicker rhizoplast and triplets; its ciliary root has two microtubules: mt1, underlying the plasma membrane, initiates at the spiral fibre; mt2, laterally attached to mt1 and nucleus, initiates in the amorphous centrosomal region. The anterior younger cilium, an immotile stub with submembrane skeleton as in Cholamonas, lacks axoneme, microtubular root, rhizoplasts and spiral fibre, but becomes the posterior one every cell cycle. The nuclear envelope donates coated vesicles directly to the Golgi, which makes kinetocyst-type extrusomes, concentrated at the cell anterior for extrusion into phagosomes. Ciliary transition region proximal hub-lattices (postulated to contain centrin) and distal nonagonal fibres are cercozoan synapomorphies, found with slight structural variation in all flagellate Cercozoa, but not in outgroups.

Multigene phylogeny and cell evolution of chromist infrakingdom Rhizaria: contrasting cell organisation of sister phyla Cercozoa and Retaria.

Infrakingdom Rhizaria is one of four major subgroups with distinct cell body plans that comprise eukaryotic kingdom Chromista. Unlike other chromists, Rhizaria are mostly heterotrophic flagellates, amoebae or amoeboflagellates, commonly with reticulose (net-like) or filose (thread-like) feeding pseudopodia; uniquely for eukaryotes, cilia have proximal ciliary transition-zone hub-lattices. They comprise predominantly flagellate phylum Cercozoa and reticulopodial phylum Retaria, whose exact phylogenetic relationship has been uncertain. Given even less clear relationships amongst cercozoan classes, we sequenced partial transcriptomes of seven Cercozoa representing five classes and endomyxan retarian Filoreta marina to establish 187-gene multiprotein phylogenies. Ectoreta (retarian infraphyla Foraminifera, Radiozoa) branch within classical Cercozoa as sister to reticulose Endomyxa. This supports recent transfer of subphylum Endomyxa from Cercozoa to Retaria alongside subphylum Ectoreta which embraces classical retarians where capsules or tests subdivide cells into organelle-containing endoplasm and anastomosing pseudopodial net-like ectoplasm. Cercozoa are more homogeneously filose, often with filose pseudopodia and/or posterior ciliary gliding motility: zooflagellate Helkesimastix and amoeboid Guttulinopsis form a strongly supported clade, order Helkesida. Cercomonads are polyphyletic (Cercomonadida sister to glissomonads; Paracercomonadida deeper). Thecofilosea are a clade, whereas Imbricatea may not be; Sarcomonadea may be paraphyletic. Helkesea and Metromonadea are successively deeper outgroups within cercozoan subphylum Monadofilosa; subphylum Reticulofilosa (paraphyletic on site-heterogeneous trees) branches earliest, Granofilosea before Chlorarachnea. Our multiprotein trees confirm that Rhizaria are sisters of infrakingdom Halvaria (Alveolata, Heterokonta) within chromist subkingdom Harosa (= SAR); they further support holophyly of chromist subkingdom Hacrobia, and are consistent with holophyly of Chromista as sister of kingdom Plantae. Site-heterogeneous rDNA trees group Kraken with environmental DNA clade 'eSarcomonad', not Paracercomonadida. Ectoretan fossil dates evidence ultrarapid episodic stem sequence evolution. We discuss early rhizarian cell evolution and multigene tree coevolutionary patterns, gene-paralogue evidence for chromist monophyly, and integrate this with fossil evidence for the age of Rhizaria and eukaryote cells, and revise rhizarian classification.

New phagotrophic euglenoid species (new genus Decastava; Scytomonas saepesedens; Entosiphon oblongum), Hsp90 introns, and putative euglenoid Hsp90 pre-mRNA insertional editing.

We describe three new phagotrophic euglenoid species by light microscopy and 18S rDNA and Hsp90 sequencing: Scytomonas saepesedens; Decastava edaphica; Entosiphon oblongum. We studied Scytomonas and Decastava ultrastructure. Scytomonas saepesedens feeds when sessile with actively beating cilium, and has five pellicular strips with flush joints and Calycimonas-like microtubule-supported cytopharynx. Decastava, sister to Keelungia forming new clade Decastavida on 18S rDNA trees, has 10 broad strips with cusp-like joints, not bifurcate ridges like Ploeotia and Serpenomonas (phylogenetically and cytologically distinct genera), and Serpenomonas-like feeding apparatus (8-9 unreinforced microtubule pairs loop from dorsal jaw support to cytostome). Hsp90 and 18S rDNA trees group Scytomonas with Petalomonas and show Entosiphon as the earliest euglenoid branch. Basal euglenoids have rigid longitudinal strips; derived clade Spirocuta has spiral often slideable strips. Decastava Hsp90 genes have introns. Decastava/Entosiphon Hsp90 frameshifts imply insertional RNA editing. Petalomonas is too heterogeneous in pellicle structure for one genus; we retain Scytomonas (sometimes lumped with it) and segregate four former Petalomonas as new genus Biundula with pellicle cross section showing 2-8 smooth undulations and typified by Biundula (=Petalomonas) sphagnophila comb. n. Our taxon-rich site-heterogeneous rDNA trees confirm that Heteronema is excessively heterogeneous; therefore we establish new genus Teloprocta for Heteronema scaphurum.

18S ribosomal RNA gene sequences of Cochliopodium (Himatismenida) and the phylogeny of Amoebozoa.

Cochliopodium is a very distinctive genus of discoid amoebae covered by a dorsal tectum of carbohydrate microscales. Its phylogenetic position is unclear, since although sharing many features with naked "gymnamoebae", the tectum sets it apart. We sequenced 18S ribosomal RNA genes from three Cochliopodium species (minus, spiniferum and Cochliopodium sp., a new species resembling C. minutum). Phylogenetic analysis shows Cochliopodium as robustly holophyletic and within Amoebozoa, in full accord with morphological data. Cochliopodium is always one of the basal branches within Amoebozoa but its precise position is unstable. In Bayesian analysis it is sister to holophyletic Glycostylida, but distance trees mostly place it between Dermamoeba and a possibly artifactual long-branch cluster including Thecamoeba. These positions are poorly supported and basal amoebozoan branching ill-resolved, making it unclear whether Discosea (Glycostylida, Himatismenida, Dermamoebida) is holophyletic; however, Thecamoeba seems not specifically related to Dermamoeba. We also sequenced the small-subunit rRNA gene of Vannella persistens, which constantly grouped with other Vannella species, and two Hartmannella strains. Our trees suggest that Vexilliferidae, Variosea and Hartmannella are polyphyletic, confirming the existence of two very distinct Hartmannella clades: that comprising H. cantabrigiensis and another divergent species is sister to Glaeseria, whilst Hartmannella vermiformis branches more deeply.

Polyubiquitin insertions and the phylogeny of Cercozoa and Rhizaria.

A single or double amino acid insertion at the monomer-monomer junction of the universal eukaryotic protein polyubiquitin is unique to Cercozoa and Foraminifera, closely related 'core' phyla in the protozoan infrakingdom Rhizaria. We screened 11 other candidate rhizarians for this insertion: Radiozoa (polycystine and acantharean radiolaria), a 'microheliozoan', and Apusozoa; all lack it, supporting suggestions that Foraminifera are more closely related to Cercozoa than either is to other eukaryotes. The insertion's size was ascertained for 12 additional Cercozoa to help resolve their basal branching order. The earliest branching Cercozoa generally have a single amino acid insertion, like all Foraminifera, but a large derived clade consisting of all Monadofilosa except Metopion, Helk-esimastix, and Cercobodo agilis has two amino acids, suggesting one doubling event and no reversions to a single amino acid. Metromonas and Sainouron, cercozoans of uncertain position, have a double insertion, suggesting that they belong in Monadofilosa. An alternative interpretation, suggested by the higher positions for Metopion and Cercobodo on Bayesian trees compared with most distance trees, cannot be ruled out, i.e. that the second insertion took place earlier, in the ancestral filosan, and was followed by three independent reversions to a single amino acid in Chlorarachnea, Metopion and Cercobodo.

Phylogeny and classification of phylum Cercozoa (Protozoa).

The protozoan phylum Cercozoa embraces numerous ancestrally biciliate zooflagellates, euglyphid and other filose testate amoebae, chlorarachnean algae, phytomyxean plant parasites (e.g. Plasmodiophora, Phagomyxa), the animal-parasitic Ascetosporea, and Gromia. We report 18S rRNA sequences of 27 culturable zooflagellates, many previously of unknown taxonomic position. Phylogenetic analysis shows that all belong to Cercozoa. We revise cercozoan classification in the light of our analysis and ultrastructure, adopting two subphyla: Filosa subphyl. nov. a clade comprising Monadofilosa and Reticulofilosa, ranked as superclasses, ancestrally having the same very rare base-pair substitution as all opisthokonts; and subphylum Endomyxa emend. comprising classes Phytomyxea (Plasmodiophorida, Phagomyxida), Ascetosporea (Haplosporidia, Paramyxida, Claustrosporida ord. nov.) and Gromiidea cl. nov., which did not. Monadofilosa comprise Sarcomonadea, zooflagellates with a propensity to glide on their posterior cilium and/or generate filopodia (e.g. Metopion; Cercomonas; Heteromitidae - Heteromita, Bodomorpha, Proleptomonas and Allantion) and two new classes: Imbricatea (with silica scales: Euglyphida; Thaumatomonadida, including Alias, Thaumatomastix) and Thecofilosea (Cryomonadida; Tectofilosida ord. nov. - non-scaly filose amoebae, e.g. Pseudodifflugia). Reticulofilosa comprise classes Chlorarachnea, Spongomonadea and Proteomyxidea (e.g. Massisteria, Gymnophrys, a Dimorpha-like protozoan). Cercozoa, now with nine classes and 17 orders (four new), will probably include many, possibly most, other filose and reticulose amoebae and zooflagellates not yet assigned to phyla.

Oxnerella micra sp. n. (Oxnerellidae fam. n.), a tiny naked centrohelid, and the diversity and evolution of heliozoa.

We describe a new tiny naked centrohelid heliozoan, Oxnerella micra, and sequenced its 18S and 28S rRNA genes. Its extremely slender axopodia have prominent extrusomes and are normally stretched across the substratum like those of many tiny granofilosean Cercozoa. Phylogenetic analysis of 18S rDNA shows that Oxnerella does not branch within any of the six known centrohelid families but very deeply in the order Pterocystida, between Choanocystidae and Pterocystidae; therefore we place it in a new family, Oxnerellidae. Oxnerella arose from ancestors with siliceous scales by losing them; as independently did Heterophryidae and Marophryidae, which replaced them by organic spicules, and Chlamydaster that is not truly naked but retains a mucilage coat and nests extremely shallowly within Pterocystidae. 28S rDNA has a group I intron. Concatenated Bayesian 18S/28S rRNA phylogeny shows centrohelids weakly as sisters to the naked non-centrohelid heliozoan Microheliella maris (Microhelida: Heliozoa). The centrohelid Marophrys marina possesses an elongation factor α-like (EFL) protein related to that of Polyplacocystis; Microheliella also has EFL. We also analysed Hsp90 and 18S rDNA sequences from 'Pinaciophora sp.' ATCC50355; they must be from a centrohelid, probably misidentified as Pinaciophora, the rDNA sequence branching deeply within Pterocystida. We reclassify two Polyplacocystis, Luffisphaera, Phaeodaria and Rotosphaerida.

Microheliella maris (Microhelida ord. n.), an ultrastructurally highly distinctive new axopodial protist species and genus, and the unity of phylum Heliozoa.

A new heliozoan, Microheliella maris, has sufficiently distinctive ultrastructure to merit a new order, Microhelida. Its 18S and 28S rRNA genes were sequenced earlier under the informal name 'marine microheliozoan'; we here sequenced its Hsp90 gene. A three-gene tree suggests that it is distantly related to centrohelids and others in chromist subkingdom Hacrobia; but it is too divergent to be placed accurately by few genes. Unlike centrohelids, its central spherical centrosome has two concentric granular shells and a dense core devoid of a trilaminar central disc. Microtubules radiate from the centrosomal shells. Unlike centrohelids, axopodia have only three microtubules, fixed basally by dense plasma membrane anchors, and bear terminal and lateral haptosome-like extrusomes. As in the heliomonad Heliomorpha, the centrosome is embedded in a nuclear cavity, and centrosomal microtubules traverse the nucleus inside cytoplasmic channels. A novel filogranular network interconnects mitochondria, ER, and plasma membrane. The microbody is attached to the nucleus and mitochondrion, which has vermicular tubular cristae. We group Microhelida and Heliomonadida, purged of dissimilar flagellates, as a new tubulicristate class Endohelea within phylum Heliozoa. Previously misassigned GenBank 18S rDNA sequences reveal Microhelida as diverse and ancient. We discuss principles underlying the biogenesis and diversity of axopodial patterns.

New genera, species, and improved phylogeny of Glissomonadida (Cercozoa).

Glissomonadida is an important cercozoan order of predominantly biflagellate gliding bacterivores found largely in soil and freshwater. Their vast diversity is largely undescribed. We studied 23 mostly newly isolated strains by light microscopy and sequenced their 18S rDNA genes; nine represent new species. For two misidentified ATCC 'Heteromita triangularis' strains, we establish novel gliding genera and species: the sandonid Mollimonas lacrima, the only glissomonad forming anterior and posterior pseudopodia, and Dujardina stenomorpha, a strongly flattened member of the new family Dujardinidae. A new strain from Oxfordshire grassland soil is the first reliably identified isolate of the virtually uniflagellate, smooth-gliding glissomonad genus, AllantionSandon, 1924. Phylogenetic analysis and cytological features reveal Allantion to be a member of Allapsidae. Sandona limna and Bodomorpha prolixa from Lake Baikal and Sandona hexamutans from volcanic Costa Rican soil are described as new species. Fifteen glissomonad strains were from grassland beside Lake Baikal. We describe two as new species of Sandona (S. heptamutans and S. octamutans); the others included strains of Sandona and Allapsa species that have already been described; and three were new species of Sandona and Allapsa but these died before being described. We discuss the ecological and evolutionary significance of these new strains.

The novel marine gliding zooflagellate genus Mantamonas (Mantamonadida ord. n.: Apusozoa).

Mantamonasis a novel genus of marine gliding zooflagellates probably related to apusomonad and planomonad Apusozoa. Using phase and differential interference contrast microscopy we describe the type species Mantamonas plasticasp. n. from coastal sediment in Cumbria, England. Cells are ∼5µm long, ∼5µm wide, asymmetric, flattened, biciliate, and somewhat plastic. The posterior cilium, on which they glide smoothly over the substratum, is long and highly acronematic. The much thinner, shorter, and almost immobile anterior cilium points forward to the cell's left. These morphological and behavioural traits suggest thatMantamonasis a member of the protozoan phylum Apusozoa. Analyses of 18S and 28S rRNA gene sequences of Mantamonas plasticaand a second genetically very different marine species from coastal sediment in Tanzania show Mantamonasas a robustly monophyletic clade, that is very divergent from all other eukaryotes. 18S rRNA trees mostly placeMantamonaswithin unikonts (opisthokonts, Apusozoa, and Amoebozoa) but its precise position varies with phylogenetic algorithm and/or taxon and nucleotide position sampling; it may group equally weakly as sister to Planomonadida, Apusomonadida or Breviata. On 28S rRNA and joint 18/28S rRNA phylogenies (including 11 other newly obtained apusozoan/amoebozoan 28S rRNA sequences) it consistently strongly groups with Apusomonadida (Apusozoa).

Phylogeny and evolution of apusomonadida (protozoa: apusozoa): new genera and species.

Apusomonadida (Apusomonas; Amastigomonas) are understudied gliding zooflagellates. We divide Amastigomonas into five genera, three new: Podomonas; Manchomonas; Multimonas. Microscopy and 18S rDNA sequences establish three new marine species (Podomonas magna; P. capensis; Multimonas media) and a new cyst-forming non-marine species from the surface of ivy leaves (Thecamonas oxoniensis). We consider the soil and freshwater Amastigomonas debruynei, caudata, and borokensis generically distinct from marine Thecamonas. We establish the new combination Multimonas marina (formerly Cercomonas or Amastigomonas). We studied by DIC microscopy and 18S rDNA sequencing three strains microscopically indistinguishable from marine Thecamonas trahens and argue that marine strains of almost identical sequence and appearance (visible largely acronematic cilia) were previously misidentified as Am. debruynei. We argue that 'Amastigomonas sp.' ATCC50062, whose 18S rRNA was sequenced previously and whose complete genome is being sequenced, is T. trahens. We include electron micrographs of T. aff. trahens, P. capensis and magna; ultrastructural cytoskeletal differences between P. capensis, Thecamonas, and Manchomonas (=Amastigomonas) bermudensis comb. n. allow novel functional interpretations of apusomonad evolution. On 18S rDNA trees Apusomonas and Manchomonas form a robust clade (Apusomonadinae), but Thecomonas trahens, T. oxoniensis, Multimonas, and Podomonas all branch deeply but unstably. Apusomonadida and Planomonas are weakly sister to opisthokonts.

Helkesimastix marina n. sp. (Cercozoa: Sainouroidea superfam. n.) a gliding zooflagellate of novel ultrastructure and unusual ciliary behaviour.

Unlike Helkesimastix faecicola and H. major, Helkesimastix marina is marine, ingests bacteria, is probably also a cannibal, and differs in cell cycle ciliary behaviour. Daughter kinetids have mirror symmetry; pre-division cilia beat asymmetrically. We sequenced its 18S rDNA and studied its ultrastructure to clarify its taxonomy. Helkesimastix (Helkesimastigidae fam. n.) differs unexpectedly radically from cercomonads, lacking their complex microtubular ciliary roots, grouping not with them but with Sainouridae within Pansomonadida. Longitudinal cortical microtubules emanate from a dense apical centrosomal plate, where a striated rhizoplast attaches the nucleus, and two very short subparallel centrioles attach by dense fibres. The marginally more posterior centriole, attached to the centrosomal plate by a dense forked fibre, bears the long 9+2 gliding posterior cilium and a microtubular root; the left-side, nucleus-attached, left centriole bears an immotile ciliary stump with abnormal axoneme of nine disorganized mainly singlet microtubules, unlike the sainourid anterior papilla. Both transitional regions have a proximal lattice, the posterior centriole with slender hub. Sainouroidea superfam. n. (Sainouridae; Helkesimastigidae) have homologous cytoskeletal geometry. Dorsal Golgi dictyosome and posterior microbody are attached to the nuclear envelope, which has slender micro-invaginations and probably a cortical lattice. Bacteria are digested posteriorly in association with numerous mitochondria with flat cristae.

Phylogeny, taxonomy, and astounding genetic diversity of glissomonadida ord. nov., the dominant gliding zooflagellates in soil (Protozoa: Cercozoa).

The cercozoan family Heteromitidae comprises morphologically rather uniform gliding zooflagellates, including Bodomorpha and Heteromita, the most ubiquitous and numerous soil protozoa. The generally used name 'Heteromita globosa' for the commonest gliding biflagellates is incorrect. 'Heteromita' Dujardin, 1841 originally contained only two probable euglenozoans and an unidentifiable flagellate, making it inapplicable to Cercozoa. Accordingly, we establish a new order Glissomonadida for Heteromitidae sensu Cavalier-Smith and Chao, 2003. We cultured over 100 glissomonad strains, sequenced their 18S rRNA genes, and studied their behaviour and morphology by differential interference contrast high definition video microscopy. Group-specific amplification and sequencing of over 450 18S rRNA genes from environmental DNA shows that one temperate grassland plot has hundreds of species, there are thousands globally, and tropical species often differ. Glissomonads are probably sisters of Pansomonadida, not Cercomonadida. In a thorough overhaul of glissomonad taxonomy we describe 29 new species, new genera Sandona, Neoheteromita, Flectomonas, Allapsa, and Teretomonas, and morphologically distinctive families: Sandonidae, Allapsidae, Bodomorphidae, and Proleptomonadidae.