Ema E. Chao

Ribosomal RNA Phylogeny of Bodonid and Diplonemid Flagellates and the Evolution of Euglenozoa

Abstract Euglenozoa is a major phylum of excavate protozoa (comprising euglenoids, kinetoplastids, and diplonemids) with highly unusual nuclear, mitochondrial, and chloroplast genomes. To improve understanding of euglenozoan evolution, we sequenced nuclear small-subunit rRNA genes from 34 bodonids (Bodo, Neobodo, Parabodo, Dimastigella-like, Rhynchobodo, Rhynchomonas, and unidentified strains), nine diplonemids (Diplonema, Rhynchopus), and a euglenoid (Entosiphon). Phylogenetic analysis reveals that diplonemids and bodonids are more diverse than previously recognised, but does not clearly establish the branching order of kinetoplastids, euglenoids, and diplonemids. Rhynchopus is holophyletic; parasitic species arose from within free-living species. Kinetoplastea (bodonids and trypanosomatids) are robustly holophyletic and comprise a major clade including all trypanosomatids and most bodonids (‘core bodonids’) and a very divergent minor one including Ichthyobodo. The root of the major kinetoplastid clade is probably between trypanosomatids and core bodonids. Core bodonids have three distinct subclades. Clade 1 has two distinct Rhynchobodo-like lineages; a lineage comprising Dimastigella and Rhynchomonas; and another including Cruzella and Neobodo. Clade 2 comprises Cryptobia/ Trypanoplasma, Procryptobia, and Parabodo. Clade 3 is an extensive Bodo saltans species complex. Neobodo designis is a vast genetically divergent species complex with mutually exclusive marine and freshwater subclades. Our analysis supports three phagotrophic euglenoid orders: Petalomonadida (holophyletic), Ploeotiida (probably holophyletic), Peranemida (paraphyletic).

Phylogeny and Classification of Cercomonadida (Protozoa, Cercozoa): Cercomonas, Eocercomonas, Paracercomonas, and Cavernomonas gen. nov.

Cercomonads (=Cercomonadida) are biflagellate gliding bacterivorous protozoa, abundant and diverse in soil and freshwater. We establish 56 new species based on 165 cultures, differential interference contrast microscopy, and 18S and ITS2 rDNA sequencing, and a new genus Cavernomonas studied by scanning electron microscopy. We fundamentally revise the phylogeny and classification of cercomonad Cercozoa. We describe 40 Cercomonas species (35 novel), six Eocercomonas (five novel), two Cavernomonas, and 18 Paracercomonas species (14 novel). We obtained additional cercomonad clade A (Cercomonas, Eocercomonas, Cavernomonas) sequences from multiple environmental DNA libraries. The most commonly cultivated genotypes are not the commonest in environmental DNA, suggesting that cercomonad ecology is far more complex than implied by laboratory cultures. Cercomonads have never been isolated from saline environments, although some species can grow in semi-saline media in the laboratory, and environmental DNA libraries regularly detect them in coastal marine sediments. The first ultrastructural study of an anaerobic cercozoan, Paracercomonas anaerobica sp. nov., a highly divergent cercomonad, shows much simpler ciliary roots than in clade A cercomonads, a ciliary hub-lattice and axosome, and mitochondria with tubular cristae, consistent with it being only facultatively anaerobic. We also describe Agitata tremulans gen. et sp. nov., previously misidentified as Cercobodo (=Dimastigamoeba) agilis Moroff.

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.

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.

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.

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 cells 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).

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.

Invalidation of Hyperamoeba by Transferring its Species to Other Genera of Myxogastria

ABSTRACT. 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 ∼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.

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.