Oramoeba fumarolia gen. nov., sp. nov., a new marine heterolobosean amoeboflagellate growing at 54 °C

An amoeba strain was isolated from marine sediment taken from the beach near a fumarole in Italy. The trophozoites of this new marine species transforms into flagellates with variable numbers of flagella, from 2 to 10. The strain forms round to oval cysts. This thermophilic amoeboflagellate grows at temperatures up to 54 °C. Molecular phylogenetic analysis of the small subunit ribosomal DNA (SSU rDNA) places the amoeboflagellate in the Heterolobosea. The closest relatives are Stachyamoeba sp. ATCC50324, a strain isolated from an ocean sample, and Vrihiamoeba italica, a recent isolate from a rice field. Like some other heterolobosean species, this new isolate has a group I intron in the SSU rDNA. Because of the unique place in the molecular phylogenetic tree, and because there is no species found in the literature with similar morphological and physiological characteristics, this isolate is considered to be a new genus and a new species, Oramoeba fumarolia gen. nov., sp. nov.     

Morphological and Molecular Characterization of a New Species of Stephanopogon,Stephanopogon pattersoni n. sp.

Stephanopogon is a taxon of multiciliated protists that is now known to belong to Heterolobosea. Small subunit ribosomal DNA (SSU rDNA) phylogenies indicate that Stephanopogon is closely related to or descended from Percolomonas, a small tetraflagellate with a different feeding structure, thus these morphologically dissimilar taxa are of ongoing evolutionary interest. A new strain of Stephanopogon, KM041, was cultured, then characterized by light microscopy, electron microscopy, and SSU rDNA sequencing. KM041 is 18–35 μm (mean 26.8 μm) long, with six main ventral ciliary rows, one ventro‐lateral ciliary row, and three anterior barbs. It closely resembles Stephanopogon minuta Lei et al. 1999 in morphology, and is very closely related to an extinct culture “S. aff. minuta”, yet is markedly dissimilar in SSU rDNA sequence from a different isolate identified as S. minuta. This confirms that there are at least two distinct lineages of S. minuta‐like cells, and we describe KM041 as a new species, Stephanopogon pattersoni n. sp. The ultrastructure of KM041 resembles that of previously studied Stephanopogon species, though it has a novel paraxonemal structure in a few cilia. We note that a sub‐basal‐body pad and bulbous axosome are unlikely to be apomorphies for the Stephanopogon–Percolomonas clade.     

Monopylocystis visvesvarai n. gen., n. sp. and Sawyeria marylandensis n. gen., n. sp.: two new amitochondrial heterolobosean amoebae from anoxic environments

Two new species of heterolobosean amoebae from anoxic environments, Monopylocystis visvesvarai and Sawyeria marylandensis, are described on the basis of light microscopy, electron microscopy, and their phylogenetic affiliation based on analyses of nuclear small-subunit ribosomal RNA gene sequences. Both species lack mitochondria but have organelles provisionally interpreted as hydrogenosomes, and neither can tolerate aerobic conditions. As their conditions of culture do not exclude all oxygen, they may be microaerophiles rather than strict anaerobes. Both species have unusual nucleolar morphologies. Monopylocystis visvesvarai, from a marine sediment, has nucleolar material distributed around the nuclear periphery. It is the first non-aerobic heterolobosean protist for which a cyst is known; the cyst is unmineralized and unornamented except for a single, raised, plugged pore. Sawyeria marylandensis, from an iron-rich freshwater stream, has nucleolar material distributed in one or two parietal masses, which persist during mitosis. In phylogenetic analyses of small-subunit rRNA gene sequences, Monopylocystis visvesvarai, Sawyeria marylandensis and Psalteriomonas lanterna converge to form a single clade of non-aerobic (anaerobic/microaerophilic) heteroloboseans.     

Biogeography,autecology, and phylogeny of Percolomonads based on newly described species

Percolomonads (Heterolobosea) are aquatic heterotrophic flagellates frequently found in saline waters up to hypersaline environments. We isolated and cultivated seven strains of percolomonad flagellates from marine waters and sediments as well as from a hypersaline inland lake in the Atacama Desert. Morphological characterizations, comprising light and scanning electron microscopy, revealed only slight differences between the strains mainly limited to the cell shape, length of flagella, and length of the ventral feeding groove. Phylogenetic analyses of the 18S and 28S rDNA genes showed the formation of three fully supported clades within the Percolomonadida: the Percolomonadidae, the Barbeliidae fam. nov. and the Lulaidae fam. nov. We describe two new families (Barbeliidae fam. nov., Lulaidae fam. nov.), a new genus (Nonamonas gen. nov.), and five new species (Percolomonas adaptabilis sp. nov., Lula levis sp. nov., Barbelia pacifica sp. nov., Nonamonas montiensis gen. et sp. nov., Nonamonas santamariensis gen. et sp. nov.). Salinity experiments showed that P. adaptabilis sp. nov. from the Atlantic was better adapted to high salinities than all other investigated strains. Moreover, comparisons of our cultivation-based approach with environmental sequencing studies showed that P. adaptabilis sp. nov. seems to be globally distributed in marine surface waters while other species seem to be more locally restricted.     

The Zooflagellates Stephanopogon and Percolomonas are a Clade (Class Percolatea: Phylum Percolozoa)

ABSTRACT. The enigmatic marine protozoan Stephanopogon was first classified with ciliate protozoa because its pellicle also has rows of cilia. As ciliates have nuclear dimorphism with separate germline and somatic nuclei, Stephanopogon with several identical nuclei was regarded as a model for a hypothetical homokaryotic ancestor of ciliates. When electron microscopy revealed radical differences from ciliates this idea was abandoned, but its evolutionary position remains controversial, affinities with three other phyla being suggested. We sequenced 18S rDNA from Stephanopogon aff. minuta and actin genes from it and Stephanopogon apogon to clarify their evolutionary position. Phylogenetic analyses of 18S rRNA nest S. aff. minuta and Stephanopogon minuta securely within the protozoan phylum Percolozoa with zooflagellates of the genus Percolomonas, their closest relatives, comprising the clade Percolatea. This supports a previous grouping of Stephanopogon (order Pseudociliatida) with Percolomonas (order Percolomonadida) as a purely zooflagellate class Percolatea within Percolozoa, in contrast to the fundamentally amoeboid Heterolobosea, which are probably ancestral to Percolatea. Stephanopogon actins evolve exceptionally fast: actin trees place them as a long branch within bikont eukaryotes without revealing their sisters. We establish Percolomonadidae fam. n. for Percolomonas, excluding Pharyngomonas kirbyi g., sp. n. and Pharyngomonas (=Tetramastix=Percolomonas) salina comb. n., which unlike Percolomonas have two anterior and two posterior cilia and a pocket‐like pharynx, like “Macropharyngomonas”, now grouped with Pharyngomonas as a new purely zooflagellate class Pharyngomonadea, within a new subphylum Pharyngomonada; this contrasts them with the revised ancestrally amoeboflagellate subphylum Tetramitia. We discuss evolution of the percolozoan cytoskeleton and different body forms.     

Molecular phylogenetic analysis places Percolomonas cosmopolitus within Heterolobosea: evolutionary implications

Percolomonas cosmopolitus is a common free-living flagellate of uncertain phylogenetic position that was placed within the Heterolobosea on the basis of ultrastructure studies. To test the relationship between Percolomonas and Heterolobosea, we analysed the primary structure of the actin and small-subunit ribosomal RNA (SSU rRNA) genes of P. cosmopolitus as well as the predicted secondary structure of the SSU rRNA. Percolomonas shares common secondary structure patterns of the SSU rRNA with heterolobosean taxa, which, together with the results of actin gene analysis, confirms that it is closely related to Heterolobosea. Phylogenetic reconstructions based on the sequences of the SSU rRNA gene suggest Percolomonas belongs to the family Vahlkampfiidae. The first Bayesian analysis of a large taxon sampling of heterolobosean SSU rRNA genes clarifies the phylogenetic relationships within this group.     

Marinamoeba thermophila, a new marine heterolobosean amoeba growing at 50 °C

Two amoeba strains were isolated from marine sediment taken at the same place with 18 months interval from a region of the sea floor heated by extended submarine hot springs and fumaroles. These thermophilic amoebae grow at temperatures up to 50 °C. Sequences of the internal transcribed spacer demonstrated that the two strains belong to the same species and are different from any genus for which sequences are known. Phylogeny using small subunit ribosomal RNA places the amoeba in the Heterolobosea. Their closest relatives are the hypersaline flagellate Pleurostomum flabellatum and the hypersaline amoeba Tulamoeba peronaphora. The freshwater amoeboflagellate genera Naegleria and Willaertia belong to the same phylogenetic clade in the Vahlkampfiidae. The new marine species does not transform into flagellates. It forms cysts, which are round to ellipsoidal with few pores. Because of their unique place in the molecular phylogenetic tree, and because there is no morphologically identical species found in the literature, these isolates are considered to be a new species and a new genus, Marinamoeba thermophila.     

Phylogenetic Position of Psalteriomonas lanterna Deduced from the SSU rDNA Sequence

ABSTRACT. The small subunit ribosomal DNA sequence (SSU rDNA) of the microaerophilic free-living amoeboflagellate Psalteriomonas lanterna has been sequenced and analyzed. The gene is 1,945 bp long and has a G + C content of 33.4%. Based upon ultrastructural studies, P. lanterna has been placed in the class Lyromonadea within the phylum Percolozoa Cavalier-Smith, 1991. However, based upon cytological characteristics, this microaerophilic free-living amoeboflagellate appears to be very primitive. It shares certain characteristics in common with some archezoans, i.e. it lacks mitochondria and dictyosomes but contains hydrogenosomes. Despite sharing these characteristics with the amitochondriate taxa, P. lanterna is not related to any of these taxa but instead to the Vahlkampfiidae. Therefore, we used primary sequence data and the secondary structure of the SSU rDNA gene to determine the placement P. lanterna in the phylogenetic tree. Our analyses showed that P. lanterna groups as a sister taxon to the Vahlkampfiidae but probably diverged from them quite early.     

Ultrastructure and phylogenetic placement within Heterolobosea of the previously unclassified, extremely halophilic heterotrophic flagellate Pleurostomum flabellatum (Ruinen 1938)

Although Pleurostomum was described almost a century ago, flagellates assigned to this taxon have been recorded only in very occasional faunistic studies of highly saline habitats, and their phylogenetic position has remained uncertain. We report the cultivation, ultrastructure, and phylogenetic relationships of Pleurostomum flabellatum isolated from a Korean saltern pond of 313 per thousand salinity. This isolate is biflagellated with a cytostomal groove, and is not distinguishable from previous accounts of P. flabellatum from saturated brines in India and Australia. Pleurostomum flabellatum shows ultrastructural features characteristic of many Heterolobosea: (1) a striated rhizoplast, (2) an absence of stacked Golgi bodies, (3) parallel basal bodies and flagella, and (4) a large number of peripheral microtubules supporting a rostrum. 18S rRNA gene phylogenies strongly confirm the affinities of P. flabellatum within Heterolobosea. Furthermore, the 18S rRNA gene of P. flabellatum has the heterolobosean-specific helix 17_1, and a group I intron in the same position as in Acrasis rosea. Within Heterolobosea, the 'amoeboflagellate' genera Naegleria and Willaertia were its closest relatives with high bootstrap support and posterior probability. P. flabellatum was observed only as a flagellate, and never as an amoeba. Since light microscopy and electron microscopy observations indicate that P. flabellatum flagellates are capable both of feeding and division, there might be no amoeba stage. Being morphologically distinct from its closest relatives and phylogenetically distant from other flagellate-only Heterolobosea, P. flabellatum cannot be moved into any previously described heterolobosean genus. Instead, we move Pleurostomum into Heterolobosea, and assign as the type species Pleurostomum salinum Namyslowski 1913, a species that closely resembles P. flabellatum. The optimal temperature for growth of P. flabellatum is 40 degrees C. Interestingly, P. flabellatum grows optimally at 300 per thousand salinity and fails to grow below 200 per thousand salinity, indicating that it is an 'extreme halophile'. The optimal salinity for growth is the highest for any eukaryote examined to date.