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.     

Phylogeny of Physarida (Amoebozoa,Myxogastria) Based on the Small‐Subunit Ribosomal RNA Gene,Redefinition of Physarum pusillum s. str. and Reinstatement of P. gravidum Morgan

Myxomycetes (also called Myxogastria or colloquially, slime molds) are worldwide occurring soil amoeboflagellates. Among Amoebozoa, they have the notable characteristic to form, during their life cycle, macroscopic fruiting bodies, that will ultimately release spores. Some 1,000 species have been described, based on the macroscopic and microscopic characteristics of their fruiting bodies. We were interested in Physarum pusillum (Berk. & M.A. Curtis) G. Lister, a very common species described with two variants, each bearing such morphological differences that they could represent two distinct species. In order to test this, we observed key characters in a large selection of specimens attributed to P.  pusillum, to its synonyms (in particular Physarum gravidum), and to related species. In addition, the small‐subunit ribosomal RNA gene was obtained from seven of these specimens. Based on these data, we provide a comprehensive phylogeny of the order Physarida (Eukaryota: Amoebozoa: Conosa: Macromycetozoa: Fuscisporidia). Morphology and phylogeny together support the reinstatement of P. gravidum Morgan 1896 with a neotype here designated, distinct from P. pusillum, here redefined.     

Life Cycles of Myxogastria Stemonitopsis typhina and Stemonitis fusca on Agar Culture

Myxogastria is a group of protozoa characterized by cellular uninucleate amoeboflagellates (myxamoebae and flagellated swarm cell), acellular multinucleate plasmodia, and stationary spore‐bearing sporocarps. The Stemonitales is a large order in the Myxogastria and contains approximately 230 species, but only 13 species have their completed life cycles observed so far. Here, we described the life cycles of two species in Stemonitales, Stemonitopsis typhina and Stemonitis fusca by culturing in water agar medium and observing the morphogenesis of their spore germination, plasmodium, and sporocarp development. The spore‐to‐spore life cycles of Ste. typhina and S. fusca were completed in approximately 67 and 12 d, respectively. Both species possessed an aphanoplasmodium. However, the spores of Ste. typhina and S. fusca germinated by the V‐shape split and pore methods, respectively. Unlike S. fusca with an evanescent peridium, Ste. typhina produced a shiny persistent peridium which was continuous with the membrane surrounding its stalk. The information will contribute to a better understanding of their taxonomy and phylogeny.     

Exclusive Gut Flagellates of Serritermitidae Suggest a Major Transfaunation Event in Lower Termites: Description of Heliconympha glossotermitis gen. nov. spec. nov.

The guts of lower termites are inhabited by host‐specific consortia of cellulose‐digesting flagellate protists. In this first investigation of the symbionts of the family Serritermitidae, we found that Glossotermes oculatus and Serritermes serrifer each harbor similar parabasalid morphotypes: large Pseudotrichonympha‐like cells, medium‐sized Leptospironympha‐like cells with spiraled bands of flagella, and small Hexamastix‐like cells; oxymonadid flagellates were absent. Despite their morphological resemblance to Pseudotrichonympha and Leptospironympha, a SSU rRNA‐based phylogenetic analysis identified the two larger, trichonymphid flagellates as deep‐branching sister groups of Teranymphidae, with Leptospironympha sp. (the only spirotrichosomid with sequence data) in a moderately supported basal position. Only the Hexamastix‐like flagellates are closely related to trichomonadid flagellates from Rhinotermitidae. The presence of two deep‐branching lineages of trichonymphid flagellates in Serritermitidae and the absence of all taxa characteristic of the ancestral rhinotermitids underscores that the flagellate assemblages in the hindguts of lower termites were shaped not only by a progressive loss of flagellates during vertical inheritance but also by occasional transfaunation events, where flagellates were transferred horizontally between members of different termite families. In addition to the molecular phylogenetic analyses, we present a detailed morphological characterization of the new spirotrichosomid genus Heliconympha using light and electron microscopy.     

Lotharella amoeboformis sp. nov.: A new species of chlorarachniophytes from Japan

The ultrastructure, morphology and life cycle of a new chlorarachniophyte alga collected from Okinawa in Japan have been studied. The life cycle of this alga consists of amoeboid, wall‐less round, coccoid and flagellated cells in culture condition; however, the coccoid and flagellate cells are very rare. The pyrenoid ultra‐structure of this alga is the same as that of a previously described species, Lotharella globosa. Since pyrenoid ultrastructure has been adopted as the main criterion for the generic classification of the chlorarachniophytes, the present alga is placed in Lotharella. However, the present alga has a dominant amoeboid cell stage and a reduced walled‐cell stage in the life cycle, while in L. globosa, the walled‐cell stage is dominant and there is no amoeboid cell stage. Therefore the present alga is described as a new species of Lotharella: Lotharella amoeboformis Ishida et Y. Hara sp. nov.     

Dietary‐driven variation effects on the symbiotic flagellate protist communities of the subterranean termite Reticulitermes grassei Clément

The ability of subterranean termites to digest lignocellulose relies not only on their digestive tract physiology, but also on the symbiotic relationships established with flagellate protists and bacteria. The objective of this work was to test the possible effect of different cellulose‐based diets on the community structure (species richness and other diversity metrics) of the flagellate protists of the subterranean termite Reticulitermes grassei. Termites belonging to the same colony were subjected to six different diets (natural diet, maritime pine wood, European beech, thermally modified European beech, cellulose powder and starvation), and their flagellate protist community was evaluated after the trials. All non‐treated sound woods produced similar flagellate protist communities that were more diverse and of high evenness (low dominance). On the contrary, flagellate protist communities from cellulose‐fed termites and starving termites were considered to be significantly different from all non‐treated woods; they were less diverse and some morphotypes became dominant as a consequence of flagellate protist communities having suffered major adaptations to these diets. The flagellate protist communities of untreated beech and thermally modified beech‐fed termites were considered to be significantly different in terms of abundance and morphotype diversity. This may be caused by a decrease in lignocellulose quality available for termites and from an interference of thermally treated wood with the chemical stability of the termite hindgut. Our study suggests that as a consequence of the strong division of labour among these protists to accomplish the intricate process of lignocellulose digestion, termite symbiotic flagellate protist communities are a dynamic assemblage able to adapt to different conditions and diets. This study is important for the community‐level alteration approach, and it is the first study to investigate the effects of thermally modified wood on the flagellate protist communities of subterranean termites.     

Ptolemeba n. gen., a Novel Genus of Hartmannellid Amoebae (Tubulinea,Amoebozoa); with an Emphasis on the Taxonomy of Saccamoeba

Hartmannellid amoebae are an unnatural assemblage of amoeboid organisms that are morphologically difficult to discern from one another. In molecular phylogenetic trees of the nuclear‐encoded small subunit rDNA, they occupy at least five lineages within Tubulinea, a well‐supported clade in Amoebozoa. The polyphyletic nature of the hartmannellids has led to many taxonomic problems, in particular paraphyletic genera. Recent taxonomic revisions have alleviated some of the problems. However, the genus Saccamoeba is paraphyletic and is still in need of revision as it currently occupies two distinct lineages. Here, we report a new clade on the tree of Tubulinea, which we infer represents a novel genus that we name Ptolemeba n. gen. This genus subsumes a clade of hartmannellid amoebae that were previously considered in the genus Saccamoeba, but whose mitochondrial morphology is distinct from Saccamoeba. In accordance with previous research, we formalize the clade as distinct from Saccamoeba. Transmission electron microscopy of our isolates illustrate that both molecularly discrete species can be further differentiated by their unique mitochondrial cristal morphology.     

Unique features of the motility and structures in the flagellate polar region of Campylobacter jejuni and other species: an electron microscopic study

Similarly to Helicobacter pylori but unlike Vibrio cholerae O1/O139, Campylobacter jejuni is non‐motile at 20°C but highly motile at ≥37°C. The bacterium C. jejuni has one of the highest swimming speeds reported (>100 μm/s), especially at 42°C. Straight and spiral bacterial shapes share the same motility. C. jejuni has a unique structure in the flagellate polar region, which is characterized by a cup‐like structure (beneath the inner membrane), a funnel shape (opening onto the polar surface) and less dense space (cytoplasm). Other Campylobacter species (coli, fetus, and lari) have similar motility and flagellate polar structures, albeit with slight differences. This is especially true for Campylobacter fetus, which has a flagellum only at one pole and a cup‐like structure composed of two membranes.     

Phylogeny and Redescription of the Testate Amoeba Diaphoropodon archeri (Chlamydophryidae,Thecofilosea, Cercozoa), De Saedeleer 1934, and Annotations on the Polyphyly of Testate Amoebae with Agglutinated Tests in the Cercozoa

The genus Diaphoropodon, Archer 1869, comprises filose amoebae with agglutinated tests made of quartz grains, diatom frustules and other particulate materials. The key trait of the genus is a hyaline theca covered with numerous 5‐ to 10‐μm‐long, hairlike rods. Based on SSU rDNA phylogeny, we show that Diaphoropodon groups closely to Lecythium, a testate amoeba genus with a flexible but naked theca. Electron microscopic images reveal that the rods of Diaphoropodon are not perforating the test but lie randomly distributed on the surface of the amoeba. Comparing fairly naked cells from our cultures with cells from the environment leads to the conclusion that these rods play a role in agglutinating the material on the test.     

Characterization of Tulamoeba bucina n. sp., an Extremely Halotolerant Amoeboflagellate Heterolobosean Belonging to the Tulamoeba–Pleurostomum Clade (Tulamoebidae n. fam.)

Most protozoans that have been cultivated recently from high salinity waters appear to be obligate halophiles. Phylogenetic analyses indicate that these species mostly represent independent lineages. Here, we report the cultivation, morphological characterization, and phylogenetic analysis of two strains (XLG1 and HLM‐8) of a new extremely halotolerant heterolobosean amoeboflagellate. This species is closely related to the obligate halophiles Tulamoeba peronaphora and Pleurostomum flabellatum, and more specifically to the former. Like Tulamoeba, the new species has a monopodial limax amoeba stage, however, its cyst stage lacks an intrusive pore plug. The flagellate stage bears a combination of a planar spiral feeding apparatus and unequal heterodynamic flagella that discriminates it from described Pleurostomum species. Strain XLG1 grows at salinities from 35‰ to 225‰. This degree of halotolerance is uncommon in protozoa, as most species showing growth in seawater are unable to grow at 200‰ salinity. The unrelatedness of most halophilic protozoa suggested that independent colonization of the hypersaline environment is more common than speciation within it. However, this study supports the idea that the Tulamoeba–Pleurostomum clade underwent an adaptive radiation within the hypersaline environment. A new species Tulamoeba bucina n. sp. is described, with Tulamoebidae n. fam. proposed for the Tulamoeba–Pleurostomum clade.     

Nucleus‐encoded mRNAs for Chloroplast Proteins GapA,PetA, and PsbO are Trans‐spliced in the Flagellate Euglena gracilis Irrespective of Light and Plastid Function

Euglena gracilis is a fresh‐water flagellate possessing secondary chloroplasts of green algal origin. In contrast with organisms possessing primary plastids, mRNA levels of nucleus‐encoded genes for chloroplast proteins in E. gracilis depend on neither light nor plastid function. However, it remains unknown, if all these mRNAs are trans‐spliced and possess spliced leader sequence at the 5′‐end and if trans‐splicing depends on light or functional plastids. This study revealed that polyadenylated mRNAs encoding the chloroplast proteins glyceraldehyde‐3‐phosphate dehydrogenase (GapA), cytochrome f (PetA), and subunit O of photosystem II (PsbO) are trans‐spliced irrespective of light or plastid function.     

Functional Ecology of the Ciliate Glaucomides bromelicola,and Comparison with the Sympatric Species Bromeliothrix metopoides

We investigated the ecology and life strategy of Glaucomides bromelicola (family Bromeliophryidae), a very common ciliate in the reservoirs (tanks) of bromeliads, assessing its response to food quality and quantity and pH. Further, we conducted competition experiments with the frequently coexisting species Bromeliothrix metopoides (family Colpodidae). In contrast to B. metopoides and many other colpodean ciliates, G. bromelicola does not form resting cysts, which jeopardizes this ciliate when its small aquatic habitats dry out. Both species form bactivorous microstomes and flagellate‐feeding macrostomes. However, only G. bromelicola has a low feeding threshold and is able to adapt to different protist food. The higher affinity to the local bacterial and flagellate food renders it the superior competitor relative to B. metopoides. Continuous encystment and excystment of the latter may enable stable coexistence of both species in their natural habitat. Both are tolerant to a wide range of pH (4–9). These ciliates appear to be limited to tank bromeliads because they either lack resting cysts and vectors for long distance dispersal (G. bromelicola) and/or have highly specific food requirements (primarily B. metopoides).     

Evidence of enhanced microbial activity in the interstitial space of branched corals: possible implications for coral metabolism

Water samples from the interstitial space of 4 Indo-Pacific coral species (Acropora sp., Echinopora horrida, Psammocora digita and Pavona clavus) and a Mediterranean coral (Cladocora cespitosa) were analysed for NO ₃ ^- +NO ₂ ^- , NH ₄ ⁺ , molybdate reactive phosphorus, bacterial and flagellate biomass and dissolved organic matter (DOM) and compared with ambient water concentrations. Higher values of NO ₃ ^- +NO ₂ ^- , bacterial and flagellate biomass were observed within the interstitial space of the corals. The lower DOM pool in the interstitium in combination with the high bacterial biomass suggests high bacterial activity and efficient substrate utilization, necessary to compensate for nanoflagellate predation. Since corals may be able to feed on bacteria, the high microbial biomass (bacterial and flagellate) may be utilized either directly as an additional heterotrophic food source, or indirectly in that microbes may act as attractants for microbe-feeding zooplankters, which in turn serve as food for the corals. The combined effect of reduced flow velocities between the coral branches and its associated fauna are probably the main factors in creating a specific environment more or less independent of the nutritive stage of the surrounding water.     

Nebela jiuhuensis nov. sp. (Amoebozoa; Arcellinida; Hyalospheniidae): A New Member of the Nebela saccifera ‐ equicalceus ‐ ansata Group Described from Sphagnum Peatlands in South‐Central China

Hyalospheniids are among the most common and conspicuous testate amoebae in high‐latitude peatlands and forest humus. These testate amoebae were widely studied as bioindicators and are increasingly used as models in microbial biogeography. However, data on their diversity and ecology are still very unevenly distributed geographically: notably, data are lacking for low‐latitude peatlands. We describe here a new species, Nebela jiuhuensis, from peatlands near the Middle Yangtze River reach of south‐central China with characteristic morphology. The test (shell) has hollow horn‐like lateral extensions also found in N. saccifera, N. equicalceus (=N. hippocrepis), and N. ansata, three large species restricted mostly to Sphagnum peatlands of Eastern North America. Mitochondrial cytochrome oxidase (COI) data confirm that N. jiuhuensis is closely related to the morphologically very similar North American species N. saccifera and more distantly to N. ansata within the N. penardiana group. These species are all found in wet mosses growing in poor fens. Earlier reports of morphologically similar specimens found in South Korea peatlands suggest that N. jiuhuensis may be distributed in comparable peatlands in Eastern Asia (China and Korea). The discovery of such a conspicuous new species in Chinese peatlands suggests that many new testate amoebae species are yet to be discovered, including potential regional endemics. Furthermore, human activities (e.g., drainage, agriculture, and pollution) have reduced the known habitat of N. jiuhuensis, which can thus be considered as locally endangered. We, therefore, suggest that this very conspicuous micro‐organism with a probably limited geographical distribution and specific habitat requirement should be considered as a flagship species for microbial biogeography as well as local environmental conservation and management.     

Sappinia sp. (Amoebozoa: Thecamoebida) and Rosculus sp. (SAR: Cercozoa) Isolated From King Penguin Guano Collected in the Subantarctic (South Georgia,Salisbury Plain) and their Coexistence in Culture

Two amoeboid organisms of the genera Sappinia Dangeard, 1896 and Rosculus Hawes, 1963 were identified in a sample containing king penguin guano. This sample, collected in the Subantarctic, enlarges the list of fecal habitats known for the presence of coprophilic amoebae. The two organisms were co‐isolated and subcultured for over 6 mo, with continuous efforts being invested to separate each one from the mixed culture. In the mixed culture, Rosculus cells were fast growing, tolerated changes in culturing conditions, formed cysts, and evidently were attracted by Sappinia trophozoites. The separation of the Rosculus strain was accomplished, whereas the Sappinia strain remained intermixed with inseparable Rosculus cells. Sappinia cell populations were sensitive to changes in culturing conditions; they improved with reduction of Rosculus cells in the mixed culture. Thick‐walled cysts, reportedly formed by Sappinia species, were not seen. The ultrastructure of both organisms was congruent with the currently accepted generic characteristics; however, some details were remarkable at the species level. Combined with the results of phylogenetic analyses, our findings indicate that the ultrastructure of the glycocalyx and the presence/absence of the Golgi apparatus in differential diagnoses of Sappinia species require a critical re‐evaluation.     

A New Heterolobosean Amoeboflagellate,Tetramitus dokdoensis n. sp., Isolated from a Freshwater Pond on Dokdo Island in the East Sea,Korea

The genus Tetramitus is a representative amoeboflagellate group within the Heterolobosea, and currently contains over a dozen species. Here, a new heterolobosean amoeboflagellate was isolated from a freshwater pond on Dokdo Island, Korea. The amoebae have eruptive pseudopodia, no uroidal filament, and a nucleus with a central nucleolus. The length and width of the amoebae are 15.5–28.0 μm and 5.4–12.6 μm, respectively. The flagellates are conical, with 4 flagella of equal length (~10 μm). There is a discrete rostrum in the subapical region of the flagellate form. The cyst has thin endo‐ and ectocyst layers and no cyst pores. The amoeba shows slow movement at 37 °C, but does not move at 42 °C under a light microscope. Phylogenies of the 18S rRNA gene and the ITS1‐5.8S rRNA gene‐ITS2 sequence show that the strain belongs to a subclade of Tetramitus that includes Tetramitus rostratus, Tetramitus waccamawensis and Tetramitus entericus, amongst others. Nonetheless, the strain is distinct from other species in both molecular phylogenetic trees. Thus the strain isolated from the Dokdo Island is proposed as a novel species, Tetramitus dokdoensis n. sp.     

Short‐term temperature change may impact freshwater carbon flux: a microbial perspective

Small freshwater bodies are abundant and economically and ecologically important on a global scale. Within these, protozoa play an important role in structuring planktonic food webs and sequestering CO₂. We hypothesized that short‐term (～20 days) fluctuations, of 2–10 °C, will significantly alter carbon flux associated with predator–prey interactions within the microbial planktonic food web. We examined the model ciliate, Urotricha farcta, which is abundant and common; it was fed the autotrophic flagellate Cryptomonas sp., which is also common. Laboratory experiments were conducted over relevant ranges: 8–24 °C; 0–2 × 10⁵ prey mL^?1. Mechanistic‐phenomenological multiple regressions were developed and fit to the data to obtain relationships for (1) growth rate and volume changes of the flagellate vs. temperature and (2) growth rates, grazing, and cell volume change of the ciliate vs. temperature and prey concentration. Responses revealed interaction between temperature and prey levels on all ciliate parameters, indicating it is inappropriate to apply simple temperature corrections (e.g. Q₁₀) to such functions. The potential impact of such temperature changes on carbon flux was illustrated using a simple ciliate–flagellate predator–prey model, with and without the top grazer, Daphnia, added. The model indicated that predator–prey pulses occurred over 20 days, with the ciliate controlling the prey population. For ciliates and prey, carbon production peaked at 20 °C and rapidly decreased above and below this maximum; differences between minimum and maximum were approximately fourfold, for both prey and ciliate, with low levels at 25–30 °C and 10–15 °C. Including literature data to parameterize, the influence of the grazer Daphnia did not alter the prediction that the ciliate may control short‐term flagellate pulses and temperature will influence these in a nonintuitive fashion.     

Ultrastructure of the Amoeboflagellate Tetramitus rostratus1

The life-cycle of the amoeboflagellate Tetramitus rostratus includes amoeboid, cyst, and flagellate stages. The ultrastructure of these three stages is illustrated, with particular emphasis on flagellate morphology. Amoeba morphology is typical of that of limax amoebas. Cysts, forming from trophic amoebas, are enclosed by a wall made up of two layers: ectocyst (ca. 70 nm), and endocyst (200 nm). The wall apparently forms from precursor material present in vesicles in the pre-cyst stage cytoplasm. Flagellate morphology is characterized by a well-defined top-shaped profile, maintained by microtubules under the plasma membrane. The flagellar apparatus or mastigont consists of four flagella, their basal bodies, sheaves of microtubules associated with two of the basal bodies, and several rhizoplasts (periodicity 20 nm). A deep, microtubule-supported, ventral invagination appears to function as a gullet. A small number of mitotic stages observed in amoeboid and flagellate individuals suggests similarity in the division process in both stages: intranuclear mitotic apparatus, nucleolus persisting through mitosis, no centrioles or basal bodies functioning as centrioles, difficulty in resolving chromosomes. The text compares ultrastructures of several amoeboflagellate organisms and evaluates the phylogenetic significance of those features common to different species. On the basis of this study, Tetramitus most closely resembles Naegleria spp.     

Testate Amoebae from a Cretaceous Forest Floor Microbiocoenosis of France

ABSTRACT. Amber‐preserved shells of testate amoebae often provide as many diagnostic features as the tests of modern taxa. Most of these well‐preserved microfossils are morphologically assignable to modern species indicating either evolutionary stasis or convergent evolution. Here we describe two Lower Cretaceous testate amoebae that are clearly distinguishable from modern species. Centropyxis perforata n. sp. and Leptochlamys galippei n. sp. possessed perforate shells that were previously unknown in these genera. They are preserved in highly fossiliferous amber pieces from the Upper Albian (ca. 100 million years old) of Archingeay/Les Nouillers (Charente‐Maritime, southwestern France). Syninclusions of soil and litter dwelling arthropods and microorganisms indicate a limnetic‐terrestrial microhabitat at the floor of a coastal conifer forest.