The Vampyrellid Amoebae (Vampyrellida, Rhizaria).

The Vampyrellida (Endomyxa, Rhizaria) is a group of free-living, predatory amoebae, which is most closely related to the Phytomyxea (plasmodiophorids and phagomyxids). It encompasses about 50 credibly described species that have a characteristic life history with the regular alternation of trophic amoebae and immobile digestive cysts. All known vampyrellid amoebae are naked and filose, but the different species display a broad morphological variety. Vampyrellids also vary greatly in their feeding habits, and range from generalist predators to specialized 'protoplast feeders' that exclusively feed on the cell contents of eukaryotic prey. They can be found in freshwater, soil and marine habitats, and appear to be globally distributed. Yet, the phenotypic diversity and ecological roles of the Vampyrellida are still poorly explored. Currently, there are eight well-recognized subclades that comprise four families (Vampyrellidae, Leptophryidae, Placopodidae and Sericomyxidae) as well as some lineages without any phenotypic information. Research on vampyrellids is challenging due to their cryptic occurrence in nature, intricate feeding habits that complicate cultivation, and a convoluted taxonomic history. Here, we review available information about cell structure, diversity, ecology, taxonomy and phylogenetics, and provide an up-to-date introduction to the Vampyrellida that may facilitate future research.

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.

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.

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.

Rhogostomidae (Cercozoa) from soils, roots and plant leaves (Arabidopsis thaliana): Description of Rhogostoma epiphylla sp. nov. and R. cylindrica sp. nov.

Cercozoa are a highly diverse protist phylum in soils and in the phyllosphere of plants. Many families are still poorly described and the vast majority of species are still unknown. Although testate amoebae are among the better-studied protists, only little quantitative information exists on the morphology, phylogeny and ecology of cercozoan Rhogostomidae. We cultured four different strains of Rhogostoma spp. isolated from Arabidopsis leaves, agricultural soil and rhizosphere soil of Ocimum basilicum and Nicotiana sp. We describe Rhogostoma epiphylla sp. nov. and R. cylindrica sp. nov. and present their morphology, studied their food spectra in food range experiments and obtained two SSU rDNA gene sequences resulting in an updated thecofilosean phylogeny. Short generation times, desiccation resistance and the ability to prey on a wide range of algae and yeasts from the phyllosphere were seen as crucial traits for the phyllosphere colonization by Rhogostoma. In contrast, the soil-dwelling R. cylindrica did not feed on eukaryotes in our experiment.

Polyphyly in the Thecate Amoeba Genus Lecythium (Chlamydophryidae, Tectofilosida, Cercozoa), Redescription of its Type Species L. hyalinum, Description of L. jennyae sp. nov. and the Establishment of Fisculla gen. nov. and Fiscullidae fam. nov.

Although testate amoebae have attracted great interest of protistologists for more than a century, some groups, especially those with a hyaline, organic test (=theca) are still poorly known. One of those is the genus Lecythium Hertwig et Lesser, 1874. Only recently Lecythium spp. were characterized by morphological and molecular means, but data on the type species Lecythium hyalinum Hertwig et Lesser, 1874, was still lacking. In this study, we screened for L. hyalinum in freshwater samples of Germany and the Netherlands. Four different isolates of L. hyalinum and one novel species were cultured and characterized by light microscopy. Phylogenetic analyses based on the small ribosomal subunit (SSU) RNA gene show that the genus Lecythium forms two robust clades, one forming a sister group to the Rhizaspididae/Pseudodifflugiidae clade (Tectofilosida), the other branching within 'Novel Clade 4' (Tectofilosida). We untangle this polyphyly by establishing Fisculla gen. nov. and the Fiscullidae fam. nov. for the former of these two clades.

Shedding Light on the Polyphyletic Thecate Amoeba Genus Plagiophrys: Transition of Some of its Species to Rhizaspis (Tectofilosida, Thecofilosea, Cercozoa) and the Establishment of Sacciforma gen. nov. and Rhogostomidae fam. nov. (Cryomonadida, Thecofilosea, Cercozoa).

For over a century testate amoebae have been a favoured group of interest for protistologists, however there is still an endless amount of unanswered questions. The genus Plagiophrys, Claparède and Lachmann 1859, is still one of the unresolved mysteries as it comprises species with high morphological diversity of which no molecular data are available. To shed light on the phylogeny and taxonomy of Plagiophrys we (a) cultured four isolates of three Plagiophrys morphospecies and provided morphological observations (b) obtained three new SSU RNA gene sequences and conducted phylogenetic analyses of the Thecofilosea and (c) did intensive literature research, showing that Plagiophrys is polyphyletic. We partially untangle this polyphyly by combining several of its species with the genus Rhizaspis, Skuja 1948. Furthermore, we establish Sacciforma gen. nov. to accommodate P. sacciformis as it groups within the formerly known Rhizaspididae, which do not comprise our isolates of Rhizaspis (and therefore were renamed Rhogostomidae) since Rhizaspis it groups with maximum support as a sister-group to the Pseudodifflugiidae.

A Bowl with Marbles: Revision of the Thecate Amoeba Genus Lecythium (Chlamydophryidae, Tectofilosida, Cercozoa, Rhizaria) Including a Description of Four New Species and an Identification Key.

Although testate amoebae have attracted interest of protistologists for more than 150 years, some groups especially those with a hyaline, organic test (= theca) are still poorly known. One of those is the genus Lecythium (Chlamydophryidae, Tectofilosida, Cercozoa, Rhizaria), first described by Hertwig and Lesser in 1874. Only old, sometimes obscure, species descriptions were available until only recently a new species of Lecythium was described and a small ribosomal subunit RNA gene (SSU) sequence was provided. To shed light on the phylogeny and taxonomy of Lecythium, we (a) cultured six isolates of five Lecythium species and provide morphological as well as ecological observations, (b) obtained six new SSU sequences and conducted phylogenetic analyses of the Tectofilosida, showing that Lecythium splits into terrestrial and freshwater clades, and (c) did an intensive literature research on testate amoebae with a theca and provide an illustrated identification key focusing on Lecythium. For the first time, the presence of cysts in the genus Lecythium is reported and we compared those to the cysts of the presumed closely related Chlamydophrys stercorea. Our results suggest that still many undescribed Lecythium species will be found in terrestrial and freshwater habitats.

A Novel Lineage of 'Naked Filose Amoebae'; Kraken carinae gen. nov. sp. nov. (Cercozoa) with a Remarkable Locomotion by Disassembly of its Cell Body.

The term 'filose amoebae' describes a highly polyphyletic assemblage of protists whose phylogenetic placement can be unpredictable based on gross morphology alone. We isolated six filose amoebae from soils of two European countries and describe a new genus and species of naked filose amoebae, Kraken carinae gen. nov. sp. nov. We provide a morphological description based on light microscopy and small subunit rRNA gene sequences (SSU rDNA). In culture, Kraken carinae strains were very slow-moving and preyed on bacteria using a network of filopodia. Phylogenetic analyses of SSU sequences reveal that Kraken are core (filosan) Cercozoa, branching weakly at the base of the cercomonad radiation, most closely related to Paracercomonas, Metabolomonas, and Brevimastigomonas. Some Kraken sequences are >99% similar to an environmental sequence obtained from a freshwater lake in Antarctica, indicating that Kraken is not exclusively soil dwelling, but also inhabits freshwater habitats.

Molecular Phylogeny of the Widely Distributed Marine Protists, Phaeodaria (Rhizaria, Cercozoa).

Phaeodarians are a group of widely distributed marine cercozoans. These plankton organisms can exhibit a large biomass in the environment and are supposed to play an important role in marine ecosystems and in material cycles in the ocean. Accurate knowledge of phaeodarian classification is thus necessary to better understand marine biology, however, phylogenetic information on Phaeodaria is limited. The present study analyzed 18S rDNA sequences encompassing all existing phaeodarian orders, to clarify their phylogenetic relationships and improve their taxonomic classification. The monophyly of Phaeodaria was confirmed and strongly supported by phylogenetic analysis with a larger data set than in previous studies. The phaeodarian clade contained 11 subclades which generally did not correspond to the families and orders of the current classification system. Two families (Challengeriidae and Aulosphaeridae) and two orders (Phaeogromida and Phaeocalpida) are possibly polyphyletic or paraphyletic, and consequently the classification needs to be revised at both the family and order levels by integrative taxonomy approaches. Two morphological criteria, 1) the scleracoma type and 2) its surface structure, could be useful markers at the family level.

Shedding light on vampires: the phylogeny of vampyrellid amoebae revisited.

With the advent of molecular phylogenetic techniques the polyphyly of naked filose amoebae has been proven. They are interspersed in several supergroups of eukaryotes and most of them already found their place within the tree of life. Although the 'vampire amoebae' have attracted interest since the middle of the 19th century, the phylogenetic position and even the monophyly of this traditional group are still uncertain. In this study clonal co-cultures of eight algivorous vampyrellid amoebae and the respective food algae were established. Culture material was characterized morphologically and a molecular phylogeny was inferred using SSU rDNA sequence comparisons. We found that the limnetic, algivorous vampyrellid amoebae investigated in this study belong to a major clade within the Endomyxa Cavalier-Smith, 2002 (Cercozoa), grouping together with a few soil-dwelling taxa. They split into two robust clades, one containing species of the genus Vampyrella Cienkowski, 1865, the other containing the genus Leptophrys Hertwig & Lesser, 1874, together with terrestrial members. Supported by morphological data these clades are designated as the two families Vampyrellidae Zopf, 1885, and Leptophryidae fam. nov. Furthermore the order Vampyrellida West, 1901 was revised and now corresponds to the major vampyrellid clade within the Endomyxa, comprising the Vampyrellidae and Leptophryidae as well as several environmental sequences. In the light of the presented phylogenetic analyses morphological and ecological aspects, the feeding strategy and nutritional specialization within the vampyrellid amoebae are discussed.

Relationships between free-living protozoa, cultivable Legionella spp., and water quality characteristics in three drinking water supplies in the Caribbean.

The study whose results are presented here aimed at identifying free-living protozoa (FLP) and conditions favoring the growth of these organisms and cultivable Legionella spp. in drinking water supplies in a tropical region. Treated and distributed water (±30°C) of the water supplies of three Caribbean islands were sampled and investigated with molecular techniques, based on the 18S rRNA gene. The protozoan host Hartmannella vermiformis and cultivable Legionella pneumophila were observed in all three supplies. Operational taxonomic units (OTUs) with the highest similarity to the potential or candidate hosts Acanthamoeba spp., Echinamoeba exundans, E. thermarum, and an Neoparamoeba sp. were detected as well. In total, 59 OTUs of FLP were identified. The estimated protozoan richness did not differ significantly between the three supplies. In supply CA-1, the concentration of H. vermiformis correlated with the concentration of Legionella spp. and clones related to Amoebozoa predominated (82%) in the protozoan community. These observations, the low turbidity (<0.2 nephelometric turbidity units [NTU]), and the varying ATP concentrations (1 to 12 ng liter(-1)) suggest that biofilms promoted protozoan growth in this supply. Ciliophora represented 25% of the protozoan OTUs in supply CA-2 with elevated ATP concentrations (maximum, 55 ng liter(-1)) correlating with turbidity (maximum, 62 NTU) caused by corroding iron pipes. Cercozoan types represented 70% of the protozoan clones in supply CA-3 with ATP concentrations of <1 ng liter(-1) and turbidity of <0.5 NTU in most samples of distributed water. The absence of H. vermiformis in most samples from supply CA-3 suggests that growth of this protozoan is limited at ATP concentrations of <1 ng liter(-1).