Search

Two marine members of the genus Vexillifera Schaeffer, 1926 (Amoebozoa, Dactylopodida) are described. Vexillifera abyssalis n. sp. originates from an abyssal sample of the Western Atlantic 4.5 km deep, which is the first unambiguous record of a deep-sea Vexillifera. The second species, V. kereti n. sp. was isolated from the soft bottom sediments of the White Sea (depth 106 m). An analysis of available data on the genus Vexillifera shows that it comprises many different species, yet they are very unevenly studied. The majority of species have only been described using light microscopy, and their phylogenetic relationships with other amoebae are unclear. However, available small-subunit (SSU) rRNA gene sequences of Vexillifera spp. form a robust, yet very heterogeneous clade in the phylogenetic tree. These species demonstrate a wide range of morphological and ultrastructural characters and originate from diverse habitats, suggesting that Vexillifera may need to be subdivided into several genera in the future. In addition to the described species, we sequenced the COI gene of original CCAP strains of Vexillifera bacillipedes, V. minutissima and Pseudoparamoeba pagei, thereby performing a phylogenetic reconstruction of the Dactylopodida based on a decent taxonomic sampling.

Mitochondrial genome sequence of Vannella croatica (Amoebozoa, Discosea, Vannellida) was obtained using pulse-field gel electrophoretic isolation of the circular mitochondrial DNA, followed by the next-generation sequencing. The mitochondrial DNA of this species has the length of 28,933 bp and contains 12 protein-coding genes, two ribosomal RNAs, and 16 transfer RNAs. Vannella croatica mitochondrial genome is relatively short compared to other known amoebozoan mitochondrial genomes but is rather gene-rich and contains significant number of open reading frames.

Vannella simplex (Amoebozoa, Discosea, Vannellida) is one of the commonest freshwater free-living lobose amoebae, known from many locations worldwide. In the present study, we describe the complete mitochondrial genome of this species. The circular mitochondrial DNA of V. simplex has 34,145obp in length and contains 27 protein-coding genes, 2 ribosomal RNAs, 16 transfer RNAs and 4 open reading frames. Mitochondiral genome of V. simplex is one of the most gene compact due to overlapping genes and reduced intergenic space. It has much in common with its closest relative, mitochondrial genome of V. croatica GenBank number MF508648. In the same time, both of them show considerable differences in length and in gene order from the next close relative – that of Neoparamoeba pemaquidensis KX611830 (deposited as Paramoeba) and even more – from other sequenced amoebozoan mitochondrial genomes. The present study confirms the opinion that the level of synteny between the mitochondrial genomes across the entire Amoebozoa clade is low. More or less considerable similarity yet was found only between members of the same clade of the genera or family level, but hardly — among more distant lineages.

Amoeboid protists are extremely abundant and diverse in natural systems where they often play outstanding ecological roles. They can be found in almost all major eukaryotic divisions, and genomic approaches are bringing major changes in our perception of their deep evolutionary relationships. At fine taxonomic levels, the generalization of barcoding is revealing a considerable and unsuspected specific diversity that can be appreciated with careful morphometric analyses based on light and electron microscopic observations. We provide examples on the difficulties and advances in amoeboid protists systematics in a selection of groups that were presented at the VIIIth ECOP/ISOP meeting in Rome, 2019. We conclude that, in all studied groups, important taxonomical rearrangements will certainly take place in the next few years, and systematics must be adapted to incorporate these changes. Notably, nomenclature should be flexible enough to integrate many new high level taxa, and a unified policy must be adopted to species description and to the establishment of types.

The genus Cunea Kudryavtsev and Pawlowski, 2015 (Amoebozoa, Dactylopodida) was initially described from the oceanic benthos: C. profundata, from over 5 km depth in the Atlantic Ocean, and C. thuwala from the Red Sea benthos at ca. 60 m depth. Both species are identical to each other in morphology (including cell coat ultrastructure), but differ significantly in the gene sequence data, including barcoding loci of small subunit ribosomal RNA and cytochrome oxidase subunit 1 gene, as well as actin. This paper describes the third species of Cunea, C. russae n. sp. isolated from a brackish water habitat without a direct connection to the ocean, a small spring of brackish water (19‰) emerging from a 246 m deep hole in the earth. This species is morphologically identical to the previous two amoebae, but differs from them significantly in the gene sequence data and ecological preferences. In particular, this species has the broadest salinity tolerance range, being able to reproduce well already at 2.5‰. It is also capable of resisting cold temperatures, like C. profundata. The data obtained suggest that the genus Cunea may comprise a significant taxonomic diversity represented by morphologically identical, but quickly diverging species with significant ecological plasticity.

The order Himatismenida (Amoebozoa, Discosea) comprises naked amoebae with an organic coat that is located on the dorsal surface of the cell. The phylogenetic relationships among deeply branching genera of the Himatismenida are unclear, as data on the species diversity of the himatismenid genera is largely restricted to the derived genus Cochliopodium. Here, we describe two new amoeba species that branch at the base of the order Himatismenida, evidenced by SSU rRNA gene and multigene analyses. Among them, a freshwater species Planopodium haveli gen. nov., sp. nov. has a dorsal cell coat consisting of flat, oval scales. This species forms a clade at the base of the Himatismenida, and the previously described Ovalopodium desertum, its closest relative, is transferred into the new genus as Planopodium desertum comb. nov. Although the two species are barely distinguishable by their sequence data, they are clearly distinct in morphology. Using this data, we can report the first evidence of a dorsal cell coat consisting of scales outside of the genus Cochliopodium. The other species has a marine origin and branches deeply, close to the root of the phylogenetic tree of Himatismenida. Based on the morphology of this amoeba, it should be described as Ovalopodium rosalinum sp. nov., a new species of the genus Ovalopodium. Analyses of the phylogenetic relationships and the ultrastructure of the deeply branching himatismenids, together with several of the newly obtained gene sequences of Parvamoeba and Cochliopodium, suggest that some elements of the dorsal cell coat of Ovalopodium may be ancestral for Himatismenida and have been partly retained in various more derived species of this clade, in particular, Cochliopodium gallicum. Although actin and Cox1 gene data do not resolve the higher-level relationships in Himatismenida, they correspond to the grouping of species within most genera.

Vannella samoroda n. sp. (Amoebozoa, Vannellida) was isolated from the mouth of the Malaya Samoroda river flowing into Elton, the largest European hypersaline lake (Russia). Among all rivers of the area, it has the highest salt content (ca. 110‰). Amoebae maintained in seawater medium with ca. 77‰ salts concentration had a set of morphological characters typical of Vannella spp.: rounded, fan-shaped, or spatulate locomotive form, floating form with bent, blunt-ended hyaline pseudopodia, and a cell coat consisting of regularly packed palisade elements and scarce simple filaments. Phylogenetic analyses based on SSU rRNA and cytochrome C oxidase subunit 1 genes show that the amoeba is most closely related to Vannella ebro Smirnov, 2001, but represents a distinct species. The clade of V. ebro and V. samoroda branches among marine species of Vannella. The studied species is the first member of the genus Vannella from a continental saline habitat described using molecular data. Interestingly, it has a broad range of salinity tolerance: cells reproduce above 18‰, while survival of a few cells regularly occurs even in highly diluted Prescott and James medium. The normal culture restores itself when PJ medium is substituted with 77‰ seawater medium even after months of experimental incubation.

Marine amebae of the genus Paramoeba (Amoebozoa, Dactylopodida) normally contain a eukaryotic endosymbiont known as Perkinsela-like organism (PLO). This is one of the characters to distinguish the genera Neoparamoeba and Paramoeba from other Dactylopodida. It is known that the PLO may be lost, but PLO-free strains of paramoebians were never available for molecular studies. Recently, we have described the first species of the genus Paramoeba which has no parasome-Paramoeba aparasomata. In this study, we present a mitochondrial genome of this species, compare it with that of Neoparamoeba pemaquidensis, and analyze the evolutionary dynamics of gene sequences and gene order rearrangements between these species. The mitochondrial genome of P. aparasomata is 46,254 bp long and contains a set of 31 protein-coding genes, 19 tRNAs, two rRNA genes, and 7 open reading frames. Our results suggest that these two mitochondrial genomes within the genus Paramoeba have rather similar organization and gene order, base composition, codon usage, the composition and structure of noncoding, and overlapping regions.

116 pages, 1 figure, 4 tables, 3 appendices, This revision of the classification of eukaryotes follows that of Adl et al., 2012 [J. Euk. Microbiol. 59(5)] and retains an emphasis on protists. Changes since have improved the resolution of many nodes in phylogenetic analyses. For some clades even families are being clearly resolved. As we had predicted, environmental sampling in the intervening years has massively increased the genetic information at hand. Consequently, we have discovered novel clades, exciting new genera and uncovered a massive species level diversity beyond the morphological species descriptions. Several clades known from environmental samples only have now found their home. Sampling soils, deeper marine waters and the deep sea will continue to fill us with surprises. The main changes in this revision are the confirmation that eukaryotes form at least two domains, the loss of monophyly in the Excavata, robust support for the Haptista and Cryptista. We provide suggested primer sets for DNA sequences from environmental samples that are effective for each clade. We have provided a guide to trophic functional guilds in an appendix, to facilitate the interpretation of environmental samples, and a standardized taxonomic guide for East Asian users, Research support was provided as follows: SMA by NSERC 249889‐2007; DB by NERC NE/H009426/1 and NE/H000887/1; MWB by NSF 1456054; FB by a Fellowship from Science for Life Laboratory and VR/2017‐04563; PC by EU‐Horizon 2020 research and innovation program through the SponGES project 679849 [...]; IC by CSF 18‐18699S; BE by RCN TaxMArc 268286/GMR; LG by ANR HAPAR (ANR‐14‐CE02‐0007); VH MK JL by ERDF; MEYS with ERC 771592 CZ 1.05/1.1.00/02.0109 BIOCEV; SK by RSF 16‐14‐10302; MK by CSF GA18‐28103S; CEL by NSF 1541510 and NIH‐AI124092; EL by CAM: 2017‐T1/AMB‐5210; and by grant 2017‐T1/AMB‐5210 from the program >Atracción de talentos> from the Consejería de Educación, Juventud y Deporte, Comunidad de Madrid; JL by ERC CZ LL1601 and OPVVV 16_019/0000759; MP by NSF DEB‐1455611; DJR by the Beatriu de Pinós postdoctoral programme of the Government of Catalonia's Secretariat for Universities and Research of the Ministry of Economy and Knowledge; CLS by the intramural research program of the National Library of Medicine, National Institutes of Health; AS by RSF 17‐14‐01391 and RFBR 16‐04‐01454 NY by NSF DEB 1557102; VZ by RFBR 16‐34‐60102 mol‐a‐dk; UniEuk and EukRef by the Gordon and Betty Moore Foundation

We isolated and described a brackish-water amoeba, Clydonella sawyeri n. sp. (Amoebozoa, Vannellida), from the littoral habitat in Kandalaksha Bay (The White Sea, northwestern Russia). Morphology of this amoeba corresponds to the initially proposed diagnosis of the genus Clydonella Sawyer, 1975, although it is sufficiently different from other described species of this genus to warrant a distinct species designation. Phylogenetic analysis based on the small-subunit rRNA gene shows that this species is closely related to the two previously studied ATCC strains identified as Clydonella sp., for which only ultrastructural and molecular data were published. Cytochrome oxidase subunit 1 (COI) gene sequence of the studied species was for the first time obtained for Clydonella. Analysis of this marker shows that this genus belongs to Vannellida and is separated from the other vannellids sampled to date. Phylogenetic analysis of the concatenated SSU rRNA and COI genes dataset yields the best resolved position of Clydonella compared to both markers when analysed separately. Based on the data presented we finally link light microscopic, ultrastructural and molecular data in a description of a single strain, which allows a refinement of the diagnosis of the genus Clydonella.

Two strains of lobose amoebae have been isolated from the deep-sea bottom sediments of the Sea of Japan (3.6km deep) and Western Atlantic Ocean (5.1km deep). Amoebae of both strains have a dactylopodial mophotype, intracellular kinetoplastid symbiont (Perkinsela-like organism) and have no microscales on the cell surface. The morphology and molecular data of the Sea of Japan strain allow us to unambiguously identify it as Neoparamoeba aestuarina (Page, 1970). At the same time, the Atlantic strain is described as a new species Neoparamoeba longipodia as it differs from other species of the genus Neoparamoeba in morphology and gene sequence data. The data presented expand the range of known habitats for the genus Neoparamoeba and permit further analysis of the phylogenetic relationships within this clade with the expanded set of molecular data.

Two new species, Ripella decalvata and R. tribonemae (Amoebozoa, Vannellida), are described and the diversity of known strains assigned to the genus analyzed. Ripella spp. are closely similar to each other in the light microscopic characters and sequences of small-subunit (SSU) ribosomal RNA gene, but differences in the cell coat structure and cytochrome oxidase (COI) gene sequences are more prominent. SSU rRNA in R. platypodia CCAP1589/2, R. decalvata and R. tribonemae demonstrates an unusual pattern of intragenomic variation. Sequencing of multiple molecular clones of this gene produced numerous sequence variants in a number of specific sites. These sites were usually terminal parts of several variable helices in all studied strains. Analysis of all known Ripella strains shows that SSU rRNA sites differing between strains of different origin are mainly restricted to these areas of the gene. There are only two sites, which differ between strains, but not within genomes. This intragenomic variability of the SSU rRNA gene, seemingly characteristic of all Ripella spp., was never reported to be so extensive in Amoebozoa. The data obtained show another example of complex organization of rRNA gene cluster in protists and emphasize caution needed when interpreting the metagenomic data based on this marker.

We present the results of an ultrastructural re-investigation of two amoebae strains that can be identified morphologically as previously described species of the genus Pellita, as well as the first molecular phylogenetic analysis of these amoebae based on SSU rRNA and actin gene sequences. The results obtained show close relationships between the genera Pellita, Gocevia, and Endostelium. These relationships are further supported by the description of Endostelium crystalliferum n. sp., which shares morphological characters simultaneously with Pellita spp. and Endostelium zonatum. The three genera form a robust clade that branches deeply within Amoebozoa, among either Flabellinia, or Longamoebia, depending on taxon sampling. The results suggest that Gocevia and Endostelium should not be included in the Himatismenida; therefore, we transfer the family Goceviidae into Pellitida. The type of cell organisation that was considered to be typical of Himatismenida (a lens-shaped cell covered dorsally with a flexible layer of organic material) has most probably evolved in some of these amoebae independently of Cochliopodiidae and Parvamoebidae. The robustness of the Pellitida clade in the molecular trees is consistent with the fine structure cytoplasmic evidence for these taxa, in particular, the presence of a centrosphere (a dictyosome-associated lamellar MTOC) in all these genera.

Two brackish water amoebae have been isolated and studied from the benthic biotopes of the Chupa Inlet (Kandalaksha Bay, northwestern Russia). Both strains can be identified as new species of the genus Paramoeba (Amoebozoa, Dactylopodida, Paramoebidae) based on light microscopical characters, structure of microscales on the cell surface and molecular evidence based on the analyses of two genes, nuclear SSU rRNA and mitochondrial cytochrome c oxidase subunit 1 (COI). Paramoeba aparasomata n. sp. is of particular interest because this amoeba is permanently lacking a symbiotic Perkinsela-like organism (PLO) present in other species of Paramoeba and Neoparamoeba. The results obtained show that scaly dactylopodial amoebae lacking PLO are not necessarily members of Korotnevella. In particular, we suggest that Korotnevella nivo Smirnov, 1997, with microscales very similar to those of Paramoeba eilhardi and the species studied here in structure, may be in fact a member of Paramoeba. Molecular data on K. nivo have to be obtained and analysed to test this hypothesis. Based on our new results we emend the diagnosis of the genus Paramoeba to make it more fit to the current phylogenetic conception.

We describe four new species of Flabellula, Leptomyxa and Rhizamoeba and publish new SSU rRNA gene and actin gene sequences of leptomyxids. Using these data we provide the most comprehensive SSU phylogeny of leptomyxids to date. Based on the analyses of morphological data and results of the SSU rRNA gene phylogeny we suggest changes in the systematics of the order Leptomyxida (Amoebozoa: Lobosa: Tubulinea). We propose to merge the genera Flabellula and Paraflabellula (the genus Flabellula remains valid by priority rule). The genus Rhizamoeba is evidently polyphyletic in all phylogenetic trees; we suggest retaining the generic name Rhizamoeba for the group unifying R. saxonica, R.matisi n. sp. and R. polyura, the latter remains the type species of the genus Rhizamoeba. Based on molecular and morphological evidence we move all remaining Rhizamoeba species to the genus Leptomyxa. New family Rhizamoebidae is established here in order to avoid paraphyly of the family Leptomyxidae. With the suggested changes both molecular and morphological systems of the order Leptomyxida are now fully congruent to each other.

I have re-investigated the light-microscopic features of Parvamoeba rugata Rogerson, 1993, type strain CCAP 1556/1. The major characters of amoebae correspond to the initial description of this species. However, one peculiarity demonstrated by the cells during adhesion to the substratum, seems to have been partly underestimated previously. At the same time it is crucial for the explanation of this species' position in the molecular phylogenetic trees and the recent system of Amoebozoa. This feature is the formation of a thin sheet of hyaloplasm on the ventral side of the cell that is used for adhesion to the substratum and locomotion, shared between Parvamoeba and members of the order Himatismenida. This explains the position of Parvamoeba as a sister clade to Cochliopodiidae in the molecular phylogenetic trees and justifies the recent inclusion of this genus into the order Himatismenida. In addition I sequenced the small-subunit ribosomal RNA of P. rugata and demonstrated that it was 99.5-99.7% similar to that of P. monoura Cole et al., 2010. This raises a question of the possible identity of these two species, however, several persistent morphological differences do not permit the unification of them, at least until more genes demonstrate identical sequences between these species.

An amoeba isolated from a weakly saline semi-desert pond in Kazakhstan (Central Asia) resembles a small Cochliopodium in the light microscope, but has a dorsal fibrous cell coat without scales. Thus it can be identified morphologically as a new species of Ovalopodium Sawyer, 1980, and it is herein named O. desertum. Phylogenetic analysis of the SSU rRNA gene sequences of the new species and four Cochliopodium spp. sequenced additionally shows that Ovalopodium desertum is a sister clade to a robustly monophyletic Cochliopodium. The close relationship between Ovalopodium and Cochliopodium is also confirmed by the analysis of SSU rRNA secondary structure showing the specific helices in the region V5 in all species of both genera. Analysis of actin gene sequences fails to resolve the position of Ovalopodium but demonstrates that Parvamoeba Rogerson, 1993 is probably related to Cochliopodium. The position of Cochliopodiidae within Amoebozoa remains unresolved, despite our efforts to resolve it using broader taxonomic sampling of Amoebozoa, testing alternative tree topologies and removing the fast-evolving sites. Among sequenced genera, Parvamoeba and Endostelium Olive et al., 1984 are probable relatives to Cochliopodiidae. Molecular trees weakly support an inclusion of the family in Flabellinia (Discosea), but more phylogenomic data are necessary to test this hypothesis.

Amoebae of the genus Korotnevella are covered with scales, the structure of which is believed to be species–specific and allows distinguishing species reliably at the morphological level. We studied members of this genus in order to assess the genetic structure of the local populations of amoebae. For the present study we isolated nine freshwater strains of Korotnevella, belonging to three species, from two locations in North-Western Russia. In order to obtain data on the population structure of these amoebae, we identified all isolates based on the light-microscopic morphology and scale structure and investigated both inter-strain and intra-strain polymorphism of Cox I and 18S rRNA genes. Results show that both genes provide congruent patterns of population structure. The Cox I gene appears to be more reliable DNA barcode while the 18S rRNA gene shows an interesting pattern of polymorphism, which may represent phylotypes of amoebae. Local population of amoebae in every studied species consists of a number of genetic lineages (phylotypes), some shared between the populations while others are unique to a local habitat.

Only two species of Cochliopodium below 20 μm in size are currently known. These are the recently described marine Cochliopodium gallicum Kudryavtsev and Smirnov, 2006 and a freshwater C. minutum West, 1901, an incompletely described species that has never been re-isolated since its initial description. In this paper, the descriptions of three independent species of similar size below 20 μm, C. minutoidum , C. kieliense, and C. maeoticum are presented. In the light microscope they all resemble the description of C. minutum, C. minutoidum being most similar to this species. However, the available description of C. minutum is inadequate to permit the assignment of any strain to this species with confidence. Therefore, a status of nomen dubium is proposed for C. minutum . Furthermore, C. minutoidum appears to be very similar to a fish endobiont identified by Dykova et al. (1998. Cochliopodium minus , a scale-bearing amoeba isolated from organs of perch Perca fluviatilis . Dis. Aquat. Org. 34, 205–210) as C. minus . Direct comparison of my strain of C. minutoidum with the type strain of C. minus suggests that it is not identical to C. minus . The obtained data enabled me to add the ultrastructural features to the diagnosis of C. minus for more precise identification of this species.

Two species of amoebae, the marine Pellita catalonica n. g., n. sp. discovered in the Ebro Delta (Spain) and Niva Bay (Denmark) and the freshwater Pellita digitata n. comb., previously known from the UK and Switzerland and now found in North-Western Russia, have a very thick (0.5–0.8 μm) cell coat consisting of a fuzzy fibrous basal layer, covered with the dense layer of complex pentagonal glycostyles. The cell coat is integrated with the cell membrane and entirely envelops the amoeba, like a typical glycocalyx. For purposes of locomotion and phagocytosis the cell produces short papilliform subpseudopodia that protrude through the cell coat and are covered solely by the cell membrane. In order to accommodate these unusual organisms we established a new family, Pellitidae n. fam., within the subclass Gymnamoebia sensu Page, 1987, order Euamoebida.

A strain of Cochliopodium isolated from grassland soil at Sourhope Research Station (Scotland, UK) was found to be identical to the strain “ Cochliopodium sp.2” studied by Bark in 1973. We name it Cochliopodium barki . It belongs to a group of species (comprising also C. minus and Cochliopodium sp. “NYS strain”) with very similar scale pattern.

This paper describes a new genus, Cunea n. g., of marine naked amoebae with two cryptic species, Cunea profundata and Cunea thuwala, isolated from distant localities in the ocean and different depths (Brazilian abyssal plain, Western Atlantic Ocean, depth >5 km and the Red Sea off the Saudi Arabian coast, depth ca. 58.7 m). Both species are very similar to each other in the set of light microscopic and ultrastructural characters and might be described as a single species, yet their genetic divergence based on 3 molecular markers (small-subunit ribosomal RNA, actin and cytochrome c oxidase subunit 1) corresponds to the level of variation typically observed between different morphospecies of Amoebozoa. In addition, the studied strains differ strongly in their temperature tolerance ranges, C. profundata isolated from the cold Atlantic deep-sea habitat being able to reproduce under lower temperatures than C. thuwala isolated from the warm Red Sea benthos. Molecular phylogenetic analysis based on SSU rRNA gene shows that the new genus robustly branches within the Dactylopodida, but forms an independent clade within this order that does not group with any of its known genera.

Microbial ecology is plagued by problems of an abstract nature. Cell sizes are so small and population sizes so large that both are virtually incomprehensible. Niches are so far from our everyday experience as to make their very definition elusive. Organisms that may be abundant and critical to our survival are little understood, seldom described and/or cultured, and sometimes yet to be even seen. One way to confront these problems is to use data of an even more abstract nature: molecular sequence data. Massive environmental nucleic acid sequencing, such as metagenomics or metatranscriptomics, promises functional analysis of microbial communities as a whole, without prior knowledge of which organisms are in the environment or exactly how they are interacting. But sequence-based ecological studies nearly always use a comparative approach, and that requires relevant reference sequences, which are an extremely limited resource when it comes to microbial eukaryotes [1]. In practice, this means sequence databases need to be populated with enormous quantities of data for which we have some certainties about the source. Most important is the taxonomic identity of the organism from which a sequence is derived and as much functional identification of the encoded proteins as possible. In an ideal world, such information would be available as a large set of complete, well-curated, and annotated genomes for all the major organisms from the environment in question. Reality substantially diverges from this ideal, but at least for bacterial molecular ecology, there is a database consisting of thousands of complete genomes from a wide range of taxa, supplemented by a phylogeny-driven approach to diversifying genomics [2]. For eukaryotes, the number of available genomes is far, far fewer, and we have relied much more heavily on random growth of sequence databases [3],[4], raising the question as to whether this is fit for purpose.

Amoebae of the genus Cochliopodium are characterized by a tectum that is a layer of scales covering the dorsal surface of the cell. A combination of scale structure, morphological features and, nowadays, molecular information allows species discrimination. Here we describe a soil species Cochliopodium plurinucleolum n. sp. that besides strong genetic divergence from all currently described species of Cochliopodium differs morphologically by the presence of several peripheral nucleoli in the nucleus. Further, we unambiguously show that the Golgi attachment associated with a dictyosome in Cochliopodium is a cytoplasmic microtubule organizing center (MTOC). Last, we provide detailed morphological and molecular information on the sister clade of C. plurinucleolum, containing C. minus, C. minutoidum, C. pentatrifurcatum and C. megatetrastylus. These species share nearly identical sequences of both, small subunit ribosomal RNA and partial Cox1 genes, and nearly identical structure of the scales. Scales of C. pentatrifurcatum differ, however, strongly from scales of the others while sequences of C. pentatrifurcatum and C. minus are nearly identical. These discrepancies urge for future sampling efforts to disentangle species characteristics within Cochliopdium and to investigate morphological and molecular patterns that allow reliable species differentiation.

Two new species of the recently described genus Stenamoeba, named S. berchidia and S. sardiniensis were isolated from a single soil sample on Sardinia, Italy. Both share morphological features characteristic to Stenamoeba and form in phylogenetic analyses together with other Stenamoeba spp. a highly supported clade within the family Thecamoebidae. The ultrastructural investigation of Stenamoeba sardiniensis revealed the presence of cytoplasmic microtubule-organizing centers (MTOCs), located close to one of several dictyosomes found inside the cell. This is the first report of cytoplasmic MTOCs among Thecamoebidae. The presence of MTOCs is now shown in five of nine orders comprising the class Discosea and potentially could be a phylogenetic marker in this group. We re-isolated Stenamoeba limacina from German soils. This strain shows a similar morphology and an almost complete SSU rDNA sequence identity with the type strain of S. limacina originating from gills of fishes, collected in Czech Republic.

Paravannella minima n. g. n. sp. (Amoebozoa, Vannellidae) isolated from a freshwater aquarium, possesses all light-microscopic and ultrastructural characteristics of the genus Vannella , being one of the smallest species among the vannellid amoebae (cell size during locomotion usually between 4.5 and 10 μm). At the same time, sequence analysis of the genes encoding for nuclear SSU rRNA, actin and mitochondrial Cox1 shows this species as the earliest-branching vannellid that appears to be sister to the rest of this clade. This is correlated with the presence of some plesiomorphic characters; in particular, the secondary structure of the hypervariable helices E23-1–E23-7 in the studied species is shared with Vannella and most of the genera of Dactylopodida. The cell coat structure of the studied species corresponds to the hypothesis that vannellid amoebae were ancestrally enclosed in a cell coat consisting of pentagonal glycostyles that have undergone multiple independent losses in various clades of Vannellidae.

Animals, plants, and fungi—the three traditional kingdoms of multicellular eukaryotic life—make up almost all of the visible biosphere, and they account for the majority of catalogued species on Earth [1]. The remaining eukaryotes have been assembled for convenience into the protists, a group composed of many diverse lineages, single-celled for the most part, that diverged after Archaea and Bacteria evolved but before plants, animals, or fungi appeared on Earth. Given their single-celled nature, discovering and describing new species has been difficult, and many protistan lineages contain a relatively small number of formally described species (Figure 1A), despite the critical importance of several groups as pathogens, environmental quality indicators, and markers of past environmental changes. It would seem natural to apply molecular techniques such as DNA barcoding to the taxonomy of protists to compensate for the lack of diagnostic morphological features, but this has been hampered by the extreme diversity within the group. The genetic divergence observed between and within major protistan groups greatly exceeds that found in each of the three multicellular kingdoms. No single set of molecular markers has been identified that will work in all lineages, but an international working group is now close to a solution. A universal DNA barcode for protists coupled with group-specific barcodes will enable an explosion of taxonomic research that will catalyze diverse applications.

Squamamoeba japonica n. g. n. sp. was isolated and described from marine bottom sediments collected at a depth of ca. 2700 m in the Sea of Japan. Trophic amoebae of this species are elongated and flattened, with a wide anterior hyaloplasm producing numerous ventral subpseudopodia for adhesion to the substratum. The cell coat consists of flat oval scales tightly packed together to form a continuous layer separated from the plasma membrane. Amoebae can form cytoplasmic projections protruding through the scale layer and having tips covered only with the plasma membrane. Small subunit ribosomal RNA gene phylogeny shows that S. japonica forms a long branch in the amoebozoan tree, robustly grouping with the marine strain ‘Pessonella’ sp. PRA-29. Morphological data available for the latter, although scarce, give additional support for the relatedness of both species. The resulting clade comprising the two taxa shows no close relationships to other Amoebozoa and seems to be a novel lineage that developed an ability to temporarily liberate local areas of the plasma membrane from the cell coat independently from Himatismenida, Trichosida, Pellitida and Dermamoeba.

Spumochlamys perforata n. sp. and Spumochlamys bryora n. sp. were isolated and described from dry epiphytic moss. The morphology and ultrastructure of both species clearly demonstrate that they belong to the genus Spumochlamys (family Microchlamyiidae). They differ from its only described member, Spumochlamys iliensis (as well as from species of Microchlamys), in the relief of the dorsal surface of the test, revealed by scanning electron microscopy, which can represent a good characteristic for species identification. They also differ in the structure of the dorsal part of the test wall (especially S. perforata). Small subunit ribosomal DNA-based molecular phylogenetic analyses show that Spumochlamys is a deeply branching lineage of the Arcellinida, without any close affinities. Actin gene sequence analysis places this genus within the Tubulinea, close to two other arcellinid lineages but without forming a monophyletic group with them. These data together strongly suggest that the lack of resolution in the arcellinid molecular phylogenies is due to serious undersampling of taxa, a limited number of sequence data, and high divergence rates in most of the species.

We isolated and described three new freshwater amoebozoan species that could be unambiguously assigned to the genus Flamella Schaeffer, 1926 by light microscopy. The phylogenetic position of the genus Flamella within the Amoebozoa was unknown, and gene sequence data were lacking. We sequenced the SSU rRNA gene of five Flamella spp., including a previously described F. aegyptia Michel et Smirnov, 1999. The phylogenetic trees inferred from these data showed, that Flamella is monophyletic and robustly branches within Amoebozoa. It belongs to a clade comprising Filamoeba spp., “Arachnula” sp., some protostelids and several SSU rRNA sequences of unidentified or uncultured eukaryotes. This clade consistently branched close to Archamoebae, Mycetozoa, Acramoeba dendroida and Multicilia marina; in contrast to the previous hypotheses, Flamella spp. did not show any relatedness either to Leptomyxida, or to Flabellinea. The ultrastructure of trophic amoebae and especially cysts of the species studied showed considerable similarity to Comandonia operculata Pernin et Pussard, 1979. We therefore suggest that Comandonia may be a junior synonym of Flamella, although more ultrastructural data about Comandonia operculata are necessary to test this hypothesis.

Spumochlamys iliensis n. g., n. sp. was isolated from a mineral pond in an arid, semi-desert region in Kazakhstan (Central Asia). This amoeba is covered with a plate-shaped, flexible organic spongious test. The thin membranous margin of this test extends ventrally to surround a flexible ventral aperture. In its morphological features and behaviour this amoeba is very similar to Microchlamys patella (Claparede and Lachmann, 1859) Cockerell, 1911, but differs from this species and from M. sylvatica Golemansky, Skarlato and Todorov, 1987 in the lack of an additional membrane separating the cell body from the test, a feature that can only be detected using electron microscopy. The presence of this membrane is considered to be a principal characteristic of the genus Microchlamys and family Microchlamyiidae; however, this conclusion was reached from the study of only two species. The data presented show a higher diversity of Microchlamys-like testate amoebae; we therefore suggest that the described species should be included in the family Microchlamyiidae with emendation of its diagnosis, but that a separate genus within this family should be established to accommodate it.

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.

Cochliopodium gallicum n. sp., isolated from cyanobacterial mats in the Camargue (France) is the smallest marine species of Cochliopodium to date. Its unusual tectum consists of flat plate-shaped scales with honeycomb-like centres, underlain by a layer of filamentous structures connected to each other in the basal and apical parts. The tectum is very fine and can be easily lost under inappropriate EM fixation. In its light-microscopical features, this species resembles Ovalopodium carrikeri Sawyer, 1980, a himatismenid that is believed to possess a scaleless, fuzzy or hairy "glycocalyx". We suggest that O. carrikeri might have been a similar species that lost scales under fixation. Our finding makes desirable a re-investigation of the genus Ovalopodium.

Amoebozoan parasites of arrow-worms (Chaetognatha) were isolated from their hosts living in plankton of the Bay of Villefranche (Mediterranean Sea). Based on the light microscopic characters, the amoebae were identified as Janickina pigmentifera (Grassi, 1881) by their limax locomotive form and due to the presence of the intracellular symbiont, Perkinsela amoebae, surrounded by a layer of pigment granules. Sequences of the 18S rRNA gene of both J. pigmentifera and its symbiont were obtained for the first time. The molecular phylogenetic analyses of 18S rRNA gene placed J. pigmentifera within the genus Neoparamoeba, a taxon also characterized by the presence of a symbiont, known as Perkinsela amoebae-like organism (PLO). The 18S rRNA gene sequence of P. amoebae from J. pigmentifera grouped with the sequences of 18S rRNA genes of PLOs from Neoparamoeba branchiphila and Neoparamoeba invadens. The first photo documentation of the light microscopic features of J. pigmentifera, such as locomotive form, the morphology of the nucleus and P. amoebae have been provided. The new results support the affinity of J. pigmentifera with the family Paramoebidae suggested previously based on the presence of PLO. In contrast to Janickina, typical members of Paramoebidae (Neoparamoeba and Paramoeba) have a flattened, dactylopodial locomotive form. This discrepancy in morphology can be explained by the obligate parasitic lifestyle of Janickina.