Phylogeny of lobose amoebae based on actin and small-subunit ribosomal RNA genes

Lobose amoebae are abundant free-living protists and important pathogenic agents, yet their evolutionary history and position in the universal tree of life are poorly known. Molecular data for lobose amoebae are limited to a few species, and all phylogenetic studies published so far lacked representatives of many of their taxonomic groups. Here we analyze actin and small-subunit ribosomal RNA (SSU rRNA) gene sequences of a broad taxon sampling of naked, lobose amoebae. Our results support the existence of a monophyletic Amoebozoa clade, which comprises all lobose amoebae examined so far, the amitochondriate pelobionts and entamoebids, and the slime molds. Both actin and SSU rRNA phylogenies distinguish two well-defined clades of amoebae, the "Gymnamoebia sensu stricto" and the Archamoebae (pelobionts + entamoebids), and one weakly supported and ill-resolved group comprising some naked, lobose amoebae and the Mycetozoa.     

Ribosomal RNA genes challenge the monophyly of the Hyalospheniidae (Amoebozoa: Arcellinida)

To date only five partial and two complete SSU rRNA gene sequences are available for the lobose testate amoebae (Arcellinida). Consequently, the phylogenetic relationships among taxa and the definition of species are still largely dependant on morphological characters of uncertain value, which causes confusion in the phylogeny, taxonomy and the debate on cosmopolitanism of free-living protists. Here we present a SSU rRNA-based phylogeny of the Hyalospheniidae including the most common species. Similar to the filose testate amoebae of the order Euglyphida the most basal clades have a terminal aperture; the ventral position of the pseudostome appears to be a derived character. Family Hyalospheniidae appears paraphyletic and is separated into three clades: (1) Heleopera sphagni, (2) Heleopera rosea and Argynnia dentistoma and (3) the rest of the species from genera Apodera, Hyalosphenia, Porosia and Nebela. Our data support the validity of morphological characters used to define species among the Hyalospheniidae and even suggest that taxa described as varieties may deserve the rank of species (e.g. N. penardiana var. minor). Finally our results suggest that the genera Hyalosphenia and Nebela are paraphyletic, and that Porosia bigibbosa branches inside the main Nebela clade.     

SSU rRNA phylogeny of Arcellinida (Amoebozoa) reveals that the largest Arcellinid genus, Difflugia Leclerc 1815, is not monophyletic

The systematics of lobose testate amoebae (Arcellinida), a diverse group of shelled free-living unicellular eukaryotes, is still mostly based on morphological criteria such as shell shape and composition. Few molecular phylogenetic studies have been performed on these organisms to date, and their phylogeny suffers from typical under-sampling artefacts, resulting in a still mostly unresolved tree. In order to clarify the phylogenetic relationships among arcellinid testate amoebae at the inter-generic and inter-specific level, and to evaluate the validity of the criteria used for taxonomy, we amplified and sequenced the SSU rRNA gene of nine taxa - Difflugia bacillariarum, D. hiraethogii, D. acuminata, D. lanceolata, D. achlora, Bullinularia gracilis, Netzelia oviformis, Physochila griseola and Cryptodifflugia oviformis. Our results, combined with existing data demonstrate the following: 1) Most arcellinids are divided into two major clades, 2) the genus Difflugia is not monophyletic, and the genera Netzelia and Arcella are closely related, and 3) Cryptodifflugia branches at the base of the Arcellinida clade. These results contradict the traditional taxonomy based on shell composition, and emphasize the importance of general shell shape in the taxonomy of arcellinid testate amoebae.     

SSU rRNA reveals a sequential increase in shell complexity among the euglyphid testate amoebae (Rhizaria: Euglyphida)

The existing data on the molecular phylogeny of filose testate amoebae from order Euglyphida has revealed contradictions between traditional morphological classification and SSU rRNA phylogeny and, moreover, the position of several important genera remained unknown. We therefore carried out a study aiming to fill several important gaps and better understand the relationships among the main euglyphid testate amoebae and the evolutionary steps that led to the present diversity at a higher level. We obtained new SSU rRNA sequences from five genera and seven species. This new phylogeny obtained shows that (1) the clade formed by species of genera Assulina and Placocista branches unambiguously at the base of the subclade of Euglyphida comprising all members of the family Trinematidae and genus Euglypha, (2) family Trinematidae (Trachelocorythion, Trinema, and Corythion) branches as a sister group to genus Euglypha, (3) three newly sequenced Euglypha species (E. cf. ciliata, E. penardi, and E. compressa) form a new clade within the genus. Since our results show that Assulina and Placocista do not belong to the Euglyphidae (unless the Trinematidae are also included in this family), we propose the creation of a new family named Assulinidae. Consequently, we give a family status to the genera Euglypha and (tentatively) Scutiglypha, which become the new family Euglyphidae. The evolutionary pattern suggested by SSU rRNA phylogeny shows a clear tendency towards increasing morphological complexity of the shell characterised by changes in the symmetry (migration of the aperture to a ventral position and/or compression of the shell) and the appearance of specialised scales at the aperture (in families Trinematidae and Euglyphidae).     

The unusually long small subunit ribosomal RNA gene found in amitochondriate amoeboflagellate Pelomyxa palustris: its rRNA predicted secondary structure and phylogenetic implication

In order to ascertain a phylogenetic position of the freshwater amitochondriate amoeboflagellate Pelomyxa palustris its small subunit (SSU) rRNA gene was amplified and sequenced. It was shown to be 3502 bp long. The predicted secondary structure of its rRNA includes at least 16 separate expansion zones located in all the variable regions (V1-V9), as well as in some conservative gene regions. Most insertions are represented by sequences of low complexity that have presumably arisen by a slippage mechanism. Relatively conservative, uniformly positioned motifs contained in regions V4 and V7, as well as in some others, made it possible to perform folding. In maximum likelihood, maximum parsimony, and neighbor-joining trees, P. palustris tends to cluster with amitochondriate and secondary lost mitochondria amoebae and amoeboflagellates Entamoeba, Endolimax nana, and Phreatamoeba balamuthi, comprising together with them and aerobic lobose amoebae Vannella, Acanthamoeba, Balamuthia, and Hartmannella a monophyletic cluster. Another pelobiont, Mastigamoeba invertens, does not belong to this cluster. No specific similarity was discovered between the SSU rRNA of P. palustris and amitochondriate taxa of 'Archezoa': Diplomonada, Parabasalia, Microsporidia. Pelomyxa palustris SSU rRNA does not occupy a basal position in the phylogenetic trees and could be ascribed to the so-called eukaryotic 'crown' group if the composition of the latter were not so sensitive to the methods of tree building. Thus, molecular and morphological data suggest that P. palustris represents a secondarily modified eukaryotic lineage.     

Molecular Phylogeny of Polychaos annulatum (Amoebozoa,Tubulinea, Euamoebida) Shows that Genus Polychaos Belongs to the Family Hartmannellidae

We have obtained a sequence of the 18S rRNA gene of the species Polychaos annulatum (Penard 1902) Smirnov et Goodkov 1998 using the isolation of a single nucleus from an amoeba cell. Attempts to amplify the 18S rRNA gene from the DNA of this species by conventional PCR were not successful, so we applied the whole genome amplification of the nuclear DNA followed by NGS sequencing. The 18S rRNA gene was found among the resulting contigs. The analysis unexpectedly shows that P. annulatum robustly groups within the family Hartmannellidae, but not Amoebidae. This finding warrants revision of the basic morphological criteria used to classify Euamoebida families and show that “proteus‐type” amoebae may belong to other families rather than Amoebidae. This makes taxonomic assignments of such species more complex and the borders between Euamoebida families more nuanced. It is getting evident that molecular data are necessary to clarify the position of species even in this most “classical” order of naked lobose amoebae.     

SSU rRNA-based phylogenetic position of the genera Amoeba and Chaos (Lobosea, Gymnamoebia): the origin of gymnamoebae revisited.

Naked lobose amoebae (gymnamoebae) are among the most abundant group of protists present in all aquatic and terrestrial biotopes. Yet, because of lack of informative morphological characters, the origin and evolutionary history of gymnamoebae are poorly known. The first molecular studies revealed multiple origins for the amoeboid lineages and an extraordinary diversity of amoebae species. Molecular data, however, exist only for a few species of the numerous taxa belonging to this group. Here, we present the small-subunit (SSU) rDNA sequences of four species of typical large gymnamoebae: Amoeba proteus, Amoeba leningradensis, Chaos nobile, and Chaos carolinense. Sequence analysis suggests that the four species are closely related to the species of genera Saccamoeba, Leptomyxa, Rhizamoeba, Paraflabellula, Hartmannella, and Echinamoeba. All of them form a relatively well-supported clade, which corresponds to the subclass Gymnamoebia, in agreement with morphology-based taxonomy. The other gymnamoebae cluster in small groups or branch separately. Their relationships change depending on the type of analysis and the model of nucleotide substitution. All gymnamoebae branch together in Neighbor-Joining analysis with corrections for among-site rate heterogeneity and proportion of invariable sites. This clade, however, is not statistically supported by SSU rRNA gene sequences and further analysis of protein sequence data will be necessary to test the monophyly of gymnamoebae.     

High Diversity of Testate Amoebae (Amoebozoa,Arcellinida) Detected by HTS Analyses in a New England Fen using Newly Designed Taxon‐specific Primers

Testate (shell‐building) amoebae, such as the Arcellinida (Amoebozoa), are useful bioindicators for climate change. Though past work has relied on morphological analyses to characterize Arcellinida diversity, genetic analyses revealed the presence of multiple cryptic species underlying morphospecies. Here, we design and deploy Arcellinida‐specific primers for the SSU‐rDNA gene to assess the community composition on the molecular level in a pilot study of two samplings from a New England fen: (1) 36‐cm horizontal transects and vertical cores; and (2) 26‐m horizontal transects fractioned into four size classes (2–10, 10–35, 35–100, and 100–300 μm). Analyses of these data show the following: (1) a considerable genetic diversity within Arcellinida, much of which comes from morphospecies lacking sequences on GenBank; (2) communities characterized by DNA (i.e. active + quiescent) are distinct from those characterized by RNA (i.e. active, indicator of biomass); (3) active communities on the surface tend to be more similar to one another than to core communities, despite considerable heterogeneity; and (4) analyses of communities fractioned by size find some lineages (OTUs) that are abundant in disjunct size categories, suggesting the possibility of life‐history stages. Together, these data demonstrate the potential of these primers to elucidate the diversity of Arcellinida communities in diverse habitats.     

Thecamoeba cosmophorea n. sp. (Amoebozoa,Discosea, Thecamoebida) — An example of sibling species within the genus Thecamoeba

A new species Thecamoeba cosmophorea n. sp. (Amoebozoa, Discosea, Thecamoebida) was isolated from leaf litter collected in the surroundings of Saint-Petersburg (Russia). This species resembles T. quadrilineata in light-microscopic morphology, but has certain morphological differences and significantly differs in 18S rRNA gene sequence. We performed a direct comparison of this newly isolated species with the Thecamoeba strain isolated from leaf litter in East Siberia (Russia) and identified as T. quadrilineata both at the morphological and the molecular level. There is no type strain of T. quadrilineata, and the type material on this species is represented with the stained slide by F.C. Page that he designated as neotype in 1977. The sequence of the 18S rRNA gene of this species deposited in the GenBank belongs to the isolate identified as T. quadrilineata by Rolf Michel in 1998 and deposited as CCAP 1583/10 strain. Hence we cannot be entirely sure that morphological and molecular data on T. quadrilineata belong to the same amoebae species. The use of molecular data for reliable species differentiation is getting obligate even within the amoebae genus Thecamoeba, which until recently was believed to be among few genera of naked lobose amoebae allowing morphological identification of species.     

A needle in a haystack: A new metabarcoding approach to survey diversity at the species level of Arcellinida (Amoebozoa: Tubulinea)

Environmental DNA-based diversity studies have increased in popularity with the development of high throughput sequencing technologies. This permits the potential simultaneous retrieval of vast amounts of molecular data from many different organisms and species, thus contributing to a wide range of biological disciplines. Environmental DNA protocols designed for protists often focused on the highly conserved small subunit of the ribosome gene, that does not permit species-level assignments. On the other hand, eDNA protocols aiming at species-level assignments allow a fine level ecological resolution and reproducible results. These protocols are currently applied to organisms living in marine and shallow lotic freshwater ecosystems, often in a bioindication purpose. Therefore, in this study, we present a species-level eDNA protocol designed to explore diversity of Arcellinida (Amoebozoa: Tubulinea) testate amoebae taxa that is based on mitochondrial cytochrome oxidase subunit I (COI). These organisms are widespread in lentic water bodies and soil ecosystems. We applied this protocol to 42 samples from peatlands, estuaries and soil environments, recovering all the infraorders in Glutinoconcha (with COI data), except for Hyalospheniformes. Our results revealed an unsuspected diversity in morphologically homogeneous groups such as Cylindrothecina, Excentrostoma or Sphaerothecina. With this protocol we expect to revolutionize the design of modern distributional Arcellinida surveys. Our approach involves a rapid and cost-effective analysis of testate amoeba diversity living in contrasted ecosystems. Therefore, the order Arcellinida has the potential to be established as a model group for a wide range of theoretical and applied studies.     

Phylogenetic divergence within the Arcellinida (Amoebozoa) is congruent with test size and metabolism type

Arcellinida (lobose testate amoebae) are abundant and diverse in many ecosystems, especially in moist to aquatic environments. Molecular phylogeny has shown that overall test morphology (e.g., spherical or elongate) is generally conserved in Arcellinida lineages, but the taxonomic value of other traits (e.g., size, ornamentation, mixotrophy/heterotrophy metabolism type) has not been systematically evaluated. Morphological and physiological traits that correspond to genetic differences likely represent adaptive traits of ecological significance. We combined high-resolution phylogenetics (NAD9-NAD7 genes) and advanced morphometrics to assess the phylogenetic signal of morphological traits of a group of elongate Difflugia species (Arcellinida). The phylogenetic analyses revealed two clades which could be reliably separated by test size and the presence/absence of mixotrophy. Differences in test size may reflect trophic level, with smaller organisms occupying lower trophic levels. In addition to having larger tests, elongate mixotrophic Difflugia are characterised by wide, flat bases and an inflation of the lower two thirds of their test. These morphological traits may provide additional volume for endosymbionts and/or increased surface area to aid light transmission. Our results showcase greater diversity within the elongate Difflugia and highlight morphological traits of ecological and evolutionary significance.     

Vannella primoblina n. sp. – an unusual species of the genus Vannella (Amoebozoa,Discosea, Vannellida) with pronounced dorsal ridges and folds

Amoebae of the order Vannellida (Amoebozoa, Discosea) have a fairly recognizable spatulate, fan-shaped or semi-circular outlines and a wide area of frontal hyaloplasm. They can be easily distinguished from the other groups of lobose amoebae even by light microscopy. The dorsal side of these amoebae is usually smooth and rarely bears ridges or folds, which are never numerous or regular. We have isolated an unusual species of vannellid amoebae, called Vannella primoblina n. sp. from a terrestrial substrate. It has well-developed dorsal relief consisting of regularly appearing folds and ridges. This amoeba superficially resembles members of the genus Thecamoeba. However, molecular analysis showed that this strain belongs to the genus Vannella. This finding indicates that dorsal folds may also be a characteristic of some species of vannellid amoebae and probably are a functional detail of the cell morphology rather than an apomorphy of Thecamoebida lineage. Overall outlines of the cell and the presence of the expanded frontal hyaline area remains the most reliable characters used to differentiate vannellid amoebae from other gymnamoebae lineages.     

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.     

PHYLOGENY OF PHAGOTROPHIC EUGLENIDS (EUGLENOZOA): A MOLECULAR APPROACH BASED ON CULTURE MATERIAL AND ENVIRONMENTAL SAMPLES1

Molecular studies based on small subunit (SSU) rDNA sequences addressing euglenid phylogeny hitherto suffered from the lack of available data about phagotrophic species. To extend the taxon sampling, SSU rRNA genes from species of seven genera of phagotrophic euglenids were investigated. Sequence analyses revealed an increasing genetic diversity among euglenid SSU rDNA sequences compared with other well‐known eukaryotic groups, reflecting an equally broad diversity of morphological characters among euglenid phagotrophs. Phylogenetic inference using standard parsimony and likelihood approaches as well as Bayesian inference and spectral analyses revealed no clear support for euglenid monophyly. Among phagotrophs, monophyly of Petalomonas cantuscygni and Notosolenus ostium, both comprising simple ingestion apparatuses, is strongly supported. A moderately supported clade comprises phototrophic euglenids and primary osmotrophic euglenids together with phagotrophs, exhibiting a primarily flexible pellicle composed of numerous helically arranged strips and a complex ingestion apparatus with two supporting rods and four curved vanes. Comparison of molecular and morphological data is used to demonstrate the difficulties to formulate a hypothesis about how the ingestion apparatus evolved in this group.     

Reinvestigation of Phryganella paradoxa (Arcellinida,Amoebozoa) Penard 1902

A major drawback in testate amoeba research is a general lack of scientific studies combining molecular approaches and classical laboratory experiments. We isolated five yet uncultured testate amoebae of the genus Phryganella Penard, 1902 from three different rivers and one pond in Germany. Based on established cultures we show their morphology, which we studied by light and electron microscopy, and present their unique feeding mode on abundant and common pennate diatoms like Nitzschia spp. and Synedra spp., whose frustules are bent and frequently, but not always, broken during the feeding process. We further obtained the first SSU rDNA sequences of strains of the family Phryganellidae, all of which contain introns. We used the sequences to confirm the taxonomic placement of the Phryganellidae in the Arcellinida (Amoebozoa), branching as a sister group to the Cryptodifflugiidae.     

Phylogenetic analysis of the Metastrongyloidea (Nematoda: Strongylida) inferred from ribosomal RNA gene sequences

Phylogenetic relationships among nematodes of the strongylid superfamily Metastrongyloidea were analyzed using partial sequences from the large-subunit ribosomal RNA (LSU rRNA) and small-subunit ribosomal RNA (SSU rRNA) genes. Regions of nuclear ribosomal DNA (rDNA) were amplified by polymerase chain reaction, directly sequenced, aligned, and phylogenies inferred using maximum parsimony. Phylogenetic hypotheses inferred from the SSU rRNA gene supported the monophyly of representative taxa from each of the 7 currently accepted metastrongyloid families. Metastrongyloid taxa formed the sister group to representative trichostrongyloid sequences based on SSU data. Sequences from either the SSU or LSU RNA regions alone provided poor resolution for relationships within the Metastrongyloidea. However, a combined analysis using sequences from all rDNA regions yielded 3 equally parsimonious trees that represented the abursate Filaroididae as polyphyletic, Parafilaroides decorus as the sister species to the monophyletic Pseudaliidae, and a sister group relationship between Oslerus osleri and Metastrongylus salmi. Relationships among 3 members of the Crenosomatidae, and 1 representative of the Skrjabingylidae (Skrjabingylus chitwoodorum) were not resolved by these combined data. However, members of both these groups were consistently resolved as the sister group to the other metastrongyloid families. These relationships are inconsistent with traditional classifications of the Metastrongyloidea and existing hypotheses for their evolution.     

Ovalopodium desertum n. sp. and the phylogenetic relationships of Cochliopodiidae (Amoebozoa)

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.     

How good are global DNA-based environmental surveys for detecting all protist diversity? Arcellinida as an example of biased representation

Metabarcoding approaches targeting microeukaryotes have deeply changed our vision of protist environmental diversity. The public repository EukBank consists of 18S v4 metabarcodes from 12,672 samples worldwide. To estimate how far this database provides a reasonable overview of all eukaryotic diversity, we used Arcellinida (lobose testate amoebae) as a case study. We hypothesised that (1) this approach would allow the discovery of unexpected diversity, but also that (2) some groups would be underrepresented because of primer/sequencing biases. Most of the Arcellinida sequences appeared in freshwater and soil, but their abundance and diversity appeared underrepresented. Moreover, 84% of ASVs belonged to the suborder Phryganellina, a supposedly species-poor clade, whereas the best-documented suborder (Glutinoconcha, 600 described species) was only marginally represented. We explored some possible causes of these biases. Mismatches in the primer-binding site seem to play a minor role. Excessive length of the target region could explain some of these biases, but not all. There must be some other unknown factors involved. Altogether, while metabarcoding based on ribosomal genes remains a good first approach to document microbial eukaryotic clades, alternative approaches based on other genes or sequencing techniques must be considered for an unbiased picture of the diversity of some groups.     

18S ribosomal RNA gene sequences of Cochliopodium (Himatismenida) and the phylogeny of Amoebozoa

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.     

Paravannella minima n. g. n. sp. (Discosea,Vannellidae) and distinction of the genera in the vannellid amoebae

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.