COI barcoding of Nebelid testate amoebae (Amoebozoa: Arcellinida): extensive cryptic diversity and redefinition of the Hyalospheniidae Schultze

We used Cytochrome Oxidase Subunit 1 (COI) to assess the phylogenetic relationships and taxonomy of Nebela sensu stricto and similar taxa (Nebela group, Arcellinida) in order to clarify the taxonomic validity of morphological characters. The COI data not only successfully separated all studied morphospecies but also revealed the existence of several potential cryptic species. The taxonomic implications of the results are: (1) Genus Nebela is paraphyletic and will need to be split into at least two monophyletic assemblages when taxon sampling is further expanded. (2) Genus Quadrulella, one of the few arcellinid genera building its shell from self-secreted siliceous elements, and the mixotrophic Hyalosphenia papilio branch within the Nebela group in agreement with the general morphology of their shell and the presence of an organic rim around the aperture (synapomorphy for Hyalospheniidae). We thus synonymise Hyalospheniidae and Nebelidae. Hyalospheniidae takes precedence and now includes Hyalosphenia, Quadrulella (previously in the Lesquereusiidae) and all Nebelidae with the exception of Argynnia and Physochila. Leptochlamys is Arcellinida incertae sedis. We describe a new genus Padaungiella Lara et Todorov and a new species Nebela meisterfeldi n. sp. Heger et Mitchell and revise the taxonomic position (and rank) of several taxa. These results show that the traditional morphology-based taxonomy underestimates the diversity within the Nebela group, and that phylogenetic relationships are best inferred from shell shape rather than from the material used to build the shell.     

Structure of a community of testate amoebae in a sphagnum dominated bog in upper sura flow (Middle Volga Territory)

Fifty nine species of testate amoebae were found in a sphagnum moss bog located in the upper Sura flow (Volga highland). The active diversity in communities of sphagnum moss biotopes was formed by 24 species. Minimal species diversity (3 species) and maximum density of organisms were noted in the driest habitat. In the habitats with the medium humidity levels, the level of species richness was similar (13–16 species) and the total density was determined by redox characteristics of the environment: under the conditions of oxygen deficit, it was two to three times lower than in an oxidized environment. The species composition specificity in sphagnum moss biotopes was determined by the degree of humidity: in the driest habitats, Arcella arenaria predominated; in the biotopes with medium humidity, Nebela tenella and Hyalosphenia elegans; and in the most humid biotopes, Hyalosphenia papilio and Heleopera sphagni. Communities of testate amoebae differed by species composition and integral characteristics at different sphagnum moss horizons: the abundance and species richness increased with depth. The proportion of living individuals in the total number of tests was 35 to 75%.     

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 testate lobose amoebae (order Arcellinida Kent, 1880) finally find their home within Amoebozoa

Testate lobose amoebae (order Arcellinida Kent, 1880) are common in all aquatic and terrestrial habitats, yet they are one of the last higher taxa of unicellular eukaryotes that has not found its place in the tree of life. The morphological approach did not allow to ascertain the evolutionary origin of the group or to prove its monophyly. To solve these challenging problems, we analyzed partial small-subunit ribosomal RNA (SSU rRNA) genes of seven testate lobose amoebae from two out of the three suborders and seven out of the 13 families belonging to the Arcellinida. Our data support the monophyly of the order and clearly establish its position among Amoebozoa, as a sister-group to the clade comprising families Amoebidae and Hartmannellidae. Complete SSU rRNA gene sequences from two species and a partial actin sequence from one species confirm this position. Our phylogenetic analyses including representatives of all sequenced lineages of lobose amoebae suggest that a rigid test appeared only once during the evolution of the Amoebozoa, and allow reinterpretation of some morphological characters used in the systematics of Arcellinida.     

18S rDNA phylogeny of lamproderma and allied genera (Stemonitales, Myxomycetes, Amoebozoa)

The phylogenetic position of the slime-mould genus Lamproderma (Myxomycetes, Amoebozoa) challenges traditional taxonomy: although it displays the typical characters of the order Stemonitales, it appears to be sister to Physarales. This study provides a small subunit (18S or SSU) ribosomal RNA gene-based phylogeny of Lamproderma and its allies, with new sequences from 49 specimens in 12 genera. We found that the order Stemonitales and Lamproderma were both ancestral to Physarales and that Lamproderma constitutes several clades intermingled with species of Diacheopsis, Colloderma and Elaeomyxa. We suggest that these genera may have evolved from Lamproderma by multiple losses of fruiting body stalks and that many taxonomic revisions are needed. We found such high genetic diversity within three Lamproderma species that they probably consist of clusters of sibling species. We discuss the contrasts between genetic and morphological divergence and implications for the morphospecies concept, highlighting the phylogenetically most reliable morphological characters and pointing to others that have been overestimated. In addition, we showed that the first part (~600 bases) of the SSU rDNA gene is a valuable tool for phylogeny in Myxomycetes, since it displayed sufficient variability to distinguish closely related taxa and never failed to cluster together specimens considered of the same species.     

Vertical micro-distribution and response to nitrogen deposition of testate amoebae in Sphagnum

Previous studies have shown the existence of a vertical micro-distribution of testate amoebae in the first centimeters of Sphagnum and their response to nutrient enrichment. In order to test the response of testate amoebae to depth and N addition in dry moss carpets recolonizing cutover peatlands, we sampled Sphagnum that had received 0, 1, 3, or 10g N m(-2) yr(-1) for three years. The mosses were cut into three segments: 0-1cm, 1-3cm and 3-5cm and analyzed for testate amoebae. The overall diversity (22 taxa) was high considering the dryness of the site, but the species richness of individual samples was low (mean 6.6). The presence of several species characteristic of wetter conditions suggests that they have a broader tolerance than usually believed and/or have a high colonization potential. Species richness increased with depth. Assulina muscorum was most abundant in the top segment, while Phryganella acropodia, Heleopera rosea and Nebela militaris were most abundant in the deepest segment. Neither the metabolism type nor the shell characteristics significantly explained the vertical distribution of species. There was no overall response of testate amoebae to N, although one species, Bullinularia indica, was significantly more abundant in the fertilized than in the control plots.     

Molecular phylogeny of the Blastocladiomycota (Fungi) based on nuclear ribosomal DNA

The Blastocladiomycota is a recently described phylum of ecologically diverse zoosporic fungi whose species have not been thoroughly sampled and placed within a molecular phylogeny. In this study, we investigated the phylogeny of the Blastocladiomycota based on ribosomal DNA sequences from strains identified by traditional morphological and ultrastructural characters. Our results support the monophyly of the Coelomomycetaceae and Physodermataceae but the Blastocladiaceae and Catenariaceae are paraphyletic or polyphyletic. The data support two clades within Allomyces with strains identified as Allomyces arbusculus in both clades, suggesting that species concepts in Allomyces are in need of revision. A clade of Catenaria species isolated from midge larvae group separately from other Catenaria species, suggesting that this genus may need revision. In the Physodermataceae, Urophlyctis species cluster with a clade of Physoderma species. The algal parasite Paraphysoderma sedebokerensis nom. prov. clusters sister to other taxa in the Physodermataceae. Catenomyces persicinus, which has been classified in the Catenariaceae, groups with the Chytridiomycota rather than Blastocladiomycota. The rDNA operon seems to be suitable for classification within the Blastocladiomycota and distinguishes among genera; however, this region alone is not suitable to determine the position of the Blastocladiomycota among other basal fungal phyla with statistical support. A focused effort to find and isolate, or directly amplify DNA from additional taxa will be necessary to evaluate diversity in this phylum. We provide this rDNA phylogeny as a preliminary framework to guide further taxon and gene sampling and to facilitate future ecological, morphological, and systematic studies.     

PHYLOGENY OF THE DUMONTIACEAE (GIGARTINALES, RHODOPHYTA) AND ASSOCIATED FAMILIES BASED ON SSU rDNA AND INTERNAL TRANSCRIBED SPACER SEQUENCE DATA

Small subunit (SSU) rDNA was sequenced for 25 species in 19 genera of the Gigartinales (Rhodophyta). As well, the internal transcribed spacer (ITS) region was sequenced, and a data matrix of 36 morphological characters was constructed for 16 species of Dumontiaceae. Phylogenetic trees were calculated from a multiple alignment of the SSU sequence data to infer relationships between species of Dumontiaceae and other gigartinalean taxa. The SSU analysis produced a polyphyletic Dumontiaceae. Notably, Acrosymphyton failed to associate with the included Gigartinales, let alone the Dumontiaceae, supporting an earlier proposal to remove it to a new family. The analyses were equivocal about the phylogenetic affinities of Dudresnaya , which clustered with the Kallymeniaceae, and the affinities of the Indo-West Pacific Gibsmithia , Kraftia , and Dasyphloea , the last-mentioned clustering with the Antarctic Gainiaceae, and these four taxa with Portieria (Rhizophyllidaceae). Further investigations are necessary to resolve relationships among these taxa. Rhodopeltis , a genus recently moved to the Dumontiaceae from the Polyideaceae, showed a weak association with the remaining northern Dumontiaceae. The final group consisted of cold-temperate Northern Hemisphere species. Phylogenetic analyses using a combination of SSU, ITS, and morphological data within this clade produced two strongly supported clades, a Dilsea / Neodilsea clade and a Cryptosiphonia / Dumontia clade. Dilsea is derived from a paraphyletic Neodilsea and may itself be polyphyletic. Atlantic and Pacific isolates of Dumontia contorta clearly showed sufficient divergence to warrant recognition as distinct species, and Dumontia alaskana , sp. nov. is proposed for the Pacific species.     

Old Lineages in a New Ecosystem: Diversification of Arcellinid Amoebae (Amoebozoa) and Peatland Mosses

Arcellinid testate amoebae (Amoebozoa) form a group of free-living microbial eukaryotes with one of the oldest fossil records known, yet several aspects of their evolutionary history remain poorly understood. Arcellinids occur in a range of terrestrial, freshwater and even brackish habitats; however, many arcellinid morphospecies such as Hyalosphenia papilio are particularly abundant in Sphagnum-dominated peatlands, a relatively new ecosystem that appeared during the diversification of Sphagnum species in the Miocene (5–20 Myr ago). Here, we reconstruct divergence times in arcellinid testate amoebae after selecting several fossils for clock calibrations and then infer whether or not arcellinids followed a pattern of diversification that parallels the pattern described for Sphagnum. We found that the diversification of core arcellinids occurred during the Phanerozoic, which is congruent with most arcellinid fossils but not with the oldest known amoebozoan fossil (i.e. at ca. 662 or ca. 750 Myr). Overall, Sphagnum and the Hyalospheniidae exhibit different patterns of diversification. However, an extensive molecular phylogenetic analysis of distinct clades within H. papilio species complex demonstrated a correlation between the recent diversification of H. papilio, the recent diversification of Sphagnum mosses, and the establishment of peatlands.     

Light variability and mixotrophy: Responses of testate amoeba communities and shell δ13C values to a peatland shading experiment

Paleoecological records suggest that growing season length and/or cloudiness may affect peatland carbon accumulation and testate amoeba-based environmental reconstructions, highlighting a need to understand how light intensity affects microbial communities. We shaded plots on two peatlands for two years to examine effects on testate amoeba communities, the relative abundance of mixotrophic and heterotrophic testate amoebae, transfer-function performance, and δ¹³C values of two species of mixotrophic testate amoebae. Surprisingly, relative abundance of mixotrophic species increased in shade, although compositional changes did not affect transfer-function performance. Shading did not affect δ¹³C values of Hyalosphenia papilio and Heleopera sphagni, which ranged from −23.5 to −19.6‰ and −23.2 to −19.2‰, respectively. These δ¹³C values were higher than those of potential food sources and lower than literature-derived values for Chlorella, the zoochlorellae inhabiting mixotrophic testate amoebae. δ¹³C values thus suggest that these mixotrophic species obtain some carbon from Chlorella, although coupled dietary and isotope studies are needed to quantify this contribution. More research is needed to assess impacts of light variability on peatland microbial communities; however, carbon sources are recorded by δ¹³C values of testate amoebae, indicating potential for studies of carbon cycling and how mixotrophy varies temporally and spatially.     

The Potential Influence of Short-term Environmental Variability on the Composition of Testate Amoeba Communities in <Emphasis Type="Italic">Sphagnum</Emphasis> Peatlands

Testate amoebae are a group of moisture-sensitive, shell-producing protozoa that have been widely used as indicators of changes in mean water-table depth within oligotrophic peatlands. However, short-term environmental variability (i.e., sub-annual) also probably influences community composition. The objective of this study was to assess the potential influence of short-term environmental variability on the composition of testate amoeba communities in Sphagnum-dominated peatlands. Testate amoebae and environmental conditions, including hourly measurements of relative humidity within the upper centimeter of the peatland surface, were examined throughout the 2008 growing season at 72 microsites within 11 peatlands of Pennsylvania and Wisconsin, USA. Relationships among testate amoeba communities, vegetation, depth to water table, pH, and an index of short-term environmental variability (EVI), were examined using nonmetric multidimensional scaling and correlation analysis. Results suggest that EVI influences testate amoeba communities, with some taxa more abundant under highly variable conditions (e.g., Arcella discoides, Difflugia pulex, and Hyalosphenia subflava) and others more abundant when environmental conditions at the peatland surface were relatively stable (e.g., Archerella flavum and Bullinularia indica). The magnitude of environmental variability experienced at the peatland surface appears to be primarily controlled by vegetation composition and density. In particular, sites with dense Sphagnum cover had lower EVI values than sites with loose-growing Sphagnum or vegetation dominated by vascular plants and/or non-Sphagnum bryophytes. Our results suggest that more environmental information may be inferred from testate amoebae than previously recognized. Knowledge of relationships between testate amoebae and short-term environmental variability should lead to more detailed and refined environmental inferences.     

Systematics of the genera of Bodotriidae (Crustacea: Cumacea)

The cumacean family Bodotriidae includes 382 species in 31 genera grouped in three subfamilies: Bodotriinae, Mancocumatinae and Vaunthompsoniinae. Generic diagnoses are based on few characters that often have overlapping states among genera, complicating the understanding of the relationships within the group. The goals of this study are to illuminate the phylogenetic relationships among the genera of the Bodotriidae using morphological characters and to review the systematics of the family. For this purpose, all species within each genus were studied from the literature to code all the variability of genera for 109 variable morphological characters. Phylogenetic analyses show that there is independent reduction of the pleopods in two clades from a plesiomorphic state of five pairs, while the number of exopods of peraeopods has been reduced gradually in more derived groups of bodotriids. The subfamily Bodotriinae is the most derived and the Vaunthompsoniinae the most basal, and is paraphyletic with the Mancocumatinae embedded within it. No discriminatory characters were found between the subfamilies Mancocumatinae and Vaunthompsoniinae and they are not clearly separated in the phylogeny. Mancocumatinae is synonymized with Vaunthompsoniinae and all the genera of the former Mancocumatinae should be included within Vaunthompsoniinae. Analyses of character evolution justify a few other taxonomic changes. All genera were redescribed based on all the coded characters and a complete list of all valid species for each genus is included. Finally, dichotomous keys for identification to genus level are provided.  © 2007 The Linnean Society of London, Zoological Journal of the Linnean Society , 2007, 151 , 1–58.     

Biogeography of testate amoebae in the soils of Mexico

We studied testate amoebae (testates) in the soils of coniferous and deciduous forests and in the wetland and aquatic habitats of Mexico. In 141 samples we found 205 taxa identified to the species or intraspecies level and 68 testate amoebae, which could not be identified to the species level. The highest species diversity of testates was found in the soils of the tropical rainforest (126 species and intraspecific taxa, including spp.) and in tropical wetlands (144 species and intraspecific taxa, including spp.). The study documented testate amoebae with a limited geographical distribution (genera Centropyxis, Cornuapyxis, Ellipsopyxis, Hoogenraadia, Planhoogenraadia, Apolimia, Certesella, Apodera, and Alocodera). We found that testate amoebae in the soils of Mexico had a high level of polymorphism and individual variability. We discuss the value of soil testate amoebae for defining biogeographical regions of Mexico.     

Recurrent ecological adaptations revealed through a molecular analysis of the secretive cophyline frogs of Madagascar

The cophyline microhylid frogs of Madagascar show a wide range of habitat specialization, ranging from terrestrial/burrowing and semi-arboreal to entirely arboreal species. The classification of these frogs is thus far mainly based upon morphological, largely osteological, characters that might be homoplastic. Using 1173 bp of DNA sequences from the mitochondrial 12S and 16S rRNA genes, we here present a molecular phylogeny for 28 species of all known genera, except for the genus Madecassophryne. The resulting maximum likelihood tree contained four major clades: one represented by the genus Anodonthyla, the second by Cophyla and Platypelis, the third by several terrestrial and semi-arboreal species of the genus Plethodontohyla, and the fourth by species of the genera Stumpffia, Plethodontohyla, and Rhombophryne. The results confirm that several cophyline lineages adapted independently to similar habitats, with multiple shifts among terrestriality and arboreality. The direction of these shifts cannot be ascertained due to unclarified relationships among the most basal lineages, but for one terrestrial species (Anodonthyla montana), it is most parsimonious to assume that it evolved from arboreal ancestors. Our results suggest that the genus Plethodontohyla is probably paraphyletic, and that the classification of this and of the genus Rhombophryne needs to be re-assessed.     

Molecular Phylogeny of Conjugating Green Algae (Zygnemophyceae,Streptophyta) Inferred from SSU rDNA Sequence Comparisons

Nuclear-encoded SSU rDNA sequences have been obtained from 64 strains of conjugating green algae (Zygnemophyceae, Streptophyta, Viridiplantae). Molecular phylogenetic analyses of 90 SSU rDNA sequences of Viridiplantae (inciuding 78 from the Zygnemophyceae) were performed using complex evolutionary models and maximum likelihood, distance, and maximum parsimony methods. The significance of the results was tested by bootstrap analyses, deletion of long-branch taxa, relative rate tests, and Kishino-Hasegawa tests with user-defined trees. All results support the monophyly of the class Zygnemophyceae and of the order Desmidiales. The second order, Zygnematales, forms a series of early-branching clades in paraphyletic succession, with the two traditional families Mesotaeniaceae and Zygnemataceae not recovered as lineages. Instead, a long-branch Spirogyra/Sirogonium clade and the later-diverging Netrium and Roya clades represent independent clades. Within the order Desmidiales, the families Gonatozygaceae and Closteriaceae are monophyletic, whereas the Peniaceae (represented only by Penium margaritaceum) and the Desmidiaceae represent a single weakly supported lineage. Within the Desmidiaceae short internal branches and varying rates of sequence evolution among taxa reduce the phylogenetic resolution significantly. The SSU rDNA-based phylogeny is largely congruent with a published analysis of the rbcL phylogeny of the Zygnemophyceae (McCourt et al. 2000) and is also in general agreement with classification schemes based on cell wall ultrastructure. The extended taxon sampling at the subgenus level provides solid evidence that many genera in the Zygnemophyceae are not monophyletic and that the genus concept in the group needs to be revised.     

A phylogenetic evaluation of a biosystematic framework: Brodiaea and related petaloid monocots (Themidaceae)

Phylogenetic analyses of plastid DNA sequences of ndhF, trnL-F intron and spacer regions, and rpl16 are presented separately and combined for 41 taxa from all 12 genera of the Themidaceae and for 20 taxa from nine related families in the higher Asparagales. The results from the combined analysis are the most resolved and provide a high level of support for the monophyly of Themidaceae. Within Themidaceae, the Milla complex of Mexico is supported as monophyletic within a paraphyletic Brodiaea complex of western North America. Four major clades are identified in each of the individual and combined analyses: (1) the Milla complex; (2) Brodiaea, Dichelostemma, and Triteleiopsis; (3) Triteleia, Bloomeria, and Muilla clevelandii; and (4) Androstephium and the other species of Muilla. These well-defined clades suggest that morphological characters (e.g., an extended perianth tube) that have been traditionally used to circumscribe the genera within the Brodiaea complex have evolved independently at least twice. In addition, common biogeographic distribution patterns (e.g., Brodiaea and Triteleia having centers of diversity in northern California and the Pacific Northwest) appear to be the result of separate evolutionary radiations.     

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.     

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.