Amoebozoan testate amoebae illuminate the diversity of heterotrophs and the complexity of ecosystems throughout geological time.

Heterotrophic protists are vital in Earth's ecosystems, influencing carbon and nutrient cycles and occupying key positions in food webs as microbial predators. Fossils and molecular data suggest the emergence of predatory microeukaryotes and the transition to a eukaryote-rich marine environment by 800 million years ago (Ma). Neoproterozoic vase-shaped microfossils (VSMs) linked to Arcellinida testate amoebae represent the oldest evidence of heterotrophic microeukaryotes. This study explores the phylogenetic relationship and divergence times of modern Arcellinida and related taxa using a relaxed molecular clock approach. We estimate the origin of nodes leading to extant members of the Arcellinida Order to have happened during the latest Mesoproterozoic and Neoproterozoic (1054 to 661 Ma), while the divergence of extant infraorders postdates the Silurian. Our results demonstrate that at least one major heterotrophic eukaryote lineage originated during the Neoproterozoic. A putative radiation of eukaryotic groups (e.g., Arcellinida) during the early-Neoproterozoic sustained by favorable ecological and environmental conditions may have contributed to eukaryotic life endurance during the Cryogenian severe ice ages. Moreover, we infer that Arcellinida most likely already inhabited terrestrial habitats during the Neoproterozoic, coexisting with terrestrial Fungi and green algae, before land plant radiation. The most recent extant Arcellinida groups diverged during the Silurian Period, alongside other taxa within Fungi and flowering plants. These findings shed light on heterotrophic microeukaryotes' evolutionary history and ecological significance in Earth's ecosystems, using testate amoebae as a proxy.

Evolution of microRNAs in Amoebozoa and implications for the origin of multicellularity.

MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression in both plants and animals. They are thought to have evolved convergently in these lineages and hypothesized to have played a role in the evolution of multicellularity. In line with this hypothesis, miRNAs have so far only been described in few unicellular eukaryotes. Here, we investigate the presence and evolution of miRNAs in Amoebozoa, focusing on species belonging to Acanthamoeba, Physarum and dictyostelid taxonomic groups, representing a range of unicellular and multicellular lifestyles. miRNAs that adhere to both the stringent plant and animal miRNA criteria were identified in all examined amoebae, expanding the total number of protists harbouring miRNAs from 7 to 15. We found conserved miRNAs between closely related species, but the majority of species feature only unique miRNAs. This shows rapid gain and/or loss of miRNAs in Amoebozoa, further illustrated by a detailed comparison between two evolutionary closely related dictyostelids. Additionally, loss of miRNAs in the Dictyostelium discoideum drnB mutant did not seem to affect multicellular development and, hence, demonstrates that the presence of miRNAs does not appear to be a strict requirement for the transition from uni- to multicellular life.

Living in the cracks: Two novel genera of Variosea (Amoebozoa) discovered on an urban sidewalk.

Biological soil crusts represent a rich habitat for diverse and complex eukaryotic microbial communities. A unique but extremely common habitat is the urban sidewalk and its cracks that collect detritus. While these habitats are ubiquitous across the globe, little to no work has been conducted to characterize protists found there. Amoeboid protists are major predators of bacteria and other microbial eukaryotes in these microhabitats and therefore play a substantial ecological role. From sidewalk crack soil crusts, we have isolated three naked amoebae with finely tapered subpseudopodia, and a simple life cycle consisting of a trophic amoeba and a cyst stage. Using a holistic approach including light, electron, and fluorescence microscopy as well as phylogenetics using the ribosomal small subunit rRNA gene and phylogenomics using 230 nuclear genes, we find that these amoeboid organisms fail to match any previously described eukaryote genus. However, we determined the amoebae belong to the amoebozoan lineage Variosea based on phylogenetics. The molecular analyses place our isolates in two novel genera forming a grade at the base of the variosean group Protosteliida. These three novel varioseans among two novel genera and species are herein named "Kanabo kenzan" and "Parakanabo toge."

Identification of new amoebae strains in rainbow trout (Oncorhynchus mykiss, Walbaum) farms affected by nodular gill disease (NGD) in Northeastern Italy.

Nodular gill disease (NGD) is an emerging condition associated with amoeba trophozoites in freshwater salmonid farms. However, unambiguous identification of the pathogens still must be achieved. This study aimed to identify the amoeba species involved in periodic NGD outbreaks in two rainbow trout (Oncorhynchus mykiss) farms in Northeastern Italy. During four episodes (February-April 2023), 88 fish were euthanized, and their gills were evaluated by macroscopic, microscopic and histopathological examination. The macroscopic and microscopic severity of the lesions and the degree of amoebae infestation were scored and statistically evaluated. One gill arch from each animal was put on non-nutrient agar (NNA) Petri dishes for amoeba isolation, cultivation and subsequent identification with SSU rDNA sequencing. Histopathology confirmed moderate to severe lesions consistent with NGD and mild to moderate amoeba infestation. The presence of amoebae was significantly correlated with lesion severity. Light microscopy of cultured amoebae strains and SSU rDNA analysis revealed the presence of a previously characterized amoeba Naegleria sp. strain GERK and several new strains: two strains from Hartmannelidae, three vannelid amoebae from the genus Ripella and cercozoan amoeba Rosculus. Despite the uncertainty in NGD etiopathogenesis and amoebae pathogenic role, identifying known and new amoebae leans towards a possible multi-aetiological origin.

A morphological and molecular reinvestigation of Janickina pigmentifera (Grassi, 1881) Chatton 1953 - an amoebozoan parasite of arrow-worms (Chaetognatha).

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.

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.

Pseudoparamoeba garorimi n. sp., with Notes on Species Distinctions within the Genus.

A new marine species of naked lobose amoebae Pseudoparamoeba garorimi n. sp. (Amoebozoa, Dactylopodida) isolated from intertidal marine sediments of Garorim Bay, Korea was studied with light and transmission electron microscopy. This species has a typical set of morphological characters for a genus including the shape of the locomotive form, type of subpseudopodia and the tendency to form the single long waving pseudopodium in locomotion. Furthermore, it has the same cell surface structures as were described for the type species, Pseudoparamoeba pagei: blister-like glycostyles with hexagonal base and dome-shaped apex; besides, cell surface bears hair-like outgrowths. The new species described here lacks clear morphological distinctions from the two other Pseudoparamoeba species, but has considerable differences in the 18S rDNA and COX1 gene sequences. Phylogenetic analysis based on 18S rDNA placed P. garorimi n. sp. at the base of the Pseudoparamoeba clade with high PP/BS support. The level of COX1 sequence divergence was 22% between P. garorimi n. sp. and P. pagei and 25% between P. garorimi n. sp. and P. microlepis. Pseudoparamoeba species are hardly distinguishable by morphology alone, but display clear differences in 18S rDNA and COX1 gene sequences.

Bog Microtopography and the Climatic Sensitivity of Testate Amoeba Communities: Implications for Transfer Function-Based Paleo-Water Table Reconstructions.

Although the use of sub-fossil testate amoebae as a proxy for raised bog hydrology in Holocene paleoecological studies is well-established, some detailed aspects of species-environment relationships remain under-researched. One such issue is the effect of bog surface microtopography on the climatic sensitivity of testate amoeba communities. Although it has been suggested that some microforms-especially hummocks-may be less sensitive to climatic forcing than others, this has rarely been objectively tested. To investigate this, subfossil testate amoebae assemblages have been examined in a series of shallow cores collected along a hummock-lawn-hollow transect from a bog in central Ireland and the resulting reconstructed water table records, dated using 210Pb, have been compared with instrumental weather data. Testate amoebae communities in the hollow microform were found to be significantly less diverse than those in the hummock and lawn, and both the hummock and lawn showed statistically significant correlations with instrumental temperature and precipitation data. Therefore, whilst the suggestion that paleoecological investigations should target intermediate bog microforms remains sound, the notion that hummock-based testate amoebae hydrological data are climatically-insensitive is challenged.

Phylogeny-wide conservation and change in developmental expression, cell-type specificity and functional domains of the transcriptional regulators of social amoebas.

BACKGROUND: Dictyostelid social amoebas self-organize into fruiting bodies, consisting of spores and up to four supporting cell types in the phenotypically most complex taxon group 4. High quality genomes and stage- and cell-type specific transcriptomes are available for representative species of each of the four taxon groups. To understand how evolution of gene regulation in Dictyostelia contributed to evolution of phenotypic complexity, we analysed conservation and change in abundance, functional domain architecture and developmental regulation of their transcription factors (TFs). RESULTS: We detected 440 sequence-specific TFs across 33 families, of which 68% were upregulated in multicellular development and about half conserved throughout Dictyostelia. Prespore cells expressed two times more TFs than prestalk cells, but stalk cells expressed more TFs than spores, suggesting that gene expression events that define spores occur earlier than those that define stalk cells. Changes in TF developmental expression, but not in TF abundance or functional domains occurred more frequently between group 4 and groups 1-3, than between the more distant branches formed by groups 1 + 2 and 3 + 4. CONCLUSIONS: Phenotypic innovation is correlated with changes in TF regulation, rather than functional domain- or TF acquisition. The function of only 34 TFs is known. Of 12 TFs essential for cell differentiation, 9 are expressed in the cell type for which they are required. The information acquired here on conserved cell type specifity of 120 additional TFs can effectively guide further functional analysis, while observed evolutionary change in TF developmental expression may highlight how genotypic change caused phenotypic innovation.

Description of Armaparvus languidus n. gen. n. sp. Confirms Ultrastructural Unity of Cutosea (Amoebozoa, Evosea).

The American Type Culture Collection (ATCC) PRA-29 isolate has a publicly available transcriptome, which has led to its inclusion in recent phylogenomic analyses. The ATCC PRA-29 isolate was originally identified and deposited as "Pessonella sp." This taxon branches robustly within the recently discovered clade Cutosea, very distantly related to the clade in which the genus Pessonella is believed to branch based on morphological data. Using detailed light and electron microscopy, we studied the morphology and ultrastructure of ATCC PRA-29 as well as other cutosean amoebae to better elucidate the morphological affinity of ATCC PRA-29 to other amoebozoans. Here, we show that ATCC PRA-29 was misidentified by the original depositor as Pessonella and name it Armaparvus languidus n. gen. n. sp. We show that a cell coat of microscales separated from the cell membrane is a unique trait found in all known cutosean amoebae. As Cutosea represents a clade at the deepest bifurcation in the amoebozoan group Evosea and because this clade is currently taxon-poor, but likely represents a major understudied group it will be important to isolate and describe more cutosean amoebae in the future.

Thecamoeba quadrilineata (Amoebozoa, Lobosa) as a new member of amphizoic amoebae-first isolation from endozoic conditions.

A free-living soil amoeba Thecamoeba quadrilineata (Carter, 1856) Lepsi, 1960 (Amoebozoa: Thecamoebidae) was isolated from endozoic conditions for the first time. Presence of amoebae was detected after 4 days following inoculation of the gut of the earthworm Lumbricus terrestris on agar plate with Escherichia coli. On the basis of our isolation, we consider T. quadrilineata as further amphizoic amoeba species. This study enlarges the range of amphizoic tendency in members of the genus Thecamoeba and stresses the need for further research on the pathogenic potential of Thecamoeba species.

Encystation: the most prevalent and underinvestigated differentiation pathway of eukaryotes.

Not long ago, protists were considered one of four eukaryote kingdoms, but recent gene-based phylogenies show that they contribute to all nine eukaryote subdomains. The former kingdoms of animals, plants and fungi are now relegated to lower ranks within subdomains. Most unicellular protists respond to adverse conditions by differentiating into dormant walled cysts. As cysts, they survive long periods of starvation, drought and other environmental threats, only to re-emerge when conditions improve. For protists pathogens, the resilience of their cysts can prevent successful treatment or eradication of the disease. In this context, effort has been directed towards understanding the molecular mechanisms that control encystation. We here firstly summarize the prevalence of encystation across protists and next focus on Amoebozoa, where most of the health-related issues occur. We review current data on processes and genes involved in encystation of the obligate parasite Entamoeba histolytica and the opportunistic pathogen Acanthamoeba. We show how the cAMP-mediated signalling pathway that controls spore and stalk cell encapsulation in Dictyostelium fruiting bodies could be retraced to a stress-induced pathway controlling encystation in solitary Amoebozoa. We highlight the conservation and prevalence of cAMP signalling genes in Amoebozoan genomes and the suprisingly large and varied repertoire of proteins for sensing and processing environmental signals in individual species.

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

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

The emerging picture of the mitochondrial protein import complexes of Amoebozoa supergroup.

BACKGROUND: The existence of mitochondria-related organelles (MROs) is proposed for eukaryotic organisms. The Amoebozoa includes some organisms that are known to have mitosomes but also organisms that have aerobic mitochondria. However, the mitochondrial protein apparatus of this supergroup remains largely unsampled, except for the mitochondrial outer membrane import complexes studied recently. Therefore, in this study we investigated the mitochondrial inner membrane and intermembrane space complexes, using the available genome and transcriptome sequences. RESULTS: When compared with the canonical cognate complexes described for the yeast Saccharomyces cerevisiae, amoebozoans with aerobic mitochondria, display lower differences in the number of subunits predicted for these complexes than the mitochondrial outer membrane complexes, although the predicted subunits appear to display different levels of diversity in regard to phylogenetic position and isoform numbers. For the putative mitosome-bearing amoebozoans, the number of predicted subunits suggests the complex elimination distinctly more pronounced than in the case of the outer membrane ones. CONCLUSION: The results concern the problem of mitochondrial and mitosome protein import machinery structural variability and the reduction of their complexity within the currently defined supergroup of Amoebozoa. This results are crucial for better understanding of the Amoebozoa taxa of both biomedical and evolutionary importance.

Longamoebia is not monophyletic: Phylogenomic and cytoskeleton analyses provide novel and well-resolved relationships of amoebozoan subclades.

Longamoebia is one of the most morphologically diverse member of Amoebozoa. It includes the human pathogen Acanthamoeba, which causes minor skin and serious eye infections as well as fatal central nervous system complications. The taxonomy and phylogeny of Longamoebia is poorly understood partly due to the growing number of molecular studies that report unsuspected affiliations of lineages with extremely different morphotypes in the group. A recent molecular study questioned the monophyly of Longamoebia. In this study, we conducted a more comprehensive phylogenomic analysis including all of putative members of Longamoebia to assess its monophyly. We conducted extensive analyses to see effects of outgroup choice, missing data, and gene and taxon sampling on resulting phylogenies. We also collected morphological characters derived from the cytoskeleton using immunocytochemistry to assess homologies of pseudopodia at a finer scale. Our phylogenomic analysis yielded a well-resolved tree of Amoebozoa and highly supported novel relationships. Discosea is recovered as a monophyletic group with all of its known taxonomic orders. However, its within-group relationships dramatically differed from those originally proposed. Our study strongly demonstrates that Longamoebia sensu Smirnov et al. (2011) is not monophyletic and an invalid taxon. Thecamoebida forms a strongly supported sister group relationship with clade Flabellinea (Dactylopodida and Vannellida), while Dermamoebida (Mayorella+Dermamoeba) form an independent branch basal to other members of Discosea. The remaining groups including members of Centramoebida form a consistently well-supported clade that was shown to form a sister group relationship with Himatismenida. This robust clade shares the unique cytoskeletal features of coiled cytoplasmic microtubule network and F-actin characters. Our analyses demonstrated that placement of unstable taxa in large-scale analysis with varying levels of missing data might be compromised by some confounding factors such as outgroup choice and gene and taxon sampling.

Lineage-Specific and Highly Derived Gene Sequences Among Amoebozoa, Revealed by the Comparative Analysis of Transcriptomes from Twelve Amoebozoan Species.

Amoebozoa represent a difficult group for traditional morphology-based taxonomy. Molecular approaches, such as gene sequencing and DNA barcoding have greatly enhanced our knowledge of the diversity of these organisms. However, metagenomic studies of Amoebozoa still did not provide as impressive results as they did among some other groups of protists. In environmental DNA surveys done on fragments of SSU rDNA gene and other traditional DNA barcodes, Amoebozoa genes normally constitute a minor part of the total gene diversity and represent only the most abundant lineages. A potential way to resolve this problem is the usage of DNA barcodes based on genes, which are unique or highly derived in this group of organisms. In the present study, we attempted to find such genes and gene families with a low level of paralogy, potentially appropriate as Amoebozoa-specific DNA barcodes. For this we re-assembled transcriptomes of 12 amoebozoan species available from the public databases and performed gene annotation and identification of orthologous genes. In our analysis Amoebozoa-specific and highly derived sequences formed 53,182 clusters of orthologs, containing from 2 to 299 proteins each. Some of these genes may be a potential target for DNA barcoding of Amoebozoa.

Diversity and Evolution of Paramoeba spp. and their Kinetoplastid Endosymbionts.

Members of the genus Paramoeba (including Neoparamoeba) (Amoebozoa) are single-celled eukaryotes of economic and ecological importance because of their association with disease in a variety of marine animals including fish, sea urchins, and lobster. Interestingly, they harbor a eukaryotic endosymbiont of kinetoplastid ancestry, Perkinsela sp. To investigate the complex relationship between Paramoeba spp. and Perkinsela sp., as well as the relationships between different Paramoeba species, molecular data was obtained for four novel isolates. We also acquired new data from the urchin pathogen P. invadens. Comprehensive molecular phylogenetic analyses were carried out using 33 newly obtained 18S rDNA sequences from the host amoebae and 16 new 18S rDNA sequences from their corresponding Perkinsela sp., together with all publicly available 18S molecular data. Intra-isolate 18S rDNA nucleotide diversity was found to be surprisingly high within the various species of Paramoeba, but relatively low within their Perkinsela sp. endosymbionts. 18S rDNA phylogenies and ParaFit co-evolution analysis revealed a high degree of congruence between the Paramoeba and Perkinsela sp. tree topologies, strongly suggesting that a single endosymbiotic event occurred in the common ancestor of known Paramoeba species, and that the endosymbionts have been inherited vertically ever since.

Mycamoeba gemmipara nov. gen., nov. sp., the First Cultured Member of the Environmental Dermamoebidae Clade LKM74 and its Unusual Life Cycle.

Since the first environmental DNA surveys, entire groups of sequences called "environmental clades" did not have any cultured representative. LKM74 is an amoebozoan clade affiliated to Dermamoebidae, whose presence is pervasively reported in soil and freshwater. We obtained an isolate from soil that we assigned to LKM74 by molecular phylogeny, close related to freshwater clones. We described Mycamoeba gemmipara based on observations made with light- and transmission electron microscopy. It is an extremely small amoeba with typical lingulate shape. Unlike other Dermamoebidae, it lacked ornamentation on its cell membrane, and condensed chromatin formed characteristic patterns in the nucleus. M. gemmipara displayed a unique life cycle: trophozoites formed walled coccoid stages which grew through successive buddings and developed into branched structures holding cysts. These structures, measuring hundreds of micrometres, are built as the exclusive product of osmotrophic feeding. To demonstrate that M. gemmipara is a genuine soil inhabitant, we screened its presence in an environmental soil DNA diversity survey performed on an experimental setup where pig cadavers were left to decompose in soils to follow changes in eukaryotic communities. Mycamoeba gemmipara was present in all samples, although related reads were uncommon underneath the cadaver.

Copromyxa laresi n. sp. (Amoebozoa: Tubulinea) and Transfer of Cashia limacoides (Page, 1967) to Copromyxa Zopf, 1885.

Five amoeboid organisms of different origin (isolated from fish organs, soil and digestive tract of earthworm) that shared light microscopical and ultrastructural features including type and arrangement of mitochondrial cristae were subjected to phylogenetic analyses based on sequences of SSU rDNA and protein coding genes (actin, cytochrome oxidase I, and eukaryotic elongation factor 2). The reconstruction of multigene phylogeny of the strains studied (i) revealed that they belong to the same single-genus Copromyxa clade; (ii) strongly supported position of Copromyxa cantabrigiensis (syn. Hartmannella cantabrigiensis) within the genus; (iii) together with comparisons of light and electron microscopy data justified reclassification of Cashia limacoides (syn. Vexillifera expectata) to Copromyxa limacoides n. comb., and (iv) justified description of a new species, Copromyxa laresi n. sp.

Vannella pentlandii n. sp., (Amoebozoa, Discosea, Vannellida) a small, cyst-forming soil amoeba.

We describe a new species of cyst-producing soil amoeba Vannella pentlandii from course pasture in the Pentland Hills, Scotland. Analysis of the 18S rDNA gene reveals that it belongs to the sub-group within the genus, presently composed of V. placida, V. epipetala and V. fimicola (the PEF group). This group share features such as longitudinal folds/ridges on the lamella (the anterior hyaline region of the trophozoite), stubby floating forms and cyst production. While each PEF species contain cyst producing strains, not all strains within these species do so. V. fimicola produces cysts on stalks leading to its former classification as a slime mould, however no such stalks were evident in the V. pentlandii, instead groups of cysts become piled on top of each other forming clumps. The encysting amoebae crawl toward each other, pushing some off the surface to form these mounds. The V. pentlandii trophozoites are of typical size for the genus but the cysts at 6.9 µm in diameter, are the smallest so far described in genus Vannella. Other cyst producing species are found in various branches within the Vannella phylogenetic tree, probably meaning that this ability was ancestral but lost in many branches (particularly in marine species), and perhaps re-gained in others.