Community structure and assembly of myxomycetes in northern Chinese forests under geographic barriers.

The study of myxomycete biogeography has a long-standing history and has consistently drawn scholarly interest. Nevertheless, studies focusing specifically on the spatial and temporal distribution patterns of myxomycete diversity are relatively limited, with even fewer investigating the mechanisms driving the generation and maintenance of myxomycete diversity. Therefore, this study selected two geographically distant sampling sites within northern Chinese forests to investigate myxomycete species composition, community structure, environmental drivers, and assembly patterns under geographic barriers. We established plots in the Altai Mountains (ALE) and the Greater Khingan Mountains (GKM), gathered bark and litter, and conducted 80-day moist chamber cultures of myxomycetes. Additionally, myxomycete specimens were collected in the field simultaneously to supplement the data set. This study collected 541 myxomycete specimens belonging to 73 species from 28 genera, spanning 12 families and eight orders. The ALE and the GKM had 20 identical species, accounting for 27% of the total species. Myxomycetes from both regions exhibited abundant occurrence 18 days after cultivation, with the quantity on bark substrates notably higher than on litter. Arcyria pomiformis and Comatricha elegans were the most common species in moist chamber cultures. Mantel test outcomes revealed that environmental factors had no significant impact on myxomycete community similarity between the two areas, aligning with findings from the neutral community model analysis, indicating a predominant influence of stochastic processes on myxomycete community structure in moist chamber cultures. This study represents the first application of a quantitative framework to analyze myxomycete community assembly cultivated in moist chambers.

On the host specificity and genetic diversity of Iodamoeba bütschlii: Observations from short amplicon-based next-generation sequencing.

Iodamoeba is a single-celled intestinal parasite, which is common in humans in certain parts of the world, and also in pigs. For the first time, we provide DNA-based evidence of goat, dromedary, fallow deer, and donkey as hosts of Iodamoeba and show that Iodamoeba-specific nucleotide sequences from these four hosts do not appear to overlap with those of humans, unlike those from pigs. We moreover show that similar strains of Iodamoeba can be found in Madagascar, Western Sahara, and Ecuador and that intra-sample diversity is typically extensive across even small fragments of DNA in both human and non-human hosts.

High above the rest: standing behaviors in the amoebae of Sappinia and Thecamoeba.

Many terrestrial microbes have evolved cell behaviors that help them rise above their substrate, often to facilitate dispersal. One example of these behaviors is found in the amoebae of Sappinia pedata, which actively lift most of their cell mass above the substrate, known as standing. This standing behavior was first described in S. pedata in the 1890s from horse dung isolates but never molecularly characterized from dung. Our study expands this understanding, revealing the first molecularly confirmed S. pedata from herbivore dung in Mississippi, USA, and describing a new species, Sappinia dangeardi n. sp., with larger trophozoite cells. Additionally, we isolated another standing amoeba, Thecamoeba homeri n. sp., from soil, exhibiting a previously unreported "doughnut shape" transient behavior. In S. dangeardi n. sp., we discovered that standing is likely triggered by substrate drying, and that actin filaments actively localize in the "stalk" to support the standing cells, as observed through confocal microscopy. While the purpose of standing behaviors has not been investigated, we hypothesize it is energetically expensive and therefore a significant evolutionary strategy in these organisms. Overall, this study emphasizes behavioral adaptations to terrestrial environments within Amoebozoa, stressing the importance of diverse laboratory conditions that replicate natural habitats.

Protozoan parasites of birds from the Tremembé formation (Oligocene of the Taubaté Basin), São Paulo, Brazil.

OBJECTIVE: To analyze the presence of protozoan parasites in bird coprolites from the Tremembé Formation (Oligocene of the Taubaté Basin). MATERIALS: Twenty avian coprolites embedded in pyrobituminous shale matrices. METHODS: Samples were rehydrated and subjected to spontaneous sedimentation. RESULTS: Paleoparasitological analyses revealed oocysts compatible with the Eimeriidae family (Apicomplexa) and one single Archamoebae (Amoebozoa) cyst. CONCLUSIONS: The present work increases the amount of information about the spread of infections throughout the Cenozoic Era and reveals that the Brazilian paleoavifauna played an important role in the Apicomplexa and Amoebozoa life cycles. SIGNIFICANCE: This is the first record of protozoans in avian coprolites from the Oligocene of Brazil. These findings can help in the interpretation of phylogenies of coccidian parasites of modern birds, as certain taxonomic characters observed in the Oligocene Protozoa characterize monophyletic groups in current molecular phylogenetic analyses. LIMITATIONS: None of the oocysts were sporulated; therefore, it is not possible to identify the morphotypes to genus or species. SUGGESTIONS FOR FURTHER RESEARCH: Our results create new perspectives related to biogeographic studies of the parasitic groups described and may improve the understanding of the temporal amplitude of parasitic evolutionary relationships between Protozoans and birds.

Arcyria similaris: A new myxomycete species from China.

A new myxomycete species, Arcyria similaris, was reported herein. The specimens were found and collected in the field on dead bark from Jingangtai National Geopark in Henan Province of China. This species has distinct and unique morphological characteristics, including dark grayish olive sporothecae that fade to smoke gray with age, shallow saucer-shaped cups with marked reticulations and thick papillae on the inner surface, a netted capillitium with many bulges, uniformly marked with low, dense, and irregular reticulations, and spores (8.0-)9.3-10.1(-10.9) µm in diameter, marked with sparse small warts and grouped prominent warts. Apart from a comprehensive morphological study, partial sequences of the nuclear 18S rDNA and elongation factor-1 alpha (EF-1α) genes were also provided in this study. This new species was described and illustrated morphologically. The specimens are deposited in the Herbarium of Fungi of Nanjing Normal University (HFNNU).

A Small Genome amidst the Giants: Evidence of Genome Reduction in a Small Tubulinid Free-Living Amoeba.

This study investigates the genomic characteristics of Echinamoeba silvestris, a small-sized amoeba within the Tubulinea clade of the Amoebozoa supergroup. Despite Tubulinea's significance in various fields, genomic data for this clade have been scarce. E. silvestris presents the smallest free-living amoeba genome within Tubulinea and Amoebozoa to date. Comparative analysis reveals intriguing parallels with parasitic lineages in terms of genome size and predicted gene numbers, emphasizing the need to understand the consequences of reduced genomes in free-living amoebae. Functional categorization of predicted genes in E. silvestris shows similar percentages of ortholog groups to other amoebae in various categories, but a distinctive feature is the extensive gene contraction in orphan (ORFan) genes and those involved in biological processes. Notably, among the few genes that underwent expansion, none are related to cellular components, suggesting adaptive processes that streamline biological processes and cellular components for efficiency and energy conservation. Additionally, our investigation into noncoding and repetitive elements sheds light on the evolution of genome size in amoebae, with E. silvestris distinguished by low percentage of repetitive elements. Furthermore, the analysis reveals that E. silvestris has the lowest mean number of introns per gene among the species studied, providing further support for its observed compact genome. Overall, this research underscores the diversity within Tubulinea, highlights knowledge gaps in Amoebozoa genomics, and positions E. silvestris as a valuable addition to genomic data sets, prompting further exploration of complexities in Amoebozoa diversity and genome evolution.

It's time to consider the Arcellinida shell as a weapon.

The shells of testate amoebae are morphologically diverse and persistent in the environment. Accordingly, the examination of the morphology and composition of shells became a standard tool in ecological, palaeoecological, and evolutionary studies. However, so far the function of the shell remains poorly understood and, although based on limited evidence, the shell was considered as a defense mechanism. Based on recent evidence, we propose that the shell of arcellinid testate amoebae is a crucial component facilitating the amoebae's attack of large prey. Accordingly, the shell is not purely protective, but must be considered also as a weapon. This change in perspective opens up numerous new avenues in protistology and will lead to a substantial change in ecological, palaeoecological, and evolutionary research.

Re-isolation and revaluation of boundaries of Thecamoeba terricola Greeff, 1886 (Amoebozoa, Discosea, Thecamoebida), with description of Thecamoeba vicaria n. sp.

We studied a large species of Thecamoeba found in a glass dish with soaked moss and obtained 18s rRNA gene sequence of this organism. Morphologically, the strain was most similar to T. terricola sensu Page, but had significant differences in cell size and nuclear morphology. A more complete similarity was found with the original description of "Amoeba terricola" by Greeff, as well as with Penard's descriptions and slides. The analysis of literature data shows that the strain described by Page in 1977 as a re-isolated T. terricola differs from the original description of this species provided by Greeff in 1866 and data by Penard published in 1902 and 1913. Based on our observations as well as on Greeff's and Penard's data, we reassessed the species boundaries of T. terricola and established T. vicaria n. sp. for the organism described by Page in 1977. The species T. terricola was defined according to its original description. The observations of amoebae on agar have shown that T. terricola cells can form the "standing amoeba" stage, previously described only for Sappinia pedata. This and some other "behaviour" features of T. terricola may be associated with living conditions in terrestrial habitats.

Omics of an Enigmatic Marine Amoeba Uncovers Unprecedented Gene Trafficking from Giant Viruses and Provides Insights into Its Complex Life Cycle.

Amoebozoa include lineages of diverse ecology, behavior, and morphology. They are assumed to encompass members with the largest genome sizes of all living things, yet genomic studies in the group are limited. Trichosphaerium, a polymorphic, multinucleate, marine amoeba with a complicated life cycle, has puzzled experts for over a century. In an effort to explore the genomic diversity and investigate extraordinary behavior observed among the Amoebozoa, we used integrated omics approaches to study this enigmatic marine amoeba. Omics data, including single-cell transcriptomics and cytological data, demonstrate that Trichosphaerium sp. possesses the complete meiosis toolkit genes. These genes are expressed in life stages of the amoeba including medium and large cells. The life cycle of Trichosphaerium sp. involves asexual processes via binary fission and multiple fragmentation of giant cells, as well as sexual-like processes involving genes implicated in sexual reproduction and polyploidization. These findings are in stark contrast to a life cycle previously reported for this amoeba. Despite the extreme morphological plasticity observed in Trichosphaerium, our genomic data showed that populations maintain a species-level intragenomic variation. A draft genome of Trichosphaerium indicates elevated lateral gene transfer (LGT) from bacteria and giant viruses. Gene trafficking in Trichosphaerium is the highest within Amoebozoa and among the highest in microbial eukaryotes.

Fifteen new species from the myxomycete genus Lycogala.

Based on a study of 255 collections from four continents and four floristic kingdoms, we describe 15 new species of the genus Lycogala. The new species, all morphologically close to L. epidendrum, L. exiguum, and L. confusum, differ from each other by the structure of the peridium and, in some cases, also by the color of the fresh spore mass and the ornamentation of the capillitium and spores. Species delimitation is confirmed by two independently inherited molecular markers, as well as previously performed tests of reproductive isolation and genetic distances. We studied authentic material of L. exiguum and L. confusum and found fresh specimens of these species, which allowed us to obtain molecular barcodes and substantiate the separation of new species from these taxa. We propose to retain the name L. epidendrum for the globally most abundant species, for which we provide a more precise description and a neotypification. Two formerly described species, L. leiosporum and L. fuscoviolaceum, we consider to be dubious. We do not recognize the species L. terrestre.

Organic preservation of vase-shaped microfossils from the late Tonian Chuar Group, Grand Canyon, Arizona, USA.

Vase-shaped microfossils (VSMs) are found globally in middle Neoproterozoic (800-730 Ma) marine strata and represent the earliest evidence for testate (shell-forming) amoebozoans. VSM tests are hypothesized to have been originally organic in life but are most commonly preserved as secondary mineralized casts and molds. A few reports, however, suggest possible organic preservation. Here, we test the hypothesis that VSMs from shales of the lower Walcott Member of the Chuar Group, Grand Canyon, Arizona, contain original organic material, as reported by B. Bloeser in her pioneering studies of Chuar VSMs. We identified VSMs from two thin section samples of Walcott Member black shales in transmitted light microscopy and used scanning electron microscopy to image VSMs. Carbonaceous material is found within the internal cavity of all VSM tests from both samples and is interpreted as bitumen mobilized from Walcott shales likely during the Cretaceous. Energy dispersive X-ray spectroscopy (EDS) and wavelength dispersive X-ray spectroscopy (WDS) reveal that VSM test walls contain mostly carbon, iron, and sulfur, while silica is present only in the surrounding matrix. Raman spectroscopy was used to compare the thermal maturity of carbonaceous material within the samples and indicated the presence of pyrite and jarosite within fossil material. X-ray absorption spectroscopy revealed the presence of reduced organic sulfur species within the carbonaceous test walls, the carbonaceous material found within test cavities, and in the sedimentary matrix, suggesting that organic matter sulfurization occurred within the Walcott shales. Our suite of spectroscopic analyses reveals that Walcott VSM test walls are organic and sometimes secondarily pyritized (with the pyrite variably oxidized to jarosite). Both preservation modes can occur at a millimeter spatial scale within sample material, and at times even within a single specimen. We propose that sulfurization within the Walcott Shales promoted organic preservation, and furthermore, the ratio of iron to labile VSM organic material controlled the extent of pyrite replacement. Based on our evidence, we conclude that the VSMs are preserved with original organic test material, and speculate that organic VSMs may often go unrecognized, given their light-colored, translucent appearance in transmitted light.

Two independent genetic markers support separation of the myxomycete Lycogala epidendrum into numerous biological species.

Lycogala epidendrum is one of the most widely known myxomycete species and the first-ever discovered representative of this group. Using 687 original DNA sequences from 330 herbarium specimens from Europe, Asia, North and Central America, and Australia, we constructed the first detailed phylogenies of the genus Lycogala, based on two independently inherited genetic markers, the ribosome small subunit 18S rRNA nuclear gene (18S rDNA) and the mitochondrial cytochrome oxidase subunit I gene (COI). In both phylogenies, L. epidendrum appeared to be a polyphyletic group, represented by numerous clades. The four other recognized species of the genus (L. confusum, L. conicum, L. exiguum, and L. flavofuscum) are scattered between branches corresponding to L. epidendrum. A barcode gap analysis revealed 60 18S rDNA phylogroups of L. epidendrum, which are distant from each other not less than from other species of the genus Lycogala. For 18 of these phylogroups with both 18S rDNA and COI sequences available, recombination patterns were analyzed to test for reproductive isolation. In contrast to the results of a simulation assuming panmixis, no crossing between ribosomal and mitochondrial phylogroups was found, thus allowing the conclusion that all tested phylogroups represent biospecies. More than one third (39.6%) of the studied specimens share a single 18S rDNA phylogroup, which we consider to be L. epidendrum s. str. This group displays the broadest geographic distribution and the highest intraspecific genetic variability. Nearly all (93.3%) of the remaining non-singleton 18S rDNA phylogroups are restricted to certain continents or even regions. At the same time, various reproductively isolated phylogroups occur sympatric at a given location.

Stenamoeba aeronauta n. sp., a new case of sibling species in the order Thecamoebida (Amoebozoa, Discosea).

A new speciesStenamoeba aeronautan. sp.was isolated from a culture of large thecamoebids during laboratory studies. Our study of this species showed almost complete morphological identity with the well-known speciesStenamoeba stenopodia. Despite the morphological similarity and proximity in the phylogenetic tree, significant differences in the sequence of the 18S rRNA gene forced us to recognize it as a new species. Known species ofStenamoebahave noticeable morphological differences, but the discovery of the new speciesshows that cryptic speciation appears in this amoeba genus as well as in many others, likeThecamoebaorVannella. In contrast with many other amoebae genera, the number of available 18S rRNA gene sequences exceeds that of morphologically described isolates. So, it is not yet possible to suggest the application of the names of monophyletic species groups, as it was recently proposed forThecamoebaspecies, since every clade ofStenamoebacontains both sequences of species with known morphology and with unknown ones.Overall, the present study further confirms that probably almost all "classical" morphospecies of amoebae may represent a cluster of a sibling species, showing remarkable differences at the molecular level.

Importance of type specimen study for understanding genus boundaries-taxonomic clarifications in Lepidoderma based on integrative taxonomy approach leading to resurrection of the old genus Polyschismium.

Type specimens of four species of Lepidoderma (Myxomycetes, Amoebozoa)-L. crassipes, L. neoperforatum, L. perforatum, and L. stipitatum-have been studied using an integrative approach including application of traditional taxonomy methods, i.e., morphological study under stereoscopic and compound microscopes, detailed analysis of micromorphological characters using scanning electron microscopy, and molecular analysis by way of Sanger sequencing of molecular markers (nuc 18S rDNA and elongation factor 1-alpha gene, EF1A). Results of the study revealed that L. crassipes is conspecific with L. tigrinum, L. stipitatum is a malformed specimen of Diderma floriforme, whereas L. perforatum and L. neoperforatum represent two well-defined morphologically and genetically separate species. Phylogeny of Physarales shows the polyphyletic character of the genus Lepidoderma. The type species of Lepidoderma clusters together with Diderma, whereas other representatives of this genus form a monophyletic, well-supported clade. The species from this clade are proposed to belong to the genus Polyschismium described by A. Corda in 1842 that is resurrected and emended here. Nine species of Lepidoderma are transferred to Polyschismium. A new key to Didymiaceae including Polyschismium is provided.

From Argentine Abyssal Plain to farmed turbot in Spain: A ubiquitous amoeba species Vannella robusta sp. nov. (Amoebozoa, Vannellida).

A strain with the characters of the genus Vannella was isolated from the water layer immediately above the deep-sea sediment collected in the south-western Atlantic Ocean, ca. 4.6 km deep. Small-subunit ribosomal RNA (SSU rRNA) and cytochrome c oxidase (Cox1) gene phylogenetic analyses showed that the new strain branches within the clade of previously isolated unnamed Vannella strains from different marine fish and invertebrate hosts. Although the SSU rRNA gene sequences of these strains show variability within 2% of all nucleotide positions without any regular pattern, the available Cox1 gene sequences from within this clade are identical. Given the morphological homogeneity of the revealed clade, all of its strains can be assigned under the same species name, and the variation of their SSU rRNA is comparable to its intragenomic variation, as shown by molecular cloning of the PCR amplicons. High variability of the SSU rRNA gene sequences within and between independently isolated morphologically identical strains in combination with highly conserved Cox1 gene sequences may be a feature in some clades of Vannella, but is not a general rule for this genus, as SSU rRNA genes conserved between different morphospecies occur in several other clades within Vannella.

Adhesion of Dictyostelium Amoebae to Surfaces: A Brief History of Attachments.

Dictyostelium amoebae adhere to extracellular material using similar mechanisms to metazoan cells. Notably, the cellular anchorage loci in Amoebozoa and Metazoa are both arranged in the form of discrete spots and incorporate a similar repertoire of intracellular proteins assembled into multicomponent complexes located on the inner side of the plasma membrane. Surprisingly, however, Dictyostelium lacks integrins, the canonical transmembrane heterodimeric receptors that dominantly mediate adhesion of cells to the extracellular matrix in multicellular animals. In this review article, we summarize the current knowledge about the cell-substratum adhesion in Dictyostelium, present an inventory of the involved proteins, and draw parallels with the situation in animal cells. The emerging picture indicates that, while retaining the basic molecular architecture common to their animal relatives, the adhesion complexes in free-living amoeboid cells have evolved to enable less specific interactions with diverse materials encountered in their natural habitat in the deciduous forest soil. Dissection of molecular mechanisms that underlay short lifetime of the cell-substratum attachments and high turnover rate of the adhesion complexes in Dictyostelium should provide insight into a similarly modified adhesion phenotype that accompanies the mesenchymal-amoeboid transition in tumor metastasis.

Diachea mitchellii: A new myxomycete species from high elevation forests in the tropical Andes of Peru.

A new species of the genus Diachea (order Physarales, Myxomycetes, Amoebozoa) is described from Peru. Relevant details on spore germination, as well as morphological and phylogenetic data, are provided. At first glance, the new species shares some morphological similarities with both D. leucopodia, type of the genus, and D. koazei, but it strikingly differs from all other species of its genus by combining a short dark stalk, with a reticulate columella, and clustered spores. Moreover, it seems to be the only species of Diachea exclusively associated with Polylepis tropical forests at elevations above 3500 m. Apart from a comprehensive morphological study of 31 specimens, we here provide phylogenetic evidence to confirm the inclusion of this species in the genus Diachea. Specifically, our phylogenetic analyses of the nuclear 18S rDNA (18S), mitochondrial 17S rDNA (17S), and elongation factor-1 alpha (EF-1α) genes show that the new species is related to D. leucopodia and D. bulbillosa. The remarkably different morphological characters distinguishing the new Diachea from all other species of its genus, along with its particular ecological preferences and geographic distribution, indicate that it is a distinct entity deserving recognition as an independent species.

Thecamoeba vumurta n. sp. (Amoebozoa, Discosea, Thecamoebida) from freshwater pond sediment - a sibling species of T. striata (Penard, 1890) Schaeffer, 1926.

Until recently, it seemed that amoebae of the genus Thecamoeba could be reliably identified using light-microscopic characters. However, recent studies show the presence of sibling species in the genus Thecamoeba, which can only be reliably distinguished based on the molecular data. Here we describe a new freshwater species, Thecamoeba vumurta n. sp., which has minor morphological differences in the light-microscopy and the cell ultrastructure with the widely distributed species Thecamoeba striata. Taken alone, these differences are hardly sufficient to justify a new species, but considerable differences in the SSU rRNA gene sequence leave no doubts that this is an independent species, forming a pair of sibling species with T. striata. The SSU gene sequence in both these species is very divergent from other thecamoebids. BLAST search identifies neither of these sequences as belonging to Thecamoeba. This study further supports the conclusion that gene sequencing is necessary for the reliable identification of Thecamoeba species. To clarify the situation with sibling species, we propose distinguishing three morphologically defined species groups within the genus Thecamoeba and using their names (instead of taxonomic species names) in case a similar species is identified without molecular studies.

Cdc42/Rac Interactive Binding Containing Effector Proteins in Unicellular Protozoans With Reference to Human Host: Locks of the Rho Signaling.

Small GTPases are the key to actin cytoskeleton signaling, which opens the lock of effector proteins to forward the signal downstream in several cellular pathways. Actin cytoskeleton assembly is associated with cell polarity, adhesion, movement and other functions in eukaryotic cells. Rho proteins, specifically Cdc42 and Rac, are the primary regulators of actin cytoskeleton dynamics in higher and lower eukaryotes. Effector proteins, present in an inactive state gets activated after binding to the GTP bound Cdc42/Rac to relay a signal downstream. Cdc42/Rac interactive binding (CRIB) motif is an essential conserved sequence found in effector proteins to interact with Cdc42 or Rac. A diverse range of Cdc42/Rac and their effector proteins have evolved from lower to higher eukaryotes. The present study has identified and further classified CRIB containing effector proteins in lower eukaryotes, focusing on parasitic protozoans causing neglected tropical diseases and taking human proteins as a reference point to the highest evolved organism in the evolutionary trait. Lower eukaryotes' CRIB containing proteins fall into conventional effector molecules, PAKs (p21 activated kinase), Wiskoit-Aldrich Syndrome proteins family, and some have unique domain combinations unlike any known proteins. We also highlight the correlation between the effector protein isoforms and their selective specificity for Cdc42 or Rac proteins during evolution. Here, we report CRIB containing effector proteins; ten in Dictyostelium and Entamoeba, fourteen in Acanthamoeba, one in Trypanosoma and Giardia. CRIB containing effector proteins that have been studied so far in humans are potential candidates for drug targets in cancer, neurological disorders, and others. Conventional CRIB containing proteins from protozoan parasites remain largely elusive and our data provides their identification and classification for further in-depth functional validations. The tropical diseases caused by protozoan parasites lack combinatorial drug targets as effective paradigms. Targeting signaling mechanisms operative in these pathogens can provide greater molecules in combatting their infections.

Balamuthia spinosa n. sp. (Amoebozoa, Discosea) from the brackish-water sediments of Nivå Bay (Baltic Sea, The Sound) - a novel potential vector of Legionella pneumophila in the environment.

We have found a new free-living amoeba species named Balamuthia spinosa n. sp. (Amoebozoa, Discosea) in the bottom sediments of the brackish-water Nivå Bay (Baltic Sea, The Sound). This species resembles members of the genus Stygamoeba morphologically and was (mis)identified as belonging to this genus during the initial investigation. However, SSU rRNA gene data show that it robustly groups with Balamuthia mandrillaris sequence among Acanthopodida and represents a new species of the genus Balamuthia. Fragments of Legionella pneumophila genome were found among the NGS contigs obtained from B. spinosa n. sp., suggesting that this species may be a vector of Legionella in the environment. We discuss a remarkable morphological and ultrastructural similarity between the genus Balamuthia and the genus Stygamoeba. In addition, our phylogenetic analysis based on the SSU rRNA gene sequences revealed a close relationship between the genera Stygamoeba and Vermistella. It is one more confirmation of the order Stygamoebida, which was formed basing on the morphological evidence. The position of these branches close to Thecamoebida clade is congruent with current phylogenomic data.