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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.

The genus Deuteramoeba is one of the six amoebae genera belonging to the best-known amoeba family - Amoebidae (Amoebozoa, Tubulinea), containing such a popular species as Amoeba proteus. However, members of other genera of the family Amoebidae are much less known, and most of the studies of their morphology and ultrastructure date back to the 1970s and 1980s. Since these "classical" species are believed to be "well studied", their morphology and fine structure rarely become a subject of re-investigation. The absence of modern morphological data may be critical when molecular data of the type strain are not available, and the only way to identify a species is by morphological comparison. For this paper, we performed an ultrastructural study of the strain CCAP 1586/1 - the type strain of the species Deuteramoeba mycophaga. Our study revealed new details of the nuclear structure, including a peripheral layer of filaments and a heterogeneous nucleolus, and provided new data on the cytoplasmic inclusions of this species. We performed a whole-genome amplification of the DNA from a single amoeba cell followed by NGS sequencing and searched for genetic evidence for the presence of a putative nuclear parasite detected in 2017, but found no evidence for the presence of Opisthosporidia.

A new species of the "proteus-type" naked amoebae (large cells with discrete tubular pseudopodia) was isolated from tree bark sample of a birch tree in the surrounding of Kislovodsk town, Russia and named Polychaos centronucleolus n. sp. (Amoebozoa, Tubulinea). Amoebae of this species have a filamentous cell coat and a nucleus with a central compact nucleolus. This type of nucleolar organization has not been previously known for the genus Polychaos. A sequence of the 18S rRNA gene of this strain was obtained using whole genome amplification of DNA from the single amoeba cell, followed by NGS sequencing. The analysis of molecular data robustly groups this species with Polychaos annulatum within the family Hartmannellidae. Our results, together with the results of our previous studies, show that the taxonomic assignment of "proteus-type" amoebae species is becoming increasingly complex, and the taxonomic characters that can be used to classify these organisms are becoming more shadowed.

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.

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

Ciliates inhabiting the intestine of horses in Iceland were investigated. The population of horses on the island has been isolated for more than 1000 years, so their hindgut ciliates were also isolated all that time. The samples were taken from 6 horses on the territory of Iceland. More than 30 species belonging to 18 genera were found. One species is a very rare.

In this detailed electron microscopic study of Paradermamoeba levis Smirnov et Goodkov 1994 (phylum Amoebozoa, class Discosea, subclass Longamoebia, order Dermamoebida, family Dermamoebidae) based on numerous fixations and studies of a large number of cells we provide the first comprehensive description of the ultrastructure of this species. P. levis possesses cytoplasmic microtubule-organizing centres (MTOCs) associated with dictyosomes of the Golgi complex. This finding adds evidence to our earlier suggestion that the presence of cytoplasmic MTOCs is a synapomorphy of the phylogenetic lineages forming the subclass Longamoebia. The so-called "supernumerary nucleus" of P. levis noted in the initial description was found to be not an individual structure but an outgrowth of the cell nucleus containing its own nucleolus. Enigmatic trichocyst-like bodies were noted in all studied strains, originating from different geographic locations. This proves that these bodies are integral parts of the cell structure, not an occasional property of the type strain. P. levis is now reliably recorded from several European locations (North-Western Russia, Croatia, Switzerland, UK) and Far East of Russia.

Until recently it seemed that amoebae of the genus Thecamoeba can be reliably identified using light-microscopic characters, like the size and shape of the cell, a characteristic pattern of folds and ridges and structure of the nucleus. However, recent molecular studies show the presence of sibling species that can be reliably distinguished based on the gene sequence data only. Here we describe a new terrestrial species, Thecamoeba foliovenanda n. sp. This species is almost identical with Thecamoeba similis in light-microscopy, which has minor differences in the ultrastructure but considerable differences in the SSU rRNA gene sequence. We investigated the light-microscopic data, as well as transmission electron-microscopic images and videorecords on the type strain of T. similis 1583/8 and performed its comparison with the new species. This study further supports the conclusion that species of the genus Thecamoeba nowadays require gene sequencing for reliable identification and species distinction.