The first arctic strain of Rhizochromulina: Morphology, ultrastructure, and position in the evolutionary tree of the order Rhizochromulinales (Heterokontophyta, Dictyochophyceae).

Rhizochromulina marina is a unicellular amoeboid alga capable of forming flagellate cells; it is a single validly named species in the genus. Besides, there are numerous environmental sequences and undescribed strains designated as Rhizochromulina sp. or R. marina. The biogeography of the genus is understudied: rhizochromulines from the Indian, Southern, and Arctic Oceans are unknown. Here, we present the description of Rhizochromulina sp. B44, which was for the first time isolated from an arctic habitat. Biofilms of this microalga grow at the bottom of a culture vessel, where neighbouring amoeboid cells form associations through a common network of pseudopodia, i.e. meroplasmodia. Pseudopodia branch, anastomose mainly during meroplasmodia formation, and are supported by microtubules that arise from the perinuclear zone. Actin filaments are localized in the cytoplasm and can be revealed only near the bases of pseudopodia. We succeeded in inducing the transformation of amoeboid cells into flagellates using a prolonged agitation of cultures. Morphological and molecular analyses revealed that the studied strain is most closely related to the type strain of R. marina. At the same time, 18S rDNA sequences of early branching-off rhizochromulinids differ significantly from Rhizochromulina sp. B44, suggesting a high divergence at the genus level.

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.

To the Origin of Fungi: Analysis of MFS Transporters of First Assembled Aphelidium Genome Highlights Dissimilarity of Osmotrophic Abilities between Aphelida and Fungi.

Aphelids are a holomycotan group, represented exclusively by parasitoids infecting algae. They form a sister lineage to Fungi in the phylogenetic tree and represent a key group for reconstruction of the evolution of Holomycota and for analysis of the origin of Fungi. The newly assembled genome of Aphelidium insullamus (Holomycota, Aphelida) with a total length of 18.9 Mb, 7820 protein-coding genes and a GC percentage of 52.05% was obtained by a hybrid assembly based on Oxford Nanopore long reads and Illumina paired reads. In order to trace the origin and the evolution of fungal osmotrophy and its presence or absence in Aphelida, we analyzed the set of main fungal transmembrane transporters, which are proteins of the Major Facilitator superfamily (MFS), in the predicted aphelid proteomes. This search has shown an absence of a specific fungal protein family Drug:H+ antiporters-2 (DAH-2) and specific fungal orthologs of the sugar porters (SP) family, and the presence of common opisthokont's orthologs of the SP family in four aphelid genomes. The repertoire of SP orthologs in aphelids turned out to be less diverse than in free-living opisthokonts, and one of the most limited among opisthokonts. We argue that aphelids do not show signs of similarity with fungi in terms of their osmotrophic abilities, despite the sister relationships of these groups. Moreover, the osmotrophic abilities of aphelids appear to be reduced in comparison with free-living unicellular opisthokonts. Therefore, we assume that the evolution of fungi-specific traits began after the separation of fungal and aphelid lineages, and there are no essential reasons to consider aphelids as a prototype of the fungal ancestor.

Diversity, Distribution, and Development of Hyperparasitic Microsporidia in Gregarines within One Super-Host.

Metchnikovellids (Microsporidia: Metchnikovellida) are poorly studied hyperparasitic microsporidia that live in gregarines inhabiting the intestines of marine invertebrates, mostly polychaetes. Our recent studies showed that diversity of metchnikovellids might be significantly higher than previously thought, even within a single host. Four species of metchnikovellids were found in the gregarines inhabiting the gut of the polychaete Pygospio elegans from littoral populations of the White and Barents Seas: the eugregarine Polyrhabdina pygospionis is the host for Metchnikovella incurvata and M. spiralis, while the archigregarine Selenidium pygospionis is the host for M. dogieli and M. dobrovolskiji. The most common species in the White Sea is M. incurvata, while M. dobrovolskiji prevails in the Barents Sea. Gregarines within a single worm could be infected with different metchnikovellid species. However, co-infection of one and the same gregarine with several species of metchnikovellids has never been observed. The difference in prevalence and intensity of metchnikovellid invasion apparently depends on the features of the life cycle and on the development strategies of individual 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.

New data on the fine structure of Deuteramoeba mycophaga CCAP 1586/1 (Amoebozoa, Tubulinea).

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.

Molecular phylogeny and new light microscopic data of Metchnikovella spiralis (Microsporidia: Metchnikovellidae), a hyperparasite of eugregarine Polyrhabdina sp. from the polychaete Pygospio elegans.

Metchnikovellids are a deep-branching group of microsporidia, parasites of gregarines inhabiting the alimentary tract of polychaetes and some other invertebrates. The diversity and phylogeny of these hyperparasites remain poorly studied. Modern descriptions and molecular data are still lacking for many species. The results of a light microscopy study and molecular data for Metchnikovella spiralis Sokolova et al., 2014, a hyperparasite of the eugregarine Polyrhabdina sp., isolated from the polychaete Pygospio elegans, were obtained. The original description of M. spiralis was based primarily on the analysis of stained preparations and transmission electron microscopy images. Here, the species description was complemented with the results of in vivo observations and phylogenetic analysis based on the SSU rRNA gene. It was shown that in this species, free sporogony precedes sac-bound sporogony, as it occurs in the life cycle of most other metchnikovellids. Spore sacs are entwined with spirally wound cords, and possess only one polar plug. Phylogenetic analyses did not group M. spiralis with M. incurvata, another metchnikovellid from the same gregarine species, but placed it as a sister branch to Amphiacantha. The paraphyletic nature of the genus Metchnikovella was discussed. The taxonomic summary for M. spiralis was emended.

Evolutionary relationships of Metchnikovella dogieli Paskerova et al., 2016 (Microsporidia: Metchnikovellidae) revealed by multigene phylogenetic analysis.

The species Metchnikovella dogieli (Paskerova et al. Protistology 10:148-157, 2016) belongs to one of the early diverging microsporidian groups, the metchnikovellids (Microsporidia: Metchnikovellidae). In relation to typical ('core') microsporidia, this group is considered primitive. The spores of metchnikovellids have no classical polar sac-anchoring disk complex, no coiled polar tube, no posterior vacuole, and no polaroplast. Instead, they possess a short thick manubrium that expands into a manubrial cistern. These organisms are hyperparasites; they infect gregarines that parasitise marine invertebrates. M. dogieli is a parasite of the archigregarine Selenidium pygospionis (Paskerova et al. Protist 169:826-852, 2018), which parasitises the polychaete Pygospio elegans. This species was discovered in samples collected in the silt littoral zone at the coast of the White Sea, North-West Russia, and was described based on light microscopy. No molecular data are available for this species, and the publicly accessible genomic data for metchnikovellids are limited to two species: M. incurvata Caullery & Mesnil, 1914 and Amphiamblys sp. WSBS2006. In the present study, we applied single-cell genomics methods with whole-genome amplification to perform next-generation sequencing of M. dogieli genomic DNA. We performed a phylogenetic analysis based on the SSU rRNA gene and reconstructed a multigene phylogeny using a concatenated alignment that included 46 conserved single-copy protein domains. The analyses recovered a fully supported clade of metchnikovellids as a basal group to the core microsporidia. Two members of the genus Metchnikovella did not form a clade in our tree. This may indicate that this genus is paraphyletic and requires revision.

Polychaos centronucleolus n. sp. - a new terrestrial species of the genus Polychaos (Amoebozoa, Tubulinea) with nontypical nuclear structure.

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.

The first case of microsporidiosis in Paramecium.

A new microsporidian species, Globosporidium paramecii gen. nov., sp. nov., from Paramecium primaurelia is described on the basis of morphology, fine structure, and SSU rRNA gene sequence. This is the first case of microsporidiosis in Paramecium reported so far. All observed stages of the life cycle are monokaryotic. The parasites develop in the cytoplasm, at least some part of the population in endoplasmic reticulum and its derivates. Meronts divide by binary fission. Sporogonial plasmodium divides by rosette-like budding. Early sporoblasts demonstrate a well-developed exospore forming blister-like structures. Spores with distinctive spherical shape are dimorphic in size (3.7 ± 0.2 and 1.9 ± 0.2 µm). Both types of spores are characterized by a thin endospore, a short isofilar polar tube making one incomplete coil, a bipartite polaroplast, and a large posterior vacuole. Experimental infection was successful for 5 of 10 tested strains of the Paramecium aurelia species complex. All susceptible strains belong to closely related P. primaurelia and P. pentaurelia species. Phylogenetic analysis placed the new species in the Clade 4 of Microsporidia and revealed its close relationship to Euplotespora binucleata (a microsporidium from the ciliate Euplotes woodruffi), to Helmichia lacustris and Mrazekia macrocyclopis, microsporidia from aquatic invertebrates.