Pinjata ruminata gen. et sp. n.-A New Member of Centrohelid Family Yogsothothidae (Haptista: Centroplasthelida) from the Brackish River.

A new genus and species of centrohelid heliozoan Pinjata ruminata from the Tuzlukkol' River (Orenburg Region of Russia) and Gor'koe Lake (Chelyabinsk Region of Russia) is studied with light- and electron microscopy. Pinjata ruminata has two types of plate scales, partially running up the sides of the axopodia. Inner plate scales (3.2-4.9 × 1.5-2.6 µm) are flat, ovate-oblong and have a broad axial thickening and a thin electron-dense border. Outer plate scales (4.2-6.7 × 1.5-3.0 µm) are concave, elongated, of irregular shape, often curved, and broadened towards one end. Roundish depressions are forming two rows on both sides of the narrow axial thickening. The cells are attached to the substratum. Molecular phylogenetic analysis based on the SSU rDNA robustly placed P. ruminata in the family Yogsothothidae. This position is confirmed with the presence of five panacanthocystid increase regions. The morphology of the new genus is in a good accordance with diagnosis of the family. The status of a genus "Heteroraphidiophrys" is discussed. Other potential findings of Pinjata from literature are analyzed. Pinjata represents the third lineage of centrohelids, characterized with the presence of only tangentially oriented plate scales. The halophilic nature of Yogsothothidae is suggested.

Yogsothoth knorrus gen. n., sp. n. and Y. carteri sp. n. (Yogsothothidae fam. n., Haptista, Centroplasthelida), with Notes on Evolution and Systematics of Centrohelids.

Two closely related new species of centrohelid heliozoans with unusual morphology were studied with light and electron microscopy. Sequences of the 18S rRNA gene were also obtained and secondary structure of 18S rRNA molecule reconstructed. The cells, covered with inner siliceous plate scales formed colonies. The entire colony was surrounded with a thick layer of external scales. Inner scales were tabulate and had a patternless surface, except for the presence of an axial rib. Outer scales had a boat-like (Yogsothoth knorrus gen. nov., sp. nov.) or pot-like (Yogsothoth carteri sp. nov.) shape with an axial rib and numerous conical papillae on the scale surface. Analysis of 18S rRNA gene sequences robustly placed the new taxa within centrohelids, but not in any existing family. Scaled Yogsothoth represents a genetically divergent closest outgroup of Acanthocystida, branching after the supposedly primary non-scaled Marophrys, and together with acanthocystids, forming the novel taxon Panacanthocystida. Reconstruction of presumptive 18S rRNA secondary structure reveals interspecific differences in expansion segments 7 and 9 of Yogsothoth. Analysis of 18S rRNA secondary structure of other centrohelids allowed identification of length increases characteristic for Panacanthocystida location and reconstruction of 18S rRNA elongation in the course of the evolution of this group.

On the phylogenetic position of the genus Raphidocystis (Haptista: Centroplasthelida) with notes on the dimorphism in centrohelid life cycle.

Centrohelid heliozoan Raphidocystis glabra was reisolated for the first time after initial description and put into a clonal culture. Its correct identification was confirmed by scanning and transmission electron microscopy of scales. The first light microscopy data from the living cells were obtained. Phylogenetic analysis of its position using 18S rDNA sequences was also performed. This species branches inside of the Polyplacocystis clade, being closely related to the strain HLO4. The latter was isolated from the same sample with R. glabra and has spicules, not siliceous scales, unlike all the other members of this clade. Using another strain isolated from this sample the co-specificity of HLO4 and R. glabra was demonstrated, and the presence of spicule-bearing stages in the life cycle of Raphidocystis was shown. Polyplacocystis revealed to be paraphyletic with Raphidocystis embedded in it. Moreover, representatives of both genera share the similar monolayered plate scales with hollow inflected margin. Thus, a family-level name Raphidocystidae for the whole clade was proposed and all the Polyplacocystis species were transferred to Raphidocystis which name has a priority. The evolution of centrohelid coverings in the light of new findings was discussed, as well as the possible wide distribution of dimorphism in centrohelid life cycles.

The Chytrid-like Parasites of Algae Amoeboradix gromovi gen. et sp. nov. and Sanchytrium tribonematis Belong to a New Fungal Lineage.

Fungi encompass, in addition to classically well-studied lineages, an ever-expanding diversity of poorly known lineages including zoosporic chytrid-like parasites. Here, we formally describe Amoeboradix gromovi gen. et sp. nov. comprising a set of closely related strains of chytrid-like parasites of the yellow-green alga Tribonema gayanum unusually endowed with amoeboid zoospores. Morphological and ultrastructural features of A. gromovi observed by light and transmission electron microscopy recall previous descriptions of Rhizophydium anatropum. A. gromovi exhibits one of the longest kinetosomes known in eukaryotes, composed of microtubular singlets or doublets. To carry out molecular phylogenetic analysis and validate the identification of different life cycle stages, we amplified 18S rRNA genes from three A. gromovi strains infecting T. gayanum cultures, single sporangia and single zoospores. Molecular phylogenetic analyses of 18S+28S rRNA concatenated genes of the type strain revealed that A. gromovi is closely related to the recently described species Sanchytrium tribonematis, another parasite of Tribonema that had been tentatively classified within Monoblepharidomycetes. However, our phylogenetic analysis with an extended taxon sampling did not show any particular affinity of Amoeboradix and Sanchytrium with described fungal taxa. Therefore, Amoeboradix gromovi and Sanchytrium tribonematis likely represent a new divergent taxon that remains incertae sedis within Fungi.

Barcoding Heliozoa: Perspectives of 18S rDNA for Distinguishing Between Acanthocystis Species.

The first application of DNA-barcoding for the centrohelids is reported. The character-rich genus Acanthocystis was chosen to compare sequence divergence and morphological similarity. Acanthocystis nichollsi, an easily identifiable and well outlined species, was isolated from four remote locations; A. costata; A. takahashii (2 strains) and A. turfacea were studied as well. Detailed light- and electron-microscopic data were obtained and a fragment of 18S rDNA (mostly V5 to V8 regions) was cloned and multiple clones were sequenced. Obtained data allowed a comparison of the level of genetic divergence between several strains of one and the same morphospecies from remote locations and several strains of different morphospecies. This analysis showed no overlap between intraspecific and interspecific divergence. Phylogenetic analysis also recovered the clades for all species correctly. The genetic divergence between individual molecular clones obtained from the same clonal culture allowed accessing the genetic structure of the local populations of heliozoans. The level of divergence inside of the morphological species suggests a possible cryptic speciation. In some subclades of A. nichollsi the sequence polymorphism caused mingling of strains on the tree. 18S rDNA was shown to be an appropriate barcode at the specific level, but the intra-strain polymorphism needs further attention.

Light- and Electron-microscopical Study of Belonocystis marina sp. nov. (Eukaryota: incertae sedis).

BelonocystisRainer, 1968 is an enigmatic protist genus, which currently lacks any supergroup affiliation. The spherical cells of this organism move on the substratum using fine non-branching pseudopodia. The cell surface is surrounded with a spiky covering. Belonocystis marina sp. nov. was studied using light- and electron microscopy. It was clearly shown that the surface structures of Belonocystis were scales, not a capsule. The new species could be distinguished by the morphology of the scales, which had a bulbous base with three "skirts" (circular latticed structures) and a spike consisting of many twisted fibrils. Each scale was associated with a short cytoplasmic outgrowth. The organic nature of these scales was confirmed by energy-dispersive X-ray microanalysis. Large multinucleated stages were discovered in the life-cycle of this organism. A survey of the cell ultrastructure revealed all the common eukaryotic organelles, including mitochondria with tubular cristae. No microtubules were detected in ultrathin sections of pseudopodia. Examination of food vacuole contents confirmed that this organism was bacterivorous. The finding of Belonocystis marina is the first record of the genus in a marine habitat. Many similarities in the scale structure and fine structure of the cell between Belonocystis and Luffisphaera were discussed.