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Heterotrophic protists Rotosphaerida, Thaumatomonadida, and centrohelid heliozoans are among the less studied silicified protists in terms of their biogeography and ecology. These organisms inhabit fresh and brackish water, and leave behind siliceous structural elements after death that are species-specific and amenable to electron microscopic analysis. This paper is the first to present data on species richness and taxonomic structure of silica-scaled heterotrophic protists-rotosphaerids, colorless free-living thaumatomonad flagellates and centrohelid heliozoans-in the large continuous water system of Siberia connecting Lake Baikal to the Kara Sea. In the study area, electron microscopy revealed 21 centrohelid heliozoan species from the genera Raphidiophrys (1), Acanthocystis (7), Choanocystis (3), Raineriophrys (2), Raphidocystis (6), and Pterocystis (2), seven rotosphaerid species from the genera Pinaciophora (3), Turriplaca (2), Rabdiophrys (1), and Pompholyxophrys (1), and one thaumatomonad flagellate species Thaumatomastix. Two species of rotosphaerids, Rabdiophrys cf. anulifera and Pinaciophora tridentata, and two species of centrohelid heliozoans, Acanthocystis cf. tubata, and A. cf. cornuta, were found in the waters of Russia for the first time. The most widespread species in fresh water from Lake Baikal to the Lower Yenissei River were Pinaciophora fluviatilis and Raineriophrys cf. fortesca. These species disappeared from the protist assemblages of the Yenissei gulf of the Kara Sea due to higher salinity, with only three species, Acanthocystis cf. mylnikovi (at 3.12‰ salinity) and A. pectinata and Raphidocystis sp. 2 (at 8‰ salinity), found in the area. The sensitivity of silica-scaled heterotrophic protists to changes in habitat parameters suggests that these microeukaryotes could be new indicator organisms.

A new strain of planktonic heliozoans (ZI172) belonging to the genus Microheliella (the sister group of Cryptista in Diaphoretickes), closely related to the only one known strain of Microheliella maris (CCAP 1945/1), was studied with light microscopy and SSU rRNA gene sequencing. Morphometric data obtained from 127 cells and based on 254 measurements showed that this strain represents the smallest heliozoan (1.66–3.42 µm, av. 2.56 µm) in diameter known to date and one of the smallest free-living eukaryotes. We also did morphometry for strain CCAP 1945/1. Its cell body size is 3.20–6.47 µm (av. 4.15 µm; n=141; m=282). The secondary structures of hairpin 15 of the SSU rRNA molecules were reconstructed for ZI172 and CCAP 1945/1 and they were compared The possible biochemical explanation for the smaller size of the ZI172 strain, which is smaller than the CCAP 1945/1 strain, is discussed, including all published electron micrographs of CCAP 1945/1. The necessary taxonomic work is also carried out. The diagnosis of Microheliella maris is amended and the new infraspecific clade Erebor is described to include ZI172. The measurements and systematics of the enigmatic heliozoan ‘Raphidiophrys’ magna O’Donoghue 1922 (non 1921; the biggest known heliozoan) are also discussed and it is transferred to the new genus Berkeleyaesol.

The majority of centrohelids bear external coverings consisting of organic spicules or siliceous scales. Cyst coverings are usually reinforced with additional layers of modified scales. The cyst wall of Raphidiophrys heterophryoidea has an unusual and complex structure. It consists of three different types of scales and includes the mosaic scale layer not known in other centrohelids. During excystment, the cyst wall fragments along the sutures of the mosaic layer. For other Raphidiophrys species, cyst coverings are not studied. The present paper describes a new Raphidiophrys species, R. elongata, belonging to the NC7 environmental clade. Trophozoites bore thin plate scales with reduced upper plate. Under starvation, cysts emerged in clonal cultures. Cyst coverings of R. elongata and R. heterophryoidea were studied in comparison with the use of FIB-SEM. Cyst wall of R. elongata was significantly thinner than in R. heterophryoidea and was formed with 3–5 layers of uniform overlapping scales. No mosaic scale layer was present. During excystment, trophozoite exited cyst shell through random fissure. Possible evolutionary events and driving forces behind the complication of cyst wall within Raphidiophrys were discussed.

The species composition of centrohelid heliozoans from freshwater habitats of different types (sphagnum bog, temporary and permanent water bodies and streams) of Southern Chile has been studied. Fourteen species (Acanthocystis nichollsi, A. pectinata, A. penardi, Pseudoraphidiophrys discoidea, P. formosa, P. veliformis, Raphidiophrys ovalis, Choanocystis perpusilla, Pterocystisfoliaceatruncata, P. pyriformis, P. quadrata, Raineriophrys erinaceoides, R. fortesca, and R. pteromorphos) are observed. Two species (Acanthocystis nichollsi and Pterocystis quadrata) are recorded in Chile for the first time. Most of these species are recorded in Southern Chile for the first time. Electron microscopy is used to investigate the external morphology of scales, having important diagnostic value. Morphological descriptions and micrographs of studied scales amplify the concept of intraspecies variability and supplement the diagnosis of species.

Centrohelid heliozoans form a monophyletic group with uncertain affinities to other groups of protists. Except for a number of naked species, they have either siliceous scales or organic spicules covering the cell. According to the phylogenetic analysis of the 18S rDNA gene, it is hypothesized that scales are the ancestral form of cell coverings, while spicules are derived structures. The present paper describes a new species of centrohelids, Raphidiophrys heterophryoidea, isolated from the inner lakes of Valamo Island (North-Western Russia). This species has tangential scales, consisting of two plates, connected with radial, sometimes branched septa. Raphidiophrys heterophryoidea also has radially oriented spindle-shaped spicules. The organic nature of spicules and the siliceous nature of scales were verified by energy-dispersive X-ray microanalysis. Such a combination of organic and siliceous skeletal elements in one heliozoan is novel for any heliozoan species. The complex bipartite structure of scales suggested placing this new species in the genus Raphidiophrys. Consequently, the diagnoses of the genus Raphidiophrys and the family Raphidiophryidae were amended taking into account the optional presence of spicules. Raphidiophrys heterophryoidea presumably represents a stage of shift from scales to spicules, which, according to the molecular phylogenetic data, occurred at least twice in centrohelid evolution.

Summary 1. The distinctive characteristic of the water column of Priest Pot in summer is its intense physical and chemical stratification. The changing dissolved oxygen tension with depth is the main factor determining which microbes live where; distinct microbial communities exploit the different microbial niches at different depths and times. 2. Most biological activity in Priest Pot, whether in sediment or water column, is driven by microorganisms. The number of protist species recorded thus far is 883. Much remains to be discovered, particularly concerning scale-bearing protists. 3. Transmission electron microscopy has been used to study the microscales from, and on, the surfaces of microbial eukaryotes living in Priest Pot. The biological material was collected from fresh samples of the water column, from the sediment surface and from the pond’s immediate surrounding habitats. ‘Subfossils’ from the superficial sediment of the pond at the end of summer were also examined. 4. A total of 220 species of scale-bearing protists have been documented in the pond, belonging to the three functional groups or ‘body plans’ of protozoa: the amoeboid protozoa, the flagellates and the ciliates. The following genera are reported and illustrated: Acanthocystis, Chrysochromulina, Chrysolepidomonas, Chrysosphaerella, Cochliopodium, Cyathobodo, Gyromitus, Korotnevella, Lepidotrachelophyllum, Luffisphaera, Mallomonas, Paraluffisphaera, Paraphysomonas, Pompholysophrys, Pseudodendromonas, Raphidiophrys, Spiniferomonas, Synura, Thuamatomastix and Trachelophyllum/Bylamellophrya.

The species composition, distribution, and morphological characters of heliozoans (Heliozoa) of fresh and marine biotopes of the European part of Russia have been studied using methods of light microscopy (LM) and scanning electron microscopy (SEM). A total of 23 species from 4 taxonomic groups have been found. Acanthocystis taurica, Polyplacocystis symmetrica, Pompholyxophris punicea, and Raphidiophrys ovalis have been found in Russia for the first time. Species Acanthocystis myriospina, Acanthocystis turfacea, and Raineriophrys fortesca have been characterized as euryhaline. The most species diversity of heliozoans has been found in sphagnum bogs. Illustrated descriptions of these species are presented.

The community characteristics of soil Sarcomastigophora in the Mayan Forest Region of the National Nature Reserve of Xiaolong Mountains was studied using the “non-flooded petri dish method” and observation in vivo from July of 2006 to April of 2007. In total 78 species were identified, including 2 unnamed species and 12 new records of soil Sarcomastigophora in China, belonging to two subphylums, five classes, 16 orders, 32 families, and 47 genera. Amoebida was the dominant group, Kinetoplastida and Euglenida were the subdominant groups, Dinoflagellida, Chaenoflagellida, Pelobiontida, Diplomonaida and Actinophyrida were the incidental group, and Bodo minimus , Bodo ovatus , Oikomonas termo and Raphidiophrys viridis were the dominant species. The result of the study showed that there were abundant species and endemic and rare species in the community of soil Sarcomastigophora, the species composition of soil Sarcomastigophora in the Mayan Forest Region of the National Nature Reserve of Xiaolong Mountains was unique. The community characteristics of soil Sarcomastigophora was discussed based on analyzing the complexity and uniqueness of the ecological environment in the Mayan Forest Region of the National Nature Reserve of Xiaolong Mountains.

Phytoplankton samples from five freshwater lakes and ponds on the island of Dominica were examined for silica-scaled chrysophytes and heliozoa using transmission electron microscopy. Three species of Synurophyceae were observed: Synura echinulata, S. petersenii and Mallomonas tonsurata; two species of Chrysophyceae: Paraphysomonas vestita and Spiniferomonas trioralis; and five species of heliozoa: Acanthocystis nichollsi, Raineriophrys erinaceoides, Raphidiophrys intermedia, Raphidocystis tubifera and Polyplacocystis marginata. The species composition was not indicative of a tropical environment, but included taxa found worldwide. All are new records for Dominica.

By recent advance in evolutionary biology, the majority of eukaryotes are classified into six eukaryotic assemblages called as "supergroups". However, several eukaryotic groups show no clear evolutionary affinity to any of the six supergroups. Centrohelida, one of major heliozoan groups, are such an unresolved lineage. In this study, we newly determined the genes encoding translation elongation factor 2 (EF2), cytosolic heat shock protein 70 (HSP70), and cytosolic heat shock protein 90 (HSP90) from the centroheliozoan Raphidiophrys contractilis. The three Raphidiophrys genes were then combined with previously determined actin, alpha-tubulin, beta-tubulin, and SSU rRNA sequences to phylogenetically analyze the position of Centrohelida in global eukaryotic phylogeny. Although the multi-gene data sets examined in this study are the largest ones including the centroheliozoan sequences, the relationships between Centrohelida and the eukaryotic groups considered were unresolved. Our careful investigation revealed that the phylogenetic estimates were highly sensitive to genes included in the multi-gene alignment. The signal of SSU rRNA and that of alpha-tubulin appeared to conflict with one another: the former strongly prefers a monophyly of Diplomonadida (e.g., Giardia), Parabasalia (e.g., Trichomonas), Heterolobosea (e.g., Naegleria), and Euglenozoa (e.g., Trypanosoma), while the latter unites Diplomonadida, Parabasalia, Metazoa, and Fungi. In addition, EF2 robustly unites Rhodophyta and Viridiplantae, while the remaining genes considered in this study do not positively support the particular relationship. Thus, it is difficult to identify the phylogenetic relatives of Centrohelida in the present study, since strong (and some are conflicting) gene-specific "signals" are predominant in the current multi-gene data. We concluded that larger scale multi-gene phylogenies are necessary to elucidate the origin and evolution of Centrohelida.

Molecular sampling of the taxonomic diversity of the living world is nowadays a task of paramount importance. Heliozoa represents one of the major eukaryotic taxa, which remain significantly underrepresented in molecular databases. The term Heliozoa was coined to embrace organisms with a rounded body and stiff pseudopodia. Despite evidences from ultrastructural studies, which conclusively show the polyphyly of selected heliozoan groups, contemporary morphological systems retain Heliozoa as a monophyletic taxon. From the perspective of reconstructing the true phylogeny of Eukaryota, molecular approaches to analyse relationships within this large protist group are evidently necessary. Phylogenetic analysis of our data shows that the four heliozoan taxa branch either independently or within different eukaryotic phyla. The actinophryids (Actinosphaerium, Actinophrys) appear as a lineage of stramenopiles, while the desmothoracids (Clathrulina, Hedriocystis) branch within “core Cercozoa”. The position of both groups is strongly supported in all analyses and is congruent with ultrastructure-based taxonomic revisions. The centrohelids (Chlamydaster, Heterophrys, Pterocystis, and Raphidiophrys) do not seem to be related to any particular eukaryotic phylum, in agreement with a recent molecular study. The taxopodid Sticholonche was found to branch between Polycystinea and Acantharea, two classes of radiolarians. Results obtained in this study suggest that the heliozoan body form cannot be used as a diagnostic argument to unite Heliozoa. Instead, we discriminate between the three heliomorphic taxa of independent origin, Actinophryida, Desmothoracida and Sticholonche, and propose the novel higher rank taxon Centrohelida. The term Heliozoa should thus be used solely in historical context.

Summary A revision of the genus Raphidiophrys ARCHER on the basis of the scale structure is performed. Two new genera are proposed. Parasphaerastrum gen. nov. includes a single species, P. marina (OSTENFELD) emend. MIKRJUKOV, surrounded by the envelope of rod-like tangential siliceous spicules. Polyplacocystis gen. nov. includes five species, two with reticulate [P. ambigua (PENARD) and P. symmetrica (PENARD)] and three with smooth [P. pallida (SCHULZE), P. marginata (SIEMENSMA) and P. pedunculata (MIKRJUKOV)] surface of scales. Six other EM-studied species [ R. capitata SIEMENSMA et ROIJACKERS, R. elegans HERTWIG et LESSER, R. intermedia PENARD, R. minuta NICHOLLS, R. Viridis ARCHER and R. ovalis (Du;RRSCHMIDT)], which have complex scales formed by two parallel plates and internal septae between them, are retained in the genus Raphidiophrys ARCHER. Only three species with plate-scales identical to those in Polyplacocystis ( R. lemani PENARD, R. tubifera PENARD, and R. glabra NICHOLLS et 0uRRSCHMIDT) stay in the genus Raphidocystis with the emended diagnosis given by NICHOLLS & DuRRSCHMIDT (1985). The new family Raphidiophryidae is proposed including the genera Parasphaerastrum , Polyplacocystis , Raphidiophrys and Raphidocystis . A key for the species of these genera is proposed.

In the present study, we isolated a species of heliozoans from a brackish pond in Shukkeien Garden, Naka-ku, Hiroshima City, Japan. Electron-microscopic observations showed that the axonemal microtubules in this heliozoan constituted a complex pattern of hexagons and triangles. By applying SDS-polyacrylamide gel electrophoresis and subsequent immunoblotting, molecular weights of α and β-tubulins were determined to be 48 and 45 kDa, respectively. X-ray microanalysis demonstrated that the numerous scales coating the cell body surface were silicic structures. Size and shape of the cell body and the scales were examined and compared with other known species of heliozoans, which led us to conclude that this is a new species belonging to the genus Raphidiophrys. This heliozoan was also found to carry out rapid axopodial contraction during food uptake at a velocity of about 1 mm/s. With reference to this characteristic contractile behavior, this new species was named Raphidiophrys contractilis.

Summary: A scanning electron microscopic observation on five species of Centroheliozoa which are common in Moscow marine aquarium systems is performed. Determination of their geographical origin is very difficult. Raphidiophrys marina , Acanthocystis wiasemskii and Choanocystis pelagica were never studied by electron microscope; the emended diagnoses for these species are suggested.

A total of 15 scale-bearing protista belonging to the genera Acanthocystis, Choanocystis, Cyathobodo, Lepidotrachelophyllum, Pterocystis, Polyplacocystis, Raineriophrys, Raphidiophrys, and Raphidocystis was observed from both lotic and lentic freshwater Nigerian sites. Three of the taxa have been previously reported from Nigeria, while 12 are new records including a description of a new species of Acanthocystis. Tropical Freshwater Biology Vol. 16 (1) 2007: pp. 1-15

A total of seven scaled protistans were observed from four freshwater sites in Nigeria. They include the holiozoan genera Acanthocystis, Polyplacocystis, Pterocystis , and Raphidiophrys . All are new records for Africa. KEY WORDS: Heliozoa, Protozoa, Acanthocystis, Polyplacocystis, Pterocystis, Raphidiophrys Tropical Freshwater Biology 11 (2002) 1-9

Summary Scales of 9 Raphidiophrys species were examined by light and electron microscopy.Most species have a doublelayered scale structure, with the upper and lower plates connected by internal radiating septa.Characteristics of shape and measurements of size and number of septa proved to be usable criteria for taxonomy within the genus.2 different colonial species, both within the original description of Raphidiophrys elegans, are recognised.A re-diagnosis of that species is given together with the description of a new colonial species, R.capitata.R.intermedia is delimited more clearly from R.elegans.Scales of R.viridis are clearly distinguished from those of R.pallida.

A simple method is described which enhances the contrast of conventionally sectioned and stained muscle tissue. After fixing and washing in phosphate buffer, dehydration of the tissue is initiated in 75% ethanol. Conventionally stained thin sections of muscle, dehydrated in this manner, are found to have approximately 35% more contrast than muscle dehydrated by the standard graded series of alcohol. There is no noticeable difference in the ultrastructural preservation of the tissue. When cacodylate buffer is substituted for phosphate in our dehydration scheme, no increase in contrast is detected. Besides lending greater contrast to object images, this method has also been used to demonstrate convincingly the presence of cross-bridges between axonemal components in, thick sections (0.5 μ m) of the centrohelidian Raphidiophrys studied in the high-voltage electron microscope. Such structures are, at best, poorly recognized in thick sections of conventionally dehydrated and stained Raphidiophrys . A rationale is proposed for the observed increase in contrast. It is based on the observation that phosphates rapidly precipitate in alcohol concentrations equal to or greater than 50%.

Summary Eleven species of the genera Acanthocystis, Raphidiophrys, Clathrulina and Pompholyxophrys are illustrated by electron microscopy from freshwater habitats in Australia.

A high-voltage electron microscope was used to study the structure of the centroplast and axonemes in the ccntrohclidian Heliozoan Raphidiophrys ambigua. In Raphidiophrys the microtubules (Mrs) of each axonemc are arranged so that they describe the apices of an array of hexagonally shaped prisms and all axonemes originate from a centroplast located near the centre of the cell. The centroplast was found to consist of 3 components: (1) a tripartite central disk, (2) a sphere of electron-opaque material, approximately 0·75μm in diameter, which immediately surrounds the central disk, and (3) a slightly less electron-opaque centroplast shell, approximately 2–3 μm in diameter, which consists primarily of hitherto undcscribed components associated with the proximal 1–2μm of each axonemal base. The new axonemal components described in this study include: (1) a 20-nm rod positioned in the centre of each hexagonal prism of Mts comprising the axonemal pattern, and (2) an electron-opaque linkage material which connects each Mt of the axonemal pattern to its 4 nearest neighbours. Both of these structures are associated with the base of each axoneme within the centroplast shell, both form crossbridges with axonemal Mts, and both terminate at the periphery of the centroplast shell once the complete axonemal Mt pattern is established. It is suggested that these structures are involved in forming and maintaining the Mt pattern seen in the axopodia of Raphidiophrys.

Microtubules are composed of 13 protofilaments which are aligned parallel to the long axis of the tubule, as can be seen in thin sections of material fixed in the presence of tannic acid. Evidence is presented based upon Markham rotations of the isolated flagellar axoneme that the bridges (spoke, nexin, dynein) must connect to certain specified protofilaments. By means of geometric considerations it is concluded that the bridges in a Centrohelida and Raphidiophrys also connect certain specified protofilaments. Two polymorphic forms of microtubules were also examined using tannic acid in the fixative, a 320 A tubule produced in Echinosphaerium by treatment with low temperature, and a vinblastine-induced microtubule crystal. In both forms, the arrangement of the subunits is not in the form of linear protofilaments. The vinblastine crystal in transverse section is made up of hexagonally packed tubules approximately 320 A in diameter. The wall of each tubule in turn is composed of two parallel protofilaments which spiral upwards, giving the appearance of a barber pole. The center-to-center separation of adjacent protofilaments is 180 A. We conclude that the tubulin heterodimer lies parallel to the axis of the protofilaments, exposing the colchicine binding site.

Sixteen taxa of the genera Raphidocystis, Raphidiophrys, and Pompholyxophrys from freshwater habitats in Canada, Chile, and New Zealand were studied by light and electron microscopy. Six taxa are described as new: Raphidocystis glabra, Raphidiophrys minuta, Raphidiophrys orbicularis ssp. orbicularis, R. orbicularis ssp. ovalis, Pompholyxophrys stellata, and P. ossea. New information on scale structure and arrangement based on scanning and transmission electron microscopy amplifies the taxonomic descriptions of Raphidiophrys ambigua, R. pallida, R. elegans, R. intermedia, R. marginata, R. symmetrica, Pompholyxophrys punicea, P. exigua, and P. ovuligera, which were previously imperfectly known by light microscopy only.

Comparative studies of axopodial microtubule pattern in 10 different centrohelidan Heliozoa belonging to the genera Acanthocystis, Raphidiophrys and Heterophrys suggest that 2 basic principles govern pattern formation in centrohelidan Heliozoa. While the larger "open" arrays with unspecified number of microtubules, e.g. in A. aculeata and R. ambigua, may result from self-linkage of additional microtubules around centroplast-nucleated "starter microtubules," the smaller "closed" arrays with specified microtubule number, e.g. in A. pectinata and H. marina, favor a template-driven linkage mechanism. The centroplast is a highly complex microtubule organizing center involved in the control of orientation, number, and diameter of the axonemes. Its shell may serve as a surface upon which the microtubule nucleating sites assemble, but how the precise positioning of these sites occurs is still open to debate. Some of the unsolved problems of microtubule pattern formation may be explained by the "linker nucleation hypothesis" which is an extension of the "gradion hypothesis" by Roth et al. It is shown how both the formation of closed arrays and the balanced lateral growth of open arrays may result from linker-induced microtubule nucleation.

The axonemes of Raphidiophrys converge near the center of the cell in an electron-opaque material, the centroplast. In order to establish whether this material acts not only to nucleate the microtubules which form the axonemes but also to give the axoneme its characteristic pattern, the microtubules were disassembled with low temperature and stages in their reformation were studied. It was shown that even though the microtubules appear to be nucleated from the centroplast, pattern formation first appeared at a distance from the centroplast. Thus, the axonemal pattern could not be attributed to any prepattern in the centroplast. Rather, the pattern appears to arise by specific interactions between tubules brought about by bridges. It was concluded that each tubule could bind to a maximum of four other tubules and that once one bridge attached to a tubule it specified the binding positions of the others, thus giving the characteristic axonemal pattern of Raphidiophrys.

The axopodial microtubule pattern of 9 centrohelidians belonging to the genera Acanthocystis, Raphidiophrys and Heterophrys, as well as the fine structure of their microtubule organizing centre, the ccntroplast, was studied to determine the rules which govern their patterns. Microtubules capable of binding a maximum of 4 linkers are arranged in regularly distorted hexagons and equilateral triangles. The number of microtubules present in each axoneme ranges from some 140 in Acanthocystis turfacea to as few as 6 in Heterophrys marina (Stock I). In the later species each axoneme contains a single hexagon of microtubules only. In other Heterophrys species, the central hexagon is surrounded by closely packed microtubules or by microtubules arranged in pentagons; only the central hexagon is anchored in the centroplast shell, whereas additional microtubules seem to originate from secondary nucleation sites somewhat distal to the centroplast. It is argued that the distortion of the basic unit hexagon (with alternate angles close to 134°and 106°) indicates that the microtubules are composed of 13 protofilaments. While in the larger Acanthocystis and Raphidiophrys species, the pattern may result from self-linkage, the arrays found in the Heterophrys species seem to favour a template-determined linkage. To explain the formation of the central hexagon in Heterophrys and balanced lateral growth in the larger microtubule arrays, a ‘linker-nucleation hypothesis’ is proposed. The assumption is made that graded conformational changes in the microtubule subunits not only specify the position where the next linker will bind, but that this linker, through linkage, becomes able to induce secondary microtubule nucleation, which will result in balanced lateral growth of the array. The application of this hypothesis to other microtubule systems, e.g. basal body formation, is discussed.