Raphidiophrys heterophryoidea sp. nov. (Centrohelida: Raphidiophryidae), the first heliozoan species with a combination of siliceous and organic skeletal elements

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.     

Three new freshwater species of centrohelid heliozoans: Acanthocystis crescenta sp. nov., A. kirilli sp. nov., and Choanocystis minima sp. nov.

Three new species of centrohelid heliozoans Acanthocystis crescenta, A. kirilli, and Choanocystis minima from two freshwater lakes of Valamo Island (North-Western Russia) were studied using light- and scanning electron microscopy. The main apomorphy of A. kirilli are slightly branched radial scales. Acanthocystis crescenta has characteristic radial scales with crescent-like structures on their tips. Cell diameter of these two species does not exceed 10 μm. Radial scales of C. minima have truncated tips and lack apical teeth; plate scales of this species are oviform, without lateral notches or central rib. The latter organism is the smallest heliozoan species known; the diameter of its body is about 3 μm. The possible domination of small heliozoans in the cryptic diversity of centrohelids is discussed.     

Siliceous scales in the centrohelid heliozoan Raphidocystis contractilis facilitate settlement to the substratum

The centrohelid heliozoan Raphidocystis contractilis has hundreds of small scales on the surface of the cell body. To understand the biological functions of the scales, comparative examinations were conducted between wild-type and scale-deficient strains that has naturally lost scales after long-term cultivation. The scale-deficient strain exhibited decreased adhesion to the substratum and had a lower sedimentation rate in water than the wild-type strain, suggesting that the scale may have the ability to attach quickly and strongly to the substratum. Percoll density gradient centrifugation showed that the scale-deficient strain had a lower density than that of the wild-type strain. In the wild-type strain, more scaled cells were observed in the higher specific gravity fractions. During the long-term culture of cells, only the cells suspended in the upper area of the flask were transferred to fresh medium. By repeating this procedure, we may have selected only cells that did not possess normal scales. In the natural environment, centrohelid heliozoans are easily flushed away if they cannot adhere strongly to the bottom. These results suggest that they use scales to ensure effective adhesion to the substratum.     

A New Species of Centrohelid Heliozoan Acanthocystis amura n. sp. Isolated From Two Remote Locations in Russia

A new species of centrohelid heliozoan Acanthocystis amura from the Amur River, the Far East of Russia was studied with light‐ and electron microscopy. Acanthocystis amura has simple oval plate scales (1.2–2.6 × 1.0–1.8 μm) with an axial thickening and a thin border as well as two types of spine scales. The spine scales of type 1 were 1.3–4.8 μm long and had four small hooks on their tips. The spine scales of type 2 were shorter, 1.0–3.6 μm long and had four teeth at the distal end. The morphology of A. amura resembles that of Acanthocystis quadrifurca. They both have similar spine scales but their plate scales are completely different. Acanthocystis valdiviense differs from the new species in absence of hook‐bearing scales. Molecular phylogeny based on 18S rDNA consistently placed A. amura into Acanthocystis nichollsi/Acanthocystis costata clade, but relationships between those three species were not resolved. Morphology of another centrohelid strain from another location (the South Urals) has been compared with that of the original strain and few minor differences in size characteristics of the scales have been revealed.     

Phylogenetic position and morphology of Raphidiophrys elongata sp. nov. (Haptista: Centroplasthelida) with notes on cyst wall structure and evolution

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.     

Discovery of a novel type of body scale in the marine dinoflagellate,Amphidinium cupulatisquama sp. nov. (Dinophyceae)

A new species of Amphidinium, A. cupulatisquama Tamura et Horiguchi, from sand samples from Ikei Island, Okinawa Prefecture in subtropical Japan, is described based on light, scanning and transmission electron microscopy and the partial sequencing of the large subunit rDNA gene. The species has a typical morphology for the genus, but is distinguished from previously described species by having a combination of the following characteristics: (i) a relatively large cell (over 30 µm in length); (ii) possessing an eyespot on the dorsal side of the cingulum; (iii) the longitudinal flagellum emerging from a point close to the cingulum; (iv) cell division taking place in the motile phase; and (v) possessing body scales. This is the third species of this genus to possess body scales. The body scales of A. cupulatisquama are uniform and cup‐shaped in side view and elliptical in face view. Their dimensions are 136.4 nm by 91.0 nm by 81.8 nm high. In side view, the scale is seen to have a thick lower half and a thin upper half. This scale type is very different from those of previously reported Amphidinium species (HG114 and HG115). The molecular tree indicated that A. cupulatisquama and the two other strains of body scale‐bearing Amphidinium are distantly related within the Amphidinium clade.     

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.     

Cellular and body scale morphology of Heterocapsa huensis sp. nov. (Peridiniales, Dinophyceae) found in Hue, Vietnam

Cellular and body scale structure of a new armored dinoflagellate Heterocapsa huensis , collected from Hue, Vietnam were investigated. Morphology of motile cell was observed by light, fluorescent and scanning electron microscopy, and body scale structure was examined by whole mounts of transmission electron microscopy. Cells of H. huensis were ellipsoid with a spherical nucleus located in the posterior and multiple pyrenoids located above the nucleus; this arrangement was similar to that of Heterocapsa pygmaea . Transmission electron microscopy revealed ultrastructure of the body scales consisted of a rounded triangular basal plate and three-dimensional ornaments. Structure of the basal plate resembles that of Heterocapsa illdefina ; however, the number of the peripheral spine is different from that of H. illdefina and this structure has never been reported from Heterocapsa species. A new Heterocapsa species, H. huensis Iwataki et Matsuoka sp. nov., is described based on positions of organelles and body scale ultrastructure.     

Further studies on Nephroselmis and its allies (Prasinophyceae). II. Mamiella gen. nov., Mamiellaceae fam. nov., Mamiellales ord. nov.

The marine prasinophycean flagellate presently known as Nephroselmis gilva has been examined, using both the type culture and material from temperate (Denmark, New Zealand) and tropical waters (Thailand). All cell surfaces are covered with unmineralized scales, two types on the body, two on the flagella including flagellar hairs. The detailed structure of the scales is described, using high power electron microscopy of detached positive–stained scales. Previously overlooked organelles within the cell include large numbers of extrusive bodies, a rare type of organelle in chlorophyll–a– and –b–containing organisms, and an eyespot. N. gilva differs profoundly from the type species of Nephroselmis , and is transferred to the new genus Mamiella. Mamiella gilva is closely related to Mantoniella squamata. Together with the genus Dolichomastix they form the new family, Mamiellaceae, a small group of marine flagellates of worldwide distribution.
The members of the new family probably represent the most primitive extant prasinophytes. When compared with other members of the class, its species stand out, particularly by the lack of small square or diamond–shaped scales on the flagella and cell body. It is suggested that the Mamiellaceae should be referred to a separate order, Mamiellales.     

THE ULTRASTRUCTURE OF PYRAMIMONAS PSEUDOPARKEAE SP. NOV. (PRASINOPHYCEAE) FROM SOUTH AFRICA1

The ultrastructure of Pyramimonas pseudoparkeae sp. nov., a member of the class Prasinophyceae occurring in tidal pools along the east, south and west coast of South Africa, is described. The cell surface is covered by three distinctive body scales whilst the flagellar surfaces possess four types of scales. The structure of these scales is described. P. pseudoparkeae resembles Pyramimonas parkeae Norris and Pearson but differs in the structure of the type 2 body scale. The symmetry and ultrastructure of the cell are described with special attention given to the flagellar apparatus. Preliminary information on the life cycle of this species is presented. This new species is compared with other closely related members of the genus Pyramimonas.     

Mallomonas wujekii (Synurophyceae), a new species from Florida, U.S.A.

A new species of Mallomonas, M. wujekii sp. nov., belonging to the Series Tonsuratae is described from a dilute, acidic locality in Florida, U.S.A. Bristle bearing domed scales are restricted to the anterior end, and scales are orientated with their longitudinal axes at a 60° to 90° angle with the longitudinal axis of the cell. Cells have three types of scales, domed anterior scales, domeless body scales and spined posterior scales. All scales possess papillae that are restricted to the shield and have one rib positioned on each end of the posterior flange. The new species is believed to be most closely related to Mallomonas tonsurata and M. galeiformis . The combination of characters of the bristles is unique among taxa of Mallomonas .     

Oxnerella micra sp. n. (Oxnerellidae fam. n.), a tiny naked centrohelid, and the diversity and evolution of heliozoa

We describe a new tiny naked centrohelid heliozoan, Oxnerella micra, and sequenced its 18S and 28S rRNA genes. Its extremely slender axopodia have prominent extrusomes and are normally stretched across the substratum like those of many tiny granofilosean Cercozoa. Phylogenetic analysis of 18S rDNA shows that Oxnerella does not branch within any of the six known centrohelid families but very deeply in the order Pterocystida, between Choanocystidae and Pterocystidae; therefore we place it in a new family, Oxnerellidae. Oxnerella arose from ancestors with siliceous scales by losing them; as independently did Heterophryidae and Marophryidae, which replaced them by organic spicules, and Chlamydaster that is not truly naked but retains a mucilage coat and nests extremely shallowly within Pterocystidae. 28S rDNA has a group I intron. Concatenated Bayesian 18S/28S rRNA phylogeny shows centrohelids weakly as sisters to the naked non-centrohelid heliozoan Microheliella maris (Microhelida: Heliozoa). The centrohelid Marophrys marina possesses an elongation factor α-like (EFL) protein related to that of Polyplacocystis; Microheliella also has EFL. We also analysed Hsp90 and 18S rDNA sequences from 'Pinaciophora sp.' ATCC50355; they must be from a centrohelid, probably misidentified as Pinaciophora, the rDNA sequence branching deeply within Pterocystida. We reclassify two Polyplacocystis, Luffisphaera, Phaeodaria and Rotosphaerida.     

Centrohelida is still searching for a phylogenetic home: analyses of seven Raphidiophrys contractilis genes

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.     

<Emphasis Type="Italic">Choanocystis antarctica</Emphasis> sp. n., a new heliozoan (Centrohelida) species from the littoral zone of King George Island,South Shetland Islands,Antarctica

The morphology of a new marine heliozoan from the sandy littoral of King George Island (the South Shetland Islands, Antarctica) is described. The species is characterized by bent or curved spicules with a spur at the shaft bend point. Similarities and differences between the new species and other representatives of the genus Choanocystis are considered.