A novel kleptoplastidic symbiosis revealed in the marine centrohelid Meringosphaera with evidence of genetic integration.

Plastid symbioses between heterotrophic hosts and algae are widespread and abundant in surface oceans. They are critically important both for extant ecological systems and for understanding the evolution of plastids. Kleptoplastidy, where the plastids of prey are temporarily retained and continuously re-acquired, provides opportunities to study the transitional states of plastid establishment. Here, we investigated the poorly studied marine centrohelid Meringosphaera and its previously unidentified symbionts using culture-independent methods from environmental samples. Investigations of the 18S rDNA from single-cell assembled genomes (SAGs) revealed uncharacterized genetic diversity within Meringosphaera that likely represents multiple species. We found that Meringosphaera harbors plastids of Dictyochophyceae origin (stramenopiles), for which we recovered six full plastid genomes and found evidence of two distinct subgroups that are congruent with host identity. Environmental monitoring by qPCR and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) revealed seasonal dynamics of both host and plastid. In particular, we did not detect the plastids for 6 months of the year, which, combined with the lack of plastids in some SAGs, suggests that the plastids are temporary and the relationship is kleptoplastidic. Importantly, we found evidence of genetic integration of the kleptoplasts as we identified host-encoded plastid-associated genes, with evolutionary origins likely from the plastid source as well as from other alga sources. This is only the second case where host-encoded kleptoplast-targeted genes have been predicted in an ancestrally plastid-lacking group. Our results provide evidence for gene transfers and protein re-targeting as relatively early events in the evolution of plastid symbioses.

Unexpected ubiquity of heart-shaped scale morphotype in Centroplasthelida (Haptista): Ancestral trait or multiple acquisitions?

Centrohelids (Haptista: Centroplasthelida) are axopodial protists with a remarkable diversity of external siliceous scale morphologies. It is believed that the last common ancestor of centrohelids had a double layer of siliceous scales composed of plate scales closer to a cell surface and spine scales radiating outwards. The characteristic morphotype of spine scales with a heart-shaped base was once believed to be a unique feature of the genus Choanocystis, as it was defined by Siemensma and Roijackers (1988). Further research revealed that this morphology is present in different and sometimes distantly related lineages: Ozanamiidae, Meringosphaeridae, and Marophryidae. Here, we report the fourth clade, Pterocystidae, which is also revealed to contain representatives having this phenotype. Cernunnos gen. nov. is erected here to place Cernunnos uralica sp. nov., Cernunnos arctica sp. nov., Cernunnos america sp. nov., and Cernunnos antarctica Tikhonenkov et Mylnikov, 2010, Gerasimova comb. nov. C. uralica was studied with scanning electron microscopy and SSU rDNA sequencing. Molecular phylogenetic analysis placed it into marine environmental clade P within Pterocystida. The ubiquity of spine scales with heart-shaped bases could be an example of parallel evolution, but taking into account the considerable similarity it is likely an ancestral trait, acquired from the last common ancestor of centrohelids.

A Genome of Temperate Enterococcus Bacteriophage Placed in a Space of Pooled Viral Dark Matter Sequences.

In the human gut, temperate bacteriophages interact with bacteria through predation and horizontal gene transfer. Relying on taxonomic data, metagenomic studies have associated shifts in phage abundance with a number of human diseases. The temperate bacteriophage VEsP-1 with siphovirus morphology was isolated from a sample of river water using Enterococcus faecalis as a host. Starting from the whole genome sequence of VEsP-1, we retrieved related phage genomes in blastp searches of the tail protein and large terminase sequences, and blastn searches of the whole genome sequences, with matches compiled from several different databases, and visualized a part of viral dark matter sequence space. The genome network and phylogenomic analyses resulted in the proposal of a novel genus "Vespunovirus", consisting of temperate, mainly metagenomic phages infecting Enterococcus spp.

Morphology and spicules elemental composition of Marophrys nikolaevi spec. nov. (Haptista: Centroplasthelida).

Marophrys is a genus of spicules-bearing centrohelids belonging to Heterophrys-like organisms (HLO's). Here Marophrys nikolaevi spec. nov. is described. Four strains were isolated from brackish waters (16-22 ppt) of the Tuzlukkol' River, the Tuzluchnoe Lake (South Urals, Russia) and the Black Sea. All the strains were characterised with light microscopy and electron microscopic study of the whole mount preparations. Molecular phylogenetic analysis has put SSU rDNA sequences, obtained for all strains, inside Marophryidae clade sister to M. marina. The organisms have a cell diameter of 4-11 µm and are surrounded with organic spicules of two types. Short (0.6-0.7 µm) and thin (0.01-0.02 µm) mostly tangentially oriented spicules form a lax sheath, surrounding the cell. Longer (3-6 µm) and thicker (0.04-0.05 µm) spicules are embedded in this sheath and are radially or obliquely oriented. Energy-dispersive X-ray analysis has shown that the spicules are purely organic. The taxonomy of marine Heterophrys-like organisms is discussed.

On the phylogenetic position of Raphidocystis pallida with some notes on its life cycle.

Raphidocystis pallida, a centrohelid heliozoan with unusually shaped tubular siliceous scales, was reisolated from Jamor river, Portugal, and studied with the use of light and electron microscopy. In enriched cultures, the cells were naked, devoid of siliceous external skeleton with the exception of several scales present in one cell. Instead, such cells were covered with a layer of rod-shaped bacteria. In clonal cultures, the cells gradually acquired siliceous coverings typical for this species and retained them in next generations. Phylogenetic position of R. pallida was clarified with SSU rDNA-based molecular phylogenetics, and its placement within the genus Raphidocystis despite unusual coverings structure was confirmed. The implications of phylogenetic placement of R. pallida and possible origins of the previously undescribed naked form were discussed.

Single cell genomics reveals plastid-lacking Picozoa are close relatives of red algae.

The endosymbiotic origin of plastids from cyanobacteria gave eukaryotes photosynthetic capabilities and launched the diversification of countless forms of algae. These primary plastids are found in members of the eukaryotic supergroup Archaeplastida. All known archaeplastids still retain some form of primary plastids, which are widely assumed to have a single origin. Here, we use single-cell genomics from natural samples combined with phylogenomics to infer the evolutionary origin of the phylum Picozoa, a globally distributed but seemingly rare group of marine microbial heterotrophic eukaryotes. Strikingly, the analysis of 43 single-cell genomes shows that Picozoa belong to Archaeplastida, specifically related to red algae and the phagotrophic rhodelphids. These picozoan genomes support the hypothesis that Picozoa lack a plastid, and further reveal no evidence of an early cryptic endosymbiosis with cyanobacteria. These findings change our understanding of plastid evolution as they either represent the first complete plastid loss in a free-living taxon, or indicate that red algae and rhodelphids obtained their plastids independently of other archaeplastids.

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.

Clypifer cribrifer gen. nov., sp. nov. (Clypiferidae fam. nov., Pterocystida, Centroplasthelida), with notes on evolution of centrohelid siliceous coverings.

A new family, genus and species of centrohelid heliozoans, Clypifer cribrifer gen. nov., sp. nov. (Clypiferidae fam. nov.), from the Gulf of Aqaba (Israel) was studied with light and electron microscopy and SSU rRNA gene sequencing. Clypifer cribrifer has only one type of scales, partially running up the sides of the axopodia. Plate scales [0.8-2.3 (av. 1.5)×0.6-1.8 (av. 1.2) µm] are flat, elliptical or circular, fenestrated with holes of irregular shape and have a marginal rim and a very short axial rib. The cell diameter is 3.9-9.6 (av. 6.0) µm. Molecular phylogenetic analysis robustly places C. cribrifer in the C4 clade for which the new family Clypiferidae is proposed here. This position is confirmed with the short sequences in the panacanthocystid increased regions. The morphology of the new genus has similarities to the genus Raphidocystis. The probability that another Clypifer species was described under a different name in the centrohelid literature is discussed. Clypiferidae represent the second lineage of Pterocystida, which are characterized by the presence of only tangentially oriented plate scales of one type. Possible ways of evolution of the centrohelid siliceous coverings are also discussed.

The long-time orphan protist Meringosphaera mediterranea Lohmann, 1902 [1903] is a centrohelid heliozoan.

Meringosphaera is an enigmatic marine protist without clear phylogenetic affiliation, but it has long been suggested to be a chrysophyte-related autotroph. Microscopy-based reports indicate that it has a worldwide distribution, but no sequence data exist so far. We obtained the first 18S rDNA sequence for M. mediterranea (identified using light and electron microscopy) from the west coast of Sweden. Observations of living cells revealed granulated axopodia and up to 6 globular photosynthesizing bodies about 2 µm in diameter, the nature of which requires further investigation. The ultrastructure of barbed undulating spine scales and patternless plate scales with a central thickening is in agreement with previous reports. Molecular phylogenetic analysis placed M. mediterranea inside the NC5 environmental clade of Centroplasthelida (Haptista) along with additional environmental sequences, together closely related to Choanocystidae. This placement is supported by similar scales in Meringosphaera and Choanocystidae. We searched the Tara Oceans 18S V9 metabarcoding dataset, which revealed four OTUs with 94.8%-98.2% similarity, with oceanic distribution similar to that based on morphological observations. The current taxonomic position and species composition of the genus are discussed. The planktonic lifestyle of M. mediterranea contradicts the view of some authors that centrohelids enter the plankton only temporarily.

The smallest known heliozoans are the Erebor lineage (nom. clad. n.) inside Microheliella maris (Eukaryota, Diaphoretickes), with the amendation of M. maris diagnosis and description of Berkeleyaesol magnus gen. nov., comb. nov. (Eukaryota, incertae sedis).

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.

Genotyping of Russian isolates of fungal pathogen Trichophyton rubrum, based on simple sequence repeat and single nucleotide polymorphism.

BACKGROUND: The Trichophyton rubrum species group consists of prevalent causative agents of human skin, nail and hair infections, including T rubrum sensu stricto and T violaceum, as well as other less well-established or debatable taxa like T soudanense, T kuryangei and T megninii. Our previous study provided limited evidence in favour of the existence of two genetic lineages in the Russian T rubrum sensu stricto population. OBJECTIVES: We aimed to study the genetic structure of the Russian population of T rubrum and to identify factors shaping this structure. METHODS: We analysed the polymorphism of 12 simple sequence repeat (SSR or microsatellite) markers and single nucleotide polymorphism in the TERG_02941 protein-coding gene in 70 T rubrum isolates and performed a phylogenomic reconstruction. RESULTS: All three types of data provided conclusive evidence that the population consists of two genetic lineages. Clustering, performed by means of microsatellite length polymorphism analysis, was strongly dependent on the number of nucleotide repeats in the 5'-area of the fructose-1,6-bisphosphate aldolase gene. Analysis of molecular variance (AMOVA) on the basis of SSR typing data indicated that 22%-48% of the variability was among groups within T rubrum. There was no clear connection of population structure with types of infection, places of geographic origin, aldolase gene expression or urease activity. CONCLUSION: Our results suggest that the Russian population of T rubrum consists of two cosmopolitan genetic lineages.

Phenotypic masquerade: Polymorphism in the life cycle of the centrohelid heliozoan Raphidiophrys heterophryoidea (Haptista: Centroplasthelida).

The life cycle of the centrohelid heliozoan Raphidiophrys heterophryoidea Zlatogursky, 2012 was studied with light and electron microscopy in clonal cultures from the type locality. The alternation of two types of trophozoites, having contrastingly different morphology, was observed. Type 1 trophozoites morphology matched the original description. Type 2 trophozoites tended to form colonies usually of 6-8 individuals, connected with cytoplasmic bridges and their cell size was noticeably bigger, namely 43-45 µm compared to 24.5 µm on average in type 1 trophozoites. Some colonies were forming stalks composed of three or four axopodia covered with scales. Spicules were lacking completely, while plate-scales differed from those of type 1 trophozoites: they had oblong-elliptical shape, larger (5.9-14.1 × 2.4-5.8 µm) size, non-branching septa always reaching scale centre, solid upper plate. The conspecificity of the two trophozoite types was confirmed with the comparison of SSU rDNA gene sequence data. Both types of trophozoites were capable of encystment and excysted individuals always were type 1 trophozoites. A new type of cyst-scales (cup-scales) was described. Transitions between cysts and the two trophozoites types were documented. The diagnosis of R. heterophryoidea was improved accordingly. The possible functions, driving forces, and taxonomic consequences of the polymorphism were discussed.

Multiple Euryhaline Lineages of Centrohelids (Haptista: Centroplasthelida) in Inland Saline Waters Revealed with Metabarcoding.

The diversity of centrohelids in inland saline waters was studied with metabarcoding for the first time. The fragment of V6-V7 regions of 18S rDNA was sequenced with newly designed primers. Obtained OTUs were identified with molecular phylogenetic analysis and comparison of the signatures in 39es9 hairpin of V7. The obtained data included some OTUs, which could be attributed to four described species, but the majority belonged to previously established or novel environmental clades. Along with some presumably marine/brackish clades and freshwater/low salinity (0-2 ppt) clades, seven presumable species demonstrating broad (from 1-2 up to 78 ppt) salinity tolerance were detected. A number of OTUs belonged to Raphidocystis contractilis, which is known from three independent findings in brackish habitats only. Thus, it was assumed that this species is stenohaline and specifically adapted to salinity 5-15 ppt. The high level of salinity tolerance was suggested for centrohelids before based on morphology, which was used to justify their cosmopolitan distribution. Later these views were criticized based on environmental sequencing, but the results of the current survey indicate, that at least some species are present at salinities from almost freshwater (1-2 ppt) to twice oceanic (78 ppt) and are presumably capable of overcoming oceanic salinity barriers for their distribution.

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.

Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes.

This revision of the classification of eukaryotes follows that of Adl et al., 2012 [J. Euk. Microbiol. 59(5)] and retains an emphasis on protists. Changes since have improved the resolution of many nodes in phylogenetic analyses. For some clades even families are being clearly resolved. As we had predicted, environmental sampling in the intervening years has massively increased the genetic information at hand. Consequently, we have discovered novel clades, exciting new genera and uncovered a massive species level diversity beyond the morphological species descriptions. Several clades known from environmental samples only have now found their home. Sampling soils, deeper marine waters and the deep sea will continue to fill us with surprises. The main changes in this revision are the confirmation that eukaryotes form at least two domains, the loss of monophyly in the Excavata, robust support for the Haptista and Cryptista. We provide suggested primer sets for DNA sequences from environmental samples that are effective for each clade. We have provided a guide to trophic functional guilds in an appendix, to facilitate the interpretation of environmental samples, and a standardized taxonomic guide for East Asian users.

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.

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.

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.