Mikrocytos mytilicoli n.sp. (Cercozoa, Mikrocytida, Mikrocytiidae) infecting the copepod Mytilicola intestinalis (Arthropoda, Cyclopoida, Mytilicolidae), a symbiont of Mytilus galloprovincialis in Galicia (NW Spain).

During a histopathological survey of Mytilus galloprovincialis in Galicia (NW Spain), microcells were observed infecting several organs of the symbiont copepod Mytilicola intestinalis. Positive results of PCR assay with specific primers for genus Mikrocytos and a clear signal of in situ hybridization with MACKINI-1 digoxigenin- labelled DNA probe (DIG-ISH) indicated a protozoan parasite of Mikrocytos genus. The ultrastructural study revealed intra and extracellular locations, polymorphic nuclei, intracellular round vesicles in the cytoplasm and absence of mitochondria. The present paper reports the characterization of the Mikrocytos sp. infecting M. intestinalis and proposes a novel species in the genus: Mikrocytos mytilicoli n. sp. A sequence of 18S-28S rDNA was obtained with 95.6% maximum identity (query cover 100%) with Mikrocytos mackini. Phylogenetic analysis showed that M. mytilicoli n. sp. and M. mackini share a common ancestor. However, comparison of the ITS1 rDNA region showed low similarity (75.8%) with M. mackini, which, combined with differences in ultrastructural details, host and geographic location, support the designation of a new species. This is the first description of a microcytid parasite of the genus Mikrocytos from a non-bivalve host.

Morphology, Ultrastructure, and Phylogeny of Two Novel Species of Ventrifissura (V. oblonga n. sp. and V. velata n. sp., Thecofilosea, Cercozoa).

Ventrifissura is a group of poorly studied heterotrophic biflagellates in the phylum Cercozoa. Despite a phylogenetic placement with only weak support and a lack of ultrastructural data, Ventrifissura was assigned to Thecofilosea. In the presented study, we established cultures of two novel species of Ventrifissura (V. oblonga n. sp. and V. velata n. sp.) isolated from coastal marine environments in Japan, and performed light and electron microscopy observations and molecular phylogenetic analysis. Transmission electron microscopy revealed that V. oblonga shares several ultrastructural characteristics with thecofilosean flagellates, including permanently condensed chromosomes, a extracellular theca, and slender extrusomes. Molecular phylogenetic analysis could not resolve the phylogenetic position, but the possibility that Ventrifissura clusters into Ventrifilosa was supported by approximately unbiased tests. Based on both morphological and phylogenetic findings, we concluded that Ventrifissura is a basal lineage of Thecofilosea.

Dinoflagellates with relic endosymbiont nuclei as models for elucidating organellogenesis.

Nucleomorphs are relic endosymbiont nuclei so far found only in two algal groups, cryptophytes and chlorarachniophytes, which have been studied to model the evolutionary process of integrating an endosymbiont alga into a host-governed plastid (organellogenesis). However, past studies suggest that DNA transfer from the endosymbiont to host nuclei had already ceased in both cryptophytes and chlorarachniophytes, implying that the organellogenesis at the genetic level has been completed in the two systems. Moreover, we have yet to pinpoint the closest free-living relative of the endosymbiotic alga engulfed by the ancestral chlorarachniophyte or cryptophyte, making it difficult to infer how organellogenesis altered the endosymbiont genome. To counter the above issues, we need novel nucleomorph-bearing algae, in which endosymbiont-to-host DNA transfer is on-going and for which endosymbiont/plastid origins can be inferred at a fine taxonomic scale. Here, we report two previously undescribed dinoflagellates, strains MGD and TGD, with green algal endosymbionts enclosing plastids as well as relic nuclei (nucleomorphs). We provide evidence for the presence of DNA in the two nucleomorphs and the transfer of endosymbiont genes to the host (dinoflagellate) genomes. Furthermore, DNA transfer between the host and endosymbiont nuclei was found to be in progress in both the MGD and TGD systems. Phylogenetic analyses successfully resolved the origins of the endosymbionts at the genus level. With the combined evidence, we conclude that the host-endosymbiont integration in MGD/TGD is less advanced than that in cryptophytes/chrorarachniophytes, and propose the two dinoflagellates as models for elucidating organellogenesis.

A novel paramyxean parasite, Marteilia tapetis sp. nov. (Cercozoa) infecting the digestive gland of Manila clam Ruditapes philippinarum from the southeast coast of Korea.

Paramyxean parasites in the genus Marteilia deteriorate digestive tissues of the host organisms, resulting in mortality of oysters, cockles, and mussels. Most reports of infection by Marteilia spp. are from Europe, while a new species of Marteilia was identified recently in Japan. Here, we report a previously unidentified species in the genus Marteilia from digestive diverticula of Manila clam Ruditapes philippinarum from the south coast of Korea. Prevalence of the parasite was low, 0.5-3.3% in the study sites. We characterized this species using light and transmission electron microscopy (TEM), and analyzed the 18S rDNA sequence. Light microscopy revealed the sporulation process from uninucleated stage to spore in the epithelial tissues of the digestive gland. TEM revealed that the parasites produced four secondary cells containing four tri-cellular spores. An electron-dense haplosporosome-like structure and striated inclusions were evident in the spore and the primary cells, respectively, while refringent granules were rarely observed in the secondary cells. Phylogenetic analyses of the 18S rDNA sequence placed this isolate in the genus Marteilia, although it is not identical to other known species in the genus. Based on morphological and molecular characters, we describe this species as Marteilia tapetis sp. nov., the second Marteilia species reported parasitizing Manila clams in Asian waters.

Two New Marine Species of Placopus (Vampyrellida, Rhizaria) That Perforate the Theca of Tetraselmis (Chlorodendrales, Viridiplantae).

Vampyrellids (Vampyrellida, Rhizaria) are a major group of predatory amoebae known primarily from freshwater and soil. Environmental sequence data indicate that there is also a considerable diversity of vampyrellids inhabiting marine ecosystems, but their phenotypic traits and ecology remain largely unexplored. We discovered algivorous vampyrellids of the filoflabellate morphotype in coastal habitats in Atlantic Canada, established cultures by single-cell isolation, and characterised three strains using light microscopy, SSU rRNA gene sequencing, feeding experiments and growth experiments at various salinities. These strains exhibit orange, discoid trophozoites with ventral filopodia, moving granules ("membranosomes"), and rolling locomotion, similar to freshwater species previously assigned to Hyalodiscus Hertwig & Lesser, but here moved to Placopus Schulze (due to homonymy with Hyalodiscus Ehrenberg). SSU rRNA gene phylogenies place our strains in two distinct positions within "lineage B3" (here referred to as Placopodidae). Based on these morphological, habitat and molecular data, we describe two new species, Placopus melkoniani sp. nov. and Placopus pusillus sp. nov., both of which feed on chlorophyte flagellates (Tetraselmis, Pyramimonas) and the cryptophyte Chroomonas. They perforate the theca of Tetraselmis to extract the protoplast, and thereby represent the first vampyrellids known to degrade the biochemically exotic cell wall of the Chlorodendrales (Chlorophyta, Viridiplantae).

Elemental composition and ultrafine structure of the skeleton in shell-bearing protists-A case study of phaeodarians and radiolarians.

Cross-sections were prepared by ultramicrotome (UM) and focused ion beam (FIB) system in order to examine the skeletal structure of ecologically and geologically important shell-bearing protists: phaeodarians and radiolarians. The elemental composition of the skeleton was clarified by the energy dispersive X-ray spectroscopy, suggesting that the skeletons of both groups are mainly made of amorphous silica (SiO2·nH2O) with other minor elements (Na, Mg, Al, Cl, K, Ca and Fe) and that these two groups have similar elemental composition, compared with other siliceous organisms (diatoms and sponges). However, the structural difference among the two groups was confirmed: phaeodarian skeletons are porous, unlike radiolarians with solid skeletons. It was also revealed that the phaeodarian skeleton contains concentric layered structure with spaces, presumably related to the ontogenetic skeleton formation. The distinction in the skeletal ultrafine structure (porous/solid and non-dense/dense) would reflect the ecological difference among the two groups and could be an effective criterion to determine whether microfossils belong to Radiolaria or Phaeodaria. The UM and FIB combined method presented in this study could be a useful approach to examine the chemical and structural characteristics of unculturable and/or rare microorganisms.

Sappinia sp. (Amoebozoa: Thecamoebida) and Rosculus sp. (SAR: Cercozoa) Isolated From King Penguin Guano Collected in the Subantarctic (South Georgia, Salisbury Plain) and their Coexistence in Culture.

Two amoeboid organisms of the genera Sappinia Dangeard, 1896 and Rosculus Hawes, 1963 were identified in a sample containing king penguin guano. This sample, collected in the Subantarctic, enlarges the list of fecal habitats known for the presence of coprophilic amoebae. The two organisms were co-isolated and subcultured for over 6 mo, with continuous efforts being invested to separate each one from the mixed culture. In the mixed culture, Rosculus cells were fast growing, tolerated changes in culturing conditions, formed cysts, and evidently were attracted by Sappinia trophozoites. The separation of the Rosculus strain was accomplished, whereas the Sappinia strain remained intermixed with inseparable Rosculus cells. Sappinia cell populations were sensitive to changes in culturing conditions; they improved with reduction of Rosculus cells in the mixed culture. Thick-walled cysts, reportedly formed by Sappinia species, were not seen. The ultrastructure of both organisms was congruent with the currently accepted generic characteristics; however, some details were remarkable at the species level. Combined with the results of phylogenetic analyses, our findings indicate that the ultrastructure of the glycocalyx and the presence/absence of the Golgi apparatus in differential diagnoses of Sappinia species require a critical re-evaluation.

Taxonomic Implications of Morphological Complexity Within the Testate Amoeba Genus Corythion from the Antarctic Peninsula.

Precise and sufficiently detailed morphological taxonomy is vital in biology, for example in the accurate interpretation of ecological and palaeoecological datasets, especially in polar regions, where biodiversity is poor. Testate amoebae on the Antarctic Peninsula (AP) are well-documented and variations in their population size have recently been interpreted as a proxy for microbial productivity changes in response to recent regional climate change. AP testate amoeba assemblages are dominated by a small number of globally ubiquitous taxa. We examine morphological variation in Corythion spp. across the AP, finding clear evidence supporting the presence of two morphospecies. Corythion constricta (Certes 1889) was identified on the AP for the first time and has potentially been previously misidentified. Furthermore, a southerly trend of decreasing average test size in Corythion dubium (Taránek 1881) along the AP suggests adaptive polymorphism, although the precise drivers of this remain unclear, with analysis hindered by limited environmental data. Further work into morphological variation in Corythion is needed elsewhere, alongside molecular analyses, to evaluate the potential for (pseudo)cryptic diversity within the genus. We advocate a parsimonious taxonomical approach that recognises genetic diversity but also examines and develops accurate morphological divisions and descriptions suitable for light microscopy-based ecological and palaeoecological studies.

Viridiuvalis adhaerens gen. et sp. nov., a novel colony-forming chlorarachniophyte.

A new chlorarachniophyte, Viridiuvalis adhaerens gen. et sp. nov. was isolated from the mucus on a coral reef from Zanpa Beach, Okinawa, Japan. The main vegetative stage of V. adhaerens consisted of unicellular coccoid cells with cell walls, although sarcinoid colonies and uniflagellate zoospores were also observed. V. adhaerens had chloroplasts with nucleomorphs and pyrenoids that were completely embedded in the chloroplast. A deep plate-like invagination of the periplastidal compartment (PPC) almost partitioned the pyrenoid and chloroplast components, which were surrounded by two membranes. The nucleomorph was positioned in the base of the invagination of the PPC. Molecular phylogenetic analyses using rRNA genes showed that V. adhaerens branched as a sister lineage of the Amorphochlora clade. The sarcinoid colony, pyrenoid embedded in the chloroplast, and nucleomorph located at the base of the deep invagination of the PPC have not been reported in other chlorarachniophytes. Based on these morphological and ultrastructural characteristics and the results of the molecular phylogenetic analyses, we propose V. adhaerens as a new genus and species of chlorarachniophyte.

Evolutionary Relationship of the Scale-Bearing Kraken (incertae sedis, Monadofilosa, Cercozoa, Rhizaria): Combining Ultrastructure Data and a Two-Gene Phylogeny.

The genus Kraken represents a distinct lineage of filose amoebae within the Cercozoa. Currently a single species, Kraken carinae, has been described. SSU rDNA phylogeny showed an affiliation to the Cercomonadida, branching with weak support at its base, close to Paracercomonas, Metabolomonas, and Brevimastigomonas. Light microscopical analyses showed several unique features of the genus Kraken, but ultrastructure data were lacking. In this study, K. carinae has been studied by electron microscopy, these data conjoined with a two-gene phylogeny were used to give more insight into the evolutionary relationship of the genus Kraken within Cercozoa. The data confirmed the absence of flagella, but also showed novel characteristics, such as the presence of extrusomes, osmiophilic bodies, and mitochondria with flat cristae. Surprising was the presence of single-tier scales which are carried by cell outgrowths, much of what is expected of the last common ancestor of the class Imbricatea. The phylogenetic analyses however confirmed previous results, indicating Kraken as a sister group to Paracercomonas in Sarcomonadea with an increased but still low support of 0.98 PP/63 BP. Based on the unique features of Kraken we establish the Krakenidae fam. nov. that we, due to contradictory results in morphology and phylogeny, assign incertae sedis, Monadofilosa.

Diversity of the Photosynthetic Paulinella Species, with the Description of Paulinella micropora sp. nov. and the Chromatophore Genome Sequence for strain KR01.

The thecate filose amoeba Paulinella chromatophora is a good model organism for understanding plastid organellogenesis because its chromatophore was newly derived from an alpha-cyanobacterium. Paulinella chromatophora was the only known photosynthetic Paulinella species until recent studies that suggested a species level of diversity. Here, we described a new photosynthetic species P. micropora sp. nov. based on morphological and molecular evidence from a newly established strain KR01. The chromatophore genome of P. micropora KR01 was fully determined; the genome was 976,991bp in length, the GC content was 39.9%, and 908 genes were annotated. A pairwise comparison of chromatophore genome sequences between strains KR01 and FK01, representing two different natural populations of P. micropora, showed a 99.85% similarity. Differences between the two strains included single nucleotide polymorphisms (SNPs) in CDSs, which resulted in 357 synonymous and 280 nonsynonymous changes, along with 245 SNPs in non-coding regions. Indels (37) and microinversions (14) were also detected. Species diversity for photosynthetic Paulinella was surveyed using samples collected from around the world. We compared our new species to two photosynthetic species, P. chromatophora and P. longichromatophora. Phylogenetic analyses using four gene markers revealed three distinct lineages of photosynthetic Paulinella species including P. micropora sp. nov.

Description of Hyalodiscus flabellus sp. nov. (Vampyrellida, Rhizaria) and Identification of its Bacterial Endosymbiont, "Candidatus Megaira polyxenophila" (Rickettsiales, Alphaproteobacteria).

The genus Hyalodiscus Hertwig and Lesser, 1874 comprises naked freshwater amoebae with a unique set of characters, namely a vibrant orange-red colour, a discoid or fan-shaped morphology, and a characteristic rolling locomotion. Some species feed on the chloroplasts of green algae and were regarded as relatives of Vampyrella Cienkowski, 1865. However, because of striking morphological differences and the lack of molecular data, the exact relationship of Hyalodiscus to vampyrellids is still obscure. Here, I describe Hyalodiscus flabellus sp. nov., a bright orange, fan-shaped amoeba feeding on Oedogonium (Chlorophyceae), which likely is a close relative of the type species H. rubicundus Hertwig and Lesser, 1874. Sequence comparisons of the SSU rRNA gene revealed that H. flabellus belongs to a deep-branching, so far uncharacterised lineage of the order Vampyrellida (Rhizaria), here defined as family Hyalodiscidae POCHE, 1913. Based on these results, the systematic position of the genus Hyalodiscus could be finally clarified, accompanied by the revision of relevant diagnoses and a taxonomic summary. Furthermore, the work reports on endosymbiotic bacteria inhabiting the cytoplasm of H. flabellus, which were identified as "Candidatus Megaira polyxenophila" (Rickettsiales, Alphaproteobacteria) using the full cycle rRNA approach with newly designed FISH probes for this widespread endosymbiotic bacterium.

Hyperspora aquatica n.gn., n.sp. (Microsporidia), hyperparasitic in Marteilia cochillia (Paramyxida), is closely related to crustacean-infecting microspordian taxa.

The Paramyxida, closely related to haplosporidians, paradinids, and mikrocytids, is an obscure order of parasitic protists within the class Ascetosporea. All characterized ascetosporeans are parasites of invertebrate hosts, including molluscs, crustaceans and polychaetes. Representatives of the genus Marteilia are the best studied paramyxids, largely due to their impact on cultured oyster stocks, and their listing in international legislative frameworks. Although several examples of microsporidian hyperparasitism of paramyxids have been reported, phylogenetic data for these taxa are lacking. Recently, a microsporidian parasite was described infecting the paramyxid Marteilia cochillia, a serious pathogen of European cockles. In the current study, we investigated the phylogeny of the microsporidian hyperparasite infecting M. cochillia in cockles and, a further hyperparasite, Unikaryon legeri infecting the digenean Meiogymnophallus minutus, also in cockles. We show that rather than representing basally branching taxa in the increasingly replete Cryptomycota/Rozellomycota outgroup (containing taxa such as Mitosporidium and Paramicrosoridium), these hyperparasites instead group with other known microsporidian parasites infecting aquatic crustaceans. In doing so, we erect a new genus and species (Hyperspora aquatica n. gn., n.sp.) to contain the hyperparasite of M. cochillia and clarify the phylogenetic position of U. legeri. We propose that in both cases, hyperparasitism may provide a strategy for the vectoring of microsporidians between hosts of different trophic status (e.g. molluscs to crustaceans) within aquatic systems. In particular, we propose that the paramyxid hyperparasite H. aquatica may eventually be detected as a parasite of marine crustaceans. The potential route of transmission of the microsporidian between the paramyxid (in its host cockle) to crustaceans, and, the 'hitch-hiking' strategy employed by H. aquatica is discussed.

Placement of the unclassified Cyranomonas australis Lee 2002 within a novel clade of Cercozoa.

Two heterotrophic flagellate strains were isolated from marine sediment samples off eastern Canada and Korea. These new isolates are indistinguishable by light microscopy from the unclassified protist Cyranomonas australis. The organisms are ovoid-shaped cells, 3.5-6µm long, laterally compressed, and somewhat flexible. They have two unequal flagella, about 1.1-2.5 times body length. Typically, the cells show a gliding motility and do not exhibit any amoeboid form or pseudopodia. 18S rDNA phylogenies clearly indicate that the isolates can be assigned to the taxon Filosa, within Cercozoa. The isolates are closest to an environmental sequence (CYSGM-16; 99% identity). Cyranomonas, CYSGM-16, and uncultured eukaryote RM1-SGM46 form a clade with strong statistical supports, here called novel clade CU (Cyranomonas plus Uncultured eukaryotes). This clade may be sister to the order Marimonadida. The novel clade CU and the Marimonadida have been detected only in marine habitats. Our findings suggest that C. australis may not belong to any previously described family within Filosa and Cercozoa.

Local and intercontinental comparisons of test morphology in the little-known testate amoeba Cyphoderia laevis Penard.

The morphology of tests ("shells") representing populations of the small testate amoeba Cyphoderia laevisPenard, 1902 (Cercozoa, Euglyphida) from Canada and Hungary was quantified. Despite often considerable variation the cross-sectional shape of tests, multivariate statistical analysis confirmed the general uniformity of test morphology within populations and between populations at both local (regional, inter- and intra-watershed) scales and over inter-continental scales (Canada and Hungary). Corrections are suggested to recently published misidentifications of C. laevis as Corythionella georgiana Nicholls. The differences between these two taxa are subtle, but significant, and are emphasized here to help preclude similar errors in the future. Schaudinnula nanaBadewitz, 2003 is shown to be a superfluous synonym for Cyphoderia laevis. Cyphoderia truncataSchulze, 1875 has some similarities to C. laevis, but its status remains unresolved. Some preliminary reservations are expressed about the classification of Cyphoderia laevis within the genus Cyphoderia owing mainly to its Corythionella-like scale structure and other characteristics of its test that appear to be outside the "usual" range found in other Cyphoderia species.

A new phylogeny and environmental DNA insight into paramyxids: an increasingly important but enigmatic clade of protistan parasites of marine invertebrates.

Paramyxida is an order of rhizarian protists that parasitise marine molluscs, annelids and crustaceans. They include notifiable pathogens (Marteilia spp.) of bivalves and other taxa of economic significance for shellfish production. The diversity of paramyxids is poorly known, particularly outside of commercially important hosts, and their phylogenetic position is unclear due to their extremely divergent 18S rDNA sequences. However, novel paramyxean lineages are increasingly being detected in a wide range of invertebrate hosts, and interest in the group is growing, marked by the first 'Paramyxean Working Group' Meeting held in Spain in February 2015. We review the diversity, host affiliations, and geographical ranges of all known paramyxids, present a comprehensive phylogeny of the order and clarify its taxonomy. Our phylogenetic analyses confirm the separate status of four genera: Paramarteilia, Marteilioides, Paramyxa and Marteilia. Further, as including M. granula in Marteilia would make the genus paraphyletic we suggest transferring this species to a new genus, Eomarteilia. We present sequence data for Paramyxa nephtys comb. n., a parasite of polychaete worms, providing morphological data for a clade of otherwise environmental sequences, sister to Marteilioides. Light and electron microscopy analyses show strong similarities with both Paramyxa and Paramyxoides, and we further discuss the validity of those two genera. We provide histological and electron microscopic data for Paramarteilia orchestiae, the type species of that genus originally described from the amphipod Orchestia; in situ hybridisation shows that Paramarteilia also infects crab species. We present, to our knowledge, the first known results of a paramyxid-specific environmental DNA survey of environmental (filtered water, sediment, etc.) and organismally-derived samples, revealing new lineages and showing that paramyxids are associated with a wider range of hosts and habitat types than previously known. On the basis of our new phylogeny we propose phylogenetic hypotheses for evolution of lifecycle and infectivity traits observed in different paramyxid genera.

Fine-structural Observations on Siliceous Scale Production and Shell Assembly in the Testate Amoeba Paulinella chromatophora.

The fine structure of shell formation was observed in P. chromatophora. Scales were formed one by one in silica deposition vesicles (SDVs) that were supported by an array of microtubules, which are probably involved in determining the shape and size of scales. The timing of silicic acid transport into an SDV was shown to be at an early stage of scale production because silicon was detected within SDVs containing immature scales. During the shell construction process, vesicles containing two types of dense materials were observed. One type of vesicle contains lower-density material and is located at the front edge of the branched, thick pseudopodium, extending from the maternal shell to the newly formed shell. The other type of vesicle, which contains higher-density material, was also observed in the thick pseudopodium. It appears that microtubules are involved in the shell construction process.

Massisteria marina has a sister: Massisteria voersi sp. nov., a rare species isolated from coastal waters of the Baltic Sea.

For many years, the genus Massisteria (Cercozoa, Leucodictyida) comprised only one species, M. marina. This small species has a biphasic life cycle and feeds through filose, radiating pseudopodia. It has a distinct swimming form and is regularly detected in association with detritus aggregates. However, environmental sequences closely related to this species indicate a larger species richness than hitherto described for the genus Massisteria. Here we provide the first report of Massisteria voersi sp. nov., investigated with microscopic and molecular methods. Several strains of this new species were isolated from brackish water at a Baltic Sea coastal monitoring station. Their characteristics are typical of the genus. Massisteria voersi differs from M. marina by smaller cell size (2.3-3 µm vs. 2.5-9 µm) and absent fused motile cells. Additionally, in contrast to M. marina, the new species lacks a paranuclear body and its kinetosomes are arranged in parallel. Both species are quite distantly related regarding their 18S rRNA gene sequences. The sparse availability of environmental sequences closely related to M. voersi as well as our preliminary results from fluorescence in situ hybridization studies suggest that this new species is a representative of low-abundance populations comprising the so-called "rare biosphere."

Vernalophrys algivore gen. nov., sp. nov. (Rhizaria: Cercozoa: Vampyrellida), a New Algal Predator Isolated from Outdoor Mass Culture of Scenedesmus dimorphus.

Microbial contamination is the main cause of loss of biomass yield in microalgal cultures, especially under outdoor environmental conditions. Little is known about the identities of microbial contaminants in outdoor mass algal cultures. In this study, a new genus and species of vampyrellid amoeba, Vernalophrys algivore, is described from cultures of Scenedesmus dimorphus in open raceway ponds and outdoor flat-panel photobioreactors. This vampyrellid amoeba was a significant grazer of Scenedesmus and was frequently associated with a very rapid decline in algal numbers. We report on the morphology, subcellular structure, feeding behavior, molecular phylogeny, and life cycle. The new amoeba resembles Leptophrys in the shape of trophozoites and pseudopodia and in the mechanism of feeding (mainly by engulfment). It possesses two distinctive regions in helix E10_1 (nucleotides 117 to 119, CAA) and E23_1 (nucleotides 522 and 523, AG) of the 18S rRNA gene. It did not form a monophyletic group with Leptophrys in molecular phylogenetic trees. We establish a new genus, Vernalophrys, with the type species Vernalophrys algivore. The occurrence, impact of the amoeba on mass culture of S. dimorphus, and means to reduce vampyrellid amoeba contamination in Scenedesmus cultures are addressed. The information obtained from this study will be useful for developing an early warning system and control measures for preventing or treating this contaminant in microalgal mass cultures.

Phylogeny, ultrastructure, and flagellar apparatus of a new Marimonad flagellate Abollifer globosa sp. nov. (Imbricatea, Cercozoa).

Abollifer is a little-known genus of marine heterotrophic flagellates with no ultrastructural and molecular information, and its taxonomic position remains uncertain. In this study, we report a new species of Abollifer, Abollifer globosa sp. nov., isolated from a seawater sample collected at Tokyo Bay. To reveal the taxonomic position and morphological characteristics of A. globosa, we performed light and electron microscopic observations and a phylogenetic analysis using small subunit ribosomal DNA sequences. A. globosa cells were 29.5 µm in length and 22.4 µm in width, oval or ovoid in shape with an apical projection. Two unequal flagella emerged from a deep subapical flagellar pit. The rim of the flagellar pit except for the ventral side was swollen. Electron microscopic observations showed that A. globosa possessed mitochondria with tubular cristae, a Golgi apparatus, microbodies, extrusomes, and many endosymbiotic bacteria. Basal bodies were arranged in parallel. The flagellar apparatus of A. globosa showed affinity with common gliding cercozoan flagellates. Our phylogenetic tree showed that A. globosa branched as the sister position of order Marimonadida (Imbricatea, Cercozoa). On the basis of the morphological and molecular phylogenetic analysis, we conclude that A. globosa is a new member of the order Marimonadida.