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Apusomonadida (apusomonads) is a group of heterotrophic biflagellates that feed on bacteria and small protists. Their diversity is not fully understood, and several major lineages remain to be identified in natural environments. Here, we report Podomonas kaiyoae n. sp., which was isolated from deep-sea sediment and can be maintained as an axenic culture. While P. kaiyoae branched within one of the major unidentified lineages, the combination of the morphological characteristics is generally similar to that of Podomonas species, but can be distinguished from that of other Podomonas species based on the cell sizes.

In an effort to broaden our understanding of the biodiversity and distribution of gregarines infecting crustaceans, this study describes two new species of gregarines, Thiriotia hyperdolphinae n. sp. and Cephaloidophora oradareae n. sp., parasitizing a deep sea amphipod (Oradarea sp.). Amphipods were collected using the ROV Hyper-Dolphin at a depth of 855 m while on a cruise in Sagami Bay, Japan. Gregarine trophozoites and gamonts were isolated from the gut of the amphipod and studied with light and scanning electron microscopy, and phylogenetic analysis of 18S rDNA. T. hyperdolphinae n. sp. was distinguished from existing species based on morphology, phylogenetic position, as well as host niche and geographic locality. While Cephaloidophora oradareae n. sp. distinguished itself from existing Cephaloidophora, based on a difference in host (Oradarea sp.), geographic location, and to a certain extent morphology. We established this new species with the understanding that a more comprehensive examination of diversity at the molecular level is necessary within Cephaloidophora. Results from the 18S rDNA molecular phylogeny showed that T. hyperdolphinae n. sp. was positioned within a clade consisting of Thiriotia spp., while C. oradareae n. sp. grouped within the Cephaloidophoridae. Still, supplemental genetic information from gregarines infecting crustaceans will be needed to better understand relationships within this group of apicomplexans. This article is protected by copyright. All rights reserved.

Although environmental DNA surveys improve our understanding of biodiversity, interpretation of unidentified lineages is limited by the absence of associated morphological traits and living cultures. Unidentified lineages of marine stramenopiles are called "MAST clades". Twenty-five MAST clades have been recognized: MAST-1 through MAST-25; seven of these have been subsequently discarded because the sequences representing those clades were found to either (1) be chimeric or (2) affiliate within previously described taxonomic groups. Eighteen MAST clades remain without a cellular identity. Moreover, the discarded "MAST-13" has been used in different studies to refer to two different environmental sequence clades. After establishing four cultures representing two different species of heterotrophic stramenopiles and then characterizing their morphology and molecular phylogenetic positions, we determined that the two different species represented the two different MAST-13 clades: (1) a lorica-bearing Bicosoeca kenaiensis and (2) a microaerophilic flagellate previously named "Cafeteria marsupialis". Both species were previously described with only light microscopy; no cultures, ultrastructural data or DNA sequences were available from these species prior to this study. The molecular phylogenetic position of three different "C. marsupialis" isolates was not closely related to the type species of Cafeteria; therefore, we established a new genus for these isolates, Cantina gen. nov.

Hemistasia phaeocysticola is a marine flagellate that preys on diatoms and dinoflagellates among others. Although its morphology and ultrastructure were previously observed and characterized, its phylogenetic position has not been analyzed using molecular sequence data. This flagellate was classified as a kinetoplastid on the basis of the presence of a kinetoplast in the mitochondrion. However, several morphological characteristics similar to those of diplonemids, a sister group of kinetoplastids, have also been noted. Herein, we report that H. phaeocysticola branches within the diplonemid clade in the phylogenetic tree reconstructed by analyzing 18S rRNA gene sequences. Its systematic placement based on this finding is also discussed.

We report the ultrastructure and phylogenetic position of a free-living heterotrophic flagellate, Tsukubamonas globosa n. gen., n. sp. This flagellate was isolated from a pond in the University of Tsukuba, Japan. Under light microscopy, the spherical vegetative cells were naked and highly vacuolated, and always swam with rotating motion. Electron microscopic observations revealed that T. globosa possessed a ventral feeding groove, which is one of the hallmark characteristics of the supergroup Excavata. The position of T. globosa was unresolved in the small subunit ribosomal RNA phylogeny. On the other hand, a multigene phylogeny using α-tubulin, β-tubulin, actin, heat shock protein 90, and translation elongation factor 2 robustly united T. globosa with members of the “Discoba” clade of Excavata, composed of jakobids, euglenozoans, and heteroloboseans, although the precise position of T. globosa in this clade remained unresolved. Our detailed morphological comparisons elucidated that T. globosa possessed a novel set of morphological features, and could not be classified into any taxa in the Discoba clade. Instead we classified T. globosa into Tsukubamonadidae n. fam. under Tsukubamonadida n. ord.

A new cercozoan flagellate Mataza hastifera n. g., n. sp. is described from a surface seawater sample collected in Tokyo Bay. Cells are 3-5 μm in diameter and have two flagella. The cells alternate between swimming and stationary states in culture. Swimming cells have a nodding motion. Phylogenetic analyses using small subunit rDNA sequences demonstrate that M. hastifera belongs to the clade comprised of only environmental sequences closely related to thecofilosean cercozoans. Ultrastructural observations reveal that M. hastifera is quite similar to members of Cryomonadida, an order in Thecofilosea, and especially to Cryothecomonas spp. The cell of M. hastifera is covered with a thin double-layered theca and possesses a cylinder-shaped extrusome, as reported from cryomonads. On the other hand, the funnel that is characteristic of cryomonads was not found in the flagellar pit of M. hastifera. Combining both morphological and molecular analyses, we conclude that M. hastifera is a new lineage in Thecofilosea and suggest that Thecofilosea may be a larger group than previously thought.

Ultrastructural and molecular phylogenetic evidence indicate that the Parabasalia consists of seven main subgroups: the Trichomonadida, Honigbergiellida, Hypotrichomonadida, Tritrichomonadida, Cristamonadida, Spirotrichonymphida, and Trichonymphida. Only five species of free-living parabasalids are known: Monotrichomonas carabina, Ditrichomonas honigbergii, Honigbergiella sp., Tetratrichomonas undula, and Pseudotrichomonas keilini. Phylogenetic analyses show that free-living species do not form a clade and instead branch in several different positions within the context of their parasitic relatives. Because the diversity of free-living parabasalids is poorly understood, the systematics of these lineages is in a significant state of disarray. In order to better understand the phylogenetic distribution of free-living parabasalids, we sequenced the small subunit rDNA from three different strains reminiscent of P. keilini; the strains were isolated from different geographical locations: (1) mangrove sediments in Japan and (2) sediments in Cyprus. These data demonstrated that the free-living parabasalids P. keilini and Lacusteria cypriaca n. g., n. sp., form a paraphyletic assemblage near the origin of a clade consisting mostly of parasitic trichomonadids (e.g. Trichomonas vaginalis). This paraphyletic distribution of similar morphotypes indicates that free-living trichomonadids represent a compelling example of morphostasis that provides insight into the suite of features present in the most recent free-living ancestor of their parasitic relatives.

Chlorarachniophytes are flagellated and/or reticulopod-forming marine algae with chlorophyll a- and b-containing plastids of secondary endosymbiotic origin. They are one of only two algal groups known to possess a "nucleomorph" (i.e. the remnant nucleus of the eukaryotic endosymbiont that donated the plastid). Apart from the recently sequenced nucleomorph genome of Bigelowiella natans, little is known about the size, structure, and composition of chlorarachniophyte nucleomorph genomes. Toward the goal of better understanding nucleomorph genome diversity, as well as establishing a phylogenetic framework with which to interpret variation in chlorarachniophyte morphology, ultrastructure, and life cycle, we are studying a wide range of chlorarachniophyte strains from public culture collections and natural habitats. We have obtained 22 new chlorarachniophyte nuclear and nucleomorph 18S rRNA gene (18S rDNA) sequences and nucleomorph genome size estimates for 14 different strains. Consistent with previous studies, all of the chlorarachniophytes examined appear to possess three nucleomorph chromosomes. However, our results suggest considerable variation in nucleomorph genome size and structure, with individual chromosome sizes ranging from approximately 90 to approximately 210 kbp, and total genome sizes between approximately 330 kbp in Lotharella amoebiformis and approximately 610 kbp in unidentified chlorarachniophyte strain CCMP622. The significance of these phylogenetic and nucleomorph karyotype data is discussed.

Summary Over the last 15 years classical culturing and environmental PCR techniques have revealed a modest number of genuinely new major lineages of protists; however, some new groups have greatly influenced our understanding of eukaryote evolution. We used culturing techniques to examine the diversity of free-living protists that are relatives of diplomonads and retortamonads, a group of evolutionary and parasitological importance. Until recently, a single organism, Carpediemonas membranifera, was the only representative of this region of the tree. We report 18 new isolates of Carpediemonas-like organisms (CLOs) from anoxic marine sediments. Only one is a previously cultured species. Eleven isolates are conspecific and were classified within a new genus, Kipferlia n. gen. The remaining isolates include representatives of three other lineages that likely represent additional undescribed genera (at least). Small-subunit ribosomal RNA gene phylogenies show that CLOs form a cloud of six major clades basal to the diplomonad-retortamonad grouping (i.e. each of the six CLO clades is potentially as phylogenetically distinct as diplomonads and retortamonads). CLOs will be valuable for tracing the evolution of diplomonad cellular features, for example, their extremely reduced mitochondrial organelles. It is striking that the majority of CLO diversity was undetected by previous light microscopy surveys and environmental PCR studies, even though they inhabit a commonly sampled environment. There is no reason to assume this is a unique situation – it is likely that undersampling at the level of major lineages is still widespread for protists.

SUMMARY A chlorarachniophycean alga, Lotharella amoebiformis, which has been classified in the genus Lotharella is placed into a new genus Amorphochlora gen. nov., based on its phylogenetic position, which has been clarified by the recently accumulated molecular phylogenetic information, and the morphological difference between the vegetative cells of the Lotharella species. Following this taxonomic treatment, a new combination Amorphochlora amoebiformis comb. nov., is proposed.