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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.

We report a new naked cercozoan flagellate, Esquamula lacrimiformis n. g., n. sp., collected from a sandy beach in Japan. Its cells were 4.5-11.3 μm in length and 3.9-8.8 μm in width and possess two unequal flagella. Cells move in a smooth gliding motion and have a trailing long posterior flagellum. Phylogenetic analyses with small and large subunit ribosomal RNA genes revealed that E. lacrimiformis forms a novel lineage within the Thaumatomonadida, the members of which are flagellates with siliceous scales. However, our light and electron microscopic observations indicated that E. lacrimiformis cells do not possess any siliceous structures. Furthermore, other morphological characteristics, such as the shape of the extrusomes and the structural arrangement of the microbody, were clearly different from those of previously described thaumatomonads. On the basis of a combination of these morphological observations and our phylogenetic analyses, we conclude that E. lacrimiformis should be treated as a new species of a new genus and placed into a new family, Esquamulidae n. fam., under Thaumatomonadida.

Elongation factor 1α (EF-1α) and elongation factor-like (EFL) proteins are considered to carry out equivalent functions in translation in eukaryotic cells. Elongation factor 1α and EFL genes are patchily distributed in the global eukaryotic tree, suggesting that the evolution of these elongation factors cannot be reconciled without multiple lateral gene transfer and/or ancestral co-occurrence followed by differential loss of either of the two factors. Our current understanding of the EF-1α/EFL evolution in the eukaryotic group Rhizaria, composed of Foraminifera, Radiolaria, Filosa, and Endomyxa, remains insufficient, as no information on EF-1α/EFL gene is available for any members of Radiolaria. In this study, EFL genes were experimentally isolated from four polycystine radiolarians (i.e. Dictyocoryne, Eucyrtidium, Collozoum, and Sphaerozoum), as well as retrieved from publicly accessible expressed sequence tag data of two acantharean radiolarians (i.e. Astrolonche and Phyllostaurus) and the endomyxan Gromia. The EFL homologs from radiolarians, foraminiferans, and Gromia formed a robust clade in both maximum-likelihood and Bayesian phylogenetic analyses, suggesting that EFL genes were vertically inherited from their common ancestor. We propose an updated model for EF-1α/EFL evolution in Rhizaria by incorporating new EFL data obtained in this study.

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.