Takashi Shiratori

Ubiquity and quantitative significance of detoxification catabolism of chlorophyll associated with protistan herbivory

Chlorophylls are essential components of the photosynthetic apparati that sustain all of the life forms that ultimately depend on solar energy. However, a drawback of the extraordinary photosensitizing efficiency of certain chlorophyll species is their ability to generate harmful singlet oxygen. Recent studies have clarified the catabolic processes involved in the detoxification of chlorophylls in land plants, but little is understood about these strategies in aquatic ecosystem. Here, we report that a variety of heterotrophic protists accumulate the chlorophyll a catabolite 132,173-cyclopheophorbide a enol (cPPB-aE) after their ingestion of algae. This chlorophyll derivative is nonfluorescent in solution, and its inability to generate singlet oxygen in vitro qualifies it as a detoxified catabolite of chlorophyll a. Using a modified analytical method, we show that cPPB-aE is ubiquitous in aquatic environments, and it is often the major chlorophyll a derivative. Our findings suggest that cPPB-aE metabolism is one of the most important, widely distributed processes in aquatic ecosystems. Therefore, the herbivorous protists that convert chlorophyll a to cPPB-aE are suggested to play more significant roles in the modern oceanic carbon flux than was previously recognized, critically linking microscopic primary producers to the macroscopic food web and carbon sequestration in the ocean.

132,173‐Cyclopheophorbide b enol as a catabolite of chlorophyll b in phycophagy by protists

Both 132,173‐cyclopheophorbide a and b enols were produced along with ingestion of green microalgae containing chlorophylls a and b by a centrohelid protist (phycophagy). The results suggest that chlorophyll b as well as chlorophyll a were directly degraded to colored yet non‐phototoxic catabolites in the protistan phycophagic process. Such a simple process by the predators makes a contrast to the much sophisticated chlorophyll degradation process of land plants and some algae, where phototoxicity of chlorophylls was cancelled through the multiple enzymatic steps resulting in colorless and non‐phototoxic catabolites.

A novel alphaproteobacterial ectosymbiont promotes the growth of the hydrocarbon-rich green alga Botryococcus braunii

Botryococcus braunii is a colony-forming green alga that accumulates large amounts of liquid hydrocarbons within the colony. The utilization of B. braunii for biofuel production is however hindered by its low biomass productivity. Here we describe a novel bacterial ectosymbiont (BOTRYCO-2) that confers higher biomass productivity to B. braunii. 16S rDNA analysis indicated that the sequence of BOTRYCO-2 shows low similarity (<90%) to cultured bacterial species and located BOTRYCO-2 within a phylogenetic lineage consisting of uncultured alphaproteobacterial clones. Fluorescence in situ hybridization (FISH) studies and transmission electric microscopy indicated that BOTRYCO-2 is closely associated with B. braunii colonies. Interestingly, FISH analysis of a water bloom sample also found BOTRYCO-2 bacteria in close association with cyanobacterium Microcystis aeruginosa colonies, suggesting that BOTRYCO-2 relatives have high affinity to phytoplankton colonies. A PCR survey of algal bloom samples revealed that the BOTRYCO-2 lineage is commonly found in Microcystis associated blooms. Growth experiments indicated that B. braunii Ba10 can grow faster and has a higher biomass (1.8-fold) and hydrocarbon (1.5-fold) yield in the presence of BOTRYCO-2. Additionally, BOTRYCO-2 conferred a higher biomass yield to BOT-22, one of the fastest growing strains of B. braunii. We propose the species name ‘Candidatus Phycosocius bacilliformis’ for BOTRYCO-2.

Group II Intron-Mediated Trans-Splicing in the Gene-Rich Mitochondrial Genome of an Enigmatic Eukaryote, Diphylleia rotans

Although mitochondria have evolved from a single endosymbiotic event, present day mitochondria of diverse eukaryotes display a great range of genome structures, content and features. Group I and group II introns are two features that are distributed broadly but patchily in mitochondrial genomes across branches of the tree of eukaryotes. While group I intron-mediated trans-splicing has been reported from some lineages distantly related to each other, findings of group II intron-mediated trans-splicing has been restricted to members of the Chloroplastida. In this study, we found the mitochondrial genome of the unicellular eukaryote Diphylleia rotans possesses currently the second largest gene repertoire. On the basis of a probable phylogenetic position of Diphylleia, which is located within Amorphea, current mosaic gene distribution in Amorphea must invoke parallel gene losses from mitochondrial genomes during evolution. Most notably, although the cytochrome c oxidase subunit (cox) 1 gene was split into four pieces which located at a distance to each other, we confirmed that a single mature mRNA that covered the entire coding region could be generated by group II intron-mediated trans-splicing. This is the first example of group II intron-mediated trans-splicing outside Chloroplastida. Similar trans-splicing mechanisms likely work for bipartitely split cox2 and nad3 genes to generate single mature mRNAs. We finally discuss origin and evolution of this type of trans-splicing in D. rotans as well as in eukaryotes.

Phylogeny and Morphology of New Diplonemids from Japan

Diplonemids were recently found to be the most species-rich group of marine planktonic protists. Based on phylogenetic analysis of 18S rRNA gene sequences and morphological observations, we report the description of new members of the genus Rhynchopus - R. humris sp. n. and R. serpens sp. n., and the establishment of two new genera - Lacrimia gen. n. and Sulcionema gen. n., represented by L. lanifica sp. n. and S. specki sp. n., respectively. In addition, we describe the organism formerly designated as Diplonema sp. 2 (ATCC 50224) as Flectonema neradi gen. n., sp. n. The newly described diplonemids share a common set of traits. Cells are sac-like but variable in shape and size, highly metabolic, and surrounded by a naked cell membrane, which is supported by a tightly packed corset of microtubules. They carry a single highly reticulated peripheral mitochondrion containing a large amount of mitochondrial DNA, with lamellar cristae. The cytopharyngeal complex and flagellar pocket are contiguous and have separate openings. Two parallel flagella are inserted sub-apically into a pronounced flagellar pocket. Rhynchopus species have their flagella concealed in trophic stages and fully developed in swimming stages, while they permanently protrude in all other known diplonemid species.

Trachyrhizium urniformis n. g., n. sp., a Novel Marine Filose Thecate Amoeba Related to a Cercozoan Environmental Clade (Novel Clade 4).

A novel cercozoan filose thecate amoeba, Trachyrhizium urniformis n. g., n. sp., was isolated from a marine sediment sample collected at Agenashiku Island, Okinawa, Japan. We performed light and electron microscopic observations, and a molecular phylogenetic analysis using the small subunit ribosomal RNA gene of the isolate. Cells of T. urniformis are spherical in shape and are covered by a thin theca possessing a wide rounded aperture. Branching and occasionally anastomosing filopodia with small granules emerge from the aperture. The granules are transported in the filopodia bidirectionally. Transmission electron microscopy showed that cells of T. urniformis possess nucleus with permanently condensed chromatin, Golgi apparatuses, microbodies, mitochondria with tubular cristae, and extrusomes. Several morphological and ultrastructural features of T. urniformis (the presence of thecae and nucleus with permanently condensed chromatin) show similarities with those of Thecofilosea. In a phylogenetic analysis, T. urniformis included in Thecofilosea with weak statistical supports and formed a clade with two sequences that constitutes a cercozoan environmental clade, novel clade 4. On the basis of morphological and ultrastructural information and the results of the phylogenetic analysis, we propose T. urniformis as a new member of class Thecofilosea.

Phylogenomics Places Orphan Protistan Lineages in a Novel Eukaryotic Super-Group

Abstract Recent phylogenetic analyses position certain “orphan” protist lineages deep in the tree of eukaryotic life, but their exact placements are poorly resolved. We conducted phylogenomic analyses that incorporate deeply sequenced transcriptomes from representatives of collodictyonids (diphylleids), rigifilids, Mantamonas, and ancyromonads (planomonads). Analyses of 351 genes, using site-heterogeneous mixture models, strongly support a novel super-group-level clade that includes collodictyonids, rigifilids, and Mantamonas, which we name “CRuMs”. Further, they robustly place CRuMs as the closest branch to Amorphea (including animals and fungi). Ancyromonads are strongly inferred to be more distantly related to Amorphea than are CRuMs. They emerge either as sister to malawimonads, or as a separate deeper branch. CRuMs and ancyromonads represent two distinct major groups that branch deeply on the lineage that includes animals, near the most commonly inferred root of the eukaryote tree. This makes both groups crucial in examinations of the deepest-level history of extant eukaryotes.

A New Deep-branching Stramenopile, Platysulcus tardus gen. nov., sp. nov.

A novel free-living heterotrophic stramenopile, Platysulcus tardus gen. nov., sp. nov. was isolated from sedimented detritus on a seaweed collected near the Ngeruktabel Island, Palau. P. tardus is a gliding flagellate with tubular mastigonemes on the anterior short flagellum and a wide, shallow ventral furrow. Although the flagellar apparatus of P. tardus is typical of stramenopiles, it shows novel ultrastructural combinations that are not applied to any groups of heterotrophic stramenopiles. Phylogenetic analysis using SSU rRNA genes revealed that P. tardus formed a clade with stramenopiles with high support. However, P. tardus did not form a subclade with any species or environmental sequences within the stramenopiles, and no close relative was suggested by the phylogenetic analysis. Therefore, we concluded that P. tardus should be treated as a new genus and species of stramenopiles and have proposed a new family, Platysulcidae fam. nov., for this phylogenetically distinct organism.

Taxonomic study of a new eustigmatophycean alga, Vacuoliviride crystalliferum gen. et sp. nov.

This study investigated the taxonomic affiliation of the algal strain nak-9, which has been reported to absorb radioactive cesium with high efficiency, using light and electron microscopy, and molecular phylogenetic analysis based on 18S ribosomal RNA gene (rDNA) sequences. This alga is spherical and coccoid, with a smooth cell wall, large vacuole, crystalline structure, reddish globule, and refractile granules (lamellate vesicles). The cells possess one to several greenish parietal chloroplasts with a bulging pyrenoid surrounded by lamellate vesicles. The chloroplasts include orderly thylakoid lamellae but no girdle lamella. Molecular phylogenetic analysis suggests that strain nak-9 is a member of the eustigmatophycean clade, which includes Goniochloris, Pseudostaurastrum, and Trachydiscus. On the basis of these results, we propose that strain nak-9 (NIES-2860) comprises a new species and new genus of the Eustigmatophyceae, Vacuoliviride crystalliferum gen. et sp. nov.

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.