Meteora sporadica, a protist with incredible cell architecture, is related to Hemimastigophora.

"Kingdom-level" branches are being added to the tree of eukaryotes at a rate approaching one per year, with no signs of slowing down.1,2,3,4 Some are completely new discoveries, whereas others are morphologically unusual protists that were previously described but lacked molecular data. For example, Hemimastigophora are predatory protists with two rows of flagella that were known since the 19th century but proved to represent a new deep-branching eukaryote lineage when phylogenomic analyses were conducted.2Meteora sporadica5 is a protist with a unique morphology; cells glide over substrates along a long axis of anterior and posterior projections while a pair of lateral "arms" swing back and forth, a motility system without any obvious parallels. Originally, Meteora was described by light microscopy only, from a short-term enrichment of deep-sea sediment. A small subunit ribosomal RNA (SSU rRNA) sequence was reported recently, but the phylogenetic placement of Meteora remained unresolved.6 Here, we investigated two cultivated Meteora sporadica isolates in detail. Transmission electron microscopy showed that both the anterior-posterior projections and the arms are supported by microtubules originating from a cluster of subnuclear microtubule organizing centers (MTOCs). Neither have a flagellar axoneme-like structure. Sequencing the mitochondrial genome showed this to be among the most gene-rich known, outside jakobids. Remarkably, phylogenomic analyses of 254 nuclear protein-coding genes robustly support a close relationship with Hemimastigophora. Our study suggests that Meteora and Hemimastigophora together represent a morphologically diverse "supergroup" and thus are important for resolving the tree of eukaryote life and early eukaryote evolution.

Rhabdamoeba marina is a heterotrophic relative of chlorarachnid algae.

Rhabdamoeba marina is a unique and poorly reported amoeba with an uncertain phylogenetic position. We successfully cultured R. marina from coastal seawater in Japan and performed a molecular phylogenetic analysis using the small subunit ribosomal RNA (SSU rRNA) gene sequence. Our phylogenetic analysis showed that R. marina branched as a basal lineage of Chlorarachnea, a group of marine photosynthetic algae belonging to the phylum Cercozoa within the supergroup Rhizaria. By comparing the ecological and morphological characteristics of R. marina with those of photosynthetic chlorarachneans and other cercozoans, we gained insight into the evolution and acquisition of plastids in Chlorarachnida.

One high quality genome and two transcriptome datasets for new species of Mantamonas, a deep-branching eukaryote clade.

Mantamonads were long considered to represent an "orphan" lineage in the tree of eukaryotes, likely branching near the most frequently assumed position for the root of eukaryotes. Recent phylogenomic analyses have placed them as part of the "CRuMs" supergroup, along with collodictyonids and rigifilids. This supergroup appears to branch at the base of Amorphea, making it of special importance for understanding the deep evolutionary history of eukaryotes. However, the lack of representative species and complete genomic data associated with them has hampered the investigation of their biology and evolution. Here, we isolated and described two new species of mantamonads, Mantamonas vickermani sp. nov. and Mantamonas sphyraenae sp. nov., for each of which we generated transcriptomic sequence data, as well as a high-quality genome for the latter. The estimated size of the M. sphyraenae genome is 25 Mb; our de novo assembly appears to be highly contiguous and complete with 9,416 predicted protein-coding genes. This near-chromosome-scale genome assembly is the first described for the CRuMs supergroup.

Molecular and morphological characterization of four new ancyromonad genera and proposal for an updated taxonomy of the Ancyromonadida.

Ancyromonads are small biflagellated protists with a bean-shaped morphology. They are cosmopolitan in marine, freshwater, and soil environments, where they attach to surfaces while feeding on bacteria. These poorly known grazers stand out by their uncertain phylogenetic position in the tree of eukaryotes, forming a deep-branching "orphan" lineage that is considered key to a better understanding of the early evolution of eukaryotes. Despite their ecological and evolutionary interest, only limited knowledge exists about their true diversity. Here, we aimed to characterize ancyromonads better by integrating environmental surveys with behavioral observation and description of cell morphology, for which sample isolation and culturing are indispensable. We studied 18 ancyromonad strains, including 14 new isolates and seven new species. We described three new and genetically divergent genera: Caraotamonas, Nyramonas, and Olneymonas, together encompassing four species. The remaining three new species belong to the already-known genera Fabomonas and Ancyromonas. We also raised Striomonas, formerly a subgenus of Nutomonas, to full genus status, on morphological and phylogenetic grounds. We studied the morphology of diverse ancyromonads under light and electron microscopy and carried out molecular phylogenetic analyses, also including 18S rRNA gene sequences from several environmental surveys. Based on these analyses, we have updated the taxonomy of Ancyromonadida.

Barthelonids represent a deep-branching metamonad clade with mitochondrion-related organelles predicted to generate no ATP.

We here report the phylogenetic position of barthelonids, small anaerobic flagellates previously examined using light microscopy alone. Barthelona spp. were isolated from geographically distinct regions and we established five laboratory strains. Transcriptomic data generated from one Barthelona strain (PAP020) were used for large-scale, multi-gene phylogenetic (phylogenomic) analyses. Our analyses robustly placed strain PAP020 at the base of the Fornicata clade, indicating that barthelonids represent a deep-branching metamonad clade. Considering the anaerobic/microaerophilic nature of barthelonids and preliminary electron microscopy observations on strain PAP020, we suspected that barthelonids possess functionally and structurally reduced mitochondria (i.e. mitochondrion-related organelles or MROs). The metabolic pathways localized in the MRO of strain PAP020 were predicted based on its transcriptomic data and compared with those in the MROs of fornicates. We here propose that strain PAP020 is incapable of generating ATP in the MRO, as no mitochondrial/MRO enzymes involved in substrate-level phosphorylation were detected. Instead, we detected a putative cytosolic ATP-generating enzyme (acetyl-CoA synthetase), suggesting that strain PAP020 depends on ATP generated in the cytosol. We propose two separate losses of substrate-level phosphorylation from the MRO in the clade containing barthelonids and (other) fornicates.

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.

Phagocytosis-like cell engulfment by a planctomycete bacterium.

Phagocytosis is a key eukaryotic feature, conserved from unicellular protists to animals, that enabled eukaryotes to feed on other organisms. It could also be a driving force behind endosymbiosis, a process by which α-proteobacteria and cyanobacteria evolved into mitochondria and plastids, respectively. Here we describe a planctomycete bacterium, 'Candidatus Uab amorphum', which is able to engulf other bacteria and small eukaryotic cells through a phagocytosis-like mechanism. Observations via light and electron microscopy suggest that this bacterium digests prey cells in specific compartments. With the possible exception of a gene encoding an actin-like protein, analysis of the 'Ca. Uab amorphum' genomic sequence does not reveal any genes homologous to eukaryotic phagocytosis genes, suggesting that cell engulfment in this microorganism is probably not homologous to eukaryotic phagocytosis. The discovery of this "phagotrophic" bacterium expands our understanding of the cellular complexity of prokaryotes, and may be relevant to the origin of eukaryotic cells.

Taxon-rich Multigene Phylogenetic Analyses Resolve the Phylogenetic Relationship Among Deep-branching Stramenopiles.

Stramenopiles are one of the major eukaryotic assemblages. This group comprises a wide range of species including photosynthetic unicellular and multicellular algae, fungus-like osmotrophic organisms and many free-living phagotrophic flagellates. However, the phylogeny of the Stramenopiles, especially relationships among deep-branching heterotrophs, has not yet been resolved because of a lack of adequate transcriptomic data for representative lineages. In this study, we performed multigene phylogenetic analyses of deep-branching Stramenopiles with improved taxon sampling. We sequenced transcriptomes of three deep-branching Stramenopiles: Incisomonas marina, Pseudophyllomitus vesiculosus and Platysulcus tardus. Phylogenetic analyses using 120 protein-coding genes and 56 taxa indicated that Pl. tardus is sister to all other Stramenopiles while Ps. vesiculosus is sister to MAST-4 and form a robust clade with the Labyrinthulea. The resolved phylogenetic relationships of deep-branching Stramenopiles provide insights into the ancestral traits of the Stramenopiles.

Horizontally-acquired genetic elements in the mitochondrial genome of a centrohelid Marophrys sp. SRT127.

Mitochondrial genomes exhibit diverse features among eukaryotes in the aspect of gene content, genome structure, and the mobile genetic elements such as introns and plasmids. Although the number of published mitochondrial genomes is increasing at tremendous speed, those of several lineages remain unexplored. Here, we sequenced the complete mitochondrial genome of a unicellular heterotrophic eukaryote, Marophrys sp. SRT127 belonging to the Centroheliozoa, as the first report on this lineage. The circular-mapped mitochondrial genome, which is 113,062 bp in length, encodes 69 genes typically found in mitochondrial genomes. In addition, the Marophrys mitochondrial genome contains 19 group I introns. Of these, 11 introns have genes for homing endonuclease (HE) and phylogenetic analyses of HEs have shown that at least five Marophrys HEs are related to those in green algal plastid genomes, suggesting intron transfer between the Marophrys mitochondrion and green algal plastids. We also discovered a putative mitochondrial plasmid in linear form. Two genes encoded in the circular-mapped mitochondrial genome were found to share significant similarities to those in the linear plasmid, suggesting that the plasmid was integrated into the mitochondrial genome. These findings expand our knowledge on the diversity and evolution of the mobile genetic elements in mitochondrial genomes.

Taming chlorophylls by early eukaryotes underpinned algal interactions and the diversification of the eukaryotes on the oxygenated Earth.

Extant eukaryote ecology is primarily sustained by oxygenic photosynthesis, in which chlorophylls play essential roles. The exceptional photosensitivity of chlorophylls allows them to harvest solar energy for photosynthesis, but on the other hand, they also generate cytotoxic reactive oxygen species. A risk of such phototoxicity of the chlorophyll must become particularly prominent upon dynamic cellular interactions that potentially disrupt the mechanisms that are designed to quench photoexcited chlorophylls in the phototrophic cells. Extensive examination of a wide variety of phagotrophic, parasitic, and phototrophic microeukaryotes demonstrates that a catabolic process that converts chlorophylls into nonphotosensitive 132,173-cyclopheophorbide enols (CPEs) is phylogenetically ubiquitous among extant eukaryotes. The accumulation of CPEs is identified in phagotrophic algivores belonging to virtually all major eukaryotic assemblages with the exception of Archaeplastida, in which no algivorous species have been reported. In addition, accumulation of CPEs is revealed to be common among phototrophic microeukaryotes (i.e., microalgae) along with dismantling of their secondary chloroplasts. Thus, we infer that CPE-accumulating chlorophyll catabolism (CACC) primarily evolved among algivorous microeukaryotes to detoxify chlorophylls in an early stage of their evolution. Subsequently, it also underpinned photosynthetic endosymbiosis by securing close interactions with photosynthetic machinery containing abundant chlorophylls, which led to the acquisition of secondary chloroplasts. Our results strongly suggest that CACC, which allowed the consumption of oxygenic primary producers, ultimately permitted the successful radiation of the eukaryotes throughout and after the late Proterozoic global oxygenation.

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.

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

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.

Pseudophyllomitus vesiculosus (Larsen and Patterson 1990) Lee, 2002, a Poorly Studied Phagotrophic Biflagellate is the First Characterized Member of Stramenopile Environmental Clade MAST-6.

There are many eukaryotic lineages that are exclusively composed of environmental sequences and lack information about which species are included. Regarding stramenopiles, at least 18 environmental lineages, known as marine stramenopiles (MAST), have been recognized. Since each MAST lineage forms deep branches in the stramenopiles, the characterization of MAST members is key to understanding the diversity and evolution of stramenopiles. In this study, we established a culture of Pseudophyllomitus vesiculosus, which is a poorly studied phagotrophic flagellate of uncertain taxonomic position. Our molecular phylogenetic analyses based on small subunit ribosomal RNA gene sequences robustly supported the inclusion of P. vesiculosus in the MAST-6 clade. Our microscopic observations indicated that P. vesiculosus shared characteristics with stramenopiles, including an anterior flagellum that exhibits sinusoidal waves and bears tubular mastigonemes. The flagellar apparatus of P. vesiculosus was also similar to that of other stramenopiles in having a transitional helix and five microtubular roots (R1-R4 and S tubules) including R2 that split into two bands. On the other hand, P. vesiculosus was distinguished from other deep-branching stramenopiles by the combination of flagellar apparatus characteristics. Based on the phylogenetic analyses and microscopic observations, we established Pseudophyllomitidae fam. nov in stramenopiles.

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.

A New Heterotrophic Cryptomonad: Hemiarma marina n. g., n. sp.

We report a new heterotrophic cryptomonad Hemiarma marina n. g., n. sp. that was collected from a seaweed sample from the Republic of Palau. In our molecular phylogenetic analyses using the small subunit ribosomal RNA gene, H. marina formed a clade with two marine environmental sequences, and the clade was placed as a sister lineage of the freshwater cryptomonad environmental clade CRY1. Alternatively, in the concatenated large and small subunit ribosomal RNA gene phylogeny, H. marina was placed as a sister lineage of Goniomonas. Light and electron microscopic observations showed that H. marina shares several ultrastructural features with cryptomonads, such as flattened mitochondrial cristae, a periplast cell covering, and ejectisomes that consist of two coiled ribbon structures. On the other hand, H. marina exhibited unique behaviors, such as attaching to substrates with its posterior flagellum and displaying a jumping motion. H. marina also had unique periplast arrangement and flagellar transitional region. On the basis of both molecular and morphological information, we concluded that H. marina should be treated as new genus and species of cryptomonads.

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.

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.

Entamoeba marina n. sp.; a New Species of Entamoeba Isolated from Tidal Flat Sediment of Iriomote Island, Okinawa, Japan.

The genus Entamoeba includes anaerobic lobose amoebae, most of which are parasites of various vertebrates and invertebrates. We report a new Entamoeba species, E. marina n. sp. that was isolated from a sample of tidal flat sediment collected at Iriomote Island, Okinawa, Japan. Trophozoites of E. marina were 12.8-32.1 µm in length and 6.8-15.9 µm in width, whereas the cysts were 8.9-15.8 µm in diam. and contained four nuclei. The E. marina cells contained a rounded nucleus with a small centric karyosome and uniformly arranged peripheral chromatin. Although E. marina is morphologically indistinguishable from other tetranucleated cyst-forming Entamoeba species, E. marina can be distinguished from them based on the combination of molecular phylogenetic analyses using SSU rDNA gene and the difference of collection sites. Therefore, we propose E. marina as a new species of the genus Entamoeba.

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.

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.