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The spacer block technique is widely used in medial fixed-bearing unicompartmental knee arthroplasty (UKA) for osteoarthritis (OA) and osteonecrosis (ON), while it is still unclear if there is any difference in the extension gap between UKAs for the two conditions. To clarify the question, the data from 87 consecutive patients (OA: 57 patients and ON: 30 patients) who underwent medial fixed-bearing UKA with the spacer block technique were retrospectively collected and analyzed. The component gap between the medial tibial osteotomy surface and the femoral trial prosthesis in extension was measured using a UKA tensor, and the preosteotomy gap was calculated in each condition. Also, pre- and postoperative coronal hip-knee-ankle (HKA) angles, the thickness of the insert, and the amount of distal femoral and proximal tibial osteotomy were measured and compared between the two groups. As a result, the mean preosteotomy gap in Group OA and Group ON was 5.4 and 3.7 mm, respectively ( p  < 0.001). The amount of change in HKA angle following UKA in Group OA and Group ON was 4.4and 3.8 degrees, respectively ( p  = 0.044), while no significant difference was found regarding pre- and postoperative HKA angle between them. Also, thinner inserts tended to be used in Group ON than in Group OA, while no significant difference in the amount of osteotomies was found between them. Since knees with OA and ON showed different biomechanical conditions at medial fixed-bearing UKA probably for the difference in the amount of remaining cartilage, the surgical technique should be carefully considered for each condition., Competing Interests: R.K. declares Consulting fees from Medacta lnternational, Arthrex, lnc., and Japan Tissue Engineering Co., Ltd, Payment or honoraria from Zimmer Biomet, Smith & Nephew KK, Johnson & Johnson KK, Japan Tissue Engineering Co., Ltd, Arthrex Japan G.K. He is Associate Editor for Knee Surgery, Sports Traumatology, Arthroscopy (KSSTA)., (Thieme. All rights reserved.)

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., (© 2023 International Society of Protistologists.)

"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 19 th century but proved to represent a new deep-branching eukaryote lineage when phylogenomic analyses were conducted. 2 Meteora sporadica 5 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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., (© 2023 The Authors. Journal of Eukaryotic Microbiology published by Wiley Periodicals LLC on behalf of International Society of Protistologists.)

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., (© 2023. Springer Nature Limited.)

Introduction: Neuronal intranuclear inclusion disease (NIID) is a neurodegenerative disorder that produces a broad spectrum of clinical conditions such as dementia, upper motor neuron involvement, extrapyramidal symptoms, and neuropathy. Some studies have reported ophthalmological conditions associated with the disease; however, the details of these conditions remain unclear., Patient Concerns: We report a 63-year-old Japanese female with cognitive decline, blurred vision, photophobia, and color blindness at 52 years of age who was diagnosed with cone dystrophy. She also had anxiety, insomnia, depression, delusions, hallucinations, a wide-based gait with short steps, and urinary incontinence., Diagnoses, Interventions, and Outcomes: Magnetic resonance imaging revealed diffuse cerebral white matter changes and subcortical hyperintensity on diffusion-weighted imaging. Skin biopsy showed p62-positive intranuclear inclusions in sweat glands. NOTCH2NLC gene analysis revealed abnormal GGC expansion; therefore, NIID was diagnosed., Conclusion: NOTCH2NLC mutation-positive NIID may be associated with retinal dystrophy. Brain magnetic resonance imaging and skin biopsy are helpful diagnostic clues, and gene analysis is crucial for accurate diagnosis and appropriate management., Competing Interests: The authors have no funding and conflicts of interest to disclose., (Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc.)

Marine phytoplankton are major primary producers, and their growth is primarily limited by nitrogen in the oligotrophic ocean environment. The haptophyte Braarudosphaera bigelowii possesses a cyanobacterial endosymbiont (UCYN-A), which plays a major role in nitrogen fixation in the ocean. However, host-symbiont interactions are poorly understood because B. bigelowii was unculturable. In this study, we sequenced the complete genome of the B. bigelowii endosymbiont and showed that it was highly reductive and closely related to UCYN-A2 (an ecotype of UCYN-A). We succeeded in establishing B. bigelowii strains and performed microscopic observations. The detailed observations showed that the cyanobacterial endosymbiont was surrounded by a single host derived membrane and divided synchronously with the host cell division. The transcriptome of B. bigelowii revealed that B. bigelowii lacked the expression of many essential genes associated with the uptake of most nitrogen compounds, except ammonia. During cultivation, some of the strains completely lost the endosymbiont. Moreover, we did not find any evidence of endosymbiotic gene transfer from the endosymbiont to the host. These findings illustrate an unstable morphological, metabolic, and genetic relationship between B. bigelowii and its endosymbiont., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Suzuki, Kawachi, Tsukakoshi, Nakamura, Hagino, Inouye and Ishida.)

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.

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., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

The difficult-to-cultivate katablepharid Hatena arenicola ingests green algae, Nephroselmis spp., and temporarily retains a Nephroselmis-derived cell compartment (kleptochloroplast), including a chloroplast within a phagocytotic vacuole. H. arenicola has a unique life history; during cell division, the Nephroselmis-derived cell compartment is only inherited by one of two daughter cells. However, the detailed morphological transition of the Nephroselmis cell to a kleptochloroplast and the mitotic process of the host cell remain unclear. Herein, we observed feeding behavior, enlargement of the Nephroselmis-derived chloroplast, and mitotic processes in H. arenicola using light and electron microscopy. During feeding behavior, H. arenicola peeled off the cell coverings and flagella of the Nephroselmis cell, which selectively accumulated in a vacuole separate to one containing a Nephroselmis cell body. An obvious nucleolus, but no heterochromatin was observed in the Nephroselmis-derived nucleus during the chloroplast-enlarging process, while compressed heterochromatin was explicitly observed in the nuclei of free-living Nephroselmis cells. The cell membrane of an ingested Nephroselmis cell disintegrated during enlargement of the Nephroselmis-derived chloroplast. The process of mitosis in H. arenicola was very similar to that of other katablepharids and cryptophytes., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

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., Competing Interests: The authors declare no competing interest.

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.

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., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Perkinsus marinus is a marine protozoan parasite that infects natural and farmed oysters, attracting attention from researchers in both fisheries and evolutionary biology. The functions of almost all cellular components and organelles are, however, poorly understood even though a draft genome sequence of P. marinus is publicly available. One of the major obstacles for a functional study of the parasite is limited experimental means for genetic manipulation: a transfection method was established in 2008, and the first drug selection system with bleomycin was reported in 2016. We here introduce the second drug-selectable marker for selection of P. marinus transfectants. The parasite growth is efficiently inhibited by puromycin (IC 50  = 4.96 μg/mL), and transfection of its resistance gene, puromycin-N-acetyl-transferase (pac), confers resistance to the drug on the parasite. Stable transfectants can be obtained within 2 months by treating with puromycin at 100 μg/mL. Furthermore, combining puromycin and bleomycin treatment can select transfectants co-expressing two marker genes. This dual-transfection method raises the possibility of using co-localization to identify the cellular localization of novel proteins in P. marinus, thereby contributing to the understanding of cellular functions and pathogenesis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

A haptonema is an elongated microtubule-based motile organelle uniquely present in haptophytes. The most notable and rapid movement of a haptonema is 'coiling', which occurs within a few milliseconds following mechanical stimulation in an unknown motor-independent mechanism. Here, we analyzed the coiling process in detail by high-speed filming and showed that haptonema coiling was initiated by left-handed twisting of the haptonema, followed by writhing to form a helix from the distal tip. On recovery from a mechanical stimulus, the helix slowly uncoiled from the proximal region. Electron microscopy showed that the seven microtubules in a haptonema were arranged mostly in parallel but that one of the microtubules often wound around the others in the extended state. A microtubule stabilizer, paclitaxel, inhibited coiling and induced right-handed twisting of the haptonema in the absence of Ca 2+ , suggesting changes in the mechanical properties of microtubules. Addition of Ca 2+ resulted in the conversion of haptonematal twist into the planar bends near the proximal region. These results indicate that switching microtubule conformation, possibly with the aid of Ca 2+ -binding microtubule-associated proteins is responsible for rapid haptonematal coiling., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Paulinella micropora is a rhizarian thecate amoeba, belonging to a photosynthetic Paulinella species group that has a unique organelle termed chromatophore, whose cyanobacterial origin is distinct from that of plant and algal chloroplasts. Because acquisition of the chromatophore was quite a recent event compared with that of the chloroplast ancestor, the Paulinella species are thought to be model organisms for studying the early process of primary endosymbiosis. To obtain insight into how endosymbiotically transferred genes acquire expression competence in the host nucleus, here we analyzed the 5' end sequences of the mRNAs of P. micropora MYN1 strain with the aid of a cap-trapper cDNA library. As a result, we found that mRNAs of 27 genes, including endosymbiotically transferred genes, possessed the common 5' end sequence of 28-33 bases that were posttranscriptionally added by spliced leader (SL) trans-splicing. We also found two subtypes of SL RNA genes encoded by the P. micropora MYN1 genome. Differing from the other SL trans-splicing organisms that usually possess poly(A)-less SL RNAs, this amoeba has polyadenylated SL RNAs. In this study, we characterize the SL trans-splicing of this unique organism and discuss the putative merits of SL trans-splicing in functional gene transfer and genome evolution., Competing Interests: The authors have declared that no competing interests exist.

Quadricilia rotundata is a heterotrophic flagellate with four flagella. However, because this species has no clear morphological characteristics or molecular data affiliating it with any known group, Q. rotundata has been treated as a protist incertae sedis, for a long time. Here, we established a clonal culture of Q. rotundata and sequenced its 18S rDNA sequence. Molecular phylogenetic analysis successfully placed Q. rotundata in an environmental clade within Cercozoa, which contributes to expand the morphological and species diversity within Cercozoa. We also discuss morphological evolution within Cercozoa based on this finding., (© 2018 The Author(s) Journal of Eukaryotic Microbiology © 2018 International Society of Protistologists.)

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., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

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., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

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.

Nonphotosynthetic plastids retain important biological functions and are indispensable for cell viability. However, the detailed processes underlying the loss of plastidal functions other than photosynthesis remain to be fully understood. In this study, we used transcriptomics, subcellular localization, and phylogenetic analyses to characterize the biochemical complexity of the nonphotosynthetic plastids of the apochlorotic diatom Nitzschia sp. NIES-3581. We found that these plastids have lost isopentenyl pyrophosphate biosynthesis and ribulose-1,5-bisphosphate carboxylase/oxygenase-based carbon fixation but have retained various proteins for other metabolic pathways, including amino acid biosynthesis, and a portion of the Calvin-Benson cycle comprised only of glycolysis/gluconeogenesis and the reductive pentose phosphate pathway (rPPP). While most genes for plastid proteins involved in these reactions appear to be phylogenetically related to plastid-targeted proteins found in photosynthetic relatives, we also identified a gene that most likely originated from a cytosolic protein gene. Based on organellar metabolic reconstructions of Nitzschia sp. NIES-3581 and the presence/absence of plastid sugar phosphate transporters, we propose that plastid proteins for glycolysis, gluconeogenesis, and rPPP are retained even after the loss of photosynthesis because they feed indispensable substrates to the amino acid biosynthesis pathways of the plastid. Given the correlated retention of the enzymes for plastid glycolysis, gluconeogenesis, and rPPP and those for plastid amino acid biosynthesis pathways in distantly related nonphotosynthetic plastids and cyanobacteria, we suggest that this substrate-level link with plastid amino acid biosynthesis is a key constraint against loss of the plastid glycolysis/gluconeogenesis and rPPP proteins in multiple independent lineages of nonphotosynthetic algae/plants., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

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., (© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.)

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., (© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.)

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., (Copyright © 2016. Published by Elsevier GmbH.)

Chlorarachniophyte algae have complex plastids acquired by the uptake of a green algal endosymbiont, and this event is called secondary endosymbiosis. Interestingly, the plastids possess a relict endosymbiont nucleus, referred to as the nucleomorph, in the intermembrane space, and the nucleomorphs contain an extremely reduced and compacted genome in comparison with green algal nuclear genomes. Therefore, chlorarachniophyte plastids consist of two endosymbiotically derived genomes, i.e., the plastid and nucleomorph genomes. To date, complete nucleomorph genomes have been sequenced in four different species, whereas plastid genomes have been reported in only two species in chlorarachniophytes. To gain further insight into the evolution of endosymbiotic genomes in chlorarachniophytes, we newly sequenced the plastid genomes of three species, Gymnochlora stellata, Lotharella vacuolata, and Partenskyella glossopodia. Our findings reveal that chlorarachniophyte plastid genomes are highly conserved in size, gene content, and gene order among species, but their nucleomorph genomes are divergent in such features. Accordingly, the current architecture of the plastid genomes of chlorarachniophytes evolved in a common ancestor, and changed very little during their subsequent diversification. Furthermore, our phylogenetic analyses using multiple plastid genes suggest that chlorarachniophyte plastids are derived from a green algal lineage that is closely related to Bryopsidales in the Ulvophyceae group.