The copy number of the eukaryotic rRNA gene can be counted comprehensively.

Gene sequence has been widely used in molecular ecology. For instance, the ribosomal RNA (rRNA) gene has been widely used as a biological marker to understand microbial communities. The variety of the detected rRNA gene sequences reflects the diversity of the microorganisms existing in the analyzed sample. Their biomass can also be estimated by applying quantitative sequencing with information on rRNA gene copy numbers in genomes; however, information on rRNA gene copy numbers is still limited. Especially, the copy number in microbial eukaryotes is much less understood than that of prokaryotes, possibly because of the large and complex structure of eukaryotic genomes. In this study, we report an alternative approach that is more appropriate than the existing method of quantitative sequencing and demonstrate that the copy number of eukaryotic rRNA can be measured efficiently and comprehensively. By applying this approach widely, information on the eukaryotic rRNA copy number can be determined, and their community structures can be depicted and compared more efficiently.

Encyclopedia of Family A DNA Polymerases Localized in Organelles: Evolutionary Contribution of Bacteria Including the Proto-Mitochondrion.

DNA polymerases synthesize DNA from deoxyribonucleotides in a semiconservative manner and serve as the core of DNA replication and repair machinery. In eukaryotic cells, there are 2 genome-containing organelles, mitochondria, and plastids, which were derived from an alphaproteobacterium and a cyanobacterium, respectively. Except for rare cases of genome-lacking mitochondria and plastids, both organelles must be served by nucleus-encoded DNA polymerases that localize and work in them to maintain their genomes. The evolution of organellar DNA polymerases has yet to be fully understood because of 2 unsettled issues. First, the diversity of organellar DNA polymerases has not been elucidated in the full spectrum of eukaryotes. Second, it is unclear when the DNA polymerases that were used originally in the endosymbiotic bacteria giving rise to mitochondria and plastids were discarded, as the organellar DNA polymerases known to date show no phylogenetic affinity to those of the extant alphaproteobacteria or cyanobacteria. In this study, we identified from diverse eukaryotes 134 family A DNA polymerase sequences, which were classified into 10 novel types, and explored their evolutionary origins. The subcellular localizations of selected DNA polymerases were further examined experimentally. The results presented here suggest that the diversity of organellar DNA polymerases has been shaped by multiple transfers of the PolI gene from phylogenetically broad bacteria, and their occurrence in eukaryotes was additionally impacted by secondary plastid endosymbioses. Finally, we propose that the last eukaryotic common ancestor may have possessed 2 mitochondrial DNA polymerases, POP, and a candidate of the direct descendant of the proto-mitochondrial DNA polymerase I, rdxPolA, identified in this study.

The First Identification of a Narnavirus in Bigyra, a Marine Protist.

Current information on the diversity and evolution of eukaryotic RNA viruses is biased towards host lineages, such as animals, plants, and fungi. Although protists represent the majority of eukaryotic diversity, our understanding of the protist RNA virosphere is still limited. To reveal untapped RNA viral diversity, we screened RNA viruses from 30 marine protist isolates and identified a novel RNA virus named Haloplacidia narnavirus 1 (HpNV1). A phylogenetic ana-lysis revealed that HpNV1 is a new member of the family Narnaviridae. The present study filled a gap in the distribution of narnaviruses and implies their wide distribution in Stramenopiles.

Podomonas kaiyoae n. sp., a novel apusomonad growing axenically.

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.

The closest lineage of Archaeplastida is revealed by phylogenomics analyses that include Microheliella maris.

By clarifying the phylogenetic positions of 'orphan' protists (unicellular micro-eukaryotes with no affinity to extant lineages), we may uncover the novel affiliation between two (or more) major lineages in eukaryotes. Microheliella maris was an orphan protist, which failed to be placed within the previously described lineages by pioneering phylogenetic analyses. In this study, we analysed a 319-gene alignment and demonstrated that M. maris represents a basal lineage of one of the major eukaryotic lineages, Cryptista. We here propose a new clade name 'Pancryptista' for Cryptista plus M. maris. The 319-gene analyses also indicated that M. maris is a key taxon to recover the monophyly of Archaeplastida and the sister relationship between Archaeplastida and Pancryptista, which is collectively called 'CAM clade' here. Significantly, Cryptophyceae tend to be attracted to Rhodophyta depending on the taxon sampling (ex., in the absence of M. maris and Rhodelphidia) and the particular phylogenetic 'signal' most likely hindered the stable recovery of the monophyly of Archaeplastida in previous studies.

An Enigmatic Stramenopile Sheds Light on Early Evolution in Ochrophyta Plastid Organellogenesis.

Ochrophyta is an algal group belonging to the Stramenopiles and comprises diverse lineages of algae which contribute significantly to the oceanic ecosystems as primary producers. However, early evolution of the plastid organelle in Ochrophyta is not fully understood. In this study, we provide a well-supported tree of the Stramenopiles inferred by the large-scale phylogenomic analysis that unveils the eukaryvorous (nonphotosynthetic) protist Actinophrys sol (Actinophryidae) is closely related to Ochrophyta. We used genomic and transcriptomic data generated from A. sol to detect molecular traits of its plastid and we found no evidence of plastid genome and plastid-mediated biosynthesis, consistent with previous ultrastructural studies that did not identify any plastids in Actinophryidae. Moreover, our phylogenetic analyses of particular biosynthetic pathways provide no evidence of a current and past plastid in A. sol. However, we found more than a dozen organellar aminoacyl-tRNA synthases (aaRSs) that are of algal origin. Close relationships between aaRS from A. sol and their ochrophyte homologs document gene transfer of algal genes that happened before the divergence of Actinophryidae and Ochrophyta lineages. We further showed experimentally that organellar aaRSs of A. sol are targeted exclusively to mitochondria, although organellar aaRSs in Ochrophyta are dually targeted to mitochondria and plastids. Together, our findings suggested that the last common ancestor of Actinophryidae and Ochrophyta had not yet completed the establishment of host-plastid partnership as seen in the current Ochrophyta species, but acquired at least certain nuclear-encoded genes for the plastid functions.

Diplonemids - A Review on "New" Flagellates on the Oceanic Block.

Diplonemids are a group of flagellate protists, that belong to the phylum Euglenozoa alongside euglenids, symbiontids and kinetoplastids. They primarily inhabit marine environments, though are also found in freshwater lakes. Diplonemids have been considered as rare and unimportant eukaryotes for over a century, with only a handful of species described until recently. However, thanks to their unprecedented diversity and abundance in the world oceans, diplonemids now attract increased attention. Recent improvements in isolation and cultivation have enabled characterization of several new genera, warranting a re-examination of all available knowledge gathered so far. Here we summarize available data on diplonemids, focusing on the recent advances in the fields of diversity, ecology, genomics, metabolism, and endosymbionts. We illustrate the life stages of cultivated genera, and summarise all reported interspecies associations, which in turn suggest lifestyles of predation and parasitism. This review also includes the latest classification of diplonemids, with a taxonomic revision of the genus Diplonema. Ongoing efforts to sequence various diplonemids suggest the presence of large and complex genomes, which correlate with the metabolic versatility observed in the model species Paradiplonema papillatum. Finally, we highlight its successful transformation into one of few genetically tractable marine protists.

Optimization of environmental DNA extraction and amplification methods for metabarcoding of deep-sea fish.

Analyses of environmental DNA (eDNA) from macroorganisms in aquatic environments have greatly advanced in recent years. In particular, eDNA metabarcoding of fish using universal PCR primers has been reported in various waters. Although pumped deep-sea water was used for eDNA metabarcoding of deep-sea fish, conventional methods only resulted in small amounts of extracted eDNA and subsequent few or no PCR amplicons. To optimize eDNA metabarcoding of deep-sea fish from pumped deep-sea water, we modified conventional procedures of eDNA extraction and PCR amplification. Here, we propose a modified eDNA extraction method, in which a filter used for eDNA sampling was shredded and incubated in microtubes for efficient lysis of eDNA sources. Total eDNA yield extracted using the modified protocol was approximately six-fold higher than that extracted by the conventional protocol. The PCR enzyme Platinum SuperFi II DNA Polymerase successfully amplified a target region of fish universal primers (MiFish) from trace amounts of eDNA extracted from pumped deep-sea water and suppressed nonspecific amplifications more effectively than the enzyme used in conventional methods. Approximately 93% of the sequence reads acquired by next generation sequencing of these amplicons were derived from fish. The improved procedure presented here provided effective eDNA metabarcoding of deep-sea fish.•A modified eDNA extraction protocol, in which a filter was shredded and incubated in microtubes, increased eDNA yields extracted from pumped deep-sea water over the conventional method.•The PCR enzyme Platinum SuperFi II DNA polymerase improved the amplification efficiency of trace amounts of MiFish objectives in eDNA extracted from pumped deep-sea water with suppressing nonspecific amplifications.•The use of Platinum SuperFi II DNA polymerase for eDNA metabarcoding using MiFish primers resulted in the acquisition of abundant sequence reads of deep-sea fish through next generation sequencing.

Structural Comparison of Diplonemid Communities around the Izu Peninsula, Japan.

Diplonemea (diplonemids) is one of the most abundant and species-rich protist groups in marine environments; however, their community structures among local and seasonal samples have not yet been compared. In the present study, we analyzed four diplonemid community structures around the Izu Peninsula, Japan using barcode sequences amplified from environmental DNA. These sequences and the results of statistical analyses indicated that communities at the same site were more similar to each other than those in the same season. Environmental variables were also measured, and their influence on diplonemid community structures was examined. Salinity, electrical conductivity, and temperature, and their correlated variables, appeared to influence the structures of diplonemid communities, which was consistent with previous findings; however, since the results obtained did not reach statistical significance, further studies are required. A comparison of each diplonemid community indicated that some lineages were unique to specific samples, while others were consistently detected in all samples. Members of the latter type are cosmopolitan candidates and may be better adapted to the environments of the studied area. Future studies that focus on the more adaptive members will provide a more detailed understanding of the mechanisms by which diplonemids are widely distributed in marine environments and will facilitate their utilization as indicator organisms to monitor environmental changes.

Development of robust models for rapid classification of microplastic polymer types based on near infrared hyperspectral images.

Hyperspectral data in the near infrared range were examined for nine common types of plastic particles of 1 mm and 100-500 µm sizes on dry and wet glass fiber filters. Weaker peak intensities were detected for small particles compared to large particles, and the reflectances were weaker at longer wavelengths when the particles were measured on a wet filter. These phenomena are explainable due to the effect of the correlation between the particle size and the absorption of infrared light by water. We constructed robust classification models that are capable of classifying polymer types, regardless of particle size or filter conditions (wet vs. dry), based on hyperspectral data for small particles measured on wet filters. Using the models, we also successfully classified the polymer type of polystyrene beads covered with microalgae, which simulates the natural conditions of microplastics in the ocean. This study suggests that hyperspectral imaging techniques with appropriate classification models allow the identification of microplastics without the time- and labor-consuming procedures of drying samples and removing biofilms, thus enabling more rapid analyses.

Engineered chlorophyll catabolism conferring predator resistance for microalgal biomass production.

Production of valuable compounds including biofuels and pharmaceutical precursors derived from microalgae has garnered significant interest. Stable production of algal biomass is essential to make the microalgal industry commercially feasible. However, one of the largest issues is severe biological contamination by predators grazing the algal biomass, resulting in the crash of outdoor cultures. In the present study, we propose a novel engineering strategy for microalgae to cope with predators. The overexpression of plant chlorophyllase (CLH) in a microalga resulted in the enhancement of resistance to the predator. This result supported our hypothesis that CLH promotes chlorophyll breakdown in the chloroplasts of the microalgae when they are digested by the predator, generating the phototoxic catabolite chlorophyllide that damages the predator. To the best of our knowledge, this is the first study to establish predator-resistant microalgae by enhancing the CLH activity.

Massive occurrence of benthic plastic debris at the abyssal seafloor beneath the Kuroshio Extension, the North West Pacific.

The abyss (3500-6500 m) covers the bulk of the deep ocean floor yet little is known about the extent of plastic debris on the abyssal seafloor. Using video imagery we undertook a quantitative assessment of the debris present on the abyssal seafloor (5700-5800 m depth) beneath the Kuroshio Extension current system in the Northwest Pacific. This body of water is one of the major transit pathways for the massive amounts of debris that are entering the North Pacific Ocean from Asia. Shallower sites (1400-1500 m depth) were also investigated for comparison. The dominant type of debris was single-use plastics - mainly bags and food packaging. The density of the plastic debris (mean 4561 items/km2) in the abyssal zone was the highest recorded for an abyssal plain suggesting that the deep-sea basin in the Northwest Pacific is a significant reservoir of plastic debris.

Plastic additives in deep-sea debris collected from the western North Pacific and estimation for their environmental loads.

Plastic waste has become a growing concern in terms of marine pollution, but little information is available on plastic debris and its possible risks of chemical additives exposure in the deep-sea. This study focused on identification of polymer type and additive concentrations in 21 plastic debris collected from deep-sea of Sagami Bay, Japan and West Pacific Ocean under the Kuroshio Extension and its recirculation gyre (KERG) zone (water depth: 1388-5819 m). Polyethylene (PE) was dominant polymer (57% of the total) in samples, followed by polyvinylchloride (PVC), epoxy resin, polyester (PES), and polypropylene. In plastic additives, bis (2-ethylhexyl) phthalate (DEHP) was detected to be contained in a PVC sheet at concentration of 48%. Butylated hydroxytoluene (BHT) was also detected in PE plastic debris with median concentration of 12,000 ng/g. PES clothes were detected to contain dyeing mixtures, 1,2,4-trichlorobenzene (1,2,4-TCB), up to 42,000 ng/g. Knowing the estimated number of plastic debris under KE current, the minimum burden of chemical additives were estimated that 720 kg of dibutyl phthalate, 570 kg of BHT, 230 kg of DEHP, and 160 kg of 1,2,4-TCB exist on the seabed of KERG zone. This result strongly suggests that enormous amount of hazardous additives lie within plastic debris on abyssal level of the ocean.

Highly Reduced Plastid Genomes of the Non-photosynthetic Dictyochophyceans Pteridomonas spp. (Ochrophyta, SAR) Are Retained for tRNA-Glu-Based Organellar Heme Biosynthesis.

Organisms that have lost their photosynthetic capabilities are present in a variety of eukaryotic lineages, such as plants and disparate algal groups. Most of such non-photosynthetic eukaryotes still carry plastids, as these organelles retain essential biological functions. Most non-photosynthetic plastids possess genomes with varied protein-coding contents. Such remnant plastids are known to be present in the non-photosynthetic, bacteriovorous alga Pteridomonas danica (Dictyochophyceae, Ochrophyta), which, regardless of its obligatory heterotrophic lifestyle, has been reported to retain the typically plastid-encoded gene for ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit (rbcL). The presence of rbcL without photosynthetic activity suggests that investigating the function of plastids in Pteridomonas spp. would likely bring unique insights into understanding the reductive evolution of plastids, their genomes, and plastid functions retained after the loss of photosynthesis. In this study, we demonstrate that two newly established strains of the non-photosynthetic genus Pteridomonas possess highly reduced plastid genomes lacking rbcL gene, in contrast to the previous report. Interestingly, we discovered that all plastid-encoded proteins in Pteridomonas spp. are involved only in housekeeping processes (e.g., transcription, translation and protein degradation), indicating that all metabolite synthesis pathways in their plastids are supported fully by nuclear genome-encoded proteins. Moreover, through an in-depth survey of the available transcriptomic data of another strain of the genus, we detected no candidate sequences for nuclear-encoded, plastid-directed Fe-S cluster assembly pathway proteins, suggesting complete loss of this pathway in the organelle, despite its widespread conservation in non-photosynthetic plastids. Instead, the transcriptome contains plastid-targeted components of heme biosynthesis, glycolysis, and pentose phosphate pathways. The retention of the plastid genomes in Pteridomonas spp. is not explained by the Suf-mediated constraint against loss of plastid genomes, previously proposed for Alveolates, as they lack Suf genes. Bearing all these findings in mind, we propose the hypothesis that plastid DNA is retained in Pteridomonas spp. for the purpose of providing glutamyl-tRNA, encoded by trnE gene, as a substrate for the heme biosynthesis pathway.

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.

Inventory and Evolution of Mitochondrion-localized Family A DNA Polymerases in Euglenozoa.

The order Trypanosomatida has been well studied due to its pathogenicity and the unique biology of the mitochondrion. In Trypanosoma brucei, four DNA polymerases, namely PolIA, PolIB, PolIC, and PolID, related to bacterial DNA polymerase I (PolI), were shown to be localized in mitochondria experimentally. These mitochondrion-localized DNA polymerases are phylogenetically distinct from other family A DNA polymerases, such as bacterial PolI, DNA polymerase gamma (Polγ) in human and yeasts, "plant and protist organellar DNA polymerase (POP)" in diverse eukaryotes. However, the diversity of mitochondrion-localized DNA polymerases in Euglenozoa other than Trypanosomatida is poorly understood. In this study, we discovered putative mitochondrion-localized DNA polymerases in broad members of three major classes of Euglenozoa-Kinetoplastea, Diplonemea, and Euglenida-to explore the origin and evolution of trypanosomatid PolIA-D. We unveiled distinct inventories of mitochondrion-localized DNA polymerases in the three classes: (1) PolIA is ubiquitous across the three euglenozoan classes, (2) PolIB, C, and D are restricted in kinetoplastids, (3) new types of mitochondrion-localized DNA polymerases were identified in a prokinetoplastid and diplonemids, and (4) evolutionarily distinct types of POP were found in euglenids. We finally propose scenarios to explain the inventories of mitochondrion-localized DNA polymerases in Kinetoplastea, Diplonemea, and Euglenida.

Genes functioned in kleptoplastids of Dinophysis are derived from haptophytes rather than from cryptophytes.

Toxic dinoflagellates belonging to the genus Dinophysis acquire plastids indirectly from cryptophytes through the consumption of the ciliate Mesodinium rubrum. Dinophysis acuminata harbours three genes encoding plastid-related proteins, which are thought to have originated from fucoxanthin dinoflagellates, haptophytes and cryptophytes via lateral gene transfer (LGT). Here, we investigate the origin of these plastid proteins via RNA sequencing of species related to D. fortii. We identified 58 gene products involved in porphyrin, chlorophyll, isoprenoid and carotenoid biosyntheses as well as in photosynthesis. Phylogenetic analysis revealed that the genes associated with chlorophyll and carotenoid biosyntheses and photosynthesis originated from fucoxanthin dinoflagellates, haptophytes, chlorarachniophytes, cyanobacteria and cryptophytes. Furthermore, nine genes were laterally transferred from fucoxanthin dinoflagellates, whose plastids were derived from haptophytes. Notably, transcription levels of different plastid protein isoforms varied significantly. Based on these findings, we put forth a novel hypothesis regarding the evolution of Dinophysis plastids that ancestral Dinophysis species acquired plastids from haptophytes or fucoxanthin dinoflagellates, whereas LGT from cryptophytes occurred more recently. Therefore, the evolutionary convergence of genes following LGT may be unlikely in most cases.

Morphological, Ultrastructural, Motility and Evolutionary Characterization of Two New Hemistasiidae Species.

Until now, Hemistasia phaeocysticola was the only representative of the monogeneric family Hemistasiidae available in culture. Here we describe two new axenized hemistasiids isolated from Tokyo Bay, Japan. Like in other diplonemids, cellular organization of these heterotrophic protists is characterized by a distinct apical papilla, a tubular cytopharynx contiguous with a deep flagellar pocket, and a highly branched mitochondrion with lamellar cristae. Both hemistasiids also bear a prominent digestive vacuole, peripheral lacunae, and paraflagellar rods, are highly motile and exhibit diverse morphologies in culture. We argue that significant differences in molecular phylogenetics and ultrastructure between these new species and H. phaeocysticola are on the generic level. Therefore, we have established two new genera within Hemistasiidae - Artemidia gen. n. and Namystynia gen. n. to accommodate Artemidia motanka, sp. n. and Namystynia karyoxenos, sp. n., respectively. A. motanka permanently carries tubular extrusomes, while in N. karyoxenos, they are present only in starving cells. An additional remarkable feature of the latter species is the presence, in both the cytoplasm and the nucleus, of the endosymbiotic rickettsiid Candidatus Sneabacter namystus.

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.

Ophirina amphinema n. gen., n. sp., a New Deeply Branching Discobid with Phylogenetic Affinity to Jakobids.

We report a novel nanoflagellate, Ophirina amphinema n. gen. n. sp., isolated from a lagoon of the Solomon Islands. The flagellate displays 'typical excavate' morphological characteristics, such as the presence of a ventral feeding groove with vanes on the posterior flagellum. The cell is ca. 4 µm in length, bears two flagella, and has a single mitochondrion with flat to discoid cristae. The flagellate exists in two morphotypes: a suspension-feeder, which bears flagella that are about the length of the cell, and a swimmer, which has longer flagella. In a tree based on the analysis of 156 proteins, Ophirina is sister to jakobids, with moderate bootstrap support. Ophirina has some ultrastructural (e.g. B-fibre associated with the posterior basal body) and mtDNA (e.g. rpoA-D) features in common with jakobids. Yet, other morphological features, including the crista morphology and presence of two flagellar vanes, rather connect Ophirina to non-jakobid or non-discobid excavates. Ophirina amphinema has some unique features, such as an unusual segmented core structure within the basal bodies and a rightward-oriented dorsal fan. Thus, Ophirina represents a new deeply-branching member of Discoba, and its mosaic morphological characteristics may illuminate aspects of the ancestral eukaryotic cellular body plan.