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.

Subunit composition of mitochondrial dehydrogenase complexes in diplonemid flagellates.

In eukaryotes, pyruvate, a key metabolite produced by glycolysis, is converted by a tripartite mitochondrial pyruvate dehydrogenase (PDH) complex to acetyl-coenzyme A, which is fed into the tricarboxylic acid cycle. Two additional enzyme complexes with analogous composition catalyze similar oxidative decarboxylation reactions albeit using different substrates, the branched-chain ketoacid dehydrogenase (BCKDH) complex and the 2-oxoglutarate dehydrogenase (OGDH) complex. Comparative transcriptome analyses of diplonemids, one of the most abundant and diverse groups of oceanic protists, indicate that the conventional E1, E2, and E3 subunits of the PDH complex are lacking. E1 was apparently replaced in the euglenozoan ancestor of diplonemids by an AceE protein of archaeal type, a substitution that we also document in dinoflagellates. Here, we demonstrate that the mitochondrion of the model diplonemid Paradiplonema papillatum displays pyruvate and 2-oxoglutarate dehydrogenase activities. Protein mass spectrometry of mitochondria reveal that the AceE protein is as abundant as the E1 subunit of BCKDH. This corroborates the view that the AceE subunit is a functional component of the PDH complex. We hypothesize that by acquiring AceE, the diplonemid ancestor not only lost the eukaryotic-type E1, but also the E2 and E3 subunits of the PDH complex, which are present in other euglenozoans. We posit that the PDH activity in diplonemids seems to be carried out by a complex, in which the AceE protein partners with the E2 and E3 subunits from BCKDH and/or OGDH.

Typical structure of rRNA coding genes in diplonemids points to two independent origins of the bizarre rDNA structures of euglenozoans.

BACKGROUND: Members of Euglenozoa (Discoba) are known for unorthodox rDNA organization. In Euglenida rDNA is located on extrachromosomal circular DNA. In Kinetoplastea and Euglenida the core of the large ribosomal subunit, typically formed by the 28S rRNA, consists of several smaller rRNAs. They are the result of the presence of additional internal transcribed spacers (ITSs) in the rDNA. Diplonemea is the third of the main groups of Euglenozoa and its members are known to be among the most abundant and diverse protists in the oceans. Despite that, the rRNA of only one diplonemid species, Diplonema papillatum, has been examined so far and found to exhibit continuous 28S rRNA. Currently, the rDNA organization has not been researched for any diplonemid. Herein we investigate the structure of rRNA genes in classical (Diplonemidae) and deep-sea diplonemids (Eupelagonemidae), representing the majority of known diplonemid diversity. The results fill the gap in knowledge about diplonemid rDNA and allow better understanding of the evolution of the fragmented structure of the rDNA in Euglenozoa. RESULTS: We used available genomic (culture and single-cell) sequencing data to assemble complete or almost complete rRNA operons for three classical and six deep-sea diplonemids. The rDNA sequences acquired for several euglenids and kinetoplastids were used to provide the background for the analysis. In all nine diplonemids, 28S rRNA seems to be contiguous, with no additional ITSs detected. Similarly, no additional ITSs were detected in basal prokinetoplastids. However, we identified five additional ITSs in the 28S rRNA of all analysed metakinetoplastids, and up to twelve in euglenids. Only three of these share positions, and they cannot be traced back to their common ancestor. CONCLUSIONS: Presented results indicate that independent origin of additional ITSs in euglenids and kinetoplastids seems to be the most likely. The reason for such unmatched fragmentation remains unknown, but for some reason euglenozoan ribosomes appear to be prone to 28S rRNA fragmentation.

Reductionist Pathways for Parasitism in Euglenozoans? Expanded Datasets Provide New Insights.

The unicellular trypanosomatids belong to the phylum Euglenozoa and all known species are obligate parasites. Distinct lineages infect plants, invertebrates, and vertebrates, including humans. Genome data for marine diplonemids, together with freshwater euglenids and free-living kinetoplastids, the closest known nonparasitic relatives to trypanosomatids, recently became available. Robust phylogenetic reconstructions across Euglenozoa are now possible and place the results of parasite-focused studies into an evolutionary context. Here we discuss recent advances in identifying the factors shaping the evolution of Euglenozoa, focusing on ancestral features generally considered parasite-specific. Remarkably, most of these predate the transition(s) to parasitism, suggesting that the presence of certain preconditions makes a significant lifestyle change more likely.

Unique Dynamics of Paramylon Storage in the Marine Euglenozoan Diplonema papillatum.

Diplonemids belong to the most diverse and abundant marine protists, which places them among the key players of the oceanic ecosystem. Under in vitro conditions, their best-known representative Diplonema papillatum accumulates in its cytoplasm a crystalline polymer. When grown under the nutrient-poor conditions, but not nutrient-rich conditions, D. papillatum synthesizes a ß-1,3-glucan polymer, also known as paramylon. This phenomenon is unexpected, as it is in striking contrast to the accumulation of paramylon in euglenids, since these related flagellates synthesize this polymer solely under nutrient-rich conditions. The capacity of D. papillatum to store an energy source in the form of polysaccharides when the environment is poor in nutrients is unexpected and may contribute to the wide distribution of these protists in the ocean.

Widespread Dominance of Kinetoplastids and Unexpected Presence of Diplonemids in Deep Freshwater Lakes.

Kinetoplastid flagellates are generally abundant in the deep sea and recently they were even found to be dominant in the hypolimnion of a deep freshwater lake. Therefore, to understand the distribution of kinetoplastids in deep freshwater lakes, we have collected vertical samples from five lakes in Japan. The abundance of kinetoplastids was enumerated by Catalyzed Reporter Deposition-Fluorescence in situ Hybridization, and the diversity was determined by 18S amplicon sequencing using universal eukaryote and kinetoplastid-specific primers. Kinetoplastids were abundant in the deep waters of all the lakes, contributing up to 53.6% of total nanoeukaryotes. Despite this significant contribution, kinetoplastids remain undetected by amplicon sequencing using universal primers that are widely used in eukaryotic diversity studies. However, they were detected with specific primers, and the communities were characterized by both ubiquitous and lake-specific unique OTUs. Oligotyping of a ubiquitous and dominant OTU revealed the presence of lake-specific sequence types (oligotypes). Remarkably, we also detected diplonemids (a sister group of kinetoplastids and considered to be specific in the marine habitat) using kinetoplastid-specific primers, showing their presence in freshwaters. Underestimation of kinetoplastids and diplonemids using universal primers indicates that euglenozoan flagellates are overlooked in diversity studies worldwide. The present study highlighted the importance of kinetoplastids in the hypolimnion of deep lakes, thereby indicating their role in material cycling in deep waters.

A single-cell genome reveals diplonemid-like ancestry of kinetoplastid mitochondrial gene structure.

Euglenozoa comprises euglenids, kinetoplastids, and diplonemids, with each group exhibiting different and highly unusual mitochondrial genome organizations. Although they are sister groups, kinetoplastids and diplonemids have very distinct mitochondrial genome architectures, requiring widespread insertion/deletion RNA editing and extensive trans-splicing, respectively, in order to generate functional transcripts. The evolutionary history by which these differing processes arose remains unclear. Using single-cell genomics, followed by small sub unit ribosomal DNA and multigene phylogenies, we identified an isolated marine cell that branches on phylogenetic trees as a sister to known kinetoplastids. Analysis of single-cell amplified genomic material identified multiple mitochondrial genome contigs. These revealed a gene architecture resembling that of diplonemid mitochondria, with small fragments of genes encoded out of order and or on different contigs, indicating that these genes require extensive trans-splicing. Conversely, no requirement for kinetoplastid-like insertion/deletion RNA-editing was detected. Additionally, while we identified some proteins so far only found in kinetoplastids, we could not unequivocally identify mitochondrial RNA editing proteins. These data invite the hypothesis that extensive genome fragmentation and trans-splicing were the ancestral states for the kinetoplastid-diplonemid clade but were lost during the kinetoplastid radiation. This study demonstrates that single-cell approaches can successfully retrieve lineages that represent important new branches on the tree of life, and thus can illuminate major evolutionary and functional transitions in eukaryotes. This article is part of a discussion meeting issue 'Single cell ecology'.

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.

Comparative molecular cell biology of phototrophic euglenids and parasitic trypanosomatids sheds light on the ancestor of Euglenozoa.

Parasitic trypanosomatids and phototrophic euglenids are among the most extensively studied euglenozoans. The phototrophic euglenid lineage arose relatively recently through secondary endosymbiosis between a phagotrophic euglenid and a prasinophyte green alga that evolved into the euglenid secondary chloroplast. The parasitic trypanosomatids (i.e. Trypanosoma spp. and Leishmania spp.) and the freshwater phototrophic euglenids (i.e. Euglena gracilis) are the most evolutionary distant lineages in the Euglenozoa phylogenetic tree. The molecular and cell biological traits they share can thus be considered as ancestral traits originating in the common euglenozoan ancestor. These euglenozoan ancestral traits include common mitochondrial presequence motifs, respiratory chain complexes containing various unique subunits, a unique ATP synthase structure, the absence of mitochondria-encoded transfer RNAs (tRNAs), a nucleus with a centrally positioned nucleolus, closed mitosis without dissolution of the nuclear membrane and nucleoli, a nuclear genome containing the unusual 'J' base (ß-D-glucosyl-hydroxymethyluracil), processing of nucleus-encoded precursor messenger RNAs (pre-mRNAs) via spliced-leader RNA (SL-RNA) trans-splicing, post-transcriptional gene silencing by the RNA interference (RNAi) pathway and the absence of transcriptional regulation of nuclear gene expression. Mitochondrial uridine insertion/deletion RNA editing directed by guide RNAs (gRNAs) evolved in the ancestor of the kinetoplastid lineage. The evolutionary origin of other molecular features known to be present only in either kinetoplastids (i.e. polycistronic transcripts, compaction of nuclear genomes) or euglenids (i.e. monocistronic transcripts, huge genomes, many nuclear cis-spliced introns, polyproteins) is unclear.

A Revised Taxonomy of Diplonemids Including the Eupelagonemidae n. fam. and a Type Species, Eupelagonema oceanica n. gen. & sp.

Recent surveys of marine microbial diversity have identified a previously unrecognized lineage of diplonemid protists as being among the most diverse heterotrophic eukaryotes in global oceans. Despite their monophyly (and assumed importance), they lack a formal taxonomic description, and are informally known as deep-sea pelagic diplonemids (DSPDs) or marine diplonemids. Recently, we documented morphology and molecular sequences from several DSPDs, one of which is particularly widespread and abundant in environmental sequence data. To simplify the communication of future work on this important group, here we formally propose to erect the family Eupelagonemidae to encompass this clade, as well as a formal genus and species description for the apparently most abundant phylotype, Eupelagonema oceanica, for which morphological information and single-cell amplified genome data are currently available.

Extreme Diversity of Diplonemid Eukaryotes in the Ocean.

The world's oceans represent by far the largest biome, with great importance for the global ecosystem [1-4]. The vast majority of ocean biomass and biodiversity is composed of microscopic plankton. Recent results from the Tara Oceans metabarcoding study revealed that a significant part of the plankton in the upper sunlit layer of the ocean is represented by an understudied group of heterotrophic excavate flagellates called diplonemids [5, 6]. We have analyzed the diversity and distribution patterns of diplonemid populations on the extended set of Tara Oceans V9 18S rDNA metabarcodes amplified from 850 size- fractionated plankton communities sampled across 123 globally distributed locations, for the first time also including samples from the mesopelagic zone, which spans the depth from about 200 to 1,000 meters. Diplonemids separate into four major clades, with the vast majority falling into the deep-sea pelagic diplonemid clade. Remarkably, diversity of this clade inferred from metabarcoding data surpasses even that of dinoflagellates, metazoans, and rhizarians, qualifying diplonemids as possibly the most diverse group of marine planktonic eukaryotes. Diplonemids display strong vertical separation between the photic and mesopelagic layers, with the majority of their relative abundance and diversity occurring in deeper waters. Globally, diplonemids display no apparent biogeographic structuring, with a few hyperabundant cosmopolitan operational taxonomic units (OTUs) dominating their communities. Our results suggest that the planktonic diplonemids are among the key heterotrophic players in the largest ecosystem of our biosphere, yet their roles in this ecosystem remain unknown.

Post-transcriptional mending of gene sequences: Looking under the hood of mitochondrial gene expression in diplonemids.

The instructions to make proteins and structural RNAs are laid down in gene sequences. Yet, in certain instances, these primary instructions need to be modified considerably during gene expression, most often at the transcript level. Here we review a case of massive post-transcriptional revisions via trans-splicing and RNA editing, a phenomenon occurring in mitochondria of a recently recognized protist group, the diplonemids. As of now, the various post-transcriptional steps have been cataloged in detail, but how these processes function is still unknown. Since genetic manipulation techniques such as gene replacement and RNA interference have not yet been established for these organisms, alternative strategies have to be deployed. Here, we discuss the experimental and bioinformatics approaches that promise to unravel the molecular machineries of trans-splicing and RNA editing in Diplonema mitochondria.

Genes in Hiding.

Unrecognizable genes are an unsettling problem in genomics. Here, we survey the various types of cryptic genes and the corresponding deciphering strategies employed by cells. Encryption that renders genes substantially different from homologs in other species includes sequence substitution, insertion, deletion, fragmentation plus scrambling, and invasion by mobile genetic elements. Cells decode cryptic genes at the DNA, RNA or protein level. We will focus on a recently discovered case of unparalleled encryption involving massive gene fragmentation and nucleotide deletions and substitutions, occurring in the mitochondrial genome of a poorly understood protist group, the diplonemids. This example illustrates that comprehensive gene detection requires not only auxiliary sequence information - transcriptome and proteome data - but also knowledge about a cell's deciphering arsenal.

Non-functional genes repaired at the RNA level.

Genomes and genes continuously evolve. Gene sequences undergo substitutions, deletions or nucleotide insertions; mobile genetic elements invade genomes and interleave in genes; chromosomes break, even within genes, and pieces reseal in reshuffled order. To maintain functional gene products and assure an organism's survival, two principal strategies are used - either repair of the gene itself or of its product. I will introduce common types of gene aberrations and how gene function is restored secondarily, and then focus on systematically fragmented genes found in a poorly studied protist group, the diplonemids. Expression of their broken genes involves restitching of pieces at the RNA-level, and substantial RNA editing, to compensate for point mutations. I will conclude with thoughts on how such a grotesquely unorthodox system may have evolved, and why this group of organisms persists and thrives since tens of millions of years.

Differential remodelling of peroxisome function underpins the environmental and metabolic adaptability of diplonemids and kinetoplastids.

The remodelling of organelle function is increasingly appreciated as a central driver of eukaryotic biodiversity and evolution. Kinetoplastids including Trypanosoma and Leishmania have evolved specialized peroxisomes, called glycosomes. Glycosomes uniquely contain a glycolytic pathway as well as other enzymes, which underpin the physiological flexibility of these major human pathogens. The sister group of kinetoplastids are the diplonemids, which are among the most abundant eukaryotes in marine plankton. Here we demonstrate the compartmentalization of gluconeogenesis, or glycolysis in reverse, in the peroxisomes of the free-living marine diplonemid, Diplonema papillatum Our results suggest that peroxisome modification was already under way in the common ancestor of kinetoplastids and diplonemids, and raise the possibility that the central importance of gluconeogenesis to carbon metabolism in the heterotrophic free-living ancestor may have been an important selective driver. Our data indicate that peroxisome modification is not confined to the kinetoplastid lineage, but has also been a factor in the success of their free-living euglenozoan relatives.