Phylogenomic position of eupelagonemids, abundant, and diverse deep-ocean heterotrophs.

Eupelagonemids, formerly known as Deep Sea Pelagic Diplonemids I (DSPD I), are among the most abundant and diverse heterotrophic protists in the deep ocean, but little else is known about their ecology, evolution, or biology in general. Originally recognized solely as a large clade of environmental ribosomal subunit RNA gene sequences (SSU rRNA), branching with a smaller sister group DSPD II, they were postulated to be diplonemids, a poorly studied branch of Euglenozoa. Although new diplonemids have been cultivated and studied in depth in recent years, the lack of cultured eupelagonemids has limited data to a handful of light micrographs, partial SSU rRNA gene sequences, a small number of genes from single amplified genomes, and only a single formal described species, Eupelagonema oceanica. To determine exactly where this clade goes in the tree of eukaryotes and begin to address the overall absence of biological information about this apparently ecologically important group, we conducted single-cell transcriptomics from two eupelagonemid cells. A SSU rRNA gene phylogeny shows that these two cells represent distinct subclades within eupelagonemids, each different from E. oceanica. Phylogenomic analysis based on a 125-gene matrix contrasts with the findings based on ecological survey data and shows eupelagonemids branch sister to the diplonemid subgroup Hemistasiidae.

Miniature RNAs are embedded in an exceptionally protein-rich mitoribosome via an elaborate assembly pathway.

The mitochondrial ribosome (mitoribosome) has diverged drastically from its evolutionary progenitor, the bacterial ribosome. Structural and compositional diversity is particularly striking in the phylum Euglenozoa, with an extraordinary protein gain in the mitoribosome of kinetoplastid protists. Here we report an even more complex mitoribosome in diplonemids, the sister-group of kinetoplastids. Affinity pulldown of mitoribosomal complexes from Diplonema papillatum, the diplonemid type species, demonstrates that they have a mass of > 5 MDa, contain as many as 130 integral proteins, and exhibit a protein-to-RNA ratio of 11:1. This unusual composition reflects unprecedented structural reduction of ribosomal RNAs, increased size of canonical mitoribosomal proteins, and accretion of three dozen lineage-specific components. In addition, we identified >50 candidate assembly factors, around half of which contribute to early mitoribosome maturation steps. Because little is known about early assembly stages even in model organisms, our investigation of the diplonemid mitoribosome illuminates this process. Together, our results provide a foundation for understanding how runaway evolutionary divergence shapes both biogenesis and function of a complex molecular machine.

Recent expansion of metabolic versatility in Diplonema papillatum, the model species of a highly speciose group of marine eukaryotes.

BACKGROUND: Diplonemid flagellates are among the most abundant and species-rich of known marine microeukaryotes, colonizing all habitats, depths, and geographic regions of the world ocean. However, little is known about their genomes, biology, and ecological role. RESULTS: We present the first nuclear genome sequence from a diplonemid, the type species Diplonema papillatum. The ~ 280-Mb genome assembly contains about 32,000 protein-coding genes, likely co-transcribed in groups of up to 100. Gene clusters are separated by long repetitive regions that include numerous transposable elements, which also reside within introns. Analysis of gene-family evolution reveals that the last common diplonemid ancestor underwent considerable metabolic expansion. D. papillatum-specific gains of carbohydrate-degradation capability were apparently acquired via horizontal gene transfer. The predicted breakdown of polysaccharides including pectin and xylan is at odds with reports of peptides being the predominant carbon source of this organism. Secretome analysis together with feeding experiments suggest that D. papillatum is predatory, able to degrade cell walls of live microeukaryotes, macroalgae, and water plants, not only for protoplast feeding but also for metabolizing cell-wall carbohydrates as an energy source. The analysis of environmental barcode samples shows that D. papillatum is confined to temperate coastal waters, presumably acting in bioremediation of eutrophication. CONCLUSIONS: Nuclear genome information will allow systematic functional and cell-biology studies in D. papillatum. It will also serve as a reference for the highly diverse diplonemids and provide a point of comparison for studying gene complement evolution in the sister group of Kinetoplastida, including human-pathogenic taxa.

18S rDNA sequence-structure phylogeny of the Euglenophyceae (Euglenozoa, Euglenida).

The phylogeny of Euglenophyceae (Euglenozoa, Euglenida) has been discussed for decades with new genera being described in the last few years. In this study, we reconstruct a phylogeny using 18S rDNA sequence and structural data simultaneously. Using homology modeling, individual secondary structures were predicted. Sequence-structure data are encoded and automatically aligned. Here, we present a sequence-structure neighbor-joining tree of more than 300 taxa classified as Euglenophyceae. Profile neighbor-joining was used to resolve the basal branching pattern. Neighbor-joining, maximum parsimony, and maximum likelihood analyses were performed using sequence-structure information for manually chosen subsets. All analyses supported the monophyly of Eutreptiella, Discoplastis, Lepocinclis, Strombomonas, Cryptoglena, Monomorphina, Euglenaria, and Colacium. Well-supported topologies were generally consistent with previous studies using a combined dataset of genetic markers. Our study supports the simultaneous use of sequence and structural data to reconstruct more accurate and robust trees. The average bootstrap value is significantly higher than the average bootstrap value obtained from sequence-only analyses, which is promising for resolving relationships between more closely related taxa.

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.

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.

Highly flexible metabolism of the marine euglenozoan protist Diplonema papillatum.

BACKGROUND: The phylum Euglenozoa is a group of flagellated protists comprising the diplonemids, euglenids, symbiontids, and kinetoplastids. The diplonemids are highly abundant and speciose, and recent tools have rendered the best studied representative, Diplonema papillatum, genetically tractable. However, despite the high diversity of diplonemids, their lifestyles, ecological functions, and even primary energy source are mostly unknown. RESULTS: We designed a metabolic map of D. papillatum cellular bioenergetic pathways based on the alterations of transcriptomic, proteomic, and metabolomic profiles obtained from cells grown under different conditions. Comparative analysis in the nutrient-rich and nutrient-poor media, as well as the absence and presence of oxygen, revealed its capacity for extensive metabolic reprogramming that occurs predominantly on the proteomic rather than the transcriptomic level. D. papillatum is equipped with fundamental metabolic routes such as glycolysis, gluconeogenesis, TCA cycle, pentose phosphate pathway, respiratory complexes, ß-oxidation, and synthesis of fatty acids. Gluconeogenesis is uniquely dominant over glycolysis under all surveyed conditions, while the TCA cycle represents an eclectic combination of standard and unusual enzymes. CONCLUSIONS: The identification of conventional anaerobic enzymes reflects the ability of this protist to survive in low-oxygen environments. Furthermore, its metabolism quickly reacts to restricted carbon availability, suggesting a high metabolic flexibility of diplonemids, which is further reflected in cell morphology and motility, correlating well with their extreme ecological valence.

18S rRNA gene sequence-structure phylogeny of the Trypanosomatida (Kinetoplastea, Euglenozoa) with special reference to Trypanosoma.

Parasites of the order Trypanosomatida are known due to their medical relevance. Despite the progress made in the past decades on understanding the evolution of this group of organisms, there are still many open questions that require robust phylogenetic markers to increase the resolution of trees. Using two known 18S rRNA gene template structures (from Trypanosoma cruzi Chagas, 1909 and Trypanosoma brucei Plimmer and Bradford, 1899), individual 18S rRNA gene secondary structures were predicted by homology modeling. Sequences and their secondary structures, automatically encoded by a 12-letter alphabet (each nucleotide with its three structural states, paired left, paired right, unpaired), were simultaneously aligned. Sequence-structure trees were generated by neighbor joining and/or maximum likelihood. The reconstructed trees allowed us to discuss not only the big picture of trypanosomatid phylogeny but also a comprehensive sampling of trypanosomes evaluated in the context of trypanosomatid diversity. The robust support (bootstrap > 75) for well-known clades and critical branches suggests that the simultaneous use of 18S rRNA sequence and secondary structure data can reconstruct robust phylogenetic trees and can be used by the trypanosomatid research community for future analysis.

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.

Single-cell genomics unveils a canonical origin of the diverse mitochondrial genomes of euglenozoans.

BACKGROUND: The supergroup Euglenozoa unites heterotrophic flagellates from three major clades, kinetoplastids, diplonemids, and euglenids, each of which exhibits extremely divergent mitochondrial characteristics. Mitochondrial genomes (mtDNAs) of euglenids comprise multiple linear chromosomes carrying single genes, whereas mitochondrial chromosomes are circular non-catenated in diplonemids, but circular and catenated in kinetoplastids. In diplonemids and kinetoplastids, mitochondrial mRNAs require extensive and diverse editing and/or trans-splicing to produce mature transcripts. All known euglenozoan mtDNAs exhibit extremely short mitochondrial small (rns) and large (rnl) subunit rRNA genes, and absence of tRNA genes. How these features evolved from an ancestral bacteria-like circular mitochondrial genome remains unanswered. RESULTS: We sequenced and assembled 20 euglenozoan single-cell amplified genomes (SAGs). In our phylogenetic and phylogenomic analyses, three SAGs were placed within kinetoplastids, 14 within diplonemids, one (EU2) within euglenids, and two SAGs with nearly identical small subunit rRNA gene (18S) sequences (EU17/18) branched as either a basal lineage of euglenids, or as a sister to all euglenozoans. Near-complete mitochondrial genomes were identified in EU2 and EU17/18. Surprisingly, both EU2 and EU17/18 mitochondrial contigs contained multiple genes and one tRNA gene. Furthermore, EU17/18 mtDNA possessed several features unique among euglenozoans including full-length rns and rnl genes, six mitoribosomal genes, and nad11, all likely on a single chromosome. CONCLUSIONS: Our data strongly suggest that EU17/18 is an early-branching euglenozoan with numerous ancestral mitochondrial features. Collectively these data contribute to untangling the early evolution of euglenozoan mitochondria.

Euglenozoa: taxonomy, diversity and ecology, symbioses and viruses.

Euglenozoa is a species-rich group of protists, which have extremely diverse lifestyles and a range of features that distinguish them from other eukaryotes. They are composed of free-living and parasitic kinetoplastids, mostly free-living diplonemids, heterotrophic and photosynthetic euglenids, as well as deep-sea symbiontids. Although they form a well-supported monophyletic group, these morphologically rather distinct groups are almost never treated together in a comparative manner, as attempted here. We present an updated taxonomy, complemented by photos of representative species, with notes on diversity, distribution and biology of euglenozoans. For kinetoplastids, we propose a significantly modified taxonomy that reflects the latest findings. Finally, we summarize what is known about viruses infecting euglenozoans, as well as their relationships with ecto- and endosymbiotic bacteria.

Phage Origin of Mitochondrion-Localized Family A DNA Polymerases in Kinetoplastids and Diplonemids.

Mitochondria retain their own genomes as other bacterial endosymbiont-derived organelles. Nevertheless, no protein for DNA replication and repair is encoded in any mitochondrial genomes (mtDNAs) assessed to date, suggesting that the nucleus primarily governs the maintenance of mtDNA. As the proteins of diverse evolutionary origins occupy a large proportion of the current mitochondrial proteomes, we anticipate finding the same evolutionary trend in the nucleus-encoded machinery for mtDNA maintenance. Indeed, none of the DNA polymerases (DNAPs) in the mitochondrial endosymbiont, a putative α-proteobacterium, seemingly had been inherited by their descendants (mitochondria), as none of the known types of mitochondrion-localized DNAP showed a specific affinity to the α-proteobacterial DNAPs. Nevertheless, we currently have no concrete idea of how and when the known types of mitochondrion-localized DNAPs emerged. We here explored the origins of mitochondrion-localized DNAPs after the improvement of the samplings of DNAPs from bacteria and phages/viruses. Past studies have revealed that a set of mitochondrion-localized DNAPs in kinetoplastids and diplonemids, namely PolIB, PolIC, PolID, PolI-Perk1/2, and PolI-dipl (henceforth designated collectively as "PolIBCD+") have emerged from a single DNAP. In this study, we recovered an intimate connection between PolIBCD+ and the DNAPs found in a particular group of phages. Thus, the common ancestor of kinetoplastids and diplonemids most likely converted a laterally acquired phage DNAP into a mitochondrion-localized DNAP that was ancestral to PolIBCD+. The phage origin of PolIBCD+ hints at a potentially large contribution of proteins acquired via nonvertical processes to the machinery for mtDNA maintenance in kinetoplastids and diplonemids.

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.

A freshwater radiation of diplonemids.

Diplonemids are considered marine protists and have been reported among the most abundant and diverse eukaryotes in the world oceans. Recently we detected the presence of freshwater diplonemids in Japanese deep freshwater lakes. However, their distribution and abundances in freshwater ecosystems remain unknown. We assessed abundance and diversity of diplonemids from several geographically distant deep freshwater lakes of the world by amplicon-sequencing, shotgun metagenomics and catalysed reporter deposition-fluorescent in situ hybridization (CARD-FISH). We found diplonemids in all the studied lakes, albeit with low abundances and diversity. We assembled long 18S rRNA sequences from freshwater diplonemids and showed that they form a new lineage distinct from the diverse marine clades. Freshwater diplonemids are a sister-group to a marine clade, which are mainly isolates from coastal and bay areas, suggesting a recent habitat transition from marine to freshwater habitats. Images of CARD-FISH targeted freshwater diplonemids suggest they feed on bacteria. Our analyses of 18S rRNA sequences retrieved from single-cell genomes of marine diplonemids show they encode multiple rRNA copies that may be very divergent from each other, suggesting that marine diplonemid abundance and diversity both have been overestimated. These results have wider implications on assessing eukaryotic abundances in natural habitats by using amplicon-sequencing alone.

Environmental determinants of the distribution of planktonic diplonemids and kinetoplastids in the oceans.

We analysed a widely used barcode, the V9 region of the 18S rRNA gene, to study the effect of environmental conditions on the distribution of two related heterotrophic protistan lineages in marine plankton, kinetoplastids and diplonemids. We relied on a major published dataset (Tara Oceans) where samples from the mesopelagic zone were available from just 32 of 123 locations, and both groups are most abundant in this zone. To close sampling gaps and obtain more information from the deeper ocean, we collected 57 new samples targeting especially the mesopelagic zone. We sampled in three geographic regions: the Arctic, two depth transects in the Adriatic Sea, and the anoxic Cariaco Basin. In agreement with previous studies, both protist groups are most abundant and diverse in the mesopelagic zone. In addition to that, we found that their abundance, richness, and community structure also depend on geography, oxygen concentration, salinity, temperature, and other environmental variables reflecting the abundance of algae and nutrients. Both groups studied here demonstrated similar patterns, although some differences were also observed. Kinetoplastids and diplonemids prefer tropical regions and nutrient-rich conditions and avoid high oxygen concentration, high salinity, and high density of algae.

Targeted integration by homologous recombination enables in situ tagging and replacement of genes in the marine microeukaryote Diplonema papillatum.

Diplonemids are a group of highly diverse and abundant marine microeukaryotes that belong to the phylum Euglenozoa and form a sister clade to the well-studied, mostly parasitic kinetoplastids. Very little is known about the biology of diplonemids, as few species have been formally described and just one, Diplonema papillatum, has been studied to a decent extent at the molecular level. Following up on our previous results showing stable but random integration of delivered extraneous DNA, we demonstrate here homologous recombination in D. papillatum. Targeting various constructs to the intended position in the nuclear genome was successful when 5' and 3' homologous regions longer than 1 kbp were used, achieving N-terminal tagging with mCherry and gene replacement of α- and ß-tubulins. For more convenient genetic manipulation, we designed a modular plasmid, pDP002, which bears a protein-A tag and used it to generate and express a C-terminally tagged mitoribosomal protein. Lastly, we developed an improved transformation protocol for broader applicability across laboratories. Our robust methodology allows the replacement, integration as well as endogenous tagging of D. papillatum genes, thus opening the door to functional studies in this species and establishing a basic toolkit for reverse genetics of diplonemids in general.

Evolution of metabolic capabilities and molecular features of diplonemids, kinetoplastids, and euglenids.

BACKGROUND: The Euglenozoa are a protist group with an especially rich history of evolutionary diversity. They include diplonemids, representing arguably the most species-rich clade of marine planktonic eukaryotes; trypanosomatids, which are notorious parasites of medical and veterinary importance; and free-living euglenids. These different lifestyles, and particularly the transition from free-living to parasitic, likely require different metabolic capabilities. We carried out a comparative genomic analysis across euglenozoan diversity to see how changing repertoires of enzymes and structural features correspond to major changes in lifestyles. RESULTS: We find a gradual loss of genes encoding enzymes in the evolution of kinetoplastids, rather than a sudden decrease in metabolic capabilities corresponding to the origin of parasitism, while diplonemids and euglenids maintain more metabolic versatility. Distinctive characteristics of molecular machines such as kinetochores and the pre-replication complex that were previously considered specific to parasitic kinetoplastids were also identified in their free-living relatives. Therefore, we argue that they represent an ancestral rather than a derived state, as thought until the present. We also found evidence of ancient redundancy in systems such as NADPH-dependent thiol-redox. Only the genus Euglena possesses the combination of trypanothione-, glutathione-, and thioredoxin-based systems supposedly present in the euglenozoan common ancestor, while other representatives of the phylum have lost one or two of these systems. Lastly, we identified convergent losses of specific metabolic capabilities between free-living kinetoplastids and ciliates. Although this observation requires further examination, it suggests that certain eukaryotic lineages are predisposed to such convergent losses of key enzymes or whole pathways. CONCLUSIONS: The loss of metabolic capabilities might not be associated with the switch to parasitic lifestyle in kinetoplastids, and the presence of a highly divergent (or unconventional) kinetochore machinery might not be restricted to this protist group. The data derived from the transcriptomes of free-living early branching prokinetoplastids suggests that the pre-replication complex of Trypanosomatidae is a highly divergent version of the conventional machinery. Our findings shed light on trends in the evolution of metabolism in protists in general and open multiple avenues for future research.

Gene fragmentation and RNA editing without borders: eccentric mitochondrial genomes of diplonemids.

Diplonemids are highly abundant heterotrophic marine protists. Previous studies showed that their strikingly bloated mitochondrial genome is unique because of systematic gene fragmentation and manifold RNA editing. Here we report a comparative study of mitochondrial genome architecture, gene structure and RNA editing of six recently isolated, phylogenetically diverse diplonemid species. Mitochondrial gene fragmentation and modes of RNA editing, which include cytidine-to-uridine (C-to-U) and adenosine-to-inosine (A-to-I) substitutions and 3' uridine additions (U-appendage), are conserved across diplonemids. Yet as we show here, all these features have been pushed to their extremes in the Hemistasiidae lineage. For example, Namystynia karyoxenos has its genes fragmented into more than twice as many modules than other diplonemids, with modules as short as four nucleotides. Furthermore, we detected in this group multiple A-appendage and guanosine-to-adenosine (G-to-A) substitution editing events not observed before in diplonemids and found very rarely elsewhere. With >1,000 sites, C-to-U and A-to-I editing in Namystynia is nearly 10 times more frequent than in other diplonemids. The editing density of 12% in coding regions makes Namystynia's the most extensively edited transcriptome described so far. Diplonemid mitochondrial genome architecture, gene structure and post-transcriptional processes display such high complexity that they challenge all other currently known systems.

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'.

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.