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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.

Kleptoplasts (kP) are distinct among photosynthetic organelles in eukaryotes (i.e., plastids) because they are routinely sequestered from prey algal cells and function only temporarily in the new host cell. Therefore, the hosts of kleptoplasts benefit from photosynthesis without constitutive photoendosymbiosis. Here, we report that the euglenozoan Rapaza viridis has only kleptoplasts derived from a specific strain of green alga, Tetraselmis sp., but no canonical plastids like those found in its sister group, the Euglenophyceae. R. viridis showed a dynamic change in the accumulation of cytosolic polysaccharides in response to light–dark cycles, and 13 C isotopic labeling of ambient bicarbonate demonstrated that these polysaccharides originate in situ via photosynthesis; these data indicate that the kleptoplasts of R. viridis are functionally active. We also identified 276 sequences encoding putative plastid-targeting proteins and 35 sequences of presumed kleptoplast transporters in the transcriptome of R. viridis . These genes originated in a wide range of algae other than Tetraselmis sp., the source of the kleptoplasts, suggesting a long history of repeated horizontal gene transfer events from different algal prey cells. Many of the kleptoplast proteins, as well as the protein-targeting system, in R. viridis were shared with members of the Euglenophyceae, providing evidence that the early evolutionary stages in the green alga-derived secondary plastids of euglenophytes also involved kleptoplasty.

The notion that mitochondria cannot be lost was shattered with the report of an oxymonad Monocercomonoides exilis, the first eukaryote arguably without any mitochondrion. Yet, questions remain about whether this extends to the entire oxymonad lineage and how this transition took place. The Oxymonadida is a group of gut endobionts taxonomically housed in the Preaxostyla which also contains free-living flagellates of the genera Trimastix and Paratrimastix. The latter two taxa harbour conspicuous mitochondrion-related organelles (MROs). Here we report high-quality genome and transcriptome assemblies of two Preaxostyla representatives, the free-living Paratrimastix pyriformis and the oxymonad Blattamonas nauphoetae. We note that P. pyriformis possesses a set of unique or ancestral features among metamonads or eukaryotes, e.g., p-cresol synthesis, NAD+ synthesis, selenium volatilization, or mercury methylation, demonstrating the biochemical versatility of this protist lineage. We performed thorough comparisons among all available genomic and transcriptomic data of Preaxostyla to further decipher the evolutionary changes towards amitochondriality, endobiosis and unstacked Golgi. Our results provide insights into the metabolic and endomembrane evolution, but most strikingly the data confirm the complete loss of mitochondria for all three oxymonad species investigated (M. exilis, B. nauphoetae, and Streblomastix strix), suggesting the amitochondriate status may be common to Oxymonadida.

Plastids, similar to mitochondria, are organelles of endosymbiotic origin, which retained their vestigial genomes (ptDNA). Their unique architecture, commonly referred to as the quadripartite (four-part) structure, is considered to be strictly conserved; however, the bulk of our knowledge on their variability and evolutionary transformations comes from studies of the primary plastids of green algae and land plants. To broaden our perspective, we obtained seven new ptDNA sequences from freshwater species of photosynthetic euglenids—a group that obtained secondary plastids, known to have dynamically evolving genome structure, via endosymbiosis with a green alga. Our analyses have demonstrated that the evolutionary history of euglenid plastid genome structure is exceptionally convoluted, with a patchy distribution of inverted ribosomal operon (rDNA) repeats, as well as several independent acquisitions of tandemly repeated rDNA copies. Moreover, we have shown that inverted repeats in euglenid ptDNA do not share their genome-stabilizing property documented in chlorophytes. We hypothesize that the degeneration of the quadripartite structure of euglenid plastid genomes is connected to the group II intron expansion. These findings challenge the current global paradigms of plastid genome architecture evolution and underscore the often-underestimated divergence between the functionality of shared traits in primary and complex plastid organelles.

Plastids, similarly to mitochondria, are organelles of endosymbiotic origin, which retained their vestigial genomes (ptDNA). Their unique architecture, commonly referred to as the quadripartite (four-part) structure, is considered to be strictly conserved; however, the bulk of our knowledge on their variability and evolutionary transformations comes from studies of the primary plastids of green algae and land plants. To broaden our perspective, we obtained seven new ptDNA sequences from freshwater species of photosynthetic euglenids – a group which obtained secondary plastids, known to have dynamically evolving genome structure, via endosymbiosis with a green alga. Our analyses have demonstrated that the evolutionary history of euglenid plastid genome structure is exceptionally convoluted, riddled with losses and multiple subsequent regains of inverted ribosomal operon (rDNA) repeats, as well as independent acquisitions of tandemly repeated rDNA copies. Moreover, we have shown that inverted repeats in euglenid ptDNA do not share their genome stabilizing property documented in chlorophytes. We hypothesize that the degeneration of the quadripartite structure of euglenid plastid genomes is connected to the group II intron expansion. These findings challenge the current global paradigms of plastid genome architecture evolution, and underscore the often-underestimated divergence between the functionality of shared traits in primary and complex plastid organelles.

teaserEuglenoids show great promise to benefit our world; as biofuels, environmental remediators, anti-cancer agents, robotics design simulators and food nutritional agents, but the absence of reference genomes currently limit realizing these benefits. The Euglena International Network (EIN) (https://euglenanetwork.org/) aims to address these challenges, and is currently seeking formative phase support and funding.Body startOf the nearly 1000 known species of euglenoids (Triemer and Zakryś, 2015), including Euglena gracilis and Rhabdomonas costata, fewer than 2 % have been explored for any level of translational potential in the past 20 years. The absence of reference genomes currently limits biotechnology applications, including the development of efficient tools for genetic manipulation in euglenoids.EIN aims to advance euglenoid science through a creative amalgam of academic institutions, national research institutes and biotechnology industry, to translate and exploit euglenoids through genome sequencing. EIN has defined goals, mobilized scientists, established a clear roadmap (Grand Challenges), connected academic and industry professionals and is currently formulating policy and partnership principles, driven by EIN Executive and Science committees. However, for EIN’s activities to be maintained and durable, long-term support is vital. We call on national and continental funding agencies and research councils, protists and algae communities, and biotechnology and pharmaceutical industries, to embrace, support and fund translational exploitation of these highly valuable organisms.

Highlights: • Generation of transcriptomes from three new Pirsoniales species for the first time. • Generation of transcriptomes from four new Developea species, enriching the clade. • Phylogenomic recovery of a monophyletic Bigyromonadea: Pirsoniales and Developea. • Bigyormonads’ ability to aggregate and form pseudopod resembles oomycete zoospores. • Report of the first case of eukaryovory in the flagellated stages of Pirsoniales. Stramenopiles are a diverse but relatively well-studied eukaryotic supergroup with considerable genomic information available (Sibbald and Archibald, 2017). Nevertheless, the relationships between major stramenopile subgroups remain unresolved, in part due to a lack of data from small nanoflagellates that make up a lot of the genetic diversity of the group. This is most obvious in Bigyromonadea, which is one of four major stramenopile subgroups but represented by a single transcriptome. To examine the diversity of Bigyromonadea and how the lack of data affects the tree, we generated transcriptomes from seven novel bigyromonada species described in this study: Develocauda condao n. gen. n. sp., Develocanicus komovi n. gen. n. sp., Develocanicus vyazemskyi n. sp., Cubaremonas variflagellatum n. gen. n. sp., Pirsonia chemainus nom. prov., Feodosia pseudopoda nom. prov., and Koktebelia satura nom. prov. Both maximum likelihood and Bayesian phylogenomic trees based on a 247 genematrix recovered a monophyletic Bigyromonadea that includes two diverse subgroups, Developea and Pirsoniales, that were not previously related based on single gene trees. Maximum likelihood analyses show Bigyromonadea related to oomycetes, whereas Bayesian analyses and topology testing were inconclusive. We observed similarities between the novel bigyromonad species and motile zoospores of oomycetes in morphology and the ability to self-aggregate. Rare formation of pseudopods and fused cells were also observed, traits that are also found in members of labyrinthulomycetes, another osmotrophic stramenopiles. Furthermore, we report the first case of eukaryovory in the flagellated stages of Pirsoniales. These analyses reveal new diversity of Bigyromonadea, and altogether suggest their monophyly with oomycetes, collectively known as Pseudofungi, is the most likely topology of the stramenopile tree.

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.

Organellar genomes often carry group II introns, which occasionally encode proteins called maturases that are important for splicing. The number of introns varies substantially among various organellar genomes, and bursts of introns have been observed in multiple eukaryotic lineages, including euglenophytes, with more than 100 introns in their plastid genomes. To examine the evolutionary diversity and history of maturases, an essential gene family among euglenophytes, we searched for their homologs in newly sequenced and published plastid genomes representing all major euglenophytes’ lineages. We found that maturase content in plastid genomes has a patchy distribution, with a maximum of eight of them present in Eutreptiella eupharyngea. The most basal lineages of euglenophytes, Eutreptiales, share the highest number of maturases, but the lowest number of introns. We also identified a peculiar convoluted structure of a gene located in an intron, in a gene within an intron, within yet another gene, present in some Eutreptiales. Further investigation of functional domains of identified maturases shown that most of them lost at least one of the functional domains, which implies that the patchy maturase distribution is due to frequent inactivation and eventual loss over time. Finally, we identified the diversified evolutionary origin of analysed maturases, which were acquired along with the green algal plastid or horizontally transferred. These findings indicate that euglenophytes’ plastid maturases have experienced a surprisingly dynamic history due to gains from diversified donors, their retention, and loss.

Dictyochophyceae (silicoflagellates) are unicellular freshwater and marine algae (Heterokontophyta, stramenopiles). Despite their abundance in global oceans and potential ecological significance, discovered in recent years, neither nuclear nor organellar genomes of representatives of this group were sequenced until now. Here, we present the first complete plastid genome sequences of Dictyochophyceae, obtained from four species: Dictyocha speculum, Rhizochromulina marina, Florenciella parvula and Pseudopedinella elastica. Despite their comparable size and genetic content, these four plastid genomes exhibit variability in their organization: plastid genomes of F. parvula and P. elastica possess conventional quadripartite structure with a pair of inverted repeats, R. marina instead possesses two direct repeats with the same orientation and D. speculum possesses no repeats at all. We also observed a number of unusual traits in the plastid genome of D. speculum, including expansion of the intergenic regions, presence of an intron in the otherwise non-intron-bearing psaA gene, and an additional copy of the large subunit of RuBisCO gene (rbcL), the last of which has never been observed in any plastid genome. We conclude that despite noticeable gene content similarities between the plastid genomes of Dictyochophyceae and their relatives (pelagophytes, diatoms), the number of distinctive features observed in this lineage strongly suggests that additional taxa require further investigation.

Cristamonadea is a large class of parabasalian protists that reside in the hindguts of wood-feeding insects, where they play an essential role in the digestion of lignocellulose. This group of symbionts boasts an impressive array of complex morphological characteristics, many of which have evolved multiple times independently. However, their diversity is understudied and molecular data remain scarce. Here we describe seven new species of cristamonad symbionts from Comatermes, Calcaritermes, and Rugitermes termites from Peru and Ecuador. To classify these new species, we examined cells by light and scanning electron microscopy, sequenced the symbiont small subunit ribosomal RNA (rRNA) genes, and carried out barcoding of the mitochondrial large subunit rRNA gene of the hosts to confirm host identification. Based on these data, five of the symbionts characterized here represent new species within described genera: Devescovina sapara n. sp., Devescovina aymara n. sp., Macrotrichomonas ashaninka n. sp., Macrotrichomonas secoya n. sp., and Macrotrichomonas yanesha n. sp. Additionally, two symbionts with overall morphological characteristics similar to the poorly-studied and probably polyphyletic ‘joeniid’ Parabasalia are classified in a new genus Runanympha n. gen.: Runanympha illapa n. sp., and Runanympha pacha n. sp., This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (RGPIN-2014-03994). JdC, VB, AK, EH and MK were supported by grants to the Centre for Microbial Diversity and Evolution from the Tula Foundation, and NATI was supported by a fellowship from NSERC. JdC was supported by a Marie Curie International Outgoing Fellowship Grant (FP7-PEOPLE-2012-IOF-331450 CAARL).

Motivation With a large number of metagenomic datasets becoming available, eukaryotic metagenomics emerged as a new challenge. The proper classification of eukaryotic nuclear and organellar genomes is an essential step toward a better understanding of eukaryotic diversity. Results We developed Tiara, a deep-learning-based approach for the identification of eukaryotic sequences in the metagenomic datasets. Its two-step classification process enables the classification of nuclear and organellar eukaryotic fractions and subsequently divides organellar sequences into plastidial and mitochondrial. Using the test dataset, we have shown that Tiara performed similarly to EukRep for prokaryotes classification and outperformed it for eukaryotes classification with lower calculation time. In the tests on the real data, Tiara performed better than EukRep in analyzing the small dataset representing eukaryotic cell microbiome and large dataset from the pelagic zone of oceans. Tiara is also the only available tool correctly classifying organellar sequences, which was confirmed by the recovery of nearly complete plastid and mitochondrial genomes from the test data and real metagenomic data. Availability and implementation Tiara is implemented in python 3.8, available at https://github.com/ibe-uw/tiara and tested on Unix-based systems. It is released under an open-source MIT license and documentation is available at https://ibe-uw.github.io/tiara. Version 1.0.1 of Tiara has been used for all benchmarks. Supplementary information Supplementary data are available at Bioinformatics online.

Euglenids represent a group of protists with diverse modes of feeding. To date, only a partial genomic sequence of Euglena gracilis and transcriptomes of several phototrophic and secondarily osmotrophic species are available, while primarily heterotrophic euglenids are seriously undersampled. In this work, we begin to fill this gap by presenting genomic and transcriptomic drafts of a primary osmotroph, Rhabdomonas costata. The current genomic assembly length of 100 Mbp is 14× smaller than that of E. gracilis. Despite being too fragmented for comprehensive gene prediction, comparison of the transcriptomic and genomic data revealed features of its introns, including several candidates for nonconventional introns. 16 % of transcripts bear a recognizable partial splice leader sequence. A set of 39,585 putative R. costata proteins were predicted from the transcriptome. Annotation of the mitochondrial core metabolism provides the first data on the facultatively anaerobic mitochondrion of R. costata, which in most respects resembles the mitochondrion of E. gracilis with certain level of streamlining. R. costata synthesises heme by a mitochondrial-cytoplasmatic C4 pathway with enzymes orthologous to those found in E. gracilis. The low percentage of green algae-affiliated genes, supports the ancestrally osmotrophic status of this species.

Several eukaryotic lineages gained the ability of photosynthesis by acquiring plastids in the events of primary endosymbiosis with cyanobacteria, or secondary endosymbiosis with plastid-bearing eukaryotes. Plastids possess genomes (ptDNA) with genetic contents considerably reduced as a result of gene losses and transfers to the host’s nucleus. Still, ptDNA encodes components of various metabolic processes, including photosynthesis. Plastid genomes usually retain quadripartite structure with two rDNA-bearing inverted repeats, but the reason for its conservation, and the consequences of its decline, have not been fully understood. As the model group to study plastid genome evolution, we chose euglenids (Euglenophyta), whose ancestor acquired the secondary plastid by endosymbiosis with a green alga. The organization of ptDNA in this lineage is rather diverse: we have shown that loss of one repeat occurred at least three times, while some species in the genus Euglena possess tandemly repeated rDNA copies. The ptDNA of euglenids is also intron-rich, but we did not confirm the previously proposed strong correlation between the prevalence of introns and quantity of maturases. Although euglenophytes are predominantly photosynthetic, a few of them lost their photosynthetic capabilities independently. Thus far, only Euglena longa has been shown to possess vestigial plastids with reduced genome; we observed that another strain lost its plastid genome completely. Currently, we are investigating the loss and retention of metabolic functions in the plastids of other non-photosynthetic euglenophytes. This, along with investigation of ptDNA structure, will bring new insights into the evolutionary processes shaping the diversity of eukaryotic plastids.

Photosynthetic euglenids (Euglenophyta) are a monophyletic group of unicellular eukaryotes characterized by the presence of plastids, which arose as the result of the secondary endosymbiosis. Many Euglenophyta plastid (pt) genomes have been characterized recently, but they represented mainly one family – Euglenaceae. Here, we report a comparative analysis of plastid genomes from eight representatives of the family Phacaceae. Newly sequenced plastid genomes share a number of features including synteny and gene content, except for genes mat2 and mat5 encoding maturases. The observed diversity of intron number and presence/absence of maturases corroborated previously suggested correlation between the number of maturases in the pt genome and intron proliferation. Surprisingly, pt genomes of taxa belonging to Discoplastis and Lepocinclis encode two inverted repeat (IR) regions containing the rDNA operon, which are absent from the Euglenaceae. By mapping the presence/absence of IR region on the obtained phylogenomic tree, we reconstructed the most probable events in the evolution of IRs in the Euglenophyta. Our study highlights the dynamic nature of the Euglenophyta plastid genome, in particular with regards to the IR regions that underwent losses repeatedly.

The small subunit ribosomal RNA (SSU rRNA) gene is a widely used molecular marker to study the diversity of life. Sequencing of SSU rRNA gene amplicons has become a standard approach for the investigation of the ecology and diversity of microbes. However, a well-curated database is necessary for correct classification of these data. While available for many groups of Bacteria and Archaea, such reference databases are absent for most eukaryotes. The primary goal of the EukRef project (eukref.org) is to close this gap and generate well-curated reference databases for major groups of eukaryotes, especially protists. Here we present a set of EukRef-curated databases for the excavate protists-a large assemblage that includes numerous taxa with divergent SSU rRNA gene sequences, which are prone to misclassification. We identified 6121 sequences, 625 of which were obtained from cultures, 3053 from cell isolations or enrichments and 2419 from environmental samples. We have corrected the classification for the majority of these curated sequences. The resulting publicly available databases will provide phylogenetically based standards for the improved identification of excavates in ecological and microbiome studies, as well as resources to classify new discoveries in excavate diversity.

Euglenophyceae are unicellular algae with the majority of their diversity known from small freshwater reservoirs. Only two dozen species have been described to occur in marine habitats, but their abundance and diversity remain unexplored. Phylogenetic studies revealed marine prasinophyte green alga, Pyramimonas parkeae, as the closest extant relative of the euglenophytes' plastid, but similarly to euglenophytes, our knowledge about the diversity of Pyramimonadales is limited. Here we explored Euglenophyceae and Pyramimonadales phylogenetic diversity in marine environmental samples. We yielded 18S rDNA and plastid 16S rDNA sequences deposited in public repositories and reconstructed Euglenophyceae reference trees. We searched high-throughput environmental sequences from the TARA Oceans expedition and Ocean Sampling Day initiative for 18S rDNA and 16S rDNA, placed them in the phylogenetic context and estimated their relative abundances. To avoid polymerase chain reaction (PCR) bias, we also exploited metagenomic data from the TARA Oceans expedition for the presence of rRNA sequences from these groups. Finally, we targeted these protists in coastal samples by specific PCR amplification of two parts of the plastid genome uniquely shared between euglenids and Pyramimonadales. All approaches revealed previously undetected, but relatively low-abundant lineages of marine Euglenophyceae. Surprisingly, some of those lineages are branching within the freshwater or brackish genera.

Our knowledge on the variability of the reduced forms of endosymbiotic organelles - mitochondria and plastids - is expanding rapidly, thanks to growing interest in peculiar microbial eukaryotes, along with the availability of the methods used in modern genomics and transcriptomics. The aim of this work is to highlight the most recent advances in understanding these organelles' diversity, physiology and evolution. We also outline the known mechanisms behind the convergence of traits between organelles which have undergone reduction independently, the importance of the earliest evolutionary events in determining the vestigial organelles' eventual fate, and a proposed classification of nonphotosynthetic plastids.

Euglenids are a well-known group of single-celled eukaryotes, with phototrophic, osmotrophic and phagotrophic members. Phagotrophs represent most of the phylogenetic diversity of euglenids, and gave rise to the phototrophs and osmotrophs, but their evolutionary relationships are poorly understood. Symbiontids, in contrast, are anaerobes that are alternatively inferred to be derived euglenids, or a separate euglenozoan group. Most phylogenetic studies of euglenids have examined the SSU rDNA only, which is often highly divergent. Also, many phagotrophic euglenids (and symbiontids) are uncultured, restricting collection of other molecular data. We generated transcriptome data for 28 taxa, mostly using a single-cell approach, and conducted the first multigene phylogenetic analyses of euglenids to include phagotrophs and symbiontids. Euglenids are recovered as monophyletic, with symbiontids forming an independent branch within Euglenozoa. Spirocuta, the clade of flexible euglenids that contains both the phototrophs (Euglenophyceae) and osmotrophs (Aphagea), is robustly resolved, with the ploeotid Olkasia as its sister group, forming the new taxon Olkaspira. Ploeotids are paraphyletic, although Ploeotiidae (represented by Ploeotia spp.), Lentomonas, and Keelungia form a robust clade (new taxon Alistosa). Petalomonadida branches robustly as sister to other euglenids in outgroup-rooted analyses. Within Spirocuta, Euglenophyceae is a robust clade that includes Rapaza, and Anisonemia is a well-supported monophyletic group containing Anisonemidae (Anisonema and Dinema spp.), ‘Heteronema II’ (represented by H. vittatum), and a clade of Neometanema plus Aphagea. Among ‘peranemid’ phagotrophs, Chasmostoma branches with included Urceolus, and Peranema with the undescribed ‘Jenningsia II’, while other relationships are weakly supported and consequently the closest sister group to Euglenophyceae remains unresolved. Our results are inconsistent with recent inferences that Entosiphon is the evolutionarily pivotal sister either to other euglenids, or to Spirocuta. At least three transitions between posterior and anterior flagellar gliding occurred in euglenids, with the phylogenetic positions and directions of those transitions remaining ambiguous.

Autotrophic euglenids (Euglenophyceae) are a common and abundant group of microbial eukaryotes in freshwater habitats. They have a limited number of features, which can be observed using light microscopy, thus species identification is often problematic. Establishing a barcode for this group is therefore an important step toward the molecular identification of autotrophic euglenids. Based on the literature, we selected verified species and used a plethora of available methods to validate two molecular markers: COI and 18S rDNA (the whole sequence and three fragments separately) as potential DNA barcodes. Analyses of the COI gene were performed based on the data set of 43 sequences (42 obtained in this study) representing 24 species and the COI gene was discarded as a DNA barcode mainly due to a lack of universal primer sites. For 18S rDNA analyses we used a data set containing 263 sequences belonging to 86 taxonomically verified species. We demonstrated that the whole 18S rDNA is too long to be a useful marker, but from the three shorter analyzed variable regions we recommend variable regions V2V3 and V4 of 18S rDNA as autotrophic euglenid barcodes due to their high efficiency (above 95% and 90%, respectively).

The presence of mitochondria and related organelles in every studied eukaryote supports the view that mitochondria are essential cellular components. Here, we report the genome sequence of a microbial eukaryote, the oxymonad Monocercomonoides sp., which revealed that this organism lacks all hallmark mitochondrial proteins. Crucially, the mitochondrial iron-sulfur cluster assembly pathway, thought to be conserved in virtually all eukaryotic cells, has been replaced by a cytosolic sulfur mobilization system (SUF) acquired by lateral gene transfer from bacteria. In the context of eukaryotic phylogeny, our data suggest that Monocercomonoides is not primitively amitochondrial but has lost the mitochondrion secondarily. This is the first example of a eukaryote lacking any form of a mitochondrion, demonstrating that this organelle is not absolutely essential for the viability of a eukaryotic cell.

116 pages, 1 figure, 4 tables, 3 appendices, This revision of the classification of eukaryotes follows that of Adl et al., 2012 [J. Euk. Microbiol. 59(5)] and retains an emphasis on protists. Changes since have improved the resolution of many nodes in phylogenetic analyses. For some clades even families are being clearly resolved. As we had predicted, environmental sampling in the intervening years has massively increased the genetic information at hand. Consequently, we have discovered novel clades, exciting new genera and uncovered a massive species level diversity beyond the morphological species descriptions. Several clades known from environmental samples only have now found their home. Sampling soils, deeper marine waters and the deep sea will continue to fill us with surprises. The main changes in this revision are the confirmation that eukaryotes form at least two domains, the loss of monophyly in the Excavata, robust support for the Haptista and Cryptista. We provide suggested primer sets for DNA sequences from environmental samples that are effective for each clade. We have provided a guide to trophic functional guilds in an appendix, to facilitate the interpretation of environmental samples, and a standardized taxonomic guide for East Asian users, Research support was provided as follows: SMA by NSERC 249889‐2007; DB by NERC NE/H009426/1 and NE/H000887/1; MWB by NSF 1456054; FB by a Fellowship from Science for Life Laboratory and VR/2017‐04563; PC by EU‐Horizon 2020 research and innovation program through the SponGES project 679849 [...]; IC by CSF 18‐18699S; BE by RCN TaxMArc 268286/GMR; LG by ANR HAPAR (ANR‐14‐CE02‐0007); VH MK JL by ERDF; MEYS with ERC 771592 CZ 1.05/1.1.00/02.0109 BIOCEV; SK by RSF 16‐14‐10302; MK by CSF GA18‐28103S; CEL by NSF 1541510 and NIH‐AI124092; EL by CAM: 2017‐T1/AMB‐5210; and by grant 2017‐T1/AMB‐5210 from the program >Atracción de talentos> from the Consejería de Educación, Juventud y Deporte, Comunidad de Madrid; JL by ERC CZ LL1601 and OPVVV 16_019/0000759; MP by NSF DEB‐1455611; DJR by the Beatriu de Pinós postdoctoral programme of the Government of Catalonia's Secretariat for Universities and Research of the Ministry of Economy and Knowledge; CLS by the intramural research program of the National Library of Medicine, National Institutes of Health; AS by RSF 17‐14‐01391 and RFBR 16‐04‐01454 NY by NSF DEB 1557102; VZ by RFBR 16‐34‐60102 mol‐a‐dk; UniEuk and EukRef by the Gordon and Betty Moore Foundation

Denis,It’s ironic that when facing a problem like this – being asked to write a piece about the way you affected us in the labs we share – our first instinct would be to come and talk to you, to ge...

Achlorophyllous unicellular microalgae of the genus Prototheca (Trebouxiophyceae, Chlorophyta) are the only known plants that cause infections in both humans and animals, collectively referred to as protothecosis. Human protothecosis, most commonly manifested as cutaneous, articular, and disseminated disease, is primarily caused by Prototheca wickerhamii, followed by Prototheca zopfii and, sporadically, by Prototheca cutis and Prototheca miyajii. In veterinary medicine, however, P. zopfii is a major pathogen responsible for bovine mastitis, which is a predominant form of protothecal disease in animals. Historically, identification of Prototheca spp. has relied upon phenotypic criteria; these were later replaced by molecular typing schemes, including DNA sequencing. However, the molecular markers interrogated so far, mostly located in the ribosomal DNA (rDNA) cluster, do not provide sufficient discriminatory power to distinguish among all Prototheca spp. currently recognized. Our study is the first attempt to develop a fast, reliable, and specific molecular method allowing identification of all Prototheca spp. We propose the mitochondrial cytb gene as a new and robust marker for diagnostics and phylogenetic studies of the Prototheca algae. The cytb gene displayed important advantages over the rDNA markers. Not only did the cytb gene have the highest discriminatory capacity for resolving all Prototheca species, but it also performed best in terms of technical feasibility, understood as ease of amplification, sequencing, and multiple alignment analysis. Based on the species-specific polymorphisms in the partial cytb gene, we developed a fast and straightforward PCR-restriction fragment length polymorphism (RFLP) assay for identification and differentiation of all Prototheca species described so far. The newly proposed method is advocated to be a new gold standard in diagnostics of protothecal infections in human and animal populations.

Although Phacus longicauda is the type species of the genus Phacus and one of the most common species among autotrophic euglenids, its correct identification is nearly impossible. Over 30 morphologically similar taxa appear in the literature, but there are no good diagnostic features to distinguish them. Using environmental sampling and whole genome amplification, we delimited species within the Phacus longicauda complex. Morphological and molecular characters were analyzed for 36 strains isolated from environmental samples (mainly from Poland). DNA was obtained from a small number of cells (20-30) isolated with a micropipette from every sample (i.e., without setting up laboratory cultures), and phylogenetic analyses were based on variation in nSSU rDNA. Apart from Phacus longicauda, three other species (Phacus circumflexus, Phacus helikoides, and Phacus tortus) were distinguished. Phacus cordata comb. nov. Zakryś et M. Łukomska and Phacus rotunda comb. nov. Zakryś et M. Łukomska had their taxonomic ranks changed and two species new to science, Phacus cristatus sp. nov. Zakryś et M. Łukomska and Phacus crassus sp. nov. Zakryś et M. Łukomska, were described. For all verified species, diagnostic descriptions were amended and epitypes designated.

Background Nuclear genes of euglenids contain two major types of introns: conventional spliceosomal and nonconventional introns. The latter are characterized by variable non-canonical borders, RNA secondary structure that brings intron ends together, and an unknown mechanism of removal. Some researchers also distinguish intermediate introns, which combine features of both types. They form a stable RNA secondary structure and are classified into two subtypes depending on whether they contain one (intermediate/nonconventional subtype) or both (conventional/intermediate subtype) canonical spliceosomal borders. However, it has been also postulated that most introns classified as intermediate could simply be special cases of conventional or nonconventional introns. Results Sequences of tubB, hsp90 and gapC genes from six strains of Euglena agilis were obtained. They contain four, six, and two or three introns, respectively (the third intron in the gapC gene is unique for just one strain). Conventional introns were present at three positions: two in the tubB gene (at one position conventional/intermediate introns were also found) and one in the gapC gene. Nonconventional introns are present at ten positions: two in the tubB gene (at one position intermediate/nonconventional introns were also found), six in hsp90 (at four positions intermediate/nonconventional introns were also found), and two in the gapC gene. Conclusions Sequence and RNA secondary structure analyses of nonconventional introns confirmed that their most strongly conserved elements are base pairing nucleotides at positions +4, +5 and +6/ -8, −7 and −6 (in most introns CAG/CTG nucleotides were observed). It was also confirmed that the presence of the 5' GT/C end in intermediate/nonconventional introns is not the result of kinship with conventional introns, but is due to evolutionary pressure to preserve the purine at the 5' end. However, an example of a nonconventional intron with GC-AG ends was shown, suggesting the possibility of intron type conversion between nonconventional and conventional. Furthermore, an analysis of conventional introns revealed that the ability to form a stable RNA secondary structure by some introns is probably not a result of their relationship with nonconventional introns. It was also shown that acquisition of new nonconventional introns is an ongoing process and can be observed at the level of a single species. In the recently acquired intron in the gapC gene an extended direct repeats at the intron-exon junctions are present, suggesting that double-strand break repair process could be the source of new nonconventional introns. Electronic supplementary material The online version of this article (doi:10.1186/s12862-016-0620-5) contains supplementary material, which is available to authorized users.

Marine alveolates (MALVs) are diverse and widespread early-branching dinoflagellates, but most knowledge of the group comes from a few cultured species that are generally not abundant in natural samples, or from diversity analyses of PCR-based environmental SSU rRNA gene sequences. To more broadly examine MALV genomes, we generated single cell genome sequences from seven individually isolated cells. Genes expected of heterotrophic eukaryotes were found, with interesting exceptions like presence of proteorhodopsin and vacuolar H +-pyrophosphatase. Phylogenetic analysis of concatenated SSU and LSU rRNA gene sequences provided strong support for the paraphyly of MALV lineages. Dinoflagellate viral nucleoproteins were found only in MALV groups that branched as sister to dinokaryotes. Our findings indicate that multiple independent origins of several characteristics early in dinoflagellate evolution, such as a parasitic life style, underlie the environmental diversity of MALVs, and suggest they have more varied trophic modes than previously thought. © 2018 International Society for Microbial Ecology All rights reserved.

Campo, Javier del ... et al.-- 8 pages, 4 figures, supplementary material https://doi.org/10.1038/s41598-017-16259-8, supplementary video https://static-content.springer.com/esm/art%3A10.1038%2Fs41598-017-16259-8/MediaObjects/41598_2017_16259_MOESM2_ESM.mov, Pseudotrichonympha is a large and structurally complex genus of parabasalian protists that play a key role in the digestion of lignocellulose in the termite hindgut. Like many termite symbionts, it has a conspicuous body plan that makes genus-level identification relatively easy, but species-level diversity of Pseudotrichonympha is understudied. Molecular surveys have suggested the diversity is much greater than the current number of described species, and that many “species” described in multiple hosts are in fact different, but gene sequences from formally described species remain a rarity. Here we describe three new species from Coptotermes and Prorhinotermes hosts, including small subunit ribosomal RNA (SSU rRNA) sequences from single cells. Based on host identification by morphology and DNA barcoding, as well as the morphology and phylogenetic position of each symbiont, all three represent new Pseudotrichonympha species: P. leei, P. lifesoni, and P. pearti. Pseudotrichonympha leei and P. lifesoni, both from Coptotermes, are closely related to other Coptotermes symbionts including the type species, P. hertwigi. Pseudotrichonympha pearti is the outlier of the trio, more distantly related to P. leei and P. lifesoni than they are to one another, and contains unique features, including an unusual rotating intracellular structure of unknown function, This work was supported by a grant (RGPIN-2014-03994) from the Natural Sciences and Engineering Research Council of Canada to PJK. JdC, YH, VB, AK, EH and MK were supported by grants to the Centre for Microbial Diversity and Evolution from the Tula Foundation, and NATI was supported by a fellowship from NSERC. JdC was supported by a Marie Curie International Outgoing Fellowship grant (FP7-PEOPLE-2012-IOF-331450 CAARL)

Members of the genus Trichonympha are among the most well-known, recognizable and widely distributed parabasalian symbionts of lower termites and the wood-eating cockroach species of the genus Cryptocercus. Nevertheless, the species diversity of this genus is largely unknown. Molecular data have shown that the superficial morphological similarities traditionally used to identify species are inadequate, and have challenged the view that the same species of the genus Trichonympha can occur in many different host species. Ambiguities in the literature, uncertainty in identification of both symbiont and host, and incomplete samplings are limiting our understanding of the systematics, ecology and evolution of this taxon. Here we describe four closely related novel species of the genus Trichonympha collected from South American and Australian lower termites: Trichonympha hueyi sp. nov. from Rugitermes laticollis, Trichonympha deweyi sp. nov. from Glyptotermes brevicornis, Trichonympha louiei sp. nov. from Calcaritermes temnocephalus and Trichonympha webbyae sp. nov. from Rugitermes bicolor. We provide molecular barcodes to identify both the symbionts and their hosts, and infer the phylogeny of the genus Trichonympha based on small subunit rRNA gene sequences. The analysis confirms the considerable divergence of symbionts of members of the genus Cryptocercus, and shows that the two clades of the genus Trichonympha harboured by termites reflect only in part the phylogeny of their hosts.

Euglenids (Excavata, Discoba, Euglenozoa, Euglenida) is a group of free-living, single-celled flagellates living in the aquatic environments. The uniting and unique morphological feature of euglenids is the presence of a cell covering called the pellicle. The morphology and organization of the pellicle correlate well with the mode of nutrition and cell movement. Euglenids exhibit diverse modes of nutrition, including phagotrophy and photosynthesis. Photosynthetic species (Euglenophyceae) constitute a single subclade within euglenids. Their plastids embedded by three membranes arose as the result of a secondary endosymbiosis between phagotrophic eukaryovorous euglenid and the Pyramimonas-related green alga. Within photosynthetic euglenids three evolutionary lineages can be distinguished. The most basal lineage is formed by one mixotrophic species, Rapaza viridis. Other photosynthetic euglenids are split into two groups: predominantly marine Eutreptiales and freshwater Euglenales. Euglenales are divided into two families: Phacaceae, comprising three monophyletic genera (Discoplastis, Lepocinclis, Phacus) and Euglenaceae with seven monophyletic genera (Euglenaformis, Euglenaria, Colacium, Cryptoglena, Strombomonas, Trachelomonas, Monomorphina) and polyphyletic genus Euglena. For 150 years researchers have been studying Euglena based solely on morphological features what resulted in hundreds of descriptions of new taxa and many artificial intra-generic classification systems. In spite of the progress towards defining Euglena, it still remains polyphyletic and morphologically almost undistinguishable from members of the recently described genus Euglenaria; members of both genera have cells undergoing metaboly (dynamic changes in cell shape), large chloroplasts with pyrenoids and monomorphic paramylon grains. Model organisms Euglena gracilis Klebs, the species of choice for addressing fundamental questions in eukaryotic biochemistry, cell and molecular biology, is a representative of the genus Euglena.

Due to their involvement in the energy metabolism, mitochondria are essential for most eukaryotic cells. Microbial eukaryotes living in low oxygen environments possess reduced forms of mitochondria, namely mitochondrion-related organelles (MROs). These do not produce ATP by oxidative phosphorylation on their membranes and some do not produce ATP at all. Still, they are indispensable because of other essential functions such as iron-sulphur (Fe-S) cluster assembly. Recently, the first microbial eukaryote with neither mitochondrion nor MRO was characterized – Monocercomonoides sp. Genome and transcriptome sequencing of Monocercomonoides revealed that it lacks all hallmark mitochondrial proteins. Crucially, the essential mitochondrial pathway for the Fe-S cluster assembly (ISC) was replaced by a bacterial sulphur mobilization (SUF) system. The discovery of such bona fide amitochondriate eukaryote broadens our knowledge about the diversity and plasticity of eukaryotic cells and provides a substantial contribution to our understanding of eukaryotic cell evolution.

The only algae which are able to inflict disease on humans and other mammals through active invasion and spread within the host tissues belong to either of two genera: Chlorella and Prototheca. Whereas Chlorella infections are extremely rare, with only two human cases reported in the literature, protothecosis is an emerging disease of humans and domestic animals, especially dairy cows. The genus Prototheca, erected by Kruger in 1894, has undergone several significant revisions, as more phenotypic, chemotaxonomic, and molecular data have become available. Due to this, a large number of Prototheca strains have been accumulated in public culture collections, over the years, where they still exist under outdated or invalid infraspecific or species names. In this study, the partial cytb gene was used as a marker to revise the taxonomy and nomenclature of a set of Prototheca strains, preserved in major algae culture repositories worldwide. Within the genus, two main lineages were observed, with a dominance of typically dairy cattle-associated (i.e. P. ciferrii, formerly P. zopfii gen. 1, the here validated P. blaschkeae, and one newly erected species, namely P. bovis, formerly P. zopfii gen. 2) and human-associated (i.e. P. wickerhamii, P. cutis, P. miyajii) species, respectively. In the former lineage, three newly described species were allocated, namely P. cookei sp. nov., P. cerasi sp. nov., and P. pringsheimii sp. nov., and the lecto- and epitypified P. zopfii species. The second, or so-called P. wickerhamii lineage, incorporated a newly proposed species of P. xanthoriae sp. nov. These protothecans were shown as the closest relatives of the photosynthetic genera, Chlorella and Auxenochlorella. The environmental species P. ulmea was synonymized with the lecto- and epitypified P. moriformis species. For circumscription and differentiation of Prototheca spp., the use of phenotypic characters, and morphology in particular, is of limited value and should rather be auxiliary to molecular marker-based approaches. As demonstrated in our previous study and corroborated in the present one, the cytb gene provides higher resolution than the conventional rDNA markers, and currently represents the most efficient barcode for the Prototheca algae.

The nuclear genomes of euglenids contain three types of introns: conventional spliceosomal introns, nonconventional introns for which a splicing mechanism is unknown (variable noncanonical borders, RNA secondary structure bringing together intron ends), and so-called intermediate introns, which combine features of conventional and nonconventional introns. Analysis of two genes, tubA and tubB, from 20 species of euglenids reveals contrasting distribution patterns of conventional and nonconventional introns--positions of conventional introns are conserved, whereas those of the nonconventional ones are unique to individual species or small groups of closely related taxa. Moreover, in the group of phototrophic euglenids, 11 events of conventional intron loss versus 15 events of nonconventional intron gain were identified. A comparison of all nonconventional intron sequences highlighted the most conserved elements in their sequence and secondary structure. Our results led us to put forward two hypotheses. 1) The first one posits that mutational changes in intron sequence could lead to a change in their excision mechanism--intermediate introns would then be a transitional form between the conventional and nonconventional introns. 2) The second hypothesis concerns the origin of nonconventional introns--because of the presence of inverted repeats near their ends, insertion of MITE-like transposon elements is proposed as a possible source of new introns.

In this paper, we describe a new photosynthetic euglenoid species, Euglenaria clepsydroides Zakryś, sp. nov., found in Poland. A large population of this species exists in a few, small, eutrophic bodies of water inside the Masurian Landscape Park (covering a part of the Masurian Lake District in Poland). The characteristic and atypical (hourglass-like) cell shape sets it well apart from the other species that have been described up to now. This atypical cell shape has so far been observed only in three species – Lepocinclis constricta, Euglena undulata and Euglena gymnodinioides – whose other morphological characteristics, such as the number and morphology of chloroplasts, the lack of mucocysts, and nuclear SSU rDNA sequence data, exclude the possibility that they could be close relatives of Euglenaria clepsydroides. On the phylogenetic tree, the new species is situated within the Euglenaria clade. While it is a sister group of the clade that includes representatives of Euglenaria anabaena, the two specie...

Euglena sanguinea (Ehrenberg 1831) was one of the first green euglenoid species described in the literature. At first, the species aroused the interest of researchers mainly due to the blood-red color of its cells, which, as it later turned out, is not a constant feature. Complicated chloroplast morphology, labeled by Pringsheim as the "peculiar chromatophore system", made the correct identification of the species difficult, which is the reason why, throughout the 20th century, new species resembling E. sanguinea were continually being named due to a lack of suitable diagnostic features to distinguish E. sanguinea. Interest in E. sanguinea has returned in recent years, following findings that the species can produce ichthyotoxins. This was followed by the need to classify E. sanguinea correctly, which was achieved through the verification of morphological and molecular data for all species similar to E. sanguinea. As the result of the analysis, the number of species sharing some morphological similarities with E. sanguinea could be reduced from 12, as described in the literature, to four, with established epitypes and updated diagnostic descriptions. The most important diagnostic features included: the presence of mucocysts (i.e., whether they were visible before and/or after staining), the number of chloroplasts, the size of the double-sheathed pyrenoids, and the presence of the large paramylon grain in the vicinity of the stigma. Moreover, sequence analysis revealed the presence of unusually long SSU rDNA sequences in E. sanguinea. Previously, SSU rDNA sequences of such length were known to be present in primary osmotrophic euglenoids.