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EukProt is a database of published and publicly available predicted protein sets selected to represent the breadth of eukaryotic diversity, currently including 993 species from all major supergroups as well as orphan taxa. The goal of the database is to provide a single, convenient resource for gene-based research across the spectrum of eukaryotic life, such as phylogenomics and gene family evolution. Each species is placed within the UniEuk taxonomic framework in order to facilitate downstream analyses, and each data set is associated with a unique, persistent identifier to facilitate comparison and replication among analyses. The database is regularly updated, and all versions will be permanently stored and made available via FigShare. The current version has a number of updates, notably ‘The Comparative Set’ (TCS), a reduced taxonomic set with high estimated completeness while maintaining a substantial phylogenetic breadth, which comprises 196 predicted proteomes. A BLAST web server and graphical displays of data set completeness are available at http://evocellbio.com/eukprot/. We invite the community to provide suggestions for new data sets and new annotation features to be included in subsequent versions, with the goal of building a collaborative resource that will promote research to understand eukaryotic diversity and diversification.

Nephridiophagids are unicellular fungi (Chytridiomycota), which infect the Malpighian tubules of insects. While most life cycle features are known, the effects of these endobionts on their hosts remain poorly understood. Here, we present results on the influence of an infection of the cockroach Blattella germanica with Nephridiophaga blattellae (Ni = Nephridiophaga-infected) on physical, physiological, and reproductive fitness parameters. Since the gut nematode Blatticola blattae is a further common parasite of B. germanica, we included double infected cockroaches (N + Ni = nematode plus Ni) in selected experiments. Ni individuals had lower fat reserves and showed reduced mobility. The lifespan of adult hosts was only slightly affected in these individuals but significantly shortened when both Nephridiophaga and nematodes were present. Ni as well as N + Ni females produced considerably less offspring than parasite-free (P-free) females. Immune parameters such as the number of hemocytes and phenoloxidase activity were barely changed by Nephridiophaga and/or nematode infections, while the ability to detoxify pesticides decreased. Quantitative proteomics from hemolymph of P-free, Ni, and N + Ni populations revealed clear differences in the expression profiles. For Ni animals, for example, the down-regulation of fatty acid synthases corroborates our finding of reduced fat reserves. Our study clearly shows that an infection with Nephridiophaga (and nematodes) leads to an overall reduced host fitness.

SummaryPhylogenomic analyses have boosted our understanding of the evolutionary trajectories of all living forms by providing continuous improvements to the tree of life1–5. Within this tree, fungi represent an ancient eukaryote group6, having diverged from the animals ∼1.35 billion years ago7. Estimates of the number of extant species range between 1.5 and 3.8 million8,9. Recent reclassifications and the discovery of the deep-branching Sanchytriomycota lineage10 have brought the number of proposed phyla to 2011; 21 if the Microsporidia are included12–14. Uncovering how the diverse and globally distributed fungi are related to each other is fundamental for understanding how their lifestyles, morphologies, and metabolic capacities evolved. To date, many of the proposed relationships among the phyla remain controversial and no phylogenomic study has examined the entire fungal tree using a taxonomically comprehensive data set and suitable models of evolution. We assembled and curated a 299-protein data set with a taxon sampling broad enough to encompass all recognised fungal diversity with available data, but selective enough to run computationally intensive analyses using best-fitting models. Using a range of reconstruction methods, we were able to resolve many contested nodes, such as a sister-relationship of Chytridiomyceta to all other non-Opisthosporidia fungi (with Chytridiomycota being sister to Monoblepharomycota + Neocallimastigomycota), a branching of Blastocladiomycota + Sanchytriomycota after the Chytridiomyceta but before other non-Opisthosporidia fungi, and a branching of Glomeromycota as sister to the Dikarya. Our most up-to-date fungal tree of life will serve as a springboard for future investigations on the evolution of fungi.

Phylogenomic analyses have boosted our understanding of the evolutionary trajectories of all living forms by providing continuous improvements to the tree of life.

The increasing number of new fungal species described from all over the world along with the use of genetics to define taxa, has dramatically changed the classification system of early-diverging fungi over the past several decades. The number of phyla established for non-Dikarya fungi has increased from 2 to 17. However, to date, both the classification and phylogeny of the basal fungi are still unresolved. In this article, we review the recent taxonomy of the basal fungi and re-evaluate the relationships among early-diverging lineages of fungal phyla. We also provide information on the ecology and distribution in Mucoromycota and highlight the impact of chytrids on amphibian populations. Species concepts in Chytridiomycota, Aphelidiomycota, Rozellomycota, Neocallimastigomycota are discussed in this paper. To preserve the current application of the genus Nephridiophaga (Chytridiomycota: Nephridiophagales), a new type species, Nephridiophaga blattellae, is proposed.

The endosymbiotic origin of plastids from cyanobacteria gave eukaryotes photosynthetic capabilities and launched the diversification of countless forms of algae. These primary plastids are found in members of the eukaryotic supergroup Archaeplastida. All known archaeplastids still retain some form of primary plastids, which are widely assumed to have a single origin. Here, we use single-cell genomics from natural samples combined with phylogenomics to infer the evolutionary origin of the phylum Picozoa, a globally distributed but seemingly rare group of marine microbial heterotrophic eukaryotes. Strikingly, the analysis of 43 single-cell genomes shows that Picozoa belong to Archaeplastida, specifically related to red algae and the phagotrophic rhodelphids. These picozoan genomes support the hypothesis that Picozoa lack a plastid, and further reveal no evidence of an early cryptic endosymbiosis with cyanobacteria. These findings change our understanding of plastid evolution as they either represent the first complete plastid loss in a free-living taxon, or indicate that red algae and rhodelphids obtained their plastids independently of other archaeplastids., The origin of primary plastids in an ancestor of Archaeplastida gave eukaryotes photosynthetic capabilities. This study used single-cell genomics and phylogenomics to infer the evolutionary origin of the plastid-lacking phylum Picozoa, a group of marine microbial heterotrophic eukaryotes, showing that they belong to the Archaeplastida and changing our understanding of plastid evolution.

Phylogenomic analyses of hundreds of protein-coding genes aimed at resolving phylogenetic relationships is now a common practice. However, no software currently exists that includes tools for dataset construction and subsequent analysis with diverse validation strategies to assess robustness. Furthermore, there are no publicly available high-quality curated databases designed to assess deep (>100 million years) relationships in the tree of eukaryotes. To address these issues, we developed an easy-to-use software package, PhyloFisher (https://github.com/TheBrownLab/PhyloFisher), written in Python 3. PhyloFisher includes a manually curated database of 240 protein-coding genes from 304 eukaryotic taxa covering known eukaryotic diversity, a novel tool for ortholog selection, and utilities that will perform diverse analyses required by state-of-the-art phylogenomic investigations. Through phylogenetic reconstructions of the tree of eukaryotes and of the Saccharomycetaceae clade of budding yeasts, we demonstrate the utility of the PhyloFisher workflow and the provided starting database to address phylogenetic questions across a large range of evolutionary time points for diverse groups of organisms. We also demonstrate that undetected paralogy can remain in phylogenomic “single-copy orthogroup” datasets constructed using widely accepted methods such as all vs. all BLAST searches followed by Markov Cluster Algorithm (MCL) clustering and application of automated tree pruning algorithms. Finally, we show how the PhyloFisher workflow helps detect inadvertent paralog inclusions, allowing the user to make more informed decisions regarding orthology assignments, leading to a more accurate final dataset., Phylogenomic analyses of hundreds of protein-coding genes aimed at resolving phylogenetic relationships is now a common practice. This article presents PhyloFisher, a community-driven tool for phylogenomic dataset construction to infer deep and shallow phylogenetic relationships among eukaryotes.

In modern oceans, eukaryotic phytoplankton is dominated by lineages with red algal-derived plastids such as diatoms, dinoflagellates, and coccolithophores. Despite the ecological importance of these groups and many others representing a huge diversity of forms and lifestyles, we still lack a comprehensive understanding of their evolution and how they obtained their plastids. New hypotheses have emerged to explain the acquisition of red algalderived plastids by serial endosymbiosis, but the chronology of these putative independent plastid acquisitions remains untested. Here, we establish a timeframe for the origin of red algal-derived plastids under scenarios of serial endosymbiosis, using Bayesian molecular clock analyses applied on a phylogenomic dataset with broad sampling of eukaryote diversity. We find that the hypotheses of serial endosymbiosis are chronologically possible, as the stem lineages of all red plastid-containing groups overlap in time. This period in the Meso- and Neoproterozoic Eras set the stage for the later expansion to dominance of red algal-derived primary production in the contemporary oceans, which profoundly altered the global geochemical and ecological conditions of the Earth., Open access funding provided by Uppsala University.

Nephridiophagids are unicellular eukaryotes that parasitize the Malpighian tubules of numerous insects. Their life cycle comprises multinucleate vegetative plasmodia that divide into oligonucleate and uninucleate cells, and sporogonial plasmodia that form uninucleate spores. Nephridiophagids are poor in morphological characteristics, and although they have been tentatively identified as early-branching fungi based on the SSU rRNA gene sequences of three species, their exact position within the fungal tree of live remained unclear. In this study, we described two new species of nephridiophagids (Nephridiophaga postici and Nephridiophaga javanicae) from cockroaches. Using long-read sequencing of the entire rDNA operon of numerous further species obtained from cockroaches and earwigs to improve the resolution of the phylogenetic analysis, we found a robust affiliation of nephridiophagids with the Chytridiomycota — a group of zoosporic fungi that comprises parasites of diverse host taxa, such as microphytes, plants, and amphibians. The presence of the same nephridiophagid species in two only distantly related cockroaches indicates their host specificity is not a strict as generally assumed.

In modern oceans, eukaryotic phytoplankton is dominated by lineages with red algal-derived plastids such as diatoms, dinoflagellates, and coccolithophores. These lineages and countless others representing a huge diversity of forms and lifestyles all belong to four algal groups: cryptophytes, ochrophytes, haptophytes, and myzozoans. Despite the ecological importance of these groups, we still lack a comprehensive understanding of their evolution and how they obtained their plastids. Over the last years, new hypotheses have emerged to explain the acquisition of red algal-derived plastids by serial endosymbiosis, but the chronology of these putative independent plastid acquisitions remains untested. Here, we have established a timeframe for the origin of red algal-derived plastids under scenarios of serial endosymbiosis, using a taxon- and gene-rich phylogenomic dataset combined to Bayesian molecular clock analyses. We find that the hypotheses of serial endosymbiosis are chronologically possible, as the stem lineages of all red plastid-containing groups overlapped in time. This period in the Meso- and Neoproterozoic Eras set the stage for the later expansion to dominance of red algal-derived primary production in the contemporary oceans, which has profoundly altered the global geochemical and ecological conditions of the Earth.

The origin of plastids was a major evolutionary event that paved the way for an astonishing diversification of photosynthetic eukaryotes. Plastids originated by endosymbiosis between a heterotrophic eukaryotic host and cyanobacteria, presumably in a common ancestor of the primary photosynthetic eukaryotes (Archaeplastida). A single origin of primary plastids is well supported by plastid evidence but not by nuclear phylogenomic analyses, which have consistently failed to recover the monophyly of Archaeplastida hosts. Importantly, plastid monophyly and nonmonophyletic hosts could be explained under scenarios of independent or serial eukaryote-to-eukaryote endosymbioses. Here, we assessed the strength of the signal for the monophyly of Archaeplastida hosts in four available phylogenomic data sets. The effect of phylogenetic methodology, data quality, alignment trimming strategy, gene and taxon sampling, and the presence of outlier genes were investigated. Our analyses revealed a lack of support for host monophyly in the shorter individual data sets. However, when analyzed together under rigorous data curation and complex mixture models, the combined nuclear data sets supported the monophyly of primary photosynthetic eukaryotes (Archaeplastida) and recovered a putative association with plastid-lacking Picozoa. This study represents an important step toward better understanding deep eukaryotic evolution and the origin of plastids. [Archaeplastida; Bayesian; chloroplast; maximum likelihood; mixture model; ortholog; outlier loci; paralog; protist.]., This work was supported by a fellowship from Science for Life Laboratory to F.B. I.I. was in part supported by a Juan de la Cierva—Incorporación postdoctoral fellowship (IJCI-2016-29566) from the Spanish Ministry of Economy and Competitiveness (MINECO).

Alveolates are a major supergroup of eukaryotes encompassing more than ten thousand free-living and parasitic species, including medically, ecologically, and economically important apicomplexans, dinoflagellates, and ciliates. These three groups are among the most widespread eukaryotes on Earth, and their environmental success can be linked to unique innovations that emerged early in each group. Understanding the emergence of these well-studied and diverse groups and their innovations has relied heavily on the discovery and characterization of early-branching relatives, which allow ancestral states to be inferred with much greater confidence. Here we report the phylogenomic analyses of 313 eukaryote protein-coding genes from transcriptomes of three members of one such group, the colponemids (Colponemidia), which unambiguously support their monophyly and position as the sister lineage to all other alveolates. Colponemid-related sequences from environmental surveys and our microscopical observations show that colponemids are not common in nature, but diverse and widespread in freshwater habitats around the world. Studied colponemids possess two types of extrusive organelles (trichocysts or toxicysts) for active hunting of other unicellular eukaryotes and potentially play an important role in microbial food webs. Colponemids have generally plesiomorphic morphology and illustrate the ancestral state of Alveolata. We further discuss their importance in understanding the evolution of alveolates as well as origin of myzocytosis and plastids.HighlightsPhylogenomics resolves Colponemidia as a sister group to all other alveolatesThe ancestor of all alveolates was a biflagellate predator feeding by phagocytosisColponemids may illuminate the ancestral states of apicomplexans, dinoflagellates, and ciliatesColponemids are geographically widespread in freshwater habitats

In modern oceans, eukaryotic phytoplankton is dominated by lineages with red algal-derived plastids such as diatoms, dinoflagellates, and coccolithophores. Despite the ecological importance of these groups and many others representing a huge diversity of forms and lifestyles, we still lack a comprehensive understanding of their evolution and how they obtained their plastids. New hypotheses have emerged to explain the acquisition of red algal-derived plastids by serial endosymbiosis, but the chronology of these putative independent plastid acquisitions remains untested. Here, we establish a timeframe for the origin of red algal-derived plastids under scenarios of serial endosymbiosis, using Bayesian molecular clock analyses applied on a phylogenomic dataset with broad sampling of eukaryote diversity. We find that the hypotheses of serial endosymbiosis are chronologically possible, as the stem lineages of all red plastid-containing groups overlap in time. This period in the Meso- and Neoproterozoic Eras set the stage for the later expansion to dominance of red algal-derived primary production in the contemporary oceans, which profoundly altered the global geochemical and ecological conditions of the Earth.

The paradox of methane oversaturation in oxygenated surface water has been described in many pelagic systems and still raises the question of the source. Temora sp. and Acartia sp. commonly dominate the surface and subsurface waters of the central Baltic Sea. It is hypothesised that their gut microbiome at least partly contributes to the methane anomaly in this ecosystem. However, the potential pathway for this methane production remains unclear. Using a microcapillary technique, we successfully overcame the challenge of sampling the gut microbiome of copepods

The flagellate protists in the hindgut of lower termites play an essential role in the digestion of lignocellulose. Most flagellate species are associated with host-specific symbionts from various bacterial lineages, which typically lack cultured representatives. In this study, we analyzed the genome of 'Candidatus Ancillula trichonymphae', an endosymbiont of Trichonympha flagellates from dry-wood termites, which represents a novel, family-level lineage of uncultured Actinobacteria encountered so far only in termite guts. The draft genome of 'Ca. A. trichonymphae' (ca. 1.48 Mbp; 95% complete) revealed a purely fermentative metabolism that is probably fueled by xylose, N-acetyl-glucosamine and glycerol 3-phosphate acquired from the flagellate host. The absence of fructose bisphosphate aldolase and the presence of a complete gene set encoding the phosphoketolase pathway underscore the sister position of the new lineage to Bifidobacteriaceae. The preservation of the pathways for the assimilation of ammonia and the synthesis of 18 amino acids and several cofactors and vitamins suggests that 'Ca. A. trichonymphae' - like other endosymbionts of termite gut flagellates - provides essential amino acids and vitamins to its host. Our findings corroborate the emerging concept that numerous lineages of unrelated flagellate endosymbionts have convergently evolved to fill similar ecological niches.

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...

8 pages, 3 figures, supporting information https://doi.org/10.1111/jeu.12701, Spores of the dinoflagellate Chytriodinium are known to infest copepod eggs causing their lethality. Despite the potential to control the population of such an ecologically important host, knowledge about Chytriodinium parasites is limited: we know little about phylogeny, parasitism, abundance, or geographical distribution. We carried out genome sequence surveys on four manually isolated sporocytes from the same sporangium, which seemed to be attached to a copepod nauplius, to analyze the phylogenetic position of Chytriodinium based on SSU and concatenated SSU/LSU rRNA gene sequences, and also characterize two genes related to the plastidial heme pathway, hemL and hemY. The results suggest the presence of a cryptic plastid in Chytriodinium and a photosynthetic ancestral state of the parasitic Chytriodinium/Dissodinium clade. Finally, by mapping Tara Oceans V9 SSU amplicon data to the recovered SSU rRNA gene sequences from the sporocytes, we show that globally, Chytriodinium parasites are most abundant within the pico/nano‐ and mesoplankton of the surface ocean and almost absent within microplankton, a distribution indicating that they generally exist either as free‐living spores or host‐associated sporangia, This work was supported by a grant from the Gordon and Betty Moore Foundation (GBMF3307) to P.J.K., T.A.R., A.Z.W., and A.E.S., and from NSERC (RGPIN‐2014–03994) to P.J.K. Ship time was supported by a grant from the David and Lucile Packard Foundation through MBARI and GBMF3788 to A.Z.W. J.dC. was supported by a Marie Curie International Outgoing Fellowship grant (FP7‐PEOPLE‐2012‐IOF-331450 CAARL), and N.O., M.K., and J.dC. were supported by a grant from the Tula Foundation to the Centre for Microbial Biodiversity and Evolution at UBC

The broad-scale tree of eukaryotes is constantly improving, but the evolutionary origin of several major groups remains unknown. Resolving the phylogenetic position of these ‘orphan’ groups is important, especially those that originated early in evolution, because they represent missing evolutionary links between established groups. Telonemia is one such orphan taxon for which little is known. The group is composed of molecularly diverse biflagellated protists, often prevalent although not abundant in aquatic environments. Telonemia has been hypothesized to represent a deeply diverging eukaryotic phylum but no consensus exists as to where it is placed in the tree. Here, we established cultures and report the phylogenomic analyses of three new transcriptome datasets for divergent telonemid lineages. All our phylogenetic reconstructions, based on 248 genes and using site-heterogeneous mixture models, robustly resolve the evolutionary origin of Telonemia as sister to the Sar supergroup. This grouping remains well supported when as few as 60% of the genes are randomly subsampled, thus is not sensitive to the sets of genes used but requires a minimal alignment length to recover enough phylogenetic signal. Telonemia occupies a crucial position in the tree to examine the origin of Sar, one of the most lineage-rich eukaryote supergroups. We propose the moniker ‘TSAR’ to accommodate this new mega-assemblage in the phylogeny of eukaryotes.

The guts of lower termites are inhabited by host-specific consortia of cellulose-digesting flagellate protists. In this first investigation of the symbionts of the family Serritermitidae, we found that Glossotermes oculatus and Serritermes serrifer each harbor similar parabasalid morphotypes: large Pseudotrichonympha-like cells, medium-sized Leptospironympha-like cells with spiraled bands of flagella, and small Hexamastix-like cells; oxymonadid flagellates were absent. Despite their morphological resemblance to Pseudotrichonympha and Leptospironympha, a SSU rRNA-based phylogenetic analysis identified the two larger, trichonymphid flagellates as deep-branching sister groups of Teranymphidae, with Leptospironympha sp. (the only spirotrichosomid with sequence data) in a moderately supported basal position. Only the Hexamastix-like flagellates are closely related to trichomonadid flagellates from Rhinotermitidae. The presence of two deep-branching lineages of trichonymphid flagellates in Serritermitidae and the absence of all taxa characteristic of the ancestral rhinotermitids underscores that the flagellate assemblages in the hindguts of lower termites were shaped not only by a progressive loss of flagellates during vertical inheritance but also by occasional transfaunation events, where flagellates were transferred horizontally between members of different termite families. In addition to the molecular phylogenetic analyses, we present a detailed morphological characterization of the new spirotrichosomid genus Heliconympha using light and electron microscopy.

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.

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.

The biodiversity of oxymonadid flagellates in termite hindguts is not fully explored. Many species have been differentiated only by morphological features, and small-celled species have been overlooked or ignored. Our analysis of the dry wood termite Neotermes jouteli by light and electron microscopy revealed the presence of two distinct morphotypes of oxymonads. The larger one matched the morphology of Oxymonas jouteli, the only oxymonad species described from this termite. Although it generally lacks the typical anterior rostellum of the genus Oxymonas, its SSU rRNA gene sequence clusters among other members of this genus, including novel phylotypes that we obtained from Incisitermes tabogae. The second morphotype was a tiny oxymonad that showed the typical traits of the genus Opisthomitus, including a pointed anterior prolongation (lappet). However, the four equal flagella were much longer than those of Opisthomitus avicularis from Kalotermes flavicollis, the only species of the genus and so far described only by light microscopy. We provide a detailed description of Opisthomitus longiflagellatus sp. nov. and demonstrate that despite ultrastructural similarities to members of the Polymastigidae, its SSU rRNA gene sequences form a separate family-level lineage with a slight affinity to the Pyrsonymphidae.

Summary Termites digest lignocellulose with the help of their symbiotic gut microbiota. In the hindgut of evolutionary lower termites, a dense community of cellulolytic flagellates sequesters wood particles from the hindgut content into their digestive vacuoles. In higher termites (family Termitidae), which possess an entirely prokaryotic microbiota, the wood particles are available for bacterial colonization. Substantial particle-associated cellulase activities have been detected in the hindgut of Nasutitermes species, but the microorganisms responsible for these activities and their potential association with the wood fibres remain to be studied. Here, we used density-gradient centrifugation to separate wood fibres and adherent bacterial cells from cells freely suspended in the hindgut fluid. In Nasutitermes corniger, the fibre fraction contained 28% of the DNA and 45% of the cellulase activity in the luminal contents (P3 region). Community fingerprinting (terminal restriction fragment length polymorphism) and pyrotag sequencing analysis of the bacterial 16S rRNA genes demonstrated that the wood fibres in the hindgut of both N. corniger and N. takasagoensis are specifically colonized by members of Fibrobacteres, the TG3 phylum, and certain lineages of Spirochaetes characteristic of the gut microbiota of wood-feeding higher termites. We propose that the loss of flagellates in higher termites provided a new niche for fibre-associated cellulolytic bacteria.

The flagellate protists in the hindgut of lower termites play an essential role in the digestion of lignocellulose. Most flagellate species are associated with host-specific symbionts from various bacterial lineages, which typically lack cultured representatives. In this study, we analyzed the genome of 'Candidatus Ancillula trichonymphae', an endosymbiont of Trichonympha flagellates from dry-wood termites, which represents a novel, family-level lineage of uncultured Actinobacteria encountered so far only in termite guts. The draft genome of 'Ca. A. trichonymphae' (ca. 1.48 Mbp; 95% complete) revealed a purely fermentative metabolism that is probably fueled by xylose, N-acetyl-glucosamine and glycerol 3-phosphate acquired from the flagellate host. The absence of fructose bisphosphate aldolase and the presence of a complete gene set encoding the phosphoketolase pathway underscore the sister position of the new lineage to Bifidobacteriaceae. The preservation of the pathways for the assimilation of ammonia and the synthesis of 18 amino acids and several cofactors and vitamins suggests that 'Ca. A. trichonymphae' - like other endosymbionts of termite gut flagellates - provides essential amino acids and vitamins to its host. Our findings corroborate the emerging concept that numerous lineages of unrelated flagellate endosymbionts have convergently evolved to fill similar ecological niches.

Summary Termite gut flagellates are colonized by host-specific lineages of ectosymbiotic and endosymbiotic bacte- ria. Previous studies have shown that flagellates of the genus Trichonympha may harbour more than one type of symbiont. Using a comprehensive approach that combined cloning of SSU rRNA genes with fluo- rescence in situ hybridization and electron micros- copy, we investigated the phylogeny and subcellular locations of the symbionts in a variety of Trichonym- pha species from different termites. The flagellates in Trichonympha Cluster I were the only species asso- ciated with 'Endomicrobia', which were located in the posterior part of the cell, confirming previous results. Trichonympha species of Cluster II from the termite genus Incisitermes (family Kalotermitidae) lacked 'Endomicrobia' and were associated with endo- symbiotic Actinobacteria, which is highly unusual. The endosymbionts, for which we suggest the name 'Candidatus Ancillula trichonymphae', represent a novel, deep-branching lineage in the Micrococcineae that consists exclusively of clones from termite guts. They preferentially colonized the anterior part of the flagellate host and were highly abundant in all species of Trichonympha Cluster II except Tricho- nympha globulosa. Here, they were outnumbered by a Desulfovibrio species associated with the cytoplas- mic lamellae at the anterior cell pole. Such symbionts are present in both Trichonympha clusters, but not in all species. Unlike the intracellular location reported for the Desulfovibrio symbionts of Trichonympha agilis (Cluster I), the Desulfovibrio symbionts of T. globulosa (Cluster II) were situated in deep invagi- nations of the plasma membrane that were clearly connected to the exterior of the host cell.

The hindgut of wood-feeding lower termites is densely colonized by a multitude of symbiotic micro-organisms. While it is well established that the eukaryotic flagellates play a major role in the degradation of lignocellulose, much less is known about the identity and function of the prokaryotic symbionts associated with the flagellates. Our ultrastructural investigations of the gut flagellate Joenia annectens (from the termite Kalotermes flavicollis) revealed a dense colonization of this flagellate by diverse ecto- and endosymbiotic bacteria. Phylogenetic analysis of the small-subunit rRNA gene sequences combined with fluorescence in situ hybridization allowed us to identify and localize the different morphotypes. Furthermore, we could show that K. flavicollis harbours two phylotypes of J. annectens that could be distinguished not only by their small-subunit rRNA gene sequences, but also by differences in their assemblages of bacterial symbionts. Each of the flagellate populations hosted phylogenetically distinct ectosymbionts from the phylum Bacteroidetes, one of them closely related to the ectosymbionts of other termite gut flagellates. A single phylotype of 'Endomicrobia' was consistently associated with only one of the host phylotypes, although not all individuals were colonized, corroborating that 'Endomicrobia' symbionts do not always cospeciate with their host lineages. Flagellates from both populations were loosely associated with a single phylotype of Spirochaetales attached to their cell surface in varying abundance. Current evidence for the involvement of Bacteroidales and 'Endomicrobia' symbionts in the nitrogen metabolism of the host flagellate is discussed.

Recent global surveys of marine biodiversity have revealed that a group of organisms known as “marine diplonemids” constitutes one of the most abundant and diverse planktonic lineages 1. Though discovered over a decade ago 2, 3, their potential importance was unrecognized, and our knowledge remains restricted to a single gene amplified from environmental DNA, the 18S rRNA gene (small subunit SSU). Here, we use single-cell genomics (SCG) and microscopy to characterize ten marine diplonemids, isolated from a range of depths in the eastern North Pacific Ocean. Phylogenetic analysis confirms that the isolates reflect the entire range of marine diplonemid diversity, and comparisons to environmental SSU surveys show that sequences from the isolates range from rare to superabundant, including the single most common marine diplonemid known. SCG generated a total of ∼915 Mbp of assembled sequence across all ten cells and ∼4,000 protein-coding genes with homologs in the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology database, distributed across categories expected for heterotrophic protists. Models of highly conserved genes indicate a high density of non-canonical introns, lacking conventional GT-AG splice sites. Mapping metagenomic datasets 4 to SCG assemblies reveals virtually no overlap, suggesting that nuclear genomic diversity is too great for representative SCG data to provide meaningful phylogenetic context to metagenomic datasets. This work provides an entry point to the future identification, isolation, and cultivation of these elusive yet ecologically important cells. The high density of nonconventional introns, however, also portends difficulty in generating accurate gene models and highlights the need for the establishment of stable cultures and transcriptomic analyses. © 2016 Elsevier Ltd

Termite gut flagellates are typically colonized by specific bacterial symbionts. Here we describe the phylogeny, ultrastructure and subcellular location of 'Candidatus Adiutrix intracellularis', an intracellular symbiont of Trichonympha collaris in the termite Zootermopsis nevadensis. It represents a novel, deep-branching clade of uncultured Deltaproteobacteria widely distributed in intestinal tracts of termites and cockroaches. Fluorescence in situ hybridization and transmission electron microscopy localized the endosymbiont near hydrogenosomes in the posterior part and near the ectosymbiont 'Candidatus Desulfovibrio trichonymphae' in the anterior part of the host cell. The draft genome of 'Ca. Adiutrix intracellularis' obtained from a metagenomic library revealed the presence of a complete gene set encoding the Wood-Ljungdahl pathway, including two homologs of fdhF encoding hydrogenase-linked formate dehydrogenases (FDHH ) and all other components of the recently described hydrogen-dependent carbon dioxide reductase (HDCR) complex, which substantiates previous claims that the symbiont is capable of reductive acetogenesis from CO2 and H2 . The close phylogenetic relationship between the HDCR components and their homologs in homoacetogenic Firmicutes and Spirochaetes suggests that the deltaproteobacterium acquired the capacity for homoacetogenesis via lateral gene transfer. The presence of genes for nitrogen fixation and the biosynthesis of amino acids and cofactors indicate the nutritional nature of the symbiosis.

A new jakobid genus has been isolated from Moroccan desert soil. The cyst-forming protist Moramonas marocensis gen. nov., sp. nov. has two anteriorly inserted flagella of which one points to the posterior cell pole accompanying the ventral feeding groove and is equipped with a dorsal vane—a feature typical for the Jakobida. It further shows a flagellar root system consisting of singlet microtubular root, left root (R1), right root (R2) and typical fibres associated with R1 and R2. The affiliation of M. marocensis to the Jakobida was confirmed by molecular phylogenetic analyses of the SSU rRNA gene, five nuclear genes and 66 mitochondrial protein-coding genes. The mitochondrial genome has the high number of genes typical for jakobids, and bacterial features, such as the four-subunit RNA polymerase and Shine–Dalgarno sequences upstream of the coding regions of several genes. The M. marocensis mitochondrial genome encodes a similar number of genes as other jakobids, but is unique in its very large genome size (greater than 264 kbp), which is three to four times higher than that of any other jakobid species investigated yet. This increase seems to be due to a massive expansion in non-coding DNA, creating a bloated genome like those of plant mitochondria.

Termite gut flagellates are colonized by host-specific lineages of ectosymbiotic and endosymbiotic bacteria. Previous studies have shown that flagellates of the genus Trichonympha may harbour more than one type of symbiont. Using a comprehensive approach that combined cloning of SSU rRNA genes with fluorescence in situ hybridization and electron microscopy, we investigated the phylogeny and subcellular locations of the symbionts in a variety of Trichonympha species from different termites. The flagellates in Trichonympha Cluster I were the only species associated with 'Endomicrobia', which were located in the posterior part of the cell, confirming previous results. Trichonympha species of Cluster II from the termite genus Incisitermes (family Kalotermitidae) lacked 'Endomicrobia' and were associated with endosymbiotic Actinobacteria, which is highly unusual. The endosymbionts, for which we suggest the name 'Candidatus Ancillula trichonymphae', represent a novel, deep-branching lineage in the Micrococcineae that consists exclusively of clones from termite guts. They preferentially colonized the anterior part of the flagellate host and were highly abundant in all species of Trichonympha Cluster II except Trichonympha globulosa. Here, they were outnumbered by a Desulfovibrio species associated with the cytoplasmic lamellae at the anterior cell pole. Such symbionts are present in both Trichonympha clusters, but not in all species. Unlike the intracellular location reported for the Desulfovibrio symbionts of Trichonympha agilis (Cluster I), the Desulfovibrio symbionts of T. globulosa (Cluster II) were situated in deep invaginations of the plasma membrane that were clearly connected to the exterior of the host cell.

The surface of many termite gut flagellates is colonized with a dense layer of bacteria, yet little is known about the evolutionary relationships of such ectosymbionts and their hosts. Here we investigated the molecular phylogenies of devescovinid flagellates (Devescovina spp.) and their symbionts from a wide range of dry-wood termites (Kalotermitidae). From species-pure flagellate suspensions isolated with micropipettes, we obtained SSU rRNA gene sequences of symbionts and host. Phylogenetic analysis showed that the Devescovina spp. present in many species of Kalotermitidae form a monophyletic group, which includes also the unique devescovinid flagellate Caduceia versatilis. All members of this group were consistently associated with a distinct lineage of Bacteroidales, whose location on the cell surface was confirmed by fluorescence in situ hybridization. The well-supported congruence of the phylogenies of devescovinids and their ectosymbionts documents a strict cospeciation. In contrast, the endosymbionts of the same flagellates ('Endomicrobia') were clearly polyphyletic and must have been acquired independently by horizontal transfer from other flagellate lineages. Also the Bacteroidales ectosymbionts of Oxymonas flagellates present in several Kalotermitidae belonged to several distantly related lines of descent, underscoring the general perception that the evolutionary history of flagellate-bacteria symbioses in the termite gut is complex.

More than 40 years ago, ten species of devescovinid flagellates were described to occur in the gut content of the termite Incisitermes marginipennis. Based on light microscopic examinations, the flagellates were then classified into the two genera Devescovina and Metadevescovina. Here, we combined molecular phylogenetic analysis of the small subunit rRNA genes of the gut flagellates with the first ultrastructural investigation of the genus Metadevescovina. Our results suggest that I. marginipennis contains only one species of devescovinid flagellates, Metadevescovina modica, which comprises three variants of the same phylotype (or =99.5% sequence similarity). Monophyly of all Metadevescovina sequences obtained from Pterotermes and Incisitermes species in this and previous studies and the absence of filamentous bacterial epibionts typical of Devescovina species (M. modica is densely colonized with spirochetes) corroborate the validity of the genus Metadevescovina and allow its differentiation from other genera of devescovinid flagellates.