Provora.

Tikhonenkov et al. introduce the Provora-a newly described, yet ancient, supergroup of unicellular protists encompassing as much genetic diversity as animals and fungi combined.

Symbiosis: A duplicated host protein controlling a nascent mutualism.

Mechanistic studies on how eukaryotes ensure vertical inheritance of beneficial intracellular prokaryotes have focused mostly on highly integrated relationships. A new study by Zakharova, Tashyreva et al. reveals how a duplicated host gene impacts symbiont inheritance in a young mutualism.

Eelgrass (Zostera spp.) associated phytomyxids are host-specific congeneric parasites and predominant eukaryotes in the eelgrass rhizosphere on a global scale.

Together with increasing environmental and anthropogenic pressures, pathogenic diseases are one of the important factors contributing to the ongoing decline of seagrass meadows worldwide; yet the diversity and ecology of the microorganisms acknowledged as seagrass parasites remain critically understudied. Here, we investigate phytomyxid parasites (Rhizaria: Endomyxa: Phytomyxea) of three different eelgrass (Zostera spp.) species found in the Northern hemisphere. We present molecular evidence that Plasmodiophora bicaudata, a long-recognized parasite of dwarf eelgrass taxa, is closely related to the novel phytomyxid recently discovered in root hairs of Zostera marina, and together they form a distinct clade within the order Phagomyxida, proposed here as Feldmanniella gen. nov. A full life cycle is systematically described in a phagomyxid representative for the first time, proving its conformity with the generalized phytomyxid life history, despite previous uncertainties. The presence of primary infection stages in nearly all collected eelgrass specimens, and subsequent analysis of amplicon sequences from a global Z. marina dataset, reveal phytomyxids to be ubiquitous and one of the predominant microeukaryotes associated with eelgrass roots on a global scale. Our discoveries challenge the current view of Phytomyxea as rare entities in seagrass meadows and suggest their generally low pathogenicity in natural ecosystems.

Microbial predators form a new supergroup of eukaryotes.

Molecular phylogenetics of microbial eukaryotes has reshaped the tree of life by establishing broad taxonomic divisions, termed supergroups, that supersede the traditional kingdoms of animals, fungi and plants, and encompass a much greater breadth of eukaryotic diversity1. The vast majority of newly discovered species fall into a small number of known supergroups. Recently, however, a handful of species with no clear relationship to other supergroups have been described2-4, raising questions about the nature and degree of undiscovered diversity, and exposing the limitations of strictly molecular-based exploration. Here we report ten previously undescribed strains of microbial predators isolated through culture that collectively form a diverse new supergroup of eukaryotes, termed Provora. The Provora supergroup is genetically, morphologically and behaviourally distinct from other eukaryotes, and comprises two divergent clades of predators-Nebulidia and Nibbleridia-that are superficially similar to each other, but differ fundamentally in ultrastructure, behaviour and gene content. These predators are globally distributed in marine and freshwater environments, but are numerically rare and have consequently been overlooked by molecular-diversity surveys. In the age of high-throughput analyses, investigation of eukaryotic diversity through culture remains indispensable for the discovery of rare but ecologically and evolutionarily important eukaryotes.

First finding of free-living representatives of Prokinetoplastina and their nuclear and mitochondrial genomes.

Kinetoplastids are heterotrophic flagellated protists, including important parasites of humans and animals (trypanosomatids), and ecologically important free-living bacterial consumers (bodonids). Phylogenies have shown that the earliest-branching kinetoplastids are all parasites or obligate endosymbionts, whose highly-derived state makes reconstructing the ancestral state of the group challenging. We have isolated new strains of unusual free-living flagellates that molecular phylogeny shows to be most closely related to endosymbiotic and parasitic Perkinsela and Ichthyobodo species that, together with unidentified environmental sequences, form the clade at the base of kinetoplastids. These strains are therefore the first described free-living prokinetoplastids, and potentially very informative in understanding the evolution and ancestral states of morphological and molecular characteristics described in other kinetoplastids. Overall, we find that these organisms morphologically and ultrastructurally resemble some free-living bodonids and diplonemids, and possess nuclear genomes with few introns, polycistronic mRNA expression, high coding density, and derived traits shared with other kinetoplastids. Their genetic repertoires are more diverse than the best-studied free-living kinetoplastids, which is likely a reflection of their higher metabolic potential. Mitochondrial RNAs of these new species undergo the most extensive U insertion/deletion editing reported so far, and limited deaminative C-to-U and A-to-I editing, but we find no evidence for mitochondrial trans-splicing.

Diversity of electron transport chains in anaerobic protists.

Eukaryotic microbes (protists) that occupy low-oxygen environments often have drastically different mitochondrial metabolism compared to their aerobic relatives. A common theme among many anaerobic protists is the serial loss of components of the electron transport chain (ETC). Here, we discuss the diversity of the ETC across the tree of eukaryotes and review hypotheses for how ETCs are modified, and ultimately lost, in protists. We find that while protists have converged to some of the same metabolism as anaerobic animals, there are clear protist-specific strategies to thrive without oxygen.

Multiple origins of obligate nematode and insect symbionts by a clade of bacteria closely related to plant pathogens.

Obligate symbioses involving intracellular bacteria have transformed eukaryotic life, from providing aerobic respiration and photosynthesis to enabling colonization of previously inaccessible niches, such as feeding on xylem and phloem, and surviving in deep-sea hydrothermal vents. A major challenge in the study of obligate symbioses is to understand how they arise. Because the best studied obligate symbioses are ancient, it is especially challenging to identify early or intermediate stages. Here we report the discovery of a nascent obligate symbiosis in Howardula aoronymphium, a well-studied nematode parasite of Drosophila flies. We have found that Haoronymphium and its sister species harbor a maternally inherited intracellular bacterial symbiont. We never find the symbiont in nematode-free flies, and virtually all nematodes in the field and the laboratory are infected. Treating nematodes with antibiotics causes a severe reduction in fly infection success. The association is recent, as more distantly related insect-parasitic tylenchid nematodes do not host these endosymbionts. We also report that the Howardula nematode symbiont is a member of a widespread monophyletic group of invertebrate host-associated microbes that has independently given rise to at least four obligate symbioses, one in nematodes and three in insects, and that is sister to Pectobacterium, a lineage of plant pathogenic bacteria. Comparative genomic analysis of this group, which we name Candidatus Symbiopectobacterium, shows signatures of genome erosion characteristic of early stages of symbiosis, with the Howardula symbiont's genome containing over a thousand predicted pseudogenes, comprising a third of its genome.

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

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

Non-photosynthetic predators are sister to red algae.

Rhodophyta (red algae) is one of three lineages of Archaeplastida1, a supergroup that is united by the primary endosymbiotic origin of plastids in eukaryotes2,3. Red algae are a diverse and species-rich group, members of which are typically photoautotrophic, but are united by a number of highly derived characteristics: they have relatively small intron-poor genomes, reduced metabolism and lack cytoskeletal structures that are associated with motility, flagella and centrioles. This suggests that marked gene loss occurred around their origin4; however, this is difficult to reconstruct because they differ so much from the other archaeplastid lineages, and the relationships between these lineages are unclear. Here we describe the novel eukaryotic phylum Rhodelphidia and, using phylogenomics, demonstrate that it is a closely related sister to red algae. However, the characteristics of the two Rhodelphis species described here are nearly opposite to those that define red algae: they are non-photosynthetic, flagellate predators with gene-rich genomes, along with a relic genome-lacking primary plastid that probably participates in haem synthesis. Overall, these findings alter our views of the origins of Rhodophyta, and Archaeplastida evolution as a whole, as they indicate that mixotrophic feeding-that is, a combination of predation and phototrophy-persisted well into the evolution of the group.

Mitochondrial complex II of plants: subunit composition, assembly, and function in respiration and signaling.

Complex II [succinate dehydrogenase (succinate-ubiquinone oxidoreductase); EC 1.3.5.1; SDH] is the only enzyme shared by both the electron transport chain and the tricarboxylic acid (TCA) cycle in mitochondria. Complex II in plants is considered unusual because of its accessory subunits (SDH5-SDH8), in addition to the catalytic subunits of SDH found in all eukaryotes (SDH1-SDH4). Here, we review compositional and phylogenetic analysis and biochemical dissection studies to both clarify the presence and propose a role for these subunits. We also consider the wider functional and phylogenetic evidence for SDH assembly factors and the reports from plants on the control of SDH1 flavination and SDH1-SDH2 interaction. Plant complex II has been shown to influence stomatal opening, the plant defense response and reactive oxygen species-dependent stress responses. Signaling molecules such as salicyclic acid (SA) and nitric oxide (NO) are also reported to interact with the ubiquinone (UQ) binding site of SDH, influencing signaling transduction in plants. Future directions for SDH research in plants and the specific roles of its different subunits and assembly factors are suggested, including the potential for reverse electron transport to explain the succinate-dependent production of reactive oxygen species in plants and new avenues to explore the evolution of plant mitochondrial complex II and its utility.

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

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

Toxin and Genome Evolution in a Drosophila Defensive Symbiosis.

Defenses conferred by microbial symbionts play a vital role in the health and fitness of their animal hosts. An important outstanding question in the study of defensive symbiosis is what determines long term stability and effectiveness against diverse natural enemies. In this study, we combine genome and transcriptome sequencing, symbiont transfection and parasite protection experiments, and toxin activity assays to examine the evolution of the defensive symbiosis between Drosophila flies and their vertically transmitted Spiroplasma bacterial symbionts, focusing in particular on ribosome-inactivating proteins (RIPs), symbiont-encoded toxins that have been implicated in protection against both parasitic wasps and nematodes. Although many strains of Spiroplasma, including the male-killing symbiont (sMel) of Drosophila melanogaster, protect against parasitic wasps, only the strain (sNeo) that infects the mycophagous fly Drosophila neotestacea appears to protect against parasitic nematodes. We find that RIP repertoire is a major differentiating factor between strains that do and do not offer nematode protection, and that sMel RIPs do not show activity against nematode ribosomes in vivo. We also discovered a strain of Spiroplasma infecting a mycophagous phorid fly, Megaselia nigra. Although both the host and its Spiroplasma are distantly related to D. neotestacea and its symbiont, genome sequencing revealed that the M. nigra symbiont encodes abundant and diverse RIPs, including plasmid-encoded toxins that are closely related to the RIPs in sNeo. Our results suggest that distantly related Spiroplasma RIP toxins may perform specialized functions with regard to parasite specificity and suggest an important role for horizontal gene transfer in the emergence of novel defensive phenotypes.

Evolutionary Biology: A New Home for the Powerhouse?

Metagenomic assemblies of oceanic datasets have unearthed novel and diverse alphaproteobacterial groups. Sophisticated phylogenetic analyses based on these metagenomes suggest that mitochondria do not descend from within Alphaproteobacteria, as typically thought, but from a still undiscovered sister lineage.

Morphological Identification and Single-Cell Genomics of Marine Diplonemids.

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.

The Earliest Stages of Mitochondrial Adaptation to Low Oxygen Revealed in a Novel Rhizarian.

Mitochondria exist on a functional and evolutionary continuum that includes anaerobic mitochondrion-related organelles (MROs), such as hydrogenosomes. Hydrogenosomes lack many classical mitochondrial features, including conspicuous cristae, mtDNA, the tricarboxylic acid (TCA) cycle, and ATP synthesis powered by an electron transport chain (ETC); instead, they produce ATP anaerobically, liberating H2 and CO2 gas in the process. However, our understanding of the evolutionary transformation from aerobic mitochondria to various MRO types remains incomplete. Here we describe a novel MRO from a cercomonad (Brevimastigomonas motovehiculus n. sp.; Rhizaria). We have sequenced its 30,608-bp mtDNA and characterized organelle function through a combination of transcriptomic, genomic, and cell biological approaches. B. motovehiculus MROs are metabolically versatile, retaining mitochondrial metabolic pathways, such as a TCA cycle and ETC-driven ATP synthesis, but also possessing hydrogenosomal-type pyruvate metabolism and substrate-level phosphorylation. Notably, the B. motovehiculus ETC is degenerate and appears to be losing cytochrome-based electron transport (complexes III and IV). Furthermore, the F1Fo ATP synthase (complex V) is unique, with the highly conserved Atpα subunit fragmented into four separate pieces. The B. motovehiculus MRO appears to be in the process of losing aerobic metabolic capacities. Our findings shed light on the transition between organelle types, specifically the early stages of mitochondrial adaptation to anaerobiosis.

Gene fusion, fission, lateral transfer, and loss: Not-so-rare events in the evolution of eukaryotic ATP citrate lyase.

ATP citrate lyase (ACL) is an enzyme critical to the generation of cytosolic acetyl-CoA in eukaryotes. In most studied organisms, ACL activity is conferred in combination by two proteins, ACLA and ACLB (dsACL); however, animals encode a single-subunit ACL (ssACL) - the result of a gene fusion event. Through phylogenetic analyses, we investigated the evolution of ACL in a broad range of eukaryotes, including numerous microbes (protists). We show that the fused form is not restricted to animals, and is instead widely distributed among eukaryotes. Furthermore, ssACL and dsACL are patchily distributed and appear to be mutually exclusive; both types arose early in eukaryotic evolution. Finally, we present several compelling hypotheses of lateral gene transfer and gene loss, along with the secondary gene fission of ssACL in Ascomycota. Collectively, our in-depth analyses suggest that a complex suite of evolutionary events, usually considered rare, has shaped the evolution of ACL in eukaryotes.

Compositional complexity of the mitochondrial proteome of a unicellular eukaryote (Acanthamoeba castellanii, supergroup Amoebozoa) rivals that of animals, fungi, and plants.

We present a combined proteomic and bioinformatic investigation of mitochondrial proteins from the amoeboid protist Acanthamoeba castellanii, the first such comprehensive investigation in a free-living member of the supergroup Amoebozoa. This protist was chosen both for its phylogenetic position (as a sister to animals and fungi) and its ecological ubiquity and physiological flexibility. We report 1033 A. castellanii mitochondrial protein sequences, 709 supported by mass spectrometry data (676 nucleus-encoded and 33 mitochondrion-encoded), including two previously unannotated mtDNA-encoded proteins, which we identify as highly divergent mitochondrial ribosomal proteins. Other notable findings include duplicate proteins for all of the enzymes of the tricarboxylic acid (TCA) cycle-which, along with the identification of a mitochondrial malate synthase-isocitrate lyase fusion protein, suggests the interesting possibility that the glyoxylate cycle operates in A. castellanii mitochondria. Additionally, the A. castellanii genome encodes an unusually high number (at least 29) of mitochondrion-targeted pentatricopeptide repeat (PPR) proteins, organellar RNA metabolism factors in other organisms. We discuss several key mitochondrial pathways, including DNA replication, transcription and translation, protein degradation, protein import and Fe-S cluster biosynthesis, highlighting similarities and differences in these pathways in other eukaryotes. In compositional and functional complexity, the mitochondrial proteome of A. castellanii rivals that of multicellular eukaryotes. BIOLOGICAL SIGNIFICANCE: Comprehensive proteomic surveys of mitochondria have been undertaken in a limited number of predominantly multicellular eukaryotes. This phylogenetically narrow perspective constrains and biases our insights into mitochondrial function and evolution, as it neglects protists, which account for most of the evolutionary and functional diversity within eukaryotes. We report here the first comprehensive investigation of the mitochondrial proteome in a member (A. castellanii) of the eukaryotic supergroup Amoebozoa. Through a combination of tandem mass spectrometry (MS/MS) and in silico data mining, we have retrieved 1033 candidate mitochondrial protein sequences, 709 having MS support. These data were used to reconstruct the metabolic pathways and protein complexes of A. castellanii mitochondria, and were integrated with data from other characterized mitochondrial proteomes to augment our understanding of mitochondrial proteome evolution. Our results demonstrate the power of combining direct proteomic and bioinformatic approaches in the discovery of novel mitochondrial proteins, both nucleus-encoded and mitochondrion-encoded, and highlight the compositional complexity of the A. castellanii mitochondrial proteome, which rivals that of animals, fungi and plants.

Data showing the compositional complexity of the mitochondrial proteome of a unicellular eukaryote (Acanthamoeba castellanii, supergroup Amoebozoa).

This article describes and directly links to 1033 Acanthamoeba castellanii mitochondrial protein sequences. Of these, 709 are supported by Mass Spectrometry (MS) data (676 nucleus-encoded and 33 mitochondrion-encoded). Two of these entries are previously unannotated mtDNA-encoded proteins, which we identify as highly divergent mitochondrial ribosomal proteins. Our analysis corrects many A. castellanii protein sequences that were incorrectly inferred previously from genomic data deposited in NCBI.

Evidence for a hydrogenosomal-type anaerobic ATP generation pathway in Acanthamoeba castellanii.

Diverse, distantly-related eukaryotic lineages have adapted to low-oxygen environments, and possess mitochondrion-related organelles that have lost the capacity to generate adenosine triphosphate (ATP) through oxidative phosphorylation. A subset of these organelles, hydrogenosomes, has acquired a set of characteristic ATP generation enzymes commonly found in anaerobic bacteria. The recipient of these enzymes could not have survived prior to their acquisition had it not still possessed the electron transport chain present in the ancestral mitochondrion. In the divergence of modern hydrogenosomes from mitochondria, a transitional organelle must therefore have existed that possessed both an electron transport chain and an anaerobic ATP generation pathway. Here, we report a modern analog of this organelle in the habitually aerobic opportunistic pathogen, Acanthamoeba castellanii. This organism possesses a complete set of enzymes comprising a hydrogenosome-like ATP generation pathway, each of which is predicted to be targeted to mitochondria. We have experimentally confirmed the mitochondrial localizations of key components of this pathway using tandem mass spectrometry. This evidence is the first supported by localization and proteome data of a mitochondrion possessing both an electron transport chain and hydrogenosome-like energy metabolism enzymes. Our work provides insight into the first steps that might have occurred in the course of the emergence of modern hydrogenosomes.

The ancient and widespread nature of the ER-mitochondria encounter structure.

Mitochondria are the result of a billion years of integrative evolution, converting a once free-living bacterium to an organelle deeply linked to diverse cellular processes. One way in which mitochondria are integrated with nonendosymbiotically derived organelles is via endoplasmic reticulum (ER)-mitochondria contact sites. The ER membrane is physically tethered to the mitochondrial outer membrane by the ER-mitochondria encounter structure (ERMES). However, to date, ERMES has only ever been found in the fungal lineage. Here, we bioinformatically demonstrate that ERMES is present in lineages outside Fungi and validate this inference by mass spectrometric identification of ERMES components in Acanthamoeba castellanii mitochondria. We further demonstrate that ERMES is retained in hydrogenosome-bearing but not mitosome-bearing organisms, yielding insight into the process of reductive mitochondrial evolution. Finally, we find that the taxonomic distribution of ERMES is most consistent with rooting the eukaryotic tree between Amorphea (Animals + Fungi + Amoebozoa) + Excavata and all other eukaryotes (Diaphoratickes).