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.

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.

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.

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.

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.

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.

Subcomplexes of ancestral respiratory complex I subunits rapidly turn over in vivo as productive assembly intermediates in Arabidopsis.

Subcomplexes of mitochondrial respiratory complex I (CI; EC 1.6.5.3) are shown to turn over in vivo, and we propose a role in an ancestral assembly pathway. By progressively labeling Arabidopsis cell cultures with (15)N and isolating mitochondria, we have identified CI subcomplexes through differences in (15)N incorporation into their protein subunits. The 200-kDa subcomplex, containing the ancestral γ-carbonic anhydrase (γ-CA), γ-carbonic anhydrase-like, and 20.9-kDa subunits, had a significantly higher turnover rate than intact CI or CI+CIII(2). In vitro import of precursors for these CI subunits demonstrated rapid generation of subcomplexes and revealed that their specific abundance varied when different ancestral subunits were imported. Time course studies of precursor import showed the further assembly of these subcomplexes into CI and CI+CIII(2), indicating that the subcomplexes are productive intermediates of assembly. The strong transient incorporation of new subunits into the 200-kDa subcomplex in a γ-CA mutant is consistent with this subcomplex being a key initiator of CI assembly in plants. This evidence alongside the pattern of coincident occurrence of genes encoding these particular proteins broadly in eukaryotes, except for opisthokonts, provides a framework for the evolutionary conservation of these accessory subunits and evidence of their function in ancestral CI assembly.

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).

Composition of the mitochondrial electron transport chain in acanthamoeba castellanii: structural and evolutionary insights.

The mitochondrion, derived in evolution from an α-proteobacterial progenitor, plays a key metabolic role in eukaryotes. Mitochondria house the electron transport chain (ETC) that couples oxidation of organic substrates and electron transfer to proton pumping and synthesis of ATP. The ETC comprises several multiprotein enzyme complexes, all of which have counterparts in bacteria. However, mitochondrial ETC assemblies from animals, plants and fungi are generally more complex than their bacterial counterparts, with a number of 'supernumerary' subunits appearing early in eukaryotic evolution. Little is known, however, about the ETC of unicellular eukaryotes (protists), which are key to understanding the evolution of mitochondria and the ETC. We present an analysis of the ETC proteome from Acanthamoeba castellanii, an ecologically, medically and evolutionarily important member of Amoebozoa (sister to Opisthokonta). Data obtained from tandem mass spectrometric (MS/MS) analyses of purified mitochondria as well as ETC complexes isolated via blue native polyacrylamide gel electrophoresis are combined with the results of bioinformatic queries of sequence databases. Our bioinformatic analyses have identified most of the ETC subunits found in other eukaryotes, confirming and extending previous observations. The assignment of proteins as ETC subunits by MS/MS provides important insights into the primary structures of ETC proteins and makes possible, through the use of sensitive profile-based similarity searches, the identification of novel constituents of the ETC along with the annotation of highly divergent but phylogenetically conserved ETC subunits.

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.

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.

An ancient fission of mitochondrial Cox1.

Many genes inherited from the alpha-proteobacterial ancestor of mitochondria have undergone evolutionary transfer to the nuclear genome in eukaryotes. In some rare cases, genes have been functionally transferred in pieces, resulting in split proteins that presumably interact in trans within mitochondria, fulfilling the same role as the ancestral, intact protein. We describe a nucleus-encoded mitochondrial protein (here named Cox1-c) in the amoeboid protist Acanthamoeba castellanii that is homologous to the C-terminal portion of conventional mitochondrial Cox1, whereas the corresponding portion of the mitochondrion-encoded A. castellanii Cox1 is absent. Bioinformatics searches retrieved nucleus-encoded Cox1-c homologs in most major eukaryotic supergroups; in these cases, also, the mitochondrion-encoded Cox1 lacks the corresponding C-terminal motif. These data constitute the first report of functional relocation of a portion of cox1 to the nucleus. This transfer event was likely ancient, with the resulting nuclear cox1-c being differentially activated across the eukaryotic domain.

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.

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.

Evidence for an early evolutionary emergence of gamma-type carbonic anhydrases as components of mitochondrial respiratory complex I.

BACKGROUND: The complexity of mitochondrial complex I (CI; NADH:ubiquinone oxidoreductase) has increased considerably relative to the homologous complex in bacteria. Comparative analyses of CI composition in animals, fungi and land plants/green algae suggest that novel components of mitochondrial CI include a set of 18 proteins common to all eukaryotes and a variable number of lineage-specific subunits. In plants and green algae, several purportedly plant-specific proteins homologous to gamma-type carbonic anhydrases (gammaCA) have been identified as components of CI. However, relatively little is known about CI composition in the unicellular protists, the characterizations of which are essential to our understanding of CI evolution. RESULTS: We have performed a tandem mass spectrometric characterization of CI from the amoeboid protozoon Acanthamoeba castellanii. Among the proteins identified were two gammaCA homologs, AcCa1 and AcCa2, demonstrating that gammaCA proteins are not specific to plants/green algae. In fact, through bioinformatics searches we detected gammaCA homologs in diverse protist lineages, and several of these homologs are predicted to possess N-terminal mitochondrial targeting peptides. CONCLUSIONS: The detection of gammaCAs in CI of Acanthamoeba, considered to be a closer relative of animals and fungi than plants, suggests that gammaCA proteins may have been an ancestral feature of mitochondrial CI, rather than a novel, plant-specific addition. This assertion is supported by the presence of genes encoding gammaCAs in the nuclear genomes of a wide variety of eukaryotes. Together, these findings emphasize the importance of a phylogenetically broad characterization of CI for elucidating CI evolution in eukaryotes.

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.

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.