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Despite the widespread occurrence of intracellular crystalline inclusions in unicellular eukaryotes, scant attention has been paid to their composition, functions, and evolutionary origins. Using Raman microscopy, we examined200 species from all major eukaryotic supergroups. We detected cellular crystalline inclusions in 77% species out of which 80% is composed of purines, such as anhydrous guanine (62%), guanine monohydrate (2%), uric acid (12%) and xanthine (4%). Our findings shifts the paradigm assuming predominance of calcite and oxalates. Purine crystals emerge in microorganisms in all habitats, e.g., in freshwater algae, endosymbionts of reef-building corals, deadly parasites, anaerobes in termite guts, or slime molds. Hence, purine biocrystallization is a general and ancestral eukaryotic process likely present in the last eukaryotic common ancestor (LECA) and here we propose two proteins omnipresent in eukaryotes that are likely in charge of their metabolism: hypoxanthine-guanine phosphoribosyl transferase and equilibrative nucleoside transporter. Purine crystalline inclusions are multifunctional structures representing high-capacity and rapid-turnover reserves of nitrogen and optically active elements, e.g., used in light sensing. Thus, we anticipate our work to be a starting point for further studies spanning from cell biology to global ecology, with potential applications in biotechnologies, bio-optics, or in human medicine.

Symbiotic relationships drive evolutionary change and are important sources of novelty. Here we demonstrate a highly structured syntrophic symbiosis between species of the anaerobic protistAnaeramoeba(Anaeramoebae, Metamonada) and bacterial ectosymbionts. We dissected this symbiosis with long-read metagenomics, transcriptomics of host and symbiont cells coupled with fluorescent in situ hybridization (FISH), and microscopy. Genome sequencing, phylogenomic analyses and FISH show that the symbionts belong to theDesulfobacteraceaeand were acquired independently in two differentAnaeramoebaspecies. We show that ectosymbionts likely reside deep within cell surface invaginations in a symbiosomal membrane network that is tightly associated with cytoplasmic hydrogenosomes. Metabolic reconstructions based on the genomes and transcriptomes of the symbionts suggest a highly evolved syntrophic interaction. Host hydrogenosomes likely produce hydrogen, acetate, and propionate that are consumed by the symbionts dissimilatory sulfate reduction, Wood-Ljungdahl and methylmalonyl pathways, respectively. Because the host genome sequences encode several vitamin B12-dependent enzymes but appear to lack the ability to biosynthesize this vitamin, we hypothesize that the symbionts supply their hosts with B12. We detected numerous lateral gene transfers from diverse bacteria toAnaeramoeba, including genes involved in oxygen defense and anaerobic metabolism. Gene families encoding membrane-trafficking components that regulate the phagosomal maturation machinery are notably expanded inAnaeramoebaspp. and may be involved in organizing and/or stabilizing the symbiosomal membrane system. Overall, the Anaeramoebae have evolved a dynamic symbiosome comprised of a vacuolar system that facilitates positioning and maintenance of sulfate-reducing bacterial ectosymbionts.

Anaerobiosis has independently evolved in multiple lineages of ciliates, allowing them to colonize a variety of anoxic and oxygen-depleted habitats. Anaerobic ciliates commonly form symbiotic relationships with various prokaryotes, including methanogenic archaea and members of several bacterial groups. The hypothesized functions of these ecto- and endosymbionts include the symbiont utilizing the ciliate's fermentative end products to increase the host's anaerobic metabolic efficiency, or the symbiont directly providing the host with energy by denitrification or photosynthesis. The host, in turn, may protect the symbiont from competition, the environment, and predation. Despite rapid advances in sampling, molecular, and microscopy methods, as well as the associated broadening of the known diversity of anaerobic ciliates, many aspects of these ciliate symbioses, including host specificity and coevolution, remain largely unexplored. Nevertheless, with the number of comparative genomic and transcriptomic analyses targeting anaerobic ciliates and their symbionts on the rise, insights into the nature of these symbioses and the evolution of the ciliate transition to obligate anaerobiosis continue to deepen. This review summarizes the current body of knowledge regarding the complex nature of symbioses in anaerobic ciliates, the diversity of these symbionts, their role in the evolution of ciliate anaerobiosis and their significance in ecosystem-level processes.

Hypoxic, sulfidic freshwater sediments typically support a diffuse consortium of distinctive ciliated protists, including caenomorphids, metopids, and odontostomatids among others. A recent resurgence of interest in these important members of sapropelic food webs has resulted in the description of many new species and an effort, still in its infancy, to characterize them from a morphologic, molecular, and metabolic standpoint and to determine their phylogenetic relationships. Their seemingly invariable association with prokaryotic endosymbionts and, less commonly, ectosymbionts has become a focus for many researchers. In this report, based on morphologic and molecular data, we describe a Brachonella species (Ciliophora, Metopida) new to science and analyze its phylogeny. We also provide a morphologic and molecular characterization of the smallest representative of the Caenomorphidae Poche, 1913, Ludio parvulus Penard, 1922. The phylogenetic analysis confirms the inclusion of this species in the Caenomorphidae.

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Background The plastid genomes of the green algal order Chlamydomonadales tend to expand their non-coding regions, but this phenomenon is poorly understood. Here we shed new light on organellar genome evolution in Chlamydomonadales by studying a previously unknown non-photosynthetic lineage. We established cultures of two new Polytoma-like flagellates, defined their basic characteristics and phylogenetic position, and obtained complete organellar genome sequences and a transcriptome assembly for one of them. Results We discovered a novel deeply diverged chlamydomonadalean lineage that has no close photosynthetic relatives and represents an independent case of photosynthesis loss. To accommodate these organisms, we establish the new genus Leontynka, with two species (L. pallida and L. elongata) distinguishable through both their morphological and molecular characteristics. Notable features of the colourless plastid of L. pallida deduced from the plastid genome (plastome) sequence and transcriptome assembly include the retention of ATP synthase, thylakoid-associated proteins, the carotenoid biosynthesis pathway, and a plastoquinone-based electron transport chain, the latter two modules having an obvious functional link to the eyespot present in Leontynka. Most strikingly, the ~362 kbp plastome of L. pallida is by far the largest among the non-photosynthetic eukaryotes investigated to date due to an extreme proliferation of sequence repeats. These repeats are also present in coding sequences, with one repeat type found in the exons of 11 out of 34 protein-coding genes, with up to 36 copies per gene, thus affecting the encoded proteins. The mitochondrial genome of L. pallida is likewise exceptionally large, with its >104 kbp surpassed only by the mitogenome of Haematococcus lacustris among all members of Chlamydomonadales hitherto studied. It is also bloated with repeats, though entirely different from those in the L. pallida plastome, which contrasts with the situation in H. lacustris where both the organellar genomes have accumulated related repeats. Furthermore, the L. pallida mitogenome exhibits an extremely high GC content in both coding and non-coding regions and, strikingly, a high number of predicted G-quadruplexes. Conclusions With its unprecedented combination of plastid and mitochondrial genome characteristics, Leontynka pushes the frontiers of organellar genome diversity and is an interesting model for studying organellar genome evolution.

The vast majority of the more than 450 described species of Parabasalia are intestinal symbionts or parasites of animals. This endobiotic life-history is presumably ancestral although the root of Parabasalia still needs to be robustly established. The half-dozen putatively free-living species thus far described are likely independently derived from endobiotic ancestors and represent the most neglected ecological group of parabasalids. Thus, we isolated and cultivated 45 free-living strains of Parabasalia obtained from a wide variety of anoxic sediments to conduct detailed morphological and SSU rRNA gene phylogenetic analyses. Sixteen species of trichomonads were recovered. Among them, we described seven new species, three new genera, two new families, and one new order. Most of the newly described species were more or less closely related to members of already described genera. However, we uncovered a new deep-branching lineage without affinity to any currently known group of Parabasalia. The newly discovered free-living parabasalids will be key taxa in comparative analyses aimed at rooting the entire lineage and deciphering the evolutionary innovations involved in transitioning between endobiotic and free-living habitats.

We investigated intestinal trichomonads in western lowland gorillas, central chimpanzees and humans cohabiting the forest ecosystem of Dzanga-Sangha Protected Area in Central African Republic, using the internal transcribed spacer (ITS) region and SSU rRNA gene sequences. Trichomonads belonging to the genus Tetratrichomonas were detected in 23% of the faecal samples and in all host species. Different hosts were infected with different genotypes of Tetratrichomonas. In chimpanzees, we detected tetratrichomonads from ‘novel lineage 2’, which was previously reported mostly in captive and wild chimpanzees. In gorillas, we found two different genotypes of Tetratrichomonas. The ITS region sequences of the more frequent genotype were identical to the sequence found in a faecal sample of a wild western lowland gorilla from Cameroon. Sequences of the second genotype from gorillas were almost identical to sequences previously obtained from an anorexic French woman. We provide the first report of the presence of intestinal tetratrichomonads in asymptomatic, apparently healthy humans. Human tetratrichomonads belonged to the lineage 7, which was previously reported in domestic and wild pigs and a domestic horse. Our findings suggest that the ecology and spatial overlap among hominids in the tropical forest ecosystem has not resulted in exchange of intestinal trichomonads among these hosts.

Bothrostoma is a genus of anaerobic ciliates in family Metopidae comprising four species, all described based solely on the morphology of living and fixed cells. Unlike other metopids, cells of Bothrostoma are not twisted anteriorly, have a flattened preoral dome, a very prominent sail-like paroral membrane, and an adoral zone of distinctive, very narrow, curved membranelles confined to a wide, non-spiraling peristome on the ventral side. We examined 20 populations of Bothrostoma from hypoxic freshwater sediments. We provide morphological characterization and 18S rRNA gene sequences of four new species, namely B. bimicronucleatum sp. nov., B. boreale sp. nov., B. kovalyovi sp. nov., and B. robustum sp. nov., as well as B. undulans (type species), B. nasutum, and B. ovale comb. nov. (original combination Metopus undulans var. ovalis Kahl, 1932). Except for B. nasutum, Bothrostoma species show low genetic variability among geographically distant populations. Intraspecific phenotypic variability might be driven by environmental conditions. In phylogenetic analyses, Bothrostoma is not closely related to Metopus sensu stricto and forms a moderately supported clade with Planometopus, here referred to as BoPl clade. The anterior axial torsion of the body, typical of other Metopidae, appears to have been lost in the last common ancestor of the BoPl clade.

Background Mitochondria and peroxisomes are the two organelles that are most affected during adaptation to microoxic or anoxic environments. Mitochondria are known to transform into anaerobic mitochondria, hydrogenosomes, mitosomes, and various transition stages in between, collectively called mitochondrion-related organelles (MROs), which vary in enzymatic capacity. Anaerobic peroxisomes were identified only recently, and their putatively most conserved function seems to be the metabolism of inositol. The group Archamoebae includes anaerobes bearing both anaerobic peroxisomes and MROs, specifically hydrogenosomes in free-living Mastigamoeba balamuthi and mitosomes in the human pathogen Entamoeba histolytica, while the organelles within the third lineage represented by Pelomyxa remain uncharacterized. Results We generated high-quality genome and transcriptome drafts from Pelomyxa schiedti using single-cell omics. These data provided clear evidence for anaerobic derivates of mitochondria and peroxisomes in this species, and corresponding vesicles were tentatively identified in electron micrographs. In silico reconstructed MRO metabolism harbors respiratory complex II, electron-transferring flavoprotein, a partial TCA cycle running presumably in the reductive direction, pyruvate:ferredoxin oxidoreductase, [FeFe]-hydrogenases, a glycine cleavage system, a sulfate activation pathway, and an expanded set of NIF enzymes for iron-sulfur cluster assembly. When expressed in the heterologous system of yeast, some of these candidates localized into mitochondria, supporting their involvement in the MRO metabolism. The putative functions of P. schiedti peroxisomes could be pyridoxal 5′-phosphate biosynthesis, amino acid and carbohydrate metabolism, and hydrolase activities. Unexpectedly, out of 67 predicted peroxisomal enzymes, only four were also reported in M. balamuthi, namely peroxisomal processing peptidase, nudix hydrolase, inositol 2-dehydrogenase, and d-lactate dehydrogenase. Localizations in yeast corroborated peroxisomal functions of the latter two. Conclusions This study revealed the presence and partially annotated the function of anaerobic derivates of mitochondria and peroxisomes in P. schiedti using single-cell genomics, localizations in yeast heterologous systems, and transmission electron microscopy. The MRO metabolism resembles that of M. balamuthi and most likely reflects the state in the common ancestor of Archamoebae. The peroxisomal metabolism is strikingly richer in P. schiedti. The presence of myo-inositol 2-dehydrogenase in the predicted peroxisomal proteome corroborates the situation in other Archamoebae, but future experimental evidence is needed to verify additional functions of this organelle.

Pelomyxa schiedti is a free-living amoeba belonging to the group Archamoebae, which encompasses anaerobes bearing mitochondrion-related organelles (MROs) – hydrogenosomes in free-living Mastigamoeba balamuthi and mitosomes in the human pathogen Entamoeba histolytica. Anaerobic peroxisomes, another adaptation to anaerobic lifestyle, were identified only recently in M. balamuthi. We found evidence for both these organelles in the single-cell-derived genome and transcriptome of P. schiedti, and corresponding vesicles were tentatively revealed in electron micrographs. In silico reconstructed MRO metabolism seems similar to that of M. balamuthi harboring respiratory complex II, electron-transferring flavoprotein, partial TCA cycle running presumably in reductive direction, pyruvate:ferredoxin oxidoreductase, [FeFe]-hydrogenases, glycine cleavage system, and sulfate activation pathway. The cell disposes with an expanded set of NIF enzymes for iron sulfur cluster assembly, but their localization remains unclear. Quite contrary, out of 67 predicted peroxisomal enzymes, only four were reported also in M. balamuthi, namely peroxisomal processing peptidase, nudix hydrolase, inositol 2-dehydrogenase, and D-lactate dehydrogenase. Other putative functions of peroxisomes could be pyridoxal 5I⍰-phosphate biosynthesis, amino acid and carbohydrate metabolism, and hydrolase activities. Future experimental evidence is necessary to define functions of this surprisingly enzyme-rich anaerobic peroxisome.Author summaryA major part of the microbial diversity cannot be cultured in isolation, and so it escapes from traditional ways of investigation. In this paper, we demonstrate the successful approach for generating good-quality genome and transcriptome drafts from a peculiar amoeba Pelomyxa schiedti using single-cell methods. P. schiedti is a member of Archamoebae clade harboring microaerobic protists including a free-living Mastigamoeba balamuthi and a human parasite Entamoeba histolytica. Mitochondria and peroxisomes represent two organelles that are most affected during adaptation to microoxic or anoxic environments. Mitochondria are known to transform to anaerobic mitochondria, hydrogenosomes, mitosomes, and various transition stages in between, all of which encompass different enzymatic capacity. Anaerobic peroxisomes have been first noticed in M. balamuthi, but their function remained unclear for now. Data obtained in this study were used for revealing the presence and for the detailed functional annotations of anaerobic derivates of mitochondria and peroxisomes in P. schiedti, which were corroborated by transmission electron microscopy.

The type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion., Bacteria use the type 2 secretion system to secrete enzymes and toxins across the outer membrane to the environment. Here the authors analyse the T2SS pathway in three protist lineages and suggest that the early mitochondrion may have been capable of secreting proteins into the cytosol.

Loxocephalids represent a nonmonophyletic assemblage of oligohymenophorean ciliates with morphological features common to both scuticociliates (mode of ontogenesis) and hymenostomes (morphology of the oral structures). In phylogenetic analyses that include both nuclear and mitochondrial molecular markers, relationships among loxocephalid ciliates are still largely unresolved. With the aim to clarify the phylogeny of the controversial order Loxocephalida Jankowski, 1980, we provide the first 18S rRNA gene sequences of three morphologically well-described ciliates currently included in the order, namely Cinetochilum margaritaceum, Zitheron hovassei, and Zitheron muscorum comb. nov. We also provide the first 18S rRNA gene sequence for the monotypic genus Urozona. Here we present improved diagnoses for these species based on updated morphologic data. Our molecular data circumscribe the family Cinetochilidae Perty, 1852 and support the exclusion of genus Cinetochilides from family Cinetochilidae; therefore, we establish a family Cinetochilididae fam. nov. We consider Urozona as incertae sedis in Oligohymenophorea at the current state of knowledge.

Background Apicomplexa is a diverse phylum comprising unicellular endobiotic animal parasites and contains some of the most well-studied microbial eukaryotes including the devastating human pathogens Plasmodium falciparum and Cryptosporidium hominis. In contrast, data on the invertebrate-infecting gregarines remains sparse and their evolutionary relationship to other apicomplexans remains obscure. Most apicomplexans retain a highly modified plastid, while their mitochondria remain metabolically conserved. Cryptosporidium spp. inhabit an anaerobic host-gut environment and represent the known exception, having completely lost their plastid while retaining an extremely reduced mitochondrion that has lost its genome. Recent advances in single-cell sequencing have enabled the first broad genome-scale explorations of gregarines, providing evidence of differential plastid retention throughout the group. However, little is known about the retention and metabolic capacity of gregarine mitochondria. Results Here, we sequenced transcriptomes from five species of gregarines isolated from cockroaches. We combined these data with those from other apicomplexans, performed detailed phylogenomic analyses, and characterized their mitochondrial metabolism. Our results support the placement of Cryptosporidium as the earliest diverging lineage of apicomplexans, which impacts our interpretation of evolutionary events within the phylum. By mapping in silico predictions of core mitochondrial pathways onto our phylogeny, we identified convergently reduced mitochondria. These data show that the electron transport chain has been independently lost three times across the phylum, twice within gregarines. Conclusions Apicomplexan lineages show variable functional restructuring of mitochondrial metabolism that appears to have been driven by adaptations to parasitism and anaerobiosis. Our findings indicate that apicomplexans are rife with convergent adaptations, with shared features including morphology, energy metabolism, and intracellularity.

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

Marine representatives of Phytomyxea (SAR: Rhizaria: Endomyxa), a peculiar class of obligate endobiotic parasites, are a greatly understudied ecological group of protists infecting many algal, diatom, and seagrass species. Very little is known about the actual diversity, ecology, and pathogenic potential of these organisms and their taxonomic treatment in many cases follows outdated morphotaxonomic concepts. Here we focused on resolving the phylogenetic relations of the phytomyxean parasites of the widespread seagrass genus Halophila. We report the first finding of Plasmodiophora halophilae, the parasite of ovate-leaf Halophila species, after more than 100 years since its original description in 1913. We provide additional information on its anatomy, morphology, distribution, and host range, together with a phylogenetic evidence that it is congeneric with the recently rediscovered species infecting the invasive seagrass Halophila stipulacea in the Mediterranean Sea. Despite the previously hypothesized affiliation of the latter to Tetramyxa, our phylogenetic analyses of the 18S rRNA gene place Tetramyxa parasitica (a parasite of brackish water phanerogams and the type species of the genus) in the freshwater/terrestrial phytomyxean order Plasmodiophorida and reveal that phytomyxids associated with Halophila spp. form a separate deep-branching clade within the class proposed here as Marinomyxa gen. nov. We further argue that M. marina infecting H. stipulacea is most likely a species-specific parasite and implies their comigration through the Suez Canal.

Oxymonads are a group of flagellates living as gut symbionts of insects or vertebrates. They have several unique features, one of them being the absence of mitochondria. Diversity of this group is seriously understudied, which is particularly true for small species from the family Polymastigidae. We isolated 34 strains of oxymonads with Polymastigidae-like morphology from 24 host species and unused cesspits and sequenced the SSU rRNA gene. Our strains formed two clades in the phylogenetic tree with Streblomastix strix branching between them. This topology was also supported by a three-gene phylogenetic analysis. Despite considerable genetic differences between the clades, light and electron microscopy revealed only subtle differences. The larger clade is considered genus Monocercomonoides and the isolates belonging here were classified into three new species (including the first potentially free-living species), two previously described species, and three unclassified lineages. The smaller clade, here described as Blattamonas gen. nov., consists of three newly described species. Concomitantly with the description of Blattamonas, we elevate the Monocercomonoides subgenus Brachymonas to the genus level. Our study shows that, despite their conserved morphology, the molecular diversity of Polymastigidae-like oxymonads is broad and represents a substantial part of the diversity of oxymonads.

Summary Discoveries of diverse microbial eukaryotes and their inclusion in comprehensive phylogenomic analyses have crucially re-shaped the eukaryotic tree of life in the 21st century.1 At the deepest level, eukaryotic diversity comprises 9–10 "supergroups." One of these supergroups, the Metamonada, is particularly important to our understanding of the evolutionary dynamics of eukaryotic cells, including the remodeling of mitochondrial function. All metamonads thrive in low-oxygen environments and lack classical aerobic mitochondria, instead possessing mitochondrion-related organelles (MROs) with metabolisms that are adapted to low-oxygen conditions. These MROs lack an organellar genome, do not participate in the Krebs cycle and oxidative phosphorylation,2 and often synthesize ATP by substrate-level phosphorylation coupled to hydrogen production.3,4 The events that occurred during the transition from an oxygen-respiring mitochondrion to a functionally streamlined MRO early in metamonad evolution remain largely unknown. Here, we report transcriptomes of two recently described, enigmatic, anaerobic protists from the genus Anaeramoeba.5 Using phylogenomic analysis, we show that these species represent a divergent, phylum-level lineage in the tree of metamonads, emerging as a sister group of the Parabasalia and reordering the deep branching order of the metamonad tree. Metabolic reconstructions of the Anaeramoeba MROs reveal many "classical" mitochondrial features previously not seen in metamonads, including a disulfide relay import system, propionate production, and amino acid metabolism. Our findings suggest that the cenancestor of Metamonada likely had MROs with more classical mitochondrial features than previously anticipated and demonstrate how discoveries of novel lineages of high taxonomic rank continue to transform our understanding of early eukaryote evolution.

We report discovery of a new lineage of anaerobic marine amoebae and amoeboflagellates, Anaeramoeba gen. nov., represented by six newly described species. The trophic form of Anaeramoeba spp. is an amoeba corresponding to the uncommon flabellate or flamellian morphotype - it is fan-shaped and produces an anterior, flattened hyaline zone and posterior hyaline projections. In contrast to other representatives of these morphotypes, cells of Anaeramoeba spp. possess acristate mitochondrion-related organelles associated with prokaryotic symbionts, and a large acentriolar centrosome. Surprisingly, two Anaeramoeba species form morphologically unique flagellates with two or four isokont, thickened flagella. Phylogenetic analyses of the SSU rRNA gene showed that Anaeramoeba spp. form a clade, which is not robustly related to any other eukaryotic lineage. We accommodate Anaeramoeba in a new family Anaeramoebidae, which we classify as Eukaryota incertae sedis.

Metopid ciliates occupy terrestrial, freshwater, and marine habitats worldwide, playing important roles as predominant consumers of bacteria, flagellates, algae, and diatoms in hypoxic environments. Metopus and Brachonella are the most species-rich metopid genera, however most of their species have not been studied by modern methods Here, we report the morphologic, morphometric and molecular characterization, and phylogeny of Metopus es and Brachonella contorta, both types of their respective genera, collected in a broad global sampling effort. Five strains of M. es and three strains of B. contorta were studied in detail, providing the first correlation of morphology, morphometrics, and 18S rRNA gene sequencing for both. We submitted 29 new 18S rRNA gene sequences to GenBank. Phylogenetic analyses yielded trees of similar topology. A strongly supported Metopus es clade is sister to the Brachonella contorta clade. Our analysis shows genus Metopus is not monophyletic. The monophyly of Brachonella cannot yet be determined due to lack of sequences for other species of this genus in molecular databases. Both species appear to have a global distribution. Metopus es was not found in Africa, probably reflecting low sampling effort. Strains of both species showed low 18S rRNA gene sequence divergence despite wide geographic separation.

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

We report the discovery of three new species of freshwater metopid ciliates, Pileometopus lynni gen. et sp. nov., Castula flexibilis gen. et sp. nov., and Longitaenia australis gen. et sp. nov. Based on morphologic features and the 18S rRNA gene phylogeny, we transfer two known species of Metopus to the new genus Castula, as C. fusca (Kahl, 1927) comb. nov. and C. setosa (Kahl, 1927) comb. nov. and another known species is herein transferred to the new genus Longitaenia, as L. gibba (Kahl, 1927) comb. nov. Pileometopus is characterized by a turbinate body shape, a dorsal field of densely spaced dikinetids, a bipartite paroral membrane, and long caudal cilia. A distinctive morphologic feature of Castula species is long setae arising over the posterior third of the body (as opposed to a terminal tuft). Longitaenia spp. are characterized by an equatorial cytostome and long perizonal ciliary stripe relative to the cell length. Based on phylogenetic analyses of 18S rRNA gene sequences, we identify and briefly discuss strongly supported clades and intraspecific genetic polymorphism within the order Metopida.

Marine phytomyxids represent often overlooked obligate biotrophic parasites colonizing diatoms, brown algae, and seagrasses. An illustrative example of their enigmatic nature is the phytomyxid infecting the seagrass Halophila stipulacea (a well-known Lessepsian migrant from the Indo-Pacific to the Mediterranean Sea). In the Mediterranean, the occurrence of this phytomyxid was first described in 1995 in the Strait of Messina (southern Italy) and the second time in 2017 in the Aegean coast of Turkey. Here we investigated, using scuba diving, stereomicroscopy, light and scanning electron microscopy, and molecular methods, whether the symbiosis is still present in southern Italy, its distribution in this region and its relation to the previous reports. From the total of 16 localities investigated, the symbiosis has only been found at one site. A seasonal pattern was observed with exceptionally high abundance (> 40% of the leaf petioles colonized) in September 2017, absence of the symbiosis in May/June 2018, and then again high infection rates (~ 30%) in September 2018. In terms of anatomy and morphology as well as resting spore dimensions and arrangement, the symbiosis seems to be identical to the preceding observations in the Mediterranean. According to the phylogenetic analyses of the 18S rRNA gene, the phytomyxid represents the first characterized member of the environmental clade “TAGIRI-5”. Our results provide new clues about its on-site ecology (incl. possible dispersal mechanisms), hint that it is rare but established in the Mediterranean, and encourage further research into its distribution, ecophysiology, and taxonomy.

Fornicata (Metamonada) is a group of Excavata living in low-oxygen environments and lacking conventional mitochondria. It includes free-living Carpediemonas-like organisms from marine habitats and predominantly parasitic/commensal retortamonads and diplomonads. Current modest knowledge of biodiversity of Fornicata limits our ability to draw a complete picture of the evolutionary history in this group. Here, we report the discovery of a novel fornicate, Iotanema spirale gen. nov. et sp. nov., obtained from fresh feces of the gecko Phelsuma madagascariensis. Our phylogenetic analyses of the small subunit ribosomal RNA gene demonstrate that I. spirale is closely related to the free-living, marine strain PCS and the Carpediemonas-like organism Hicanonectes teleskopos within Fornicata. Iotanema spirale exhibits several features uncommon to fornicates, such as a single flagellum, a highly reduced cytoskeletal system, and the lack of the excavate ventral groove, but shares these characters with the poorly known genus Caviomonas. Therefore, I. spirale is accommodated within the family Caviomonadidae, which represents the third known endobiotic lineage of Fornicata. This study improves our understanding of character evolution within Fornicata when placed within the molecular phylogenetic context.

Archamoebae is an understudied group of anaerobic free-living or endobiotic protists that constitutes the major anaerobic lineage of the supergroup Amoebozoa. Hitherto, the phylogeny of Archamoebae was based solely on SSU rRNA and actin genes, which did not resolve relationships among the main lineages of the group. Because of this uncertainty, several different scenarios had been proposed for the phylogeny of the Archamoebae. In this study, we present the first multigene phylogenetic analysis that includes members of Pelomyxidae, and Rhizomastixidae. The analysis clearly shows that Mastigamoebidae, Pelomyxidae and Rhizomastixidae form a clade of mostly free-living, amoeboid flagellates, here called Pelobiontida. The predominantly endobiotic and aflagellated Entamoebidae represents a separate, deep-branching lineage, Entamoebida. Therefore, two unique evolutionary events, horizontal transfer of the nitrogen fixation system from bacteria and transfer of the sulfate activation pathway to mitochondrial derivatives, predate the radiation of recent lineages of Archamoebae. The endobiotic lifestyle has arisen at least three times independently during the evolution of the group. We also present new ultrastructural data that clarifies the primary divergence among the family Mastigamoebidae which had previously been inferred from phylogenetic analyses based on SSU rDNA.

Deep sequencing of prokaryotic 16S rDNA regularly reveals thousands of microbial species thriving in many common habitats. It is still unknown how this huge microbial diversity, including many potentially competing organisms, may persist at a single site. One of plausible hypotheses is that a large number of spatially separated microcommunities exist within each complex habitat. Smaller subset of the species may exist in each microcommunity and actually interact with each other. We sampled two groups of microbial stalactites growing at a single acidic mine drainage outlet as a model of multiplicated, low-complexity microhabitat. Samples from six other sites were added for comparison. Both tRFLP and 16S rDNA pyrosequencing showed that microbial communities containing 6 to 51 species-level operational taxonomic units (OTU) inhabited all stalactites. Interestingly, most OTUs including the highly abundant ones unpredictably alternated regardless of physical and environmental distance of the stalactites. As a result, the communities clustered independently on sample site and other variables when using both phylogenetic dissimilarity and OTU abundance metrics. Interestingly, artificial communities generated by pooling the biota of several adjacent stalactites together clustered by the locality more strongly than when the stalactites were analyzed separately. The most probable interpretation is that each stalactite contains likely random selection from the pool of plausible species. Such degree of stochasticity in assembly of extremophilic microbial communities is significantly greater than commonly proposed and requires caution when interpreting microbial diversity.

SUMMARYTo address the molecular diversity and occurrence of pathogenic species of the genusEntamoebaspp. in wild non-human primates (NHP) we conducted molecular-phylogenetic analyses onEntamoebafrom wild chimpanzees living in the Issa Valley, Tanzania. We compared the sensitivity of molecular [using a genus-specific polymerase chain reaction (PCR)] and coproscopic detection (merthiolate-iodine-formaldehyde concentration) ofEntamoebaspp. We identifiedEntamoebaspp. in 72 chimpanzee fecal samples (79%) subjected to species-specific PCRs for sixEntamoebaspecies/groups (Entamoeba histolytica, Entamoeba nuttalli, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba coliandEntamoeba poleckiST2). We recorded threeEntamoebaspecies:E. coli(47%),E. dispar(16%),Entamoeba hartmanni(51%). Coproscopically, we could only distinguish the cysts of complexE. histolytica/dispar/moshkovskii/nuttalliandE. coli. Molecular prevalence of entamoebas was higher than the prevalence based on the coproscopic examination. Our molecular phylogenies showed that sequences ofE. disparandE. colifrom Issa chimpanzees are closely related to sequences from humans and other NHP from GenBank. The results showed that wild chimpanzees harbourEntamoebaspecies similar to those occurring in humans; however, no pathogenic species were detected. Molecular-phylogenetic methods are critical to improve diagnostics of entamoebas in wild NHP and for determining an accurate prevalence ofEntamoebaspecies.

Oxygen plays a crucial role in energetic metabolism of most eukaryotes. Yet adaptations to low-oxygen concentrations leading to anaerobiosis have independently arisen in many eukaryotic lineages, resulting in a broad spectrum of reduced and modified mitochondrion-related organelles (MROs). In this study, we present the discovery of two new class-level lineages of free-living marine anaerobic ciliates, Muranotrichea, cl. nov. and Parablepharismea, cl. nov., that, together with the class Armophorea, form a major clade of obligate anaerobes (APM ciliates) within the Spirotrichea, Armophorea, and Litostomatea (SAL) group. To deepen our understanding of the evolution of anaerobiosis in ciliates, we predicted the mitochondrial metabolism of cultured representatives from all three classes in the APM clade by using transcriptomic and metagenomic data and performed phylogenomic analyses to assess their evolutionary relationships. The predicted mitochondrial metabolism of representatives from the APM ciliates reveals functional adaptations of metabolic pathways that were present in their last common ancestor and likely led to the successful colonization and diversification of the group in various anoxic environments. Furthermore, we discuss the possible relationship of Parablepharismea to the uncultured deep-sea class Cariacotrichea on the basis of single-gene analyses. Like most anaerobic ciliates, all studied species of the APM clade host symbionts, which we propose to be a significant accelerating factor in the transitions to an obligately anaerobic lifestyle. Our results provide an insight into the evolutionary mechanisms of early transitions to anaerobiosis and shed light on fine-scale adaptations in MROs over a relatively short evolutionary time frame.

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

Arguments based on cell energetics favour the view that a mitochondrion capable of oxidative phosphorylation was a prerequisite for the evolution of other features of the eukaryotic cell including increased volume, genome size and, eventually, phagotrophy. Contrary to this we argue that: (i) extant amitochondriate eukaryotes possess voluminous phagotrophic cells with large genomes; (ii) picoeukaryotes demonstrate that phagotrophy is feasible at prokaryotic cell sizes; and (iii) the assumption that evolution of complex features requires extra ATP, often mentioned in this context, is unfounded and should not be used in such considerations. We claim that the diversity of cell organisations and functions observed today in eukaryotes gives no reason to postulate that a mitochondrion must have preceded phagocytosis in eukaryogenesis.