Article

First multigene analysis of Archamoebae (Amoebozoa: Conosa) robustly reveals its phylogeny and shows that Entamoebidae represents a deep lineage of the group

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Abstract

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.

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... The name Archamoebae was introduced by Cavalier-Smith in 1983 [27] and is currently used as a class within the Amoebozoa group [28]. The Archamoebae is currently composed of four main clades, the entamoebae, pelomyxids, mastigamoebids and Rhizomastix, with Tricholimax sometimes treated as a fifth clade or as an incertae sedis genus. ...
... These species are known to form resilient uninucleate cysts in the environment and to infect the host Malpighian tubules and midgut [12]. The conditions The name Archamoebae was introduced by Cavalier-Smith in 1983 [27] and is currently used as a class within the Amoebozoa group [28]. The Archamoebae is currently composed of four main clades, the entamoebae, pelomyxids, mastigamoebids and Rhizomastix, with Tricholimax sometimes treated as a fifth clade or as an incertae sedis genus. ...
... The Archamoebae is currently composed of four main clades, the entamoebae, pelomyxids, mastigamoebids and Rhizomastix, with Tricholimax sometimes treated as a fifth clade or as an incertae sedis genus. Nevertheless, phylogenetic analysis splits the group into entamoebids, with the genus Entamoeba, and pelobionts (pelomyxids, mastigamoebids and Rhizomastix) [28]. ...
Article
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As the insects for food and feed industry grows, a new understanding of the industrially reared insect microbiome is needed to better comprehend the role that it plays in both maintaining insect health and generating disease. While many microbiome projects focus on bacteria, fungi or viruses, protists (including microsporidia) can also make up an important part of these assemblages. Past experiences with intensive invertebrate rearing indicate that these parasites, whilst often benign, can rapidly sweep through populations, causing extensive damage. Here, we review the diversity of microsporidia and protist species that are found in reared insect hosts and describe the current understanding of their host spectra, life cycles and the nature of their interactions with hosts. Major entomopathogenic parasite groups with the potential to infect insects currently being reared for food and feed include the Amoebozoa, Apicomplexa, Ciliates, Chlorophyta, Euglenozoa, Ichtyosporea and Microsporidia. However, key gaps exist in the understanding of how many of these entomopathogens affect host biology. In addition, for many of them, there are very limited or even no molecular data, preventing the implementation of molecular detection methods. There is now a pressing need to develop and use novel molecular tools, coupled with standard molecular diagnostic methods, to help unlock their biology and predict the effects of these poorly studied protist parasites in intensive insect rearing systems.
... Archamoebae are free-living or parasitic members of the group Amoebozoa, occurring under anaerobic or microaerobic conditions (Ptáčková et al., 2013;Zadrobílková et al., 2015;Panek et al., 2016;Kang et al., 2017). Archamoebae are highly unusual protists lacking mitochondria, peroxisomes and morphologically differentiated dictyosomes. ...
... In the other Archamoebae, microtubular cytoskeleton is mostly represented by the elements of the flagellar apparatus (Brugerolle, 1982, Chavez et al., 1986, Simpson et al.,1997, Walker at al., 2001, Ptáčková et al., 2013, Zadrobílková et al., 2015, 2016, Panek et al., 2016. All archamoebae without exception have radial microtubules in the rootlet system, which start from the lateral surface of the kinetosome in one or several layers. ...
... In Ludmila Chistyakova, Mariia Berdieva, Victor Tsarev and Alexander Frolov Mastigamoeba spp. and M. hylae these microtubules are involved in the formation of the karyomastigont (Brugerolle, 1982, Simpson et al.,1997, Walker at al., 2001, Chistyakova et al., 2012, Panek et al., 2016. In Mastigella spp. ...
... The base of the flagellum gives rise to a cone of microtubules that connect to the nucleus. Molecular phylogenies divide mastigamoebids into two clades, "A," containing large species with a broad flagellar apparatus (e.g., Mastigamoeba balamuthi), and "B," containing small species with a narrow flagellar apparatus and trailing pseudopodia (e.g., Mastigamoeba simplex) as well as Endolimax and Iodamoeba (Ptáčková et al. 2013;Pánek et al. 2016). Members of Endolimax and Iodamoeba have entirely lost the flagellar apparatus and were historically classified within entamoebids. ...
... Mastigina is a poorly known genus, with few sightings and no molecular data, currently classified as incertae sedis (Pánek et al. 2016) though likely to be a member of the mastigamoebids. It has many similarities to Mastigamoeba but has a limax body shape where pseudopodia emerge only at the anterior or posterior ends. ...
... Tricholimax hylae, a large multinucleate amoeba with a short, nonfunctional flagellum, is endobiotic in the hindgut of frog tadpoles. The phylogenetic placement of Tricholimax is unknown, in the absence of molecular data, so it is classified as Archamoebae incertae sedis (Pánek et al. 2016); nonetheless, it shows considerable similarity to Mastigella and Pelomyxa. Its identity has historically been confused with that of Mastigina. ...
Chapter
Members of the Archamoebae comprise free-living and endobiotic amoeboid flagellates, amoeboflagellates, and amoebae, with distinctive hyaline cytoplasm and bulging pseudopodia. They live in anoxic or microoxic habitats and are anaerobes, lacking typical mitochondria, as well as Golgi stacks, plastids, and normal peroxisomal microbodies. They have a distinctive flagellar apparatus present in all flagellated members of the group. Life cycles of individual species can include flagellates, amoebae of various sizes, and cysts. In recent years, the group has been divided into five separate families, Mastigamoebidae, Entamoebidae, Pelomyxidae, Tricholimacidae, and Rhizomastixidae, whose interrelationships have not been completely resolved. Here, we clarify the composition of these groups and the circumscription of genera in the Archamoebae.
... The base of the flagellum gives rise to a cone of microtubules that connect to the nucleus. Molecular phylogenies divide mastigamoebids into two clades, "A," containing large species with a broad flagellar apparatus (e.g., Mastigamoeba balamuthi), and "B," containing small species with a narrow flagellar apparatus and trailing pseudopodia (e.g., Mastigamoeba simplex) as well as Endolimax and Iodamoeba (Ptáčková et al. 2013;Pánek et al. 2016). Members of Endolimax and Iodamoeba have entirely lost the flagellar apparatus and were historically classified within entamoebids. ...
... Mastigina is a poorly known genus, with few sightings and no molecular data, currently classified as incertae sedis (Pánek et al. 2016) though likely to be a member of the mastigamoebids. It has many similarities to Mastigamoeba but has a limax body shape where pseudopodia emerge only at the anterior or posterior ends. ...
... Tricholimax hylae, a large multinucleate amoeba with a short, nonfunctional flagellum, is endobiotic in the hindgut of frog tadpoles. The phylogenetic placement of Tricholimax is unknown, in the absence of molecular data, so it is classified as Archamoebae incertae sedis (Pánek et al. 2016); nonetheless, it shows considerable similarity to Mastigella and Pelomyxa. Its identity has historically been confused with that of Mastigina. ...
Chapter
Members of the Archamoebae comprise free-living and endobiotic amoeboid flagellates, amoeboflagellates, and amoebae, with distinctive hyaline cytoplasm and bulging pseudopodia. They live in anoxic or microoxic habitats and are anaerobes, lacking typical mitochondria, as well as Golgi stacks, plastids, and normal peroxisomal microbodies. They have a distinctive flagellar apparatus present in all flagellated members of the group. Life cycles of individual species can include flagellates, amoebae of various sizes, and cysts. In recent years, the group has been divided into five separate families, Mastigamoebidae, Entamoebidae, Pelomyxidae, Tricholimacidae, and Rhizomastixidae, whose interrelationships have not been completely resolved. Here, we clarify the composition of these groups and the circumscription of genera in the Archamoebae.
... For instance, it is possible to conclude that LCAA had a flagellate state in its life history (Spiegel 1991(Spiegel , 2011Spiegel et al. 1995;Lahr et al. 2011b;Adl et al. 2012;Yubuki and Leander 2013) as this character must have been present in the Last Eukaryote Common Ancestor (LECA; Goodenough and Heitman 2014). Relatively few amoebozoan lineages have a flagellate state, and all confirmed flagellate taxa are found in Evosea ( fig. 3, see supplementary table S1, Supplementary Material online ;Spiegel 1991;Spiegel et al. 1995;Mikrjukov and Mylnikov 1998;Smirnov et al. 2011;Adl et al. 2012;Ptackova et al. 2013;Berney et al. 2015;Zadrobilkova et al. 2015;P anek et al. 2016). ...
... These nearly universal features of flagella are consistent with their shared evolutionary history with LECA; thus, the presence of a flagellate state in LCAA. Flagellates in Archamoebae have a less complex flagellar apparatus compared with those within Variosea and Eumycetozoa (Ptackova et al. 2013;Zadrobilkova et al. 2015;P anek et al. 2016), and careful work has yet to conclude what homologies exist between the flagellar rootlets in Archamoebae and those of the more complex flagellar apparatuses in Variosea and Eumycetozoa. Some superficial comparisons have been made (Cavalier-Smith 1998;Cavalier-Smith et al. 2004. ...
... The best-fitting available model for ML analyses was LG þ C4 þ C60 þ F with class weights optimized from the data set. We used this model to estimate the "posterior mean site frequencies" using the PHYLOBAYES tree as a guide tree (using the exchangeabilities from the LG matrix; Wang et al. 2014;P anek et al. 2016) followed by tree-searching and bootstrapping (http://www.iqtree.org/doc/Complex-Models/; last accessed December 20, 2016). ...
Article
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Amoebozoa is the eukaryotic supergroup sister to Obazoa, the lineage that contains the animals and Fungi, as well as their protistan relatives, and the breviate and apusomonad flagellates. Amoebozoa is extraordinarily diverse, encompassing important model organisms and significant pathogens. Although amoebozoans are integral to global nutrient cycles and present in nearly all environments, they remain vastly understudied. We present a robust phylogeny of Amoebozoa based on broad representative set of taxa in a phylogenomic framework (325 genes). By sampling 61 taxa using culture-based and single-cell transcriptomics, our analyses show two major clades of Amoebozoa, Discosea and Tevosa. This phylogeny refutes previous studies in major respects. Our results support the hypothesis that the last common ancestor of Amoebozoa was sexual and flagellated, it also may have had the ability to disperse propagules from a sporocarp-type fruiting body. Overall, the main macroevolutionary patterns in Amoebozoa appear to result from the parallel losses of homologous characters of a multiphase life cycle that included flagella, sex, and sporocarps rather than independent acquisition of convergent features.
... Archamoebae represents a clade of microaerophilic protists nested within a broader group of predominantly aerobic amoebozoans [19,20] represented, e.g., by Dictyostelium discoideum (Eumycetozoa), known to bear a classical aerobic mitochondrion [21], or by their more distant amoebozoan relative Acanthamoeba castellanii (Centramoebida) with mitochondria potentially adapted to periods of anaerobiosis and exhibiting a highly complex proteome [12,22]. Small to almost inconspicuous MROs have been characterized in two Archamoebae, the parasitic E. histolytica and the free-living M. balamuthi. ...
... Pelomyxa is a free-living archamoeba that is distantly related to both M. balamuthi and E. histolytica [19], and so it represents a valuable point for tracing the evolution of anaerobic adaptations. Pelomyxa schiedti was previously isolated from freshwater sediments of lake Skadar in Albania [27]. ...
Article
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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.
... Entamoebids lack flagella, whereas a single flagellum exists in most pelobionts. The flagellum, when present, has a standard axoneme except for the for the lack of outer dynein arms, and the basal body is often associated with a cone of microtubules covering the nucleus (Walker et al. 2001;P anek et al. 2016). Phylogenetic analyses revealed that Archamoebae clustered with the aerobic taxa Eumycetozoa and Variosea in the Conosa group (also called Evosea) and that the last common Conosa ancestor (LCCA) was clearly an aerobic free-living protist (P anek et al. 2016;Kang et al. 2017). ...
... The flagellum, when present, has a standard axoneme except for the for the lack of outer dynein arms, and the basal body is often associated with a cone of microtubules covering the nucleus (Walker et al. 2001;P anek et al. 2016). Phylogenetic analyses revealed that Archamoebae clustered with the aerobic taxa Eumycetozoa and Variosea in the Conosa group (also called Evosea) and that the last common Conosa ancestor (LCCA) was clearly an aerobic free-living protist (P anek et al. 2016;Kang et al. 2017). Therefore, the Conosa group provides an interesting opportunity to trace the history of parasitism from the aerobic LCCA via the last common anaerobic ancestor of Archamoebae (LCAA) to parasitic Entamoeba. ...
Article
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The transition of free-living organisms to parasitic organisms is a mysterious process that occurs in all major eukaryotic lineages. Parasites display seemingly unique features associated with their pathogenicity; however, it is important to distinguish ancestral preconditions to parasitism from truly new parasite-specific functions. Here, we sequenced the genome and transcriptome of anaerobic free-living M. balamuthi and performed phylogenomic analysis of four related members of the Archamoebae, including Entamoeba histolytica, an important intestinal pathogen of humans. We aimed to trace gene histories throughout the adaptation of the aerobic ancestor of Archamoebae to anaerobiosis and throughout the transition from a free-living to a parasitic lifestyle. These events were associated with massive gene losses that, in parasitic lineages, resulted in a reduction in structural features, complete losses of some metabolic pathways, and a reduction in metabolic complexity. By reconstructing the features of the common ancestor of Archamoebae, we estimated preconditions for the evolution of parasitism in this lineage. The ancestor could apparently form chitinous cysts, possessed proteolytic enzyme machinery, compartmentalized the sulfate activation pathway in mitochondrion-related organelles, and possessed the components for anaerobic energy metabolism. After the split of Entamoebidae, this lineage gained genes encoding surface membrane proteins that are involved in host-parasite interactions. In contrast, gene gains identified in the M. balamuthi lineage were predominantly associated with polysaccharide catabolic processes. A phylogenetic analysis of acquired genes suggested an essential role of lateral gene transfer in parasite evolution (Entamoeba) and in adaptation to anaerobic aquatic sediments (Mastigamoeba).
... protists (Pánek et al., 2016) Phaeodactylum tricornutum, a diatom alga, ...
... similarly identified a large number of genes encoding identical amino acid sequences, albeit by PCR (Lahr et al., 2011). The fact that these three organisms are members of the Amoebozoa (Pánek et al., 2016) suggests that this might be a general feature of amoeboid genomes and marks a departure from the otherwise similar genomic organisation of cytoskeletal elements in plants (see section 1.3.2). ...
Thesis
During the evolution of gene families, functional diversification of proteins often follows gene duplication. However, some gene families expand while preserving protein sequence. Why would a cell need to maintain multiple copies of the same gene? In this thesis I have addressed this question for an actin gene family containing 17 genes encoding an identical protein in the social amoeba Dictyostelium discoideum. Using bioinformatics I identified several highly conserved sequence elements as potential regulatory motifs, yet found that gene expression patterns during development are broadly similar across the gene family. Turning to live cell imaging I showed that family members display different transcription dynamics, with strong 'bursty' behaviours contrasted by more steady, continuous transcriptional activity. By switching promoters I showed that different dynamics are directly determined by endogenous promoter sequences, rather than genomic context. I have explored how cell-to-cell variability in gene expression introduced by bursty transcription propagates to resultant cytoplasmic mRNA and protein and showed that population variance of these molecules is reduced compared to nascent transcription. Finally, I generated cell lines with up to 6 genes knocked out and showed that these cells potentially display a minor defect in growth. Overall these data suggest that expanded gene families are utilised not only to generate sufficient protein for normal cell physiology, but also to enable both robustness and responsiveness to a range of stimuli regulating the expression of essential genes.
... The genus Pelomyxa belongs to Archamoebae and its representatives inhabit hypoxic freshwater environments [1][2][3]. Species of Pelomyxa, which may reach several millimeters in size, possess numerous non-motile flagella with an aberrant axonemal structure [4,5] and were originally considered to lack elementary cell organelles such as Golgi bodies and mitochondria. Early morphological studies of Pelomyxa palustris have shown the presence of microbody-like granules that might represent a mitochondriarelated organelle (MRO) [6], and in Pelomyxa schiedti such MRO was recently characterized using single-cell genomic and transcriptomic approaches [7]. ...
Article
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Pelomyxa is a genus of anaerobic amoebae that live in consortia with multiple prokaryotic endosymbionts. Although the symbionts represent a large fraction of the cellular biomass, their metabolic roles have not been investigated. Using single-cell genomics and transcriptomics, we have characterized the prokaryotic community associated with P. schiedti , which is composed of two bacteria, Candidatus Syntrophus pelomyxae (class Deltaproteobacteria ) and Candidatus Vesiculincola pelomyxae (class Clostridia ), and a methanogen, Candidatus Methanoregula pelomyxae. Fluorescence in situ hybridization and electron microscopy showed that Ca . Vesiculincola pelomyxae is localized inside vesicles, whereas the other endosymbionts occur freely in the cytosol, with Ca . Methanoregula pelomyxae enriched around the nucleus. Genome and transcriptome-based reconstructions of the metabolism suggests that the cellulolytic activity of P. schiedti produces simple sugars that fuel its own metabolism and the metabolism of a Ca . Vesiculincola pelomyxae, while Ca . Syntrophus pelomyxae energy metabolism relies on degradation of butyrate and isovalerate from the environment. Both species of bacteria and the ameba use hydrogenases to transfer the electrons from reduced equivalents to hydrogen, a process that requires a low hydrogen partial pressure. This is achieved by the third endosymbiont, Ca . Methanoregula pelomyxae, which consumes H 2 and formate for methanogenesis. While the bacterial symbionts can be successfully eliminated by vancomycin treatment without affecting the viability of the amoebae, treatment with 2-bromoethanesulfonate, a specific inhibitor of methanogenesis, killed the amoebae, indicating the essentiality of the methanogenesis for this consortium.
... Entamoebidae is a predominantly endobiotic and aflagellated group representing a separate, deep-branching lineage of Archamoebae. 15 Entamoeba is a unique genus typified by the enteric pathogen of humans, Entamoeba histolytica. One of the unique aspects of this group is that its members, including E. histolytica, lack mitochondria and have a complex taxonomic relationship with other Archamoebae. ...
Article
The amoeba Malpighamoeba mellificae is the etiologic agent of amoebic (amoeba) disease of Western honey bees (Apis mellifera). M. mellificae damages the Malpighian tubules, which is believed to weaken and kill the host bee. Here, the authors describe the detection of this organism in a honey bee colony in the Yukon Territory, Canada. The Malpighian tubules of 14% (7/50) of the adult worker bees were discolored dark brown. Fifteen bees screened using conventional polymerase chain reaction for the 18S gene of M. mellificae were positive for the pathogen. Histologically, the lumens of Malpighian tubules were packed with amoebae, causing dilation of the tubules and attenuation and loss of the tubular epithelium. This phylogenetic analysis places M. mellificae in a new clade, a sister group to the Entamoebidae. This work provides a foundation for further investigation into the distribution, prevalence, and pathology associated with M. mellificae infection.
... Most Pelomyxa spp. have immotile or slow-moving flagella uninvolved in cell locomotion (Frolov, 2011;Ptáčková et al., 2013;Chistyakova et al., 2013Chistyakova et al., , 2020aPanek et al., 2016, Walker et al., 2017. The cytoplasm of most pelomyxae has a "foamy" appearance due to the abundance of optically empty vacuoles, the so-called "structural vacuoles" (Andresen et al., 1968;Goodkov and Seravin, 1991;Chistyakova et al., 2013). ...
Article
We described Pelomyxa doughnuta sp. nov. and examined it with the use of light, electron and immunofluorescence microscopy as well as cytochemical methods. The cells of P. doughnuta sp. nov. are usually binuclear, although cells with one, three or four nuclei are sometimes found in the population. A unique feature of the new species is a dense capsule around the nucleus. It consists of a continuous layer of glycogen 5-20 µm thick. The tubulin cytoskeleton is mainly represented by perinuclear microtubules. P. doughnuta sp. nov. has a filamentous glycocalyx and strongly reduced components of flagellar apparatus. Obligate prokaryotic endocytobionts of two morphotypes are present in the cytoplasm.
... Also for cells with low RNA content, there can be a problem with unspecific amplification due to changed balance between the concentration of oligos and mRNA of the cell [15]. The potential problem with lysis is addressed by using freezethaw cycles in − 80°C chilled isopropanol, which previously have been reported as a successful lysis procedure [16,17]. Besides the improved lysis we already know can be crucial, we test modifications of Smart-seq2 to maximise costefficiency and minimise artifacts during cDNA synthesis. ...
Article
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Background: Most diversity in the eukaryotic tree of life is represented by microbial eukaryotes, which is a polyphyletic group also referred to as protists. Among the protists, currently sequenced genomes and transcriptomes give a biased view of the actual diversity. This biased view is partly caused by the scientific community, which has prioritized certain microbes of biomedical and agricultural importance. Additionally, some protists remain difficult to maintain in cultures, which further influences what has been studied. It is now possible to bypass the time-consuming process of cultivation and directly analyze the gene content of single protist cells. Single-cell genomics was used in the first experiments where individual protists cells were genomically explored. Unfortunately, single-cell genomics for protists is often associated with low genome recovery and the assembly process can be complicated because of repetitive intergenic regions. Sequencing repetitive sequences can be avoided if single-cell transcriptomics is used, which only targets the part of the genome that is transcribed. Results: In this study we test different modifications of Smart-seq2, a single-cell RNA sequencing protocol originally developed for mammalian cells, to establish a robust and more cost-efficient workflow for protists. The diplomonad Giardia intestinalis was used in all experiments and the available genome for this species allowed us to benchmark our results. We could observe increased transcript recovery when freeze-thaw cycles were added as an extra step to the Smart-seq2 protocol. Further we reduced the reaction volume and purified the amplified cDNA with alternative beads to test different cost-reducing changes of Smart-seq2. Neither improved the procedure, and reducing the volumes by half led to significantly fewer genes detected. We also added a 5' biotin modification to our primers and reduced the concentration of oligo-dT, to potentially reduce generation of artifacts. Except adding freeze-thaw cycles and reducing the volume, no other modifications lead to a significant change in gene detection. Therefore, we suggest adding freeze-thaw cycles to Smart-seq2 when working with protists and further consider our other modification described to improve cost and time-efficiency. Conclusions: The presented single-cell RNA sequencing workflow represents an efficient method to explore the diversity and cell biology of individual protist cells.
... Moreover, oxygen-sensitive enzymes such as hydrogenases are present in M. balamuthi hydrogenosomes and in the cytosol, producing hydrogen from both compartments (19). Archamoebae belongs to the group Conosa with the sister lineage Mycetozoa that inhabits aerobic niches and contains oxygen-respiring mitochondria and standard peroxisomes (20,21). Thus, it is likely that hydrogenosomes in M. balamuthi evolved from aerobic mitochondria via secondary adaptation to anaerobic environments, and we expected that an anaerobic lifestyle would lead to the loss of peroxisomes, as observed in other anaerobic protists. ...
Article
The adaptation of eukaryotic cells to anaerobic conditions is reflected by substantial changes to mitochondrial metabolism and functional reduction. Hydrogenosomes belong among the most modified mitochondrial derivative and generate molecular hydrogen concomitant with ATP synthesis. The reduction of mitochondria is frequently associated with loss of peroxisomes, which compartmentalize pathways that generate reactive oxygen species (ROS) and thus protect against cellular damage. The biogenesis and function of peroxisomes are tightly coupled with mitochondria. These organelles share fission machinery components, oxidative metabolism pathways, ROS scavenging activities, and some metabolites. The loss of peroxisomes in eukaryotes with reduced mitochondria is thus not unexpected. Surprisingly, we identified peroxisomes in the anaerobic, hydrogenosome-bearing protist Mastigamoeba balamuthi . We found a conserved set of peroxin (Pex) proteins that are required for protein import, peroxisomal growth, and division. Key membrane-associated Pexs ( Mb Pex3, Mb Pex11, and Mb Pex14) were visualized in numerous vesicles distinct from hydrogenosomes, the endoplasmic reticulum (ER), and Golgi complex. Proteomic analysis of cellular fractions and prediction of peroxisomal targeting signals (PTS1/PTS2) identified 51 putative peroxisomal matrix proteins. Expression of selected proteins in Saccharomyces cerevisiae revealed specific targeting to peroxisomes. The matrix proteins identified included components of acyl-CoA and carbohydrate metabolism and pyrimidine and CoA biosynthesis, whereas no components related to either β-oxidation or catalase were present. In conclusion, we identified a subclass of peroxisomes, named “anaerobic” peroxisomes that shift the current paradigm and turn attention to the reductive evolution of peroxisomes in anaerobic organisms.
... The ML tree corresponding to Fig. 3 had substantially lower support for many bipartitions and a less accurate topology (not shown), not only with respect to planomonads but also in wrongly placing Cutosea within Discosea making Discosea seem paraphyletic. Our CAT GTR 51-protein tree was markedly superior for Amoebozoa than a tree using the slightly less accurate CAT Poisson algorithm for only seven proteins that wrongly placed Cutosea within Conosa and Tubulinea within Discosea (Panek et al. 2016), though that tree more correctly placed Archamoebae as sisters of M y c e t o z o ap e r h a p s b e c a u s e i t i n c l u d e d e i g h t Archamoebae, not just one. ...
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Palaeontologically, eubacteria are > 3× older than neomura (eukaryotes, archaebacteria). Cell biology contrasts ancestral eubacterial murein peptidoglycan walls and derived neomuran N-linked glycoprotein coats/walls. Misinterpreting long stems connecting clade neomura to eubacteria on ribosomal sequence trees (plus misinterpreted protein paralogue trees) obscured this historical pattern. Universal multiprotein ribosomal protein (RP) trees, more accurate than rRNA trees, are taxonomically undersampled. To reduce contradictions with genically richer eukaryote trees and improve eubacterial phylogeny, we constructed site-heterogeneous and maximum-likelihood universal three-domain, two-domain, and single-domain trees for 143 eukaryotes (branching now congruent with 187-protein trees), 60 archaebacteria, and 151 taxonomically representative eubacteria, using 51 and 26 RPs. Site-heterogeneous trees greatly improve eubacterial phylogeny and higher classification, e.g. showing gracilicute monophyly, that many ‘rDNA-phyla’ belong in Proteobacteria, and reveal robust new phyla Synthermota and Aquithermota. Monoderm Posibacteria and Mollicutes (two separate wall losses) are both polyphyletic: multiple outer membrane losses in Endobacteria occurred separately from Actinobacteria; neither phylum is related to Chloroflexi, the most divergent prokaryotes, which originated photosynthesis (new model proposed). RP trees support an eozoan root for eukaryotes and are consistent with archaebacteria being their sisters and rooted between Filarchaeota (=Proteoarchaeota, including ‘Asgardia’) and Euryarchaeota sensu-lato (including ultrasimplified ‘DPANN’ whose long branches often distort trees). Two-domain trees group eukaryotes within Planctobacteria, and archaebacteria with Planctobacteria/Sphingobacteria. Integrated molecular/palaeontological evidence favours negibacterial ancestors for neomura and all life. Unique presence of key pre-neomuran characters favours Planctobacteria only as ancestral to neomura, which apparently arose by coevolutionary repercussions (explained here in detail, including RP replacement) of simultaneous outer membrane and murein loss. Planctobacterial C-1 methanotrophic enzymes are likely ancestral to archaebacterial methanogenesis and β-propeller-α-solenoid proteins to eukaryotic vesicle coats, nuclear-pore-complexes, and intraciliary transport. Planctobacterial chaperone-independent 4/5-protofilament microtubules and MamK actin-ancestors prepared for eukaryote intracellular motility, mitosis, cytokinesis, and phagocytosis. We refute numerous wrong ideas about the universal tree.
... Also for cells with low RNA content, there can be a problem with unspecific amplification due to changed balance between the concentration of oligos and mRNA of the cell [13]. The potential problem with lysis is addressed by using freeze-thaw cycles in -80 °C chilled isopropanol, which previously have been reported as a successful lysis procedure [14,15]. Besides the improved lysis we already know can be crucial, we test modifications of Smart-seq2 that could make the protocol more cost-efficient or reduce the generation of artifacts during cDNA synthesis. ...
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Most diversity in the eukaryotic tree of life are represented by microbial eukaryotes, which is a polyphyletic group also referred to as protists. Among the protists, currently sequenced genomes and transcriptomes give a biased view of the actual diversity. This biased view is partly caused by the scientific community, which has prioritized certain microbes of biomedical and agricultural importance. Additionally, it is challenging to establish protist cultures, which further influence what has been studied. It is now possible to bypass the time-consuming process of cultivation and directly analyze the gene content of single protist cells. Single-cell genomics was used in the first experiments where individual protists cells were genomically explored. Unfortunately, single-cell genomics for protists are often associated with low genome recovery and the assembly process can be complicated because of repetitive intergenic regions. Sequencing repetitive sequences can be avoided if single-cell transcriptomics is used, which only targets the part of the genome that is transcribed. In this study we test different modifications of Smart-seq2, a single-cell RNA sequencing protocol originally developed for mammalian cells, to establish a robust and more cost-efficient workflow for protists. The diplomonad Giardia intestinalis was used in all experiments and the available genome for this species allowed us to benchmark our results. We could observe increased transcript recovery when freeze-thaw cycles were added as an extra step to the Smart-seq2 protocol. Further we tried reducing the reaction volume and purifying with alternative beads to test different cost-reducing changes of Smart-seq2. Neither improved the procedure, and cutting the volumes by half actually led to significantly fewer genes detected. We also added a 5′ biotin modification to our primers and reduced the concentration of oligo-dT, to potentially reduce generation of artifacts. Except adding freeze-thaw cycles and reducing the volume, no other modifications lead to a significant change in gene detection. Therefore, we suggest adding freeze-thaw cycles to Smart-seq2 when working with protists and further consider our other modification described to improve cost and time-efficiency.
... Entamoeba is a member of the Entamoebidae, a deep lineage within the Archamoebae (Pánek et al., 2016). Entamoeba species use pseudopodia for locomotion and lack flagella, a morphologically identifiable Golgi apparatus, peroxisomes and canonical mitochondria (Loftus et al., 2005;Ptáčková et al., 2013). ...
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The genus Entamoeba comprises mostly gut parasites and commensals of invertebrate and vertebrate animals including humans. Herein, we report a new species of Entamoeba isolated from the gut of Asian swamp eels (Monopterus albus) in northern Thailand. Morphologically, the trophozoite is elongated and has a single prominent pseudopodium with no clear uroid. The trophozoite is actively motile, 30-50 μm in length and 9-13 μm in width. Observed cysts were uninucleate, ranging in size from 12.5-17.5 μm in diameter. Chromatin forms a fine, even lining along the inner nuclear membrane. Fine radial spokes join the karyosome to peripheral chromatin. Size, host and nucleus morphology set our organism apart from other members of the genus reported from fish. The SSU rRNA gene sequences of the new isolates are the first molecular data of an Entamoeba species from fish. Phylogenetic analysis places the new organism as sister to Entamoeba invadens. Based on the distinct morphology and SSU rRNA gene sequence we describe it as a new species, Entamoeba chiangraiensis.
... In addition, we provide the Bayesian tree from which the posterior probabilities were obtained (Supporting information S3). Our tree was rooted with Variosea according to recent phylogenies (Cavalier-Smith et al. 2015, Pánek et al. 2016. We excluded the long-branched Archamoebae. ...
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Myxogastria (also called Myxomycetes or plasmodial slime‐moulds) are mostly known through their usually conspicuous fruiting bodies. Another unifying trait is the presence of a facultative flagellate stage along with the obligate amoeboid stage. Here we show with two‐gene phylogenies (SSU rRNA and EF‐1alpha genes) that the incertae sedis, non‐flagellate Echinosteliopsis oligospora belongs to the dark‐spore clade (Fuscisporidia) of the Myxogastria. In addition, we confirm that Echinostelium bisporum, firstly described as a protostelid, belongs to the Echinosteliida, which are divided into three major clades and are paraphyletic to the remaining Fuscisporidia. This article is protected by copyright. All rights reserved.
... In additon to the Bayesian analyses, we employed C-series models [34] that account for heterogeneous site-specific features of sequence evolution in the phylogenomic dataset under a maximum-likelihood (ML) framework in IQ-TREE v1.3.3 [38]. The best-fitting model available under ML analyses that we were capable of running with computational constraints was LG + Γ4 + C20 + F with class weights optimized from the dataset using the exchangeabilities from the LG Q-Matrix (LG + Γ4 + FMIX (empirical, C20pi1-C20pi10)) [39,40]. Topological support was estimated from 1,000 ultrafast ML bootstrap (ML BS) replicates in IQ-TREE. ...
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Background Acanthamoebidae is a "family" level amoebozoan group composed of the genera Acanthamoeba, Protacanthamoeba, and very recently Luapeleamoeba. This clade of amoebozoans has received considerable attention from the broader scientific community as Acanthamoeba spp. represent both model organisms and human pathogens. While the classical composition of the group (Acanthamoeba + Protacanthamoeba) has been well accepted due to the morphological and ultrastructural similarities of its members, the Acanthamoebidae has never been highly statistically supported in single gene phylogenetic reconstructions of Amoebozoa either by maximum likelihood (ML) or Bayesian analyses. Results Here we show using a phylogenomic approach that the Acanthamoebidae is a fully supported monophyletic group within Amoebozoa with both ML and Bayesian analyses. We also expand the known range of morphological and life cycle diversity found in the Acanthamoebidae by demonstrating that the amoebozoans "Protostelium" arachisporum, Dracoamoeba jormungandri n. g. n. sp., and Vacuolamoeba acanthoformis n.g. n.sp., belong within the group. We also found that "Protostelium" pyriformis is clearly a species of Acanthamoeba making it the first reported sporocarpic that is, an amoebae that individually form a walled dormant propagule elevated by non- cellular stalks. Our phylogenetic analyses recover a fully supported Acanthamoebidae composed of five genera. Two of these genera (Acanthamoeba and Luapeleameoba) have members that are sporocarpic. Conclusions Our results provide high statistical support for an Acanthamoebidae that is composed of five distinct genera. This study increases the known morphological diversity of this group, show that the life cycle of Acanthamoeba can include spore-bearing stages. This further illustrates the widespread nature of spore-bearing stages across the tree of Amoebozoa.
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Plants must rearrange the network of complex carbohydrates in their cell walls during normal growth and development. To accomplish this, all plants depend on proteins called expansins that nonenzymatically loosen noncovalent bonding between cellulose microfibrils. Surprisingly, expansin genes have more recently been found in some bacteria and microbial eukaryotes, where their biological functions are largely unknown. Here, we reconstruct a comprehensive phylogeny of microbial expansin genes. We find these genes in all eukaryotic microorganisms that have structural cell wall cellulose, suggesting expansins evolved in ancient marine microorganisms long before the evolution of land plants. We also find expansins in an unexpectedly high diversity of bacteria and fungi that do not have cellulosic cell walls. These bacteria and fungi inhabit varied ecological contexts, mirroring the diversity of terrestrial and aquatic niches where plant and/or algal cellulosic cell walls are present. The microbial expansin phylogeny shows evidence of multiple horizontal gene transfer events within and between bacterial and eukaryotic microbial lineages, which may in part underlie their unusually broad phylogenetic distribution. Overall, expansins are unexpectedly widespread in bacteria and eukaryotes, and the contribution of these genes to microbial ecological interactions with plants and algae has probbaly been underappreciated.
Chapter
The eukaryote pangenome has a chimeric structure encompassing genes unique to eukaryotes (~41%), and genes of bacterial (~50% of total) and archaeal ancestry (~9%). Crucially, bacterial sequences far exceed the set acquired from the proteobacterial ancestor of mitochondria. Extant eukaryotic lineages share a common ancestor (LECA) that had all the fundamental traits of eukaryotes including the mitochondrion. The current debate about eukaryote origins revolves around two competing scenarios. The fusion model posits that the eukaryotes derive from the “fusion” of an archaeon and a bacterium, and that the acquisition of the mitochondrion was pivotal to the evolution of other eukaryotic traits. The neomuran model maintains that the archaea and eukaryotes are sister groups devived from a bacterial ancestor, and that fundamental eukaryotic traits including phagocytosis were already in place before the evolution of the mitochondrion by endosymbiosis. Eukaryote placement within the archaea in phylogenomic analysis supports the fusion scenario. The predominance of bacterial sequences in the eukaryote pangenome, the bacterial stereochemistry of eukaryote membrane lipids, and similar trajectories in mitochondrial and chloroplast evolution favour the neomuran scenario. Phylogenomic analysis resolves two major eukaryotic domains, the Amorphea and Diaphoretika, with traditional Excavata being probably paraphyletic. The root of the eukaryote tree remains elusive. Paleontological evidence and molecular clock analysis date the eukaryote lineage to at least 1.5 GYA, the concestor of extant eukaryotes to about 1.2 GYA, and major extant lineages to 900 MYA or less. The chapter includes a review of mitochondrial properties and of locomotor organelles in bacteria, archaea and eukaryotes.
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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 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 Exavata, 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. This article is protected by copyright. All rights reserved.
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Pelomyxa palustris is a giant anaerobic/microaerobic amoeba, characterized by a number of exceptional cytological and physiological features, among them the presumed absence of energy producing organelles and the presence of endosymbiotic bacteria. These endosymbionts have been previously distinguished as: a large rectangular-shaped Gram-variable rod with a central cleft; a slender Gram-negative rod; and a slender Gram-positive rod. Using DNA extracted from P. palustris cysts, we have obtained three SSU rRNA gene sequences. We have determined that these sequences are affiliated to three different prokaryotic genera: Methanosaeta (a methanogenic archaea), Syntrophorhabdus (a syntrophic Gram-negative bacteria) and Rhodococcus (an aerobic chemoorganotrophic Gram-positive bacteria). To our knowledge, it is the first time that Syntrophorhabdus has been described as an endosymbiont in association with a methanogen. Strikingly, no traces of Methanobacterium formicicum could be detected, despite this methanogen had allegedly been isolated from trophozoites of P. palustris. It seems that the host and the endosymbionts have established a multipartite syntrophic consortium resembling to some extent those found in sewage treatment plants.
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We used culture-based and culture-independent approaches to discover diversity and ecology of anaerobic jakobids (Excavata: Jakobida), an overlooked, deep-branching lineage of free-living nanoflagellates related to Euglenozoa. Jakobids are among a few lineages of nanoflagellates frequently detected in anoxic habitats by PCR-based studies, however only two strains of a single jakobid species have been isolated from those habitats. We recovered 712 environmental sequences and cultured 21 new isolates of anaerobic jakobids that collectively represent at least ten different species in total, from which four are uncultured. Two cultured species have never been detected by environmental, PCR-based methods. Surprisingly, culture-based and culture-independent approaches were able to reveal a relatively high proportion of overall species diversity of anaerobic jakobids—60 or 80%, respectively. Our phylogenetic analyses based on SSU rDNA and six protein-coding genes showed that anaerobic jakobids constitute a clade of morphologically similar, but genetically and ecologically diverse protists—Stygiellidae fam. nov. Our investigation combines culture-based and environmental molecular-based approaches to capture a wider extent of species diversity and shows Stygiellidae as a group that ordinarily inhabits anoxic, sulfide- and ammonium-rich marine habitats worldwide.
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Morphology of a pelobiont Pelomyxa paradoxa Penard, 1902 was investigated at light- and electron-microscopical levels. Locomoting cells are cigar-shaped. The cells produce many hyaline pseudopodia of digital and conical form at lateral sides of the body. The organism has a pronounced hyaline bulbous uroid with broad peripheral zone of hyaloplasm and many conical hyaline villi. There is a thin layer of amorphous glycocalix at the cell surface. "Structure" and food vacuoles of different size are very abundant in the endoplasm. Two different species of prokaryote endocytobionts are peculiar for P. paradoxa. Uninucleate stage dominates in the life cycle of P. paradoxa. Usually there are no more than 10-12 nuclei in multinucleate forms of P. paradoxa. Pelomyxae nuclei are closely surrounded by thick multilaminar layer and additionally by one more layer, which is formed by small vesicles with electron-dense content. Several irregular-shaped nucleoli are situated at the nucleus periphery. Inside the nucleoli, and sometimes directly in nucleoplasm the small round bodies are revealed, these bodies being formed by tightly packed electron-dense fibrils. Many non-motile flagellae are located mainly in the uroidal zone of the cell. Pronounced lateral root and 50-60 radial microtubules originate from the electrone-dense muft around the kinetosome. All elements of the rootlet system of flagella are limited by peripheral layers of cytoplasm. P. paradoxa occupy an intermediate position between two groups of species of Pelomyxa genus--P. gruberi + P. prima and P. palustris + P. stagnalis + P. belewski, which differ greatly by the organization of their flagella basal apparatus.
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Significance The root of eukaryote phylogeny formally represents the last eukaryotic common ancestor (LECA), but its position has remained controversial. Using new genome sequences, we revised and expanded two datasets of eukaryotic proteins of bacterial origin, which previously yielded conflicting views on the eukaryotic root. Analyses using state-of-the-art phylogenomic methodology revealed that both expanded datasets now support the same root position. Our results justify a new nomenclature for the two main eukaryotic groups and provide a robust phylogenetic framework to investigate the early evolution of the eukaryotic cell.
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Lateral gene transfer (LGT) is an important mechanism of evolution for protists adapting to oxygen-poor environments. Specifically, modifications of energy metabolism in anaerobic forms of mitochondria (e.g., hydrogenosomes) are likely to have been associated with gene transfer from prokaryotes. An interesting question is whether the products of transferred genes were directly targeted into the ancestral organelle or initially operated in the cytosol and subsequently acquired organelle-targeting sequences. Here, we identified key enzymes of hydrogenosomal metabolism in the free-living anaerobic amoebozoan Mastigamoeba balamuthi and analyzed their cellular localizations, enzymatic activities, and evolutionary histories. Additionally, we characterized 1) several canonical mitochondrial components including respiratory complex II and the glycine cleavage system, 2) enzymes associated with anaerobic energy metabolism, including an unusual D-lactate dehydrogenase and acetyl CoA synthase, and 3) a sulfate activation pathway. Intriguingly, components of anaerobic energy metabolism are present in at least two gene copies. For each component, one copy possesses an mitochondrial targeting sequence (MTS), whereas the other lacks an MTS, yielding parallel cytosolic and hydrogenosomal extended glycolysis pathways. Experimentally, we confirmed that the organelle targeting of several proteins is fully dependent on the MTS. Phylogenetic analysis of all extended glycolysis components suggested that these components were acquired by LGT. We propose that the transformation from an ancestral organelle to a hydrogenosome in the M. balamuthi lineage involved the lateral acquisition of genes encoding extended glycolysis enzymes that initially operated in the cytosol and that established a parallel hydrogenosomal pathway after gene duplication and MTS acquisition.
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Large phylogenomics data sets require fast tree inference methods, especially for maximum-likelihood (ML) phylogenies. Fast programs exist, but due to inherent heuristics to find optimal trees, it is not clear whether the best tree is found. Thus, there is need for additional approaches that employ different search strategies to find ML trees and that are at the same time as fast as currently available ML programs. We show that a combination of hill-climbing approaches and a stochastic perturbation method can be time-efficiently implemented. If we allow the same CPU time as RAxML and PhyML, then our software IQ-TREE found higher likelihoods between 62.2% and 87.1% of the studied alignments, thus efficiently exploring the tree-space. If we use the IQ-TREE stopping rule, RAxML and PhyML are faster in 75.7% and 47.1% of the DNA alignments and 42.2% and 100% of the protein alignments, respectively. However, the range of obtaining higher likelihoods with IQ-TREE improves to 73.3–97.1%. IQ-TREE is freely available at http://www.cibiv.at/software/iqtree.
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Amoebozoa is a key phylum for eukaryote phylogeny and evolutionary history, but its phylogenetic validity has been questioned since included species are very diverse: amoebo-flagellate slime-moulds, naked and testate amoebae, and some flagellates. 18S rRNA gene trees have not firmly established its internal topology. To rectify this we sequenced cDNA libraries for seven diverse Amoebozoa and conducted phylogenetic analyses for 109 eukaryotes (17-18 Amoebozoa) using 60-188 genes. We conducted Bayesian inferences with the evolutionarily most realistic site-heterogeneous CAT-GTR model and maximum likelihood analyses. These unequivocally establish the monophyly of Amoebozoa, showing a primary dichotomy between the previously contested subphyla Lobosa and Conosa. Lobosa, the entirely non-flagellate lobose amoebae, are robustly partitioned into the monophyletic classes Tubulinea, with predominantly tube-shaped pseudopodia, and Discosea with flattened cells and different locomotion. Within Conosa 60/70-gene trees with very little missing data show a primary dichotomy between the aerobic infraphylum Semiconosia (Mycetozoa and Variosea) and secondarily anaerobic Archamoebae. These phylogenetic features are entirely congruent with the most recent major amoebozoan classification emphasising locomotion modes, pseudopodial morphology, and ultrastructure. However, 188-gene trees where proportionally more taxa have sparser gene-representation weakly place Archamoebae as sister to Macromycetozoa instead, possibly a tree reconstruction artefact of differentially missing data.
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Current sampling of genomic sequence data from eukaryotes is relatively poor, biased, and inadequate to address important questions about their biology, evolution, and ecology; this Community Page describes a resource of 700 transcriptomes from marine microbial eukaryotes to help understand their role in the world's oceans.
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Although many NGS read pre-processing tools already existed, we could not find any tool or combination of tools which met our requirements in terms of flexibility, correct handling of paired-end data, and high performance. We have developed Trimmomatic as a more flexible and efficient pre-processing tool, which could correctly handle paired-end data. The value of NGS read pre-processing is demonstrated for both reference-based and reference-free tasks. Trimmomatic is shown to produce output which is at least competitive with, and in many cases superior to, that produced by other tools, in all scenarios tested. Trimmomatic is licensed under GPL V3. It is cross-platform (Java 1.5+ required) and available from http://www.usadellab.org/cms/index.php?page=trimmomatic CONTACT: usadel@bio1.rwth-aachen.de SUPPLEMENTARY INFORMATION: Manual and source code are available from http://www.usadellab.org/cms/index.php?page=trimmomatic.
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Emerging methods for the accurate quantification of gene expression in individual cells hold promise for revealing the extent, function and origins of cell-to-cell variability. Different high-throughput methods for single-cell RNA-seq have been introduced that vary in coverage, sensitivity and multiplexing ability. We recently introduced Smart-seq for transcriptome analysis from single cells, and we subsequently optimized the method for improved sensitivity, accuracy and full-length coverage across transcripts. Here we present a detailed protocol for Smart-seq2 that allows the generation of full-length cDNA and sequencing libraries by using standard reagents. The entire protocol takes ∼2 d from cell picking to having a final library ready for sequencing; sequencing will require an additional 1-3 d depending on the strategy and sequencer. The current limitations are the lack of strand specificity and the inability to detect nonpolyadenylated (polyA(-)) RNA.
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Two species of amoebae, capable of living and multiplying in the absence of oxygen, were isolated from anaerobic marine sediments. Vahlkampfia anaerobica does not have typical mitochondria (but has organelles covered by a double membrane) and it harbours endocytic bacteria. The nuclear envelope of V. anaerobica is associated with the cisternae of the rough endoplasmic reticulum which is a remarkable feature of this species. Vannella peregrinia has double membraned organelles with internal tubular structures, which morphologically resemble mitochondria. Its cell coat consists of glycostyles, which differ in dimensions from those of other Vannella spp.; it needs further investigation.
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The archamoebae form a small clade of anaerobic/microaerophilic flagellates or amoebae, comprising the pelobionts (mastigamoebids and pelomyxids) and the entamoebae. It is a member of the eukaryotic supergroup Amoebozoa. We examined 22 strains of 13 species of Mastigamoeba, Pelomyxa and Rhizomastix by light-microscopy and determined their SSU rRNA gene sequences. The SSU rRNA gene sequences of Pelomyxa palustris and Mastigella commutans in GenBank are shown to belong to P. stagnalis and Mastigamoeba punctachora, respectively. Five new species of free-living archamoebae are described: Mastigamoeba abducta, M. errans, M. guttula, M. lenta, and Rhizomastix libera spp. nov. A species of Mastigamoeba possibly living endosymbiotically in Pelomyxa was identified. Rhizomastix libera, the first known free-living member of that genus, is shown to be an archamoeba. R. libera possesses an ultrastructure unique within archamoebae: a rhizostyle formed from a modified microtubular cone and a flagellum with vanes. While many nominal species of pelobionts are extremely hard to distinguish by light microscopy, transient pseudopodial characters are worthy of further investigation as taxonomic markers.
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The morphology of a new species of pelobionts Pelomyxa flava was studied by light and electron microscopy. The envelopes of P. flava are consist of a plasma membrane with a thick layer of weakly structured glycocalyx on its outer surface. Numerous flagella are often located at the apices of short conical pseudopodia. Kinetosomes of flagella reach length of 0.9 μm and are hollow with a pronounced central filament. The rootlet system is represented by three groups of microtubules: the radial, basal, and the microtubules of lateral root. The transitory zone is short and does not reach beyond the level of the cell surface; the axoneme is characterized by an unstable set of microtubules. Trophic stages of the P. flava life cycle are represented by binuclear cells; plasmotomy is performed at the tetranuclear stage. Nuclei have a granular structure. Fibrillar nuclear bodies are revealed in the karyoplasm. The nuclear envelope has a complex organization; on its surface, the outer membrane has a layer of electron-dense material that contacts with short microtubules located single-row at the surface of the nuclear envelope. Vesicles and cisterns of endoplasmic reticulum are located away from microtubules and are derivatives of the nuclear envelope. In the P. flava endoplasm, the presence of structural and digestive vacuoles and glycogen granules was found. Three types of prokaryotic cytobionts were revealed. Large multimembranous organelles reaching 5 μm in diameter were described for the first time. Peculiarities of the morphology and biology of P. flava compared to the previously studied Pelomyxa species are discussed. Keywordspelobionts–morphology– Pelomyxa flava –ultrastructure
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Iodamoeba is the last genus of obligately parasitic human protist whose phylogenetic position is unknown. Iodamoeba small subunit ribosomal DNA sequences were obtained using samples from three host species, and phylogenetic analyses convincingly placed Iodamoeba as a sister taxon to Endolimax. This clade in turn branches among free-living amoeboflagellates of the genus Mastigamoeba. Two Iodamoeba ribosomal lineages (RL1 and RL2) were detected whose sequences differ by 31%, each of which is found in both human and nonhuman hosts.
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Mitochondrion-related organelles, mitosomes and hydrogenosomes, are found in a phylogenetically broad range of organisms. Their components and functions are highly diverse. We have previously shown that mitosomes of the anaerobic/microaerophilic intestinal protozoan parasite Entamoeba histolytica have uniquely evolved and compartmentalized a sulfate activation pathway. Although this confined metabolic pathway is the major function in E. histolytica mitosomes, their physiological role remains unknown. In this study, we examined the phenotypes of the parasites in which genes involved in the mitosome functions were suppressed by gene silencing, and showed that sulfate activation in mitosomes is important for sulfolipid synthesis and cell proliferation. We also demonstrated that both Cpn60 and unusual mitochondrial ADP/ATP transporter (mitochondria carrier family, MCF) are important for the mitosome functions. Immunoelectron microscopy demonstrated that the enzymes involved in sulfate activation, Cpn60, and mitochondrial carrier family were differentially distributed within the electron dense, double membrane-bounded organelles. The importance and topology of the components in E. histolytica mitosomes reinforce the notion that they are not "rudimentary" or "residual" mitochondria, but represent a uniquely evolved crucial organelle in E. histolytica.
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Massively parallel sequencing of cDNA has enabled deep and efficient probing of transcriptomes. Current approaches for transcript reconstruction from such data often rely on aligning reads to a reference genome, and are thus unsuitable for samples with a partial or missing reference genome. Here we present the Trinity method for de novo assembly of full-length transcripts and evaluate it on samples from fission yeast, mouse and whitefly, whose reference genome is not yet available. By efficiently constructing and analyzing sets of de Bruijn graphs, Trinity fully reconstructs a large fraction of transcripts, including alternatively spliced isoforms and transcripts from recently duplicated genes. Compared with other de novo transcriptome assemblers, Trinity recovers more full-length transcripts across a broad range of expression levels, with a sensitivity similar to methods that rely on genome alignments. Our approach provides a unified solution for transcriptome reconstruction in any sample, especially in the absence of a reference genome.
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Iron-sulphur (Fe-S) clusters have long been recognized as essential and versatile cofactors of proteins involved in catalysis, electron transport and sensing of ambient conditions. Despite the relative simplicity of Fe-S clusters in terms of structure and composition, their synthesis and assembly into apoproteins is a highly complex and coordinated process in living cells. Different biogenesis machineries in both bacteria and eukaryotes have been discovered that assist Fe-S-protein maturation according to uniform biosynthetic principles. The importance of Fe-S proteins for life is documented by an increasing number of diseases linked to these components and their biogenesis.
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The continuous accumulation of sequence data, for example, due to novel wet-laboratory techniques such as pyrosequencing, coupled with the increasing popularity of multi-gene phylogenies and emerging multi-core processor architectures that face problems of cache congestion, poses new challenges with respect to the efficient computation of the phylogenetic maximum-likelihood (ML) function. Here, we propose two approaches that can significantly speed up likelihood computations that typically represent over 95 per cent of the computational effort conducted by current ML or Bayesian inference programs. Initially, we present a method and an appropriate data structure to efficiently compute the likelihood score on 'gappy' multi-gene alignments. By 'gappy' we denote sampling-induced gaps owing to missing sequences in individual genes (partitions), i.e. not real alignment gaps. A first proof-of-concept implementation in RAXML indicates that this approach can accelerate inferences on large and gappy alignments by approximately one order of magnitude. Moreover, we present insights and initial performance results on multi-core architectures obtained during the transition from an OpenMP-based to a Pthreads-based fine-grained parallelization of the ML function.
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Archezoan protists are though to represent lineages that diverged from other eukaryotes before acquisition of the mitochondrion and other organelles. The parasite Entamoeba histolytica was originally included in this group. Ribosomal RNA based phylogenies, however, place E. histolytica on a comparatively recent branch of the eukaryotic tree, implying that its ancestors had these structures. In this study, direct evidence for secondary loss of mitochondrial function was obtained by isolating two E. histolytica genes encoding proteins that in other eukaryotes are localized in the mitochondrion: the enzyme pyridine nucleotide transhydrogenase and the chaperonin cpn60. Phylogenetic analysis of the E. histolytica homolog of cpn60 confirmed that it is specifically related to the mitochondrial lineage. The data suggest that a mitochondrial relic may persist in this organism. Similar studies are needed in archezoan protists to ascertain which, if any, eukaryotic lineages primitively lack mitochondria.
Chapter
Flagellated protozoa are important in two biological disciplines. In evolutionary biology, flagellates are critical to understanding the origins of eukaryotic cells and their diversification as protists and subsequently as plants, animals, and fungi. Flagellated protozoa also play a key role in aquatic ecosystems, where they regulate bacterial numbers and control the remineralization of nutrients. The aim of this volume is to provide a synthesis of information on these organisms. Chapters deal with the organization, diversity, ecology, and maintenance of free-living flagellates. Each chapter is written by a recognized authority in their field. The book will be of interest to protozoologists, protistologists, evolutionary biologists, and ecologists dealing with aquatic or soil ecosystems.
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A revised six-kingdom system of life is presented, down to the level of infraphylum. As in my 1983 system Bacteria are treated as a single kingdom, and eukaryotes are divided into only five kingdoms: Protozoa, Animalia, Fungi, Plantae and Chromista. Intermediate high level categories (superkingdom, subkingdom, branch, infrakingdom, superphylum, subphylum and infraphylum) are extensively used to avoid splitting organisms into an excessive number of kingdoms and phyla (60 only being recognized). The two 'zoological' kingdoms, Protozoa and Animalia, are subject to the International Code of Zoological Nomenclature, the kingdom Bacteria to the International Code of Bacteriological Nomenclature, and the three 'botanical' kingdoms (Plantae, Fungi, Chromista) to the International Code of Botanical Nomenclature. Circumscriptions of the kingdoms Bacteria and Plantae remain unchanged since Cavalier-Smith (1981). The kingdom Fungi is expanded by adding Microsporidia, because of protein sequence evidence that these amitochondrial intracellular parasites are related to conventional Fungi, not Protozoa. Fungi are subdivided into four phyla and 20 classes; fungal classification at the rank of subclass and above is comprehensively revised. The kingdoms Protozoa and Animalia are modified in the light of molecular phylogenetic evidence that Myxozoa are actually Animalia, not Protozoa, and that mesozoans are related to bilaterian animals. Animalia are divided into four subkingdoms: Radiata (phyla Porifera, Cnidaria, Placozoa, Ctenophora), Myxozoa, Mesozoa and Bilateria (bilateral animals: all other phyla). Several new higher level groupings are made in the animal kingdom including three new phyla: Acanthognatha (rotifers, acanthocephalans, gastrotrichs, gnathostomulids), Brachiozoa (brachiopods and phoronids) and Lobopoda (onychophorans and tardigrades), so only 23 animal phyla are recognized. Archezoa, here restricted to the phyla Metamonada and Trichozoa, are treated as a subkingdom within Protozoa, as in my 1983 six-kingdom system, not as a separate kingdom. The recently revised phylum Rhizopoda is modified further by adding more flagellates and removing some 'rhizopods' and is therefore renamed Cercozoa. The number of protozoan phyla is reduced by grouping Mycetozoa and Archamoebae (both now infraphyla) as a new subphylum Conosa within the phylum Amoebozoa alongside the subphylum Lobosa, which now includes both the traditional aerobic lobosean amoebae and Multicilia. Haplosporidia and the (formerly microsporidian) metchnikovellids are now both placed within the phylum Sporozoa. These changes make a total of only 13 currently recognized protozoan phyla, which are grouped into two subkingdoms: Archezoa and Neozoa; the latter is modified in circumscription by adding the Discicristata, a new infrakingdom comprising the phyla Percolozoa and Euglenozoa). These changes are discussed in relation to the principles of megasystematics, here defined as systematics that concentrates on the higher levels of classes, phyla, and kingdoms. These principles also make it desirable to rank Archaebacteria as an infrakingdom of the kingdom Bacteria, not as a separate kingdom. Archaebacteria are grouped with the infrakingdom Posibacteria to form a new subkingdom, Unibacteria, comprising all bacteria bounded by a single membrane. The bacterial subkingdom Negibacteria, with separate cytoplasmic and outer membranes, is subdivided into two infrakingdoms: Lipobacteria, which lack lipopolysaccharide and have only phospholipids in the outer membrane, and Glycobacteria, with lipopolysaccharides in the outer leaflet of the outer membrane and phospholipids in its inner leaflet. This primary grouping of the 10 bacterial phyla into subkingdoms is based on the number of cell-envelope membranes, whilst their subdivision into infrakingdoms emphasises their membrane chemistry; definition of the negibacterial phyla, five at least partly photosynthetic, relies chiefly on photosynthetic mechanism and cell-envelope structure and chemistry corroborated by ribosomal RNA phylogeny. The kingdoms Protozoa and Chromista are slightly changed in circumscription by transferring subphylum Opalinata (classes Opalinea, Proteromonadea, Blastocystea cl. nov.) from Protozoa into infrakingdom Heterokonta of the kingdom Chromista. Opalinata are grouped with the subphylum Pseudofungi and the zooflagellate Developayella elegans (in a new subphylum Bigyromonada) to form a new botanical phylum (Bigyra) of heterotrophs with a double ciliary transitional helix, making it necessary to abandon the phylum name Opalozoa, which formerly included Opalinata. The loss of ciliary retronemes in Opalinata is attributed to their evolution of gut commensalism. The nature of the ancestral chromist is discussed in the light of recent phylogenetic evidence.
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The genus Entamoeba includes anaerobic lobose amoebae, most of which are parasites of various vertebrates and invertebrates. We report a new Entamoeba species, E. marina n. sp. that was isolated from a sample of tidal flat sediments collected at Iriomote Island, Okinawa, Japan. Trophozoites of E. marina were 12.8-32.1 μm in length and 6.8-15.9 μm in width, whereas the cysts were 8.9-15.8 μm in diameter and contained four nuclei. The E. marina cells contained a rounded nucleus with a small centric karyosome and uniformly arranged peripheral chromatin. Although E. marina is morphologically indistinguishable from other tetranucleated cyst-forming Entamoeba species, E. marina can be distinguished from them based on the combination of molecular phylogenetic analyses using SSU rDNA gene and the difference of collection site. Therefore, we propose E. marina as a new species of the genus Entamoeba. This article is protected by copyright. All rights reserved.
Article
The genus Rhizomastix is a poorly-known group of amoeboid heterotrophic flagellates living as intestinal commensals of insects, amphibians or reptiles, and as inhabitants of organic freshwater sediments. Eleven Rhizomastix species have been described so far, but DNA sequences from only a single species have been published. Recently, phylogenetic analyses confirmed a previous hypothesis that the genus belongs to the Archamoebae; however, its exact position therein remains unclear. In this study we cultured nine strains of Rhizomastix, both endobiotic and free-living. According to their light-microscopic morphology and SSU rRNA and actin gene analyses, the strains represent five species, of which four are newly described here: R. bicoronata sp. nov., R. elongata sp. nov., R. vacuolata sp. nov. and R. varia sp. nov. In addition, R. tipulae sp. nov., living in the intestine of crane flies, is separated from the type species, R. libera. We also examined the ultrastructure of R. elongata sp. nov., which revealed that it is more complicated than the previously-described R. libera. Our data show that either the endobiotic lifestyle of some Rhizomastix species has arisen independently from other endobiotic archamoebae, or the free-living members of this genus represent a secondary switch from the endobiotic lifestyle. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Article
Amoebae able to form cytoplasmic networks or displaying a multiply branching morphology remain very poorly studied. We sequenced the small-subunit ribosomal RNA gene of 15 new amoeboid isolates, 14 of which are branching or network-forming amoebae (BNFA). Phylogenetic analyses showed that these isolates all group within the poorly-known and weakly-defined class Variosea (Amoebozoa). They are resolved into six lineages corresponding to distinct new morphotypes; we describe them as new genera Angulamoeba (type species Angulamoeba microcystivorans n. gen., n. sp.; and A. fungorum n. sp.), Arboramoeba (type species Arboramoeba reticulata n. gen., n. sp.), Darbyshirella (type species Darbyshirella terrestris n. gen., n. sp.), Dictyamoeba (type species Dictyamoeba vorax n. gen., n. sp.), Heliamoeba (type species Heliamoeba mirabilis n. gen., n. sp.), and Ischnamoeba (type species Ischnamoeba montana n. gen., n. sp.). We also isolated and sequenced four additional variosean strains, one belonging to Flamella, one related to Telaepolella tubasferens, and two members of the cavosteliid protosteloid lineage. We identified a further 104 putative variosean environmental clone sequences in GenBank, comprising up to 14 lineages that may prove to represent additional novel morphotypes. We show that BNFA are phylogenetically widespread in Variosea and morphologically very variable, both within and between lineages.
Article
Thirty five species of free-living anaerobic flagellates from freshwater and coastal sediments from Danish and Australian sites are reported. These belong to the genera Ancyromonas, Barthelona n. gen., Bodo, Cafeteria, Carpediemonas, Cercomonas, Chilomastix, Chilomonas, Dimastigella, Goniomonas, Heteromita, Jakoba Mastigamoeba, Monotrichomonas n. gen., Paraphysomonas, Percolomonas, Pseudotrichomonas, Quasibodo n. gen., Rhabdomonas, Rhynchobodo, Rhynchomonas, Salpingoeca, Spumella, Trepomonas and Trimastix. Six new species are described, Barthelona vulgaris, Jakoba incarcerata, Mastigamoeba punctachora, Monotrichomonas carabina, Quasibodo laughtoni and Trimastix inaequalis. The composition of the communities encountered under anoxic conditions overlaps with communities observed in aerobic environments and includes species of heteroloboseids, kinetoplastids, euglenids, jakobids, stramenopiles, cercomonads, cryptomonads and choanoflagellates — all of which are mitochondriate. About half of the flagellates observed lack classical mitochondria, or belong to known amitochondriate groups. These taxa are assignable to the pelobionts, retortamonads, diplomonads, trichomonads, and to the genera Trimastix and Carpediemonas. Some taxa observed during this study have no clear identity and further study is necessary. This work confirms the existence of many poorly understood anaerobic flagellates, the study of which could increase our understanding of the pattern of mitochondrial gain and/or loss among extant eukaryotes, as well as the operation of anoxic ecosystems.
Article
Members of the archamoebae comprise free-living and endobiotic amoeboid flagellates and amoebae that live in anoxic/microoxic habitats. Recently, the group has been divided into four separate families, Mastigamoebidae, Entamoebidae, Pelomyxidae, and Rhizomastixidae, whose interrelationships have not been completely resolved. There still are several key members of the archamoebae, notably the genus Mastigella, from which sequence data are missing. We established 12 strains of 5 species of Mastigella and Pelomyxa in culture, examined their morphology and determined their actin gene sequences. In addition, we examined the ultrastructure of three strains and determined and analyzed SSU rDNA sequences of two strains. Our data strongly suggest that Mastigella is specifically related to Pelomyxa, and it is transferred into the family Pelomyxidae. Surprisingly, Mastigella is likely paraphyletic with Pelomyxa forming its internal branch. The two genera share several morphological features that point to their common evolutionary history. Three new species of Mastigella are described: M. erinacea sp. nov., M. rubiformis sp. nov. and M. ineffigiata sp. nov. Copyright © 2014 Elsevier GmbH. All rights reserved.
Article
Summary Flagellation ofPhysarum polycephalum amoebae (Myxomycete) involves the formation around the two kinetosomes of a flagellar apparatus leading to a modification in the shape of the amoeba and its nucleus. A tridimensional ultrastructural model of the flagellar apparatus is proposed, based upon observation of the isolated nucleo-flagellar apparatus complex. The flagellar apparatus is composed of a non-microtubular structure (the posterior para-kinetosomal structure), five microtubular arrays and two flagella: a long anterior flagellum and a short flagellum directed backwards. The asymmetry of the flagellar apparatus is due mainly to the presence of the posterior para-kinetosomal structure on the right side of the posterior kinetosome and of the two asymmetrical microtubular arrays 3 and 4. Thus, the flagellar apparatus is right-handed. This asymmetry implies also some spatial constraints on two other microtubular arrays (2 and 5). Except in the case of the microtubular array 1 which links the proximal end of the anterior kinetosome to the nuclear membrane, the number of microtubules of each microtubular array seems to be well defined: 39, 5–6, 7–9, and 2+2 for the microtubular arrays 2, 3, 4, and 5 respectively. All the elements of the nucleo-flagellar apparatus complex are linked either directly or indirectly through bridges. Furthermore, the microtubules which composed the microtubular array 3 are linked through bridges while the microtubules of the microtubular arrays 2, 3, and 4 seem to be linked through a reticulate material. All these spatial relationships lead to a great cohesion of the nucleo-flagellar apparatus complex which appears to be a well defined structure. This suggests thatPhysarum amoebal flagellation can be a promising system to study the morphogenesis of an eucaryotic cell.
Article
The taxonomic position of the uniciliate, unicentriolar zooflagellate Phalansterium is problematic; its distinctive ultrastructure with a pericentriolar microtubular cone placed it in its own order and suggested phenotypic closeness to the eukaryote cenancestor. We sequenced the 18S rRNA of a unicellular Phalansterium. Phylogenetic analysis shows that it belongs to Amoebozoa, decisively rejecting a postulated relationship with the cercozoan Spongomonas; Phalansterium groups with Varipodida ord. nov. (Gephyramoeba/Filamoeba) or occasionally Centramoebida emend. (Acanthamoebidae/Balamuthiidae fam. nov.), centrosomes of the latter suggesting flagellate ancestors. We also studied Phalansterium solitarium cyst ultrastructure; unlike previously studied P. solitarium, this strain has pentagonally symmetric walls like P. consociatum. We also sequenced 18S rRNA genes of further isolates of Hyperamoeba, an aerobic unicentriolar amoeboflagellate with conical microtubular skeleton; both group strongly with myxogastrid Mycetozoa. However, the four Hyperamoeba strains do not group together, suggesting that Hyperamoeba are polyphyletic derivatives of myxogastrids that lost fruiting bodies independently. We revise amoebozoan higher-level classification into seven classes, establishing Stelamoebea cl. nov. for Protosteliida emend. plus Dictyosteliida (biciliate former ‘protostelids’ comprise Parastelida ord. nov. within Myxogastrea), and new subphylum Protamoebae to embrace Variosea cl. nov. (Centramoebida, Phalansteriida, Varipodida), Lobosea emend., Breviatea cl. nov. for ‘Mastigamoeba invertens’ and relatives, and Discosea cl. nov. comprising Glycostylida ord. nov. (vannellids, vexilliferids, paramoebids, Multicilia), Dermamoebida ord. nov. (Thecamoebidae) and Himatismenida. We argue that the ancestral amoebozoan was probably unikont and that the cenancestral eukaryote may have been also.
Article
The architecture of eukaryotic cells is underpinned by complex arrrays of microtubules that stem from an organizing center referred to as the MTOC. With few exceptions, MTOCs consist of two basal bodies that anchor flagellar axonemes and different configurations of microtubular roots. Variations in the structure of this cytoskeletal system, also referred to as the "flagellar apparatus", reflect phylogenetic relationships and provide compelling evidence for inferring the overall tree of eukaryotes. However, reconstructions and subsequent comparisons of the flagellar apparatus are challenging, because these studies require sophisticated microscopy, spatial reasoning, and detailed terminology. In an attempt to understand the unifying features of MTOCs and broad patterns of cytoskeletal homology across the tree of eukaryotes, we present a comprehensive overview of the eukaryotic flagellar apparatus within a modern molecular phylogenetic context. Specifically, we used the known cytoskeletal diversity within major groups of eukaryotes to infer the unifying features (ancestral states) for the flagellar apparatus in the Plantae, Opisthokonta, Amoebozoa, Stramenopiles, Alveolata, Rhizaria, Excavata, Cryptophyta, Haptophyta, Apusozoa, Breviata, and Collodictionidae. We then mapped these data onto the tree of eukaryotes in order to trace broad patterns of trait changes during the evolutionary history of the flagellar apparatus. This synthesis suggests that (1) the most recent ancestor of all eukaryotes already had a complex flagellar apparatus, (2) homologous traits associated with the flagellar apparatus have a punctate distribution across the tree of eukaryotes, and (3) streamlining (trait losses) of the ancestral flagellar apparatus occurred several times independently in eukaryotes. © 2013 The Authors. The Plant Journal © 2013 Blackwell Publishing Ltd.
Article
I discuss how different feeding modes and related cellular structures map onto the eukaryote evolutionary tree. Centrally important for understanding eukaryotic cell diversity are Loukozoa: ancestrally biciliate phagotrophic protozoa possessing a posterior cilium and ventral feeding groove into which ciliary currents direct prey. I revise their classification by including all anaerobic Metamonada as a subphylum and adding Tsukubamonas. Loukozoa, often with ciliary vanes, are probably ancestral to all protozoan phyla except Euglenozoa and Percolozoa and indirectly to kingdoms Animalia, Fungi, Plantae, and Chromista. I make a new protozoan phylum Sulcozoa comprising subphyla Apusozoa (Apusomonadida, Breviatea) and Varisulca (Diphyllatea; Planomonadida, Discocelida, Mantamonadida; Rigifilida). Understanding sulcozoan evolution clarifies the origins from them of opisthokonts (animals, fungi, Choanozoa) and Amoebozoa, and their evolutionary novelties; Sulcozoa and their descendants (collectively called podiates) arguably arose from Loukozoa by evolving posterior ciliary gliding and pseudopodia in their ventral groove. I explain subsequent independent cytoskeletal modifications, accompanying further shifts in feeding mode, that generated Amoebozoa, Choanozoa, and fungi. I revise classifications of Choanozoa, Conosa (Amoebozoa), and basal fungal phylum Archemycota. I use Choanozoa, Sulcozoa, Loukozoa, and Archemycota to emphasize the need for simply classifying ancestral (paraphyletic) groups and illustrate advantages of this for understanding step-wise phylogenetic advances.
Article
A new species of pelobiont, Mastigamoeba schizophrenia n. sp., is described using light and electron microscopy. Three forms were observed: flagellates, aflagellated amoebae and cysts. This species is distinguished by having adhering paired nuclei. Most cells have a single pair of nuclei but large amoebae may have several pairs. The flagellar apparatus consists of a single basal body, connected to the most anterior nucleus by a cone of microtubules. The basal body is composed of doublets of microtubules. A single ribbonlike microtubular root originates alongside the basal body. Like other pelobionts, the cell has a comparatively simple endomembrane system which includes endoplasmic reticulum but lacks mitochondria, dictyosomes, contractile vacuole and other elements found in many eukaryotic cells. The taxonomy of the pelobionts is discussed, the genus Phreatamoeba is synonymised with Mastigamoeba, and arguments for a deep-branching position for the pelobionts among eukaryotes are discussed.
Article
The morphology of Mastigamoeba aspera, a type species of the genus Mastigamoeba Schulze, 1875, has been investigated at the light- and electron-microscopical level. Motile individuals are oval or peach-shaped. Motile flagella is situated at the anterior end of uninucleate cells. During locomotion, the surface of mastigamoebes forms many conical or finger-shaped hyaline pseudopodia, wereas bulbous uroid is often formed at the posterior end of the cell. Micropopulations of M. aspera consist of uninucleate flagellate forms as well as multinucleate aflagellate ones. There is a thick layer ofglycocalix on the cell surface where many rod-shaped bacterial ectobionts live. The nucleus is vesicular with spherical central nucleolus. The flagellar apparatus of M. aspera is connected with nucleus to form so called kariomastigont. A single kinetosome is associated with many radial microtubules and a lateral root. A distinct microtubule organization centre (MTOC) is situated at the basal part of the kinetosome. Microtubules of the nuclear cone are connected with the MTOC. This microtubules take part in the formation of kariomastigont. The axoneme has a standart set of microtubules 9(2)+2. Digestive vacuoles are the main component of the cytoplasm of M. aspera. Beside, many light-difracted granules and glycogen bodies were found in the cells. Mitochondria, dictyosomes of the Golgi apparatus and microbodies were not revealed in the cytoplasm of M. aspera.
Article
Great numbers of a new ameba have been collected from freshwater, lagooning, disposal tanks for citrus pulp wastes. The ameba is morphologically similar to the marine Flamella magnifica (Schaeffer, 1926), and clearly belongs to the genus. It differs sufficiently in the formation and characters of locomotor organelles, and in habitat, to be considered a separate species, Flamella citrensis n. sp.
Article
A revised classification of the naked amoebae is proposed on the basis of a synthesis of many kinds of information presently available for taxonomic purposes above the species level. These amoebae, constituting the subclass Gymnamoebia within the class Lobosea, superclass Rhizopoda, include not only strictly lobose amoebae but also those with more or less filose subpseudopodia produced from a broader hyaline lobe. The subclass is divided into the orders Amoebida, Schizopyrenida, and Pelobiontida, and suborders are recognized within the order Amoebida. Although the Gymnamoebia are undoubtedly heterogeneous and polyphyletic and the proposed classification is intended chiefly as a practical system with a logical basis, there are a few suggestions of natural relationships.
Article
Hyperamoeba dachnaya, a new taxon affiliated with Hyperamoeba flagellata, is described from freshwater anaerobic sediments. Organisms exist as amoeboid flagellates, amoebae and cysts. The flagellates of H. dachnaya are distinguished from those of H. flagellata by larger size and a longer anteriorly-directed finger-shaped pseudopodium. H. dachnaya cysts have bilaminar walls with connectives between the outer and inner layers appearing to be T-shaped spines, while cyst walls of H. flagellata are smooth. H. dachnaya has two basal bodies and a single anteriorly-directed flagellum emerging from the anterior one (BB1). A fibrillar rootlet with a microtubule organising centre (MTOC; ‘R1’) arises from a diaphragm-shaped basal plate at the base of BB1; a short striated fibre (SSF) covers the dorsal side of BB1 and initiates microtubular rootlets ‘R2’ and ‘R3’ and the fibrillar bridge leading to microtubular rootlet ‘R4’. ‘R5’ is composed of 2 microtubules splitting from R4 and 2 others initiating by BB1. The posterior basal body is associated with a fibrillar posterior parakinetosomal structure. A long striated fibre arises in association with the right end of the SSF. A Golgi apparatus is associated with the flagellar apparatus. The flagellar apparatus is present internally in amoebae. All three stages have mitochondria with tubular cristae and an electron-dense central body.Analysis of small subunit ribosomal RNA sequences shows H. dachnaya branching with the myxogastrids within the mycetozoa, but polyphyletic with a nominal Hyperamoeba sp. isolate that is morphologically similar to H. flagellata. We reject the suggestion that H. flagellata and this Hyperamoeba sp. isolate of Zaman et al. (1999) are conspecific. The study reveals extensive similarities between Hyperamoeba and the flagellated stages of myxogastrids.
Article
Eighty-seven isolates of amebae assigned to the genus Entamoeba have been studied by riboprinting (restriction enzyme polymorphism analysis of polymerase chain reaction amplified small subunit ribosomal RNA genes). Twenty-four distinct patterns were obtained, most of which corresponded to previously described species. In three species (Entamoeba coli, Entamoeba gingivalis and Entamoeba moshkovskii) intraspecific variation was detected that led to the grouping of isolates into ‘ribodemes’ (populations of amebae that share the same riboprint pattern). The riboprint data were used to estimate genetic distances among and within species for the construction of phylogenetic trees based on parsimony and distance analyses. The trees obtained with the two methods are largely congruent. In some cases the estimated distances between species were greater than the upper limit recommended for the fragment comigration method of analysis indicating unusually deep branches within this genus. However, it appears that those species producing cysts with eight nuclei, those producing cysts with one nucleus, and those producing cysts with four nuclei form morphologically based groups that are supported by the riboprint data. The oral parasite Entamoeba gingivalis, which does not encyst, clusters with the third group indicating secondary loss of this ability.
Article
A new amoeba, isolated from well water in Gambia, West Africa, is described and named Phreatamoeba balamuthi n. g., n. sp. Requiring anaerobic conditions for growth, it is easily cultured monoxenically with Escherichia coli or axenically in complex, undefined organic media. Three phenotypes have been observed in the life cycle: an amoeba, a flagellate, and a cyst. The amoeba moves by monopodia, is predominantly multinucleate, and varies from 11 to 160 μm in length. The flagellate has a single flagellum and is from 6 to 50 μm long. The cyst is surrounded by a resistant wall that lacks pores and ranges from 9 to 18 μm in diameter. The transformation from amoeba to flagellate can be induced nutritionally, the exact inducing factor(s) being unknown. Sexual reproduction has not been observed.
Article
During the last decade Entamoeba moshkovskii has become relevant given its capacity to infect humans, especially when considering that it is morphologically indistinguishable from E. histolytica. For a long time, E. moshkovskii was considered as a free living amoeba, but in the last decade it has been demonstrated that E. moshkovskii can infect humans and can be found more frequently in regions where amebiasis shows high prevalence values, becoming a challenge to differentiate it from the E. histolytica/E. dispar complex. Recently there have been studies that raise the possibility that E. moshkovskii could be a pathogenic species, as there are reports in different countries that associated this infection with gastrointestinal symptoms even though others have described it as a non pathogenic species. For this reasons, both clinical and epidemiological studies are required.
Article
The ultrastructural appearances of Mastigamoeba punctachora, Mastigamoeba simplex and Mastigella commutansare described. All three species have electron-dense membrane-bounded bodies, suggestive of mitochondrial homologues. All species have a single basal body giving rise to conical arrays of microtubules and to a single ribbon-like microtubular root. The proximal portion of the root is associated with a sheet of dense material. All species have ‘9+2’ flagellar axonemes, with basal bodies composed of triplets of microtubules, but axonemal outer dynein arms appear to be absent. All have a cylinder at the base of the transition zone. The transition zone of Mastigamoeba punctachora is elongate and also contains a column of electron-dense material. Ultrastructural data are compiled and analysed to assess two alternative views that the pelobionts are a primitive group of eukaryotes and the source of the other eukaryotes, or that they are related to eumycetozoan slime moulds.
Article
In phylogenetic analyses of molecular sequence data, partitioning involves estimating independent models of molecular evolution for different sets of sites in a sequence alignment. Choosing an appropriate partitioning scheme is an important step in most analyses because it can affect the accuracy of phylogenetic reconstruction. Despite this, partitioning schemes are often chosen without explicit statistical justification. Here, we describe two new objective methods for the combined selection of best-fit partitioning schemes and nucleotide substitution models. These methods allow millions of partitioning schemes to be compared in realistic time frames and so permit the objective selection of partitioning schemes even for large multilocus DNA data sets. We demonstrate that these methods significantly outperform previous approaches, including both the ad hoc selection of partitioning schemes (e.g., partitioning by gene or codon position) and a recently proposed hierarchical clustering method. We have implemented these methods in an open-source program, PartitionFinder. This program allows users to select partitioning schemes and substitution models using a range of information-theoretic metrics (e.g., the Bayesian information criterion, akaike information criterion [AIC], and corrected AIC). We hope that PartitionFinder will encourage the objective selection of partitioning schemes and thus lead to improvements in phylogenetic analyses. PartitionFinder is written in Python and runs under Mac OSX 10.4 and above. The program, source code, and a detailed manual are freely available from www.robertlanfear.com/partitionfinder.
Article
The archezoan phylum Archamoebae Cavalier-Smith, 1983 is here modified by adding a new order Phreatamoebida (presently containing only Phreatamoeba) and removing the family Entamoebidae. Entamoebidae are instead tentatively placed as a class Entamoebea together with the classes Heterolobosea, Percolomonadea and Pseudociliatea in the new protozoan phylum Percolozoa Cavalier-Smith, 1991. Thus emended the phylum Archamoebae is more homogeneous; it is more distinguished from the other two phyla of the primitively amitochondrial kingdom and superkingdom Archezoa (i.e. Metamonada and Microsporidia) by having kinetids with only a single flagellum and basal body and a flagellar root consisting of a cone of evenly spaced microtubules. This unikont character of the archamoebae suggests that they may be ancestral to the tetrakont Metamonada, from which the non-flagellate Microsporidia possibly evolved. Higher eukaryotes (superkingdom Metakaryota) probably evolved from a tetrakont metamonad by the symbiotic origin of mitochondria and peroxisomes. If so, the Archamoebae are the most primitive extant phylum of eukaryotes; if molecular phylogenetic studies confirm this idea, Archamoebae will deserve intensive study, which could reveal much about the origin of the eukaryote condition and also establish what is truly universal among eukaryotes. Archamoebae, like other Archezoa, lack mitochondria and peroxisomes and have no obvious Golgi dictyosomes. Their evolutionary significance is discussed and a detailed classification is presented in which the two earlier classes are merged into a single one: Pelobiontea Page, 1976 stat. nov., containing two orders Mastigamoebida Frenzel, 1892 (Syn. Rhizo-Flagellata Kent, 1880 non Rhizomastigida auct.) (including Mastigamoeba, Mastigina, Mastigella, Pelomyxa and probably a few other genera, which have one or more flagella or cilia (motile or immotile, 9 + 2 or otherwise) in the amoeboid trophic phase), and Phreatamoebida ord. nov. (including only Phreatamoeba in the new family Phreatamoebidae, which has alternating phases of non-flagellate amoebae and uniflagellate cells). Mastigamoebida are divided into three families: Mastigamoebidae Goldschmidt, 1907; Mastigellidae fam. nov.; Pelomyxidae Schulze, 1877. Archamoebae may be uni- or multi-nucleate and either gut parasites or (more usually) free-living in soil, freshwater, or marine habitats. Some can form cysts that would probably fossilize; the earliest (1450 My old) smooth-walled fossil cells large enough to be probable eukaryotes might therefore be archamoebal cysts.
Article
Specimens of Pelomyxa palustris from five collecting sites had numerous nonmotile flagella. The structures are called flagella because of morphological similarities to flagella and because P. palustris has affinities with amoeboid flagellates. Flagella were photographed on living cells and studied by transmission and scanning electron microscopy. From 64 to 742 flagella per cell were estimated from scanning electron microscopy of ten cells 204 to 1269 micron in length. The nonmotile flagella arise from basal granules which were, in one strain, surrounded by radiating electron-dense microtubules. This strain also had excess axonemal microtubules. Abundant cytoplasmic microtubules were arranged in several different patterns. In about half of the P. palustris cells in which nuclei were studied, microtubules were either apposed to the nuclear membrane in a parallel alignment (with some also radiating) or radiating from the nuclear membrane (with none parallel). Bacteria associated with nuclei were of three characteristic types: Gram-negative rods, Gram-positive rods, and large rods. All nuclei within a given trophozoite had similar perinuclear features. Recent proposals for separation of Pelomyxa to its own phylum (based on its proposed primitive, unique nature) can not be justified. Pelomyxa is a complex, highly specialized organism adapted to live in a specific fresh-water environment. Mastigamoebid amoeboid flagellates of the genera Mastigamoeba, Mastigella, Mastigina, and possibly Dinamoeba are placed with Pelomyxa within the order Pelobiontida Page, 1976, emend., containing two families. Pelomyxidae Schulze, 1877, and Mastigamoebidae Goldschmidt, 1907.
Article
A new amoeba, isolated from well water in Gambia, West Africa, is described and named Phreatamoeba balamuthi n. g., n. sp. Requiring anaerobic conditions for growth, it is easily cultured monoxenically with Escherichia coli or axenically in complex, undefined organic media. Three phenotypes have been observed in the life cycle: an amoeba, a flagellate, and a cyst. The amoeba moves by monopodia, is predominantly multinucleate, and varies from 11 to 160 microns in length. The flagellate has a single flagellum and is from 6 to 50 microns long. The cyst is surrounded by a resistant wall that lacks pores and ranges from 9 to 18 microns in diameter. The transformation from amoeba to flagellate can be induced nutritionally, the exact inducing factor(s) being unknown. Sexual reproduction has not been observed.
Article
The ultrastructure of the flagellar apparatuses of four species of protostelids is described. All four species have the same three major groups of rootlet microtubules in common, microtubule arrays (MTA) 2, 3, and 4. Variation is found in the number of centrioles per flagellar apparatus, presence or absence of two other microtubule arrays, MTA 1 and MTA 5, types of connectives linking the centrioles to the MTAs, nature of the transitional elements of the flagella, and the association of the flagellar apparatus with the nucleus. It is concluded on the basis of this study and comparison with earlier studies on protostelids, myxomycetes, and other amoeboflagellates that the flagellate species of protostelids are monophyletic. The significance of this to the phylogeny of protostelids and related mycetozoans is discussed.
Article
Eighty-seven isolates of amebae assigned to the genus Entamoeba have been studied by riboprinting (restriction enzyme polymorphism analysis of polymerase chain reaction amplified small subunit ribosomal RNA genes). Twenty-four distinct patterns were obtained, most of which corresponded to previously described species. In three species (Entamoeba coli, Entamoeba gingivalis and Entamoeba moshkovskii) intraspecific variation was detected that led to the grouping of isolates into 'ribodemes' (populations of amebae that share the same riboprint pattern). The riboprint data were used to estimate genetic distances among and within species for the construction of phylogenetic trees based on parsimony and distance analyses. The trees obtained with the two methods are largely congruent. In some cases the estimated distances between species were greater than the upper limit recommended for the fragment comigration method of analysis indicating unusually deep branches within this genus. However, it appears that those species producing cysts with eight nuclei, those producing cysts with one nucleus, and those producing cysts with four nuclei form morphologically based groups that are supported by the riboprint data. The oral parasite Entamoeba gingivalis, which does not encyst, clusters with the third group indicating secondary loss of this ability.