Phylogenetic position of Lophomonas striata Bütschli (Parabasalia) from the hindgut of the cockroach Periplaneta americana

Lophomonas striata is a multiflagellate parabasalid commensal in the hindgut of the omnivorous cockroaches Blatta orientalis and Periplaneta americana. Its closest relatives were traditionally thought to include similar multiflagellate parabasalids with a single flagellar area that degenerates during mitosis, such as Joenia and Kofoidia. However, molecular phylogenetic analyses have shown that "lophomonads" are not monophyletic. We have determined the SSU rRNA sequence of L. striata and we find that it branches sister to the Trichonymphida with strong support. This is surprising because all other lophomonads sampled to date branch within the Cristamonadida, and the order Trichonymphida (e.g. Trichonympha, Pseudotrichonympha, and Hoplonympha) is both morphologically coherent and monophyletic in SSU rRNA phylogenies. Trichonymphida, unlike the lophomonads, share a bilateral symmetry, in which their multiple flagella occur in two (or sometimes four) regions, and instead of degenerating upon mitosis, half of the flagella are passed to each daughter cell. The single apical flagellar region characteristic of lophomonads is therefore either plesiomorphic or it has arisen multiple times in parabasalids; our phylogenetic analyses and available ultrastructural evidence suggest the latter. Our results also suggest that parabasalid gut symbionts may have been vertically transmitted in cockroaches before the common ancestor of Cryptocercus and termites.     

Diversification of the Microtubule System in the Early Stage of Eukaryote Evolution: Elongation Factor 1α and α-Tubulin Protein Phylogeny of Termite Symbiotic Oxymonad and Hypermastigote Protists

The symbiotic protists of the lower termite have been regarded as a model of early-branched eukaryotes because of their simple cellular systems and morphological features. However, cultivation of these symbiotic protists is very difficult. For this reason, these interesting protists have not been well characterized in terms of their molecular biology. In research on these organisms which have not yet been cultivated, we developed a method for retrieving specific genes from a small number of cells, through micromanipulation without axenic cultivation, and we obtained EF-1 alpha and alpha-tubulin genes from members of the Hypermastigida--the parabasalid protist Trichonympha agilis and the oxymonad protists Pyrsonympha grandis and Dinenympha exilis--from the termite Reticulitermes speratus gut community. Results of phylogenetic analysis of the amino acid sequences of both proteins, EF-1 alpha and alpha-tubulin, indicate that the hypermastigid, parabasalid, and oxymonad protists do not share a close common ancestor. In addition, although the EF-1 alpha phylogeny indicates that these two groups of protists branched at an early stage of eukaryotic evolution, the alpha-tubulin phylogeny indicates that these protists can be assigned to two diversified clades. As shown in a recent investigation of alpha-tubulin phylogeny, eukaryotic organisms can be divided into three classes: an animal--parabasalids clade, a plant--protists clade, and the diplomonads. In this study, we show that parabasalids, including hypermastigids, can be classified as belonging to the animal--parabasalids clade and the early-branching eukaryote oxymonads can be classified as belonging to the plant--protists clade. Our findings suggest that these protists have a cellular microtubule system that has diverged considerably, and it seems that such divergence of the microtubule system occurred in the earliest stage of eukaryotic evolution.     

Molecular phylogeny of parabasalids based on small subunit rRNA sequences, with emphasis on the Trichomonadinae subfamily

We determined small subunit ribosomal DNA sequences from three parabasalid species, Trichomitus batrachorum strain R105, Tetratrichomonas gallinarum, and Pentatrichomonas hominis belonging to the Trichomonadinae subfamily. Unrooted molecular phylogenetic trees inferred by distance, parsimony, and likelihood methods reveal four discrete clades among the parabasalids. The Trichomonadinae form a robust monophyletic group. Within this subfamily T. gallinarum is closely related to Trichomonas species as supported by morphological data, with P. hominis and Pseudotrypanosoma giganteum occupying basal positions. Our analysis does not place T. batrachorum within the Trichomonadinae. Trichomitus batrachorum (strains R105 and BUB) and Hypotrichomonas acosta form a well-separated cluster, suggesting the genus Trichomitus is polyphyletic. The emergence of T. batrachorum precedes the Trichomonadinae-Tritrichomonadinae dichotomy, emphasizing its pivotal evolutionary position among the Trichomonadidae. A third cluster unites the Devescovinidae and the Calonymphidae. The fourth clade contains the three hypermastigid sequences from the genus Trichonympha, which exhibit the earliest emergence among the parabasalids. The addition of these three new parabasalid species did not however resolve ambiguities regarding the relative branching order of the parabasalid clades. The phylogenetic positions of Tritrichomonas faetus, Monocercomonas sp., Dientamoeba fragilis, and the unidentified Reticulitermes flavipes gut symbiont 1 remain unclear.     

Phylogenetic position of parabasalid symbionts from the termite Calotermes flavicollis based on small subunit rRNA sequences.

Small subunit rDNA genes were amplified by polymerase chain reaction using specific primers from mixed-population DNA obtained from the whole hindgut of the termite Calotermes flavicollis. Comparative sequence analysis of the clones revealed two kinds of sequences that were both from parabasalid symbionts. In a molecular tree inferred by distance, parsimony and likelihood methods, and including 27 parabasalid sequences retrieved from the data bases, the sequences of the group II (clones Cf5 and Cf6) were closely related to the Devescovinidae/Calonymphidae species and thus were assigned to the Devescovinidae Foaina. The sequence of the group I (clone Cf1) emerged within the Trichomonadinae and strongly clustered with Tetratrichomonas gallinarum. On the basis of morphological data, the Monocercomonadidae Hexamastix termitis might be the most likely origin of this sequence.     

Inheritance and diversification of symbiotic trichonymphid flagellates from a common ancestor of termites and the cockroach Cryptocercus

Cryptocercus cockroaches and lower termites harbour obligate, diverse and unique symbiotic cellulolytic flagellates in their hindgut that are considered critical in the development of social behaviour in their hosts. However, there has been controversy concerning the origin of these symbiotic flagellates. Here, molecular sequences encoding small subunit rRNA and glyceraldehyde-3-phosphate dehydrogenase were identified in the symbiotic flagellates of the order Trichonymphida (phylum Parabasalia) in the gut of Cryptocercus punctulatus and compared phylogenetically to the corresponding species in termites. In each of the monophyletic lineages that represent family-level groups in Trichonymphida, the symbionts of Cryptocercus were robustly sister to those of termites. Together with the recent evidence for the sister-group relationship of the host insects, this first comprehensive study comparing symbiont molecular phylogeny strongly suggests that a set of symbiotic flagellates representative of extant diversity was already established in an ancestor common to Cryptocercus and termites, was vertically transmitted to their offspring, and subsequently became diversified to distinct levels, depending on both the host and the symbiont lineages.     

Molecular phylogenetic position of the genera Stephanonympha and Caduceia (Parabasalia) inferred from nuclear small subunit rRNA gene sequences

Nuclear small subunit (SSU) rRNA gene sequences were obtained by polymerase chain reaction from trichomonad symbionts of termites that belong to the Devescovinidae (Caduceia versatilis) and polymastigont Calonymphidae (Stephanonympha nelumbium). The unidentified SSU rRNA sequence Nk3, previously obtained from the termite Neotermes koshunensis, has also been shown to derive from a Stephanonympha sp. by in situ hybridization. These sequences were analysed in a broad phylogeny including nearly all identified parabasalid sequences available in the databases, and some as yet unidentified sequences likely deriving from the new order Cristamonadida (Devescovinidae, Calonymphidae, and hypermastigids Lophomonadida). A global phylogeny of parabasalids reveals a partial agreement between the clades identified in this work and the last classification of this phylum into four orders. However, this classification is still incongruent with our data and new taxonomic considerations are proposed. The analysis confirms the monophyly of the Cristamonadida and separates this order into two groups: the first unites nearly all the Devescovinidae including Caduceia and the Calonymphidae Coronympha and Metacoronympha, whereas the second group is composed of a few Devescovinidae, Lophomonadida, and Calonymphidae such as Stephanonympha. Caduceia is closely related to Devescovina, corroborating the marked morphological similarity between these two genera whereas Stephanonympha groups together with the Calonymphidae Snyderella and Calonympha. These data also confirm the polyphyly of the families Devescovinidae and Calonymphidae and support the arrangement of the axostyle-pelta complexes as a valuable character for taxonomic considerations within the Calonymphidae.     

Molecular phylogeny of parabasalids inferred from small subunit rRNA sequences,with emphasis on the Devescovinidae and Calonymphidae (Trichomonadea)

Small subunit rRNA sequences were obtained by polymerase chain reaction from trichomonad symbionts of termites that belong to the polymastigont Calonymphidae, including Snyderella tabogae, Calonympha grassii, and Metacoronympha senta. The yet-unidentified sequence Nk9 previously obtained from the termite Neotermes koshunensis, has also been shown to derive from the Devescovinidae Devescovina sp. by in situ hybridization. These new sequences were analyzed by distance, parsimony, and likelihood methods in a broad phylogeny including all identified parabasalid sequences available in databases. All analyses revealed the emergence of a very well supported Devescovinidae/Calonymphidae group but showed an unexpected dichotomy of the Calonymphidae represented by the "Coronympha" and "Calonympha" groups. It strongly suggests that the polymastigont state observed in the Calonymphidae might be explained by at least two independent evolutionary events. In a second phylogenetic analysis, some yet-unidentified parabasalid sequences likely deriving from the Devescovinidae/Calonymphidae taxa, were added to our data set. This analysis confirmed the polyphyly of the Calonymphidae. A tentative identification is proposed for each of these sequences, and hypotheses on the origin of the Devescovinidae and Calonymphidae are discussed. Tritrichomonas foetus or a close relative might be the best candidate for the ancestor of the Devescovinidae, fairly consistent with morphology-based hypotheses. Regarding the Calonymphidae, the origin of the "Coronympha" group might be found within the Devescovinidae, related to Foaina, whereas the "Calonympha" group may directly descend from Tritrichomonas or related species.     

Organization and composition of the striated roots supporting the Golgi apparatus,the so-called parabasal apparatus,in parabasalid flagellates

Summary— In parabasalid flagellates, trichomonads and hypermastigids, the stack of cisternae of the Golgi apparatus are supported by striated roots attached to the basal bodies of flagella forming the so-called parabasal apparatus. Monoclonal antibodies raised for several trichomonad species, Monocercomonas, Trichomonas and Tetratrichomonas, label the parabasal fibre in immunofluorescence or immunogold staining and protein bands in immunoblotting. Several antibodies cross-react between trichomonad species, and one of them labels the homologous parabasal fibre in the hypermastigids: Trichonympha, Joenia, Pseudotrichonympha and Holomastigotoides. Considering the molecular mass range of the labelled proteins (100–135 kDa) and the lack of antibody cross-reactivity with the striated root proteins (centrin, assemblin, kinetodesmal protein, ciliary root proteins of epithelial ciliated cells) of other organisms, these proteins recognized by these antibodies seem to represent a new class of protein forming striated roots. The occurrence and significance of parabasal organization in eukaryogenesis is discussed.     

Phylogenetic position of the trichomonad parasite of turkeys, Histomonas meleagridis (Smith) Tyzzer, inferred from small subunit rRNA sequence.

The phylogenetic position of the trichomonad, Histomonas meleagridis was determined by analysis of small subunit rRNAs. Molecular trees including all identified parabasalid sequences available in data bases were inferred by distance, parsimony, and likelihood methods. All reveal a close relationship between H. meleagridis, and Dientamoeba fragilis. Moreover, small subunit rRNAs of both amoeboid species have a reduced G + C content and increased chain length relative to other parabasalids. Finally, the rRNA genes from H. meleagridis and D. fragilis share a recent common ancestor with Tritrichomonasfoetus, which exhibits a more developed cytoskeleton. This indicates that Histomonas and Dientamoeba secondarily lost most of the typical trichomonad cytoskeletal structures and hence, do not represent primitive morphologies. A global phylogeny of parabasalids revealed significant discrepancies with morphology-based classifications, such as the polyphyly of most of the parabasalid families and classes included in our study.     

Wolbachia endosymbionts responsible for various alterations of sexuality in arthropods.

Rickettsia-like maternally inherited bacteria have been shown to be involved in a variety of alterations of arthropod sexuality, such as female-biased sex ratios, parthenogenesis, and sterility of crosses either between infected males and uninfected females or between infected individuals (cytoplasmic incompatibility). We have characterized several of these microorganisms through partial sequences of the small (16S) and large (23S) subunit ribosomal DNA. All the symbionts identified, which include several cytoplasmic incompatibility microorganisms, several endosymbionts of terrestrial isopods, and symbionts of two thelytokous Trichogramma wasp species, belong to a monophyletic group of related symbionts, some of which have previously been detected in several insects exhibiting cytoplasmic incompatibility. Three molecular lineages can be identified on the basis of 16S as well as 23S sequences. Although they are only known as endocellular symbionts, Wolbachia spread by horizontal transfer across host lineages as evidenced by their diversification which occurred long after that of their hosts, and by the non-congruence of the phylogenetic relationships of symbionts and their hosts. Indeed, symbionts of two different lineages have been found in the same host species, whereas closely related endosymbionts are found in distinct insect orders. Isopod endosymbionts form a separate lineage, and they can determine feminization as well as cytoplasmic incompatibility. The ability to determine cytoplasmic incompatibility, found in all lineages, is probably ancestral to this group.     

Identification and characterization of ectosymbionts of distinct lineages in Bacteroidales attached to flagellated protists in the gut of termites and a wood-feeding cockroach

Bacterial attachments to nearly the entire surface of flagellated protists in the guts of termites and the wood-feeding cockroach Cryptocercus are often observed. Based on the polymerase chain reaction-amplified 16S rRNA gene sequences, we investigated the phylogenetic relationships of the rod-shaped, attached bacteria (ectosymbionts) of several protist species from five host taxa and confirmed their identity by fluorescence in situ hybridizations. These ectosymbionts are affiliated with the order Bacteroidales but formed three distinct lineages, each of which may represent novel bacterial genera. One lineage consisted of the closely related ectosymbionts of two species of the protist genus Devescovina (Cristamonadida). The second lineage comprised three phylotypes identified from the protist Streblomastix sp. (Oxymonadida). The third lineage included ectosymbionts of the three protist genera Hoplonympha, Barbulanympha and Urinympha in the family Hoplonymphidae (Trichonymphida). The ultrastructural observations indicated that these rod-shaped ectosymbionts share morphological similarities of their cell walls and their point of attachment with the protist but differ in shape. Elongated forms of the ectosymbionts appeared in all the three lineages. The protist cells Streblomastix sp. and Hoplonympha sp. display deep furrows and vane-like structures, but these impressive structures are probably evolutionarily convergent because both the host protists and their ectosymbionts are distantly related.     

Symbiosis, morphology, and phylogeny of Hoplonymphidae (Parabasalia) of the wood-feeding roach Cryptocercus punctulatus

Anaerobic cellulolytic flagellate protists of the hindguts of lower termites and the wood-feeding cockroach Cryptocercus are essential to their host's ability to digest lignocellulose. Many have bacteria associated with their surfaces and within cytoplasmic vesicles-likely important symbioses as suggested by molecular and other data. Some of the most striking examples of these symbioses are in the parabasalid family Hoplonymphidae, but little or no data exist on the structural aspects of their symbioses, their relationships with bacteria through different life-cycle stages, or their diversity and phylogenetic relationships in Cryptocercus. We investigated these areas in the hoplonymphid genera Barbulanympha and Urinympha from Cryptocercus punctulatus using light and electron microscopy, and analysis of small subunit rRNA. Microscopy reveals variation in density of bacterial surface symbionts related to life-cycle stage, a glyococalyx possibly important in bacterial adhesion and/or metabolite exchange, and putative viruses associated with bacterial surface symbionts. Patterning of surface bacteria suggests protists emerging from the resistant (dormant) stage are colonized by a small population of bacterial cells, which then divide to cover their surface. Additionally, cytoplasmic protrusions from the protist are covered by bacteria. Phylogenetic analysis rejects the monophyly of Hoplonymphidae, suggesting multiple origins or losses of these bacterial symbioses.     

Acidovorax-like symbionts in the nephridia of earthworms

Dense accumulations of bacteria in the excretory organs, nephridia, were first described more than 75 years ago in members of the annelid family Lumbricidae (earthworms). These nephridial symbionts were assumed to play a role in the degradation of proteins in the excretory fluid for nitrogen recycling. In the present study, the phylogenetic affiliation of the nephridial bacteria of the earthworms Lumbricus terrestris, Aporrectodea tuberculata, Octolasion lacteum and Eisenia foetida was resolved. The 16S rRNA gene sequences of the symbionts formed a monophyletic cluster within the genus Acidovorax. Similarity between symbiont sequences from different host species was 95.5-97.6%, whereas similarity was> 99% between symbiont sequences from individuals of the same species. Densely packed bacteria were detected in the ampulla of the nephridia by fluorescence in situ hybridization (FISH) using Acidovorax-specific oligonucleotide probes. No other bacterial cells could be found by FISH, although a few sequences other than Acidovorax had been found by PCR and cloning. These results suggest that the Acidovorax-earthworm symbiosis is a stable, host-specific association that has evolved from a common bacterial ancestor. Given the close phylogenetic relationship of the symbionts to proteolytic, free-living Acidovorax species, they may indeed play a role in protein degradation during nitrogen excretion by earthworms.     

Cryptic diversity of free-living parabasalids, Pseudotrichomonas keilini and Lacusteria cypriaca n. g., n. sp., as inferred from small subunit rDNA sequences

Ultrastructural and molecular phylogenetic evidence indicate that the Parabasalia consists of seven main subgroups: the Trichomonadida, Honigbergiellida, Hypotrichomonadida, Tritrichomonadida, Cristamonadida, Spirotrichonymphida, and Trichonymphida. Only five species of free-living parabasalids are known: Monotrichomonas carabina, Ditrichomonas honigbergii, Honigbergiella sp., Tetratrichomonas undula, and Pseudotrichomonas keilini. Phylogenetic analyses show that free-living species do not form a clade and instead branch in several different positions within the context of their parasitic relatives. Because the diversity of free-living parabasalids is poorly understood, the systematics of these lineages is in a significant state of disarray. In order to better understand the phylogenetic distribution of free-living parabasalids, we sequenced the small subunit rDNA from three different strains reminiscent of P. keilini; the strains were isolated from different geographical locations: (1) mangrove sediments in Japan and (2) sediments in Cyprus. These data demonstrated that the free-living parabasalids P. keilini and Lacusteria cypriaca n. g., n. sp., form a paraphyletic assemblage near the origin of a clade consisting mostly of parasitic trichomonadids (e.g. Trichomonas vaginalis). This paraphyletic distribution of similar morphotypes indicates that free-living trichomonadids represent a compelling example of morphostasis that provides insight into the suite of features present in the most recent free-living ancestor of their parasitic relatives.     

Pea aphid symbiont relationships established by analysis of 16S rRNAs

The pea aphid (Acyrthosiphon pisum Harris) harbors two morphologically distinct procaryotic intracellular symbionts. The genes for the 16S rRNA from these symbionts have been cloned and sequenced. Comparisons with sequences of 16S rRNAs from selected procaryotes indicate that the two symbionts are evolutionarily distinct from each other and are members of the gamma-3 subdivision of the class Proteobacteria. One of the symbionts is a member of the family Enterobacteriaceae, while the other constitutes a lineage distinct from these organisms. Both symbionts appear to have only one copy of their rRNA operon.     

Primary symbiont of the ancient scale insect family Coelostomidiidae exhibits strict cophylogenetic patterns

Bacterial symbionts play a critical role in the physiology, ecology and evolution of a diverse range of insects. Such symbionts with unknown roles in the ecology and evolution of their hosts have been reported from archaeococcoid scale insects of family Coelostomidiidae. We examine in detail the bacterial community associated with the remaining species of this family, and calculate the cophylogenetic relationship between the hosts and their symbionts. The 28S ribosomal RNA (rRNA) and mitochondrial cytochrome oxidase I genes were used to reconstruct the host phylogeny while the 16S rRNA gene was used for the bacterial phylogeny. Three well-supported clades were detected within the phylogeny of the monophyletic family Coelostomidiidae. Besides the known symbionts, a novel Sodalis-like symbiont was detected from three of the species. The primary bacteriome inhabiting B-symbiont (Bacteroidetes; ‘Candidatus Hoataupuhia coelostomidicola’) was widespread across the host family. Cophylogenetic comparison using Jungles-based reconciliation analysis and ParaFit statistical test revealed a strongly congruent phylogeny of this symbiont with the host family, with no host-switches and few losses and duplications. A similar pattern was observed across a relatively unrelated neococcoid family that exhibits a different physiology and symbiont community, besides a related Bacteroidetes symbiont. We reconfirm that the B-symbiont is a primary symbiont, owing to its strongly congruent evolution with the host and its bacteriome-inhabiting nature. Our analysis affirms recent suggestions that the Bacteroidetes-affiliated symbionts may have driven the hyper-diversification of scale insects worldwide.     

Evidence for phylogenetic congruence among sulfur-oxidizing chemoautotrophic bacterial endosymbionts and their bivalve hosts

Sulfur-oxidizing chemoautotrophic (thioautotrophic) bacteria are now known to occur as endosymbionts in phylogenetically diverse bivalve hosts found in a wide variety of marine environments. The evolutionary origins of these symbioses, however, have remained obscure. Comparative 16S rRNA sequence analysis was used to investigate whether thioautotrophic endosymbionts are monophyletic or polyphyletic in origin and to assess whether phylogenetic relationships inferred among these symbionts reflect those inferred among their hosts. 16S rRNA gene sequences determined for endosymbionts from nine newly examined bivalve species from three families (Vesicomyidae, Lucinidae, and Solemyidae) were compared with previously published 16S rRNA sequences of thioautotrophic symbionts and free-living bacteria. Distance and parsimony methods were used to infer phylogenetic relationships among these bacteria. All newly examined symbionts fall within the gamma subdivision of the Proteobacteria, in clusters containing previously examined symbiotic thioautotrophs. The closest free-living relatives of these symbionts are bacteria of the genus Thiomicrospira. Symbionts of the bivalve superfamily Lucinacea and the family Vesicomyidae each form distinct monophyletic lineages which are strongly supported by bootstrap analysis, demonstrating that host phylogenies inferred from morphological and fossil evidence are congruent with phylogenies inferred for their respective symbionts by molecular sequence analysis. The observed congruence between host and symbiont phylogenies indicates shared evolutionary history of hosts and symbiont lineages and suggests an ancient origin for these symbioses. Correspondence to: D.L. Distel     

Molecular evolution inferred from small subunit rRNA sequences: what does it tell us about phylogenetic relationships and taxonomy of the parabasalids?

The Parabasala are a primitive group of protists divided into two classes: the trichomonads and the hypermastigids. Until recently, phylogeny and taxonomy of parabasalids were mainly based on the comparative analysis of morphological characters primarily linked to the development of their cytoskeleton. Recent use of molecular markers, such as small subunit (SSU) rRNA has led to now insights into the systematics of the Parabasala and other groups of prolists. An updated phylogeny based on SSU rRNA is provided and compared to that inferred from ultrastructural data. The SSU rRNA phylogeny contradicts the dogma equating simple characters with pumitive characters. Hypermastigids, possessing a hyperdeveloped cytoskeleton, exhibit the most basal emergence in the parabasalid lineage. Other observations emerge from the SSU rRNA analysis, such as the secondary loss of some cytoskeleton structures in all representatives of the Monocercomonadidae, the existence of secondarily free living taxa (reversibility of parasitism) and the evidence against the co-evolution of the endobiotic parabasalids and their animal hosts. According to phylogenies based on SSU rRNA, all the trichomonad families are not monophyletic groups, putting into question the validity of current taxonomic assignments. The precise branching order of some taxa remains unclear, but this issue can possibly be addressed by the molecular analysis of additional parabasalids. The goal of such additional analyses would be to propose, in a near future, a revision of the taxonomy of this group of protists that takes into account both molecular and morphological data.