Long branch attraction effects and the status of "basal eukaryotes": phylogeny and structural analysis of the ribosomal RNA gene cluster of the free-living diplomonad Trepomonas agilis

The three taxa emerging at the base of the eukaryotic ribosomal RNA phylogenetic tree (Diplomonadida, Microspora, and Parabasalia) include a wide array of parasitic species. and some free-living organisms that appear to be derived from a parasitic ancestry. The basal position of these taxa, which lack mitochondria, has recently been questioned. I sequenced most of the ribosomal RNA gene cluster of the free-living diplomonad Trepomonas agilis and a secondary structure model was reconstructed for the SSU rRNA. I conducted a RASA matrix analysis to identify, independently from tree reconstruction, putative long branch attraction effects in the data matrix. The results show that each of the basal clades and the euglenozoan clade act, indeed, as long branches and may have been engaged in a process of accelerated rate of evolution. A nucleotide signature analysis was conducted in the conserved regions for positions defining the three great domains of life (Eubacteria, Archea, and Eukaryota). For the three basal taxa, this analysis showed the presence of a significant number of different non-eukaryotic nucleotides. A precise study of the nature and location of these nucleotides led to conclusions supporting the results of the RASA analysis. Altogether, these findings suggest that the basal placement of these taxa in the SSU ribosomal RNA phylogenetic tree is artifactual, and flawed by long branch attraction effects.     

Morphology and molecular phylogeny of Pseudotrichonympha hertwigi and Pseudotrichonympha paulistana (Trichonymphea, parabasalia) from neotropical rhinotermitids

Pseudotrichonympha is a large hypermastigote parabasalian found in the hindgut of several species of rhinotermitid termites. The genus was discovered more than 100 years ago, and although over a dozen species have since been described, this represents only a small fraction of its likely diversity: the termite genera from which Pseudotrichonympha is known are all species rich, and in most cases their hindgut symbionts have not been examined. Even formally described species are mostly lacking in detailed microscopic data and/or sequence data. Using small subunit ribosomal RNA gene sequences and light and scanning electron microscopy we describe here the morphology and molecular phylogenetic position of two Pseudotrichonympha species: the type species for the genus, Pseudotrichonympha hertwigi from Coptotermes testaceus (described previously in line drawing only), and Pseudotrichonympha paulistana from Heterotermes tenuis (described previously based on light microscopy only).     

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.     

Polymorphic Insertions and Deletions in Parabasalian Enolase Genes

Insertions and deletions in gene sequences have been used as characters to infer phylogenetic relationships and, like any character, the information they contain varies in utility between different levels of evolution. In one case, the absence of two otherwise highly conserved deletions in the enolase genes of parabasalian protists has been interpreted as a primitive characteristic that suggests these were among the first eukaryotes. Here, semi-environmental 3-RACE was used to sample enolases from parabasalia in the hindgut of the termite Zootermopsis angusticolis to examine the conservation of this character within the parabasalia. Parabasalian homologues were found to be polymorphic for these deletions, and the phylogeny of parabasalian enolases shows that the deletion-possessing genes branch within deletion-lacking genes (i.e., they did not form two clearly distinct groups). Phylogenetic incongruence was detected in the carboxy-terminal third of the sequence (in the region of the deletions), but there is no unambiguous evidence for recombination. The polymorphism of this character discredits these deletions as strong evidence for the early origin of parabasalia, although the complex distribution makes it impossible to state whether parabasalian enolases were ancestrally like those of other eukaryotes. These observations stress the importance of strong corroborating evidence when considering insertion and deletion data, and raises some interesting questions about the apparent variation in degree of conservation of these deletions between different eukaryotic groups.     

Flagellar and cytoskeletal systems in amitochondrial flagellates: Archamoeba,Metamonada and Parabasala

Summary The hypothesis that protists without mitochondria, the so-called Archezoa of Cavalier-Smith, are primitive has received some support from rRNA sequence studies on Microsporidia and Diplomonadida. In spite of the lack of mitochondria the archezoan groups of protists show considerable differences in their organization: mastigont and cytoskeletal system, mitosis, Golgi apparatus, hydrogenosomes. This paper examines the characters of the flagellar apparatus and its associated cytoskeleton to obtain clues used for phylogenetic consideration on the three cited groups of flagellates. Archamoebae of the Pelobiontida order comprising families such as Pelomyxidae and Mastigamoebidae share common features: a rudimentary mastigont system composed of only one basal body giving rise to a poorly motile flagellum and a basal body associated microtubular cone capping the nucleus. No Golgi apparatus has been detected.Metamonada, comprising three orders: Retortamonadida, Diplomonadida, and Oxymonadida, have been tentatively assembled on the basis of the absence of mitochondria, Golgi apparatus, and basal body arrangement. They all have four basal bodies arranged in two pairs with always one recurrent flagellum generally included in a cytostomal depression. The recurrent basal body/flagellum is in relation to recurrent microtubular fibers. However, they display marked differences in their cytoskeletal system and fiber ultrastructure indicating a distant evolutionary relationship. The presence of a corset of microtubules in retortamonads and three microtubular fibers are distinguished in diplomonads, as well as a paracrystalline preaxostyle and axostyle in oxymonads are features that lend support to these groups being highly divergent.Parabasala, comprising the orders Trichomonadida and Hypermastigida, is a monophyletic group with a set of homologous features such as the presence of the same arrangement of four basic basal bodies, the parabasal apparatus (striated fibre supporting Golgi), the microtubular pelta-axostyle complex, the external mitotic apparatus (crypto-pleuro-mitosis), the hydrogenosomes. These three phyla appear distantly related, the Parabasala being a homogeneous group, perhaps also the Pelobiontida, while the Metamonada is heterogeneous and composed of three evolutionary lineages. Additional information such as rRNA and protein sequence data could contribute to a better understanding of the phylogenetic relationships among these groups.Abbreviations EM electron microscopy - MTOC microtubule organizing centre - PF parabasal fibre     

Comparative cytological study of four species in the genera Holomastigotes and Uteronympha n. comb. (Holomastigotidae, Parabasalia), symbiotic flagellates of termites

Cytological features observed using light, immunofluorescence, and electron microscopy of the type species Holomastigotes elongatum were compared with Holomastigotes lanceolata and to Holomastigotes flexuosum n. sp. The comparison was extended to Spirotrichonymphella pudibunda and to Uteronympha africana n. gen. n. sp., in order to present the common features of the Holomastigotidae (Spirotrichonymphida). All these species have anterior basal bodies bearing microfibrillar or striated rootlets that are reduced or absent posterior to the nucleus. An axostylar trunk is present in Holomastigotes elongatum and Holomastigotes lanceolata, whereas the axostylar microtubules do not extend posterior to the nucleus in Holomastigotes flexuosum, Spirotrichonymphella, and Uteronympha. Uteronympha africana has specific features, such as a transverse plaque inside the columella from which arise microtubules capping the nucleus, and as in Spirotrichonympha the striated lamina is present all along the flagellar lines. Uteronympha africana has ability to endocytose wood particles in addition to the osmotrophic feeding that occurs in all the Holomastigotidae.