Vladimír Hampl

The revised classification of eukaryotes.

This revision of the classification of eukaryotes, which updates that of Adl et al. [J. Eukaryot. Microbiol. 52 (2005) 399], retains an emphasis on the protists and incorporates changes since 2005 that have resolved nodes and branches in phylogenetic trees. Whereas the previous revision was successful in re‐introducing name stability to the classification, this revision provides a classification for lineages that were then still unresolved. The supergroups have withstood phylogenetic hypothesis testing with some modifications, but despite some progress, problematic nodes at the base of the eukaryotic tree still remain to be statistically resolved. Looking forward, subsequent transformations to our understanding of the diversity of life will be from the discovery of novel lineages in previously under‐sampled areas and from environmental genomic information.

Phylogenomic analyses support the monophyly of Excavata and resolve relationships among eukaryotic “supergroups”

Nearly all of eukaryotic diversity has been classified into 6 suprakingdom-level groups (supergroups) based on molecular and morphological/cell-biological evidence; these are Opisthokonta, Amoebozoa, Archaeplastida, Rhizaria, Chromalveolata, and Excavata. However, molecular phylogeny has not provided clear evidence that either Chromalveolata or Excavata is monophyletic, nor has it resolved the relationships among the supergroups. To establish the affinities of Excavata, which contains parasites of global importance and organisms regarded previously as primitive eukaryotes, we conducted a phylogenomic analysis of a dataset of 143 proteins and 48 taxa, including 19 excavates. Previous phylogenomic studies have not included all major subgroups of Excavata, and thus have not definitively addressed their interrelationships. The enigmatic flagellate Andalucia is sister to typical jakobids. Jakobids (including Andalucia), Euglenozoa and Heterolobosea form a major clade that we name Discoba. Analyses of the complete dataset group Discoba with the mitochondrion-lacking excavates or “metamonads” (diplomonads, parabasalids, and Preaxostyla), but not with the final excavate group, Malawimonas. This separation likely results from a long-branch attraction artifact. Gradual removal of rapidly-evolving taxa from the dataset leads to moderate bootstrap support (69%) for the monophyly of all Excavata, and 90% support once all metamonads are removed. Most importantly, Excavata robustly emerges between unikonts (Amoebozoa + Opisthokonta) and “megagrouping” of Archaeplastida, Rhizaria, and chromalveolates. Our analyses indicate that Excavata forms a monophyletic suprakingdom-level group that is one of the 3 primary divisions within eukaryotes, along with unikonts and a megagroup of Archaeplastida, Rhizaria, and the chromalveolate lineages.

Construction and bootstrap analysis of DNA fingerprinting-based phylogenetic trees with the freeware program FreeTree: application to trichomonad parasites.

The Win95/98/NT program FreeTree for computation of distance matrices and construction of phylogenetic or phenetic trees on the basis of random amplified polymorphic DNA (RAPD), RFLP and allozyme data is presented. In contrast to other similar software, the program FreeTree (available at http://www.natur.cuni.cz/~flegr/programs/freetree or http://ijs.sgmjournals.org/content/vol51/issue3/) can also assess the robustness of the tree topology by bootstrap, jackknife or operational taxonomic unit-jackknife analysis. Moreover, the program can be also used for the analysis of data obtained in several independent experiments performed with non-identical subsets of taxa. The function of the program was demonstrated by an analysis of RAPD data from 42 strains of 10 species of trichomonads. On the phylogenetic tree constructed using FreeTree, the high bootstrap values and short terminal branches for the Tritrichomonas foetus/suis 14-strain branch suggested relatively recent and probably clonal radiation of this species. At the same time, the relatively lower bootstrap values and long terminal branches for the Trichomonas vaginalis 20-strain branch suggested more ancient radiation of this species and the possible existence of genetic recombination (sexual reproduction) in this human pathogen. The low bootstrap values and the star-like topology of the whole Trichomonadidae tree confirm that the RAPD method is not suitable for phylogenetic analysis of protozoa at the level of higher taxa. It is proposed that the repeated bootstrap analysis should be an obligatory part of any RAPD study. It makes it possible to assess the reliability of the tree obtained and to adjust the amount of collected data (the number of random primers) to the amount of phylogenetic signals in the RAPD data of the taxon analysed. The FreeTree program makes such analysis possible.

A Eukaryote without a Mitochondrial Organelle

The presence of mitochondria and related organelles in every studied eukaryote supports the view that mitochondria are essential cellular components. Here, we report the genome sequence of a microbial eukaryote, the oxymonad Monocercomonoides sp., which revealed that this organism lacks all hallmark mitochondrial proteins. Crucially, the mitochondrial iron-sulfur cluster assembly pathway, thought to be conserved in virtually all eukaryotic cells, has been replaced by a cytosolic sulfur mobilization system (SUF) acquired by lateral gene transfer from bacteria. In the context of eukaryotic phylogeny, our data suggest that Monocercomonoides is not primitively amitochondrial but has lost the mitochondrion secondarily. This is the first example of a eukaryote lacking any form of a mitochondrion, demonstrating that this organelle is not absolutely essential for the viability of a eukaryotic cell.

Critical taxonomic revision of Parabasalids with description of one new genus and three new species.

We propose a new classification of Parabasalia which is congruent with both ultrastructural and molecular-phylogenetic studies. We identify six main parabasalid lineages and give them the rank of class: Hypotrichomonadea, Trichomonadea, Tritrichomonadea, Cristamonadea, Trichonymphea, and Spirotrichonymphea. Trichomonadea is characterized by a single mastigont and by the absence of both a comb-like structure and an infrakinetosomal body. Most representatives also possess a lamelliform undulating membrane. Trichomonadea is divided into two monophyletic orders, Trichomonadida (family Trichomonadidae; with a B-type costa) and Honigbergiellida (families Honigbergiellidae, Hexamastigidae and Tricercomitidae; without a costa). The class Tritrichomonadea, with a single order Tritrichomonadida, is ancestrally characterized by a single mastigont with four flagella, and both a comb-like structure and an infrakinetosomal body. The morphologically most complex representatives (family Tritrichomonadidae) possess in addition a rail-type undulating membrane, an A-type costa, and a suprakinetosomal body. These last three characters are absent in families Monocercomonadidae and Simplicimonadidae. The remaining tritrichomonadids, Dientamoebidae, have undergone reductive evolution. Cristamonads (Cristamonadea) are morphologically derived from tritrichomonads. Because we are unable to determine morphologically homogenous monophyletic lineages within cristamonads, we classify all cristamonads into a single family, Lophomonadidae. Hypotrichomonadea, comprising the genera Trichomitus and Hypotrichomonas, resembles Tritrichomonadea by an A-type costa, and by the presence of a comb-like structure in the mastigont. However, they do not possess an infrakinetosomal body, and are not specifically related to Tritrichomonadea in molecular-phylogenetic analyses. Moreover, unlike Tritrichomonadea, Hypotrichomonadea possesses a lamelliform undulating membrane. The remaining parabasalids are of complex morphology and belong to the classes Trichonymphea and Spirotrichonymphea. A new parabasalid genus, Simplicimonas (Tritrichomonadea), and three new species, Tetratrichomonas undula, Hexamastix coercens and Simplicimonas similis, are described.

Cattle pathogen tritrichomonas foetus (Riedmüller, 1928) and pig commensal Tritrichomonas suis (Gruby & Delafond, 1843) belong to the same species.

Abstract A number of reports suggest that the sexually transmitted pathogen of cattle, Tritrichomonas foetus, and a gastrointestinal commensal of pigs, Tritrichomonas suis, are very similar and may be co-specific. A conclusive review of the taxonomic and nomenclatural status of these species has not been presented so far. Toward this end, we reexamined and compared porcine and bovine trichomonads with regard to their morphology, pathogenic potential, and DNA polymorphism. Using light and electron microscopy, no distinguishing features between T. foetus and T. suis strains were found in size, general morphology, and karyomastigont structure. Both bovine and porcine trichomonads showed pathogenic potential in the subcutaneous mouse assays and did not separate into distinct groups according to strain virulence. Three DNA fingerprinting methods (i.e. RFLP, RAPD, and PCR-based analysis of variable-length DNA repeats) that produce species-specific DNA fragment patterns did not distinguish between the bovine and porcine strains. Sequencing of a variable 502-bp DNA fragment as well as comparison of 16S rRNA gene sequences did not reveal species-specific differences between the cattle and porcine strains. Therefore, we conclude that T. foetus and T. suis belong to the same species. To prevent confusion that may arise from T. foetus–T. suis synonymy, we propose to suppress the older name suis and maintain its accustomed junior synonym foetus as a nomen protectum for both cattle and porcine trichomonads. The case has been submitted to the International Commision on Zoological Nomenclature for ruling under its plenary power.

Comparison of European Trichobilharzia species based on ITS1 and ITS2 sequences

Schistosomes are parasites of considerable medical and veterinary importance and, therefore, all aspects of their biology have been intensively studied. In contrast, our knowledge of species of the largest genus, Trichobilharzia, is insufficient. Because morphological characterization of Trichobilharzia species provides a limited number of criteria for species determination, molecular data are required. In the present paper, we sequenced internal transcribed spacers ITS1 and ITS2, and 5.8S ribosomal RNA (rRNA) genes of 3 European Trichobilharzia species (T. regenti, T. szidati and T. franki). We showed that ITS1 and ITS2 sequences can be used in species identification. Repetitive elements were found in ITS1 of all 3 Trichobilharzia species; their number and length varied depending on the species. Phylogenetic analysis showed that the visceral T. franki is more related to the nasal T. regenti, than to the visceral T. szidati. The newly designed primer, which is specific for T. regenti, might be used as a tool for diagnosis of this potential pathogen.

Validity reassessment of Trichobilharzia species using Lymnaea stagnalis as the intermediate host

The systematics within the genus Trichobilharzia is complicated. After the description of the type species Trichobilharzia ocellata, the name was routinely used for nearly all European findings of ocellate furcocercariae. T. ocellata was also described from North America and Japan. However, the identity of T. ocellata remains questionable. Comparison of data from the literature showed differences among various T. ocellata isolates and led us to the conclusion that the North American and the Japanese findings are not identical with European T. ocellata. In addition, the description of T. szidati corresponds with the recently reported European T. ocellata isolates. Sequence analysis of the ITS region confirmed that they are identical.

Cattle Pathogen Tritrichomonas foetus () and Pig Commensal Tritrichomonas suis () Belong to the Same SpeciesRiedmüller, 1928Gruby & Delafond, 1843

Abstract A number of reports suggest that the sexually transmitted pathogen of cattle, Tritrichomonas foetus, and a gastrointestinal commensal of pigs, Tritrichomonas suis, are very similar and may be co-specific. A conclusive review of the taxonomic and nomenclatural status of these species has not been presented so far. Toward this end, we reexamined and compared porcine and bovine trichomonads with regard to their morphology, pathogenic potential, and DNA polymorphism. Using light and electron microscopy, no distinguishing features between T. foetus and T. suis strains were found in size, general morphology, and karyomastigont structure. Both bovine and porcine trichomonads showed pathogenic potential in the subcutaneous mouse assays and did not separate into distinct groups according to strain virulence. Three DNA fingerprinting methods (i.e. RFLP, RAPD, and PCR-based analysis of variable-length DNA repeats) that produce species-specific DNA fragment patterns did not distinguish between the bovine and porcine strains. Sequencing of a variable 502-bp DNA fragment as well as comparison of 16S rRNA gene sequences did not reveal species-specific differences between the cattle and porcine strains. Therefore, we conclude that T. foetus and T. suis belong to the same species. To prevent confusion that may arise from T. foetus–T. suis synonymy, we propose to suppress the older name suis and maintain its accustomed junior synonym foetus as a nomen protectum for both cattle and porcine trichomonads. The case has been submitted to the International Commision on Zoological Nomenclature for ruling under its plenary power.

Peptidases of trematodes.

Among human and veterinary parasitic diseases the trematodiases (e.g. schistosomiasis, fascioliasis) represent a problem of global importance with vast social, economic and public health impacts, especially in developing countries. Therefore, host-parasite (host-trematode) interactions represent a key topic in many research laboratories, and modern approaches and technologies allow us to study the molecular basis of these interactions. As a consequence, key molecules produced by trematodes in order to ensure parasite invasion and survival within a hosts can be characterized. Trematode peptidases certainly belong to such molecules; as they are indispensable biocatalysts in a number of basal biological processes in trematodes (e.g. tissue invasion/migration, nutrition, immune evasion or other host-parasite interactions). Schistosoma mansoni cercarial elastase (CE) (penetration enzyme), cathepsin B (CB) (mainly nutrition enzyme) and Fasciola hepatica cathepsin L (CL) (nutrition, immune evasion enzyme) are probably the most studied trematode peptidases with well-characterized critical functions. Due to the importance of peptidases in host-parasite interactions they are considered to be promising targets for the development of novel chemotherapeutic drugs and vaccines against a number of trematodiases, including schistosomiasis, fascioliasis, paragonimiasis and opisthorchiasis. The present chapter summarizes the data on the biochemical and molecular features of the major trematode peptidases, and describes their role in trematode biology and host-parasite interactions based on proteolysis (peptidolysis).