Charles University
Faculty of Science
Department of Parasitology
Vladimír Hampl
Ph.D. thesis
EVOLUTION OF METAMONADA
Thesis supervisor: Doc. RNDr. Jaroslav Flegr, CSc.
Thesis advisor: Prof. RNDr. Jaroslav Kulda, CSc.
Introduction
Parabasalids, diplomonads, retortamonads and oxymonads are protistan groups
with uncertain phylogenetic affiliation. In outgroup-rooted trees they mostly appear
among the first eukaryotic branches, but this position is questioned and ascribed to the
action of long-branch attraction artefact. Cavalier-Smith (2003) classifies these groups
together with genera Trimastix and Carpediemonas to a broadly defined phylum
Metamonada, a member of eukaryotic megagroup Excavata. There are no diagnostic
characters for Metamonada sensu Cavalier-Smith (2003) and their definition is rather
complex: Ancestrally tetrakont anaerobic flagellates with a tendency for multiplication of
karyomastigont; recurrent flagellum ancestrally bears vanes and passes through a ventral
groove. There is growing evidence that parabasalids, diplomonads and Carpediemonas
share an exclusive common ancestor. This evidence is based on multiple gene
phylogenies and sharing genes that were acquired in a single event of horizontal gene
transfer. Oxymonads and Trimastix are indisputably closely related to each other in the
18S rRNA tree, but their affiliation to other metamonads is uncertain.
The evolution within the individual metamonad groups was traditionally depicted as
proceeding from simple to complex forms – from monocercomonadids to hypermatigotes
in parabasalids, from unizoic to diplozoic genera in diplomonads, and from simple
polymastigids to “higher” oxymonad families in oxymonads. In diplomonads and
oxymonads these evolutionary hypotheses have never been tested using molecular
methods, but in parabasalids molecular trees refuted the simple-to-complex scenario.
Molecular trees showed that the most basal parabasalid linage formed a very derived
trichonymphids. Monocercomonadids appeared to be non-monophyletic and simple
morphology of some of them is apparently secondary. Many other parabasalian taxa are
very probably non-monophyletic and despite a few recent revisions, the parabasalian
classification is still not in accordance with the phylogenetic trees.
Aims of the thesis
1. Elucidate the phylogenetic position of retortamonads and oxymonads.
2. Reconstruct the internal phylogeny of diplomonads with a special respect to the
position of unizoic enteromonads.
3. Critically analyse the rooting a relationships within parabasalids with the effort to
minimize the effect of tree construction artefacts on topology reconstruction.
4. Confirm parabasalid affiliation of the genus Cochlosoma and determine its
position in parabasalid tree.
5. Investigate molecular diversity of the polyphyletic family Monocercomonadidae
by determining phylogenetic positions of new isolates.
6. Find tree-congruent morphological characters in representatives of family
Monocercomonadiae that could be used in the future revision of parabasalid
classification.
Results
1. We sequenced 18S rRNA gene for the genus Retortamonas and demonstrated that
they form a robust clade (100% bootstrap) with diplomonads.
Analysis of nine-gene concatenate (---tubulin, EF-1, EF-2, cytHSP70,
HSP90 and ubiquitin) supported the existence of Metamonada sensu CavalierSmith (2003) including oxymonads. Because the Metamonada clade branched
weakly with euglenozoans, the tree supported also the monophyly of the proposed
megagroup Excavata. Detailed inspection of phylogenetic signal in individual
genes, however, revealed that the concatenation of α- and β-tubulin recovered a
tree, which was in statistically significant conflict with the existence of
metamonad clade. As the two genes are in minority we regard their phylogeny
less reliable, however, we intend to confirm the monophyly of Metamonada by
using more genes.
2. We sequenced 18S rRNA gene for enteromonad genus Trimitus. The sequence of
the unizoic Trimitus robustly branched within diplomonadid clade above three
diplomonad branches. This topology questioned the dominant view that
diplomonad genera evolved from unizoic ancestors in one duplication event,
because more duplication or reduction events must be evoked to explain the tree.
3. We analysed the internal phylogeny of parabasalids using the 16S rRNA
sequences and applied methods to minimize the effect of LBA artefact. Most
clades remained unaffected by employing of these methods but one
(Trichonymphida, Hexamastix, putative Tricercomitus) split, indicating that it was
artificially held by LBA. We also examined whether the LBA underlies the
rooting on the trichonymphid branch. The exclusion method showed that LBA
may be involved, but did not suggest any reliable alternative position. Again, the
resolution must await other well-sampled gene phylogenies.
4. We confirmed the parabasalid affiliation of protist Cochlosoma anatis. The
analyses suggested that its sister taxon is probably the genus Pentatrichomonas
from family Trichomonadidae. Taxonomic consequences were drawn from this
finding: family Cochlosomatidae was abandoned, family Trichomonadidae was
split into three monophyletic families (Trichomonadidae, Tritrichomonadidae and
Trichomitidae) and Cochlosoma was moved to the amended family
Trichomonadidae.
5. We gathered and sequenced 22 new isolates of genus Monocercomonas and two
isolates of genus Hexamastix. Monocercomonadidae sequences formed four
distinct clades in the tree (our sequences occupied two of them) and their
polyphyly was statistically significant. The genus Monocercomonas appeared
polyphyletic, which led us to establish a new genus Honigberiella gen. nov.
6. We determined four characters that discriminate the monocercomonadidae clades
and correspond with their position in the tree. These characters are – shape of the
axostyle trunk and the presence or absence of infrakinetosomal body, comb-like
structure and marginal lamella. We suggest that some higher taxa can be defined
using these characters in the future systematic revision of parabasalids.
Vladimír Hampl
CURRICULUM VITAE
ADDRESS:
TELEPHONE:
FAX:
E-MAIL:
Department of Parasitology
Faculty of Science
Charles University
Viničná 7
120 00 - PRAGUE 2
Czech Republic
(#420)221951816
(#420)224919704
vlada@natur.cuni.cz
PERSONAL DATA:
Birth date:
Birthplace:
January 10, 1976
Zábřeh na Moravě, Czech Republic
EDUCATION:
1994 – 1999: Master degree in Biology, Faculty of Science Charles University, thesis:
RAPD method and its application for the study of the polymorphism of
trichomonads.
1999 –
Postgraduate study of Parasitology, Faculty of
Science, Charles University, thesis: Evolution of
Metamonada.
2001,2003: Visiting student at laboratory of Prof. Martin T. Embley.
Natural History Museum, London, UK.
List of publications
Papers:
Hampl V, Pavlíček A, Flegr J: Construction and bootstrap analysis of DNA
fingerprinting-based phylogenetic trees with a freeware program FreeTree: Application to
trichomonad parasites. Int J Syst Evol Microbiol 2001 51: 731-735.
Hampl V, Vaňáčová Š, Kulda J, Flegr J: Concordance between genetic and phenotypic
similarities of Trichomonas vaginalis strains. BMC Evolutionary Biology 2001 1:11.
Dvořák J, Vaňáčová Š, Hampl V, Flegr J, Horák P: Comparison of European
Trichobilharzia species based on ITS1 and ITS2 sequences. Parasitology 2002 124: 307313
Silberman JD, Simpson AGB, Kulda J, Cepicka I, Hampl V, Johnson PJ, Roger AJ:
Retortamonad flagellates are closely related to diplomonads – implications for the history
of mitochondrial function in eukaryote evolution. Mol Biol Evol 2002 19(5):777-786.
Tachezy J, Tachezy R, Hampl V, Šedinová M, Vaňáčová Š, Vrlík M, vanRanst M, Flegr
J, Kulda J: Cattle Pathogen Tritrichomonas foetus (Riedmüller, 1928) and Pig Comensal
Tritrichomonas suis (Gruby & Delafond, 1843) Belong to the Same Species. J Euk
Microbiol 2002 49:(2)154-163.
Hampl V, Cepicka I, Flegr J, Tachezy J, Kulda J: Critical analysis of the topology and
rooting of the parabasalian 16S rRNA tree. Mol Phylogenet Evol 2004 32:711-723.
Kostka M, Hampl V, Cepicka I, Flegr J: The phylogenetic position of Protoopalina
intestinalis based on 18S rRNA gene. Mol Phylogenet Evol 2004 33: 220-224.
Rudolfová J, Hampl V, Bayssade-Dufour C a Horák P: Validity reassessment of
Trichobilharzia spp. developing in Lymnaea stagnalis. Parasitol res 2005 95(2):79-89.
Kolísko M, Cepicka I, Hampl V, Kulda J, Flegr J: Molecular phylogeny of enteromonads
based on SSU rDNA of Trimitus sp.: the challenge for the present models of diplomonad
evolution. Int J Syst Evol Microbiol, Int J Syst Evol Microbiol 2005 55:1729-1733.
Hampl V, Horner DS, Dyal P, Kulda J, Flegr J, Coombs GH and Embley TM: Inference
of phylogenetic position of oxymonads based on 9 genes: Support for monophyly of
Metamonada and Excavata. Mol Biol Evol, in press.
Hampl V, Vrlík M, Kulda J, Tachezy J: Affiliation of Cochlosoma to trichomonads
confirmed by phylogenetic analysis of small subunit rRNA gene. Int J Syst Evol
Microbiol, in press.
Kolarova L, Rudolfova J, Hampl V, Skirnisson K: Allobilharzia visceralis gen. nov., sp.
Nov. (Schistosomatidae - Trematoda) from Cygnus cygnus (L.) (Anatidae). Parasitol Int,
in press.
Cepicka I, Hampl V, Kulda J, Flegr J. New evolutionary lineages, unexpected diversity
and host specificity in the parabasalid genus Tetratrichomonas. Sent to Mol Phylogenet
Evol
Hampl V, Cepicka I, Flegr J, Tachezy J, Kulda J: Morphological and molecular diversity
of monocercomonadid genera Monocercomonas, Hexamastix and Honigbergiella gen.
nov. In preparation.
Book translation:
Carl Zimmer: Parasite Rex (2000). Translated - Hampl V, Harantová O: Vládce parazit
(Paseka 2005).
Selected abstracts:
Hampl V, Vaňáčová Š, Flegr J: Concordance between genetic and phenotypic
similarities of Trichomonas vaginalis strains. 13th Meeting of the Society for Evolutionary
Protistology. České Budějovice, Czech Republic.
Hampl V, Horner DS, Dyal P, Kulda J, Flegr J, Embley M: Another blow to the
Archezoa hypothesis:Phylogenetic relationships of Oxymonad flagellates. Molecular
Evolution 2002. Sorrento, Italy.
Hampl V, Cepicka I, Kulda J, Flegr J, Tachezy J: Polarity of anagenesis of motility
apparatus and repeated switches of life strategies in trichomonad flagellates. 8th Meeting
of PhD students in evolutionary biology 2002. Lohja. Finland.
Hampl V, Horner DS, Dyal P, Kulda J, Flegr J, Foster R, Embley M: Inference of
phylogenetic position of oxymonads based on 9 genes: Support for monophyly of
Metamonada and Excavata. 12th International Congress of Protozoology 2005.
Guangzhou, China.
Examination committee:
Prof. RNDr. Petr Volf, CSc. (head)
Charles University, Prague
Doc. RNDr. Václav Hypša, CSc.
University of South Bohemia, České Budějovice
Doc. RNDr. Julius Lukeš, CSc.
University of South Bohemia, České Budějovice
RNDr. Hana Kabíčková, CSc.
Klinlab, s.r.o., Prague
RNDr. Eva Nohýnková, Ph.D.
Charles University, Prague
Prof. RNDr. Jiří Vávra, Dr.Sc.
Charles University, Prague
Reviewers:
Doc. RNDr. Julius Lukeš, CSc.
University of South Bohemia, České Budějovice
Joel B. Dacks, Ph.D.
University of Calgary, Calgary
The thesis will be defended on Monday, 12th December, 2005, 10:00 at the Department
of Parasitology, Charles University, Viničná 7, Prague 2, Czech Republic.