Guy Brugerolle

The New Higher Level Classification of Eukaryotes with Emphasis on the Taxonomy of Protists

Abstract. This revision of the classification of unicellular eukaryotes updates that of Levine et al. (1980) for the protozoa and expands it to include other protists. Whereas the previous revision was primarily to incorporate the results of ultrastructural studies, this revision incorporates results from both ultrastructural research since 1980 and molecular phylogenetic studies. We propose a scheme that is based on nameless ranked systematics. The vocabulary of the taxonomy is updated, particularly to clarify the naming of groups that have been repositioned. We recognize six clusters of eukaryotes that may represent the basic groupings similar to traditional “kingdoms.” The multicellular lineages emerged from within monophyletic protist lineages: animals and fungi from Opisthokonta, plants from Archaeplastida, and brown algae from Stramenopiles.

Diversity, Nomenclature, and Taxonomy of Protists

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Phylogeny of trichomonads based on partial sequences of large subunit rRNA and on cladistic analysis of morphological data.

ABSTRACT. Several domains of large subunit rRNA from nine trichomonad species have been sequenced. Molecular phylogenies obtained with parsimony and distance methods demonstrate the trichomonads are a monophyletic group which branches very early in the eukaryotic tree. the topology of the trees is in general agreement with traditional views on evolutionary and systematic relationships of trichomonads. A clear dichotomy is noted between the subfamily Trichomonadinae and the subfamily Tritrichomonadinae. In the latter subfamily, a second division separates the “Tritrichomonas muris‐type” species from the “Tritrichomonas augusta‐type” ones. Previous evolutionary schemes in which the Monocercomonadidae were regarded as the most “primitive” and the Trichomonadidae as more “evolved” are not in agreement with our molecular data. the emergence of Monocercomonas and Hypotrichomonas at the base of the Tritrichomonas lineage suggests a secondary loss of some cytoskeletal structures, the costa and undulating membrane in these genera. This is corroborated by the early branching position of Trichomitus. which possesses a costa and an undulating membrane and has usually been placed among the Trichomonadidae on the basis of cytological characters. A cladistic analysis was applied to the available morphological characters in order to produce a hierarchical grouping of the taxa reflecting their morphological diversity. Supplementary key words. Evolution, molecular phylogeny, morphological cladistic analysis.

Cryptophagus subtilis: a new parasite of cryptophytes affiliated with the Perkinsozoa lineage

An unknown parasitic protist is described in the free-living cryptophyte Chilomonas paramaecium. The biflagellated sporozoite has an elongated shape with an apical complex and a central nucleus. The pair of flagella are inserted at the base of the pseudo-conoid, and two microtubular roots situated underneath the plasma membrane are attached to each basal body. The apical complex comprises a set of 10-14 micronemes and a train of dumb-bell vesicles which converge toward the tip of the pseudo-conoid. Micronemes are outlined by a four-microtubule fibre and the train of vesicles is associated with a three microtubule in a rake-like fibre. The mitochondrial network has few cristae and a fairly reduced dense matrix. A large vacuole containing microfilamentous material is connected to the nuclear envelope and the Golgi body is situated close to the nucleus. The life cycle begins with the adhesion of the anterior tip, followed by the penetration of the flagellated sporozoite in a specialized region at the base of Chilomonas flagella. The trophont grows within the host cytoplasm, and after 4-5 successive nuclear mitoses, the trophozoites/sporozoites bud on the surface, while they acquire flagella, micronemes and other apical structures. The mitosis is closed with an external spindle lacking a paradesmosis. There is no sporangium, but a resting-cyst was sometimes observed inside the host. This parasite is phylogenetically affiliated to the Perkinsozoa lineage comprising Perkinsus and Parvilucifera. The new genus Cryptophagus is created for this parasite.

Identification of the ectosymbiotic bacteria of Mixotricha paradoxa involved in movement symbiosis

Mixotricha paradoxa, a trichomonad from the hindgut of the Australian termite Mastotermes darwiniensis Froggatt, is a rare example of a movement symbiosis between eukaryotic and prokaryotic microorganisms. The surface of Mixotricha paradoxa is covered with spirochaetes and a rod-shaped bacterium. The four flagella at the anterior end seem only to alter the direction of movement, while the ectosymbiotic spirochaetes propel the flagellate cells. Based on a 16S rDNA sequence analysis after a semi-specific PCR, and subsequent fluorescence in situ hybridization applying helper oligonucleotides and a denaturing step of the 16S rRNA, three different spirochaete clones could be clearly identified on the surface of the protozoal cells. They belonged to the Treponema cluster. The rod shaped bacterium showed highest 16S rDNA sequence similarity to species related to Bacteroides. Due to its low phylogenetic relationship to its nearest relatives in the database, it should represent a so far undescribed species.

Colpodella vorax: ultrastructure, predation, life-cycle, mitosis, and phylogenetic relationships

Summary The flagellar apparatus of Colpodella vorax comprises two widely separated basal bodies/flagella linked by a pluri-lamellar connector, very short basal bodies and a slightly longer transitional zone. The anterior flagellum arises from a flagellar pit: its basal body is connected to a root of three apically oriented microtubules. Two roots are connected to the basal body of the posterior flagellum: the oblique root composed of a ribbon of 6–7 microtubules is directed towards the anterior flagellar pit and the rostrum, the posterior root of two microtubules is short and directed towards the rear of the cell. The apical complex is composed of a C-ring of interlinked microtubules forming a pseudo-conoid which arises at the apex, as do the associated rhoptries and micronemes. The pellicle is composed of the endoplasmic reticulum beneath the plasma-membrane and of widely separated microtubules arising subapically and present only in the anterior part of the cell. For predation on Bodo and Spumella flagellates, C. vorax attaches itself to the prey by its anterior portion; the pseudo-conoid transforms into a ring of microtubules encircling the attachment zone. The cytoplasm of the prey is aspirated and drawn into a large posterior food vacuole. After feeding the cell encysts and divides, producing four flagellate cells; the mitosis is of the semi-open type similar to that of the apicomplexan Diplauxis hatti. These observations on C. vorax contribute to the ultrastructural definition of the genus Colpodella , which also includes C. gonderi and C. edax (= C. angusta ). The species Spiromonas perforans differs from Colpodella in its flagellar apparatus, apical complex, the presence of dinoflagellate-type trichocysts and predatory behaviour. Colpodella is also distinct from the genera Perkinsus , Parvilucifera and Cryptophagus which are bona fide alveolate protists.

Scale formation in chrysomonad flagellates.

Events of scale morphogenesis and secretion were compared at the ultrastructural level in Synura, Mallomonas, and Paraphysomonas. These three genera were chosen for several reasons: Synura generates several types of scales with a controlled shape and position along the cell body, in Mallomonas the baseplate of the scale is built independently of the spine, and in Paraphysomonas there is no plastid. In all cases the silicified scale and spine are constructed and molded in a scale-forming vesicle before being secreted. The scale-vesicles are apposed on the outer surface of the periplastic reticulum (PER) that surrounds both the nucleus and plastids in Synura and Mallomonas, or on branches of the perinuclear reticulum in Paraphysomonas. Fine structure as well as staining of glycoconjugates reveal that the membrane of the scale-vesicles and that of the reticulum are different, even though they both play a role in scale formation. Scale-vesicles come from the Golgi apparatus and, guided by microtubules, reach the synthesis zone that is restricted to the outer surface of one plastid on Synura and Mallomonas. The formation of scale ornaments, such as the spine and the hull, needs a supplement of membrane surface or a local synthesis of membrane by the appressed PER. The PER branches grow and deform the scale-forming vesicle. They contain a fibrons network that was suspected to play a role in scale molding as well as in protein transport. Certain parts of the outer surface of the scale-vesicle were covered with fibrous material arranged in various networks; some were only present in the first steps of scale formation. It is supposed that these microfibrils and thick filaments control the molding of the scale-forming vesicle and probably also the molecular organization of the membrane vesicle itself.

Ultrastructure of Trimastix convexa hollande, an amitochondriate anaerobic flagellate with a previously undescribed organization

Summary Trimastix convexa is a free-living flagellate with four flagella and found in anaerobic habitats. The light microscopical appearance resembles that of Percolomonas and Tetramitus and some retortamonads, but the flagellate is shown to have an ultrastructural identity that is distinct from that of other quadriflagellate protists such as the Heterolobosea, retortamonads, diplomonads, oxymonads and trichomonads. The cell has a ventral groove bordered on the left by a microtubular root associated with a striated fibre, and on the right by a microtubular root. There are no other major non-microtubular roots or microtubular roots except a poorly developed microtubular dorsal system. The groove contains a recurrent modified flagellum with a vane reminiscent of retortamonads. The cell contains a Golgi apparatus and has hydrogenosome-like organelles but no mitochondria. On the basis of this information, we are unable to assign this species to any of the genera in which it has been previously described, and consequently classify it as Protista incertae sedis.

Collodictyon triciliatum and Diphylleia rotans (=Aulacomonas submarina) form a new family of flagellates (Collodictyonidae) with tubular mitochondrial cristae that is phylogenetically distant from other flagellate groups.

Comparative electron microscopic studies of Collodictyon triciliatum and Diphylleia rotans (=Aulacomonas submarina) showed that they share a distinctive flagellar transitional zone and a very similar flagellar apparatus. In both species, the basic couple of basal bodies and flagella #1 and #2 are connected to the dorsal and ventral roots, respectively. Collodictyon triciliatum has two additional basal bodies and flagella, #3 and #4, situated on each side of the basic couple, each of which also bears a dorsal root. The horseshoe-shaped arrangement of dictyosomes, mitochondria with tubular cristae and the deep ventral groove are very similar to those of Diphylleia rotans. These two genera have very specific features and are placed in a new family, Collodictyonidae, distinct from other eukaryotic groups. Electron microscopic observation of mitotic telophase in Diphylleia rotans revealed two chromosomal masses, surrounded by the nuclear envelope, within the dividing parental nucleus, as in the telophase stage of the heliozoan Actinophrys and the helioflagellate Dimorpha. Spindle microtubules arise from several MTOCs outside the nucleus, and several microtubules penetrate within the dividing nucleus, via pores at the poles. This semi-open type of orthomitosis is reminiscent of that of actinophryids. The SSU rDNA sequence of Diphylleia rotans was compared with that of all the eukaryotic groups that have a slow-evolving rDNA. Diphylleia did not strongly assemble with any group and emerged in a very poorly resolved part of the eukaryotic phylogenetic tree.

Ultrastructure of Joenina pulchella Grassi, 1917 (Protista, Parabasalia), a reassessment of evolutionary trends in the parabasalids, and a new order Cristamonadida for devescovinid, calonymphid and lophomonad flagellates

Abstract The parabasalids include parasites (e.g. trichomonads) as well as many hypermastigid flagellates which live in termites and other wood-eating insects and contribute to the cellulose-digesting capacity of those animals. A hypermastigid, Joenina pulchella Grassi, is shown to have a “flagellar area” composed of 1300 flagella, including three privileged basal bodies which have homologues in the trichomonads. The cytoskeleton includes preaxostylar fibres, two parabasal fibres and two atractophores with the parabasal fibres subdividing to form many parabasals. The microtubular rows of the pelta-axostyle system surround the flagellar area and converge towards a multispiralled axostylar trunk. On the basis of similarities of ultrastructure, joeniids and devescovinids are argued to be members of the same clade. Projoenia Lavette is in the sister group to Devescovina Foa and gives rise to the series Placojoenia Radek, Joenia Grassi, Joenina Grassi. Projoenia has a “flagellar area” as in the joeniids, but also a recurrent flagellum with a paraxonemal fibre and a cresta as does Devescovina. Projoenia has a parabasal fibre twisted around the axostyle, as well as a multispiralled axostyle. In Placojoenia, Joenia and Joenina the recurrent flagellum is absent or reduced to the basal body as is the cresta; the parabasal apparatus becomes multibranched. The classical Hypermastigida is in need of major revision. Parabasalids such as Lophomonadidae, Joeniidae, Deltotrichonymphidae, and possibly Rhizonymphidae and Kofoidiidae, collectively the lophomonads, have conserved the trichomonad/devescovinid organization and have a trichomonad-like morphogenesis involving only the privileged basal bodies and attached fibres. They can be distinguished from the rest of hypermastigids and should be classified with the Devescovinidae and Calonymphidae in a large clade — the Cristamonadida (new order). The remaining hypermastigids (the Trichonymphina and Spirotrichonymphina) have a rostrum which separates in two hemi-rostra at division, form a sister group to all other parabasalids and are not closely related to the remainder of the hypermastigids.