Paul Kugrens

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.

Chloroplast iron-sulfur cluster protein maturation requires the essential cysteine desulfurase CpNifS

NifS-like proteins provide the sulfur (S) for the formation of iron-sulfur (Fe-S) clusters, an ancient and essential type of cofactor found in all three domains of life. Plants are known to contain two distinct NifS-like proteins, localized in the mitochondria (MtNifS) and the chloroplast (CpNifS). In the chloroplast, five different Fe-S cluster types are required in various proteins. These plastid Fe-S proteins are involved in a variety of biochemical pathways including photosynthetic electron transport and nitrogen and sulfur assimilation. In vitro, the chloroplastic cysteine desulfurase CpNifS can release elemental sulfur from cysteine for Fe-S cluster biogenesis in ferredoxin. However, because of the lack of a suitable mutant allele, the role of CpNifS has not been studied thus far in planta. To study the role of CpNifS in Fe-S cluster biogenesis in vivo, the gene was silenced by using an inducible RNAi (interference) approach. Plants with reduced CpNifS expression exhibited chlorosis, a disorganized chloroplast structure, and stunted growth and eventually became necrotic and died before seed set. Photosynthetic electron transport and carbon dioxide assimilation were severely impaired in the silenced plant lines. The silencing of CpNifS decreased the abundance of all chloroplastic Fe-S proteins tested, representing all five Fe-S cluster types. Mitochondrial Fe-S proteins and respiration were not affected, suggesting that mitochondrial and chloroplastic Fe-S assembly operate independently. These findings indicate that CpNifS is necessary for the maturation of all plastidic Fe-S proteins and, thus, essential for plant growth.

AN ULTRASTRUCTURAL SURVEY OF CRYPTOMONAD PERIPLASTS USING QUICK-FREEZING FREEZE FRACTURE TECHNIQUES

A quick‐freezing technique for freeze fracturing was used to determine periplast plate types in 20 cryptomonads. With this technique cells are frozen so rapidly that major artifacts are eliminated. We propose that periplast plates are attached to the cell membrane by intramembrane particles (IMPs), consequently plate shapes are outlined by IMP distribution in fractured membranes. Round to oval, sometimes slightly angular, plates occur in Cryptomonas ovata, Cryptomonas tetrapyrenoidosa, Cryptomonas parapyrenoidifera, Cryptomonas obovata, Cryptomonas erosa and two unidentified species of Cryptomonas; large rectangular plates occur in Chroomonas pochmannii, Chroomonas coerulea and Hemiselmis sp.; small rectangular plates were found in Cryptomonas sp. (Strain SDB); square to slightly rounded plates occur in Cryptomonas chrysoidea and a single continuous plate or sheet, perforated by ejectisome pores, was observed in Cryptomonas caudata, Cryptomonas rostratiformis, Cryptomonas marssonii, Cryptomonas platyuris, Cryptomonas curvata, Cryptomonas ozolini, Chilomonas paramecium and Rhodomonas sp. Oval and square plates are described for the first time in Cryptomonas. Plate IMPs may be morphologically modified in size and shape, depending upon their location in relation to the plate, the plate ridges, and ejectisome chambers. Conformational changes in plate shapes, to form hexagons or polygons, may be induced when cells are subjected to fixation, desiccation, cryoprotectants or centrifugation.

ULTRASTRUCTURE OF TETRASPOROGENESIS IN THE PARASITIC RED ALGA LEVRINGIELLA GARDNERI (SETCHELL) KYLIN12

Ultrastructural studies on tetraspore formation in Levringiella gardneri revealed that 3 stages may be recognized during their formation. The youngest stage consists of a uninucleate tetraspore mother cell with synaptonemal complexes present during early prophase of meiosis I. Mitochondria are aggregated around the nucleus, dictyosome activity is low, and chloroplasts occur in the peripheral cytoplasm. A 4‐nucleate tetraspore mother cell is formed prior to tetrahedral cell cleavage, and an increase in the number of chloroplasts and mitochondria occurs. Small straight‐profiled dictyosomes secrete vesicles into larger fibrous vesicles or contribute material to the developing tetraspore wall. During the second stage of tetraspore formation, striated vesicles form within endoplasmic reticulum, semicircular profiled dictyosomes secrete vesicles for fibrous vesicles or wall material, and starch formation increases. The final stage is characterized by the disappearance of striated vesicles, presence of straight, large dictyosomes which secrete cored vesicles, and an abundance of starch grains. Cleavage is usually complete at this stage and the tetraspore wall consists of a narrow outer layer of fibrillar material and an inner, electron transparent layer. These spores are surrounded by a tetrasporangial wall which was the original wall surrounding the tetraspore mother cell.

Cell form and surface patterns in Chroomonas and Cryptomonas cells (Cryptophyta) as revealed by scanning electron microscopy

Fifteen freshwater cryptomonad species were freeze‐dried and examined with the scanning electron microscope. Surveys of cell surfaces revealed four general cell types. Chroomonas type cells lack a furrow but possess a shallow vestibular depression where the flagella are inserted. The presence of a gullet could not be detected. Cryptomonas spp. displayed three morphological types, all lacking gullets. The first type of Cryptomonas has a simple, shallow furrow with ridges that apparently can close to form a raphe but an oval opening or stoma remains at the posterior end and an opening from the vestibulum is formed at the anterior end. The second Cryptomonas type consists of a complex furrow with furrow ridges and folds that extend almost two‐thirds of the cell length. A sloma is present in the central region of the closed furrow. The folds apparently can separate thereby exposing the underlying furrow. The third type of Cryptomonas possesses a simple, non‐closing furrow. At the anterior end there is a vestibular ligule which extends from the dorsalleft side of the cell and covers the region of the vestibulum where the contractile vacuole discharges.

THE ULTRASTRUCTURE OF CARPOSPOROGENESIS IN THE MARINE HEMIPARASITIC RED ALGA ERYTHROCYSTIS SACCATA1

The ultrastructure of carposporogenesis for Erythrocystis saccata is described. The fusion and gonimoblast cells contain few organelles, and chloroplasts are in a proplastid state, with pit plugs between gonimoblast cells dissolving early in development. Carpospore development may be separated into 3 stages, the first stage being characterized by the appearance of straight‐profiled dictyosomes, fibrous vesicles, and an increase of discoid thylakoids within the chloroplasts. During the second, stage the dictyosomes assume a curved profile and striped vesicles are formed by the endoplasmic reticulum. The third stage is initiated by the disappearance of striped vesicles and the appearance of straight‐profiled dictyosomes secreting vesicles with cores. Mature carpospores consist of many cored vesicles, fibrous vesicles, and floridean starch grains. A single wall layer surrounds each carpospore since the carposporangial wall becomes incorporated into a mucilaginous matrix surrounding the spores.

Systematics of the Enigmatic Kathablepharids, Including EM Characterization of the Type Species, Kathablepharis phoenikoston, and New Observations on K. remigera comb. nov.

The colorless flagellate Kathablepharis has consisted of five species based on light microscopic studies, and the ultrastructure of the type species, Kathablepharis phoenikoston, is described for the first time. The heterotrophic, marine flagellate Leucocryptos consisted of two species, but additional ultrastructural details for one of these, Kathablepharis remigera comb. nov. (= Leucocryptos remigera Vørs), indicates that it should be transferred to Kathablepharis. The cellular structure of these two species is similar to previously studied kathablepharids. However, there is variation in the feeding apparatus and cytoskeleton. The feeding apparatus of both species has a cytostome, a cytostomal ring, and cytopharyngeal rings. The cytoskeleton consists of inner microtubular arrays and outer or sub-pellicular microtubular arrays. In addition, several features of the flagellar apparatus are described for K. phoenikoston and K. remigera. The ultrastructure of these two species is compared with other kathablepharids to evaluate their taxonomy and phylogeny. We classify Kathablepharis and Leucocryptos in the family Kathablepharididae incertae sedis.

KATABLEPHARIS OVALIS, A COLORLESS FLAGELLATE WITH INTERESTING CYTOLOGICAL CHARACTERISTICS1

Katablepharis ovalis Skuja, isolated from an impoundment in Colorado, has a cell covering composed of two layers over the cell body and flagella. The outer component of the cell covering contains 25‐nm‐diameter hexagonal scales arranged in rows. The inner component of the cell covering is composed of a layer of interwoven microfibrils. The inner component of the cell covering is joined to the plasma membrane by one or more attachment strips that always occur outside, and along, one of the microtubular groups of the outer array. The attachment strips resemble hemidesmosomes and are composed of rows of electron‐dense material, 12 nm apart, that protrude through the plasma membrane into the extracellular space, to attach to the inner wall. The two flagella are inserted subapically into a raised area of the cell. The flagella do not have any fibrillar or tubular hairs and are covered only by the two‐layered cell covering. The cell has an inner and outer array of microtubules, both of which are spindle‐shaped, arising at the anterior end of the cell and continuing into the posterior end of the cell. A single large Golgi apparatus occurs in the anterior cytoplasm. The nucleus is in the center of the cell. Two rows of large ejectisomes occur posterior to the area of flagellar attachment. Smaller ejectisomes occur under the plasma membrane in the posterior and medial areas of the cell. Each ejectisome is composed of a single body containing a spirally wound, tapered ribbon. On discharge, the ejectisome ribbon rolls inward, creating a tubular structure. The possible relationship between Katablepharis, the green algae, and the cryptophytes is discussed.

ULTRASTRUCTURE OF SPERMATIAL DEVELOPMENT IN THE PARASITIC RED ALGAE LEVRINGIELLA GARDNERI AND ERYTHROCYSTIS SACCATA12

Morphologically, the development of spermatia in Levringiella gardneri and Erythrocystis saccata is identical, although cytologically several differences are evident. Mature spermatia contain 1 or 2 large spermatial vesicles that contain fibrous material, several small mitochondria, some proplastids, and are surrounded by a wall, either single‐layered as in Erythrocystis or triple‐layered as in Levringiella. Spermatial vesicles are formed by aggregations of endoplasmic reticulum in Levringiella, whereas concentric membrane bodies and dictyosomes may be involved in Erythrocystis. In addition to being fibrillar, the contents of the vesicle assume a convoluted appearance in Levringiella. Several spermatia are formed per mother cell and are connected by small pit connections which rupture to allow spermatial release from the spermatangial branch.

Origin and Evolution of Kinesin-Like Calmodulin-Binding Protein

Kinesin-like calmodulin-binding protein (KCBP), a member of the Kinesin-14 family, is a C-terminal microtubule motor with three unique domains including a myosin tail homology region 4 (MyTH4), a talin-like domain, and a calmodulin-binding domain (CBD). The MyTH4 and talin-like domains (found in some myosins) are not found in other reported kinesins. A calmodulin-binding kinesin called kinesin-C (SpKinC) isolated from sea urchin (Strongylocentrotus purpuratus) is the only reported kinesin with a CBD. Analysis of the completed genomes of Homo sapiens, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and a red alga (Cyanidioschyzon merolae 10D) did not reveal the presence of a KCBP. This prompted us to look at the origin of KCBP and its relationship to SpKinC. To address this, we isolated KCBP from a gymnosperm, Picea abies, and a green alga, Stichococcus bacillaris. In addition, database searches resulted in identification of KCBP in another green alga, Chlamydomonas reinhardtii, and several flowering plants. Gene tree analysis revealed that the motor domain of KCBPs belongs to a clade within the Kinesin-14 (C-terminal motors) family. Only land plants and green algae have a kinesin with the MyTH4 and talin-like domains of KCBP. Further, our analysis indicates that KCBP is highly conserved in green algae and land plants. SpKinC from sea urchin, which has the motor domain similar to KCBP and contains a CBD, lacks the MyTH4 and talin-like regions. Our analysis indicates that the KCBPs, SpKinC, and a subset of the kinesin-like proteins are all more closely related to one another than they are to any other kinesins, but that either KCBP gained the MyTH4 and talin-like domains or SpKinC lost them.