Pyramimonas tychotreta, sp. nov. (prasinophyceae), a new marine species from antarctica: light and electron microscopy of the motile stage and notes on growth rates

An undescribed marine prasinophyte, Pyramimonas tychotreta, sp. nov., was isolated from a water sample collected near the ice edge in the Weddell Sea (Antarctica) and is characterized by means of light and electron microscopy. This is the second described Antarctic species in the genus and it possesses a cell ultrastructure typical for members of the subgenus Vestigifera McFadden. The quadriflagellated cells measure 8–12 μm in length and 6–7 μm in width and are equipped with seven types of organic scales that cover the flagella and cell body. The scale floor of the box scales is ornamented by quadrants of parallel striations running perpendicular to one another. The scale floor is further characterized by a number of randomly positioned perforations. The wall of the box scales may be solid or possess up to five perforations. The base of the crown scales is square with rounded corners. It is formed of two crossed ribs, the extremities of which are interconnected by a peripheral rib. Four upright arms, attached to the peripheral rib in positions slightly offset from its junction with the cross ribs, join up with the distal extremity of a central upright strut. Each arm possesses two spines. The limuloid scales are cross‐striped by 10–12 ribs. Some details of the flagellar apparatus are briefly reported. Pyramimonas tychotreta is compared with other species of the genus. Experiments were conducted to study the response of growth rate to variations in temperature and salinity in the clonal culture. The best growth rate (0.45 divisions·24 h ^{? 1}) was found at 4.6° C; growth ceased at temperatures in excess of 12° C. Growth in salinities ranging from 15 to 35 psu was similar, but was arrested at 10 psu. These studies suggest that P. tychotreta as a cold stenotherm and euryhaline taxon. New observations are presented on the geographic distribution of previously described species of Pyramimonas Schmarda from the Northern Foxe Basin, Canada.     

LIGHT AND ELECTRON MICROSCOPY OF A PUNCTATE SPECIES OF PYRAMIMONAS,P. MUCIFERA SP. NOV. (PRASINOPHYCEAE)1

Pyramimonas mucifera sp. nov., a punctate species of the genus, is unusual both behaviorally and at the fine structural level. It forms two distinct populations in culture, one benthic and one planktonic. Planktonic forms are more conventional for the genus, but benthic forms are found in loosely packed mucilage, have flagellar rather than ciliary beating of the flagella, and display a higher degree of metaboly. Ultrastructurally this species is unusual in that it has a unique scale complement and the cells contain numerous muciferous vesicles, leaving only small pockets of cytoplasm containing the usual organelles. This species has a 3–1 type flagellar apparatus but has an additional fibrillar band, a 4–3-2–3 microtubular root system and a flexible synistosome. The discovery of a mucilage-producing species of Pyramimonas draws attention to possible links with other prasinophytes (Halosphaera) and green algae of questionable affiliation (Oltmannsiellopsis and Hafniomonas). It also provides a model of the primitive pyramimonad.     

Diversity of swimming behavior in Pyramimonas (Prasinophyceae)

High‐speed videography and inverted light microscope observations of cultures of several Pyramimonas Schmarda species (Pyramimonas chlorina Sym et Pienaar, Pyramimonas disomata McFadden Hill et Wetherbee, Pyramimonas gelidicola McFadden Wether‐bee et Moestrup, Pyramimonas mantoniae Moestrup et Hill, Pyramimonas melkonianii Sym et Pienaar, Pyramimonas mitra Moestrup et Hill, Pyramimonas moestrupii McFadden, Pyramimonas mucifera Sym et Pienaar, Pyramimonas nephroidea McFadden, Pyramimonas orientalis McFadden Hill et Wetherbee, Pyramimonas parkeae Norris et Pearson, Pyramimonas propulsa Moestrup et Hill) revealed swimming behavior to be much more diverse than originally reported for the genus. This diversity shows gradation between extremes of recurved flagellar beating and non‐biphasic ciliary beating. Comparison with the behavior of presumably more primitive members of the Pyramimonadales (Cymbomonas tetramitformis Schiller and Pterosperma cristatum Schiller) leads to the conclusion that the former condition is primitive and the gradation may assist in reconstructing phylogenetic relationships within the genus Pyramimonas, particularly as it is consistent with phylogenies derived from ultrastructural and molecular data.     

FLAGELLAR HAIRS IN PRASINOPHYTES (CHLOROPHYTA): ULTRASTRUCTURE AND DISTRIBUTION ON THE FLAGELLAR SURFACE1

The flagellar hair ultrastructure of 16 strains of species of the prasinophycean genera Mantoniella, Mamiella, Pseudoscourfieldia, Nephroselmis, Tetraselmis, Scherffelia, Pterosperma, and Pyraminonas was examined in detail by whole-mount electron microscopy. The flagellar hairs of all genera displayed a high degree of ultrastructural complexity that was completely conserved within each strain. In all strains, flagellar hairs occurred on the sides of the flagella (lateral hairs); in several strains, special flagellar hairs also were found on the flagellar tips (tip hairs; absent in the Chlorodendrales and in Nephroselmis). Two groups of lateral hairs were distinguished: 1) T-hairs (“Tetraselmis-type” flagellar hairs), characterized by a smooth, tubular shaft of ca. 15 nm diameter and an overall length of 0.5–1.3 μm, and 2) P_t-hairs (“Pterosperma-type lateral flagellar hairs”), which were considerably longer (ca. 1.5–5.4 μm), characterized by a thick shaft of ca. 30 nm diameter, which was covered with a layer of regularly spaced small particles of ca. 10 nm diameter. In both groups of flagellar hairs, a strain-specific number of subunits (1–101) in linear arrangement was attached to the distal end of the shaft. Tip hairs were either structurally related to T-hairs (Mamiellales, Pseudoscourfieldia) or represented a separate group, P_t-hairs (“Pterosperma-type flagellar tip hairs”; Pterosperma, Pyramimonas). In four genera (Mantoniella, Mamiella, Pseudoscourfieldia, Nephroselmis), both groups of lateral hairs occurred together on the same cell. Interestingly in these taxa the P_t-hairs were exclusively attached to the shorter immature flagella (no. 2), but, in contrast, in Mantoniella and Pseudoscourfieldia the tip hairs were restricted to the longer mature flagellum (no. 1). Thus, flagella of different developmental status differ in their hair-scale complement. The occurrence, distribution, and ultrastructure of flagellar hairs can be used to identify and classify prasinophytes at all taxonomic levels.     

The external morphology of the type species of Pyramimonas (P. tetrarhynchus Schmarda) by electron microscopy

Pyramimonas tetrarhynchus has been grown in culture and its external features examined using direct preparations and sections. The flagellar and body scales are described and compared with those of previously investigated species.     

Observations on the microanatomy of the type species of Pyramimonas (P. tetrarhynchus Schmarda)

Salient features of microanatomy of Pyramimonas tetrarhynchus , type species of the genus, are described and illustrated. The species is shown to possess a flagellar scale reservoir comparable to that previously described for a marine species (P. amylifera) though somewhat simpler. The body scales are shown to arise directly from Golgi eisternae as in P. amylifera. Other features discussed include the flagellar roots and certain aspects of the mode of liberation of scales to the exterior. Participation by endoplasmic reticulum in the latter process is shown to be probable but there is no precise explanation forthcoming for the elaborate scale arrangement encountered both on the flagella and on the cell. Participation by cytoplasmic tubules is nevertheless effectively excluded for the latter.     

Light and electron microscopical studies onHafniomonas gen. nov. (Chlorophyceae,Volvocales), a genus resemblingPyramimonas (Prasinophyceae)

Based on light and electron microscopical studies ofPyramimonas reticulata the genusPyramimonas is shown to contain a number of unrelated flagellates.P. reticulata andP. montana are transferred to the new genusHafniomonas, cells of which differ fromPyramimonas in shape, in the absence of scales and hairs on the body and flagellar surfaces, in details of the chloroplast, the position of the nucleus, the Golgi apparatus, the internal structure of the flagellar apparatus, and in cell division. The prasinophytePyramimonas contains a characteristic association of a large microbody and a rhizoplast, situated on the nuclear surface. A similar association is being found in an increasing number of prasinophycean flagellates, but is absent inHafniomonas, which is considered related to chlorophycean rather than prasinophycean flagellates. The phylogenetic position ofHafniomonas is discussed, based in particular on details of the unique flagellar apparatus.     

The fine structure of Synura sphagnicola (Korsh.) Korsh. (Chrysophyceae)

S. sphagnicola resembles other species of Synura previously described by electron microscopy in most features of structure but differs in possessing pyrenoids and up to five cylindrical stacks of smooth cisternae which occur between the pyrenoids and leucosin vesicles. Each stack is surrounded by a tubular cisterna which bears ribosomes on its distal face but there are no clear permanent connections between this and the chloroplast ER. Other features apparently unique to this species previously known from light microscopy are described. These include the axial position of the chloroplasts; the peripheral position of the leucosin vesicles; and the loose attachment of the scales. The structure of the body scales is described for the first time from sections. The flagellar scales are formed in the swollen edges of the Golgi cisternae and appear to pass to the cell surface in large vesicles.     

The flagellar apparatus of the scaly green flagellatePyramimonas obovata: Absolute configuration

Summary The flagellar apparatus of the quadriflagellate scaly green algaPyramimonas obovata has been studied in detail and the absolute configuration of the flagellar apparatus has been determined. The flagellar root system is cruciate (4-2-4-2-system). 18 major basal body associated fibrous structures connect the four basal bodies with each other. Each basal body is linked to an adjacent basal body by a unique set of connecting fibres, i.e., the flagellar apparatus does not exhibit 180° rotational symmetry. The flagellar apparatus ofPyramimonas obovata is compared with that of quadriflagellate motile cells of theChlorophyceae sensu Stewart andMattox and the phylogenetic relationships are discussed.     

Life history stages of Pyramimonas tychotreta (Prasinophyceae, Chlorophyta), a marine flagellate from the Ross Sea, Antarctica

During a summer cruise to the Ross Sea (Antarctica) areas of snow‐covered sea ice were red‐coloured due to high concentrations of the recently described Pyramimonas tychotreta Daugbjerg. Light microscopy of living material revealed that the population was comprised of quadriflagellate motile cells and thick‐walled cysts. The red colour was due to large numbers of secondary carotenoid‐containing granules, positioned in the periphery of motile cells and cysts. Mature cysts also contained numerous starch grains and lipid droplets. Cells from a red‐coloured field sample turned green overnight as the secondary carotenoids disappeared when cells were placed in low light conditions. The sample then exhibited the typical grass‐green colour of motile cells observed in water samples from the area. Under reduced light motile cells showed strong positive phototaxis. The encystment process involved the asexual transformation of quadriflagellate cells into cysts. A single type of square cyst scale, with perforated floors and walls, replaced the body scales of motile cells. A marked extension, often ending in a hook was at each corner of the cyst scales. Germinating cysts produced four motile cells. Electron microscopy showed the cyst wall to be tri‐layered, with a thin, electron‐dense inner layer, a thick middle layer and a thin outer layer. Sea ice samples with dense populations of motile cells and cyst stages also contained elongate uniflagel‐late cells. These cells were covered with box scales, foot‐print scales, an underlayer of pentagonal scales, limuloid scales and flagellar hair scales identical to those present on the quadriflagellate stage. We tentatively suggest that the uniflagellate stage represents a gamete and its presence implies the occurrence of sexual reproduction. Although, fusion of gametes was not observed, a biflagellate cell with a larger volume was seen which may have been a zygote. How this stage fits into of the life history remains to be explained.     

Taxonomy of Pyramimonas obovata and other observations on the subgenus Vestigifera of Pyramimonas (Prasinophyceae, Chlorophyta)

A vestigiferan species commonly referred to as Pyramimonas obovata N. Carter has been redescribed as P. melkonianii sp, nov. Characters of this species and a further six (P. disomata Butcher ex McFadden, Hill et Wetherbee, P. mantoniae Moestrup et Hill, P. mitra Moestrup et Hill. P. moestrupii McFadden, P. aff. nephroidea McFadden, P. orientalis Butcher ex McFadden, Hill et Wetherbee) isolated from South African waters are used to define further the subgenus Vestigifera McFadden. This includes a unique chloroplast shape and basal hyaline region with stellate or cruciform vacuoles, a transitional plate-like structure in the flagellum, and a different microtubular root system. The proximal set of basal body connectives were found to be remarkably symmetrical and like those of the subgenus Trichocystis McFadden, and a duct fibre was found associated with the Id root in all currently investigated species. The validity of the larger body (box and crown) scales as taxonomic markers at a fine level is also questioned.     

PHYLOGENY OF THE GENUS PYRAMIMONAS (PRASINOPHYCEAE,CHLOROPHYTA) INFERRED FROM THE rbcL GENE1

A 1089-basepair fragment (approx. 75%) of the large subunit of the chloroplast-encoded gene, ribulose-1,5-bis-phosphate carboxylase/oxygenase (rbcL), was sequenced from 16 species of the genus Pyramimonas Schmarda. Electron microscopic and biochemical studies of Pyramimonas, one of the most morphologically diverse genera within the potential sister groups to the chlorophyll a- and b-containing plants, suggest that this genus consists of at least four separate subgenera. Using the homologous sequence of rbcL from Cymbomonas tetramitiformis Schiller (Halosphaeraceae) as an outgroup and applying the maximum likelihood method, we show that the inferred topology is congruent with traditional delimitations of the taxa based on observations of periplast, internal ultrastructure, and biochemical features. A bootstrap analysis also supports division at the subgeneric level; however, the low bootstrap support associated with the deep nodes precludes resolution of these branches. A maximum likelihood relative rate test revealed that the rbcL gene in these single-celled green flagellates has a heterogeneous rate of substitution. The rbcL gene in species of the subgenus Pyramimonas has evolved at an accelerated rate relative to that of congenerics.     

ULTRASTRUCTURE OF CHRYSODIDYMUS SYNUROIDEUS (SYNUROPHYCEAE)1

A clonal isolate of Chrysodidymus synuroideus Prowse was derived form a Sphagnum bog in northern Wisconsin and maintained in culture for over 3 years. Cultured colonies consisted almost exclusively of two cells attached at the posterior, each cell bearing two unequal flagella. Correlative light and electron microscopic observations revealed that colonies composed of smaller, ovate cells represented more recent products of cell division, while colonies of elongate cells were more mature. These results support previous taxonomic conclusions, based on light microscopic observations, of field-collected specimens and body scale ultrastructure, that Chrysodidymus is a valid genus, and that two species described by Prowse on the basis of cell size differences, should be merged. In addition, ultrastructural studies of cultured Chrysodidymus demonstrated that this genus is a member of the Synurophyceae on the basis of characters related to flagellar morphology, basal body arrangement, and cytoskeletal ultrastructure. Chrysodidymus synuroideus resembles Synura sphagnicola in body scale structure, the presence of distinctive linear or clavate scales on both flagella, a relatively loose scale case, and acidophilic habital, Unlike S. sphagnicola, Chrysodidymus has no Pyrennoids, Peripheral (rather than axial) plastids, and a single posterior storage vesicle (rather than two peripheral storage vesicles).     

FINE STRUCTURE OF CELL DIVISION IN PYRAMIMONAS PARKEAE NORRIS AND PEARSON (CHLOROPHYTA,PRASINOPHYCEAE)1

Cell division in Pyramimonas parkeae is described and compared with some other green algae. The first indication of mitosis is division of the chloroplast, accompanied by growth of a prominent microbody, followed by replication of the 4 basal bodies. Also closely timed with this is the replication of the Golgi and other components of the basal body complex. Two basal body complexes separate, each taking a position at either pole of the nucleus which has migrated to a characteristic position just beneath the plasmalemma of a broadened and flattened flagellar pit. Cytokinesis is accomplished by the fusion of ducts and vesicles with the simultaneous release of scales to the newly formed exterior. Cells swim throughout division.     

Fine structure of Pyramimonas octopus sp. nov., an octoflagellated benthic species of Pyramimonas (Prasinophyceae), with some observations on its ecology

A new octoflagellated species of Pyramimonas is described from three localities in Denmark. It is characterized by its ecology, being a marine psammophilic species associated with sand grains, and by details of the scaly covering on the cell surface. In the scale cover there are similarities to P. amylifera and to P. tetrarhynchus. P. octopus is distinguished by the possession of circular body scales of a type not previously found in the genus.
The general fine structure of the new species is described with emphasis on scale structure, the internal structure of the cell body and the flagella.     

Heterocapsa psammophila sp. nov. (Peridiniales, Dinophyceae), a new sand-dwelling marine dinoflagellate

A new armored dinoflagellate species, Heterocapsa psammophila Tamura, Iwataki et Horiguchi sp. nov. is described from Kenmin‐no‐hama beach, Hiroshima, Japan using light and electron microscopy. This dinoflagellate possesses the typical thecal plate arrangement of the genus Heterocapsa, Po, cp, 5′, 3a, 7′′, 6c, 5s, 5′′′, 2′′′′; and the 3‐D body scales of Heterocapsa on the plasma membrane. The cell shape is ovoidal. The spherical nucleus and the pyrenoid are situated in the hypotheca and the epitheca, respectively. The ultrastructure of H. psammophila is typical of dinoflagellates and the pyrenoid is invaginated by cytoplasmic tubules. H. psammophila is distinguished from all other hitherto‐described Heterocapsa species by the cell shape, the relative position of the nucleus and pyrenoid and the structure of the body scale. The habitat and behavior of this new species in culture suggest that the organism is truly a sand‐dwelling species.     

COMPARATIVE ANALYSIS OF THE DINOFLAGELLATE FLAGELLAR APPARATUS IV. GYMNODINIUM ACIDOTUM1

Gymnodinium acidotum Nygaard is a freshwater dinoflagellate that is known to harbor a cryptomonad endosymbiont whose chloroplasls give the organism an overall blue-green color. The ultrastructure of G. acidotum was examined with particular attention being given to the three dimensional nature of the flagellar apparatus. The fiagellar apparatus is composed of two functional basal bodies that are slightly offset and lie at an angle of approximately 90° to one another. As in other dinoflagellates the transverse basal body is associated with a striated, fibrous root that extends from the proximal end of the basal body to the transverse flagellar opening. At least one microtubular root extends from the proximal end of the transverse basal body, and a multi-membered longitudinal microtubular root is associated with the longitudinal basal body. The most striking feature of the flagellar apparatus of G. acidotum is the large fibrous connective that extends from the region of the proximal ends of the basal bodies to the cingulum on the dorsal side of the cell. A similar structure has been reported from only one other dinoflagellate, Amphidinium cryophilum Wedemayer, Wilcox, and Graham. The presence of this structure as well as similarities in external morphology suggest thai these two species may be more closely related to each other than either is to other gymnodinioid taxa. The taxonomic importance of dinoflagellate flagellar apparatus components is discussed.     

AN ADDITIONAL PUNCTATE SPECIES OF PYRAMIMONAS, P. FORMOSA, SP. NOV., AND ITS IMPACT ON THE SUBGENERA PUNCTATAE AND PYRAMIMONAS (PRASINOPHYCEAE, CHLOROPHYTA)

A new punctate species of Pyramimonas Schmarda from South Africa is described. This species is somewhat anomalous and, like P. chlorina Sym et Pienaar (subgenus Pyramimonas McFadden), has characters that bridge the divide between the subgenera Pyramimonas and Punctatae McFadden. Features of these two species, together with the lack of exclusive character sets for either subgenus derived from other species of these subgenera, lead to the conclusion that Punctatae now should be subsumed formally into the subgenus Pyramimonas.     

THE FINE STRUCTURE OF THE FLAGELLAR APPARATUS OF CARTERIA1,2

The fine structure of the flagellar apparatus of 5 species of the green quadriflagellate alga Carteria is described. The 5 species can be morphologically separated into 2 groups on the bases of cell shape and ultrastructure of the pyrenoid and flagellar apparatus. Group I cells are spherical, possess many pyrenoid thylakoids, and retain a flagellar apparatus similar to that of Chlamydomonas reinhardi. The flagellar bases are oriented at approximately 90° to one another, have distal and proximal fibers, and are associated with 4 cruciately arranged microtubule bands. Cells of group II are ellipsoid, possess few pyrenoid thylakoids, and show a complex system of microtubule bands and sigmoid-shaped, electron dense rods which extend between opposite pairs of basal bodies. The basal bodies of group II cells are directed inward in a circular pattern rather than outward as in group I cells. Unlike Chlamydomonas, the distal fiber of the Carteria species is nonstriated. The proximal fiber is striated, and both distal and proximal fibers are composed of 60–80 Å diameter microfibrils.