MORPHOLOGY AND MOLECULAR ANALYSES OF THREE TOXIC SPECIES OF GAMBIERDISCUS (DINOPHYCEAE): G. PACIFICUS, SP. NOV., G. AUSTRALES, SP. NOV., AND G. POLYNESIENSIS, SP. NOV.

Three new dinoflagellate species, Gambierdiscus polynesiensis, sp. nov., Gambierdiscus australes, sp. nov., and Gambierdiscus pacificus, sp. nov., are described from scanning electron micrographs. The morphology of the three new Gambierdiscus species is compared with the type species Gambierdiscus toxicus Adachi et Fukuyo 1979, and two other species: Gambierdiscus belizeanus Faust 1995 and Gambierdiscus yasumotoi Holmes 1998. The plate formula is: Po, 3′, 7", 6C, 8S, 5‴, 1p, 2". Culture extracts of these three new species displayed both ciguatoxin- and maitotoxin-like toxicities. The following morphological characteristics differentiated each species. 1) Cells of G. polynesiensis are 68–85 μm long and 64–75 μm wide, and the cell’s surface is smooth. They are identified by a large triangular apical pore plate (Po), a narrow fish-hook opening surrounded by 38 round pores, and a large, broad posterior intercalary plate (1p) wedged between narrow postcingular plates 2‴ and 4‴. Plate 1p occupies 60% of the width of the hypotheca. 2) Cells of G. australes also have a smooth surface and are 76–93 μm long and 65–85 μm wide in dorsoventral depth. They are identified by the broad ellipsoid apical pore plate (Po) surrounded by 31 round pores and a long and narrow 1p plate wedged between postcingular plates 2‴ and 4‴. Plate 1p occupies 30% of the width of the hypotheca. 3) Cells of G. pacificus are 67–77 μm long and 60–76 μm wide in dorsoventral depth, and its surface is smooth. They are identified by the four-sided apical pore plate (Po) surrounded by 30 round pores. A short narrow 1p plate is wedged between the wide postcingular plates 2‴ and 4‴. Plate 1p occupies 20% of the width of the hypotheca. These three newly described species were also characterized by isozyme electrophoresis and DNA sequencing of the D8–D10 region of their large subunit (LSU) rRNA genes. The consistency between species designations based on SEM microscopy and classification inferred from biochemical and genetic heterogeneities was examined among seven isolates of Gambierdiscus. Their classification into four morphospecies was not consistent with groupings inferred from isozyme patterns. Three molecular types could be distinguished based on the comparison of their LSU rDNA sequences. Although G. toxicus TUR was found to be more closely related to G. pacificus, sp. nov. than to other G. toxicus strains, the molecular classification was able to discriminate G. polynesiensis, sp. nov. and G. australes, sp. nov. from G. toxicus. These results suggest the usefulness of the D8–D10 portion of the Gambierdiscus LSU rDNA as a valuable taxonomic marker.     

PROROCENTRUM BELIZEANUM,PROROCENTRUM ELEGANS,AND PROROCENTRUM CARIBBAEUM,THREE NEW BENTHIC SPECIES (DINOPHYCEAE) FROM A MANGROVE ISLAND,TWIN CAYS,BELIZE1

Three new benthic dinoflagellate species, Prorocentrum belizeanum, Prorocentrum elegans, and Prorocentrum caribbaeum, from mangrove floating detritus are described from scanning electron micrographs. Species were identified based on shape, size, surface micromorphology, ornamentation of thecal plates, and architecture of the periflagellar area and intercalary band. Cells of P. belizeanum are round to slightly oval with a cell size of 55–60 μm long and 50–55 μm wide. Areolae are round and numerous (853–1024 per valve) and range from 0.66 to 0.83 μm in size. The periflagellar area of P. belizeanum is a broad V-shaped depression; it accommodates a flagellar and an auxiliary pore and a flared, curved apical collar. The intercalary band of P. belizeanum is horizontally striated. Prorocentrum elegans is a small species 15–20 μm long and 10–14 μm wide, with an ovate cell shape. The thecal surface is smooth. Two sizes of valve pores were recognized: large, round pores (20–22 per valve) arranged in a distinct pattern and smaller pores situated in an array along the intercalary band. The periflagellar area is V-shaped; it accommodates an uneven sized flagellar pore, an auxiliary pore, and an angled protuberant flagellar plate. The intercalary band is transversely striated. It is a bloom-forming species. Prorocentrum caribbaeum cells are heart-shaped with a rounded anterior end and a pointed posterior end. Cells range from 40 to 45 μm long and 30 to 35 μm wide. Thecal surface has two different-sized pores: large, round pores (145–203 per valve) arranged perpendicularly from the posterior margins, and small, round pores unevenly distributed on the thecal surface. The periflagellar area is ornate. It is V-shaped with a curved apical collar located next to the auxiliary pore; a smaller protuberant apical plate is adjacent to the flagellar pore. The intercalary band is transversely striated and sinuous. Cells are active swimmers.     

MORPHOLOGY AND LIFE CYCLE EVENTS IN PYROPHACUS STEINII (SCHILLER) WALL ET DALE (DINOPHYCEAE)

Cells of Pyrophacus steinii (Schiller) Wall et Dale are round and lens shaped and have an anteroposteriorly compressed theca. The epitheca has a truncated, conical horn and a hexagonally shaped apical pore plate with two arched slits positioned off center. The cingulum is equatorial, narrow, and deep. The hypotheca is flat. The sulcus is narrow, slightly curved, and recessed and does not reach the cell's antapex. The plate formula in these specimens of P. steinii is Po, 8', Oa, 13", 13C, 12"', 3p, 3"", and 8S with a difference in the number of precingular (13") and postcingular (12"') plates. No additional posterior intercalary plates were present (Oap). Pregametic stages of P. steinii were observed during cell division via binary fission, with formation of two cells and multiple division with formation of four and eight cells. These newly formed cells were pale in color and were enclosed in double-layered hyaline membrane. Gametes with gymnodinoid morphology were observed within the parental cells. Planozygotes are large and round and enclosed in double-layered hyaline membrane. Mature cell forms are brown with a microgranular cytoplasm, storage bodies, and a red accumulation body. The hypnozygote exhibits triple-layered hyaline membrane, irregularly shaped and comparable with bulbous processes of Tuberculodinium vancampoae Rossigol resting cysts. Division within a hypnocyst of P. steinii involves shedding the parental theca and the development and emergence of two daughter cells with the size and morphology of pregametic cells.     

A FURTHER SEM STUDY OF MARINE BENTHIC DINOFLAGELLATES FROM A MANGROVE ISLAND,TWIN CAYS,BELIZE, INCLUDING PLAGIODINIUM BELIZEANUM GEN. ET SP. NOV.1

This study indicates that bilaterally flattened, armored, benthic dinoflagellates are more diverse in morphology than previously known. A new species, Plagiodinium belizeanum Faust et Balech gen. et. sp. nov., is described in floating detritus from Twin Cays, Belize, mangrove habitats. Plagiodinium belizeanum cells are small, with dimensions of 26.5–30.5 μm in length, 20–24.5 μm in width, and 6.5–8.5 μm in depth. Cells are oblong and bilaterally compressed with a posteriorly located, spherical nucleus, many chloroplasts, and spherical starch granules. The epitheca descends ventrally, is cap-shaped, and is composed of five plates and a very small platelet provisionally named P₀ situated in the center. The epitheca is narrowly oval in apical view with a pointed truncated ventral side and a rounded dorsal side. The cingulum is composed of five plates. The hypotheca is constructed of five posteriorly elongated postcingular plates and one antapical plate. The sulcus is very short and narrow, comprised of five very small plates. The thecal plate arrangement of P. belizeanum is P₀, 5′, O″, 5C, 5″′, 1″″, 5S. No lists are present. Thecal plates have a smooth surface with small and irregularly scattered pores. The intercalary band is smooth on outer cell surface and broadly striated on its inner surface. We conclude that P. belizeanum represents a new, benthic, peridinioid, armored genus, Plagiodinium gen. nov. The taxonomic position of P. belizeanum sp. nov. is compared to related sand-dwelling and bilaterally flattened benthic dinoflagellates.     

MORPHOLOGIC DETAILS OF SIX BENTHIC SPECIES OF PROROCENTRUM (PYRROPHYTA) FROM A MANGROVE ISLAND,TWIN CAYS,BELIZE, INCLUDING TWO NEW SPECIES1

Two new dinoflagellate species, Prorocentrum hoffmannianum and Prorocentrum ruetzlerianum, and four known species, Prorocentrum emarginatum Fukuyo 1981, Prorocentrum mesicanum Tafall 1942, Prorocentrum concavum Fukuyo 1981, and Prorocentrum lima (Ehr.) Dodge 1975, from floating detritus and sediments in a subtropical mangrove island, Twin Cays, Belize, Central America are described from scanning electron micrographs. Differences in the following characters of surface micromorphology separated the species: ornamentation of thecal plates (shape, size, and number of valve pores and areolae) and the architecture of the periflagellar area and intercalary band.     

FURTHER SEM STUDY OF MARINE DINOFLAGELLATES: THE GENUS OSTREOPSIS (DINOPHYCEAE)1

This paper presents a comprehensive examination of the taxonomy of the genus Ostreopsis Schmidt. The morphology of six species of marine dinoflagellates, Ostreopsis siamensis Schmidt 1902. Ostreopsis lenticularis Fukuyo 1981, Ostreopsis ovata Fukuyo 1981, Ostreopsis heptagona Norris, Bomber, et Balech 1985, Ostreopsis mascarenensis Quod 1994, and Ostreopsis labens Faust et Morton 1995 from three geographical regions (Japan, Southwest Indian Ocean, and the Caribbean) and three marine habitats (sand, water column, and macroalgal surfaces) are described from scanning electron micrographs. Differences in the following morphological characteristics differentiated the species: cell shape and size, and ornamentation of the epitheca, cingulum, and hypotheca. The thecal plate formula of the six Ostreopsis species is P_o, 3′, 7″, 6C, 6S?, V_p, R_p, 5′″, 1p, 2″″, with differences in thecal plate size and shape. The cingulum in ventral view has two prominent structures: a ventral plate (V_p) with a ventral pore (V_o) and a ridged plate (R_p) that distinguishes Ostreopsis species from any other dinoflagellate taxa. This paper also includes ecological and toxicity information regarding the six Ostreopsis species.     

OBSERVATIONS ON THE MORPHOLOGY AND SEXUAL REPRODUCTION OF COOLIA MONOTIS (DINOPHYCEAE)1

The surface morphology of the dinoflagellate Coolia monotis Meunier was compared with the surface morphology of Ostreopsis, The apical pore of C. monotis is similar in architecture to that of Ostreopsis but considerably longer (12 μm) than in O. heptagona (8–9 μm) and O. ovata (6–7 μm). A ventral pore in C. monotis is located on the right ventral margin between apical plate l′ and precingular plate 6″ and is similar in appearance and location to the ventral pore of O. ovata. The longitudinal flagellum (20 μm) in C. monotis is longer than in O. ovata (12 μ). Although Coolia and Ostreopsis appear to be distinctly different and should remain as two separate genera, they appear to be related. Cells of C. monotis divided by binary fission. Doubling time was 3–4 days in the logarithmic phase of growth at 23°C, 12:12 h L:D, 30–90 μE-m^?2·s^?1, and a salinity of 36%. Cultures reached cell densities of 2.5 × 10³ cells·L^?1 after 15 days of growth. The sexual process in C. monotis occurred in Erdschreiber's medium when Danish soil extract was substituted with mangrove sediment extract under the culture conditions described above. Gamete fusion produced large biflagellated planozygotes (70–75 μm diam). Planozygote maturation involved cytoplasmic reorganization, loss of motility, development of a spherical shape (80–90 μm diam), and two to three orange accumulation bodies. The cells at this stage appeared to be thin-walled cysts. Further development included reorganization of cyst contents, emergence of non-motile gametes, and development of chloroplasts, sulcus, and girdle. The nucleus of the newly formed cells occupied 50% or more of the total cell volume. Meiosis occurred in the cyst, but nuclear cyclosis was not observed. Four daughter cells were produced within 36–48 h, and motile gametes developed. The gametes exhibited sexuality for 2 months and completed the sexual life cycle by going through a thin-walled cyst stage.     

PHYLOGENETIC ANALYSIS OF NINE SPECIES OF PROROCENTRUM (DINOPHYCEAE) INFERRED FROM 18S RIBOSOMAL DNA SEQUENCES, MORPHOLOGICAL COMPARISONS, AND DESCRIPTION OF PROROCENTRUM PANAMENSIS, SP. NOV.

Sequences of 18S rRNA genes were obtained from eight species of Prorocentrum Ehrenberg: P. minimum (Pavillard) Schiller, P. mexicanum Osorio Tafall, P. emarginatum Fukuyo, P. lima (Ehrenberg) Dodge, P. arenarium Faust, P. maculosum Faust, P. concavum Fukuyo, and P. panamensis, sp. nov. Prorocentrum panamensis is a new species of tropical dinoflagellate isolated from a benthic coral reef on the Pacific coast of Panama and described here using scanning electron microscopy. Cells are heart shaped, 46–52 μm long and 43–46 μm wide. The valve surfaces are areolate except in the central area. Pores of 0.15 μm in diameter are scattered in areolae, mainly around the periphery of the cell. The right valve has a specific ovoid depression with numerous appressed pores; we named this structure the sieve-like depression. The periflagellar area is nearly ovoid, located in a shallow depression, and almost equally set into both valves. It is unornamented (no apical expansion) but has numerous depressions in platelets. The flagellar and auxiliary pores are different in size and shape. The intercalary band is transversally striated. Phylogenetic relationships of gonyaulacoid, peridinioid, gymnodinioid, and prorocentroid dinoflagellates were inferred from complete 18S rDNA sequences. Two distinct phylogenetic analyses are presented for armored and unarmored Dinophyceae in an attempt to make the phylogenetic relationships between these different kinds of organisms clearer. The Prorocentrales appear to have a common origin, although two groups of Prorocentrum spp. are apparent. The first group includes benthic, symmetrical species (P. lima, P. arenarium, P. maculosum, and P. concavum). The second group contains planktonic and bentho-planktonic species (P. micans Ehrenberg, P. minimum, P. mexicanum, and P. panamensis sp. nov.). Genetic distances between species within these two groups were high; however, the divergence between the two groups seems to have occurred late in dinoflagellate evolution. In addition, the bentho-planktonic P. emarginatum appeared distantly related to both groups; however,its 18S rDNA sequence shares specific nucleotide substitutions with the two groups, suggesting an older origin of this species compared to the others. A morphological interpretation of this phylogenetic analysis is made on the basis of the specific structure of the periflagellar area. Finally, genetic data and morphological observations support the hypothesis that the genus Prorocentrum is rather heterogeneous; several species could be considered to constitute distinct genera.