FURTHER EVIDENCE FOR A MEMBRANE-BOUND ENDOSYMBIONT WITHIN THE DINOFLAGELLATE PERIDINIUM FOLIACEUM1

The fine structure of the binucleate, fucoxanthin-containing dinoflagellate Peridinium foliaceum (Stein) Biechler was re-examined for evidence of an endosymbiout. The eucaryotic nucleus, chloroplasts and associated ribosome-dense cytoplasm were separated by a single invaginating membrane from the rest of the dinoflagellate cell. The triple membrane-enclosed eyespot, mesocaryotic nucleus, trichocysts and accumulation bodies resided in the dinoflagellate cytoplasm. These observations suggest that P. foliaceum contains a membrane-bound endosymbiont, similar to that already described for the closely related species. P. balticum (Levander) Lemmermann.     

NUCLEAR BASIC PROTEINS FROM THE BINUCLEATE DINOFLAGELLATE PERIDINIUM FOLIACEUM (PYRROPHYTA)1

Histories of the endosymbiont nucleus of the binucleate dinoflagellate Peridinium foliaceum Stein were prepared from isolated nuclei and analyzed by peptide mapping, ammo acid composition, and two-dimensional gel electrophoresis. Using these criteria, we identified the presence of two HI-like histories and the core histones H3, H2A, H2B, and H4. These histones are similar but not identical to those of the endosymbiont nucleus of the bi-nucleate dinoflagellate Peridinium balticum Levander.     

Serial replacement of a diatom endosymbiont in the marine dinoflagellate Peridinium quinquecorne (Peridiniales, Dinophyceae)

To infer the phylogeny of both the host and the endosymbiont of Peridinium quinquecorne Abé, the small subunit (SSU) ribosomal DNA (rDNA) from the host and two genes of endosymbiont origin (plastid‐encoded rbcL and nuclear‐encoded SSU rDNA) were determined. The phylogenetic analysis of the host revealed that the marine dinoflagellate P. quinquecorne formed a clade with other diatom‐harbouring dinoflagellates, including Kryptoperidinium foliaceum (Stein) Lindeman, Durinskia baltica (Levander) Carty et Cox and Galeidinium rugatum Tamura et Horiguchi, indicating a single endosymbiotic event for this lineage. Phylogenetic analyses of the endosymbiont in these organisms revealed that the endosymbiont of P. quinquecorne formed a clade with a centric diatom (SSU data indicated it to be closely related to Chaetoceros), whereas the endosymbionts of other three dinoflagellates formed a clade with a pennate diatom. The discrepancy between the host and the endosymbiont phylogenies suggests a secondary replacement of the endosymbiont from a pennate to a centric diatom in P. quinquecorne.     

GALEIDINIIUM RUGATUM GEN. ET SP. NOV. (DINOPHYCEAE), A NEW COCCOID DINOFLAGELLATE WITH A DIATOM ENDOSYMBIONT1

A new sand‐dwelling dinoflagellate from Palau, Galeidinium rugatum Tamura et Horiguchi gen. et sp. nov., is described. The life cycle of this new alga consists of a dominant nonmotile phase and a brief motile phase. The motile cell transforms itself directly into the nonmotile cell after swimming for a short period, and cell division takes place in the nonmotile phase. The nonmotile cell possesses a dome‐like cell covering, which is wrinkled and equipped with a transverse groove on the surface. The cell has 10–20 chloroplasts and a distinct eyespot. The motile cell is Gymnodinium‐like in shape. The dinoflagellate possesses an endosymbiotic alga to which the chloroplasts belong and which is separated from the host (dinoflagellate) cytoplasm by a unit membrane. The endosymbiont cytoplasm also possesses its own eukaryotic nucleus and mitochondria. The eyespot is surrounded by triple membranes and is located in the host cytoplasm. Photosynthetic pigment analysis, using HPLC, revealed that G. rugatum possesses fucoxanthin as the principal accessory pigment instead of peridinin. The rbcL tree showed that G. rugatum is monophyletic with Durinskia baltica (Levander) Carty et Cox and Kryptoperidinium foliaceum (Stein) Lindemann and that this clade is closely related to the pennate diatom, Cylindrotheca sp. The endosymbiont of G. rugatum is therefore shown to be a diatom. Phylogenetic analysis based on small subunit rDNA sequences demonstrated that G. rugatum, D. baltica, and K. foliaceum, all of which are known to harbor an endosymbiont of diatom origin, are closely related.     

Nuclease Digestion of Chromatin from the Eukaryotic Algae Olisthodiscus luteus,Peridinium balticum,and Crypthecodinium cohnii 1, 2

Chromatin from a uninucleate dinoflagellate, Crypthecodinium cohnii, a binucleate dinoflagellate, Peridinium balticum, and a chromophyte, Olisthodiscus luteus, was examined by nuclease digestion and the results were compared to those from vertebrates. Gel analysis of the products of staphylococcal (micrococcal) nuclease digestion revealed a DNA repeat unit of 220(±5) base pairs for O. luteus and 215(±5) for P. balticum. Limit digestion gave a core particle of 140 base pairs, revealing that these longer repeat sizes are due to longer linker regions. No repeating subunit structure was found upon electrophoresis of digests of C. cohnii nuclei. Examination of the DNA fragments produced by DNAse I digestion of nuclei isolated from P. balticum and O. luteus showed the same ladder of ten base multiples as seen in chromatin from other eukaryotes. Examination of the kinetics of digestion by DNAse II of Peridinium chromatin revealed less susceptibility when compared to DNAse I digestions while 70% of Olisthodiscus chromatin and 35% of C. cohnii chromatin was sensitive to DNAse II. These data, taken together with previous results from Euglena, indicate that while algal chromatin is similar to that of higher eukaryotes in regard to DNAse I and II action, it differs in that the linker DNA is longer. In addition, the Hl-like histone from O. luteus and P. balticum is located in the linker DNA as in higher eukaryotes.     

FERTILIZATION AND ZYGOTE DEVELOPMENT IN THE BINUCLEATE DINOFLAGELLATE PERIDINIUM BALTICUM (PYRRHOPHYTA)

Peridinium balticum (Pyrrhophyta) exists as a symbiosis between a nonphotosynthetic dinoflagellate host and a chlorophyll c-containing alga. It is hypothesized that P. balticum is an evolutionary link between primitive nonphotosynthetic and advanced photosynthetic dinoflagellates. This study documents pre- and postfertilization events of sexual reproduction in this unusual dinoflagellate for the first time. Light microscopy and TEM observations showed that gametes resemble vegetative cells except in the organization of their chloroplasts. Fusion of gametes occurred in a specific orientation, i.e., apical to sulcal area. The presence of an intact membrane between fusing pairs prior to plasmogamy was suggestive of enzymatic digestion of plates during fertilization. Rupture of this membrane triggers plasmogamy and karyogamy of the host, followed by that of the algal symbiont. A discussion of the cellular processes involved in gamete formation, fertilization, and zygote development is presented. The results of this investigation demonstrate that a synchronous sexual reproduction cycle has evolved for the symbiont and its dinoflagellate host.     

Isolation and Characterization of Carotenoid-rich Lipid Globules from Peridinium foliaceum

Carotenoid-rich oil globules were isolated from the cytoplasm of the binucleate dinoflagellate, Peridinium foliaceum. These orange globules were collected from ruptured cells by ultracentrifugation on a sucrose density gradient, and checked for purity by electron microscopy. The osmiophilic globules were assayed for lipid (including pigment) and protein content. The lipid to protein ratio was 1.39:1, with a calculated density of the globules of 1.05 grams per cubic centimeter. The lipids were composed of hydrocarbon, wax ester (phytyl ester), triglyceride, and polar (no phospholipid) fractions. The biochemical composition indicated that the globules function as a reservoir of energy-rich components in the cell. Microspectrophotometric observations were consistent with pigment analyses which demonstrated that the globules were carotenoid-rich. In addition to β-carotene, γ-carotene, and canthaxanthin, the carotenogenic precursors: phytoene, phytofluence, ζ-carotene and β-zeacarotene were isolated from the globules. Corrected fluorescence maxima of phytoene and phytofluene in hexane were recorded at 340 and 490 nanometers, respectively. Carotenes constituted 3.3% of the total oil globule lipid. The possibility of an extraplastidic carotenogenic enzyme system in P. foliaceum is discussed.     

EFFECTS OF ULTRAVIOLET‐A RADIATION AND NUTRIENT AVAILABILITY ON THE CELLULAR COMPOSITION OF PHOTOPROTECTIVE COMPOUNDS IN GLENODINIUM FOLIACEUM (DINOPHYCEAE)1

The photoprotective response in the dinoflagellate Glenodinium foliaceum F. Stein exposed to ultraviolet‐A (UVA) radiation (320–400 nm; 1.7 W · m²) and the effect of nitrate and phosphate availability on that response have been studied. Parameters measured over a 14 d growth period in control (PAR) and experimental (PAR + UVA) cultures included cellular mycosporine‐like amino acids (MAAs), chls, carotenoids, and culture growth rates. Although there were no significant effects of UVA on growth rate, there was significant induction of MAA compounds (28 ± 2 pg · cell^?1) and a reduction in chl a (9.6 ± 0.1 pg · cell^?1) and fucoxanthin (4.4 ± 0.1 pg · cell^?1) compared to the control cultures (3 ± 1 pg · cell^?1, 13.3 ± 3.2 pg · cell^?1, and 7.4 ± 0.3 pg · cell^?1, respectively). In a second investigation, MAA concentrations in UVA‐exposed cultures were lower when nitrate was limited (P < 0.05) but were higher when phosphate was limiting. Nitrate limitation led to significant decreases (P < 0.05) in cellular concentration of chls (chl c₁, chl c₂, and chl a), but other pigments were not affected. Phosphate availability had no effect on final pigment concentrations. Results suggest that nutrient availability significantly affects cellular accumulation of photoprotective compounds in G. foliaceum exposed to UVA.     

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF PHYTOPLANKTON PIGMENTS USING A POLYMERIC REVERSED-PHASE C18 COLUMN1

A high-performance liquid chromatographic (HPLC) method is described that allows improved resolution of several chemotaxonomically significant phytoplankton pigments. The protocol, which employs two pumps and a modified Mantoura and Llewellyn (1983) solvent system, can be easily adapted for many HPLC systems currently in use. The most unique aspect of the method is the use of a polymeric C₁₈ reversed phase HPLC column (VYDAC 201TP). In comparison to the monomeric C₁₈ columns typically used in the characterization of phytoplankton pigments, polymeric C₁₈ columns offer superior selectivity for structurally similar compounds. The protocol was evaluated for the ability to resolve most of the phytoplankton pigments of diagnostic importance using algal cultures from nine classes. Pigment pairs that were resolved by the method include a) lutein and zeaxanthin, b) neoxanthin and 19′-hexanoyloxyfucoxanthin, and c) α-carotene and β-carotene, and partial resolution of chlorophyll c₁ and chlorophyll c₂.     

Dinophyte chloroplasts and phylogeny - A review

Dinophytes acquired chloroplasts obviously early in evolution and later lost them multiple times. Most families and genera contain both photosynthetic and heterotrophic species. Chloroplasts enveloped by three membranes with thylakoids in stacks of three, containing peridinin as the main pigment, are regarded as the original dinophyte plastids. Pyrenoids are generally present. Stigmata, if present, are usually parts of the chloroplast or are modified original plastids. The form II type RUBISCO found in the dinophytes is unique for eukaryotes, otherwise known only in some anaerobic bacteria. It is disputed whether the original dinophyte chloroplasts are derived from a prokaryotic or an eukaryotic endosymbiosis. Various dinoflagellates contain aberrant chloroplasts. Glenodinium foliaceum and Peridinium balticum have a single complete endosymbiont, originally a pcnnate diatom. Podolampas bipes houses several dictyophycean symbiont cells. The “symbionts” of Lepidodiniurn viride and Gymnodinium chlorophorum are highly reduced prasinophyte cells. The chloroplasts of Gymnodinium mikimotoi have aberrant pigments (fucoxanthin derivatives, no peridinin) and fine structure. The dinoflagellate hosts do not seem to contain any parts of the former endosymbiont except the chloroplasts. Photosynthetic Dinophysis species have cryptophycean-like chloroplasts, whereas symbiotic cyanobacteria are found in other members of the Dinophysiales, e.g., Ornithocercus. Various dinophytes, e.g. Gymnodinium aeruginosum, use kleptochloroplasts from ingested cryptophytes transiently for photosynthesis. Original or secondarily acquired chloroplasts can only be used for phylogenetic considerations in exceptionally cases: it seems unlikely that the Prorocentrales have evolved from the Dinophysiales because all Prorocentrales possess original dinoflagellate chloroplasts, whereas no member of the Dinophysiales has such chloroplasts.     

IDENTITY OF THE ENDOSYMBIONT OF PERIDINIUM FOLIACEUM (PYRROPHYTA): ANALYSIS OF THE rbcLS OPERON1

Extant chromophytic algae have been suggested to have originated via the engulfment of a photo synthetic alga by a colorless protist. The dinoflagellate Peridinium foliaceum (Stein) Biecheler contains a reduced chlorophyll c–containing endosymbiont and, thus, represents an evolutionary intermediate stage in the establishment of chloroplasts. Although the exact phylogenetic relationship of the symbiont to extant algal species is unknown, it had been suggested that the P. foliaceum symbiont was either a diatom or a chrysophyte. Identification of the closest living relative of the P. foliaceum symbiont would provide a free-living model system with which the photosynthetic symbiont could be compared. Nucleotide sequence analysis of rbcL and rbcS (encoding the large and small subunits ofribulose-1,5-bisphosphate carboxylase/oxygenase) by the P. foliaceum symbiont was performed to provide insights into its identity. Cloned restriction fragments from a chloroplast DNA library were screened, and clones encoding the rbcLS operon were sequenced. Parsimony phylogenetic analysis was performed for each gene. Our data strongly suggest that the symbiont originated from a photosynthetic diatom.     

Kryptoperidinium foliaceum blooms in South Carolina: a multi-analytical approach to identification

Observations following the discovery of Kryptoperidinium foliaceum blooms in South Carolina (SC), USA, suggest that a multi-analytical approach, using a standard, minimal set of criteria, should be adopted for determining dinoflagellate species identity and taxonomic placement. A combination of morphological, molecular, and biochemical analyses were used to determine the identity of this “red tide” dinoflagellate, first documented in SC waters in the spring of 1998. Results from thecal plate tabulations (based on scanning electron and epifluorescence microscopy), gene sequence data, species-specific PCR probe assays, and microalgal pigment profiles were analyzed and compared to reference cultures of K. foliaceum. Comparative data showed marked inconsistencies among the K. foliaceum reference culture isolates. In addition, the SC bloom isolate was shown to be mononucleate, contrary to previous reports for K. foliaceum, suggesting a more transient endosymbiotic association than previously considered.     

FREEZE-FRACTURE STUDY OF THE SINGLE MEMBRANE BETWEEN HOST CELL AND ENDOCYTOBIONT IN THE DINOFLAGELLATES GLENODINIUM FOLIACEUM AND PERIDINIUM BALTICUM1

The dinoflagellates Glenodinium foliaceum Stein and Peridinium balticum (Levander) Lemmermann harbor a chrysophytic endocytobiont which is bounded by only a single membrane. This unique membrane is of particular interest because it could correspond to an intermediate stage in the evolution of “complex” plastids found in many Plastids of this type are surrounded by three or membranes instead of the usual two. With freeze-fracture techniques, we show that the single membrane in P. balticum has a pronounced polarity with respect to the distribution of intramembrane particles (IMPs) on the two corresponding fracture faces. The inner face exhibited more IMPs than the outer. We suggest that this stdedness identifies the separating membrane as the plasma membrane of the endocytobiont. A symbiontophoric vacuole with a separate membrane apparently is lacking. In the endocytobiosis of G. foliaccum, the single membrane separating host and endocylobiont exhibits a symmetrical particle partition. Nevertheless, from the size distribution of the IMPs it appears likely that this membrane, too, corresponds to the plasma membrane of the symbiont.     

PIGMENTS,FATTY ACIDS,AND STEROLS OF THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM1

Cultures and field samples of the toxic dinoflagellate Gymnodinium catenatum Graham from Tasmania, Australia, were analyzed for pigment, fatty acid, and sterol composition. Gymnodinium catenatum contained the characteristic pigments of photosynthetic dinoflagellates, including chlorophyll a, chlorophyll c₂, and the carotenoids peridinin, dinoxanthin, diadinoxanthin, diatoxanthin, and β,β-carotene. In midlogarithmic and early stationary phase cultures, the chlorophyll a content ranged 50–72 pg · cell^?1, total lipids 956–2084 pg · cell^?1, total fatty acids 426–804 pg · cell^?1, and total sterols 8–20 pg · cell^?1. The major fatty acids (in order of decreasing abundance) were 16:0, 22:6(n-3), and 20:5(n-3) (collectively 65–70% of the total fatty acids), followed by 16:1(n-7), 18:2(n-6), and 14:0. This distribution is characteristic of most dinoflagellates, except for the low abundance (<3%) of the fatty acid 18:5(n-3), considered by some authors to be a marker for dinoflagellates. The three major sterols were 4α-methyl-5α-cholest-7-en-3β-ol, 4α,23,24-trimethyl-5α-cholest-22E-en-3β-ol (the dinoflagellate sterol, dinosterol), and 4α,23,24-trimethyl-5α-cholest-7-en-3β-ol. These three sterols comprised about 75% of the total sterols in both logarithmic and early stationary phase cultures, and they were also found in high proportions (22–25%) in natural dinoflagellate bloom samples. 4-Desmethyl sterols, which are common in most microalgae, were only present in trace amounts in G. catenatum. The chemotaxonomic affinities of G. catenatum and the potential for using specific signature lipids for monitoring toxic dinoflagellate blooms are discussed.     

The Monophyletic Origin of the Peridinin‐, and Fucoxanthin‐Containing Dinoflagellate Plastid Through Tertiary Replacement

The dinoflagellates contain diverse plastids of uncertain origin. To determine the origin of the peridinin‐ and fucoxanthin‐containing dinoflagellate plastid, we sequenced the plastid‐encoded psaA, psbA, and rbcL genes from various red and dinoflagellate algae. The psbA gene phylogeny, which was made from a dataset of 15 dinoflagellates, 22 rhodophytes, five cryptophytes, seven haptophytes, seven stramenopiles, two chlorophytes, and a glaucophyte as the outgroup, supports monophyly of the peridinin‐, and fucoxanthin‐containing dinoflagellates, as a sister group to the haptophytes. The monophyletic relationship with the haptophytes is recovered in the psbA + psaA phylogeny, with stronger support. The rubisco tree utilized the ‘Form I’ red algal type of rbcL and included fucoxanthin‐containing dinoflagellates. The dinoflagellate + haptophyte sister relationship is also recovered in this analysis. Peridinium foliaceum is shown to group with the diatoms in all the phylogenies. Based on our analyses of plastid sequences, we postulate that: (1) the plastid of peridinin‐, and fucoxanthin‐containing dinoflagellates originated from a common ancestor; (2) the ancestral dinoflagellate acquired its plastid from a haptophyte though a tertiary plastid replacement; (3) ‘Form II’ rubisco replaced the ancestral rbcL after the divergence of the peridinin‐, and fucoxanthin‐containing dinoflagellates; and (4) we confirm that the plastid of P. foliaceum originated from a Stramenopiles endosymbiont.     

LIPOPHILIC PIGMENTS FROM CYANOBACTERIAL (BLUE-GREEN ALGAL) AND DIATOM MATS IN HAMELIN POOL,SHARK BAY,WESTERN AUSTRALIA1

Lipophilic pigments were examined in microbial mat communities dominated by cyanobacteria in the intertidal zone and by diatoms in the subtidal and sublittoral zones of Hamelin Pool, Shark Bay, Western Australia. These microbial mats have evolutionary significance because of their similarity to lithified stromatolites from the Proterozoic and Early Paleozoic eras. Fucoxanthin, diatoxanthin, diadinoxanthin, β-carotene, and chlorophylls a and c characterized the diatom mats, whereas cyanobacterial mats contained myxoxanthophyll zeaxanthin, echinenone, β-carotene, chlorophyll a and, in some cases, sheath pigment. The presence of bacteriochlorophyll a with in the mats suggest a close association of photosynthetic bacteria with diatoms and cyanobacteria. The high carotenoids: chlorophyll a ratios (0.84–2.44 wt/wt) in the diatom mats suggest that carotenoids served a photoprotective function in this high light environment. By contrast, cyanobacterial sheath pigment may have largely supplanted the photoprotective role of carotenoids in the intertidal mats.     

EFFECT OF LOW TEMPERATURE ON THE STEREOISOMER COMPOSITION OF β-CAROTENE IN THE HALOTOLERANT ALGA DUNALIELLA BARDAWIL (CHLOROPHYTA)1

Dunaliella bardawil Ben-Amotz & Avron, a β-carotene-accumulating halotolerant alga, was analyzed for the effect of growth temperatures on its pigment content and on the stereoisomeric composition of β-carotene by the use of advanced liquid chromatography and photodiode array detection. Decreasing culture temperature from 30° to 10°C increased the β-carotene content twofold and the ratio of 9-cis to all-trans β-carotene fourfold, with no significant changes in the other cell pigments. The variation of total β-carotene content by temperature was correlated with the integral irradiance received by the algal culture during a cell division cycle, whereas the 9-cis stereoisomer increased over the amount expected by that integration. The massive accumulation of 9-cisβ-carotene within the β-carotene globules is interpreted as indicating that the oily 9-cis stereoisomer protects against the crystallization of all-trans β-carotene at low temperatures.     

Localised uptake of63nickel into dinoflagellate chromosomes: An autoradiographic study

Summary The uptake of⁶³Ni into cells of the binucleate dinoflagellateGlenodinium foliaceum was investigated using insoluble compound light and electron microscope autoradiography. Cells labelled over a period of 2 hours showed active uptake throughout the whole population, with an increase in mean cell grain count when the labelling period was extended to 4 hours and 24 hours. The mean grain count did not vary with type of fixation (glutaraldehyde, paraformaldehyde or acetic alcohol) suggesting that retention of⁶³ Ni is not a specific fixation-binding artefact. At light microscope level, silver grains were not localised to any major cell component, but with the greater resolution of electron microscope autoradiography, a high degree of localisation was demonstrated in the typical dinoflagellate (dinocaryotic) nucleus-which contained about 83% of the cell label (cytoplasm 16%, supernumerary nucleus 1%). Silver grain distribution within the dinocaryotic nucleus was consistent with some degree of localisation to the condensed chromatin.The autoradiographic results corroborate previous X-ray microanalytical data which demonstrated high levels of transition metals in dinoflagellate nuclei. The distinction between the two types of nucleus inGlenodinium is further emphasised, giving additional support to the concept of a separate phyllogenetic origin of the supernumerary nucleus.