Sterols of the ‘dinotom’ Durinskia baltica (Dinophyceae) are of dinoflagellate origin

‘Dinotoms’ are a relatively small group of dinoflagellates with aberrant tertiary plastids of diatom origin, thus differing from the majority of photosynthetic dinoflagellates which possess the carotenoid pigment peridinin and have secondary plastids of red algal origin. As part of our laboratory's continuing efforts to examine such unusual dinoflagellates in the search for clues to the evolution of their lipid compositions, we have examined the sterol composition of the dinotom Durinskia baltica. As such, we here compared its sterols to those of the previously examined dinotom, Kryptoperidinium foliaceum, more broadly to other photosynthetic, peridinin-containing dinoflagellates, and to the diatom genus Nitzschia, which is the presumed ancestor of the D. baltica dinotom plastid. Sterols are ringed lipids, common to eukaryotes, thought to reinforce phospholipid bilayers. Many peridinin-containing dinoflagellates have sterol compositions which are enriched by the presence of cholesterol (cholest-5-en-3β-ol) and 4α-methyl-substituted sterols such as dinosterol (4α,23,24-trimethyl-5α-cholest-22E-en-3β-ol); this has also been found to be true for K. foliaceum despite its aberrant plastid ancestry. Our objective was to determine if this is also true for D. baltica as only the second dinotom to have its sterols characterized in detail, and to determine if there is any indication of prominent sterols which are uncommon to dinoflagellates, possibly originating from the diatom endosymbiont, as has been demonstrated previously with K. foliaceum and D. baltica chloroplast-associated galactolipids of clear diatom origin. Our results demonstrate that like K. foliaceum, the major sterols of D. baltica are cholesterol, dinosterol, and other 4α-methyl-substituted sterols common to dinoflagellates. Although there were a number of minor sterols, none were found with obvious origin from the diatom endosymbiont, indicating that most originated with the dinoflagellate host itself, most likely before acquisition of the diatom tertiary plastid.     

STRUCTURAL ORGANIZATION OF PLASTID DNA IN TWO ANOMALOUSLY PIGMENTED DINOFLAGELLATES1

The structural organization of DNA in the plastids of two anomalously pigmented dinoflagellates, Glenodinium foliaceum Stein and Gyrodinium aureolum Hulburt, was determined using the DNA-specific fluorochrome DAPI and correlated with TEM observations. The plastids of G. foliaceum were found to possess both a peripheral DNA ring and isolated point nucleoids. This arrangement was shown to be similar to that of the diatom Asterionella formosa Hass. and may be characteristic of the Bacillariophyceae. G. aureolum exhibited a novel distribution of plastid. DNA as one or two beaded bands, whereas the plastids of the similarly pigmented haptophyte, Emiliania huxleyi (Lohm.) Hay & Mohler, possessed scattered point nucleoids. These findings support the idea that G. foliaceum harbours an endosymbiotic diatom, but suggest that the plastids of G. aureolum and E. huxleyi are unrelated. The use of plastid DNA configuration as a phylogenetic marker is considered.     

PIGMENTS OF THE DINOFLAGELLATE PERIDINIUM BALTICUM AND ITS PHOTOSYNTHETIC ENDOSYMBIONT1

An examination of the pigments of the binucleate dinoflagellate Peridinium balticum (Levander) Lemmerman revealed the presence of chlorophylls a, c₁ and c₂ and the carotenoids: fucoxanthin (most abundant), diadinoxanthin, diatoxanthin, an unidentified fucoxanthin-like xanthophyll, β-carotene, γ-carotene and astaxanthin. A comparison of the pigments of P. balticum and P. foliaceum (Stein) Biecheler, also a binucleate dinoflagellate, demonstrated similar compositions. However P. balticum lacked the β-carotene precursors (e.g. phytoene) which accumulated outside the chloroplast in P. foliaceum. This study indicates that P. balticum and P. foliaceum are closely related; each species is a heterotrophic dinoflagellate with a photosynthetic endosymbiont taxonomically affiliated with the Chrysophyta (Chrysophyceae or Bacillariophyceae).     

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.     

THE EVOLUTION OF ELONGATE SHAPE IN DIATOMS1

Diatoms have been classified historically as either centric or pennate based on a number of features, cell outline foremost among them. The consensus among nearly every estimate of the diatom phylogeny is that the traditional pennate diatoms (Pennales) constitute a well‐supported clade, whereas centric diatoms do not. The problem with the centric–pennate classification was highlighted by some recent analyses concerning the phylogenetic position of Toxarium, whereby it was concluded that this “centric” diatom independently evolved several pennate‐like characters including an elongate, pennate‐like cell outline. We performed several phylogenetic analyses to test the hypothesis that Toxarium evolved its elongate shape independently from Pennales. First, we reanalyzed the original data set used to infer the phylogenetic position of Toxarium and found that a more thorough heuristic search was necessary to find the optimal tree. Second, we aligned 181 diatom and eight outgroup SSU rDNA sequences to maximize the juxtapositioning of similar primary and secondary structure of the 18S rRNA molecule over a much broader sampling of diatoms. We then performed a number of phylogenetic analyses purposely based on disparate sets of assumptions and found that none of these analyses supported the conclusion that Toxarium acquired its pennate‐like outline independently from Pennales. Our results suggest that elongate outline is congruent with SSU rDNA data and may be synapomorphic for a larger, more inclusive clade than the traditional Pennales.     

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.     

EVOLUTIONARY ANALYSES OF THE NUCLEAR‐ENCODED PHOTOSYNTHETIC GENE psbO FROM TERTIARY PLASTID‐CONTAINING ALGAE IN DINOPHYTA1

Although the dinophytes generally possess red‐algal‐derived secondary plastids, tertiary plastids originating from haptophyte and diatom ancestors are recognized in some lineages within the Dinophyta. However, little is known about the nuclear‐encoded genes of plastid‐targeted proteins from the dinophytes with diatom‐derived tertiary plastids. We analyzed the sequences of the nuclear psbO gene encoding oxygen‐evolving enhancer protein from various algae with red‐algal‐derived secondary and tertiary plastids. Based on our sequencing of 10 new genes and phylogenetic analysis of PsbO amino acid sequences from a wide taxon sampling of red algae and organisms with red‐algal‐derived plastids, dinophytes form three separate lineages: one composed of peridinin‐containing species with secondary plastids, and the other two having haptophyte‐ or diatom‐derived tertiary plastids and forming a robust monophyletic group with haptophytes and diatoms, respectively. Comparison of the N‐terminal sequences of PsbO proteins suggests that psbO genes from a dinophyte with diatom‐derived tertiary plastids (Kryptoperidinium) encode proteins that are targeted to the diatom plastid from the endosymbiotic diatom nucleus as in the secondary phototrophs, whereas the fucoxanthin‐containing dinophytes (Karenia and Karlodinium) have evolved an additional system of psbO genes for targeting the PsbO proteins to their haptophyte‐derived tertiary plastids from the host dinophyte nuclei.     

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.     

PHYLOGENETIC DISTRIBUTION OF THE MAJOR DIATOM LIGHT-HARVESTING PIGMENT-PROTEIN DETERMINED BY IMMUNOLOGICAL METHODS 1

Monospecific, polyclonal antibodies raised against the apoprotein of the major light-harvesting pigment-protein of Phaeodactylum tricornutum Bohlin UTEX 646 were used to determine (1) whether this complex was common to the class Bacillariophyceae, whose members contain chlorophylls a and c and fucoxanlhin; (2) whether antigenically-related apoproteins were present in other chlorophyll c-containing groups, and (3) whether there was immunological homology with the light-hanvsting chlorophyll a/b protein of similar photosynthetic function in the Chlorophyta and vascular plants. We have used protein blotting techniques to show that antibodies against the two P. tricornutum light-harvesting complex polypeptides cross-reacted with one or two polypeptides of similar molecular weight (17–21 kD) in all ten diatom species examined, representing two orders and six families. No cross-reactivity was obtained with total membrane polypeptides from isolated representatives of three chromophyte algal divisions (Chrysophyta, Cryptophyta, Pyrrophyta), all of which contained chlorophyll c. No cross-reactivity was observed with membrane Polypeptides isolated from members of two classes of Chlorophte algae. These data suggest that the Bacillariophyceae may be monophyletic, and that the primary structure of the diatom light-harvesting complex is not closely related to pigment-protein complexes with similar function in other chlorophyll c-containing unicellular algal groups. Lastly, it may be possible to use the antibodies to the diatom light-harvesting polypeptides as specific markers for diatoms in natural phytoplankton assemblages.     

VARIABILITY IN THE PRIMARY SITE OF PHOTOSYNTHETIC DAMAGE IN SYMBIODINIUM SP. (DINOPHYCEAE) EXPOSED TO THERMAL STRESS1

Exposure to elevated temperature is known to cause photosynthetic inhibition in the coral symbiont Symbiodinium sp. Through the use of the artificial electron acceptor, methyl viologen, this study identified how reduced photosynthetic capacity occurs as a result of inhibition up‐ and/or downstream of ferredoxin in Symbiodinium sp. in hospite and in culture. Heterogeneity between coral species and symbiont clades was identified in the thermal sensitivity of photosynthesis in the symbionts of the scleractinian corals Stylophora pistillata and Pocillopora damicornis, as well as among Symbiodinium cultures of clades A, B, and C. The in hospite symbionts of S. pistillata and the cultured clade C Symbiodinium both exhibited similar patterns in that their primary site of thermal inhibition occurred downstream of ferredoxin at 32°C. In contrast, the primary site of thermal inhibition occurred upstream of ferredoxin in clades A and B at 32°C, while at 34°C, all samples showed combined up‐ and downstream inhibition. Although clade C is common to both P. damicornis and S. pistillata, the manner of thermal inhibition was not consistent when observed in hospite. Results showed that there is heterogeneity in the primal site of thermal damage in Symbiodinium among coral species and symbiont clades.     

COMPARATIVE PHYSIOLOGICAL STUDY OF MARINE DIATOMS AND DINOFLAGELLATES IN RELATION TO IRRADIANCE AND CELL SIZE. II. RELATIONSHIP BETWEEN PHOTOSYNTHESIS,GROWTH, AND CARBON/CHLOROPHYLL a RATIO1,2

Photosynthetic rates, growth rates, cell carbon, cell protein, and chlorophyll a content of two diatom and two dinoflagellate species were measured. The microalgae were chosen to have one small and one large species from each phylogenetic group; the two size categories differed from each other by 1.5 orders of magnitude in terms of cell carbon or cell protein. The cultures for the experiments were grown under continuous light at an irradiance high enough for the light-saturation of growth for all four species. The four species were found to have similar maximum photosynthetic rates per unit chlorophyll a. The diatom species showed lower carbon/chlorophyll a ratios and higher photosynthetic rates per unit carbon than the dinoflagellates. The higher growth rates of the diatoms were shown to be related to their higher photosynthetic rates per unit carbon. The ecological significance of the physiological difference between these two groups of microalgae is discussed.     

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.     

THE INFLUENCE OF SUBMERGED MACROPHYTES ON SEDIMENTARY DIATOM ASSEMBLAGES1

Submerged macrophytes are a central component of lake ecosystems; however, little is known regarding their long‐term response to environmental change. We have examined the potential of diatoms as indicators of past macrophyte biomass. We first sampled periphyton to determine whether habitat was a predictor of diatom assemblage. We then sampled 41 lakes in Quebec, Canada, to evaluate whether whole‐lake submerged macrophyte biomass (BiomEpiV) influenced surface sediment diatom assemblages. A multivariate regression tree (MRT) was used to construct a semiquantitative model to reconstruct past macrophyte biomass. We determined that periphytic diatom assemblages on macrophytes were significantly different from those on wood and rocks (ANOSIM R = 0.63, P < 0.01). A redundancy analysis (RDA) of the 41‐lake data set identified BiomEpiV as a significant (P < 0.05) variable in structuring sedimentary diatom assemblages. The MRT analysis classified the lakes into three groups. These groups were (A) high‐macrophyte, nutrient‐limited lakes (BiomEpiV ≥525 μg · L^?1; total phosphorus [TP] <35 μg · L^?1; 23 lakes); (B) low‐macrophyte, nutrient‐limited lakes (BiomEpiV <525 μg · L^?1; TP <35 μg · L^?1; 12 lakes); and (C) eutrophic lakes (TP ≥35 μg · L^?1; six lakes). A semiquantitative model correctly predicted the MRT group of the lake 71% of the time (P < 0.001). These results suggest that submerged macrophytes have a significant influence on diatom community structure and that sedimentary diatom assemblages can be used to infer past macrophyte abundance.     

IDENTIFICATION OF A NOVEL CYTOCHROME P450 cDNA,CYP97E1, FROM THE MARINE DIATOM SKELETONEMA COSTATUM BACILLARIOPHYCEAE1

Here we report identification of a 2269‐base pair full‐length cDNA, CYP97E1, encoding a novel cytochrome P450 protein from the marine diatom Skeletonema costatum. The CYP97E1 protein contains 659 amino acids (Mr 74,200) and is the largest P450 isoform described to date. Our BLAST homology search and parsimony analysis showed that CYP97E1 shared high sequence identity (>40%) and genetic relatedness, respectively, with the CYP97B isoforms from different plant species. CYP97E1 was predicted by PSORT (a protein localization site prediction program) to be a cytosolic protein. Northern hybridization analysis indicated that CYP97E1 expression in S. costatum was not significantly affected by 2,4‐dichlorophenol, suggesting that CYP97E1 may not be involved in 2,4‐dichlorophenol detoxification in this diatom.     

MICROAUTORADIOGRAPHIC STUDIES OF THE MARINE PHYCOBIONTS RHIZOSOLENIA AND RICHELIA1

Microautoradiography was used to study photosynthetic and heterotrophic activities in cells of the diatom Rhizosolenia styliformis var. longispina containing the nitrogen-fixing, endophytic, blue-green alga Richelia intracellularis. In field studies with ¹⁴C-labeled bicarbonate, the blue-green alga appeared to be actively photosynthesizing but the diatom did not. Neither Richelia nor Rhizosolenia assimilated, ³H-labeled acetate, glucose, or an amino acid mixture. The diatom lacked cytoplasmic streaming and appeared, to be in a senescent condition. Implications of these observations for the nature of this algal association are discussed.     

FIRST INDUCED PLASTID GENOME MUTATIONS IN AN ALGA WITH SECONDARY PLASTIDS: psbA MUTATIONS IN THE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) REVEAL CONSEQUENCES ON THE REGULATION OF PHOTOSYNTHESIS1

Diatoms play a crucial role in the biochemistry and ecology of most aquatic ecosystems, especially because of their high photosynthetic productivity. They often have to cope with a fluctuating light climate and a punctuated exposure to excess light, which can be harmful for photosynthesis. To gain insight into the regulation of photosynthesis in diatoms, we generated and studied mutants of the diatom Phaeodactylum tricornutum Bohlin carrying functionally altered versions of the plastidic psbA gene encoding the D1 protein of the PSII reaction center (PSII RC). All analyzed mutants feature an amino acid substitution in the vicinity of the Q_B‐binding pocket of D1. We characterized the photosynthetic capacity of the mutants in comparison to wildtype cells, focusing on the way they regulate their photochemistry as a function of light intensity. The results show that the mutations resulted in constitutive changes of PSII electron transport rates. The extent of the impairment varies between mutants depending on the proximity of the mutation to the Q_B‐binding pocket and/or to the nonheme iron within the PSII RC. The effects of the mutations described here for P. tricornutum are similar to effects in cyanobacteria and green microalgae, emphasizing the conservation of the D1 protein structure among photosynthetic organisms of different evolutionary origins.     

The dinoflagellates <Emphasis Type="Italic">Durinskia baltica</Emphasis> and <Emphasis Type="Italic">Kryptoperidinium foliaceum</Emphasis> retain functionally overlapping mitochondria from two evolutionarily distinct lineages

Background  

The dinoflagellates Durinskia baltica and Kryptoperidinium foliaceum are distinguished by the presence of a tertiary plastid derived from a diatom endosymbiont. The diatom is fully integrated with the host cell cycle and is so altered in structure as to be difficult to recognize it as a diatom, and yet it retains a number of features normally lost in tertiary and secondary endosymbionts, most notably mitochondria. The dinoflagellate host is also reported to retain mitochondrion-like structures, making these cells unique in retaining two evolutionarily distinct mitochondria. This redundancy raises the question of whether the organelles share any functions in common or have distributed functions between them.     

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.     

AUXOSPORE FORMATION AND THE MORPHOLOGY OF THE INITIAL CELL OF THE MARINE ARAPHID DIATOM GEPHYRIA MEDIA (BACILLARIOPHYCEAE)1

Recent studies have led to a rapid increase in knowledge of auxospore formation in diatoms. However, these studies have been limited to centric and raphid pennate diatoms, and there is still very little information for the araphid pennate diatoms. Using LM and SEM, we studied the development of the auxospore and the initial cell of the marine epiphytic diatom Gephyria media Arnott. Auxospores were bipolar and curved in side view, as in many other pennate diatoms. SEM revealed many transverse perizonial bands, all of which were incomplete rings. There was an elongate, sprawling, silicified structure beneath the ventral suture of the transverse perizonial bands. This structure is presumably equivalent to the longitudinal perizonial band in other pennate diatoms, although we could not determine the homologous relationship between the two features. Scales were found both in the inner wall of the perizonium and around the primary perizonial bands. The presence or absence of scales may be of phylogenetic significance in diatoms, only during the final stages of auxospore formation because scales are found in early spherical stages. The distinctive finger‐like structures observed throughout all stage of G. media have not been observed before in the other diatom taxa.