OLIGONUCLEOTIDE PRIMERS FOR THE DETECTION OF BIOLUMINESCENT DINOFLAGELLATES REVEAL NOVEL LUCIFERASE SEQUENCES AND INFORMATION ON THE MOLECULAR EVOLUTION OF THIS GENE1

Bioluminescence is reported in members of 18 dinoflagellate genera. Species of dinoflagellates are known to have different bioluminescent signatures, making it difficult to assess the presence of particular species in the water column using optical tools, particularly when bioluminescent populations are in nonbloom conditions. A “universal” oligonucleotide primer set, along with species and genus‐specific primers specific to the luciferase gene were developed for the detection of bioluminescent dinoflagellates. These primers amplified luciferase sequences from bioluminescent dinoflagellate cultures and from environmental samples containing bioluminescent dinoflagellate populations. Novel luciferase sequences were obtained for strains of Alexandrium cf. catenella (Whedon et Kof.) Balech and Alexandrium fundyense Balech, and also from a strain of Gonyaulax spinifera (Clap. et Whitting) Diesing, which produces bioluminescence undetectable to the naked eye. The phylogeny of partial luciferase sequences revealed five significant clades of the dinoflagellate luciferase gene, suggesting divergence among some species and providing clues on their molecular evolution. We propose that the primers developed in this study will allow further detection of low‐light‐emitting bioluminescent dinoflagellate species and will have applications as robust indicators of dinoflagellate bioluminescence in natural water samples.     

Light and electron microscopic observations of feeding behavior, nutrition, and reproduction in laboratory cultures of Thalassicolla nucleata

T. nucleata is a skeletonless, single-celled radiolarian commonly found in ocean surface water. Specimens were cultured in the laboratory in a seawater--antibiotic medium. Feeding experiments show that T. nucleata is omnivorous with preference for crustacea, colorless flagellates, and occasional diatoms. Pigmented algae bearing organic thecae are occasionally consumed, but Dunaliella sp. is rejected. Rhizopodial activity during algal predation is compared to activity during crustacean predation, and evidence for differentiation of function among rhizopodia is presented. Electron microscopic observations and evidence from cytochalasin B treatment support the hypothesis that microfilaments mediate organized rhizopodial streaming. Reproduction is by isospores which resemble those in other spumellarian Radiolaria. Survival time in laboratory culture indicates that T. nucleata has a life span of at least 3 weeks. These findings are discussed in relation to the unique requirements imposed by a pelagic habitat on survival of a single-celled floating organism.     

Durinskia baltica (Dinophyceae), a newly recorded species and genus from China, and its systematics

A freshwater dinoflagellate was identified as Durinskia baltica (Levander) Carty & Cox by morphological characteristics,with the plate formula:Po,x,4',2a,6',5c,4s,5',2'.Durinskia was a newly recorded dinoflagellate genus for China with two anterior intercalary plates and six characteristic precingular plates.Partial sequences of the small and large subunit ribosomal DNA and internal transcribed spacer sequences for the dinoflagellate cells were obtained from field samples.Molecular phylogenetic results indicated Durinskia species could cluster into a monophyletic group,which were distinct from Peridinium species.According to morphological and molecular evidence,it was agreed that the genus Durinskia was separated from the genus Peridinium,which could be a polyphyletic group.In addition,D.baltica was an infrequent diatom-harboring dinoflagellate which was known to possess an endosymbiotic diatom or diatom-like alga.The phylogenetic analyses indicated that D.baltica had a close affinity with Peridiniopsis penardii and P.niei,common freshwater bloom-forming species in China.     

Phylogenetic position of the copepod-infesting parasite Syndinium turbo (Dinoflagellata, Syndinea)

Sequences were determined for the nuclear-encoded small subunit (SSU) rRNA and 5.8S rRNA genes as well as the internal transcribed spacers ITS1 and ITS2 of the parasitic dinoflagellate genus Syndinium from two different marine copepod hosts. Syndinium developed a multicellular plasmodium inside its host and at maturity free-swimming zoospores were released. Syndinium plasmodia in the copepod Paracalanus parvus produced zoospores of three different morphological types. However, full SSU rDNA sequences for the three morphotypes were 100% identical and also their ITS1-ITS2 sequences were identical except for four base pairs. It was concluded that the three morphotypes belong to a single species that was identified as Syndinium turbo, the type species of the dinoflagellate subdivision Syndinea. The SSU rDNA sequence of another Syndinium species infecting Corycaeus sp. was similar to Syndinium turbo except for three base pairs and the ITS1-ITS2 sequences of the two species differed at 34-35 positions. Phylogenetic analyses placed Syndinium as a sister taxon to the blue crab parasite Hematodinium sp. and both parasites were affiliated with the so-called marine alveolate Group II. This corroborates the hypothesis that marine alveolate Group II is Syndinea.     

Dinoflagellate phylogeny as inferred from heat shock protein 90 and ribosomal gene sequences

Background

Interrelationships among dinoflagellates in molecular phylogenies are largely unresolved, especially in the deepest branches. Ribosomal DNA (rDNA) sequences provide phylogenetic signals only at the tips of the dinoflagellate tree. Two reasons for the poor resolution of deep dinoflagellate relationships using rDNA sequences are (1) most sites are relatively conserved and (2) there are different evolutionary rates among sites in different lineages. Therefore, alternative molecular markers are required to address the deeper phylogenetic relationships among dinoflagellates. Preliminary evidence indicates that the heat shock protein 90 gene (Hsp90) will provide an informative marker, mainly because this gene is relatively long and appears to have relatively uniform rates of evolution in different lineages.

Methodology/Principal Findings

We more than doubled the previous dataset of Hsp90 sequences from dinoflagellates by generating additional sequences from 17 different species, representing seven different orders. In order to concatenate the Hsp90 data with rDNA sequences, we supplemented the Hsp90 sequences with three new SSU rDNA sequences and five new LSU rDNA sequences. The new Hsp90 sequences were generated, in part, from four additional heterotrophic dinoflagellates and the type species for six different genera. Molecular phylogenetic analyses resulted in a paraphyletic assemblage near the base of the dinoflagellate tree consisting of only athecate species. However, Noctiluca was never part of this assemblage and branched in a position that was nested within other lineages of dinokaryotes. The phylogenetic trees inferred from Hsp90 sequences were consistent with trees inferred from rDNA sequences in that the backbone of the dinoflagellate clade was largely unresolved.

Conclusions/Significance

The sequence conservation in both Hsp90 and rDNA sequences and the poor resolution of the deepest nodes suggests that dinoflagellates reflect an explosive radiation in morphological diversity in their recent evolutionary past. Nonetheless, the more comprehensive analysis of Hsp90 sequences enabled us to infer phylogenetic interrelationships of dinoflagellates more rigorously. For instance, the phylogenetic position of Noctiluca, which possesses several unusual features, was incongruent with previous phylogenetic studies. Therefore, the generation of additional dinoflagellate Hsp90 sequences is expected to refine the stem group of athecate species observed here and contribute to future multi-gene analyses of dinoflagellate interrelationships.     

SMALL SUBUNIT rDNA SEQUENCE ANALYSIS OF SYMBIOTIC DINOFLAGELLATES FROM SEVEN SCLERACTINIAN CORALS IN A TAHITIAN LAGOON

The diversity of symbiotic dinoflagellates from reef-building corals collected in the lagoon of Tahiti (South Pacific ocean) was investigated by using a molecular approach. Populations of symbionts (strains or species) of 7 coral species ( Fungia scutaria , F. paumotensis Stutchbury, Pavona cactus Forskål, Leptastrea transversa Kluzinger, Pocillopora verrucosa Ellis and Solender, Montastrea curta Dana, and Acropora formosa Dana) were delimited by phylogenetic analysis of small subunit rDNA sequences. Coral P. verrucosa harbored 2 populations of symbiont SSU rDNA sequences that may correspond to two different Symbiodinium species. Corals F. scutaria and M. curta also seemed to contain two different Symbiodinium species. SSU rDNA dinoflagellate sequences from P. cactus , L. transversa , F. scutaria , F. paumotensis , and P. verrucosa were in the same phylogenetic cluster and showed low variability. For these distantly related coral species, dinoflagellate strains from the same species, rDNA paralogues from the same strain, or closely related Symbiodinium species could not be distinguished because monophyletic subgroups were not observed. SSU rDNA dinoflagellate sequences from A. formosa and M. curta were clearly different from the other Symbiodinium sequences and may represent specific species. This molecular approach highlighted a greater diversity of symbiotic dinoflagellates from corals in South Pacific ( Symbiodinium groups A, B, and C) than that observed in the rest of the Pacific ocean ( Symbiodinium group C). The diversity of symbiotic associations in a restricted area of the lagoon of Tahiti may reflect the complexity of interactions between species of Symbiodinium and corals.     

Phylogenetic analyses indicate that the 19'Hexanoyloxy-fucoxanthin-containing dinoflagellates have tertiary plastids of haptophyte origin

The three anomalously pigmented dinoflagellates Gymnodinium galatheanum, Gyrodinium aureolum, and Gymnodinium breve have plastids possessing 19'-hexanoyloxy-fucoxanthin as the major carotenoid rather than peridinin, which is characteristic of the majority of the dinoflagellates. Analyses of SSU rDNA from the plastid and the nuclear genome of these dinoflagellate species indicate that they have acquired their plastids via endosymbiosis of a haptophyte. The dinoflagellate plastid sequences appear to have undergone rapid sequence evolution, and there is considerable divergence between the three species. However, distance, parsimony, and maximum-likelihood phylogenetic analyses of plastid SSU rRNA gene sequences place the three species within the haptophyte clade. Pavlova gyrans is the most basal branching haptophyte and is the outgroup to a clade comprising the dinoflagellate sequences and those of other haptophytes. The haptophytes themselves are thought to have plastids of a secondary origin; hence, these dinoflagellates appear to have tertiary plastids. Both molecular and morphological data divide the plastids into two groups, where G. aureolum and G. breve have similar plastid morphology and G. galatheanum has plastids with distinctive features.     

Phylogeny of Dinoflagellate Plastid Genes Recently Transferred to the Nucleus Supports a Common Ancestry with Red Algal Plastid Genes

It is generally accepted that peridinin-containing dinoflagellate plastids are derived from red alga, but whether they are secondary plastids equivalent to plastids of stramenopiles, haptophytes, or cryptophytes, or are tertiary plastids derived from one of the other secondary plastids, has not yet been completely resolved. As secondary plastids, plastid gene phylogeny should mirror that of nuclear genes, while incongruence in the two phylogenies should be anticipated if their origin was as tertiary plastids. We have analyzed the phylogeny of plastid-encoded genes from Lingulodinium as well as that of nuclear-encoded dinoflagellate homologues of plastid-encoded genes conserved in all other plastid genome sequences. Our analyses place the dinoflagellate, stramenopile, haptophyte, and cryptophyte plastids firmly in the red algal lineage, and in particular, the close relationship between stramenopile plastid genes and their dinoflagellate nuclear-encoded homologues is consistent with the hypothesis that red algal-type plastids have arisen only once in evolution.     

Molecular organization of dinoflagellate ribosomal DNA: evolutionary implications of the deduced 5.8 S rRNA secondary structure

The 5.8 S rRNA gene of Prorocentrum micans, a primitive dinoflagellate, has been cloned and its 159 base pairs (bp) have been sequenced along with the two flanking internal transcribed spacers (ITS 1 and 2), respectively, 212 and 195 bp long. Nucleotide sequence homologies between several previously published 5.8 S rRNA gene sequences including those from another dinoflagellate, an ascomycetous yeast, protozoans, a higher plant and a mammal have been determined by sequence alignment. Two prokaryotic 5'-ends of the 23 S rRNA gene have been compared owing to their probable common origin with eucaryotic 5.8 S rRNA genes. Several nucleotides are distinctive for dinoflagellates when compared with either typical eucaryotes or procaryotes. This is consistent with an early divergence of the dinoflagellate lineage from the typical eucaryotes. The secondary structure of dinoflagellate 5.8 S rRNA molecules fits the model of Walker et al. (1983). Conserved nucleotides which distinguish dinoflagellate 5.8 S rRNA from that of other eucaryotes are located in specific loops which are assumed to play a structural role in the ribosome. A 5.8 S rRNA phylogenetic tree which is proposed, based on sequence data, supports our initial assumption of the dinoflagellates.     

When Naked Became Armored: An Eight-Gene Phylogeny Reveals Monophyletic Origin of Theca in Dinoflagellates

The dinoflagellates are a diverse lineage of microbial eukaryotes. Dinoflagellate monophyly and their position within the group Alveolata are well established. However, phylogenetic relationships between dinoflagellate orders remain unresolved. To date, only a limited number of dinoflagellate studies have used a broad taxon sample with more than two concatenated markers. This lack of resolution makes it difficult to determine the evolution of major phenotypic characters such as morphological features or toxin production e.g. saxitoxin. Here we present an improved dinoflagellate phylogeny, based on eight genes, with the broadest taxon sampling to date. Fifty-five sequences for eight phylogenetic markers from nuclear and mitochondrial regions were amplified from 13 species, four orders, and concatenated phylogenetic inferences were conducted with orthologous sequences. Phylogenetic resolution is increased with addition of support for the deepest branches, though can be improved yet further. We show for the first time that the characteristic dinoflagellate thecal plates, cellulosic material that is present within the sub-cuticular alveoli, appears to have had a single origin. In addition, the monophyly of most dinoflagellate orders is confirmed: the Dinophysiales, the Gonyaulacales, the Prorocentrales, the Suessiales, and the Syndiniales. Our improved phylogeny, along with results of PCR to detect the sxtA gene in various lineages, allows us to suggest that this gene was probably acquired separately in Gymnodinium and the common ancestor of Alexandrium and Pyrodinium and subsequently lost in some descendent species of Alexandrium.     

Phylogenetic incongruence between dinoflagellate endosymbionts (Symbiodinium) and their host foraminifera (Sorites): small-subunit ribosomal RNA gene sequence evidence

Phototrophic dinoflagellate zooxanthellae commonly occur as endosymbionts in many planktic and certain benthic foraminifera (soritids). Many taxonomic issues and specific identities of foraminiferal dinoflagellates are not yet resolved. To assess taxonomic affinities among other dinoflagellates, we have determined the complete nucleotide sequence of the small-subunit rRNA coding region from Symbiodinium sp., an endosymbiotic dinoflagellate of the larger foraminifer Sorites orbiculus. The poly merase chain reaction was adopted for the in vitro amplification of ribosomal DNA, utilizing primers complementary to conserved regions. PCR-amplified DNA was directly sequenced and the sequence was aligned to all complete 18S-rDNA dinoflagellate sequences currently available through GenBank. Apicomplexan, ciliate, chromistacean, and rhodophycean sequences were added to infer across-kingdom phylogenetic relationships. Phylogenetic analysis of aligned nucleotide sequences produced a single most parsimonious tree (generated by the branch and bound method of PAUP). The inferred phylogeny indicates that the dinoflagellate extracted from the foraminifer Sorites orbiculus is a sister taxon to the symbiont present in the larger foraminifera Marginopora kudakajimaensis, but only distantly related to the dinoflagellate isolated from the soritid Amphisorus hemprichii. The sequence heterogeneity demonstrates a high degree of genetic diversity among Symbiodinium-like zooxanthellae and re-emphasizes that they are a variety of distinct entities.The inferred molecular phylogenetic relationships among symbiotic dinoflagellates are not congruent with the foraminiferal phylogeny based on cladistic methodology. The lack of correlation between the evolutionary history of dinoflagellate symbionts and their foraminiferal hosts argues against co-evolution. This lack of co-evolution implies that flexible recombinations among hosts and symbionts are evolutionarily favorable over permanently associated lineages, at least in these benthic foraminifera.     

Genetic polymorphism in Gymnodinium galatheanum chloroplast DNA sequences and development of a molecular detection assay

Nuclear and chloroplast-encoded small subunit ribosomal DNA sequences were obtained from several strains of the toxic dinoflagellate Gymnodinium galatheanum. Phylogenetic analyses and comparison of sequences indicate that the chloroplast sequences show a higher degree of sequence divergence than the nuclear homologue. The chloroplast sequences were chosen as targets for the development of a 5'--3' exonuclease assay for detection of the organism. The assay has a very high degree of specificity and has been used to screen environmental water samples from a fish farm where the presence of this dinoflagellate species has previously been associated with fish kills. Various hypotheses for the derived nature of the chloroplast sequences are discussed, as well as what is known about the toxicity of the species.     

Yihiella yeosuensis gen. et sp. nov. (suessiaceae,dinophyceae), a novel dinoflagellate isolated from the coastal waters of Korea

A small (7–11 μm long) dinoflagellate with thin amphiesmal plates was isolated into culture from a water sample collected in coastal waters of Yeosu, southern Korea, and examined by LM, SEM, and TEM, and molecular analyses. The hemispheric episome was smaller than the hyposome. The nucleus was oval and situated from the central to the episomal region of the cell. A large yellowish‐brown chloroplast was located at the end of the hyposome, and some small chloroplasts extended into the periphery of the episome. The dinoflagellate had a single elongated apical vesicle (EAV) and a type E eyespot, which are key characteristics of the family Suessiaceae. Unlike other genera in this family, it had two long furrow lines, one on the episome and the other on the hyposome, and encircling the dorsal, and lateral sides of the cell body. The pyrenoid lacked starch sheaths, but tubular invaginations into the pyrenoid matrix from the cytoplasm were observed. In the TEM, the dinoflagellate was observed to have cable‐like structures (CLSs) near the eyespot but so far not observed in other dinoflagellates. The SSU rDNA sequences examined were 1.2%–5.1% different from those of other genera in the family Suessiaceae, whereas the LSU (D1‐D3) rDNA sequences of this dinoflagellate were 15.1%–31.5% different. The dinoflagellate lacked a 51‐bp fragment in domain D2 of the LSU rDNA, but it had an ~100‐bp fragment in domain D2. This feature has been found previously only in the genera Leiocephalium and Polarella, two other genera of the Suessiaceae. The molecular phylogeny and sequence divergence based on SSU, and LSU rDNA indicate that the Korean dinoflagellate holds a taxonomically distinctive position and we consider it to be a new species in a new genus in the family Suessiaceae, named Yihiella yeosuensis gen. et sp. nov.     

Origins of plastids and glyceraldehyde-3-phosphate dehydrogenase genes in the green-colored dinoflagellate Lepidodinium chlorophorum

The dinoflagellate Lepidodinium chlorophorum possesses "green" plastids containing chlorophylls a and b (Chl a+b), unlike most dinoflagellate plastids with Chl a+c plus a carotenoid peridinin (peridinin-containing plastids). In the present study we determined 8 plastid-encoded genes from Lepidodinium to investigate the origin of the Chl a+b-containing dinoflagellate plastids. The plastid-encoded gene phylogeny clearly showed that Lepidodinium plastids were derived from a member of Chlorophyta, consistent with pigment composition. We also isolated three different glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes from Lepidodinium-one encoding the putative cytosolic "GapC" enzyme and the remaining two showing affinities to the "plastid-targeted GapC" genes. In a GAPDH phylogeny, one of the plastid-targeted GapC-like sequences robustly grouped with those of dinoflagellates bearing peridinin-containing plastids, while the other was nested in a clade of the homologues of haptophytes and dinoflagellate genera Karenia and Karlodinium bearing "haptophyte-derived" plastids. Since neither host nor plastid phylogeny suggested an evolutionary connection between Lepidodinium and Karenia/Karlodinium, a lateral transfer of a plastid-targeted GapC gene most likely took place from a haptophyte or a dinoflagellate with haptophyte-derived plastids to Lepidodinium. The plastid-targeted GapC data can be considered as an evidence for the single origin of plastids in haptophytes, cryptophytes, stramenopiles, and alveolates. However, in the light of Lepidodinium GAPDH data, we need to closely examine whether the monophyly of the plastids in the above lineages inferred from plastid-targeted GapC genes truly reflects that of the host lineages.     

COMPLEX GENE STRUCTURE OF THE FORM II RUBISCO IN THE DINOFLAGELLATE PROROCENTRUM MINIMUM (DINOPHYCEAE)1

We analyzed the unusually complex organization of the nuclear‐encoded (form II) RUBISCO gene in the dinoflagellate Prorocentrum minimum (Parvillard) Schiller by intensive genomic DNA and cDNA sequencing and Western blotting. Over 10 transcribed units (TUs) were detected, which varied dramatically in their 3′ untranslated region. Each TU appeared to contain four tandem copies of the RUBISCO coding region (1.46 kb each; coding unit, or CU) interspersed by a 63‐bp spacer; the four CUs in each TU were cotranscribed and apparently cotranslated to a tetrameric polyprotein that may undergo successive cleavage steps to yield mature RUBISCO. By means of real‐time PCR analysis, it was estimated that each of the P. minimum genome harbored 148±16 CUs. Although nucleotide sequences varied by 1%–9% among the detected CUs, their inferred amino acid sequences were essentially identical. Our results suggest that the complex structure of Pmrbc has been derived from extensive and repeated gene duplications, an evolutionary process that has also been observed for other dinoflagellate genes.     

Molecular diversity of alveolates associated with neritic north atlantic radiolarians

Seven species of polycystine radiolarians and one phaeodarian species were investigated in order to determine the diversity of their associate organisms and their species specificity. Twelve partial 18S ribosomal DNA (rDNA) sequences were obtained showing a high diversity of associates, both within spumellarian and nassellarian radiolarians and among species. Two of the sequences obtained are highly similar to Scrippsiella, a dinoflagellate genus already reported as a symbiont of polycystine radiolarians. Nine of the new 18S rDNA sequences group with various alveolates. Some of these groups include parasites, such as the lethal endoparasite Amoebophrya, while others consist of non-annotated novel organisms found worldwide in various types of marine environments. We also obtained a sequence from a bacillariophytan highly similar to the 18S rDNA of the diatom species Diatoma tenue, which may derive from radiolarian food. Additionally, this is the first study to report on a phaeodarian associate.