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.     

Effects of macroalgae Ulva pertusa (Chlorophyta) and Gracilaria lemaneiformis (Rhodophyta) on growth of four species of bloom-forming dinoflagellates

The effects of fresh thalli and culture medium filtrates from two species of marine macroalgae, Ulva pertusa Kjellm (Chlorophyta) and Gracilaria lemaneiformis (Bory) Dawson (Rhodophyta), on growth of marine microalgae were investigated in co-culture under controlled laboratory conditions. A selection of microalgal species were used, all being identified as bloom-forming dinoflagellates: Prorocentrum donghaiense Lu sp., Alexandrium tamarense (Lebour) Balech, Amphidinium carterae Hulburt and Scrippsiella trochoide (Stein) Loeblich III. Results showed that the fresh thalli of either U. pertusa or G. lemaneiformis significantly inhibited the microalgal growth, or caused mortality at the end of the experiment. However, the overall effects of the macroalgal culture filtrates on the growth of the dinoflagellates were species-specific (inhibitory, stimulatory or none) for different microalgal species. Results indicated an allelopathic effect of macroalga on the co-cultured dinoflagellate. We then took P. donghaiense as an example to further assess this hypothesis. The present study was carried out under controlled conditions, thereby excluded the fluctuation in light and temperature. Nutrient assays showed that nitrate and phosphate were almost exhausted in G. lemaneiformis co-culture, but remained at enough high levels in U. pertusa co-culture, which were well above the nutrient limitation for the microalgal growth, when all cells of P. donghaiense were killed in the co-culture. Daily f/2 medium enrichment greatly alleviated the growth inhibition on P. donghaiense in G. lemaneiformis co-culture, but could not eliminate it. Other environmental factors, such as carbonate limitation, bacterial presence and the change of pH were also not necessary for the results. We thus concluded that allelopathy was the most possible reason leading to the negative effect of U. pertusa on P. donghaiense, and the combined roles of allelopathy and nutrient competition were essential for the effect of G. lemaneiformis on P. donghaiense.     

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.     

Roles of mixotrophy in blooms of different dinoflagellates: Implications from the growth experiment

Studies over the last two decades suggested that mixotrophy could be an important adaptive strategy for some bloom-forming dinoflagellates. In the coastal waters adjacent to the Changjiang River estuary in the East China Sea, recurrent blooms of dinoflagellates Prorocentrum donghaiense, Karenia mikimotoi and Alexandrium catenella started to appear from the beginning of the 21 century, but roles of mixotrophy in the formation of dinoflagellate blooms were not well understood. In the current study, mixotrophy-based growth of four selected bloom-causative dinoflagellate species, i.e. K. mikimotoi, A. catenella, P. donghaiense and Prorocentrum micans, were studied. Dinoflagellates were co-cultured with different prey organisms, including bacterium Marinobacter sp., microalgae Isochrysis galbana and Hemiselmis virescens, under a variant of nutrient conditions. It was found that growth of dinoflagellate K. mikimotoi was significantly promoted with the presence of prey organisms. Growth of P. donghaiense and P. micans was only slightly improved. For A. catenella, the addition of prey organisms has no effects on the growth, while both of the two prey microalgae I. galbana and H. virescens were killed, probably by allelochemicals released from A. catenella. There was no apparent relationship between nutrient conditions and the mixotrophy-based growth of the tested dinoflagellates. Based on the results of the growth experiment, it is implicated that mixotrophy may play different roles in the growth and bloom of the four dinoflagellate species. It can be an important competitive strategy for K. mikimotoi. For the two Prorocentrum species and A. catenella, however, the role of mixotrophy is much limited. They may depend more on other competitive strategies, such as phototrophy-based growth and allelopathic effect, to prevail in the phytoplankton community and form blooms.     

Combined heat shock protein 90 and ribosomal RNA sequence phylogeny supports multiple replacements of dinoflagellate plastids

Dinoflagellates harbour diverse plastids obtained from several algal groups, including haptophytes, diatoms, cryptophytes, and prasinophytes. Their major plastid type with the accessory pigment peridinin is found in the vast majority of photosynthetic species. Some species of dinoflagellates have other aberrantly pigmented plastids. We sequenced the nuclear small subunit (SSU) ribosomal RNA (rRNA) gene of the "green" dinoflagellate Gymnodinium chlorophorum and show that it is sister to Lepidodinium viride, indicating that their common ancestor obtained the prasinophyte (or other green alga) plastid in one event. As the placement of dinoflagellate species that acquired green algal or haptophyte plastids is unclear from small and large subunit (LSU) rRNA trees, we tested the usefulness of the heat shock protein (Hsp) 90 gene for dinoflagellate phylogeny by sequencing it from four species with aberrant plastids (G. chlorophorum, Karlodinium micrum, Karenia brevis, and Karenia mikimotoi) plus Alexandrium tamarense, and constructing phylogenetic trees for Hsp90 and rRNAs, separately and together. Analyses of the Hsp90 and concatenated data suggest an ancestral origin of the peridinin-containing plastid, and two independent replacements of the peridinin plastid soon after the early radiation of the dinoflagellates. Thus, the Hsp90 gene seems to be a promising phylogenetic marker for dinoflagellate phylogeny.     

Molecular evidence that plastids in the toxin-producing dinoflagellate genus Dinophysis originate from the free-living cryptophyte Teleaulax amphioxeia

Some species of the dinoflagellate genus Dinophysis form red tides and are toxin producers with a great environmental impact. The dinoflagellates as a group display high plastid diversity. Several cases indicate that plastids have been replaced. In the case of the genus Dinophysis, the plastids show characteristics of a plastid originating from a cryptophyte. Recent molecular evidence showed that the plastid indeed originates from a cryptophyte, but the source could not be identified to species or genus level. The data presented here show that both a 799 bp region of the psbA gene and 1,221 bp region of the 16S rRNA gene from Dinophysis spp. are identical to the same loci in Teleaulax amphioxeia SCCAP K434. This strongly indicates that the plastid was acquired recently in Dinophysis and may be a so-called kleptoplastid, specifically originating from a species of Teleaulax.     

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.     

Growth and competition of several harmful dinoflagellates under different nutrient and light conditions

Three near-shore type harmful dinoflagellates, Prorocentrum minimum, Prorocentrum donghaiense and Karlodinium veneficum, and one off-shore dinoflagellate, Karenia brevis, were grown in laboratory monocultures and mixed batch cultures. The dinoflagellate cultures were grown on treatments of two ambient nitrogen (N):phosphorus (P) ratios; two N substrates (nitrate and urea) and two light intensities. The microalgae Rhodomonas and Synechococcus were also added in separate treatments to the mixed culture treatments as potential food sources. All tested species grew well on both N substrates. In mixed culture, P. minimum outgrew K. veneficum, and P. donghaiense outgrew K. brevis in most treatments reaching higher growth rates and higher biomass. However, when a third algae, Rhodomonas, was added, the growth of P. minimum was inhibited relative to that of K. veneficum. In contrast, when grown with K. brevis, the growth rate of P. donghaiense was not significantly affected by the addition of potential prey. K. brevis had a longer growth phase, and kept growing after P. donghaiense reached stationary phase, suggesting better adaptation of K. brevis to low inorganic nutrient conditions. The growth of K. brevis was also significantly limited in the low light treatment. K. veneficum overgrew P. minimum in the presence of Rhodomonas, a potential nutrient source. The growth rates of both K. brevis and P. donghaiense were reduced with the presence of Synechococcus. In addition to nutrient competition, mixotrophy and allelopathy were likely mechanisms in determining the dominant species.     

Proliferating cell nuclear antigen (PCNA) as a marker of cell proliferation in the marine dinoflagellate <Emphasis Type="BoldItalic">Prorocentrum donghaiense</Emphasis> Lu and the green alga <Emphasis Type="BoldItalic">Dunaliella salina</Emphasis> Teodoresco

Proliferating cell nuclear antigen (PCNA) was detected in Prorocentrum donghaiense Lu and Dunaliella salina Teodoresco by enhanced chemiluminescence techniques with one mono-antibody. The observed band detected on western blots of P. donghaiense and D. salina had a molecular weight of 36–33 kDa rather than a PCNA-like protein with a size of 55 kDa reported in the dinoflagellates Crypthecodinium cohnii Biecheler and Gymnodinium catenatum Brav. The abundance of PCNA proteins was growth-stage dependent. Whole-cell immunoflurescence labeling showed that the PCNA antibody specifically stained the target proteins in P. donghaiense and D. salina, and PCNA is only present in the nucleus during the cell cycle. Synchronized cells of P. donghaiense show a cell cycle specific expression pattern with the highest expression in S phase and little expression in the G₁ and G₂/M phases. The results demonstrated that the PCNA-like proteins could be a marker for the estimation of marine phytoplankton growth rates. The different sizes of the PCNA-like proteins observed in dinoflagellates could be related to the variety of dinoflagellate chromosomal structure.     

Hypervariable regions (V1–V9) of the dinoflagellate 18S rRNA using a large dataset for marker considerations

DNA sequencing methods have been used for the molecular taxonomic discrimination of dinoflagellate protists, particularly using partial 18S rRNA sequences. This study evaluated the taxonomic discrimination power of rRNA gene hypervariable regions (V1 to V9) in dinoflagellates from a large dataset. These included 77 dinoflagellate species (9 orders, 17 families, 40 genera). The complete 18S rRNA sequences of the dinoflagellates ranged from 1,787 to 1,813?bp in length, and consisted of eight V regions with a total combined length of 678 to 699?bp. Regions longer than 100?bp were recoded for V2, V4, and V8 regions; high nucleotide divergences were detected in V1, V2, and V4 regions. Statistic tests showed that the divergences of individual V regions were significantly different (t-test, P?<?0.05) compared with the complete 18S rRNA. The V2 region showed the highest score (83.5%) for PI sites. Moreover, intra-genus DNA similarities of the V2 were considerably low (<93%). Neighbor-joining analyses showed that phylogenetic resolution in the V2–V4 region was 1.32-fold higher than that of the complete 18S rRNA. These results demonstrate that V2 has the highest taxonomic resolving power within the 18S rRNA gene of dinoflagellates, suggesting the V2 and adjacent regions (e.g., V1 to V4) may be the best for marker considerations.     

Phylogenetic relations of the dinoflagellate Gymnodinium baicalense from Lake Baikal

Freshwater dinoflagellates still remain poorly studied by modern biological methods. This lack of knowledge prevents us from understanding the evolution and colonization patterns of these ecologically important protists. Gymnodinium baicalense is the most abundant, and possibly endemic, planktonic dinoflagellate from the ancient Lake Baikal. This dinoflagellate species blooms in the spring under the ice. This study analyzed the origin of this Baikalian dinoflagellate using three markers (two ribosomal and one mitochondrial DNA). It was found that this species is a true member of the order Gymnodiniales and has close relatives in the glacial melt waters of the Arctic Ocean. It seems that G. baicalense has diversified relatively recently from the arctic marine gymnodinioids. These results shed light on dinoflagellate biogeography and their colonizations in Lake Baikala biodiversity hotspot.     

Origin of the plastid in the anomalously pigmented dinoflagellate Gymnodinium mikimotoi (Gymnodiniales, Dinophyta) as inferred from phylogenetic analysis based on the gene encoding the large subunit of form I-type RuBisCO

The dinoflagellate Gymnodinium mikimotoi Miyake et Kominami ex Oda possesses an anomalously pigmented plastid which contains 19′‐hexanoyloxyfucoxanthin, 19′‐butanoyloxyfucoxanthin and fucoxanthin instead of peridinin as the major carotenoids. Previously, we have shown that the plastid of G. mikimotoi belongs to the rhodoplast lineage as inferred from phylogenetic analyses based on the amino acid sequences deduced from psbA and psaA and the nucleotide sequence of the plastid small subunit ribosomal RNA. Furthermore, in the present study, we cloned and sequenced an additional representative plastid gene, rbcL, encoding the large subunit of ribulose 1–5 bisphosphate carboxylase/oxygenase (RuBisCO LSU) from G. mikimotoi. The amino acid sequence deduced from the rbcL gene of G. mikimotoi apparently revealed the conventional form I RuBisCO LSU, which is present in most photosynthetic organisms, and not the divergent form II existing in typically pigmented dinofl age Nates with plastids containing peridinin as the main carotenoid. This finding supports the hypothesis that the origins of the plastids in G. mikimotoi and peridinin‐type dinoflagellates are not related to each other. Molecular phylogenetic analysis based on the amino acid sequence deduced from the rbcL gene further showed that the plastid of G. mikimotoi belongs to the rhodoplast lineage. In particular, G. mikimotoi clustered with haptophytes in the phylogenetic tree. From this result, two hypotheses with respect to the origin of the plastid in G. mikimotoi can be proposed: G. mikimotoi may have engulfed a haptophyte‐like cell (tertiary symbiosis) or englulfed a rhodophyte‐like cell that was closely related to the origin of the plastid in the haptophyte (secondary symbiosis).     

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.     

Anucleated cryptophyte vestiges in the gonyaulacalean dinoflagellates Amylax buxus and Amylax triacantha (Dinophyceae)

Cryptophyte vestiges showing selective digestion of nuclei were found in the gonyaulacalean dinoflagellates Amylax buxus (Balech) Dodge and Amylax triacantha (Jörgensen) Sournia. They emitted bright yellow‐orange fluorescence (590‐nm emission) under epifluorescent microscopy and possessed U‐shaped plastids, suggesting the vestiges were active in photosynthesis. Under transmission electron microscopy, the plastid was characterized by a loose arrangement of two to three thylakoid stacks and included a stalked pyrenoid, as in the cryptophyte genus Teleaulax. Indeed, molecular data based on the plastid small‐subunit rRNA gene demonstrated that the vestiges in Amylax originated from Teleaulax amphioxeia. The stolen plastid (kleptoplastids) in Dinophysis is also derived from this cryptophyte species. However, in sharp contrast to Dinophysis, the plastid of the vestige in Amylax was surrounded by a double layer of plastid endoplasmic reticulum, and within the periplastidal area, a nucleomorph was retained. The vestiges also possessed mitochondria with characteristic plate‐like cristae, but lost the cell‐surface structure. The phagocytotic membrane of the dinoflagellates seemed to surround the cryptophytes right after the incorporation, but the membrane itself would probably be digested eventually. Remarkably, only one cryptophyte cell among 14 vestiges in a cell of A. buxus had a nucleus. This is the first recording of possible kleptoplastidy in gonyaulacalean dinoflagellates, and documents the strategy of a dinoflagellate involving the selective elimination of the cryptophyte nucleus.     

Rate Variation as a Function of Gene Origin in Plastid-Derived Genes of Peridinin-Containing Dinoflagellates

Peridinin-pigmented dinoflagellates contain secondary plastids that seem to have undergone more nearly complete plastid genome reduction than other eukaryotes. Many typically plastid-encoded genes appear to have been transferred to the nucleus, with a few remaining genes found on minicircles. To understand better the evolution of the dinoflagellate plastid, four categories of plastid-associated genes in dinoflagellates were defined based on their history of transfer and evaluated for rate of sequence evolution, including minicircle genes (presumably plastid-encoded), genes probably transferred from the plastid to the nucleus (plastid-transferred), and genes that were likely acquired directly from the nucleus of the previous plastid host (nuclear-transferred). The fourth category, lateral-transferred genes, are plastid-associated genes that do not appear to have a cyanobacterial origin. The evolutionary rates of these gene categories were compared using relative rate tests and likelihood ratio tests. For comparison with other secondary plastid-containing organisms, rates were calculated for the homologous sequences from the haptophyte Emiliania huxleyi. The evolutionary rate of minicircle and plastid-transferred genes in the dinoflagellate was strikingly higher than that of nuclear-transferred and lateral-transferred genes and, also, substantially higher than that of all plastid-associated genes in the haptophyte. Plastid-transferred genes in the dinoflagellate had an accelerated rate of evolution that was variable but, in most cases, not as extreme as the minicircle genes. Furthermore, the nuclear-transferred and lateral-transferred genes showed rates of evolution that are similar to those of other taxa. Thus, nucleus-to-nucleus transferred genes have a more typical rate of sequence evolution, while those whose history was wholly or partially within the dinoflagellate plastid genome have a markedly accelerated rate of evolution. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Debashish Battacharya]     

Genome size‐dependent pcna gene copy number in dinoflagellates and molecular evidence of retroposition as a major evolutionary mechanism

Proliferating cell nuclear antigen (PCNA) plays critical roles in eukaryotic DNA replication and replication‐associated processes. It is typically encoded by one or two gene copies (pcna) in eukaryotic genomes. Recently reported higher copy numbers of pcna in some dinoflagellates raised a question of how this gene has uniquely evolved in this phylum. Through real‐time PCR quantification, we found a wide range of pcna copy number (2–287 copies) in 11 dinoflagellate species (n = 38), and a strong positive correlation between pcna copy number and genome size (log₁₀–log₁₀ transformed). Intraspecific pcna diverged up to 21% and are dominated by nonsynonymous substitutions, indicating strong purifying selection pressure on and hence functional necessity of this gene. By surveying pcna copy numbers in eukaryotes, we observed a genome size threshold at 4 pg DNA, above which more than two pcna copies are found. To examine whether retrotransposition is a mechanism of pcna duplication, we measured the copy number of retroposed pcna, taking advantage of the 22‐nt dinoflagellate‐specific spliced leader (DinoSL) capping the 5′ end of dinoflagellate nuclear‐encoded mRNAs, which would exist in the upstream region of a retroposed gene copy. We found that retroposed pcna copy number increased with total pcna copy number and genome size. These results indicate co‐evolution of dinoflagellate pcna copy number with genome size, and retroposition as a major mechanism of pcna duplication in dinoflagellates. Furthermore, we posit that the demand of faithful replication and maintenance of the large dinoflagellate genomes might have favored the preservation of the retroposed pcna as functional genes.