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.     

Molecular data and the evolutionary history of dinoflagellates

We have sequenced small-subunit (SSU) ribosomal RNA (rRNA) genes from 16 dinoflagellates, produced phylogenetic trees of the group containing 105 taxa, and combined small- and partial large-subunit (LSU) rRNA data to produce new phylogenetic trees. We compare phylogenetic trees based on dinoflagellate rRNA and protein genes with established hypotheses of dinoflagellate evolution based on morphological data. Protein-gene trees have too few species for meaningful in-group phylogenetic analyses, but provide important insights on the phylogenetic position of dinoflagellates as a whole, on the identity of their close relatives, and on specific questions of evolutionary history. Phylogenetic trees obtained from dinoflagellate SSU rRNA genes are generally poorly resolved, but include by far the most species and some well-supported clades. Combined analyses of SSU and LSU somewhat improve support for several nodes, but are still weakly resolved. All analyses agree on the placement of dinoflagellates with ciliates and apicomplexans (=Sporozoa) in a well-supported clade, the alveolates. The closest relatives to dinokaryotic dinoflagellates appear to be apicomplexans, Perkinsus, Parvilucifera, syndinians and Oxyrrhis. The position of Noctiluca scintillans is unstable, while Blastodiniales as currently circumscribed seems polyphyletic. The same is true for Gymnodiniales: all phylogenetic trees examined (SSU and LSU-based) suggest that thecal plates have been lost repeatedly during dinoflagellate evolution. It is unclear whether any gymnodinialean clades originated before the theca. Peridiniales appear to be a paraphyletic group from which other dinoflagellate orders like Prorocentrales, Dinophysiales, most Gymnodiniales, and possibly also Gonyaulacales originated. Dinophysiales and Suessiales are strongly supported holophyletic groups, as is Gonyaulacales, although with more modest support. Prorocentrales is a monophyletic group only in some LSU-based trees. Within Gonyaulacales, molecular data broadly agree with classificatory schemes based on morphology. Implications of this taxonomic scheme for the evolution of selected dinoflagellate features (the nucleus, mitosis, flagella and photosynthesis) are discussed.     

A Three-Gene Dinoflagellate Phylogeny Suggests Monophyly of Prorocentrales and a Basal Position for <Emphasis Type="Italic">Amphidinium</Emphasis> and <Emphasis Type="Italic">Heterocapsa</Emphasis>

Many outstanding questions about dinoflagellate evolution can potentially be resolved by establishing a robust phylogeny. To do this, we generated a data set of mitochondrial cytochrome b (cob) and mitochondrial cytochrome c oxidase 1 (cox1) from a broad range of dinoflagellates. Maximum likelihood, maximum parsimony, and Bayesian methods were used to infer phylogenies from these genes separately and as a concatenated alignment with and without small subunit (SSU) rDNA sequences. These trees were largely congruent in topology with previously published phylogenies but revealed several unexpected results. Prorocentrum benthic and planktonic species previously placed in different clusters formed a monophyletic group in all trees, suggesting that the Prorocentrales is a monophyletic group. More strikingly, our analyses placed Amphidinium and Heterocapsa as early splits among dinoflagellates that diverged after the emergence of O. marina. This affiliation received strong bootstrap support, but these lineages exhibited relatively long branches. The approximately unbiased (AU-) test was used to assess this result using a three-gene (cob + cox1 + SSU rDNA) DNA data set and the inferred tree. This analysis showed that forcing Amphidinium or Heterocapsa to relatively more derived positions in the phylogeny resulted in significantly lower likelihood scores, consistent with the phylogenies. The position of these lineages needs to be further verified. Reviewing Editor: Dr. Martin Kreitman     

MITOCHONDRIAL DNA PHYLOGENY OF THE SYMBIOTIC DINOFLAGELLATES (SYMBIODINIUM,DINOPHYTA)1

Symbiotic dinoflagellates belonging to the genus Symbiodinium (Freudenthal) are found worldwide in association with shallow‐water tropical and subtropical marine invertebrates. Most phylogenetic studies of Symbiodinium have used nuclear rRNA (nrDNA) genes to infer relationships among members of the genus. In this report, we present the first phylogeny of Symbiodinium based on DNA sequences from a mitochondrial protein‐coding gene (cytochrome oxidase subunit I [cox1]). Two principal groups, one comprised of Symbiodinium clade A and the second encompassing Symbiodinium clades B/C/D/E/F, are strongly supported in the cox1 phylogeny. Relationships within Symbiodinium clades B/C/D/E/F, however, are less well resolved compared with phylogenies inferred from nrDNA and chloroplast large subunit (cp23S)‐rDNA genes. Statistical tests between alternative tree topologies verified, with an exception being the position of one controversial member of Symbiodinium clade D, that relationships inferred from cox1 are congruent with those inferred from nrDNA and cp23S‐rDNA. Taken together, the relationships between the major Symbiodinium clades are robust, and there appears to be no evidence of hybridization or differential introgression of nuclear and plastid genomes between clades.     

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.     

Revised dinoflagellate phylogeny inferred from molecular analysis of large-subunit ribosomal RNA gene sequences

The nucleotide sequence analysis of the PCR products corresponding to the variable large-subunit rRNA domains D1, D2, D9, and D10 from ten representative dinoflagellate species is reported. Species were selected among the main laboratory-grown dinoflagellate groups: Prorocentrales, Gymnodiniales, and Peridiniales which comprise a variety of morphological and ecological characteristics. The sequence alignments comprising up to 1,000 nucleotides from all ten species were employed to analyze the phylogenetic relationships among these dinoflagellates. Maximum parsimony and neighbor joining trees were inferred from the data generated and subsequently tested by bootstrapping. Both the D1/D2 and the D9/D10 regions led to coherent trees in which the main class of dinoflagellates, Dinophyceae, is divided in three groups: prorocentroid, gymnodinioid, and peridinioid. An interesting outcome from the molecular phylogeny obtained was the uncertain emergence of Prorocentrum lima. The molecular results reported agreed with morphological classifications within Peridiniales but not with those of Prorocentrales and Gymnodiniales. Additionally, the sequence comparison analysis provided strong evidence to suggest that Alexandrium minutum and Alexandrium lusitanicum were synonymous species given the identical sequence they shared. Moreover, clone Gg1V, which was determined Gymnodinium catenatum based on morphological criteria, would correspond to a new species of the genus Gymnodinium as its sequence clearly differed from that obtained in G. catenatum. The sequence of the amplified fragments was demonstrated to be a valuable tool for phylogenetic and taxonomical analysis among these highly diversified species. Correspondence to: J. M. Bautista     

STRUCTURE AND EVOLUTION OF THE RDNA INTERNAL TRANSCRIBED SPACER (ITS) REGION 2 IN THE SYMBIOTIC DINOFLAGELLATES (SYMBIODINIUM,DINOPHYTA)1

Internal transcribed spacer (ITS) regions of the eukaryotic rDNA operon are integral to the correct processing and maturation of rRNAs. To further understand the evolution of this region, we elucidated the secondary structure of ITS2 from representatives of the eight divergent clades of Symbiodinium Freud., a large genus of dinoflagellate endosymbionts occurring in association with zooxanthellate marine protists and invertebrates. Symbiodinium ITS2 molecules folded into one of two distinct conformations. One conformation, the “four‐fingered hand” model, has been described from a wide variety of eukaryotes, including free‐living dinoflagellates. A monophyletic assemblage comprising several Symbiodinium clades shared an unusual conformation, a five‐stem model previously known only from drosopholids, indicating that it arose in the common ancestor to this “superclade” of Symbiodinium. Several conserved features were identified in the ITS2 secondary structures, including a pyrimidine–pyrimidine bulge and a highly conserved 11 bp sequence motif, that correspond to known processing sites in other eukaryotes. Lastly, the ITS2 structural data are discussed in the context of Symbiodinium evolution, phylogenetics, and ecology.     

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.     

Dinoflagellate-targeted PCR reveals highly abundant and diverse communities of parasitic dinoflagellates in and near Zhubi Reef,South China Sea

While diversity of symbiodiniacean dinoflagellates has been a focus of coral reef ecological research, information on the diversity of planktonic dinoflagellates in reef ecosystems remains limited. We used dinoflagellate-targeted PCR to investigate dinoflagellate diversity for a coral reef ecosystem. In the summer of 2007, plankton samples were collected from a lagoon, atoll, and open sea area of Zhubi Reef in the Nansha Islands, South China Sea. Sequencing of dinoflagellate-specific SSU rDNA clone libraries from samples in each of these habitats revealed high diversity and numerous novel dinoflagellate lineages. Gymnodiniales were most abundantly represented in all three water areas. Lagoon assemblages were co-dominated by Syndiniales and Gonyaulacales, the atoll by Gonyaulacales and Peridiniales, and the open sea by Syndiniales and Prorocentrales taxa. Species in the Syndiniales (group II) genus Amoebophrya were represented by eight new sequences and 13 previously described clades and were dominated by species reported to infect Gymnodiniales, Gonyaulacales, Peridiniales, and Prorocentrales taxa. And Amoebophrya were particularly abundant and diverse in the lagoon. Our results suggest that Amoebophrya probably play an important role in regulating dynamics of dinoflagellate assemblages in the Zhubi Reef coral ecosystem. In contrast, the few symbiodiniacean taxa detected occurred only in the open sea, suggesting planktonic aposymbiotic Symbiodiniaceae rarely occur in the reef ecosystem. We demonstrate the usefulness of a dinoflagellate-specific molecular technique for profiling dinoflagellate communities, and uncover diversity and the potential importance of parasitic lineages in a coral reef ecosystem.

     

mRNA EDITING AND SPLICED‐LEADER RNA TRANS‐SPLICING GROUPS OXYRRHIS,NOCTILUCA, HETEROCAPSA,AND AMPHIDINIUM AS BASAL LINEAGES OF DINOFLAGELLATES1

Identification of novel dinoflagellate taxa through molecular analysis is hindered by lack of well‐defined basal lineages. To address this issue, we attempted to reassess the phylogenetic status of Oxyrrhis marina Dujard. as well as other potentially basal taxa. The analysis was based on two newly established premises: (1) editing density of mitochondrial cob and cox1 mRNA increases from basal to later diverging lineages; (2) nuclear‐encoded mRNA in dinoflagellates is trans‐spliced to receive a 22 bp spliced leader (SL) at the 5′‐end. We analyzed these two genetic traits in O. marina, Noctiluca scintillans (Macartney) Kof. et Swezy, Heterocapsa triquetra (Ehrenb.) F. Stein, H. rotundata (Lohmann) Ge. Hansen, Amphidinium carterae Hulburt, and A. operculatum Clap. et J. Lachm. Surprisingly, no editing was detected in cob and cox1 mRNAs in these lineages, except for a small number of editing events in Amphidinium. However, nuclear‐encoded mRNAs in these species contained the SL sequence at the 5′‐end, indicative of SL RNA trans‐splicing. These findings, together with the recent cob‐cox1‐18S rRNA three‐gene phylogeny, suggest the following: (1) O. marina is a basal dinoflagellate; (2) Heterocapsa, Amphidinium, and Noctiluca likely are also early diverging lineages of dinoflagellates, and the position of Heterocapsa is inconsistent with literature and needs further investigation; and (3) the presence of the 22 bp SL and mitochondrial (mt) mRNA editing can be considered a landmark of dinoflagellate splits.     

Phylogenetic analyses of the dinoflagellate Noctiluca scintillans based on beta-tubulin and Hsp90 genes

The noctilucid dinoflagellate Noctiluca scintillans is an unarmed heterotrophic protist that inhabits the world's oceans and is sometimes responsible for harmful red tides. The phylogenetic position of the noctilucids has been widely disputed because of two alternative views based on morphological characters and phylogenetic analyses using SSU rDNA. Specifically, noctilucids are either placed in a basal position within the dinoflagellates or they are seen as evolutionarily recent derivations descended from unarmored dinoflagellates in the order Gymnodiniales. Thus, the precise relationship of noctilucids to other dinoflagellates is still uncertain. In this study, we isolated β-tubulin and heat shock protein 90 genes from N. scintillans to examine this relationship further. The deduced amino acid sequences share commonly substituted amino acids and a deletion with other dinoflagellates, but not with Perkinsus marinus or other alveolates. Although Hsp90 analysis did not give robust support, β-tubulin analysis including an AU test, as well as combined analysis of these two amino acid sequences showed that N. scintillans is the next earliest branch after Oxyrrhis marina, within the dinoflagellates. Given the phylogenetic position of N. scintillans, its extremely specialized diploid trophont, and the primitive dinoflagellate-like characteristics of its haploid zoospore, we propose that noctilucids are a possible evolutionary link between ancestral diploid dinoflagellates and haploid core dinoflagellates. This implies that the transition from diploidy to haploidy in trophonts probably occurred via neoteny of a noctilucid-like zoospore.     

CORRESPONDENCE BETWEEN COLD TOLERANCE AND TEMPERATE BIOGEOGRAPHY IN A WESTERN ATLANTIC SYMBIODINIUM (DINOPHYTA) LINEAGE1

Many corals form obligate symbioses with photosynthetic dinoflagellates of the genus Symbiodinium Freudenthal (1962). These symbionts vary genotypically, with their geographical distribution and abundance dependent upon host specificity and tolerance to temperature and light variation. Despite the importance of these mutualistic relationships, the physiology and ecology of Symbiodinium spp. remain poorly characterized. Here, we report that rDNA internal transcribed spacer region 2 (ITS2) defined Symbiodinium type B2 associates with the cnidarian hosts Astrangia poculata and Oculina arbuscula from northerly habitats of the western Atlantic. Using pulse‐amplitude‐modulated (PAM) fluorometry, we compared maximum photochemical efficiency of PSII of type B2 to that of common tropical Symbiodinium lineages (types A3, B1, and C2) under cold‐stress conditions. Symbiont cultures were gradually cooled from 26°C to 10°C to simulate seasonal temperature declines. Cold stress decreased the maximum photochemical efficiency of PSII and likely the photosynthetic potential for all Symbiodinium clades tested. Cultures were then maintained at 10°C for a 2‐week period and gradually returned to initial conditions. Subsequent to low temperature stress, only type B2 displayed rapid and full recovery of PSII photochemical efficiency, whereas other symbiont phylotypes remained nonfunctional. These findings indicate that the distribution and abundance of Symbiodinium spp., and by extension their cnidarian hosts, in temperate climates correspond significantly with the photosynthetic cold tolerance of these symbiotic algae.     

HOST‐SPECIALIST LINEAGES DOMINATE THE ADAPTIVE RADIATION OF REEF CORAL ENDOSYMBIONTS

Bursts in species diversification are well documented among animals and plants, yet few studies have assessed recent adaptive radiations of eukaryotic microbes. Consequently, we examined the radiation of the most ecologically dominant group of endosymbiotic dinoflagellates found in reef‐building corals, Symbiodinium Clade C, using nuclear ribosomal (ITS2), chloroplast (psbA^ncr), and multilocus microsatellite genotyping. Through a hierarchical analysis of high‐resolution genetic data, we assessed whether ecologically distinct Symbiodinium, differentiated by seemingly equivocal rDNA sequence differences, are independent species lineages. We also considered the role of host specificity in Symbiodinium speciation and the correspondence between endosymbiont diversification and Caribbean paleo‐history. According to phylogenetic, biological, and ecological species concepts, Symbiodinium Clade C comprises many distinct species. Although regional factors contributed to population‐genetic structuring of these lineages, Symbiodinium diversification was mainly driven by host specialization. By combining patterns of the endosymbiont's host specificity, water depth distribution, and phylogeography with paleo‐historical signals of climate change, we inferred that present‐day species diversity on Atlantic coral reefs stemmed mostly from a post‐Miocene adaptive radiation. Host‐generalist progenitors spread, specialized, and diversified during the ensuing epochs of prolonged global cooling and change in reef‐faunal assemblages. Our evolutionary reconstruction thus suggests that Symbiodinium undergoes “boom and bust” phases in diversification and extinction during major climate shifts.     

Hyperdiversity of Genes Encoding Integral Light-Harvesting Proteins in the Dinoflagellate Symbiodinium sp

The superfamily of light-harvesting complex (LHC) proteins is comprised of proteins with diverse functions in light-harvesting and photoprotection. LHC proteins bind chlorophyll (Chl) and carotenoids and include a family of LHCs that bind Chl a and c. Dinophytes (dinoflagellates) are predominantly Chl c binding algal taxa, bind peridinin or fucoxanthin as the primary carotenoid, and can possess a number of LHC subfamilies. Here we report 11 LHC sequences for the chlorophyll a-chlorophyll c ₂-peridinin protein complex (acpPC) subfamily isolated from Symbiodinium sp. C3, an ecologically important peridinin binding dinoflagellate taxa. Phylogenetic analysis of these proteins suggests the acpPC subfamily forms at least three clades within the Chl a/c binding LHC family; Clade 1 clusters with rhodophyte, cryptophyte and peridinin binding dinoflagellate sequences, Clade 2 with peridinin binding dinoflagellate sequences only and Clades 3 with heterokontophytes, fucoxanthin and peridinin binding dinoflagellate sequences.     

POTENTIAL UTILITY OF MITOCHONDRIAL CYTOCHROME B AND ITS MRNA EDITING IN RESOLVING CLOSELY RELATED DINOFLAGELLATES: A CASE STUDY OF PROROCENTRUM (DINOPHYCEAE)1

Difficulties often occur in separating closely related dinoflagellate species. In this study, the potential utility of mitochondrial cytochrome b (cob) gene sequence and mRNA editing characteristics was assessed using Prorocentrum Ehrenberg as a model. The cob sequences and the patterns of their mRNA editing were analyzed for several Prorocentrum taxa. Results revealed little difference in cob sequence and mRNA editing characteristics between geographic populations of P. minimum (Pavillard) Schiller, while a notable difference was detected between different species (P. minimum and P. micans Ehrenberg). Furthermore, these P. minimum populations consistently formed a tight cluster on phylogenetic trees inferred from cob sequences as well as mRNA editing characteristics, whereas different Prorocentrum species were well separated, with a genetic distance of 0.0042±0.0024 for the former and 0.0141±0.0012 for the latter (P<0.01; two‐tailed t‐test). When the analysis was applied to the case of P. donghaiense Lu et Goebel and CCMP1517 strain of P. dentatum Stein, no differences were detected between these two taxa with respect to cob mRNA editing pattern and only small differences equivalent to those between P. minimum populations were detected in terms of cob sequence. On the cob sequence‐ and editing‐based phylogenetic trees, P. donghaiense and P. dentatum CCMP1517 consistently clustered together at a position sister to P. minimum. The results suggest that cob, combined with its mRNA editing, can potentially be a useful delineator of Prorocentrum species, and that P. donghaiense and P. dentatum CCMP1517 are most likely the same species and both are closely related to P. minimum.     

FLOW‐CYTOMETRIC CHARACTERIZATION OF THE CELL‐SURFACE GLYCANS OF SYMBIOTIC DINOFLAGELLATES (SYMBIODINIUM SPP.)1

Symbiodinium spp. dinoflagellates are common symbionts of marine invertebrates. The cell‐surface glycan profile may determine whether a particular Symbiodinium is able to establish and maintain a stable symbiotic relationship. To characterize this profile, eight Symbiodinium cultures were examined using eight glycan‐specific fluorescent lectin probes. Confocal imaging and flow‐cytometric analysis were used to determine significant levels of binding of each probe to the cell surface. No significant variation in glycan profile was seen within each Symbiodinium culture, either over time or over growth phase. No cladal trends in glycan profile were found, but of note, two different Symbiodinium cultures (from clades A and B) isolated from one host species had very similar profiles, and two other cultures (from clades B and F) from different host species had identical profiles. Two lectin probes were particularly interesting: concanavalin A (ConA) and Griffonia simplicifolia‐II (GS‐II). The ConA probe showed significant binding to all Symbiodinium cultures, suggesting the widespread presence of cell‐surface mannose residues, while the GS‐II probe, which is specific for glycans possessing N‐acetyl groups, showed significant binding to six of eight Symbiodinium cultures. Other probes showed significant binding to the following percentage of Symbiodinium cultures examined: wheat germ agglutinin (WGA), 37.5%; peanut agglutinin (PNA), 50%; Helix pomatia agglutinin (HPA), 50%; phytohemagglutinin‐L (PHA‐L), 62.5%; soybean agglutinin (SBA), 50%; and Griffonia simplicifolia‐IB₄ (GS‐IB₄), 12.5%. This study highlights the complexity of cell‐surface glycan assemblages and their potential role in the discrimination of different dinoflagellate symbionts by cnidarian hosts.     

PHYLOGENETIC ANALYSIS OF A FREE‐LIVING STRAIN OF SYMBIODINIUM ISOLATED FROM JIAOZHOU BAY,P.R. CHINA1

Recently, the isolation of a free‐living strain of symbiotic dinoflagellate belonging to the genus Symbiodinium was reported. Although the specimen procured and characterization from Jiaozhou Bay, P.R. China is a Symbiodinium spp., the manner in which this isolate was classified is inconsistent with the currently used and accepted Symbiodinium cladal nomenclature. To avert unnecessary confusion in the field, I place this important scientific contribution into the proper context and state of Symbiodinium research.     

Latitudinal and intracolony ITS-rDNA sequence variation in the symbiotic dinoflagellate genus Symbiodinium (Dinophyceae) in Zoanthus sansibaricus (Anthozoa: Hexacorallia)

We sequenced the internal transcribed spacer of ribosomal DNA (ITS‐rDNA) of Symbiodinium spp. (Freudenthal) from conspecific Zoanthus sansibaricus (Carlgren) colonies along a latitudinal gradient in Japan. Phylogenetic analysis reveals that Zoanthus in the two northern sites of Kokubu and Sakurajima harbor exclusively Symbiodinium subclade C1, whereas Yakushima Zoanthus harbors Symbiodinium subclades C1 and C15, and southernmost Amami Zoanthus Symbiodinium subclades A1 and C1, indicating holobiont flexibility. Individual Zoanthus colonies associated exclusively with one single subclade, but unexpectedly there was small variation between Symbiodinium ITS‐rDNA clone sequences obtained from within individual Zoanthus colonies. There was also a large deletion in the ITS‐2/28S rDNA boundary region in one clone sequence, and another large deletion in the 5.8S rDNA region in another clone. Our intracolony sequence heterogeneity might be a result of the presence of multiple copies of the ITS‐rDNA region within individual Symbiodinium genomes, or result from the possible presence of closely related Symbiodinium genotypes in the host.     

SYMBIODINIUM (PYRRHOPHYTA) GENOME SIZES (DNA CONTENT) ARE SMALLEST AMONG DINOFLAGELLATES1

Using flow cytometric analysis of fluorescence, we measured the genome sizes of 18 cultured “free‐living” species and 29 Symbiodinium spp. isolates cultured from stony corals, gorgonians, anemones, jellyfish, and giant clams. Genome size directly correlated with cell size, as documented previously for most eukaryotic cell lines. Among the smallest of dinoflagellates, Symbiodinium spp. (6–15 μm) possessed the lowest DNA content that we measured (1.5–4.8 pg·cell^?1). Bloom‐forming or potentially harmful species in the genera Alexandrium, Karenia, Pfiesteria, and Prorocentrum possessed genomes approximately 2 to 50 times larger in size. A phylogenetic analysis indicated that genome/cell size has apparently increased and decreased repeatedly during the evolution of dinoflagellates. In contrast, genome sizes were relatively consistent across distantly and closely related Symbiodinium spp. This may be the product of intracellular host habitats imposing strong selective pressures that have restricted symbiont size.     

Implications of complete nuclear large subunit ribosomal RNA molecules from the harmful unarmored dinoflagellate Cochlodinium polykrikoides (Dinophyceae) and relatives

The D1/D2 domains of large subunit (LSU) rDNA have commonly been used for phylogenetic analyses of dinoflagellates; however, their properties have not been evaluated in relation to other D domains due to a deficiency of complete sequences. This study reports the complete LSU rRNA gene sequence in the causative unarmored dinoflagellate Cochlodinium polykrikoides, a member of the order Gymnodiniales, and evaluated the segmented domains and secondary structures when compared with its relatives. Putative LSU rRNA coding regions were recorded to be 3433 bp in length (49.0% GC content). A secondary structure predicted from the LSU and 5.8S rRNAs and parsimony analyses showed that most variation in the LSU rDNA was found in the 12 divergent (D) domains. In particular, the D2 domain was the most informative in terms of recent evolutional and taxonomic aspects, when compared with both the phylogenetic tree topologies and molecular distance (approximately 10 times higher) of the core LSU. Phylogenetic analysis was performed with a matrix of LSU DNA sequences selected from domains D2 to D4 and their flanking core sequences, which showed that C. polykrikoides was placed on the same branch with Akashiwo sanguinea in the “GPP” complex, which is referred to the gymnodinioid, peridinioid and prorocentroid groups. A broad phylogeny showed that armored and unarmored dinoflagellates were never clustered together; instead, they were clearly divided into two groups: the GPP complex and Gonyaulacales. The members of Gymnodiniales were always interspersed with peridinioid, prorocentroid and dinophysoid forms. This supports previous findings showing that the Gymnodiniales are polyphyletic. This study highlights the proper selection of LSU rDNA molecules for molecular phylogeny and signatures.