Macroalgal-associated dinoflagellates belonging to the genus Symbiodinium in Caribbean reefs

Coral-algal symbiosis has been a subject of great attention during the last two decades in response to global coral reef decline. However, the occurrence and dispersion of free-living dinoflagellates belonging to the genus Symbiodinium are less documented. Here ecological and molecular evidence is presented demonstrating the existence of demersal free-living Symbiodinium populations in Caribbean reefs and the possible role of the stoplight parrotfish (Sparisoma viride) as Symbiodinium spp. dispersers. Communities of free-living Symbiodinium were found within macroalgal beds consisting of Halimeda spp., Lobophora variegata, Amphiroa spp., Caulerpa spp. and Dictyota spp. Viable Symbiodinium spp. cells were isolated and cultured from macroalgal beds and S. viride feces. Further identification of Symbiodinium spp. type was determined by length variation in the Internal Transcribed Spacer 2 (ITS2, nuclear rDNA) and length variation in domain V of the chloroplast large subunit ribosomal DNA (cp23S-rDNA). Determination of free-living Symbiodinium and mechanisms of dispersal is important in understanding the life cycle of Symbiodinium spp.     

Molecular phylogeny, evolutionary rates, and divergence timing of the symbiotic dinoflagellate genus Symbiodinium

Symbiotic dinoflagellates belonging to the genus Symbiodinium are found in association with a wide variety of shallow-water invertebrates and protists dwelling in tropical and subtropical coral-reef ecosystems. Molecular phylogeny of Symbiodinium, initially inferred using nuclear ribosomal genes, was recently confirmed by studies of chloroplastic and mitochondrial genes, but with limited taxon sampling and low resolution. Here, we present the first complete view of Symbiodinium phylogeny based on concatenated partial sequences of chloroplast 23S-rDNA (cp23S) and nuclear 28S-rDNA (nr28S) genes, including all known Symbiodinium lineages. Our data produced a well resolved phylogenetic tree and provide a strong statistical support for the eight distinctive clades (A-H) that form the major taxa of Symbiodinium. The relative-rate tests did not show particularly high differences between lineages and both analysed markers. However, maximum likelihood ratio tests rejected a global molecular clock. Therefore, we applied a relaxed molecular clock method to infer the divergence times of all extant lineages of Symbiodinium, calibrating its phylogenetic tree with the fossil record of soritid foraminifera. Our analysis suggests that Symbiodinium originated in early Eocene, and that the majority of extant lineages diversified since mid-Miocene, about 15 million years ago.     

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.     

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.     

Molecular genetic evidence that dinoflagellates belonging to the genus Symbiodinium freudenthal are haploid

Microscopic and cytological evidence suggest that many dinoflagellates possess a haploid nuclear phase. However, the ploidy of a number of dinoflagellates remains unknown, and molecular genetic support for haploidy in this group has been lacking. To elucidate the ploidy of symbiotic dinoflagellates belonging to the genus Symbiodinium, we used five polymorphic microsatellites to examine populations harbored by the Caribbean gorgonians Plexaura kuna and Pseudopterogorgia elisabethae; we also studied a series of Symbiodinium cultures. In 690 out of 728 Symbiodinium samples in hospite (95% of the cases) and in all 45 Symbiodinium cultures, only a single allele was recovered per locus. Statistical testing of the Symbiodinium populations harbored by P. elisabethae revealed that the observed genotype frequencies deviate significantly from those expected under Hardy-Weinberg equilibrium. Taken together, our results confirm that, in the vegetative life stage, members of Symbiodinium, both cultured and in hospite, are haploid. Furthermore, based on the phylogenetics of the dinoflagellates, haploidy in vegetative cells appears to be an ancestral trait that extends to all 2,000 extant species of these important unicellular protists.     

Symbiodinium diversity among host clionaid sponges from Caribbean and Pacific reefs: Evidence of heteroplasmy and putative host-specific symbiont lineages

Among the Porifera, symbiosis with Symbiodinium spp. (i.e., zooxanthellae) is largely restricted to members of the family Clionaidae. We surveyed the diversity of zooxanthellae associated with sponges from the Caribbean and greater Indo-Pacific regions using chloroplast large subunit (cp23S) domain V sequences. We provide the first report of Clade C Symbiodinium harbored by a sponge (Cliona caesia), and the first report of Clade A Symbiodinium from an Indo-Pacific sponge (C. jullieni). Clade A zooxanthellae were also identified in sponges from the Caribbean, which has been reported previously. Sponges that we examined from the Florida Keys all harbored Clade G Symbiodinium as did C. orientalis from the Indo-Pacific, which also supports earlier work with sponges. Two distinct Clade G lineages were identified in our phylogenetic analysis; Symbiodinium extracted from clionaid sponges formed a monophyletic group sister to Symbiodinium found in foraminiferans. Truncated and 'normal' length variants of 23S rDNA sequences were detected simultaneously in all three morphotypes of C. varians providing the first evidence of chloroplast-based heteroplasmy in a sponge. None of the other sponge species examined showed evidence of heteroplasmy. As in previous work, length variation in cp23S domain V sequences was found to correspond in a highly precise manner to finer resolution of phylogenetic topology among Symbiodinium clades. On a global scale, existing data indicate that members of the family Clionaidae that host zooxanthellae can form symbiotic associations with at least four Symbiodinium clades. The majority of sponge hosts appear to harbor only one cladal type of symbiont, but some species can harbor more than one clade of zooxanthellae concurrently. The observed differences in the number of partners harbored by sponges raise important questions about the degree of coevolutionary integration and specificity of these symbioses. Although our sample sizes are small, we propose that one of the Clade G lineages identified in this study is comprised of sponge-specialist zooxanthellae. These zooxanthellae are common in Caribbean sponges, but additional work in other geographic regions is necessary to test this idea. Sponges from the Indo-Pacific region harbor zooxanthellae from Clades A, C, and G, but more sponges from this region should be examined.     

Sinophysis and Pseudophalacroma are distantly related to typical Dinophysoid dinoflagellates (Dinophysales, Dinophyceae)

Dinophysoid dinoflagellates are usually considered a large monophyletic group. Large subunit and small subunit (SSU) rDNA phylogenies suggest a basal position for Amphisoleniaceae (Amphisolenia,Triposolenia) with respect to two sister groups, one containing most Phalacroma species plus Oxyphysis and the other Dinophysis,Ornithocercus, Dinophysoid dinoflagellates are usually considered a large monophyletic group. Large subunit and small subunit (SSU) rDNA phylogenies suggest a basal position for Amphisoleniaceae (Amphisolenia,Triposolenia) with respect to two sister groups, one containing most Phalacroma species plus Oxyphysis and the other Dinophysis,Ornithocercus, Histioneis,Citharistes and some Phalacroma species. We provide here new SSU rDNA sequences of Pseudophalacroma (pelagic) and Sinophysis (the only benthic dinophysoid genus). Molecular phylogenies support that they are very divergent with respect to the main clade of Dinophysales. Additional molecular markers of these two key genera are needed to elucidate the evolutionary relations among the dinophysoid dinoflagellates. Histioneis,Citharistes and some Phalacroma species. We provide here new SSU rDNA sequences of Pseudophalacroma (pelagic) and Sinophysis (the only benthic dinophysoid genus). Molecular phylogenies support that they are very divergent with respect to the main clade of Dinophysales. Additional molecular markers of these two key genera are needed to elucidate the evolutionary relations among the dinophysoid dinoflagellates.     

SMALL-SUBUNIT RIBOSOMAL DNA SEQUENCE ANALYSES AND A RECONSTRUCTION OF THE INFERRED PHYLOGENY AMONG SYMBIOTIC DINOFLAGELLATES (PYRROPHYTA)1

The small-subunit ribosomal RNA genes (SSU rDNA) from the four symbiotic dinoflagellates, Symbiodinium corculorum Trench isolated from the bivalve mollusc Corculum cardissa (from Belau, Western Caroline Is.), S. meandrinae Trench, from the scleractinian coral Meandrina meandrites (from famaica, W.I.), Gloeodinium viscum Banaszak et al. from the hydrocoral Millepora dichotoma (from the Gulf of Aqaba), and Amphidinium belauense Trench from the acoel flatworm Haplodiscus sp. (from Belau) have been amplified by the polymerase chain reaction, cloned, and sequenced. Following alignment of these complete sequences to homologous sequences from six other dinoflagellates, eight api-complexans, six ciliates, six chromophytes and oomycetes, three ascomycetes, two rhodophytes, two chlorophytes, and two myxomycetes (with Physarum polycephalum as the outgroup), phylogenetic reconstruction was conducted using Fitch and Margoliash distance, DNA maximum likelihood, and Wagner parsimony methods, with bootstrap resampling. All methods generated trees with similar topologies. The inferred “across Kingdom” phylogeny reemphasizes previous reports that show that the dinoflagellates, the apicomplexans, and the ciliates share a common ancestry and that the dinoflagellates are distantly related to the chromophyte-oömycete lineage. The evidence supports the concept of a polyphyletic origin of dinoflagellate-invertebrate symbioses, as symbiotic dinoflagellates represent seven genera in at least four orders. The three symbiotic species, S. corculorum, S. meandrinae, and S. pilosum, consistent with their morphological and biochemical similarities, cluster most closely. Symbiodinium pulchrorum Trench, the symbiontfrom the Hawaiian sea anemone Aiptasia pulchella, is more distantly related to them. Gloeodinium viscum is not closely related to the Symbiodinium species. Amphidinium carterae (free-living) and A. belauense (symbiotic) also appear to be distantly related to Symbiodinium. Some symbionts (e.g. S. corculorum, S. pilosum) from distant geographic locations (the Indo-Pacific and Caribbean, respectively) were found to be very closely related, whereas S. pulchrorum and S. corculorum from the Pacific were found to be distantly related. Analyses of 10 additional symbiotic and nonsymbiotic dinoflagellates, using partial SSU rDNA sequences to generate a tentative dinoflagellate phylogeny, indicate that members of the genus Symbiodinium cluster with most of the other (free-living) dinoflagellates in the genus Gymnodinium. The genus Amphidinium, as represented by A. carterae and A. belauense, appear to be distantly related to the other members of the Gymnodiniaceae. This analysis, combined with morphological and biochemical data, indicates that the symbionts S. pulchrorum (from Aiptasia pulchella) and S. bermudense Trench (from Aiptasia tagetes) from the Indo-Pacific and Caribbean, respectively, are very closely related but are not identical.     

Phylogenetic relationships among zooxanthellae (Symbiodinium) associated to excavating sponges (Cliona spp.) reveal an unexpected lineage in the Caribbean

Phylogenetic relationships of symbiotic dinoflagellate lineages, distributed in all tropical and subtropical seas, suggest strategies for long distance dispersal but at the same time strong host specialization. Zooxanthellae (Symbiodinium: Dinophyta), which are associated to diverse shallow-water cnidarians, also engage in symbioses with some sponge species of the genus Cliona. In the Caribbean, zooxanthellae-bearing Cliona has recently become abundant due to global warming, overfishing, and algae abundance. Using molecular techniques, the symbionts from five excavating species (Clionacaribbaea, C. tenuis, C. varians, C. aprica and C. laticavicola) from the southern and southwestern Caribbean were surveyed. Several DNA sequence regions were used in order to confirm zooxanthellae identity; 18S rDNA, domain V of chloroplast large subunit (cp23S), internal transcribed spacer 2 (ITS2), and ITS2 secondary structure. Sequence analyses corroborated the presence of three zooxanthellae clades: A, B, and G. Presence of clades A and B in common boring sponges of the Caribbean fit with the general pattern of the province. The discovery of clade G for the first time in any organism of the Atlantic Ocean leads us to consider this unusual finding as a phylogenetic relict through common ancestors of sponge clades or an invasion of the sponge from the Indo-Pacific.     

Measuring rDNA diversity in eukaryotic microbial systems: how intragenomic variation, pseudogenes, and PCR artifacts confound biodiversity estimates

Molecular approaches have revolutionized our ability to study the ecology and evolution of micro-organisms. Among the most widely used genetic markers for these studies are genes and spacers of the rDNA operon. However, the presence of intragenomic rDNA variation, especially among eukaryotes, can potentially confound estimates of microbial diversity. To test this hypothesis, bacterially cloned PCR products of the internal transcribed spacer (ITS) region from clonal isolates of Symbiodinium, a large genus of dinoflagellates that live in symbiosis with many marine protists and invertebrate metazoa, were sequenced and analysed. We found widely differing levels of intragenomic sequence variation and divergence in representatives of Symbiodinium clades A to E, with only a small number of variants attributed to Taq polymerase/bacterial cloning error or PCR chimeras. Analyses of 5.8S-rDNA and ITS2 secondary structure revealed that some variants possessed base substitutions and/or indels that destabilized the folded form of these molecules; given the vital nature of secondary structure to the function of these molecules, these likely represent pseudogenes. When similar controls were applied to bacterially cloned ITS sequences from a recent survey of Symbiodinium diversity in Hawaiian Porites spp., most variants (approximately 87.5%) possessed unstable secondary structures, had unprecedented mutations, and/or were PCR chimeras. Thus, data obtained from sequencing of bacterially cloned rDNA genes can substantially exaggerate the level of eukaryotic microbial diversity inferred from natural samples if appropriate controls are not applied. These considerations must be taken into account when interpreting sequence data generated by bacterial cloning of multicopy genes such as rDNA.     

Cohesive molecular genetic data delineate species diversity in the dinoflagellate genus Symbiodinium

The diversity of symbiotic dinoflagellates ( Symbiodinium ) in pocilloporid corals originating from various reef habitats surrounding Heron Island, southern Great Barrier Reef, was examined by targeting ribosomal, mitochondrial, and chloroplast genes using six methods that analyse for sequence differences. The ability of each of 13 genetic analyses to characterize eight ecologically distinct Symbiodinium spp. was dependent on the level of conservation of the gene region targeted and the technique used. Other than differences in resolution, phylogenetic reconstructions using nuclear and organelle gene sequences were complementary and when combined produced a well-resolved phylogeny. Analysis of the ribosomal internal transcribed spacers using denaturing gradient gel electrophoresis fingerprinting in combination with sequencing of dominant bands provided a precise method for rapidly resolving and characterizing symbionts into ecologically and evolutionarily distinct units of diversity. Single-stranded conformation polymorphisms of the nuclear ribosomal large subunit (D1/D2 domain) identified the same number of ecologically distinct Symbiodinium spp., but profiles were less distinctive. The repetitive sequencing of bacterially cloned ITS2 polymerase chain reaction amplifications generated numerous sequence variants that clustered together according to the symbiont under analysis. The phylogenetic relationships between these clusters show how intragenomic variation in the ribosomal array diverges among closely related eukaryotic genomes. The strong correlation between phylogenetically independent lineages with different ecological and physiological attributes establishes a clear basis for assigning species designations to members of the genus Symbiodinium .     

造礁石珊瑚与其共生藻(Symbiodinium)共生研究进展

对造礁石珊瑚与其共生藻共生研究现状及其在全球变化下的适应能力进行较全面的综述.造礁石珊瑚与遗传和生理功能独特的共生藻组成内共生关系是成功演化的范例.近年来对珊瑚共生体的分子系统学研究表明共生藻遗传多样性极为丰富,当前认为共生藻属至少包括8个(A-H)各自包含亚系群的世系或系群.珊瑚-共生藻共生功能体对诸如全球变化引起的海水温度上升等环境变化十分敏感.由于珊瑚以及珊瑚礁面临气候变化的严峻挑战,对珊瑚与其共生藻共生关系和共生功体适应能力的研究将是未来重要的研究领域之一.     

Nuclear 28S rDNA phylogeny supports the basal placement of Noctiluca scintillans (Dinophyceae; Noctilucales) in dinoflagellates

Noctiluca scintillans (Macartney) Kofoid et Swezy, 1921 is an unarmoured heterotrophic dinoflagellate with a global distribution, and has been considered as one of the ancestral taxa among dinoflagellates. Recently, 18S rDNA, actin, α-, β-tubulin, and Hsp90-based phylogenies have shown the basal position of the noctilucids. However, the relationships of dinoflagellates in the basal lineages are still controversial. Although the nuclear rDNA (e.g. 18S, ITS-5.8S, and 28S) contains much genetic information, DNA sequences of N. scintillans rDNA molecules were insufficiently characterized as yet. Here the author sequenced a long-range nuclear rDNA, spanning from the 18S to the D5 region of the 28S rDNA, of N. scintillans. The present N. scintillans had a nearly identical genotype (>99.0% similarity) compared to other Noctiluca sequences from different geographic origins. Nucleotide divergence in the partial 28S rDNA was significantly high (p<0.05) as compared to the 18S rDNA, demonstrating that the information from 28S rDNA is more variable. The 28S rDNA phylogeny of 17 selected dinoflagellates, two perkinsids, and two apicomplexans as outgroups showed that N. scintillans and Oxyrrhis marina formed a clade that diverged separately from core dinoflagellates.     

基因序列在蚜虫分子系统发育研究中的应用

总结了核基因和线粒体基因在半翅目蚜虫分子系统发育研究中的应用。核基因中EF-1α应用最广泛,适用于探讨属级及属以上的问题; 核rDNA在蚜虫中应用较少,18S rDNA适用于探讨科级以上高级阶元的问题;LWO是新近在蚜虫中开发使用的一个新基因。线粒体基因中,COⅠ/COⅡ使用最多,12S rDNA/16S rDNA、ND1、Cyt b以及F-ATP6均有应用,探讨的问题从属、种级到科级不等。核基因和线粒体基因间以及不同线粒体基因间的联合分析在解决不同层次的问题中均有应用。建议不断尝试新基因以找出适合蚜虫类群的“标准基因”。并对未来蚜虫分子系统发育研究趋势进行了展望。     

Phylogenetic Identification of Symbiotic Dinoflagellates via Length Heteroplasmy in Domain V of Chloroplast Large Subunit (cp23S)—Ribosomal DNA Sequences

A protocol that takes advantage of length heteroplasmy in domain V of chloroplast large subunit (cp23S)–ribosomal DNA to identify members of the symbiotic dinoflagellate genus Symbiodinium is presented. This protocol is highly specific for Symbiodinium, can provide intercladal and intracladal identification of a particular Symbiodinium isolate, and can detect multiple Symbiodinium chloroplast genotypes simultaneously in the same isolate, making his technique attractive for a variety of research questions. We used this technique to characterize variation among Symbiodinium populations associated with a range of phylogenetically diverse and geographically discrete hosts. We also examined symbiont variation within a single host, the Caribbean gorgonian Pseudopterogorgia elisabethae, from 9 sites in the Bahamas, and we report a previously undocumented level of symbiont specificity for particular members of Symbiodinium clade B in this gorgonian. Current address of Scott R. Santos: Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ, 85721, U.S.A.     

Isolation and characterization of microsatellite loci in Symbiodinium B1/B184, the dinoflagellate symbiont of the Caribbean sea fan coral, Gorgonia ventalina

Here we report primers targeting 10 microsatellite loci of dinoflagellates in the genus Symbiodinium (clade B1/B184) symbiotic with the Caribbean sea fan coral, Gorgonia ventalina. Primers were tested on 12 Symbiodinium B1/B184 cultures, as well as 40 genomic DNA extracts of G. ventalina tissue samples. All loci were polymorphic with allelic richness ranging from 4-16. Gene diversity ranged from 0.15 to 0.91. These primers provide powerful tools for examining the fine-scale population structure and dynamics of Symbiodinium within a single host species.     

Biogeography of two species of Symbiodinium (Freudenthal) inhabiting the intertidal sea anemone Anthopleura elegantissima (Brandt)

We have analyzed the genetic profiles of dinoflagellate populations obtained from the Pacific coast sea anemone Anthopleura elegantissima (Brandt) at collection sites from Washington to California. Genetic differences within the symbiont populations of California anemones have been uncovered by restriction length polymorphism (RFLP) analysis of the small subunit (SSU) and large subunit (LSU) ribosomal RNA genes, and by denaturing gradient gel electrophoresis (DGGE) of the internal transcribed spacer region 2 (ITS 2). The existence of two Symbiodinium species is substantiated by sequence analysis of the variable regions V1, V2, and V3 of the SSUrDNA, which also establishes their phylogenetic relatedness to other members of the genus Symbiodinium. Anemones from Washington and Oregon harbor a single dinoflagellate species, for which we propose the name S. muscatinei sp. nov. At these northern locations, S. muscatinei either exists alone or co-occurs with the Chlorella-like green algal symbiont. Our results indicate that S. muscatinei co-occurs with a second dinoflagellate, S. californium, in mixed populations in central and southern California. We suggest that the geographic distribution of these dinoflagellates is related to the temperature cline created by latitude.     

Zooxanthellactone, a novel gamma-lactone-type oxylipine from dinoflagellates of Symbiodinium sp.: structure, distribution, and biological activity

A novel fatty acid derivative named zooxanthellactone (ZL) was isolated from several strains of symbiotic microalgae, dinoflagellates of the genus Symbiodinium. The metabolite is structurally related to docosahexaenoic acid (DHA) and seems to be biosynthesized by oxidation and subsequent lactonization. The absolute stereochemistry was determined from the specific rotation of the perhydro derivative. The distribution of ZL within several Symbiodinium isolates was quantitatively analyzed by HPLC techniques and suggested a relationship between the productivity of this metabolite and the Symbiodinium phylogeny. The cytotoxicity of ZL was evaluated by using human squamous cell carcinoma cell lines in comparison with that of DHA and other common fatty acids, suggesting that the long unsaturated chain was important rather than the gamma-lactone moiety.