Differential antioxidant response between two Symbiodinium species from contrasting environments

High sea surface temperature accompanied by high levels of solar irradiance is responsible for the disruption of the symbiosis between cnidarians and their symbiotic dinoflagellates from the genus Symbiodinium. This phenomenon, known as coral bleaching, is one of the major threats affecting coral reefs around the world. Because an important molecular trigger to bleaching appears related to the production of reactive oxygen species (ROS), it is critical to understand the function of the antioxidant network of Symbiodinium species. In this study we investigated the response of two Symbiodinium species, from contrasting environments, to a chemically induced oxidative stress. ROS produced during this oxidative burst reduced photosynthesis by 30 to 50% and significantly decreased the activity of superoxide dismutase. Lipid peroxidation levels and carotenoid concentrations, especially diatoxanthin, confirm that these molecules act as antioxidants and contribute to the stabilization of membrane lipids. The comparative analysis between the two Symbiodinium species allowed us to highlight that Symbiodinium sp. clade A temperate was more tolerant to oxidative stress than the tropical S. kawagutii clade F. These differences are very likely a consequence of adaptation to their natural environment, with the temperate species experiencing conditions of temperature and irradiance much more variable and extreme.     

Validation and description of Symbiodinium microadriaticum,the type species of Symbiodinium (Dinophyta)

It has been 55 years since Hugo Freudenthal described Symbiodinium microadriaticum (Dinophyceae), the type species of this large and important dinoflagellate genus found commonly in mutualistic symbiosis with cnidarians, other invertebrates, and certain protists. However, no type specimen was designated by Freudenthal, thus S. microadriaticum was invalid, as was Symbiodinium and every species subsequently assigned to the genus. The original culture was lost, but since 1979, a different culture, CCMP2464/rt‐061, had been considered to represent S. microadriaticum. From this culture, a preserved specimen is herein designated the holotype of S. microadriaticum, validating the binomial and Symbiodinium. All binary designations previously considered to belong in Symbiodinium also are validated herein.     

Separate Introns Gained within Short and Long Soluble Peridinin-Chlorophyll a-Protein Genes during Radiation of Symbiodinium (Dinophyceae) Clade A and B Lineages

Here we document introns in two Symbiodinium clades that were most likely gained following divergence of this genus from other peridinin-containing dinoflagellate lineages. Soluble peridinin-chlorophyll a-proteins (sPCP) occur in short and long forms in different species. Duplication and fusion of short sPCP genes produced long sPCP genes. All short and long sPCP genes characterized to date, including those from free living species and Symbiodinium sp. 203 (clade C/type C2) are intronless. However, we observed that long sPCP genes from two Caribbean Symbiodinium clade B isolates each contained two introns. To test the hypothesis that introns were gained during radiation of clade B, we compared sPCP genomic and cDNA sequences from 13 additional distinct Caribbean and Pacific Symbiodinium clade A, B, and F isolates. Long sPCP genes from all clade B/B1 and B/B19 descendants contain orthologs of both introns. Short sPCP genes from S. pilosum (A/A2) and S. muscatinei (B/B4) plus long sPCP genes from S. microadriaticum (A/A1) and S. kawagutii (F/F1) are intronless. Short sPCP genes of S. microadriaticum have a third unique intron. Symbiodinium clade B long sPCP sequences are useful for assessing divergence among B1 and B19 descendants. Phylogenetic analyses of coding sequences from four dinoflagellate orders indicate that introns were gained independently during radiation of Symbiodinium clades A and B. Long sPCP introns were present in the most recent common ancestor of Symbiodinium clade B core types B1 and B19, which apparently diverged sometime during the Miocene. The clade A short sPCP intron was either gained by S. microadriaticum or possibly by the ancestor of Symbiodinium types A/A1, A3, A4 and A5. The timing of short sPCP intron gain in Symbiodinium clade A is less certain. But, all sPCP introns were gained after fusion of ancestral short sPCP genes, which we confirm as occurring once in dinoflagellate evolution.     

SYMBIODINIUM NATANS SP. NOV.: A “FREE‐LIVING” DINOFLAGELLATE FROM TENERIFE (NORTHEAST‐ATLANTIC OCEAN)1

We examined a free‐living Symbiodinium species by light and electron microscopy and nuclear‐encoded partial LSU rDNA sequence data. The strain was isolated from a net plankton sample collected in near‐shore waters at Tenerife, the Canary Islands. Comparing the thecal plate tabulation of the free‐living Symbiodinium to that of S. microadriaticum Freud., it became clear that a few but significant differences could be noted. The isolate possessed two rather than three antapical plates, six rather than seven to eight postcingular plates, and finally four rather than five apical plates. The electron microscopic study also revealed the presence of an eyespot with brick‐shaped contents in the sulcal region and a narrow anterior plate with small knob‐like structures. Bayesian analysis revealed the free‐living Symbiodinium to be a member of the earliest diverging clade A. However, it did not group within subclade A_I (=temperate A) or any other subclades within clade A. Rather, it occupied an isolated position, and this was also supported by sequence divergence estimates. On the basis of comparative analysis of the thecal plate tabulation and the inferred phylogeny, we propose that the Symbiodinium isolate from Tenerife is a new species (viz. S. natans). To elucidate further the species diversity of Symbiodinium, particularly those inhabiting coral reefs, we suggest combining morphological features of the thecal plate pattern with gene sequence data. Indeed, future examination of motile stages originating from symbiont isolates will demonstrate if this proves a feasible way to identify and characterize additional species of Symbiodinium and thus match ribotypes or clusters of ribotypes to species.     

Flexible associations between Pocillopora corals and Symbiodinium limit utility of symbiosis ecology in defining species

Corals in the genus Pocillopora are the primary framework builders of eastern tropical Pacific (ETP) reefs. These corals typically associate with algal symbionts (genus Symbiodinium) in clade C and/or D, with clade D associations having greater thermal tolerance and resistance to bleaching. Recently, cryptic "species" delineations within both Pocillopora and Symbiodinium have been suggested, with host–symbiont specificity used as a supporting taxonomic character in both genera. In particular, it has been suggested that three lineages of Pocillopora (types 1–3) exist in the ETP, of which type 1 is the exclusive host of heat-tolerant Symbiodinium D1. This host specificity has been used to support the species name "Symbiodinium glynni" for this symbiont. To validate these host–symbiont relationships and their taxonomic utility, we identified Pocillopora types and their associated Symbiodinium at three sites in the ETP. We found greater flexibility in host–symbiont combinations than previously reported, with both Pocillopora types 1 and 3 able to host and be dominated by Symbiodinium in clade C or D. The prevalence of certain combinations did vary among sites, showing that a gradient of specificity exists which may be mediated by evolutionary relationships and environmental disturbance history. However, these results limit the utility of apparent host–symbiont specificity (which may have been a result of undersampling) in defining species boundaries in either corals or Symbiodinium. They also suggest that a greater diversity of corals may benefit from the thermal tolerance of clade D symbionts, affirming the need to conserve Pocillopora across its entire geographic and environmental range.     

Antigenic variation in mucilage secreted by members of the genus Symbiodinium (Dinophyceae)

Symbiodinium reside intracellularly in a complex symbiosome (host and symbiont‐derived) within cnidarian hosts in a specific host‐symbiont association. Symbiodinium is a diverse genus with variation greater than other dinoflagellate orders. In this paper, our investigation into specificity examines antigenic variation in the algal mucilage secretions at the host‐symbiont interface. Cultured Symbiodinium from a variety of clades were labeled with one of two antibodies to symbiont mucilage (PC3, developed using a clade B alga cultured from Aiptasia pallida; BF10, developed using a clade F alga cultured from Briareum sp.). The labeling was visualized with a fluorescent marker and examined with epifluorescence and confocal microscopy. PC3 antigen was found in cultured Symbiodinium from clades A and B, but not clades C, D, E and F. The correlation between labeling and clade may account for some of the specificity between host and symbiont in the field. Within clades A and B there was variation in the amount of label present. BF10 antigen was more specific and only found in cultures of the same cp23S‐rDNA strain the antibody was created against. These results indicate that the mucilage secretions do vary both qualitatively and quantitatively amongst Symbiodinium strains. Since the mucilage forms the host‐symbiont interface, variation in its molecular composition is likely to be the source of any signals involved in recognition and specificity.     

cDNA cloning and phylogenetic and expression analyses of actin in symbiotic dinoflagellates (Symbiodinium spp.)

Three cDNAs encoding actins were identified in two culturable strains (clades A and F) of the symbiotic dinoflagellates Symbiodinium spp. In a molecular phylogenetic analysis these actin sequences formed a monophyletic group with known dinoflagellate actins, remote from Syact-p that had been isolated from a clade A Symbiodinium strain (HG39). One of the newly identified actin sequences (SyAct-F1) was the most closely related to partial actin cDNA sequences (named AGfact-p and AFcact-p) isolated from adult colonies of two reef corals (Galaxea fascicularis and Favites chinensis) that were inhabited by Symbiodinium spp., suggesting the possibility that the latter two were from the symbionts. Partial AFcact-p sequences could be amplified by PCR using genomic DNA prepared from a symbiotic adult colony of F. chinensis as the template, but not from planula larvae in which zooxanthellae could not be detected, also arguing for the origin of AFcact-p in the symbiont. An expression analysis showed that the levels of the SyAct-A1 mRNA were comparable in symbiotic and non-symbiotic states, and also in motile and non-motile phases in a cultured condition, suggesting its usefulness as a constitutively expressed control gene in expression analysis of Symbiodinium mRNAs.     

The distribution of the thermally tolerant symbiont lineage (Symbiodinium clade D) in corals from Hawaii: correlations with host and the history of ocean thermal stress

Spatially intimate symbioses, such as those between scleractinian corals and unicellular algae belonging to the genus Symbiodinium, can potentially adapt to changes in the environment by altering the taxonomic composition of their endosymbiont communities. We quantified the spatial relationship between the cumulative frequency of thermal stress anomalies (TSAs) and the taxonomic composition of Symbiodinium in the corals Montipora capitata, Porites lobata, and Porites compressa across the Hawaiian archipelago. Specifically, we investigated whether thermally tolerant clade D Symbiodinium was in greater abundance in corals from sites with high frequencies of TSAs. We recovered 2305 Symbiodinium ITS2 sequences from 242 coral colonies in lagoonal reef habitats at Pearl and Hermes Atoll, French Frigate Shoals, and Kaneohe Bay, Oahu in 2007. Sequences were grouped into 26 operational taxonomic units (OTUs) with 12 OTUs associated with Montipora and 21 with Porites. Both coral genera associated with Symbiodinium in clade C, and these co‐occurred with clade D in M. capitata and clade G in P. lobata. The latter represents the first report of clade G Symbiodinium in P. lobata. In M. capitata (but not Porites spp.), there was a significant correlation between the presence of Symbiodinium in clade D and a thermal history characterized by high cumulative frequency of TSAs. The endogenous community composition of Symbiodinium and an association with clade D symbionts after long‐term thermal disturbance appear strongly dependent on the taxa of the coral host.     

Sibling species of mutualistic Symbiodinium clade G from bioeroding sponges in the western Pacific and western Atlantic oceans

Dinoflagellates in the genus Symbiodinium associate with a broad array of metazoan and protistian hosts. Symbiodinium‐based symbioses involving bioeroding sponge hosts have received less attention than those involving popular scleractinian hosts. Certain species of common Cliona harbor high densities of an ecologically restricted group of Symbiodinium, referred to as Clade G. Clade G Symbiodinium are also known to form stable and functionally important associations with Foraminifera and black corals (Antipatharia) Analyses of genetic evidence indicate that Clade G likely comprises several distinct species. Here, we use nucleotide sequence data in combination with ecological and geographic attributes to formally describe Symbiodinium endoclionum sp. nov. obtained from the Pacific boring sponge Cliona orientalis and Symbiodinium spongiolum sp. nov. from the congeneric western Atlantic sponge Cliona varians. These species appear to be part of an adaptive radiation comprising lineages of Clade G specialized to the metazoan phyla Porifera and Cnidaria, which began prior to the separation of the Pacific and Atlantic Oceans.     

Microsatellite allele sizes alone are insufficient to delineate species boundaries in Symbiodinium

Symbiodinium are a diverse group of unicellular dinoflagellates that are important nutritional symbionts of reef‐building corals. Symbiodinium putative species (‘types’) are commonly identified with genetic markers, mostly nuclear and chloroplast encoded ribosomal DNA regions. Population genetic analyses using microsatellite loci have provided insights into Symbiodinium biogeography, connectivity and phenotypic plasticity, but are complicated by: (i) a lack of consensus criteria used to delineate inter‐ vs. intragenomic variation within species; and (ii) the high density of Symbiodinium in host tissues, which results in single samples comprising thousands of individuals. To address this problem, Wham & LaJeunesse (2016) present a method for identifying cryptic Symbiodinium species from microsatellite data based on correlations between allele size distributions and nongeographic genetic structure. Multilocus genotypes that potentially do not recombine in sympatry are interpreted as secondary ‘species’ to be discarded from downstream population genetic analyses. However, Symbiodinium species delineations should ideally incorporate multiple physiological, ecological and molecular criteria. This is because recombination tests may be a poor indicator of species boundaries in Symbiodinium due to their predominantly asexual mode of reproduction. Furthermore, discontinuous microsatellite allele sizes in sympatry may be explained by secondary contact between previously isolated populations and by mutations that occur in a nonstepwise manner. Limitations of using microsatellites alone to delineate species are highlighted in earlier studies that demonstrate occasional bimodal distributions of allele sizes within Symbiodinium species and considerable allele size sharing among Symbiodinium species. We outline these issues and discuss the validity of reinterpretations of our previously published microsatellite data from Symbiodinium populations on the Great Barrier Reef (Howells et al. 2013).     

CONSPECIFICITY AND INDO-PACIFIC DISTRIBUTION OF SYMBIODINIUM GENOTYPES (DINOPHYCEAE) FROM GIANT CLAMS

We previously reported the occurrence of genetically‐diverse symbiotic dinoflagellates (zooxanthellae) within and between 7 giant clam species (Tridacnidae) from the Philippines based on the algal isolates' allozyme and random amplified polymorphic DNA (RAPD) patterns. We also reported that these isolates all belong to clade A of the Symbiodinium phylogeny with identical 18S rDNA sequences. Here we extend the genetic characterization of Symbiodinium isolates from giant clams and propose that they are conspecific. We used the combined DNA sequences of the internal transcribed spacer (ITS)1, 5.8S rDNA, and ITS2 regions (rDNA‐ITS region) because the ITS1 and ITS2 regions evolve faster than 18S rDNA and have been shown to be useful in distinguishing strains of other dinoflagellates. DGGE of the most variable segment of the rDNA‐ITS region, ITS1, from clonal representatives of clades A, B, and C showed minimal intragenomic variation. The rDNA‐ITS region shows similar phylogenetic relationships between Symbiodinium isolates from symbiotic bivalves and some cnidarians as does 18S rDNA, and that there are not many different clade A species or strains among cultured zooxanthellae (CZ) from giant clams. The CZ from giant clams had virtually identical sequences, with only a single nucleotide difference in the ITS2 region separating two groups of isolates. These data suggest that there is one CZ species and perhaps two CZ strains, each CZ strain containing individuals that have diverse allozyme and RAPD genotypes. The CZ isolated from giant clams from different areas in the Philippines (21 isolates, 7 clam species), the Australian Great Barrier Reef (1 isolate, 1 clam species), Palau (8 isolates, 7 clam species), and Okinawa, Japan (1 isolate, 1 clam species) shared the same rDNA‐ITS sequences. Furthermore, analysis of fresh isolates from giant clams collected from these geographical areas shows that these bivalves also host indistinguishable clade C symbionts. These data demonstrate that conspecific Symbiodinium genotypes, particularly clade A symbionts, are distributed in giant clams throughout the Indo‐Pacific.     

Horizontal transmission of Symbiodinium cells between adult and juvenile corals is aided by benthic sediment

Of all reef-building coral species, 80–85 % initially draw their intracellular symbionts (dinoflagellates of the genus Symbiodinium) from the environment. Although Symbiodinium cells are crucial for the growth of corals and the formation of coral reefs, little is known about how corals first encounter free-living Symbiodinium cells. We report how the supply of free-living Symbiodinium cells to the benthos by adult corals can increase the rate of horizontal symbiont acquisition for conspecific recruits. Three species of newly settled aposymbiotic (i.e., symbiont-free) corals were maintained in an open aquarium system containing: sterilized sediment and adult coral fragments combined; adult coral fragments alone; sterilized sediment alone; or seawater at Heron Island, Great Barrier Reef, Australia. In all instances, the combination of an adult coral and sediment resulted in the highest symbiont acquisition rates by juvenile corals (up to five-fold greater than seawater alone). Juvenile corals exposed to individual treatments of adult coral or sediment produced an intermediate acquisition response (<52 % of recruits), and symbiont acquisition from unfiltered seawater was comparatively low (<20 % of recruits). Additionally, benthic free-living Symbiodinium cells reached their highest densities in the adult coral + sediment treatment (up to 1.2 × 10⁴ cells mL⁻¹). Our results suggest that corals seed microhabitats with free-living Symbiodinium cells suitable for many coral species during the process of coral recruitment.

     

Molecular identification of symbiotic dinoflagellates in Pacific corals in the genus <Emphasis Type="Italic">Pocillopora</Emphasis>

This study focused on the association between corals of the genus Pocillopora, a major constituent of Pacific reefs, and their zooxanthellae. Samples of P. meandrina, P. verrucosa, P. damicornis, P. eydouxi, P. ligulata and P. molokensis were collected from French Polynesia, Tonga, Okinawa and Hawaii. Symbiodinium diversity was explored by looking at the 28S and ITS1 regions of the ribosomal DNA. Most zooxanthellae were found to belong to clade C, sub-clade C1, with little differentiation between populations. Interestingly, individuals of P. damicornis harbored sub-clade C1, clade D and clade A, depending on location. The symbiotic association of P. damicornis with its zooxanthellae may be somewhat more flexible than those of other Pocillopora species.     

Many corals host thermally resistant symbionts in high-temperature habitat

Physiologically distinct lines of dinoflagellate symbionts, Symbiodinium spp., may confer distinct thermal tolerance thresholds on their host corals. Therefore, if a coral can alternately host distinct symbionts, changes in their Symbiodinium communities might allow corals to better tolerate increasing environmental temperatures. However, researchers are currently debating how commonly coral species can host different symbiont types. We sequenced chloroplast 23 s rDNA from the Symbiodinium communities of nine reef-building coral species across two thermally distinct lagoon pools separated by ~500 m. The hotter of these pools reaches 35°C in the summer months, while the other pool’s maximum temperature is 1.5°C cooler. Across 217 samples from nine species, we found a single haplotype in both Symbiodinium clades A and D, but four haplotypes in Symbiodinium clade C. Eight of nine species hosted a putatively thermally resistant member of clade D Symbiodinium at least once, one of which hosted this clade D symbiont exclusively. Of the remaining seven that hosted multiple Symbiodinium types, six species showed higher proportions of the clade D symbiont in the hotter pool. Average percentage rise in the frequency of the clade D symbiont from the hotter to cooler pool was 52% across these six species. Even though corals hosted members of both the genetically divergent clades D and C Symbiodinium, some showed patterns of host–symbiont specificity within clade C. Both Acropora species that hosted clade C exclusively hosted a member of sub-clade C2, while all three Pocillopora species hosted a member of sub-clade C1 (sensu van Oppen et al. 2001). Our results suggest that coral–algal symbioses often conform to particular temperature environments through changes in the identity of the algal symbiont.     

Relationship between the dinoflagellate cyst Spiniferites pachydermus and Gonyaulax ellegaardiae sp. nov. from Izmir Bay,Turkey, and molecular characterization

Here, we established the cyst‐motile stage relation‐ship for Spiniferites pachydermus through incubation of cysts with a characteristically microreticulate/perforate surface isolated from Izmir Bay in the eastern Aegean Sea of the eastern Mediterranean. The morphology of the motile stage was similar to Gonyaulax spinifera but had a different size, overhang, displacement and reticulations. Based on the distinct morphology of the cyst and morphological differences in motile cells, we assigned S. pachydermus from Izmir Bay to the new species Gonyaulax ellegaardiae. We elucidate the phylogenetic relationship of G. ellegaardiae through large and small subunit ribosomal DNA and show that it forms a clade with other species that belong to the G. spinifera complex.     

Symbiont diversity in scleractinian corals from tropical reefs and subtropical non-reef communities in Taiwan

We examined zooxanthellae diversity in scleractinian corals from southern Taiwan and the Penghu Archipelago, a tropical coral reef and a subtropical non-reefal community, respectively. Zooxanthellae diversity was investigated in 52 species of scleractinian corals from 26 genera and 13 families, using restriction fragment length polymorphism (RFLP), and phylogenetic analyses of the nuclear small-subunit ribosomal DNA (nssrDNA) and large-subunit ribosomal DNA (nlsrDNA). RFLP and phylogenetic analyses of nuclear-encoded ribosomal RNA genes showed that Symbiodinium clade C was the dominant zooxanthellae in scleractinian corals in the seas around Taiwan; Symbiodinium clade D was also found in some species. Both Symbiodinium clade C and D were found in colonies of seven species of scleractinian corals. Symbiodinium clade D was associated with corals that inhabit either shallow water or the reef edge in deep water, supporting the hypothesis that Symbiodinium clade D is a relatively stress-tolerant zooxanthellae found in marginal habitats.Communicated by Biological Editor H.R. Lasker     

Morphological and phylogenetic analyses of Pythium species in South Africa

The genus Pythium is important in agriculture, since it contains many plant pathogenic species, as well as species that can promote plant growth and some that have biocontrol potential. In South Africa, very little is known about the diversity of Pythium species within agricultural soil, irrigation and hydroponic systems. Therefore, the aim of the study was to characterise a selection of 85 Pythium isolates collected in South Africa from 1991 through to 2007. The isolates were characterised morphologically as well as through sequence and phylogenetic analyses of the internal transcribed spacer regions (ITS) and the 5.8S gene of the nuclear ribosomal DNA. Phylogenetic analyses showed that the isolates represented ten of the 11 published Pythium clades [Lévesque & De Cock, 2004. Molecular phylogeny and taxonomy of the genus Pythium. Mycological Research 108: 1363–1383]. Characterisation of isolates in clade D and J suggested that the phylogenetic concept of Pythium acanthicum and Pythium perplexum respectively, needs further investigation in order to enable reliable species identification within these clades. Our phylogenetic analyses of Pythium species in clade B also showed that species with globose sporangia group basal within this clade, and are not dispersed within the clade as previously reported. The 85 South African isolates represented 34 known species, of which 20 species have not been reported previously in South Africa. Additionally, three isolates (PPRI 8428, 8300 and 8418) were identified that may each represent putative new species, Pythium sp. WJB-1 to WJB-3.