Expanding the population genetic perspective of cnidarian‐Symbiodinium symbioses

The modern synthesis was a seminal period in the biological sciences, establishing many of the core principles of evolutionary biology that we know today. Significant catalysts were the contributions of R.A. Fisher, J.B.S. Haldane and Sewall Wright (and others) developing the theoretical underpinning of population genetics, thus demonstrating adaptive evolution resulted from the interplay of forces such as natural selection and mutation within groups of individuals occupying the same space and time (i.e. a population). Given its importance, it is surprising that detailed population genetic data remain lacking for numerous organisms vital to many ecosystems. For example, the coral reef ecosystem is well recognized for its high biodiversity and productivity, numerous ecological services and significant economic and societal values (Moberg & Folke 1999; Cinner 2014). Many coral reef invertebrates form symbiotic relationships with single‐celled dinoflagellates within the genus Symbiodinium Freudenthal (Taylor 1974), with hosts providing these (typically) intracellular symbionts with by‐products of metabolism and in turn receiving photosynthetically fixed carbon capable of meeting hosts’ respiratory demands (Falkowski et al. 1984; Muscatine et al. 1984). Unfortunately, the health and integrity of the coral reef ecosystem has been significantly and negatively impacted by onslaughts like anthropogenic eutrophication and disease in addition to global climate change, with increased incidences of ‘bleaching’ events (characterized as the loss of photosynthetic pigments from the algal cell or massive reduction of Symbiodinium density from hosts’ tissue) and host mortality leading to staggering declines in geographic coverage (Bruno & Selig 2007) that have raised questions on the viability of this ecosystem as we know it (Bellwood et al. 2004; Parmesan 2006). One avenue towards anticipating the future of the coral reef ecosystem is by developing a broader and deeper understanding of the current genotypic diversity encompassed within and between populations of their keystone species, the scleractinian corals and dinoflagellate symbionts, as they potentially possess functional variation (either singularly or in combination) that may come under selection due to the ongoing and rapid environmental changes they are experiencing. However, such studies, especially for members of the genus Symbiodinium, are sparse. In this issue, Baums et al. (2014) provide a significant contribution by documenting the range‐wide population genetics of Symbiodinium ‘fitti’ (Fig. 1 ) in the context of complementary data from its host, the endangered Caribbean elkhorn coral Acropora palmata (Fig. 1 ). Notable results of this study include a single S. ‘fitti’ genotype typically dominates an individual A. palmata colony both spatially and temporally, gene flow among coral host populations is a magnitude higher to that of its symbiont populations, and the partners possess disparate patterns of genetic differentiation across the Greater Caribbean. The implications of such findings are discussed herein.     

The effects of Symbiodinium (Pyrrhophyta) identity on growth,survivorship, and thermal tolerance of newly settled coral recruits

For many coral species, the obligate association with phylogenetically diverse algal endosymbiont species is dynamic in time and space. Here, we used controlled laboratory inoculations of newly settled, aposymbiotic corals (Orbicella faveolata) with two cultured species of algal symbiont (Symbiodinium microadriaticum and S. minutum) to examine the role of symbiont identity on growth, survivorship, and thermal tolerance of the coral holobiont. We evaluated these data in the context of Symbiodinium photophysiology for 9 months post‐settlement and also during a 5‐d period of elevated temperatures Our data show that recruits that were inoculated with S. minutum grew significantly slower than those inoculated with S. microadriaticum (occasionally co‐occurring with S. minutum), but that there was no difference in survivorship of O. faveolata polyps infected with Symbiodinium. However, photophysiological metrics (?Fv/F′m, the efficiency with which available light is used to drive photosynthesis and α, the maximum light utilization coefficient) were higher in those slower growing recruits containing S. minutum. These findings suggest that light use (i.e., photophysiology) and carbon acquisition by the coral host (i.e., host growth) are decoupled, but did not distinguish the source of this difference. Neither Symbiodinium treatment demonstrated a significant negative effect of a 5‐d exposure to temperatures as high as 32°C under low light conditions similar to those measured at settlement habitats.     

Genetics and Morphology Characterize the Dinoflagellate Symbiodinium voratum,n. sp., (Dinophyceae) as the Sole Representative of Symbiodinium Clade E

Dinoflagellates in the genus Symbiodinium are ubiquitous in shallow marine habitats where they commonly exist in symbiosis with cnidarians. Attempts to culture them often retrieve isolates that may not be symbiotic, but instead exist as free‐living species. In particular, cultures of Symbiodinium clade E obtained from temperate environments were recently shown to feed phagotrophically on bacteria and microalgae. Genetic, behavioral, and morphological evidence indicate that strains of clade E obtained from the northwestern, southwestern, and northeastern temperate Pacific Ocean as well as the Mediterranean Sea constitute a single species: Symbiodinium voratum n. sp. Chloroplast ribosomal 23S and mitochondrial cytochrome b nucleotide sequences were the same for all isolates. The D1/D2 domains of nuclear ribosomal DNA were identical among Western Pacific strains, but single nucleotide substitutions differentiated isolates from California (USA) and Spain. Phylogenetic analyses demonstrated that S. voratum is well‐separated evolutionarily from other Symbiodinium spp. The motile, or mastigote, cells from different cultures were morphologically similar when observed using light, scanning, and transmission electron microscopy; and the first complete Kofoidian plate formula for a Symbiodinium sp. was characterized. As the largest of known Symbiodinium spp., the average coccoid cell diameters measured among cultured isolates ranged between 12.2 (± 0.2 SE) and 13.3 (± 0.2 SE) μm. Unique among species in the genus, a high proportion (approximately 10–20%) of cells remain motile in culture during the dark cycle. Although S. voratum occurs on surfaces of various substrates and is potentially common in the plankton of coastal areas, it may be incapable of forming stable mutualistic symbioses.     

The Occurrence of Mixed Infections of Symbiodinium (Dinoflagellata) within Individual Hosts

Coral reef ecosystems depend on symbiosis between dinoflagellates of the genus Symbiodinium Freudenthal and their various hosts. The physiological characteristics associated with a particular lineage or species of Symbiodinium can determine a host's susceptibility to harmful bleaching. Therefore, the threat posed by global climate change on a host may be reduced if it can switch or shuffle its dominant algal symbiont type. An important prerequisite to this potential to switch or shuffle is the ability to host multiple alternative dominant symbiont genotypes. To examine the distribution of this trait, we review reports of mixed Symbiodinium infections in corals and nonscleractinian hosts from a phylogenetic perspective. Hosts showing evidence of mixed infection are broadly distributed across the most deeply divergent host lineages, including foraminifera, mollusks, sponges, and cnidarians. The occurrence of mixed infections is also broadly distributed across most clades of scleractinian corals. Individual colonies of certain well‐studied cosmopolitan coral genera, such as Acropora, Montastraea, and Pocillopora, yield many reports of mixed infection, while other genera, such as Porites, do not. We further discuss mixed Symbiodinium infections in the context of evolutionary ecology theory. Selection pressures that affect the prevalence of mixed infection may be exerted by variation in host environment, host ontogeny, symbiont transmission strategy, host regulation of symbiont populations, availability of free‐living symbiont lineages, competition between symbiont lineages, and niche partitioning of the internal host environment.     

PHYLOGENETIC POSITION OF SYMBIODINIUM (DINOPHYCEAE) ISOLATES FROM TRIDACNIDS (BIVALVIA), CARDIIDS (BIVALVIA), A SPONGE (PORIFERA), A SOFT CORAL (ANTHOZOA), AND A FREE-LIVING STRAIN

The genus Symbiodinium is the commonly observed symbiotic dinoflagellate (zooxanthellae) that forms mutual associations with various marine invertebrates. Numerous studies have revealed that the genus is comprised of a group of diverse taxa, and information on the phylogenetic relationships among the genus’ members is increasing. In this study, small subunit (SSU) ribosomal RNA (ssrRNA) gene sequences were determined for 15 more Symbiodinium strains from 12 relatively unstudied host taxa (Indo-Pacific tridacnids, cardiids, sponge, and soft coral), 1 hitherto unreported free-living Symbiodinium strain, and 4 other Symbiodinium strains from four other host taxa (Indo-Pacific zoanthid, foraminifer, jellyfish, and mid-Pacific hard coral). Their respective phylogenetic positions were inferred, and strains that are either closely related to or distinct from previously reported Symbiodinium taxa were revealed. The cultured Symbiodinium strains isolated from individuals of six species of tridacnids and three species of cardiids all had identical ssrRNA gene sequences, are closely related to S. microadriaticum Freudenthal, and are indistinguishable from the RFLP Type A strain previously reported. However, the ssrRNA gene sequences of clam symbionts that were obtained via gene cloning were different from those of the cultured isolates and represent strains that are close to the RFLP Type C strains. The Symbiodinium-like dinoflagellate from the Indo-Pacific sponge Haliclona koremella De Laubenfels is distinct from any of the Symbiodinium taxa studied and may be similar to the symbiont previously isolated from the stony coral Montipora patula Quelch. The isolates from the soft coral Sarcophyton glaucum Quoy et Gaimard and from the zoanthid Zoanthus sp. are both very closely related to S. pilosum Trench et Blank. The free-living Symbiodinium isolate is very closely related to the symbiont isolated from the Indo-Pacific foraminifer Amphisorus hemprichii Ehrenberg, which in turn is distinct from the Red Sea strain isolated from a similar host. Theisolate from Cassiopeia sp. is different from S. microadriaticum F., the type species harbored by Cassiopeia xamachana Bigelow, and is instead very closely related to S. pulchrorum Trench isolated from a sea anemone. The symbiont from the stony coral M. verrucosa Lamarck is a sister taxon to the symbionts isolated from the foraminifera Marginopora kudakajimensis Gudmundsson and Sorites orbiculus Forskål. These data suggest that polymorphic symbioses extend from cnidarians to some bivalve, foraminifer, and jellyfish host species.     

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).     

Do introduced endosymbiotic dinoflagellates ‘take’ to new hosts?

In a recent communication by Stat and Gates (Biol Invasions 10: 579–583, 2008), discovery of a symbiotic combination involving the coral Acropora cytherea and the dinoflagellate endosymbiont, Symbiodinium A1 (Symbiodinium microadriaticum, Freudenthal sensu stricto) in the Northwest Hawaiian Islands was interpreted to be the result of a ‘recent’ introduction. While introductions of symbiotic dinoflagellates have occurred and are occurring, the authors’ conclusion was made without sufficient information about the geographic range and host specificity exhibited by A1. The only direct genetic analysis of symbionts from the putative host vector, a jellyfish in the genus Cassiopeia sp., from Kaneohe Bay on the Island of Oahu, found that it contained a different symbiont species, A3. Furthermore, Stat and Gates (Biol Invasions 10: 579–583, 2008) did not consider the importance of host-symbiont specificity in preventing the establishment of a foreign symbiont species. In comparison to A. cytherea, A. longicyathus on the southern most Great Barrier Reef also hosts Symbiodinium A1 and a closely related endemic, A1a. Instead of assuming that A. cytherea has an unnatural association, a practical explanation is that long-term ecological and evolutionary processes influenced by local environments underlie the unusual, but not unprecedented finding of a Pacific acroporid associating with Clade A Symbiodinium spp.     

Exploring Symbiodinium diversity and host specificity in Acropora corals from geographical extremes of Western Australia with 454 amplicon pyrosequencing

Scleractinian corals have demonstrated the ability to shuffle their endosymbiotic dinoflagellate communities (genus Symbiodinium) during periods of acute environmental stress. This has been proposed as a mechanism of acclimation, which would be increased by a diverse and flexible association with Symbiodinium. Conventional molecular techniques used to evaluate Symbiodinium diversity are unable to identify genetic lineages present at background levels below 10%. Next generation sequencing (NGS) offers a solution to this problem and can resolve microorganism diversity at much finer scales. Here we apply NGS to evaluate Symbiodinium diversity and host specificity in Acropora corals from contrasting regions of Western Australia. The application of 454 pyrosequencing allowed for detection of Symbiodinium operational taxonomic units (OTUs) occurring at frequencies as low as 0.001%, offering a 10 000‐fold increase in sensitivity compared to traditional methods. All coral species from both regions were overwhelmingly dominated by a single clade C OTU (accounting for 98% of all recovered sequences). Only 8.5% of colonies associated with multiple clades (clades C and D, or C and G), suggesting a high level of symbiont specificity in Acropora assemblages in Western Australia. While only 40% of the OTUs were shared between regions, the dominance of a single OTU resulted in no significant difference in Symbiodinium community structure, demonstrating that the coral‐algal symbiosis can remain stable across more than 15° of latitude and a range of sea surface temperature profiles. This study validates the use of NGS platforms as tools for providing fine‐scale estimates of Symbiodinium diversity and can offer critical insight into the flexibility of the coral‐algal symbiosis.     

Spatio-Temporal Analyses of Symbiodinium Physiology of the Coral Pocillopora verrucosa along Large-Scale Nutrient and Temperature Gradients in the Red Sea

Algal symbionts (zooxanthellae, genus Symbiodinium) of scleractinian corals respond strongly to temperature, nutrient and light changes. These factors vary greatly along the north-south gradient in the Red Sea and include conditions, which are outside of those typically considered optimal for coral growth. Nevertheless, coral communities thrive throughout the Red Sea, suggesting that zooxanthellae have successfully acclimatized or adapted to the harsh conditions they experience particularly in the south (high temperatures and high nutrient supply). As such, the Red Sea is a region, which may help to better understand how zooxanthellae and their coral hosts successfully acclimatize or adapt to environmental change (e.g. increased temperatures and localized eutrophication). To gain further insight into the physiology of coral symbionts in the Red Sea, we examined the abundance of dominant Symbiodinium types associated with the coral Pocillopora verrucosa, and measured Symbiodinium physiological characteristics (i.e. photosynthetic processes, cell density, pigmentation, and protein composition) along the latitudinal gradient of the Red Sea in summer and winter. Despite the strong environmental gradients from north to south, our results demonstrate that Symbiodinium microadriaticum (type A1) was the predominant species in P. verrucosa along the latitudinal gradient. Furthermore, measured physiological characteristics were found to vary more with prevailing seasonal environmental conditions than with region-specific differences, although the measured environmental parameters displayed much higher spatial than temporal variability. We conclude that our findings might present the result of long-term acclimatization or adaptation of S. microadriaticum to regionally specific conditions within the Red Sea. Of additional note, high nutrients in the South correlated with high zooxanthellae density indicating a compensation for a temperature-driven loss of photosynthetic performance, which may prove promising for the resilience of these corals under increase of temperature increase and eutrophication.     

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.     

Antioxidant plasticity and thermal sensitivity in four types of Symbiodinium sp.

Warmer than average summer sea surface temperature is one of the main drivers for coral bleaching, which describes the loss of endosymbiotic dinoflagellates (genus: Symbiodinium) in reef‐building corals. Past research has established that oxidative stress in the symbiont plays an important part in the bleaching cascade. Corals hosting different genotypes of Symbiodinium may have varying thermal bleaching thresholds, but changes in the symbiont's antioxidant system that may accompany these differences have received less attention. This study shows that constitutive activity and up‐regulation of different parts of the antioxidant network under thermal stress differs between four Symbiodinium types in culture and that thermal susceptibility can be linked to glutathione redox homeostasis. In Symbiodinium B1, C1 and E, declining maximum quantum yield of PSII (F_v/F_m) and death at 33°C were generally associated with elevated superoxide dismutase (SOD) activity and a more oxidized glutathione pool. Symbiodinium F1 exhibited no decline in F_v/F_m or growth, but showed proportionally larger increases in ascorbate peroxidase (APX) activity and glutathione content (GSx), while maintaining GSx in a reduced state. Depressed growth in Symbiodinium B1 at a sublethal temperature of 29°C was associated with transiently increased APX activity and glutathione pool size, and an overall increase in glutathione reductase (GR) activity. The collapse of GR activity at 33°C, together with increased SOD, APX and glutathione S‐transferase activity, contributed to a strong oxidation of the glutathione pool with subsequent death. Integrating responses of multiple components of the antioxidant network highlights the importance of antioxidant plasticity in explaining type‐specific temperature responses in Symbiodinium.     

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.

     

Symbiodinium (Dinophyceae) diversity in reef‐invertebrates along an offshore to inshore reef gradient near Lizard Island,Great Barrier Reef

Despite extensive work on the genetic diversity of reef invertebrate‐dinoflagellate symbioses on the Great Barrier Reef (GBR; Australia), large information gaps exist from northern and inshore regions. Therefore, a broad survey was done comparing the community of inshore, mid‐shelf and outer reefs at the latitude of Lizard Island. Symbiodinium (Freudenthal) diversity was characterized using denaturing gradient gel electrophoresis fingerprinting and sequencing of the ITS2 region of the ribosomal DNA. Thirty‐nine distinct Symbiodinium types were identified from four subgeneric clades (B, C, D, and G). Several Symbiodinium types originally characterized from the Indian Ocean were discovered as well as eight novel types (C1kk, C1LL, C3nn, C26b, C161a, C162, C165, C166). Multivariate analyses on the Symbiodinium species diversity data showed a strong link with host identity, consistent with previous findings. Of the four environmental variables tested, mean austral winter sea surface temperature (SST) influenced Symbiodinium distribution across shelves most significantly. A similar result was found when the analysis was performed on Symbiodinium diversity data of genera with an open symbiont transmission mode separately with chl a and PAR explaining additional variation. This study underscores the importance of SST and water quality related variables as factors driving Symbiodinium distribution on cross‐shelf scales. Furthermore, this study expands our knowledge on Symbiodinium species diversity, ecological partitioning (including host‐specificity) and geographic ranges across the GBR. The accelerating rate of environmental change experienced by coral reef ecosystems emphasizes the need to comprehend the full complexity of cnidarian symbioses, including the biotic and abiotic factors that shape their current distributions.     

Molecular signatures of host specificity linked to habitat specialization in Exaiptasia sea anemones

Rising ocean temperatures associated with global climate change induce breakdown of the symbiosis between coelenterates and photosynthetic microalgae of the genus Symbiodinium. Association with more thermotolerant partners could contribute to resilience, but the genetic mechanisms controlling specificity of hosts for particular Symbiodinium types are poorly known. Here, we characterize wild populations of a sea anemone laboratory model system for anthozoan symbiosis, from contrasting environments in Caribbean Panama. Patterns of anemone abundance and symbiont diversity were consistent with specialization of holobionts for particular habitats, with Exaiptasia pallida/S. minutum (ITS2 type B1) abundant on vertical substrate in thermally stable, shaded environments but E. brasiliensis/Symbiodinium sp. (ITS2 clade A) more common in shallow areas subject to high temperature and irradiance. Population genomic sequencing revealed a novel E. pallida population from the Bocas del Toro Archipelago that only harbors S. minutum. Loci most strongly associated with divergence of the Bocas‐specific population were enriched in genes with putative roles in cnidarian symbiosis, including activators of the complement pathway of the innate immune system, thrombospondin‐type‐1 repeat domain proteins, and coordinators of endocytic recycling. Our findings underscore the importance of unmasking cryptic diversity in natural populations and the role of long‐term evolutionary history in mediating interactions with Symbiodinium.     

Molecular Features and mRNA Expression of the Receptor for Activated C Kinase 1 from Symbiodinium microadriaticum ssp. microadriaticum During Growth and the Light/Dark cycle

Two genes of the RACK1 homolog from the photosynthetic dinoflagellate Symbiodinium microadriaticum ssp. microadriaticum (SmicRACK1), termed SmicRACK1A and SmicRACK1B, were found tandemly arrayed and displayed a single synonymous substitution (T/C) encoding threonine. They included two exons of 942 bp each, encoding 313 amino acids with seven WD‐40 repeats and two PKC‐binding motifs. The protein theoretical mass and pI were 34,200 Da and 5.9, respectively. SmicRACK1 showed maximum identities with RACK1 homologs at the amino acid and nucleotide level, respectively, of 92 and 84% with S. minutum, and phylogenetic analysis revealed clustered related RACK1 sequences from the marine dinoflagellates S. minutum, Heterocapsa triquetra, Karenia brevis, and Alexandrium tamarense. Interestingly, light‐dependent regulatory elements were found both within the 282 bp SmicRACK1A promotor sequence, and within an intergenic sequence of 359 nucleotides that separated both genes, which strongly suggest light‐related functions. This was further supported by mRNA accumulation analysis, which fluctuated along the light and dark phases of the growth cycle showing maximum specific peaks under either condition. Finally, qRT‐PCR analysis revealed differential SmicRACK1 mRNA accumulation with maxima at 6 and 20 d of culture. Our SmicRACK1 characterization suggests roles in active growth and proliferation, as well as light/dark cycle regulation in S. microadriaticum.     

Deep-Sequencing Method for Quantifying Background Abundances of Symbiodinium Types: Exploring the Rare Symbiodinium Biosphere in Reef-Building Corals

The capacity of reef-building corals to associate with environmentally-appropriate types of endosymbionts from the dinoflagellate genus Symbiodinium contributes significantly to their success at local scales. Additionally, some corals are able to acclimatize to environmental perturbations by shuffling the relative proportions of different Symbiodinium types hosted. Understanding the dynamics of these symbioses requires a sensitive and quantitative method of Symbiodinium genotyping. Electrophoresis methods, still widely utilized for this purpose, are predominantly qualitative and cannot guarantee detection of a background type below 10% of the total Symbiodinium population. Here, the relative abundances of four Symbiodinium types (A13, C1, C3, and D1) in mixed samples of known composition were quantified using deep sequencing of the internal transcribed spacer of the ribosomal RNA gene (ITS-2) by means of Next Generation Sequencing (NGS) using Roche 454. In samples dominated by each of the four Symbiodinium types tested, background levels of the other three types were detected when present at 5%, 1%, and 0.1% levels, and their relative abundances were quantified with high (A13, C1, D1) to variable (C3) accuracy. The potential of this deep sequencing method for resolving fine-scale genetic diversity within a symbiont type was further demonstrated in a natural symbiosis using ITS-1, and uncovered reef-specific differences in the composition of Symbiodinium microadriaticum in two species of acroporid corals (Acropora digitifera and A. hyacinthus) from Palau. The ability of deep sequencing of the ITS locus (1 and 2) to detect and quantify low-abundant Symbiodinium types, as well as finer-scale diversity below the type level, will enable more robust quantification of local genetic diversity in Symbiodinium populations. This method will help to elucidate the role that background types have in maximizing coral fitness across diverse environments and in response to environmental change.     

Different strategies of energy storage in cultured and freshly isolated Symbiodinium sp.

The endosymbiotic relationship between cnidarians and Symbiodinium is critical for the survival of coral reefs. In this study, we developed a protocol to rapidly and freshly separate Symbiodinium from corals and sea anemones. Furthermore, we compared these freshly‐isolated Symbiodinium with cultured Symbiodinium to investigate host and Symbiodinium interaction. Clade B Symbiodinium had higher starch content and lower lipid content than those of clades C and D in both freshly isolated and cultured forms. Clade C had the highest lipid content, particularly when associated with corals. Moreover, the coral‐associated Symbiodinium had higher protein content than did cultured and sea anemone‐associated Symbiodinium. Regarding fatty acid composition, cultured Symbiodinium and clades B, C, and D shared similar patterns, whereas sea anemone‐associated Symbiodinium had a distinct pattern compared coral‐associated Symbiodinium. Specifically, the levels of monounsaturated fatty acids were lower than those of the saturated fatty acids, and the level of polyunsaturated fatty acids (PUFAs) were the highest in all examined Symbiodinium. Furthermore, PUFAs levels were higher in coral‐associated Symbiodinium than in cultured Symbiodinium. These results altogether indicated that different Symbiodinium clades used different energy storage strategies, which might be modified by hosts.     

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.