The dynamics of microbial partnerships in the coral Isopora palifera

Both bacteria and algal symbionts (genus Symbiodinium), the two major microbial partners in the coral holobiont, respond to fluctuations in the environment, according to current reports; however, little evidence yet indicates that both populations have any direct interaction with each other in seasonal fluctuation. In this study, we present field observations of a compositional change in bacteria and Symbiodinium in the coral Isopora palifera in three separate coral colonies following monthly sampling from February to November in 2008. Using massively parallel pyrosequencing, over 200 000 bacterial V6 sequences were classified to build the bacterial community profile; in addition, the relative composition and quantity of Symbiodinium clades C and D were determined by real-time PCR. The results showed that coral-associated bacterial and Symbiodinium communities were highly dynamic and dissimilar among the tagged coral colonies, suggesting that the effect of host specificity was insignificant. The coral-associated bacterial community was more diverse (Shannon index up to 6.71) than previous estimates in other corals and showed rapid seasonal changes. The population ratios between clade C and D groups of Symbiodinium varied in the tagged coral colonies through the different seasons; clade D dominated in most of the samples. Although significant association between bacteria and symbiont was not detected, this study presents a more detailed picture of changes in these two major microbial associates of the coral at the same time, using the latest molecular approaches.     

Impact of Light and Temperature on the Uptake of Algal Symbionts by Coral Juveniles

The effects of temperature and light on the breakdown of the coral-Symbiodinium symbiosis are well documented but current understanding of their roles during initial uptake and establishment of symbiosis is limited. In this study, we investigate how temperature and light affect the uptake of the algal symbionts, ITS1 types C1 and D, by juveniles of the broadcast-spawning corals Acropora tenuis and A. millepora. Elevated temperatures had a strong negative effect on Symbiodinium uptake in both coral species, with corals at 31°C showing as little as 8% uptake compared to 87% at 28°C. Juveniles in high light treatments (390 µmol photons m⁻² s⁻¹) had lower cell counts across all temperatures, emphasizing the importance of the light environment during the initial uptake phase. The proportions of the two Symbiodinium types taken up, as quantified by a real time PCR assay using clade C- and D-specific primers, were also influenced by temperature, although variation in uptake dynamics between the two coral species indicates a host effect. At 28°C, A. tenuis juveniles were dominated by C1 Symbiodinium, and while the number of D Symbiodinium cells increased at 31°C, they never exceeded the number of C1 cells. In contrast, juveniles of A. millepora had approximately equal numbers of C1 and D cells at 28°C, but were dominated by D at 30°C and 31°C. This study highlights the significant role that environmental factors play in the establishment of coral-Symbiodinium symbiosis and provides insights into how potentially competing Symbiodinium types take up residence in coral juveniles.     

Functional significance of dinitrogen fixation in sustaining coral productivity under oligotrophic conditions

Functional traits define species by their ecological role in the ecosystem. Animals themselves are host–microbe ecosystems (holobionts), and the application of ecophysiological approaches can help to understand their functioning. In hard coral holobionts, communities of dinitrogen (N₂)-fixing prokaryotes (diazotrophs) may contribute a functional trait by providing bioavailable nitrogen (N) that could sustain coral productivity under oligotrophic conditions. This study quantified N₂ fixation by diazotrophs associated with four genera of hermatypic corals on a northern Red Sea fringing reef exposed to high seasonality. We found N₂ fixation activity to be 5- to 10-fold higher in summer, when inorganic nutrient concentrations were lowest and water temperature and light availability highest. Concurrently, coral gross primary productivity remained stable despite lower Symbiodinium densities and tissue chlorophyll a contents. In contrast, chlorophyll a content per Symbiodinium cell increased from spring to summer, suggesting that algal cells overcame limitation of N, an essential element for chlorophyll synthesis. In fact, N₂ fixation was positively correlated with coral productivity in summer, when its contribution was estimated to meet 11% of the Symbiodinium N requirements. These results provide evidence of an important functional role of diazotrophs in sustaining coral productivity when alternative external N sources are scarce.     

Do clades of symbiotic dinoflagellates in scleractinian corals of the Gulf of Eilat (Red Sea) differ from those of other coral reefs?

The symbiotic association between corals and zooxanthellae has been a major contributing factor in the success of reef-building corals. Most of these endocellular microalgal symbionts belong to the dinoflagellate genus Symbiodinium. However, considerable genetic diversity was revealed within this taxon, as is evident in the several clades of Symbiodinium found in association with hermatypic corals all over the world. The coral reefs of Eilat (Aqaba), where winter temperature minima of 21 °C are close to threshold values that prevent reef development, are among the northernmost reefs in the world. Furthermore, due to the circulation patterns of the Gulf, the extremely high evaporation, and lack of any riverine inputs, the Gulf's waters are highly saline (40.5‰). In spite of the extreme location, a high diversity of coral species has been reported in this area. In this study, using PCR, we specifically amplified zooxanthellae 18S ribosomal DNA from symbionts of 11 coral species, and analyzed it with respect to RFLP and DNA sequence.Of the several clades described from the same coral hosts in other localities, only A and C were found in the present study. Symbiodinium populations in the host examined from Eilat were different relative to other parts of the world. This distribution is discussed in relation to reproduction strategy: broadcasting versus brooding. Based on our results, we suggest that clade A is transferred through a closed system. As mass bleaching in the Gulf has never been observed, we suggest that the adaptive mechanisms presumably favoring clade diversity were not yet significant in our relatively cool area.     

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.     

Corals shed bacteria as a potential mechanism of resilience to organic matter enrichment

Understanding the mechanisms of resilience of coral reefs to anthropogenic stressors is a critical step toward mitigating their current global decline. Coral–bacteria associations are fundamental to reef health and disease, but direct observations of these interactions remain largely unexplored. Here, we use novel technology, high-speed laser scanning confocal microscopy on live coral (Pocillopora damicornis), to test the hypothesis that corals exert control over the abundance of their associated bacterial communities by releasing (‘shedding'') bacteria from their surface, and that this mechanism can counteract bacterial growth stimulated by organic inputs. We also test the hypothesis that the coral pathogen Vibrio coralliilyticus can evade such a defense mechanism. This first report of direct observation with high-speed confocal microscopy of living coral and its associated bacterial community revealed a layer (3.3–146.8 μm thick) on the coral surface where bacteria were concentrated. The results of two independent experiments showed that the bacterial abundance in this layer was not sensitive to enrichment (5 mg l⁻¹ peptone), and that coral fragments exposed to enrichment released significantly more bacteria from their surfaces than control corals (P<0.01; 35.9±1.4 × 10⁵ cells cm⁻² coral versus 1.3±0.5 × 10⁵ cells cm⁻² coral). Our results provide direct support to the hypothesis that shedding bacteria may be an important mechanism by which coral-associated bacterial abundances are regulated under organic matter stress. Additionally, the novel ability to watch this ecological behavior in real-time at the microscale opens an unexplored avenue for mechanistic studies of coral–microbe interactions.     

The Roles and Interactions of Symbiont,Host and Environment in Defining Coral Fitness

Background

Reef-building corals live in symbiosis with a diverse range of dinoflagellate algae (genus Symbiodinium) that differentially influence the fitness of the coral holobiont. The comparative role of symbiont type in holobiont fitness in relation to host genotype or the environment, however, is largely unknown. We addressed this knowledge gap by manipulating host-symbiont combinations and comparing growth, survival and thermal tolerance among the resultant holobionts in different environments.

Methodology/Principal Findings

Offspring of the coral, Acropora millepora, from two thermally contrasting locations, were experimentally infected with one of six Symbiodinium types, which spanned three phylogenetic clades (A, C and D), and then outplanted to the two parental field locations (central and southern inshore Great Barrier Reef, Australia). Growth and survival of juvenile corals were monitored for 31–35 weeks, after which their thermo-tolerance was experimentally assessed. Our results showed that: (1) Symbiodinium type was the most important predictor of holobiont fitness, as measured by growth, survival, and thermo-tolerance; (2) growth and survival, but not heat-tolerance, were also affected by local environmental conditions; and (3) host population had little to no effect on holobiont fitness. Furthermore, coral-algal associations were established with symbiont types belonging to clades A, C and D, but three out of four symbiont types belonging to clade C failed to establish a symbiosis. Associations with clade A had the lowest fitness and were unstable in the field. Lastly, Symbiodinium types C1 and D were found to be relatively thermo-tolerant, with type D conferring the highest tolerance in A. millepora.

Conclusions/Significance

These results highlight the complex interactions that occur between the coral host, the algal symbiont, and the environment to shape the fitness of the coral holobiont. An improved understanding of the factors affecting coral holobiont fitness will assist in predicting the responses of corals to global climate change.     

Change in algal symbiont communities after bleaching,not prior heat exposure,increases heat tolerance of reef corals

Mutualistic organisms can be particularly susceptible to climate change stress, as their survivorship is often limited by the most vulnerable partner. However, symbiotic plasticity can also help organisms in changing environments by expanding their realized niche space. Coral–algal (Symbiodinium spp.) symbiosis exemplifies this dichotomy: the partnership is highly susceptible to ‘bleaching’ (stress‐induced symbiosis breakdown), but stress‐tolerant symbionts can also sometimes mitigate bleaching. Here, we investigate the role of diverse and mutable symbiotic partnerships in increasing corals' ability to thrive in high temperature conditions. We conducted repeat bleaching and recovery experiments on the coral Montastraea cavernosa, and used quantitative PCR and chlorophyll fluorometry to assess the structure and function of Symbiodinium communities within coral hosts. During an initial heat exposure (32 °C for 10 days), corals hosting only stress‐sensitive symbionts (Symbiodinium C3) bleached, but recovered (at either 24 °C or 29 °C) with predominantly (>90%) stress‐tolerant symbionts (Symbiodinium D1a), which were not detected before bleaching (either due to absence or extreme low abundance). When a second heat stress (also 32 °C for 10 days) was applied 3 months later, corals that previously bleached and were now dominated by D1a Symbiodinium experienced less photodamage and symbiont loss compared to control corals that had not been previously bleached, and were therefore still dominated by Symbiodinium C3. Additional corals that were initially bleached without heat by a herbicide (DCMU, at 24 °C) also recovered predominantly with D1a symbionts, and similarly lost fewer symbionts during subsequent thermal stress. Increased thermotolerance was also not observed in C3‐dominated corals that were acclimated for 3 months to warmer temperatures (29 °C) before heat stress. These findings indicate that increased thermotolerance post‐bleaching resulted from symbiont community composition changes, not prior heat exposure. Moreover, initially undetectable D1a symbionts became dominant only after bleaching, and were critical to corals' resilience after stress and resistance to future stress.     

Local adaptation constrains the distribution potential of heat-tolerant Symbiodinium from the Persian/Arabian Gulf

The symbiotic association of corals and unicellular algae of the genus Symbiodinium in the southern Persian/Arabian Gulf (PAG) display an exceptional heat tolerance, enduring summer peak temperatures of up to 36 °C. As yet, it is not clear whether this resilience is related to the presence of specific symbiont types that are exclusively found in this region. Therefore, we used molecular markers to identify the symbiotic algae of three Porites species along >1000 km of coastline in the PAG and the Gulf of Oman and found that a recently described species, Symbiodinium thermophilum, is integral to coral survival in the southern PAG, the world''s hottest sea. Despite the geographic isolation of the PAG, we discovered that representatives of the S. thermophilum group can also be found in the adjacent Gulf of Oman providing a potential source of thermotolerant symbionts that might facilitate the adaptation of Indian Ocean populations to the higher water temperatures expected for the future. However, corals from the PAG associated with S. thermophilum show strong local adaptation not only to high temperatures but also to the exceptionally high salinity of their habitat. We show that their superior heat tolerance can be lost when these corals are exposed to reduced salinity levels common for oceanic environments elsewhere. Consequently, the salinity prevailing in most reefs outside the PAG might represent a distribution barrier for extreme temperature-tolerant coral/Symbiodinium associations from the PAG.     

<Emphasis Type="Italic">Symbiodinium</Emphasis> associations with diseased and healthy scleractinian corals

Despite recent advances in identifying the causative agents of disease in corals and understanding the impact of epizootics on reef communities, little is known regarding the interactions among diseases, corals, and their dinoflagellate endosymbionts (Symbiodinium spp.). Since the genotypes of both corals and their resident Symbiodinium contribute to colony-level phenotypes, such as thermotolerance, symbiont genotypes might also contribute to the resistance or susceptibility of coral colonies to disease. To explore this, Symbiodinium were identified using the internal transcribed spacer-2 region of ribosomal DNA from diseased and healthy tissues within individual coral colonies infected with black band disease (BB), dark spot syndrome (DSS), white plague disease (WP), or yellow blotch disease (YB) in the Florida Keys (USA) and the US Virgin Islands. Most of the diseased colonies sampled contained B1, B5a, or C1 (depending on host species), while apparently healthy colonies of the same coral species frequently hosted these types and/or additional symbiont diversity. No potentially “parasitic” Symbiodinium types, uniquely associated with diseased coral tissue, were detected. Within most individual colonies, the same dominant Symbiodinium type was detected in diseased and visually healthy tissues. These data indicate that specific Symbiodinium types are not correlated with the infected tissues of diseased colonies and that DSS and WP onset do not trigger symbiont shuffling within infected tissues. However, few diseased colonies contained clade D symbionts suggesting a negative correlation between hosting Symbiodinium clade D and disease incidence in scleractinian corals. Understanding the influence of Symbiodinium diversity on colony phenotypes may play a critical role in predicting disease resistance and susceptibility in scleractinian corals.     

Light microenvironment and single-cell gradients of carbon fixation in tissues of symbiont-bearing corals

Recent coral optics studies have revealed the presence of steep light gradients and optical microniches in tissues of symbiont-bearing corals. Yet, it is unknown whether such resource stratification allows for physiological differences of Symbiodinium within coral tissues. Using a combination of stable isotope labelling and nanoscale secondary ion mass spectrometry, we investigated in hospite carbon fixation of individual Symbiodinium as a function of the local O₂ and light microenvironment within the coral host determined with microsensors. We found that net carbon fixation rates of individual Symbiodinium cells differed on average about sixfold between upper and lower tissue layers of single coral polyps, whereas the light and O₂ microenvironments differed ~15- and 2.5-fold, respectively, indicating differences in light utilisation efficiency along the light microgradient within the coral tissue. Our study suggests that the structure of coral tissues might be conceptually similar to photosynthetic biofilms, where steep physico-chemical gradients define form and function of the local microbial community.The quantity and quality of solar radiation are arguably the most important environmental resources that affect the structure and function of photosynthetic communities in both terrestrial and aquatic environments. Sunlight is of key importance for symbiont-bearing corals, driving the symbiotic interaction between the coral animal and its photosynthetic microalgae of the genus Symbiodinium (Roth, 2014). Light attenuation through the water mass and over the reef matrix has a fundamental role in structuring morphology, function and distribution of corals and their symbiotic algae with depth (Falkowski et al., 1990). Recent studies on the optical properties of corals have shown that light is also a highly stratified resource at the level of individual coral polyps and tissue layers (Wangpraseurt et al., 2014). Steep light gradients exist within the polyp tissues of some corals and light can attenuate by more than an order of magnitude within tissues, that is, comparable to the attenuation that can occur in open oceanic waters between the surface and >25 m of water depth (Kirk, 1994; Wangpraseurt et al., 2012). In this study, we investigated whether such light gradients within coral tissues are correlated with a stratification of Symbiodinium physiology in hospite.We used fibre-optic and electrochemical microsensors together with stable isotopic labelling and nanoscale secondary ion mass spectrometry (NanoSIMS) to estimate single-cell carbon fixation rates across light gradients within coral tissues. We collected several fragments of Favites sp. from the Heron Island reef flat (152°69'' E, 20°299'' S), Great Barrier Reef, Australia. Fragments were cultured under a downwelling photon irradiance (400–700 nm) of ~100 μmol photons per m² per s (12/12 h cycle), in aerated seawater (25 °C, salinity 33). Photosynthesis-irradiance curves for the investigated corals were determined with an imaging pulse amplitude modulated fluorometer (I-PAM, Walz GmbH, Effeltrich, Germany; Ralph et al., 2005). Values for saturating irradiance, E_max, and irradiance at onset of saturation, E_k, were ~350 μmol photons per m² per s and ~160 μmol photons per m² per s, respectively (data not shown). These values are typical for healthy corals kept under moderate irradiance (Ralph et al., 2005). To ensure incubations at irradiance levels where photosynthesis and irradiance correlated linearly, that is, on the linearly increasing part of the P vs I curve, all experiments were performed at ~80 μmol photons per m² per s (12/12 h cycle). Microsensor measurements of scalar irradiance (tip size ~60 μm; Lassen et al., 1992) and O₂ concentration (OX-50, tip size 50 μm, Unisense A/S, Aarhus, Denmark) were performed within the polyp and coenosarc tissues of corals as described previously (Figures 1a and b; Wangpraseurt et al., 2012). After microsensor measurements, corals were incubated with ¹³C-bicarbonate (Supplementary Text S1). NanoSIMS imaging was then applied on coral tissue sections, as described by Pernice et al. (2014) to quantify the assimilation of dissolved inorganic carbon into individual Symbiodinium cells across polyp (oral and aboral) and coenosarc tissues of corals. Briefly, corals were incubated in small aquaria with 2 mm NaH¹³CO₃ in artificial sea water (recipe adapted from Harrison et al., 1980). After 24 h of isotopic incubation, coral fragments were sampled, chemically fixed and processed for NanoSIMS analyses (see Kopp et al., 2013; Pernice et al., 2012, 2014; and Supplementary Text S1,Supplementary Figure S1).Open in a separate windowFigure 1Internal microenvironment and single-cell ¹³C assimilation by Symbiodinium cells within Favites sp. (a) Representative measurement locations indicating connecting tissue (c, coenosarc; white circle) and polyp tissue (p; red circle). Scale bar is 0.5 cm. (b) Schematic diagram of the vertical arrangement of the polyp tissue structure (not drawn to scale). The coral tissue consists of oral and aboral gastrodermal tissues that contain photosymbiont cells (~10 μm in diameter). The two tissue layers are separated by a flexible gastrodermal cavity and the entire mean polyp tissue thickness was 1150 μm (±385 s.d., n=8) as determined by microsensor profiles. The NanoSIMS images (c–e) show the ¹³C/¹²C isotopic ratio for Symbiodinium cells in coenosarc tissue (c), the upper oral polyp tissue (d) and in the lowest layer of aboral polyp tissue (e). Scale bars are 10 μm. The colour scale of the NanoSIMS images is in hue saturation intensity ranging from 220 in blue (which corresponds to natural ¹³C/¹²C isotopic ratio of 0.0110) to 1000 in red (which corresponds to ¹³C/¹²C isotopic ratio of 0.05, ~4.5 times above the natural ¹³C/¹²C isotopic ratio). Quantification of ¹³C enrichment of individual Symbiodinium cells was obtained by selecting regions of interest that were defined in Open_MIMS (http://nrims.harvard.edu/software/openmims) by drawing the contours of the Symbiodinium cells directly on the NanoSIMS images. (f) Mean enrichment measured in Symbiodinium cells by NanoSIMS, in coenosarc tissue (in white, n=33), in upper oral polyp tissue (in grey, n=25), in the lowest layer of polyp tissue (in turquoise, n=17) and in the control treatment (n=20). Bars in the histograms indicate the s.e.m. enrichment quantified for the different whole Symbiodinium cells for each tissue category. Microsensor measurements of (g) scalar irradiance and (h) O₂ performed along depth gradients within the polyp tissue (mean±s.d., n=4). Measurements were averaged for the first 100 μm from the tissue surface (oral) and the last 100 μm from the skeleton (aboral). The oral and aboral depth was defined through gentle touching of the microsensor tip at the surface of the coral tissue and skeleton, respectively.Our combined approach of using NanoSIMS and microsensors within the tissue of corals provides, to the best of our knowledge, the first evidence for physiological differences of individual Symbiodinium cells in hospite in relation to the local microenvironmental conditions across different coral tissue layers, that is, oral vs aboral parts of polyp and coenosarc. Quantitative analysis based on tissue sections from different coral tissue layers showed that mean incorporation of ¹³C-bicarbonate by individual Symbiodinium cells was up to 6.5-fold higher in the upper oral polyp and coenosarc tissues compared with the lowermost layer of polyp tissues (δ¹³C: 1609±147‰, n=25 for Symbiodinium cells in upper oral polyp tissue; 1696±205‰, n=33 for Symbiodinium cells in coenosarc tissue and 246±82‰, n=17 for Symbiodinium cells in the lowest aboral layer of polyp tissue). Although the sample sizes in this study are small and the ¹³C signal is heterogeneous within individual Symbiodinium cells (because of carbon fixation hotspots in specific compartments; Supplementary Figure S2; Kopp et al., 2015), the magnitude of the difference in mean ¹³C incorporation between the aboral part of the polyp and the two other parts of coral tissue was clear and statistically significant (one-way analysis of variance (ANOVA) F_2,75=15.91; P<0.0001; 6.5-fold increase in polyp oral vs aboral polyp tissue, Fischer''s least significant difference (LSD) P<0.0001; 6.9-fold increase in coenosarc vs aboral polyp tissue Fischer''s LSD P<0.0001; and no significant difference between oral polyp vs coenosarc tissue, Fischer''s LSD P=0.718; Figure 1c–f; Supplementary Table S1). The internal microenvironment within the corresponding polyp tissues was highly stratified with respect to light and O₂ (Figures 1g and h). Scalar irradiance decreased about 15-fold from the surface to the bottom of the polyp tissues. Gradients of O₂ were less steep but still significant, with an approximate reduction in O₂ concentration by about 2.5 times (Figure 1; Supplementary Table S2; ANOVA F_1,6= 16.4; P=0.006).These results suggest that coral tissues are vertically stratified systems that affect the physiological activity of their symbionts along a fine-scale microenvironmental gradient. The presence and role of microscale heterogeneity has hitherto largely been ignored in the field of coral symbiosis research, while much is known for other photosynthetic tissues. For instance, for terrestrial plant leaves and for aquatic photosynthetic biofilms, it is known that the photosynthetic unit can adapt to microenvironmental light gradients, where chloroplasts/phototrophs harboured in low-light niches show increased photosynthetic quantum efficiencies at low light levels (Terashima and Hikosaka, 1995; Al-Najjar et al., 2012). Although the steady-state O₂ concentration values reported here are a function of the different metabolic processes of the coral holobiont (that is, Symbiodinium photosynthesis and the combined respiration by the coral host, Symbiodinium and microbes), the NanoSIMS approach allowed us to separate ¹³C fixation of Symbiodinium from the host metabolic activity. Our study provides the first experimental evidence from carbon fixation measurements that Symbiodinium cells can adapt to optical microniches in coral tissues. The 15-fold reduction in irradiance with depth in the coral tissue led only to an ~6.5-fold reduction in net carbon fixation suggesting enhanced light-harvesting efficiency or a reduced P/R ratio for Symbiodinium harboured in aboral tissues. Although such enhanced efficiency under low light often reflects the adaptation of the photosynthetic apparatus (for example, an increase in light-harvesting complexes (Walters, 2005) and reduced cell respiration (Givnish, 1988), it might additionally be the result of physiologically distinct populations or clades of Symbiodinium. Several studies have revealed remarkable genetic and physiological diversities among different Symbiodinium clades (Loram et al., 2007; Stat et al., 2008; Baker et al., 2013; Pernice et al., 2014). Although Favites sp. corals from Southern Great Barrier Reef are generally reported in association with one specific Symbiodinium type (clade C3; Tonk et al., 2013), Symbiodinium diversity within the microenvironment of these common corals could have been overlooked and such physiological diversity could further provide selective advantage to different genotypes in microenvironments within coral tissue. Coral tissues might thus exhibit similar characteristics to photosynthetic biofilms where steep physico-chemical microgradients give rise to different pheno- and ecotypes of phototrophs along those gradients (Musat et al., 2008; Ward et al., 1998).These first experiments were performed under sub-saturating irradiance of ~80 μmol photons per m² per s. Earlier studies showed that the local scalar irradiance in upper vs deeper tissue layers relates to the incident photon irradiance in a linear fashion such that at stressful incident irradiance levels of, for example, 2000 μmol photons per m² per s, light levels in the lowermost polyp tissue layers are ~200 μmol photons per m² per s (Wangpraseurt et al., 2012), still representing optimal conditions for photosynthesis. We thus consider it likely that excess irradiance triggering photoinhibition in oral tissues is unlikely to cause photoinhibition of Symbiodinium in aboral polyp tissues. The internal light field is species specific and in some thin-tissued, branching corals such as Pocillopora damicornis, intra-tissue light attenuation is not very pronounced (Wangpraseurt et al., 2012; Szabó et al., 2014). The ability to harbour Symbiodinium cells in low-light niches might be an important resilience factor for thick-tissued corals, such as massive faviids, during and after coral bleaching. Our study gives first insights to the functional diversity of Symbiodinium along microscale gradients in coral tissue and underscores the importance of considering such heterogeneity in studies linking symbiont diversity and coral physiology responses to environmental stress factors.     

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.     

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.     

Genetic diversity of free-living <Emphasis Type="Italic">Symbiodinium</Emphasis> in the Caribbean: the importance of habitats and seasons

Although reef corals are dependent of the dinoflagellate Symbiodinium, the large majority of corals spawn gametes that do not contain their vital symbiont. This suggests the existence of a pool of Symbiodinium in the environment, of which surprisingly little is known. Reefs around Curaçao (Caribbean) were sampled for free-living Symbiodinium at three time periods (summer 2009, summer 2010, and winter 2010) to characterize different habitats (water column, coral rubble, sediment, the macroalgae Halimeda spp., Dictyota spp., and Lobophora variegata, and the seagrass Thalassia testudinum) that could serve as environmental sources of symbionts for corals. We detected the common clades of Symbiodinium that engage in symbiosis with Caribbean coral hosts A, B, and C using Symbiodinium-specific primers of the hypervariable region of the chloroplast 23S ribosomal DNA gene. We also discovered clade G and, for the first time in the Caribbean, the presence of free-living Symbiodinium clades F and H. Additionally, this study expands the habitat range of free-living Symbiodinium as environmental Symbiodinium was detected in T. testudinum seagrass beds. The patterns of association between free-living Symbiodinium types and habitats were shown to be complex. An interesting, strong association was seen between some clade A sequence types and sediment, suggesting that sediment could be a niche where clade A radiated from a free-living ancestor. Other interesting relationships were seen between sequence types of Symbiodinium clade C with Halimeda spp. and clades B and F with T. testudinium. These relationships highlight the importance of some macroalgae and seagrasses in hosting free-living Symbiodinium. Finally, studies spanning beyond a 1-yr cycle are needed to further expand on our results in order to better understand the variation of Symbiodinium in the environment through time. All together, results presented here showed that the great diversity of free-living Symbiodinium has a dynamic distribution across habitats and time.     

The distribution of mycosporine-like amino acids (MAAs) and the phylogenetic identity of symbiotic dinoflagellates in cnidarian hosts from the Mexican Caribbean

A survey of 54 species of symbiotic cnidarians that included hydrozoan corals, anemones, gorgonians and scleractinian corals was conducted in the Mexican Caribbean for the presence of mycosporine-like amino acids (MAAs) in the host as well as the Symbiodinium fractions. The host fractions contained relatively simple MAA profiles, all harbouring between one and three MAAs, principally mycosporine-glycine followed by shinorine and porphyra-334 in smaller amounts. Symbiodinium populations were identified to sub-generic levels using PCR-DGGE analysis of the Internal Transcribed Spacer 2 (ITS2) region. Regardless of clade identity, all Symbiodinium extracts contained MAAs, in contrast to the pattern that has been found in cultures of Symbiodinium, where clade A symbionts produced MAAs whereas clade B, C, D, and E symbionts did not. Under natural conditions between one and four MAAs were identified in the symbiont fractions, mycosporine-glycine (λ_max = 310 nm), shinorine (λ_max = 334 nm), porphyra-334 (λ_max = 334 nm) and palythine (λ_max = 320 nm). One sample also contained mycosporine-2-glycine (λ_max = 331 nm). These data suggest that Symbiodinium is restricted to producing five MAAs and there also appears to be a defined order of appearance of these MAAs: mycosporine-glycine followed by shinorine (in one case mycosporine-2-glycine), then porphyra-334 and palythine. Overall, mycosporine-glycine was found in highest concentrations in the host and symbiont extracts. This MAA, unlike many other MAAs, absorbs within the ultraviolet-B range (UVB, 280-320 nm) and is also known for moderate antioxidant properties thus potentially providing protection against the direct and indirect effects of UVR. No depth-dependent changes could be identified due to a high variability of MAA concentrations when all species were included in the analysis. The presence of at least one MAA in all symbiont and host fractions analyzed serves to highlight the importance of MAAs, and in particular the role of mycosporine-glycine, as photoprotectants in the coral reef environment.     

Host–symbiont recombination versus natural selection in the response of coral–dinoflagellate symbioses to environmental disturbance

Mutualisms between reef-building corals and endosymbiotic dinoflagellates are particularly sensitive to environmental stress, yet the ecosystems they construct have endured major oscillations in global climate. During the winter of 2008, an extreme cold-water event occurred in the Gulf of California that bleached corals in the genus Pocillopora harbouring a thermally ‘sensitive’ symbiont, designated Symbiodinium C1b-c, while colonies possessing Symbiodinium D1 were mostly unaffected. Certain bleached colonies recovered quickly while others suffered partial or complete mortality. In most colonies, no appreciable change was observed in the identity of the original symbiont, indicating that these partnerships are stable. During the initial phases of recovery, a third species of symbiont B1_Aiptasia, genetically identical to that harboured by the invasive anemone, Aiptasia sp., grew opportunistically and was visible as light-yellow patches on the branch tips of several colonies. However, this symbiont did not persist and was displaced in all cases by C1b-c several months later. Colonies with D1 were abundant at inshore habitats along the continental eastern Pacific, where seasonal turbidity is high relative to offshore islands. Environmental conditions of the central and southern coasts of Mexico were not sufficient to explain the exclusivity of D1 Pocillopora in these regions. It is possible that mass mortalities associated with major thermal disturbances during the 1997–1998 El Niño Southern Oscillation eliminated C1b-c holobionts from these locations. The differential loss of Pocillopora holobionts in response to thermal stress suggests that natural selection on existing variation can cause rapid and significant shifts in the frequency of particular coral–algal partnerships. However, coral populations may take decades to recover following episodes of severe selection, thereby raising considerable uncertainty about the long-term viability of these communities.