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Reef-building corals in the genus Porites are one of the most important constituents of Indo-Pacific reefs. Many species within this genus tolerate abnormally warm water and exhibit high specificity for particular kinds of endosymbiotic dinoflagellates that cope with thermal stress better than those living in other corals. Still, during extreme ocean heating, some Porites exhibit differences in their stress tolerance. While corals have different physiological qualities, it remains unknown whether the stability and performance of these mutualisms is influenced by the physiology and genetic relatedness of their symbionts. We investigated two ubiquitous Pacific reef corals, Porites rus and Porites cylindrica, from warmer inshore and cooler offshore reef systems in Palau. While these corals harbored a similar kind of symbiont in the genus Cladocopium (within the ITS2 C15 subclade), rapidly evolving genetic markers revealed evolutionarily diverged lineages corresponding to each Porites species living in each reef habitat. Furthermore, these closely related Cladocopium lineages were differentiated by their densities in host tissues, cell volume, chlorophyll concentration, gross photosynthesis, and photoprotective pathways. When assessed using several physiological proxies, these previously undifferentiated symbionts contrasted in their tolerance to thermal stress. Symbionts within P. cylindrica were relatively unaffected by exposure to 32℃ for 14 days, whereas P. rus colonies lost substantial numbers of photochemically compromised symbionts. Heating reduced the ability of the offshore symbiont associated with P. rus to translocate carbon to the coral. By contrast, high temperatures enhanced symbiont carbon assimilation and delivery to the coral skeleton of inshore P. cylindrica. This study indicates that large physiological differences exist even among closely related symbionts, with significant implications for thermal susceptibility among reef-building Porites.

Warming and nutrient limitation are stressors known to weaken the health of microalgae. In situations of stress, access to energy reserves can minimize physiological damage. Because of its widespread requirements in biochemical processes, iron is an important trace metal, especially for photosynthetic organisms. Lowered iron availability in oceans experiencing rising temperatures may contribute to the thermal sensitivity of reef-building corals, which rely on mutualisms with dinoflagellates to survive. To test the influence of iron concentration on thermal sensitivity, the physiological responses of cultured symbiotic dinoflagellates (genus Breviolum; family Symbiodiniaceae) were evaluated when exposed to increasing temperatures (26 to 30°C) and iron concentrations ranging from replete (500 pM Fe') to limiting (50 pM Fe') under a diurnal light cycle with saturating radiance. Declines in photosynthetic efficiency at elevated temperatures indicated sensitivity to heat stress. Furthermore, five times the amount of iron was needed to reach exponential growth during heat stress (50 pM Fe' at 26-28°C vs. 250 pM Fe' at 30°C). In treatments where exponential growth was reached, Breviolum psygmophilum grew faster than B.minutum, possibly due to greater cellular contents of iron and other trace metals. The metal composition of B.psygmophilum shifted only at the highest temperature (30°C), whereas changes in B.minutum were observed at lower temperatures (28°C). The influence of iron availability in modulating each alga's response to thermal stress suggests the importance of trace metals to the health of coral-algal mutualisms. Ultimately, a greater ability to acquire scarce metals may improve the tolerance of corals to physiological stressors and contribute to the differences in performance associated with hosting one symbiont species over another.

Within microeukaryotes, genetic variation and functional variation sometimes accumulate more quickly than morphological differences. To understand the evolutionary history and ecology of such lineages, it is key to examine diversity at multiple levels of organization. In the dinoflagellate family Symbiodiniaceae, which can form endosymbioses with cnidarians (e.g., corals, octocorals, sea anemones, jellyfish), other marine invertebrates (e.g., sponges, molluscs, flatworms), and protists (e.g., foraminifera), molecular data have been used extensively over the past three decades to describe phenotypes and to make evolutionary and ecological inferences. Despite advances in Symbiodiniaceae genomics, a lack of consensus among researchers with respect to interpreting genetic data has slowed progress in the field and acted as a barrier to reconciling observations. Here, we identify key challenges regarding the assessment and interpretation of Symbiodiniaceae genetic diversity across three levels: species, populations, and communities. We summarize areas of agreement and highlight techniques and approaches that are broadly accepted. In areas where debate remains, we identify unresolved issues and discuss technologies and approaches that can help to fill knowledge gaps related to genetic and phenotypic diversity. We also discuss ways to stimulate progress, in particular by fostering a more inclusive and collaborative research community. We hope that this perspective will inspire and accelerate coral reef science by serving as a resource to those designing experiments, publishing research, and applying for funding related to Symbiodiniaceae and their symbiotic partnerships.

Coral bleaching is the single largest global threat to coral reefs worldwide. Integrating the diverse body of work on coral bleaching is critical to understanding and combating this global problem. Yet investigating the drivers, patterns, and processes of coral bleaching poses a major challenge. A recent review of published experiments revealed a wide range of experimental variables used across studies. Such a wide range of approaches enhances discovery, but without full transparency in the experimental and analytical methods used, can also make comparisons among studies challenging. To increase comparability but not stifle innovation, we propose a common framework for coral bleaching experiments that includes consideration of coral provenance, experimental conditions, and husbandry. For example, reporting the number of genets used, collection site conditions, the experimental temperature offset(s) from the maximum monthly mean (MMM) of the collection site, experimental light conditions, flow, and the feeding regime will greatly facilitate comparability across studies. Similarly, quantifying common response variables of endosymbiont (Symbiodiniaceae) and holobiont phenotypes (i.e., color, chlorophyll, endosymbiont cell density, mortality, and skeletal growth) could further facilitate cross-study comparisons. While no single bleaching experiment can provide the data necessary to determine global coral responses of all corals to current and future ocean warming, linking studies through a common framework as outlined here, would help increase comparability among experiments, facilitate synthetic insights into the causes and underlying mechanisms of coral bleaching, and reveal unique bleaching responses among genets, species, and regions. Such a collaborative framework that fosters transparency in methods used would strengthen comparisons among studies that can help inform coral reef management and facilitate conservation strategies to mitigate coral bleaching worldwide. published

The interaction of reef-building coral and symbiotic dinoflagellates (family: Symbiodiniaceae) is of critical importance to organismal performance and ecosystem functioning. A standard biometric commonly used to evaluate coral physiology and health status, is coral biomass. Within-colony Symbiodiniaceae assemblages typically range from 1 to 6 million cells cm−2; however, no direct measurements have quantified the overall biomass of these dense symbiont populations. We use direct gravimetric measurements to determine the mass of individual Symbiodiniaceae cells and investigate the ratio of symbiotic dinoflagellate biomass to coral holobiont biomass in five species of scleractinian corals: Acropora cervicornis, A. palmata, Orbicella annularis O. faveolata, and Siderastrea radians from the Florida Keys, FL, USA (25.119°N, 80.302°W). Genetic identification of symbiotic dinoflagellates revealed a single, dominant population of symbiotic dinoflagellates among each scleractinian species, suggesting that the corals did not change symbionts during this study. The overall contribution of the symbionts to holobiont biomass ranged from ~ 5 to 15%, and remained relatively constant throughout the winter and summer for all species with the exception of the Acroporids. This relatively small biomass of symbionts is in contrast to the millions of cells cm−2 which are present in these corals, and that enhance calcification, recycle nutrients, and supply nutrition to their reef-building hosts.

Improving coral reef conservation requires heightened understanding of the mechanisms by which coral cope with changing environmental conditions to maintain optimal health. We used a long-term (10 month) in situ experiment with two phylogenetically diverse scleractinians (Acropora palmata and Porites porites) to test how coral-symbiotic algal interactions changed under real-world conditions that were a priori expected to be beneficial (fish-mediated nutrients) and to be harmful, but non-lethal, for coral (fish + anthropogenic nutrients). Analyzing nine response variables of nutrient stoichiometry and stable isotopes per coral fragment, we found that nutrients from fish positively affected coral growth, and moderate doses of anthropogenic nutrients had no additional effects. While growing, coral maintained homeostasis in their nutrient pools, showing tolerance to the different nutrient regimes. Nonetheless, structural equation models revealed more nuanced relationships, showing that anthropogenic nutrients reduced the diversity of coral-symbiotic algal interactions and caused nutrient and carbon flow to be dominated by the symbiont. Our findings show that nutrient and carbon pathways are fundamentally "rewired" under anthropogenic nutrient regimes in ways that could increase corals' susceptibility to further stressors. We hypothesize that our experiment captured coral in a previously unrecognized transition state between mutualism and antagonism. These findings highlight a notable parallel between how anthropogenic nutrients promote symbiont dominance with the holobiont, and how they promote macroalgal dominance at the coral reef scale. Our findings suggest more realistic experimental conditions, including studies across gradients of anthropogenic nutrient enrichment as well as the incorporation of varied nutrient and energy pathways, may facilitate conservation efforts to mitigate coral loss.

High sea surface temperatures often lead to coral bleaching wherein reef-building corals lose significant numbers of their endosymbiotic dinoflagellates (Symbiodiniaceae). These increasingly frequent bleaching events often result in large scale coral mortality, thereby devasting reef systems throughout the world. The reef habitats surrounding Palau are ideal for investigating coral responses to climate perturbation, where many inshore bays are subject to higher water temperature as compared with offshore barrier reefs. We examined fourteen physiological traits in response to high temperature across various symbiotic dinoflagellates in four common Pacific coral species, Acropora muricata, Coelastrea aspera, Cyphastrea chalcidicum and Pachyseris rugosa found in both offshore and inshore habitats. Inshore corals were dominated by a single homogenous population of the stress tolerant symbiont Durusdinium trenchii, yet symbiont thermal response and physiology differed significantly across coral species. In contrast, offshore corals harbored specific species of Cladocopium spp. (ITS2 rDNA type-C) yet all experienced similar patterns of photoinactivation and symbiont loss when heated. Additionally, cell volume and light absorption properties increased in heated Cladocopium spp., leading to a greater loss in photo-regulation. While inshore coral temperature response was consistently muted relative to their offshore counterparts, high physiological variability in D. trenchii across inshore corals suggests that bleaching resilience among even the most stress tolerant symbionts is still heavily influenced by their host environment.

High sea surface temperatures often lead to coral bleaching wherein reef-building corals lose significant numbers of their endosymbiotic dinoflagellates (Symbiodiniaceae). These increasingly frequent bleaching events often result in large scale coral mortality, thereby devasting reef systems throughout the world. The reef habitats surrounding Palau are ideal for investigating coral responses to climate perturbation, where many inshore bays are subject to higher water temperature as compared with offshore barrier reefs. We examined fourteen physiological traits in response to high temperature across various symbiotic dinoflagellates in four common Pacific coral species, Acropora muricata, Coelastrea aspera, Cyphastrea chalcidicum and Pachyseris rugosa found in both offshore and inshore habitats. Inshore corals were dominated by a single homogenous population of the stress tolerant symbiont Durusdinium trenchii, yet symbiont thermal response and physiology differed significantly across coral species. In contrast, offshore corals harbored specific species of Cladocopium spp. (ITS2 rDNA type-C) yet all experienced similar patterns of photoinactivation and symbiont loss when heated. Additionally, cell volume and light absorption properties increased in heated Cladocopium spp., leading to a greater loss in photo-regulation. While inshore coral temperature response was consistently muted relative to their offshore counterparts, high physiological variability in D. trenchii across inshore corals suggests that bleaching resilience among even the most stress tolerant symbionts is still heavily influenced by their host environment.

Evolutionary rescue of populations depends on their ability to produce phenotypic variation that is heritable and adaptive. DNA mutations are the best understood mechanisms to create phenotypic variation, but other, less well‐studied mechanisms exist. Marine benthic foundation species provide opportunities to study these mechanisms because many are dominated by isogenic stands produced through asexual reproduction. For example, Caribbean acroporid corals are long lived and reproduce asexually via breakage of branches. Fragmentation is often the dominant mode of local population maintenance. Thus, large genets with many ramets (colonies) are common. Here, we observed phenotypic variation in stress responses within genets following the coral bleaching events in 2014 and 2015 caused by high water temperatures. This was not due to genetic variation in their symbiotic dinoflagellates (Symbiodinium “fitti”) because each genet of this coral species typically harbours a single strain of S. “fitti”. Characterization of the microbiome via 16S tag sequencing correlated the abundance of only two microbiome members (Tepidiphilus, Endozoicomonas) with a bleaching response. Epigenetic changes were significantly correlated with the host's genetic background, the location of the sampled polyps within the colonies (e.g., branch vs. base of colony), and differences in the colonies’ condition during the bleaching event. We conclude that long‐term microenvironmental differences led to changes in the way the ramets methylated their genomes, contributing to the differential bleaching response. However, most of the variation in differential bleaching response among clonemates of Acropora palmata remains unexplained. This research provides novel data and hypotheses to help understand intragenet variability in stress phenotypes of sessile marine species.

Evolutionary rescue of populations depends on their ability to produce phenotypic variation that is heritable and adaptive. DNA mutations are the best understood mechanisms to create phenotypic variation, but other, less well-studied mechanisms exist. Marine benthic foundation species provide opportunities to study these mechanisms because many are dominated by isogenic stands produced through asexual reproduction. For example, Caribbean acroporid corals are long lived and reproduce asexually via breakage of branches. Fragmentation is often the dominant mode of local population maintenance. Thus, large genets with many ramets (colonies) are common. Here, we observed phenotypic variation in stress response within genets following the coral bleaching events in 2014-and 2015 caused by high water temperatures. This was not due to genetic variation in their symbiotic dinoflagellates (Symbiodinium ‘fitti’) because each genet of this coral species typically harbors a single strain of S. ‘fitti’. Characterization of the microbiome via 16S tag sequencing did not provide evidence for a central role of microbiome variation in determining bleaching response. Instead, epigenetic changes were significantly correlated with the host’s genetic background, the position of the sampled polyps within the colonies (e.g. branch versus base of colony), and differences in the colonies’ condition during the bleaching event. We conclude that microenvironmental differences in growing conditions led to long-term changes in the way the ramets methylated their genomes contributing to, but not fully explaining, the differential bleaching response. This research provides novel data to understanding intra-genet variability in stress phenotypes of sessile marine species.

Recently, the Caribbean reef-building coral Orbicella faveolata was listed as “threatened” under the U.S. Endangered Species Act. Despite attention to this species’ conservation, the extent of geographic variation within O. faveolata warrants further investigation. O. faveolata is unusual in that it can simultaneously harbor multiple genetically distinct and co-dominant species of endosymbiotic dinoflagellates in the genus Symbiodinium. Here, we investigate the geographic and within-colony complexity of Symbiodinium-O. faveolata associations from Florida Keys, USA; Exuma Cays, Bahamas; Puerto Morelos, Mexico; and Carrie Bow Cay, Belize. We collected coral samples along intracolony axes, and Symbiodinium within O. faveolata samples was analyzed using the nuclear ITS2 region and chloroplast 23S rDNA genotyping. O. faveolata associated with species of Symbiodinium in clades A (type A3), B (B1 and B17), C (C3, C7, and C7a), and D (D1a/Symbiodinium trenchii). Within-colony distributions of Symbiodinium species correlated with light availability, cardinal direction, and depth, resulting in distinct zonation patterns of endosymbionts within a host. Symbiodinium species from clades A and B occurred predominantly in the light-exposed tops, while species of clade C generally occurred in the shaded sides of colonies or in deeper-water habitats. Furthermore, geographic comparisons of host–symbiont associations revealed regional differences in Symbiodinium associations. Symbiodinium A3 was detected in Mesoamerican coral colonies, but not in colonies from the Florida Keys or Bahamas. Likewise, Symbiodinium B17 was unique to Mesoamerican O. faveolata, whereas Symbiodinium B1 was found at all localities sampled. However, using cp23S genotyping paired with ITS2 analysis revealed geographically endemic haplotypes among Symbiodinium clades A, B, and C. Since Symbiodinium spatial heterogeneity among this coral species is greater than most corals, a question arises as to whether all western Atlantic populations of O. faveolata should be considered equally “threatened”? Alternatively, geographically and spatially distinct coral–symbiont associations may benefit from specialized management protocols.

Community dynamics and physiology of Symbiodinium associated with Orbicella ( = Montastraea) faveolata were examined before, during, and after a thermally induced coral bleaching event in Puerto Morelos, Mexico. We combined microsampling molecular genotyping with in situ pulse-amplitude modulated fluorometry to correlate colony variability of Symbiodinium population identities and the phenomena of partial coral bleaching. Pigmented nonbleached portions of O. ( = M.) faveolata were compared with bleached portions of the same colony. During bleaching, maximum quantum yield of photosystem II (PSII; Fv : Fm) was significantly lower and highly variable (range 0.110 to 0.680) compared with previous summers in which coral bleaching was absent (range 0.516 to 0.661) and recovery (range 0.480 to 0.716). Differential susceptibility to environmental perturbation of Fv : Fm corresponded to distinct genetic identities of Symbiodinium. Analysis of ribosomal deoxyribonucleic acid (rDNA) internal transcribed spacer 2 (ITS2) revealed regions of the coral colonies that had phylotype A3 prior to bleaching were more resistant to the bleaching perturbation than adjacent bleaching-prone patches that harbored phylotypes B17 and C7. During environmental perturbation, regions of the colonies containing predominantly Symbiodinium phylotypes A3 or D1a retained significantly higher Fv : Fm values than adjacent regions with phylotypes B17 and C7. Following bleaching, rapid recovery of symbiotic algal densities greatly exceeded normal seasonal oscillations. During recovery we document shifts in Symbiodinium populations and increase prevalence of Symbiodinium types A3 and D1a, phylotypes known to have enhanced thermal tolerances. Thermal tolerance of Symbiodinium spp. influences the changes of coral—Symbiodinium communities during disturbance events and the dynamics of coral—Symbiodinium repopulation.

Although apicomplexans are a widely recog- nized and important parasitic group, little is known about those associated with invertebrates, such as reef-building scleractinian corals. To resolve the potential impact of api- complexans on coral health, it is first necessary to further describe this group of putative parasites and determine their prevalence among host species. Here, it was hypothesized that apicomplexan prevalence would vary seasonally, simi- lar to what occurs in other marine apicomplexans as well as some coral symbionts. To test this, Caribbean scleractinian species Porites astreoides, Montastraea (=Orbicella) annu- laris, M.( =O.) faveolata ,a ndSiderastrea siderea were sampled seasonally from two reefs each in the Florida Keys and the Bahamas for 9- and 5.5-year periods, respectively. Utilizing a PCR-based screening assay, apicomplexan DNA was detected from most Floridian (80.1 %: n = 555/693) and Bahamian (90.7 %: n = 311/343) coral tissue samples collected over these multi-year periods. Furthermore, api- complexan DNA was detected from nearly all (98.7 %: n = 78/79) single polyps sampled at multiple locations within six M. faveolata colonies, indicating little to no in- tracolonial variation in the screening assay. Mixed-model logistic regression was utilized to determine the effects of season, host species, and reef on apicomplexan prevalence. The model identified a significant seasonal effect, with the highest apicomplexan prevalence occurring during fall. There also was a large effect of host species, with apicom- plexan prevalence significantly lower among S. siderea colonies relative to the other species. While reef did not have a significant effect in the full model, there was a sig- nificant difference in apicomplexan prevalence between Floridian and Bahamian reefs for S. siderea, implying regional differences in this host species. Despite seasonal and species-specific differences in prevalence, apicomplex- ans are ubiquitous constituents of these particular sclerac- tinian coral species from Florida and the Bahamas.

Coral reefs of the Florida Keys typically experience seasonal temperatures of 20–31 °C. Deviation outside this range causes physiological impairment of reef-building corals, potentially leading to coral colony death. In January and February 2010, two closely spaced cold fronts, possibly driven by an unusually extreme Arctic Oscillation, caused sudden and severe seawater temperature declines in the Florida Keys. Inshore coral reefs [e.g., Admiral Reef (ADM)] experienced lower sustained temperatures (i.e.

Scleractinian coral evolution is often characterized by alternating patterns of lineage diversification and fusion, thus leading to reticulate evolution. Although this pattern is hypothesized in many coral lineages, including the Montastraea annularis species complex, it is not known what effects cladogenesis and hybridization have on the symbioses between corals and their endosymbi- otic dinoflagellates (genus Symbiodinium). To explore this, the genetic diversity of M. faveolata and M. annularis in the Upper Florida Keys, USA, and Exuma Cays, The Bahamas, was examined using a mtDNA intergenic region. The host genotypic data were then analyzed in relation to the diversity of the corals' Symbiodinium communities as determined by internal transcribed spacer region 2 (ITS2) and 3 microsatellite markers specific to Symbiodinium Clade B. M. faveolata and M. annularis in the Upper Florida Keys were genetically distinct from one another while these coral species in the Exuma Cays shared mtDNA haplotypes. These findings suggest possible regional differences in the degree of intergressive hybridization between M. faveolata and M. annularis. When Symbiodinium diversity was examined, Montastraea spp. from both regions shared Symbiodinium ITS2 genotypes; however, host-symbiont specificity was observed using higher resolution microsatellite markers. Specifically, M. faveolata and M. annularis from the Upper Florida Keys all harbored genetically dis- tinct multilocus Clade B genotypes, whereas these 2 coral species in the Exuma Cays shared Clade B genotypes. Consequently, the degree of fine-scale specificity between Symbiodinium Clade B geno- types and Montastraea spp. appears to be governed by the degree of genetic distinction, and possi- bly hybridization, between these host 'species'.

Denaturing gradient gel electrophoresis (DGGE) is commonly utilized to identify and quantify microbial diversity, but the conditions required for different electrophoretic systems to yield equivalent results and optimal resolution have not been assessed. Herein, the influence of different DGGE system configuration parameters on microbial diversity estimates was tested using Symbiodinium, a group of marine eukaryotic microbes that are important constituents of coral reef ecosystems. To accomplish this, bacterial clone libraries were constructed and sequenced from cultured isolates of Symbiodinium for the ribosomal DNA internal transcribed spacer 2 (ITS2) region. From these, 15 clones were subjected to PCR with a GC clamped primer set for DGGE analyses. Migration behaviors of the resulting amplicons were analyzed using a range of conditions, including variation in the composition of the denaturing gradient, electrophoresis time, and applied voltage. All tests were conducted in parallel on two commercial DGGE systems, a C.B.S. Scientific DGGE-2001, and the Bio-Rad DCode system. In this context, identical nucleotide fragments exhibited differing migration behaviors depending on the model of apparatus utilized, with fragments denaturing at a lower gradient concentration and applied voltage on the Bio-Rad DCode system than on the C.B.S. Scientific DGGE-2001 system. Although equivalent PCR–DGGE profiles could be achieved with both brands of DGGE system, the composition of the denaturing gradient and application of electrophoresis time × voltage must be appropriately optimized to achieve congruent results across platforms.

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

We propose ‘the moving target hypothesis’ to describe the aetiology of a contemporary coral disease that differs from that of its historical disease state. Hitting the target with coral disease aetiology is a complex pursuit that requires understanding of host and environment, and may lack a single pathogen solution. White pox disease (WPX) affects the Caribbean coral Acropora palmata . Acroporid serratiosis is a form of WPX for which the bacterial pathogen ( Serratia marcescens ) has been established. We used long-term (1994–2014) photographic monitoring to evaluate historical and contemporary epizootiology and aetiology of WPX affecting A. palmata at eight reefs in the Florida Keys. Ranges of WPX prevalence over time (0–71.4%) were comparable for the duration of the 20-year study. Whole colony mortality and disease severity were high in historical (1994–2004), and low in contemporary (2008–2014), outbreaks of WPX. Acroporid serratiosis was diagnosed for some historical (1999, 2003) and contemporary (2012, 2013) outbreaks, but this form of WPX was not confirmed for all WPX cases. Our results serve as a context for considering aetiology as a moving target for WPX and other coral diseases for which pathogens are established and/or candidate pathogens are identified. Coral aetiology investigations completed to date suggest that changes in pathogen, host and/or environment alter the disease state and complicate diagnosis.

Genotypic characterization of Symbiodinium symbionts in hard corals has routinely involved coring, or the removal of branches or a piece of the coral colony. These methods can potentially underestimate the complexity of the Symbiodinium community structure and may produce lesions. This study demonstrates that microscale sampling of individual coral polyps provided sufficient DNA for identifying zooxanthellae clades by RFLP analyses, and subclades through the use of PCR amplification of the ITS-2 region of rDNA and denaturing-gradient gel electrophoresis. Using this technique it was possible to detect distinct ITS-2 types of Symbiodinium from two or three adjacent coral polyps. These methods can be used to intensely sample coral-symbiont population/communities while causing minimal damage. The effectiveness and fine scale capabilities of these methods were demonstrated by sampling and identifying phylotypes of Symbiodinium clades A, B, and C that co-reside within a single Montastraea faveolata colony.

White pox disease (WPD) affects the threatened elkhorn coral, Acropora palmata . Owing in part to the lack of a rapid and simple diagnostic test, there have been few systematic assessments of the prevalence of acroporid serratiosis (caused specifically by Serratia marcescens ) versus general WPD signs. Six reefs in the Florida Keys were surveyed between 2011 and 2013 to determine the disease status of A. palmata and the prevalence of S. marcescens . WPD was noted at four of the six reefs, with WPD lesions found on 8 to 40% of the colonies surveyed. S. marcescens was detected in 26.9% (7/26) of the WPD lesions and in mucus from apparently healthy colonies both during and outside of disease events (9%; 18/201). S. marcescens was detected with greater frequency in A. palmata than in the overlying water column, regardless of disease status ( P = 0.0177). S. marcescens could not be cultured from A. palmata but was isolated from healthy colonies of other coral species and was identified as pathogenic pulsed-field gel electrophoresis type PDR60. WPD lesions were frequently observed on the reef, but unlike in prior outbreaks, no whole-colony death was observed. Pathogenic S. marcescens was circulating on the reef but did not appear to be the primary pathogen in these recent WPD episodes, suggesting that other pathogens or stressors may contribute to signs of WPD. Results highlight the critical importance of diagnostics in coral disease investigations, especially given that field manifestation of disease may be similar, regardless of the etiological agent.

Coral bleaching involves the loss of symbiotic algae (zooxanthellae) from reef corals and other cnidarians during periods of environmental stress, particularly elevated temperature. In this study we compared the thermal bleaching responses of the zoanthid Palythoa caribaeorum from three populations along the southeast coast of Florida. Winter (2002–2003) and summer (2003) samples from three geographically separate sites were experimentally exposed to increased temperatures and the loss of zooxanthellae was measured. Population densities of zooxanthellae were analyzed and their genetic identity determined using PCR-DGGE analysis of the internal transcribed spacer region 2. The results showed that samples of P. caribaeorum from reefs that experienced the smallest range in annual seawater temperature released the most zooxanthellae. Seasonal comparisons revealed that winter samples lost more zooxanthellae than summer samples. P. caribaeorum harbored two genetic types of zooxanthellae, C1 and D1a. Individual colonies contained populations of only C1 or D1a, or combinations of C1 and D1a. However, these genotypic patterns did not relate latitudinal distribution nor to differences in experimental thermal tolerance.

Coral bleaching, in which corals become visibly pale and typically lose their endosymbiotic zooxanthellae (Symbiodinium spp.), increasingly threatens coral reefs worldwide. While the proximal environmental triggers of bleaching are reasonably well understood, considerably less is known concerning physiological and ecological factors that might exacerbate coral bleaching or delay recovery. We report a bleaching event in Belize during September 2004 in which Montastraea spp. corals that had been previously grazed by corallivorous parrotfishes showed a persistent reduction in symbiont density compared to intact colonies. Additionally, grazed corals exhibited greater diversity in the genetic composition of their symbiont communities, changing from uniform ITS2 type C7 Symbiodinium prior to bleaching to mixed assemblages of Symbiodinium types post-bleaching. These results suggest that chronic predation may exacerbate the influence of environmental stressors and, by altering the coral-zooxanthellae symbiosis, such abiotic-biotic interactions may contribute to spatial variation in bleaching processes.

This report documents the extent to which coral colonies show fluctuations in their associations with different endosymbiotic dinoflagellates. The genetic identity of Symbiodinium from six coral species [Acropora palmata (Lamarck), A. cervicornis (Lamarck), Siderastrea siderea (Ellis and Solander), Montastrea faveolata (Ellis and Solander), M. annularis (Ellis and Solander), and M. franksi (Gregory)] was examined seasonally over five years (1998 and 2000–2004) in the Bahamas and Florida Keys at shallow (1 to 4 m) fore-reef/patch reef sites and at deeper fore-reef (12–15 m) locations. Symbionts were identified genetically using denaturing gradient gel electrophoresis (DGGE) fingerprinting of the internal transcribed spacer region 2 (ITS2) of ribosomal RNA gene loci. Repetitive sampling from most labeled colonies from the Bahamas and the Florida Keys showed little to no change in their dominant symbiont. In contrast, certain colonies of M. annularis and M. franksi from the Florida Keys exhibited shifts in their associations attributed to recovery from the stresses of the 1997–1998 El Nino southern oscillation (ENSO) event. Over several years, a putatively stress-tolerant clade D type of Symbiodinium was progressively replaced in these colonies by symbionts typically found in M. annularis and M. franksi in Florida and at other Caribbean locations. Greater environmental fluctuations in Florida may explain the observed changes among some of the symbioses. Furthermore, symbiotic associations were more heterogeneous at shallow sites, relative to deep sites. The exposure to greater environmental variability near the surface may explain the higher symbiont diversity found within and between host colonies.

Increased sea-surface temperatures linked to warming climate threaten coral reef ecosystems globally. To better understand how corals and their endosymbiotic dinoflagellates (Symbiodinium spp.) respond to environmental change, tissue biomass and Symbiodinium density of seven coral species were measured on various reefs approximately every four months for up to thirteen years in the Upper Florida Keys, United States (1994-2007), eleven years in the Exuma Cays, Bahamas (1995-2006), and four years in Puerto Morelos, Mexico (2003-2007). For six out of seven coral species, tissue biomass correlated with Symbiodinium density. Within a particular coral species, tissue biomasses and Symbiodinium densities varied regionally according to the following trends: Mexico≥Florida Keys≥Bahamas. Average tissue biomasses and symbiont cell densities were generally higher in shallow habitats (1-4 m) compared to deeper-dwelling conspecifics (12-15 m). Most colonies that were sampled displayed seasonal fluctuations in biomass and endosymbiont density related to annual temperature variations. During the bleaching episodes of 1998 and 2005, five out of seven species that were exposed to unusually high temperatures exhibited significant decreases in symbiotic algae that, in certain cases, preceded further decreases in tissue biomass. Following bleaching, Montastraea spp. colonies with low relative biomass levels died, whereas colonies with higher biomass levels survived. Bleaching- or disease-associated mortality was also observed in Acropora cervicornis colonies; compared to A. palmata, all A. cervicornis colonies experienced low biomass values. Such patterns suggest that Montastraea spp. and possibly other coral species with relatively low biomass experience increased susceptibility to death following bleaching or other stressors than do conspecifics with higher tissue biomass levels.