The future of coral reefs: a microbial perspective

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1 Review The future of coral reefs: a microbial perspective Tracy D. Ainsworth 1, Rebecca Vega Thurber 2 and Ruth D. Gates 3 1 ARC Centre of Excellence for Coral Reef Studies, James Cook University, Townsville, QLD 4810, Australia 2 Florida International University, Department of Biological Sciences, Biscayne Bay Campus, North Miami, FL 33181, USA 3 Hawaii Institute for Marine Biology, SOEST, University of Hawaii, HI 96744, USA Microbial communities respond and quickly adapt to disturbance and have central roles in ecosystem function. Yet, the many roles of coral-associated microbial communities are not currently accounted for in predicting future responses of reef ecosystems. Here, we propose that a clearer understanding of coral-associated microbial diversity and its interaction with both host and environment will identify important linkages occurring between the microbial communities and macroecological change. Characterizing these links is fundamental to understanding coral reef resilience and will improve our capacity to predict ecological change. Microbial communities and the future of coral reefs The future state of coral reefs will be determined by the ability of the ecosystem to respond to increasing environmental disturbances while remaining a coral dominated system [1,2]. Complex microbial communities are known to be extremely important members of many ecosystems [3 5], and also has significant influence over coral reef ecosystems. In terrestrial ecosystems, microbes have pivotal roles in, and sustain important linkages between, aboveground and belowground processes [6]. In these ecosystems, the microbial community is extremely sensitive to environmental change, and following disturbance, these communities remain altered and functionally different for long periods of time. It is believed that these microbial community dynamics are as important as plant communities in predictive modeling of ecosystem change [7]. In coral reef ecosystems the ways in which microbial communities function are likely to be as complex, diverse and as important to whole ecosystem function as those of other ecosystems. The coral reef provides a structurally and environmentally complex array of habitats, which support a broad microbial diversity that influences both host physiology and ultimately ecosystem processes. Microbial processes and metabolisms strongly influence biogeochemical and ecological processes within the reef environment, such as food webs, organism life cycles, and chemical and nutrient cycling; all of which are fundamental to ecosystem stability. Microbial processes are also key drivers of the many factors that influence the resilience of coral reef ecosystems, such as larval recruitment, colonization, and overall species diversity. For example, chemical cues from the benthic microbial communities influence the settlement of larvae of many keystone species, including corals Corresponding author: Ainsworth, T.D. (tracy.ainsworth@jcu.edu.au). and sea urchins [8,9]. Also, endosymbiosis between corals and the eukaryotic dinoflagellate genus Symbiodinium is responsible for the evolutionary success of stony corals in the shallow tropics and the long-term survival of the coral reef ecosystem [10,11]. Given their established roles in ecosystem functioning, it is surprising how little research has been aimed at understanding the linkages between the broader microbial ecology (communities of bacteria, Achaea, viruses and fungi) and macroecological shifts on reefs. Only recently have the role of microbial diversity and host microbe interactions in the response of reef ecosystems to environmental change been explored. Although we recognize that complex microbial interactions probably exist for all reef organisms, here we focus specifically on the microbial interactions with stony corals as a rationale for incorporating microbial ecology into future studies investigating coral reef resilience. We argue that by incorporating microbial ecology into studies of coral reef ecological change, we will improve our capacity to predict and model the consequences of environmental disturbances on coral reefs. Glossary Biofilm: an aggregation or community of microbes growing on an environmental surface (for examples see [8,71]). Coral Holobiont: the holobiont is the collective community of coral host and its metazoan, protist, and microbial symbionts (see [11]). Coral microbiome: the collective genome of the coral-associated (symbiotic and non-symbiotic) micro-organisms. Keystone species: a species that is crucial to the structure of the ecosystem. Koch s postulates: a system to determine causation of disease by a specific microbe (etiological agent). Microbiome: the collective genome of microorganisms or microbial assemblage (Bacteria, Archaea, and protists) associated with any system such as the body of an animal, a water or soil sample, or an entire ocean. Microbial community: an assemblage or population of microbes Metagenomics: the study of nucleic acids from a given source, including environmental samples. The technique is usually applied to determine both the composition (diversity) and capabilities (function) of a community of organisms (for review [72]). Next generation sequencing: rapid high-throughput sequencing of large sample sets (for reviews [73,74]). Resilience: the capacity of an ecosystem to absorb disturbance (for reviews see [1,75,76]). Scleractinia: the order Scleractinina (within the Anthozoans) are the stony or hard corals. Symbiosis: a close interaction or association between evolutionarily distinct organisms (for review of marine invertebrate symbiosis see [10]) /$ see front matter ß 2009 Elsevier Ltd. All rights reserved. doi: /j.tree Available online 16 December

2 Identification of the coral holobiont and the coral reef microbiome During the 1990s, it became increasingly apparent that marine microbial diversity was orders of magnitude higher than previously believed. Fuhrman and Campbell [12] indicated that upwards of 95% of marine bacterioplankton could not be cultured. Following this, Rohwer and colleagues [11] successfully applied culture-independent techniques to describe the microbial communities associated with the Caribbean coral Montastraea franksi. Using variations in the third hypervariable region (V3) and sequencing of the 16S rrna gene, it was found that approximately 30% of coral-associated bacterial phylotypes were similar to Cyanobacteria and a-proteobacteria species. By contrast, the cultivable bacteria were found to be dominated by the Pseudoalteromonads and Vibrio spp. and members of the Cytophaga Flavobacter Flexibacter Bacteriodes group (CFBs). This study revealed that the diversity of bacteria on corals was significantly greater than previously believed and demonstrated that culturing methods do not accurately reflect the composition or diversity of the microbial populations living on coral reefs. Coral-associated microbial communities have subsequently been shown to be highly diverse >50% of the ribotypes were less than 63% similar to those in the database [11]. Individual coral species have a unique and stable microbial fingerprint that is distinct from the water column [13,14]. However, microbial diversity [15] and specific coral microbe associations [16] have also been shown to vary between reef locations and also along the length of coral host branches [13]. The specificity of coral microbial associations is evidently more complex than current estimates predict and is probably strongly influenced by host and environmental variability. Coral-specific microbial communities are hypothesized to have important physiological and ecological roles on coral reefs. For example, microbial-mediated nitrogen fixation on corals was first proposed in 1987 [17]. It has since been demonstrated that some coral species contain intracellular bacteria that are capable of nitrogen fixation [18]. This occurs during periods when host intracellular oxygen concentrations are low; conditions which are driven by the endosymbiotic eukaryotic dinoflagellate photosynthesis [18,19]. Other reports have found that coral-associated Archaea conduct ammonium oxidization and nitrogenous waste removal for the coral host [20]. The coral microbiota is further believed to provide a protective role for their coral hosts through the production of antibiotics and the occupation of physical space and competition with invading or opportunistic pathogens [21]. This overall model that corals exist in a multipartite symbiosis with both endosymbiotic dinoflagellate and resident microbiota (bacteria and Archaea) has been termed the coral holobiont [11,17,18,20,21]. Yet, the current opinion that members of the coral-associated microbial communities have both beneficial and negative roles in coral and reef ecosystem function are in fact supported by little direct evidence. This is because of limitations in both the technological approaches to document microbial community composition and the overall complexity of the coral reef microbiome. Initially the field of coral reef microbiology was dominated by culture based identification of bacterial pathogens [22 25] and specifically the application of Koch s postulates [22 25]. Although these studies raised awareness of the potential importance of microbes on reefs, these approaches did not address the complexity of the coral microbial communities or the nature of host microbe interactions. The identification of single etiological agents associated with patterns of coral mortality is constrained by several important microbial and coral phenomena. The vast majority of marine microbes require complex nutritional and/or physical conditions that have yet to be replicated in culture conditions. In addition, microbes often exist in interdependent assemblages functioning as single metabolic units, scenarios that cannot be replicated in monoculture conditions. Also, microbes often exist in highly specific relationships with their hosts, and the absence of key host factors inhibits culture. Furthermore, the signs of coral disease are inherently vague [26,27] and degraded coral tissues are rapidly colonized within the marine environment, significantly altering both the microbial community and its interaction with the host. By not acknowledging these limitations in the application of culture based studies to coral microbial ecology, we limit our understanding of the complex role of coral microbial communities in reef stability, their responses to climate change, and their role in coral reef responses. Finally, studies of microbial diversity have relied on phylogenetic markers to infer the metabolic potential of the community. However, microbes with almost identical ssrrna genes can have vastly different physiology, and subsequently, have very different roles in ecosystem function (Box 1). These factors need to be considered in investigations of the significance and roles of microbial communities in ecological change on coral reefs. Many recent studies are now providing compelling evidence that coral microbial associations are more complex Box 1. Overcoming the limitations of phylogenetic markers to characterize microbial community function. Phylogenetic markers, such as the small subunit of the ribosome, are useful for characterizing the diversity of the microbial community but reveal little about the metabolic capabilities of organisms. Physiological differences among phylogenetically related taxa can occur owing to gene duplications, insertions and deletions, as well as through horizontal gene transfer events such as plasmid acquisition or the incorporation of viral genes. An example of this phenomenon is the cyanobacteria Prochlorococcus. Strains of this bacteria are found in almost all tropical oligotrophic habitats, but vary in abundance with latitude and depth [77]. These strains have almost identical 16S sequences (97% similarity), but differ physiologically [78]. Termed ecotypes, each Prochlorococcus strain is spectrally tuned to reside within a given light regime [79]. High light-adapted Prochlorococcus are abundant at the sea surface and have remarkably small genomes encoding only 1700 proteins. Low light-adapted Prochlorococcus are abundant at depths of 200 m and have larger genomes that encode for 2300 genes [78]. Similar variations in genome size can be found in Vibrio splendidus where 70% of the 206 strains that make up a single Vibrio splendidus ribotype cluster have differences in genome size even though the 16S genes of these strains are < 1% divergent [80]. In this case, the size variations range from 4.5 kb to 5.6 kb and are not driven by the presence of bacterial plasmids [80]. Perhaps more importantly, it has been shown that the presence of a single gene variant can alter both the microbial host range and the interactive status of a microbial symbiosis from benign or beneficial to pathogenic [81]. Metagenomics enables the functional capacity of microbial communities to be characterized in an ecologically relevant setting. 234

3 than previously estimated and are significantly influenced by factors specific to both the physical [15,16] and the host environment [13,18,18,18,28]. Only by addressing the true complexity of the microbial habitat and the strong influence of environmental and host factors can we understand the role of microbial communities in coral reefs ecosystems and incorporate these communities into predictive models of future ecosystem stability. Habitat complexity and microbial diversity Coral reefs are formed as a result of calcium carbonate deposition by scleractinian corals, and these vast biological structures form complex habitats for an enormous diversity of marine life (Figure 1), including microbial communities. Factors, such as temperature, light and water flow, vary significantly over these complex structures and influence both the host and the microbial communities [29 31]. The scleractinian coral hosts exhibit highly plastic morphologies (both within and among coral colonies) in response to the environment, modifying the microbial environment [32 36]. Therefore, just as the reef provides a multitude of niches for macro-organisms, each coral colony contains numerous microhabitats for an array of microbial communities (Figure 2, Box 2). Microhabitats found on and within coral structures support diverse microbial communities and influence microbial function within the coral holobiont. For example, in branching corals, branch tips are exposed to higher light intensities and more rapid water flow than are the bases of the branch. These differences result in rapid host tissue growth and low endosymbiotic dinoflagellate (Symbiodinium spp.) densities at the tip, and comparatively slow host tissue growth and high endosymbiotic dinoflagellate densities at the base of the branch [37]. The variability in endosymbiont density occurs on extremely small scales within the coral colony [38,39], strongly influences host growth and calcification [40], and the surface environment [27,28]. Fang and colleagues demonstrated a metabolic Figure 1. The complex coral reef structure. The coral reef environment is a structurally complex system with extensive species (both within and between genera) and habitat diversity that supports an array of microbial life. diffusion gradient from the base of a coral branch (dense endosymbionts) to the rapidly growing tip (without endosymbionts), which facilitates rapid metabolism, growth and calcification in the host [40]. Kuhl et al. [39] showed that coral tissue surface O 2 saturation fluctuates daily from >250% air saturation following light exposure to <2% air saturation following dark exposure, and that ph of the coral surface rapidly varies from 8.5 (during light conditions) to 7.3 (during dark conditions). Furthermore, Ulstrup et al. [38] demonstrate that oxygen saturation of the tissue surface varies significantly between the coral polyp and connective tissue between polyps (cenosarc), and that variability in oxygen saturation is further influenced by light availability of the environment (e.g. in bright and shaded areas) on the reef. These differences in microhabitats within the coral colony and within the reef environment will also impact members of the microbial community. Comparisons of the microbes from the coral surface mucus layer (or surface biofilm), the intracellular spaces within host tissues, and the skeletal matrix show that each community is distinct. Rohwer et al. [13] demonstrated that the diversity of bacterial communities associated with coral varied across the length of the coral branch. The internal environment of the coral host is also impacted by abiotic parameters, such as light penetration to the skeleton, and these factors have been shown to influence the endosymbiotic dinoflagellate populations [41] and also the endolithic microbial communities within the coral skeleton [42,43]. However, the means by which the extent of microenvironment variability and the resultant coral microbial ecology is ultimately manifested in the physiology and performance of the coral reef is yet to be explored. In other systems, microenvironmental variability significantly impacts both the host organism s physiology and ecosystem function. For example, biofilms and microbial associations of plant surfaces are strongly adapted to, and influenced by, small-scale environmental variability. Factors such as surface chemistry, nutrient availability, and water saturation, which are specific to plant microhabitats, such as leaf surfaces, stems and shoots, influence the microbial associations within each habitat [44]. Patchy or non-uniform environmental factors further influence plant root growth and formation, and, in turn, also influence the composition of microbial communities of both the root nodules and the surrounding soil [45]. Corals similarly create complex and heterogeneous microhabitats for microbial life, and the microbial diversity of these habitats is potentially as significant to host physiology as it is in other systems. Microhabitat partitioning strongly influences the composition of microbial communities, the contribution of the microbial community to host physiology (as a multipartite symbiotic entity or holobiont), and the role of the host within the ecosystem [11]. The idea that the coral holobiont, or any other organism in the reef ecosystem, represents a stable and uniform habitat vastly over-simplifies these complex and dynamic systems, and is likely to lead to misinterpretations of experimental data and the role of microbes in coral stress, bleaching and disease [46 48]. Understanding microbial diversity and the interaction of this diversity within a highly complex and variable host environment is essential 235

4 Figure 2. The complexity of the coral reef structure results in many microhabitats. The host structure provides various microhabitats for microbial colonization within the coral colony, coral polyps and coral tissues. Each microbial compartment on the reef is influenced by physical and biological environmental conditions that vary in time and space. Environmental variability through the water column (dark blue arrow) is related to reef depth and reefal position. Biological variability along the branch axis (brown arrow) is related to environmental (light and water flow) and biological factors (colony openness, endosymbiotic dinoflagellate density, respiration and photosynthesis). Variability along the branch apical to the basel axes (green arrow) is related to the variable 3-dimension structure of host, polvp density, and niche environments for microbial colonization and biofilm formation. to understanding the role of microbial communities in coral responses to environmental change. Environmental influences on the coral microbiome Environmental changes to reef ecosystems are now considered an unavoidable feature of coral reefs [49]. These worldwide declines in tropical reefs are linked to a variety of environmental problems, many of which are predicted to increase in severity in the future [2,50]. The impact of these problems, particularly thermal stress (which results in mass coral bleaching), on the physiology of corals and their eukaryotic microbial endosymbionts (Symbiodinium spp.) has been widely investigated in recent years (reviewed in [51]). However, the physiological changes occurring within the coral host in response to environmental stressors will also alter other host microbe interactions. The effects of environmental stress on the host and its associated microbial communities include: loss of mucus from the coral surface, reduced mucus supply to the coral surface mucus layer, changes in nutrient availability, impaired endosymbiotic dinoflagellate function, and also host tissue breakdown [52 56]. Environmental stressors that impact host physiology also change the microbial environment and subsequently the microbiome. Bourne et al demonstrated that changes in microbial communities are evident in bleached coral colonies when compared to healthy coral in the months before bleaching and in coral following recovery [49]. The authors are rightly cautious in attributing pathogenicity to specific microbial changes, as environmental changes will impact the coral holobiont in a multitude of ways. Microbial metabolism and physiology are directly affected by the same environmental disturbances that alter host metabolism, and these changes will alter the 236

5 Box 2. Host partitioning of the microhabitat Typically, hundreds to thousands of individual polyps form a single coral colony and form niche environments in which microbial communities can establish and impact the host physiology. Regions such as the coral tentacles, mouth and gut harbor microbial communities as a result of feeding, and host-specific interactions occur within the surface mucus layer and within the coral tissues and exposed coral skeleton. Similar to surface microbial communities in other systems, the coral surface mucus layer forms a microbial biofilm which is strongly influenced by the coral and the photosynthetic endosymbiotic dinoflagellates (Symbiodinium spp.) The host environment and surrounding seawater provide and sustains nourishment to the mucus layer (reviewed in reference [56]) and its associated microbial communities [21]. However, intracellular microbial symbionts other than the endosymbiotic dinoflagellates (Symbiodinium spp.) are rarely observed in situ. Some bacterial endosymbioses of coral host tissues have been proposed to occur within the coral gastroderm (or endoderm) a tissue layer that also houses the endosymbiotic dinoflagellates [19,25,27,82]. In addition, Lesser et al. [18,28] have also provided evidence for endosymbiotic cyanobacteria in epithelial tissues of some, but not all, color morphs of Montastrea cavernosa in the Caribbean. The calcium carbonate skeletal matrix of reef-forming corals harbors diverse microbial communities, supporting photosynthetic algae and cyanobacteria within the endolithic layer, which is visible as a green layer below the coral tissues in many massive coral species. Other members of the endolithic microfauna include microalgae, fungi, cyanobacteria and bacteria. interaction between the coral host and the microbial community within each microhabitat. Although this has not yet been studied in corals, the impacts of environmental changes on ecosystem function via direct and indirect effects on the microbial communities have been documented in other systems. For example, soil plant microbe interactions are considered important drivers of plant community ecology [57] and these are influenced by environmental change. The community of ammonia-oxidizing bacteria within grasslands responds to environmental changes such as increased atmospheric CO 2, temperature and precipitation. These feedback onto the ecosystem by significantly altering nutrient cycling [58]. Invasive plant species alter the soil microbial community structure and function and, ultimately, the soil environment, compared with native species [59,60]. Interestingly, recent work by Stat and Gates [61,62] report one endosymbiotic dinoflagellate clade (subspecies) is an invasive (non-native) symbiont of corals on Hawaiian reefs and is linked to increased host susceptibility to disease and mortality. In such examples, both direct and indirect effects of environmental change and stress on microbial community structure and function result in consequences that are evident within the ecosystem. Documenting the complexity of microbial community and holobiont responses to environmental stress, and their subsequent role in macroecological change, is therefore fundamental for predicting reef change. Documenting the responses of the coral reef microbiome Metagenomic approaches overcome the limitations of previous diversity and culture-based studies and provide a means to assess the complex roles that microbes play in virtually any ecosystem. This approach allows simultaneous assessment of coral-associated microbial diversity, microhabitat and functional responses of the microbiome. Metagenomics uses high throughput sequencing to characterize entire microbial assemblages or metagenomes within a given system using phylogenetic markers (e.g. ssrna, RecA, DNA polymerase) to provide insight into the diversity of the community in combination with the identification of protein encoding gene sequences to identify the potential metabolic pathways available within the community. This technology has been applied to microbial and viral consortia from a variety of environments, experimental scenarios [63 66] and temporal and spatial scales (Figure 3) [67]. Studies using metagenomics methods have also begun to unveil the functional roles of unique microbial communities [67]. Wegley et al. [20], found that coral microbial communities significantly contribute to coral holobiont protein and nitrogen cycling. Another recent metagenomic study has found that significant shifts in the functional capacity of the coral-associated microbiome occur when the coral is exposed to abiotic stress [68]. A microbial community (all organisms less than 8 mm) present on Pacific corals exposed to thermal, ph, nutrient, and carbon stress shifted from a community with few virulence genes to one with an abundance of genes involved in pathogenesis. Therefore, this study hypothesized that small, albeit significant, metabolic changes are much more important than changes in the structure of a microbial community when determining the ability of a system to adapt to local environmental shifts. Such a hypothesis could be extrapolated to whole ecosystems. For example, Dinsdale et al. [69] examined the microbial communities (<0.45 mm) from waters adjacent to four central Pacific islands. They found significant differences in both the abundance and metabolic capacity of the microbial populations with location [69]. Although the islands were different oceanographically, the dramatic difference in microbial metabolic function indicated that proximity to human populations and local disturbance significantly influenced microbial diversity and metabolism and as a result influenced coral reef health. The information acquired using these large-scale sequencing approaches provides compelling evidence that microbial communities are highly complex, environmentally and temporally dynamic, and extremely significant in terms of host animal physiology and ecosystem function. These results are provocative and provide ample rationale for incorporating metagenomic analyses and a microbial community perspective into studies and predictive models of ecological change on not just coral reef systems, but all ecosystems. We acknowledge that these approaches currently have limitations, including high costs and requirements for additional computational infrastructure. Rapid advances in technology are already reducing cost and bioinformatic tools for the analysis of metagenomic are improving very rapidly. Future directions In considering the future sustainability of coral reefs worldwide, it is fundamental to evaluate and understand the complex roles that microbial communities have in the processes that govern ecological change in these 237

6 Figure 3. The application of metagenomics to coral microbial ecology. The application of metagenomics to the coral microbiome allows scientists to explore microbial diversity and the reef system metabolic capacity simultaneously. ecosystems. Whereas a range of hypotheses related to the role of microbial communities on coral reefs have been presented in the literature, few have been tested. To begin to address this gap, we pose a series of questions that once addressed, will provide insight into the roles of microbes and host microbe interactions in large-scale ecosystem processes on coral reefs. 1. When are coral microbial partnerships established and how are they maintained throughout host life cycles? 2. In what ways do environmental changes influence the host microbe interactions? 3. Are specific host microbe associations indicative of coral reef health? 4. Do microbial community changes and host microbe interactions influence coral recovery and mortality following disturbance? 5. Can changes in microbial community composition mitigate ecosystem affects from environmental stress and at what scales? 6. Do microbial community host interactions influence the ability of the host to withstand stressors? 7. Can we incorporate microbial communities into predictive models of coral reef ecosystem change? Prosser [70] recently stated that quantitative information on the links between microbial community 238

7 Figure 4. Microbial function impacts on ecosystem stability. Linkages between micro-ecology (microbial community dynamics) and macro-ecology on coral reefs (coral community dynamics). structure, populations and activities will allow predictions on the impacts of climate change to microbial contributions to ecosystem processes. In determining the future of coral reefs worldwide we need to understand and incorporate the complex role of microbial communities (Figure 4) as systems that can respond to environmental change within an ecological time frame and influence ecological outcomes. The application of metagenomics provides a means to answer many key questions that define the relationship between microbial communities and environmentally driven macro-ecological change on reefs. Ultimately, this microbial perspective will improve our ability to accurately predict the resilience of specific reefs and contribute to the conservation of these important ecosystems. Acknowledgements We thank Dr William Leggat and Dr Andrew Baird for extremely valuable editorial assistance. The authors also thank funding sources including; Edwin W. Pauley Foundation, Hawaii EPSCOR; The National Science Foundation (OCE to RDG); The National Science Foundation research starter grant (IOS to RLVT); and The Australian Research Council, Australian Postdoctoral Fellowship (DP to TDA). This manuscript represents Hawaii Institute for Marine Biology contribution number References 1 Nystrom, M. and Folke, C. (2001) Spatial resilience of coral reefs. Ecosystems 4, Hughes, T.P. et al. (2003) Climate change, human impacts, and the resilience of coral reefs. Science 301, Balser, T. et al. (2006) Bridging the gap between micro - and macroscale perspectives on the role of microbial communities in global change ecology. Plant and Soil 289, Gutknecht, J.et al. (2006) Linking soil process and microbial ecology in freshwater wetland ecosystems. Plant and Soil 289, Schimel, J. et al. (2007) Microbial stress-response physiology and its implications for ecosystem function. Ecology 88, Wardle,D.A. et al. (2004) Ecological linkages between aboveground and belowground biota. Science 304, Allison, S.D. and Martiny, J.B.H. (2008) Resistance, resilience, and redundancy in microbial communities. Proc. Natl. Acad. Sci. U. S. A. 105, Webster, N.S. et al. (2004) Metamorphosis of a Scleractinian coral in response to microbial biofilms. Appl.Environ.Microbiol.70, Huggett, M.J. et al. (2008) Recruitment of the sea urchin Heliocidaris erythrogramma and the distribution and abundance of inducing bacteria in the field. Aquat. Microb. Ecol. 53, Yellowlees, D. et al. (2008) Metabolic interactions between algal symboints and invertebrate hosts. Plant Cell Environ. 31, Rohwer, F. et al. (2001) Diversity of bacteria associated with the Caribbean coral Montastraea franksi. Coral Reefs 20, Fuhrman, J.A. and Campbell, L. (1998) Microbial microdiversity. Nature 393, Rohwer, F. et al. (2002) Diversity and distribution of coral-associated bacteria. Mar. Ecol. Prog. Ser. 243, Frias-Lopez, J. et al. (2002) Partitioning of bacterial communities between seawater and healthy, black band biseased, and dead coral surfaces. Appl. Environ. Microbiol. 68, Justin, R.S. et al. (2005) Spatial dynamics of virus-like particles and heterotrophic bacteria within a shallow coral reef system. Mar. Ecol. Prog. Ser. 288, Raechel, A.L. et al. (2009) Diversities of coral-associated bacteria differ with location, but not species, for three acroporid corals on the Great Barrier Reef. FEMS Microbiol. Ecol. 68, Williams, W.M. et al. (1987) Nitrogen fixation (acetylene reduction) associated with the living coral Acropora variabilis. Mar. Biol. 94, Lesser, M.P. et al. (2004) Discovery of symbiotic nitrogen-fixing cyanobacteria in corals. Science 305, Kvennefors, E. and Roff, G. (2009) Evidence of cyanobacteria-like endosymbionts in Acroporid corals from the Great Barrier Reef. Coral Reefs 28, Wegley, L. et al. (2007) Metagenomic analysis of the microbial community associated with the coral Porites astreoides. Environ. Micro. 9, Ritchie, K.B. (2006) Regulation of microbial populations by coral surface mucus and mucus-associated bacteria. Mar. Ecol. Prog. Ser. 322, Garrett, P. and Ducklow, H. (1975) Coral diseases in Bermuda. Nature 253,

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