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Microbial symbionts frequently localize within specific body structures or cell types of their multicellular hosts. This spatiotemporal niche is critical to host health, nutrient exchange, and fitness. Measuring host-microbe metabolite exchange has conventionally relied on tissue homogenates, eliminating dimensionality and dampening analytical sensitivity. We have developed a mass spectrometry imaging workflow for a soft- and hard-bodied cnidarian animal capable of revealing the host and symbiont metabolome in situ, without the need for a priori isotopic labelling or skeleton decalcification. The mass spectrometry imaging method provides critical functional insights that cannot be gleaned from bulk tissue analyses or other presently available spatial methods. We show that cnidarian hosts may regulate microalgal symbiont acquisition and rejection through specific ceramides distributed throughout the tissue lining the gastrovascular cavity. The distribution pattern of betaine lipids showed that once resident, symbionts primarily reside in light-exposed tentacles to generate photosynthate. Spatial patterns of these metabolites also revealed that symbiont identity can drive host metabolism., (© 2023 Chan et al.)

Genetic signatures caused by demographic and adaptive processes during past climatic shifts can inform predictions of species' responses to anthropogenic climate change. To identify these signatures in Acropora tenuis , a reef-building coral threatened by global warming, we first assembled the genome from long reads and then used shallow whole-genome resequencing of 150 colonies from the central inshore Great Barrier Reef to inform population genomic analyses. We identify population structure in the host that reflects a Pleistocene split, whereas photosymbiont differences between reefs most likely reflect contemporary (Holocene) conditions. Signatures of selection in the host were associated with genes linked to diverse processes including osmotic regulation, skeletal development, and the establishment and maintenance of symbiosis. Our results suggest that adaptation to post-glacial climate change in A. tenuis has involved selection on many genes, while differences in symbiont specificity between reefs appear to be unrelated to host population structure., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Physiological performance, disease and bleaching prevalence are often patchy within individual coral colonies. These responses are largely influenced by coral-associated microbes, but how the coral microbiome changes over small spatial scales has never been quantified before. We performed a high-resolution quantification of the spatial scale of microbial species turnover (β-diversity) within skeletons of boulder-forming Porites corals. We found very strong prokaryotic species turnover across spatial scales ranging from 4 mm to 2 m within individual colonies, possibly resulting from dispersal limitation and microbial interactions. The microalgal community was more homogeneously distributed, which is likely due to these photosymbionts actively boring through limestone. Our findings highlight unprecedented levels of intra-colony heterogeneity in the skeletal microbiome, which has implications for the experimental design of coral microbiome studies and for our understanding of coral resilience.

Symbiodinium, the dinoflagellate photosymbiont of corals, is posited to become more susceptible to viral infections when heat-stressed. To investigate this hypothesis, we mined transcriptome data of a thermosensitive and a thermotolerant type C1 Symbiodinium population at ambient (27 °C) and elevated (32°C) temperatures. We uncovered hundreds of transcripts from nucleocytoplasmic large double-stranded DNA viruses (NCLDVs) and the genome of a novel positive-sense single-stranded RNA virus (+ssRNAV). In the transcriptome of the thermosensitive population only, +ssRNAV transcripts had remarkable expression levels in the top 0.03% of all transcripts at 27 °C, but at 32 °C, expression levels of +ssRNAV transcripts decreased, while expression levels of anti-viral transcripts increased. In both transcriptomes, expression of NCLDV transcripts increased at 32 °C, but thermal induction of NCLDV transcripts involved in DNA manipulation was restricted to the thermosensitive population. Our findings reveal that viruses infecting Symbiodinium are affected by heat stress and may contribute to Symbiodinium thermal sensitivity.

Understanding how pathogens maintain their virulence is critical to developing tools to mitigate disease in animal populations. We sequenced and assembled the first draft genome of Roseofilum reptotaenium AO1, the dominant cyanobacterium underlying pathogenicity of the virulent coral black band disease (BBD), and analyzed parts of the BBD-associated Geitlerinema sp. BBD_1991 genome in silico . Both cyanobacteria are equipped with an adaptive, heritable clustered regularly interspaced short palindromic repeats (CRISPR)-Cas defense system type I-D and have potential virulence genes located within several prophage regions. The defense system helps to prevent infection by viruses and mobile genetic elements via identification of short fingerprints of the intruding DNA, which are stored as templates in the bacterial genome, in so-called "CRISPRs." Analysis of CRISPR target sequences (protospacers) revealed an unusually high number of self-targeting spacers in R. reptotaenium AO1 and extraordinary long CRIPSR arrays of up to 260 spacers in Geitlerinema sp. BBD_1991. The self-targeting spacers are unlikely to be a form of autoimmunity; instead these target an incomplete lysogenic bacteriophage. Lysogenic virus induction experiments with mitomycin C and UV light did not reveal an actively replicating virus population in R. reptotaenium AO1 cultures, suggesting that phage functionality is compromised or excision could be blocked by the CRISPR-Cas system. Potential prophages were identified in three regions of R. reptotaenium AO1 and five regions of Geitlerinema sp. BBD_1991, containing putative BBD relevant virulence genes, such as an NAD-dependent epimerase/dehydratase (a homolog in terms of functionality to the third and fourth most expressed gene in BBD), lysozyme/metalloendopeptidases and other lipopolysaccharide modification genes. To date, viruses have not been considered to be a component of the BBD consortium or a contributor to the virulence of R. reptotaenium AO1 and Geitlerinema sp. BBD_1991. We suggest that the presence of virulence genes in potential prophage regions, and the CRISPR-Cas defense systems are evidence of an arms race between the respective cyanobacteria and their bacteriophage predators. The presence of such a defense system likely reduces the number of successful bacteriophage infections and mortality in the cyanobacteria, facilitating the progress of BBD.

Reef-building corals possess a range of acclimatisation and adaptation mechanisms to respond to seawater temperature increases. In some corals, thermal tolerance increases through community composition changes of their dinoflagellate endosymbionts (Symbiodinium spp.), but this mechanism is believed to be limited to the Symbiodinium types already present in the coral tissue acquired during early life stages. Compelling evidence for symbiont switching, that is, the acquisition of novel Symbiodinium types from the environment, by adult coral colonies, is currently lacking. Using deep sequencing analysis of Symbiodinium rDNA internal transcribed spacer 2 (ITS2) PCR amplicons from two pocilloporid coral species, we show evidence consistent with de novo acquisition of Symbiodinium types from the environment by adult corals following two consecutive bleaching events. Most of these newly detected symbionts remained in the rare biosphere (background types occurring below 1% relative abundance), but one novel type reached a relative abundance of ~33%. Two de novo acquired Symbiodinium types belong to the thermally resistant clade D, suggesting that this switching may have been driven by consecutive thermal bleaching events. Our results are particularly important given the maternal mode of Symbiodinium transmission in the study species, which generally results in high symbiont specificity. These findings will cause a paradigm shift in our understanding of coral-Symbiodinium symbiosis flexibility and mechanisms of environmental acclimatisation in corals.

Corals rely on photosynthesis by their endosymbiotic dinoflagellates (Symbiodinium spp.) to form the basis of tropical coral reefs. High sea surface temperatures driven by climate change can trigger the loss of Symbiodinium from corals (coral bleaching), leading to declines in coral health. Different putative species (genetically distinct types) as well as conspecific populations of Symbiodinium can confer differing levels of thermal tolerance to their coral host, but the genes that govern dinoflagellate thermal tolerance are unknown. Here we show physiological and transcriptional responses to heat stress by a thermo-sensitive (physiologically susceptible at 32 °C) type C1 Symbiodinium population and a thermo-tolerant (physiologically healthy at 32 °C) type C1 Symbiodinium population. After nine days at 32 °C, neither population exhibited physiological stress, but both displayed up-regulation of meiosis genes by ≥ 4-fold and enrichment of meiosis functional gene groups, which promote adaptation. After 13 days at 32 °C, the thermo-sensitive population suffered a significant decrease in photosynthetic efficiency and increase in reactive oxygen species (ROS) leakage from its cells, whereas the thermo-tolerant population showed no signs of physiological stress. Correspondingly, only the thermo-tolerant population demonstrated up-regulation of a range of ROS scavenging and molecular chaperone genes by ≥ 4-fold and enrichment of ROS scavenging and protein-folding functional gene groups. The physiological and transcriptional responses of the Symbiodinium populations to heat stress directly correlate with the bleaching susceptibilities of corals that harbored these same Symbiodinium populations. Thus, our study provides novel, foundational insights into the molecular basis of dinoflagellate thermal tolerance and coral bleaching., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Connectivity underpins the persistence and recovery of marine ecosystems. The Great Barrier Reef (GBR) is the world's largest coral reef ecosystem and managed by an extensive network of no-take zones; however, information about connectivity was not available to optimize the network's configuration. We use multivariate analyses, Bayesian clustering algorithms and assignment tests of the largest population genetic data set for any organism on the GBR to date (Acropora tenuis, >2500 colonies; >50 reefs, genotyped for ten microsatellite loci) to demonstrate highly congruent patterns of connectivity between this common broadcast spawning reef-building coral and its congener Acropora millepora (~950 colonies; 20 reefs, genotyped for 12 microsatellite loci). For both species, there is a genetic divide at around 19°S latitude, most probably reflecting allopatric differentiation during the Pleistocene. GBR reefs north of 19°S are essentially panmictic whereas southern reefs are genetically distinct with higher levels of genetic diversity and population structure, most notably genetic subdivision between inshore and offshore reefs south of 19°S. These broadly congruent patterns of higher genetic diversities found on southern GBR reefs most likely represent the accumulation of alleles via the southward flowing East Australia Current. In addition, signatures of genetic admixture between the Coral Sea and outer-shelf reefs in the northern, central and southern GBR provide evidence of recent gene flow. Our connectivity results are consistent with predictions from recently published larval dispersal models for broadcast spawning corals on the GBR, thereby providing robust connectivity information about the dominant reef-building genus Acropora for coral reef managers., (© 2016 John Wiley & Sons Ltd.)

Black band disease (BBD) is a common disease of reef-building corals with a worldwide distribution that causes tissue loss at a rate of up to 3 cm/day. Critical for a mechanistic understanding of the disease's aetiology is the cultivation of its proposed pathogen, filamentous cyanobacteria (genus Roseofilum). Here, we optimise existing protocols for the isolation and cultivation of Roseofilum cyanobacteria using a new strain from the central Great Barrier Reef. We demonstrate that the isolation of this bacterium via inoculation onto agar plates was highly effective with a low percentage agar of 0.6% and that growth monitoring was most sensitive with fluorescence measurements of chlorophyll-a (440/685 nm). Cell growth curves in liquid and solid media were generated for the first time for this cyanobacterium and showed best growth rates for the previously untested L1-medium (growth rate k = 0.214 biomass/day; doubling time t gen = 4.67 days). Our results suggest that the trace metals contained in L1-medium maximise biomass increase over time for this cyanobacterium. Since the newly isolated Roseofilum strain is genetically closest to Pseudoscillatoria coralii, but in terms of pigmentation and cell size closer to Roseofilum reptotaenium, we formally merge the two species into a single taxon by providing an emended species description, Roseofilum reptotaenium (Rasoulouniriana) Casamatta emend. Following this optimized protocol is recommended for fast isolation and cultivation of Roseofilum cyanobacteria, for growth curve generation in strain comparisons and for maximisation of biomass in genetic studies.

Abundant bioinformatics resources are available for the study of complex microbial metagenomes, however their utility in viral metagenomics is limited. HoloVir is a robust and flexible data analysis pipeline that provides an optimized and validated workflow for taxonomic and functional characterization of viral metagenomes derived from invertebrate holobionts. Simulated viral metagenomes comprising varying levels of viral diversity and abundance were used to determine the optimal assembly and gene prediction strategy, and multiple sequence assembly methods and gene prediction tools were tested in order to optimize our analysis workflow. HoloVir performs pairwise comparisons of single read and predicted gene datasets against the viral RefSeq database to assign taxonomy and additional comparison to phage-specific and cellular markers is undertaken to support the taxonomic assignments and identify potential cellular contamination. Broad functional classification of the predicted genes is provided by assignment of COG microbial functional category classifications using EggNOG and higher resolution functional analysis is achieved by searching for enrichment of specific Swiss-Prot keywords within the viral metagenome. Application of HoloVir to viral metagenomes from the coral Pocillopora damicornis and the sponge Rhopaloeides odorabile demonstrated that HoloVir provides a valuable tool to characterize holobiont viral communities across species, environments, or experiments.

The current lack of understanding of the genetic basis underlying environmental stress tolerance in reef-building corals impairs the development of new management approaches to confronting the global demise of coral reefs. On the Great Barrier Reef (GBR), an approximately 51% decline in coral cover occurred over the period 1985-2012. We conducted a gene-by-environment association analysis across 12° latitude on the GBR, as well as both in situ and laboratory genotype-by-phenotype association analyses. These analyses allowed us to identify alleles at two genetic loci that account for differences in environmental stress tolerance and antioxidant capacity in the common coral Acropora millepora. The effect size for antioxidant capacity was considerable and biologically relevant (32.5 and 14.6% for the two loci). Antioxidant capacity is a critical component of stress tolerance because a multitude of environmental stressors cause increased cellular levels of reactive oxygen species. Our findings provide the first step toward the development of novel coral reef management approaches, such as spatial mapping of stress tolerance for use in marine protected area design, identification of stress-tolerant colonies for assisted migration, and marker-assisted selective breeding to create more tolerant genotypes for restoration of denuded reefs.

Within populations of brooding sessile corals, sperm dispersal constitutes the mechanism by which gametes interact and mating occurs, and forms the first link in the network of processes that determine specieswide connectivity patterns. However, almost nothing is known about sperm dispersal for any internally fertilizing coral. In this study, we conducted a parentage analysis on coral larvae collected from an area of mapped colonies, to measure the distance sperm disperses for the first time in a reef-building coral and estimated the mating system characteristics of a recently identified putative cryptic species within the Seriatopora hystrix complex (ShA; Warner et al. 2015). We defined consensus criteria among several replicated methods (COLONY 2.0, CERVUS 3.0, MLTR v3.2) to maximize accuracy in paternity assignments. Thirteen progeny arrays indicated that this putative species produces exclusively sexually derived, primarily outcrossed larvae (mean t(m) = 0.999) in multiple paternity broods (mean r(p) = 0.119). Self-fertilization was directly detected at low frequency for all broods combined (2.8%), but comprised 23% of matings in one brood. Although over 82% of mating occurred between colonies within 10 m of each other (mean sperm dispersal = 5.5 m ± 4.37 SD), we found no evidence of inbreeding in the established population. Restricted dispersal of sperm compared to slightly greater larval dispersal appears to limit inbreeding among close relatives in this cryptic species. Our findings establish a good basis for further work on sperm dispersal in brooding corals and provide the first information about the mating system of a newly identified and abundant cryptic species., (© 2016 John Wiley & Sons Ltd.)

Disease is an increasing threat to reef-building corals. One of the few identified pathogens of coral disease is the bacterium Vibrio coralliilyticus. In Vibrio cholerae, infection by a bacterial virus (bacteriophage) results in the conversion of non-pathogenic strains to pathogenic strains and this can lead to cholera pandemics. Pathogenicity islands encoded in the V. cholerae genome play an important role in pathogenesis. Here we analyse five whole genome sequences of V. coralliilyticus to examine whether virulence is similarly driven by horizontally acquired elements. We demonstrate that bacteriophage genomes encoding toxin genes with homology to those found in pathogenic V. cholerae are integrated in V. coralliilyticus genomes. Virulence factors located on chromosomal pathogenicity islands also exist in some strains of V. coralliilyticus. The presence of these genetic signatures indicates virulence in V. coralliilyticus is driven by prophages and other horizontally acquired elements. Screening for pathogens of coral disease should target conserved regions in these elements.

Zooxanthellate corals (i.e. those harbouring Symbiodinium) are the main builders of the world's shallow-water marine coral reefs. They represent intimate diverse symbioses between coral animals, single-celled photosynthetic dinoflagellates (Symbiodinium spp.), other microscopic eukaryotes, prokaryotes and viruses. Crabs and other crustaceans, worms, sponges, bivalves and hydrozoans, fishes, sea urchins, octopuses and sea stars are itinerant members of these 'rainforests of the sea'. This review focuses on the biodiversity of scleractinian coral animals and their best studied microscopic epi- and endosymbionts. In relation to coral-associated species diversity, Symbiodinium internal transcribed spacer region sequence types tally 10(2) -10(3) or up to ~15 different operational taxonomic units (OTUs, or putative species at the 97% sequence identity level; this cut-off was chosen based on intragenomic sequence diversity observed in monoclonal cultures) and prokaryotes (mostly bacterial) total 10(2) -10(4) OTUs. We analysed all publically accessible 16S rRNA gene sequence data and found Gammaproteobacteria were extremely abundant, followed by Alphaproteobacteria. Notably, Archaea were poorly represented and 'unassigned OTUs' were abundant in data generated by high-throughput DNA sequencing studies of corals. We outline and compare model systems that could be used in future studies of the coral holobiont. In our future directions, we recommend a global coral sampling effort including substantial attention being paid to method of coral tissue acquisition, which compartments (mucus, tissue, skeleton) to explore, broadening the holobiont members considered and linking biodiversity with functional investigations., (© 2015 John Wiley & Sons Ltd.)

Bleaching episodes caused by increasing seawater temperatures may induce mass coral mortality and are regarded as one of the biggest threats to coral reef ecosystems worldwide. The current consensus is that this phenomenon results from enhanced production of harmful reactive oxygen species (ROS) that disrupt the symbiosis between corals and their endosymbiotic dinoflagellates, Symbiodinium. Here, the responses of two important antioxidant defence components, the host coenzyme Q (CoQ) and symbiont plastoquinone (PQ) pools, are investigated for the first time in colonies of the scleractinian coral, Acropora millepora, during experimentally-induced bleaching under ecologically relevant conditions. Liquid chromatography-mass spectrometry (LC-MS) was used to quantify the states of these two pools, together with physiological parameters assessing the general state of the symbiosis (including photosystem II photochemical efficiency, chlorophyll concentration and Symbiodinium cell densities). The results show that the responses of the two antioxidant systems occur on different timescales: (i) the redox state of the Symbiodinium PQ pool remained stable until twelve days into the experiment, after which there was an abrupt oxidative shift; (ii) by contrast, an oxidative shift of approximately 10% had occurred in the host CoQ pool after 6 days of thermal stress, prior to significant changes in any other physiological parameter measured. Host CoQ pool oxidation is thus an early biomarker of thermal stress in corals, and this antioxidant pool is likely to play a key role in quenching thermally-induced ROS in the coral-algal symbiosis. This study adds to a growing body of work that indicates host cellular responses may precede the bleaching process and symbiont dysfunction.

Reef-building corals form close associations with organisms from all three domains of life and therefore have many potential viral hosts. Yet knowledge of viral communities associated with corals is barely explored. This complexity presents a number of challenges in terms of the metagenomic assessments of coral viral communities and requires specialized methods for purification and amplification of viral nucleic acids, as well as virome annotation. In this minireview, we conduct a meta-analysis of the limited number of existing coral virome studies, as well as available coral transcriptome and metagenome data, to identify trends and potential complications inherent in different methods. The analysis shows that the method used for viral nucleic acid isolation drastically affects the observed viral assemblage and interpretation of the results. Further, the small number of viral reference genomes available, coupled with short sequence read lengths might cause errors in virus identification. Despite these limitations and potential biases, the data show that viral communities associated with corals are diverse, with double- and single-stranded DNA and RNA viruses. The identified viruses are dominated by double-stranded DNA-tailed bacteriophages, but there are also viruses that infect eukaryote hosts, likely the endosymbiotic dinoflagellates, Symbiodinium spp., host coral and other eukaryotes in close association., (© 2015 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Unravelling the contributions of local anthropogenic and seasonal environmental factors in suppressing the coral immune system is important for prioritizing management actions at reefs exposed to high levels of human activities. Here, we monitor health of the model coral Acropora millepora adjacent to a high-use and an unused reef-based tourist platform, plus a nearby control site without a platform, over 7 months spanning a typical austral summer. Comparisons of temporal patterns in a range of biochemical and genetic immune parameters (Toll-like receptor signalling pathway, lectin-complement system, prophenoloxidase-activating system and green fluorescent protein-like proteins) among healthy, injured and diseased corals revealed that corals exhibit a diverse array of immune responses to environmental and anthropogenic stressors. In healthy corals at the control site, expression of genes involved in the Toll-like receptor signalling pathway (MAPK p38, MEKK1, cFos and ATF4/5) and complement system (C3 and Bf) was modulated by seasonal environmental factors in summer months. Corals at reef platform sites experienced additional stressors over the summer, as evidenced by increased expression of various immune genes, including MAPK p38 and MEKK1. Despite increased expression of immune genes, signs of white syndromes were detected in 31% of study corals near tourist platforms in the warmest summer month. Evidence that colonies developing disease showed reduced expression of genes involved in the complement pathway prior to disease onset suggests that their immune systems may have been compromised. Responses to disease and physical damage primarily involved the melanization cascade and GFP-like proteins, and appeared to be sufficient for recovery when summer heat stress subsided. Overall, seasonal and anthropogenic factors may have interacted synergistically to overwhelm the immune systems of corals near reef platforms, leading to increased disease prevalence in summer at these sites.

Coral reefs surrounding the islands lying close to the coast are unique to the Great Barrier Reef (GBR) in that they are frequently exposed to disturbance events including floods caused by cyclonic rainfall, strong winds and occasional periods of prolonged above-average temperatures during summer. In one such group of islands in the southern GBR, the Keppel Island archipelago, climate-driven disturbances frequently result in major coral mortality. Whilst these island reefs have clearly survived such dramatic disturbances in the past, the consequences of extreme mortality events may include the loss of genetic diversity, and hence adaptive potential, and a reduction in fitness due to inbreeding, especially if new recruitment from external sources is limited. Here we examined the level of isolation of the Keppel Island group as well as patterns of gene flow within the Keppel Islands using 10 microsatellite markers in nine populations of the coral, Acropora millepora. Bayesian cluster analysis and assignment tests indicated gene flow is restricted, but not absent, between the outer and inner Keppel Island groups, and that extensive gene flow exists within each of these island groups. Comparison of the Keppel Island data with results from a previous GBR-wide study that included a single Keppel Island population, confirmed that A. millepora in the Keppel Islands is genetically distinct from populations elsewhere on the GBR, with exception of the nearby inshore High Peak Reef just north of the Keppel Islands. We compared patterns of genetic diversity in the Keppel Island populations with those from other GBR populations and found them to be slightly, but significantly lower, consistent with the archipelago being geographically isolated, but there was no evidence for recent bottlenecks or deviation from mutation-drift equilibrium. A high incidence of private alleles in the Keppel Islands, particularly in the outer islands, supports their relative isolation and contributes to the conservation value of the archipelago. The lack of evidence for genetic erosion, in combination with our observation that the North Keppel Island population samples collected in 2002 and 2008, respectively, exhibited a pairwise genetic distance of zero, supports previous published work indicating that, following bleaching, Acropora corals in the Keppel Islands predominantly recover from regrowth of small amounts of remaining live tissue in apparently dead coral colonies. This is likely supplemented by recruitment of larvae from genetically similar, less disturbed populations at nearby reefs, particularly following extreme flood events.

Increasing physical damage on coral reefs from predation, storms and anthropogenic disturbances highlights the need to understand the impact of injury on the coral immune system. In this study, we examined the regulation of the coral immune response over 10 days following physical trauma artificially inflicted on in situ colonies of the coral Acropora aspera, simultaneously with bacterial colonization of the lesions. Corals responded to injury by increasing the expression of immune system-related genes involved in the Toll-like and NOD-like receptor signalling pathways and the lectin-complement system in three phases (<2, 4 and 10 days post-injury). Phenoloxidase activity was also significantly upregulated in two phases (<3 and 10 days post-injury), as were levels of non-fluorescent chromoprotein. In addition, green fluorescent protein expression was upregulated in response to injury from 4 days post-injury, while cyan fluorescent protein expression was reduced. No shifts in the composition of coral-associated bacterial communities were evident following injury based on 16S rRNA gene amplicon pyrosequencing. Bacteria-specific fluorescence in situ hybridization also showed no evidence of bacterial colonization of the wound or regenerating tissues. Coral tissues showed near-complete regeneration of lesions within 10 days. This study demonstrates that corals exhibit immune responses that support rapid recovery following physical injury, maintain coral microbial homeostasis and prevent bacterial infestation that may compromise coral fitness., (© 2015 John Wiley & Sons Ltd.)

Background: The advent of next-generation sequencing has brought about an explosion of single nucleotide polymorphism (SNP) data in non-model organisms; however, profiling these SNPs across multiple natural populations still requires substantial time and resources., Results: Here, we introduce two cost-efficient quantitative High Resolution Melting (qHRM) methods for measuring allele frequencies at known SNP loci in pooled DNA samples: the "peaks" method, which can be applied to large numbers of SNPs, and the "curves" method, which is more labor intensive but also slightly more accurate. Using the reef-building coral Acropora millepora, we show that both qHRM methods can recover the allele proportions from mixtures prepared using two or more individuals of known genotype. We further demonstrate advantages of each method over previously published methods; specifically, the "peaks" method can be rapidly scaled to screen several hundred SNPs at once, whereas the "curves" method is better suited for smaller numbers of SNPs., Conclusions: Compared to genotyping individual samples, these methods can save considerable effort and genotyping costs when relatively few candidate SNPs must be profiled across a large number of populations. One of the main applications of this method could be validation of SNPs of interest identified in population genomic studies.

Mounting evidence of cryptic species in a wide range of taxa highlights the need for careful analyses of population genetic data sets to unravel within-species diversity from potential interspecies relationships. Here, we use microsatellite loci and hierarchical clustering analysis to investigate cryptic diversity in sympatric and allopatric (separated by 450 km) populations of the widespread coral Seriatopora hystrix on the Great Barrier Reef. Structure analyses delimited unique genetic clusters that were confirmed by phylogenetic and extensive population-level analyses. Each of four sympatric yet distinct genetic clusters detected within S. hystrix demonstrated greater genetic cohesion across regional scales than between genetic clusters within regions (<10 km). Moreover, the magnitude of genetic differentiation between different clusters (>0.620 G"ST ) was similar to the difference between S. hystrix clusters and the congener S. caliendrum (mean G"ST 0.720). Multiple lines of evidence, including differences in habitat specificity, mitochondrial identity, Symbiodinium associations and morphology, corroborate the nuclear genetic evidence that these distinct clusters constitute different species. Hierarchical clustering analysis combined with more traditional population genetic methods provides a powerful approach for delimiting species and should be regularly applied to ensure that ecological and evolutionary patterns interpreted for single species are not confounded by the presence of cryptic species., (© 2015 John Wiley & Sons Ltd.)

Background: The symbiosis between corals and the dinoflagellate alga Symbiodinium is essential for the development and survival of coral reefs. Yet this fragile association is highly vulnerable to environmental disturbance. A coral's ability to tolerate temperature stress depends on the fitness of its resident symbionts, whose thermal optima vary extensively between lineages. However, the in hospite population genetic structure of Symbiodinium is poorly understood and mostly based on analysis of bulk DNA extracted from thousands to millions of cells. Using quantitative single-cell PCR, we enumerated DNA polymorphisms in the symbionts of the reef-building coral Pocillopora damicornis, and applied a model selection approach to explore the potential for recombination between coexisting Symbiodinium populations., Results: Two distinct Symbiodinium ITS2 sequences (denoted C100 and C109) were retrieved from all P. damicornis colonies analysed. However, the symbiont assemblage consisted of three distinct Symbiodinium populations: cells featuring pure arrays of ITS2 type C109, near-homogeneous cells of type C100 (with trace ITS2 copies of type C109), and those with co-dominant C100 and C109 ITS2 repeats. The symbiont consortia of some colonies consisted almost entirely of these putative C100 × C109 recombinants., Conclusions: Our results are consistent with the occurrence of sexual recombination between Symbiodinium types C100 and C109. While the multiple-copy nature of the ITS2 dictates that the observed pattern of intra-genomic co-dominance may be a result of incomplete concerted evolution of intra-genomic polymorphisms, this is a less likely explanation given the occurrence of homogeneous cells of the C109 type. Conclusive evidence for inter-lineage recombination and introgression in this genus will require either direct observational evidence or a single-cell genotyping approach targeting multiple, single-copy loci.

The genetic enhancement of wild animals and plants for characteristics that benefit human populations has been practiced for thousands of years, resulting in impressive improvements in commercially valuable species. Despite these benefits, genetic manipulations are rarely considered for noncommercial purposes, such as conservation and restoration initiatives. Over the last century, humans have driven global climate change through industrialization and the release of increasing amounts of CO2, resulting in shifts in ocean temperature, ocean chemistry, and sea level, as well as increasing frequency of storms, all of which can profoundly impact marine ecosystems. Coral reefs are highly diverse ecosystems that have suffered massive declines in health and abundance as a result of these and other direct anthropogenic disturbances. There is great concern that the high rates, magnitudes, and complexity of environmental change are overwhelming the intrinsic capacity of corals to adapt and survive. Although it is important to address the root causes of changing climate, it is also prudent to explore the potential to augment the capacity of reef organisms to tolerate stress and to facilitate recovery after disturbances. Here, we review the risks and benefits of the improvement of natural and commercial stocks in noncoral reef systems and advocate a series of experiments to determine the feasibility of developing coral stocks with enhanced stress tolerance through the acceleration of naturally occurring processes, an approach known as (human)-assisted evolution, while at the same time initiating a public dialogue on the risks and benefits of this approach.

The reduction in coral cover on many contemporary tropical reefs suggests a different set of coral community assemblages will dominate future reefs. To evaluate the capacity of reef corals to persist over various time scales, we examined coral community dynamics in contemporary, fossil, and simulated future coral reef ecosystems. Based on studies between 1987 and 2012 at two locations in the Caribbean, and between 1981 and 2013 at five locations in the Indo-Pacific, we show that many coral genera declined in abundance, some showed no change in abundance, and a few coral genera increased in abundance. Whether the abundance of a genus declined, increased, or was conserved, was independent of coral family. An analysis of fossil-reef communities in the Caribbean revealed changes in numerical dominance and relative abundances of coral genera, and demonstrated that neither dominance nor taxon was associated with persistence. As coral family was a poor predictor of performance on contemporary reefs, a trait-based, dynamic, multi-patch model was developed to explore the phenotypic basis of ecological performance in a warmer future. Sensitivity analyses revealed that upon exposure to thermal stress, thermal tolerance, growth rate, and longevity were the most important predictors of coral persistence. Together, our results underscore the high variation in the rates and direction of change in coral abundances on contemporary and fossil reefs. Given this variation, it remains possible that coral reefs will be populated by a subset of the present coral fauna in a future that is warmer than the recent past.

Reef-building corals comprise multipartite symbioses where the cnidarian animal is host to an array of eukaryotic and prokaryotic organisms, and the viruses that infect them. These viruses are critical elements of the coral holobiont, serving not only as agents of mortality, but also as potential vectors for lateral gene flow, and as elements encoding a variety of auxiliary metabolic functions. Consequently, understanding the functioning and health of the coral holobiont requires detailed knowledge of the associated viral assemblage and its function. Currently, the most tractable way of uncovering viral diversity and function is through metagenomic approaches, which is inherently difficult in corals because of the complex holobiont community, an extracellular mucus layer that all corals secrete, and the variety of sizes and structures of nucleic acids found in viruses. Here we present the first protocol for isolating, purifying and amplifying viral nucleic acids from corals based on mechanical disruption of cells. This method produces at least 50% higher yields of viral nucleic acids, has very low levels of cellular sequence contamination and captures wider viral diversity than previously used chemical-based extraction methods. We demonstrate that our mechanical-based method profiles a greater diversity of DNA and RNA genomes, including virus groups such as Retro-transcribing and ssRNA viruses, which are absent from metagenomes generated via chemical-based methods. In addition, we briefly present (and make publically available) the first paired DNA and RNA viral metagenomes from the coral Acropora tenuis.

Understanding levels of connectivity among scleractinian coral populations over a range of temporal and spatial scales is vital for managing tropical coral reef ecosystems. Here, we use multilocus microsatellite genotypes to assess the spatial genetic structure of two molecular operational taxonomic units (MOTUs, types α and β) of the widespread coral Pocillopora damicornis on the Great Barrier Reef (GBR) and infer the extent of connectivity on spatial scales spanning from local habitat types to latitudinal sectors of the GBR. We found high genetic similarities over large spatial scales spanning > 1000 km from the northern to the southern GBR, but also strong genetic differentiation at local scales in both MOTUs. The presence of a considerable number of first-generation migrants within the populations sampled (12% and 27% for types α and β, respectively) suggests that genetic differentiation over small spatial scales is probably a consequence of stochastic recruitment from different genetic pools into recently opened up spaces on the reef, for example, following major disturbance events. We explain high genetic similarity among populations over hundreds of kilometres by long competency periods of brooded zooxanthellate larvae and multiple larval release events each year, combined with strong longshore currents typical along the GBR. The lack of genetic evidence for predominantly clonal reproduction in adult populations of P. damicornis, which broods predominantly asexually produced larvae, further undermines the paradigm that brooded larvae settle close to parent colonies shortly after the release., (© 2013 John Wiley & Sons Ltd.)

Understanding connectivity of coral populations among and within reefs over ecologically significant timescales is essential for developing evidence-based management strategies, including the design of marineprotected areas. Here, we present the first assessment of contemporary connectivity among populations of two Molecular Operational Taxonomic Units (MOTUs) of the brooding coral Pocillopora damicornis. We used individual-based genetic assignment methods to identify the proportions of philopatric and migrant larval recruits, settling over 12 months at sites around Lizard Island (northern Great Barrier Reef [GBR]) and over 24 months at sites around the Palms Islands (central GBR). Overall, we found spatially and temporally variable rates of self-recruitment and dispersal, demonstrating the importance of variation in local physical characteristics in driving dispersal processes. Recruitment patterns and inferred dispersal distances differed between the two P. damicornis MOTUs, with type α recruits exhibiting predominantly philopatric recruitment, while the majority of type β recruits were either migrants from identified putative source populations or assumed migrants based on genetic exclusion from all known populations. While P. damicornis invests much energy into brooding clonal larvae, we found that only 15% and 7% of type α and type β recruits, respectively, were clones of sampled adult colonies or other recruits, challenging the hypothesis that reproduction is predominantly asexual in this species on the GBR. We explain high rates of self-recruitment and low rates of clonality in these MOTUs by suggesting that locally retained larvae originate predominantly from spawned gametes, while brooded larvae are mainly vagabonds., (© 2013 John Wiley & Sons Ltd.)

Background: Contemporary coral reef research has firmly established that a genomic approach is urgently needed to better understand the effects of anthropogenic environmental stress and global climate change on coral holobiont interactions. Here we present KEGG orthology-based annotation of the complete genome sequence of the scleractinian coral Acropora digitifera and provide the first comprehensive view of the genome of a reef-building coral by applying advanced bioinformatics., Description: Sequences from the KEGG database of protein function were used to construct hidden Markov models. These models were used to search the predicted proteome of A. digitifera to establish complete genomic annotation. The annotated dataset is published in ZoophyteBase, an open access format with different options for searching the data. A particularly useful feature is the ability to use a Google-like search engine that links query words to protein attributes. We present features of the annotation that underpin the molecular structure of key processes of coral physiology that include (1) regulatory proteins of symbiosis, (2) planula and early developmental proteins, (3) neural messengers, receptors and sensory proteins, (4) calcification and Ca2+-signalling proteins, (5) plant-derived proteins, (6) proteins of nitrogen metabolism, (7) DNA repair proteins, (8) stress response proteins, (9) antioxidant and redox-protective proteins, (10) proteins of cellular apoptosis, (11) microbial symbioses and pathogenicity proteins, (12) proteins of viral pathogenicity, (13) toxins and venom, (14) proteins of the chemical defensome and (15) coral epigenetics., Conclusions: We advocate that providing annotation in an open-access searchable database available to the public domain will give an unprecedented foundation to interrogate the fundamental molecular structure and interactions of coral symbiosis and allow critical questions to be addressed at the genomic level based on combined aspects of evolutionary, developmental, metabolic, and environmental perspectives.

The dinoflagellate photosymbiont Symbiodinium plays a fundamental role in defining the physiological tolerances of coral holobionts, but little is known about the dynamics of these endosymbiotic populations on coral reefs. Sparse data indicate that Symbiodinium populations show limited spatial connectivity; however, no studies have investigated temporal dynamics for in hospite Symbiodinium populations following significant mortality and recruitment events in coral populations. We investigated the combined influences of spatial isolation and disturbance on the population dynamics of the generalist Symbiodinium type C2 (ITS1 rDNA) hosted by the scleractinian coral Acropora millepora in the central Great Barrier Reef. Using eight microsatellite markers, we genotyped Symbiodinium in a total of 401 coral colonies, which were sampled from seven sites across a 12-year period including during flood plume-induced coral bleaching. Genetic differentiation of Symbiodinium was greatest within sites, explaining 70-86% of the total genetic variation. An additional 9-27% of variation was explained by significant differentiation of populations among sites separated by 0.4-13 km, which is consistent with low levels of dispersal via water movement and historical disturbance regimes. Sampling year accounted for 6-7% of total genetic variation and was related to significant coral mortality following severe bleaching in 1998 and a cyclone in 2006. Only 3% of the total genetic variation was related to coral bleaching status, reflecting generally small (8%) reductions in allelic diversity within bleached corals. This reduction probably reflected a loss of genotypes in hospite during bleaching, although no site-wide changes in genetic diversity were observed. Combined, our results indicate the importance of disturbance regimes acting together with limited oceanographic transport to determine the genetic composition of Symbiodinium types within reefs., (© 2013 John Wiley & Sons Ltd.)

Knowledge of the degree to which corals undergo physiological acclimatization or genetic adaptation in response to changes in their thermal environment is crucial to the success of coral reef conservation strategies. The potential of corals to acclimatize to temperatures exceeding historical thermal regimes was investigated by reciprocal transplantation of Acropora millepora colonies between the warm central and cool southern regions of the Great Barrier Reef (GBR) for a duration of 14 months. Colony fragments retained at native sites remained healthy, whereas transplanted fragments, although healthy over initial months when temperatures remained within native thermal regimes, subsequently bleached and suffered mortality during seasonal temperature extremes. Corals hosting Symbiodinium D transplanted to the southern GBR bleached in winter and the majority suffered whole (40%; n=20 colonies) or partial (50%) mortality at temperatures 1.1 degrees C below their 15-year native minimum. In contrast, corals hosting Symbiodinium C2 transplanted to the central GBR bleached in summer and suffered whole (50%; n=10 colonies) or partial (42%) mortality at temperatures 2.5 degrees C above their 15-year native maximum. During summer bleaching, the dominant Symbiodinium type changed from C2 to D within corals transplanted to the central GBR. Corals transplanted to the cooler, southern GBR grew 74-80% slower than corals at their native site, and only 50% of surviving colonies reproduced, at least partially because of cold water bleaching of transplants. Despite the absence of any visual signs of stress, corals transplanted to the warmer, central GBR grew 52-59% more slowly than corals at their native site before the summer bleaching (i.e., from autumn to spring). Allocation of energy to initial acclimatization or reproduction may explain this pattern, as the majority (65%) of transplants reproduced one month earlier than portions of the same colonies retained at the southern native site. All parameters investigated (bleaching, mortality, Symbiodinium type fidelity, reproductive timing) demonstrated strong interactions between genotype and environment, indicating that the acclimatization potential of A. millepora populations may be limited by adaptation of the holobiont to native thermal regimes.

Scleractinian corals occur in symbiosis with a range of organisms including the dinoflagellate alga, Symbiodinium, an association that is mutualistic. However, not all symbionts benefit the host. In particular, many organisms within the microbial mucus layer that covers the coral epithelium can cause disease and death. Other organisms in symbiosis with corals include the recently described Chromera velia, a photosynthetic relative of the apicomplexan parasites that shares a common ancestor with Symbiodinium. To explore the nature of the association between C. velia and corals we first isolated C. velia from the coral Montipora digitata and then exposed aposymbiotic Acropora digitifera and A. tenuis larvae to these cultures. Three C. velia cultures were isolated, and symbiosis was established in coral larvae of both these species exposed to all three clones. Histology verified that C. velia was located in the larval endoderm and ectoderm. These results indicate that C. velia has the potential to be endosymbiotic with coral larvae., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Background: Knowledge of genetic markers that are correlated to stress tolerance may improve spatial mapping of reef vulnerability and can inform restoration efforts, including the choice of genotypes for breeding and reseeding. In this manuscript we present two methods for screening transcriptome data for candidate genetic markers in two reef building corals, Acropora millepora and Pocillopora damicornis (types α and β). In A. millepora, Single Nucleotide Polymorphisms (SNPs) were pre-selected by targeting genes believed to be involved in the coral thermal stress responses. In P. damicornis (type α and β), SNPs showing varying allele frequencies between two populations from distinct environments were pre-selected. Allele frequencies at nine, five and eight of the pre-selected SNP loci were correlated against gradients of water clarity and temperature in a large number of populations along the Great Barrier Reef., Results: A significant correlation between environmental category and SNP allele frequency was detected in up to 55% of the tested loci, which is an exceptional success rate for these types of tests. In P. damicornis, SNP allele frequencies of β-hexosaminidase and Elongation factor 1-α were significantly correlated to temperature in type α and to temperature and/or water clarity respectively in type β. Type α also showed a correlation between water clarity and SNP allele frequency in a gene of unknown function. In A. millepora, allele frequencies at five (β-gamma crystallin, Galaxin, Ubiquitin, Ligand of Numb X2 and Thioredoxin) SNP loci showed significant correlations., Conclusions: After validation of these candidate loci through laboratory or field assessment of relative stress tolerance of colonies harbouring different alleles, it is anticipated that a proportion of these markers may represent the first coral candidate Quantitative Trait Loci for environmental stress tolerance and provide an important genetic tool that can be incorporated into spatial management decisions and restoration efforts of coral reefs. One pertinent example would be to combine spatial data of tolerant populations with genetic connectivity and thus identify high priority conservation reefs and implement targeted coral husbandry and active restoration efforts that use locally- and stress-adapted genotypes.

Pocillopora damicornis (Linnaeus, 1758; Scleractinia, Pocilloporidae) has recently been found to comprise at least five distinct genetic lineages in Eastern Australia, some of which likely represent cryptic species. Due to similar and plastic gross morphology of these lineages, field identification is often difficult. Here we present a quick, cost effective genetic assay as well as three novel microsatellite markers that distinguish the two most common lineages found on the Great Barrier Reef. The assay is based on PCR amplification of two regions within the mitochondrial putative control region, which show consistent and easily identifiable fragment size differences for the two genetic lineages after Alu1 restriction enzyme digestion of the amplicons.

To build new tools for the continued protection and propagation of coral from the Great Barrier Reef (GBR), an international group of coral and cryopreservation scientists known as the Reef Recovery Initiative joined forces during the November 2011 mass-spawning event. The outcome was the creation of the first frozen bank for Australian coral from two important GBR reef-building species, Acropora tenuis and Acropora millepora. Approximately 190 frozen samples each with billions of cells were placed into long-term storage. Sperm cells were successfully cryopreserved, and after thawing, samples were used to fertilize eggs, resulting in functioning larvae. Additionally, developing larvae were dissociated, and these pluripotent cells were cryopreserved and viable after thawing. Now, we are in a unique position to move our work from the laboratory to the reefs to develop collaborative, practical conservation management tools to help secure Australia's coral biodiversity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In sessile modular marine invertebrates, chimeras can originate from fusions of closely settling larvae or of colonies that come into contact through growth or movement. While it has been shown that juveniles of brooding corals fuse under experimental conditions, chimera formation in broadcast spawning corals, the most abundant group of reef corals, has not been examined. This study explores the capacity of the broadcast spawning coral Acropora millepora to form chimeras under experimental conditions and to persist as chimeras in the field. Under experimental conditions, 1.5-fold more larvae settled in aggregations than solitarily, and analyses of nine microsatellite loci revealed that 50 per cent of juveniles tested harboured different genotypes within the same colony. Significantly, some chimeric colonies persisted for 23 months post-settlement, when the study ended. Genotypes within persisting chimeric colonies all showed a high level of relatedness, whereas rejecting colonies displayed variable levels of relatedness. The nearly threefold greater sizes of chimeras compared with solitary juveniles, from settlement through to at least three months, suggest that chimerism is likely to be an important strategy for maximizing survival of vulnerable early life-history stages of corals, although longer-term studies are required to more fully explore the potential benefits of chimerism.

The coral holobiont is a complex assemblage of organisms spanning a diverse taxonomic range including a cnidarian host, as well as various dinoflagellate, prokaryotic and acellular symbionts. With the accumulating information on the molecular diversity of these groups, binomial species classification and a reassessment of species boundaries for the partners in the coral holobiont is a logical extension of this work and will help enhance the capacity for comparative research among studies. To aid in this endeavour, we review the current literature on species diversity for the three best studied partners of the coral holobiont (coral, Symbiodinium, prokaryotes) and provide suggestions for future work on systematics within these taxa. We advocate for an integrative approach to the delineation of species using both molecular genetics in combination with phenetic characters. We also suggest that an a priori set of criteria be developed for each taxonomic group as no one species concept or accompanying set of guidelines is appropriate for delineating all members of the coral holobiont., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

Background: Corals, like many other marine invertebrates, lack a mature allorecognition system in early life history stages. Indeed, in early ontogeny, when corals acquire and establish associations with various surface microbiota and dinoflagellate endosymbionts, they do not efficiently distinguish between closely and distantly related individuals from the same population. However, very little is known about the molecular components that underpin allorecognition and immunity responses or how they change through early ontogeny in corals., Methodology/principal Findings: Patterns in the expression of four putative immune response genes (apextrin, complement C3, and two CELIII type lectin genes) were examined in juvenile colonies of Acropora millepora throughout a six-month post-settlement period using quantitative real-time PCR (qPCR). Expression of a CELIII type lectin gene peaked in the fourth month for most of the coral juveniles sampled and was significantly higher at this time than at any other sampling time during the six months following settlement. The timing of this increase in expression levels of putative immune response genes may be linked to allorecognition maturation which occurs around this time in A. millepora. Alternatively, the increase may represent a response to immune challenges, such as would be involved in the recognition of symbionts (such as Symbiodinium spp. or bacteria) during winnowing processes as symbioses are fine-tuned., Conclusions/significance: Our data, although preliminary, are consistent with the hypothesis that lectins may play an important role in the maturation of allorecognition responses in corals. The co-expression of lectins with apextrin during development of coral juveniles also raises the possibility that these proteins, which are components of innate immunity in other invertebrates, may influence the innate immune systems of corals through a common pathway or system. However, further studies investigating the expression of these genes in alloimmune-challenged corals are needed to further clarify emerging evidence of a complex innate immunity system in corals.

Effective management of reef corals requires knowledge of the extent to which populations are open or closed and the scales over which genetic exchange occurs, information which is commonly derived from population genetic data. Such data are sparse for Great Barrier Reef (GBR) corals and other organisms, with the studies that are available being mostly based on a small number of sampling locations spanning only part of the GBR. Using 11 microsatellite loci, we genotyped 947 colonies of the reef-building coral Acropora millepora from 20 sites spanning almost the full length of the GBR (∼12° of latitude and ∼1550 km). The results show a major divide between the southernmost central to southern offshore populations and all other sampled populations. We interpret this divide as a signature of allopatric divergence in northern and southern refugia during the Pleistocene glaciations, from which the GBR was subsequently recolonized. Superimposed on this pattern is a cross-shelf genetic division, as well as a separation of inshore populations south of the Cape Clifton Front at ∼21.5-22°S. Most inshore populations north of this, as well as mid-shelf populations in the northern and far northern GBR, are open, exchanging recruits frequently. In contrast, inshore populations south of the Cape Clifton Front and offshore populations in the central and southern GBR are largely self-seeding, at least within the spatial resolution that was achieved given our sampling intensity. Populations that have been impacted by recent disturbance events causing extensive coral mortality show no evidence of reduced genetic diversity., (© 2011 Blackwell Publishing Ltd.)

Background: Divergent natural selection across environmental gradients has been acknowledged as a major driver of population and species divergence, however its role in the diversification of scleractinian corals remains poorly understood. Recently, it was demonstrated that the brooding coral Seriatopora hystrix and its algal endosymbionts (Symbiodinium) are genetically partitioned across reef environments (0-30 m) on the far northern Great Barrier Reef. Here, we explore the potential mechanisms underlying this differentiation and assess the stability of host-symbiont associations through a reciprocal transplantation experiment across habitats ('Back Reef', 'Upper Slope' and 'Deep Slope'), in combination with molecular (mtDNA and ITS2-DGGE) and photo-physiological analyses (respirometry and HPLC)., Results: The highest survival rates were observed for native transplants (measured 14 months after transplantation), indicating differential selective pressures between habitats. Host-symbiont assemblages remained stable during the experimental duration, demonstrating that the ability to "shuffle" or "switch" symbionts is restricted in S. hystrix. Photo-physiological differences were observed between transplants originating from the shallow and deep habitats, with indirect evidence of an increased heterotrophic capacity in native deep-water transplants (from the 'Deep Slope' habitat). Similar photo-acclimatisation potential was observed between transplants originating from the two shallow habitats ('Back Reef' and 'Upper Slope'), highlighting that their genetic segregation over depth may be due to other, non-photo-physiological traits under selection., Conclusions: This study confirms that the observed habitat partitioning of S. hystrix (and associated Symbiodinium) is reflective of adaptive divergence along a depth gradient. Gene flow appears to be reduced due to divergent selection, highlighting the potential role of ecological mechanisms, in addition to physical dispersal barriers, in the diversification of scleractinian corals and their associated Symbiodinium.

The photobiology of two reef corals and the distribution of associated symbiont types were investigated over a depth gradient of 0-60 m at Scott Reef, Western Australia. Pachyseris speciosa hosted mainly the same Symbiodinium C type similar to C3 irrespective of sampling depth. By contrast, Seriatopora hystrix hosted predominantly Symbiodinium type D1a or D1a-like at shallow depths while those in deeper water were dominated by a Symbiodinium C type closely related to C1. The photosynthesis/respiration (P/R) ratio increased consistently with depth at the two sampling times (November 2008 and April 2009) for P. speciosa and in November 2008 only for S. hystrix, suggesting a reduction in metabolic energy expended for every unit of energy obtained from photosynthesis. However, in April 2009, shallow colonies of S. hystrix exhibited decreased P/R ratios down to depths of approximately 23 m, below which the ratio increased towards the maximum depth sampled. This pattern was mirrored by changes in tissue biomass determined as total protein content. The depth of change in the direction of the P/R ratio correlated with a shift from Symbiodinium D to C-dominated colonies. We conclude that while photobiological flexibility is vital for persistence in contrasting light regimes, a shift in Symbiodinium type may also confer a functional advantage albeit at a metabolic cost with increased depth.

Approximately one quarter of zooxanthellate coral species have a depth distribution from shallow waters (<30 m) down to mesophotic depths of 30-60 m. The deeper populations of such species are less likely to be affected by certain environmental perturbations, including high temperature/high irradiance causing coral bleaching. This has led to the hypothesis that deep populations may serve as refuges and a source of recruits for shallow reef habitats. The extent of vertical connectivity of reef coral species, however, is largely unquantified. Using 10 coral host microsatellite loci and sequences of the host mtDNA putative control region, as well as ribosomal DNA (rDNA) ITS2 sequences of the coral's algal endosymbionts (Symbiodinium), we examine population structure, connectivity and symbiont specificity in the brooding coral Seriatopora hystrix across a depth profile in both northwest (Scott Reef) and northeast Australia (Yonge Reef). Strong genetic structuring over depth was observed in both regions based on the microsatellite loci; however, Yonge Reef exhibited an additional partitioning of mtDNA lineages (associated with specific symbiont ITS2 types), whereas Scott Reef was dominated by a single mtDNA lineage (with no apparent host-symbiont specificity). Evidence for recruitment of larvae of deep water origin into shallow habitats was found at Scott Reef, suggesting that recovery of shallow water habitats may be aided by migration from deep water refuges. Conversely, no migration from the genetically divergent deep slope populations into the shallow habitats was evident at Yonge Reef, making recovery of shallow habitats from deeper waters at this location highly unlikely., (© 2011 Blackwell Publishing Ltd.)

We report an accurate multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay, capable of reproducing gene expression profiles from 16 target genes [12 genes of interest (GOIs) and four reference genes (RGs)] in Acropora millepora, a common reef-building model coral species. The 12 GOIs have known or putative roles in the coral bleaching response, yet the method is not restricted to this particular assay and gene set. The procedure is based on the Beckman Coulter (Fullerton, CA, USA) GenomeLab™ GeXP Genetic Analysis System and bridges the gap between quantitative real-time PCR (qPCR) expression analysis of a single or a small number of genes and microarray gene expression surveys of thousands of genes. Despite large variation among biological replicates, the majority of GOIs were up-regulated (up to 4000%) in most colonies during a laboratory-based thermal stress experiment. Two genes, Nf-kβ2 and MnSod, were consistently up-regulated in all colonies tested, and we therefore propose these as candidate markers useful for population-level evaluations of thermal stress. Our assay provides an important new tool for coral bleaching studies; because of the lower cost, labour and amount of cDNA required compared with singleplex qPCR, population-level studies with large biological replication are feasible., (© 2010 Blackwell Publishing Ltd.)

Background: The Symbiodinium community associated with scleractinian corals is widely considered to be shaped by seawater temperature, as the coral's upper temperature tolerance is largely contingent on the Symbiodinium types harboured. Few studies have challenged this paradigm as knowledge of other environmental drivers on the distribution of Symbiodinium is limited. Here, we examine the influence of a range of environmental variables on the distribution of Symbiodinium associated with Acropora millepora collected from 47 coral reefs spanning 1,400 km on the Great Barrier Reef (GBR), Australia., Methodology/principal Findings: The environmental data included Moderate Resolution Imaging Spectroradiometer (MODIS) satellite data at 1 km spatial resolution from which a number of sea surface temperature (SST) and water quality metrics were derived. In addition, the carbonate and mud composition of sediments were incorporated into the analysis along with in situ water quality samples for a subset of locations. Analyses were conducted at three spatio-temporal scales [GBR (regional-scale), Whitsunday Islands (local-scale) and Keppel Islands/Trunk Reef (temporal)] to examine the effects of scale on the distribution patterns. While SST metrics were important drivers of the distribution of Symbiodinium types at regional and temporal scales, our results demonstrate that spatial variability in water quality correlates significantly with Symbiodinium distribution at local scales. Background levels of Symbiodinium types were greatest at turbid inshore locations of the Whitsunday Islands where SST predictors were not as important. This was not the case at regional scales where combinations of mud and carbonate sediment content coupled with SST anomalies and mean summer SST explained 51.3% of the variation in dominant Symbiodinium communities., Conclusions/significance: Reef corals may respond to global-scale stressors such as climate change through changes in their resident symbiont communities, however, management of local-scale stressors such as altered water quality is also necessary for maintenance of coral-Symbiodinium associations.

Background: Lipids in reef building corals can be divided into two classes; non-polar storage lipids, e.g. wax esters and triglycerides, and polar structural lipids, e.g. phospholipids and cholesterol. Differences among algal endosymbiont types are known to have important influences on processes including growth and the photobiology of scleractinian corals yet very little is known about the role of symbiont types on lipid energy reserves., Methodology/principal Findings: The ratio of storage lipid and structural lipid fractions of Scott Reef corals were determined by thin layer chromatography. The lipid fraction ratio varied with depth and depended on symbiont type harboured by two corals (Seriatopora hystrix and Pachyseris speciosa). S. hystrix colonies associated with Symbiodinium C1 or C1/C# at deep depths (>23 m) had lower lipid fraction ratios (i.e. approximately equal parts of storage and structural lipids) than those with Symbiodinium D1 in shallow depths (<23 m), which had higher lipid fraction ratios (i.e. approximately double amounts of storage relative to structural lipid). Further, there was a non-linear relationship between the lipid fraction ratio and depth for S. hystrix with a modal peak at ∼23 m coinciding with the same depth as the shift from clade D to C types. In contrast, the proportional relationship between the lipid fraction ratio and depth for P. speciosa, which exhibited high specificity for Symbiodinium C3 like across the depth gradient, was indicative of greater amounts of storage lipids contained in the deep colonies., Conclusions/significance: This study has demonstrated that Symbiodinium exert significant controls over the quality of coral energy reserves over a large-scale depth gradient. We conclude that the competitive advantages and metabolic costs that arise from flexible associations with divergent symbiont types are offset by energetic trade-offs for the coral host.

Coral bleaching is a major threat to coral reefs worldwide and is predicted to intensify with increasing global temperature. This study represents the first investigation of gene expression in an Indo-Pacific coral species undergoing natural bleaching which involved the loss of algal symbionts. Quantitative real-time polymerase chain reaction experiments were conducted to select and evaluate coral internal control genes (ICGs), and to investigate selected coral genes of interest (GOIs) for changes in gene expression in nine colonies of the scleractinian coral Acropora millepora undergoing bleaching at Magnetic Island, Great Barrier Reef, Australia. Among the six ICGs tested, glyceraldehyde 3-phosphate dehydrogenase and the ribosomal protein genes S7 and L9 exhibited the most constant expression levels between samples from healthy-looking colonies and samples from the same colonies when severely bleached a year later. These ICGs were therefore utilised for normalisation of expression data for seven selected GOIs. Of the seven GOIs, homologues of catalase, C-type lectin and chromoprotein genes were significantly up-regulated as a result of bleaching by factors of 1.81, 1.46 and 1.61 (linear mixed models analysis of variance, P < 0.05), respectively. We present these genes as potential coral bleaching response genes. In contrast, three genes, including one putative ICG, showed highly variable levels of expression between coral colonies. Potential variation in microhabitat, gene function unrelated to the stress response and individualised stress responses may influence such differences between colonies and need to be better understood when designing and interpreting future studies of gene expression in natural coral populations.

Background: Coral reefs are hotspots of biodiversity, yet processes of diversification in these ecosystems are poorly understood. The environmental heterogeneity of coral reef environments could be an important contributor to diversification, however, evidence supporting ecological speciation in corals is sparse. Here, we present data from a widespread coral species that reveals a strong association of host and symbiont lineages with specific habitats, consistent with distinct, sympatric gene pools that are maintained through ecologically-based selection., Methodology/principal Findings: Populations of a common brooding coral, Seriatopora hystrix, were sampled from three adjacent reef habitats (spanning a approximately 30 m depth range) at three locations on the Great Barrier Reef (n = 336). The populations were assessed for genetic structure using a combination of mitochondrial (putative control region) and nuclear (three microsatellites) markers for the coral host, and the ITS2 region of the ribosomal DNA for the algal symbionts (Symbiodinium). Our results show concordant genetic partitioning of both the coral host and its symbionts across the different habitats, independent of sampling location., Conclusions/significance: This study demonstrates that coral populations and their associated symbionts can be highly structured across habitats on a single reef. Coral populations from adjacent habitats were found to be genetically isolated from each other, whereas genetic similarity was maintained across similar habitat types at different locations. The most parsimonious explanation for the observed genetic partitioning across habitats is that adaptation to the local environment has caused ecological divergence of distinct genetic groups within S. hystrix.

The persistence of tropical coral reefs is threatened by rapidly increasing climate warming, causing a functional breakdown of the obligate symbiosis between corals and their algal photosymbionts (Symbiodinium) through a process known as coral bleaching. Yet the potential of the coral-algal symbiosis to genetically adapt in an evolutionary sense to warming oceans is unknown. Using a quantitative genetics approach, we estimated the proportion of the variance in thermal tolerance traits that has a genetic basis (i.e. heritability) as a proxy for their adaptive potential in the widespread Indo-Pacific reef-building coral Acropora millepora. We chose two physiologically different populations that associate respectively with one thermo-tolerant (Symbiodinium clade D) and one less tolerant symbiont type (Symbiodinium C2). In both symbiont types, pulse amplitude modulated (PAM) fluorometry and high performance liquid chromatography (HPLC) analysis revealed significant heritabilities for traits related to both photosynthesis and photoprotective pigment profile. However, quantitative real-time polymerase chain reaction (qRT-PCR) assays showed a lack of heritability in both coral host populations for their own expression of fundamental stress genes. Coral colony growth, contributed to by both symbiotic partners, displayed heritability. High heritabilities for functional key traits of algal symbionts, along with their short clonal generation time and high population sizes allow for their rapid thermal adaptation. However, the low overall heritability of coral host traits, along with the corals' long generation time, raise concern about the timely adaptation of the coral-algal symbiosis in the face of continued rapid climate warming.