A Phylogeny-Informed Analysis of the Global Coral-Symbiodiniaceae Interaction Network Reveals that Traits Correlated with Thermal Bleaching Are Specific to Symbiont Transmission Mode.

The complex network of associations between corals and their dinoflagellates (family Symbiodiniaceae) are the basis of coral reef ecosystems but are sensitive to increasing global temperatures. Coral-symbiont interactions are restricted by ecological and evolutionary determinants that constrain partner choice and influence holobiont response to environmental stress; however, little is known about how these processes shape thermal resilience of the holobiont. Here, we built a network of global coral-Symbiodiniaceae associations, mapped species traits (e.g., symbiont transmission mode and biogeography) and phylogenetic relationships of both partners onto the network, and assigned thermotolerance to both host and symbiont nodes. Using network analysis and phylogenetic comparative methods, we determined the contribution of species traits to thermal resilience of the holobiont, while accounting for evolutionary patterns among species. We found that the network shows nonrandom interactions among species, which are shaped by evolutionary history, symbiont transmission mode (horizontally transmitted [HT] or vertically transmitted [VT] corals) and biogeography. Coral phylogeny, but not Symbiodiniaceae phylogeny, symbiont transmission mode, or biogeography, was a good predictor of thermal resilience. Closely related corals have similar Symbiodiniaceae interaction patterns and bleaching susceptibilities. Nevertheless, the association patterns that explain increased host thermal resilience are not generalizable across the entire network but are instead unique to HT and VT corals. Under nonstress conditions, thermally resilient VT coral species associate with thermotolerant phylotypes and limit their number of unique symbionts and overall symbiont thermotolerance diversity, while thermally resilient HT coral species associate with a few host-specific symbiont phylotypes.IMPORTANCE Recent advances have revealed a complex network of interactions between coral and Symbiodiniaceae. Specifically, nonrandom association patterns, which are determined in part by restrictions imposed by symbiont transmission mode, increase the sensitivity of the overall network to thermal stress. However, little is known about the extent to which coral-Symbiodiniaceae network resistance to thermal stress is shaped by host and symbiont species phylogenetic relationships and host and symbiont species traits, such as symbiont transmission mode. We built a frequency-weighted global coral-Symbiodiniaceae network and used network analysis and phylogenetic comparative methods to show that evolutionary relatedness, but not transmission mode, predicts thermal resilience of the coral-Symbiodiniaceae holobiont. Consequently, thermal stress events could result in nonrandom pruning of susceptible lineages and loss of taxonomic diversity with catastrophic effects on community resilience to future events. Our results show that inclusion of the contribution of evolutionary and ecological processes will further our understanding of the fate of coral assemblages under climate change.

Uncovering the role of Symbiodiniaceae assemblage composition and abundance in coral bleaching response by minimizing sampling and evolutionary biases.

BACKGROUND: Biodiversity and productivity of coral-reef ecosystems depend upon reef-building corals and their associations with endosymbiotic Symbiodiniaceae, which offer diverse functional capabilities to their hosts. The number of unique symbiotic partners (richness) and relative abundances (evenness) have been hypothesized to affect host response to climate change induced thermal stress. Symbiodiniaceae assemblages with many unique phylotypes may provide greater physiological flexibility or form less stable symbioses; assemblages with low abundance phylotypes may allow corals to retain thermotolerant symbionts or represent associations with less-suitable symbionts. RESULTS: Here we demonstrate that true richness of Symbiodiniaceae phylotype assemblages is generally not discoverable from direct enumeration of unique phylotypes in association records and that cross host-species comparisons are biased by sampling and evolutionary patterns among species. These biases can be minimized through rarefaction of richness (rarefied-richness) and evenness (Probability of Interspecific Encounter, PIE), and analyses that account for phylogenetic patterns. These standardized metrics were calculated for individual Symbiodiniaceae assemblages composed of 377 unique ITS2 phylotypes associated with 123 coral species. Rarefied-richness minimized correlations with sampling effort, while maintaining important underlying characteristics across host bathymetry and geography. Phylogenetic comparative methods reveal significant increases in coral bleaching and mortality associated with increasing Symbiodiniaceae assemblage richness and evenness at the level of host species. CONCLUSIONS: These results indicate that the potential flexibility afforded by assemblages characterized by many phylotypes present at similar relative abundances does not result in decreased bleaching risk and point to the need to characterize the overall functional and genetic diversity of Symbiodiniaceae assemblages to quantify their effect on host fitness under climate change.

Bleaching response of coral species in the context of assemblage response.

Caribbean coral reefs are declining due to a mosaic of local and global stresses, including climate change-induced thermal stress. Species and assemblage responses differ due to factors that are not easily identifiable or quantifiable. We calculated a novel species-specific metric of coral bleaching response, taxon-α and -ß, which relates the response of a species to that of its assemblages for 16 species over 18 assemblages. By contextualizing species responses within the response of their assemblages, the effects of environmental factors are removed and intrinsic differences among taxa are revealed. Most corals experience either a saturation response, overly-sensitive to weak stress (α > 0) but under-responsive compared to assemblage bleaching (ß < 1), or a threshold response, insensitive to weak stress (α < 0) but over-responsive compared to assemblage bleaching (ß > 1). This metric may help reveal key factors of bleaching susceptibility and identify species as targets for conservation.

Skeletal light-scattering accelerates bleaching response in reef-building corals.

BACKGROUND: At the forefront of ecosystems adversely affected by climate change, coral reefs are sensitive to anomalously high temperatures which disassociate (bleaching) photosynthetic symbionts (Symbiodinium) from coral hosts and cause increasingly frequent and severe mass mortality events. Susceptibility to bleaching and mortality is variable among corals, and is determined by unknown proportions of environmental history and the synergy of Symbiodinium- and coral-specific properties. Symbiodinium live within host tissues overlaying the coral skeleton, which increases light availability through multiple light-scattering, forming one of the most efficient biological collectors of solar radiation. Light-transport in the upper ~200 µm layer of corals skeletons (measured as 'microscopic' reduced-scattering coefficient, µ'(S,m)), has been identified as a determinant of excess light increase during bleaching and is therefore a potential determinant of the differential rate and severity of bleaching response among coral species. RESULTS: Here we experimentally demonstrate (in ten coral species) that, under thermal stress alone or combined thermal and light stress, low-µ'(S,m) corals bleach at higher rate and severity than high-µ'(S,m) corals and the Symbiodinium associated with low-µ'(S,m) corals experience twice the decrease in photochemical efficiency. We further modelled the light absorbed by Symbiodinium due to skeletal-scattering and show that the estimated skeleton-dependent light absorbed by Symbiodinium (per unit of photosynthetic pigment) and the temporal rate of increase in absorbed light during bleaching are several fold higher in low-µ'(S,m) corals. CONCLUSIONS: While symbionts associated with low-[Formula: see text] corals receive less total light from the skeleton, they experience a higher rate of light increase once bleaching is initiated and absorbing bodies are lost; further precipitating the bleaching response. Because microscopic skeletal light-scattering is a robust predictor of light-dependent bleaching among the corals assessed here, this work establishes µ'(S,m) as one of the key determinants of differential bleaching response.

Modulation of light-enhancement to symbiotic algae by light-scattering in corals and evolutionary trends in bleaching.

Calcium carbonate skeletons of scleractinian corals amplify light availability to their algal symbionts by diffuse scattering, optimizing photosynthetic energy acquisition. However, the mechanism of scattering and its role in coral evolution and dissolution of algal symbioses during "bleaching" events are largely unknown. Here we show that differences in skeletal fractal architecture at nano/micro-lengthscales within 96 coral taxa result in an 8-fold variation in light-scattering and considerably alter the algal light environment. We identified a continuum of properties that fall between two extremes: (1) corals with low skeletal fractality that are efficient at transporting and redistributing light throughout the colony with low scatter but are at higher risk of bleaching and (2) corals with high skeletal fractality that are inefficient at transporting and redistributing light with high scatter and are at lower risk of bleaching. While levels of excess light derived from the coral skeleton is similar in both groups, the low-scatter corals have a higher rate of light-amplification increase when symbiont concentration is reduced during bleaching, thus creating a positive feedback-loop between symbiont concentration and light-amplification that exposes the remaining symbionts to increasingly higher light intensities. By placing our findings in an evolutionary framework, in conjunction with a novel empirical index of coral bleaching susceptibility, we find significant correlations between bleaching susceptibility and light-scattering despite rich homoplasy in both characters; suggesting that the cost of enhancing light-amplification to the algae is revealed in decreased resilience of the partnership to stress.

Fetal-juvenile origins of point mutations in the adult human tracheal-bronchial epithelium: absence of detectable effects of age, gender or smoking status.

Allele-specific mismatch amplification mutation assays (MAMA) of anatomically distinct sectors of the upper bronchial tracts of nine nonsmokers revealed many numerically dispersed clusters of the point mutations C742T, G746T, G747T of the TP53 gene, G35T of the KRAS gene and G508A of the HPRT1 gene. Assays of these five mutations in six smokers have yielded quantitatively similar results. One hundred and eighty four micro-anatomical sectors of 0.5-6x10(6) tracheal-bronchial epithelial cells represented en toto the equivalent of approximately 1.7 human smokers' bronchial trees to the fifth bifurcation. Statistically significant mutant copy numbers above the 95% upper confidence limits of historical background controls were found in 198 of 425 sector assays. No significant differences (P=0.1) for negative sector fractions, mutant fractions, distributions of mutant cluster size or anatomical positions were observed for smoking status, gender or age (38-76 year). Based on the modal cluster size of mitochondrial point mutants, the size of the adult bronchial epithelial maintenance turnover unit was estimated to be about 32 cells. When data from all 15 lungs were combined the log2 of nuclear mutant cluster size plotted against log2 of the number of clusters of a given cluster size displayed a slope of approximately 1.1 over a range of cluster sizes from approximately 2(6) to 2(15) mutant copies. A parsimonious interpretation of these nuclear and previously reported data for lung epithelial mitochondrial point mutant clusters is that they arose from mutations in stem cells at a high but constant rate per stem cell doubling during at least ten stem cell doublings of the later fetal-juvenile period. The upper and lower decile range of summed point mutant fractions among lungs was about 7.5-fold, suggesting an important source of stratification in the population with regard to risk of tumor initiation.

Accurately quantifying low-abundant targets amid similar sequences by revealing hidden correlations in oligonucleotide microarray data.

Microarrays have enabled the determination of how thousands of genes are expressed to coordinate function within single organisms. Yet applications to natural or engineered communities where different organisms interact to produce complex properties are hampered by theoretical and technological limitations. Here we describe a general method to accurately identify low-abundant targets in systems containing complex mixtures of homologous targets. We combined an analytical predictor of nonspecific probe-target interactions (cross-hybridization) with an optimization algorithm that iteratively deconvolutes true probe-target signal from raw signal affected by spurious contributions (cross-hybridization, noise, background, and unequal specific hybridization response). The method was capable of quantifying, with unprecedented specificity and accuracy, ribosomal RNA (rRNA) sequences in artificial and natural communities. Controlled experiments with spiked rRNA into artificial and natural communities demonstrated the accuracy of identification and quantitative behavior over different concentration ranges. Finally, we illustrated the power of this methodology for accurate detection of low-abundant targets in natural communities. We accurately identified Vibrio taxa in coastal marine samples at their natural concentrations (<0.05% of total bacteria), despite the high potential for cross-hybridization by hundreds of different coexisting rRNAs, suggesting this methodology should be expandable to any microarray platform and system requiring accurate identification of low-abundant targets amid pools of similar sequences.

Distributions of five common point mutants in the human tracheal-bronchial epithelium.

The mutations C742T, G746T, G747T in the TP53 gene and G35T in the KRAS gene have been repeatedly found in sectors of human tumors by direct DNA sequencing. The mutation G508A in the HPRT1 gene has been repeatedly found among peripheral T lymphocytes by clonal expansion under selective conditions. To discover if these mutations also occur frequently in normal tissues from which tumors arise, we have developed and validated allele-specific mismatch amplification mutation assays (MAMA) for each mutation. Reconstruction experiments demonstrated linearity in the range of 9-3000 mutant alleles among 3 x 10(6) wild-type alleles. The cumulative distributions of all negative controls established robust detection limits (P<0.05) of 34-125 mutants per 10(6) copies assayed depending on the mutation. One hundred and seventy-seven micro-anatomical samples of approximately (0.5-6)x10(6) tracheal-bronchial epithelial cells from nine non-smokers were assayed representing en toto the equivalent of approximately 1.6 human bronchial trees to the fifth bifurcation. Statistically significant mutant copy numbers were found in 257 of 463 assays. Clusters of mutant copies ranged from 10 to 1000 in 239/257 positive samples. As all five point mutations were detected at mutant fractions of >10(-5) in two or more lungs, we infer that they are mutational hotspots generated in lung epithelial stem cells. As the cancer-associated mutations did not differ in cluster size distribution from the HPRT1 mutation, we infer that none of the mutations conferred a growth advantage to somatic heterozygous clusters or maintenance turnover units. Specific mutants appeared in very large copy numbers, 1000-35,000, in 18/257 positive assays. Various hypotheses to account for the observed cluster size distributions are offered.

Diversity and dynamics of a north atlantic coastal Vibrio community.

Vibrios are ubiquitous marine bacteria that have long served as models for heterotrophic processes and have received renewed attention because of the discovery of increasing numbers of facultatively pathogenic strains. Because the occurrence of specific vibrios has frequently been linked to the temperature, salinity, and nutrient status of water, we hypothesized that seasonal changes in coastal water bodies lead to distinct vibrio communities and sought to characterize their level of differentiation. A novel technique was used to quantify shifts in 16S rRNA gene abundance in samples from Barnegat Bay, N.J., collected over a 15-month period. Quantitative PCR (QPCR) with primers specific for the genus Vibrio was combined with separation and quantification of amplicons by constant denaturant capillary electrophoresis (CDCE). Vibrio populations identified by QPCR-CDCE varied between summer and winter samples, suggesting distinct warm-water and year-round populations. Identification of the CDCE populations by cloning and sequencing of 16S rRNA genes from two summer and two winter samples confirmed this distinction. It further showed that CDCE populations corresponded in most cases to approximately 98% rRNA similarity groups and suggested that the abundance of these follows temperature trends. Phylogenetic comparison yielded closely related cultured and often pathogenic representatives for most sequences, and the temperature ranges of these isolates confirmed the trends seen in the environmental samples. Overall, this suggests that temperature is a good predictor of the occurrence of closely related vibrios but that considerable microdiversity of unknown significance coexists within this trend.

Divergence and redundancy of 16S rRNA sequences in genomes with multiple rrn operons.

The level of sequence heterogeneity among rrn operons within genomes determines the accuracy of diversity estimation by 16S rRNA-based methods. Furthermore, the occurrence of widespread horizontal gene transfer (HGT) between distantly related rrn operons casts doubt on reconstructions of phylogenetic relationships. For this study, patterns of distribution of rrn copy numbers, interoperonic divergence, and redundancy of 16S rRNA sequences were evaluated. Bacterial genomes display up to 15 operons and operon numbers up to 7 are commonly found, but approximately 40% of the organisms analyzed have either one or two operons. Among the Archaea, a single operon appears to dominate and the highest number of operons is five. About 40% of sequences among 380 operons in 76 bacterial genomes with multiple operons were identical to at least one other 16S rRNA sequence in the same genome, and in 38% of the genomes all 16S rRNAs were invariant. For Archaea, the number of identical operons was only 25%, but only five genomes with 21 operons are currently available. These considerations suggest an upper bound of roughly threefold overestimation of bacterial diversity resulting from cloning and sequencing of 16S rRNA genes from the environment; however, the inclusion of genomes with a single rrn operon may lower this correction factor to approximately 2.5. Divergence among operons appears to be small overall for both Bacteria and Archaea, with the vast majority of 16S rRNA sequences showing <1% nucleotide differences. Only five genomes with operons with a higher level of nucleotide divergence were detected, and Thermoanaerobacter tengcongensis exhibited the highest level of divergence (11.6%) noted to date. Overall, four of the five extreme cases of operon differences occurred among thermophilic bacteria, suggesting a much higher incidence of HGT in these bacteria than in other groups.

Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by 'reconditioning PCR'.

Although it has been recognized that PCR amplification of mixed templates may generate sequence artifacts, the mechanisms of their formation, frequency and potential elimination have not been fully elucidated. Here evidence is presented for heteroduplexes as a major source of artifacts in mixed-template PCR. Nearly equal proportions of homoduplexes and heteroduplexes were observed after co-amplifying 16S rDNA from three bacterial genomes and analyzing products by constant denaturing capillary electrophoresis (CDCE). Heteroduplexes became increasingly prevalent as primers became limiting and/or template diversity was increased. A model exploring the fate of cloned heteroduplexes during MutHLS-mediated mismatch repair in the Escherichia coli host demonstrates that the diversity of artifactual sequences increases exponentially with the number of both variable nucleotides and of original sequence variants. Our model illustrates how minimization of heteroduplex molecules before cloning may reduce artificial genetic diversity detected during sequence analysis by clone screening. Thus, we developed a method to eliminate heteroduplexes from mixed-template PCR products by subjecting them to 'reconditioning PCR', a low cycle number re-amplification of a 10-fold diluted mixed-template PCR product. This simple modification to the protocol may ensure that sequence richness encountered in clone libraries more closely reflects genetic diversity in the original sample.