Symbiodinium population genetics: testing for species boundaries and analysing samples with mixed genotypes

Population genetic markers are increasingly being used to study the diversity, ecology and evolution of Symbiodinium, a group of eukaryotic microbes that are often mutualistic with reef‐building corals. Population genetic markers can resolve individual clones, or strains, from samples of host tissue; however, samples may comprise different species that may confound interpretations of gene flow and genetic structure. Here, we propose a method for resolving species from population genetic data using tests for genetic recombination. Assigning individuals to genetically recombining populations prior to further analyses avoids critical errors in the interpretation of gene flow and dispersal. To demonstrate the effectiveness of the approach, we first apply this method to a simulated data set. We then use the method to resolve two species of host generalist Symbiodinium that commonly co‐occur in reef‐building corals collected from Indo‐West Pacific reefs. We demonstrate that the method is robust even when some hosts contain genotypes from two distinct species. Finally, we examine population genetic data sets from two recently published papers in Molecular Ecology. We show that each strongly supports a two species interpretation, which significantly changes the original conclusions presented in these studies. When combined with available phylogenetic and ecological evidence, the use of population genetic data offers a robust method for unambiguously delimiting morphologically cryptic species.     

Restricted gene flow and local adaptation highlight the vulnerability of high‐latitude reefs to rapid environmental change

Global climate change poses a serious threat to the future health of coral reef ecosystems. This calls for management strategies that are focused on maximizing the evolutionary potential of coral reefs. Fundamental to this is an accurate understanding of the spatial genetic structure in dominant reef‐building coral species. In this study, we apply a genotyping‐by‐sequencing approach to investigate genome‐wide patterns of genetic diversity, gene flow, and local adaptation in a reef‐building coral, Pocillopora damicornis, across 10 degrees of latitude and a transition from temperate to tropical waters. We identified strong patterns of differentiation and reduced genetic diversity in high‐latitude populations. In addition, genome‐wide scans for selection identified a number of outlier loci putatively under directional selection with homology to proteins previously known to be involved in heat tolerance in corals and associated with processes such as photoprotection, protein degradation, and immunity. This study provides genomic evidence for both restricted gene flow and local adaptation in a widely distributed coral species, and highlights the potential vulnerability of leading‐edge populations to rapid environmental change as they are locally adapted, reproductively isolated, and have reduced levels of genetic diversity.     

Mitochondrial and nuclear genes suggest that stony corals are monophyletic but most families of stony corals are not (Order Scleractinia, Class Anthozoa, Phylum Cnidaria)

Modern hard corals (Class Hexacorallia; Order Scleractinia) are widely studied because of their fundamental role in reef building and their superb fossil record extending back to the Triassic. Nevertheless, interpretations of their evolutionary relationships have been in flux for over a decade. Recent analyses undermine the legitimacy of traditional suborders, families and genera, and suggest that a non-skeletal sister clade (Order Corallimorpharia) might be imbedded within the stony corals. However, these studies either sampled a relatively limited array of taxa or assembled trees from heterogeneous data sets. Here we provide a more comprehensive analysis of Scleractinia (127 species, 75 genera, 17 families) and various outgroups, based on two mitochondrial genes (cytochrome oxidase I, cytochrome b), with analyses of nuclear genes (ss-tubulin, ribosomal DNA) of a subset of taxa to test unexpected relationships. Eleven of 16 families were found to be polyphyletic. Strikingly, over one third of all families as conventionally defined contain representatives from the highly divergent "robust" and "complex" clades. However, the recent suggestion that corallimorpharians are true corals that have lost their skeletons was not upheld. Relationships were supported not only by mitochondrial and nuclear genes, but also often by morphological characters which had been ignored or never noted previously. The concordance of molecular characters and more carefully examined morphological characters suggests a future of greater taxonomic stability, as well as the potential to trace the evolutionary history of this ecologically important group using fossils.     

Positive genetic associations among fitness traits support evolvability of a reef‐building coral under multiple stressors

Climate change threatens organisms in a variety of interactive ways that requires simultaneous adaptation of multiple traits. Predicting evolutionary responses requires an understanding of the potential for interactions among stressors and the genetic variance and covariance among fitness‐related traits that may reinforce or constrain an adaptive response. Here we investigate the capacity of Acropora millepora, a reef‐building coral, to adapt to multiple environmental stressors: rising sea surface temperature, ocean acidification, and increased prevalence of infectious diseases. We measured growth rates (weight gain), coral color (a proxy for Symbiodiniaceae density), and survival, in addition to nine physiological indicators of coral and algal health in 40 coral genets exposed to each of these three stressors singly and combined. Individual stressors resulted in predicted responses (e.g., corals developed lesions after bacterial challenge and bleached under thermal stress). However, corals did not suffer substantially more when all three stressors were combined. Nor were trade‐offs observed between tolerances to different stressors; instead, individuals performing well under one stressor also tended to perform well under every other stressor. An analysis of genetic correlations between traits revealed positive covariances, suggesting that selection to multiple stressors will reinforce rather than constrain the simultaneous evolution of traits related to holobiont health (e.g., weight gain and algal density). These findings support the potential for rapid coral adaptation under climate change and emphasize the importance of accounting for corals’ adaptive capacity when predicting the future of coral reefs.     

New insights into the dynamics between reef corals and their associated dinoflagellate endosymbionts from population genetic studies

The mutualistic symbioses between reef‐building corals and micro‐algae form the basis of coral reef ecosystems, yet recent environmental changes threaten their survival. Diversity in host‐symbiont pairings on the sub‐species level could be an unrecognized source of functional variation in response to stress. The Caribbean elkhorn coral, Acropora palmata, associates predominantly with one symbiont species (Symbiodinium ‘fitti’), facilitating investigations of individual‐level (genotype) interactions. Individual genotypes of both host and symbiont were resolved across the entire species’ range. Most colonies of a particular animal genotype were dominated by one symbiont genotype (or strain) that may persist in the host for decades or more. While Symbiodinium are primarily clonal, the occurrence of recombinant genotypes indicates sexual recombination is the source of this genetic variation, and some evidence suggests this happens within the host. When these data are examined at spatial scales spanning the entire distribution of A. palmata, gene flow among animal populations was an order of magnitude greater than among populations of the symbiont. This suggests that independent micro‐evolutionary processes created dissimilar population genetic structures between host and symbiont. The lower effective dispersal exhibited by the dinoflagellate raises questions regarding the extent to which populations of host and symbiont can co‐evolve during times of rapid and substantial climate change. However, these findings also support a growing body of evidence, suggesting that genotype‐by‐genotype interactions may provide significant physiological variation, influencing the adaptive potential of symbiotic reef corals to severe selection.     

The “Naked Coral” Hypothesis Revisited – Evidence for and Against Scleractinian Monophyly

The relationship between Scleractinia and Corallimorpharia, Orders within Anthozoa distinguished by the presence of an aragonite skeleton in the former, is controversial. Although classically considered distinct groups, some phylogenetic analyses have placed the Corallimorpharia within a larger Scleractinia/Corallimorpharia clade, leading to the suggestion that the Corallimorpharia are “naked corals” that arose via skeleton loss during the Cretaceous from a Scleractinian ancestor. Scleractinian paraphyly is, however, contradicted by a number of recent phylogenetic studies based on mt nucleotide (nt) sequence data. Whereas the “naked coral” hypothesis was based on analysis of the sequences of proteins encoded by a relatively small number of mt genomes, here a much-expanded dataset was used to reinvestigate hexacorallian phylogeny. The initial observation was that, whereas analyses based on nt data support scleractinian monophyly, those based on amino acid (aa) data support the “naked coral” hypothesis, irrespective of the method and with very strong support. To better understand the bases of these contrasting results, the effects of systematic errors were examined. Compared to other hexacorallians, the mt genomes of “Robust” corals have a higher (A+T) content, codon usage is far more constrained, and the proteins that they encode have a markedly higher phenylalanine content, leading us to suggest that mt DNA repair may be impaired in this lineage. Thus the “naked coral” topology could be caused by high levels of saturation in these mitochondrial sequences, long-branch effects or model violations. The equivocal results of these extensive analyses highlight the fundamental problems of basing coral phylogeny on mitochondrial sequence data.     

Genetic signatures through space,time and multiple disturbances in a ubiquitous brooding coral

The predominance of self‐recruitment in many reef‐building corals has fundamental and complex consequences for their genetic diversity, population persistence and responses to climate change. Knowledge of genetic structure over local scales needs to be placed within a broad spatial context, and also integrated with genetic monitoring through time to disentangle these consequences. Here, we examined patterns of genetic diversity over multiple spatio‐temporal scales across tropical Australia in the ubiquitous brooding coral, Seriatopora hystrix. We also analysed complimentary environmental and demographic data to elucidate the seascape drivers of these patterns. Large genetic differences were detected between the east vs. west coasts of Australia. In northwest Australia, geographic differentiation dominated genetic structure over multiple scales. However, three sympatric lineages were detected at the largest offshore reef system (Scott Reef). Similar to the differences observed among putative species in eastern Australia, these lineages were associated with different levels of wave exposure. Local genetic structure within the Scott Reef system was relatively stable over 10 years, but temporal differences were observed that reflected small but important genetic changes over a few generations during recovery after severe bleaching. These results highlight the importance of self‐recruitment together with occasional longer distance connectivity for the persistence of a metapopulation across spatially and temporally variable environments. Our multidimensional research provides a foundation for further long‐term genetic monitoring to inform conservation strategies and highlights that sampling scales, ecological effects and cryptic diversity are important considerations to develop realistic understanding of the evolutionary resilience of corals.     

Regional‐scale dominance of non‐framework building corals on Caribbean reefs affects carbonate production and future reef growth

Coral cover on Caribbean reefs has declined rapidly since the early 1980's. Diseases have been a major driver, decimating communities of framework building Acropora and Orbicella coral species, and reportedly leading to the emergence of novel coral assemblages often dominated by domed and plating species of the genera Agaricia, Porites and Siderastrea. These corals were not historically important Caribbean framework builders, and typically have much smaller stature and lower calcification rates, fuelling concerns over reef carbonate production and growth potential. Using data from 75 reefs from across the Caribbean we quantify: (i) the magnitude of non‐framework building coral dominance throughout the region and (ii) the contribution of these corals to contemporary carbonate production. Our data show that live coral cover averages 18.2% across our sites and coral carbonate production 4.1 kg CaCO₃ m^?2 yr^?1. However, non‐framework building coral species dominate and are major carbonate producers at a high proportion of sites; they are more abundant than Acropora and Orbicella at 73% of sites; contribute an average 68% of the carbonate produced; and produce more than half the carbonate at 79% of sites. Coral cover and carbonate production rate are strongly correlated but, as relative abundance of non‐framework building corals increases, average carbonate production rates decline. Consequently, the use of coral cover as a predictor of carbonate budget status, without species level production rate data, needs to be treated with caution. Our findings provide compelling evidence for the Caribbean‐wide dominance of non‐framework building coral taxa, and that these species are now major regional carbonate producers. However, because these species typically have lower calcification rates, continued transitions to states dominated by non‐framework building coral species will further reduce carbonate production rates below ‘predecline’ levels, resulting in shifts towards negative carbonate budget states and reducing reef growth potential.     

Protein expression and genetic structure of the coral Porites lobata in an environmentally extreme Samoan back reef: does host genotype limit phenotypic plasticity?

The degree to which coral reef ecosystems will be impacted by global climate change depends on regional and local differences in corals’ susceptibility and resilience to environmental stressors. Here, we present data from a reciprocal transplant experiment using the common reef building coral Porites lobata between a highly fluctuating back reef environment that reaches stressful daily extremes, and a more stable, neighbouring forereef. Protein biomarker analyses assessing physiological contributions to stress resistance showed evidence for both fixed and environmental influence on biomarker response. Fixed influences were strongest for ubiquitin‐conjugated proteins with consistently higher levels found in back reef source colonies both pre and post‐transplant when compared with their forereef conspecifics. Additionally, genetic comparisons of back reef and forereef populations revealed significant population structure of both the nuclear ribosomal and mitochondrial genomes of the coral host (F_ST = 0.146 P < 0.0001, F_ST = 0.335 P < 0.0001 for rDNA and mtDNA, respectively), whereas algal endosymbiont populations were genetically indistinguishable between the two sites. We propose that the genotype of the coral host may drive limitations to the physiological responses of these corals when faced with new environmental conditions. This result is important in understanding genotypic and environmental interactions in the coral algal symbiosis and how corals may respond to future environmental changes.     

Draft genome of an iconic Red Sea reef fish,the blacktail butterflyfish (Chaetodon austriacus): current status and its characteristics

Butterflyfish are among the most iconic of the coral reef fishes and represent a model system to study general questions of biogeography, evolution and population genetics. We assembled and annotated the genome sequence of the blacktail butterflyfish (Chaetodon austriacus), an Arabian region endemic species that is reliant on coral reefs for food and shelter. Using available bony fish (superclass Osteichthyes) genomes as a reference, a total of 28 926 high‐quality protein‐coding genes were predicted from 13 967 assembled scaffolds. The quality and completeness of the draft genome of C. austriacus suggest that it has the potential to serve as a resource for studies on the co‐evolution of reef fish adaptations to the unique Red Sea environment, as well as a comparison of gene sequences between closely related congeneric species of butterflyfish distributed more broadly across the tropical Indo‐Pacific.     

Gene expression under chronic heat stress in populations of the mustard hill coral (Porites astreoides) from different thermal environments

Recent evidence suggests that corals can acclimatize or adapt to local stress factors through differential regulation of their gene expression. Profiling gene expression in corals from diverse environments can elucidate the physiological processes that may be responsible for maximizing coral fitness in their natural habitat and lead to a better understanding of the coral's capacity to survive the effects of global climate change. In an accompanying paper, we show that Porites astreoides from thermally different reef habitats exhibit distinct physiological responses when exposed to 6 weeks of chronic temperature stress in a common garden experiment. Here, we describe expression profiles obtained from the same corals for a panel of 9 previously reported and 10 novel candidate stress response genes identified in a pilot RNA‐Seq experiment. The strongest expression change was observed in a novel candidate gene potentially involved in calcification, SLC26, a member of the solute carrier family 26 anion exchangers, which was down‐regulated by 92‐fold in bleached corals relative to controls. The most notable signature of divergence between coral populations was constitutive up‐regulation of metabolic genes in corals from the warmer inshore location, including the gluconeogenesis enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase and the lipid beta‐oxidation enzyme acyl‐CoA dehydrogenase. Our observations highlight several molecular pathways that were not previously implicated in the coral stress response and suggest that host management of energy budgets might play an adaptive role in holobiont thermotolerance.     

Nearshore coral growth declining on the Mesoamerican Barrier Reef System

Anthropogenic global change and local stressors are impacting coral growth and survival worldwide, altering the structure and function of coral reef ecosystems. Here, we show that skeletal extension rates of nearshore colonies of two abundant and widespread Caribbean corals (Siderastrea siderea, Pseudodiploria strigosa) declined across the Belize Mesoamerican Barrier Reef System (MBRS) over the past century, while offshore coral conspecifics exhibited relatively stable extension rates over the same temporal interval. This decline has caused nearshore coral extension rates to converge with those of their historically slower growing offshore coral counterparts. For both species, individual mass coral bleaching events were correlated with low rates of skeletal extension within specific reef environments, but no single bleaching event was correlated with low skeletal extension rates across all reef environments. We postulate that the decline in skeletal extension rates for nearshore corals is driven primarily by the combined effects of long‐term ocean warming and increasing exposure to higher levels of land‐based anthropogenic stressors, with acute thermally induced bleaching events playing a lesser role. If these declining trends in skeletal growth of nearshore S. siderea and P. strigosa continue into the future, the structure and function of these critical nearshore MBRS coral reef systems is likely to be severely impaired.     

Functional genomic analysis of corals from natural CO2‐seeps reveals core molecular responses involved in acclimatization to ocean acidification

Little is known about the potential for acclimatization or adaptation of corals to ocean acidification and even less about the molecular mechanisms underpinning these processes. Here, we examine global gene expression patterns in corals and their intracellular algal symbionts from two replicate population pairs in Papua New Guinea that have undergone long‐term acclimatization to natural variation in pCO₂. In the coral host, only 61 genes were differentially expressed in response to pCO₂ environment, but the pattern of change was highly consistent between replicate populations, likely reflecting the core expression homeostasis response to ocean acidification. Functional annotations highlight lipid metabolism and a change in the stress response capacity of corals as key parts of this process. Specifically, constitutive downregulation of molecular chaperones was observed, which may impact response to combined climate change‐related stressors. Elevated CO₂ has been hypothesized to benefit photosynthetic organisms but expression changes of in hospite Symbiodinium in response to acidification were greater and less consistent among reef populations. This population‐specific response suggests hosts may need to adapt not only to an acidified environment, but also to changes in their Symbiodinium populations that may not be consistent among environments, adding another challenging dimension to the physiological process of coping with climate change.     

The draft genome of Actinia tenebrosa reveals insights into toxin evolution

Sea anemones have a wide array of toxic compounds (peptide toxins found in their venom) which have potential uses as therapeutics. To date, the majority of studies characterizing toxins in sea anemones have been restricted to species from the superfamily, Actinioidea. No highly complete draft genomes are currently available for this superfamily, however, highlighting our limited understanding of the genes encoding toxins in this important group. Here we have sequenced, assembled, and annotated a draft genome for Actinia tenebrosa. The genome is estimated to be approximately 255 megabases, with 31,556 protein‐coding genes. Quality metrics revealed that this draft genome matches the quality and completeness of other model cnidarian genomes, including Nematostella, Hydra, and Acropora. Phylogenomic analyses revealed strong conservation of the Cnidaria and Hexacorallia core‐gene set. However, we found that lineage‐specific gene families have undergone significant expansion events compared with shared gene families. Enrichment analysis performed for both gene ontologies, and protein domains revealed that genes encoding toxins contribute to a significant proportion of the lineage‐specific genes and gene families. The results make clear that the draft genome of A. tenebrosa will provide insight into the evolution of toxins and lineage‐specific genes, and provide an important resource for the discovery of novel biological compounds.     

Lineage‐specific plasmid acquisition and the evolution of specialized pathogens in Bacillus thuringiensis and the Bacillus cereus group

Bacterial plasmids can vary from small selfish genetic elements to large autonomous replicons that constitute a significant proportion of total cellular DNA. By conferring novel function to the cell, plasmids may facilitate evolution but their mobility may be opposed by co‐evolutionary relationships with chromosomes or encouraged via the infectious sharing of genes encoding public goods. Here, we explore these hypotheses through large‐scale examination of the association between plasmids and chromosomal DNA in the phenotypically diverse Bacillus cereus group. This complex group is rich in plasmids, many of which encode essential virulence factors (Cry toxins) that are known public goods. We characterized population genomic structure, gene content and plasmid distribution to investigate the role of mobile elements in diversification. We analysed coding sequence within the core and accessory genome of 190 B. cereus group isolates, including 23 novel sequences and genes from 410 reference plasmid genomes. While cry genes were widely distributed, those with invertebrate toxicity were predominantly associated with one sequence cluster (clade 2) and phenotypically defined Bacillus thuringiensis. Cry toxin plasmids in clade 2 showed evidence of recent horizontal transfer and variable gene content, a pattern of plasmid segregation consistent with transfer during infectious cooperation. Nevertheless, comparison between clades suggests that co‐evolutionary interactions may drive association between plasmids and chromosomes and limit wider transfer of key virulence traits. Proliferation of successful plasmid and chromosome combinations is a feature of specialized pathogens with characteristic niches (Bacillus anthracis, B. thuringiensis) and has occurred multiple times in the B. cereus group.     

Caribbean massive corals not recovering from repeated thermal stress events during 2005–2013

Massive coral bleaching events associated with high sea surface temperatures are forecast to become more frequent and severe in the future due to climate change. Monitoring colony recovery from bleaching disturbances over multiyear time frames is important for improving predictions of future coral community changes. However, there are currently few multiyear studies describing long‐term outcomes for coral colonies following acute bleaching events. We recorded colony pigmentation and size for bleached and unbleached groups of co‐located conspecifics of three major reef‐building scleractinian corals (Orbicella franksi, Siderastrea siderea, and Stephanocoenia michelini; n = 198 total) in Bocas del Toro, Panama, during the major 2005 bleaching event and then monitored pigmentation status and changes live tissue colony size for 8 years (2005–2013). Corals that were bleached in 2005 demonstrated markedly different response trajectories compared to unbleached colony groups, with extensive live tissue loss for bleached corals of all species following bleaching, with mean live tissue losses per colony 9 months postbleaching of 26.2% (±5.4 SE) for O. franksi, 35.7% (±4.7 SE) for S. michelini, and 11.2% (±3.9 SE) for S. siderea. Two species, O. franksi and S. michelini, later recovered to net positive growth, which continued until a second thermal stress event in 2010. Following this event, all species again lost tissue, with previously unbleached colony species groups experiencing greater declines than conspecific sample groups, which were previously bleached, indicating a possible positive acclimative response. However, despite this beneficial effect for previously bleached corals, all groups experienced substantial net tissue loss between 2005 and 2013, indicating that many important Caribbean reef‐building corals will likely suffer continued tissue loss and may be unable to maintain current benthic coverage when faced with future thermal stress forecast for the region, even with potential benefits from bleaching‐related acclimation.     

Prospects on the evolutionary mitogenomics of plants: A case study on the olive family (Oleaceae)

The mitogenome is rarely used to reconstruct the evolutionary history of plants, contrary to nuclear and plastid markers. Here, we evaluate the usefulness of mitochondrial DNA for molecular evolutionary studies in Oleaceae, in which cases of cytoplasmic male sterility (CMS) and of potentially contrasted organelle inheritance are known. We compare the diversity and the evolution of mitochondrial and chloroplast genomes by focusing on the olive complex and related genera. Using high‐throughput techniques, we reconstructed complete mitogenomes (ca. 0.7 Mb) and plastomes (ca. 156 kb) for six olive accessions and one Chionanthus. A highly variable organization of mitogenomes was observed at the species level. In olive, two specific chimeric genes were identified in the mitogenome of lineage E3 and may be involved in CMS. Plastid‐derived regions (mtpt) were observed in all reconstructed mitogenomes. Through phylogenetic reconstruction, we demonstrate that multiple integrations of mtpt regions have occurred in Oleaceae, but mtpt regions shared by all members of the olive complex derive from a common ancestor. We then assembled 52 conserved mitochondrial gene regions and complete plastomes of ten additional accessions belonging to tribes Oleeae, Fontanesieae and Forsythieae. Phylogenetic congruence between topologies based on mitochondrial regions and plastomes suggests a strong disequilibrium linkage between both organellar genomes. Finally, while phylogenetic reconstruction based on plastomes fails to resolve the evolutionary history of maternal olive lineages in the Mediterranean area, their phylogenetic relationships were successfully resolved with complete mitogenomes. Overall, our study demonstrates the great potential of using mitochondrial DNA in plant phylogeographic and metagenomic studies.     

New Species of Closely Related Endosymbiotic Dinoflagellates in the Greater Caribbean have Niches Corresponding to Host Coral Phylogeny

Symbiotic dinoflagellates in the genus Breviolum (formerly Symbiodinium Clade B) dominate coral communities in shallow waters across the Greater Caribbean. While some formally described species exist, mounting genetic, and ecological evidence indicate that numerous more comprise this genus, many of which are closely related. To test this, colonies of common reef‐building corals were sampled across a large geographical range. Phylogenetic and population genetic markers then used to examine evolutionary divergence and delineate boundaries of genetic recombination. Three new candidate species were distinguished by fixed differences in nucleotide sequences from nuclear and chloroplast DNA. Population connectivity was evident within each lineage over thousands of kilometers, however, substantial genetic structure persisted between lineages co‐occurring within sampling locations, signifying reproductive isolation. While geographically widespread with overlapping distributions, each species is ecologically distinct, exhibiting specific mutualisms with phylogenetically distinct coral hosts. Moreover, significant differences in mean cell sizes provide some morphological evidence substantiating formal species distinctions. In providing evidence that satisfies the biological, phylogenetic, ecological, and morphological species concepts, we classify and formally name Breviolum faviinorum n. sp., primarily associated with Caribbean corals belonging to the Caribbean subfamily Faviinae; B. meandrinium n. sp., associated with corals belonging to the family Meandrinidae; and B. dendrogyrum n. sp., a symbiont harbored exclusively by the threatened coral Dendrogyra cylindrus. These findings support the primary importance of niche diversification (i.e. host habitat) in the speciation of symbiotic dinoflagellates.