The evolution of facultative symbiosis in stony corals.

Stony corals (order: Scleractinia) differ in growth form and structure. While stony corals have gained the ability to form their aragonite skeleton once in their evolution, the suite of proteins involved in skeletogenesis is different for different coral species. This led to the conclusion that the organic portion of their skeleton can undergo rapid evolutionary changes by independently evolving new biomineralization-related proteins. Here, we used liquid chromatography-tandem mass spectrometry to sequence skeletogenic proteins extracted from the encrusting temperate coral Oculina patagonica. We compare it to the previously published skeletal proteome of the branching subtropical corals Stylophora pistillata as both are regarded as highly resilient to environmental changes. We further characterized the skeletal organic matrix (OM) composition of both taxa and tested their effects on the mineral formation using a series of overgrowth experiments on calcite seeds. We found that each species utilizes a different set of proteins containing different amino acid compositions and achieve a different morphology modification capacity on calcite overgrowth. Our results further support the hypothesis that the different coral taxa utilize a species-specific protein set comprised of independent gene co-option to construct their own unique organic matrix framework. While the protein set differs between species, the specific predicted roles of the whole set appear to underline similar functional roles. They include assisting in forming the extracellular matrix, nucleation of the mineral and cell signaling. Nevertheless, the different composition might be the reason for the varying organization of the mineral growth in the presence of a particular skeletal OM, ultimately forming their distinct morphologies.

Astrangia poculata is a temperate scleractinian coral that exists in facultative symbiosis with the dinoflagellate alga Breviolum psygmophilum across a range spanning the Gulf of Mexico to Cape Cod, Massachusetts. Our previous work on metabolic thermal performance of Virginia (VA) and Rhode Island (RI) populations of A. poculata revealed physiological signatures of cold (RI) and warm (VA) adaptation of these populations to their respective local thermal environments. Here, we used whole-transcriptome sequencing (mRNA-Seq) to evaluate genetic differences and identify potential loci involved in the adaptive signature of VA and RI populations. Sequencing data from 40 A. poculata individuals, including 10 colonies from each population and symbiotic state (VA-white, VA-brown, RI-white, and RI-brown), yielded a total of 1,808 host-associated and 59 algal symbiont-associated single nucleotide polymorphisms (SNPs) post filtration. Fst outlier analysis identified 66 putative high outlier SNPs in the coral host and 4 in the algal symbiont. Differentiation of VA and RI populations in the coral host was driven by putatively adaptive loci, not neutral divergence (Fst = 0.16, p = 0.001 and Fst = 0.002, p = 0.269 for outlier and neutral SNPs respectively). In contrast, we found evidence of neutral population differentiation in B. psygmophilum (Fst = 0.093, p = 0.001). Several putatively adaptive host loci occur on genes previously associated with the coral stress response. In the symbiont, three of four putatively adaptive loci are associated with photosystem proteins. The opposing pattern of neutral differentiation in B. psygmophilum, but not the A. poculata host, reflects the contrasting dynamics of coral host and algal symbiont population connectivity, dispersal, and gene by environment interactions.

Coral reefs are the biodiversity hot spots of the oceans, but have suffered from increasing environmental stresses caused principally by anthropogenic global warming. The keystone species of coral reefs are scleractinian corals, which maintain obligatory symbiotic relationships with photosynthetic dinoflagellates or zooxanthellae. Understanding cellular and molecular mechanisms of symbiosis is therefore essential for future preservation of coral reefs. To date, however, almost no single-cell level experimental systems have been devised to illuminate such mechanisms. To this end, our previous study established stable in vitro cell culture lines, including IVB5, originating from planula larvae of the stony coral, Acropora tenuis. Here, we show that soon after mixture with the zooxanthella, Breviolum minutum, flattened amorphous cells with endodermal properties exhibited elevated locomotor activity using filopodia and lamellipodia and interacted with zooxanthellae. Several minutes thereafter, coral cells began to endocytose B. minutum, and in vitro symbiosis was accomplished within 30 minutes. Nearly a half of the coral cells had incorporated algal cells within 24 hours in a reproducible manner. Coral cells that harbored zooxanthellae gradually became round and less mobile, and the zooxanthellae sometimes settled in vacuole-like structures in coral cell cytoplasm. This symbiotic state was maintained for at least a month. The IVB5 line of A. tenuis therefore provides an experimental system to explore cellular and molecular mechanisms involved in establishment of coral symbiosis at the single-cell level, results of which may be useful for future preservation of coral reefs.

Coral reefs are the biodiversity hot spots of the oceans, but have suffered from increasing environmental stresses caused principally by anthropogenic global warming. The keystone species of coral reefs are scleractinian corals, which maintain obligatory symbiotic relationships with photosynthetic dinoflagellates or zooxanthellae. Understanding cellular and molecular mechanisms of symbiosis is therefore essential for future preservation of coral reefs. To date, however, almost no single-cell level experimental systems have been devised to illuminate such mechanisms. To this end, our previous study established stable in vitro cell culture lines, including IVB5, originating from planula larvae of the stony coral, Acropora tenuis. Here, we show that soon after mixture with the zooxanthella, Breviolum minutum, flattened amorphous cells with endodermal properties exhibited elevated locomotor activity using filopodia and lamellipodia and interacted with zooxanthellae. Several minutes thereafter, coral cells began to endocytose B. minutum, and in vitro symbiosis was accomplished within 30 minutes. Nearly a half of the coral cells had incorporated algal cells within 24 hours in a reproducible manner. Coral cells that harbored zooxanthellae gradually became round and less mobile, and the zooxanthellae sometimes settled in vacuole-like structures in coral cell cytoplasm. This symbiotic state was maintained for at least a month. The IVB5 line of A. tenuis therefore provides an experimental system to explore cellular and molecular mechanisms involved in establishment of coral symbiosis at the single-cell level, results of which may be useful for future preservation of coral reefs.

Thermal acclimation and light-harvesting complex expression in Symbiodinium

The resilience of coral reefs in oligotrophic, (sub)tropical oceans is largely due to the symbiotic relationship between scleractinian corals and Symbiodiniaceae algae, which enables efficient internal nutrient recycling. Investigating the history of this coral symbiosis can provide insights into its role in sustaining the health of both present and future coral reefs. The isotopic composition of organic nitrogen (15N/14N or δ15N) bound within coral skeletons has been utilized to trace the existence of symbiosis in fossil corals, suggesting that coral symbiosis dates back to at least 210 million years ago. The basis of this proxy is that symbiotic corals are expected to exhibit lower δ15N compared to their non-symbiotic (aposymbiotic) counterparts within the same environments, owing to internal nitrogen recycling between the coral host and algal symbiont, and reduced leakage of low-δ15N ammonium into seawater. However, this hypothesis has not been adequately tested in contemporary settings. In a laboratory experiment, we examined the δ15N differences between the symbiotic and aposymbiotic branches within the same genetic backgrounds of the facultatively symbiotic coral Oculina arbuscula under well-fed conditions. Across five different genotypes in two separate experiments, symbiotic branches consistently showed lower δ15N than their aposymbiotic counterparts. These findings corroborate the use of δ15N as a proxy for identifying coral symbiosis in the past, particularly when multiple species of corals coexisted in the same environments.

ABSTRACTThe class Hexacorallia, encompassing stony corals and sea anemones, plays a critical role in marine ecosystems. Coral bleaching, the disruption of the symbiosis between stony corals and zooxanthellate algae, is driven by seawater warming and further exacerbated by pathogenic microbes. However, how pathogens, especially viruses, contribute to accelerated bleaching remains poorly understood. Here the model sea anemone Nematostella vectensis is used to explore these dynamics by creating a transgenic line with a reporter gene regulated by sequences from two RIG‐I‐like receptor genes involved in antiviral responses. Under heat stress, the reporter genes showed significant upregulation. Further, transcriptomes from N. vectensis, Exaiptasia diaphana and the stony coral Stylophora pistillata were analysed to reveal stress‐induced activation of a set of bona fide immune‐related genes conserved between the three species. Population‐specific differences in stress‐induced transcriptional responses of immune‐related genes were evident in both Nematostella and Stylophora, depending on geographic origin. In Exaiptasia, the presence of zooxanthellae also influenced stress‐induced immune gene expression. To test whether the viruses themselves contribute to this immune response under stress, we subjected N. vectensis to heat stress and measured the transcription dynamics of resident viruses as well as selected antiviral genes. While the antiviral genes responded within hours of heat stress, viral gene expression was already upregulated within 30 min, suggesting that their increase might be contributing to the elevated immune response under stress, and consequentially, the further demise of organismal homeostasis. These findings highlight the interplay between environmental stress, viruses, immune responses and symbiotic states in Hexacorallia.

Symbiosis and coloniality are ecologically important traits for corals of the order Scleractinia. Symbiotic (zooxanthellate) species are highly successful in shallow waters of tropical and subtropical seas and most of them are colonial. On the other hand, azooxanthellate species present wide distribution ranges and expand to the deep-sea at more than 6,000 m depth. These are mostly solitary, with only few species colonial that form extensive deep reefs. Each ecologically distinctive group encompasses half of the biodiversity of the order and they are not grouped into differentiated monophyletic clades. Paleontologists and evolutionary biologists have debated for decades whether modern scleractinian corals have evolved from symbiotic or colonial ancestors and how these traits have evolved and being involved in the diversification process in corals. Previous comparative analyses throw evidence in favor of coevolution of these characters and toward repetitive loss of symbiosis and coloniality. Nevertheless, the discovery of the origin of the group deep in the Paleozoic, with a deep divergent clade composed of only azooxanthellate corals has questioned these findings. With this work, we disentangle the patterns in the evolution of symbiosis and coloniality, testing if they are correlated and if they follow a gradual or episodic mode of evolution. To this end, we first produce the most complete time-calibrated phylogenetic tree for the order Scleractinia, including new sequences of never-before sampled species and genera. These novel sequences contribute to alleviate the current molecular under sampling of azooxanthellate species. Incorporating phylogenetic uncertainty, we obtained strong evidence in favor of a correlated and episodic model of evolution. This model led to the inference of an azooxanthellate and solitary most recent ancestor of scleractinians. Transition rates between the four different combinations of the two traits showed that while coloniality is gained and lost multiple times, symbiosis first appears around 282 Ma and is never lost. Also, coloniality seems to have appeared before symbiosis in azooxanthellate lineages. Thus, azooxanthellate corals, and especially colonial lineages, have been acting as a source of biodiversity for shallow zooxanthellate coral communities, highlighting the uniqueness of shallow and deep species and the need to preserve them.

Los corales son metazoos del phylum Cnidaria que se consideran el producto de una interaccion mutualista entre el coral y algas unicelulares simbioticas del genero Symbiodinium conocidas como zooxantelas; aunque tambien mantienen asociaciones con bacterias, hongos y arqueas, formado un consorcio colaborativo denominado el ?holobionte coralino? (Rohwer et al., 2001; Knowlton y Rohwer, 2003). Los arrecifes de coral son probablemente uno de los primeros ecosistemas marinos en sufrir las consecuencias del cambio climatico y del aumento de las temperaturas de los oceanos (Wilkinson, 2008). El blanqueamiento de los corales (ruptura de la simbiosis existente entre los corales y sus zooxantelas) como consecuencias del cambio climatico ha aumentado tanto en su frecuencia como severidad en las ultimas decadas (Hoegh-Guldberg, 1999). El coral Oculina patagonica De Angelis, 1908 es un coral scleractinio introducido en el Mar Mediterraneo que fue detectado por primera vez en 1966 en la costa de Liguria en Italia, cerca del puerto de Savona (Zibrowius, 1974) y en 1973 3n el puerto de Alicante (Zibrowius and Ramos, 1983). Hoy en dia, esta especie esta ampliamente distribuida a traves de todo el mar Mediterraneo, debido al intenso trafico maritimo de este mar (Zibrowius, 1992) y al incremento de actividades antropogenicas (Serrano et al., 2013; Salomidi et al., 2013), asi como a su habilidad para subsistir bajo diferentes condiciones ambientales (Armoza-Zvuloni et al., 2012). El blanqueamiento de O. patagonica ha sido ampliamente estudiado, aunque existe una gran controversia sobre la naturaleza de su causa principal, debido a que existen diferentes hipotesis sobre el papel que juegan los microorganismos en este fenomeno. Este proceso fue observado por primera vez en las costas de Israel en el verano de 1993 y mediante la aplicacion de los postulados de Koch se demostro que el agente causante era Vibrio shilonii (=Vibrio mediteranei) (Kushmaro et al., 1996, 1997); siendo el aumento de la temperatura del mar el factor ambiental que lo desencadenaba (Kushmaro et al., 1998). Estudios posteriores sugirieron que V. meditarranei no estaba involucrado o al menos no era la causa principal del blanqueamiento anual de O.patagonica en el Mediterraneo oriental (Ainsworth et al., 2008), sugiriendo que las bacterias juegan un papel secundario en el blanqueamiento. El objetivo principal de esta investigacion es mejorar el conocimiento sobre la respuesta del coral O. patagonica y su comunidad microbiana ante diferentes condiciones ambientales, asi como esclarecer el papel de los Vibrios y los factores ambientales en los eventos de blanqueamiento, particularmente en el actual escenario de calentamiento global. Para lograr este objetivo el estudio se estructurara en dos partes, la primera centrada en las caracteristicas biologicas y ecologicas del coral, estudiando su distribucion espacial y ?nivel de invasion? en la Comunidad Valenciana, asi como su tasa crecimiento y blanqueamiento en diferentes condiciones ambientales. En la segunda parte se caracterizara la comunidad microbiana asociada a O. patagonica (zooxantellas y bacterias), asi como los cambios de la misma con los factores ambientales. En esta tesis doctoral se resalta el hecho de que uno de los mayores riesgos para O. patagonica es el cambio climatico y el consecuente aumento de temperatura del agua, asi como la necesidad de estudiar los cambios producidos a todos los niveles del holobionte. Se ha demostrado la existencia de una relacion entre los cambios de la microbiota de O. patagonica y el aumento de temperatura, asi como la presencia de posibles Vibrios patogenos y el blanqueamiento. Por tanto, se reafirma que la relacion entre Vibrios patogenos y las nfermedades en corales puede incrementarse como consecuencia del cambio climatico

The extensive human-mediated modifications of shallow coastal habitats drastically alter selection regimes and may assist alien invasions. The preferential presence of a non-indigenous scleractinian coral (Oculina patagonica) on anthropogenic hard substrata was investigated in a highly disturbed coastal area, along the eastern Saronikos Gulf (Aegean Sea, Eastern Mediterranean). Although the species occurred on both natural and anthropogenic substrata at similar frequencies, its abundance was substantially higher on the latter. The species was present all along the shallow (0.5–5 m) infralittoral zone of the studied coastline, and its percent cover even exceeded 50 % at a site of anthropogenic hard substratum. The occupancy of the species declined with distance from a highly disturbed industrialized/urbanized area (Athens metropolitan coastal front and the port of Piraeus). Space availability as a result of habitat modification appears to have been an important factor enhancing the coral’s abundance in this area. The ongoing degradation of the coastal zone, as a combined effect of coastal pollution, proliferation of artificial substrata and overgrazing seems to be paving the way to this new invasion in the Aegean Sea.

Reproductive characteristics and steroid levels in the scleractinian coral Oculina patagonica inhabiting contaminated sites along the Israeli Mediterranean coast

Certain stony corals can alternate between a calcifying colonial form and noncalcifying solitary polyps, supporting the hypothesis that corals have survived through geologic timescale periods of unfavorable calcification conditions. However, the mechanisms enabling this biological plasticity are yet to be identified. Here we show that incubation of two coral species (Pocillopora damicornis and Oculina patagonica) under reduced pH conditions (pH 7.2) simulating past ocean acidification induce tissue-specific apoptosis that leads to the dissociation of polyps from coenosarcs. This in turn leads to the breakdown of the coenosarc and, as a consequence, to loss of coloniality. Our data show that apoptosis is initiated in the polyps and that once dissociation between polyp and coenosarc terminates, apoptosis subsides. After reexposure of the resulting solitary polyps to normal pH (pH 8.2), both coral species regenerated coenosarc tissues and resumed calcification. These results indicate that regulation of coloniality is under the control of the polyp, the basic modular unit of the colony. A mechanistic explanation for several key evolutionarily important phenomena that occurred throughout coral evolution is proposed, including mechanisms that permitted species to survive the third tier of mass extinctions.

Globally, species are migrating in an attempt to track optimal isotherms as climate change increasingly warms existing habitats. Stony corals are severely threatened by anthropogenic warming, which has resulted in repeated mass bleaching and mortality events. Since corals are sessile as adults and with a relatively old age of sexual maturity, they are slow to latitudinally migrate, but corals may also migrate vertically to deeper, cooler reefs. Herein we describe vertical migration of the Mediterranean coral Oculina patagonica from less than 10 m depth to > 30 m. We suggest that this range shift is a response to rapidly warming sea surface temperatures on the Israeli Mediterranean coastline. In contrast to the vast latitudinal distance required to track temperature change, this species has migrated deeper where summer water temperatures are up to 2 °C cooler. Comparisons of physiology, morphology, trophic position, symbiont type, and photochemistry between deep and shallow conspecifics revealed only a few depth-specific differences. At this study site, shallow colonies typically inhabit low light environments (caves, crevices) and have a facultative relationship with photosymbionts. We suggest that this existing phenotype aided colonization of the mesophotic zone. This observation highlights the potential for other marine species to vertically migrate.

Nitrogen is one of the limiting nutrients for coral growth and primary productivity. Therefore, the capacity of different associations between corals and their algal symbionts (Symbiodiniaceae) to efficiently exploit the available nitrogen sources will influence their distribution and abundance. Recent studies have advanced our understanding of nitrogen assimilation in reef-building scleractinian (hard) coral-Symbiodiniaceae symbioses. However, the nutrient metabolism of other coral taxa, such as Alcyoniina (soft corals), remains underexplored. Using stable isotope labeling, we investigated the assimilation of dissolved nitrogen (i.e., ammonium, nitrate, and free amino acids) by multiple species of soft and hard corals sampled in the Gulf of Aqaba in shallow (8–10 m) and mesophotic (40–50 m) reefs. Our results show that dissolved nitrogen assimilation rates per tissue biomass were up to 10-fold higher in hard than in soft coral symbioses for all sources of nitrogen. Although such differences in assimilation rates could be linked to the Symbiodiniaceae density, Symbiodiniaceae species, or the C:N ratio of the host and algal symbiont fractions, none of these parameters were different between the two coral taxa. Instead, the lower assimilation rates in soft coral symbioses might be explained by their different nutritional strategy: whereas soft corals may obtain most of their nitrogen via the capture of planktonic prey by the coral host (heterotrophic feeding), hard corals may rely more on dissolved nitrogen assimilation by their algal symbionts to fulfill their needs. This study highlights different nutritional strategies in soft and hard coral symbioses. A higher reliance on heterotrophy may help soft corals to grow in reefs with higher turbidity, which have a high concentration of particles in suspension in seawater. Further, soft corals may benefit from lower dissolved nitrogen assimilation rates in areas with low water quality.

Coastal oceans are increasingly eutrophic, warm and acidic through the addition of anthropogenic nitrogen and carbon, respectively. Among the most sensitive taxa to these changes are scleractinian corals, which engineer the most biodiverse ecosystems on Earth. Corals’ sensitivity is a consequence of their evolutionary investment in symbiosis with the dinoflagellate alga, Symbiodinium. Together, the coral holobiont has dominated oligotrophic tropical marine habitats. However, warming destabilizes this association and reduces coral fitness. It has been theorized that, when reefs become warm and eutrophic, mutualistic Symbiodinium sequester more resources for their own growth, thus parasitizing their hosts of nutrition. Here, we tested the hypothesis that sub-bleaching temperature and excess nitrogen promotes symbiont parasitism by measuring respiration (costs) and the assimilation and translocation of both carbon (energy) and nitrogen (growth; both benefits) within Orbicella faveolata hosting one of two Symbiodinium phylotypes using a dual stable isotope tracer incubation at ambient (26 °C) and sub-bleaching (31 °C) temperatures under elevated nitrate. Warming to 31 °C reduced holobiont net primary productivity (NPP) by 60% due to increased respiration which decreased host %carbon by 15% with no apparent cost to the symbiont. Concurrently, Symbiodinium carbon and nitrogen assimilation increased by 14 and 32%, respectively while increasing their mitotic index by 15%, whereas hosts did not gain a proportional increase in translocated photosynthates. We conclude that the disparity in benefits and costs to both partners is evidence of symbiont parasitism in the coral symbiosis and has major implications for the resilience of coral reefs under threat of global change.