Symbiodiniaceae diversity varies by host and environment across thermally distinct reefs.

Endosymbiotic dinoflagellates (Symbiodiniaceae) influence coral thermal tolerance at both local and regional scales. In isolation, the effects of host genetics, environment, and thermal disturbances on symbiont communities are well understood, yet their combined effects remain poorly resolved. Here, we investigate Symbiodiniaceae across 1300 km in Australia's Coral Sea Marine Park to disentangle these interactive effects. We identified Symbiodiniaceae to species-level resolution for three coral species (Acropora cf humilis, Pocillopora verrucosa, and Pocillopora meandrina) by sequencing two genetic markers of the symbiont (ITS2 and psbAncr), paired with genotype-by-sequencing of the coral host (DArT-seq). Our samples predominantly returned sequences from the genus Cladocopium, where Acropora cf humilis affiliated with C3k, Pocillopora verrucosa with C. pacificum, and Pocillopora meandrina with C. latusorum. Multivariate analyses revealed that Acropora symbionts were driven strongly by local environment and thermal disturbances. In contrast, Pocillopora symbiont communities were both partitioned 2.5-fold more by host genetic structure than by environmental structure. Among the two Pocillopora species, the effects of environment and host genetics explained four times more variation in symbionts for P. meandrina than P. verrucosa. The concurrent bleaching event in 2020 had variable impacts on symbiont communities, consistent with patterns in P. verrucosa and A. cf humilis, but not P. meandrina. Our findings demonstrate how symbiont macroscale community structure responses to environmental gradients depend on host species and their respective population structure. Integrating host, symbiont, and environmental data will help forecast the adaptive potential of corals and their symbionts amidst a rapidly changing environment.

Formal recognition of host-generalist species of dinoflagellate (Cladocopium, Symbiodiniaceae) mutualistic with Indo-Pacific reef corals.

The existence of widespread species with the capacity to endure diverse, or variable, environments are of importance to ecological and genetic research, and conservation. Such "ecological generalists" are more likely to have key adaptations that allow them to better tolerate the physiological challenges of rapid climate change. Reef-building corals are dependent on endosymbiotic dinoflagellates (Family: Symbiodiniaceae) for their survival and growth. While these symbionts are biologically diverse, certain genetic types appear to have broad geographic distributions and are mutualistic with various host species from multiple genera and families in the order Scleractinia that must acquire their symbionts through horizontal transmission. Despite the considerable ecological importance of putative host-generalist symbionts, they lack formal species descriptions. In this study, we used molecular, ecological, and morphological evidence to verify the existence of five new host-generalist species in the symbiodiniacean genus Cladocopium. Their geographic distribution and prevalence among host communities corresponds to prevailing environmental conditions at both regional and local scales. The influence that each species has on host physiology may partially explain regional differences in thermal sensitivities among coral communities. The potential increased prevalence of a generalist species that endures environmental instability is a consequential ecological response to warming oceans. Large-scale shifts in symbiont dominance could ensure reef coral persistence and productivity in the near term. Ultimately, these formal designations should advance scientific communication and generate informed research questions on the physiology and ecology of coral-dinoflagellate mutualisms.

Calcification rates of a massive and a branching coral species were unrelated to diversity of endosymbiotic dinoflagellates.

BACKGROUND: To explore the possibility that endosymbiotic dinoflagellates (Symbiodiniaceae) are associated with coral calcification rates, we investigated the diversity of symbiotic algae in coral colonies with different calcification rates within massive and branching corals (Porites australiensis and Acropora digitifera). METHODS AND RESULTS: Genotyping symbiotic algae from colonies with different calcification rates revealed that all the colonies of both species harbored mainly Cladocopium (previously clade C of Symbiodinium). The Cladocopium symbionts in P. australiensis were mainly composed of C15 and C15bn, and those in A. digitifera of C50a and C50c. We did not detect clear relationships between symbiont compositions and calcification rates within the two coral species. CONCLUSIONS: Our results suggest that different coral calcification rates within species may be attributed to genetic factors of coral hosts themselves and/or within symbiont genotypes.

Transcriptomes of Giant Sea Anemones from Okinawa as a Tool for Understanding Their Phylogeny and Symbiotic Relationships with Anemonefish.

The relationship between anemonefish and sea anemones is one of the most emblematic examples of mutualistic symbiosis in coral reefs. Although this is a textbook example, the major aspects of this symbiosis are still not fully understood in mechanistic terms. Moreover, since studies of this relationship have usually been focused on anemonefish, much less is known about giant sea anemones, their similarities, their phylogenetic relationships, and their differences at the molecular level. Since both partners of the symbiotic relationship are important, we decided to explore this well-known phenomenon from the perspective of giant sea anemones. Here, we report reference transcriptomes for all seven species of giant sea anemones that inhabit fringing reefs of Okinawa (Japan) and serve as hosts for six species of local anemonefish. Transcriptomes were used to investigate their phylogenetic relations, genetic differences and repertoires of nematocyte-specific proteins. Our data support the presence of three distinct groups corresponding to three genera: Entacmaea, Heteractis, and Stichodactyla. The basal position among the three groups belongs to Entacmaea, which was the first to diverge from a common ancestor. While the magnitude of genetic difference between the representatives of Entacmaea and Stichodactyla is large, intra-specific variation within Stichodactyla is much smaller and seems to result from recent speciation events. Our data reconfirms that Heteractis magnifica belongs to the genus Stichodactyla, despite an overall morphological similarity with representatives of the genus Heteractis. The availability of reference transcriptomes will facilitate further research into the fascinating relationship between sea anemones and anemonefish.

Different functional traits among closely related algal symbionts dictate stress endurance for vital Indo-Pacific reef-building corals.

Reef-building corals in the genus Porites are one of the most important constituents of Indo-Pacific reefs. Many species within this genus tolerate abnormally warm water and exhibit high specificity for particular kinds of endosymbiotic dinoflagellates that cope with thermal stress better than those living in other corals. Still, during extreme ocean heating, some Porites exhibit differences in their stress tolerance. While corals have different physiological qualities, it remains unknown whether the stability and performance of these mutualisms is influenced by the physiology and genetic relatedness of their symbionts. We investigated two ubiquitous Pacific reef corals, Porites rus and Porites cylindrica, from warmer inshore and cooler offshore reef systems in Palau. While these corals harbored a similar kind of symbiont in the genus Cladocopium (within the ITS2 C15 subclade), rapidly evolving genetic markers revealed evolutionarily diverged lineages corresponding to each Porites species living in each reef habitat. Furthermore, these closely related Cladocopium lineages were differentiated by their densities in host tissues, cell volume, chlorophyll concentration, gross photosynthesis, and photoprotective pathways. When assessed using several physiological proxies, these previously undifferentiated symbionts contrasted in their tolerance to thermal stress. Symbionts within P. cylindrica were relatively unaffected by exposure to 32℃ for 14 days, whereas P. rus colonies lost substantial numbers of photochemically compromised symbionts. Heating reduced the ability of the offshore symbiont associated with P. rus to translocate carbon to the coral. By contrast, high temperatures enhanced symbiont carbon assimilation and delivery to the coral skeleton of inshore P. cylindrica. This study indicates that large physiological differences exist even among closely related symbionts, with significant implications for thermal susceptibility among reef-building Porites.

Cladocopium community divergence in two Acropora coral hosts across multiple spatial scales.

Many broadly-dispersing corals acquire their algal symbionts (Symbiodiniaceae) "horizontally" from their environment upon recruitment. Horizontal transmission could promote coral fitness across diverse environments provided that corals can associate with divergent algae across their range and that these symbionts exhibit reduced dispersal potential. Here we quantified community divergence of Cladocopium algal symbionts in two coral host species (Acropora hyacinthus, Acropora digitifera) across two spatial scales (reefs on the same island, and between islands) across the Micronesian archipelago using microsatellites. We find that both hosts associated with a variety of multilocus genotypes (MLG) within two genetically distinct Cladocopium lineages (C40, C21), confirming that Acropora coral hosts associate with a range of Cladocopium symbionts across this region. Both C40 and C21 included multiple asexual lineages bearing identical MLGs, many of which spanned host species, reef sites within islands, and even different islands. Both C40 and C21 exhibited moderate host specialization and divergence across islands. In addition, within every island, algal symbiont communities were significantly clustered by both host species and reef site, highlighting that coral-associated Cladocopium communities are structured across small spatial scales and within hosts on the same reef. This is in stark contrast to their coral hosts, which never exhibited significant genetic divergence between reefs on the same island. These results support the view that horizontal transmission could improve local fitness for broadly dispersing Acropora coral species.

The dispersant Corexit 9500 and (dispersed) oil are lethal to coral endosymbionts.

Endosymbionts (Symbiodiniaceae) play a vital role in the health of corals. Seawater pollution can harm these endosymbionts and dispersants used during oil spill cleanup can be extremely toxic to these organisms. Here, we examined the impact of oil and a specific dispersant, Corexit-9500, on two representative endosymbionts - Symbiodinium and Cladocopium - from the Southwestern endemic coral Mussismilia braziliensis. The survival and photosynthetic potential of the endosymbionts decreased dramatically after exposure to the dispersant and oil by ~25 % after 2 h and ~50 % after 7 days. Low concentrations of dispersant (0.005 ml/l) and dispersed oil (Polycyclic Aromatic Hydrocarbons, 1132 µg/l; Total Petroleum Hydrocarbons, 595 µg/l) proved highly toxic to both Symbiodinium and Cladocopium. These levels triggered a reduction in growth rate, cell size, and cell wall thickness. After a few hours of exposure, cellular organelles were damaged or destroyed. These acute toxic effects underline the fragile nature of coral endosymbionts.

Toxicological effects of oxybenzone on the growth and bacterial composition of Symbiodiniaceae.

Oxybenzone, a common ultraviolet (UV) filter, is a growing environmental concern due to its ecotoxicological effects. However, the responses of Symbiodiniaceae and their bacterial communities to oxybenzone are largely unknown. In this study, the effects of oxybenzone on Effrenium voratum and Cladocopium goreaui were investigated. The results revealed that sensitivity of Symbiodiniaceae to oxybenzone was species-dependent. 50 µg L-1 of oxybenzone significantly impacted the cell density of C. goreaui, causing a 36.73% decrease. When oxybenzone concentration increased to 500 µg L-1 and 5000 µg L-1, cell division was completely suppressed; meanwhile, chl-a content declined to zero. Compared to C. goreaui, E. voratum had higher resistance to oxybenzone. There was no significant difference in cell density between 50 µg L-1 group and control group. At higher dosage of oxybenzone (500 µg L-1 and 5000 µg L-1), the cell density declined 32.02% and 45.45% compared to the control group, respectively. Additionally, we revealed that the diversity and structure of bacterial community were affected by oxybenzone. Briefly, 500 µg L-1 and 5000 µg L-1 of oxybenzone altered the diversity of bacterial community in C. goreau. Furthermore, the relative abundances of Costertonia, Roseitalea, Rhodopirellula, and Roseobacter were negatively affected by oxybenzone ranging 50 µg L-1 to 5000 µg L-1. Compare to C. goreaui, the bacterial community composition associated with E. voratum was more stable. As revealed by KEGG pathway analysis, oxybenzone affected energy metabolism and inhibited the metabolism of cofactors and vitamins in C. goreaui, while 5000 µg L-1 of oxybenzone significantly altered the carbohydrate metabolism, membrane transport and amino acid metabolism in E. voratum. The changes of bacterial composition may contribute to the variation in algal growth. These results indicated that oxybenzone pollution could injury Symbiodiniaceae, even threaten coral reef ecosystems.

Improved Cladocopium goreaui Genome Assembly Reveals Features of a Facultative Coral Symbiont and the Complex Evolutionary History of Dinoflagellate Genes.

Dinoflagellates of the family Symbiodiniaceae are crucial photosymbionts in corals and other marine organisms. Of these, Cladocopium goreaui is one of the most dominant symbiont species in the Indo-Pacific. Here, we present an improved genome assembly of C. goreaui combining new long-read sequence data with previously generated short-read data. Incorporating new full-length transcripts to guide gene prediction, the C. goreaui genome (1.2 Gb) exhibits a high extent of completeness (82.4% based on BUSCO protein recovery) and better resolution of repetitive sequence regions; 45,322 gene models were predicted, and 327 putative, topologically associated domains of the chromosomes were identified. Comparison with other Symbiodiniaceae genomes revealed a prevalence of repeats and duplicated genes in C. goreaui, and lineage-specific genes indicating functional innovation. Incorporating 2,841,408 protein sequences from 96 taxonomically diverse eukaryotes and representative prokaryotes in a phylogenomic approach, we assessed the evolutionary history of C. goreaui genes. Of the 5246 phylogenetic trees inferred from homologous protein sets containing two or more phyla, 35-36% have putatively originated via horizontal gene transfer (HGT), predominantly (19-23%) via an ancestral Archaeplastida lineage implicated in the endosymbiotic origin of plastids: 10-11% are of green algal origin, including genes encoding photosynthetic functions. Our results demonstrate the utility of long-read sequence data in resolving structural features of a dinoflagellate genome, and highlight how genetic transfer has shaped genome evolution of a facultative symbiont, and more broadly of dinoflagellates.

Metatranscriptomic Analysis of Corals Inoculated With Tolerant and Non-Tolerant Symbiont Exposed to High Temperature and Light Stress.

Algal symbionts of corals can influence host stress resistance; for example, in the Pacific Ocean, whereas Cladocopium (C-type) is generally dominant in corals, Durusdinium (D-type) is found in more heat-resistant corals. Thus, the presence of D-type symbiont likely increases coral heat tolerance, and this symbiotic relationship potentially provides a hint to increase the stress tolerance of coral-algal symbioses. In this study, transcriptome profiles of Cladocopium- and Durusdinium-harboring Acropora solitaryensis (C-coral and D-coral, respectively) and algal photosystem functioning (F v /F m ) under bleaching conditions (high temperature and light stress) were compared. Stress treatment caused algal photoinhibition that the F v /F m value of Symbiodiniaceae was immediately reduced. The transcriptome analysis of corals revealed that genes involved in the following processes were detected: endoplasmic reticulum (ER) stress, mitophagy, apoptosis, endocytosis, metabolic processes (acetyl-CoA, chitin metabolic processes, etc.), and the PI3K-AKT pathway were upregulated, while DNA replication and the calcium signaling pathway were downregulated in both C- and D-corals. These results suggest that unrepaired DNA and protein damages were accumulated in corals under high temperature and light stress. Additionally, some differentially expressed genes (DEGs) were specific to C- or D-corals, which includes genes involved in transient receptor potential (TRP) channels and vitamin B metabolic processes. Algal transcriptome analysis showed the increased expression of gene encoding photosystem and molecular chaperone especially in D-type symbiont. The transcriptome data imply a possible difference in the stress reactions on C-type and D-type symbionts. The results reveal the basic process of coral heat/light stress response and symbiont-type-specific coral transcriptional responses, which provides a perspective on the mechanisms that cause differences in coral stress tolerance.

Identification of coral endosymbionts of Veedhalai and Mandapam coasts of Palk Bay, India using small subunit rDNA.

Coral endosymbionts act as a bio-indicator of coral ecosystem under extreme environmental conditions. The health of the coral ecosystem depends on the endosymbiont cell density of the coral hosts. Therefore, it is of interest to analyze ten coral fragments found to be under the genera Acropora, Favites, Favia, and Porites collected at various locations from Veedhalai to Mandapam, southeast coast of India during January 2019 to March 2019. The zooxanthellae cell count ranged between 4.08 (Porites sp.9) and 13.75x105 cells cm2 -1 (Favites sp.3). This indicates the health of the corals in the region. The genus (clade) level identification of endosymbionts was detected using the host excluding primers of small subunit DNA (nssrDNA). Bidirectional sequencing of 18S nrDNA gene (SSU) of all ten coral fragments show that the Veedhalai corals is associated with the genus Durusdinium (Clade D) but the corals of Mandapam is associated with the genera, Cladocopium (Clade C) and Durusdinium (Clade D). It is known that the thermal stress has negative impact on coral reef ecosystem of the world. The dominance of the genus Durusdinium in the scleractinian corals of Palk Bay may be due to frequent exposure to thermal stress. This thermotolerant endosymbionts is opportunistic. Thus, the corals of Veedhalai and Mandapam coasts, Palk Bay, India are necessarily packed with thermotolerant endosymbionts enabling conservation.

Insights on the biochemical and cellular changes induced by heat stress in the Cladocopium isolated from coral Mussismilia braziliensis.

Corals are treatened by global warming. Bleaching is one immediate effect of global warming, resulting from the loss of photosynthetic endosymbiont dinoflagellates. Understanding host-symbiont associations are critical for assessing coral's habitat requirements and its response to environmental changes. Cladocopium (formerly family Symbiodiniaceae clade C) are dominant endosymbionts in the reef-building coral, Mussismilia braziliensis. This study aimed to investigate the effect of temperature on the biochemical and cellular features of Cladocopium. Heat stress increased oxygen (O2) and decreased proteins, pigments (Chla + Chlc2), hexadecanoic acid- methyl ester, methyl stearate, and octadecenoic acid (Z)- methyl ester molecules. In addition, there was an increase in neutral lipids such as esterified cholesterol and a decrease in free fatty acids that may have been incorporated for the production of lipid droplets. Transmission electron microscopy (TEM) demonstrated that Cladocopium cells subjected to heat stress had thinner cell walls, deformation of chloroplasts, and increased lipid droplets after 3 days at 28°C. These findings indicate that thermal stress negatively affects isolated Cladocopium spp. from Mussismilia host coral.

Unraveling the metabolic effects of benzophenone-3 on the endosymbiotic dinoflagellate Cladocopium goreaui.

As a well-known pseudo-persistent environmental pollutant, oxybenzone (BP-3) and its related organic ultraviolet (UV) filters have been verified to directly contribute to the increasing mortality rate of coral reefs. Previous studies have revealed the potential role of symbiotic Symbiodiniaceae in protecting corals from the toxic effects of UV filters. However, the detailed protection mechanism(s) have not been explained. Here, the impacts of BP-3 on the symbiotic Symbiodiniaceae Cladocopium goreaui were explored. C. goreaui cells exhibited distinct cell growth at different BP-3 doses, with increasing growth at the lower concentration (2 mg L-1) and rapid death at a higher concentration (20 mg L-1). Furthermore, C. goreaui cells showed a significant BP-3 uptake at the lower BP-3 concentration. BP-3 absorbing cells exhibited elevated photosynthetic efficiency, and decreased cellular carbon and nitrogen contents. Besides, the derivatives of BP-3 and aromatic amino acid metabolism highly responded to BP-3 absorption and biodegradation. Our physiological and metabolic results reveal that the symbiotic Symbiodiniaceae could resist the toxicity of a range of BP-3 through promoting cell division, photosynthesis, and reprogramming amino acid metabolism. This study provides novel insights into the influences of organic UV filters to coral reef ecosystems, which urgently needs increasing attention and management.

The seasonal investigation of Symbiodiniaceae in broadcast spawning, Acropora humilis and brooding, Pocillopora cf. damicornis corals.

The density and diversity of Symbiodiniaceae associated with corals can be influenced by seasonal changes . This study provided the first annual investigation of Symbiodiniaceae density and diversity associated with Acropora humilis and Pocillopora cf. damicornis corals in the Gulf of Thailand using both zooxanthellae cell count and next-generation sequencing (ITS-1, ITS-2 regions) techniques, respectively. The results from this study indicated that zooxanthellae cell densities in both coral species differ significantly. The number of zooxanthellae was negatively correlated with the physical environment variable (light intensity). The diversity within A. humilis consisted of two genera, Cladocopium (Cspc_C3: 56.39%, C3w: 33.62%, C93type1: 4.42% and Cspf: 3.59%) and a small amount of Durusdinium (D1: 1.03%) whereas P. cf. damicornis was found to be 100% associated with Durusdinium (D1: 95.58%, D6: 1.01% and D10: 2.7%) suggesting that each coral species may select their appropriate genus/species of Symbiodiniaceae in response to local environmental stressors. The results of this study provided some information on the coral-Symbiodiniaceae relationship between seasons, which may be applied to predict the potential adaptation of corals in localized reef environments.

Lipidomes of phylogenetically different symbiotic dinoflagellates of corals.

The structural base of all membranes of symbiotic dinoflagellates (SD) is composed of glycolipids and betaine lipids, whereas triacylglycerols (TG) constitute an energy reserve and are involved in biosynthesis of glycolipids. Since data on the SD lipidome and the host's influence on symbionts' lipidome are scanty, we analyzed and compared the lipidomes of SD isolated from the zoantharian Palythoa tuberculosa and the alcyonarian Sinularia heterospiculata. A sequencing of nuclear gene regions showed that both cnidarians hosted the dinoflagellates Cladocopium sp. (subclades C1 and C3), but the zoantharian also contained the dinoflagellates Durusdinium trenchii (clade D). The presence of the thermotolerant D. trenchii resulted in a higher unsaturation of mono- and digalactosyldiacylglycerols (MGDG and DGDG), but a lower unsaturation of sulfoquinovosyldiacylglycerol (SQDG). The same features were earlier described for same SD from a reef-building coral. Hence, the profile of glycolipid molecules, which form SD thylakoid membranes, seems to be species-specific and does not depend on the host's taxonomic position. In contrast, the betaine lipid molecular species profile of diacylglyceryl-3-O-carboxyhydroxymethylcholine (DGCC), which forms SD cell membranes, can be influenced by the host. The profiles of the TG molecular species from freshly isolated SD have been determined for the first time. These molecular species can be divided on the basis of the acyl group in sn-2 position. The TG with 16:0 acyl group in sn-2 position may enrich total TG of a cnidarian colony and originate from SD cytoplasm. In contrast, TG 18:3/18:4/18:3 may be biosynthetically related with DGDG and concentrated in SD plastoglobules. Our data may be useful for further investigations of natural and technogenic variations in microalgal lipids and symbiont-host interactions in marine ecosystems.

Response of Coral Reef Dinoflagellates to Nanoplastics under Experimental Conditions Suggests Downregulation of Cellular Metabolism.

Plastic products contribute heavily to anthropogenic pollution of the oceans. Small plastic particles in the microscale and nanoscale ranges have been found in all marine ecosystems, but little is known about their effects upon marine organisms. In this study, we examine changes in cell growth, aggregation, and gene expression of two symbiotic dinoflagellates of the family Symbiodiniaceae, Symbiodinium tridacnidorum (clade A3), and Cladocopium sp. (clade C) under exposure to 42-nm polystyrene beads. In laboratory experiments, the cell number and aggregation were reduced after 10 days of nanoplastic exposure at 0.01, 0.1, and 10 mg/L concentrations, but no clear correlation with plastic concentration was observed. Genes involved in dynein motor function were upregulated when compared to control conditions, while genes related to photosynthesis, mitosis, and intracellular degradation were downregulated. Overall, nanoplastic exposure led to more genes being downregulated than upregulated and the number of genes with altered expression was larger in Cladocopium sp. than in S. tridacnidorum, suggesting different sensitivity to nano-plastics between species. Our data show that nano-plastic inhibits growth and alters aggregation properties of microalgae, which may negatively affect the uptake of these indispensable symbionts by coral reef organisms.

Depth Influences Symbiodiniaceae Associations Among Montastraea cavernosa Corals on the Belize Barrier Reef.

In Belize, shallow populations (10 and 16 m) of the coral species Montastraea cavernosa from the back reef and reef crest are genetically differentiated from deeper populations on the fore reef and reef wall (25 and 35 m). Like many species of scleractinian corals, M. cavernosa has an obligate symbiosis with dinoflagellate microalgae from the family Symbiodiniaceae. Here, we describe the Symbiodiniaceae taxa found within previously sampled and genotyped M. cavernosa populations along a depth gradient on the Belize Barrier Reef by implementing high-throughput sequencing of the ITS2 region of Symbiodiniaceae ribosomal DNA and the SymPortal analysis framework. While Symbiodiniaceae ITS2 type profiles across all sampling depths were almost entirely (99.99%) from the genus Cladocopium (formerly Symbiodinium Clade C), shallow (10 and 16 m) populations had a greater diversity of ITS2 type profiles in comparison to deeper (25 and 35 m) populations. Permutational multivariate analysis of variance (PERMANOVA) confirmed significant differences in ITS2 type profiles between shallow and deep sample populations. Overall Symbiodiniaceae communities changed significantly with depth, following patterns similar to the coral host's population genetic structure. Though physiological differences among species in the cosmopolitan genus Cladocopium are not well-described, our results suggest that although some members of Cladocopium are depth-generalists, shallow M. cavernosa populations in Belize may harbor shallow-specialized Symbiodiniaceae not found in deeper populations.

Monoclonal Culture and Characterization of Symbiodiniaceae C1 Strain From the Scleractinian Coral Galaxea fascicularis.

The symbiosis between cnidarian hosts and photosynthetic dinoflagellates of the family Symbiodiniaceae (i.e., zooxanthellae) provides the energy foundation of coral reef ecosystems in oligotrophic waters. The structure of symbiont biota and the dominant species of algal symbiont partly shape the environmental adaptability of coral symbiotes. In this study, the algal symbiont cells were isolated from the tentacles of Galaxea fascicularis, a hermatypic coral with obvious differentiation in heat resistance, and were cultured in vitro with an improved L1 medium. An algal monoclonal cell line was established using separated algal culture drops and soft agar plating method, and named by GF19C1 as it was identified as Cladocopium sp. C1 (Symbiodiniaceae) based on its ITS1, ITS2, and the non-coding region of the plastid psbA minicircle (psbA ncr ) sequences. Most GF19C1 cells were at the coccoid stage of the gymnodinioid, their markedly thickened (ca. two times) cell wall suggests that they developed into vegetative cysts and have sexual and asexual reproductive potential. The average diameter of GF19C1 cells decreased significantly, probably due to the increasing mitotic rate. The chloroplasts volume density of GF19C1 was significantly lower than that of their symbiotic congeners, while the surface area density of thylakoids relative to volumes of chloroplasts was not significantly changed. The volume fraction of vacuoles increased by nearly fivefold, but there was no significant change in mitochondria and accumulation bodies. Light-temperature orthogonal experiments showed that, GF19C1 growth preferred the temperature 25 ± 1°C (at which it is maintained post-isolation) rather than 28 ± 1°C under the light intensity of 42 ± 2 or 62 ± 2 µmol photons m-2 s-1, indicating an inertia for temperature adaptation. The optimum salinity for GF19C1 growth ranged between 28-32 ppt. The monoclonal culture techniques established in this study were critical to clarify the physiological and ecological characteristics of various algal symbiont species, and will be instrumental to further reveal the roles of algal symbionts in the adaptive differentiation of coral-zooxanthellae holobionts in future studies.

Photosynthetic capacity of the endosymbiotic dinoflagellate Cladocopium sp. is preserved during digestion of its jellyfish host Mastigias papua by the anemone Entacmaea medusivora.

The sea anemone Entacmaea medusivora (Actiniaria, Anthozoa) commonly feeds on the golden jellyfish Mastigias papua (Rhizostomeae, Scyphozoa) which harbours an endosymbiotic dinoflagellate of the genus Cladocopium (Symbiodiniaceae). In this study, we monitored the photosynthetic activity of the endosymbiotic microalgae while their host jellyfish were ingested and digested by starved medusivorous anemones. By analyzing the photosynthetic yield of photosystem II, we observed that Cladocopium cells remain photosynthetically competent during the whole digestion process, thus confirming the exceptional resistance of Symbiodiniaceae to digestive enzymes. In the gastric cavity of E. medusivora, Cladocopium cells release oxygen, which could broadly stimulate the gastric microbiotic flora of the sea anemone. Ultimately, E. medusivora is not able to retain Cladocopium cells more than few days and physiologically-unaltered cells are therefore expelled in faecal pellets. The potential contribution of E. medusivora to maintain a reservoir of Cladocopium symbionts and its role in the life cycle of M. papua is discussed.

Thermal and Herbicide Tolerances of Chromerid Algae and Their Ability to Form a Symbiosis With Corals.

Reef-building corals form an obligate symbiosis with photosynthetic microalgae in the family Symbiodiniaceae that meet most of their energy requirements. This symbiosis is under threat from the unprecedented rate of ocean warming as well as the simultaneous pressure of local stressors such as poor water quality. Only 1°C above mean summer sea surface temperatures (SSTs) on the Great Barrier Reef (GBR) can trigger the loss of Symbiodiniaceae from the host, and very low concentrations of the most common herbicide, diuron, can disrupt the photosynthetic activity of microalgae. In an era of rapid environmental change, investigation into the assisted evolution of the coral holobiont is underway in an effort to enhance the resilience of corals. Apicomplexan-like microalgae were discovered in 2008 and the Phylum Chromerida (chromerids) was created. Chromerids have been isolated from corals and contain a functional photosynthetic plastid. Their discovery therefore opens a new avenue of research into the use of alternative/additional photosymbionts of corals. However, only two studies to-date have investigated the symbiotic nature of Chromera velia with corals and thus little is known about the coral-chromerid relationship. Furthermore, the response of chromerids to environmental stressors has not been examined. Here we tested the performance of four chromerid strains and the common dinoflagellate symbiont Cladocopium goreaui (formerly Symbiodinium goreaui, ITS2 type C1) in response to elevated temperature, diuron and their combined exposure. Three of the four chromerid strains exhibited high thermal tolerances and two strains showed exceptional herbicide tolerances, greater than observed for any photosynthetic microalgae, including C. goreaui. We also investigated the onset of symbiosis between the chromerids and larvae of two common GBR coral species under ambient and stress conditions. Levels of colonization of coral larvae with the chromerid strains were low compared to colonization with C. goreaui. We did not observe any overall negative or positive larval fitness effects of the inoculation with chromerid algae vs. C. goreaui. However, we cannot exclude the possibility that chromerid algae may have more important roles in later coral life stages and recommend this be the focus of future studies.