Abstract Experimentally evolved, heat-tolerant algal symbionts (heat-evolved; HE) offer a promising means of enhancing coral holobiont thermotolerance under rapidly warming oceans. However, translating their benefits into restoration practices requires scalable delivery methods. Coral tissue fusion may provide one such pathway by facilitating HE symbiont transfer to wild corals; however, its feasibility remains largely untested. As an initial test, we paired adult isografts of Galaxea fascicularis and Psammocora columna hosting HE Cladocopium proliferum (SS8) with chemically bleached, SS8-naive recipients. Fusion was first observed after three days in G. fascicularis and nine days in P. columna . In both species, fusion was followed by increased pigmentation and photochemical efficiency at the recipient’s fusion interface relative to distal tissue and unfused controls. After ∼50 days, SS8 was detected at low levels (<3.5%) in 15/19 fused G. fascicularis recipients, although detection was also common among unfused horizontal-transmission controls maintained in the same water column (13/18). These findings provide the first empirical evidence that conspecific coral tissue fusion is associated with localised physiological recovery and can coincide with HE symbiont acquisition, while highlighting the need to distinguish tissue-mediated transfer from background horizontal transmission. Fusion may therefore represent a complementary pathway for beneficial symbiont delivery in assisted-evolution frameworks.

Heat-tolerant coral stock could supplement populations with tolerance-conferring alleles to combat rising sea surface temperatures and marine heat waves. We tested coral selective breeding and Symbiodiniaceae experimental evolution independently and in tandem as interventions for generating heat-tolerant stock. Broodstock from two sites were ranked using Symbiodiniaceae photochemical efficiency under rapid heat stress and crossed to produce offspring from heat-tolerant colonies (top 25th percentile) and control offspring. Offspring were inoculated with heat-evolved or wild-type symbionts and exposed to 28°C (ambient) or 32°C (elevated) for 2 months. Selective breeding using rapid assays enhanced Davies but not Moore Reef recruit survival and growth at 32°C, suggesting that this method does not universally generate heat-tolerant coral due to genetics, maternal effects, and/or acclimation. Heat-evolved symbionts enhanced survival and bleaching resilience at 32°C but reduced growth at 28°C. Combining interventions yielded additive benefits, no enhancement, or resulted in one intervention diminishing the other's impact. These results demonstrate assisted evolution's potential while cautioning against generalizing its outcomes.

Experimental evolution under elevated temperatures has generated heat-evolved (HE) strains of Symbiodiniaceae that enhance coral bleaching tolerance. However, the biomolecular mechanisms underlying this resilience remain poorly understood. We conducted a laboratory heat-stress experiment and applied synchrotron-based Fourier transform infrared (FTIR) microspectroscopy to examine physiological and biomolecular responses of HE (strain: SS8) and wild-type (strain: WT10) Cladocopium proliferum to thermal stress across three physiological contexts: in hospite, expelled, and cultured. In hospite, both strains exhibited heat-induced increases in free amino acids, phosphorylated compounds, and lipids, coupled with reduced protein content - hallmarks of cellular stress. SS8, however, showed a dampened response overall, in line with an improved thermotolerance based on holobiont phenotypes. Expelled and in hospite cells shared broadly similar biomolecular profiles, though expelled cells of both strains responded less strongly - indicating expulsion may relieve host-imposed stress. Cultured cells differed from in hospite and expelled cells but showed similar strain-specific trends. WT10 responded strongly to heat stress - displaying depleted amino acids, phosphorylated metabolites, and disrupted lipid balance - whereas SS8 mounted a relatively muted metabolic response. These findings support the potential of HE symbionts for reef restoration, highlight the importance of physiological context in assessing Symbiodiniaceae thermotolerance, and the utility of single-cell FTIR microspectroscopy.

Symbiodiniacean dinoflagellates are fundamental to the functioning of coral reefs, underpinning primary production, nutrient cycling, and calcification through intimate intracellular symbioses with corals and other marine invertebrates. The identity and functional traits of these endosymbionts strongly influence host physiology, particularly thermal tolerance and stress resilience. Despite their ecological importance, Symbiodiniaceae have not yet been characterized within a formal functional ecology framework. Trait-based functional ecology enables standardized comparative analysis using metrics including functional diversity (richness, evenness, and divergence) and redundancy, which are critical for assessing ecosystem stability and vulnerability. Progress on this front requires elucidating clearly defined traits for Symbiodiniaceae. Here, we propose a standardized functional trait framework for these organisms. We identify key conceptual and methodological barriers that have hindered the integration of Symbiodiniaceae into formal descriptions of functional ecology, including unresolved species boundaries, limited trait standardization, and the context-dependent expression of traits in hospite versus in vitro. Building on principles from trait-based ecology, supported by empirical data and experimental measurements, we define and propose a set of 19 functional traits categorized into nine core functions: photosynthesis, photoprotection, cellular growth, population growth, energy reserves and composition, symbiotic relationship, nitrogen assimilation, ecological plasticity, and thermal tolerance. These traits capture fundamental dimensions of algal symbiont performance, including resource acquisition, stress tolerance, metabolic allocation, and host interaction, providing a foundation for calculating functional diversity metrics. Integrating Symbiodiniaceae into a functional trait framework will improve our capacity to assess functional redundancy, vulnerability, and resilience of coral reefs, ultimately strengthening forecasts of reef persistence under ongoing climate change.

The intimate relationship between scleractinian corals and endosymbiotic dinoflagellates (Symbiodiniaceae) drives primary production and calcification in reef ecosystems. Despite the extensive research focused on coral-Symbiodiniaceae relationships at shallow depths, only a few studies have assessed the symbiont diversity at mesophotic depths. Here, we examined the Symbiodiniaceae diversity associated with nine putative mesophotic Leptoseris species in Australian reefs using the ITS2 and COI marker regions. Amplicon sequencing revealed an almost exclusive association with the genus Cladocopium . While the COI region retrieved four distinct haplotypes, 17 ITS2 profiles grouped into five clusters were identified. Across the Symbiodiniaceae profiles, 24% were found across wide depth ranges, although 53% were only observed at mesophotic depths. Despite a certain degree of host-symbiont specificity, geography and depth also contributed significantly to the composition of Symbiodiniaceae communities. These findings provide much-needed insights into the diversity of Symbiodiniaceae associations with mesophotic Leptoseris , and emphasize the need for further research to establish to what extent lower mesophotic habitats in the Indo-Pacific harbor unique versus generalist endosymbiont associations.

Oxylipins are oxygenated products of fatty acids proposed to exert a regulatory role in cnidarian-dinoflagellate symbiosis; however, this has not been investigated in detail. We integrated physiological measurements and molecular phenotyping with comparative transcriptome mining to examine how the symbiotic cnidarian model, the sea anemone Aiptasia (i.e. Exaiptasia diaphana), and its dinoflagellate symbiont Breviolum minutum respond to symbiosis and thermal stress. We performed lipidomics in combination with the quantification of oxylipins, including octadecanoids, eicosanoids, and docosanoids derived from C18, C20, and C22 fatty acids, respectively, and reconstructed their putative biosynthetic routes through cross-phylogenetic protein sequence homology. Relative to aposymbiotic, symbiotic anemones were enriched with omega-3 fatty acids and downstream octadecanoids of symbiont origin, consistent with inter-partner metabolite flux. Cytochrome P450-derived eicosanoids and docosanoids increased up to 300-fold in symbiotic versus aposymbiotic anemones. Under elevated temperature, anemones showed minor changes in their physiology and lipid profiles; however, the symbiont fraction displayed multiple signatures of stress. In comparison, aposymbiotic anemones showed a 50% reduction in protein abundance as well as structural and storage lipids, while simultaneously accumulating oxylipins linked to inflammation and oxidative stress. Our findings report novel oxylipins that have not been previously observed in dinoflagellates. We identified regulatory pathways that are conserved across cnidarians and higher metazoans, advancing our understanding of cnidarian-dinoflagellate symbiosis and its response to warming climate. We are targeting specific oxylipins and signalling pathways for further research that may aid molecular intervention strategies for selective breeding and assisted evolution to enhance coral resilience in warming oceans.

Anthropogenic climate change has driven many coral reef ecosystems to the brink of collapse as extreme temperature events cause widespread bleaching and mortality. Interventions that boost coral resilience are being developed to help restore reefs, and one such intervention is the manipulation of a coral’s symbiotic algae (which greatly affects their host’s physiology). New experimentally evolved symbiont strains have shown promise in increasing their host’s thermal tolerance, but how they will perform in juvenile corals when other strains are present is unclear. This study investigates the uptake and establishment of experimentally evolved Cladocopium proliferum and Durusdinium trenchii in mixed inocula and describes the effects hosting multiple symbiont strains has on the growth and survival of juvenile corals. Our results reveal that while both strains in the mixed culture inocula were taken up, most recruits were dominated by D. trenchii. Corals hosting D. trenchii over C. proliferum also displayed faster growth despite previous studies showing the opposite. We show that the costs and benefits of hosting mixed communities are highly dependent on the identities of the individual strains therein and suggest the compatibility of symbiont–symbiont–host assemblages should be assessed before being used for restoration.

Aim: Understanding local adaptation in species along environmental gradients provides insight for how species can and will adapt to increasing climate-driven marine heatwaves. Here, the population structure of two closely related Pocillopora corals was evaluated across environmentally heterogeneous reef gradients to understand spatially variable adaptive potential of vulnerable species. Location: Australia's Coral Sea and Great Barrier Reef Marine Parks span a ca. 1300 km latitudinal gradient across a 1.6 degrees C range in Maximum Monthly Mean temperatures. Method: A total of 255 colonies of Pocillopora verrucosa and 188 colonies of Pocillopora cf. meandrina were prepared and genotyped by sequencing using Illumina NovaSeq. Initial filtering and population statistics were conducted for each species. Correlations between genetic structure and environmental variables were assessed using partial redundancy analyses. The specific effects of environmental (including temperature, which was also analysed separately) and spatial variables were quantified to understand the drivers of local adaptation and how this varies across taxa and regions. Results: Both species exhibited gene flow between the two reef systems (Coral Sea and Great Barrier Reef), indicating connectivity across similar to 12 degrees of latitude and longitude. Population structure differed between species: P. cf. meandrina displayed pronounced genetic subdivision with two ancestral lineages partitioned along a north-south axis, while P. verrucosa maintained one dominant lineage across most reefs. In both species, population structure was shaped by ancestral lineage and thermal history. Additionally, P. cf. meandrina showed genetic structure correlated with sea current velocity, whereas P. verrucosa correlated with sea surface temperature. Main Conclusions: Closely related taxa can exhibit distinct population genetic patterns and environmental responses, reflecting different capacities for local adaptation. These results highlight the importance of incorporating multiple taxa and environmental variables when predicting responses to environmental change. Conservation strategies relying solely on environmental data may suffice for some species but not others.

Coral bleaching is a widespread stress response of reef-building corals to elevated sea temperatures, resulting in the loss of symbiotic algae and often leading to coral death and reef degradation. Although coral bleaching occurs globally, not all reefs, species, colonies, or polyps bleach equally. Understanding intra-colony bleaching heterogeneity is crucial to anticipate the extent of coral loss at 2°C warming and harness variability to inform restorative interventions. Partially bleached coral colonies are commonly documented yet rarely tracked to determine whether they reflect ecologically distinct heterogeneity (e.g., in thermal tolerance) or eventually bleach completely. Focusing on bleaching that appears restricted to certain areas within a coral colony, we examine its putative basis in the spatial variability of the holobiont. A coral's three-dimensional structure creates mosaics of microenvironments. Adaptations to these microenvironments are underpinned by intra-colony differences in Symbiodiniaceae association, microbiome assemblage, and nutritional status, giving rise to microhabitats. Genetic mosaicism and epigenetic changes further contribue to intra-colony phenotypic heterogeneity. We pinpoint methodologies to align spatially restricted bleaching to different forms of coral surface heterogeneity, examine the common assumption that coral fragments represent entire colonies, and illuminate implications for coral biology and restoration.

Traditional coral reef restoration methods often fail to consider rising sea-surface temperatures driven by climate change. The introduction of experimentally heat-evolved algal symbionts into corals offers a promising solution by enhancing coral holobiont thermotolerance in a relatively short timeframe. However, the scalability of this approach remains a key challenge. Coral expulsion of viable symbiont cells may provide a passive pathway for upscaling this intervention by facilitating the widespread transmission of heat-evolved symbionts and their physiological benefits across coral reefs. Here, we investigated the expulsion and horizontal transmission dynamics of heat-evolved Cladocopium proliferum (strain SS8) in the scleractinian coral Galaxea fascicularis. First, we assessed the 24-hour symbiont expulsion dynamics of three colonies of G. fascicularis hosting SS8 in addition to homologous symbionts. SS8 was detected in the expelled symbiont community of all colonies, with diel peaks in mitotic index and photochemical efficiency observed at night and the majority of expelled cells appearing morphologically intact. Second, we tested whether expelled SS8 could be acquired by chemically bleached adult G. fascicularis fragments in a custom-designed multi-lane raceway experiment. After 55 days of exposure to an SS8-expelling G. fascicularis donor, SS8 was detected at background levels (≤0.06%) in 11.1% of recipient fragments (5/45). These findings provide the first empirical evidence that viable, heat-evolved symbionts can be expelled and acquired by bleached adult corals, highlighting a potential natural pathway for the scaling up of this intervention to enhance coral thermal resilience.

Pocilloporid corals and their Symbiodiniaceae symbionts have co-evolved. Host–symbiont associations might be driven by adaptation to distinct ecological niches. Here, we used single nucleotide polymorphisms (SNPs) to examine host population structure, characterised Symbiodiniaceae associations in shaded and exposed areas of coral using Internal Transcribed Spacer (ITS2) metabarcoding, and identified photobiological phenotypes of Pocillopora acuta colonies from two acidic, deoxygenated and highly variable temperature mangrove environments versus two adjacent reef locations. We found two genetic clusters of P. acuta with evidence of potential hybrid individuals. Limited admixture suggests low levels of gene flow between the reef and mangrove sites. Each of the two lineages was predominantly associated with either reef or mangrove habitats, with distinct dominant symbionts (Cladocopium (reef) and Durusdinium (mangrove)), each with different photobiological strategies. Hybrid individuals exhibited greatest heterogeneity in ITS2 profiles compared to the two other populations. Our results provide evidence that the two lineages are part of a known species complex as suggested by population structure and morphological differences. The genetic distinctiveness of the sampled populations emphasises the unique diversity within the extreme environments. Consequently, conservation efforts should aim to minimise additional anthropogenic impacts at these sites.

The heat tolerance of corals is largely determined by their microbial photosymbionts (Symbiodiniaceae, colloquially known as zooxanthellae). Therefore, manipulating symbiont communities may enhance the ability of corals to survive summer heatwaves. Although heat-tolerant and -sensitive symbiont species occur in nature, even corals that harbour naturally tolerant symbionts have been observed to bleach during summer heatwaves. Experimental evolution (i.e., laboratory selection) of Symbiodiniaceae cultures under elevated temperatures has been successfully used to enhance their upper thermal tolerance, both in vitro and, in some instances, following their reintroduction into corals. In this review, we present the state of this intervention and its potential role within coral reef restoration, and discuss the next critical steps required to bridge the gap to implementation.

The thermal tolerance of symbiodiniacean photo-endosymbionts largely underpins the thermal bleaching resilience of their cnidarian hosts such as corals and the coral model Exaiptasia diaphana. While variation in thermal tolerance between species is well documented, variation between conspecific strains is understudied. We compared the thermal tolerance of three closely related strains of Breviolum minutum represented by two internal transcribed spacer region 2 profiles (one strain B1-B1o-B1g-B1p and the other two strains B1-B1a-B1b-B1g) and differences in photochemical and non-photochemical quenching, de-epoxidation state of photopigments, and accumulation of reactive oxygen species under rapid short-term cumulative temperature stress (26-40 degrees C). We found that B. minutum strains employ distinct photoprotective strategies, resulting in different upper thermal tolerances. We provide evidence for previously unknown interdependencies between thermal tolerance traits and photoprotective mechanisms that include a delicate balancing of excitation energy and its dissipation through fast relaxing and state transition components of non-photochemical quenching. The more thermally tolerant B. minutum strain (B1-B1o-B1g-B1p) exhibited an enhanced de-epoxidation that is strongly linked to the thylakoid membrane melting point and possibly membrane rigidification minimizing oxidative damage. This study provides an in-depth understanding of photoprotective mechanisms underpinning thermal tolerance in closely related strains of B. minutum. Closely related strains of Breviolum minutum , the dinoflagellate endosymbiont of a sea anemone, have distinct upper thermal tolerances and respond to heat stress by differentially activating a range of photoprotective mechanisms.

Summary Coral thermal bleaching resilience can be improved by enhancing photosymbiont thermal tolerance via experimental evolution. While successful for some strains, selection under stable temperatures was ineffective at increasing the thermal threshold of an already thermo‐tolerant photosymbiont (Durusdinium trenchii). Corals from environments with fluctuating temperatures tend to have comparatively high heat tolerance. Therefore, we investigated whether exposure to temperature oscillations can raise the upper thermal limit of D. trenchii. We exposed a D. trenchii strain to stable and fluctuating temperature profiles, which varied in oscillation frequency. After 2.1 yr (54–73 generations), we characterised the adaptive responses under the various experimental evolution treatments by constructing thermal performance curves of growth from 21 to 31°C for the heat‐evolved and wild‐type lineages. Additionally, the accumulation of extracellular reactive oxygen species, photophysiology, photosynthesis and respiration rates were assessed under increasing temperatures. Of the fluctuating temperature profiles investigated, selection under the most frequent oscillations (diurnal) induced the greatest widening of D. trenchii's thermal niche. Continuous selection under elevated temperatures induced the only increase in thermal optimum and a degree of generalism. Our findings demonstrate how differing levels of thermal homogeneity during selection drive unique adaptive responses to heat in a coral photosymbiont.

Interspecific hybridisation increases genetic diversity and has played a significant role in the evolution of corals in the genus Acropora. In vitro fertilisation can be used to increase the frequency of hybridisation among corals, potentially enhancing their ability to adapt to climate change. Here, we assessed the field performance of hybrids derived from the highly cross-fertile coral species Acropora sarmentosa and Acropora florida from the Great Barrier Reef. Following outplanting to an inshore reef environment, the 10-month survivorship of the hybrid offspring groups was intermediate between that of the purebred groups, although not all pairwise comparisons were statistically significant. The A. florida purebreds, which had the lowest survivorship, were significantly larger at 10 months post-deployment compared to the other three groups. The four offspring groups harboured the same intracellular photosymbiont communities (Symbiodiniaceae), indicating that observed performance differences were due to the coral host and not photosymbiont communities. The limited differences in the performance of the groups and the lack of outbreeding depression of the F1 hybrids in the field suggest that interspecific hybridisation may be a useful method to boost the genetic diversity, and as such increase the adaptive capacity, of coral stock for restoration of degraded and potentially genetically eroded populations.

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 psbA ncr ), 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.

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.