Introduction

Coral reefs are one of the most diverse and valuable ecosystems on Earth, providing significant ecological, economic, and societal benefits1,2. Coral reefs have rapidly declined worldwide due to climate change and human activities, resulting in reduced ecosystem services3. Recent predictions highlighted that continued warming could lead to significant coral loss4. Corals rely on the symbiotic relationship with the dinoflagellate photosynthetic microalgae of the family Symbiodiniaceae5,6. The microalgae cells live inside the coral host and provide the required energy to build the calcium carbonate skeletons, exerting a crucial role in the holobiont5. Prolonged exposure to elevated temperatures can break this relationship, leading to bleaching (i.e., the loss of the coral-associated Symbiodiniaceae7, which is also followed by the loss of the algae pigments and is indicative of coral health decline) and, ultimately, coral death8. Marine heatwaves, characterized by periods of extremely high sea surface temperature that can span thousands of kilometres, have become more frequent, contributing to four global coral bleaching events9. These conditions challenge corals’ ability to maintain their symbiotic relationship, often resulting in widespread coral mortality10. Hence, efforts are underway to identify coral species and populations with enhanced heat stress resistance for restoration and conservation efforts5,11. Understanding how corals respond to temperature changes, as well as their capacity for long-term acclimation and adaptation, is essential for developing effective strategies to mitigate the impacts of these increasingly common marine heatwaves. The thermal threshold of corals (the maximum temperature that a coral can tolerate) has been studied for a variety of species across many locations and appears to commonly align with local long-term thermal summer maxima11,12. Thus, while absolute thermal thresholds differ, corals typically exhibit bleaching under prolonged heat stress exceeding only 1–2 °C above their maximum summer mean temperature (i.e., MMM)13,14. The exposure to repeated warming events (e.g. heatwaves) may enhance thermal tolerance, improving projections of coral survival in successive warming events15,16. In addition, the variability of thermal regimes experienced by corals was shown to affect their thermal tolerance so that higher daily or seasonal thermal fluctuations positively affect thermal resilience and thus may provide a higher adaptive capacity of tolerance to heat stress17,18. In recent years, the development of standardized short-term acute heat stress assays18 has allowed the experimental assessment of coral thermal tolerance (and resilience), as well as their connection to the prevailing local and/or regional thermal regime19.

Coral physiology (e.g. photosynthetic efficiency, calcification, symbiont density) has been demonstrated to vary seasonally20,21,22,23. However, the extent of seasonal variation has been shown to be species-specific20. More importantly, whether physiological acclimation is affecting seasonal thermal thresholds, in particular in the framework of standardized stress testing (in the form of acute thermal assays, e.g. CBASS) is currently unresolved. Such information may also aid to better predict responses to changes in temperature due to climate change24,25. Thus, it is key to assess whether coral thermal tolerance thresholds change over time, based on seasonal thermal fluctuations, and how these changes potentially affect the capacity of corals to respond to changes in temperature.

The Red Sea is considered an underwater laboratory because of its particular features and extreme thermal regimes experienced by the organisms inhabiting it. Corals in the central Red Sea are exposed to temperatures exceeding 30 °C in summer, while the coldest temperatures recorded are around 24 °C18,26,27. The natural thermal variation experienced by corals in this region suggests mechanisms of acclimation and/or adaptation for the different species to cope with this fluctuation year-round. Here, we experimentally assess the thermal tolerance threshold of two coral species using standardized acute thermal stress assays18, complemented by in situ monitoring of the photochemical efficiency of the coral-associated Symbiodiniaceae community (Fv/Fm) as a response to seasonal changes in temperature in the central Red Sea.

Results and discussion

Colonies of P. verrucosa and Acropora spp. were examined during different periods of the year, encompassing all four seasons in Al Fahal Reef (central Red Sea) to determine whether coral thermal threshold varies as a function of the in situ temperature. The in situ photosynthetic efficiency (Fv/Fm) of both species varied significantly with seawater temperature (Fig. 1, Supplementary Table 1), exhibiting an unimodal thermal response curve. The maximum Fv/Fm performance for both species was recorded between November and December 2021 when in situ daily mean seawater temperatures were 28-29 °C, in agreement with previous results from the same region26,28,29. Minimum Fv/Fm values were recorded during the temperature peak in the summer of 2021 and 2022, when seawater temperatures were above 31 °C. The thermal optima (Topt) was 28.62 °C (95% CI [28.49, 28.73]) for Acropora spp. and 28.60 °C (95% CI [28.25, 28.79]) for P. verrucosa, with no statistically significant differences in the thermal performance curve between both species (Supplementary Table 1). The overall Topt estimated considering the combined thermal performance curves for P. verrucosa and Acropora spp. was 28.36 °C (95% CI [28.18, 28.58]). The trend of high Fv/Fm values in winter and low Fv/Fm in summer has also been reported previously for colonies of Stylophora pistillata and Favia favus from the Gulf of Aqaba22. In this case, the temperature at which the maximum performance was measured was around 23-25 °C, lower than that reported in our study. This difference is not surprising due to the lower ambient seawater temperatures in the northern Red Sea compared to the central Red Sea11.

Fig. 1: Relationship between in situ photosynthetic efficiency (Fv/Fm) and in situ mean seawater temperature for Acropora spp. and P. verrucosa.
figure 1

The transparent points represent coral colonies. Bright colored points represent the mean and error bars show the standard error of the mean (s.e.m). Lines denote the thermal performance curve estimated by the Sharpe-Schoofield model and the shadow area around the fitting line is the 95% confidence interval estimated using bootstrapping method. The number of experiments included were nP.verrucosa = 12 and nAcropora spp. = 7. The number of biological replicates included in each experiment is given in Supplementary Table 6.

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To assess species-specific differences in thermal tolerance over seasons, we experimentally determined the thermal thresholds for each species at different sampling periods. Thermal thresholds were estimated by calculating the temperature at which the photosynthetic efficiency rate (Fv/Fm) is reduced to 50% (based on the ecotoxicological term ED50)18,30. ED50 estimated using photosynthetic efficiency rates (Fv/Fm) has been demonstrated as a standardized proxy for coral thermotolerance and it captures differences in the susceptibility of corals to bleach18. We determined the thermal threshold of two different species at different times of the year including in situ temperatures ranging from 24.5 to 32 °C. We observed fluctuations in the estimated ED50 values during the various sampling periods. The shape of the performance curves based on experimental Fv/Fm rates over assay temperatures differed markedly between the two coral species, indicating a species-specific response to increasing temperature (Fig. 2). P. verrucosa exhibited different thermal response curves at different sampling times: corals sampled during the coldest period exhibited a gradual and continuous decrease in Fv/Fm at increasing experimental temperatures, whereas corals sampled in the warmer period maintained their Fv/Fm values up to a threshold and then dropped drastically (Fig. 2). Differences in the shape of the thermal performance curves, similar to those observed here for P. verrucosa colonies, have been previously reported11. Although our study cannot pinpoint the exact mechanisms behind the changes in the shape of the performance curves, it appears to be related to the colonies’ exposure to a gradual increase in temperature over time. The ED50 values estimated for P. verrucosa increased from 35.42 ± 0.53 °C in colder months to 38.10 ± 0.54 °C in warmer periods (August 2021), corresponding to the peak in seawater temperature.

Fig. 2: Thermal performance curves for each CBASS experiment.
figure 2

Changes in Fv/Fm with assay temperature for each CBASS experiment performed using P. verrucosa (A) and Acropora spp. (B). Dots represent the relationship between Fv/Fm and experimental temperature for each CBASS run. Lines reflect the log-logistic model fitted to each experiment (nP. verrucosa = 10 & nAcropora spp = 5). Color code represents in situ temperature at the time of sample collection for the CBASS experiment. The dots’ shape and lines represent the different seasons: winter (January to March), Spring (April to June), Summer (July to September) and Autumn (October to December). The details on the biological replicates sampled in each experiment are given in Supplementary Table 6.

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On the other hand, Acropora spp. had a more stable pattern, sustaining Fv/Fm to a threshold before exhibiting a sharp decline (Fig. 2). The fact that different corals exhibit different shapes in their thermal performance curves after being exposed to thermal stress might suggest differential susceptibility to warming11. The ED50 range for Acropora spp. (36.76 ± 0.68 °C to 37.73 ± 1.02 °C) was narrower (~1 °C) than that of P. verrucosa (~3 °C), despite both being exposed to similar seasonal temperature ranges (P. verrucosa: 24.5-32 °C, Acropora spp.: 24.5-31.4) and experimental conditions (30–39 °C). A quantitative comparison of thermal thresholds indicated that ED50 of P. verrucosa significantly differed between warmer and colder periods, while Acropora spp. showed no significant changes (Table 1). Differences in the thermal threshold of around 1 °C between summer and winter seasons for Pocillopora damicornis have been previously reported using a different experimental approach, consisting on 5-day tank experiments using corals located on the Great Barrier Reef31. However, here we observe differences up to 3 °C using a standardized comparison. These findings suggest that different acclimation mechanisms to environmental conditions may occur in the same area, depending on the species and even different genotypes, which is evidenced by the high variability observed between biological replicates (Supplementary Figs. 1 and 2).

Table 1 Experimentally estimated thermal threshold for each species at each sampling time
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To investigate whether prevailing seawater temperature had an effect on the observed variability of the coral thermal thresholds, we studied the relationship between the changes in ED50 and the average seawater temperature measured in situ at the sampling time. P. verrucosa thermal threshold values (ED50) showed a flat response until Topt was reached (28.36 °C), followed by a steep linear correlation with the increase of the seawater temperature (Fig. 3A, Supplementary Table 2, linear mixed-effect model: p < 0.001). Although the pattern was similar to P. verrucosa, no significant increase was observed in the ED50 for Acropora spp. (Fig. 3B, Supplementary Table 3, linear mixed-effect model: p = 0.08), suggesting a more stable thermal threshold across different seasons and a different species strategy to respond to temperature change. Some variation in thermal tolerance has been previously reported for P. damicornis31; however, establishing a clear relationship with the in situ temperature has not been previously possible due to the lack of year-round coral monitoring data. Although the identification of the mechanism driving the differential response between species is out of the scope of this work, the seasonal changes observed here suggest that some coral species (e.g. genus Pocillopora) might be capable of acclimatizing to gradual changes in temperature in the short-term.

Fig. 3: Linking the thermal tolerance threshold of the corals with in situ performance and seawater temperature.
figure 3

Changes in the thermal tolerance threshold (ED50) with seawater temperature at the time of sampling for P. verrucosa (A) and Acropora spp. (B). The gray dots correspond to the individual colonies, and the fitting line is the result of the linear regression considering in situ temperature above Topt. The gray area represents the 95% confidence interval (CI). Full dots correspond to the mean ED50 of each experiment and the error bars to the standard error of the mean (s.e.m.). We found that ED50 was positively correlated with in situ temperature (Linear mixed-effect model, p < 0.001) for P. verrucosa, while this relationship was positive but not significant for Acropora spp (Linear mixed-effect model, p = 0.08) (Supplementary Tables 2 and 3). Relationship between in situ photosynthetic efficiency, Fv/Fm, and ED50 for P. Verrucosa (C) and Acropora spp. (D). The gray dots correspond to the individual replicates corresponding to the experiments performed at in situ temperatures above the Topt of the coral. The solid line corresponds to the linear model fitting, and the gray area represents the 95% CI. The full dots represent the average ED50 and Fv/Fm and the error bars correspond to the s.e.m of each variable. We found that ED50 was negatively correlated with in situ Fv/Fm in P. verrucosa (Linear mixed-effect model, p = 0.01), but not in Acropora spp (Linear mixed-effect model, p = 0.94) (Supplementary Table 4 and 5). The number of experiments included were nP.verrucosa = 8 and nAcropora spp. = 4. The number of biological replicates included in each experiment is given in Supplementary Table 6.

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The species included in this study exhibited different experimentally derived standardized thermal tolerance thresholds (ED50s), surpassing (~7 °C) the maximum monthly mean (MMM) of the region (MMM AL Fahal reef = 30.75 °C) in summer. This offset between ED50 and local MMM is typically observed and aligns with what has been previously shown in the area11,30,32. Importantly, the fact that the thermal threshold is not a fixed value (for a given species and location), underlines the significance of characterizing seasonal variations in coral thermal tolerance. This understanding aids in predicting responses to temperature changes throughout the year, which is crucial when abrupt temperature shifts occur (e.g. heatwaves events). Some coral species, like P. verrucosa, show a gradual thermotolerance increase, aiding to cope with summer heat (~32–34 °C) and adapting to seasonal temperature variations, including winter cold stress (~20–24 °C)27,29. The decline in P. verrucosa thermal threshold during winter may be an acclimation strategy against cold stress. While cold stress data are limited compared to warm stress, corals have shown paling during winter months when temperatures drop below thermal optima27,29. Our findings suggest that the short-term thermal threshold flexibility of P. verrucosa could offer an advantage for coping with temperature changes.

We then explored the link between thermal thresholds (ED50) and in situ Fv/Fm. Despite a similar performance of the studied coral species (Fig. 1), we observed a significant inverse correlation between P. verrucosa in situ Fv/Fm and estimated ED50 (Fig. 3C, Supplementary Table 4, linear mixed-effect model: p = 0.01), while Acropora spp. exhibited no correlation (Fig. 3D, Supplementary Table 5, linear mixed-effect model: p = 0.94). These results, in agreement with the previous results reported here, suggest distinct strategies for coping with changing environmental conditions.

The flexibility of P. verrucosa might stem from modifying thermal thresholds in response to rising temperatures, potentially due to metabolic compensation processes33, indicating short-term acclimation to temperature. Differential responses to disparate environmental conditions have been shown in Pocillopora and Acropora genera34. Additionally, Acropora spp. has been shown to exhibit higher susceptibility after surpassing their thermal threshold12, as was also evident in our study from the marked drop in the dose-response curve (Fig. 2). The recovery after bleaching has been observed to be faster in P. verrucosa compared to Acropora spp.12. The species-specific responses reported here could also be aligned with the previously observed differences in microbiome flexibility between Pocillopora and Acropora species25,34. Pocillopora spp. maintain a stable microbiome, even under stress, whereas Acropora spp. harbor a more flexible bacterial community in response to various impacts, sites, and environmental conditions. Differences in microbiome flexibility could potentially readjust species-specific performance traits (growth, respiration, photosynthetic efficiency) to better cope with seasonal temperature changes.

The reduction in coral performance (photosynthetic efficiency) during acute stress assessments has been correlated with in situ warming events and local temperature oscilation18,19. In addition, Fv/Fm derived ED50 has been demonstrated to reveal differences in the thermal threshold of the coral colonies even at a genotypic level using short-term acute stress experimental levels and be comparable with long-term heat stress experiment11,18,30. However, other physiological parameters may also be suitable for consistently comparing coral performance and thermal tolerance, which is still under study. Similarly to the rest of the performance rates, Fv/Fm rates are likely to be affected by temperature changes in an unimodal way, as we observe here and in other studies31. The performance increases up to an optimum and then exhibits a rapid decrease once the optimum has been surpassed. Therefore, it is not surprising that precisely above the Topt we observe a stronger relationship between thermal threshold and in situ temperature. The decrease in the performance observed in this part of the performance curve associated with the increase in the thermal tolerance of P. verrucosa species is indicative of a possible re-adjustment in the metabolic fluxes to compensate for the energy needed to acclimatize to the in situ environmental variation increasing the thermal threshold accordingly. These compensation mechanisms have been previously reported in microbes33,35 and are likely playing a role here. However, more specific analyses are required to confirm the mechanisms. Changes in abiotic conditions other than seawater temperature, such as light intensity and water quality, have also been reported to affect the performance of the symbionts21,22. While the effects of light cannot be entirely dismissed in our observations, the strong correlation with temperature suggests it is the primary driver of the patterns we have observed. Although our study did not explore the underlying mechanisms, we hypothesize that coral species may have evolved to cope with environmental stress. These strategies are likely linked to microbial flexibility, and short-term acclimation patterns, which are significant for their ability to adapt to climate change.

This study highlights the utility and the need of using standardized methods, such as CBASS, to identify which coral species or genotypes exhibit acclimation to seasonal variations12,18,30. The ability of a coral species or genotype to acclimate seasonally may differ depending on their physiological and genetic underpinnings, in addition to Symbiodiniaceae and bacterial assemblage25. These variables have also been demonstrated to differ between heat tolerant and heat susceptible colonies and their capacity to recover36. Therefore, the identification of coral species or genotypes that show short-term (seasonal) acclimatization of thermal tolerance thresholds could potentially be used as an indicator of which coral species are more sensitive to ocean warming.

To conclude, our study highlights distinct seasonal fluctuations in thermal tolerance among two coral species in the Red Sea. P. verrucosa displayed a response reflective of prevailing seawater variations, possibly resulting from metabolic adjustments in response to gradual temperature shifts. Conversely, Acropora spp. maintained a more consistent thermal threshold, potentially supported by higher flexibility and, therefore, adaptation level of the associated microbiome25,34, likely selected from the seasonally different microbial communities available in the surrounding seawater. These findings imply that coral species may respond differently to a changing climate because it may be easier for some coral species, likely the most sensitive, to adapt and respond to gradual temperature changes, slowly enhancing their resilience. However, these species might become more vulnerable to stress and bleaching under acute thermal stress conditions. Future investigations should explore the mechanisms underlying these short-term responses to environmental changes within the holobiont, such as the host gene expression and microbiome shifts.

Methods

Study site and sample collection

Coral colonies from P. verrucosa and Acropora spp. at 8 m deep from the Coral Probiotic Village (CPV) located in Al Fahal Reef, central Red Sea (N 22.29634° E38.95914°)37 were considered for this study. The study included colonies monitored and sampled from August 2021 to February 2023. As part of different experiments, P. verrucosa colonies and Acropora spp. colonies were studied at different sampling times (see Supplementary Table 6 for detailed information about the colonies, biological replicates, and dates. The data corresponding to the experiments PvN has been previously published in Delgadillo-Ordoñez et al.38 corresponding to non-treated P. verrucosa colonies.). The taxonomic identification of the Acropora species included in the study was not feasible due to the challenges of the species identification for this genus39. It is unclear whether colonies from different species were sampled, but for comparative analysis, we considered them as Acropora spp. complex, as they behave similarly in their responses to temperature, which is supported by their dose–response curves. All colonies were visually healthy during the different experiments. All data included in the study at each sampling time were collected on the same day. The temperature was monitored using multiparameter CTDs (Ocean Seven 310 Multiparameter CTD, Idronaut) deployed in two locations within the experimental area (Supplementary Fig. 3). CTDs were placed on metal frames deployed on the sea bottom (at 7–8 m deep) and exchanged every 2–3 weeks to retrieve the data, to cover seasonal variations. The average seawater temperature measured on the day each experiment was conducted was used as in situ seawater temperature.

Colonies used for thermal threshold assessment were sampled by SCUBA diving at least 3 m apart from each other to minimize the chance of sampling clonal genotypes (see Supplementary Table 6 for details regarding sampling time, species, and biological replication). Fragments of about 5 cm were sampled using sterile gloves and pliers and stored in individual sterile opaque collection bags (Whirl-Pak®) filled with seawater and transported in a cooler box filled with seawater to the wet laboratory where they were subjected to short-term thermal stress assays on the same day of collection. All the fieldwork and sampling procedures were conducted in compliance with 22IBEC003 permits and KAUST’s regulations.

In situ photosynthetic efficiency

The photosynthetic efficiency of the coral-associated Symbiodiniaceae community was assessed through the maximum quantum yield of PSII photochemistry, Fv/Fm. Fv/Fm is a non-invasive parameter that measures the photosynthetic efficiency of the coral symbiont and is widely used as a proxy for coral health18,22,31,36. It can detect stress signs before the coral colony exhibits tissue loss and bleaching40.

A diving PAM system (Diving PAM II, Walz) with a red-emitting diode (LED; peak at 655 nm) was used in situ to perform the measurements for all the experiments except CRG. For this experiment, a blue-emitting diode (LED; peak at 475 nm) was used. PAM data was collected after sunset, at least 30 min after complete darkness, to ensure the corals were dark-adapted, and, therefore, the full photochemical dissipation of the reaction centers. The diving-PAM settings details used in the experiments were: measuring light intensity = 6; gain = 2; and damping = 4.

Short-term thermal stress assay

Fragments from different colonies were sampled for Acropora spp. and P. verrucosa genotypes (colonies) from August 2021 to August 2022 (see details from each sampling time in Supplementary Table 6). Fragments were subjected to short-term thermal stress assays using the standardized Coral Bleaching Automated Stress System (CBASS)18,30. The fragments were transported in seawater to the wet lab facility of the Coastal and Marine Resources Core Lab (CMOR, KAUST). The CBASS system consisted of four 10 L flow-through aquaria supplied with seawater collected from the site a day before the assays. Each tank was configured using a different temperature regime and the light setting of the dimmable 165 W full spectrum LED aquarium lights (Galaxyhydro) was adjusted to ~600 µmol photons m−2 s−1 using an LI-193 Spherical Underwater Quantum Sensor (LI-COR). The light level selected was above the minimum light levels suggested by Grottoli et al.41. but below levels that could cause photodamage to the corals according to the daily range expected for coral reef systems in the Red Sea. The lights followed a 12:12 hr day: night cycle. The temperature of each tank was controlled using an ITC-310T-B (Inkbird) thermostat connected to an IceProbe Thermoelectric chiller (Nova Tec) and 200 W titanium aquarium heaters (Schego). HOBO Pendant Temperature Data Loggers were placed to record the temperature every 10 min in each tank during the experiment. From each colony, four fragments from each colony were sampled and distributed in the tanks to be exposed to each of the four different temperature treatments (Control = 30 °C, medium = 33 °C, high = 36 °C, extreme = 39 °C). The four temperature treatment conditions were as follows. The baseline (control) tank was maintained at 30 °C (corresponding to the MMM in Al Fahal reef) for the entire duration of the experiment. The other three tanks were heated to 33 °C, 36 °C, and 39 °C, respectively, over a period of 3 h. The respective temperatures were held for 3 h and then decreased to 30 °C over the course of 1 h. All corals were kept at 30 °C overnight until the following morning, completing the 18 h short-term thermal stress assay18,30. Lights were turned off after 7 h from the start of the experiment (following the heat stress and subsequent ramping down to the control temperature) to ensure complete darkness. Corals were maintained in dark conditions for 1 h, and, after this period, we measured the dark-adapted maximum quantum yield (Fv/Fm) of photosystem II of all coral fragments in all temperature treatments using a pulse amplitude modulated (PAM) fluorometer (WALZ).

Characterization of the coral thermal performance (F v /F m)

To quantitatively evaluate the changes in the coral performance as a function of seawater temperature, we used a generalized additive mixed-effect model (GAMM) that was fit for each species using the function uGamm from the R package MuMIn42. The full model included Fv/Fm as the response variable, and the following fixed effects and smoothing terms: species and smoothing term on species and seawater temperature. We chose this model to consider the shape of each curve in the species comparison.

We then estimated the thermal optima (Topt) by fitting the four parameters Sharpe-Schoolfield function to the unimodal response exhibited by each species28. We additionally fitted the model to all the data to obtain an overall Topt parameter.

$${\mathrm{ln}}\left(b\left(T\right)\right)={E}_{a}\left(\frac{1}{k{T}_{c}}-\frac{1}{{kT}}\right)+{\mathrm{ln}}\left(b\left({T}_{c}\right)\right)-{\mathrm{ln}}\left(1+{e}^{{E}_{h}\left(\frac{1}{k{T}_{h}}-\frac{1}{{kT}}\right)}\right)$$
(1)

where \(b\) is the metabolic rate (mg O2 l−1 h−1), k is Boltzmann’s constant (8.62 × 10−5 eV K−1), \({E}_{a}\) is the activation energy (eV), indicative of the steepness of the slope leading up to the thermal optima, T is temperature in Kelvin (K), \({E}_{h}\) is the deactivation energy which characterizes temperature-induced decrease in rates above \({T}_{h}\) where half the enzymes have become non-functional and \(b\left(\right.\)Tc) is rate normalized to an arbitrary reference temperature, here Tc = 28 °C (+273.15), where no low or high temperature inactivation is experienced. All in situ Fv/Fm data were fitted to Eq. 1 using the R function “nls_multstart”43. By differentiating Eq. 1 and solving for the global maxima an optimum temperature can be estimated using the following expression

$${T}_{{opt}}=\frac{{E}_{h}{T}_{h}}{{E}_{h}+k{T}_{h}{\mathrm{ln}}\left(\frac{{E}_{h}}{{E}_{a}}-1\right)}$$
(2)

Bootstrapping approach using residual resampling was selected to estimate the 95% confidence intervals around the predictions using the R function rTPC43. The number of experiments included were nP.verrucosa = 12 and nAcropora spp. = 7. The number of biological replicates included in each experiment is given in Supplementary Table 6.

Thermal tolerance threshold estimation

We used the experimental Fv/Fm data collected during the short-term thermal stress assay.

Temperature tolerance thresholds were determined for each species, and sampling time as the mean (across all genotypes) temperature at which photosynthetic efficiency dropped to 50% of the value at baseline temperatures, defined as the Effective Dose 50 or ED5044 using the dose–response curve (DRC) package in R45. Statistical differences among experiment-specific including individual genotypes ED50s (biological replicates, see details about the number of replicates per experiment and species in Supplementary Table 6) as the response variable and experiment ID as a factor (nP.verrucosa = 10; nAcropora spp. = 5) were assessed using Kruskal-Wallis non-parametric test and Dunn test (Holm correction) was used for multiple comparisons between experiments for each of the species considered independently. The number of biological replicates included in each experiment is given in Supplementary Table 6.

Linking thermal tolerance threshold with environmental seawater temperature

To assess if the changes in the ED50 observed were related to the seawater temperature variation over time we used a linear mixed-effect model using R function “lmer” from R package “lme4”46, treating biological replicates (colony) nested to each experiment ID as a random effect of the intercept. In doing so, we accounted for different experiments and the non-independence of the replicates within the same experiment. The model was fitted above the Topt estimated using both species combined as they did not show a significant difference in the thermal performance. ED50 was included in the model as a response variable and seawater temperature as a predictor variable. We fit the same model to each species separately to ensure that the differences in the number of experiments and replicates per species did not affect the model. We performed model selection using likelihood-ratio tests. The number of experiments included were nP.verrucosa = 8 and nAcropora spp. = 4. The number of biological replicates included in each experiment is given in Supplementary Table 6.

Impact of the thermal tolerance threshold on coral performance

A linear mixed-effect model was applied to study the correlation between the thermal tolerance threshold of the corals and the performance in situ (Fv/Fm). To perform the analysis we used the R function “lmer” from R package “lme4”46, treating biological replicates (different colonies) nested to each experiment as a random effect of the intercept to account for different experiments and the non-independence of the replicates within the same experiment. The model was fitted above the Topt estimated using both species combined as they did not show a significant difference in the thermal performance. In situ Fv/Fm was included in the model as a response variable and ED50 as a predictor variable. We fit the same model to each species separately to ensure that the differences in the number of experiments and replicates per species did not affect the model. We performed model selection using likelihood-ratio tests. The number of experiments included were nP.verrucosa = 8 and nAcropora spp. = 4. The number of biological replicates included in each experiment is given in Supplementary Table 6.

Statistics and reproducibility

Generated data was analyzed using R program (R Core Team) with a sample size or replication of at least 3. The replicates sampling size for individual experiments and the biological replicates included within each experiment are included in the respective figures and tables legend. Detailed information about the experiment performed, associated metadata and biological replicates included in each experiment is given in Supplementary Table 6.