Heron Reef (23°26′32.66″S, 151°54′55.53″E) is part of the Mackay Capricorn management area of the Great Barrier Reef Marine Park in the Southern Great Barrier Reef (Australia). Remotely-sensed weather information was extracted from the GIOVANNI online system for satellite derived climate data, which is maintained by NASA (http://disc.sci.gsfc.nasa.gov/giovanni). Monthly area-averaged data between 2003 and 2016 collected by the Moderate Resolution Imaging Spectroradiometer (MODIS-aqua) were used in this study, including PAR at the ocean surface (Einstein m⁻² d⁻¹), and daytime sea surface temperature (SST, °C). Satellite data were retrieved for Heron Reef using the bounding box function to define the area of interest. GPS co-ordinates of the bounding box (151.949, −23.463, 151.977, −23.443) were selected to match the location of relay poles RP1 and RP6 (spaning an area of ca. 8 km²) of the Heron Reef Integrated Marine Observing System (IMOS; Bainbridge et al., 2010). Supporting in situ information was retrieved from the Heron Reef IMOS network from all eight relay poles (RP1-8) that record high-resolution physical data, including above-water PAR (daily maxima and daily averages from RP8, μmol photons m⁻² s⁻¹) and sea surface water temperature at 1–2 m depth (RP1-8, daily maxima and daily averages, °C). This data is freely available under a Creative Commons 3.0 license (https://creativecommons.org/licenses/by/3.0/au/deed.en) via the Australian Institute of Marine Science (www.aims.gov.au), and was accessed on 18/10/2016 using the database provided rule-based quality control for eliminating outliers or errors.

Further environmental data that relate directly to the optical clarity of the water at Heron Reef (Michael et al., 2012) were accessed from the Bureau of Meteorology (BOM) Heron Island weather station (http://bom.gov.au) which is maintained by the Australian Government. Data for wind speed (km h⁻¹), including; maximum daily gusts; wind speed at 09:00 h and 15:00 h; during the experimental periods in spring and summer (described below) were retrieved from BOM. All available data for total rainfall data (mm) at Heron Island were also retrieved from BOM between 1959 and 2007. The diffuse attenuation coefficient (K_d) of downwelling irradiance at 490 nm, used to approximate seasonal changes in turbidity (Suggett et al., 2012), was collected from the same area of interest from MODIS (as above) during the experimental periods in spring and summer.

The Heron Reef system experiences significant seasonal variability in physical conditions (Kline et al., 2015), and therefore seasonal MODIS (remotely sensed) and IMOS (in situ) data were specifically considered for periods coinciding with our experiments (Figure 1). The first experiment was conducted between 28th October and 5th November (2015), which we refer to as “spring.” The second experiment was conducted between 6th and 16th March (2016); whilst this second period is regarded as post-peak of the summer season, we refer to it as “summer” for the purposes of this study. When strictly considering monthly averaged temperature pooled through 2003 and 2015 (Figure 1), the MODIS data indicate that November and March are typically near equal in sea surface temperature (SST) at 26.4 (±0.2 SE) and 25.9 (±0.2) °C, respectively, while the interceding months contain the annual maximum temperatures. However, the in situ IMOS data for SST pooled through 2008 and 2015 with higher temporal resolution indicate that the MODIS data likely over-estimate the actual SST here by ca. 1.2°C in November, and under-estimate by ca. 0.6°C in March (Figure 1). Strictly considering monthly averaged PAR (Figure 1), the monthly average irradiance of 665.3 (±20.3) μmol photons m⁻² s⁻¹ coincide with the spring experiment in November, but following this month steadily decreases, reaching 457.7 (±13.9) μmol photons m⁻² s⁻¹ in March. This trend is consistent across both MODIS and IMOS data sets. Thus, our two experimental periods contrast “spring” and “summer” as seasonal priming through (i) increases in temperature, and (ii) decreases in light availability.

Key photophysiological parameters of interest in this study (described in further detail in sections below) are known to acclimate in as few as 5–10 days (Anthony and Hoegh-Guldberg, 2003; Roth, 2014). Thus, we utilized the high-temporal resolution (daily) IMOS system for the 14 days pre-experiment (for both spring and summer) to capture the environment under which the photochemical pathways have primed, i.e., the environmental “acclimation history.” The environmental conditions immediately following this during the experiment are referred to as “ambient.” The summer period of this study in 2016 was conducted directly after Heron Reef was downgraded from “Bleach-Watch” status (low thermal stress for all of February) in the Coral Reef Watch models produced by the National Ocean and Atmospheric Administration (NOAA, http://coralreefwatch.noaa.gov/satellite/index.php).

Ten coral species were used in this study (Table 1); Acropora aspera; Acropora digitifera; Acropora formosa; Acropora millepora; Pocillopora damicornis; Montipora digitata; Isopora palifera; Stylophora pistillata; Porites cylindrica; and Porites lutea (N = 4 per species, <2 m depth). All are considered common on the Great Barrier Reef (Madin et al., 2016) and are typically abundant on the shallow reef-flat and/or reef-crest of Heron Reef. These corals are typically dominated by Symbiodinium that broadly fall within clade C (LaJeunesse et al., 2003), and span a range of thermal tolerances that have previously been characterized in studies at Heron Reef (Table 1). Notably, corals containing Symbiodinium C15 (e.g., M. digitata, P. cylindrica, and P. lutea) are expected to be significantly more tolerant to transient heat stress than other species (Fitt et al., 2009; Fisher et al., 2012). Only coral colonies that visually appeared healthy (free of lesions or abnormal pigmentation) were selected for collection. No immediate visual signs of bleaching (i.e., significant paling of coral colonies) was observed on the reef during the spring or summer collections. Collected corals were transferred to Heron Island Research Station (The University of Queensland, Australia) and each colony was split into halves (~6 × 2 cm). All species (except P. lutea fragments which were mounted in a glass petri dish) were secured onto glass microscope slides using Selleys Epoxy (Selleys Pty Ltd., Australia). Coral collections were performed under the conditions of the Great Barrier Reef Marine Park Authority permits, numbered G15/37922.1 and G15/37538.1

Coral fragments were allowed to recover and acclimate for 5 days prior to experimentation in an unfiltered flow-through aquaria system under 50% of ambient light (using a neutral density shade-cloth) and a continuous flow of ambient lagoon seawater. This was deemed necessary (especially in spring) as the corals would otherwise experience artificially extreme light-doses on a daily basis due to the limited depth (and size) of the holding tanks. After acclimation, corals were distributed to 8 replicate aquaria (20 L, N = 1 fragment of each species per tank) also under 50% shaded natural light in a custom built open flow through system utilizing ambient lagoon seawater (minimum flow through of 0.5 L min⁻¹). The water temperature within the system was recorded every 10 min with temperature loggers (HOBO pendant data loggers, Onset Computer Corp., Bourne, MA, USA) and/or temperature buttons (Thermochron, OnSolution, NSW, Australia). This same acclimation protocol and experimental system was used in both spring and summer.

PSII photochemistry of coral-hosted Symbiodinium cells was measured via chlorophyll a fluorescence kinetics using a Pulse Amplitude Modulated (PAM) fluorometer (Diving PAM, Walz GmbH, Effeltrich, Germany, settings: MI: 12, Gain: 12, SI: 12, SW: 0.8 s, LC-INT: 3) with a glass optical fiber. Light response curves (applying actinic light to coral tissues incrementally in steps of increasing irradiance) are classified according to the time duration of each light step. Rapid light curves [RLC, sensu Ralph and Gademann (2005); Serôdio et al. (2005)] apply light steps of short duration (e.g., 10, 20, 30 s, Perkins et al., 2010) and are widely used in coral photobiology, largely to maximize sample replication while underwater (e.g., Warner et al., 2010). Whilst RLCs provide limited time for photoprotective mechanisms to be maximally induced, steady-state light curves (SSLC) are longer in duration (e.g., 3.5 min of each light step) and are designed to allow the induction of heat-dissipation mechanisms to reach completion. However, assuming a strong diel profile of PSII photochemical efficiency in corals on shallow reef environments (Hill and Ralph, 2005), replicates (or species) may enter different states of acclimation if not sampled rapidly and thus SSLCs are not practical for comparisons of multiple field samples. Furthermore, as coral tissues migrate in response to high-light (Levy et al., 2003; Wangpraseurt et al., 2017b), the assumption that the optical properties related to the structure of the sample remain constant (Serôdio et al., 2006) may be violated during the long-duration of the SSLC and the retrieved fluorescence signals may become error-prone (as seen for corals in Lichtenberg et al., 2016). Suggett et al. (2015) demonstrated that RLCs (20 s light steps) and SSLCs (3.5 min light steps) generally resolve similar light utilization patterns across diverse taxa of Symbiodinium in culture, however it is unknown if Symbiodinium cells organized into the tissue layers of corals will show similar patterns (although both RLCs and SSLCs are equally limited in this regard). With these factors considered (and discussed in sections below), the use of RLCs are preferred in the present study to conduct all measurements within a narrow time-frame, and we interpret the findings within the limitations of the method. Importantly, we do not interpret the findings as absolute rates of electron transport in photosynthesis (Enríquez and Borowitzka, 2010; Warner et al., 2010) and we do not utilize the RLC as a substitute for a SSLC (Perkins et al., 2010). RLCs with 8 actinic light steps were conducted on low-light acclimated corals during the first 1.5 h of the morning light period (N = 3 technical replicates per fragment for N = 4 biological replicates) to minimize the effect of night-time reduction of the plastoquinone pool (Hill and Ralph, 2005). Actinic light levels (calibrated against a factory calibrated LI-192 quantum sensor, Li-Cor, Lincoln, NB, USA) of the RLC were 0, 115, 168, 228, 335, 450, 666, 954, 1,356 μmol photons m⁻² s⁻¹, and 0, 98, 162, 240, 325, 480, 610, 971, 1,350 μmol photons m⁻² s⁻¹ in spring and summer, respectively, and were delivered in 20 s intervals. All RLC measurements were conducted over 4-days, with one replicate per species per day. Distributing the RLCs over a 4-day experimental period was necessary to complete the technical replicates for each fragment within the naturally low-light acclimated state immediately following sunrise.

All RLCs were initiated with a dark measurement to provide the minimum and maximum dark acclimated fluorescence yields, F_o and F_m, respectively, and all subsequent steps measure the minimum, and maximum fluorescence yields under actinic light, F′ and Fm′, respectively. These parameters are then used to calculate the maximum (F_v/F_m = [F_m –F_o]/F_m) and effective (Fq′/Fm′ = [Fm′ –F′]/Fm′) photochemical efficiency. Whilst the latter term is often denoted as Φ_PSII, we use Fq′/Fm′ to be consistent with Hennige et al. (2008) and Suggett et al. (2012). A light-response curve for Fq′/Fm′ was thus generated for each RLC and then fit to a model that characterizes the light-dependent quantum efficiency of PSII using least squares non-linear regression (Equation 1), described in Hennige et al. (2008).

Briefly, this model provides an estimate of the minimum saturating irradiance (E_k μmol photons m⁻² s⁻¹) which describes the transition between the light-limited and the light-saturated states of PSII (Hennige et al., 2008). Equation (1) also derives Fq′/Fm′ _(max) (dimensionless) which is an alternate estimate of the maximum PSII photochemical efficiency, which can differ from the dark-acclimated maximum quantum yield of photosystem II (F_v/F_m) depending on light history, see Suggett et al. (2012).

To calculate the poise of the photosystem and the activity of pathways of excitation energy transfer other than fluorescence, the extent of light dependant photochemical ([1 – C], Equation 2) and non-photochemical quenching ([1 – Q], Equation 3) parameters were both derived from each RLC light step across seasons and species as follows.

where [1 – C] is the same as qP referred to in previous studies (Hennige et al., 2008; Oxborough et al., 2012), which assumes zero connectivity (C) between adjacent reaction centers and represents the proportion of excitation energy used to drive photochemistry as the fraction of open reaction centers. [1 – Q] describes the dynamic non-photochemical quenching (Q) that is equivalent to the excitation pressure over PSII (Iglesias-Prieto et al., 2004; Suggett et al., 2015). Use of [1 – Q] is preferred for our study over other calculations of non-photochemical quenching, [e.g., NPQ = (F_m –Fm′)/Fm′], since the product of [1 – C] and [1 – Q] account for the total energetic dissipation (effective photochemical efficiency) normalized to taxonomic differences in maximum photochemical efficiency, i.e., (Fq′/Fm′)/(F_v/F_m) (see Suggett et al., 2015). These parameters decrease over the course of the RLC as they become active or are “utilized.”

While all corals were collected from the same light environment (<2 m depth), and care was taken to avoid species at Heron Reef that are characterized as having comparatively thick tissue layers (e.g., Platygyra spp. or Pavona spp.), it is possible that differences in host pigmentation (Salih et al., 2000), alteration of light fields due to host macrostructure (Enríquez et al., 2005, 2017; Terán et al., 2010; Wangpraseurt et al., 2014), or even the aspect of the colony that fragments were removed from Brown et al. (2000), could potentially introduce variation in the acclimation states of the fragments. Thus, parameters generated from the RLC described below were normalized to E_k to eliminate this source of variability (as per Hennige et al., 2008; Suggett et al., 2012). Examining parameters as E/E_k rather than E allows the differences in photoacclimation to be viewed independent of how photosynthetically usable radiation varies between species, and provides an indication of how mechanisms associated with regulating light-limited (below E_k) or light-saturated (above E_k) PSII are preferentially modified to optimize toward E_k (Hennige et al., 2008).

Both [1 – C] and [1 – Q] were analyzed against the normalized values of E/E_k and assessed with non-linear regression using a standard one phase decay model in Graphpad Prism (version 6). Replicates tests (Graphpad Prism version 6) that test for an inadequate model fit were performed on every curve and confirmed that the one-phase decay model adequately described all quenching curves for all species across both seasons (p > 0.05), except for A. millepora [1 – C] in spring which did not fit the one phase decay model and was instead fit to a linear model (R² = 0.76). For every curve (each representing the mean of 4 replicates) the values of [1 – C] and [1 – Q] where E/E_k = 1 (i.e., at the minimum saturating irradiance) were interpolated.

Following the completion of the RLC described above in the non-stressed condition, half of the experimental tanks (N = 4) were subjected to changes in temperature to induce transient thermal stress, and the remaining experimental tanks (N = 4) were unchanged and served as ambient controls with continuous flow of ambient lagoon seawater. Our approach aimed to maximize synchronicity and equivalency of the ramping profiles in spring and summer (Figure S1) and reach equivalent end-points in temperature. In spring, temperature was increased from 26°C in 1°C increments daily to reach the target treatment temperature of 32°C, and then was held for a further 3 days. 32°C was selected as an environmentally relevant upper limit as the single warmest day recorded by the IMOS network (measured across all RP) reached a maximum of 32.55°C (21st February 2009). In the summer experiment, the ambient lagoon was naturally fluctuating between 26 and 28°C on a diel basis and thus to synchronize the profile with spring, treatment tanks were allowed to fluctuate for the first day under which spring ramped from 26 to 27°C, and then the profiles were synchronized from 28 to 32°C and increased in 1°C increments daily (Figure S1). Temperature was increased by pre-heating continuous flow-through lagoon seawater in a separate sump (200 L) with two custom built, thermostat controlled (±0.1°C) submersible heaters (300 W). Treatment water was pumped from this sump to aquaria at a flow rate matching the flow-through of ambient aquaria. During the temperature ramp and maintenance at 32°C, dark-acclimated maximum quantum yield of photosystem II (F_v/F_m) was measured twice every day for every coral fragment to monitor for changes in photochemical efficiency (as per Oliver and Palumbi, 2011; Suggett et al., 2012); once in the first hour following sunrise (ca. 07:00 h local time) and once again in the evening during the first hour following sunset (ca. 19:00 h local time; N = 3 technical replicates per fragment). An inverted exponential function (Equation 4, as per Vieira et al., 2009) was used to describe the decrease in F_v/F_m through time as follows.

where F_v/F_m is expressed as % (%F_v/F_m, relative to untreated fragments), F_v/F_{m (0)} = %F_v/F_m prior to thermal stress (i.e., x = 0), x = time (d), S is a parameter related to the length of the period during which %F_v/F_m remains stable, and k (d⁻¹) is the rate constant that quantifies the rate of %F_v/F_m decrease. The minimum (typically reached at the final measurement) dark-acclimated maximum quantum yield of photosystem II reached during exposure to heat stress, will be referred to as %F_v/F_m(min).

Environmental variables were compared between seasons using unpaired two tailed t-tests, assuming unequal variances which were accounted for with Welch's correction. This same statistical test was used to analyse the pooled photoacclimation response (E_k and Fq′/Fm′ _(max)) of all coral species when comparing between spring and summer. To identify specific differences across seasons and species two-way ANOVA was used to investigate the interactions between season (two levels) and species (10 levels) on derived values of E_k and Fq′/Fm′ _(max), with Sidak's multiple comparisons post hoc tests to locate significant differences. All statistical tests were performed in Graphpad Prism (version 6) against an α of 0.05. To identify functional groupings of corals based on all derived components of the light response curves and thermal tolerance of PSII function, cluster analysis and multidimensional scaling (MDS) were performed with E_k; Fq′/Fm′ _(max); [1 – C]: [1 – Q] at E/E_k = 1; and %F_v/F_m(min), with PRIMER-E v6.1 (PRIMER-E Ltd, Ivybridge, Devon, UK). All values were standardized using a square root transformation using PRIMER-E and correlations were drawn based on Pearsons distance. An analysis of similarity (ANOSIM) was used to determine whether the corals separated by MDS ordination significantly differed between seasons.

Ambient SST during the experiments in spring (2015) and summer (2016, IMOS in situ) closely matched the typical SST for October-November and February-March (2009–2016; Table 2). SST increased significantly from spring through summer as expected; daily averages were 24.2°C (±0.7) and 26.9°C (±0.3) during the spring and summer experiments, respectively. Maximum ambient temperatures reached across these periods were 27.3 and 30.2°C, respectively (Table 2, Figure S2). Ambient PAR for spring and summer were also consistent with previous years (Table 2, Figure S2), whereby ambient daily average PAR during spring was 541.5 (±148.2) μmol photons m⁻² s⁻¹ and was significantly lower during the summer at 442.8 (±142.9) μmol photons m⁻² s⁻¹ (unpaired two tailed t-test with Welch's correction, t₍₄₃₎ = 3.10 and t₍₃₈₎ = 2.4, respectively, p < 0.05). Maximum daily PAR values during these periods were 2091.3 and 2004 μmol photons m⁻² s⁻¹, respectively. This pattern appears to be driven by consistent levels of PAR (i.e., days to weeks) in spring, whereas summer months were frequently punctuated by multiple events of comparatively low PAR (i.e., storm events, Figure S2). Indeed, the acclimation history directly preceding the summer experiment included a significant weather event that lowered the average daily and average maximum PAR to 124 and 560.3 μmol photons m⁻² s⁻¹, respectively (Figure S2). This pattern of decreasing PAR from spring through summer appears seasonally typical and supported by long-term trends gathered from MODIS (2003–2016; Figure 1).

Further physical changes that play a significant role in modulating in-water PAR include the diffuse attenuation coefficient (K_d) of downwelling irradiance, which is used to approximate changes in seawater turbidity (Suggett et al., 2012), elevated wind speeds [which are known to increase K_d at Heron Reef (Michael et al., 2012)], and monthly rainfall. Heron Reef is subjected to significantly faster wind speeds and nearly three-fold greater rainfall in summer (Figure S2). However, this more extreme weather of summer is not reflected in seasonal differences in the remotely-sensed K_d(490) retrieved from the MODIS instrument (Figure S3).

Eqn. 1 produced a robust description of the light dependant response of Fq′/Fm′ across species and seasons (R² ca. 0.9), except for A. aspera and M. digitata in spring (R² = 0.76 and 0.65, respectively) although values for E_k and Fq′/Fm′ _(max) were still within the range retrieved for all species. Significant seasonal changes in E_k (Figure 2) were evident for all species. In spring, the pooled average of E_k across all 10 species was 534.1 (± 67.3) μmol photons m⁻² s⁻¹, and in summer decreased >2-fold to 227.5 (± 28.5) μmol photons m⁻² s⁻¹ [unpaired two tailed t-test with Welch's correction, t₍₉₎ = 17.77, p < 0.01]. These values of minimum saturating irradiance are thus consistent with the differences in acclimation history between spring and summer experiments (Table 2, Figure S2). A two-way ANOVA of species × season revealed significant effects of each factor [F_{(9, 59)} = 2.835, p < 0.01, and F_{(1, 59)} = 345.2, p < 0.0001, respectively] and Sidak's multiple comparisons tests confirmed that all comparisons of species from spring to summer had significantly lower E_k (all multiple comparison tests p < 0.05). The largest change in E_k between seasons was for A. aspera decreasing from 652.9 (±75.4) μmol photons m⁻² s⁻¹ in spring to 244 (±10.4) μmol photons m⁻² s⁻¹ in summer. In contrast, the smallest changes in E_k were observed for P. cylindrica, decreasing from 442.8 (±47.8) μmol photons m⁻² s⁻¹ to 218.5 (±8.9) μmol photons m⁻² s⁻¹ between seasons.

Values for the derived maximum PSII photochemical efficiency Fq′/Fm′ _(max) (dimensionless) also differed significantly between seasons and when averaged across all species increased from 0.68 (±0.01) in spring to 0.77 (±0.02) in summer [Figure S4, unpaired two tailed t-test with Welch's correction, t₍₁₂₎ = 4.94, p < 0.001]. This is supported by the measured values of F_v/F_m which also increase across seasons (Figure S5). A two-way ANOVA of species × season revealed a significant interaction between factors [F_{(9, 59)} = 2.147, p < 0.05]; however, Sidak's multiple comparisons tests confirmed that this was not driven by a consistent response across all species (Figure S4). For example, whilst M. digitata, P. lutea, P. cylindrica, and S. pistillata, exhibited significant changes in E_k, no corresponding significant shift in Fq′/Fm′ _(max) was observed (Figure S4). However, all other species demonstrated a shift in Fq′/Fm′ _(max) with an increase in summer (Sidak's multiple comparisons tests p < 0.05), and the largest change in Fq′/Fm′ _(max) of 0.14 (dimensionless) between seasons was observed in A. digitifera. Overall, these general complimentary responses for E_k and Fq′/Fm′ _(max) are consistent with acclimation to the decrease of light availability (Hennige et al., 2008; Suggett et al., 2012) that appears typical between spring and summer, including our 2015/16 sampling season (above, Table 2).

Values derived for [1 – C] at E/E_k = 1 pooled across all species differed significantly between seasons [unpaired two tailed t-test with Welch's correction, t₍₁₀₎ = 4.675, p < 0.001], decreasing from 0.79 (±0.02) in spring to 0.72 (±0.01 SE) in summer. Similarly, the derived [1 – Q] at E/E_k = 1 pooled across all species also differed significantly between seasons [unpaired two tailed t-test with Welch's correction, t₍₁₃₎ = 7.56, p < 0.001], increasing from 0.79 (±0.02) in spring to 0.94 (±0.01) in summer. As such, at the light intensity at which electron transfer rates “saturate” (E/E_k = 1), corals generally shifted toward increased reliance of photochemical over non-photochemical quenching from spring into summer as light availability decreased. In fact, three distinct quenching patterns previously described for Symbiodinium isolates (Suggett et al., 2015) were observed in spring. “Normal quenching,” where [1 – C] and [1 – Q] are utilized equally, P. damicornis and A. digitifera; preferential photochemical quenching, where the ratio of [1 – C] to [1 – Q] < 1 for P. lutea, P. cylindrica, S. pistillata, and M. digitata; and preferential non-photochemical quenching, where the ratio of [1 – C] to [1 – Q] > 1 for A. aspera, A. formosa, A. millepora, and I. palifera. However, all coral species switched to preferential utilizing [1 – C] in summer (Figure 3). The largest seasonal shift in the ratio of [1 – C] to [1 – Q] at E/E_k = 1 was for A. formosa (1.23 in spring, decreased to 0.73 in summer), whereas the smallest shift was for S. pistillata (0.88 in spring, decreased to 0.79 in summer).

Corals subjected to transient heat stress typically exhibited comparable %F_v/F_m(min) across spring and summer, except for P. damicornis and S. pistillata (Figures 4E,H). S. pistillata suffered total dysfunction in spring, i.e., chlorophyll fluorescence was below the levels of detection (not able to measure F_o), representing the strongest reaction of all species to transient heat stress. In contrast, S. pistillata was less sensitive in summer with %F_v/F_m(min) reaching 41.12 (±13.91) relative to ambient fragments; as such, S. pistillata was the only species to show significantly enhanced thermal tolerance from spring to summer. P. damicornis exhibited an opposite response whereby %F_v/F_m(min) reached 75.31 (±8.85) relative to ambient fragments in spring, but had a heightened sensitivity in summer [%F_v/F_m(min) was reduced to 19.91 (±8.89) relative to ambient fragments].

PSII photochemical efficiency of M. digitata and P. lutea was tolerant to transient heat stress across both seasons (Figures 4F,I). However, in summer, P. lutea fragments raised to 32°C demonstrated increased diel variability (i.e., morning vs. evening %F_v/F_m) relative to ambient fragments. All other coral species exhibited compromised PSII photochemical efficiency via heat stress for both seasons. Parameters retrieved by the inverted exponential function fit to changes in %F_v/F_m (Equation 4, Table S1) indicate that PSII photochemical efficiency for multiple species declined earlier in summer than in spring (i.e., S_summer < S_spring), however, the rate of decline (once it began) was comparatively more rapid in spring than in summer (i.e., k_summer < k_spring). For example, for A. aspera, S (dimensionless) was 5.18 (±3.26) in spring, but declined to 1.64 (±0.53) in summer, whereas k (d⁻¹) declined from 0.94 (±0.40) d⁻¹ to 0.58 (±0.06) d⁻¹ from spring to summer (Table S1, Figure 4A). Despite these minor seasonal differences in kinetics of PSII photochemical efficiency, %F_v/F_m(min) for A. aspera after 3 d at 32°C was not significantly different across seasons [83.33 (±8.43) in spring vs. 75.76 (±4.19) in summer]. This same pattern of an earlier, more gradual decline in PSII photochemical efficiency during the summer experiment was also seen in A. digitifera, A. millepora, and P. cylindrica (Table S1). A. formosa and I. palifera were more sensitive to transient heat stress than the other Acroporid species, and %F_v/F_m(min) reached 44.63 (±25.81) and 50.75 (±23.43) % for the two species respectively in spring compared to 56.83 (±19.87) and 60.88 (±20.53) % in summer. Additionally, A. formosa and I. palifera demonstrated high inter-fragment variability. For example, the %F_v/F_m of I. palifera exposed to transient heat stress in spring ranged from 0.62 to 0.15 (dimensionless) across all four replicates.

All of the derived photophysiological parameters, including E_k, Fq′/Fm′ _(max), [1 – C] and [1 – Q] at E/E_k = 1, as well as %F_v/F_m(min), for both spring and summer, were analyzed using MDS to examine functional differences across species. ANOSIM further confirmed the significant separation of photobiology across seasons (Figure 5, global R = 0.88, p < 0.001), whereby separation of seasonal clusters was driven primarily by the RLC-derived parameters (horizontal vectors of E_k, Fq′/Fm′_(max), [1 – C] and [1 – Q], Figure 5). Within each season, the ordinations of individual species did not cluster exclusively according to growth form, host taxonomy, or known symbiont associations (Table 1). For example, in spring, three distinct genera (Acropora, Porites, and Montipora) cluster together at 97% similarity. Species within each season were thus largely ordinated according to the response of PSII photochemical efficiency to heat stress (vertical vector of %F_v/F_m(min)) with the most thermally tolerant species (M. digitata and P. lutea) clustered together at 97% similarity in both seasons. S. pistillata was the most dissimilar to all other species (<80% similarity) and this is primarily driven by the complete dysfunction in response to temperature [i.e., %F_v/F_m(min) = 0], rather than differences in the non-stressed photobiology (above). For species that survived the transient heat stress (i.e., excluding S. pistillata), the only RLC parameter demonstrating a significant linear relationship with PSII photochemical efficiency under stress was the extent of light-dependant dynamic quenching (Figures 5B,D). Specifically, a negative linear relationship between %F_v/F_m(min) and [1 – C] at E/E_k = 1 (R² = 0.48, p < 0.05), and a positive linear relationship between %F_v/F_m(min) and [1 – Q] at E/E_k = 1 (R² = 0.50, p < 0.05), indicating that species inherently primed to preferentially employ greater photochemical over non-photochemical quenching during non-stressed conditions also have PSII that are more thermally tolerant. However, this linear relationship was absent in summer (Figure 5).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.