Chemical bleaching severity determines uptake of heat-evolved Symbiodiniaceae in corals

Chemical bleaching severity determines uptake of heat-evolved Symbiodiniaceae in corals | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 26 February 2026 V1 Latest version Share on Chemical bleaching severity determines uptake of heat-evolved Symbiodiniaceae in corals Authors : Corinne E. Allen 0009-0004-5406-1914 [email protected] , Nadine M. Boulotte , Wing Yan Chan , Matthew Nitschke , and Madeleine J.H. van Oppen Authors Info & Affiliations https://doi.org/10.22541/au.177213271.14545774/v1 257 views 170 downloads Contents Abstract Discussion Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Coral reefs are increasingly threatened by marine heatwaves, driving efforts to enhance coral thermal resilience. One promising approach involves engineering coral symbioses by introducing heat-evolved (HE) algal symbionts into corals. To use this intervention with adult corals, the native symbiont community needs to be depleted, typically via chemical bleaching, prior to inoculation with HE symbionts. However, host recovery and community assembly dynamics in mildly bleached corals remain poorly understood. In this study, a dose-dependent chemical bleaching (CB) experiment was conducted using menthol and diuron (DCMU) to understand how different levels of CB impact host health and acquisition of HE symbionts ( Cladocopium and Durusdinium ). We monitored coral mortality, change in area, pigmentation, and photochemical efficiency through bleaching and twelve weeks of recovery and found that impacts on host health were proportional to bleaching level, although there were strong genotypic effects. Shuffling to the previously low abundance native Durusdinium was also proportional to CB treatment, and only fragments from the severe treatment experienced significant uptake of HE symbionts. Furthermore, significant uptake was not detected until 12 weeks post-deployment, suggesting delayed niche establishment and proliferation of these symbionts. Findings from this study provide insight into symbiont community assembly processes, informing the optimisation and scaling of symbiont engineering and manipulation interventions that enhance coral resilience. Chemical bleaching severity determines uptake of heat-evolved Symbiodiniaceae in corals Authors: Corinne E. Allen* 1,2 : [email protected] Nadine M. Boulotte 2 : [email protected] Wing Yan Chan 2,3 : [email protected] Matthew R. Nitschke 2,4 [email protected] Madeleine J.H. van Oppen 1,2 : [email protected] *Corresponding author Affiliations: 1 School of Biosciences, The University of Melbourne, Parkville, Victoria, Australia 2 Australian Institute of Marine Science, Townsville, Queensland, Australia 3 Department of Biochemistry and Pharmacology, Bio21 Institute of Molecular Science and Biotechnology, The University of Melbourne, Parkville, Victoria, Australia 4 School of Biological Sciences, Victoria University of Wellington, New Zealand Short tunning title: Bleaching severity drives symbiont uptake Abstract Coral reefs are increasingly threatened by marine heatwaves, driving efforts to enhance coral thermal resilience. One promising approach involves engineering coral symbioses by introducing heat-evolved (HE) algal symbionts into corals. To use this intervention with adult corals, the native symbiont community needs to be depleted, typically via chemical bleaching, prior to inoculation with HE symbionts. However, host recovery and community assembly dynamics in mildly bleached corals remain poorly understood. In this study, a dose-dependent chemical bleaching (CB) experiment was conducted using menthol and diuron (DCMU) to understand how different levels of CB impact host health and acquisition of HE symbionts ( Cladocopium and Durusdinium ). We monitored coral mortality, change in area, pigmentation, and photochemical efficiency through bleaching and twelve weeks of recovery and found that impacts on host health were proportional to bleaching level, although there were strong genotypic effects. Shuffling to the previously low abundance native Durusdinium was also proportional to CB treatment, and only fragments from the severe treatment experienced significant uptake of HE symbionts. Furthermore, significant uptake was not detected until 12 weeks post-deployment, suggesting delayed niche establishment and proliferation of these symbionts. Findings from this study provide insight into symbiont community assembly processes, informing the optimisation and scaling of symbiont engineering and manipulation interventions that enhance coral resilience. Keywords: symbiosis, coral reefs, experimental evolution, community assembly Coral survival is intrinsically tied to the symbiotic relationship they form with Symbiodiniaceae, a genetically and functionally diverse group of microalgae (LaJeunesse et al., 2018). These photosymbionts provide the coral host with up to 95% of their daily energetic (Davies, 1984; Falkowski et al., 1984; Muscatine & Porter, 1977); however, prolonged heat stress can lead to a breakdown of the partnership, resulting in loss of symbionts from the coral tissue in a phenomenon known as coral bleaching (Brown, 1997; Glynn, 1991). Some Symbiodiniaceae genera, such as Durusdinium , are naturally heat-tolerant and have been shown to enhance thermal tolerance in corals by 1-2°C (Berkelmans & van Oppen, 2006; Hoadley et al., 2019; Rowan, 2004; Silverstein et al., 2015; Stat & Gates, 2011). However, many coral species in the Indo-Pacific and on the Great Barrier Reef primarily associate with Cladocopium (LaJeunesse et al., 2003; Loh et al., 2001; van Oppen et al., 2001), which can be more sensitive to heat (LaJeunesse et al., 2010). As marine heatwaves and their associated mass bleaching events have become more frequent and severe over the last few decades (Oliver et al., 2018), there is concern that corals may not be able to keep up with the rapid pace of climate change (Hoegh-Guldberg, 1999; Lachs et al., 2024). Interventions aimed at enhancing coral thermal resilience may help maximise the likelihood of coral reef survival over the next few decades while we tackle global carbon emissions. The manipulation of algal symbionts targeted at developing more heat tolerant communities can be achieved through pre-conditioning (i.e., stress-hardening) of corals. In this approach, corals are thermally bleached to sublethal levels and then allowed to recover naturally. While this method may contribute to thermal tolerance by regulating relevant genes within the coral host, corals will often recover with higher proportions of the more naturally heat-tolerant Durusdinium symbiont (Baker, 2001; Buzzoni et al., 2023; Cunning et al., 2015, 2018; Rodriguez-Casariego et al., 2022; Silverstein et al., 2015). The degree to which this symbiont ‘shuffling’ occurs is dependent on disturbance severity and duration, as well as recovery conditions (Cunning et al., 2015). Symbiont manipulation approaches harnessing the concept of symbiont shuffling are being widely explored in the Caribbean, where both lab- and field-based nursery research efforts are underway to outplant pre-conditioned corals that may be more resilient to future conditions (Buzzoni, 2020; DeMerlis et al., 2022). Another strategy takes advantage of corals’ ability to ‘switch’ their symbiotic communities by introducing novel symbionts. This can be achieved by bleaching adult corals and subsequently inoculating them with symbiont cultures, including naturally heat-tolerant or heat-evolved (HE) symbionts (Chan et al., 2023a; Scharfenstein et al., 2022). Inoculations with HE symbionts may be advantageous, as HE Cladocopium proliferum has been shown to shift the upper thermal limit of corals without imposing the growth tradeoffs typically seen with naturally heat-tolerant Durusdinium (Chan et al., 2023a; Jones & Berkelmans, 2010; Little et al., 2004; Quigley et al., 2023). Furthermore, HE Durusdinium may enhance coral thermal tolerance beyond that of native (wild type) Durusdinium (Scharfenstein et al. 2024), although in hospite data are still lacking. Application of this intervention to adult corals is important, as deployment of adult coral fragments (via asexual propagation) has been the most widely used reef restoration approach to date (Rinkevich et al., 2005; Lirman and Schopmeyer, 2016; Young et al., 2011) and adult fragments typically reach reproductive maturity faster than recruits (Rapuano et al., 2023). However, inoculation of adult corals with cultured symbionts to date has only been done by chemically bleaching corals to >95% symbiont removal (Chan et al., 2023a; Scharfenstein et al., 2022). The rationale behind this approach is that in the near absence of remnant symbiont populations, competition for resources (e.g., light, space and nutrients) within the symbiotic niche is reduced and the nutrient deprived state of the host is augmented, increasing the likelihood of exogenous symbiont uptake. However, chemically bleaching corals to this level has an inherent impact on coral health. Menthol is a cyclic terpene alcohol that has anesthetic effects on the coral host (Haeseler et al., 2002) and contributes to inhibition of photosystem II in the Symbiodiniaceae (Wang et al., 2012, 2017). Diuron, or 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), is an herbicide that also affects Symbiodiniaceae by inhibiting their photosystem II complex (Metz et al., 1986; Negri et al., 2005; Wang et al., 2017). If applied to corals for too long, a threshold may be reached where the corals become too starved to recover and the risk of mortality greatly increases (Hoegh-Guldberg, 1999; Jones, 2008). Indeed, previous experiments in our group employing severe bleaching (i.e., 12 days of chemical exposure) (unpublished) have experienced total mortality in up to 20% of experimental fragments and partial mortality in ~75% of experimental fragments. If this approach is to be used as a viable and efficient restoration strategy, mortality induced by CB could be a major limitation. A potential solution to this is to reduce bleaching severity, allowing more native symbionts to persist while still creating space for novel symbionts. However, this introduces more intense competition between remnant and introduced symbionts. If a large population of native symbionts remains, they may exhibit priority effects, outcompeting novel symbionts due to their already established dominance within the coral host (Buzzoni et al., 2023; Debray et al., 2022; McIlroy et al., 2019). It remains to be seen whether HE symbionts can compete with homologous symbionts to occupy vacant microenvironments within the host, which is critical information for optimising the engineering and manipulation of coral symbiont communities. To address this knowledge gap, we used a dose dependent CB experiment to investigate the extent of bleaching on host health and the uptake of HE symbionts. This study provides essential knowledge on how to maximise symbiont diversity within a coral while minimising the risk of mortality induced by CB. Such information will be essential when considering the scalability and implementation of this approach into reef restoration practices. Six colonies of Platygyra cf. daedalea were collected from Falcon Reef (18°46.083’S, 146°32.161’E) in the central region of the GBR at depths of 2-6 m on January 19, 2023 (collection permit: G22/46479.1) and transported back to aquarium facilities in the National Sea Simulator (SeaSim) at the Australian Institute of Marine Science (AIMS) (Cape Cleveland, Australia). After 2 weeks, the colonies were fragmented (n=156 fragments/colony; n=936 total) into ~1.5 x 1.5 cm pieces using a using a Gryphon AquaSaw XL, Model C40-CR Custom, fitted with a 42” Gryphon Diamond Band Saw Blade. All fragments were superglued onto a 2 x 2 cm aragonite plug (Frag Plugs Aragonite Large; OW100LCFP, Aquasonic, Wauchope, Australia) and placed onto custom built PVC trays. After fragmentation, fragments were allowed to recover and grow for Methods section 1.1), after which they were moved to experimental tanks (300 L) for an 8-week acclimation period. During this time, fragments were taken off their seasonal temperature profile and ramped up 0.1°C/day for three weeks until they reached 28°C, which they were held at for the remainder of the experiment. This temperature was chosen as it is closer to what the heat-evolved Symbiodiniaceae were grown at and would ease transition to deployment following the experiment. Hydra 64HD lights (Aqua Illumination, Bethlehem, PA, USA) were used for illumination at an intensity of 82-180 µmol/m 2 /s. The photoperiod was 06:00-18:00, with a 2-hour ramp up (06:00-08:00) and ramp down (16:00-18:00). Fragments were fed daily with a mixture of Artemia nauplii and rotifers, and tanks were cleaned once every week to remove any filamentous algae. These conditions remained the same throughout the duration of the experiment. Experimental design Fragments (n=936) were assigned to one of four bleaching treatments based on rounds of chemical exposure: a) severe bleaching (3 rounds of exposure); b) medium bleaching (2 rounds of exposure); c) low bleaching (1 round of exposure); and d) control (0 rounds of exposure) (Figure 1). Each round consisted of three consecutive days of exposure to menthol (M2772, Sigma-Aldrich, St. Louis, MO, USA) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU; D2425, Sigma-Aldrich), followed by 3 consecutive days of no exposure. Briefly, for each day of exposure, menthol and DCMU were added to 60 L of water at final concentrations of 0.38 mM and 0.13 µM, respectively (Scharfenstein et al., 2022), kept in the spiked sweater for 8 hours (8:30-16:30), and then returned to flow-through for 16 hours (16:30-8:30; overnight) (Supplementary Methods 1.2; Figure S1A). Following CB, fragments were assigned to one of the three symbiont treatments: inoculation with HE C. proliferum (SCF055-01.08; SS8), inoculation with HE D. trenchii (SCF086.01.04; SSD), or no re-inoculation where corals were allowed to recover naturally (negative control; NC) (Figure 1). This led to 10 treatments total (3 CB treatments x 3 symbiont treatments, plus a control treatment). Each treatment comprised n = 90 fragments (n = 15 fragments/colony ID) except for the control, which consisted of n = 126 fragments (n = 20-23 fragments/colony ID). Inoculations were completed overnight (16:30-8:30; 16 hours) in 5 L of water at 10 4 cells/mL for seven consecutive nights (Supplementary Methods 1.3-1.4). Following inoculation, fragments were allowed to recover in their respective inoculation tanks for twelve weeks (Figure 1; Figure S1B). Each week, photos were taken of all coral fragments to monitor their pigmentation throughout the bleaching and recovery periods. All photos were taken on a high-resolution DSLR camera (Nikon D810) with a strobe attached (Godox V860II) under fixed settings (Manual, F8, 1/160, sRGB mode, RAW photo) at a camera height of 42 cm. Photos were taken at the same time (~9:00-10:00am) every week along with a photo of calibration reflectance standards (Calibrite ColorChecker; Spectralon) for standardisation of light and colour. Image calibration and analysis was conducted in ImageJ, with the greyscale standards used to linearise RGB values of the coral fragments using a polynomial function (Supplementary Methods 1.5). Two-dimensional surface area was also obtained from images for all fragments prior to the experiment starting and after 12 weeks of recovery in ImageJ using an in-image scale reference. Photochemical efficiency ( F v / F m ) of fragments was measured using the Walz Diving Pulse Amplitude Modulation Fluorometer (DIVING-PAM II) with a 5 mm fiber optic cable and the following settings: Gain 2, Measuring Intensity 4, Measuring Frequency 4, Damping 2, and Saturating Intensity 0. These settings were chosen to capture the majority of fragments on both ends of the bleaching spectrum and remained the same for the entirety of the experiment to allow for comparisons across time points. Measurements were taken at six time points: prior to CB, post-inoculation, and 4, 8, and 12-weeks post-inoculation. The same fragments were measured at each time point (n = 3 fragments/colony ID/treatment; n = 180), with two measurements taken from each fragment: one on the top and one on the side. Prior to measurement, the fragments were low-light acclimated (~5 μmol m −2 s −1 ) for 10 minutes. Measurements of all fragments occurred on the same day from 06:30-09:00, with treatment order randomised. Coral fragments were sampled at four time points: prior to CB (n = 5 fragments/colony ID; n = 30), post re-inoculation (n = 3 fragments/colony ID/bleaching treatment; n = 72), 1 month post re-inoculation (n = 3 fragments/colony ID/treatment; n = 180), and 3 months post re-inoculation (n = 2-15 fragments/colony ID/treatment; n = 561 fragments). Sample sizes at 3 months post re-inoculation were variable due to a) mortality of some colonies and b) dual purpose of these samples for field deployments. Generally, fragments sampled at one time point were not sampled at a subsequent time point, except for those sampled at 1 month post re-inoculation, which were also sampled at 3 months post re-inoculation. At each sampling time point, two small (~1 mm) samples were taken from each fragment, one from the top and one from the side. This aimed to capture the symbiont diversity on the light-exposed and shaded sides of the fragments. Both samples were pooled, rinsed with sterile seawater to remove loosely attached (i.e., free-living) symbionts and other undesirable algal species. All samples were snap frozen in liquid nitrogen and stored at -80°C until DNA extraction. DNA was extracted from the pooled samples using a modified Wayne’s method (Wilson et al., 2002) with an added bead-beating step. Symbiont identification was performed using the internal transcribed spacer-2 region of ribosomal DNA (ITS2 rDNA). To amplify the region, the SYM_VAR_5.8S2 and SYM_VAR_REV primers (Hume et al., 2018) were used with custom sequencing adaptors. Detailed polymerase chain reaction (PCR) and library preparation protocols can be found in the Supplementary Methods 1.6. All samples were split across two sequencing libraries, with the first library containing the first three time points, and the second library containing the final time point. Both libraries were sequenced on an Illumina NextSeq platform (2 x 300 bp) at the Walter and Eliza Hall Institute (WEHI) in Melbourne, Australia. Libraries were demultiplexed and cutadapt was used to trim adaptors from raw sequences (Martin, 2011). The fastq files were then submitted to SymPortal (symportal.org) for ITS2 profiling (Hume et al., 2019). Due to the multicopy nature of the ITS2 marker, it can be difficult to differentiate between intragenomic and intergenomic variability. However, the SymPortal framework is able to assess patterns of intragenomic variation and match sequences to “defining intragenomic variants” (DIVs) to create profiles, representative of putative Symbiodiniaceae taxa (Hume et al., 2019). The ITS2 sequence and profile abundance data was imported into R where it was analysed. Upon subtracting the DIVs in the no template controls (average read depth: 758; primarily C21) from each of the corresponding samples, any samples containing <5 DIVs and having <100 reads were removed (n = 21 samples). This filtered dataset was used in the downstream symbiont community analyses. A kmer-based approach (k = 7) was used to calculate a pairwise sequence similarity matrix using the kmer package (Wilkinson, 2018). These distances were incorporated into a hierarchical clustering analysis using the upgma function in the phangorn package (Schliep et al., 2017; Schliep, 2011) to generate a phylogenetic tree of ITS2 sequences. This tree, along with the information on sequence abundance and relatedness within each sample, was then used to generate weighted UniFrac distances between all fragment samples (Lozupone & Knight, 2005). Diagnostic profiles of HE C. proliferum (SCF055-01.08), HE D. trenchii (SCF086.01.04), native Cladocopium , and native Durusdinium were created by pooling internal references from multiple library runs (Figure S2). To determine the relative abundance of each strain in the coral fragments, the contribution of each DIV from the inoculum profile to the total read count was calculated. Where there were overlapping signals (i.e., DIVs) with native symbionts, they were disentangled using the r package deconvR (Gunduz et al., 2025) and the pre-established reference profiles (Supplementary Methods 1.7). Statistical analysis All analyses were run in RStudio version 4.4.3 (R Core Team, 2021). For each physiological response variable (i.e., mortality, change in area, pigmentation, and F v / F m ), candidate models included all main effects and interaction terms between CB treatment, colony ID, and side of colony (side only relevant for F v / F m ). Since controls did not receive symbiont inoculations, symbiont treatment was evaluated in a second set of models that excluded the control CB treatment. These models included fixed effects of CB treatment, symbiont treatment, colony ID, side (for F v / F m ), and their interactions. Tank replicate was included as a random effect in all models, unless otherwise stated. Colony ID was chosen as a fixed effect rather than random as it a) allowed for direct assessment of the effect of colony on each response variable, and b) improved model fit (as assessed using Akaike Information Criteria (AIC)). Model selection was guided by AIC and Bayesian Information Criterion (BIC) and Likelihood Ratio Tests, ensuring that only terms improving model fit were retained. Model assumptions were evaluated using simulation-based residual diagnostics (DHARMa). The significance of factors in each statistical model is reported using partial F -tests (LMMs) or Type II Wald χ²-tests (GLMs/GLMMs) via ANOVA, with significance determined at α < 0.05. Differences between factor levels were assessed by conducting post-hoc interaction analyses using emmeans (Lenth, 2025). Models for each response variable are summarised below. Mortality was assessed using a general linear model ( glm ) with a binomial family and logit link function. No random effect was included in this model, as negligible variance resulted in model singularity. Change in area (i.e., growth or partial mortality) was assessed by using log-transformed changes in area between the initial and final time points. The log-transformed values adjust for differences in starting size and show improved normality. Only fragments with remaining tissue at the end of the experiment were incorporated in this analysis. Reflectance values were standardised using decostand from the r package vegan (Oskansen et al., 2025) and run through a principal component analysis (PCA). As the PC1 axis explained ~95% of the variance (primarily driven by the Red channel), the distance fragments travelled along the PC1 axis between time points was used to explore shifts in pigmentation. Pigmentation at six major time points (post-bleaching, post-inoculation, 2 weeks post-inoculation, and 1-, 2-, and 3-months post-inoculation) was analysed using generalised linear mixed models ( glmmTMB ). PC1 values were shifted to be positive and models were fit using a Tweedie distribution with a log link and a dispersion parameter of CB treatment to address heteroscedasticity. Symbiont photochemical efficiency ( F v / F m ) measurements at five time points (pre-bleaching, post-inoculation, and 1-, 2-, and 3-months post-inoculation) were assessed using beta regression models ( glmmTMB ) with a logit link and a dispersion parameter modeled as a function of CB treatment to address heteroscedasticity. An additional random intercept for individual fragment was incorporated in these models to account for paired top/side measurements. Measurements with minimum fluorescence ( F o ) readings <100 were excluded, as these may reflect noise and can produce F v / F m values that are unreliable. This led to an exclusion of 8% (n = 203) of readings (n = 174 top and n = 29 side), mostly from the post-bleaching time point. Correlations between traits (i.e., photochemical efficiency, pigmentation loss, and change in area) across colony IDs were assessed using ggpairs from the GGally r package (Schloerke et al., 2025). Pairwise Pearson correlation coefficients were calculated for each trait combination grouped by colony ID. For this analysis, only F v / F m values taken from the top of the fragment were used to better compare against pigmentation loss since photos were only taken of the tops of fragments. All change in area measurements (log-transformed) were included (including corals experiencing total mortality) to capture both partial and total mortality within this analysis. Differences in symbiont community composition across CB treatments, time points, and colonies, were assessed by performing a permutational multivariate analysis of variance (PERMANOVA) on the weighted UniFrac distances between samples ( adonis2 ). Each sample’s distance to its group centroid was then measured using betadisper as a proxy for beta diversity dispersion, followed by permutation tests and Tukey HSD post-hoc comparisons to identify significant differences between groups. To explore patterns in uptake of HE symbionts, we used a two-part modelling approach. First, we assessed the likelihood of uptake using generalised linear models ( glm ) with a binomial family, with CB treatment and colony ID as fixed effects. Of the fragments that exhibited successful uptake, we then ran beta-regression models ( glmmTMB ) with a logit link to examine the effect of CB treatment and colony ID on relative abundance of HE symbionts. All models were run separately for HE D. trenchii and HE C. proliferum at both post-inoculation time points where uptake was detected (1 month and 3 months). To assess variability in uptake within each CB treatment x colony ID group, we independently fit binomial logistic regression models using three predictors: change in area (log-transformed), pigmentation lost during CB, and initial fragment size. To increase power within each of the models, uptake was defined as the presence of either HE symbiont. Models were only fit in CB treatment x colony ID levels where there was variability in uptake (i.e., fragments with and without uptake) and all predictors were run together. No fragments experienced mortality until seven weeks after chemical exposure (ten weeks into the experiment), but 7.7% of the coral fragments (n = 72) experienced total mortality within the remaining six weeks (Figure 2A). There was a significant effect of CB treatment (χ² = 124.36, p < 0.0001; Table S2), with severely bleached fragments reaching 21% mortality (n = 56 fragments). In contrast, only 5% of fragments (n = 13) from the medium treatment and 1% (n = 3) from the low treatment experienced mortality, and there was no mortality experienced in the control treatment (Figure 2B). Mortality did not vary by symbiont inoculation treatment (Figure 2C) but was influenced by coral colony (χ²=168.67, p < 0.0001; Figure 2D), with one colony in particular (colony E) contributing to most of the mortality experienced in all treatments. Change in area of coral fragments was influenced by CB treatment (χ² = 553.92, p < 0.0001), colony ID (χ² = 633.20, p < 0.0001), and their interaction (χ² = 240.71, p < 0.0001) (Table S3). Over the sixteen-week experimental period, fragments from the control and low CB treatments experienced net growth, with average log changes in area of 0.42 and 0.17, corresponding to percent changes in area of 54% and 21%, respectively (Figure 3A, Figure S3). In contrast, the medium and severe CB treatment largely experienced net declines in area (i.e., partial mortality), with average log changes of -0.12 (-6% change in area) and -0.53 (-35% change in area), respectively. Post-hoc comparisons revealed that differences between all CB treatments were statistically significant (all p < 0.0001). There were also consistent colony-level differences across treatments that became more pronounced as bleaching severity increased (Figure 3B). For instance, colonies A and E experienced the lowest amount of growth across all treatments, with colony A becoming significantly different from all other colonies in the medium and severe CB treatments (all p < 0.05), while colonies B, C, and F maintained higher growth throughout all treatments. Upon looking at symbiont treatment within the low, medium, and severe bleaching treatments, the effect accounted for minimal variation when compared with CB treatment and colony ID and was not significant (Figure 3C; Table S3). Bleaching While there was no difference in the pigmentation of corals prior to CB, corals that were chemically bleached experienced shifts in their pigmentation relative to the controls (Figure 4A). These shifts can be quantified by distance travelled along the PC1 axis, with more positive distance travelled corresponding to more loss of pigmentation (i.e., more bleaching). There were significant differences in the distances travelled between CB treatments (χ² = 7643.45, p < 0.0001), with corals in the severely bleached treatment travelling an average of nearly three times as much distance as corals in the control treatment (Control = -0.02; Low = 0.03; Medium = 0.18; Severe = 0.26) (Figure 4B). There was also a significant effect of colony ID on how much fragments bleached (χ² = 528.24, p < 0.0001), with colonies B and D experiencing significantly more bleaching than all other colonies (p < 0.0001) (Figure S4B). Colony F retained the most pigment, and there were no significant differences between colonies A, C, and E. Recovery Following inoculation, there were significant effects of CB treatment, colony ID, and their interaction at all recovery time points (all p < 0.0001) (Table S4). Low and medium bleached corals started regaining pigmentation immediately following inoculations; however, the severely bleached corals did not start regaining pigmentation until 1-month post-inoculation (Figure 4C; Figure S4A). All CB treatments remained significantly different from each other until ~8 weeks post-inoculation, when the low treatment became indistinguishable from the controls. The medium and severe treatments never reached control pigmentation levels during the 12-week recovery period, with the exception of colony F (Figure S4A, B). Symbiont treatment also significantly influenced pigmentation in the early and mid-recovery stages (all p < 0.01) (Table S4). While there was variability immediately following inoculation, fragments that were inoculated with HE C. proliferum or HE D. trenchii pigmented significantly faster than those that were not (NC) after 4 weeks post-inoculation. At later stages of recovery, fragments inoculated with HE. D. trenchii trended towards higher pigmentation levels than those inoculated with HE C. proliferum ; however, these differences were not significant. By 12 weeks post-inoculation, pigmentation did not differ among symbiont treatments. PSII maximum quantum yield (F v /F m ) Bleaching Symbiont photochemical efficiency ( F v / F m ) after CB was influenced by CB treatment (χ² = 627.52, p < 0.0001), colony ID (χ² = 14.54, p = 0.006), and side of fragments (χ² = 7.00, p = 0.008) (Table S5). Overall, increased chemical exposure was associated with lower photochemical efficiency, with low, medium, and severe CB treatments experiencing 14%, 23%, and 31% reductions in F v / F m from pre-bleaching levels, respectively (averaged over tops and sides). Post-hoc comparisons revealed that all of these were significantly different from each other and from the controls (all p < 0.0001). The control, medium, and severe CB treatments had no significant differences in F v / F m values between tops and sides of fragments; however, the low treatment saw significantly lower values on the tops of fragments (z.ratio = 4.43, p < 0.0001), suggesting the presence of a bleaching landscape across the surface of fragments in this treatment (Figure 5A). There was also an interaction between CB treatment and coral colony (χ² = 53.44, p < 0.0001), with some colonies experiencing up to 25% lower F v / F m values than others within the same CB treatment. Notably, colonies C and F exhibited distinct responses across all bleaching treatments (all p 0.4). (Figure 5C). Colony B had no data in the severe treatment at the post-inoculation time point due to all F values falling below the threshold value (99). Recovery Photochemical efficiency gradually improved over time, but there were significant effects of CB treatment, side, and colony ID (all p < 0.01), as well as interactive effects of CB treatment and colony ID (all p < 0.01) and CB treatment and side (all p < 0.05) at all recovery time points (Table S5). By 4 weeks post-inoculation, fragments in the low CB treatment had functionally recovered to control levels, whereas it took 8 weeks for medium fragments to functionally recover. On the other hand, neither the tops or sides of fragments from the severe CB treatment functionally recovered to control levels by 12 weeks (Figure 5B). Symbiont treatment was only a significant factor at 4-weeks post-inoculation, when fragments inoculated with HE D. trenchii had significantly higher values than fragments that weren’t inoculated (NC treatment) (z.ratio = -2.897, p = 0.01). Trait correlations across colonies Across most colonies, photochemical efficiency measurements and distance travelled along the PC1 axis were negatively correlated (r = -0.65, p < 0.0001; Figure 6A), with more pigmentation loss corresponding with lower F v / F m values. Additionally, pigmentation loss and change in area were typically negatively correlated (r = -0.40, p < 0.0001; Figure 6B), with more bleaching leading to higher partial or total mortality (Figure 6B). The only metrics positively correlated with each other across most colonies were photochemical efficiency and change in area (r = 0.48, p < 0.0001; Figure 6C). However, the strength and direction of these relationships varied by colony ID. For instance, colony B exhibited the highest amount of pigmentation loss (0.23) but neutral changes in area (-0.01), whereas colonies A and E exhibited the least amount of pigmentation loss (0.13 and 0.11, respectively) but some of the most amount of tissue mortality (-0.54 and -0.65) and lowest F v / F m values (Figure 6B, C). In contrast, colony D displayed more linear trade-off trajectories, with intermediate levels of pigmentation loss and F v / F m values (0.17 and 0.58, respectively) corresponding to intermediate levels of tissue loss (-0.30). ITS2 sequencing yielded 498 unique sequences across 820 samples after quality filtering. The average per-sample depth was 10,577 reads, although this varied by time point (Table S1). The majority of DIVs belonged to the genus Cladocopium (69.8%), followed by Durusdinium (27.7%), and Symbiodinium (2.4%). The native Cladocopium found in fragments primarily belonged to one of several profiles from the C21 lineage: a) C21-C21d-C21e-C21af; b) C21-C3-C21d-C3at-C21e-C3db; or c) C21/C21en-C3-C3ed-C21d-C21e (hereafter referred to as ‘C21’; Figure S2); whereas the native Durusdinium profile was D1/D4-D2-D4c-D6 (Figure S2). Initial symbiont communities Prior to CB, all sampled fragments (n = 30) were dominated (> 99%) by native Cladocopium (i.e., C21 profiles) (Figure 7). Many of the fragments sampled also harboured background abundances of Durusdinium (0.01 - 0.74%), except for six fragments where Cladocopium was the only symbiont detected. There were no significant differences in community composition between colonies according to the permutational analysis of variance (PERMANOVA,\(R^{2}\) = 0.27, F = 1.78, p = 0.068). Post-inoculation symbiont communities Due to low symbiont abundances left in corals following bleaching, ten samples from this time point were removed as a result of low read depths. The symbiont communities of the remaining fragments (n = 61) still largely resembled the pre-bleaching communities (Figure 7). Thirty-three fragments harboured only Cladocopium , nineteen contained background Durusdinium (0.01 - 3.9%), and nine fragments had moderate to high amounts of Durusdinium (7.1 - 92%). Permutational analysis of variance confirmed that there was a significant difference between pre-bleaching and post-inoculation communities (PERMANOVA, \(R^{2}\) = 0.04, F = 3.37, p = 0.031) and between CB treatments (PERMANOVA, \(R^{2}\ \)= 0.14, F = 9.09, p = 0.001). Pairwise comparisons revealed that this difference only occurred in the severely bleached fragments (all p < 0.01). We did not have the statistical power to determine any differences between colonies due to the removal of samples. Post-inoculation communities (1 month) Four weeks after inoculation, only 8.5% (n = 9) of sampled, inoculated fragments (n = 106) harboured detectable levels of HE symbionts. HE D. trenchii was the more prevalent of the two, occurring in 13% (n = 7) of the fragments inoculated with HE D. trenchii (n = 54) at relative abundances of 0.4 - 42.3%. There were no significant differences in the number of fragments with HE D. trenchii or HE C. proliferum uptake between CB treatments or colony ID at this time point (Table S6). However, shuffling to native Durusdinium was an important cause of symbiont community changes, with 57% of corals harbouring Durusdinium at relative abundances of 0.01 - 100%. This was heavily influenced by both CB treatment and colony F (p < 0.0001), with colony F from the severe CB treatment experiencing the most shuffling in terms of quantity of fragments and the magnitude of change. Post-inoculation communities (3 months) While apparent uptake of HE symbionts was low at four weeks post-inoculation, at twelve weeks post-inoculation 34% (n = 68) of the sampled fragments inoculated with HE D. trenchii (n = 198) harboured it in relative abundances ranging from 0.4 - 99.9%. Of the sampled fragments inoculated with HE C. proliferum (n = 208), the inoculum was detected in 30% (n = 63) in relative abundances of 0.2 - 99.5%. There were significant effects of CB treatment (HE D. trenchii : χ² = 89.85, p < 0.0001; HE C. proliferum : χ² = 88.17, p < 0.0001) and colony ID (HE D. trenchii : χ²= 56.83, p < 0.0001; HE C. proliferum : χ² = 49.20, p < 0.0001) on the number of fragments with successful acquisition (Table S6), with severely bleached fragments experiencing the most uptake (n = 88), followed by the medium (n = 39) and then the low treatment (n = 4). Similarly, there were significant effects of CB treatment (HE D. trenchii : χ² = 40.73, p < 0.0001; HE C. proliferum : χ² = 25.54, p < 0.0001) and colony ID (HE D. trenchii : χ² = 25.41, p < 0.0001; HE C. proliferum : χ² = 62.65, p < 0.0001) on the relative abundance of HE symbionts within fragments (Table S6), with severely bleached fragments experiencing the highest abundances in their tissues. Patterns in uptake While CB treatment and colony ID explained broad patterns in HE symbiont uptake, there was still variability in the number of fragments exhibiting uptake within each bleaching treatment x colony ID level. However, none of the predictors tested (change in area, pigmentation loss, and initial size) consistently explained this variation (Figure S5). Change in area had a significant negative effect only in colony B within the severe CB treatment (p = 0.042). Furthermore, pigmentation loss significantly predicted a decrease in probability of uptake for colony F (p = 0.042). Initial size did not significantly explain uptake variability in any treatment or colony combination. Overall community diversity Overall community diversity compared to pre-bleaching diversity was higher for the severe CB treatment at all time points (all p < 0.001) (Figure S6). While the medium treatment had marginally higher diversity at 12 weeks-post bleaching than prior to CB treatment (p = 0.079), this was not significant. There were also no significant differences in community diversity detected in the low CB treatment or controls at any time point (all p > 0.90). These patterns in community dispersion were associated with more pronounced shuffling to Durusdinium as CB intensified (\(R^{2}\) = 0.60, p < 0.0001). Discussion As efforts to scale and implement coral reef interventions become increasingly urgent, there is a need to optimise strategies to minimise costs and risks while maximising ecological benefits. For symbiont manipulation interventions involving adult corals, this includes finding ways to balance symbiont uptake with host health. This study found that there are tradeoffs between the two outcomes and decisions about which chemical bleaching severity to use should be made based on experimental and restoration goals. We found coral health was proportionally impacted by length of chemical exposure, with overall performance ranking as no CB > low CB > medium CB > severe CB across all metrics monitored. These results further support other studies finding CB to be a quick, reliable, and effective way to remove symbionts from cnidarians, with symbiont loss strongly related to dosage (time and/or concentration) (Matthews et al., 2015; Wang et al., 2012). Recovery trajectories (determined by F v / F m values and pigmentation) also differed between CB treatments. Fragments from the low and medium treatments functionally recovered to control levels in as little as four and eight weeks, respectively; however, fragments from the severe treatment had still not fully recovered by twelve weeks. Interestingly, symbiont treatment was not found to play a significant role in total or partial mortality within the timeframe of this experiment, suggesting that the effects of CB were stronger than the effect of receiving symbionts under ambient conditions. However, because mortality continued to increase towards the end of the experiment, it is possible that significant effects of receiving an inoculum may have played out over longer timescales. Furthermore, symbiont effects on mortality may be stronger under elevated temperatures. However, we did find that inoculated corals generally regained pigment more quickly than non-inoculated ones, indicating quicker symbiont repopulation and recovery where symbionts were introduced, despite these being heterologous symbionts. This has been observed in early life stages, where larvae or recruits inoculated with symbionts experience quicker establishment, earlier pigmentation, and higher survival than those not inoculated (Hazraty-Kari et al., 2022; Suzuki et al., 2013). Furthermore, fragments inoculated with Durusdinium re-pigmented slightly faster than those inoculated with Cladocopium , consistent with the idea that Durusdinium is particularly effective at proliferating within bleached or uninhabited tissue (Cavailles et al., 2025; Claar et al., 2020; Cunning et al., 2015; Ivory et al., 2025; McIlroy et al., 2019, 2020). In addition to CB treatment effects, strong colony (i.e., genotypic) patterns were evident in bleaching responses, growth, and mortality. For instance, some colonies displayed much lower photochemical efficiency values than others. Furthermore, some colonies experienced no difference in photochemical efficiencies between the medium and severe treatments at the post-inoculation time point. These results suggest that some colonies have different thresholds for stress tolerance (Humanes et al., 2022) and/or limited capacities to handle stress once a critical threshold is reached. This was further confirmed by the trait tradeoff analysis, where some colonies displayed high mortality despite experiencing less bleaching. Host genotype is known to play a role in coral bleaching response (Cunning et al., 2016; Dilworth et al., 2021; Drury et al., 2017; Fuller et al., 2020; Klepac et al., 2023), post-bleaching mortality (Ladd et al., 2017; Sakai et al., 2019), physiological plasticity (Drury & Lirman, 2021; Million et al., 2022), growth (Bowden-Kerby & Carne, 2012; Drury et al., 2017; Grasso, 2016), and many other physiological traits (Drury, 2020). Given that the colonies in this study came from the same environment and similar depths and there were no differences in symbiont community compositions prior to CB, the variable responses observed here are likely attributed to genetic differences between them. These could be related to differences in regulatory gene expression pathways (Thomas et al., 2018) or variation in baseline energy reserves that influence the ability to withstand and recover from stress (Rodrigues & Grottoli, 2007; see ’Nutritional implications of chemical bleaching’). Regardless, these results highlight the importance of incorporating genetic diversity into restoration efforts (Baums et al., 2019, 2022; Bowden-Kerby & Carne, 2012). Chemical bleaching ultimately causes a breakdown of symbiosis, triggering a cascade of effects that results in the loss of Symbiodiniaceae from the coral host. This in turn reduces photosynthate supply and alters host metabolism (Wang et al., 2012), with the magnitude of bleaching determining the extent of symbiont loss and decline in photosynthetic activity (Cunning et al., 2015). Although corals can survive periods of low photosynthetic productivity (28-114 days) by drawing on lipid reserves (Davies, 1991), severely bleached corals in this study experienced ~100 days of reduced photosynthetic activity and would have been approaching their upper threshold of energetic reserves (Anthony et al., 2007; Rodrigues & Grottoli, 2007). This is consistent with the large amount of partial mortality seen in the severe CB treatment and the delayed onset of total mortality seen towards the end of the experiment. These dynamics may also explain some of the genotypic patterns seen in this study (e.g., higher mortality of colony E), as lipid content and energy reserves have been shown to vary among genotypes (Bay et al., 2013; Schoepf et al., 2013). Mitigation of CB impacts could potentially be achieved through nutritional interventions before or after the bleaching phase; for instance, Wang et al. (2012) found that supplementing corals with nutrients (e.g., glycerol, free amino acid mixture, and vitamins) aided in the physiological and biochemical performance of bleached corals. Future studies could build on this by using lipidomics or metabolomics to characterise the components and/or mechanisms of the coral energy budget being affected by CB and design targeted diets to directly offset such impacts and improve coral survival. The differences in photochemical efficiencies observed between tops and sides of fragments represent a ‘bleaching landscape’, shaped by the fragment’s three-dimensional structure and associated microenvironments (Hill et al., 2004; Voolstra et al., 2025). The tops of fragments, which received more light, had lower photochemical efficiencies than the more shaded sides or walls, suggesting potential additive effects of chemical exposure and light stress. However, this spatially heterogeneous bleaching response was only significant in the low CB treatment, indicating a ‘transitory’ state of bleaching, beyond which chemical exposure outweighs effects of light and may lead to whole-fragment bleaching (Voolstra et al., 2025). These patterns may have affected symbiont community trajectories. While we pooled samples for this study in order to maximise sample sizes and can thus not differentiate between spatial gradients in symbiont communities, there are many other studies that have demonstrated spatial distributions or landscapes of symbionts driven by light patterns (Garren et al., 2006; Kemp et al., 2015; Rowan et al., 1997; Toller et al., 2001; Ulstrup et al., 2023) and available niche space (McCauley et al., 2023). Thus, we urge other studies to take samples from multiple microenvironments of a fragment or colony when sampling for symbiont community composition in order to capture potential spatial heterogeneity (Lewis et al., 2022). In the literature, uptake of new symbionts is often denoted as ‘symbiont switching’ (Baker et al., 2004; Buddemeier & Fautin, 1993; Fautin & Buddemeier, 2004). While this has been documented during heat stress tests and bleaching events (Boulotte et al., 2016; Lewis & Coffroth, 2004; Quigley et al., 2022), this study provides the first known direct evidence of it occurring in mildly bleached corals. Successful establishment in a new host requires overcoming several barriers, including coming into contact with the host, successfully evading the host’s immune system, and rapidly establishing an intracellular niche (Albright et al., 2022; Bright & Bulgheresi, 2010; Kaltenpoth et al., 2025; Raina et al., 2019). Given the inoculation dose and frequency used in this experiment (7 rounds of 10 4 cells/ml) and the fact that C. proliferum and D. trenchii are generalist symbionts known to associate with Platygyra spp. in experiments (Scharfenstein et al., 2022; Ivory et al., 2025) and in nature (Jia et al., 2023; Wang et al., 2019; Wong et al., 2016), the first two barriers were likely not the limiting ones. However, niche establishment may be hindered in mildly bleached corals due to the presence of residual native symbionts. These symbionts may exhibit priority effects (Chase, 2003; Debray et al., 2022; Fukami, 2015; Kong et al., 2025), where the established community can prevent or inhibit new or later arrivals through occupation of nutritional niches (Gause, 1934), modification of the physical or chemical environment (Chappell et al., 2022; Peterson, 1984), or employment of density-dependent strategies (Eitam et al., 2005; Fraser et al., 2015; Waters et al., 2013). Such effects are common in ecological communities (Stroud et al., 2024) and have been observed in many organisms, including juvenile octocorals (McIlroy et al., 2019). As the residual symbionts in this study were already integrated into the host’s cellular structure and nutrient sharing pathways, they may have had a competitive advantage, making it difficult for new symbionts to establish themselves. This is supported by the delay in proliferation of HE symbionts observed in the severe treatment. If HE symbionts require up to 12 weeks to establish and proliferate when >95% of all other symbionts are removed, it is unlikely that they would have time to establish and compete against remaining native symbionts in mildly bleached corals to achieve significant abundances within the tissue. Slow colonisation of heterologous symbionts has been observed in inoculation studies done on Aiptasia (Gabay et al., 2018; Medrano et al., 2019; Schoenberg & Trench, 1980; Tivey et al., 2022; Tsang Min Ching et al., 2022), with success improving over time (Tivey at al., 2022; Tsang Min Ching et al., 2022). For instance, Tivey et al. (2022) found a density threshold for Durusdinium proliferation within Aiptasia at 3 weeks, beyond which proliferation was much higher. While this study didn’t include samples taken between 4 weeks and 12 weeks and so it is possible that the threshold lies somewhere within this window, we suggest waiting until at least 3 months post re-inoculation to determine whether uptake was successful. While not as significant as in the severe treatment, there was evidence of HE symbionts present in low and medium bleached corals. While each fragment was sampled in two locations to maximise the chance of capturing community diversity, these samples were quite small (~1 mm 2 ). Given the bleaching landscapes present in mildly bleached corals, localised uptake may have occurred in spatially isolated parts of the colony, unsampled microhabitats, or at levels below detection limits. Under favorable conditions, these symbionts may be able to proliferate to higher abundances or different regions within the fragments (Bay et al., 2016; Buzzoni et al., 2023; Silverstein et al., 2015). Continued monitoring, particularly under heat stress, will be essential to determine whether this can occur, in which case mildly bleaching corals may be able to promote health of corals and still provide benefits of HE symbionts. Even though uptake was low in the medium treatment, the overall symbiont diversity was higher following CB than it was prior to bleaching. This suggests that mild disturbances can promote shifts in composition, potentially by bringing background symbionts forward during recovery. Interestingly, this wasn’t seen in the low CB treatment, suggesting there is a threshold of disturbance that needs to occur for symbiont shuffling to take place. This is further supported by the consistently higher diversity in the severe CB treatment across all time points. The observed beta diversity is likely driven by increases in background abundances of other Cladocopium DIVs or Durusdinium , as fragments had limited opportunity to acquire other symbionts. Our results corroborate other studies that find severe bleaching (thermally or chemically induced) promotes symbiont community shifts toward native Durusdinium . Cunning et al. (2015) similarly reported that only high thermal bleaching severity induced a shift in dominance from Breviolum to Durusdinium , while lower bleaching levels actually reduced Durusdinium abundance. Although the corals in this study initially had background levels of Durusdinium and thus did not experience any significant decreases, the largest increases in Durusdinium were observed in the severe CB treatment. Interestingly, where shuffling to native Durusdinium was the greatest, uptake of HE symbionts was low. As mentioned before, this may be due to Durusdinium being particularly effective at proliferating within bleached or uninhabited tissue, where competition for resources (i.e., light and nutrients) is minimal and opportunistic expansion is allowed (McIlroy et al., 2019; Ivory et al., 2025). Interestingly, HE Durusdinium appeared to be excluded when native Durusdinium was present, which may provide further evidence for priority effects within these corals. Alternatively, this could represent the consequence of niche overlap between the Durusdinium strains, as observations of intra-genus co-occurrence of Durusdinium strains or species are rare. There was also a genotypic effect in shuffling patterns, with one colony in particular (colony F) experiencing the greatest shifts to Durusdinium despite all colonies harbouring similar communities prior to CB. Host genetics can be responsible for regulating physiological flexibility in the abundance and diversity of symbionts and microbes (Glasl et al., 2019; Manzello et al., 2019; Tortorelli et al., 2020). This underscores the complexity of host-symbiont-environment interactions and suggests that deciphering genotype x genotype x environment (GxGxE) interactions is critically important in understanding symbiont community trajectories and adaptive capacity under future climate change scenarios (Million et al., 2022). In summary, the results here show that more severe chemical bleaching of corals maximises the chances of successful HE symbiont acquisition, but at the cost of greater impacts to host health and recovery. This tradeoff underscores the importance of determining restoration goals (i.e., thermal tolerance vs. growth) when designing and implementing interventions. If the desired goal is to outplant corals that are in good health with slightly elevated symbiont diversity, then bleaching corals to more moderate levels (i.e., 2 rounds or 6 days of exposure) is recommended. However, if the priority is to enhance thermal tolerance in the short term, severe chemical bleaching (i.e., 3 rounds or 9 days of exposure) may be warranted to maximise the establishment and relative abundance of HE or heat-tolerant symbionts. Alternatively, researchers or practitioners could apply severe bleaching but incorporate more genotypes, buffering against mortality by diversifying the starting material. Regardless, we recommend incorporating host genetic diversity into restoration interventions given the strong colony effects on bleaching and recovery. The increase in some metrics (e.g., mortality and HE symbiont proliferation) towards the end of this experiment also underscores the importance of long-term monitoring to fully understand host and symbiont community trajectories. 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We pay our respects to their elders both past and present and extend that respect to all Aboriginal and Torres Strait Islanders. We thank the National Sea Simulator staff, especially Matthew Salmon, Jarvis Aland, Grant Milton, and Steve Green for their technical support in experimental setup. We also thank Catalina Parra Velazquez for her help in experimental maintenance, and Eve Hinchliffe, Annika Withers, and Mathilda Bates for their assistance in DNA extractions. This research was supported by the Reef Restoration and Adaptation Program (funded by the partnership between the Australian Government’s Reef Trust and the Great Barrier Reef Foundation), the Paul G. Allen Family Foundation (PGAFF), the Australian Institute of Marine Science, the University of Melbourne, and an Australian Research Council (ARC) Laureate Fellowship FL180100036 to MJHvO. CEA was also supported by the Dr. Albert Shimmins Fund and the American Australian Association, MRN was supported by a Royal Society Te Apārangi Marsden Fast Start grant (21-VUW-021), and WYC was supported by ARC Discovery Early Career Researcher Award DE240100317 and the Westpac Research Fellowship. Conflict of interests None declared. Data availability Raw sequences of the ITS2 Symbiodiniaceae datasets will be made available in GenBank (BioSample accession: XXXX; BioProject ID: PRJNAXXXX). The data files and associated code that support the findings of this study will be made openly available on Github (https://github.com/corinneallen/bleaching_levels). Author contributions Conceptualisation: Corinne E. Allen, Wing Yan Chan, Matthew R. Nitschke, Madeleine J.H. van Oppen Investigation: Corinne E. Allen, Nadine M. Boulotte Data curation: Corinne E. Allen, Nadine M. Boulotte Validation: Corinne E. Allen, Nadine M. Boulotte Formal analysis: Corinne E. Allen, Matthew R. Nitschke Visualisation: Corinne E. Allen Writing – Original draft: Corinne E. Allen Writing – Review and editing: Corinne E. Allen, Nadine M. Boulotte, Wing Yan Chan, Matthew R. Nitschke, Madeleine J.H. van Oppen Supervision: Wing Yan Chan, Matthew R. Nitschke, Madeleine J.H. van Oppen Funding acquisition: Madeleine J.H. van Oppen Figure legends Figure 1. Overview of experimental design. Fragments were chemically bleached to one of three levels, based on rounds of exposure they received (1 round = 3 days exposure, 3 days recovery). Following chemical bleaching, fragments were inoculated with heat-evolved (HE) C. proliferum , HE D. trenchii , or not inoculated (NC). Inoculations were performed every day for 1 week at 10,000 cell/mL, after which fragments were allowed to recover in their individual tanks for 12 weeks. Throughout the experiment, several metrics were monitored, including survival, pigmentation, growth, photochemical efficiency, and symbiont community composition. Figure 2. A) Percent survival of all fragments through the chemical bleaching and recovery phases of the experiment. The dashed line represents the point at which mortality was first seen (10 weeks into the experiment; 7 weeks post-bleaching). B) Proportion of fragment mortality within each chemical bleaching treatment at 12 weeks post-inoculation. C) Proportion of fragment mortality by symbiont treatment within each chemical bleaching treatment. HECp: heat-evolved C. proliferum ; HEDt: heat-evolved D. trenchii ; NC: negative control. D) Fragment mortality by specific colony within each chemical bleaching treatment. The horizontal dashed line represents the starting biomass for each colony (n = 45 fragments/colony/chemical bleaching treatment). Figure 3. Change in area over fifteen weeks for each A) chemical bleaching treatment and B) colony ID within chemical bleaching treatment. The horizontal dashed line represents no change in area; above the line represents growth and below the line represents partial mortality. Figure 4. A) Plots showing changes in pigmentation of fragments from pre-bleaching to post-bleaching. Each dot represents an individual fragment and fill colours represent chemical bleaching treatments. Movement to the right along the PC1 axis represents a loss in pigmentation, whereas movement to the left represents a gain in pigmentation. B) Changes in pigmentation of fragments from pre-bleaching to post-bleaching for each colony ID and chemical bleaching treatment. Images represent a fragment from each of the chemical bleaching treatments at the end of the chemical exposure. C) Time series showing recovery in pigmentation. Figure 5. A) Photos from the top and side of a coral fragment showcasing the bleaching landscape present across the surface of a fragment. B) Photochemical efficiencies of tops and sides of coral fragments at each time point by chemical bleaching treatment (n = 90/chemical bleaching treatment; n = 30 for controls). C) Photochemical efficiencies of fragments within each colony ID (n = 15/colony/chemical bleaching treatment; n = 5/colony for controls). Figure 6. Trait trade-offs among pigmentation loss (PC1 distance travelled), photochemical efficiency ( F v / F m ), and change in area (log-transformed). Each dot represents the mean value of a colony ID for that trait combination, with colour indicating the strength and direction of the within-colony Pearson’s correlation coefficients (r). Blue hues represent negative correlations, whereas pink hues represent positive correlations. Figure 7. Composition of symbiont communities in fragments sampled at the pre-bleach (n = 5/colony), post-inoculation (n = 3/colony/chemical bleaching treatment), 1-month post-inoculation (n = 3/colony/chemical bleaching x symbiont treatment), and 3-month post-inoculation time point (n = 2-15/colony/ chemical bleaching x symbiont treatment). Symbionts are indicated by colour corresponding to their native or heat-evolved genus, with shades of the same colour representing ITS2 DIVs of the same native or heat-evolved genus. Note: fragments are ordered by sample similarity and not by sample name; therefore, sample orders differ between treatments and individual lines do not match up going down the column. Photos of fragments next to the symbiont plots provide examples of coral fragments at each of the sampling time points for each chemical bleaching treatment. Image (ble_figure_2.png) is missing or otherwise invalid. Image (ble_figure_3.png) is missing or otherwise invalid. Image (ble_figure_4.png) is missing or otherwise invalid. Image (ble_figure_5.png) is missing or otherwise invalid. Image (ble_figure_6.png) is missing or otherwise invalid. Image (ble_figure_7.png) is missing or otherwise invalid. Information & Authors Information Version history V1 Version 1 26 February 2026 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords community ecology ecological experiment experimental evolution laboratory marine method development multiple sequencing Authors Affiliations Corinne E. Allen 0009-0004-5406-1914 [email protected] The University of Melbourne View all articles by this author Nadine M. Boulotte Australian Institute of Marine Science View all articles by this author Wing Yan Chan The University of Melbourne View all articles by this author Matthew Nitschke Australian Institute of Marine Science View all articles by this author Madeleine J.H. van Oppen The University of Melbourne View all articles by this author Metrics & Citations Metrics Article Usage 257 views 170 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Corinne E. Allen, Nadine M. Boulotte, Wing Yan Chan, et al. Chemical bleaching severity determines uptake of heat-evolved Symbiodiniaceae in corals. Authorea . 26 February 2026. DOI: https://doi.org/10.22541/au.177213271.14545774/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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