Wyatt C. Million

Deoxygenation, the global loss of biologically available oxygen (O ₂ ) from the world’s oceans, presents a pervasive and intensifying threat for reef-building corals that will require coordinated efforts to understand and manage. However, recent expansion of coral deoxygenation experimentation has produced methodological gaps, limiting collective knowledge generation. Here, we show that growing divergence in this field arises primarily from differences in focal phenotypes, deoxygenation exposures, and experimental execution that together prevent biological from technical variation from being disentangled between studies. To address this, we propose a series of recommendations to establish the foundation for a future standardized framework for coral deoxygenation methodology. Such efforts are critical to establish a robust consensus of scientific data to support effective management of reef deoxygenation. Waiting decades for diverging methodologies to prompt a call for standardization may be imprudent given the urgent threat of deoxygenation and the abundant methodological divergence present in coral deoxygenation experimentation.

Accelerating ocean deoxygenation driven by anthropogenic warming, eutrophication, and water column stratification poses a global threat to coral reef ecosystems. Several large-scale coral mortality events have been documented on reefs in response to periods of severe hypoxia, wherein environmental dissolved oxygen (DO) levels are insufficient for maintaining physiological homeostasis and supporting coral health. Although adult corals have the capacity for oxyregulation, the extent to which such capacity is affected by reproduction or during coral larval life stages remains poorly understood. Here, we examined oxygen consumption patterns of eight coral species and the offspring of four of these species using closed system respirometry to characterise oxyregulatory performance before and after spawning. Adult corals exhibited a significant increase in oxyregulatory capacity, exerting their maximum regulation capacity (Pcmax) at a lower DO partial pressure (pO2) one week following spawning relative to the 1–2 days preceding gamete release, indicating heightened vulnerability of gravid colonies to hypoxia. Coral larvae also exhibited capacity for oxyregulation, albeit at a lowered capacity (i.e., less regulation possible above oxyconformity based on total positive regulation, Tpos) compared to adults, and larval performance did not correlate with conspecific adult responses. These results highlight ontogenetic and reproductive-state oxyregulation variability, suggesting critical variation in metabolic responses to DO availability across life stages. Characterising coral life stage-specific responses to altered DO availability provides important physiological context for understanding how coral reef organisms may respond to ongoing and future ocean deoxygenation.

Dinoflagellates of the family Symbiodiniaceae are main symbionts of diverse marine animals. A large diversity of Symbiodiniaceae also occur beyond the bounds of their multicellular hosts, occupying environmental niches on coral reefs. The link between spatial diversity at ecosystem scale to microhabitats of Symbiodiniaceae within the coral holobiont are largely unknown. Using ITS2 amplicon sequencing, we compared Symbiodiniaceae communities across four environments (seawater, near-reef and distant sediments, and turf algae mats) and two coral microhabitats (tissue and mucus) on a coral reef in the Red Sea. Analysis of ITS2 sequences revealed that coral and environmental habitats were both dominated by the genera Symbiodinium , Cladocopium , and Durusdinium , but environmental habitats additionally harbored Fugacium , Gerakladium , and Halluxium . Each environmental habitat harbored a distinct Symbiodiniaceae community, with 14-27 % exclusive ITS2 sequences. Nonetheless, 17 ITS2 sequences were shared among all habitat types and were variants defining nearly half of the ITS2 type profiles used to further resolve Symbiodiniaceae identity of coral-based communities. Tissues and surface mucus layers of 49 coral colonies from 17 genera had largely identical Symbiodiniaceae communities. Together with the large difference between environmental Symbiodiniaceae communities and those in the mucus, our results indicate a clear barrier between host-associated and environmental Symbiodiniaceae communities marked by only few shared complete type profiles under normal conditions. It remains to be determined how Symbiodiniaceae community dynamics between coral microhabitats and environmental reservoirs change during coral bleaching events. Monitoring coral colonies after mucus sampling confirmed its suitability for repeated long-term monitoring of coral-associated Symbiodiniaceae communities.

As environments are rapidly reshaped due to climate change, phenotypic plasticity plays an important role in the ability of organisms to persist and is considered an especially important acclimatization mechanism for long-lived sessile organisms such as reef-building corals. Often, this ability of a single genotype to display multiple phenotypes depending on the environment is modulated by changes in gene expression, which can vary in response to environmental changes via two mechanisms: baseline expression and expression plasticity. We used transcriptome-wide expression profiling of eleven genotypes of common-gardened Acropora cervicornis to explore genotypic variation in the expression response to thermal and acidification stress, both individually and in combination. We show that the combination of these two stressors elicits a synergistic gene expression response, and that both baseline expression and expression plasticity in response to stress show genotypic variation. Additionally, we demonstrate that frontloading of a large module of coexpressed genes is associated with greater retention of algal symbionts under combined stress. These results illustrate that variation in the gene expression response of individuals to climate change stressors can persist even when individuals have shared environmental histories, affecting their performance under future climate change scenarios.

The sensitivity of reef-building coral to elevated temperature is a function of their symbiosis with dinoflagellate algae in the family Symbiodiniaceae. Changes in the composition of the endosymbiont community in response to thermal stress can increase coral thermal tolerance. Consequently, this mechanism is being investigated as a human-assisted intervention for rapid acclimation of coral in the face of climate change. Successful establishment of novel symbioses that increase coral thermal tolerance have been demonstrated in laboratory conditions; however, it is unclear how long these heterologous relationships persist in nature. Here, we test the persistence of a novel symbiosis between Acropora palmata and Durusdinium spp. from Mote Marine Laboratory’s ex situ nursery by outplanting clonal replicates (ramets) of five A. palmata host genotypes to natural reefs in the lower Florida Keys. Amplicon sequencing analysis of ITS2-type profiles revealed that the majority of surviving ramets remained dominated by Durusdinium spp. two years after transplantation. However, 15% of ramets, including representatives of all genotypes, exhibited some degree of symbiont shuffling or switching at six of eight sites, including complete takeover by site-specific strains of the native symbiont, Symbiodinium fitti. The predominant long-term stability of the novel symbiosis supports the potential effectiveness of symbiont modification as a management tool. Although, the finding that 6–7 year-old coral can alter symbiont community composition in the absence of bleaching indicates that Symbiodiniaceae communities are indeed capable of great flexibility under ambient conditions.

Genotype-by-environment interactions (GxE) indicate that variation in organismal traits cannot be explained by fixed effects of genetics or site-specific plastic responses alone. For tropical coral reefs experiencing dramatic environmental change, identifying the contributions of genotype, environment, and GxE on coral performance will be vital for both predicting persistence and developing restoration strategies. We quantified the impacts of G, E, and GxE on the morphology and survival of the endangered coral, Acropora cervicornis , through an in situ transplant experiment exposing common garden (nursery)-raised clones of ten genotypes to nine reef sites in the Florida Keys. By fate-tracking outplants over one year with colony-level 3D photogrammetry, we uncovered significant GxE on coral size, shape, and survivorship, indicating that no universal winner exists in terms of colony performance. Rather than differences in mean trait values, we found that individual-level morphological plasticity is adaptive in that the most plastic individuals also exhibited the fastest growth and highest survival. This indicates that adaptive morphological plasticity may continue to evolve, influencing the success of A. cervicornis and resulting reef communities in a changing climate. As focal reefs are active restoration sites, the knowledge that variation in phenotype is an important predictor of performance can be directly applied to restoration planning. Taken together, these results establish A. cervicornis as a system for studying the ecoevolutionary dynamics of phenotypic plasticity that also can inform genetic- and environment-based strategies for coral restoration.

Genotype-by-environment interactions (GxE) indicate that variation in organismal traits cannot be explained by fixed effects of genetics or site-specific plastic responses alone. For tropical coral reefs experiencing dramatic environmental change, identifying the contributions of genotype, environment, and GxE on coral performance will be vital for both predicting persistence and developing restoration strategies. We quantified the impacts of G, E, and GxE on the morphology and survival of the endangered coral, A. cervicornis , through an in situ transplant experiment exposing common garden (nursery) raised clones of ten genotypes to nine reef sites in the Florida Keys. By fate-tracking outplants over one year with colony-level 3D photogrammetry, we uncovered significant GxE on coral size and survivorship indicating that no universal winner exists in terms of colony performance. Moreover, the presence of GxE also implies the existence of intraspecific variation in phenotypic plasticity. Rather than differences in mean trait values, we find that individual-level morphological plasticity is adaptive in that the most plastic individuals also exhibited the fastest growth and highest survival. This indicates that adaptive morphological plasticity may continue to evolve, influencing the success of A. cervicornis and resulting reef communities in a changing climate. As focal reefs are active restoration sites, the knowledge that variation in phenotype is an important predictor of performance can be directly applied to restoration planning. Taken together, these results establish A. cervicornis as a system for studying the eco-evolutionary dynamics of phenotypic plasticity that also can inform genetic- and environment-based strategies for coral restoration.

The rapid loss of reef-building corals owing to ocean warming is driving the development of interventions such as coral propagation and restoration, selective breeding and assisted gene flow. Many of these interventions target naturally heat-tolerant individuals to boost climate resilience, but the challenges of quickly and reliably quantifying heat tolerance and identifying thermotolerant individuals have hampered implementation. Here, we used coral bleaching automated stress systems to perform rapid, standardized heat tolerance assays on 229 colonies of Acropora cervicornis across six coral nurseries spanning Florida's Coral Reef, USA. Analysis of heat stress dose–response curves for each colony revealed a broad range in thermal tolerance among individuals (approx. 2.5°C range in F _v /F _m ED50), with highly reproducible rankings across independent tests ( r = 0.76). Most phenotypic variation occurred within nurseries rather than between them, pointing to a potentially dominant role of fixed genetic effects in setting thermal tolerance and widespread distribution of tolerant individuals throughout the population. The identification of tolerant individuals provides immediately actionable information to optimize nursery and restoration programmes for Florida's threatened staghorn corals. This work further provides a blueprint for future efforts to identify and source thermally tolerant corals for conservation interventions worldwide.

The ability to quantify changes in the structural complexity of reefs and individual coral colonies that build them is vital to understanding, managing, and restoring the function of these ecosystems. However, traditional methods for quantifying coral growth in situ fail to accurately quantify the diversity of morphologies observed both among and within species that contribute to topographical complexity. Three-dimensional (3D) photogrammetry has emerged as a powerful tool for the quantification of reefscape complexity but has yet to be broadly adopted for quantifying the growth and morphology of individual coral colonies. Here we debut a high-throughput method for colony-level 3D photogrammetry and apply this technique to explore the relationship between linear extension and other growth metrics in Acropora cervicornis. We fate-tracked 156 individual coral transplants to test whether initial growth can be used to predict subsequent patterns of growth. We generated photographic series of fragments in a restoration nursery immediately before transplanting to natural reef sites and re-photographed coral at 6 months and 1 year post-transplantation. Photosets were used to build 3D models with Agisoft Metashape, which was automated to run on a high-performance computing system using a custom script to serially process models without the need for additional user input. Coral models were phenotyped in MeshLab to obtain measures of total linear extension (TLE), surface area, volume, and volume of interstitial space (i.e., the space between branches). 3D-model based measures of TLE were highly similar to by-hand measurements made in the field (r = 0.98), demonstrating that this method is compatible with established techniques without additional in water effort. However, we identified an allometric relationship between the change in TLE and the volume of interstitial space, indicating that growth in higher order traits is not necessarily a linear function of growth in branch length. Additionally, relationships among growth measures weakened when comparisons were made across time points, implying that the use of early growth to predict future performance is limited. Taken together, results show that 3D photogrammetry is an information rich method for quantifying colony-level growth and its application can help address contemporary questions in coral biology.

This protocol describes the process of phenotyping branching coral using the 3D model editing software, MeshLab. MeshLab is a free, straightforward software to analyze 3D models of corals that is especially useful in its ability to import color from Agisoft Metashape models. This protocol outlines the steps used by the Kenkel lab to noninvasively phenotype Acropora cervicornis colonies for total linear extension (TLE), surface area, volume, and volume of interstitial space. We incorporate Agisoft Metashape markers with our Tomahawk scaling system (see Image Capture Protocol) in our workflow which is useful for scaling and to improve model building. Other scaling objects can be used, however these markers provide a consistent scale that do not obstruct the coral during image capture. MeshLab measurements of TLE have been groundtruthed to field measures of TLE. 3D surface area and volume have not yet been compared to traditional methods of wax dipping, for surface area, and water displacement, for volume. However, in tests with shapes of known dimensions, i.e. cubes, MeshLab produced accurate measures of 3D surface area and volume when compared to calculated surface area and volume. For directions to photograph coral for 3D photogrammetry see our Image Capture Protocol. For a walkthrough and scripts to run Agisoft Metashape on the command line, see https://github.com/wyattmillion/Coral3DPhotogram. These protocols, while created for branching coral, can be applied to 3D models of any coral morphology or any object really. Our goal is to make easy-to-use protocols using accessible softwares in the hopes of creating a standardized method for 3D photogrammetry in coral biology. Go to http://www.meshlab.net/#download to download the appropriate software for your operating system. P. Cignoni, M. Callieri, M. Corsini, M. Dellepiane, F. Ganovelli, G. Ranzuglia MeshLab: an Open-Source Mesh Processing Tool Sixth Eurographics Italian Chapter Conference, page 129-136, 2008