Environmental stability and phenotypic plasticity benefit the cold-water coral *Desmophyllum dianthus* in an acidified fjord

Scleractinian cold-water corals (CWCs) are crucial ecosystem engineers, creating complex three-dimensional habitats in cold, deep waters, similar to tropical coral reefs. They support high biodiversity and serve as nursery grounds for various species. Their distribution is influenced by factors like carbonate chemistry, temperature, salinity, oxygen, food availability, and substrate. Like tropical corals, CWCs utilize adaptive mechanisms to cope with environmental variability, making them vulnerable to rapid anthropogenic changes, including ocean warming, acidification, and deoxygenation. Previous research on CWC responses to temperature, pH, and aragonite saturation (Ωarag) has mainly relied on laboratory studies under controlled conditions. These studies often overlook the natural environmental variability experienced by corals in their habitats. While some in situ studies exist, they rarely consider seasonal or small-scale environmental heterogeneity. Available data suggests that temperature, salinity, oxygen, and pH vary significantly due to tides, internal waves, and advection, indicating a less uniform environment than previously assumed. The limited in situ data restricts our ability to assess CWC coping mechanisms under fluctuating conditions. The physiological response to variable conditions can differ from responses to constant conditions, as seen in phytoplankton and mussels. This highlights that laboratory experiments with stable conditions may not accurately reflect CWC performance in natural environments. Organisms from variable environments may exhibit enhanced tolerance to change, as demonstrated in some tropical coral species which are more resistant to heat stress in thermally variable environments. Conversely, corals from stable environments might be less resilient to climate change. Understanding how populations are adapted to local conditions and their responses to future changes is critical. To assess CWC resilience, understanding their physiological performance under current in situ conditions and environmental variability is paramount. Long-term studies of in situ oceanographic conditions are crucial, and studying species in diverse environments helps us understand their acclimatization and adaptation potential. Acclimatization represents physiological changes in response to environmental shifts without genetic changes (phenotypic plasticity), while adaptation involves genetic changes over generations. Reciprocal transplantation experiments are valuable tools for distinguishing between long-term adaptation and short-term acclimatization. They can reveal adaptation if performance depends on origin and not environment, local adaptation if performance depends on both, or acclimatization if performance depends on the environment regardless of origin. While reciprocal transplantations often show local adaptation, sometimes native organisms are not better adapted than transplanted organisms. Though studies exist on tropical corals transplanted to contrasting environments, only a few reciprocal transplantations have been conducted on CWCs. This study aimed to investigate the physiological performance and acclimatization potential of CWCs to changing in situ environmental conditions. A year-long reciprocal transplantation experiment with the CWC *Desmophyllum dianthus* was conducted in Comau Fjord (Chile). This fjord offers contrasting environments: low salinity, high pH and oxygen in surface waters, and contrasting conditions at depth. Corals were collected from opposing ends of horizontal and vertical environmental gradients and reciprocally transplanted to study physiological responses. Coral fitness traits were evaluated every 3–4 months, focusing on calcification, respiration, and tissue composition. The goal was to understand acclimatization and adaptation by characterizing environmental differences, measuring coral parameters, and correlating environmental conditions with *D. dianthus* performance.

The literature review section extensively cites previous research on cold-water coral (CWC) biology, ecology, and physiology. It highlights the existing knowledge on CWC distribution patterns, their sensitivity to environmental changes like ocean warming and acidification, and the limitations of laboratory studies compared to in situ observations. It discusses previous work on reciprocal transplantation experiments in both tropical and cold-water corals, emphasizing the importance of understanding acclimatization versus adaptation. Key publications referenced include Roberts et al. (2009) on the biology and geology of deep-sea coral habitats, studies on the physiological responses of CWCs to changing temperature and pH (e.g., Georgian et al., 2016; Gori et al., 2016; Form & Riebesell, 2012), findings on the impact of ocean acidification on CWC calcification (e.g., Rodolfo-Metalpa et al., 2015; Hennige et al., 2015), and studies utilizing in situ growth rate measurements (e.g., Jantzen et al., 2013). The review also draws upon research concerning the effects of environmental variability on organismal performance, including works comparing responses to constant versus fluctuating conditions (e.g., Bernhardt et al., 2018; Morash et al., 2018). The studies on tropical corals' responses to thermal variability (e.g., Oliver & Palumbi, 2011; Palumbi et al., 2014) are also compared with cold-water species, emphasizing the differences in responses and the lack of equivalent studies for CWCs. Finally, the literature reviewed also covers the importance of local adaptation and phenotypic plasticity in marine invertebrates (e.g., Hereford, 2009; Sanford & Kelly, 2011).

This study employed a year-long reciprocal transplantation experiment in the Comau Fjord, Chile, utilizing the cold-water coral *Desmophyllum dianthus*. The fjord's stratification provides a natural gradient in environmental conditions (shallow, aragonite-saturated; deep, aragonite-undersaturated). Six shallow stations (~20 m depth) and one deep station (~300 m depth) were established. **Environmental Data Collection:** Water temperature was continuously monitored at each station using Tidbit v2 loggers. Salinity, oxygen, pH, and total alkalinity (TA) and dissolved inorganic carbon (DIC) were measured seasonally using a CTD and discrete water samples. These data were used to calculate aragonite saturation (Ωarag) and other carbonate chemistry parameters using CO2SYS software. **Coral Collection and Transplantation:** Scientific SCUBA divers collected *D. dianthus* colonies from shallow stations. A remotely operated vehicle (ROV) was used for deep-water collection. Corals were carefully cleaned and fixed onto labeled polyamide screws on plastic plates. These plates were mounted on holders affixed to the fjord walls for shallow corals and a metal rack for deep corals, allowing for repeated sampling. Corals were reciprocally transplanted between shallow stations (horizontal gradient) and between shallow and deep stations (vertical gradient). **Coral Physiology Measurements:** Two subsets of corals were used. The 'experimental corals' were repeatedly sampled for calcification and respiration rate measurements. The 'tissue corals' were sampled for biomass analysis. Calcification rates were measured using the buoyant weighing technique, measuring buoyant weight before and after growth periods. Respiration rates were determined via closed-cell incubations at a standardized temperature (12.75 °C). The tissue-covered surface area of the corals was measured to normalize both calcification and respiration rates. **Statistical Analysis:** Linear mixed-effect models (LMMs) were used to analyze the relationships between calcification and respiration rates and factors like depth, season, station, and transplantation status. A multifactorial analysis using linear models was conducted to determine the correlation between calcification rates and environmental parameters (temperature variability, temperature, pH, Ωarag, salinity, and oxygen concentration), with model selection based on the Akaike Information Criterion (AIC).

The study revealed several key findings: 1. **Environmental Variability:** Significant seasonal and spatial variability was observed in water temperature, salinity, oxygen, pH, and Ωarag. Shallow waters exhibited higher variability, especially in summer and autumn. Deep waters showed lower variability and consistently lower temperatures, oxygen, pH, and Ωarag. 2. **Coral Performance:** Corals at the deep station (300 m) showed significantly higher calcification and respiration rates compared to shallow stations. In shallow waters, calcification rates were higher at the fjord mouth than at the head, and respiration rates showed variability across stations and seasons. 3. **Rapid Acclimatization:** Following transplantation, both native and novel (cross-transplanted) corals quickly adjusted their calcification rates to the new environmental conditions of each station. No significant differences in respiration rates were observed between native and novel corals at any station. 4. **Environmental Drivers of Calcification:** Multifactorial analysis indicated that mean seasonal temperature and temperature variability were the most important factors explaining variation in calcification rates (adjusted R2 = 0.555). Calcification rates were highest at the deep station, which had the lowest temperature variability, revealing an inverse relationship between calcification and environmental variability. The lowest aragonite saturation levels did not negatively impact coral calcification; indeed, the highest rates were observed in the aragonite undersaturated conditions of the deep station. 5. **Tissue Coverage:** Shallow corals (particularly at stations B, C, and Es) had lower tissue coverage than deep corals, suggesting reduced somatic growth and increased energy expenditure to defend against endolithic photoautotrophic organisms. Deep corals maintained tissue coverage after transplantation to shallow waters. 6. **Energetic Trade-off?:** The observed differences in coral performance between shallow and deep waters might reflect differences in food availability and energy allocation. While shallow waters have potentially higher zooplankton abundance, they also have higher environmental variability, possibly reducing feeding opportunities and increasing energetic costs. In contrast, the more stable conditions in deeper waters might lead to higher energy availability for both calcification and growth. This idea is supported by the six-fold greater investment into biomass increase in deep waters compared to shallow waters.

This study challenges the assumption that low aragonite saturation is a major constraint on CWC growth and distribution. While the deep station exhibited aragonite undersaturation, corals there showed the highest calcification and respiration rates, suggesting other factors outweigh the impact of carbonate chemistry. The inverse relationship between calcification and environmental variability highlights the significance of considering high-frequency fluctuations of abiotic and biotic factors, which have been largely neglected in previous research. This study underscores the remarkable phenotypic plasticity of *D. dianthus*, allowing rapid acclimatization to new environments. The observed differences in somatic growth might suggest local adaptation, although further investigation is needed. The results imply a trade-off between energy allocation to calcification and somatic growth in relation to environmental stability and food availability. While shallow corals may invest more energy into reproduction given their seasonal reproductive cycle, deep corals might invest more energy into somatic growth and biomass.

This study demonstrates the strong phenotypic plasticity and remarkable adaptability of *Desmophyllum dianthus*. The fittest corals were found in the stable, aragonite-undersaturated deep waters, highlighting the importance of environmental stability over aragonite saturation in determining CWC performance. Environmental variability, particularly temperature fluctuations, negatively impacts calcification rates, suggesting that future studies should incorporate environmental variability into their design rather than relying solely on constant conditions. This research emphasizes the need for further investigation into the relative roles of various factors in shaping CWC distribution and resilience in a changing ocean. Future research should focus on examining other physiological traits, long-term effects of transplantation, and understanding the interplay between environmental variability, food availability, and energy allocation in CWCs.

The study focused primarily on calcification and respiration rates as indicators of fitness. Further investigations into other physiological traits (e.g., biochemical markers, gene expression) could provide more comprehensive insights into adaptation and acclimatization. The study's duration (one year) might not fully capture long-term adaptation processes. The impact of biological factors (e.g., predation, competition) and specific components of food availability (e.g., prey size, energy density) were not fully addressed. Although the study controlled for the negative effects of bioerosion on shallow corals, quantifying specific food availability at each station would be beneficial to better understand the influence of food intake on the physiology of cold-water corals. It is important to note the standardization of the respiration measurements at a specific temperature which might introduce some uncertainty to the interpretation of this variable.