Coral reefs are one of the most important ecosystems in the marine environment, as they are highly diverse and productive ecosystems (Connell, 1978; Moberg and Folke, 1999). The high productivity of coral reef systems is based on a mutualistic symbiosis between unicellular dinoflagellates (Symbiodiniaceae) and different invertebrate reef species, including corals, sea anemones and sponges among many others (Trench, 1993; Coffroth and Santos, 2005; Decelle et al., 2018).

The success of this symbiosis depends on complementary intracellular solute exchange such as nutrients and carbon compounds. The symbionts can provide up to 95% of photosynthetically fixed organic carbon in the form of glycerol, glucose and alanine to the host (Muscatine and Porter, 1977; Burriesci et al., 2012; Davy et al., 2012). In exchange the host offers protection against environmental stresses (e.g., herbivory) and delivers inorganic nitrogen (Davies, 1984; Steen and Muscatine, 1984). Despite their immense ecological importance, coral reefs are diminishing worldwide due to various environmental stressors (Hoegh-Guldberg et al., 2007; Hughes et al., 2018, 2020). There are several factors, such as low salinity, water pollution, unusually high or low water temperatures or high solar irradiance causing coral bleaching (Van Oppen and Lough, 2018). When, for instance, the local temperature exceeds the upper physiological threshold of the host, the symbiosis collapses and “bleaching” occurs as a result of the loss of algal endosymbionts (Glynn and D’Croz, 1990). This impact has already been observed globally, with an increasing frequency of these “bleaching events” due to more frequent El-Nino events, particularly in recent years (Glynn, 1993; Eakin et al., 2010; Hughes et al., 2017, 2018, 2020). However, the probability of bleaching depends either on the host characteristics (Baird et al., 2009), on the symbiont ecophysiological traits (Rowan, 2004) or on the prevailing environmental conditions (Hoegh-Guldberg, 1999; Anthony et al., 2009). The flexibility of the coral host, in particular, to associate with different photosymbiont strains at the same time could be critical to cope with rapid environmental fluctuations (Baker, 2001; Berkelmans and Van Oppen, 2006; Abrego et al., 2008; Lewis et al., 2019; Qin et al., 2019).

Several studies suggested that the host might be able to acclimate to higher water temperatures by switching to more temperature-tolerant symbiont species or strains from the local environment or by distributing temperature-tolerant symbionts to make them more abundant (Buddemeier and Fautin, 1993; Baker, 2003; Fautin and Buddemeier, 2004; Correa and Baker, 2011; Cunning et al., 2018). Nevertheless, some investigations revealed that altered host-symbiont pairings often return to their original composition when sufficient time elapsed after a bleaching event (Thornhill et al., 2006; Sampayo et al., 2008). For example, this was found in several coral species at the Great Barrier Reef after a bleaching event in 2002. During bleaching, corals harbored the same Symbiodiniaceae as before and after the thermal stress events (Stat et al., 2009). This suggests that not all symbiotic strains are beneficial to the respective host (Stat et al., 2008; Starzak et al., 2014; Gabay et al., 2018) or that the competitive advantage varies with the environment (Thornhill et al., 2006; Jones et al., 2008; LaJeunesse et al., 2009). The Symbiodiniaceae are highly diverse and combine at least seven different genera, which were recently taxonomically revised (LaJeunesse et al., 2018). The six genera are Breviolum, Cladocopium, Durusdinium, Effrenium, Fugacium, and Gerakladium. Members of the Symbiodiniaceae have a different sensitivity to solar radiation and temperature fluctuations. The underlying mechanisms can be explained by biochemical and molecular processes of the photosynthetic machinery (Takahashi et al., 2008). The key pigments peridinin, chlorophyll a and c2 are integrated into the chloroplast membrane as the two major antennas a-chlorophyll c2-peridinin protein complex (acpPC) and peridinin-chlorophyll a-binding protein (PCP) (Brown et al., 1999; Takahashi et al., 2008; Niedzwiedzki et al., 2014; Hennige et al., 2019).

High irradiances and temperatures are factors contributing to the phenomenon of coral bleaching. High temperature contributes to an increased production of harmful reactive oxygen molecules (ROS), such as hydrogen peroxide (H₂O₂), singlet oxygen ¹O₂ or superoxide O₂. ROS originate as a metabolic byproduct and are primarily produced by photosystem I and in parts of photosystem II (Szabó et al., 2020). ROS are chemically highly reactive and hence can, for example, mutate DNA, denature proteins and oxidize lipids as well as cell membranes (Takahashi et al., 2008, 2009; Venn et al., 2008; Lesser et al., 2010). As a result of ROS production and the cellular cascade, this can lead to a disruption of communication and interaction between the host and symbiont. ROS production is considered as the key element for the bleaching process (Weis, 2008; Baird et al., 2009; Szabó et al., 2020). Evidence suggests that the ROS leakage varies between species, as well as depending on temperature and salinity (Gegner et al., 2019). Various species of the Symbiodiniaceae acclimate their photosynthetic performance to different light conditions (Iglesias-Prieto and Trench, 1994; Hennige et al., 2009; Suggett et al., 2015). With elevated temperatures, various Symbiodiniaceae did not grow (Lesser, 1996), while other investigations reported the opposite (Sakami, 2000; Karim et al., 2015; Klueter et al., 2017). Karim et al. (2015), for example, that growth and photochemical efficiency of PSII remained unchanged between 25 and 30°C in various Symbiodiniaceae genera. At temperatures above 33°C, however, the thermal tolerance was exceeded. Further reports point to different genotypes of Symbiodiniaceae which are tolerant to specific temperatures and radiation conditions (Robison and Warner, 2006). Consequently, the tolerance width and upper survival temperature varies from species to species and genotype to genotype (Berkelmans and Van Oppen, 2006; Van Oppen et al., 2009). The genetic identity and ecophysiological capability of Symbiodiniaceae species play a major role in acclimation and adaptation to thermal stress. Several studies identified significant differences in heat tolerance both within a single Symbiodiniaceae genus (Díaz-Almeyda et al., 2017; Bayliss et al., 2019) and between genera (Grégoire et al., 2017). In addition, there are several species, such as Durusdinum trenchii (Rowan, 2004; Jones et al., 2008; Bellantuono et al., 2019) and Cladocopium thermophilum (formerly Class C, ITS2-“Golf C3”) (Hume et al., 2015) which are considered thermo-tolerant. A genetic subtype of ITS2-D1a (LaJeunesse et al., 2018) within Clade D, gives coral hosts a 1–1.5°C increase in thermal tolerance (Rowan, 2004; Berkelmans and Van Oppen, 2006). A culture of Cladocopium C1 in a laboratory selection exhibited better photophysiology and growth at high temperature (31°C) compared to wild type cells (Chakravarti et al., 2017). However, alternating heat-resistant host-symbiont assemblages are associated with metabolic “costs,” since the growth rates of the corals often decrease (Abrego et al., 2008).

The objective of this study was to examine the photosynthetic performance [net primary production], hyperthermal tolerance, acclimation capacity of several genotypes (strains), species, and genera of Symbiodiniaceae using oxygen optodes. Based on previous studies (Rowan, 2004; Jones et al., 2008; Bellantuono et al., 2019), in which Durusdinium trenchii exhibited a pronounced heat tolerance, we hypothesized that D. trenchii outperforms the other strains in terms of photosynthetic efficiency as no loss of photosynthetic function occurs at higher temperature. A total of nine algal strains belonging to four genera and four species of Symbiodiniaceae were comparatively examined under controlled manipulative conditions: Breviolum minutum (4 strains), Breviolum psygmophilum (2 strains), D. trenchii (one strain), Effrenium voratum (one strain), and Symbiodinium linucheae (one strain).

The susceptibility of corals is influenced by a variety of abiotic factors such as increased light and temperature conditions, and the physiological and genetic diversity of the endosymbionts strongly contribute to different response patterns of the host (Berkelmans and Van Oppen, 2006; Robison and Warner, 2006; Baird et al., 2009). In the present study the experiments focused on the physiological tolerance and plasticity of genetically different Symbiodiniaceae strains in response to elevated temperatures for a better understanding of their photosynthetic traits as a function of light and temperature.

The Symbiodiniaceae used were grown as unialgal cultures under identical and controlled conditions for at least 1 year, which might explain relatively similar alpha, I_c and I_k values derived from the respective PI curves. Symbiodiniaceae species acclimate their photosynthetic performance to various light levels (Iglesias-Prieto and Trench, 1994; Anthony and Hoegh-Guldberg, 2003; Roth, 2014). The applied light conditions under which the cultures were grown are comparable to the shady in vivo conditions inside the coral tissue (Anthony et al., 2005). Furthermore, Symbiodiniaceae maximize light absorption and utilization under low light conditions by increasing the concentration of photosynthetic pigments thereby improving photosynthetic efficiency (Anthony and Hoegh-Guldberg, 2003).

The photosynthetic optimum under the prevailing environmental conditions is represented by the maximum rate of photosynthesis (NPP_max) (Raven and Geider, 2003). The Symbiodiniaceae cultures investigated in this study revealed different type-specific eco-physiological response patterns regarding NPP_max and respiration. These patterns are mainly caused by type-specific traits rather than as a result of the stable cultivation conditions. The data displayed a similar NPP_max for various genera (MAC 703, CCMP 2556, and CCMP 421), which was distinct to both genotypes of Breviolum psygmophilum MAC HIAp and 1046. Furthermore, the NPP_max of Symbiodinium linucheae SSA 01 was much lower in contrast to the other eight strains. This strain is also considered “heat tolerant” (Swain et al., 2017; LaJeunesse et al., 2018; Gegner et al., 2019) but might not be so beneficial for the coral host (Stat et al., 2008; Herrera et al., 2020). Although only minor photoinhibition was detected in the studied strains we cannot completely exclude an experimental effect as longer exposure to high irradiances could have resulted in a stronger photoinhibitory response. The data from this study, clearly indicate that SSA 01 has a much lower photosynthetic rate compared to the other strains than, for example, strain B. psygmophilum (1046), which had the highest average photosynthetic rates per chlorophyll a. Therefore, SSA 01 likely transfers less important photosynthetically fixed organic carbon to the host (Stat et al., 2008). The photosynthetic rates of the present study are in agreement with data of Grégoire et al. (2017), who used two identical genotypes for their experiments. The photosynthesis performance between the species were different under the same cultivation conditions (Iglesias-Prieto and Trench, 1994), pointing to genotypic traits. Previous studies indicate an important role of the genetic identity of Symbiodiniaceae strains as well as their respective eco-physiological capabilities in acclimation and adaptation to thermal stress in the reef environment (Baker, 2003; Abrego et al., 2008; Frade et al., 2008a, b). Both the endosymbiont and the host are affected by elevated temperatures. Host genetics have been identified as an important trait in the response of the symbionts (Cunning et al., 2015; Howells et al., 2016). In order to determine how the genetic identity of the symbiont is affected by elevated temperatures, the cultures were exposed to different thermal stress scenarios.

The results of this study indicated a clear species-specific threshold of upper temperature tolerance, in the range of 30°–40°C for all tested species, and the Symbiodiniaceae cultures demonstrated the strongest stress response with a reduced or an inhibition of photosynthesis and strongly increased respiration rate. At 35°C, only five of nine species showed a reduced but measurable photosynthesis (Figure 4). Above 35°C, none of the strains exhibited any photosynthetic activity. It can be assumed that the Symbiodiniaceae were not dead due to the continuing respiratory activity. Moreover, the results of the Symbiodiniaceae cultures showed clearly a tolerance to short-term cold stress at 10°C and the most efficient photosynthesis performance was measured on average at 25°C (Figure 4). The different Symbiodiniaceae species are classified based on a variation in thermal tolerances, which is also reflected in the results of this study (Robison and Warner, 2006; Suggett et al., 2008; Díaz-Almeyda et al., 2017; Bellantuono et al., 2019). While any oxygen production at 35°C was not traceable in the isolates SSB 01, CCMP 2556, SSA 01, and CCMP 421, the other strains still showed a reduced activity. Durusdinium trenchii is described as heat tolerant species (Bellantuono et al., 2019) but demonstrated in culture a similar pattern as the other tested isolates up to 34°C. The hypothesis that D. trenchii is more heat tolerant than the other tested strains could not be confirmed in this experiment. Thus, all tested Symbiodiniaceae exhibited quite similar temperature tolerance with an optimal photosynthetic performance between 20° and 26°C. In addition, the strains MAC 703 and SSA 01 showed an “optimal” photosynthetic capacity up to 32°C. The upper temperature tolerance for photosynthesis of the investigated strains was between 34 and 35°C, that of respiration even higher. Consequently, it is reasonable to assume that the increased consumption of oxygen under elevated temperatures might favor anoxic conditions within the holobiont, which can cause symbiont loss and bleaching. The results of the present study are comparable to those of other studies, which demonstrated a decrease in the maximum quantum yield of PSII and the rate of gross photosynthesis under heat stress in different zooxanthellae in culture. Impairment of photosynthesis was mainly measured at 32–34°C in cultivated zooxanthellae (Iglesias-Prieto et al., 1992; Warner et al., 1996; Iglesias-Prieto and Trench, 1997; Jones et al., 1998; Brown et al., 1999). In Symbiodinium microadriaticum, gradual inhibition of photosynthesis occurred at temperatures above 30°C, followed by complete inhibition at higher temperatures of 34–36°C (Iglesias-Prieto et al., 1992). The reason why so many zooxanthellae exhibit a similar upper temperature tolerance for photosynthesis might be related to the fact that dinoflagellates have a Type II Rubisco, which differs in many properties including heat sensitivity from Type I, the dominant form in other algal groups (Tabita et al., 2008).

In the second temperature experiment, it became clear that Breviolum minutum SSB 01 was much more susceptible to enhanced temperatures starting at 30°C. This demonstrates different temperature thresholds between strains of B. minutum, also within the genus Breviolum the species-specific tolerances differ. This is in line with another study, where closely related Breviolum species revealed significant functional variation against elevated temperature (Bayliss et al., 2019; Herrera et al., 2020). The different genotypes exhibited decreasing photosynthetic activity at elevated temperatures, which could be an important feature for the strength of the relationship between the symbiont and the host. Some studies concluded that thermo-tolerance is not species- or clade-specific but is widespread and diverse among members of the genus Symbiodinium (Suggett et al., 2008; Díaz-Almeyda et al., 2017). Considering the photosynthetic machinery of coral symbionts as very sensitive to changes in the environment, it was observed that exposure of Symbiodiniaceae to light and temperature caused an impairment of the photosynthetic apparatus by PSII photoinhibition (Iglesias-Prieto et al., 1992). The level of photoinhibition is partly determined by elevated temperatures or light, as these factors can accelerate PSII photodamage and inhibit repair processes (Brown et al., 1999; Takahashi et al., 2009). But other studies report that the temperature-induced inhibition of the dark reactions (Calvin-cycle) in zooxanthellae does not exclusively contribute to coral bleaching, while the concomitant ROS formation could be the main trigger (Hill et al., 2014).

The results of Mansour et al. (2018) found intra- and interspecific differences in the melting points of thylakoid membranes of Symbiodiniaceae species. Such process could explain the inhibition of photosynthetic oxygen production under elevated temperatures during the experiments. A still ongoing respiration was measured at higher temperatures, indicating that the repair mechanisms are working and that the experimental cooling time was too short to be used as proxy for recovery. However, in the experiment 20–34°C the thermal capacity of the photosynthetic membrane was probably not exceeded and hence significant differences between the strain-specific response patterns could be measured. A certain, genetically determined temperature threshold must be reached to harm the photosynthetic activity. The loss of photosynthetic function at extremely high temperatures occurs in a short time period in isolated Symbiodiniaceae cultures. The results of this study clearly indicated that the physiological properties of Symbiodiniaceae at elevated temperatures vary even within the same taxon, i.e., genotypic differentiation even on taxonomically lower rank levels plays a key role for temperature acclimation/adaptation, with consequences for the endosymbiont-host interactions (Bayliss et al., 2019). However, the physiological temperature response patterns of Symbiodiniaceae might differ between culture conditions and in symbiosis as documented for D. trenchii (Bellantuono et al., 2019). These authors compared in hospite and free-living transcriptomes, and reported strong alteration of transcriptional activity in hospite under increased temperature conditions, indicating that symbiotic interactions elicited an exacerbated stress response compared to free-living cells.

Although mainly vegetative (clonal) reproduction occurs within the Symbiodiniaceae, mutation rates can be high, resulting in new variants of individual genotypes. These genetic variations are further increased or maintained by transposons, retrotransposons, tandem repeats, or recombination during sexual reproduction (Shoguchi et al., 2013). Even hosts harboring only a single symbiont genotype can quickly accumulate genetic variation due to this high mutation rate in Symbiodiniaceae (van Oppen et al., 2011). Natural selection is likely to lead to a higher temperature tolerance in the symbiont population, since many temperature tolerance traits are inherited (Császár et al., 2010; Quigley et al., 2016). This selection in the symbiont population also strengthens the holobiont, allowing the host to survive periods of warming (Chakravarti and Van Oppen, 2018). Nevertheless, previous work suggests that the physiology of symbionts actually differs in culture and host (Ralph et al., 2001; Bhagooli and Hidaka, 2003; Howells et al., 2012; Chakravarti et al., 2017; Ravelo and Conaco, 2018).

Intact relationships between host and symbionts are essential for the ecological health of coral reefs, as they represent one of the most important ecosystems in tropical marine waters. Coral reefs provide many ecosystem services such as high biodiversity and productivity, habitat, fishery, water quality and biogeochemical cycling, and coastal protection (Moberg and Folke, 1999), which are negatively influenced by anthropogenic global warming.

In this study we demonstrate a necessity for a comprehensive comparative eco-physiological analysis at intra-specific taxonomic level to fully understand the underlying genetic traits responsible for the variation in thermotolerance. The results clearly demonstrate that there are significant eco-physiological differences among the tested species but also within a species, regarding light affinity and temperature tolerance. Although the short-term responses shown here do not necessarily reflect the long-term response of corals in the reef, this study highlights that symbionts play an important role in the response of holobionts to temperature increases within their average summer maxima. The results showed clearly that B. minutum (SSB 01) is much more sensitive to heat in comparison to the strain B. minutum (MAC 703) and to the species S. linucheae (SSA 01). Furthermore, S. linucheae (SSA 01) exhibited a lower photosynthetic rate, suggesting that this strain in symbiosis probably transfers less photosynthetically fixed organic carbon to the host (Stat et al., 2008). Therefore, future studies in which specific symbiont genotypes are introduced into the same host (e.g., Starzak et al., 2014; Hoadley et al., 2015; Herrera et al., 2020) are required to determine the role of host and symbiont in the response patterns. The different response patterns suggest that in symbiosis with a host, the carbon supply from the symbiont to the host might be affected by light and temperature. Presumably, thermal stress leads to an unbalanced bi-directional flow of metabolites between host and endosymbiont which should be further investigated in future studies.

The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author/s.

VR and UK planned and designed the ecophysiological experiments. VR conducted the ecophysiological characterization of the algal strains, and processed the data. MR-L and VR planned molecular analysis, which was conducted by VR. VR wrote the first draft of the manuscript, which was edited by MR-L and UK. All authors edited and approved the final version of this manuscript.

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