There has been considerable work demonstrating that different genera or species of algal symbionts affect performance of their cnidarian hosts and the response of the holobiont to increased temperature 45,57−64. As in previous research 37,38, we found similar effects of genetic diversity at a lower taxonomic level—different genotypes within the species Symbiodinium microadriaticum have different physiological responses to temperature and differentially affect host fitness responses to temperature. The algal-cnidarian mutualism is largely based on the amount of photosynthetically-derived sugar that the algae provide to their host, often providing up to 95% of the nutrition to the host in nutrient-poor water 65,66. Here we found that which algal genotype has the greatest potential to provide benefits to the host depends on temperature, as the response of respiration and photosynthesis to temperature differs among algal genotypes. Algal genotypes also have different effects on the developmental timing and fitness of Cassiopea xamachana hosts. Understanding the ecological dynamics of this holobiont in response to increasing ocean temperatures will require understanding selection on and evolutionary dynamics of the algal symbionts.
Variation in algal physiology in response to temperature
We measured algal traits that are likely to affect thermal tolerance of the holobiont 41: photosynthesis, respiration, and growth rates. Previous work has identified tremendous variation in thermotolerance of these traits among and within species in the family Symbiodiniaceae 40–42,67−69. We found similar levels of variation in thermotolerance within a species. Despite variation in growth rates at different temperatures, the fact that all genotypes had positive growth rates at temperatures ranging from 26° to 32°C, suggests broad thermal tolerance in Symbiodinium microadriaticum. However, despite this high level of tolerance relative to other species, four of the five S. microadriaticum genotypes in our study showed decreased rates of respiration and photosynthesis with increased temperature, indicating variation in the amount of nutrients that could be supplied to hosts. The genotypes with the highest growth and photosynthetic rates at 26°C did not have the highest growth and photosynthetic rates at 30°C or 32°C (Figs. 2, 3). These trends suggest that, as ocean temperatures rise, the particular genotypes with the highest relative fitness that experience positive selection will differ among temperatures, resulting in selection for more heat tolerant genotypes at higher temperatures. There is evidence for rapid evolution in other Symbiodiniaceae species 46,47, so evolution of symbionts on ecological time scales may occur commonly in cnidarian-algal symbioses and provide the potential for evolutionary rescue of these important mutualisms 19,70.
Whether differences in thermal tolerance among algal genotypes allow for temperature to impose selection on thermal tolerance depends on whether such variation exists within the same algal population. Our results serve as a proof of concept that such dynamics are possible in cnidarian-algal mutualisms. However, the genetic diversity in our experiment arises from S. microadriaticum genotypes collected from the Pacific, Atlantic, and Indian Oceans, and from multiple host species (Table 1). Whether such variation also exists within a population remains to be seen. The lack of any relationship between genetic similarity and trait similarity across our global sample though, suggests that even in closely related populations of algae, there may still be sufficient trait variation upon which selection can act. Whether such variation need exist within a single polyp, a population of polyps, or in the water column, will depend on how often hosts exchange symbionts with the water column and how far algae travel in currents during the oceanic phase of their life cycle.
The effect of temperature on symbiont growth rate not only varied between genotypes, but also between experimental rounds (Fig. 3, Fig. S1). Both rounds of the experiment were conducted in seemingly identical controlled conditions in growth chambers. However, the length of the experiment differed between rounds. The six fewer days of growth in the second round resulted in many cultures still in their exponential phase of growth, before they had reached a carrying capacity (Fig. S1). Slight variation in pipetting could result in differences in cell densities during exponential growth. Logistic growth curves fit less well in the second round, relative to the first, suggesting that the estimates of maximum growth rate in the first round are more precise. Overall, the variation in growth rates between genotypes suggests that populations of S. microadriaticum exhibit varying responses to temperature stress, suggesting sufficient opportunity for selection. The amount of time that cultures spend in an exponential growth phase versus the amount of time they spend close to their carrying capacity could affect selection on growth rate in vitro. If the goal of artificial selection is to select for or against growth rates, then the population dynamics in vitro and the timing at which cultures are refreshed may be important.
Host response to temperature is dependent on symbiont genotype
Algal genotype had a significant effect on the response to temperature of a number of the fitness components of the host. Cassiopea xamachana can respond to changes in temperature through changes in growth rate and respiration rate 71,72, budding rate 73, and developmental timing 71,74. Our results confirm that infection, budding, and developmental timing are affected by temperature, but importantly, the responses to temperature are also affected by the genotype of the algal symbionts. As the genetic composition of the symbiont community changes, the capacity for the holobiont to respond to changes in temperature is likely to change as well.
Developmental rate is an important component of fitness, with faster development times potentially resulting in higher reproductive output via sexual reproduction 75,76. However, investment in sexual reproduction potentially trades-off with investment in asexual reproduction via budding. Budding asexually produces more polyps, but strobilation and ephyra production eventually allow for sexual reproduction in mature jellies. Because Cassiopea, once infected, shifts energy from asexual budding to strobilation 77, the polyps that developed fastest produced the most ephyrae, but the fewest buds, resulting in a significant, albeit weak, correlation between ephyrae and buds (Kendall’s Tau = -0.14, P = 0.003). This suggests multiple pathways to increased fitness, but only one of them involves algal symbionts. Although algal symbionts are required for the host to complete its life cycle, a longer time spent without algal symbionts is likely to result in greater asexual reproduction via budding (Fig. 8a) and may eventually pay off with higher overall capacity for sexual reproduction by more polyps in the future.
Across the three temperatures, polyps hosting either FLCass or KB8 generally developed faster than polyps hosting any of the other three symbiont genotypes, which resulted in a higher proportion of polyps reaching all three stages of development and ultimately greater ephyra production (Fig. 7). For polyps hosting these two symbiont genotypes, increased temperature decreased the time to strobilation and ephyra release, with more rapid ephyra production at higher temperatures. Subsequently, polyps hosting either FLCass or KB8 at the two higher temperatures were the only groups that produced more than one ephyra. In contrast, polyps hosting CCMP2458 and RT362 produced the fewest ephyra at 26°C, but also produced many buds at that temperature, likely due to fewer polyps being infected at 26°C. As temperature increased, polyps hosting those two genotypes showed increased infection rates and ephyra production, but decreased bud production. Polyps hosting CCMP2464 also had higher rates of infection and lower bud production with increased temperature, however this response did not result in an increase of ephyra production, which remained consistently low, relative to polyps hosting other symbiont genotypes, across temperatures (Fig. 8). Ultimately, determining the effects of each algal genotype on lifetime fitness of the host will depend on the survival rate of polyps produced via budding, ephyra production by those polyps, and successful sexual reproduction by adult jellies. However, the variable temperature responses of each host-symbiont combination suggests that genetic variation of the symbiont is likely to play an important role in lifetime reproductive success.
Interestingly, the magnitude of algal trait responses to temperature in vitro was not a good predictor of the magnitude of the response of hosts to temperature. For example, net photosynthesis of FLCass decreased sharply in response to increased temperature in vitro (Fig. 2), but temperature had little effect on the time to infection or number of buds produced by hosts (Figs. 4, 8). Polyps infected with FLCass actually produced more ephyra with increasing temperature (Fig. 6), despite the decrease in potential benefits provided by the symbionts, as measured in vitro (Fig. 2). We found little evidence that traits measured in vitro (respiration, photosynthesis, growth rate) were correlated with any aspect of host fitness at 26°C (P > 0.18), 30°C (P > 0.27), or 32°C (P > 0.14), with two exceptions: at 30°C, in vitro growth rate was positively correlated with bud production (Mantel r = 0.64, P = 0.033) and at 32°C, respiration rate was positively correlated with bud production (Mantel r = 0.84, P = 0.017). Given the large number of possible potential correlations, we are cautious about giving too much weight to these lone two significant correlations, but future research could investigate why these correlations might only exist at these temperatures. Overall, these results demonstrate the difficulty of predicting holobiont fitness and temperature response based on in vitro traits of symbionts and suggest that individual interactions between hosts and symbionts can produce unique holobiont responses, which may be of more importance for understanding holobiont performance 37,38.
The lack of correlation between symbiont traits measured in vitro and host fitness responses to temperature does not mean that these trait measurements are irrelevant though. Species in the family Symbiodiniaceae spend a portion of their life cycle in the ambient environment 78, where genetic and trait variation and selection may differ from selection in hospite. The evolution of symbiont populations in the ambient environment could influence host fitness as Cassiopea and many cnidarian species take up environmental symbiont populations every generation, or show changes in algal communities following bleaching events 59,79. Additionally, because species in the family Symbiodiniaceae have a relatively high mutation rate 70, they could continue to accumulate variation, even in hospite. The effects of different selection pressures on populations of symbionts in vitro versus in hospite is important for understanding the eco-evolutionary dynamics of these mutualisms.
Implications for evolutionary rescue via mutualists
As corals experience massive bleaching events and die-offs 26, there has been a large focus on the potential for the holobiont to be rescued by hosting more thermally tolerant symbionts 19,34,45,47,58,70,80. Although thermally tolerant symbionts can be acquired via exchange with the ambient environment 79,81,82, cnidarian-algal mutualisms are often very specific and switching to alternate taxa may not be feasible 83,84. Early polyp stages of Cassiopea can be flexible in symbiont uptake and host several species, but strobilation only occurs with a smaller subset of taxa 53,56,64. Cassiopea xamachana adults almost exclusively host Symbiodinium microadriaticum and polyps will preferentially take up homologous strains when offered homologous and heterologous strains simultaneously 56.
Our results suggest the potential for evolution of symbiont populations that could occur within or outside the host. However, because in vitro traits do not explain holobiont fitness well, from a conservation standpoint, it may not be productive to conduct selection experiments on symbionts in vitro, but rather to focus on selection on holobionts and consider evolution in this community context 23. Algal symbionts have a high mutation rate, so selection could act on favorable mutations and result in evolution of thermal tolerance in hospite 70. If algal thermal tolerance evolves in the ambient environment, or in populations of algae in hospite that are later expelled, then symbiont switching could play a role in acquiring temperature tolerant genotypes while maintaining the specificity of the mutualism. If the evolution of thermal tolerance in algal populations confers increased holobiont thermal tolerance, then perhaps the holobiont could experience evolutionary rescue via association with symbionts. Fully answering this question will require tracking lifetime fitness of the host and quantifying benefits and costs of hosting thermally tolerant symbionts. Hosting thermally-tolerant can come with costs at less stressful temperatures 85–87, so understanding lifetime fitness is critical. The genetic composition and evolution of algal symbiont populations seem likely to play a significant role in the response of cnidarians to rising ocean temperatures associated with climate change.