Genetic variation in Breviolum antillogorgium, a coral reef symbiont, in response to temperature and nutrients

Abstract Symbionts within the family Symbiodiniaceae are important on coral reefs because they provide significant amounts of carbon to many different reef species. The breakdown of this mutualism that occurs as a result of increasingly warmer ocean temperatures is a major threat to coral reef ecosystems globally. Recombination during sexual reproduction and high rates of somatic mutation can lead to increased genetic variation within symbiont species, which may provide the fuel for natural selection and adaptation. However, few studies have asked whether such variation in functional traits exists within these symbionts. We used several genotypes of two closely related species, Breviolum antillogorgium and B. minutum, to examine variation of traits related to symbiosis in response to increases in temperature or nitrogen availability in laboratory cultures. We found significant genetic variation within and among symbiont species in chlorophyll content, photosynthetic efficiency, and growth rate. Two genotypes showed decreases in traits in response to increased temperatures predicted by climate change, but one genotype responded positively. Similarly, some genotypes within a species responded positively to high‐nitrogen environments, such as those expected within hosts or eutrophication associated with global change, while other genotypes in the same species responded negatively, suggesting context‐dependency in the strength of mutualism. Such variation in traits implies that there is potential for natural selection on symbionts in response to temperature and nutrients, which could confer an adaptive advantage to the holobiont.

Individual genetic variation in phenotypes provides the raw material for natural selection, which can lead to evolutionary rescue of populations from stressful conditions that would otherwise lead to local extinction (Gomulkiewicz & Holt, 1995). Mounting evidence suggests that genetic variation among individuals within a host species may affect bleaching response (Carilli, Donner, & Hartmann, 2012;Dixon et al., 2015;Kenkel et al., 2013;Pineda et al., 2013;Polato et al., 2010). Similarly, limited evidence suggests that, given sufficient genetic variation, symbionts can adapt to local conditions (Chakravarti et al., 2017;Chakravarti & van Oppen, 2018;Howells et al., 2012).
Evidence suggests that symbiont populations can adapt to changes in temperature. Symbiont genotypes from warmer reefs performed better and promoted higher growth rates in hosts exposed to higher temperatures (Howells et al., 2012). Thermal tolerance traits can be highly heritable in symbiont populations, indicating that changes in symbiont performance following natural selection are likely to be passed on to the next generation after sexual reproduction (Császár, Ralph, Frankham, Berkelmans, & van Oppen, 2010;Quigley, Willis, & Bay, 2016). In fact, invasion of exapted symbiont genotypes into the Persian/Arabian Gulf resulted in strong selection that led to the dominance of Cladocopium thermophilum (formerly clade C, ITS2-"Gulf C3") genotypes throughout the Gulf (Hume et al., 2016). Similarly, stresstolerant Durusdinium trenchii has spread through the Caribbean Sea (Pettay, Wham, Smith, Iglesias-Prieto, & LaJeaunesse, 2015).
In a recent laboratory study, Chakravarti et al. (2017) found that Cladocopium C1 cultures subjected to laboratory selection at high temperature (31°C) had better photophysiology and growth at high temperature compared to wild-type cells, suggesting that at least in some symbiont types, variation could allow a response to selection.
Here, we examine functional trait variation within the newly erected genus Breviolum (LaJeunesse et al., 2018) to examine whether genetic variation that could give rise to evolutionary rescue exists within species. Using several genotypes, we quantified functional traits that are most likely to affect the strength of interactions with the host. We ask whether the traits of different genotypes respond differently to increases in temperature or changes in the local nutrient environment to better understand the capacity of these populations to evolve in response to global change.

| Source of symbionts
We isolated two symbiont species within the genus Breviolum (B1-ITS2 type) from the octocoral host, Antillogorgium bipinnata, from two locations within the Florida Keys (Looe Key and Tennessee Reef). We maintained the cultures in the Buffalo Undersea Reef Research Culture Collection for one to six years. Briefly, we collected ~3 cm from each of five host colonies; we preserved 1 cm in 95% ethanol for later molecular analysis of the dominant symbiont type within the host and ground the remaining 2 cm in 2 ml of filtered seawater (FSW). We poured the resultant slurry through a series of mesh filters (125, 74, and 20 µm) to remove larger pieces of host tissue and sclerites. We brought the homogenate to 10 ml with FSW and spun at 800 rpm for 5 min. The pellet was washed two more times with FSW and then resuspended in 1.0 ml of F/2 media (Guillard & Ryther, 1962). We added aliquots of 20 or 50 µl to each of six 50-ml flasks with 30 ml of F/2 media. We maintained cultures under 40 W cool white lights with a 14:10-hr light:dark cycle at 26°C and examined every 4-7 days for growth over a three-month period.
We transferred new Breviolum growth immediately to fresh media.
Once growth was established, we transferred cultures to fresh media monthly and maintained cultures under the same conditions for three to nine years before imposing temperature and nutrient treatments and measuring traits.
In total, we identified seven distinct genotypes among our cultures (Table 1). Molecular analysis revealed that a subset of these were the symbiont Breviolum antillogorgium, the dominant symbiont within the host (Parkinson & Coffroth, 2015). Given that symbionts representative of the host are notoriously difficult to isolate in culture (LaJeunesse, 2002;Santos, Taylor, & Coffroth, 2001), the ability to isolate the dominant symbiont from this octocoral host makes A. bipinnata ideal for studying symbiont traits in culture that are also relevant for interactions with the host. A second symbiont species, Breviolum minutum, was represented by some cultures (Table 1), and we used these to examine both within-and between-species variation in functional traits. All cultures came from different hosts, except 08-0689.4 and 08-0689.6, which came from the same host.

| Molecular analysis
To determine symbiont species and genotype, symbiont DNA was extracted from each culture following the protocols of Coffroth, Lasker, Diamond, Bruenn, and Bermingham (1992). Species identification was based on sequence analysis of the flanking region of the B7Sym15 microsatellite and the chloroplast 23S rDNA (Parkinson & Coffroth, 2015) following the protocols of Thornhill, Xiang, Pettay, and Santos (2013) and Santos et al. (2002), respectively. PCR products were directly sequenced (TACGen, Richmond, CA), aligned in  Figure S1), so after 39 days of growth, we used a wellmixed 5 ml sample from each culture to quantify three performance traits (cell growth, photosynthetic efficiency, and chlorophyll concentration), as described below.  (Table 1). Cultures were initiated in 75-cm 3 Cell Culture Flasks (NEST ® ) at initial cell densities of 10 3 cells/ml.

| Experiment 2: Genotype responses to nitrogen
Cultures were maintained for 35 days. Each week we removed 10 ml of media and replaced it with media of the assigned nitrogen concentration, in order to maintain the nitrogen treatments.
Although we did not quantify nitrogen levels in this experiment, changes in nitrate levels over one week (~1-5 mg/L) were considerably less than the differences among treatments (25, 75, and 150 mg/L). Before replacing media each week, we measured cell densities and used these to estimate population growth rate of cells in each culture. At the end of the experiment, we measured the same performance traits as in Experiment 1.

| Performance measurements
At the end of each experiment, we mixed each culture well before removing 5 ml that we used to measure three performance traits: provides an estimate of photosynthetic efficiency, where a decrease in dark-adapted quantum yield (Fv/Fm) of photosystem II measured in the same organism in response to a treatment reflects a stress response to that treatment (Suggett et al., 2008). Fv/Fm may vary with cell size (Maxwell & Johnson, 2000;Suggett et al., 2015), but here we used two closely related species of similar cell size (B. antillogorgium: 7.1-8.1 µm, Parkinson & Coffroth, 2015 and B. minutum: 6.5-8.5 µm, LaJeunesse, Parkinson, & Reimer, 2012). (c) We used the same sample as above to measure in vivo chlorophyll a (Chl a) fluorescence on a Trilogy Laboratory Fluorometer (Turner Designs).
As the Fluorometer has an upper limit in readable in vivo Chl a, 50% dilutions were used for samples that exceeded that limit by replacing 1.25 ml of sample with 1.25 ml of F/2 media. Chlorophyll concentrations were quantified as relative fluorescence units (RFU) and used to compare relative differences in Chl a concentrations between treatments, standardized by cell density.

| Statistical analysis
We used trait data from each experiment in a principal component analysis (PCA) to visualize differences between genotypes in multidimensional space, using "princomp" in R version 3.3.2. For the PCA, we used performance traits measured at 26°C and normal F/2 nitrogen levels in each experiment, so that all traits were measured in the same environmental conditions. All traits were converted to z-scores to meet assumptions of normality.
To examine differences in traits among genotypes and differential responses of genotypes to temperature, we used generalized linear models to examine treatment effects on each trait separately.
Models included temperature, genotype, and their interactions as fixed factors. We used sample size-corrected Akaike information criterion (AICc) and backwards stepwise selection to choose the best-fit model for each variable. We examined differences among genotypes in response to nitrogen concentrations using similar analyses with nitrogen concentration as a fixed factor. We quantified the effects of each factor on: population growth rate, quantum yield, and chlorophyll concentration per cell.  Figure S2). B. antillogorgium is a host-specialist of Antillogorgia (Parkinson & Coffroth, 2015). B. minutum is the common symbiont in the anemone, Exaiptasia, and is most likely a transient species that can occur on the surface of or inside Antillogorgia, but is rarely the dominant symbiont type of this host. Microsatellite analysis yielded two to four alleles among the five microsatellite loci examined resulting in the identification of multiple distinct genotypes of Breviolum (Supporting Information Figure S2, Table S1).

| Genetic variation in Breviolum
Most genotypes occupied unique spaces in multidimensional space ( Figure 1). The first PC axis explained 54% of the variation in trait data and was primarily associated with chlorophyll per cell.
The second PC axis explained 30% of the variation in trait data and was more associated with quantum yield and population growth rate.

| Effects of temperature on traits
Temperature had a significant effect on the performance of

| Effects of nutrients on traits
The growth rates of genotypes were significantly different from one another (F 4,37 = 2.92, p = 0.034), though there was no evidence that species were more different than genotypes (Figure 3a). Nitrogen had no significant effect on growth rates (F 2,37 = 1.84, p = 0.174), and the Genotype*Nitrogen interaction was not a part of the best-fit model (ΔAICc = 9.81).
Tukey post hoc tests revealed that one B. minutum genotype

| D ISCUSS I ON
Although taxonomists continue to partition the genetic variation in the family Symbiodiniaceae into genera and ultimately species (Coffroth & Santos, 2005;Hume et al., 2015;Jeong et al., 2014; LaJeunesse, Lee, Gil-Agudelo, Knowlton, & Jeong, 2015;LaJeunesse et al., 2018LaJeunesse et al., , 2012Parkinson & Coffroth, 2015), our study reveals that significant functional variation exists among and within two and nitrogen. Traits that are likely to affect the strength of the symbiont's relationship with hosts, such as photosynthetic efficiency or growth rate, decreased at higher temperature or nitrogen concentrations in some genotypes, but were unaffected or increased in other genotypes. This suggests that the relationship between symbionts and hosts depends on the specific genetic composition of the symbiont population. Further, this variation implies that these symbiont traits have the potential to evolve in response to selective pressures of increased ocean temperatures associated with climate change, or in response to nitrogen concentrations within the host or in the water column, and that selection will be environment-dependent.
The host (Antillogorgia bipinnata) from which the symbionts in this study were collected typically harbors one dominant symbiont, though others may be present at abundances too low to detect with our methodology, which can detect approximately 10-1,000 cells in a sample (Santos et al., 2003). However, it is noteworthy that other hosts can maintain a number of genotypes, species, and even genera within a host (Howells et al., 2009;Quigley et al., 2014;Rowan & Knowlton, 1995). Interestingly, two Breviolum genotypes (08-0689.4 and 08-0689.6) that were isolated from the same host, but were different species, had similar traits and similar responses to temperature in Experiment 1. Conversely, that were identified as the same genotype based on our molecular data, showed significant variation in the traits that we measured, suggesting that they are likely different genotypes not detected by our microsatellites.
As clonal reproduction predominates within this symbiont family, most variation in Breviolum genotypes is likely the result of mutations, though genetic variation may be further increased or maintained by transposons, retrotransposons, tandem repeats, or recombination during sexual reproduction (Shoguchi et al., 2013). Because of the high mutation rate in Symbiodiniaceae (van Oppen et al., 2011), even hosts that initially harbor a single symbiont genotype may quickly accumulate genetic variation. Natural selection occurs when some genotypes outperform other genotypes under different environmental conditions. As many temperature tolerance traits are heritable (Császár et al., 2010;Quigley et al., 2016), natural selection is likely to lead to evolution of temperature tolerance in the symbiont population. Selection on the symbiont population could result in evolutionary rescue of the holobiont, allowing hosts to persist through periods of higher temperature (Baskett, Gaines, & Nisbet, 2009;Chakravarti & van Oppen, 2018;van Oppen, Oliver, Putnam, & Gates, 2015;van Oppen et al., 2011).
As with temperature, we found variation in the functional response of Breviolum genotypes to nitrogen concentrations, which may affect how populations of symbionts respond to eutrophication in the water column, or to different nitrogen environments within a host. In the case of both temperature and nitrogen, such functional variation could be indicative of existing niche partitioning allowing for genotypic coexistence, similar to niche partitioning among species (Chase & Leibold, 2003). Differences among genotypes could maintain genetic diversity in natural populations of Breviolum and other species within the Symbiodiniaceae, similar to results in other systems (salt marshes [Proffitt, Travis, & Edwards, 2003], sea grass communities [Reusch, Ehlers, Hammerli, & Worm, 2005], arboreal communities [Schweitzer et al., 2004], and plant-insect interactions [Johnson & Agrawal, 2005]).
An important consideration to whether the temperature-or nitrogen-tolerant genotypes in this experiment would lead to holobiont adaptation is how well performance in laboratory cultures relates to performance in a host (Moran & Sloan, 2015). The traits we have measured are likely to be important for interactions with a host, but the exact effects are difficult to predict. For example, high growth rates may be beneficial to hosts as they allow the host to acquire large symbiont populations quickly, or recover from bleaching in a short time. However, if genotypes with high growth rates do not supply the host with adequate carbon, the relationship may be more parasitic and the symbiosis more likely to break down (Cunning & Baker, 2013). Previous work suggests symbiont physiology indeed differs in culture and in hosts (Bhagooli & Hidaka, 2003;Chakravarti et al., 2017;Howells et al., 2012;Ralph, Gademann, & Larkum, 2001).
For symbiont evolution to result in holobiont adaptation, the symbionts must not only evolve in response to changing environmental conditions, but also continue a mutually beneficial relationship with the host. Although Chakravarti et al. (2017) found evidence for thermal adaptation in vitro, thermally selected strains of Cladocopium C1 had less of an effect when introduced into the host. Work to determine whether symbiont genotype responses to temperature in culture and in hosts differ quantitatively or qualitatively is ongoing.
Differing amounts and types of nitrogen available in vitro and in hospite may also affect the outcome of host-symbiont interactions. Nitrogen concentrations in F/2 media are quite high, and it is unlikely that nitrogen was limiting, even in our low-nitrogen treatment. This could be one reason why we did not observe significant differences in growth rates among nitrogen treatments, on average. Experimental nutrient conditions ranged from 0.4-2.4 µM nitrate L −1 , whereas waters surrounding most coral reefs are very low in dissolved inorganic nitrogen with measures of <1 µM/L (Fiore, Jarett, Olson, & Lesser, 2010;Tanaka, Miyajima, Koike, Hayashibara, & Ogawa, 2007). Corals may supplement nitrogen available to symbionts by as little as 0.264 µmol N cm −1 day −1 , an amount that exceeds the growth needs of the algae (Falkowski, Dubinsky, Muscatine, & Mccloskey, 1993;Rees, 1991). Further, we manipulated nitrate, which many algae, including those in the Symbiodiniaceae, can use, but most of the nitrogenous waste produced by hosts is in the form of ammonium and is a preferred source of nitrogen (Grover, Maguer, Allemand, & Ferrier-Pages, 2003). Some portion of the nitrate in cultures was likely reduced to other forms of nitrogen, although the extent to which this occurred was not quantified in this experiment. Bacteria also likely play in role in the abundance and forms of nitrogen available, and we do not yet know whether genotype traits differ because of specific genetic differences in the algae, or if different algal genotypes harbor different bacterial communities.
Although future experiments should explore different quantities or forms of nitrogen, growing symbionts in culture at such low concentrations can be difficult, or at best, time consuming. Regardless, this work suggests that we are unlikely to understand the performance and response of hosts to eutrophication or other aspects of global change without accounting for genetic differences in the symbiont population.
The surprising amount of genetic and functional trait variation observed within and among these symbiont species, coupled with the short generation times of these organisms, suggests that populations of symbionts have the capacity to evolve over ecologically relevant time scales. Though hosts may evolve in response to global change, the rapid evolutionary potential of the symbionts with shorter generation times may be a faster route to adaptation for the holobiont. For example, Chakravarti and van Oppen (2018) found that symbiont populations grown at high temperatures began to outperform wild-type symbionts in terms of growth rate and photosynthetic efficiency in as little 40-70 asexual generations. The potential for this group of symbionts to evolve offers some hope to the future of coral reefs. Evolutionary rescue may be an important mechanism by which species persist in the face of global change (Gomulkiewicz & Holt, 1995). Beyond evolution in the wild, others have called for assisted evolution by developing temperature-tolerant strains of corals of critical conservation concern (Chakravarti & van Oppen, 2018;van Oppen et al., 2015).
Though our research suggests that variation within species exists, allowing some scope for natural selection, the success of introducing adapted strains in natural populations will also require successful infection of hosts with those strains, growth inside the host, and the adapted symbionts must increase the fitness of the holobiont.

ACK N OWLED G M ENTS
We are grateful for laboratory assistance from Louie Buccella, Leah Bandak, Emma Collosi, DJ Valint, and Katherine Wong. We appreciate the comments of D. Suggett and three anonymous reviewers on a previous draft of this manuscript. This work was supported by collaborative grants from the National Science Foundation to MAC and CPt (OCE-1559286 and OCE-1559105) and funding from the California State University Thesis Support Program to SLJB and ZRS.

CO N FLI C T O F I NTE R E S T
None declared.