Temperature‐mediated acquisition of rare heterologous symbionts promotes survival of coral larvae under ocean warming

Abstract Reef‐building corals form nutritional symbioses with endosymbiotic dinoflagellates (Symbiodiniaceae), a relationship that facilitates the ecological success of coral reefs. These symbionts are mostly acquired anew each generation from the environment during early life stages (“horizontal transmission”). Symbiodiniaceae species exhibit trait variation that directly impacts the health and performance of the coral host under ocean warming. Here, we test the capacity for larvae of a horizontally transmitting coral, Acropora tenuis, to establish symbioses with Symbiodiniaceae species in four genera that have varying thermal thresholds (the common symbiont genera, Cladocopium and Durusdinium, and the less common Fugacium and Gerakladium). Over a 2‐week period in January 2018, a series of both no‐choice and four‐way choice experiments were conducted at three temperatures (27, 30, and 31°C). Symbiont acquisition success and cell proliferation were measured in individual larvae. Larvae successfully acquired and maintained symbionts of all four genera in no‐choice experiments, and >80% of larvae were infected with at least three genera when offered a four‐way choice. Unexpectedly, Gerakladium symbionts increased in dominance over time, and at high temperatures outcompeted Durusdinium, which is regarded as thermally tolerant. Although Fugacium displayed the highest thermal tolerance in culture and reached similar cell densities to the other three symbionts at 31°C, it remained a background symbiont in choice experiments, suggesting host preference for other symbiont species. Larval survivorship at 1 week was highest in larvae associated with Gerakladium and Fugacium symbionts at 27 and 30°C, however at 31°C, mortality was similar for all treatments. We hypothesize that symbionts that are currently rare in corals (e.g., Gerakladium) may become more common and widespread in early life stages under climate warming. Uptake of such symbionts may function as a survival strategy in the wild, and has implications for reef restoration practices that use sexually produced coral stock.


| INTRODUC TI ON
Coral bleaching events are increasing in severity, duration, and frequency globally with recovery windows shortening between heating events; for example, Australia's Great Barrier Reef in 2016Thiault et al., 2020), Kāneʻohe Bay, O'ahu, Hawai'i in 2014(Matsuda et al., 2020Ritson-Williams & Gates, 2020), Mo'orea, French Polynesia in 2002 (Penin et al., 2007) and 2019 (Burgess et al., 2021), and the Florida Keys in 2014 and 2015 (Fisch et al., 2019). In order to replenish reefs following mortality events, the recovery and persistence of reefs depend on reproduction, recruitment, and survival of new coral offspring (Doropoulos et al., 2015). However, bleaching compromises the reproductive capacity of surviving corals (Baird & Marshall, 2002;Howells et al., 2016;Johnston et al., 2020;Ward et al., 2002), making the survival of available offspring even more critical for reef persistence Ward et al., 2002). Growth and survival of early coral life stages through the recruitment bottleneck is contingent upon the successful establishment of symbiosis with algal endosymbionts (family: Symbiodiniaceae) that provide coral nutrition through photosynthetically fixed carbon (Aranda et al., 2016;Harii et al., 2010). However, the influence of thermal stress on the timing and success of establishing symbiosis and subsequent proliferation of the symbiont population within the host is not well understood.
Corals acquire their algal symbionts through three strategies: vertical transmission: directly from the parent colony; horizontal transmission: from the surrounding environment (Cumbo et al., 2013); or mixed-mode transmission: a combination of the two (Quigley et al., 2018). Horizontal transmission is the most widespread mode of acquisition for reef-building corals Fadlallah, 1983), and symbioses are established either in larval or post-settlement stages (i.e., recruits). Coral larvae primarily utilize endogenous energy reserves (i.e., lipids) during development, however, symbionts may provide an additional source of nutrition (Harii et al., 2007).
Associating with algal symbionts at the larval stage can have fitness consequences, such as a higher settlement rate in Lobactis scutaria (Schwarz et al., 1999) and greater survival in Acropora yongei (Suzuki et al., 2013). However, these effects are variable and species specific-for example, establishment of symbiosis had no effect on settlement in Platygyra acuta (Ng et al., 2019), and even reduced survival in Orbicella faveolata larvae (Hartmann et al., 2018).
As molecular techniques have achieved higher resolution, symbionts from less common genera have recently been identified in low proportions as subdominant or background coral symbionts in some species (Boulotte et al., 2016;Cunning et al., 2015;Qin et al., 2019;Stat et al., 2013). For example, Gerakladium has been found with Stylophora pistillata , Merulina ampliata (G3: 2.3% relative abundance), Platygyra daedalea (G3: 3.3% relative abundance), Porites lutea (G3: 1.9% relative abundance), and Hydnophora exesa (Qin et al., 2019). Fugacium is the dominant symbiont in only a single hermatypic coral, Alveopora japonica (F-Ajap) (De Palmas et al., 2015), and has also been found in background symbioses, for example, with P. lutea (<0.1% relative abundance; Qin et al., 2019) (Teschima et al., 2019). However, the influence of background symbionts on holobiont performance is still largely unknown. Symbiodiniaceae species display differential functional performance and nutritional characteristics, and therefore it is critically important to understand how symbiosis dynamics impact survival during coral early life history. Symbiodiniaceae provide photosynthetic products to the host (Muscatine & Cernichiari, 1969), but the quality and quantity of translocated photosynthates varies among symbiont species, which can have important implications for performance (Cantin et al., 2009;Stat et al., 2008). Coral flexibility in algal symbiont association is a potentially beneficial trait, allowing the host to respond to the current environmental conditions by selecting the most appropriate symbiont(s); however, there may also be negative impacts to the host. Association with Durusdinium could be an adaptive response to warming oceans because Durusdinium species often confer greater thermal tolerance to their hosts than Cladocopium (Berkelmans & van Oppen, 2006;Glynn et al., 2001;Manzello et al., 2019;Rowan, 2004;Silverstein et al., 2015; however, see Abrego et al., 2008). For example, Montipora capitata associates with both Cladocopium and Durusdinium symbionts (Stat et al., 2011) and exhibits physiological trade-offs between these partners (Wall et al., 2020). Durusdinium confers higher coral thermal tolerance on its host, but it is also known to provide less nutrition (carbon translocation) to the host than Cladocopium (Cantin et al., 2009), which may affect host performance during ambient temperature conditions (Jones & Berkelmans, 2010Wall et al., 2020).
There is evidence of symbiotic flexibility in larval and recruit stages, where early life stages have been found to associate with more or different types of symbionts than adults (Abrego et al., 2009b;Gómez-Cabrera et al., 2008;Little et al., 2004). For example, Acropora tenuis larvae and juveniles can be dominated by Durusdinium, Cladocopium, or a mixture, despite most adult colonies being dominated by Cladocopium (Little et al., 2004;Yamashita et al., 2021). Recently, low levels of Fugacium sp. were found in A. tenuis juveniles, which has not yet been observed in adults (Quigley et al.,

K E Y W O R D S
climate change, coral, digital droplet PCR (ddPCR), larvae, Symbiodiniaceae, symbiont choice experiments, symbiosis, thermal stress 2016). After symbionts are incorporated into the host's gastrodermal cells (i.e., "infection"), there is a "winnowing" period during which the symbiont community stabilizes and reflects adult symbiotic associations (Gómez-Cabrera et al., 2008;Rodriguez-Lanetty et al., 2006). Early life history symbiosis flexibility therefore provides a window of opportunity for symbiotic flexibility to provide survival advantages. It is critical to understand how symbiotic flexibility in early life stages is affected by, and mediates response to, climate change driven ocean warming, and whether rare symbionts may present benefits and/or trait trade-offs.
In laboratory settings, inoculation experiments have evaluated the capacity for different symbionts to associate with and proliferate within coral larvae (Bay et al., 2011;Cumbo et al., 2013;Schwarz et al., 1999). The majority of these studies were no-choice experiments where corals were exposed to a single symbiont species (Rodriguez-Lanetty et al., 2006;Schwarz et al., 1999;Weis et al., 2001;Yakovleva et al., 2009) and demonstrated that symbionts found naturally in symbiosis with adult corals (i.e., homologous) have greater infectivity and proliferation than those that are not naturally dominant (i.e., heterologous). Multi-genus infections can provide further insights on symbiont infection under competition and have been studied in other cnidarian-Symbiodiniaceae models, including sea anemones (Gabay et al., 2019;Herrera et al., 2020) and an octocoral (McIlroy et al., 2019); however, no studies to date have examined infection dynamics of more than two Symbiodiniaceae genera in scleractinian corals.
Association with rare symbionts with enhanced thermal thresholds could be a product of host adaptive responses, competition among symbionts, or early life history acquisition of symbionts that are maintained at low abundances. The use of next generation sequencing has resulted in an increase in our capacity to identify rare background symbionts in corals (Silverstein et al., 2012), and has provided evidence of symbiont assemblages changing (i.e., "switching" or "shuffling") in response to heat stress (Boulotte et al., 2016;Cunning et al., 2018;Rouzé et al., 2017; but see Stat et al., 2009).
Although rare, there is evidence that coral-Symbiodiniaceae partnerships change when a new symbiont becomes available. Durusdinium trenchii was recently introduced into the Caribbean and there has been an increase in dominance of this symbiont species over the past few decades in many coral species in the Caribbean (e.g., species here; Pettay et al., 2015). Durusdinium trenchii confers increased bleaching resistance to the host over conspecifics associated with historic partners (Manzello et al., 2019;Rowan et al., 1997). Coralalgal associations in adult corals also vary across environmental gradients, including marginal and extreme environments. For example, Goniastrea aspera, which associates with Cladocopium in "optimal" sites, forms symbiosis with Durusdinium in mangroves, characterized by high temperature and light conditions as well as high turbidity (Hennige et al., 2010). These differences in symbiont assemblages by environment may also impact fitness and performance under future climate change related stressors. Given variation in symbiotic relationships across environmental conditions, it is critical to evaluate how the environment drives symbiosis formation in early life stages.
Here, we investigated symbiont infection dynamics in four-way choice and no-choice experiments and tracked implications for coral larval survival under thermal stress. We hypothesized that larvae exposed to higher temperatures would (1) associate with a greater proportion of thermally tolerant Durusdinium than thermally sensitive Cladocopium, and (2) exhibit reduced algal cell proliferation over time accompanied by higher larval mortality. We exposed larvae of A. tenuis, a broadcast spawner and horizontal transmitter, to four species of Symbiodiniaceae in no-choice and four-way choice experiments over a 14 day period at 27, 30, and 31°C. Acropora tenuis is a common reef-building coral on the Great Barrier Reef, whose adult colonies mostly associate with Symbiodiniaceae in the genus Cladocopium (van Oppen et al., 2001). Juveniles can associate with Symbiodinium, Cladocopium (C1 predominantly, and other C-types), and/or Durusdinium (D, D1, D1a, and other D-types) (Little et al., 2004;Quigley et al., 2017;Quigley, Alvarez Roa, et al., 2020;Quigley, Randall, et al., 2020); Fugacium and Gerakladium have also been detected at background levels in juveniles (Quigley et al., 2017;Quigley, Alvarez Roa, et al., 2020;Quigley, Randall, et al., 2020). The four genera used here vary in their thermal tolerance (Fugacium > Gerakladium > Durusdinium > Cladocopium; Chakravarti et al., 2017;Chakravarti & van Oppen, 2018), but whether resilience is also conferred to the host after infection has not been studied previously.
Our results demonstrate that, surprisingly, when all four symbionts were offered to larvae in equal proportions, rare heterologous symbionts were as, or more, successful relative to the common, homologous species in infecting and proliferating in the coral host across temperatures. Simulator (SeaSim) research facility (Townsville, Australia). After spawning on the evening of the full moon, gametes were pooled and fertilized as described in Chakravarti et al. (2019). Larvae were reared in ten 420 L flow-through tanks (0.4 μm filtered sea water, FSW) at ambient temperature (27°C) for 10 weeks until the present experiment commenced ( Figure S1). Acropora tenuis larvae remained competent (e.g., swimming, not deformed) despite their extended maintenance in tanks. While coral larvae can begin to settle days to weeks post-spawning, the upper length of duration they are able to remain in the water column is not well known. A study of another acroporid, Acropora latistella, and four other species on the Great Barrier Reef found that after an initial drop in survivorship during the first month, there was low mortality until 100 days, with some surviving past 200 days (Graham et al., 2008). In A. tenuis specifically, Graham et al. (2013) observed a median survivorship of 57 days, at which point larvae were observed to successfully settle.

| Larval collection and Symbiodiniaceae culture
Additionally, Nishikawa et al. (2003) observed that when provided substrate at 60 days post-spawning, settlement occurred up to 9 days later, highlighting the long duration that A. tenuis larvae can survive and settle. Lastly, another study used larvae from the same pool as this experiment within the same time frame and observed high larval survival and symbiont acquisition (Buerger et al., 2020).
Four Symbiodiniaceae species in four genera were used in the experiment. These symbiont cultures were originally isolated from four coral species from the Great Barrier Reef and subsequently grown from a monoclonal culture and described in Chakravarti and van Oppen (2018) and Chakravarti et al. (2017) (Table 1). Cultures include Cladocopium C1 acro (previously referred to as Cladocopium goreaui; Beltrán et al., 2021), Durusdinium sp., Fugacium kawagutii, and Gerakladium sp., formerly types C1, D1, F1, and G3, respectively, and hereafter referred to solely by their generic epithets. In vitro tolerance for each culture was determined previously for temperatures where cultures experienced positive growth and was as F I G U R E 1 Adult Acropora tenuis colony (a) fluorescent microscopy of an Acropora tenuis larva (b) (red: Symbiodiniaceae; green: green fluorescent protein). (c) Acropora tenuis larvae and algal cultures were acclimated at 27, 30, or 31°C. Symbiont acquisition treatments included no-choice (Cladocopium, Durusdinium, Fugacium, or Gerakladium; 3x per temperature) and four-way choice (equal mix of all four species). A single jar of aposymbiotic larvae was kept at each temperature. Photo: Hannah Epstein (a), S. Matsuda (b)  Cultures were kept at 27°C in a Steridium E500 growth chamber on a 14:10 h light/dark cycle at 65 ± 10 μmol photons m −2 s −1 (Sylvania FHO24W/T5/865 fluorescent tubes). The Cladocopium C1 acro symbiont culture has been previously used successfully in infection studies of larval A. tenuis (Buerger et al., 2020), and Acropora spathula (Quigley, Alvarez Roa, et al., 2020;Quigley, Randall, et al., 2020),

| Experimental design
Larvae in this experiment were exposed to Symbiodiniaceae cul- counted on a hemocytometer and the algae were added to larval jars to achieve a final density of 3 × 10 4 cells/ml. To achieve high initial exposure densities, larvae were inoculated in 100 ml of 0.2 µm FSW for 72 h, thereafter the volume was increased to 400 ml with 50% water changes via pipette every 2-3 days for the duration of the experiment.

| Sampling for in hospite Symbiodiniaceae cell density
Symbiodiniaceae cell densities in A. tenuis larvae were measured on days 3, 7, and 14 during the inoculation experiment; this time frame was chosen because previous A. tenuis acquisition studies have reported 100% infection rates by days 3-6 (Bay et al., 2011;Yamashita et al., 2014). Cell densities were measured by sampling 20 larvae per jar, however, this sample size was reduced at later time points as numbers declined due to mortality, which is discussed below. Larvae were rinsed twice in 0. cell density (cells per larvae) during the experiment. Relative abundance comparisons were analyzed using ddPCR data (described below). Symbiont densities measured by hemocytometer and/or epifluorescence did not differ significantly from those measured by ddPCR ( Figure S2; Table S1).

| DNA extraction and ddPCR
Genomic DNA was extracted from individual larvae using the protocol outlined in (Wilson et al., 2002). Briefly, samples were placed in an extraction buffer (100 mm Tris pH 9.0, 100 mm EDTA, 100 mm NaCl, 1% SDS, and MilliQ water) with ProteinaseK (20 mg/ml), bead beaten (sterile glass beads, 710-1180 µm, Sigma-Aldrich G1152; MP Biomedicals FastPrep-24 4 m/s, 20 s), and incubated (55°C 2 h); 5 m KOAc was added and samples were incubated on ice (30 min) and subsequently centrifuged at 25,000 g-force for 15 min. Incubation was followed by an RNAse A treatment, ethanol cleanup, and resuspension in DNAse-free water. We used previously developed primers (Table S2). Primers for Cladocopium and Durusdinium were developed by Cunning and Baker (2012) and amplified the actin gene, and primers targeting ITS2 were used for Gerakladium (Meistertzheim et al., 2019) and Fugacium (forward: Meistertzheim et al., 2019;reverse: this study, modified from Meistertzheim et al., 2019). ITS2 reverse primer for Fugacium were modified using ITS2 sequences (Table S2) from the cultures used in the present study. Target gene copy number (described below) is estimated and accounted for using DNA extracted from counted cells, which normalizes potential variation in copy number among taxa and between different marker genes.
All primer pairs were tested on DNA extractions from cultures of all four species using ddPCR to ensure primers were specific to the target species. In these trials there was no evidence of crossamplification (Table S3).

| Symbiont population proliferation
We examined symbiont cell density (cells per larva) for each symbiont species in larvae exposed to no-choice and four-way choice experiments that were successfully infected; larvae that were not infected were removed. Cell densities were log 10 transformed to meet normality assumptions. First, we used a linear mixed effect model to test the effects of treatment (no-choice and four-way choice treatment), temperature, time, and their interactions on cell densities. Then, for each species, we compared proliferation (increase over time) when alone (the no-choice treatment) to proliferation when in competition (four-way choice).

| Interspecies competitive dynamics
The four-way choice treatment allowed us to examine competitive dynamics between Symbiodiniaceae species. Because ddPCR reactions for each species primer pair were run individually (not multiplexed), there were occasions when reactions for all species in a larva were not successful (i.e., a failed ddPCR, different from an instance of zero symbiont acquisition in a successful ddPCR). Therefore, for all relative abundance models, only larvae where ddPCR reactions were successful for all four species were included. We conducted Kruskal-Wallis non-parametric tests at each time point to examine the effects of temperature on the number of species that infected larvae. Next, we used linear mixed effects models to test the effects of Symbiodiniaceae species, temperature, time, and their interactions on relative abundance, which was arcsine square root transformed to meet normality assumptions of analyses.

| Symbiont community structure
We used multivariate analysis to analyze the effect of temperature treatment and time on symbiont community structure in the fourway choice treatment. Here, we visualized the variation in symbiont community relative abundance (arcsine square root transformed) among larvae using nonmetric multidimensional scaling (nMDS) based on a Bray-Curtis dissimilarity matrix and evaluation of main effects using a two-way PERMANOVA (999 permutations).

| Acquisition success: How does temperature and symbiont treatment (no-choices and the four-way choice) influence symbiont cell densities within larvae over time?
Acquisition success (i.e., the presence of ≥1 cell in a larva) over the course of the experiment differed between symbiont treatments (p < .01; Table S4; Figure 2a) but was not influenced by temperature (p = .72) or time (p = .33) or their interactions. Acquisition success in no-choice treatments was not uniformly higher in homologous Cladocopium than heterologous (Durusdinium, Fugacium, and Gerakladium) symbionts and was high in the four-way choice experiment across temperature and time (>92%). Across all temperature treatments, larvae exposed to either Cladocopium or Fugacium symbionts only had the lowest mean acquisition success on day 3 ( Figure 2a). However, by day 14, >75% of the larvae at 27 and 30°C were infected in all treatments except the no-choice Fugacium, which was significantly lower with a mean of 47% (27°C) and 67% (30°C).
The only group that showed a difference in acquisition success over time was no-choice Cladocopium. In the no-choice Cladocopium treatment at 27°C, mean acquisition was 56% on day 3, which increased to 100% after 14 days. A similar pattern occurred at 30°C, however, at 31°C, only 30% of larvae established symbioses by day 14.
To understand how infection dynamics change under competition, we compared acquisition success for each species in no-choice against their performance in the four-way choice ( Figure S3). Acquisition success was not influenced by competition in any temperature treatments or symbiont species (all species p > .05; Table S5).

| Symbiont proliferation: How does symbiont cell proliferation in larvae differ with temperature?
There were significant effects of symbiont choice treatment (no-choices or four-way choice), temperature, time, and their interactions on symbiont cell densities in larvae that had acquired symbionts (p ≤ .01; Table 2 and Table S6). Temperature did not affect cell densities within any symbiont choice treatment on day 3 (post hoc, p > .05; Figure S4). Between days 3 and 7, mean cell densi-  Figure S4). Interestingly, no-choice Gerakladium was the only treatment to display significant increases in cell density between days 3 and 7 at all temperature treatments (post hoc, p < .01; Figure S4).
To understand how symbiont choice influenced cell proliferation, we compared cell densities over the course of the experiment for each symbiont species between no-choice and four-way choice treatments across the range of temperatures (Table S7; Figure S5).
There were higher mean cell densities in no-choice Cladocopium at 27°C on day 7 and day 14 (post hoc, p < .01) compared to the fourway choice. There were also higher cell densities in the no-choice Durusdinium as compared to the four-way choice on day 7 at 27°C.
There was a significant interaction of choice and time (p < .01, Table S7) on cell proliferation in Fugacium and Gerakladium species.
Gerakladium was the only species that had higher cell densities on

| Relative Abundance of species and mixed assemblage dynamics
To understand the competitive dynamics of symbiont acquisition in mixed assemblages we examined the symbiont community compo-   Figure 5) and there was no difference in dispersion between groups (Table S10). There was a species-specific pattern of relative abundance (p < .01), which was further modulated by temperature (p < .01) and time (p < .01; Table 4 and Table S11).

| Larval survival: How did symbiont choice treatment and temperature influence larval survival over time?
Larval survivorship decreased over the course of the experiment (p < .01) with the lowest survivorship in larvae exposed to the high temperature treatments (p < .01; Table S12; Figure 6). Larvae in all symbiont treatments suffered >50% mortality by day 7 of the experiment and by day 14, there was <13% survivorship across all treatments (Figure 6). At 31°C on day 14, there was 100% mortality of larvae exposed to no-choice Durusdinium and the four-way choice treatments. Survivorship in the aposymbiotic control treatments was lower than or equal to survivorship in the symbiont acquisition treatments ( Figure 6, Table S13). The decrease in survivorship across symbiont choice treatments as well as the aposymbiotic control is consistent with other symbiont infection and larval studies that demonstrate high larval mortality (Graham et al., 2008;Randall & Szmant, 2009a;Schnitzler et al., 2012).  Figure 6). Here, larvae infected with heterologous Fugacium and Gerakladium species consistently displayed higher mean survivorship than those infected with Cladocopium and Durusdinium species, which are common symbionts of A. tenuis at the larval and juvenile stages (Little et al., 2004;Quigley, Alvarez Roa, et al., 2020;Quigley, Randall, et al., 2020;Yorifuji et al., 2017). Under warming oceans, symbionts normally present at low relative

| Larval survivorship and symbiont recognition
Summer heat waves occurring during the larval stage will further reduce larval stock and subsequently reduce recruitment (Randall & Szmant, 2009b). Our observations confirm that larvae are negatively impacted by ocean warming; after 2 weeks, elevated temperatures of 30°C moderately reduced survival, with severe mortality at 31°C.
These findings suggest that like adult and juvenile A. tenuis (Shitaoka et al., 2021;Yorifuji et al., 2017), larvae cannot tolerate elevated temperatures beyond 30°C (Shitaoka et al., 2021;Yorifuji et al., 2017). Furthermore, the majority of larvae in the four-way choice acquisition treatment were infected with a mixed symbiont community (≥3 species per larva) and had a higher mean survivorship after 1 week at 27 and 30°C than those from the no-choice Cladocopium or Durusdinium treatments.

Symbiont identity had the most influence on survivorship 1 week after inoculation at 27 and 30°C. Interestingly, larvae inoculated with
Fugacium under no-choice had the highest survival, but the lowest cell densities compared to all other treatments. It is possible that the combination of Fugacium's high thermal tolerance and low cell densities led to increased larval survivorship, because adult coral colonies with lower symbiont densities have also been observed to have lower bleaching susceptibility (Cunning & Baker, 2012). Other studies have shown increased mortality in symbiotic as compared to aposymbiotic larva at both ambient (Hartmann et al., 2018) and elevated temperatures (Hartmann et al., 2018;Schnitzler et al., 2012), which could be attributed to an increase in oxidative stress (Hartmann et al., 2018), but this may come with other developmental costs (i.e., reduced settlement; Schwarz et al., 1999). Here, we found that survival was not reduced with acquisition of Cladocopium and Durusdinium symbionts, but there was a trade-off between acquisition and survival in larvae infected with rare symbionts. Previous work has demonstrated the complexity in the relationship between symbiont acquisition and coral fitness in A. tenuis; for example, Abrego et al. (2008) found that inoculation with Cladocopium resulted in higher thermal tolerance while others (Yorifuji et al., 2017;Yuyama & Higuchi, 2014) found that Durusdinium conferred enhanced tolerance. However, there is evidence suggesting that the A. tenuis population studied in Abrego et al. (2008) from Magnetic Island, GBR, could be a distinct species (Cooke et al., 2020). Therefore, it is possible that associations with other symbiont species or combinations of symbiont species may be more beneficial at the larval life stage.
Acropora tenuis larvae exhibit the capacity to host mixed symbiont assemblages (≥2 species) (Cumbo et al., 2013), and we provide evidence that A. tenuis can form symbioses that can include rare symbiont species in high proportions. Scleractinian corals, including A. tenuis, exhibit greater flexibility in algal partnerships in early life stages than adults (Abrego et al., 2009a;Gómez-Cabrera et al., 2008;Coffroth et al., 2001;Little et al., 2004). This flexibility has been linked to host selectivity, availability of a diversity of symbionts, environment factors, and symbiont opportunism during acquisition periods. However, the benefits of hosting different mixed assemblages may be variable (Howe-Kerr et al., 2020;Putnam et al., 2012).
For example, Quigley et al. (2016) found higher mortality in groups of A. tenuis juveniles with greater symbiont diversity after 25 days, and attributed this to both the low abundance of specific taxa (i.e., A3 and D1) and the negative consequences of random uptake, resulting in suboptimal taxa (i.e., F) that may outcompete higher performers. In contrast, our results in larvae show higher survivorship in the four-way choice treatment at 14 days, where larvae had high relative abundances of Fugacium and Gerakladium. This difference could be related to symbiont competition resulting in a trade-off in juveniles that may not occur in the comparatively shorter larval stage. Although survival benefits were small, these differences were significant and as climate change stressors increase, even marginal gains in survival are important to consider. Here, survivorship after 1 week is influenced the most, and as temperatures and time increase, this effect becomes smaller. However, even the small but significant differences, for example in no-choice Fugacium on day 7 at 30°C, could make the difference for more larvae surviving long enough to settle, which would need to be tested, along with if those symbionts are beneficial at the juvenile stage or not. With the growing trend of annual bleaching on the reef, and the huge impact those events are having on recruitment , differences of 5%-10% could make difference or provide a conservation strategy, if also found to be beneficial at later life stages, for those working in management. The winnowing period for A. tenuis closes on the scale of days (Bay et al., 2011) to years (Abrego et al., 2009a;Gómez-Cabrera et al., 2008;Rodriguez-Lanetty et al., 2006). Host requirements may best be met by a mixed assemblage of symbionts during the larval stage that then may be winnowed out as a strategy to maintain the species that best match the environment and host genotype downstream. However, it is important to note that despite evidence for a longer length of the symbiont winnowing period observed in A. tenuis juveniles, it is possible that the long aposymbiotic larval duration here could influence symbiont specificity, although there are currently no studies testing whether specificity changes over time in larvae.

| Background symbionts may be more competitive in warming oceans, if available
Gerakladium was a successful and common symbiont partner when available to A. tenuis larvae in a laboratory setting. Gerakladium (subclade G3: MH229354.1) is not a known dominant symbiont in any reef-building coral, and therefore, less is known about its functional traits in hospite (e.g., nutritional translocation, micronutrient requirements). While Gerakladium is found predominantly in symbiosis with sponges (Strehlow et al., 2021) and Foraminifera (Pochon & Gates, 2010), and has been observed in soft coral  and black coral (Bo et al., 2011), it has also been detected at background levels in symbiosis with a variety of reef-building corals (Boulotte et al., 2016;Chakravarti & van Oppen, 2018;LaJeunesse et al., 2010;van Oppen et al., 2005;Quigley et al., 2017;Stat et al., 2013;Thomas et al., 2014). Gerakladium is also found in the water column Fujise et al., 2020) and in sediments Fujise et al., 2020) and on macroalgae (Fujise et al., 2020), suggesting it could be an available partner for A. tenuis larvae on the GBR; however, it is usually present at low abundances (including G3;Quigley, Alvarez Roa, et al., 2020;Quigley, Randall, et al., 2020). However, selectivity is not related to symbiont abundance in the environmental pool (Quigley et al., 2017). A study in the Central West Coast of India, which experienced multiple years of bleaching temperatures, found that despite the symbiont population in the surrounding water containing high abundances of both Durusdinium and Gerakladium, corals had higher relative abundances of Durusdinium (Mote et al., 2021). Despite symbiont assemblages in corals being dominated by Durusdinium, Gerakladium was also present in higher abundances (10.2 ± 11.7%) than typically found as a background symbiont (Mote et al., 2021). The growing number of observations of Gerakladium as a background coral symbiont in a variety of coral species could be a result of increased sampling and molecular methods, or due to an increase in the frequency Gerakladium is taken up by corals that might relate to environmental change or stress. Continued monitoring of symbiont assemblages at all life stages, especially on highly impacted reefs, will be necessary to elucidate the degree to which corals are associating with more rare symbiont types.
Like Gerakladium, Fugacium is not a common dominant coral symbiont. It is predominantly found in symbiosis with Foraminifera (Pochon et al., 2001) and has also been reported at background levels in reef-building corals (De Palmas et al., 2015;LaJeunesse, 2001;Rodriguez-Lanetty et al., 2003), and only as the dominant symbiont in A. japonica, which is found in temperate environments (De Palmas et al., 2015;LaJeunesse, 2001;Rodriguez-Lanetty et al., 2003). It has been detected in very low abundance on the GBR (F1; Quigley, Alvarez Roa, et al., 2020;Quigley, Randall, et al., 2020 Durusdinium . In addition, cell surface recognition mechanisms, such as a glycan-lectin interactions, could limit the uptake of specific symbiont types (Kuniya et al., 2015;Wood-Charlson et al., 2006) or their persistence in the symbiosis .
There is evidence that smaller symbionts are more readily acquired than larger ones, and therefore size differences between symbiont species here could have contributed to differences in  (Hume et al., 2016). Coral symbiont studies have been predominantly conducted in adults and juveniles and it is difficult to assess symbiont acquisition at the larval stage in the field. Further compounding this is whether symbionts acquired at the larval stage are retained into the juvenile stage, when they would more likely be observed. Given that A. tenuis may switch from a mixed genera symbiont community at the juvenile stage to a single genus as adults (Abrego et al., 2009a), it is also possible that symbionts acquired at the larval stage differ from those in juveniles. While we may observe selection for thermotolerant homologous symbionts, we cannot rule out the possibility for heterologous symbionts to become more beneficial. Fugacium and Gerakladium have been found in environmental pools and, critically, our findings show that A. tenuis larvae can acquire these symbionts at high densities even in the presence of the homologous Cladocopium and another common heterologous symbiont (Durusdinium). This supports our hypothesis that shifts toward rare or background symbionts may occur in coral larvae under future climate change conditions and warrants further study.

| Optimizing symbiosis manipulation strategies for assisted evolution
Symbiosis manipulation has been suggested as a possible mechanism of human assisted evolution to aid in coral restoration practices under increasingly warmer conditions (van Oppen et al., 2015). If heterologous symbionts have the capacity to confer higher thermal tolerance than homologous partners, they may be beneficial partners given our rapidly warming climate. Furthermore, inoculating larvae with Fugacium and Gerakladium may provide phenotypic benefits, even over short periods of time if the symbionts will be replaced by homologous symbionts at a later stage, which has relevance to reef restoration initiatives using sexually produced coral stock. In this context, both Fugacium and Gerakladium are worth consideration for enhancing climate resilience of coral early life stages in interventions that depend on the rearing of sexually produced coral stock, however, caution should be taken, and further experiments considered before any field implementation occurs. In the present study, there was higher survivorship of larvae infected with Fugacium and Gerakladium (Fugacium > Gerakladium) and in larvae offered a four-way symbiont choice. Our observations at the larval stage point to a new direction in evaluating approaches to symbiont optimization strategies. Acropora larvae typically become competent to settle after ~5 days (Graham et al., 2013;Harii et al., 2007). Therefore, survival rates measured at 1 week are ecologically relevant as larvae that have not been swept off the reef or have been transported to another reef on ocean currents will have attached to the substratum and metamorphosed into a coral primary polyp. The impacts on survivorship here were small, but significant. Increasing survivorship under moderately elevated temperatures can enhance the pool of larvae available for settlement and recruitment, which is becoming more important as recruit stocks have shown signs of severe decline in the wake of repeat bleaching events .
Our results warrant further research to examine whether symbioses with these species can be established at a higher frequency in the laboratory, whether they are temporally stable, and phenotypic implications beyond the larval stage. Considerations should include, first, the impact of mixed assemblages on larval settlement and post-settlement survival. Second, further research should examine whether winnowing will remove these partners from the assemblage or, alternatively, whether symbiosis with heterologous symbionts, such as Gerakladium, could persist into the adult stage. Third, it is important to consider nutritional properties of the symbiont and whether there is a trade-off between thermal tolerance and capacity to meet the host's energetic needs. Together, these considerations will improve our understanding of the role of currently unusual symbionts in driving host thermal tolerance and fitness. Because both of the strains of Fugacium and Gerakladium tested here were isolated from cnidarians from the GBR and are present on the GBR (Fujise et al., 2020), this form of assisted evolution would not introduce foreign symbionts to the environment.

ACKNOWLEDGMENTS
We are grateful to Carlos Alvarez Roa for his support at the Symbiont

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data and R scripts can be found in Zenodo at doi: https://zenodo. org/badge/ lates tdoi/22756 2725