Increasingly frequent and severe thermal stress events are the predominant threat to reef ecosystems in the Anthropocene. Thermal stress perturbs the photosynthetic mechanism and can result in loss or expulsion of the dinoflagellate algae in the process known as bleaching [1]. While persistent bleaching can result in coral death due to the loss of autotrophic carbon contributed by the symbionts [2], bleaching can also precipitate an acclimatory change in the composition of the endosymbiont community that can subsequently increase coral thermal tolerance [3, 4]. For coral hosting multiple symbiont types, changes in their relative abundance, or “shuffling”, can facilitate survival and recovery [3]. The ability for adult coral to acquire completely new symbiont types following bleaching was also recently confirmed [5]. Although this capacity for flexibility is not universal [6], is influenced by environmental cues, and physiological trade-offs [7], symbiont shuffling and/or switching has been proposed as an important target for human-assisted evolution [8]. Successful establishment of novel symbioses that increase bleaching tolerance have been demonstrated in laboratory conditions [9]; however, it is unclear how long these heterologous relationships persist, especially in natural reef environments. In an essential test of the utility of this approach, we investigate the long-term stability of a heterologous coral symbiosis in the context of reef restoration.

Acropora palmata were once one of the dominant habitat builders of shallow Caribbean reefs [10]. Unprecedented demographic declines since the mid-1970’s have resulted in extensive propagation efforts in both land and ocean-based nursery systems to maintain existing genetic diversity and produce biomass for outplanting efforts aimed at restoring ecosystem structure and function [11]. Naturally occurring populations of A. palmata are highly specific in their symbiosis with Symbiodinium fitti, formerly ITS2-type A3 [12, 13]. Single nucleotide polymorphism genotyping of ex situ nursery-reared lines of A. palmata, and work by Gantt et al. (in press) [14] revealed the dominance of Durusdinium spp. and presence of Cladocopium in Mote A. palmata genotypes (Fig. S1A, Supplementary Methods, GenBank Accessions: ON455821–ON455829) [14]. This provided a unique opportunity to test the maintenance of this heterologous symbiotic relationship after outplanting back to the reef environment.

In April 2018, we transplanted three clonal replicates (ramets) of five exclusively ex situ nursery-reared A. palmata genotypes to nine reefs in the lower Florida Keys (n = 27 ramets per genet) to evaluate the long-term stability of the Durusdinium association on natural reefs using periodic phenotypic monitoring and ITS2 amplicon sequencing for symbiont typing using the SymPortal pipeline [15] (Supplementary Methods, Fig. 1). A random sub-sampling of 10–12 ramets per genet confirmed that no Symbiodinium fitti were detectable prior to outplanting in most genotypes (Supplementary Results, Supplementary Table 3 and Supplementary Fig. 2 and Fig. 2A). Four genets were exclusively dominated by a Durusdinium ITS2 type, whereas genet 13-XK showed consistent co-infection with a Cladocopium ITS2 type composed of three co-occurring C1 amplicon sequence variants at T0 (Fig. 2A). Four sequence variants (0.1% of reads) matching to an S. fitti type were identified in one ramet of genet 13-XK at T0 (Supplementary Table 3). An ordination of T0 samples confirmed a significant effect of genotype (p < 0.001) with 13-XK driving divergence (Fig. 2B).

Fig. 1: Experimental setup showing genotypes subsampled at time point 0 (T0), the locations of outplanting sites, the locations of water quality monitoring stations, and the genotype array strategy at each site.
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All surviving corals were sampled at time point 24 (T24).

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Fig. 2: Relative abundance and principal coordinates analysis (PCoA) plots showing that symbiont community did not significantly change after 2 years of outplanting.
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A The relative abundance plot shows the ITS2 symbiont profiles of each individual coral grouped by genotype that was subsampled at T0. Each bar represents an individual and each is labeled with either their ex situ nursery ID or their outplant site ID. Color represents symbiont type. B The relative abundance plot shows the ITS2 symbiont profiles of each individual coral grouped by genotype that was sampled at T24. Each bar represents an individual. Samples are grouped by site, then genotype, and then frag id. Asterisks (*) identify individuals that were also sampled at T0. Color represents symbiont type. C A PCoA plot that shows that symbiont profiles did not significantly change after 2 years of being outplanted. Color represents coral genotype and shape represents site. The ex situ nursery and T0 is included as a site.

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No bleaching was observed during quarterly site visits, but site-specific mortality was evident, with 100% ramet mortality at Bahia Honda following 18 months (Supplementary Methods, Table S2). At the remaining eight sites, Durusdinium remained the dominant ITS2 profile after 2 years of outplanting (T24, Fig. 2B, C). Surviving co-infected 13-XK ramets also shuffled to Durusdinium dominance across sites (Fig. 2C) and a PERMANOVA confirmed genotype was no longer a significant factor (p = 0.764). Novel symbiont types were detected at T24, with site-specific patterns (PERMANOVA, p < 0.031, Fig. 2C, Supplementary Table 3 and Supplementary Fig. 3). The homologous symbiont Symbiodinium fitti (A3) was observed at four sites, with ITS2-type profiles indicating potential sub-species or strain-level differences between Dave’s Ledge, Eastern Dry Rocks, Looe Key, and Maryland Shoals (Fig. 2C and Supplementary Table 3). Two ramets of 13-X7 acquired Cladocopium types which were unobserved at T0, including one apparently novel strain (C66) although their relative abundance remained low (Fig. 2C and Supplementary Table 3). A Gerakladium (G) type profile was also detected in ramets of two different genets at two different sites, also at low relative abundance (Fig. 2C and Supplementary Table 3).

The general long-term stability of the heterologous symbiosis between A. palmata and Durusdinium spp. on natural reefs supports the potential effectiveness of symbiont modification as a management intervention, although the ethical implications and long-term ramifications remain to be considered. Infection of coral larvae from mass spawning events with thermally tolerant symbionts and outplanting them to degraded reefs could also be a restoration tool [16]. However, the occasional (re)acquisition of homologous, site-specific strains of Symbiodinium fitti indicates that the temporal dynamics of symbiosis establishment in coral may be more complicated than previously thought. While corals can uptake symbionts in their larval phase, the current consensus is that the majority of symbionts are acquired after settlement. New recruits and juvenile corals can form a variety of novel symbiont associations [17], but this diversity is generally pruned to one numerically dominant taxon per genus over the first few years of life [18]. Consensus suggests that changes to the dominant symbiont taxon in adult coral occur in response to bleaching-level stress with reversion to the dominant symbiont profile in the absence of bleaching stress [3, 5, 19].

The A. palmata used in this study were produced using individual larvae from batch crosses in 2013/14. Although physically small, at the time of outplanting, experimental ramets were 4–5 years old, which under field conditions is the age of sexual maturity for this species [20]. The finding that adult corals can shuffle or switch their symbionts in situ in the absence of bleaching suggests that acquisition and winnowing may be dependent on other factors rather than a simple age-dependent process. It is also possible that Symbiodiniaceae communities are indeed capable of greater flexibility under ambient conditions [21]. However, the stability of the heterologous symbiosis in most individuals suggests that infection with a novel symbiont type as a recruit and maintenance till maturity could produce a stable symbiosis.