Using naturally occurring climate resilient corals to construct bleaching-resistant nurseries

Heat Tolerance, Microclimate, Symbiont, and Biomarkers Across Species.

To evaluate heat stress performance of parent colonies, we used a standardized stress assay (20), and measured chlorophyll loss of heated branches compared with controls in small, computer controlled temperature tanks. We denote the reaction to this experimental stress as “heat tolerance” and the response to naturally experienced heating experienced later in the study as “natural bleaching.” To determine species-specific maximum stress temperatures, we tested all species at half-degree intervals from 32 °C to 37 °C. We then determined the temperature at which 50% of the bleaching response occurred (denoted T50; 34 °C for A. hyacinthus, 35 °C for A. gemmifera and P. damicornis, and 36 °C for P. cylindrica). We then tested four fragments from each of the 20 parental colonies for each species in August 2014 for heat tolerance at the T50 temperature, compared with four paired fragments in the control tank. As expected, P. cylindrica showed the greatest heat tolerance in this rapid test: fragments ranged from losing 50% of their original chlorophyll to losing none (Fig. 1, Lower), despite experiencing the warmest experimental heating temperature. The other three species showed lower levels of heat tolerance but also showed marked variation among parental colonies (Fig. 1, Lower). The five least tolerant parent colonies lost two to three times more chlorophyll than the five most tolerant colonies.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 1.

(Upper) Forty corals from the HVP (red border) and 40 from the MVP (orange border) colored by time spent above 31 °C from December 2014 to April 2015 (austral summer). (Lower) Thermal tolerance (relative chlorophyll retained after a single day, species-specific heat stress) tends to be higher in corals from the HVP. Corals are arranged in ascending order of chlorophyll retention for the four species. Error bars represent SD across four paired replicates per colony.

Individual temperature loggers showed that parent colonies ranged from spending 2.4–11.3% of time above 31 °C (Fig. 1, Upper). Colonies from a warmer section of the back reef [the highly variable pool (HVP)] spent on average 8% of time above 31 °C compared with 4% of the time for colonies from a less variable section [the moderately variable pool (MVP)]. Across all colonies, time spent above 31 °C was also highly correlated to time spent above 32 °C to 34 °C (R² > 0.85). Heat tolerance was higher for colonies that originated in the HVP versus the MVP (Fig. 1, Lower). For example, of the five most heat-tolerant parent colonies for all four species, 75% originated from the HVP (binomial probability, P = 0.0206). Heat tolerance of a parent colony was also positively correlated to time that colony spent above 31 °C for all species, but not significantly for any one species.

Parent colonies of P. cylindrica only hosted genus Cladocopium symbionts. We tested for subtypes of symbionts by mapping transcriptomes to known ITS2 regions. The species A. gemmifera, and A. hyacinthus hosted symbiont species Cladocopium C3k and Durusdinium trenchii (formerly clade D1): P. damicornis, hosted Cladocopium C1d and D. trenchii, though all colonies tended to be dominated by one type or the other (Dataset S1). Within genera of corals, we found no evidence of multiple subclades in either Cladocopium or Durusdinium in any parent. Nearly half of the colonies for all three Acropora and Pocillopora species were dominated by less heat resilient Cladocopium clones. Overall 70% of the corals with the highest heat tolerance hosted the heat-tolerant symbiont D. trenchii rather than clones from genus Cladocopium (binomial P = 0.0214). We also mapped transcriptome data against full symbiont genomes and found 576 SNPs within the Acropora and Pocillopora parental colonies. These SNPs confirmed the distinctions between species Cladocopium C3k and Cladocopium C1d subclades, but they also suggested hidden variation in genus Durusdinium between corals that did not appear in the ITS2 data (see Materials and Methods). This approach may open up the low-diversity D. trenchii to more fine scale genetic analysis, suggesting more research is necessary to establish genera-specific associations with D. trenchii.

For A. hyacinthus and A. gemmifera, we used de novo transcriptomes to map published biomarkers that have been shown to be associated with thermal tolerance in coral for each parent. A subset of the Bay and Palumbi (11) candidate heat-tolerance alleles was tested for the species in which they were characterized, A. hyacinthus, and the congener A. gemmifera. We evaluated the proportion of the more tolerant alleles found across these sites for each parent colony. For A. hyacinthus, parents ranged from hosting 13–59% of the more tolerant alleles and in A. gemmifera, parents ranged from hosting 87–96% of the more tolerant alleles. There was a significant positive correlation between the density of tolerant alleles and measured heat tolerance among A. hyacinthus colonies, but this relationship was dominated by the differences in allele proportions between pools. For coral parent colonies compared within pools, we found no significant correlations between alleles and heat tolerance. For A. gemmifera, the high level of tolerant alleles correlates with higher heat tolerance in this species compared with A. hyacinthus, but there was low statistical power to detect trends within A. gemmifera. Jin et al. (13) identified the C70S236 marker to be correlated with bleaching tolerance in the Australian A. millepora. Our tests showed that this marker was homozygous for the more tolerant allele in nearly all parent colonies in Samoan A. hyacinthus and A. gemmifera.

Growth and Survival in Common Garden Before Bleaching.

In the common garden nursery, we measured growth by buoyant weight of each replicate nubbin from August 2014 to April 2015, when the first signs of bleaching occurred. Species differed in relative growth rate, with the branching Acropora growing by three- to fivefold during this period compared with two- to threefold growth in P. cylindrica (SI Appendix, Fig. S1A). The lowest net growth was recorded for P. damicornis but this was due to consistent predation of branches by resident corallivores.

Pool of origin was an important predictor of growth for two species. In P. cylindrica, nubbins from parent colonies grew more slowly when they were from the warmer HVP (88% vs. 114% growth, P < 0.0001, t test). The opposite trend was seen in A. hyacinthus, where nubbins from parent colonies grew more slowly when they were from the less variable MVP (144% vs. 188% growth, P = 0.007, t test). Origin was not a significant factor in growth of A. gemmifera or P. damicornis during this prebleaching period.

Symbiont genus played a significant role in growth for A. hyacinthus (ANOVA P = 0.004): colonies hosting primarily D. trenchii symbionts and switchers tended to grow faster than those hosting primarily genus Cladocopium. Colonies of A. hyacinthus with D. trenchii grew by 295% compared with 249% for those with Cladocopium. Though we saw the same relationship in A. gemmifera (274% growth with Durusdinium vs. 225% with Cladocopium) this difference was not significant, largely because of the small number of colonies hosting Cladocopium. By contrast, in P. damicornis, colony growth was higher for colonies with Cladocopium symbionts (188% vs. 156%, ANOVA P = 0.0007). Symbionts were not variable in P. cylindrica.

For all species but P. cylindrica, identity of the parent colony played a very strong role in determining overall growth rate. There was about a twofold difference in growth across the 20 colonies of each Acropora species, with relatively low variance among growth rates of the 10 replicates of each colony (average coefficient of variation ∼25% of the mean, SI Appendix, Fig. S1A). As a result, a significant proportion of the variance in growth among clones in the nursery was explained by parent identity (ANOVA P < 7e-10).

We found that survival was most affected by conditions of the transplant site. In particular, predation by small benthic reef fish at the transplant site in Sili Reef caused the most mortality, particularly in P. damicornis and A. hyacinthus (SI Appendix, Fig. S1B). By April 2015, survival was 94% for A. gemmifera, 97% for P. cylindrica, 67% for A. hyacinthus, and 54% for P. damicornis.

Bleaching and Recovery in the 2015 and 2017 Events.

Eight months after the establishment of the nursery, the 2015 El Niño brought temperatures reaching above 35 °C to back reef areas of American Samoa, causing bleaching in both the parental populations and the nursery nubbins (Figs. 2 and 3). Bleaching occurred again in 2017, 31 mo after establishment (SI Appendix, Fig. S2). Monitoring 10 nubbins per parent allowed us to evaluate the way replicate nubbins from different parents reacted to a natural bleaching event. In both events, bleaching was variable within species, allowing us to test the value of our proxies of thermal tolerance, origin, biomarkers, and microclimate measured for parent colonies to predict nursery results.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 2.

(A) Average temperature in the HVP, the MVP, and the transplant site in Sili with observed natural bleaching marked in the red box. The gray line marks the expected threshold bleaching temperature. (B) Visual bleaching severity of nubbins in the nursery partitioned using the three simplest proxies for predicted resilience. Labels on the predicted resilient or vulnerable stocks axis are as follows: (A, in blue lettering) corals from the HVP; (B, in blue lettering) top 10 experimental stress performers; (C in blue lettering) hottest extreme microclimates and the corresponding predicted vulnerable stocks: (a) corals from the MVP; (b) bottom 10 experimental stress performers; and (c) coolest extreme microclimates.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 3.

Correlations between the average bleaching severity of replicate nursery nubbins in Sili versus the average bleaching severity of their parent genotypes in their original habitats. Photograph demonstrates severely bleached A. gemmifera next to nonbleached nubbins.

After the 2015 bleaching event, visual bleaching of nubbins was less severe for P. cylindrica (average bleaching was 12%), moderate for P. damicornis (32%), and severe for A. hyacinthus (39%) and A. gemmifera (43%). However, there was also a great deal of variation within species. Even after 8 mo of growth side by side in common garden settings, bleaching of nubbins was 1.9–2.5 times higher when they originated from parents in the MVP compared with the HVP (Figs. 2B and 4, two-way ANOVA F = 67.1, P = 10⁻¹²). This result shows that common garden growth of 200–500% and 8 mo of acclimatization did not erase the differences in heat tolerance among parent colonies.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 4.

Average percent bleaching across four species in the Sili nursery after the 2015 natural bleaching event. Bleaching of nubbins that came from parents from the HVP (red) was two- to threefold lower than for nubbins with parents from the MVP (orange).

Because we have data on three bleached species, from 10 parental colonies from each of two pools, and 10 replicates each, we tested how much variation in bleaching is partitioned by parent, origin, and symbiont. Different colonies, even from the same pool, had nubbins that bleached very differently than those of other colonies (Fig. 4, two-way ANOVA, F = 21.9, P = 10⁻¹⁸). For example, A. hyacinthus colony SU03 had nubbins that bleached 80–100% (mean 92%, n = 10), whereas conspecific SU08 had nubbins that bleached only 20–40% (mean 23%, n = 8). These colonies were from the same pool but had different symbionts (SI Appendix, Fig. S1A).

To tease apart the relationship between pool of origin, symbiont, and nubbin bleaching, we used both general linear models (GLMs) and multiple linear regression. For both analyses (implemented in R), pool of origin was a significant factor in bleaching after accounting for symbiont (SI Appendix, Tables S1 and S2). Symbiont was separately significant in the multiple linear regression but only marginally so in the general linear model analysis. These results suggest that bleaching in the nursery was affected by both the pool of origin of the parent and to a slightly lesser degree by the parent’s symbiont type: the combination of these effects explains much of the complex variation of nubbins in their bleaching patterns (SI Appendix, Fig. S1A).

This variation was consistent with heat resistance we previously measured for these parents. Bleaching of nubbins in the common garden in 2015 was higher when they were derived from parents with higher heat tolerance. The correlation was significant for the two Acropora species (P = 0.006 and P = 0.032) and was in the same direction but nonsignificant for P. damicornis (P = 0.18) (SI Appendix, Fig. S3).

Similar results were recorded after the 2017 bleaching event. The two Acropora species bleached, whereas P. cylindrica did not (predation-based mortality between 2015 and 2017 was too high to score bleaching in P. damicornis). In both Acropora species, bleaching was less severe for parents from the HVP than the MVP, and for parents scored as more heat resistant in 2014 (SI Appendix, Fig. S2). This second event was less drastic in A. gemmifera than the first bleaching.

Parent Versus Nursery Bleaching.

In addition, by monitoring parents during the bleaching event, we could compare bleaching in nursery nubbins and parents. Even though they experienced high water temperatures on different reefs in different months, clones and corresponding nursery replicates showed a strong and significant correlation in the 2015 bleaching for the three species that bleached (Fig. 3; from Pearson’s correlation: P. damicornis P = 8.89e-5, A. gemmifera P = 8.82e-8, A. hyacinthus P = 4.14e-4; P. cylindrica colonies did not bleach in their original environments). For example, A. gemmifera clone GE_J bleached 80% in its native habitat and the nursery replicates from this colony bleached an average of 94%. By contrast, GE_C did not bleach in its native habitat and none of its replicates in the transplant bleached more than 20%. The strong correlation from clone to corresponding nursery replicates and low SD (<10%) across nubbins indicate stability in the bleaching response.

Acclimatization in Common Garden Settings.

Acclimatization to overall lower heat tolerance was also apparent in our study. The bleaching-inducing temperatures experienced in the nursery site were relatively mild compared with the environmental history experienced by parent colonies (SI Appendix, Fig. S4). Nevertheless, nursery replicates at Sili bleached more (average 11%) than their parents did in their original habitat. Despite this small loss of bleaching tolerance in a milder environment (SI Appendix, Fig. S3), the rank order of bleaching remained highly correlated between clones in their home environment and corresponding nursery replicates (Fig. 3).

Symbiont Switching During Bleaching.

For the two Acropora species, we observed symbiont shifts to occur in 11 of the 40 parent colonies, all shifting from Cladocopium-dominated to Durusdinium-dominated symbiont populations. For nubbins, lower bleaching was seen for parent colonies that switched to genus Durusdinium (52% vs. 88% for A. gemmifera P = 0.01, 16% vs. 80% for A. hyacinthus, P = 0.008) than for parents that retained Cladocopium.

Variation in symbionts allowed a limited test of the expected tradeoff between increased growth in corals with genus Cladocopium and increased heat tolerance in corals with genus Durusdinium (21). Transplant corals from the MVP with genus Durusdinium symbionts bleached less than the MVP transplants with genus Cladocopium symbionts. In addition, clones that switched from genus Cladocopium to genus Durusdinium showed much less bleaching than colonies that started with genus Cladocopium and did not switch (Fig. 4). However, the impact of symbiont type on growth was negligible. Within the MVP, parent colonies with genus Cladocopium did not produce nubbins that grew more quickly than those with genus Durusdinium, as might be expected if the tradeoff between growth and bleaching were strong (15).

Correlations Between Bleaching and Simple Predictive Proxies.

All of our simple environmental and physiological proxies allowed the identification of parental corals producing nubbins that bleached substantially less in both the 2015 and 2017 bleaching events (Fig. 2 and SI Appendix, Fig. S2). In the 2015 event, all three environmental and physiological proxies significantly explained the bleaching response (linear model P values <0.01). Origin (HVP vs. MVP) and symbiont were the proxies that performed best, explaining 21% of the response. Microhabitat and stress performance explained 11% and 8%, respectively after removing the effect of origin. Biomarkers proved effective at predicting tolerance for the species in which they were characterized (A. hyacinthus) (SI Appendix, Fig. S5), but were not effective in other species.

Limited Tradeoffs in Growth in a Resilient Nursery.

Host-driven bleaching tolerance is predicted to come with potential tradeoffs (22). For P. cylindrica, the parents that bleached less tended to grow at slower rates under prebleaching conditions (August 2014–December 2014), as expected by tradeoff predictions [Pearson’s P = 0.005, correlation coefficient (cor) = 0.6]. In the period after bleaching (April 2015–August 2015), this tradeoff persisted for P. cylindrica (Pearson’s P = 0.008, cor = 0.6). For A. gemmifera, a similar but weaker trend was also seen (Pearson’s P = 0.05, cor = 0.4), and was largely explained by high growth and high bleaching for colonies from the MVP. However, in the period after bleaching, the trend reversed and coral from more tolerant parents grew faster than coral from less tolerant parents, dominated by low growth in those that were bleached (Pearson’s P = 0.02, cor = −0.5). By contrast, we did not see significant trends in A. hyacinthus or P. damicornis between growth and bleaching.

Diversity in Nurseries Before and After Bleaching.

We constructed our nurseries with an even mix of species and colonies, so they began the experiment with high Shannon–Weaver diversity [measured as H′ = ∑p_i*ln(p_i) based on the average size of nubbins and the number surviving across the 80 parents]. Predation on P. damicornis, larger growth of P. cylindrica, and A. gemmifera, and differential bleaching among species and clones led to a decline in overall species and clonal diversity over the course of the experiment (H′ = 4.37 → 4.13 out of a maximum of 4.38). However, the decline was less among the HVP clones (4.31 → 4.20) than among the MVP clones (4.41 → 4.05). In particular, diversity declined among the MVP clones after the 2015 bleaching event (4.29 → 4.05) but dropped much less among the HVP clones (4.23 → 4.21).