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10.1371/journal.pone.0309719
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Assisted sexual coral recruits show high thermal tolerance to the 2023 Caribbean mass bleaching event
Thermal tolerance in coral assisted recruits
https://orcid.org/0000-0003-0883-5064
Miller Margaret W. Conceptualization Data curation Formal analysis Project administration Writing – original draft 1 *
Mendoza Quiroz Sandra Conceptualization Data curation Investigation Writing – review & editing 1 2
Lachs Liam Data curation Formal analysis Writing – review & editing 3
https://orcid.org/0000-0002-6667-3983
Banaszak Anastazia T. Conceptualization Project administration Resources Writing – review & editing 2
Chamberland Valérie F. Investigation Writing – review & editing 1 4 5
Guest James R. Methodology Resources Writing – review & editing 3
https://orcid.org/0000-0002-9810-9850
Gutting Alexandra N. Investigation Writing – review & editing 6
Latijnhouwers Kelly R. W. Investigation Writing – review & editing 1 4 5
Sellares-Blasco Rita I. Resources Supervision Writing – review & editing 7
https://orcid.org/0009-0006-7222-6238
Virdis Francesca Investigation Resources Writing – review & editing 8
Villalpando Maria F. Data curation Investigation Writing – review & editing 7
Petersen Dirk Conceptualization Funding acquisition Writing – review & editing 1
1 SECORE International, Miami, FL, United States of America
2 Unidad Académica de Sistemas Arrecifales, Universidad Nacional Autónoma de México, Puerto Morelos, Quintana Roo, México
3 School of Natural and Environmental Sciences, Newcastle University, Newcastle upon Tyne, United Kingdom
4 CARMABI Foundation, Willemstad, Curaçao
5 Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands
6 The Nature Conservancy, St. Croix, US Virgin Islands
7 Fundación Dominicana de Estudios Marinos (FUNDEMAR), Bayahíbe, Dominican Republic
8 Reef Renewal Foundation Bonaire (RRFB), Bonaire, Caribbean Netherlands
Thuesen Erik V. Editor
Evergreen State College, UNITED STATES OF AMERICA
Competing Interests: The authors have declared that no competing interests exist.

* E-mail: m.miller@secore.org
18 9 2024
2024
19 9 e030971910 4 2024
16 8 2024
© 2024 Miller et al
2024
Miller et al
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Assisted sexual coral propagation, resulting in greater genet diversity via genetic recombination, has been hypothesized to lead to more adaptable and, hence, resilient restored populations compared to more common clonal techniques. Coral restoration efforts have resulted in substantial populations of ‘Assisted sexual Recruits’ (i.e., juvenile corals derived from assisted sexual reproduction; AR) of multiple species outplanted to reefs or held in in situ nurseries across many locations in the Caribbean. These AR populations provided context to evaluate their relative resilience compared to co-occurring coral populations during the 2023 marine heat wave of unprecedented duration and intensity that affected the entire Caribbean. Populations of six species of AR, most ranging in age from 1–4 years, were surveyed across five regions during the mass bleaching season in 2023 (Aug-Dec), alongside co-occurring groups of corals to compare prevalence of bleaching and related mortality. Comparison groups included conspecific adult colonies as available, but also the extant co-occurring coral assemblages in which conspecifics were rare or lacking, as well as small, propagated coral fragments. Assisted sexual recruits had significantly lower prevalence of bleaching impacts (overall pooled ~ 10%) than conspecific coral populations typically comprised of larger colonies (~ 60–100% depending on species). In addition, small corals derived from fragmentation (rather than sexual propagation) in two regions showed bleaching susceptibility intermediate between AR and wild adults. Overall, AR exhibited high bleaching resistance under heat stress exposure up to and exceeding Degree Heating Weeks of 20°C-weeks. As coral reefs throughout the globe are subject to increasingly frequent and intense marine heatwaves, restoration activities that include sexual reproduction and seeding can make an important contribution to sustain coral populations.

Builders Initiative 2022-4883 Petersen Dirk http://dx.doi.org/10.13039/100001780 Ocean Foundation CBF EbA2 #027 Petersen Dirk BMUV IKI Coral Carib 2023 Sellares-Blasco Rita I. http://dx.doi.org/10.13039/100007430 National Fish and Wildlife Foundation 0318.20.069532 CONAHCYT 425888 https://orcid.org/0000-0002-6667-3983
Banaszak Anastazia T. Quintana Roo State Government (MX) 2021 Z4 https://orcid.org/0000-0002-6667-3983
Banaszak Anastazia T. Funding support was received from The Builders Initiative (to DP;https://www.buildersinitiative.org/), the Caribbean Biodiversity Fund (to DP and RSB via the Ocean Foundation; https://caribbeanbiodiversityfund.org/), The National Fish and Wildlife Foundation (Award ID0318.20.069532 to J.Ward, The Nature Conservancy; nfwf.org), BMUV IKI Coral Carib 2023 (to RSB; https://www.international-climate-initiative.com/en/), Consejo Nacional de Humanidades, Ciencias y Tecnologías (Project #425888 to ATB; https://conahcyt.mx/), State government of Quintana Roo, Mexico, (Project # 2021 Z4 to ATB) These funders played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data AvailabilityAll relevant data are within the manuscript and its Supporting Information files.
Data Availability

All relevant data are within the manuscript and its Supporting Information files.
==== Body
pmcIntroduction

The rapid pace of coral population and species declines combined with the inadequacy of actions to address their root causes (e.g., greenhouse gas emissions and local pollution) is driving increased interest and investment in active coral restoration globally. While coral restoration alone is not a solution to the current coral crisis, it may ‘buy time’ for crucial efforts to address environmental degradation [1]. Asexual propagation (i.e., through fragmentation) has been widely utilized in fast-growing, branching species for nearly two decades as a means of rapidly producing corals for transplantation [2, 3]. In contrast, sexual propagation (or breeding) has only more recently been widely pursued due to additional challenges involving timing, multiple steps required and resultant need for additional expertise and facilities in this process [4, 5]. High natural fecundity of spawning corals provides great potential for upscaling while genetic recombination during sexual reproduction provides the expectation of higher adaptive potential to rapidly changing environments in resulting restored populations [6]. In the Caribbean, the major reef-building species (Acropora spp., Orbicella spp., and to a lesser extent, brain corals) are broadcast spawners whose natural sexual recruitment has remained poor to non-existent [7–10] and hence have long been key targets for breeding and assisted recruitment efforts [11–13].

Corals generally experience very high mortality rates in the period immediately after settlement, being particularly vulnerable to competition and predation [e.g., 14, 15], and highly susceptible to disease [16]. Meanwhile, previous work has suggested a general pattern that juvenile corals (often estimated simply as small colonies in wild populations) are less susceptible to heat-related bleaching. For example, Brandt [17] found large colonies of Colpophyllia natans were more susceptible to thermal bleaching, succumbing at an accumulated heat stress level of 3–4 Degree Heating Weeks (DHW, units°C-weeks; [18]) whereas small colonies did not bleach until 9–10°C-weeks. It has been hypothesized that juvenile corals may be more resistant to heat stress due to a number of mechanisms such as living in shaded environments, different mass transfer rates, or more flexible symbiont associations.

Throughout the Caribbean region, 2023 was a record-breaking year in terms of heat stress with accumulation in coral reef habitats of not just the highest on record (Fig 1), but in many cases four times the previous highest record and lasting for one to two months longer than normal seasonal patterns [19]. The relatively long duration of efforts toward coral restoration via breeding and assisted recruitment in this region [11–13] thus provided an opportunity to evaluate the resistance of sexually propagated juveniles under extreme levels of thermal stress. We sought to quantify the bleaching response of Assisted Recruits (AR; derived from coral restoration activities involving sexual propagation and seeding) of six reef-building coral species across five regions spanning the Caribbean basin, alongside that of various comparison groups of corals, including co-occurring ‘wild’ assemblages, conspecific corals at parental sites (from which gametes were collected), and small colonies propagated via fragmentation.

10.1371/journal.pone.0309719.g001 Fig 1 A) Satellite-derived estimates of heat stress exposure (Degree Heating Weeks, as of 1 Oct 2023; from https://coralreefwatch.noaa.gov/data/5km/v3.1_op/image/daily/dhw/png/2023/10/ct5km_dhw_v3.1_caribbean_20231001.png) across the Caribbean with the estimated maxima for the five study regions (MEX: Puerto Morelos, Mexico; DR: Bayahíbe, Dominican Republic; STX: St. Croix, US Virgin Islands; CUR: Curaçao; BON: Bonaire). B) Image of bleaching-resistant Assisted Recruit (red circle) adjacent to bleaching or recently dead adult conspecific (white circle) of Acropora palmata (Dominican Republic; photo M.Villalpando).

Materials & methods

Surveys were conducted of cohorts of assisted coral recruits created in five regions between 2011 and 2022 (most between 2019–2022), all of which were derived from coral restoration programs implementing sexual propagation and seeding (Table 1). In some regions, these cohorts consisted of individually tagged corals that were surveyed repeatedly as part of performance monitoring. In other cases, surveys were conducted in areas where unmarked AR had been outplanted haphazardly but were recognizable because they were attached to artificial settlement substrates. All were in areas where natural recruitment of these spawning species is rare or absent. Each colony was visually scored as having one of four conditions: ‘healthy’ coloration, ‘pale’, fully ‘bleached’ (appearing completely white over at least a portion of the live colony surface), or recently dead (whole-colony mortality with stark white skeleton, and/or recorded mortality of tagged individuals within the previous four months). Timing of surveys was different in each location, but was within four weeks of estimated peak heat stress for that location based on NOAA’s Coral Reef Watch satellite-derived heat stress estimates (https://coralreefwatch.noaa.gov/product/vs/data.php) as described in Table 1.

10.1371/journal.pone.0309719.t001 Table 1 Information for the surveyed Assisted Recruits (AR; derived from coral breeding efforts for restoration) and various comparison populations (WC: Wild conspecific adults at the same site as AR; PAR: Parental site conspecifics; WA: Wild adult assemblage at the same site as AR; WJ: Wild juvenile assemblage at same site as AR; F: small colonies derived from fragment (asexual) propagation) across the five regions (MEX: Puerto Morelos, Mexico; DR: Bayahibe, Dominican Republic; STX: St. Croix, US Virgin Islands; CUR: Curacao; BON: Bonaire).

Maximum Degree Heating Weeks (DHW, units°C-weeks) estimated from nearest NOAA Coral Reef Watch ‘virtual stations’, https://coralreefwatch.noaa.gov/product/vs/data.php). Ofav: Orbicella faveolata; Oann: O. annularis; Dlab: Diploria labyrinthiformis; Apal: Acropora palmata; Cnat: Colpophyllia natans; Pstr: Pseudodiploria strigosa.

Region/Site	Focal Species (AR/PAR)	Population	Depth (m)	Lat	Lon	Survey Date (2023)	Max DHW (Timing)	
MEX/Jardines	Ofav, Oann,
Dlab, Pstr	AR, WA, WJ
PAR(Dlab)	6	20.8314	-86.8745	23–31 Aug
26 Sep (WJ)	>25 (Sept/Oct)	
MEX/Acuario	Dlab	AR, WA, WJ	12	20.8059	-86.8790	19 Sept	
MEX/Bonanza	Dlab	PAR	9	20.9641	-86.8073	11 Aug	
MEX/Limones	Apal	PAR (2022)*	2.5	20.9885	-86.7972	30 Aug	
MEX/Bocana	Apal	PAR (2011–15)*	6	20.8740	-86.8513	25–30 Aug	
MEX/Picudas	Apal	AR (2011–15)*, WC	3	20.8833	-86.8483	30 Aug	
MEX/Punta Maroma	Apal	AR (2022)*, WA,WJ	3–5	20.7156	-86.9741	11–12 Sept	
STX/Channel Rock	Dlab,
Pstr	AR
AR, WC	8	17.7667	-64.5946	30 Nov
7 Nov	19 (Late Oct)	
STX/Cane Bay East	Dlab	PAR	7.3	17.7742
	-64.8116
	31 Oct	
STX/Deep End Beach	Pstr	PAR	4	17.7614	-64.6693
	2 Nov	
STX/Cane Bay West	Pstr	WC	10	17.7736	-64.8137
	31 Oct	
CUR/
CARMABI	Cnat	AR, WC
	5–10	12.1213	-68.9697	20 Nov
	22 (Nov)
	
BON/Buddy’s	Cnat
Ofav/Oann	AR
F	5	12.1712	-68.2891	28 Nov	22 (Nov)	
DR/CRIB site	Dlab	AR,WA,WJ	3–4	18.3715	-68.8478	4 Dec	23 (Late Oct/ early Nov)	
DR/Sombrero	Apal	AR,WA,WJ, F,	1–2	18.3706	-68.8467	4 Dec	
*Gametes for AR of Acropora palmata in MEX were collected at separate parental sites in different time frames

In each location, one or more ‘comparison’ populations were also surveyed, scoring each colony with the same aforementioned health status categories. Because of the rarity of conspecifics in many current Caribbean reef assemblages, we sampled a variety of different comparison populations of co-occurring corals among the different regions to maximize inclusion of conspecifics. In a few cases, ‘wild’ conspecific adults (WC) were present in the same reef site as the outplanted AR and were targeted for scoring via directed search. This was considered the most appropriate comparison when both species and macro-environmental exposure were matched at the same site. In other regions (Mexico and Dominican Republic) we conducted standardized surveys (AGRRA method as described in [20] or [21]) of the extant ‘wild’ adult (WA; > 4 cm diameter) and ‘wild’ juvenile (WJ; < 4cm) assemblages in the reef surrounding the outplanted AR. Four cm diameter was used as a demographic cutoff, consistent with widespread monitoring protocols (e.g., AGRRA, Lang et al. 2012; NOAA’s National Coral Reef Monitoring Program, https://ncrmp.coralreef.noaa.gov/pages/ncrmp-data#BenthicSection), but we acknowledge that different species (and different individual colonies) may mature at somewhat smaller or larger sizes. These surveys were intended to provide comparisons within the same macro-environmental exposure, but these assemblages were generally mis-matched to the AR species. In several cases, surveys of conspecific colonies at parental sites (PAR) were also undertaken; i.e., gamete collection sites from which the AR were bred, providing within-species comparison but at different sites (hence, different macro-environmental exposures). Lastly, in two regions, small colonies propagated by fragmentation (F) that were co-located with AR either in an in situ nursery (Bonaire, heterospecific comparison colonies) or within the same outplant site (Dominican Republic, conspecifics) were also surveyed.

All pooled comparison populations were compared to AR populations qualitatively (i.e. visually via stacked bar graphs). However, we restricted our statistical analyses to conspecific comparisons within the six species used for restoration (AR species listed in Table 1) to remove potential confounding effects of differential species susceptibility. That is, data were subsetted from all comparison population types to include only individuals of these six species (i.e., 303 observations of 1176 total Comparison observations). Statistical analyses were run to assess whether the severity of bleaching responses differed between AR versus conspecific colonies from Comparison populations (fixed factor with two levels, herein referred to as AR-Comp). We tested the effect of this fixed factor (Assisted Recruit vs. Comparison; AR-Comp) on the ordinal health status scores (0: healthy; 1: pale; 2: bleached; 3: recently dead) by species using a cumulative link model (CLM; ordinal regression, R-package: ordinal) in the form: Status ~ AR-Comp * Species, with species-specific effects computed using least-squares means.

Given the complexity of possible confounding factors in this dataset (multiple regions, sites, types of comparison population, and survey methods, with each factor combination being represented unequally by species), including them as random effects in the CLM was not compatible with the modelling framework. Therefore, we conducted two further statistical models to test if the CLM results were robust to possible confounding factors. First, we converted the ordinal health status response into a Bleaching and Mortality Index (BMI), with scores set to equidistant values between 0 (healthy) and 1 (recently dead), and ran a Generalized Linear Model (GLM) with a binomial error distribution equivalent to the CLM (form: BMI ~ AR-Comp * Species). The assumption of numerical equidistance between health status categories was tested by comparing the CLM and GLM results. Second, the species-specific effect of AR-Comp on BMI was tested using a Generalized Linear Mixed-effect Model (GLMM; R-package: glmmTMB) with binomial error distribution, accounting for differences among regions (5-level random effect), sites (14-level random effect), and comparison population types (6-level random effect) by fitting random intercepts for each (BMI ~ AR-Comp * Species + (1|Region) + (1|Site) + (1|Population.type)). Notably, the species that showed statistically significant difference in BMI between AR and other colonies (i.e. significant AR-Comp effect) in the CLM were also statistically significant in the GLM and GLMM, although with larger uncertainties (and hence higher P values, S1 Table). Therefore, all reported P values in the remainder of the manuscript refer to the GLMM. Lastly, we visually compared the frequency of bleaching states in the AR populations to subsets of the various comparison populations described above to explore specific hypotheses for increased thermal tolerance of AR.

Results and discussion

The pooled sample of all AR was over three times more likely to appear visually healthy (i.e., normal coloration) during the 2023 marine heatwave, with a 90% prevalence of healthy-colored AR (695/767), in comparison to only 24% (286/1176 colonies) in the pooled sample of all other colonies (Fig 2A). However, this summary pools unbalanced comparisons among species, sites (including likely differential macro-environmental exposure including thermal stress), and colony origin (e.g., fragment propagation vs. ‘wild’ colonies). Substantial differences are also apparent within species (Fig 2B), and confirmed by a generalized linear mixed effect model which does account for variability in bleaching due to possible confounding effects of region, site, and comparison population type. These GLMM analyses confirm that AR for each species except O. faveolata showed significantly greater resistance compared to conspecific Comparison corals (GLMM post-hoc comparisons, P values < 0.05, Fig 2C). O. faveolata AR do appear significantly more heat resistant in the CLM (ordinal) model (S1 Table). However, when accounting for additional confounding factors such as location, comparison population type, etc. in the GLMM, increased uncertainty due to limited observations (AR were from a single site in Mexico while available Comparison colonies were only Fragments from Bonaire) yields larger confidence intervals and hence lack of significance in the AR-Comp bleaching comparison (Fig 2C).

10.1371/journal.pone.0309719.g002 Fig 2 Bleaching and mortality responses for the six species propagated for restoration.

A) Cumulative colony condition for Assisted Recruits (AR; derived from coral breeding efforts for restoration) and all comparison population types pooled across sites. Sample size for each group is given above the stacked bars. B) Subsets of data from A) that are parsed by the six species for which AR existed. C) Differences in Bleaching and Mortality Index (BMI) between AR and conspecific comparison colonies. Estimates of the mean BMI (points) and SE (bars) are based on a binomial GLMM in the form: BMI ~ AR-Comp * Species, accounting for differences among regions, sites, and comparison population type (random intercepts). Statistical significance of pairwise comparisons by species are shown for P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).

It is worth noting that most (65%) of the comparison colonies that were scored in systematic surveys of coral assemblages surrounding AR outplants (in the Dominican Republic (2 sites) and Mexico (3 sites)) were comprised of distinct species (Porites spp, Agaricia spp, and Siderastrea siderea). The pooled frequency of colonies with bleaching signs in these extant assemblages was 78% for adults (>4 cm; 170/767) and 60% for juveniles (<4 cm; 58/144); so similar susceptibility for adults and higher susceptibility for juveniles than generally observed in the six AR species. The relative dearth of these six hermaphroditic spawning, reef building species, and the virtual absence of their natural recruits are compelling reasons that restoration is needed for these species; the increased bleaching resistance of AR compared to naturally occurring juveniles of primarily weedy species makes sexual propagation an important method of restoration.

One caveat relates to the potential artifact of comparing (whole colony) mortality between large and small colonies, especially in the context of multiple stressors. Small colonies (including AR) might succumb to lethal conditions (such as bleaching) more quickly than large colonies. Therefore, bleaching impact as whole-colony mortality of AR colonies might be underestimated (relative to larger, slower-dying colonies) in one-off surveys because they may have died sooner and are no longer detectable due to overgrowth. This potential underestimate is at least partially offset as many of the AR scored in the study were individually tagged and therefore their mortality over the previous monitoring interval (up to 4 months) was not overlooked. Conversely, the bleaching mortality, particularly of small colonies might have been overestimated due to confounding mortality from disease. Stony coral tissue loss disease (SCTLD) was indeed at high prevalence during the time of our surveys on reefs in Curaçao and Bonaire though data reported from Bonaire were from a nursery setting where active disease was not concurrently observed. Active SCTLD was observed in surveyed colonies in Curaçao, with two C. natans (conspecific) adults showing signs of active disease (but no bleaching).

Is bleaching resistance a general characteristic of small and/or young colonies?

Results presented here are consistent with previous reports that juvenile corals are generally more bleaching resistant to moderate heat exposures (i.e., DHW of ~4–10°C-weeks) than adult corals. For example, Speare et al. [22] found that heatwave-induced mortality was substantially lower for smaller size classes from the 2019 marine heatwave in Moorea which reached DHWs of 5–6°C-weeks. Similarly, Brandt [17] reported reduced bleaching susceptibility in small corals (<10 cm diameter) compared to larger conspecifics, but only up to a DHW of 9°C-weeks, after which all size classes converged at >75% bleaching prevalence. During the 2020 mass bleaching event in eastern Australia, Burn et al. [23] also found that patterns of relative bleaching susceptibility were dependent on the overall intensity of reef-wide mass bleaching among sites (used as a proxy of a reef’s DHW exposure). For example, in eight of ten taxa, juveniles were less likely to bleach than adults, but this pattern disappeared at sites with extremely high bleaching prevalence (>81% of colonies bleached). Although these authors do not provide DHW exposures across their sampling sites, NOAA satellite data suggests the highest levels experienced in this region in 2020 were in the range of 13–15°C-weeks (https://coralreefwatch.noaa.gov/data/5km/v3.1_op/image/daily/dhw/png/2020/03/ct5km_dhw_v3.1_gbr_20200315.png), substantially lower than that experienced in the Caribbean 2023 event reported here. Thus, our study adds to previous reports by 1) providing observations on known, sexual recruits (rather than presumed recruits based on small size) of multiple species from restoration activities and 2) documenting patterns in the midst of a regional thermal stress event of unprecedented severity and duration, with DHW exposures estimated between 19 and 25°C-weeks and durations in excess of five months across all study sites (Table 1).

While the AR in our study showed strong resistance to bleaching (90% of 771 colonies with healthy coloration), extant wild juvenile assemblages sampled at outplant sites (largely comprised of different species from those used for restoration) showed variable responses; relatively high tolerance in the Dominican Republic (80% of 25 colonies healthy) and much less tolerance in Mexico (only 32% of 119 colonies healthy). Colony size data were not systematically collected in our study (only available for a relatively small subset of conspecific colonies), but those available showed no significant relationship between colony size and BMI (S1 Fig), with the exception of A. palmata comparison populations where the anomalous trend showed better health status for larger colony sizes (S1 Fig).

In the two regions where we surveyed nearby small coral colonies propagated for restoration via fragmentation, they fared worse than the AR, in line with the fact that these fragments, although small in size, are not true juveniles. In Bonaire, Colpophyllia natans AR showed no bleaching, whereas small propagated fragments of Orbicella spp. (2–4 cm in diameter) co-located in a field nursery showed substantial prevalence of heat stress (only 22% of 143 colonies with healthy coloration). However, AR of Orbicella also showed slightly higher bleaching susceptibility than AR of C. natans (Fig 2A and 2B). Meanwhile, in a conspecific comparison, slightly larger (5–10 cm) fragment-originated colonies of A. palmata outplanted to Sombrero reef in the Dominican Republic showed intermediate levels of tolerance (between AR and other adults with 55% of 11 colonies appearing healthy; Fig 3A).

10.1371/journal.pone.0309719.g003 Fig 3 Summary of bleaching and mortality responses of subsets of data relating to specific hypotheses.

A) A. palmata Assisted Recruits (AR) compared to small A. palmata colonies propagated by fragmentation at the same site in Dominican Republic. B) One cohort of adult A. palmata derived from AR in Mexico compared to its parental site. C) Comparing juvenile AR with colonies from their parental sites (PAR) for three species where the comparison was possible (Acropora palmata from Mexico, Diploria labyrinthiformis pooled from Mexico and St. Croix, and Pseudodiploria strigosa from St. Croix) is not consistent with the hypothesis that thermal tolerance is inherited. Sample size for each group is given above the stacked bar.

It is possible that the high bleaching resistance of sexually propagated juvenile colonies recorded in our study is lost when they reach adult stages later in life. Our dataset includes one cohort of >10 year-old (i.e., reproductive adult) colonies derived from AR of A. palmata in Mexico [13]. In contrast to the overall sample of AR and to the conspecific juvenile AR in the same location, this set of colonies was highly affected by the heat stress with none retaining healthy coloration and 32% (of 19 colonies) having already succumbed to bleaching-related mortality at the time of survey (Fig 3B). Adult colonies from their parental population also showed high bleaching related mortality (Fig 3B, 74% of 19 colonies). In comparison, a cohort of <2 year-old A. palmata AR outplanted in Mexico and their parent population showed the typical juvenile resistance (96% of 97 colonies healthy) and adult sensitivity (44% of 27 colonies healthy, Fig 3C). Together these observations suggest that thermal tolerance of coral AR is an ontogenic characteristic that diminishes with age.

Overall, some degree of bleaching resistance appears likely in both assisted and wild juvenile corals, but less so for small corals propagated through fragmentation (as they are derived from adult colonies). Nonetheless, on Caribbean reefs where juveniles of most reef-building coral species are virtually absent (and likely to be increasingly rare in other reef areas around the globe as global warming proceeds, e.g., [24]), interventions to assist coral recruitment can be an important tool to buffer population impacts (via persistence of young colonies) from heatwave-induced bleaching.

Why are recruits more bleaching resistant?

This study was not designed to resolve the different mechanism(s) driving the emergent pattern of widespread bleaching resistance in AR. Several hypotheses are posed in the literature that may (partially) account for the observed pattern. Mumby [25] had proposed that wild recruits suffered less from a mass bleaching event due to their natural preference for settling in cryptic habitats, protecting them from exacerbating light stress. It is indeed possible that the AR in the current study experienced less light than comparison colonies. Although they were on artificial substrates, these substrates were outplanted by wedging them into reef crevices, and AR survive preferentially in grooves or other cryptic features of designed substrates [26, 27]. Another theory is that because juveniles are not engaging in gametogenesis that they enjoy greater physiological buffering from energy reserves than reproductive adults that are devoting energy to egg production [28]. Meanwhile, generally small colony size may simply help alleviate physiological stress by providing for greater mass-transfer across the colony surface [29]. These latter two hypotheses are consistent with the subsets of our dataset showing higher susceptibility of the small sample of adult AR of A. palmata in Mexico (Fig 3B) which are both of large size and reproductive [13]. The small fragment-based colonies surveyed in the present study (< 10 cm) are likely not reproductive and showed intermediate susceptibility.

The sexual recombination of traits in populations of larval recruits has been hypothesized to confer greater adaptability to restored populations [6, 30], compared to restoration interventions based on fragmentation of fixed genotype(s). Although the resistance of AR to bleaching found in this study could be considered consistent with this hypothesis, in four paired comparisons where we surveyed a given cohort of AR and colonies at the parental site from which that cohort of AR were bred (P. strigosa in St. Croix, D. labyrinthiformis in St. Croix and Mexico; and two A. palmata corhorts in Mexico), all four parental populations showed high bleaching susceptibility (Fig 3B and 3C). This high susceptibility among parental populations is generally not consistent with the possibility that genetic inheritance and/or recombination from resistant parental genotypes accounts for offspring resistance.

The presence of resistant symbiont types is another plausible mechanism for observed resistance of AR. Recruits of all the species reported here acquire symbionts via horizontal transmission, i.e., from their environment rather than by maternal inheritance. This is understood to be a flexible process [31] with multiple potential sources for transmission. For example, P. strigosa recruits exposed to natal reef sediment and coral host fragments in laboratory tanks contained more diverse Symbiodinaceae communities than adult conspecifics at their natal site [32]. Additionally, outplanted AR of O. faveolata in the Florida Keys hosted complements of symbionts that were, at least initially, distinct from both their parental population and from co-occurring conspecific adults [33]. AR of Acropora palmata reared in land-based propagation efforts are capable of maintaining symbiosis predominantly with heat-tolerant Durisdinium [34] over multiple years after outplanting which could constitute an added intervention, though performance during bleaching events has yet to be tested. This enhanced diversity implies flexibility in the symbiont community composition of young corals that may confer the capacity for rapid adjustment to heat-tolerant assemblages in the case of environmental extremes.

Conclusion

As previously hypothesized, sexually-propagated juvenile corals of six important reef-building species of less than five years of age were highly resistant to bleaching and related mortality during and within a month following the extreme marine heatwave in July-November 2023 (DHWs up to 25°C-weeks). Although bleaching resistance may wane with age, seeding can make an important contribution to coral population persistence assuming some assisted recruits reach reproductive maturity. As coral reefs throughout the globe are hit by increasingly frequent and intense marine heatwaves, restoration activities that include sexual reproduction and seeding can make an important contribution to sustain coral populations.

Supporting information

S1 Fig Associations between colony size and Bleaching and Mortality Index (BMI) for each species used in restoration, with the negative trend for wild A. palmata being the only statistically significant trend.

Note that colony size was not consistently recorded across the data set.

(TIF)

S1 Data (CSV)

S1 Table Model results testing for species-specific differences in bleaching responses between assisted recruits and other comparison colonies (model form: Response ~ AR-Comparison * Species), such that a negative estimate is a worse health status.

The cumulative link model (CLM) uses ordinal health status scores as the response variable, the GLM and GLMM use the Bleaching and Mortality Index as the response variable, and the GLMM in addition accounts for variations in response due to region, site, or population type (proxy of census method).

(DOCX)

The dedicated staff and partners all of the authors’ institutions that were involved in propagation and monitoring are gratefully acknowledged for enabling this study. Specifically, R. Tecalco Renería, E. Avila Pech, T. Doblado Speck, S. Tuijten, N. Le Trocquer, M. Davies, D. Gonzalez, R. Maronde, S. Orndorff, M. Alperstein, S. Bideau contributed to data collection. M.V. Grosso-Becerra is also gratefully acknowledged for programmatic contributions.

10.1371/journal.pone.0309719.r001
Decision Letter 0
Thuesen Erik V. Academic Editor
© 2024 Erik V. Thuesen
2024
Erik V. Thuesen
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version0
24 Jun 2024

PONE-D-24-14553Assisted sexual coral recruits show high thermal tolerance to the 2023 Caribbean mass bleaching eventPLOS ONE

Dear Dr. Miller,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Both reviewers have made good suggestions to improve the manuscript. In particular, please note the comments of Reviewer #2 regarding the need for more explicit caveats regarding the comparisons between restored and natural specimens. Your data sets should meet the assumptions of the statistical tests. If not, please revise those analyses. Please make sure that all of your data are fully available. See #3 below. There appear to be gaps of data (missing rows) in your Excel sheets. Please check those.

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Reviewer #2: No

**********

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Reviewer #1: Yes

Reviewer #2: No

**********

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Reviewer #1: Yes

Reviewer #2: No

**********

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Reviewer #2: Yes

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Reviewer #1: Miller et al. have submitted a very compelling study indicating that larval recruits may be more bleaching tolerant than larger, conspecifics (or comparative populations). Although there are challenges within the data set, the authors do a substantial job working with the data that are available. This could, of course, lead to a more targeted study that controls for species, colony size, and environmental conditions to see how ubiquitous these results may be and when the ontogenetic shift may result in the loss of this trait.

The statistical approach is strong and comprehensively tackles it in several ways ensuring that the caveats are accounted for in the best way possible. The team also represents a breadth of expertise and a diverse geography.

I have listed some minor comments and suggestions below. There are no major issues to address.

Abstract

Line 43: why not ASR to capture all the words as in Koch et al.?

Line 50: change ‘in which’ to ‘when’ to better convey this occurred as a replacement when conspecifics were rare

Line 53: ‘hypothesized mechanisms were explored’ – the follow up sentence doesn’t convey a mechanism, just explores another type of coral and bleaching susceptibility that could be related to size or production source…think reword this to reflect different types of corals explored or something like that

Introduction

Line 69: I think its worth mentioning that there are additional steps AND it’s harder to accomplish AND there was a lot of R&D needed to do it even with the species we can use today.

Line 74/75: ‘long been’ is used twice in the same sentence…recommend altering one.

Line 77: change to ‘…juveniles have shown vulnerability…’

Lines 81-84: This could also be a result of differences in mass transfer and not related to juvenile vs adult life stage, which I think would be worth mentioning Effects of coral colony morphologies on mass transfer and susceptibility to thermal stress | Coral Reefs (springer.com)

Methods

Line 115: change to ‘but within four weeks of estimated peak heat stress’

Lines 134/135: change to ‘most appropriate comparison as both species and environmental exposure was matched at the same site’

Line 149: I am unsure of what ‘to remove the possible influence of assemblage structure on emergent trends’ means here

Line 149: there is a ‘Firstly’ here, but then on line 157 there is another ‘First’ and a ‘Secondly’ on line 162…seems like we need a different beginning to the sentence

Results and Discussion

Line 181: change ‘are’ to ‘were’

Line 182: change ‘retain’ to ‘retained’

Line 183: change ‘is’ to ‘was’

Line 203: something wrong with this sentence ending. Either words should be deleted or there are words missing.

Line 208: SCTLD, when spelled out, is not capitalized

Line 231: remove the word ‘significantly’ as there were no statistical tests comparing these

Line 246-253: I think this is the coolest conclusion, where you can control for size and still compare juveniles vs asexual frags. But, figure 3 seem more compelling and the text is sort of confusing as you are comparing CNAT vs Orbicella vs Apal. The figures though suggest you have within species comparisons.

Line 288/289: change to ‘AR survive preferentially in grooves…’

Line 312/313: change to ‘in the lab host more diverse…’

Section around Line 315/316, could add another interesting study, Long-term maintenance of a heterologous symbiont association in Acropora palmata on natural reefs | The ISME Journal | Oxford Academic (oup.com)

Reviewer #2: The manuscript by Miller et al describes the impacts of the 2023 mass coral bleaching event on corals generated through assisted reproduction deployed at 5 locations throughout the Caribbean region over the last ~12 years. They compare these impacts to reference populations at each site based on availability of conspecifics or co-occurring coral populations. While I think it is very important to document the impacts of the bleaching on natural and restored coral populations, and I really appreciate the effort the authors have made to find suitable comparison groups, my main concern is that I don’t think the comparisons are valid in some cases, given (1) species-level differences in heat tolerance, and (2) ontogenetic/size differences in heat tolerance. Given that it was nearly impossible to find similarly-sized natural conspecifics in the same locations as the outplants, the finding that ‘assisted sexual recruits had significantly lower prevalence of bleaching than conspecific corals’ (line 51/52) isn’t fair to make without clear and upfront caveats that the size-frequency distributions of AR and conspecific corals didn’t overlap (i.e. Fig S1).

I still believe that these data are important to publish and the lack of natural juvenile corals in these populations shouldn’t preclude it. I also appreciate that the authors were transparent that the summary analyses make ‘unbalanced comparisons among species, sites and colony origin’ (line 179), but it then begs the question – what’s the value of trying to make direct statistical comparisons? I think the text needs to be even more explicit about these caveats and the statistical comparisons should be limited in favour of simply reporting the observational data and then discussing the results and proposing mechanisms, potential implications and meaning, contextualized in the caveats.

My other concern is that these results are couched in the argument that AR populations should have greater resilience due to higher genotypic diversity via genetic recombination (line 40-41) and that their performance is compared to the ‘predicted resistance of sexually propagated juveniles’ (line 90). However, no genetic data for these outplanted populations are presented and there is very little evidence to date that demonstrates that restored coral populations are more genetically diverse than natural ones (although perhaps if no conspecifics exist at the site then by default it’s true). But, it is quite possible that inbreeding depression could also occur, reducing fitness. While there are great references and resources that can be used to consider ways to maximize adaptive potential (i.e. the Baums et al reference), I don’t think it’s supported here by genetic data. Were there specific ‘predictions’ (line 90) made beyond that the AR corals would be ‘more’ heat tolerant? Without a directly comparable reference population, it is difficult to evaluate whether this ‘resistance’ is in line with that ‘predicted’ so I would use caution in interpreting these results in the context of heat resistance/resilience.

Finally, I felt that the manuscript introduction was short and at times the introduction and discussion lacked continuity. I have pointed to some specific examples below that warrant attention.

Minor comments:

- Line 40: I suggest deleting the first sentence of the abstract. Firstly, it felt disjointed with the rest of the abstract and didn’t flow very well. Secondly, AR ‘could’ but not necessarily ‘would’ lead to more resilient restored populations as inbreeding depression could actually reduce resilience.

- Line 56 (And others): I found the use of the term ‘dosage’ in this context to be odd and maybe not needed.

- Line 59 (And 325): I suggest defining ‘demographic buffer’ here and in the conclusion. I’m not sure why maintaining high densities of juveniles (that are not yet reproductive) that become susceptible to bleaching as they grow would be beneficial, and reduce variance in vital rates (a la Hilde et al. 2020 TREE).

- Line 65: suggest ‘could’ rather that ‘should buy time’

- Line 74-75: suggest editing so ‘long been’ isn’t used twice in once sentence

- Line 75: I think a bridging sentence may be useful here to link the 1st and 2nd paragraphs. I also think the 2nd paragraph could benefit from additional examples and/or a discussion of potential mechanisms for why juveniles may be more (or less) susceptible than adults to various stressors. I understand it is unpacked in the discussion further, but I think the implications of being more or less susceptible to heat should be introduced here. The end of the 2nd paragraph could also benefit from a concluding statement.

- Line 91: I think this aim should make it clear what the susceptibility of AR is being compared to.

- Figure 1 caption: suggest changing ‘illustration’ to ‘image’ in (B).

- Table 2: fix shading formatting for CUR/CARMABI line

- Line 136: I recognize the need to identify some threshold to delineate adult and juvenile, but is there a justification for why >4 cm diameter was considered ‘adult’? Typically 5 cm is used as a cut-off, but even then it’s likely that colonies of many taxa aren’t yet reproductively mature at that size. Do you have a reference and/or justification for this?

- Line 147: I was confused by the statement that ‘a subset of data including only the six species used for restoration’ were used in the analyses because, according to table 1, there were wild assemblages at the sites used in some comparisons (table 1). So, were the wild populations limited to only those 6 taxa?

- Line 151: I’m not too familiar with the cumulative link model but a quick search indicated that it was a form of ordinal regression? Perhaps a brief justification/explanation of this approach would be useful since I don’t think it is that common of an analysis. Also, please report which program and packages were used for the analysis.

- Line 183: suggest changing ‘difference is’ to ‘difference was’ and checking for past tense throughout results and discussion

- Line 197-201: I am confused by this sentence. Is it suggesting that while within-species comparisons are most appropriate, most taxa around AR outplants were weedy so within-species comparisons were not possible? Also the sentence appears to be unfinished. Please edit.

- Paragraph of line 204: I think this paragraph requires more discussion. Does the point regarding SCTLD suggest that whole-colony mortality from disease may have been confused with bleaching-related mortality, and that this could disproportionately affect smaller colonies? Please expand and clarify this point.

- Line 217: suggest finishing this sentence by adding ‘…than adult colonies.’

- Line 266: suggest changing ‘this set of colonies were’ to ‘this set of colonies was’

**********

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Reviewer #1: Yes: Erinn Muller

Reviewer #2: No

**********

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10.1371/journal.pone.0309719.r002
Author response to Decision Letter 0
Submission Version1
2 Aug 2024

Reviewer #1: Miller et al. have submitted a very compelling study indicating that larval recruits may be more bleaching tolerant than larger, conspecifics (or comparative populations). Although there are challenges within the data set, the authors do a substantial job working with the data that are available. This could, of course, lead to a more targeted study that controls for species, colony size, and environmental conditions to see how ubiquitous these results may be and when the ontogenetic shift may result in the loss of this trait.

The statistical approach is strong and comprehensively tackles it in several ways ensuring that the caveats are accounted for in the best way possible. The team also represents a breadth of expertise and a diverse geography.

I have listed some minor comments and suggestions below. There are no major issues to address.

Abstract

Line 43: why not ASR to capture all the words as in Koch et al.?

Koch et al. 2022 define the ASR acronym for Assisted Sexual Reproduction (the process, not the recruits themselves), which is somewhat different. Hence, we will retain the term AR (to refer to offspring, rather than the process of reproduction) for this paper.

Line 50: change ‘in which’ to ‘when’ to better convey this occurred as a replacement when conspecifics were rare

‘In which’ has been retained but this sentence has been revised. The full coral assemblages around the outplants were sampled as comparison populations to incorporate the same ecological exposure conditions, not just as replacements ‘when’ conspecifics were rare.

Line 53: ‘hypothesized mechanisms were explored’ – the follow up sentence doesn’t convey a mechanism, just explores another type of coral and bleaching susceptibility that could be related to size or production source…think reword this to reflect different types of corals explored or something like that

This sentence has been deleted

Introduction

Line 69: I think its worth mentioning that there are additional steps AND it’s harder to accomplish AND there was a lot of R&D needed to do it even with the species we can use today.

This sentence has been revised/elaborated

Line 74/75: ‘long been’ is used twice in the same sentence…recommend altering one.

Done

Line 77: change to ‘…juveniles have shown vulnerability…’

This sentence has been deleted

Lines 81-84: This could also be a result of differences in mass transfer and not related to juvenile vs adult life stage, which I think would be worth mentioning Effects of coral colony morphologies on mass transfer and susceptibility to thermal stress | Coral Reefs (springer.com)

This possibility (and reference) is already mentioned in the discussion (now ln 326-327)

Methods

Line 115: change to ‘but within four weeks of estimated peak heat stress’

Done

Lines 134/135: change to ‘most appropriate comparison as both species and environmental exposure was matched at the same site’

Done

Line 149: I am unsure of what ‘to remove the possible influence of assemblage structure on emergent trends’ means here

Sentence has been revised

Line 149: there is a ‘Firstly’ here, but then on line 157 there is another ‘First’ and a ‘Secondly’ on line 162…seems like we need a different beginning to the sentence

Corrected

Results and Discussion

Line 181: change ‘are’ to ‘were’

Line 182: change ‘retain’ to ‘retained’

Line 183: change ‘is’ to ‘was’

These were corrected

Line 203: something wrong with this sentence ending. Either words should be deleted or there are words missing.

Corrected

Line 208: SCTLD, when spelled out, is not capitalized

Corrected

Line 231: remove the word ‘significantly’ as there were no statistical tests comparing these

Done

Line 246-253: I think this is the coolest conclusion, where you can control for size and still compare juveniles vs asexual frags. But, figure 3 seem more compelling and the text is sort of confusing as you are comparing CNAT vs Orbicella vs Apal. The figures though suggest you have within species comparisons.

In the Bonaire in situ nursery, we did not have conspecific comparisons; AR were C.natans and fragments were Orbicella spp. We have new emphasized this in the text. Meanwhile, in the Dominican Republic there was indeed a conspecific comparison, which is why we chose to only depict these data in Fig 3.

Line 288/289: change to ‘AR survive preferentially in grooves…’

Done

Line 312/313: change to ‘in the lab host more diverse…’

Done

Section around Line 315/316, could add another interesting study, Long-term maintenance of a heterologous symbiont association in Acropora palmata on natural reefs | The ISME Journal | Oxford Academic (oup.com)

This reference has now been included

Reviewer #2: The manuscript by Miller et al describes the impacts of the 2023 mass coral bleaching event on corals generated through assisted reproduction deployed at 5 locations throughout the Caribbean region over the last ~12 years. They compare these impacts to reference populations at each site based on availability of conspecifics or co-occurring coral populations. While I think it is very important to document the impacts of the bleaching on natural and restored coral populations, and I really appreciate the effort the authors have made to find suitable comparison groups, my main concern is that I don’t think the comparisons are valid in some cases, given (1) species-level differences in heat tolerance, and (2) ontogenetic/size differences in heat tolerance. Given that it was nearly impossible to find similarly-sized natural conspecifics in the same locations as the outplants, the finding that ‘assisted sexual recruits had significantly lower prevalence of bleaching than conspecific corals’ (line 51/52) isn’t fair to make without clear and upfront caveats that the size-frequency distributions of AR and conspecific corals didn’t overlap (i.e. Fig S1).

All statistical comparisons in the paper are restricted to con-specific comparisons (hence controlling for species-specific variation in heat tolerance). The cited sentence in the abstract has been modified to read ‘Assisted sexual recruits had significantly lower prevalence of bleaching impacts (overall pooled ~ 10%) than conspecific coral populations typically comprised of larger colonies (~ 60-100% depending on species).’

I still believe that these data are important to publish and the lack of natural juvenile corals in these populations shouldn’t preclude it. I also appreciate that the authors were transparent that the summary analyses make ‘unbalanced comparisons among species, sites and colony origin’ (line 179), but it then begs the question – what’s the value of trying to make direct statistical comparisons? I think the text needs to be even more explicit about these caveats and the statistical comparisons should be limited in favour of simply reporting the observational data and then discussing the results and proposing mechanisms, potential implications and meaning, contextualized in the caveats.

We only include one statistical analysis (and another supporting test without random effects in supplement), and it accounts for these biases. That addition of random effects turns a significant AR-Wild comparison for O. faveolata (see ordinal CLM) into a non-significant comparison (as all the difference is explained by Site (Bonaire/Mexico).

In any statistical analysis, one cannot include all possible covariates of the response variable. While there are very strong arguments against pooling all species together, there are more nuanced relationships between colony size and location versus bleaching. In this analysis we did not compare AR of one species to wild colonies of a different species, always restricting analyses within a single species. Notably, in our statistical comparison (Fig. 2B) the generalised linear mixed effect model does account for variability in bleaching due to possible confounding effects of region, site, and comparison population type (a proxy for census method). Importantly, if we do not account for any of these possible confounding factors, we even see a significant difference in tolerance for Orbicella faveolata, but once these confounding effects are included in the model, it increases the uncertainty (confidence intervals) in the AR-Wild bleaching comparison (Fig. 2B) and that group is no longer significantly different. For O. faveolata, that is because the two comparison groups come from two different countries (assisted corals from Bonaire vs wild corals from Mexico).

My other concern is that these results are couched in the argument that AR populations should have greater resilience due to higher genotypic diversity via genetic recombination (line 40-41) and that their performance is compared to the ‘predicted resistance of sexually propagated juveniles’ (line 90). However, no genetic data for these outplanted populations are presented and there is very little evidence to date that demonstrates that restored coral populations are more genetically diverse than natural ones (although perhaps if no conspecifics exist at the site then by default it’s true). But, it is quite possible that inbreeding depression could also occur, reducing fitness. While there are great references and resources that can be used to consider ways to maximize adaptive potential (i.e. the Baums et al reference), I don’t think it’s supported here by genetic data. Were there specific ‘predictions’ (line 90) made beyond that the AR corals would be ‘more’ heat tolerant? Without a directly comparable reference population, it is difficult to evaluate whether this ‘resistance’ is in line with that ‘predicted’ so I would use caution in interpreting these results in the context of heat resistance/resilience.

Acknowledged that we are not presenting any genetic data (our statements were simply based on general principles of sexual recombination as articulated in Baums et al. 2019). We have moderated the wording in both of these sections, avoiding ‘prediction’ terminology.

Inbreeding depression certainly could occur, but is generally an unlikely outcome in single-generation breeding among large populations of unrelated individual (which have historically been the case for the species in question) as long as general precautions are taken (e.g., incorporating as many parents as possible in larval cohorts and not outplanting offspring adjacent to parents; Baums et al. 2019). Inbreeding depression becomes a much greater concern in breeding among ‘small’ populations. We are likely reaching a point where certain Caribbean coral populations are reaching ‘small’ status (e.g., A.palmata or D.cylindrus in Florida) and require much more careful breeding within genetic management plans to avoid – and such planning is currently underway for the Florida population of A.palmata (Rodriguez-Clark et al. 2023) but is beyond the scope for this paper.

Finally, I felt that the manuscript introduction was short and at times the introduction and discussion lacked continuity. I have pointed to some specific examples below that warrant attention.

Minor comments:

- Line 40: I suggest deleting the first sentence of the abstract. Firstly, it felt disjointed with the rest of the abstract and didn’t flow very well. Secondly, AR ‘could’ but not necessarily ‘would’ lead to more resilient restored populations as inbreeding depression could actually reduce resilience.

Sentence modified

- Line 56 (And others): I found the use of the term ‘dosage’ in this context to be odd and maybe not needed.

Changed to ‘exposure’ throughout

- Line 59 (And 325): I suggest defining ‘demographic buffer’ here and in the conclusion. I’m not sure why maintaining high densities of juveniles (that are not yet reproductive) that become susceptible to bleaching as they grow would be beneficial, and reduce variance in vital rates (a la Hilde et al. 2020 TREE).

The term ‘demographic buffer’ has been removed from the manuscript and the intended meaning re-phrased.

- Line 65: suggest ‘could’ rather that ‘should buy time’

Revised wording here

- Line 74-75: suggest editing so ‘long been’ isn’t used twice in once sentence

Done

- Line 75: I think a bridging sentence may be useful here to link the 1st and 2nd paragraphs. I also think the 2nd paragraph could benefit from additional examples and/or a discussion of potential mechanisms for why juveniles may be more (or less) susceptible than adults to various stressors. I understand it is unpacked in the discussion further, but I think the implications of being more or less susceptible to heat should be introduced here. The end of the 2nd paragraph could also benefit from a concluding statement.

A sentence has been included here articulating hypotheses for bleaching resistnace

- Line 91: I think this aim should make it clear what the susceptibility of AR is being compared to.

This sentence now include reference to the Comparison population types.

- Figure 1 caption: suggest changing ‘illustration’ to ‘image’ in (B).

Done

- Table 2: fix shading formatting for CUR/CARMABI line

We are not sure about the meaning here. Shading in the table was intended to help the reader distinguish the rows related to different regions. Hence, the rows for St. Croix and Bonaire are shaded, those for MEX, CUR/CARMABI, and the DR are not.

- Line 136: I recognize the need to identify some threshold to delineate adult and juvenile, but is there a justification for why >4 cm diameter was considered ‘adult’? Typically 5 cm is used as a cut-off, but even then it’s likely that colonies of many taxa aren’t yet reproductively mature at that size. Do you have a reference and/or justification for this?

The 4 cm cutoff was chosen to be consistent with widespread monitoring protocols that are widely applied in the Caribbean (e.g. AGRRA and NOAAs national monitoring program). A sentence has been added to articulate this (Ln 145-148).

- Line 147: I was confused by the statement that ‘a subset of data including only the six species used for restoration’ were used in the analyses because, according to table 1, there were wild assemblages at the sites used in some comparisons (table 1). So, were the wild populations limited to only those 6 taxa?

This paragraph has been substantially revised to clarify that we collected data on all species in wild assemblages, and qualitatively compared the entire sample, while statistical modeling and comparisons were limited to within-species.

- Line 151: I’m not too familiar with the cumulative link model but a quick search indicated that it was a form of ordinal regression? Perhaps a brief justification/explanation of this approach would be useful since I don’t think it is that common of an analysis. Also, please report which program and packages were used for the analysis.

This is a common Ordinal regression method. The R-packages are now specified for each analysis

- Line 183: suggest changing ‘difference is’ to ‘difference was’ and checking for past tense throughout results and discussion

Done

- Line 197-201: I am confused by this sentence. Is it suggesting that while within-species comparisons are most appropriate, most taxa around AR outplants were weedy so within-species comparisons were not possible? Also the sentence appears to be unfinished. Please edit.

Incomplete sentence has been revised. This paragraph emphasizes the important contribution of AR in extant Caribbean coral communities, given the rarity/absence of natural recruitment by reef-building species.

- Paragraph of line 204: I think this paragraph requires more discussion. Does the point regarding SCTLD suggest that whole-colony mortality from disease may have been confused with bleaching-related mortality, and that this could disproportionately affect smaller colonies? Please expand and clarify this point.

Yes, the caveat was intended to acknowledge that full colony bleaching mortality estimates might have been affected by artifacts – specifically, small colonies may die more quickly (and hence be undetectable after colonization) than large colonies, thus underestimating bleaching impact on AR relative to larger colonies. Conversely, SCTLD mortality might have been lumped with bleaching mortality, hence inflating it, in our data s

Attachment Submitted filename: ReviewerResponse.docx

10.1371/journal.pone.0309719.r003
Decision Letter 1
Thuesen Erik V. Academic Editor
© 2024 Erik V. Thuesen
2024
Erik V. Thuesen
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version1
14 Aug 2024

PONE-D-24-14553R1

Assisted sexual coral recruits show high thermal tolerance to the 2023 Caribbean mass bleaching event

PLOS ONE

Dear Dr. Miller,

Thank you for submitting your revised manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. PLOS ONE does not copy-edit manuscripts. Therefore, we invite you to submit a revised version that addresses the two minor points below. 

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10.1371/journal.pone.0309719.r004
Author response to Decision Letter 1
Submission Version2
15 Aug 2024

Requested corrections have been made

Attachment Submitted filename: ReviewResponse.pdf

10.1371/journal.pone.0309719.r005
Decision Letter 2
Thuesen Erik V. Academic Editor
© 2024 Erik V. Thuesen
2024
Erik V. Thuesen
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version2
19 Aug 2024

Assisted sexual coral recruits show high thermal tolerance to the 2023 Caribbean mass bleaching event

PONE-D-24-14553R2

Dear Dr. Miller,

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Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

10.1371/journal.pone.0309719.r006
Acceptance letter
Thuesen Erik V. Academic Editor
© 2024 Erik V. Thuesen
2024
Erik V. Thuesen
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
23 Aug 2024

PONE-D-24-14553R2

PLOS ONE

Dear Dr. Miller,

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on behalf of

Dr. Erik V. Thuesen

Academic Editor

PLOS ONE
==== Refs
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