PLoS ONE plos plosone PLOS ONE 1932-6203 Public Library of Science San Francisco, CA USA 10.1371/journal.pone.0290479 PONE-D-23-24872 Research Article Physical sciencesChemistryChemical reactionsBleaching Biology and life sciencesDevelopmental biologyModes of reproductionSexual reproductionSpawning Biology and life sciencesPhysiologyReproductive physiologyEggs Biology and life sciencesMarine biologyCoral reefs Earth sciencesMarine and aquatic sciencesMarine biologyCoral reefs Earth sciencesMarine and aquatic sciencesReefsCoral reefs Biology and life sciencesPhysiologyReproductive physiologyEggsBird eggs Biology and life sciencesCell biologyCellular typesAnimal cellsGerm cellsSperm Biology and life sciencesCell biologyCellular typesAnimal cellsGerm cellsOva Biology and life sciencesMarine biologyCorals Earth sciencesMarine and aquatic sciencesMarine biologyCorals Parental effects provide an opportunity for coral resilience following major bleaching events Coral sexual reproduction after bleaching https://orcid.org/0000-0002-0475-1368 Lenz Elizabeth A. Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Software Validation Visualization Writing – original draft Writing – review & editing 1 Donahue Megan J. Conceptualization Formal analysis Supervision Writing – review & editing 2 Gates Ruth D. Conceptualization Funding acquisition Methodology Resources Supervision 2 Putnam Hollie M. Conceptualization Funding acquisition Methodology Writing – review & editing 3 van der Steeg Eveline Data curation Methodology Project administration Writing – review & editing 4 Padilla-Gamiño Jacqueline L. Conceptualization Formal analysis Writing – original draft Writing – review & editing 5 * University of Hawaiʻi Sea Grant College Program, University of Hawaiʻi at Mānoa, Honolulu, HI, United States of America Hawaiʻi Institute of Marine Biology, University of Hawaiʻi at Mānoa, Kāneʻohe, HI, United States of America Department of Biological Science, University of Rhode Island, Kingston, Kingston, RI, United States of America School of Natural and Environmental Science, Newcastle University, Newcastle, United Kingdom School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA, United States of America Fujimura Atsushi Editor University of Guam, GUAM

The authors have declared that no competing interests exist.

 * E-mail: jpgamino@uw.edu 7 1 2025 2025 20 1 e0290479 7 8 2023 17 9 2024 2025 Lenz et al 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.

Identifying processes that promote coral reef recovery and resilience is crucial as ocean warming becomes more frequent and severe. Sexual reproduction is essential for the replenishment of coral populations and maintenance of genetic diversity; however, the ability for corals to reproduce may be impaired by marine heatwaves that cause coral bleaching. In 2014 and 2015, the Hawaiian Islands experienced coral bleaching with differential bleaching susceptibility in the species Montipora capitata, a dominant reef-building coral in the region. We tested the hypothesis that coral bleaching resistance enhances reproductive capacity and offspring performance by examining the reproductive biology of colonies that bleached and recovered (B) and colonies that did not bleach (NB) in 2015 in the subsequent spawning seasons. The proportion of colonies that spawned was higher in 2016 than in 2017. Regardless of parental bleaching history, we found eggs with higher abnormality and bundles with fewer eggs in 2016 than 2017. While reproductive output was similar between B and NB colonies in 2016, survivorship of offspring that year were significantly influenced by the parental bleaching history (egg donor × sperm donor: B × B, B × NB, NB × B, and NB × NB). Offspring produced by NB egg donors had the highest survivorship, while offspring from previously bleached colonies had the lowest survivorship, highlighting the negative effects of bleaching on parental investment and offspring performance. While sexual reproduction continues in M. capitata post-bleaching, gametes are differentially impacted by recovery time following a bleaching event and by parental bleaching resistance. Our results demonstrate the importance of identifying bleaching resistant individuals during and after heating events. This study further highlights the significance of maternal effects through potential egg provisioning for offspring survivorship and provides a baseline for human-assisted intervention (i.e., selective breeding) to mitigate the effects of climate change on coral reefs.

 http://dx.doi.org/10.13039/100000192 National Oceanic and Atmospheric Administration A/AS-1 https://orcid.org/0000-0002-0475-1368 Lenz Elizabeth University of Hawai'i Sea Grant College Program NA22OAR4170108 NOAA Office of Sea Grant, Department of Commerce UNIHI-SEAGRANT-4941 This paper is funded in part by a grant from the National Oceanic and Atmospheric Administration, Project A/AS-1; which is sponsored by the University of Hawai'i Sea Grant College Program, SOEST, under Institutional Grant No. NA22OAR4170108 from NOAA Office of Sea Grant, Department of Commerce. The views expressed herein are those of the author(s) and do not necessarily reflect the views of NOAA or any of its subagencies. UNIHI-SEAGRANT-4941. Data Availability The data is currently held in a public repository on GitHub: https://github.com/ealenz/Mcap-BNB-Reproduction-SelectiveBreeding. Introduction

Ocean warming caused by anthropogenic greenhouse gas emissions is one of the primary threats to the function of shallow tropical coral reefs [1,2]. Prolonged warming above the local thermal threshold for bleaching coupled with high irradiances can cause severe coral bleaching [3], the disruption of the nutritional symbiosis between the coral host and its unicellular dinoflagellates, Symbiodiniaceae (formerly, Symbiodinium spp.) [4]. This can subsequently result in increased rates of disease transmission [5] and mortality [6] along with reduced calcification rates and reproductive capacity in corals [7,8]. Continual declines in coral cover are predicted given the range of local and global disturbances simultaneously acting on coral reefs, with warming ranked as the most severe [9–11]. Identifying sources of resilience in coral reef ecosystems, such as locating exceptional coral genotypes that can thrive under extreme warming or temperature fluctuations, will be key in maintaining and restoring reefs for the future.

Differential bleaching susceptibility [12–14] during a thermal stress event illustrates biological variation within populations that may serve as a source of resilience and an opportunity for selection through reproductive success [15,16]. Thermal tolerance and capacity to recover after bleaching are important factors that influence sexual reproduction, recruitment, and success of future generations to adapt [7,8,17,18]. Successful sexual reproduction and recruitment are essential in maintaining coral populations [19], repopulating disturbed coral reefs [20–23], and enhancing genetic diversity within populations to overcome selective pressures [24,25]. However, parental investment in gametogenesis is energetically costly [26] and for corals reproductive cycles may exceed six to ten months [27,28]. Therefore, prolonged environmental stress can drive prioritization of energetic investment into basic metabolic function and repair, at the expense of growth and sexual reproduction [29–31]. Importantly, this tradeoff in energetic investment is likely to depend on the susceptibility and severity of coral bleaching, with greater energy available for reproduction in corals resistant to bleaching [32].

Previous studies have identified some of the way coral bleaching can impact aspects of sexual reproduction [8,33] and dampen recruitment [34,35]. For example, after the 1987 coral bleaching event in the Caribbean, Orbicella annularis recovered from bleaching by metabolizing tissue biomass, but did not complete gametogenesis in the following months, whereas colonies that had not bleached of the same species were able to develop and release gametes [7]. Similarly, during the 1998 bleaching event on the Great Barrier Reef, bleached corals showed high variation in reproduction compared to colonies resistant to bleaching nearby that experienced the same thermal stress. For acroporid species, reproductive polyps were more common in colonies that did not bleach, with larger eggs at higher densities per polyp than colonies that bleached and recovered [30]. More resolution is needed to better understand the impact and extent of coral bleaching events on the early life cycles of coral, from the stress event through recruitment.

Given logistical complexities and challenges, most studies have primarily investigated gametogenesis in the life cycle of coral with some understanding of cross-generational effects (i.e., parental, carry-over, or transgenerational effects) following major bleaching events. The impacts of coral bleaching may last for months to years after the initial thermal stress [36], and can manifest in life stages downstream such as fertilization [37–39], larval development, and recruitment [34,35,40]. Between the 2005 and 2010 bleaching events in Panama, Levitan et al. (2014) found that thermally tolerant Orbicella franksi recovered the capacity to produce and release gametes more quickly (within 3 to 5 years) than the more thermally sensitive O. annularis. While these studies demonstrate a range of responses in sexual reproductive biology and ecology during recovery post bleaching (i.e., gametogenesis and recruitment), few studies have followed both the intra- and intergenerational impacts of bleaching. Recent marine heatwaves eliciting differential coral bleaching of M. capitata in Hawaiʻi provide an opportunity to compare the impacts of parental bleaching history on coral reproduction and offspring performance during recovery and offer potential insight on coral resilience [15,41,42].

Coral reefs in the subtropical waters of Hawaiʻi were largely naive to global bleaching events [43–45] with bleaching events first recorded in the Main Hawaiian Islands in 1996 and then in the Northwestern Hawaiian Islands in 2002 [43–45]. However, the Hawaiian Archipelago experienced “the blob” heatwave, followed by an El Niño that resulted in severe back-to-back coral bleaching in 2014 and 2015 (Fig 1A) [46,47]. During these consecutive bleaching events, degree heating weeks (DHW) in the Main Hawaiian Islands exceeded 8 weeks by September in both years [46,47]. In Kāneʻohe Bay (Oʻahu, Hawaiʻi), ~70% of corals on the shallow reefs (< 2 m depth) bleached and exhibited 13–22% mortality in 2014 and 2015 [46,48–50]. During both events in Kāneʻohe Bay, colonies of the dominant reef-building coral, Montipora capitata, visibly bleached or remained pigmented during prolonged heat stress (Fig 1B). Despite widespread bleaching, approximately 70% of M. capitata that bleached in 2014 and 2015 were considered recovered by the following December and January based on visual coloration [12,14,15,46,51–53].

10.1371/journal.pone.0290479.g001 <p>A) Temperature data from 2010 to 2017 (NOAA Buoy Moku oʻ Loe, HI Station ID: 1612480) illustrate historical patterns and identify years of bleaching events in Oʻahu, Hawaiʻi. The bleaching threshold between 30 to 31°C of corals in Kāneʻohe Bay (Coles et al., 2018) is shown in the shaded red, thermometers indicate the 2014 and 2015 bleaching events and the spawning corals indicate the spawning seasons. B) An image depicting the tagged bleached (left) and nonbleached (right) parental colonies in response to the 2015 heat stress in Kāneʻohe Bay.</p> </caption> <graphic mimetype="image" position="float" xlink:href="info:doi/10.1371/journal.pone.0290479.g001" xlink:type="simple"/> </fig> <p><italic>M</italic>. <italic>capitata</italic> demonstrates relatively high tolerance against multiple local and global stressors [<xref ref-type="bibr" rid="pone.0290479.ref054">54</xref>,<xref ref-type="bibr" rid="pone.0290479.ref055">55</xref>], with varied sensitivity among individual colonies and their traits measured under elevated temperature [<xref ref-type="bibr" rid="pone.0290479.ref015">15</xref>,<xref ref-type="bibr" rid="pone.0290479.ref051">51</xref>], such as survivorship [<xref ref-type="bibr" rid="pone.0290479.ref049">49</xref>], growth [<xref ref-type="bibr" rid="pone.0290479.ref045">45</xref>], and biomass composition [<xref ref-type="bibr" rid="pone.0290479.ref045">45</xref>,<xref ref-type="bibr" rid="pone.0290479.ref053">53</xref>,<xref ref-type="bibr" rid="pone.0290479.ref054">54</xref>,<xref ref-type="bibr" rid="pone.0290479.ref056">56</xref>–<xref ref-type="bibr" rid="pone.0290479.ref058">58</xref>]. Reproductive effort of <italic>M</italic>. <italic>capitata</italic>, particularly oocyte characteristics and spawning, has shown little response to warming [<xref ref-type="bibr" rid="pone.0290479.ref036">36</xref>,<xref ref-type="bibr" rid="pone.0290479.ref059">59</xref>]. This reproductive response may have contributed to its ecological success along the fringing and patch reefs of Kāneʻohe Bay in the past. However, percent of motile sperm from <italic>M</italic>. <italic>capitata</italic> declined from 80–90% in 2011 to 40.5% in 2015, corresponding with the consecutive bleaching events in Kāneʻohe Bay [<xref ref-type="bibr" rid="pone.0290479.ref036">36</xref>]. For <italic>M</italic>. <italic>capitata</italic>, oogenesis can begin as early as July, which means that early egg development may cooccur with severe, prolonged warming events (July-October), and later egg development continues when corals are recovering from these events (November-August). This could create a strain on energetic resources when corals are compromised during a substantial fraction of the typical gametogenic cycle [<xref ref-type="bibr" rid="pone.0290479.ref060">60</xref>,<xref ref-type="bibr" rid="pone.0290479.ref061">61</xref>]. Therefore, tracking <italic>M</italic>. <italic>capitata</italic> through subsequent spawning seasons after bleaching events can reveal the reproductive capacity of this species as ocean temperature continues to increase.</p> <p>In this study, we examined cross-generation plasticity (i.e., parental effects) to determine how parental response to environmental events influence reproduction [<xref ref-type="bibr" rid="pone.0290479.ref062">62</xref>]. We measured the reproductive biology of <italic>M</italic>. <italic>capitata</italic> for two spawning seasons (2016 and 2017) following bleaching events (2014 and 2015). We tested the following hypotheses: (i) that parental bleaching history [bleached (B) and nonbleached (NB)] would affect reproductive performance in subsequent spawning seasons and (ii) intentional crosses of gametes from parent colonies of differential bleaching history would influence offspring success (<xref ref-type="fig" rid="pone.0290479.g002">Fig 2A</xref>). In 2016, we tested the second hypothesis and quantified the downstream effects of parental bleaching history from gamete release to settlement of the offspring in parent colonies that did and did not bleach during the 2015 warming event (<xref ref-type="fig" rid="pone.0290479.g002">Fig 2B</xref>). This study was designed to assess the impacts of consecutive bleaching events on the early stages within the coral life cycle and selective processes already occurring in nature while also testing basic breeding techniques as an intervention strategy for coral restoration to maintain genetic diversity and promote resilience.</p> <fig id="pone.0290479.g002" position="float"> <object-id pub-id-type="doi">10.1371/journal.pone.0290479.g002</object-id> <label>Fig 2</label> <caption> <title>Experimental design of the study.

A) Bleached and nonbleached colonies were tagged in October 2015 at the peak of the bleaching event. Bleached colonies in this experiment recovered by January 2016. Total reproductive output and gamete collections were measured during the 2016 and 2017 spawning seasons. Months of the spawning season differ between years because of the different timing of the new moon in 2016 and 2017. B) Selective breeding matrix illustrating the crossing of egg and sperm donors conducted in July 2016 based on parental bleaching history. Colored squares indicate the cross of individuals attempted and solid black circles indicate successful fertilization. Offspring from these crosses were used to measure survivorship of larvae and settlers and settlement.

 Materials and methods 2.1 Selecting parent colonies and spawning events

Montipora capitata is a hermaphroditic broadcast spawner and its reproductive cycle, spawning dynamics, and early life stages have been extensively studied at the Hawai‘i Institute of Marine Biology (HIMB) located in Kāneʻohe Bay, on the windward side of Oʻahu, Hawaiʻi, USA [27,60,61,63–65]. In Hawaiʻi, oogenesis begins a 9–10 month period as early as July and as late as October, while spermatogenesis begins the following April to May, ca. 1 month prior to the first spawning event in May or June [28], creating the potential for differential effects of bleaching on oocytes and sperm. Symbiodiniaceae are vertically transferred from M. capitata parent colonies into eggs prior to the formation of the egg-sperm bundles, which are released during spawning [63]. Spawning in M. capitata extends over three, consecutive lunar months between May and September for 3 to 5 consecutive nights between 20:45 and 22:30 hrs, starting on the night of the new moon [27,60]. The second and third nights are when the largest spawning events most commonly occur [60].

During the peak of the 2015 bleaching event in Hawaiʻi, ten pairs of colonies (30–100 cm diameter) of M. capitata were identified and tagged as bleached (B) and nonbleached (NB) along the leeward side of the reef surrounding HIMB (21’26.09 N, 157’47.47’ W) on 20 October 2015 (Fig 3C). These colonies remained in the field until retrieved three days prior to the new moon of the spawning months in 2016 (June, July, and August) and 2017 (May, June, and July) (Fig 3A). To examine reproductive performance of B and NB colonies of M. capitata, parent colonies were collected by removing the entire colony from the reef, or by breaking large fragments (30–40 cm in diameter) from tagged colonies using a hammer and chisel. These collections were first completed on 4 and 5 June 2016. Of the twenty colonies tagged, seven colonies that had not bleached and eight colonies that had bleached and recovered were alive and used for the study. The other five colonies not recovered had either died or were missing from the reef. The fifteen colonies were transported to the wet laboratory at HIMB in 20L buckets filled with seawater from Kāneʻohe Bay at an ambient temperature of ~28 to 29°C. Colonies were randomly allocated to two ~1,300L shaded outdoor flow-through tanks [55,66]. Both tanks had sand-filtered seawater delivered at a flow rate of ~6L minute-1 and a circulation pump (700 gph Magnetic Drive, Danner Manufacturing Inc. Islandia, NY, USA). Irradiance and temperature within each tank were recorded every fifteen minutes with a cosine corrected photosynthetically active radiation (PAR) sensor (Odyssey PAR loggers, Dataflow Systems Ltd, Christchurch, NZ) calibrated to a Licor 192SA sensor, and a temperature logger (Hobo™ Water Temp Pro v2 resolution ± 0.2°C, Onset Computer Corporation, Bourne, MA, USA). Three to five days after each spawning event, colonies were returned to the original field site by attaching them to a fixed rack with cable ties and retrieved two days before the next new moon of the spawning season.

10.1371/journal.pone.0290479.g003 Reproductive traits measured from the same parent colonies in <xref ref-type="fig" rid="pone.0290479.g002">Fig 2</xref> during 2016 and 2017 spawning seasons following the 2015 bleaching event.

A) Proportion of spawning each night in 2016 and 2017 spawning seasons from parent colonies that had bleached and not bleached. “0” indicates the night of the new moon. Mean (± SE) values for B) reproductive output normalized to planar surface area, C) egg volume, D) number of eggs per bundle, and E) percent egg abnormality measured from bleached and nonbleached parents in 2016 and 2017.

 2.2 Sexual reproduction

Starting one night prior to the new moon, M. capitata parent colonies were monitored for seven nights. During each night of spawning, colonies were isolated at 19:30 in individual containers filled with ambient seawater from the flow-through tanks. When spawning occurred, M. capitata released egg-sperm bundles into the water column between 20:45 and 22:30 with peak spawning typically expected on the second night of the new moon [27,59,60]. Spawning activity of individual colonies was monitored each night and recorded as “spawn” or “no spawn”. For the spawning colonies, we quantified the total volume of gametes released, number of eggs per bundle, and egg quality (i.e., area and abnormality).

Sterilized disposable pipets (2 mL) were used to gently collect all egg-sperm bundles at the water surface from each individual colony to avoid cross contamination or prematurely breaking the egg-sperm bundles. We preserved 3–5 egg-sperm bundles per colony per night to quantify the number of eggs per bundle, egg volume for size, and abnormality. Each egg-sperm bundle was placed in a 2 mL microcentrifuge tube and allowed to break up in 0.1 mL of seawater and for the eggs to hydrate for 2 hrs before preserved in zinc fixative (1:4 Z-fix, Sigma-Aldrich Inc. to 0.2 μm filtered seawater FSW). Preserved eggs from each bundle were photographed using an Olympus SZX7 dissecting microscope equipped with an Olympus America camera (SN: BH039933-H); from photographs, we counted the number of eggs per bundle and measured the egg diameter using ImageJ2 software (Schneider et al., 2012). Egg volume was calculated using the equation for a sphere with the measured egg diameter of spherical eggs. We also recorded the proportion of abnormal (irregular) eggs packaged within each bundle [36,63]. Remaining egg-sperm bundles from each colony were placed into individual 50 mL Falcon tubes to quantify the total volume of gametes of each colony per night. Annual reproductive output per colony was estimated by summing the spawn volume across the entire spawning season, normalized to planar surface area of the colony using Fiji software [67].

 2.3 Fertilization success and Offspring

To compare offspring performance of bleached and nonbleached parents, we isolated the egg-sperm bundles from each parental colony that released more than 1 mL of spawn volume on the nights of 5 and 6 July 2016 (peak spawning) and placed egg-sperm bundles from each colony into a separate 50 mL falcon tube. Within one hour of the bundle breaking apart, eggs floated to the surface and sperm sank to the bottom. Sperm were pipetted from the bottom of the tube, and eggs were rinsed twice with 0.2 μm filtered seawater (FSW). Sperm from each colony was placed in separate 50 mL falcon tubes and later used to fertilize eggs from specific colonies. Nine colonies had adequate spawn volume to include in crosses, and thirty individual crosses were made from gametes based on parental bleaching history to generate four cross-types (egg donor × sperm donor): B × B (n = 8), B × NB (n = 4), NB × B (n = 4), and NB × NB (n = 7) (Fig 2B). For fertilization, the eggs (1 mL) were in a concentration of ~106 sperm mL-1 (by visual inspection) within a 50 mL falcon tube [68]. Thirty minutes after sperm and eggs were mixed, each cross type of fertilized eggs was transferred into individual 1 L conical tanks filled with UV-sterilized 1-μm FSW to avoid polyspermy. For M. capitata, self-fertilization is extremely rare [68,69]. To estimate fertilization success, three subsamples of 20–30 eggs were collected from each conical after approximately 3-hrs (i.e., when initial cleave stages are expected [70,71]), placed in a 2-μL microcentrifuge tube, and preserved in Z-fix (1:4 Z-fix to FSW). Remaining embryos in the conical tanks developed, and slow flow rate of FSW was introduced to mitigate potential effects of montiporic acid [65]. Five days post-fertilization, 10–15 larvae per conical tank were placed in a 10 mL well-plate filled with 5 mL of FSW with a chip of crustose coralline algae to track settlement through time; FSW was exchanged every other day. The proportion of planulae and settlers were examined on days 7, 28, and 53 post-fertilization while the total number of offspring alive were counted on days 6, 7, 28, 53, and 59 post-fertilization to estimate survivorship probability curves.

 2.4 Statistical analysis

All analyses were conducted in R (R Core Team, 2014; v. 3.5.1). We used a generalized linear mixed effects model to determine the effects of bleaching history on spawning activity, number of eggs per bundle, and egg abnormality of the 8 B and 7 NB parental colonies observed (glmer in lme4) [72] with a binomial (spawn/no spawn and proportion of abnormal eggs) and poisson (eggs per bundle count) response. Bleaching history (B/NB) and year (2016/2017) were included as fixed effects, and spawning month (1/2/3) and colony ID were included as random effects. To analyze total reproductive output and egg size, we used linear mixed effects models (lme in lme4) [72] with bleaching history and year as fixed effects, and colony ID as a random effect. Analysis of variance (ANOVA) tables were generated using type II sum of squares (Anova in car) [73]. Post-hoc analyses were conducted to further explore significant main effects and interactions. We utilized the emmeans package [74] to calculate and compare the estimated marginal means (EMMs), which represent the predicted means of the response variable for each level of the fixed effects, adjusted for the other covariates in the model. Pairwise comparisons between the levels of the fixed effects were then performed using Tukey’s Honest Significant Difference (HSD) test to adjust for multiple comparisons. This approach allowed us to identify significant differences between specific treatment groups, while accounting for the variability associated with random effects.

To test the effects of parental bleaching history on offspring performance, we first analyzed the proportion of eggs fertilized using generalized linear mixed effects models with cross-type as a fixed effect and the egg donor and sperm donor as random effects. The proportion of eggs reaching each developmental stage (2-cell, 4-cell, 8-cell, and 16-cell), the Kruskal-Wallace test was applied as the dataset did not meet the assumption of normality. For post-hoc analysis, we performed the Dunn’s test for multiple pairwise comparisons to determine which specific cross-types differed. To analyze the proportion of larvae that settled at 7, 28, and 59-days post-fertilization, we used a generalized linear mixed effects model with cross-type and day (7, 28, and 59-d post-fertilization) as fixed effects and colony ID of egg donor and sperm donor as random effects. Lastly, we generated survivorship estimate curves to visualize offspring fate by cross-type with ggsurvplot of the census over time (i.e., days 6, 7, 23, 27, 28, 53, and 59 post-fertilization) (survfit in survminer) [75]. Cox proportional hazards (CPH) model was used to analyze the effects of cross, egg donor, and sperm donor individually on offspring survivorship (coxph in survminer) [75]. Dispersion parameters were inspected through a simulation-based approach (DHARMa package) [76].

 Results 3.1 Sexual reproduction and egg traits

All fifteen colonies observed in this study released egg-sperm bundles one or more nights in both years (Fig 3A). When spawning was observed, colonies began releasing egg-sperm bundles between 20:20 and 21:32 hrs and ended between 20:30 and 22:15 hrs. Parental bleaching history did not affect the occurrence of spawning (P = 0.619) and had no interactive effect with year (P = 0.982). The proportion of colonies releasing gametes significantly differed by year (P < 0.001) which may largely be due to some spawning in 2016 compared to no spawning in 2017 during the third month (August). In 2017, the proportion of colonies participating in spawning events was 36% lower than in 2016. In both years, the second month of the spawning season had the highest proportion of colonies spawning.

In 2016, the spawning season following consecutive bleaching events, colonies that bleached and recovered had 22.5% higher mean total reproductive output than colonies that did not bleach, although this was not statistically significant (Fig 3B; Table 1; P = 0.076). There was no effect of year and no interaction between bleaching history and year on reproductive output (Fig 3B; Table 1; P ≥ 0.560). Individual egg volume ranged from 0.032 to 0.099 mm3 and did not differ by parental bleaching history, year, or by their interaction (Fig 3C; Table 1; P ≥ 0.462). The number of eggs per bundle from both bleached and nonbleached parental colonies ranged from 2 to 29, and mean eggs per bundle for all colonies examined was 13.3% less in 2016 than in 2017 (Fig 3D; Table 1; P = 0.017). Eggs per bundle did not differ by parental bleaching history (Fig 3D; P = 0.249). There were 79.5% more eggs with irregularities in 2016 than in 2017 (P < 0.001) with no difference by bleaching history (Fig 3E; Table 1; P = 0.292).

10.1371/journal.pone.0290479.t001 Statistical summary of Type II Wald χ<sup>2</sup> test of generalized linear mixed effects model and linear mixed effect models testing the fixed effects of spawning year and parent history of bleaching susceptibility on sexual reproduction.
Response Variables Fixed Effects χ2 df P-value Post-hocSummary
Colony-level Spawning Bleaching History 0.248 1 0.619
(0 = no spawn / 1 = spawn) Year 22.479 1 < 0.001 2016 > 2017
  Bleaching History * Year 0.001 1 0.982
Total Reproductive OutputLog transformed Bleaching History 3.155 1 0.076
Year 0.339 1 0.560
Bleaching History * Year 0.097 1 0.756
Egg Volume Bleaching History 0.108 1 0.742
Year 0.541 1 0.462
Bleaching History * Year 0.225 1 0.635
Eggs per Bundle Bleaching History 1.332 1 0.249
Year 5.656 1 0.017 2016 < 2017
Bleaching History * Year 1.408 1 0.235
Egg Abnormality Bleaching History 1.109 1 0.292
Year 191.259 1 <0.001 2016 > 2017
Bleaching History * Year 0.035 1 0.852

Significance indicated in bold text.

 3.2 Fertilization, survivorship, and settlement

While reproduction continued in the colonies examined, we found that cross-type did have an effect on fertilization, embryonic development, and percent larval survivorship (Fig 4; Table 2). Specifically, fertilization success in the NB × NB cross-type was higher than the B × NB and NB × B cross-types (Fig 4B; Table 2; post-hoc P = 0.002 and 0.010, respectively) but not B × B (post-hoc P = 0.163). The fertilization success in cross-type B × B also did not differ between NB × B (post-hoc P = 0.250), but was higher than B × NB (post-hoc P = 0.047). Cell division advanced beyond the 2-cell stage more quickly for within cross-types (B × B and NB × NB) than between cross-types (B × NB and NB × B) at 3-h post-fertilization. Embryos from both B × B and NB × NB cross-types reached the 16-cell stage at 3-h post fertilization, whereas embryos from B×NB and NB × B crosses developed at a slower rate and only reached the 4-cell stage (Fig 4C, Table 2).

10.1371/journal.pone.0290479.g004 Offspring performance from selected crosses.

A) Images of fertilized eggs and embryos (scale bar = 500 μm), planula larvae (scale bar = 500 μm), and settlement (1 mm). Mean ± SE. B) proportion of eggs fertilized by cross-type, C) proportion of cell division after 3-h fertilization D) proportion of motile larvae and E) settlers during five timepoints over a 59-d period, and F) survivorship estimate curves by cross over seven timepoints between 6 and 59-d with the figure embedded comparing the survivorship curves of offspring from bleached and nonbleached egg donors.

 10.1371/journal.pone.0290479.t002 Statistical summary of Type II Wald χ<sup>2</sup> test of generalized linear mixed effects model testing the fixed effects of cross-type (NB × NB, NB × B, B × NB, B × B) on the proportion of fertilized embryos and summary of the Kruskal-Wallace test for the cellular development (2-Cell, 4-Cell, 8-Cell, and 16-Cell).

Statistical summary of Type II Wald χ2 test of generalized linear mixed effects model testing the fixed effects of cross-type on larval survivorship, and settlement over three timepoints post-fertilization.

Response Variable Effect χ2 df P-value Post-hoc Summary
Embryonic Development Fertilization Cross 16.334 3 0.001 B×B > B×NBNB×NB > B×NB, NB×B
2-Cell 34.071 3 <0.001 B×B = NB×NBNB×B = B×NB
4-Cell 21.729 3 <0.001 B×B = NB×NB, NB×BNB×B = B×NB
8-Cell 33.882 3 <0.001 B×B = NB×NBB×NB = NB×B
16-Cell 20.445 3 <0.001 B×B = NB×NB, B×NB, NB×BNB×B = B×NB
Response Variable Fixed Effects
Larval SurvivalSquare-root transformed Cross 20.915 3 <0.001 B×B ≠ NB×B
Days Post-Fertilization 178.595 2 <0.001
Cross * Days Post-Fertilization 7.174 6 0.305
Larval Settlement Cross 7.623 3 0.055
Days Post-Fertilization 17.214 2 <0.001
Cross * Days Post-Fertilization 9.066 6 0.170

Significance indicated in bold text.

Percent larval survivorship and settlement varied by cross-type, driven by egg donor bleaching history (Fig 4D and 4E; Table 3). Offspring developed from eggs from previously B egg donors had lower survivorship than those from NB egg donors. NB egg donors had a significant effect on the proportion of larvae survival (Fig 4E; P < 0.001). However, no difference was found in offspring survivorship from bleached or nonbleached sperm donors (P = 0.992). Overall, percent mortality from the initial to final time point (i.e., day 5 to 59) were 92.5% for B × B, 87.8% for B × NB, 85.6% for NB × B, and 77.3% for NB × NB (Fig 4F).

10.1371/journal.pone.0290479.t003 Summary of Cox proportional hazards analysis of coral offspring survival influenced by the fixed effects: Cross-type, dam, and sire over time with model average estimates of the hazard ratio (with 95% confidence intervals; Cross (NB × NB, NB × B, B × NB, B × B): df = 3 or egg/sperm donor (NB vs. B): df = 1; <italic>n</italic> = 1,318; number of events = 560) for five timepoints (day 6, 7, 28, 53, and 59).
Fixed effect Hazard ratio z P-value
Cross 0.90 (0.84–0.96) -3.071 0.002
Egg Donor 0.77 (0.65–0.91) -3.068 0.002
Sperm Donor 1.00 (0.80–1.24) -0.010 0.992

Significance indicated in bold text.

 Discussion

Here, we demonstrate the influences of marine heatwaves on coral reproductive capacity and parental effects in spawning seasons following major bleaching events. It is noteworthy that the unprecedented, consecutive warming events in 2014 and 2015 in Kāneʻohe Bay, Hawaiʻi influenced the reproductive capacity of M. capitata regardless of parental bleaching response. When comparing the first spawning season following the 2015 bleaching event to the second, M. capitata colonies had fewer eggs packaged within the egg-sperm bundles released. While average egg volume did not differ between years, the egg abnormality was higher in 2016 than in 2017 regardless of parental bleaching history. Parental colonies that bleached and did not bleach had similar reproductive output, number of eggs per bundle and egg abnormality. However, delayed beneficial maternal effects were observed in offspring from parents resistant to bleaching. These results demonstrate that although M. capitata has the energetic capacity to continue reproduction despite bleaching response, cross-generational impacts occur (Byrne et al., 2020), with possible ecological consequences downstream.

4.1 Reproductive capacity after bleaching events

M. capitata appears to maintain reproductive resilience, as well as recovery with time, after consecutive marine heatwaves and coral bleaching events, as evidenced by continuing synchronous broadcast spawning and production of viable eggs and sperm. These results are consistent with prior studies examining the influence of environmental and biological factors on M. capitata gametogenesis and spawning in Kāneʻohe Bay [28,59]. For instance, Padilla-Gamiño et al. [28] found similar rates of gametogenesis along a strong sedimentation gradient. Further, Cox [59] found no differences in reproductive output, eggs per bundle, and egg size between B and NB parents in the spawning season immediately following the 2004 mild warming event. Resilience in M. capitata may be due to its capacity to maintain energetic stability under stress [53], here evident by the completion of gametogenesis even at the cost of producing fewer eggs per bundle with higher proportion of irregularity in shape in 2016 than in 2017. One hypothesis to explain similar reproductive traits in bleached and nonbleached parents, is that after the thermal stress (Septemebr-October), there is still time for the colonies to recover (~5–6 months) and develop gametes that can be released during the spawning season (May-August) [14,53,60,63]. Furthermore, Rodrigues & Padilla-Gamino [77] found that M. capitata colonies that bleached allocated 10% more carbon to gametes despite bleaching by limiting the allocation of carbon to adult tissues, with 50–80% less carbon allocated to bleached compared to non‑bleached colonies. Compared to other species, M. capitata prioritizes gametogenesis at the expense of the adult colony. Maintaining egg traits such as size and biochemical composition would serve as an advantageous strategy to ensure ecological fitness of parents and their developing offspring [61,78,79]. For example, there may be an optimal egg size that needs to be achieved to ensure successful fertilization [80,81]. It is notable that the relationship between egg size and number of eggs per bundle in our study has shifted from prior studies; we found 10–12 eggs per bundle in 2016–2017 compared to 15–18 eggs per bundle in studies and egg size was 11% larger in our study than previous studies [59,60]. This apparent tradeoff in reproductive effort suggests plasticity in response to environmental changes and emphasizes the need for long-term studies to detect changes in sexual reproduction [14,35,36]. While further examination of egg traits, such as total lipid content and composition of lipid classes, was beyond the scope of this study due to limited material available, larger egg volume could be beneficial in storing lipids and carbohydrates as well as increased surface area for slower sperm to fertilize eggs in the water column.

High inter- and intraspecific variation in thermal tolerance contribute to reproductive consequences after bleaching events [8,17,33]. For example, there were no differences in percent reproductive polyps between bleached and nonbleached colonies of acroporid species at Heron Island on the Great Barrier Reef after the 1998 bleaching event [82]. Baird and Marshall [8] found that the bleaching response of Acropora millepora did not influence fecundity, whereas the bleaching response of Acropora hyacinthus strongly influenced the completion of gametogenesis. It is important to emphasize that although reproductive capacity after bleaching events can be greatly suppressed, there are species and populations that are resistant and/or more able to recover from bleaching [7,8,17,39,59,82]. Distinctive populations carrying resilient individuals are critical to identify and protect, particularly if they are successful in continuing sexual reproduction to replenish impacted neighboring reefs [83,84]. Coral reproductive modes and strategies have evolved to withstand environmental fluctuations and severe selective pressures, but the question of how much thinning can a population withstand without complete collapse remains.

 4.2 Parental effects on fertilization and offspring survivorship

We demonstrate parental effects, or cross-generational plasticity, in M. capitata, with parent cross-type having an effect on fertilization and embryonic development with maternal effects apparent in offspring survivorship. Fertilization success differed by cross-type which may be due to gametic compatibility [85]. Such compatibility could be driven by gamete-recognition proteins that mediate fertilization through chemoattraction, binding, and fusion of egg and sperm [85–87]. Furthermore, high gamete compatibility may explain the advanced rate in cell division during embryogenesis in offspring from NB × NB and B × B cross-types. The lack of compatibility observed in crosses between B × NB and B × NB could potentially result from lineage crossing. However, in our study, we were unable to analyze the genetic composition of the parent organisms and we could not determine if they belonged to distinct parental lineages. Future studies should take into account parental lineages to better understand gamete compatibility, inheritance patterns and traits that can lead to increased genetic diversity or novel offspring phenotypes.

Egg-sperm compatibility has been observed as a mechanism for pre-zygotic isolation to select for populations that are likely to succeed under intense environmental pressures, such as temperature [88–91]. With regards to sperm selection, Henley et al. [92] demonstrated sperm motility in M. capitata is strained with a severe decline that may be associated with damaged mitochondria in response to heat stress. Eggs from parent colonies that were resistant to bleaching had offspring with notably higher survivorship regardless of the sperm donor bleaching history [42]. More pronounced benefits of nonbleached egg donors support previous work of maternal provisioning in coral offspring [93–95]. Previous studies have demonstrated that beneficial cross-generational plasticity can occur from maternal effects observed in offspring survivorship. Benefits of maternal effects could be associated with phenotypic traits that help overcome hurdles created by thermal stress such as energetic provisioning through lipid reserves stored in the eggs and larvae [61,96,97], mitochondria [96], or vertical transmission of Symbiodinaceae from the parent into the eggs [64,94,97].

M. capitata houses the endosymbionts Cladocopium spp. and Durusdinium spp., formerly Clade C and D, respectively. It has been shown that M. capitata colonies associate with Durusdinium spp. in more challenging environments such as high light and variable thermal regimes [61,98,99]. After a bleaching event, there was a rise in the relative proportion of the heat-tolerant symbiont Durusdinium spp. in M. capitata colonies across most areas in Kāneʻohe Bay [98]. However, despite this increase, the overall composition of Symbiodiniaceae symbionts remained largely unchanged, and distinct regions of the bay retained their pre-bleaching compositions. In M. capitata, these symbionts are vertically transferred to the eggs creating offspring with different assemblages [64] that could confer different physiological attributes to the offspring. For example, Little et al. [100] found that Acropora juveniles grew faster when infected with Cladocopium spp. than Durusdinium spp. (formerly clade C and D, respectively)and Abrego et al. (2008) showed enhanced physiological tolerance and higher 14C photosynthate incorporation in juveniles infected with Cladocopium spp. (clade C1). Padilla-Gamiño et al. [64] showed that Cladocopium spp. is more likely to be transferred to M. capitata eggs, but further research is needed to better understand transfer mechanisms, and how different symbionts influence survival, tolerance and/or tradeoffs in larvae and juveniles.

 4.3 Interventions for thermal tolerance

Research on coral reefs has become greatly focused on identifying human interventions (i.e., assisted evolution) that support biological persistence and resilience against anthropogenic stressors [101–103]. Developing effective interventions to implement has become increasingly urgent to protect shallow-dwelling coral reef ecosystems [103]. Current strategies proposed to overcome bottlenecks in early life history include identifying genetic adaptation [104], environmental hardening through non-genetic or epigenetic mechanisms [105–110], manipulation of Symbiodiniaceae symbionts [41,111,112], cryopreservation for coral conservation [113], and selective breeding [94,95,114].

Human interventions, such as selective breeding in coral sexual propagation, has been proposed as one of the viable options to maintain genetic diversity and increase resilience in restoration efforts [15,102,103,105,115,116]; however, feasibility to potentially scale up efforts remain limited and costly without full understanding of tradeoffs [117,118]. Our study supports the potential for selective breeding and environmental hardening to have positive fitness consequences. In our study, bleaching in M. capitata did not severely disrupt reproductive output or egg traits measured (size and abnormality), but the use of eggs from NB colonies in the intentional crossing of gametes produced offspring with higher settlement and survivorship, while bleached corals had higher overall fecundity to balance reduced survivorship and settlement. These results are important to maximize restoration efforts through selective breeding by identifying candidate colonies in the natural environment or through manipulated stress tests and performing crosses using the gametes of resilient colonies. We encourage further research to test the efficacy and trade-offs of human-assisted evolution, particularly selective propagation and environmental hardening, designed to increase coral resistance that would ensure the continuation of coral reefs confronted by global climate change.

We would like to thank members of the Gates Coral Laboratory for their technical support and advice, especially Jen Davidson and Dr. James Guest. We are grateful to Mary Hagedorn, Amy Moran, and Peter Marko for their feedback on the manuscript, the many volunteers especially Dyson Chee, Megan Buras, Katie Allen, Shayne Fabian, and Kat McPherson and the security Greg Miranda and Moses at Moku o Loʻe who ensured safety and the success of this research. We dedicate this research to Dr. Ruth D. Gates and her infectious enthusiasm that pushed assisted evolution to the forefront of coral biology–you rock!

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Producing coral offspring with cryopreserved sperm: A tool for coral reef restoration. Sci Rep. 2017;7(1):14432. doi: 10.1038/s41598-017-14644-x 29089578 Chan WY, Peplow LM, Menéndez P, Hoffmann AA, van Oppen MJH. Interspecific hybridization may provide novel opportunities for coral reef restoration. Front Mar Sci. 2018;5:157. Epstein N, Bak RPM, Rinkevich B. Applying forest restoration principles to coral reef rehabilitation. Aquat Conserv Mar Freshw Ecosyst. 2003;13(5):387–395. Hancock JR, Barrows AR, Roome TC, Huffmyer AS, Matsuda SB, Munk NJ, et al. Coral husbandry for ocean futures: leveraging abiotic factors to increase survivorship, growth, and resilience in juvenile Montipora capitata. Mar Ecol Prog Ser. 2021;657:123–133. Edwards AJ, Guest JR, Heyward AJ, Villanueva RD, Baria MV, Bollozos ISF, et al. Direct seeding of mass-cultured coral larvae is not an effective option for reef rehabilitation. Mar Ecol Prog Ser. 2015;525:105–116. Chamberland VF, Petersen D, Guest JR, Petersen U, Brittsan M, Vermeij MJ. New seeding approach reduces costs and time to outplant sexually propagated corals for reef restoration. Sci Rep. 2017;7(1):18076. doi: 10.1038/s41598-017-17555-z 29273761 10.1371/journal.pone.0290479.r001 Decision Letter 0 Fujimura Atsushi Academic Editor 2025 Atsushi Fujimura 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 Version 0

5 Nov 2023

PONE-D-23-24872Parental effects provide an opportunity for coral resilience following major bleaching eventsPLOS ONE

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Reviewer #1: Reviewer comments on lines following "~".

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Introduction:

“Coral bleaching is known to impact sexual reproduction (Baird & Marshall, 2002; Fisch et al.,2019) and recruitment (Hughes et al. 2019; Price et al., 2019)… colonies that bleached and recovered (Ward et al., 2002).”

~ This reads like regurgitated info. I recommend adding a concluding sentence to this paragraph that highlights what you want the reader to understand about the importance of your project from the inclusion of this paragraph in the intro.

~ There is differential bleaching observed dramatically across individuals so close to each other on the same reef. Has microsatellite work been done on these colonies? Is there a possibility of cryptic species/lineages across the bleaching susceptible and resistant corals here?

~ The authors talk a lot in the intro about how bleached individuals do not produce as many eggs as individuals that did not bleach for several coral species, but the authors findings (and included previous work on M. capitata) do not support this. Why include so many examples of this information in the intro then? Maybe just say concisely in discussion.

“This reproductive response may contribute to its ecological success along the fringing and patch reefs of Kāneʻohe Bay (Kolinski, 2004).”

~ This statement is contradictory to some of the authors’ explanations of their results and statements in the discussion. I would consider including this association with different wording. Maybe say “This reproductive response may have previously contributed..” since you are talking about historical characteristics that may have been beneficial to the reproductive efforts of this species, but the authors are now highlighting how these characteristics may no longer be beneficial for some individuals in this paper.

“This study was designed to assess selective processes in nature confronted by climate change while also exploring breeding techniques as an intervention strategy for coral restoration to maintain genetic diversity.”

~ I like this statement that tries to summarize the importance of this study at the end of the intro, but the wording is a bit confusing. Rework this sentence for more clarity.

Methods:

2.1 Selecting parent colonies and spawning events

~ The first paragraph of this section reads like it should be included in the introduction, instead of in the methods. Also, you highlight that Symbiodiniaceae symbionts are provided by the mothers to the eggs. Does this study assess dominant symbiont association of the mothers and/or larvae produced? That would help a lot with connecting impacts observed to more and less beneficial symbiont associations for this species.

2.3 Fertilization success and larval survivorship

“Five days post-fertilization, 10-15 larvae per conical tank were placed in a 10-mL well-plate filled with 5-mL of FSW”

~ Hyphens should be removed between 10 and mL, along with 5 and mL.

Results:

3.1 Sexual reproduction and egg traits

“The proportion of colonies releasing gametes significantly differs by year (P < 0.001). In 2017, the proportion of colonies participating in spawning events was 36% lower than in 2016.”

~ It would help the reader to have the actual number of colonies that spawned for each group and each year listed here (or maybe in a table). Can also be in parentheses stated in a sentence.

~Also, this looks like some legacy effect of heat stress across B and NB since both had less spawning individuals in 2017. Would the findings for egg production and frequency of abnormal eggs not also represent possible legacy effects from the bleaching events? Might be an important consideration for how long it actually takes corals to fully “recover” from bleaching, whether or not they were visibly bleached.

“In 2016, the spawning season following consecutive bleaching events, colonies that bleached and recovered had 22.5% higher mean total reproductive output than colonies that did not bleach, although this was not statistically significant”

~ What explanation do the authors have for this since this seems contrary to bleached individuals being less fit than individuals that did not bleach.

~ Was any data collected to make sure the bleached and unbleached colonies were not in fact cryptic lineages/species? If not, then the incompatibility in BxNB and NBxB crosses could be a product of lineage crossing rather than the listed reasons by the authors. Indicating the authors might not be comparing apples to apples. This option should also be included in the manuscript to explain the differences in results if there was no testing of these colonies to eliminate this possibility.

3.2 Fertilization, survivorship, and settlement

“There were no differences between BxB and the other three cross-types (P ≥ 0.342).”

~ the font changes here, make sure this is formatted correctly

Cell division was faster for within than between type crosses.

~ Cryptic lineages could explain this as well.

Egg donor’s bleaching history affects Percent swimming larvae and settlement of larvae. Along with lower survivorship of larvae from bleached donors. History of parent did not affect sperm.

BxB had highest mortality(~92%), then BxNB, and finally NBxNB (~77%) over 59 days.

~Differences in egg quality? lipid class analyses of the eggs from B and NB would greatly benefit the impact and explanation of findings in this paper, due to the observed impacts of B and NB eggs on larvae survival. This would allow comparison of egg components, which might be driving differences in larval survival since eggs were of comparable volumes across B and NB groups and the results could be compared to previous measures of egg lipids from cited papers within this manuscript. If samples are available, the authors might consider adding these results.

Discussion:

“These results demonstrate the impacts of marine heatwaves and coral bleaching on reproductive capacity in the following spawning season with the capacity to recover in subsequent nonbleaching years.”

~ I agree with this statement by the authors, but I think it should also be emphasized here that similar impacts to egg production were observed whether or not the colonies were visibly bleached (B or NB) in this study. This supports that corals that are not bleached are also suffering some of the same impacts (i.e., the number of spawning individuals, number of eggs produced per bundle, number of abnormal eggs, etc.) as bleached corals after bleaching events, even if they are better positioned to survive and recover than bleached individuals.

4.1 Reproductive capacity after bleaching events:

~ Authors should define what is meant by “recover” here. Colonies after bleaching events are more susceptible to disease for more than a year after and can have reduced energy reserves for longer than that. If you consider these part of “recovery” of the coral, then ~5-6 months is not long enough to consider the corals as recovered. The authors also note in the intro that “The impacts of coral bleaching may last for months to years after the initial thermal stress” and cite a paper indicating 3 to 5 years for coral recovery, both of these statements contradict the statement made in the discussion of ~3 to 5 months for recovery. The authors need to be consistent in how they define “recovery” throughout the manuscript.

“Maintaining egg traits such as size and biochemical composition would serve as an advantageous strategy... long-term studies to detect changes in sexual reproduction (Levitan et al., 2014; Hagedorn et al., 2016; Price et al., 2019; Schlesinger et al., 2019).”

~ Why would eggs be larger than before? Authors should explain their thoughts on this finding more.

~ Also, this might be a good place to highlight that the plasticity utilized by B and NB may have been equally beneficial for B and NB before but that now egg quality might be suffering for B mothers due to continued impacts.

4.2 Parental effects on fertilization and offspring survivorship:

“In this study, bleached colonies had similar fertilization success to nonbleached colonies, the thermally tolerant individuals will likely have a stronger selective advantage as warming continues to weed out thermally sensitive adults and their offspring (Drury et al., 2022).”

~ I do not understand this statement, authors need to clarify more here. How can the B and NB colonies having similar fertilization success mean there is stronger selective advantage for NB adults and offspring? I understand why the authors are trying to link in natural selection towards more resilient individuals, but that is contrary to the results discussed in the beginning of this sentence. Especially since similar fertilization, number of eggs, and volume of eggs were found across both these groups for both years following the bleaching events. Rework this statement for clarity.

“Beneficial cross-generational plasticity through maternal effects observed in offspring survivorship may be attributed to energetic provisioning through lipid reserves stored in the eggs and larvae (Jones et al., 2011; Padilla-Gamiño et al., 2013; Rivest et al., 2017), mitochondria (Dixon 15 et al., 2015), or vertical transmission of Symbiodinaceae from the parent into the eggs (Jones et al., 2010;Padilla-Gamiño et al., 2012; Quigley et al., 2016).”

~ This statement should be fleshed out more and clarified further rather than just being a list of semi-unrelated traits. For example, what is meant by “Mitochondria” in this list because it does not look like it is connected to energy reserves.

“M. capitata houses the endosymbionts Cladocopium spp. and Durusdinium spp., formerly Clade C and D, respectively.”

~ It is outdated and improper to list genera levels of specific symbiont-host associations. Since only one coral species is assessed in the current study the specific symbiont species that associate with this coral in this area should be listed instead (i.e., Durusdinium trenchii instead of “Durusdinium spp.”)

~ Also, Padilla-Gamiño et al., 2013 does not actually identify the symbionts in M. capitata so it would be more correct to cite Padilla-Gamiño et al. 2012 in PLoSOne instead, or better yet de Souza et al. 2022 since that has more recent and specific symbiont identifications for M. capitata in Kāne‘ohe Bay.

“In M. capitata, these symbionts are vertically transferred to the eggs creating offspring with different assemblages (Padilla-Gamiño et al., 2013) that could confer different physiological attributes to the offspring.”

~ This should also be Padilla-Gamiño et al., 2012 PLoSOne that looked at symbionts in eggs, rather than 2013.

“For example, Little et al. (2004) found that Acropora juveniles grew faster when infected with clade C than clade D, and Abrego et al. (2008)”

~ Same comment about symbiont names here. Look up the specific genera for these symbiont-host associations. It is not appropriate to call the symbionts by the antiquated “Clades” designations, also the authors should be consistent with how they reference the symbionts throughout the manuscript rather than changing between genera names and clade names.

“Our results suggest that nonbleached colonies have higher ecological fitness than their bleached counterparts and may benefit the maintenance of genetic diversity while guiding populations towards higher thermal tolerance in a warming ocean.”

~ Authors discuss the importance of the photosynthetic endosymbiont to the host and that this symbiont is vertically transmitted to the larvae by the mother. After disturbance the dominant Symbiodiniaceae community members can shift rapidly back to a previous non-disturbed community state (sometimes within a few months to a year, see references below this comment). It is important to know what symbionts were at play in B and NB parents (and the sampled larvae) before making any statements about what impacts these may have had on the larvae survivorship in the current study.

Were the symbionts ever identified for the parent colonies or subset of larvae to determine if any claims in this paragraph were relevant to the corals and findings of this study? If not, then the concluding sentence of this paragraph should be removed since no data collected by the authors corroborates the dominant symbiont of the mothers or larvae.

• Elder H, Million WC, Bartels E, Krediet CJ, Muller EM, Kenkel CD (2022) Long-term maintenance of a heterologous symbiont association in Acropora palmata on natural reefs. The ISME Journal

• Thornhill DJ, LaJeunesse TC, Kemp DW, Fitt WK, Schmidt GW (2006) Multi-year, seasonal genotypic surveys of coral-algal symbioses reveal prevalent stability or post-bleaching reversion. Marine Biology 148:711-722

4.3 Interventions for thermal tolerance:

“manipulation of coral-algal endosymbiosis”

~ the authors have not referenced the Symbiodiniaceae as “algae” yet in this manuscript or defined the use of “algae” to mean Symbiodiniaceae. For consistency in text and lack of confusion with readers this should be changed to reflect the terms used to reference the Symbiodiniaceae symbionts throughout the manuscript.

“Human interventions applying selective breeding in coral sexual propagation has been proposed as one of the viable options to maintain genetic diversity and increase resilience in restoration efforts (Barott et al., 2021; Epstein et al., 2003; van Oppen et al., 2015, 2017; NASEM, 2019; Hancock et al., 2021); however, feasibility to potentially scale up efforts remain limited and costly without full understanding of tradeoffs (Edwards et al., 2015; Chamberland et al., 2017).”

~ Need commas here like "Human interventions, such as selective breeding in coral sexual propagation, has been..."

~ The way this is written it sounds here like only thermally resistant colonies (NB colonies in this paper) should be considered for selective breeding, but thermal resilience is not the only trait corals need to survive anthropogenic climate change, which also causes increased disease prevalence and ocean acidification along with other factors. Since B adults have also been successful thus far, it is likely they have a different trait necessary for acclimating well to their home reefs, like disease resistance, greater trophic plasticity, or that they utilize different trade-offs to deal with thermal stress. The data here does not necessarily support selective breeding for thermal tolerance with individuals that do not bleach, but instead the preference for using eggs from NB parents when creating crosses following bleaching events. Therefore, the authors should consider making less definitive statements here.

Figures:

Figure 3: Reproductive output was higher in bleached colonies and had fewer abnormalities than NB colonies, how do the authors explain this in relation to their statements that the bleached colonies are less fit in the discussion?

Figure 4: Panel B should include x-axis labels for clarity. It is confusing to look at a separate panel for the axis labels, especially since only those two panels in the figure share labels.

Reviewer #2: PLOS ONE Review

General comments

Lenz and colleagues present results from experiments spanning multiple years to investigate the potential for multigenerational effects of thermal stress on the coral Montipora capitata. They found that while thermal stress impaired aspects of reproduction regardless of if a colony bleached or not, parental bleaching history was an important driver of offspring success. These findings supporting the concept that bleaching-resistant colonies could be targeted for selective breeding to produce offspring that to support the restoration of more thermally-tolerant coral populations. The authors do an excellent job of highlighting the importance of their findings and avoid overextending their conclusions beyond the scope of their study. Their findings are particularly timely given the increasing severity, prevalence, and mortality caused by thermal stress on coral reefs across the globe. I have relatively minor comments that are articulated below that I recommend addressing before the ms is published.

There are no line numbers on the submission, thus I have not included them below but did my best to describe where in the ms I am referring to by referencing paragraphs within sections.

Introduction: The introduction is well written and provides a thorough yet concise overview of how thermal stress can impact coral reproduction and population dynamics, while also highlighting the knowledge gap this paper addresses (intra- and intergenerational impacts of bleaching).

Paragraph (henceforth, P) 4: The sentence regarding high recruitment in Mo’orea after the 2016 thermal stress is incomplete/lacking details compared to the other examples. The examples presented in P3 and P4 provide clear links between thermal stress/bleaching and reproduction in corals. However, the Mo’orea example suggests that the minor 2016 bleaching event may have had the potential to affect reproductive output, but provides no concrete information that some corals were reproductively compromised. How it is currently written, one could conclude that the minor bleaching in 2016 did not impact reproduction. Suggest revising to better leverage this example for the introduction.

P5: Sentence with “~70% of reef corals on shallow reefs” – is there a reason that these corals are distinguished as reef corals? If not, remove to avoid confusion, or if there is an important distinction between reef corals and other corals, provide details for clarity.

Methods

P2: Do the authors have any bleaching history of the colonies used in this study during 2014? If so, that would be worthwhile to include here or as supplemental information.

P5: For quantification of larval survivorship – was this recorded as the number of larvae remaining in an individual well-plate, or did this take into account the number of settlers (which is needed to quantify larval survivorship)? For example, if a well-plate started with 10 larvae, and 5 had settled after 7 days and 5 remained swimming, was this recorded as 100% survivorship (5 out of the 5 larvae remaining) or 50% survivorship (5 of the initial 10 placed in the well-plate)? This should be clarified in the methods section.

P6: (second sentence) – revise for clarity. Mentions using a generalized mixed effects model twice in the same sentence, but is only referring to one model.

P7: The survivorship curve analysis includes days 23 and 27, but these days are not mentioned in the previous section regarding when the number of offspring alive were counted (days 6, 7, 28, 53, and 59).

P7: Was the effect of fragmentation included in any of the models to test if fragmenting impacted reproduction? Given the reported size of the fragments, there is not reason to believe there would be any impact, but this would be worth mentioning somewhere in the ms.

Results

Discussion: The discussion is well written and thorough. The authors did a nice job of placing their findings in the context of previous work (e.g. reduction in no. of eggs/bundle), proposing potential hypotheses for the patterns they observed (and supporting evidence), expanding the discussion to broader concepts of population ecology, and explaining how these results can be used to inform interventions like selective breeding.

Figures

Figure 2: Suggest choosing a color palette that facilitates distinguishing between B and NB colonies. I can understand why these were chosen to look like healthy (NB) vs. bleached (B) colonies, but the colors are difficult to quickly distinguish.

Figure 3: 3A -Lines are not appropriate for this type of plot – these data would be more clearly presented as a bar plot since each night of spawning is a discrete event, not continuous as the lines connecting the dots imply.

3B-E: suggest adding a legend for the B and NB colors even though it is on the x-axis labels (and hidden in the 2016 panel of Figure 3A.

Figure 4B: Add x-axis labels to this plot, it is difficult to follow them up from Fig 4C.

4C: Error bars on stacked bar plots make for very busy figures that are difficult to interpret and take away from the data being shown. Suggest individual bars for each of the developmental stages for each cross – this would make the difference in developmental stages easily observable by looking at Fig 4C.

4D-F: Similar to Fig 4, these colors are similar and thus hard to distinguish, especially given the that these are small panels within a 6-panel figure. Suggest selecting a different color scheme.

Reviewer #3: I enjoyed reading "Parental effects provide an opportunity for coral resilience following major bleaching events". I think the manuscript is sound and comprehensive look at colony fecundity, well-written, and almost ready for publication. My only reservation is some aspects of the discussion don't quite jive with the findings which I will detail below. However, I think this is good work and after a tidy up of some claims in the discussion, warrants publication. Despite my concerns with the discussion, I think the summary in the last paragraph "In our study... to balance reduced survivorship and settlement" to be a perfect summary of the findings.

Major concerns

*To me, the main result is that parental bleaching history did not significantly affect colony fecundity, the size of eggs, or eggs per bundle. If true, this runs counter to a lot of the discussion which emphasizes how bleaching affects reproductive capacity. For example on page 12, authors claims that this study "demonstrates the impact of bleaching on reproductive capacity" but the stated evidence doesn't show sig differences between B/NB (egg volume, eggs/bundle, abnormalities). Moreover, this carries over to the fertilization, survival, and settlement result where there seem to be no differences between larvae from B and NB colonies. On pg 15, authors claim that NB colonies have higher fitness, but I don't see how the results of this study support that for the reasons above plus seemingly minor effects on survival (I didn't see the magnitude of maternal effect on survival mentioned... seems small).

*Consider making maternal effects a larger focus of the discussion if this was the main sig result (and few outright B/NB differences).

*Discussion seems to be a bit fast and loose when describing insignificant effects. A focus on effect size could help support your claims. For example on pg 12, authors claim that 'maternal effects are demonstrated from fertilization through survival' but the maternal effect on fertilization is insignificant and the effect on survival seems to be quite small (Fig 4E inset). Same issue with "colonies have higher fitness" claim on pg 15.

*The sig. difference in proportion colonies spawned 2016 vs 2017 seems to be largely due to the third month, which is a light spawn in 2016 and no spawn in 2017. There seems to be little difference in months 1 and 2 between years. This may affect how the effects of 2015 bleaching are interpreted

*Where is there a significant maternal effect of NB/B? Do analyses need to be redesigned to test for maternal NB/B versus paternal NB/B?

Minor concerns

*Not sure if this is due to journal format, but Tables were not included in the submission which made it difficult to evaluate some claims

*I doubt it will change the conclusions, but technically, proportions and counts shouldn't use gaussian error distribution in linear mixed model

*pg11 Results section 3.2 p value 0.342 is in a different font

*Consider shrinking the discussion section on interventions

* Could be due to PDF conversion, but figures were blurry

* pg 12 please add 'for example' to describe what "delayed beneficial maternal effects might be" (you give more details later). maybe maternal effects need to be earlier in the disc. and a bigger part of the manuscript.

* What led to 2016 vs 2017 differences in abnormality?

* What could have led to B*B and NB*NB having faster development?

*Settlement rates seem a bit low. Is settlement typically <50% for this species?

**********

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Submitted filename: Review_PONE-D-23-24872.docx

Submitted filename: PLOSONE_Review.docx

 10.1371/journal.pone.0290479.r002 Author response to Decision Letter 0 Submission Version 1

2 Apr 2024

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“This work was supported by funding from Paul G. Allen Family Foundation to RG and the National Science Foundation Graduate Research Fellowships to EAL. We would like to thank members of the Gates Coral Laboratory for their technical support and advice, especially Jen Davidson and Dr. James Guest, we are grateful to Mary Hagedorn, Amy Moran, and Peter Marko for their feedback on the manuscript, the many volunteers especially Dyson Chee, Megan Buras, Katie Allen, Shayne Fabian, and Kat McPherson and the security Greg Miranda and Moses at Moku o Loʻe who ensured safety and the success of this research. We dedicate this research to Dr. Ruth D. Gates and her infectious enthusiasm that pushed human-assisted evolution to the forefront of coral biology – you rock!”

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Response 3: Thank you for the clarification, we have removed information regarding our funders from the acknowledgements and would like to include the following as our Funding Statement:

“This work was supported by funding from Paul G. Allen Family Foundation to RDG and the National Science Foundation Graduate Research Fellowships to EAL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

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Response 4: Data repository is on GitHub

https://github.com/ealenz/Mcap-BNB-Reproduction-SelectiveBreeding

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Response 6: These images were produced and provided by the lead author of the paper. I, Elizabeth Lenz, give permission for these images to be used for the publication.

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Response 7: Thank you, we have included the tables as part of the main manuscript.

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Response 8: We reviewed our reference list and ensured it is updated.

Additional Editor Comments:

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Response 9: and have included the following:

“Dr. Ruth D. Gates passed away before the submission of the final version of this manuscript. Dr. Elizabeth A. Lenz accepts responsibility for the integrity and validity of the data collected and analyzed."

Please also add a "†" symbol next to the author's name in the author list and include a note that this author is deceased.

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Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Partly

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Reviewer #2: Yes

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Reviewer #2: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

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Direct quotes being addressed from manuscript are in "".

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Response: Thank you for providing clear instructions and guidance on the feedback provided. We greatly appreciate the clarity and thoughtfulness behind the comments.

Introduction:

“Coral bleaching is known to impact sexual reproduction (Baird & Marshall, 2002; Fisch et al.,2019) and recruitment (Hughes et al. 2019; Price et al., 2019)… colonies that bleached and recovered (Ward et al., 2002).”

~ This reads like regurgitated info. I recommend adding a concluding sentence to this paragraph that highlights what you want the reader to understand about the importance of your project from the inclusion of this paragraph in the intro.

Response: We appreciate the feedback on providing a stronger concluding sentence to help contextualize the study for the readers and provide stronger understanding.

LN 81-93: “Previous studies have identified some of the way coral bleaching can impact aspects of sexual reproduction (Baird & Marshall, 2002; Fisch et al., 2019) and dampen recruitment (Hughes et al. 2019; Price et al., 2019). For example, after the 1987 coral bleaching event in the Caribbean, Orbicella annularis recovered from bleaching by metabolizing tissue biomass, but did not complete gametogenesis in the following months, whereas colonies that had not bleached of the same species were able to develop and release gametes (Szmant & Gassman, 1990). Similarly, during the 1998 bleaching event on the Great Barrier Reef, bleached corals showed high variation in reproduction compared to colonies resistant to bleaching nearby that experienced the same thermal stress. For acroporid species, reproductive polyps were more common in colonies that did not bleach, with larger eggs at higher densities per polyp than colonies that bleached and recovered (Ward et al., 2002). More resolution is needed to better understand the impact and extent of coral bleaching events on the early life cycles of coral, from the stress event through recruitment.”

~ There is differential bleaching observed dramatically across individuals so close to each other on the same reef. Has microsatellite work been done on these colonies? Is there a possibility of cryptic species/lineages across the bleaching susceptible and resistant corals here?

Response: We did not conduct microsatellites on these colonies and cannot conclude a pattern of cryptic species/lineages within the samples. However, there is evidence that colonies more vulnerable to bleaching have clade C symbionts and that corals more resistant to bleaching are dominated by thermotolerant clade D (Cunning et al. 2016). Since 2014, former members of the Gates Coral Lab, including Drs. Raphael Ritson Williams, Christopher Wall, Hollie Putnman, Ross Cunning, Shayle Matsuda, Katie Barott and now the Coral Resilience Lab at the Hawaiʻi Institute of Marine Biology continue to conduct studies to understand and explain the mechanisms that drives the differential bleaching responses to marine heatwaves in Kāneʻohe Bay. This mechanism behind this differential bleaching phenomenon remains unknown.

~ The authors talk a lot in the intro about how bleached individuals do not produce as many eggs as individuals that did not bleach for several coral species, but the authors findings (and included previous work on M. capitata) do not support this. Why include so many examples of this information in the intro then? Maybe just say concisely in discussion.

Response: The intent of the introduction was to provide a thorough overview of what has been observed in the past, particularly with regards to corals in the acroporidae family. We believe it is worthwhile to keep as is and that a hypothesis for the difference in results could be attributed to acclimation over the last decade as waters have warmed.

“This

Submitted filename: 4. Response to Reviewers Final.docx

 10.1371/journal.pone.0290479.r003 Decision Letter 1 Fujimura Atsushi Academic Editor 2025 Atsushi Fujimura 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 Version 1

5 Jun 2024

PONE-D-23-24872R1Parental effects provide an opportunity for coral resilience following major bleaching eventsPLOS ONE

Dear Dr. Lenz,

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. Please see the comments below from one of the reviewers.

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Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

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Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: Maybe they were lost in the format of my response, but several comments were not addressed in the authors response. I list them here as suggestions that may improve the manuscript.

Major concerns:

*Discussion seems to be a bit fast and loose when describing insignificant effects. A focus on effect size could help support your claims. For example on pg 12, authors claim that 'maternal effects are demonstrated from fertilization through survival' but the maternal effect on fertilization is insignificant and the effect on survival seems to be quite small (Fig 4E inset).

*The sig. difference in proportion colonies spawned 2016 vs 2017 seems to be largely due to the third month, which is a light spawn in 2016 and no spawn in 2017. There seems to be little difference in months 1 and 2 between years. This may affect how the effects of 2015 bleaching are interpreted

*Where is there a significant maternal effect of NB/B? Do analyses need to be redesigned to test for maternal NB/B versus paternal NB/B?

Minor concerns:

*I doubt it will change the conclusions, but technically, proportions and counts shouldn't use gaussian error distribution in linear mixed model

**********

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Reviewer #3: No

**********

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 10.1371/journal.pone.0290479.r004 Author response to Decision Letter 1 Submission Version 2

3 Sep 2024

Reviewer #3: Maybe they were lost in the format of my response, but several comments were not addressed in the authors response. I list them here as suggestions that may improve the manuscript.

RESPONSE: We apologize for missing reviewer 3 comments in the first round of our response to reviewers. We appreciate the opportunity to address them as we had not seen Reviewer 3’s comments prior.

Major concerns:

*Discussion seems to be a bit fast and loose when describing insignificant effects. A focus on effect size could help support your claims. For example on pg 12, authors claim that 'maternal effects are demonstrated from fertilization through survival' but the maternal effect on fertilization is insignificant and the effect on survival seems to be quite small (Fig 4E inset).

RESPONSE: We appreciate the comment and in response we have revised the results and discussion. We have added the following language to the results:

LN 328-338: “While reproduction continued in the colonies examined, we found that cross-type did have an effect on fertilization, embryonic development, and percent larval survivorship (Figure 4; Table 2). Specifically, fertilization success in the NB × NB cross-type was higher than the B × NB and NB × B cross-types (Figure 4B; Table 2; post-hoc P = 0.002 and 0.010, respectively) but not B × B (post-hoc P = 0.163). The fertilization success in cross-type B × B also did not differ between NB × B (post-hoc P = 0.250), but was higher than B × NB (post-hoc P = 0.047).. Cell division advanced beyond the 2-cell stage more quickly for within cross-types (B × B and NB × NB) than between cross-types (B × NB and NB × B) at 3-h post-fertilization. Embryos from both B × B and NB × NB cross-types reached the 16-cell stage at 3-h post fertilization, whereas embryos from B×NB and NB × B crosses developed at a slower rate and only reached the 4-cell stage (Figure 4C, Table 2).”

We also added the following to the Discussion:

LN 420-422” “We demonstrate parental effects, or cross-generational plasticity, in M. capitata, with parent cross-type having an effect on fertilization and embryonic development with maternal effects apparent in offspring survivorship.”

*The sig. difference in proportion colonies spawned 2016 vs 2017 seems to be largely due to the third month, which is a light spawn in 2016 and no spawn in 2017. There seems to be little difference in months 1 and 2 between years. This may affect how the effects of 2015 bleaching are interpreted

RESPONSE: We understand the reviewer’s concern that there is little difference between the first two months of the years compared and impact may be driven by the lack of spawning in the third month. We have added the following to the results:

LN 307-311: “The proportion of colonies releasing gametes significantly differed by year (P < 0.001) which may largely be due to some spawning in 2016 compared to no spawning in 2017 during the third month (August). In 2017, the proportion of colonies participating in spawning events was 36% lower than in 2016. In both years, the second month of the spawning season had the highest proportion of colonies spawning.”

*Where is there a significant maternal effect of NB/B? Do analyses need to be redesigned to test for maternal NB/B versus paternal NB/B?

RESPONSE: Montipora capitata are hermaphroditic colonies, see Figure 2B for matrix of crosses. We identified an effect of NB and B for egg donors in the larval survivorship in the Summary of Cox proportional hazards see Figure 4F. We found that larval survivorship was influenced by the bleaching status of the egg donors. Offspring from non-bleached (NB) corals exhibited higher survival rates.

Minor concerns:

*I doubt it will change the conclusions, but technically, proportions and counts shouldn't use gaussian error distribution in linear mixed model

RESPONSE: We greatly appreciate the reviewer pointing out this error in our analysis and we have revised the methods to apply glmer to the proportion and count data. Where assumptions of normality were not met, we applied the Kruskal-Wallace test for the embryonic development variables. Please see our revised methods, results, and tables.

LN 265-297: “All analyses were conducted in R (R Core Team, 2014; v. 3.5.1). We used a generalized linear mixed effects model to determine the effects of bleaching history on spawning activity, number of eggs per bundle, and egg abnormality of the 8 B and 7 NB parental colonies observed (glmer in lme4: Bates et al. 2015) with a binomial (spawn/no spawn and proportion of abnormal eggs) and poisson (eggs per bundle count) response. Bleaching history (B/NB) and year (2016/2017) were included as fixed effects, and spawning month (1/2/3) and colony ID were included as random effects. To analyze total reproductive output and egg size, we used linear mixed effects models (lme in lme4: Bates et al. 2015) with bleaching history and year as fixed effects, and colony ID as a random effect. Analysis of variance (ANOVA) tables were generated using type II sum of squares (Anova in car: Fox and Weisberg 2011).Post-hoc analyses were conducted to further explore significant main effects and interactions. We utilized the emmeans package (Lenth, 2020) to calculate and compare the estimated marginal means (EMMs), which represent the predicted means of the response variable for each level of the fixed effects, adjusted for the other covariates in the model. Pairwise comparisons between the levels of the fixed effects were then performed using Tukey’s Honest Significant Difference (HSD) test to adjust for multiple comparisons. This approach allowed us to identify significant differences between specific treatment groups, while accounting for the variability associated with random effects.

To test the effects of parental bleaching history on offspring performance, we first analyzed the proportion of eggs fertilized using generalized linear mixed effects models with cross-type as a fixed effect and the egg donor and sperm donor as random effects. The proportion of eggs reaching each developmental stage (2-cell, 4-cell, 8-cell, and 16-cell), the Kruskal-Wallace test was applied as the dataset did not meet the assumption of normality. For post-hoc analysis, we performed the Dunn’s test for multiple pairwise comparisons to determine which specific cross-types differed. To analyze the proportion of larvae that settled at 7, 28, and 59-days post-fertilization, we used a generalized linear mixed effects model with cross-type and day (7, 28, and 59-d post-fertilization) as fixed effects and colony ID of egg donor and sperm donor as random effects. Lastly, we generated survivorship estimate curves to visualize offspring fate by cross-type with ggsurvplot of the census over time (i.e., days 6, 7, 23, 27, 28, 53, and 59 post-fertilization) (survfit in survminer; Kassambara et al., 2017). Cox proportional hazards (CPH) model was used to analyze the effects of cross, egg donor, and sperm donor individually on offspring survivorship (coxph in survminer; Kassambara et al., 2017). Dispersion parameters were inspected through a simulation-based approach (DHARMa package: Hartig, 2019).”

 10.1371/journal.pone.0290479.r005 Decision Letter 2 Fujimura Atsushi Academic Editor 2025 Atsushi Fujimura 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 Version 2

18 Sep 2024

Parental effects provide an opportunity for coral resilience following major bleaching events

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 10.1371/journal.pone.0290479.r006 Acceptance letter Fujimura Atsushi Academic Editor 2025 Atsushi Fujimura 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.

26 Sep 2024

PONE-D-23-24872R2

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