DOI: 10.5061/dryad.cz8w9gj9m
title: Negative parental and offspring environmental effects of macroalgae on coral recruitment are linked with alterations in the coral larval microbiome

description: The persistence of reef-building corals is threatened by macroalgal
 competitors leading to a major demographic bottleneck in coral
 recruitment. Whether parental effects exist under coral-algal competition
 and whether they influence offspring performance via microbiome
 alterations represent major gaps in our understanding of the mechanisms by
 which macroalgae may hinder coral recovery. We investigated the diversity,
 variability and composition of the microbiome of adults and larvae of the
 coral Pocillopora acuta and surrounding benthic substrate on algal-removed
 and algal-dominated bommies. We then assessed the relative influence of
 parental and offspring environmental effects on coral recruitment
 processes by reciprocally exposing coral larvae from two parental origins
 (algal-removed and algal-dominated bommies) to algal-removed and
 algal-dominated environmental conditions. Dense macroalgal assemblages
 impacted the microbiome composition of coral larvae. Larvae produced by
 parents from algal-dominated bommies were depleted in putative beneficial
 bacteria and enriched in opportunistic taxa. These larvae had a
 significantly lower survival compared to larvae from algal-removed bommies
 regardless of environmental conditions. In contrast, algal-induced
 parental and offspring environmental effects interacted to reduce the
 survival of coral recruits. Together our results demonstrate negative
 algal-induced parental and offspring environmental effects on coral
 recruitment that could be mediated by alterations in the offspring
 microbiome.
description: Manipulative field experiment on Mo'orea, French Polynesia
 to test the influence of macroalgae on the microbiome of adults and larvae
 of the coral Pocillopora acuta, and surrouding benthic substrate.
 Microbiome data are 16s rRNA gene metabarcoding data and sequenced on a
 Illumina NovaSeq 6000 system. Lab-based and field-based coral recruitment
 experiments were conducted on larval survival, larval settlement and
 post-settlement survival.
description: # Negative parental and offspring environmental effects of macroalgae on
 coral recruitment are linked with alterations in the coral larval
 microbiome
 [https://doi.org/10.5061/dryad.cz8w9gj9m](https://doi.org/10.5061/dryad.cz8w9gj9m) Manipulative field experiment in Mo'orea, French Polynesia conducted between June and December 2020 to investigate competitive mechanisms of macroalgae against corals. The presence/absence of macroalgae was manipulated on coral bommies assigned to two algal treatments : i) algal-removed (N = 4 bommies) and ii) algal-dominated (N = 4 bommies). Colonies of the coral *Pocillopora acuta* (N = 6 colonies per bommies) and tiles (N = 11 tiles per bommies with 2 extra in case of loss) were transplanted on each bommie. Microbiomes of coral adults (N = 72 samples with 3 fragments x 3 colonies x 4 bommies x 2 algal treatment), their larvae (N = 14 samples)  and microbenthic substrates (N = 48 samples with 2 surfaces x 3 tiles x 4 bommies x 2 algal treatments) were studied from 16S rRNA gene metabarcoding (NoveSeq). Coral recruitment experiments on larval survival, settlement and post-settlement survival were conducted testing the effects of parental origins and offspring environmental conditions. Coral larvae and recruits from two parental origins (algal-removed and algal-dominated bommies) were reciprocally exposed to algal-removed and algal-dominated environmental conditions. For survival and settlement experiments, 12 replicated jars per combination of factors: i) algal-removed parental origin x algal-removed seawater and tile (i.e., hereafter “offspring environment”), ii) algal-removed parental origin x algal-dominated offspring environment, iii) algal-dominated parental origin x algal-removed offspring environment, and iv) algal-dominated parental origin x algal-dominated offspring environment. Larval survival was assessed over 6 days. Settlement was recorded 24h after larval deployment. After 24h, the number of settled larvae were counted on the exposed (upperside), cryptic and vertical surfaces of the tiles and individually mapped. After the initial mapping, tiles with settled larvae were returned to their tagged field positions and reexamined at days 7 and 21 to estimate early post-settlement survival ## Description of the data and file structure Available data and scripts are separated into three groups : benthic community composition, microbiome analysis and recruitment experiments. #### Benthic community composition For this analysis, we have submitted the raw benthic cover data (**data_bommie_raw.xlsx**) and script (**bommies benthic cover_chap2.R**). 5 photo-quadrats (20 x 20 m) per bommie of the two algal treatment were taken. For each photograph, the benthic category of 25 random points were assigned. Benthic categories included: bare substrate (i.e., absence of macroorganisms but presumably colonized by microalgae), CCA, cyanobacteria, living hard coral, algal turf (i.e., mixed species assemblages of filamentous algae &lt; 1 cm in height) and macroalgae (upright and anatomically complex algae with canopy height &gt; 1 cm). Within the macroalgae category, 8 genera or species were recorded: *Amansia rhodantha, Chnoospora spp.*, *Dictyota bartayresiana*, *Halimeda spp., Lobophora spp., Padina boryana, Sargassum pacificum* and *Turbinaria ornata.*  In the data, **data_bommie_raw.xlsx** :  * *month* : censored months with june before algal removal and subsequent months after algal removal. For this study, bommies were monitored in August and November, so please only consider these two months here. * *image* : info of the photo-quadrats with the name of the bommie and the number of the photo-quadrats * *trmt:* algal treatment * "algae" for algal-dominated bommies *  "control" for algal-removed bommies * *name* : name of the benthic categories * *nb_point:* number of points recorded of the benthic category * *per_cov* : Percent cover (%) of each benthic category. For each category, the percent cover corresponded to the number of points of that category divided by the total number of identifiable points in the quadrat multiplied by 100 for percent data. For each image, the percent cover sums to 100. * *tot_pt* and *ref_pt* correspond to the total number of points in the quadrats The script, **bommies_benthic_cover_chap2.R**, is divided into three successive numbered sections. Annotations are provided throughout the script for 1) data curation, 2) benthic community structure and 3) detailed benthic community composition. It allows to reproduce figure S3. #### Microbiome analysis We have submitted our raw sequence table with count abundance data of each amplicon sequence variant or ASVs (**seqtab.rds**), taxonomy table (**taxa_chap2.rds**) and sample metadata file (**sampleMetadata.xlsx**) for implementation in the phyloseq R package. Already filtered microbiome data can be accessioned from the file "**ps.filt.rds**" to be directly used for statistical analysis . A single script (**microbiome_clean.Rmd**) allows to repeat the microbiome results and figures 1 and 2. In sample metadata, **sampleMetadata.xlsx** :  * *sample* : sample name * *sampleType* : The type of samples, whether they are blank samples ("blank") or biological samples ("sample") * *sampleGroup* : The group of samples * blank = blank samples which are extraction blanks (i.e., swab, filters, extraction kit, PCR) * sub = tile substrate samples * coral = coral samples * larva = larvae samples * tank = planulation tank samples * *trmt* : algal treatment  * ctrl = algal-removed * ma = algal-dominated * N/A = for blank samples this variable is not applicable * *grp* : variable distinguising the two faces of the tiles, the coral and larval samples * exp = exposed (upperside) of the tiles * cryp = cryptic (underside) of the tiles * coral = coral samples * larva = larval samples * N/A = for blank and tank samples this variable is not applicable * *var*: combined variable of *grp and* trmt * exp_ma = exposed surfaces of tiles from algal-dominated bommies * exp_ctrl = exposed surfaces of tiles from algal-removed bommies * cryp_ma = cryptic surfaces of tiles from algal-dominated bommies * cryp_ctrl = cryptic surfaces of tiles from algal-removed bommies * coral_ma = coral samples of colonies transplanted on algal-dominated bommies * coral_ctrl = coral samples of colonies transplanted on algal-removed bommies * larva_ma = larval samples of brooded by parents transplanted on algal-dominated bommies * larva_ctrl = larval samples of brooded by parents transplanted on algal-removed bommies * N/A = for blank and tank samples this variable is not applicable * *bommie* : identity of the experimental bommies where the samples originated from. Not applicable for blank and tank samples ("N/A") * *bom_pair* : bommies were mapped as pairs on the field * *colony* : identity of the colonies transplanted on bommies. Not applicable for the samples that are not coral samples ("N/A") * *side* : surface of the tiles, either "exposed" or "cryptic" * *tile* : identity of the tiles with the 1st number corresponding to the bommie and the 2nd to the tile The script, **microbiome_clean.Rmd,**  is divided into 7 successive numbered sections. Annotations are provided throughout the script for 1) construction of the phyloseq object, 2) cleaning and checking the metabarcoding data, 3) alpha-diversity analysis with figure1 a and b, 4) beta-diversity analysis with figure 1c and d, 5) microbiome composition with figure 1e and f, 6) random forest analysis  with figure 2a and 7) differential abundance analysis with ANCOM-BC and figure 2b and c.  #### Recruitment experiments We have submitted one dataset for the larval survival experiment (**survival_d6.xlsx**) and a second dataset for larval settlement and post-settlement survival experiments (**settlement_data.xlsx**) along with a single script (**recruitment_final.R**) for statistics and figure 3. In the data, **survival_d6.xlsx** :  * *ID_pot* : identity of the jar in which were larvae were deployed (N = 48 jars with12 replicated jars per combination of factors) * *trmt_larve* : parental origin of the larvae * ctrl = algal-removed bommies * ma = algal-dominated bommies * *trmt_eau* : algal-treatment of the tested water * bbl_c = algal-removed seawater * bbl_ma  = algal-dominated seawater * *tank* : number of the tank (water crate) in which jars were randomly placed * *day_start* : date of the experiment 18th december 2020 * *nb_swim* : number of swimming larvae * *nb_mlf* : number of metamorphosed floating larvae * *nb_settled* :  number of settled larvae * *nb_subtesting*: number of larvae testing bottom of the jar * *nb_motionless* : number of motion less larvae, even after three gentle pipetting * *nb_dead* : number of dead larvae with visible tissue degradation * *day* : day of the record, here T6 that is 6th and final day of the survival experiment * *nb_tot* : total number of larvae counted in the jar. A total of 10 larvae were added initially * *dead_tot* : total number of dead larvae * *percent* : percentage of living larvae In the data, **settlement_data.xlsx**:  * *ID_pot* : identity of the jar in which were larvae were deployed (N = 48 jars with12 replicated jars per combination of factors) * *trmt_larve* : parental origins of the larvae * ctrl =  algal-removed * ma = algal-dominated * *ID_tiles*: identity tag of the tiles * *ID_bommie* : identity of the experimental bommies where the tiles originated from * *trmt_ma* : algal treatment of the bommies * ctrl = algal-removed * ma = algal-dominated * *tank* : number of the tank (water crate) in which jars were randomly placed * *day_start* : date of the experiment 20th december 2020 * *nb_exp* : number of living settlers on exposed surfaces of the tiles * *nb_cryp* : number of living settlers on cryptic surfaces of the tiles * *nb_side* : number of living settlers on vertical surfaces of the tiles * *nb_glass* : number of living settlers on the jar's glass * *nb_swim* : number of swimming larvae * *nb_mlf* : number of metamorphosed floating larvae * *nb_tot_rec* : total number of settlers on tiles. 10 larvae were initially added in each jar * *nb_tot* : total number of larvae and settlers * *day* : censored day * T0 : at 24h after deployment to assess settlement rates * T7 : 7 days after outplanting on bommies * T21 : 21 days after outplanting on bommies In nb_exp, nb_cryp and nb_side, "N/A" values indicate that for T7 and T21 tiles were lost in the field so data was absent In nb_glass, nb_swim, nb_mlf and nb_tot, "N/A" values indicate that data is not applicable for T7 and T21, as these measures are only applicable to the settlement experiment at T0. The script, **recruitment_final.R**, is divided into 4 successive numbered sections. Annotations are provided throughout the script for 1) larval survival analysis, 2) settlement analysis, 3) post-settlement survival analysis and 4) creation of figure 3. ## Code/Software All scripts were coded on R software (version 4.2.3)