DOI: 10.5061/dryad.1g1jwsv65
title: Holobiont traits shape climate change responses in cryptic coral lineages

description: As ocean warming threatens reefs worldwide, identifying corals with
 adaptations to higher temperatures is critical for conservation.
 Genetically distinct but morphologically similar (i.e., cryptic) coral
 populations can be specialized to extreme habitats and thrive under
 stressful conditions. These corals often associate with locally beneficial
 microbiota (Symbiodiniaceae photobionts and bacteria), obscuring the main
 drivers of thermal tolerance. Here, we leverage a holobiont (massive
 Porites) with high fidelity for C15 photobionts to investigate adaptive
 variation across classic (“typical” conditions) and extreme reefs
 characterized by higher temperatures and light attenuation. We uncovered
 three cryptic lineages that exhibit limited micro-morphological variation;
 one lineage dominated classic reefs (L1), one had more even distributions
 (L2), and a third was restricted to extreme reefs (L3). L1 and L2 were
 more closely related to populations ~4300 km away, suggesting that some
 lineages are widespread. All corals harbored Cladocopium C15 photobionts;
 L1 and L2 shared a photobiont pool that differed in composition between
 reef types, yet L3 mostly harbored unique photobiont strains not found in
 the other lineages. Assemblages of bacterial partners differed among reef
 types in lineage-specific ways, suggesting lineages employ distinct
 microbiome regulation strategies. Analysis of light harvesting capacity
 and thermal tolerance revealed adaptive variation underpinning survival in
 distinct habitats: L1 had the highest light absorption efficiency and
 lowest thermal tolerance, suggesting it is a classic reef specialist. L3
 had the lowest light absorption efficiency and the highest thermal
 tolerance, showing that it is an extreme reef specialist. L2 had
 intermediate light absorption efficiency and thermal tolerance, suggesting
 that is a generalist lineage. These findings reveal diverging holobiont
 strategies to cope with extreme conditions. Resolving coral lineages is
 key to understanding variation in thermal tolerance among coral
 populations, can strengthen our understanding of coral evolution and
 symbiosis, and support global conservation and restoration efforts.
description: <strong>Materials and Methods</strong>
 <strong>Site selection and sample
 collection</strong> Three extreme and three
 paired classic sites were selected in Chelbacheb, Palau. Extreme sites
 were shallow semi-enclosed lagoons with high water retention, increased
 light attenuation, and distinct assemblages of massive <em>Porites
 </em>lineages compared to classic sites (Rivera et al., 2022; van
 Woesik et al., 2012). Between 2010 and 2018, these sites had naturally
 elevated water temperatures year-round (up to 2°C) compared to nearby
 classic sites. Seasonal temperature ranges at extreme sites were ~29-32
 °C, compared to ~28-31 °C at classic sites (Rivera et al., 2022). Sites
 were selected to ensure that, aside from the contrast in terms of
 temperature between classic and extreme sites, variability was minimal:
 they were all comparable in terms of depth (1 - 6 m), proximity to land
 (~10 - 20 m from land), and wave exposure (calm, on the leeward side of
 the archipelago or in a lagoon). Water temperatures
 were measured every 30 minutes using triplicate temperature loggers at
 each site (3-4 m depth) between November 2021 and May 2022, and light
 levels were measured with duplicate loggers for 16 days in April 2022
 (Onset, Wareham, USA). Temperature and light data from each site were then
 averaged across replicate loggers. Colonies resembling the gross
 morphology of <em>Porites lobata</em> Dana, 1846 were tagged
 at all sites in November 2021 in a transect along the shoreline. All
 colonies were sampled between 1 – 6 m depth, with the majority between 3 -
 4 m. All selected colonies were at least 1 - 5 m apart to reduce the risk
 of sampling clone mates while maximizing the probability that the colonies
 were exposed to similar conditions within a site. Targeted colonies were
 also relatively small in size (30 – 50 cm) to facilitate transportation to
 aquarium facilities for further analyses and experiments. The total area
 over which corals were collected was 250 - 500
 m<sup>2</sup> per site. Tissue samples were taken from the
 center of each colony and immediately stored in ethanol at -20℃ (2x2 cm
 samples; <em>n</em> = 90 total, 15/site). An additional 22
 colonies were sampled in April 2022 (<em>n</em> = 2-7 per
 site).  <strong>Characterization of microbial
 communities</strong> Characterization of
 photobiont and bacterial communities associated with each lineage was
 conducted on samples collected in November 2021 using ITS-2 and 16S
 sequencing, respectively. Raw ITS-2 reads were processed by Symportal
 (Hume et al., 2019) to produce defining intragenomic sequence variant
 (DIV) profiles for each coral colony (final <em>n</em> = 73).
 All colonies were dominated by C15 DIVs. Quality filtering, denoising,
 merging, and taxonomy assignments of 16S rRNA gene reads were conducted
 with DADA2 against the <em>Silva </em>v. 138.1 database (Quast
 et al., 2012), and contaminant reads were removed in Phyloseq (McMurdie
 & Holmes, 2013) (final <em>n</em> = 48). 
 <strong>Analyses of holobiont optical
 traits</strong> To characterize differences in
 holobiont structural and optical traits, we quantified polyp densities and
 light-harvesting characteristics in a subset of coral colonies. A total of
 20 tagged colonies of known lineage and reef type were transported to
 Boston University in May 2023, fragmented, and then acclimated for 63 days
 in aquariums. Reflectance (R) between 400 and 750 nm was measured
 (<em>sensu</em> Enríquez et al., 2005; Vásquez-Elizondo et
 al., 2017). Chlorophyll <em>a</em> densities were quantified
 and the specific absorption coefficient of
 Chlrophyll <em>a</em> (Chl<em>a</em>;
 a*<sub>chla</sub>), a measure of the light absorption
 efficiency of the holobiont, was estimated following Enríquez et al.,
 (2005).  <strong>Thermal challenge
 experiment</strong> A 25-day common garden heat
 challenge experiment was conducted to test for differences in thermal
 tolerance between the three <em>Porites</em> lineages
 (<em>n</em> = 24). L1 colonies were collected from classic
 reefs and L2 and L3 colonies were collected from extreme reefs. We
 initially aimed to include L1 and L2 colonies from both extreme and
 classic reefs, but were unsuccessful. Two cores were extracted from each
 colony; one core was assigned to the control treatment, and the other to
 the heat treatment. Mean temperatures in control tanks
 (<em>n </em>= 3 tanks per treatment) were maintained at 29.5°C
 ± 0.1°C. Temperatures in the heat treatment tanks were ramped by ~3°C over
 seven days, followed by a 12-day hold. On day 19, temperatures were
 increased by an additional ~1°C to simulate an extreme thermal stress
 event until day 25. All cores were inspected daily for mortality. Maximum
 PSII photochemical efficiency (Fv/Fm) was measured daily or semi-daily
 following &gt;90 minutes of dark incubation. All fragments were also
 photographed with a color standard at six timepoints to measure changes in
 coloration (paling), quantified as the intensity in the gray channel using
 ImageJ (sensu McLachlan & Grottoli, 2021). To control for
 differences in gray intensity at the start of the experiment, we also
 calculated and analyzed relative changes in gray intensity (D grey
 intensity; values in heat - values in control for each colony at each
 timepoint).
description: # Holobiont traits shape climate change responses in cryptic coral
 lineages
 [https://doi.org/10.5061/dryad.1g1jwsv65](https://doi.org/10.5061/dryad.1g1jwsv65) ## Description of the data and file structure This submission contains 9 datafiles from a study in Chelbacheb (the Rock Islands of Palau) studying the genetic diversity, microbial partners, physiology, and thermal tolerance of massive *Porites* inhabiting extreme and non-extreme (classic) reefs. Datafiles 1 and 2 contain temperature and light data at six study sites (N=3 extreme, N=3 classic sites) in Chelbacheb, respectively. Datafiles 3 and 4 contain data on dinoflagellate symbiont and bacterial diversity associated with colonies of massive* Porites* corals at these sites, respectively. Datasets 5 and 6 contain data on polyp densities and light harvesting mechanisms of these corals, respectively. Datasets 7-9 contain data from a thermal challenge experiment with the coral colonies: Dataset 7 contains colony mortality rates; dataset 8 contains the maximum quantum yield of photosystem II (Fv/Fm); dataset 9 contains data on colony coloration. ### Files and variables #### File: Supplementary\_Datafile\_4\_16s-seq.rds **Description:** 16S rRNA gene amplicon sequencing data of bacterial communities associated with colonies of* *massive* Porites* in Chelbacheb. #### File: Supplementary\_Datafile\_9\_paling.csv **Description:**  ##### Variables | Date | Date of measurement | | :-------- | :----------------------------------------------------------------------------------------------- | | Day | Day in experiment | | ID | Colony ID. Numbers are equivalent to numbers in the sample name in other supplementary datafiles | | Treatment | Temperature treatment, one of two: C (Control), H (Heat) | | Bin | Replicate aquarium number, one of three: 1, 2, 3 | | Site | Site name, one of six: Ngerur, Outer Taoch, Ngelsibel, Risong, Taoch, Mecherchar | | Type | Reef type, one of two: Classic or Extreme | | Lineage | Assigned lineage, one of three: L1, L2, L3 | | Color | Intensity in the grey channel, relative to white standard in the same image, unitless | #### File: Supplementary\_datafile\_1\_temperatures.csv **Description:** Temperature data from six study sites in Chelbacheb. ##### Variables | Date\_time | String with combined date and time stamp of temperature measurement | | :--------- | :------------------------------------------------------------------------------- | | Site | Site name, one of six: Ngerur, Outer Taoch, Ngelsibel, Risong, Taoch, Mecherchar | | Type | Reef type, one of two: Classic or Extreme | | Temp1 | Temperature reading from logger 1 in degrees Celsius | | Temp2 | Temperature reading from logger 2 in degrees Celsius | | Temp3 | Temperature reading from logger 3 in degrees Celsius | | Mean\_temp | Mean temperature across the three readings in degrees Celsius | #### File: Supplementary\_Datafile\_5\_polypdensity.csv **Description:** Density of polyps on colonies of massive* Porites* in Chelbacheb. ##### Variables | Date | Date of measurement | | :-------- | :------------------------------------------------------------------------------------------------ | | ID  | Colony ID. Numbers are equivalent to numbers in the sample name in other supplementary datafiles. | | Site | Site name, one of six: Ngerur, Outer Taoch, Ngelsibel, Risong, Taoch, Mecherchar | | Type | Reef type, one of two: Classic or Extreme | | Lineage | Assigned lineage, one of three: L1, L2, L3 | | M\_number | Measurement number. Three replicate measurements were conducted for each colony. | | Polyps | Calculated number of polyps mm^-2. | #### File: Supplementary\_Datafile\_2\_light.csv **Description:** Light intensity data from six study sites in Chelbacheb. ##### Variables | Date\_time | String with combined date and time stamp of light intensity measurement | | :----------- | :------------------------------------------------------------------------------- | | Site | Site name, one of six: Ngerur, Outer Taoch, Ngelsibel, Risong, Taoch, Mecherchar | | Type | Reef type, one of two: Classic or Extreme | | Light..lux | Light intensity reading from logger 1 in lux | | Light..lux.1 | Light intensity reading from logger 2 in lux | | Mean | Mean light intensity across the two readings in lux | #### File: Supplementary\_Datafile\_7\_mortalityrates.csv **Description:** Mortality rates of coral colonies in the thermal challenge experiment per genetic lineage. ##### Variables | Days | Number of days in the thermal challenge experiment until an "event". Event meaning colony death or end of the experiment | | :------ | :--------------------------------------------------------------------------------------------------------------------------------- | | Lineage | Assigned lineage, one of three: L1, L2, L3 | | col\_id | Colony number. Note that these are unique numbers for the experiment, not the same as colony numbers in other supplementary files. | | Status | Live or dead; Live = 1, Dead = 2  | #### File: Supplementary\_Datafile\_6-opticaltraits.csv **Description:** Light harvesting traits of colonies of massive* Porites* in Chelbacheb. ##### Variables | Coral.ID | Colony ID. Numbers are equivalent to numbers in the sample name in other supplementary datafiles. Letters after the coral ID indicate the nubbin number | | :---------------- | :------------------------------------------------------------------------------------------------------------------------------------------------------ | | Lineage | Assigned lineage, one of three: L1, L2, L3 | | Type | Reef type, one of two: Classic or Extreme | | Site | Site name, one of six: Ngerur, Outer Taoch, Ngelsibel, Risong, Taoch, Mecherchar | | Abs\_630 | Absorbance at 630 nm, unitless | | Abs\_663 | Absorbance at 663 nm, unitless | | ug.chla.in.0.3.ml | Density in µg of Chlorophyll a in 0.3 ml subsample | | ug.chlc.in.0.3.ml | Density in µg of Chlorophyll c in 0.3 ml subsample | | ug.chla.ml.1 | Calculated density of Chlorophyll a in µg ml-1 | | ug.chlc.ml.1 | Calculated density of Chlorophyll c in µg ml-1 | | cm2 | Size of the colony in cm2 | | ug.chla.cm.2 | Density of chlorophyll a in µg cm-2 coral surface | | ug.chlc.cm.2 | Density of chlorophyll c in µg cm-2 coral surface | | mg.chla.m.2 | Density of chlorophyll a in mg m-2 coral surface | | mg.chlc.m.2 | Density of chlorophyll c in mg m-2 coral surface | | astar\_host\_Chla | Chl*a*-specific absorption coefficient (a\*Chl*a*, m2 mg chl*a*-1)  | #### File: Supplementary\_Datafile\_8\_FvFm.csv **Description:** Maximum quantum yield of photosystem II (Fv/Fm) measured in coral colonies during the thermal stress experiment. ##### Variables | Date | Date of measurement | | :-------- | :----------------------------------------------------------------------------------------------- | | Day | Day in experiment | | ID | Colony ID. Numbers are equivalent to numbers in the sample name in other supplementary datafiles | | Treatment | Temperature treatment, one of two: C (Control), H (Heat) | | Bin | Replicate aquarium number, one of three: 1, 2, 3 | | ITS2 | ITS2 DIV as determined using ITS-2 rDNA gene sequencing and processing with Symportal | | Site | Site name, one of six: Ngerur, Outer Taoch, Ngelsibel, Risong, Taoch, Mecherchar | | Type | Reef type, one of two: Classic or Extreme | | Lineage | Assigned lineage, one of three: L1, L2, L3 | | Fv.Fm.1 | Photosynthetic efficiency (Fv/Fm) reading 1, unitless | | Fv.Fm.2 | Photosynthetic efficiency (Fv/Fm) reading 2, unitless | | Fv.Fm.3 | Photosynthetic efficiency (Fv/Fm) reading 3, unitless | | Average | Mean Fv/Fm, unitless | | Average2 | Mean Fv/Fm/100, unitless | #### File: Supplementary\_Datafile\_3\_its2-seq.csv **Description:** Defining intragenomic variant Symbiodiniaceae type, based on processing of ITS-2 sequences by Symportal. ##### Variables | Sample | Sample name | | :--------- | :------------------------------------------------------------------------------- | | Site | Site name, one of six: Ngerur, Outer Taoch, Ngelsibel, Risong, Taoch, Mecherchar | | Lineage | Assigned lineage, one of three: L1, L2, L3 | | Type | Reef type, one of two: Classic or Extreme | | DIV | Defining intragenomic variant (DIV), as assigned by Symportal | | Reads | Total number of reads per DIV for a given sample | | Rel\_reads | Relative number of reads per DIV for a given sample | ## Code/software **Software** All data were analyzed in R v4.1.2 (R Core Team, 2019). Code for the analyses (including required packages) have been deposited on Github: [https://github.com/grupstra-lab/Holobiont-specialization-in-massive-porites-lineages](https://github.com/grupstra-lab/Holobiont-specialization-in-massive-porites-lineages).