This dataset contains host genomic and associated microbiome sequence data generated to investigate patterns of cryptic lineage structure, symbiotic diversity, and microbial community composition in reef-building corals (Porites spp.) across environmental gradients. Samples were collected from three classic (typical temperatures) and three extreme (higher temperatures and light attenuation) reef sites in Palau, and processed using high-throughput sequencing approaches to characterize (1) host population genomic variation and (2) taxonomic  diversity of coral-associated microbial communities (Symbiodiniaceae and bacteria). Host genomic data were generated using a 2bRAD approach and analyzed to resolve lineage differentiation. Microbiome communities were characterized through amplicon sequencing, enabling assessment of microbial assemblages associated with distinct host lineages and environmental conditions.

Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • BCO-DMO Processing Description
• Related Publications
• Parameters
• Instruments
• Project Information
• Funding

Colonies resembling the gross morphology of Porites lobata Dana, 1846 were tagged at six sites, at the Rock Islands of Palau, in November 2021 in a transect along the shoreline (N=15 per site, 90 colonies total). All colonies were sampled using a hammer and chisel between 1 and 6 meters (m) depth, with the majority between 3 and 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 centimeters (cm)) to facilitate transportation to aquarium facilities for further analyses and experiments. The total area over which corals were collected was 250-500 square meters (m²) per site. Tissue samples were taken from the center of each colony, immediately fixed in ethanol, and stored at -20 degrees Celsius (°C) (2 × 2 cm samples).

Tissue samples from all coral colonies were crushed with a sterile razor blade, and DNeasy Blood and Tissue kits (Qiagen) were used to isolate DNA from the resulting homogenate according to the manufacturer's instructions, with one modification: the lysis step was conducted overnight. Isolated DNA was then cleaned with a Zymo Clean and Concentrator kit (Zymo Research, CA). DNA was quantified using a Qubit fluorometer (Invitrogen), standardized to 25 nanograms per microliter (ng μL⁻¹), prepared for 2b-RAD sequencing according to (Wang et al., 2012), and sequenced across one lane of Illumina HiSeq 2500 using single-end 50 bp sequencing at the Tufts University Core Facility (TUCF) Genomics. Five technical replicates were included in the library preparation to aid the downstream identification of clonemates.

Photobiont communities were characterized in samples through sequencing of the internal transcribed spacer region 2 (ITS2) region using SYM_VAR_5.8S2 and SYM_VAR_REV primers (Hume et al., 2015, 2018). The PCR profile included 26 cycles of 95 °C for 40 seconds, 59 °C for 2 minutes, 72 °C for 1 minute and a final extension of 72 °C for 7 minutes. A negative control was included in the initial amplification but failed to amplify, so it was not included in downstream library preparations. Successful amplifications were cleaned using the GeneJET PCR Purification kit (ThermoFisher Scientific) and a second PCR was conducted to attach Illumina MiSeq dual barcodes to the PCR product before samples were pooled. Volumes for pooling were based on the visualization of barcoded sample band intensity on a 1% agarose gel. This pool was cleaned using the GeneJET PCR Purification kit, gel extracted, and submitted for sequencing as described below.

To characterize bacterial communities, the V4 region of the 16S rRNA gene was amplified from the same samples via PCR using Hyb515f (Parada et al., 2016) and Hyb806R (Apprill et al., 2015) primers and the following PCR profile: 35 cycles of 95 °C for 40 seconds, 65 °C for 2 minutes, 72 °C for 1 minute and a final extension of 7 minutes. Subsequent PCR amplification, cleaning, dual-barcoding, and gel extraction followed the same protocol described for ITS2 with the inclusion of three negative controls, which were also submitted for sequencing. ITS2 and 16S pools were quantified and combined in a 1:3 ratio, respectively. Libraries were sequenced together on Illumina MiSeq (paired-end 250 bp) at Tufts University Core Facility (TUCF) Genomics.

2bRAD Sequencing:
Raw 2bRAD reads were deduplicated and trimmed using the FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit). Reads under 25 base pairs (bp) in length or with quality scores <15 were discarded. Following Rippe et al. (2021), photobiont reads were removed by discarding reads that mapped to concatenated Symbiodiniaceae genomes (Symbiodinium (Aranda et al., 2016), Breviolum (Shoguchi et al., 2013), Cladocopium (Dougan, 2020), and Durusdinium with Bowtie2 v2.4.2 (Langmead & Salzberg, 2012). The remaining host reads were then mapped to the Porites lobata genome (Noel et al., 2023). Genotyping was performed using ANGSD v0.923 (Korneliussen et al., 2014). Filters that were used across all analyses included loci that were present in ≥ 80% of individuals, and a minimum read depth of 6 across all samples. Triallelic sites were removed. Reads had a minimum quality of 25, minimum mapping quality of 20 with a strand bias p-value of 1e-5 and a heterozygosity bias p-value of 1e-5. Clones were detected using hierarchical clustering based on pairwise identity by state (IBS) with an additional minor allele frequency (MAF) filter of 0.05. Technical replicates provided the clone detection threshold, and one member of each clone pair was removed for downstream analyses.

A total of 75 samples remained after quality control and technical replicate removal. These libraries were selected for further population genomic analyses due to a higher proportion of the genome covered compared to reduced RAD samples. For all population genomic analyses an additional MAF filter of 0.05 was added, with the exception of site frequency spectrum (SFS) based analyses. Admixture was estimated using NGSadmix; admixture plots were then created using a custom R script (https://github.com/z0on/2bRAD_denovo/blob/master/admixturePlotting_v5.R). Principal Component Analysis (PCA) was conducted using a covariance matrix based on single-read resampling calculated in ANGSD. Admixture results were visualized using the K with the least cross validation error reported from ADMIXTURE. These analyses demonstrated the presence of three distinct lineages amongst our six sampling sites. F_ST was estimated between pairs of lineages using ANGSD before and after outlier loci were removed using Bayescan (https://doi.org/10.1534/genetics.108.092221, Foll & Gaggiotti, 2008).

Microbial community sequencing:
Photobiont communities were characterized through sequencing of the internal transcribed spacer region 2 (ITS2) region using SYM_VAR_5.8S2 and SYM_VAR_REV primers (Hume et al., 2015, 2018). The PCR profile included 26 cycles of 95 °C for 40 seconds, 59 °C for 2 minutes, 72 °C for 1 minute and a final extension of 72 °C for 7 minutes. A negative control was included in the initial amplification but failed to amplify, so it was not included in downstream library preparations. Successful amplifications were cleaned using the GeneJET PCR Purification kit (ThermoFisher Scientific) and a second PCR was conducted to attach Illumina MiSeq dual barcodes to the PCR product before samples were pooled. Volumes for pooling were based on the visualization of barcoded sample band intensity on a 1% agarose gel. This pool was cleaned using the GeneJET PCR Purification kit, gel extracted, and submitted for sequencing as described below.

To characterize bacterial communities, the V4 region of the 16S rRNA gene was amplified from the same samples via PCR using Hyb515f (Parada et al., 2016) and Hyb806R (Apprill et al., 2015) primers and the following PCR profile: 35 cycles of 95 °C for 40 seconds, 65 °C for 2 minutes, 72 °C for 1 minute and a final extension of 7 minutes. Subsequent PCR amplification, cleaning, dual-barcoding, and gel extraction followed the same protocol described for ITS2 with the inclusion of three negative controls, which were also submitted for sequencing. ITS2 and 16S pools were quantified and combined in a 1:3 ratio, respectively. Libraries were sequenced together on Illumina MiSeq (paired-end 250 bp) at Tufts University Core Facility (TUCF) Genomics.

Sequences with adaptor contamination were removed and raw 16S and ITS-2 sequences were separated based on primer sequences using bbduk following Bove et al. (2023). Raw ITS-2 reads were processed by Symportal (Hume et al., 2019) to produce defining intragenomic sequence variant (DIV) profiles for each coral colony. Two samples with <1,000 reads were removed, as well as one outlier sample with >1 million reads; the remaining samples (n = 73) had an average of ~5,500 reads per sample (min: 1,116; max: 16,931). All samples were dominated by one of ten Cladocopium C15 types, and four samples hosted low abundances of Symbiodinium A3 sequences.

- Imported original file "BCO_DMO_sequencing data_final.csv" into the BCO-DMO system.
- Converted Date field to YYYY-MM format.
- Renamed fields to comply with BCO-DMO naming conventions.
- Saved the final file as "996942_v1_cryptic_porites_lineage.csv".

- Imported the supplemental file "Supplementary Datafile 3_its2-seq.csv" into the BCO-DMO system.
- Converted Date field to YYYY-MM format.
- Saved the final file as "996942_v1_supplemental_div_data.csv".

Coverage: Palauan coral reefs

NSF Award Abstract:
Coral reefs host thousands of marine species, help protect coastlines from storm damage, generate tourism, and house fish used for human consumption. However, corals are vulnerable to increasing water temperatures, which can lead to coral death. One way for reefs to survive in warming oceans is for corals that are well-suited to warmer waters to repopulate reefs that have less temperature-tolerant individuals. For this strategy to succeed, however, the more temperature-tolerant corals need to be able to disperse to and survive in these different environments. This project takes advantage of reef systems in the Pacific nation of Palau that naturally experience a wide range in temperatures across short geographic distances. Using cutting-edge ecological and genomic techniques, the team of investigators is directly testing whether young corals from Palau’s warmest reefs can successfully be carried by ocean currents to Palau’s currently cooler reefs and subsequently survive and thrive in these habitats. Given the relevance of this research for the local ecology, the team is disseminating results to the Palauan government through a written report in conjunction with Palauan scientists who are interning with the team, and to the Palauan people through public presentations. As part of this work, the investigators are maintaining a blog and are organizing a music-lecture series combining dance, music, and science to promote awareness of the coral reef crisis across English and Spanish-speaking communities in the US. Results from this project are informing restoration and conservation practices of the Coral Conservation Consortium as well as other efforts worldwide.

A major question in evolutionary biology is how plasticity and adaptation interact to influence survival under novel environments. Understanding these processes is increasingly important as rising temperatures associated with climate change influence species globally. For marine organisms with pelagic larval phases, including reef-building corals, the post-settlement period constitutes a critical bottleneck for adaptation and plasticity, with the added complexity that the conditions experienced and time spent as larvae can incur carryover effects. This project leverages reefs in Palau that span a steep environmental gradient to study how environmental variation drives selection and plasticity and to examine if dispersal between reefs limits success across habitats due to carryover effects. The investigators are testing the overarching hypothesis that corals from warmer and more variable environments are adapted to warmer temperatures and exhibit increased plasticity, but that dispersal between reefs incurs a fitness cost. The team integrates field and molecular techniques to: 1) investigate the degree of selection occurring on warmer and more variable reefs, 2) test whether corals transplanted to more variable environments improve their thermal tolerance through developmental plasticity, and 3) examine whether delays in metamorphosis required for dispersal across reefs comes at a fitness cost due to carryover effects.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.