DOI: 10.5061/dryad.0vt4b8h4t
title: Data for: Genetic structuring and species boundaries in the Atlantic stony coral Favia (Scleractinia, Faviidae)

description: Scleractinian corals are the main modern builders of coral reefs, which
 are major hot spots of marine biodiversity.
 Southern Atlantic reef corals are understudied compared to their Caribbean
 and Indo-Pacific counterparts and many hypotheses about their population
 dynamics demand further testing. We employed thousands of single
 nucleotide polymorphisms (SNPs) recovered via ezRAD to characterize
 genetic population structuring and species boundaries in the
 amphi-Atlantic hard coral genus Favia. Coalescent-based
 species delimitation (BFD* - Bayes Factor Delimitation) recovered
 F. fragum and F. gravida as separate species. Although our results agree
 with depth-related genetic structuring in F. fragum, they did not support
 incipient speciation of the “tall” and “short”
 morphotypes. The preferred
 scenario also revealed a split between two main
 lineages of F. gravida, one from Ascension Island and the other
 from Brazil. The Brazilian lineage is further divided into a
 species that occurs throughout the Northeastern coast and another that
 ranges from the Abrolhos Archipelago to the state of
 Espírito Santo. BFD* scenarios were corroborated by analyses of
 SNP matrices with varying levels of missing data and by
 a speciation-based delimitation approach (DELINEATE). Our results
 challenge current notions about Atlantic reef corals
 because they uncovered surprising genetic diversity in
 Favia and rejected the long-standing hypothesis that
 Abrolhos Archipelago may have served as a Pleistocenic refuge during the
 last glaciations. 
description: DNA was extracted from tissue samples
 of <em>F. fragum</em> using the Omega E.Z.N.A Tissue DNA kit
 and Invitrogen PureLink Genomic DNA kit. Extractions were purified using
 1.8X AmPureXP magnetic beads. DNA quality was assessed via electrophoresis
 in 1.5% agarose gel, ensuring that only high molecular weight DNA was
 carried over to the digestion step. Quantification was performed using a
 Qubit 2 Fluorometer and the dsDNA High Sensitivity Assay kit. Libraries
 were prepared following the ezRAD protocol (Toonen et al. 2013). Briefly,
 samples were digested using the enzyme DpnII (New England Biolabs), in
 50μL reactions containing 5μL DpnII NEB 10X Buffer, 2 units of DpnII, and
 200-1000ng of DNA. Digestions were incubated at 37°C for 3 hours, then
 heat-inactivated for 20 minutes at 65°C, purified using 1.8X AMPureXP
 beads, run in 1.5% agarose gels and considered successful if producing a
 smeared band. Library preparation began with the KAPA HyperPrep Library
 kit (Roche Sequencing Store) following Knapp et al. (2016) with minor
 modifications. DNA samples were end-repaired and a-tailed, and then
 adapter ligation was performed using IDT xGen Stubby Adapters and Unique
 Dual Index (UDI) primer pairs. These index-ligated products were size
 selected with Mag-Bind Magnetic Beads (Omega Bio-Tek) targeting fragments
 in a 350-700bp range in two steps, with DNA:bead ratios of 1:0.6 and
 1:0.2, respectively. Samples were amplified using six to ten PCR cycles
 with KAPA HiFi Hotstart Ready-mix (Omega Bio-Tek) and purified using 1:1
 DNA:AMPure XP beads. Libraries were validated using Qubit dsDNA HS kit,
 Agilent 2100 Bioanalyzer, and qPCR, and then pooled and sequenced as
 paired-end (2x150bp) reads on the Illumina HiSeq 4000 sequencer at the
 Research Technology Support Facility (RTSF) Genomics Core, at Michigan
 State University. We
 assessed raw read quality with FASTQC (Andrews, 2010) and subsequent
 processing was performed with the bioinformatic pipeline dDocent (Puritz,
 Hollenbeck, & Gold, 2014).   First, universal adapters and reads with
 Phred &lt;30 were excluded from further analyses. We randomly chose
 three individuals from each population to create the <em>de
 novo</em> assembly reference onto which all samples would be mapped.
 We used dDocent scripts ReferenceOpt.sh and RefMapOpt.sh to select the
 optimal combination of parameters for the <em>de
 novo</em> assembly, aiming at maximizing the number of paired
 mapping reads while minimizing the number of mismatched reads. We set the
 minimum level of similarity among sequences in the same cluster, or
 clustering threshold (<em>c</em>), to 90%, the minimum within
 individual coverage level (<em>k1</em>) to 3, and the minimum
 number of individuals sharing a read
 (<em>k2</em>)<em> </em>to 4. Within dDocent,
 sequences from all individuals were mapped against the reduced
 representation reference and SNPs were called with FreeBayes (Garrison
 & Marth, 2012). The resulting raw VCF file was filtered using
 VCFTools (Danecek et al. 2011). We applied a minimum quality filter of 30
 and a minimum mean depth of 3, then removed individuals with more than 55%
 missing loci. To assess the influence of missing data on the results we
 created three subsets with 0, 10 and 20% missing loci. Finally, we removed
 indels, filtered out loci with minor allele frequencies lower than 0.01
 and kept a single SNP per locus in downstream analyses.
description: # Title of dataset: **Genetic structuring and species boundaries in the
 Atlantic stony coral *Favia* (Scleractinia, Faviidae)** Authors: Carolina
 de Lima Adam, Robert J. Toonen, David B. Carlon, Carla Zilberberg, Marcos
 Soares Barbeitos 
 Corresponding author information: * Carolina de Lima Adam *
 Current Institution address: University of Oregon * Email:
 [carolinaladam@gmail.com](mailto:carolinaladam@gmail.c) This README file
 describes the data collection and the data package accompanying the above
 publication. Date of collection: *F. gravida* (Ascension Island - 6
 individuals; Brazil - 47 individuals); *F. fragum* (Caribbean - 16
 individuals) ## File list 1. TotalRawSNPs.vcf 2. favia_coral_20missing.vcf
 3. popmap.csv 4. *.R files: ## Description of individual files
 TotalRawSNPs.vcf * Variant Call File (VCF) with raw single nucleotide
 polymorphisms (SNP) for each individual in the dataset * File obtained
 with dDocent with the following parameters: * Clustering threshold *c =
 90%* * Minimum within individual coverage level *k1 = 3* * Minimum number
 of individuals sharing a read *k2 = 4* favia_coral_20missing.vcf * Subset
 of TotalRawSNPs.vcf after filtering with
 *[vcftools](https://vcftools.github.io/index.html)* * Filters applied: *
 Minimum quality Phred 30 (*vcftools --minQ 30*), minimum mean depth 3
 (*vcftools min-mean-DP 3*) * Removed individuals with more than 55%
 missing data * *vcftools --missing-ind* (generates *out.imiss* file) *
 *awk '$5 &gt; 0.55' out.imiss | cut -f1 &gt; lowDP.indv* *
 *vcftools --remove lowDP.indv* * Loci with more than 20% missing data were
 removed (*vcftools --max-missing 0.8*) * Indels (insertions and deletions)
 were removed (*vcftools remove-indels*) * Loci with minor allele frequency
 lower than 0.01 were removed (*vcftool --maf 0.01*) * Only one SNP per
 locus was retained (*vcftools --thin 500*) popmap.csv: * tab-delimited
 file designating the sampling site for each sample in the VCF file sNMF.R:
 * Script to run
 [sNMF](http://membres-timc.imag.fr/Olivier.Francois/snmf/index.htm)
 analysis (Fast and Efficient Estimation of Individual Ancestry
 Coefficients) fst_heatmap.R: * Script to estimate pairwise Fst (Fixation
 index) between *Favia* individuals using the VCF file as input and to plot
 a heatmap based on the obtained values pca_dapc.R: * Script to perform
 Principal Components Analysis (PCA) and Discriminant Analysis of Principal
 Components (DAPC) using the VCF file as input