Projeto de pesquisa

In the largest effort to generate the first comprehensive data on the genetic diversity and connectivity of all shallow-water scleractinian species found at the Southwestern Atlantic, 18 scleractinian corals of which 15 are zooxanthellate (Agaricia agaricites, A. fragilis, Favia gravida, Madracis decactis, Meandrina brasiliensis, Montastraea cavernosa, Mussismilia braziliensis, M. harttii, M. hispida, M. leptophylla, Porites astreoides, P. branneri, Scolymia wellsi, Siderastrea stellata, Stephanocoenia intersepta) and three are azooxanthellate (Astrangia rathbuni, A. solitaria, Phyllangia americana), and three calcified hydrozoans (Millepora alcicornis, M. braziliensis, M. nitida), will be sampled at 12 localities along the Brazilian coast and oceanic islands, according to the species occurrence (see supplementary material 1). For each species/locality, seven juveniles and seven adult colonies will be sampled at shallow and also at deep (> 30 m depth) waters, and preserved in CHAOS solution (4M Guanidine thiocyanate, 0,1% N-Laurilsarcosina, 10 mM Tris-HCl pH 8,0, 0,1M 2-Mercaptoethanol) (Fukami et al., 2004).
Total DNA will be extracted using DNeasy Tissue and Blood Kit (QIAGEN) and DNA quality will be checked on 2% agarose gel and spectrophotometer. After DNA extraction, cleaning, and quality check (see Genome section), genome digestion will follow the ezRAD protocol (Toonen et al., 2013; Knapp et al., 2016). Each sample will be digested using restriction enzymes, either DpnII or a combination of MboI and Sau3AI in 50 μl reactions following the manufacturer’s protocols with three hours incubation at 37 °C and 20 minutes at 65 °C. Subsequently, samples will be cleaned with Ampure XP beads in a 1:1.8 (DNA:beads) ratio, and the KAPA HyperPrep kit (Roche) will be used for sizeselected (250-400bp) library construction (see Knapp et al., 2016). PCR steps will follow the manufacturer’s recommended number of cycles and cycling conditions. For each lane of the Illumina HiSeq 2000, a total of 96 individuals (libraries individually barcoded) will be pooled and sequenced. Sequenced fragments will be trimmed from the 3’ end and low-quality reads (Phred score = 33) will be removed from further analysis. Reads without an exact match to the barcode and also to the restriction site will also be filtered out. After removing the barcode and restriction site from all remaining reads, the population genomic analysis/single nucleotide polymorphism (SNP) discovery and genotyping will be performed using Python scripts and the Stacks software (Catchen et al., 2013). Putative SNPs are going to be selected based on minimum depth coverage. The genetic diversity of each population/depth will be estimated using Arlequin (Excoffier and Lischer, 2010). Pairwise AMOVA will be applied to estimate fixation indexes and population structure according to Weir (1996). Genetic clusters will be analyzed using a Bayesian approach implemented in Structure 2.2 (Falush et al., 2007) and visualized using Structure Harvester (Earl and VonHoldt, 2012). Population structure will be estimated based on two approaches: considering only outlier loci (Fst outliers - potential under selection); and considering only neutral loci.
Although potential pitfalls have been indicated (Bierne et al., 2011), Fst outliers will be searched by comparing AMOVA Fst results – loci with high standard deviation; scans using Bayescan (Foll and Gaggiotti, 2008); and by applying a modified Fdist method (Beaumont and Nichols, 1996) implemented in the Arlequin loci under selection option (Excoffier et al., 2009; Excoffier and Lischer, 2010). The identification of the detected outlier loci will be carried out using the Blast search, with matching sequences classified using KEGG assignments (Kanehisa et al., 2008).