Raw reads were trimmed and filtered of adapter sequences, low-complexity sequences and low-quality (Q<15) bases using BBDuk in the BBTools package (Bushnell, 2014). Processed reads were mapped to the repeat-masked E. affinis complex (Atlantic clade) draft reference genome using BWA-MEM v.0.7.17 (Li, 2013). Paired-end reads that did not align concordantly with BWA-MEM were aligned as single-end reads using NextGenMap v.0.5.5 (Sedlazeck et al., 2013) to aid in the alignment of diverged sequences. The combined read-mapping procedure achieved a mean mapping rate of 95.09 ± 1.42%. Duplicate reads were removed using Picard v.2.18.27 (Soifer, 2022) and regions around insertions or deletions were realigned using GATK v.3.8 (Van der Auwera & O'Connor, 2020). SAMtools v.1.3.1(Li et al., 2009) was used to convert BAM files into mpileup format after removing low-quality alignments and bases (Q<20). Sites within 3 bp (base pair) of an insertion or deletion were removed and the filtered mpileup was converted to sync format using PoPoolation2 (Kofler, Pandey, Schlotterer, 2011). The R package poolfstat v.1.0 (Gautier et al., 2021) was used to detect bi-allelic SNPs with a global minor allele frequency >0.05, at least four reads were required for a base call, and a minimum of 20 and a maximum of 200 total read counts were required for all populations. In total, 6,635,765 SNPs passed these filters when considering all nine populations. A total of 7,565,621 and 5,323,780 SNPs were called for the Atlantic and Gulf clades, respectively.

Software
(See also Related Publications section below)

BBTools v38.33 (Bushnell, 2014)
BWA-MEM v0.7.17 (Li, 2013)
NextGenMap v0.5.5 (Sedlazeck, 2013)
Picard v2.18.27 (Soifer, 2022)
GATK v3.8 (Van der Auwera & O'Connor, 2020)
SAMtools v1.3.1 (Li et al., 2009)
PoPoolation v1.2.2 (Kofler et al., 2011)
PoPoolation2 v1.013 (Kofler, Pandey, Schlotterer, 2011)
poolfstat v.1.0 (Gautier et al., 2021)
TreeMix v1.13 (Pickrell & Pritchard, 2012)
phytools v.0.6 (Revell, 2011)
BayeScan 2, BayeScan 3 (Foll & Gaggiotti, 2008)
BayPass v2.1 (Gautier, 2015)
BetaScan2 (Siewert & Voight, 2020)
GenMap (Pockrandt et al., 2020)
edgeR (Robinson et al, 2010)
BedTools v2.28 (Quinlan & Hall, 2010)
BEDOPS (Neph et al., 2012)
Gowinda (Kofler & Schlotterer, 2012)
custom code (Stern, 2022 for https://github.com/TheDBStern/poolseq_utils)

BCO-DMO Processing
- Converted date to Y-M-D format
- Separated latitude and longitude into separate columns and converted to decimal degrees
- Added a column for BioProject
- Added dates of collection based on Supplemental File information
- Sorted by Collection_Date