A detailed description of the Materials and Methods is provided in supplementary materials and methods, Supplementary Material online; here, we give a brief overview of the data set and the basic methods used. The 2014 DrosEU data set represents the most comprehensive spatiotemporal sampling of European D. melanogaster populations to date (fig. 1 and supplementary table S1, Supplementary Material online). It comprises 48 samples of D. melanogaster collected from 32 geographical locations across Europe at different time points in 2014 through a joint effort of 18 research groups. Collections were mostly performed with baited traps using a standardized protocol (see supplementary materials and methods, Supplementary Material online). From each collection, we pooled 33–40 wild-caught males. We used males as they are more easily distinguishable morphologically from similar species than females. Despite our precautions, we identified a low level of D. simulans contamination in our sequences; we computationally filtered these sequences from the data prior to further analysis (see Supplementary Material online). To sequence these samples, we extracted DNA and barcoded each sample, and sequenced the ∼40 flies per sample as a pool (Pool-Seq; Schlötterer et al. 2014 (link)), as paired-end fragments on a Illumina NextSeq 500 sequencer at the Genomics Core Facility of Pompeu Fabra University. Samples were multiplexed in five batches of ten samples, except for one batch of eight samples (supplementary table S1, Supplementary Material online). Each multiplexed batch was sequenced on four lanes at ∼50× raw coverage per sample. The read length was 151 bp, with a median insert size of 348 bp (range 209–454 bp). Our genomic data set is available under NCBI Bioproject accession PRJNA388788. Sequences were processed and mapped to the D. melanogaster reference genome (v.6.12) and reference sequences from common commensals and pathogens. Our bioinformatic pipeline is available at https://github.com/capoony/DrosEU_pipeline (last accessed May 22, 2020). To call SNPs, we developed custom software (PoolSNP; see supplementary materials and methods, Supplementary Material online; https://github.com/capoony/PoolSNP, last accessed May 22, 2020), using stringent heuristic parameters. In addition, we obtained genome sequences from African flies from the Drosophila Genome Nexus (DGN; http://www.johnpool.net/genomes.html, last accessed May 22, 2020; see supplementary table S14, Supplementary Material online, for SRA accession numbers). We used data from 14 individuals from Rwanda and 40 from Siavonga (Zambia). We mapped these data to the D. melanogaster reference genome using the same pipeline as for our own data above, and built consensus sequences for each haploid sample by only considering alleles with >0.9 allele frequencies. We converted consensus sequences to VCF and used VCFtools (Danecek et al. 2011 (link)) for downstream analyses. Additional steps in the mapping and variant calling pipeline and further downstream analyses of the data are detailed in supplementary materials and methods, Supplementary Material online.