Sampling and analytical procedures:
The Eurytemora affinis copepods used in the laboratory natural selection experiment were collected from Kiel Canal in Kiel, Germany (latitude = 54° 19' 59.88"N, longitude = 10° 9' 0"W) in 2017 (approximately 1000 copepods) and on May 30, 2018 (85 gravid females and 40 juveniles). Wild E. affinis populations were collected from eight locations in the Baltic Sea using bongo and WP2 nets with 100 μm mesh in 2019 (see related dataset 878322). The two collections of copepods were mixed and maintained at 15 PSU to increase population size and acclimate to laboratory conditions. Two samples of adult copepods (25 male and 25 female each) from the mixed culture were collected for pooled whole-genome sequencing (Pool-seq) to represent the starting population for the laboratory natural selection experiment and capture variance in starting SNP frequency. The culture was then split into 14 equally sized beakers. Control lines (N = 4) were maintained for the duration of the experiment in 15 PSU water made with Instant Ocean, along with Primaxin (20 milligrams per liter) to avoid bacterial infection. The control lines were fed the marine alga Rhodomonas salina every three to four days with the water changed weekly. The ten treatment lines were exposed to decreasing salinity over the first six generations until they reached 0 PSU (Lake Michigan water, ~300 µS/cm conductivity), and then maintained at 0 PSU for four additional generations.

Beginning at generation two, salinity declination proceeded at each generation as follows: 5 PSU, 1 PSU, 0.1 PSU, 0.01 PSU, 0 PSU. The generation number was monitored by assuming a generation time of approximately three weeks. Treatment lines were fed a 50:50 mixture of R. salina and the freshwater alga R. minuta at 5 PSU and only R. minuta at 1 PSU and below.

Individual adult copepods (N = 50; 25 male and 25 female) were collected for sequencing from each laboratory selection line at generations six (after one generation at 0 PSU in the treatment lines) and ten (after five generations at 0 PSU in the treatment lines). Sampled copepods from each line were pooled and their DNA was extracted using the DNeasy Blood and Tissue Extraction kit (Qiagen, Inc.). Paired-end whole-genome sequencing libraries were prepared using the Nextera DNA kit (Illumina Inc.) and sequenced on four lanes of Illumina Hi-Seq 4000 and one lane of Illumina NovaSeq 6000 at the University of Chicago Genomics Facility, generating an average of approximately 117 million paired-end (100 bp) reads per pool.