Study objectives
The goal of this study was to explore evolutionary shifts in gene expression between ancestral saline and freshwater invading populations of the Eurytemora affinis (copepod) species complex on a genome-wide scale.

To explore mechanisms of freshwater adaptation and distinguish between adaptive (evolutionary) and acclimatory (plastic) responses to salinity change, laboratory experiments were conducted using both ancestral saline and derived freshwater populations of Eurytemora affinis. Then RNA-seq data -- Illumina short paired-end (PE) reads (101 base pairs) of 10 freshwater and 12 saline E.affinis samples -- were used to answer the following questions:

(1) What are the patterns of evolutionary shifts in gene expression between the ancestral saline and freshwater invading populations?
(2) What are the plastic (acclimatory) changes in gene expression between salinities (0 PSU vs. 15 PSU conditions) within each of the saline and freshwater populations?
(3) Are the magnitude and direction of plasticity in gene expression correlated with evolutionary responses?
(4) Has plasticity in gene expression evolved following freshwater invasions?

Collection of ancestral populations
The copepods were collected using a plankton net mesh size of 50 μm in diameter, from a depth of 1-4 meters from near the shore. The freshwater copepods were collected in April-May 2006 by throwing the plankton net off the dock in Racine Harbor, Lake Michigan in Wisconsin, USA (42.729444 N, 87.778889 W). The saline copepods were collected by small boats near the shore in Baie de L'Isle Verte, St. Lawrence marsh, Quebec, Canada (48.003889 N, 69.425278 W) in May-June 2006. Collected samples were transported to the laboratory where Eurytemora affinis individuals were identified and sampled under the microscope.

Laboratory cultures and experiments
Four inbred lines of Eurytemora affinis (two each from the two populations) were generated through full-sibling mating for 30 generations. Two independent saltwater inbred lines (SW1 and SW2) were derived from the ancestral saline population in Baie de L’Isle Verte (Canada) and reared at their native salinity of 15 PSU. The two freshwater inbred lines (FW1 and FW2) were derived from the freshwater invading population in Racine Harbor (USA) and reared in Lake Michigan water (0 PSU, conductivity 300 μS/cm). In addition, reciprocal F1 crosses between freshwater and saline inbred lines were created and reared to test for allele-specific expression by comparing gene expression in parental lines and their F1 crosses.

Two replicate common-garden reaction norm experiments, each consisting of a 2 × 2 factorial design, were performed to compare patterns of the gene expression of the FW and SW inbred lines (see Materials and Methods in Posavi et al. 2020). Total RNA from whole bodies of 50 copepods (25 females and 25 males) per sample was extracted using Trizol reagent (Ambion RNA) and Qiagen RNeasy Mini Kit for purification (Qiagen cat. no. 74104). Extracted and purified RNA samples were stored at -80 degrees Celsius until sequencing. The strand-specific Illumina RNA-seq libraries (Parkhomchuk et al., 2009) of polyA purified mRNA were constructed using the TruSeq RNA Sample Prep kit (Illumina). Three biological replicates per inbred line were sequenced using the Illumina HiSeq 2000 platform in the Institute for Genome Sciences at the University of Maryland School of Medicine and generated 101-bp-long paired-end read data.

These data have important implications for understanding the evolutionary and physiological mechanisms of range expansions by some of the most widespread invaders in aquatic habitats.

Problem report
One replicate of the FW1 inbred line was excluded because of bacterial infection

Additional information
~ Detailed methods, results, and figures can be found in Posavi et al. (2020) (see Related Publications section).
~ The sequence data can be viewed under NCBI BioProject PRJNA278152 (see Related Datasets).