Sampling and analytical procedures:

 Wild adults were caught at spawning time using seine nets at Jekyll Island, Georgia (GA; 3103N, 8126W) and Patchogue, New York (NY; 4045N, 7300W) in spring 2017. Individuals were transported live to the Rankin Seawater Facility at the University of Connecticuts Avery Point campus. A full reciprocal crossing design was set up by strip-spawning multiple males and females in batches onto mesh screens submerged in plastic dishes in seawater. We created reciprocal F1 crosses: NY♀ x NY♂ (NY), NY♀ x GA♂ (NYxGA), GA♀ x NY♂ (GAxNY), and GA♀ x GA♂ (GA). Fertilized eggs were kept in 20L rearing containers placed in large temperature-controlled water baths at constant salinity (30psu) and photoperiod (15L:9D). We split the fertilized eggs of each pure cross (NY and GA) into four batches, and hatched and reared two batches per cross at 20C and two batches at 26C. The hybrid crosses were each split into two batches, with one batch for each crossing direction incubated at either 20C or 26C. The two temperatures, 20C and 26C, were chosen to reflect the common rearing temperatures at both parental spawning locations (NY and GA), respectively. Individuals were reared to an approximate total length of 30mm, with the rearing durations differing between populations and temperature regimes. Most individuals from the GAxNY cross died at 26C and all at 20C and thus we could not include these crosses in present study. From the remaining crosses, we randomly selected 6-8 individuals for RNA-sequencing.

Total RNA was extracted from whole larvae (n=42) using the ZymoResearch Direct-zol Miniprep RNA plus kit. Whole larvae were homogenized in Trizol using a pestle prior to RNA extraction. During the extraction, an optional in-column DNAse I treatment step was performed to remove traces of genomic DNA from the sample, and samples were eluted in 50l of RNAse-free water and stored at -70C. RNA quantity was determined using the HS Assay kit for the Qubit 3.0 fluorometer (Life Technologies, Carlsbad, CA) and quality was assessed using a Fragment Analyzer (Agilent, Santa Clara, CA) at the Cornell University Biotechnology Resource Centre. RIN values ranged from 5.3 to 8.3, with an average RIN of 6.9. RNA-seq libraries were prepared at BGI Genomics using the stranded Illumina TruSeq mRNA sequencing kit with Poly-A selection.

Instruments:

 Each library was sequenced to an average of 37.1M 2x150bp paired-end reads ( 0.194M s.d.) using an Illumina HiSeq 4000 sequencer at BGI Genomics.

Location:
The larvae reared in the laboratory were F1 offspring of parents collected either in :

1) Jekyll Island, Georgia (31.02,-81.43), or
2) Patchogue, New York (40.75,-73.00)