In April 2012, we collected 100 adult oysters (80-100 mm shell length) from 3-5 separate reefs at each of 6 sites: St. Augustine, FL (FL-1; 30.0224, -81.3287), Jacksonville, FL (FL-2; 30.4446, -81.4199), Sapelo Island, GA (GA/SC-1; 31.4777, -81.2726), Ace Basin, SC (GA/SC-2; 32.4846, -80.6001), Masonboro, NC (NC-1; 34.1510, -77.8551), and Middle Marsh, NC (NC-2; 34.6951, -76.6183). They were held in flowing seawater tanks or suspended in cages from docks in their home region for 2-3 weeks until 30 oysters from each site could be tested and certified as disease free. The remaining 70 oysters were then shipped on ice to a single hatchery facility in Florida (Research Aquaculture Inc., Tequesta, FL; 26.9607, -80.0931) at the end of April.

The adult oysters from each site were used as the broodstock to produce 6 separate site-specific "cohorts" (one cohort per site). From their arrival at the hatchery, the adult oysters were held for 2 weeks until they were ready to spawn under the same conditions in separate flow-through seawater systems to prevent cross-contamination. All families were manually spawned (i.e., strip spawned) on May 7 (see details below). Because the original FL-1 family did not produce many offspring, the remaining broodstock oysters from this site were spawned on June 1 using the same process. Due to variation in ripeness and sex, the number of oysters spawned and the ratio of males to females varied across broodstock (Table 1 of Hughes et al., 2019), though our broodstock numbers for each cohort are comparable to those commonly used in hatchery settings (30-60 individuals; Morvezen et al. 2016).

The broodstock oysters from each source site were strip spawned, sexed, and fertilized on the same day by a team of 7 people, who each had a specific job to perform: shucking the animals, sampling and preparing tissue for microscopic analysis of sex, identifying the sex, stripping the male sperm, stripping the female eggs, mixing the sperm and eggs after all of the animals from a particular source were stripped, overseeing the process and keeping track of broodstock source. We sanitized equipment between individuals and again between broodstock sources. Stripping was done by broodstock source independently and quickly so that the sperm and eggs would remain viable, and all viable sperm and eggs were used. During the gamete mixing process, the eggs from all females and the sperm from all males were first pre-mixed and then combined to ensure equal access of gametes to one another. We allowed 30-60 minutes for fertilization; once 75-90% of the eggs were fertilized, they were moved to larval tanks. All larvae were retained except for minimal numbers of individuals in each cohort that did not grow or had improper development. Larval culture occurred in 60-gallon conical tanks utilizing a flow-through seawater system with Banjo screens that is commercially used in multiple bivalve hatcheries (e.g., Taylor Shellfish in WA; Cherrystones in VA).

Over a period of 3 days the week of May 28, oysters were sieved on a 250-micron sieve and settled on crushed oyster cultch in a recirculating flow-through system. The week of June 11, once they reached 800 microns in size, they were moved into a nursery facility compliant with state regulations, again under flow-through seawater conditions (salinity = 32 ppt, temperature = 30ºC). In the hatchery and nursery stages, the oysters were fed a mixed diet of T. isochrysis, Chaetocerous gracilis, and Tetraselmis via a constantly running peristaltic pump. Although growth was similar during the larval culture phase, some cohorts produced more juvenile oysters ("spat") than others during settlement, despite following the same procedures for all. To maintain consistency in their growing conditions, we selected a random sample of each cohort to yield similar total abundances across cohorts on June 18. At the end of June (June 27) at approximately 4mm in size, the 6 cohorts were transferred to a common flow-through facility at the Whitney Marine Biological Laboratory in St. Augustine, FL. To assess genetic diversity within and between oyster cohorts produced in the hatchery, 50 individuals were haphazardly collected from each juvenile cohort prior to the start of the field experiments and preserved at -80ᵒC for genetic analysis. This sample size is sufficient to estimate allele frequencies accurately (Hale et al. 2012).

To extract DNA, we ground each tissue sample with a pestle, and used the tissue centrifugation protocol from the Omega Bio-Tek E-Z 96 Tissue DNA Kit. We determined genetic diversity and population structure using 12 highly variable microsatellite loci developed for C. virginica: Cvi9, Cvi11, and Cvi13 from Brown et al. (2000); Cvi1i24b, Cvi2i23, Cvi2j24, and Cvi2k14 from Reece et al. (2004); Cvi4313E-VIMS from Carlsson and Reece (2007); and RUCV1, RUCV66, RUCV73, and RUCV74 from Wang and Guo (2007). We amplified four loci in each multiplexed polymerase chain reaction (PCR) using the Qiagen Type-It Microsatellite PCR Kit. Each 10 l reaction consisted of 1 l DNA template, 5 l 2X type-it multiplex master mix (Qiagen), 2.4 l water, and 0.2 l each 10 M primer. Using a T100 thermal cycler (Bio-Rad), PCR cycling conditions included initial activation/denaturation at 95ᵒC for 5 min, followed by 28 cycles of 95ᵒC for 30 sec, 60ᵒC for 90 sec, and 72ᵒC for 30 sec, and final extension at 60ᵒC for 30 min. PCR products were separated on a 3730xl Genetic Analyzer (Applied Biosystems) with the internal size standard GeneScan 500 LIZ (Applied Biosystems), and fragment analysis was performed using GeneMarker version 2.6 (SoftGenetics).

We created panels for each multiplexed reaction in GeneMarker, which included bins that were assigned manually for all alleles; the same panels were used to score all samples, and the alignment of the panels was checked prior to each analysis to account for any run-to-run variation and to identify any new alleles. We used these panels to do a preliminary first assignment of alleles based on peak position and bin position, but every sample was then scored manually for all loci to examine signal intensity, to confirm the presence/absence of alleles, and to identify any reruns. A subset of samples was then rerun (at least 15% per multiplex PCR reaction) and manually scored again to confirm any uncertain allele calls and account for any genotyping error.