Dataset: DNA microsatellite alleles from flowering shoots and seeds collected Curlew Beach in Nahant, MA and Niles Beach in Gloucester, MA in 2014

SCUBA was used to sample Zostera marina in late summer 2014 from two coastal eelgrass meadows in the Gulf of Maine, USA, separated by approximately 48 km: Curlew Beach in Nahant, MA (hereafter CB) and Niles Beach in Gloucester, MA (hereafter NI). Samples were collected from three depths at each site: the center of the meadow (mid), and approximately 5 m from the inshore and offshore edges (shallow and deep, respectively); depth of our shallow, mid and deep samples was approximately 1, 3 and 5 m MLLW, respectively. At each depth, 8-10 flowering shoots. separated by at least 2 meters, were collected; from each of these focal flowering shoots, a leaf tissue sample was harvested along with ~8-15 developing seeds from within a single spathe on the highest and youngest rhipidia, or cluster of inflorescences (n = ~250 seeds per site). Leaf tissue was preserved in silica or frozen until DNA extraction; seeds were frozen at -80C.

Molecular methods:
DNA was extracted from leaf tissue by grinding each sample with a Retsch mixer mill MM400 and using the Omega Bio-Tek E.Z.N.A.® Plant DNA Kit. DNA from seeds was extracted with Chelex 100 (Bio-Rad). Individual seeds were ground by hand in microfuge tubes with micropestles after removing the seed coat; samples were then incubated at 55°C for 8-24 h, gently mixed, heated to 98°C for 10 min, vortexed, centrifuged at 14,000 rpm for 5 min, and the supernatant stored at -20°C until PCR.

Each leaf sample was genotyped using 12 microsatellite loci developed for Zostera marina, multiplexed in three 11 ul PCR reactions. Each reaction consisted of 1 ul DNA template, 5 μl 2X Type-It multiplex master mix (Qiagen), and 0.25 ul of each 10 uM primer. PCR cycling conditions included initial activation/denaturation at 95°C for 5 min, followed by 28 cycles of 95°C for 30 sec, 60°C for 90 sec, and 72°C for 30 sec, and final extension at 60°C for 30 min. PCR products were separated on a 3730xl Genetic Analyzer (Applied Biosystems) at the Yale University DNA Analysis Facility, and fragment analysis was performed using GeneMarker version 2.6 (SoftGenetics). Nine of the 12 markers used for adult shoots proved useful for analyzing seed diversity. ZMC-12075 was eliminated due to a pattern of stutter around peaks that made scoring paternal alleles unreliable; two other markers (GA-3, CT-12) showed low allelic diversity in the adult populations and thus low power to discriminate among fathers.