We surveyed a 41 km-long transect of the Belize Barrier reef, centered at Carrie Bow Cay, by SCUBA to conduct a genetic parentage study of the reef fish Elactinus lori. All underwater sampling was conducted using SCUBA at an average (±SD) depth of 16.03 ± 2.19 m. A waypoint was recorded from the boat at the beginning and end of every collection dive, with the midpoint of each dive taken as the location for all individuals sampled on that dive. To collect settlers, we sampled ~ 100 individuals every kilometer. Individuals were collected from the outsides of sponges using slurp guns and placed them in plastic bags. At the surface, settlers were anesthetized with MS-222. For adults, we collected non-lethal tissue samples at three regions along the transect (n ≈ 1,000 per region). Each adult was collected with a slurp gun and restrained in a net; we took a small tissue sample from the caudal fin using scissors. All tissue was stored in 95% EtOH. At each adult collection sponge, we also measured: sponge depth (m, using dive computers), number of tubes per sponge, and length of largest sponge tube (nearest cm, using a tape measure).

For genetic analyses, DNA was extracted using a HotSHOT protocol; fragments were amplified using the Type-It Microsatellite PCR Kit (Qiagen) and screened on an ABI 3730 automated sequencer.

Otoliths were extracted from the 120 settlers that were assigned to parents. Otoliths were dissected, cleared of tissue, immersed in oil for 2-7 days, and rings were counted under a 50× oil immersion lens

Further details on all methods can be found in D’Aloia et al. (2015), PNAS.