made from polyester silk (Silkessence, Jo Ann’s Fabrics www. joann.com, Fig. 1). These nets were pillow-shaped and approx- imately 1 m long and wide with a nylon synch cord at the bottom and a 10 cm-wide funnel hot-glued to the top of the 10 cm tapered net. Straddling the funnel tip was a 100 ml urine cup lid with a 2.5 cm hole glued in place. This allowed urine cups to be attached and removed during the collection process. The net was held buoyant by a small float (8 cm in diameter, 2.5 cm thick) so the urine cup remained parallel to the water surface, allowing the buoyant bundles to float upwards through the inverted funnel into the 100 ml urine cup. Thirty to 45 min before the anticipated onset of spawning (,2100 hr), divers cinched the nets to a coral branch (typically at a depth of 1 to 5 m) on a given colony. These nets were placed only on colonies that were ‘setting’, meaning bundles were visible on the coral’s surface. After ,45 min of release of egg/sperm bundles, collection cups were removed from the stationary lid while held inverted underwater to keep the bundles in place, recapped with a spare lid and then transferred to shore within 30 min. For the A. palmata study, egg-sperm bundles were transferred to 50 ml conical tubes to reduce the volume of water, and the eggs and sperm were separated by gentle agitation of the tubes. The eggs floated in the tube, whereas sperm were suspended throughout. Concentrated spermatozoa were removed Preserving and Banking Coral to a separate 50 ml plastic tube and maintained in the original salt water until cell concentration was determined. In parallel, eggs were serially washed (at least 36, or until the rinse water was clear and the eggs floated and moved unencumbered at the top of the tube) with 50 ml rinses of 0.2 mm-filtered seawater (FSW) at room temperature (typically 28 to 30uC). Unless stated otherwise, all other described solutions were made using 0.2 mm- FSW (1,000 mOsm) generated by passing raw seawater through a Nalgene Steri-cup filtration system. Fungia scutaria adults were collected from various shallow reef flats in Kaneohe Bay and then maintained in shallow running seawater tables at the Hawaii Institute for Marine Biology, University of Hawaii. We adhered to the gamete collection and larvae rearing methods of Krupp [38] and Schwarz et al. [39]. Briefly, F. scutaria spawns over 2 to 3 nights for multiple months each summer (June to September), ,2 nights after a full moon [38]. Two hr before spawning onset (,1800 hr), adults were placed into individual 2 L plastic bowls filled with seawater, and individual disposable pipettes (Fisher Scientific) were placed in each bowl to minimize cross contamination of gametes between bowls. On the basis of observing a uniform milky cloud from the mouth of individual coral specimens, spermatozoa were immedi- ately aspirated using a plastic, disposable pipette and held in Figure 1. Scientific SECORE divers placing several collection nets on a large A. palmata colony prior to spawning in the evening. A) A. palmata setting, note the pink egg/sperm bundles resting on the surface of the brown colony prior to release into the water column. B) Scientific divers placing the specially-designed nets on a colony to collect egg/sperm bundles. C) Egg/sperm bundles were collected in the cup at the top of the net. Photos contributed courtesy of the Pittsburgh Zoo & PPG Aquarium, Photo 2010 Paul A. Selvaggio. doi:10.1371/journal.pone.0033354.g001 PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e33354