The third experiment ran from April to June of 2021. Each acetate sheet containing a single settler was glued directly to the cap of a 15 ml centrifuge tube for deployment in the field. The PVC poles were arranged in a transect that ran parallel to the shore (Figure 1c). There were four treatments: (1) alone (one focal colony); (2) far (a focal colony with nearest neighbour 1 m away); (3) near (a focal colony with nearest neighbour 15 cm away); and (4) both (a focal colony with one neighbour 15 cm away and another colony 1 m away).
Each treatment had four replicates, one in each of four spatial blocks (8 colonies × 4 spatial blocks = 32 total colonies). Within each block, the order of the treatment was randomized. Within a treatment, all colonies were unrelated (i.e., offspring of different mother colonies). Each focal colony was
placed on a transect line parallel to the shore. The distance between spatial blocks was 10 m. Neighbouring colonies were placed perpendicular from the transect line at their allocated distances, and the direction of those neighbours from the focal colony (either towards or away from the shore) was randomized for each replicate.
Prior to deployment, the surrounding seagrass was searched to ensure no B. neritina colonies were present. During deployment, the poles and surrounding benthos were monitored every 4 days and any non-experimental colonies were removed to minimize the contribution of sperm from non-experimental colonies. Experimental colonies remained in the field for 14 days, which is sufficient time to exchange sperm, fertilize eggs, for embryos to develop, and for larvae to brood. All 32 colonies were collected from the field after 14 days and transported back to the lab, where survival and the number of zooids per colony were measured. All colonies were then placed into individual glass bowls (one colony per bowl) with 250 ml of FSW and a roughened acetate sheet was floated on the surface for larvae to settle upon. Each day, the colonies were placed under a hanging LED light for approximately 8 hr to induce larval release. Every 4 days, the sheets were removed and replaced. To measure reproductive output, the number of settlers from each colony was counted every 4 days for 20 days (i.e., six occasions). No new offspring were released after 20 days.