Figure 5: Schematic of light gradient experiment to determine minimum integrated light requirements for seagrass survival. Two species will be tested - Zostera and Halodule. A bank of overhead Halogen lights of increasing wattage will provide light. Plants in one of the two tubs will be subject to additional light stress at one month intervals by shading for 2, 5, & 10 days.

Pilot experiments on temperate Zostera marina demonstrated that this species can tolerate up to 30-45 days of aphotic conditions before 100% mortality occurred. Furthermore, seedlings (the most vulnerable and critical life-phase) were able to persist in extremely low light conditions (1% surface irradiance) for up to three months (90 days) - this is a very encouraging finding, demonstrating the robustness of these plants. The tropical species, Halodule wrightii however, experienced total mortality in less than 28 days in complete darkness. These inter-specific differences in tolerance to light deprivation found in the pilot studies were complicated by the ability of different individuals to withstand light deprivation over a range of durations.

A light gradient (seven levels: 0, 5, 10, 35, 70 150, 300 mmol photons m-2s-1) will be created in a large indoor tank under controlled environmental (light, air temperature) conditions (Fig. 5). The light field will be mapped at 5cm intervals using a LiCor Scalar (4-p) sensor at monthly intervals to determine possible reductions in irradiance with aging of the Halogen lamps and differential fouling of the water column. The lights will be placed on a timer to create a 12:12 L:D environment resulting in integrated irradiances of: 0, 0.2, 0.5, 1.5, 3, 7, 12 mol photons m-2 day-1 respectively. Water temperature will be monitored using Stowaway Tidbit Temperature Loggers (www.OnsetComp.com) at 30 min intervals at both ends of the light gradient, to account for potential warming of the water as a result of heat radiated from the halogen lamps. Water quality data will be collected with a YSI 6000 series multi-parameter water-quality probe (www.YSI.com). To create additional light stress mimicking natural light attenuation events (i.e., storms, blooms), subsets of plants in one of two tubs in each light level will be subject to different periods of light deprivation at monthly intervals, by shading them with black plastic sheeting. Three plants each will be subject to 2 days (1/15), 5 days (1/6), and 10 days (1/3) per month of light deprivation compared to the their control counterparts not subject to additional deprivation.

Two tubs per light level will be planted with nine Zostera seedlings, or nine Halodule plants (3x3 arrangement) and placed into the seven light gradient treatments after 10 days recovery from transplant stress. Initial measurements will be taken on the day the plants are placed into the light gradient tank, and weekly thereafter until the experiment is terminated. Weekly measurements taken on each plant will include (Table 1): #shoots, total # leaves, longest leaf length, and photosynthetic efficiency (Fv/Fm and other PEA parameters). Biweekly mature leaves from a random subset of plants will be collected to measure width and leaf reflectance spectrum. Monthly, three plants each will be marked and then harvested after two weeks growth to determine plastochrone interval using the method of Short and Duarte (2001). Lab analyses of seagrass leaf samples collected at biweekly intervals will include: chlorophyll extraction, tissue nutrient content (C:N:P), N-index = %N/leaf biomass (F. Short, pers.comm.), tissue carbohydrate content (an index of storage reserves), and other pertinent biochemical parameters, using standard analytical methods.