Project Title: Density-Dependent Effects on Grazing and Success for Seed Generated Seagrass (Zostera marina L.) Plants.
Principal Investigator(s): Scott W. Nixon, Stephen Granger, Brian Maynard, and malia Schwartz.

Accomplishments
Scheduled Tasks:

1. As proposed in our last progress report, the final six months of this project focused on monitoring germination success during the first trials of our seeding sled. During the fall of 2000, we used the prototype sled to seed two aquaria, three sites in Narragansett Bay and one site in Ninigret Pond, a coastal lagoon. The formulation of the gel/seed mixture and the rate at which the sediment was seeded remained constant between test plots to allow for the comparison of success between sites.
2. We also proposed the construction of a pamphlet entitled "Growing Eelgrass from Seed- A Practical Guide to the Collection and Storage of Seeds for the Propagation and Restoration of Zostera marina L". The drafted manual contains data, numerous photographs and illustrations enabling the reader to recognize the various stages of sexual development in eelgrass and to select the optimum time for harvesting reproductive shoots. Methods for separating the seeds from their attendant shoots and storage of the seed stock are also described and illustrated by text and photographs.
3. Finally, we proposed the preparation of a manuscript that would include results from the density experiment conducted during the second year of funding. The relationships of seeding density to germination success, lateral shoot development, and biomass are fundamental to seagrass growth models that are designed to estimate the rate of bed recovery during restoration efforts, and for colonization of unvegetated sediments within a bed.

Progress on Tasks

1. Germination success of the seeds was determined by the appearance of a healthy seedling. Our previous work indicated that April is the month when seed germination is nearly complete and lateral shoot production is just beginning. Surveys documenting seed germination conducted later than April require destructive sampling to distinguish lateral shoots from a single seedling propagated from seed. Therefore, during April 2001, seedlings were counted in test plots seeded by the sled, and in hand-planted controls plots, located at each of the test sites. Control plots, seeded at the same density and depth as the test plots, were included to document the effect of the gel on seed germination and seedling success.
2. We have completed the construction of the seed collection pamphlet, including text, illustrations, photographs and data. We have negotiated a the cost of printing 2,000 6.5 inch by 8.5 inch pamphlets with a local printer. Presently, we are completing suggested changes from our internal review and working on specific layouts for the text and photographs.
3. While we have completed the necessary review of literature concerning past seagrass modeling efforts, we have not begun the construction of a manuscript at this time. We observed that the relationship of seed density to germination success, lateral shoot development and plant biomass was responsive to the organic content in marine sediments. We have recently received funding from CICEET to measure seed germination and seedling success in a relatively large-scale restoration effort in Narragansett Bay. As it was our original intention to apply these relationships developed in aquaria, to estimate the growth and expansion potential in newly seeded beds or in unvegetated areas within a bed. The comparison of our observations in aquaria experiments with those taken at the restoration site, adding validity to the development of a growth model. As a result, we have decided to wait until we have completed our field observations at the restoration site before advancing model.

Difficulties Encountered
The first tests of our seeding sled revealed some potential complications from the use of gel as a suspension media for the seeds. Shortly after seeding test plots in the aquaria, we noticed that the furrows darkened and developed a fungal mat. We determined this was caused by the enhanced respiration created by the organic content of the gel and developing seeds. While short periods of anoxia have not proven detrimental to eelgrass seeds in the past (and may even be an environmental stimulus for germination), there is evidence that persistent anoxia can be lethal to developing seeds. The fungal mat remained for a few months and diminished during the colder winter months, possibly reducing germination rates (see below).

Anticipated Success in Meeting Project Objectives in Scheduled Project Period
We do not anticipate any difficulties in meeting our project objectives for this reporting period.

Preliminary Results
In late April of 2001, divers counted the emerging seedlings in each of four field test sites along with those in controlled aquarium tank trials. The results are documented in the tables below (Tables 1 and 2). Generally lower germination was observed in the machine- planted seeds. As stated earlier, we feel that this lower success may be attributed to an increase in sediment respiratory demand due to gelatin used in the seed injection process. A reduction in the gelatin/seed ratio, or the use of non-organic gelling agents in future plantings, may help to improve germination rates. The Hope Island location displayed the lowest success of all field sites. Facing southwest, this site has a relatively long fetch, and is subjected to severe southwest winds during much of the year. Although success was lower at this location, it is worth noting that the only seedlings were located in the machine planted plots, indicating promise for machine seeding success in restoration locations with higher energetics.

Tasks and activities for next reporting period

Tasks for the next reporting period
The US Navy has funded our group for a large-scale restoration using seeding technologies developed with CICEET funding. They have dredged 0.25 acres of an existing eelgrass bed at Mc Allister Point, Rhode Island. We have been contracted to reseed the dredged area, and this will be the first large-scale test of the seeding machine. We are in the process of collecting and holding over 1.5 million seeds for this effort, 3 times that collected in previous years. Our seeding operations should begin in early October 2001, after making modifications to the seeding sled, scaling up from a 4-tine to an 8-tine model. The new sled will also be outfitted with a larger gel pump that will be capable of pumping the gelatin needed to fill the extra 4 furrows.

As a second task for this fall, we will also investigate new gelling substances for application with the seeding machine. As mentioned earlier, we believe the increased respiratory load on the sediments caused by the gelatin may have had a negative effect on seed germination. We would like to conduct tests using commercially available non-organic gelling agents such as sodium bentonite. These gelling agents are used in terrestrial seeding applications and may be transferable to our technique. Preliminary tests will be conducted this fall to investigate general characteristics of the material and its suitability for use with the seeding machine.

Finally, we will design a series of experiments to measure the effects of gelatin on the growth of seedlings. We observed increased biomass in the seedlings propagated from the seed/gel mixture in low organic sediments. This increased biomass may indicate that the gelatin is having a fertilizing effect on the seedlings. Previous research has shown that seedlings grown in high organic sediment (1.5 % TOM) will have significantly higher biomass at the end of one growing season when compared to those grown in lower organic sediments (0.5 % TOM). As time and resources were limited this year, we were unable to measure this conclusively; therefore designing and conducting a series of experiments to investigate the effect of gelatin on the emerging seedlings could give us valuable information.

Work plan to accomplish tasks
We are scheduled to begin seeding a site at McAllister Point in early October of this year. Flowering plant collection is complete and seed extraction will continue through mid- September; harvested seeds are being held in flow-through seawater tanks until planting. Our next task is the delineation of the restoration area and installation of buoys around its perimeter. Divers will take sediment cores from the area to measure sediment grain size and organic carbon; this information will aid us in determining the rate of seed delivery to the sediment. Once seeding has been completed we will return to the site during February-March 2002 and monitor early seedling emergence and again later in the spring.

We have contacted a provider of sodium bentonite (Texas Sodium Bentonite Inc., Comanche, TX) who will provide test samples and allow us to investigate the feasibility of this material for use in mechanized eelgrass reseeding. We will determine the consistency and viscosity of the material, along with its reaction to seawater. If the material appears promising, we will conduct tests in our aquarium. We will seed a series of sediment-lined trays with and without gelatin to test the effect on seedling growth. The trays will be held in flow-through seawater tanks at ambient irradiance, temperature, and salinity through the winter. Germination will be monitored in May of 2002. The seedlings will be held through one growing season then sampled in September 2002, to measure above and below ground biomass.

Concerns or difficulties
We are currently in the early stages of the McAllister Point restoration project and of documenting of our efforts as part of our CICEET grant. We are encouraged by our progress in reengineering the seeding sled to increase the number of seeds delivered to the sediment. We have also been successful at increasing the scale of harvesting and storing eelgrass seed. We are confident that the eelgrass seeding technology developed from aquaria experiments and small test plots can be successfully transferred to a large-scale restoration.

Expenditures
Our expenditures for this project have been within our anticipated budget. We do not foresee any unexpected costs within the next reporting period.