Field Sites and Methods

Study Locations

Field collection were made of 20 individuals from 10 populations of S. alterniflora and 10 populations of J. roemerianus across the northern Gulf Coast. Collection sites ranged from Lake Pontchartrain, LA to Pensacola Bay, FL. Living plants were maintained as separate populations at Mississippi State University (MSU).

Field collection was made of up 25 individuals from 10 populations of smooth cordgrass and 10 populations of black needlerush across the northern Gulf Coast (Figure 1). Locations were accessed primarily from the water, requiring use of a small vessel for a number of collection sites. At each collection location, researchers obtained vegetative material (single slips/rhizomes to secure genetic uniformity of resulting plant) from discrete plants (individuals with multiple shoots along a common root/rhizome) that were separated by at least 0.6m (2ft) using a “sharp-shooter” shovel to remove approx 0.25 cu ft (0.5x0.5ft area x 1ft depth) plugs from the marsh soil (total approx. 2 cu yards). Holes were backfilled with sand to facilitate rapid revegetation by the surrounding plants. Samples were placed in individually labelled gallon Ziploc^® bags and placed upright in a 100 qt cooler for transport. GPS coordinates of each collection location were recorded on a Garmin 76c (Table 1) and points downloaded in Google Earth compatible KML format for visualization. On return to the GCRL, plants were maintained sub-irrigated in labelled 1 gallon plastic pots (date and site collected) and grown in a 1:1 sand:topsoil mix (Figure 2).

Test samples of both plant species were sent to MSU for analysis during summer 2016 and results were successful. DNA from the tissue samples sent were amplified for ribosomal ITS using microsatellites. We found that S. alternifloraamplified better than J. roemerianus.

At the end of the collection season in 2016 and after a period of recovery of between 2-6 weeks all plant samples were transferred to MSU on 30 Aug 2016 for genetic analysis and cross fertilization studies. A datasheet of all samples and collection locations/dates accompanied the individually tagged samples.

During inspection of the plants in the MSU greenhouse, the vast majority seemed to be in excellent condition and were ready for DNA extractions. The senescent growth of the S. alterniflora was cut back in the late fall. The plants were maintained in the MSU greenhouse under ambient photoperiod. During inspection of the plants in the MSU greenhouse, the vast majority seemed to be in excellent condition and have been used for DNA extractions. The plants were moved to larger and cooler a new greenhouse, with more space and better temperature controls in May 2017. MSU researchers built three large ponds with circulating water in this greenhouse, with plenty of space for the plants. They then fertilized and encouraged the bulking up of the plants to start experimental crosses and selfs when they bloomed in fall 2017 and spring 2018 respectively.

Living tissue from each plant will then be harvested for DNA “fingerprinting” via chloroplast sequence (cpDNA) variation and nuclear microsatellite analyses to estimate nuclear genetic variability and outcrossing rates within each sampled population. Independent of molecular analyses all plants will be assessed for their potential to produce seed. Seed production by population will allow assessment of the relationship of population diversity to seed production.