EVALUATING THE EFFECTS OF SALT ACCLIMATION ON THE GROWTH AND SURVIVAL OF SPARTINA ALTERNIFLORA A Thesis Submitted to the Graduate Faculty of Nicholls State University in Partial Fulfillment of the Requirements for the Degree Master of Science in Marine and Environmental Biology By Lisa Ann Breaux B.S., Nicholls State University, 2009 Fall 2011 1
ABSTRACT Smooth cordgrass Spartina alterniflora is an important and dominant species in salt marsh habitats along the Gulf and Atlantic coasts. Smooth cordgrass is often used for coastal restoration because it stabilizes sediment and reduces erosion rates, can withstand fluctuating salinity and water levels and are an important nursery habitat for many species. Commercial producers typically culture smooth cordgrass in freshwater (salinity < 1 ppt) and the LDNR currently requires salt acclimation prior to transplantation for restoration. The purpose of this study was to determine the necessity of acclimating cultured smooth cordgrass plants prior to transplantation. By comparing survival and growth of two smooth cordgrass ecotypes ( Vermilion and wild type) that have been acclimated to either 0, 5, 10, or 15 ppt prior to being transplanted to either 0, 10, 20, or 30 ppt. Vertical extension (cm) and number of new stems of two ecotypes were compared at the end of a standard LDNR acclimation period. At the end of the acclimation period, plant height was similar among acclimation salinities for the wild type, but height of Vermilion was greater at 5 ppt than 0 and 15 ppt. Number of new stems was greater at 5 ppt than 10 ppt for the wild type, but was similar among acclimation salinities for Vermilion. Stem diameter was similar among acclimation salinities for each ecotype. Comparison between ecotypes at the end of the acclimation period revealed the wild type to have greater height increase (at 0, 10, and 15 ppt), and greater stem diameter (at 0 and 15 ppt) than Vermilion. However, Vermilion produced greater number of stems than the wild type at 10 ppt at the end of the acclimation period. During the grow out period, acclimation effects on height revealed a greater height change for wild type plants acclimated at 15 ppt than 0 and 5 ppt. There was no difference in height change for 2
Vermilion among acclimation salinities during the grow out period. Acclimation salinity did not affect the number of new stems or the belowground biomass for either ecotype during the grow out period. Comparison between ecotypes revealed greater change in height for Vermilion acclimated at 5 ppt for the grow out period and greater number of new stems for Vermilion acclimated at 10 and 15 ppt during the grow out. Comparison of belowground biomass between ecotypes was similar among acclimation salinities. Results from this study suggest salt-acclimation prior to transplanting is not necessary for early survival and growth, and indicates the feasibility of using non-vermilion ecotypes for future restoration efforts. 3
ACKNOWLEDGEMENTS This research project would not have been possible without the help and guidance of many people. I would first like to thank my advisor Dr. Quenton Fontenot for his constant guidance and support during my time at Nicholls State University. Sincere appreciation goes to my committee members, Dr. Allyse Ferrara, Dr. Rajkumar Nathaniel, and Mr. Gary Fine for their help with this project. I would also like to thank Nicholls State University for their use of equipment, McNeese State University and the Natural Resource Conservation Service for funding to complete this project, the Plant Materials Center for their use of equipment, Rockefeller Wildlife Refuge for their help in the field and the Louisiana Native Plant Initiative. A sincere appreciation also goes to many wonderful graduate students at Nicholls State University who willingly helped with set up and data collection: Rachel Ianni, Michele Felterman, Alyssa Sanders, Emily Rombach, Taren Manley, Kent Bolfrass, Stephen Byrne and Daniel Davies. Help of friends outside of the university is also greatly appreciated, and a special thanks goes to Jade Smith for her sincere desire to help and enthusiasm for the work. Most importantly, I would like to thank my family for their lifelong love, encouragement, and confidence in me. I would especially like to thank my parents, Kevin and Deborah Breaux, for their desire to be involved in helping with my data collections. Lastly, I would like to thank Donald Spahr, Jr. for his motivation, love, and support throughout every day of this project, which has inspired me more than I could have ever hoped for. 4
TABLE OF CONTENTS Certificate... i Abstract...ii Acknowledgements iii Table of Contents... iv List of Figures.v List of Tables..vii List of Abbreviations..viii Introduction. Methods.. Results. Discussion... Literature Cited... Appendix I.. Appendix II. Appendix III Biographical Sketch... Curriculum Vitae... 5
LIST OF FIGURES Figure 1. Mean (±SE) salinity for each salt acclimation treatment for the entire acclimation treatment prior to the greenhouse grow out period....21 Figure 2. Salinity for each acclimation treatment for the entire acclimation treatment prior to the field grow out period.. Figure 3. Mean (± SE) height (cm) for wild (a) and Vermilion (b) smooth cordgrass acclimated at 0, 5, 10, or 15 ppt. Graph (c) represents a comparison between ecotypes for each acclimation salinity.. Figure 4. Mean (± SE) number of stems per pot for wild (a) and Vermilion (b) smooth cord grass acclimated at 0, 5, 10, or 15 ppt. Graph (c) represents a comparison between ecotypes for each acclimation salinity. Figure 5. Mean (± SE) stem diameter (mm) for wild (a) and Vermilion (b) smooth cordgrass acclimated at 0, 5, 10, or 15 ppt. Graph (c) represents a comparison between ecotypes for each acclimation salinity.. Figure 6. Mean (±SE) change in height for wild (a) and Vermilion (b) smooth cordgrass acclimated to 0, 5, 10, and 15 ppt salinities at the end of the grow out period. Graph (c) represents a comparison between ecotypes for each acclimation salinity.. Figure 7. Mean (±SE) change in height for wild (a) and Vermilion (b) smooth cordgrass acclimated to 0, 5, 10, and 15 ppt salinities for the grow out period. Means with a similar letter are not different. Graph (c) is a comparison of the two ecotypes for each acclimation salinity Figure 8. Mean (±SE) change in height for wild type smooth cordgrass over time 6
Figure 9. End of grow out mean (±SE) change in height for Vermilion smooth cordgrass over time Figure 10. Mean (±SE) number of new stems produced during the grow out at each acclimation salinity for wild type (a) and Vermilion (b). Graph c is a comparison of the two ecotypes at each acclimation salinity (wild type = white bars and Vermilion = black bars). Figure 11. Mean (± SE) number of new stems for Vermilion (top) and common type (bottom) plants grown out at either 0, 10, 20, or 30 ppt over 20, 40, and 60 days Figure 12. Comparison of mean (± SE) number of new stems for Vermilion (black bars) and wild type (white bars) at day 40 (a) and day 60 (b) of the grow out... Figure 13. Mean (±SE) dry weight (g) of below ground biomass at the end of the greenhouse grow out for wild type (a) and Vermilion (b) acclimated to salinities of either 0, 5, 10, or 15 ppt. Graph (c) is a comparison between ecotypes Figure 14. Mean (±SE) dry weight (g) of below ground biomass at the end of the greenhouse grow out for wild type (a) and Vermilion (b) grown out at salinities of either 0, 10, 20, or 30 ppt. Graph (c) is a comparison between ecotypes.. Figure 15. Figure 16. Mean (±SE) height for plants acclimated to 0 or 15 ppt and grown out at Rockefeller Wildlife Refuge for 20, 40, and 63 days.. Mean (± SE) survival of smooth cordgrass acclimated to 0 or 15 ppt and grown out at Rockefeller Wildlife Refuge for 20, 40, and 63 days 7
LIST OF TABLES Table 1. Mean (±SE) number of new stems for Vermilion and common type smooth cordgrass at grow out salinities of 0, 10, 20, and 30 ppt for 20, 40, and 60 days 8
LIST OF ABBREVIATIONS Carbon dioxide Catfish Lake ecotype Celsius Centimeter Day Hours Kilogram Kilometer Liter Louisiana Department of Natural Resources Milliliter Millimeter Mississippi River Gulf Outlet Natural Resource Conservation Service Number Parts per thousand Plant Materials Center Potassium Potassium chloride Sodium Sodium Chloride Standard Error United States Geological Survey Vermilion cultivar Year CO2 wild C cm d hrs kg km L LDNR ml mm MRGO NRCS No ppt PMC K KCl Na NaCl SE USGS V yr 9
INTRODUCTION Louisiana s coastal wetlands are home to one of the world s most productive and diverse ecosystems, yet they are experiencing rapid and extensive conversion of land to water. Some of the largest commercial fisheries in the United States are supported by Louisiana s coastal wetlands (USGS 2011), which also provide habitat for neotropical birds and waterfowl. Wetlands also help to regulate water levels within watersheds and improve water quality through assimilation of excess nutrients from pollutants such as sewage and agricultural drainage (Knight 1992). Approximately half of Louisiana s population resides along the coast (USGS 2011) and are protected from storms and floods by coastal wetlands. The wetlands function as a natural buffer against storm surges, thus reducing flood and storm damage to urban areas. The milieu for the unique Cajun culture of south Louisiana is also provided by its wetlands, making them not only valued in the context of the environmental and economic services they provide, but also in the context of cultural heritage. While Louisiana makes claims over 30% of the United States coastal marsh, it has experienced extensive land loss accounting for approximately 90% of the total coastal land loss in the continental United States (Couvillion et al. 2011). The loss rate has been estimated to be approximately 61.3 km 2 per year (Barras et al. 2003), which may cause Louisiana s coast to be reduced by another 1300-2000 km 2 by 2050 (Barras et al. 2003). One-third of the Louisiana coast will have vanished if that loss rate continues. The Barataria-Terrebonne basin, which spans approximately 1.7 million hectares between the Atchafalaya River and the Mississippi River, is experiencing the majority of Louisiana s coastal land loss. Critical habitat for many coastal species, including the white shrimp 10
(Litopenaeus setiferus), brown shrimp (Farfantepenaeus aztecus), and blue crab (Callinectes sapidus) are lost in conjunction with the land. These species, along with many others, spend part of their life cycle in the salt marshes where they can find food and shelter. Continued coastal land loss will have severe impacts on the people, wildlife, energy infrastructure, and fisheries of coastal Louisiana (USGS 2011). A combination of factors contributes to coastal land loss, including both natural and human induced factors. One of the biggest factors affecting wetland loss is the gradual subsidence of land below the level adequate to support vegetation. When combined with sea level rise, subsidence is an even greater threat to land loss. National Ocean Survey tide gage records show that Louisiana is experiencing the highest relative sea level rise throughout the Gulf Coast, at a rate at 1.04 cm/yr for Grand Isle (Penland and Ramsey 1990). The global relative sea level rise rate (0.12 cm/yr), in comparison with the 1.04 cm/yr rate in Grand Isle, indicates a 10 times faster rise in sea level in Louisiana than in many other places of the world (Penland and Ramsey 1990). Erosion of land is a consequence of subsidence, but is only a minor cause of land loss compared to subsidence. Although subsidence is a natural process, the rate of subsidence is often enhanced by human activities. Madison Bay is a known hotspot for wetland loss in Louisiana. According to Morton et al. (2003), two-thirds of the permanent flooding of the Madison Bay area in the 1960 s to 1970 was caused by subsidence and the other third by subsequent erosion. The cause of the rapid subsidence is attributed to human actions, specifically the acceleration of hydrocarbon production that may have led to fault activation and as a result, increased subsidence (Morton et al. 2003). 11
Accretion occurs naturally as river water brings sediments and nutrients to the wetlands, but the hydrologic modifications along the Mississippi River have stopped the natural land building process that would otherwise occur during annual flooding of the river. Before the Mississippi River was leveed off, the river would change course and deposit sediment to create new land. With the current levees in place, the natural process of land accretion no longer offsets subsidence. Sediment discharge to the Mississippi delta has decreased by 70% since 1860 due to dam building (Day et al. 2008). In the case of canal dredging, the Mississippi River Gulf Outlet (MRGO) provides an example of canal dredging that resulted in an extensive amount of wetland loss. The MRGO was meant to provide a shorter route from the Gulf of Mexico to the New Orleans Industrial Canal, but saltwater intrusion, wetland loss, and greater storm surges into New Orleans were unintended consequences (Shaffer 2009). Natural sedimentation processes cannot occur at rates fast enough to offset the impacts induced by humans. The rapidly deteriorating wetlands of coastal Louisiana are priceless in their value to the culture and heritage of the people. Due to the complexity of the situation, wise management practices should be based on a multi-scale understanding of these biologically complex ecosystems (Day et al. 2008). Recovery of coastal wetlands after disturbance is often naturally slow, but replanting vegetation can accelerate the recovery time for marsh restoration. Current efforts to restore coastal areas experiencing land loss involve the creation of new land and revegetation of land to stabilize the soil and prevent further loss, subsequently creating habitat for many species (Knutson et al. 1981). Spartina alterniflora (smooth cordgrass) is commonly used due to its salt tolerance, 12
vigorous spreading, and strong belowground rhizome growth ability, which aids in the prevention of soil erosion and also in the reclamation of land. Smooth cordgrass is the dominant salt marsh plant along the tidal wetlands of the Gulf and Atlantic coasts (Biber and Caldwell 2008). Growth of smooth cordgrass primarily occurs by vegetative propagation, which leads to the formation of dense monospecific stands. The dense stands of vegetation provide critical habitat for many ecologically and economically important coastal species (Minello et al. 2003), and contribute a significant amount of detritus to the salt marsh system (Wainright et al. 2000). Ramets are connected by belowground rhizomes that grow a short distance from the parent plant and eventually sprout new stems. As stems grow, they emerge in a circular fashion, spreading in diameter until meeting another stand. Pieces of plants may break off and float to another area for colonization. Floating seeds may also germinate after reaching a suitable habitat. The plant shoots consist of aerenchyma cells, which act as conduits for oxygen movement (Armstrong 1991) as well as conduits for carbon (Gallagher and Plumley 1979). The aerenchyma tissue allows atmospheric oxygen to diffuse into the plant, which is then transported to the roots by a suction created from the anoxic soils surrounding the roots. Microorganisms break down the dead plant material for the shrimp, crab, and fish to consume as a main carbon and energy source (Wainright et al. 2000). Nutrients from decaying plant material are eventually cycled back into the system as fertilizer for the next season s growth. The extensive root system of smooth cordgrass binds and traps sediment, and is a valuable aspect of using smooth cordgrass for coastal land reclamation (Partridge 1987). 13
Previous studies have found sedimentation accretion rates by smooth cordgrass to be as high as 13 mm/year (Simenstad and Thom 1995). Smooth cordgrass can grow in areas permanently inundated, accumulating sediment and creating habitat for other species such as ribbed mussels (Geukensia demissa; Partridge 1987). The combination of sediment accumulation and species colonization gradually increases land levels at the edge of the marsh. Accretion also coincides with new outward growth along the marsh edge, where smooth cordgrass grows in its tallest forms. However, without a source of sediment, accretion cannot occur. Ecotypes are considered genetic variations of a species determined by surrounding environmental conditions, and whose existence is crucial to the ability of vegetation to adapt to its habitat (McMillan 1960). Although smooth cordgrass primarily grows by vegetative propagation, environmental conditions vary along the coast and different ecotypes have been found in coastal populations (Proffit 2003). Seliskar et al. (2002) suggests that different ecotypes may display different ecosystem functions, such as primary production, necessary for living organisms (Seliskar et al. 2002). In a study by Seliskar et al. (2002), differences in detritus production among three genotypes of smooth cordgrass were noted. The differences in detritus production by smooth cordgrass can influence the amount of production in the marsh system. To possibly maintain genetic diversity, more than one ecotype of smooth cordgrass should be used in restoration. Currently the Louisiana Department of Natural Resources (LDNR) requires the use of Spartina alterniflora cv. Vermilion for all LDNR re-vegetation projects. The decision to release the Vermilion cultivar was based on a study by the Natural Resource Conservation Service (NRCS) in 1989. The NRCS 14
compared vigor among 90 individual collections of smooth cordgrass throughout the Gulf of Mexico basin and found the Vermilion ecotype to have a high survival rate and consistency among soil types (Soil Conservation Service 1989). Among the variables observed were transplant survival, productivity, heat tolerance, and pest resistance. Smooth cordgrass grows well in freshwater, but is usually outcompeted by other species in freshwater habitats (USDA, NRCS 2000). The ability of smooth cordgrass to withstand fluctuating salinity and water levels allows the species to maintain dominance in the salt marsh system (Huckle et al. 2000). Smooth cordgrass is able to alter its physiological mechanisms in response to heightened salinity levels to maintain internal homeostasis. Smooth cordgrass uses four mechanisms to regulate osmotic pressure from salinity fluxes. Salt exclusion at the roots is due to a selectively permeable membrane, which allows for uptake of K + over Na +. Salt accumulation occurs by storage of excess NaCl in vacuoles (Glenn et al. 1999). However, accumulation requires active transport, which is energy intensive. For this reason, accumulation is only used when metabolic processes are at their maximum rate as during the height of the growing season. Smooth cordgrass can also excrete NaCl that was stored in vacuoles via salt secreting glands present in the leaves (Bradley and Morris 1991). Smooth cordgrass is also able to use Na + for osmotic adjustment in the shoots to acclimate to salinity fluctuations (Vasquez et al. 2006). Solutes such as proline and glycine betaine are increased in the plant cells, thus altering the osmotic pressure and reducing the uptake of Na + (Ashraf and Foolad 2007). The C4 properties of smooth cordgrass also allow for a higher rate of CO2 assimilation, resulting in a reduction of the amount of saline water taken up by the roots (Bradley and Morris 1991). 15
Successful restoration by vegetation depends on variable site characteristics such as soil characteristics, nutrient availability, elevation, wave climate, and salinity (Roland and Douglass 2005). Smooth cordgrass can tolerate a wide range of environmental conditions and salinities from 0 to 60 ppt, with an optimal range between 10 and 20 ppt (Landin 1991). Haines and Dunn (1976) reported maximum growth rates of smooth cordgrass to occur at or below 20ppt. Height was also found to be inversely related to salinity, as height decreased from creek banks to salt marshes (Nestler 1976). Maximum heights of smooth cordgrass were found to reach 3 m near creeks, yet maximum height was only 10 cm furthest from creeks where soil salinity was high (Howes et al. 1981). Wave climate also depends on a number of factors, such as wind speed, fetch, orientation of the shoreline, and water depth (Broome et al. 1986). Roland and Douglass (2005) demonstrated that increasing wave levels corresponded to decreasing vegetation along the shoreline. Smooth cordgrass vegetation along a shoreline has been demonstrated to decrease as wave levels increased (Roland and Douglass 2005) with an upper limit of wave energy at a median wave height of approximately 0.1m to 0.2 m for 80% of the time. Allen and Webb (1982) demonstrated significant washout of smooth cordgrass transplants within one month when planted in an area unprotected from wave action, yet the addition of a fixed breakwater system protected the plants and resulted in a significant difference in initial plant survival and densities after two months The LDNR currently requires plants of smooth cordgrass to be salt acclimated prior to transplantation in the field. Plants are required to be salt acclimated to a minimum of 10 ppt at increments no greater than 5 ppt per week. The salt acclimation must occur at 10 ppt (or maximum acclimation salinity) for at least 14 consecutive days 16
before transplantation. However, survival rates of up to 94% have been recorded for smooth cordgrass transplanted to coastal areas without salt acclimation (unpublished data, Q. Fontenot). The purpose of this research is to determine if salt acclimation of smooth cordgrass plants prior to transplantation results in greater survival and growth rates compared to plants not salt acclimated, while incorporating different ecotypes into the study. It was hypothesized that survival and growth of two smooth cordgrass ecotypes are not affected by salinity exposure regimes. It was also hypothesized that survival and growth of two smooth cordgrass ecotypes are similar regardless of salinity exposure regimes. The goal was to compare survival and growth of two smooth cordgrass ecotypes acclimated to salinities of 0, 5, 10, or 15 ppt and transplanted to 0, 10, 20, or 30 ppt in a greenhouse or to a location along the coast. The objectives included: 1. Compare survival and growth of two ecotypes ( Vermilion and wild type) acclimated to 0, 5, 10, or 15 ppt within a greenhouse at the end of the acclimation period. 2. Compare the vertical extension, number of stems, and below-ground biomass of two ecotypes ( Vermilion and wild type) acclimated to 0, 5, 10, or 15 ppt within a greenhouse at the end of the acclimation period. 3. Compare survival of two ecotypes ( Vermilion and wild type) acclimated to 0, 5, 10, or 15 ppt that have been transplanted within a greenhouse to 0, 10, 20, or 30 ppt after 20, 40, and 60 days. 4. Compare the vertical extension, number of stems, and belowground biomass of two ecotypes ( Vermilion and wild type) acclimated to 0, 5, 10, or 15 ppt 17
that have been transplanted within a greenhouse to 0, 10, 20, or 30 ppt after 20, 40, and 60 days. 5. Compare the survival of two ecotypes ( Vermilion and wild type) acclimated to 0 or 15 ppt within a greenhouse that have been transplanted to Rockefeller Wildlife Refuge after 20, 40, and 60 days. 6. Compare the vertical extension of the two ecotypes ( Vermilion and wild type) acclimated to 0 or 15 ppt within a greenhouse that have been transplanted to Rockefeller Wildlife Refuge after 20, 40, and 60 days. 18
Methods Specimen collection Specimens of the Vermilion cultivar S. alterniflora cv. Vermilion were collected from the Natural Resource Conservation Service Plant Materials Center (NRCS-PMC) in Galliano, Louisiana on 1 September 2010 and specimens of S. alterniflora wild type were collected from Catfish Lake(29 23'26.00"N 90 16'40.69"W) in Lafourche Parish on 2 September 2010. The Vermilion cultivar of Spartina alterniflora will be referred to as Vermilion type and the Catfish Lake collection will be referred to as the wild type. The newly collected plants were maintained in burlap sacks or plastic tubs with just enough water to cover the roots for transport to the Nicholls State University Farm in Thibodaux, LA. The plants were maintained in plastic tubs with fresh water until transplanting into 2.84 L trade-gallon pots. Individual stems of Vermilion were potted on 2-3 September 2010 and individual stems of wild type were potted on 4-5 September 2010 using a general-purpose medium (Premier Pro-mix Bx/ Mycorise Pro, Quakertown, PA) composed of sphagnum peat moss (80-85%/vol), endomycorrhize, perlite, vermiculite, macro and micronutrients. All stems were trimmed to approximately 60mm from tip to beginning of root before potting. Immediately following potting, all plants were transported to a 0.74 x 3.66 m raceway filled with approximately 13 cm of fresh water in a greenhouse. A total of 12 raceways were used and each raceway contained 32 Vermilion and 32 wild type specimens that were individually labeled. As new shoots emerged from the soil, original stems were trimmed to the soil so all growth consisted of new stems before the beginning of the acclimation period. The plants were then maintained in freshwater for 22 weeks 19
until the beginning of the salt acclimation on 10 February 2011. Scotts Peters Professional Water Soluble Fertilizer 20-10-20 Peat-Lite Special was added to the raceways weekly using a 1:100 ratio (2,400 ml per 22.7 L). Acclimation for Greenhouse Grow Out On 10 February 2011, all plants were trimmed to 18 cm, corresponding to the maximum stem height of the shortest plant among the 768 plants. Diameter (mm) of the largest stem and number of stems per pot were recorded for each plant. Stem diameter was measured with a digital caliper approximately 2.5 cm from the top of the soil. Stem height was measured from the top of the soil to the tip of the longest stem for each plant. Based on the LDNR smooth cordgrass acclimation protocol (DNR plant specifications, Section 02960), salinity was increased by 5 ppt in weekly intervals beginning on 10 February 2011. To begin the acclimation process, 3 trays were left at 0 ppt and the salinity in the other 9 trays was increased to 5 ppt using Instant Ocean Sea Salt (United Pet Group, Cincinnati, OH). Salinity was measured daily with a handheld temperature-oxygen-conductivity-salinity meter (YSI Model 85, Yellow Springs, OH) and adjusted to maintain target salinity (Figure 1). Seven days later, 3 of the 9 trays at 5 ppt were kept at 5 ppt and the salinity of the other 6 trays was increased to 10 ppt. Seven days later, 3 of the 6 trays at 10 ppt were left at 10 ppt and the remaining 3 trays was increased to 15 ppt and left at 15ppt for the final 14 days of the acclimation period (Figure 1). On 10 March 2011, the end of the 35 d acclimation period, height (cm) of the 20
Figure 1. Mean (±SE) salinity for each salt acclimation treatment for the entire acclimation treatment. Three replicates of each acclimation salinity were used. 21
tallest stem, diameter (mm) of the largest stem, and number of stems per pot were measured and recorded for each plant. Analysis of variance (alpha=0.05) was then used to compare the response variables among the acclimation salinities for each ecotype, and then the response variables were compared between ecotypes for each acclimation salinity. Tukeys or Duncans post hoc analysis was used to delineate differences among treatments if needed. Greenhouse Grow Out Methods At the end of the acclimation period, plants from each acclimation salinity were transferred to raceways with salinities of 0, 10, 20, or 30 ppt and allowed to grow out for 60 days. Salinity (ppt) was recorded daily and adjusted using the method previously discussed to maintain target salinities. There were three replicates for each treatment combination and each replicate consisted of eight plants. Growth for each plant was determined by measuring the change in stem height (cm), stem diameter (mm), and number of stems from day 0 (end of acclimation, beginning of grow out) to days 20, 40, and 60 after transfer. Plants were fertilized once per week using a 1:100 ratio (2400 ml per 22.7 L water) of Scotts Professional Water Soluble Fertilizer 20-10-20 Peat-Lite Special. At the end of the grow out period, below ground biomass was determined. Between 13 May 2011 and 15 May 2011, two plants from each treatment replicate were removed from pots, above ground growth was trimmed to the top of the soil, and soil was then rinsed thoroughly from the roots and rhizomes. All below ground materials were then weighed and placed into brown paper bags, and then maintained in a freezer to prevent root decomposition until transport to the Plant Materials Center (PMC) in 22
Galliano, LA. On 24 May 2011, all 192 bags of roots were placed in a 60 ºC forced air oven at the PMC and dried for approximately 50 hrs at 60 ºC. After the drying time was completed, roots were removed from the oven and weighed again to measure dry weight. Repeated measures ANOVA was used to determine if there was an interaction between the acclimation salinity and the grow out salinity for each response variable. Repeated measured ANOVA was also used to determine if there was a time and acclimation salinity or time and grow out salinity interaction. If there were no interactions, then data were pooled and analyzed with ANOVA to compare below ground biomass among the treatment combinations. Tukeys post hoc analysis was used to delineate differences among treatments if needed. Field Acclimation Methods On 8 June 2011, plants that were maintained at 0 ppt for the entire greenhouse acclimation and grow out periods were separated into individual stems and repotted into Dee pots (6.4 cm diameter by 25 cm deep) using the same potting medium (Premier Promix Bx/ Mycorise Pro, Quakertown, PA) used for the initial potting. A total of 130 stems per ecotype were re-potted and maintained within two raceways filled with approximately 13 cm of water in the greenhouse at the Nicholls State University farm until the end of the acclimation period. Each raceway held 65 Vermilion and 65 wild type stems. Stems were trimmed to 18 cm on 9 June 2011 and measurements of stem diameter (mm) and number of stems were recorded. Acclimation methods were carried out beginning 9 June 2011 according to the LDNR acclimation protocol for smooth cordgrass. One raceway was maintained at 0 ppt and the other raceway was raised to 5 ppt using Instant Ocean Sea Salt (United Pet Group, Cincinnati, OH). The raceway 23
beginning at 0 ppt was maintained at 0 ppt for the entire acclimation and the other raceway at 5 ppt was maintained at 5 ppt for 7 days. After 7 days, the raceway was then raised to 10 ppt and maintained and 10 ppt for 7 days, until finally being raised to 15 ppt and maintained at 15 ppt for 14 days (Figure 2). Field Grow Out Methods On 7 July 2011, plants were transferred to Rockefeller Wildlife Refuge in Cameron Parish, LA (29º40 8 N 92º45 91 W) at the end of a no wake zone. The site is located approximately 3.2 km from the Gulf of Mexico where salinity levels are slightly elevated (22.13 ± 3.44 ppt from day 0 to end of the grow out). This site was chosen because of the elevated salinity levels and tidal influences, which contribute to periodic oxygenation of the plant roots. Plants were kept in pots until immediately before planting. The experiment included 4 treatments replicated 3 times in a randomized complete block design. Twelve blocks of 20 plants each were planted with 1 m spacing between plots and 0.3 m centers within plots. Treatment combinations include Vermilion acclimated to 15 ppt or not acclimated (0 ppt) and wild type acclimated to 15 ppt or not acclimated (0ppt). A shovel was used to make a hole and plants were removed from pots and transplanted into the ground. The depth of the hole was fixed so that the top surface of the root ball was even or slightly below (not greater than 1 cm) ground level. The mud was firmly pressed back around the stems to ensure the planting hole was tightly closed around the plants and stems remained erect after planting. Firmly packing the mud also ensured the absence of air pockets, which may have increased the likelihood of stems washing away with the tides. Measurements of height (cm), number of stems, and salinity (ppt) were 24
Figure 2. Daily salinity for each salt acclimation treatment for the entire acclimation period prior to the field grow out period. 25
recorded on days 20, 40, and 63 after planting. Salinity at the site was recorded at days 0, 20, 40, and 63 and to determine mean (±SE) salinity over the time of the grow out period. Repeated measures ANOVA was used to determine if there was an interaction between the acclimation salinity and the grow out salinity for each response variable. Repeated measured ANOVA was also used to determine if there was a time and acclimation salinity or time and grow out salinity interaction. If there were no interactions, then data were pooled and analyzed with ANOVA. Percent data were arcsine-square root transformed prior to analysis. Tukeys post hoc analysis was used to delineate treatment differences if needed. 26
Results Greenhouse Acclimation Period - Height There was no difference in height among the acclimation salinities for the wild type (Figure 3a). However, Vermilion plants acclimated to 5 ppt had a greater height than plants acclimated to 0 or 15 ppt, but was not different than 10 ppt (Figure 3b). Comparison of height between the ecotypes revealed that wild type plants had a greater height than Vermilion plants when acclimated to 0, 10, and 15 ppt. At acclimation salinity of 5 ppt, there was no difference in mean height between the ecotypes (Figure 3c). Greenhouse Acclimation Period Number of New Stems For the wild type plants, number of new stems for plants acclimated at 5 ppt was greater than 10 ppt (Figure 4a). For Vermilion plants, there was no difference in the number of new stems among acclimation salinities (Figure 4b). Comparison between the ecotypes revealed that Vermilion plants had a greater number of new stems than the wild type plants at 10 ppt (Figure 4c). Greenhouse Acclimation Period - Stem Diameter For the wild type, stem diameter for plants acclimated to salinities of 5 and 10 were larger than plants acclimated to 0 ppt (Figure 5a). However, there was no difference in stem diameter among acclimation salinities for the Vermilion plants (Figure 5b). Comparison between ecotypes revealed that wild type plants had a greater mean stem diameter than Vermilion plants at 15 ppt (p = 0.0403; Figure 5c). 27
Figure 3. Mean (± SE) height (cm) for wild (a) and Vermilion (b) smooth cordgrass acclimated at 0, 5, 10, or 15 ppt. Means with a similar letter are not different. Graph c is comparison of the two ecotypes for each acclimation salinity. Black bars represent Vermilion and white bars represent wild. Asterisks indicate a difference between the ecotypes for that acclimation salinity 28
Figure 4. Mean (± SE) number of stems per pot for wild (a) and Vermilion (b) smooth cord grass acclimated at 0, 5, 10, or 15 ppt. Means with a similar letter are not different. Graph c is comparison of the two ecotypes for each acclimation salinity. Black bars represent Vermilion and white bars represent wild. Asterisks indicate a difference between the ecotypes for that acclimation salinity. 29
Stem Diameter (mm) 1.2 1 0.8 0.6 0.4 0.2 0 a 0 5 10 Stem Diameter (mm) 1.2 1 0.8 0.6 0.4 0.2 0 b 0 5 10 Stem Diameter (mm) 1.2 1 0.8 0.6 0.4 0.2 0 c * * 0 5 10 Figure 5. Mean (± SE) stem diameter (mm) for wild (a) and Vermilion (b) smooth cordgrass acclimated at 0, 5, 10, or 15 ppt. Graph c is a comparison of the two ecotypes for each acclimation salinity. Black bars represent Vermilion and white bars represent wild. Asterisks indicate a difference between the ecotypes for that acclimation salinity. 30
There was no difference between ecotypes at 0, 5, and 10 ppt (Figure 5c). Greenhouse Grow Out Period - Interactions There was no acclimation salinity by grow out salinity by time interaction. Therefore, acclimation salinities did not affect the change in height over the grow out period, and the main effects (acclimation salinity and grow out salinity) were evaluated separately. For acclimation salinity effect on change in height for the grow out period, data were pooled across all grow out salinities. For grow out salinity effects on change in height for the grow out period, data were pooled across all acclimation salinities. Greenhouse Grow Out Period- Acclimation Effects on Height For the wild type, there was no time by acclimation interaction so ANOVA was used to compare the overall mean value for the acclimation effect on height for the grow out period. ANOVA detected a difference in change in height among treatments for the grow out period (F3,140=5.54, p=0.0013), and Tukeys post hoc determined that plants acclimated at 15 ppt had a greater change in height than plants acclimated at 0 ppt and 5 ppt during the grow out period (Figure 6a). For Vermilion, there was no time by acclimation interaction (Wilks Lambda=0.697 F6,62=2.04, p=0.073), so ANOVA was used to compare the overall mean value for the acclimation effect on height for the grow out period. There was no difference in change in height for plants acclimated at 0, 5, 10, or 15 ppt during the grow out period (F3,140=1.45, p=0.2298; Figure 6b). Comparison between the ecotypes with ANOVA detected that the wild type grew more than the 31
Figure 6. Mean (±SE) change in height for wild (a) and Vermilion (b) smooth cordgrass acclimated to 0, 5, 10, and 15 ppt salinities for the grow out period. Means with a similar letter are not different. Graph c is a comparison of the two ecotypes for each acclimation salinity. Black bars represent Vermilion and white bars represent wild. Asterisk indicates a difference between ecotypes for that acclimation salinity. Values represent mean values over days 20, 40, and 60. 32
Vermilion at 5 ppt acclimation salinity (F1,70=9.19, p=0.0034; Figure 6c). Greenhouse Grow Out Period-Acclimation Effects on Change in Number of New Stems There was no interaction between time and acclimation salinity for the wild type (Wilks Lambda=0.779, F6,62=1.38, p=0.239) or Vermilion (Wilks Lambda=0.833, F6,62=0.99, p=0.439), so ANOVA was used to compare the overall mean value for the effect of acclimation salinity on new stems produced during the grow out period. There was no difference in number of new stems for wild type (F3,140=0.03, p=0.992) or Vermilion (F3,140=0.31, p=0.817) among the acclimation salinities (Figures 7a and 7b). Comparison between ecotypes with ANOVA revealed that Vermilion grew more stems during the grow out period when acclimated at 10 ppt (F1,70=4.60, p=0.036) and at 15 ppt (F1,70=5.71, p=0.0195), compared to the wild type (Figure 7c). Greenhouse Grow Out Period- Grow Out Salinity Effect on Height For the wild type, there was a time by grow out interaction (Wilks Lambda=0.646, F6,62=2.52, p=0.0299), so treatment differences were compared for each day. There was no difference at day 20 (F3,44=0.81, p=0.495) or day 40 (F3,44=2.26, p=0.095), but at day 60 the plants grown out in 0, 10, or 20 ppt outperformed those grown out in 30 ppt salinity (F3,44=3.38, p=0.027; Figure 8). For Vermilion, there was no time by grow out interaction (Wilks Lambda=0.724, F6,62=1.81, p=0.111), so I pooled the acclimation treatments to determine grow out effects. There was a difference in height change among the grow out salinities 33
Figure 7. Mean (±SE) number of new stems produced during the grow out at each acclimation salinity for wild type (a) and Vermilion (b). Graph c is a comparison of the two ecotypes at each acclimation salinity. Black bars represent Vermilion and white bars represent wild. Asterisk indicates a difference between ecotypes for that acclimation salinity. 34
Figure 8. Mean (±SE) change in height for wild type smooth cordgrass over time. Means with a same letter are not different. 35
(F3,140=3.94, p=0.0098) delineated by Tukeys post hoc analysis (Figure 9). Plants grown out in 0 ppt and 10 ppt outperformed those grown out at 20 and 30 ppt. Greenhouse Grow Out Period- Grow Out Salinity effect on Number of New Stems For the wild type, there was an interaction between time and grow out salinity (Wilks Lambda=0.5479, F6,62=3.63, p=0.0038) for the number of new stems, so treatments were compared for each day (Figure 10a). Number of new stems was similar among grow out salinities at day 20, but at day 40 plants grown out in 10 ppt grew more new stems than plants grown out in 20 or 30 ppt. At day 60, plants grown out in 0, 10, and 20 ppt all grew more new stems than plants grown out in 30 ppt (Figure 10a). For Vermilion, there was also an interaction between time and grow out salinity for the number of new stems, so treatments were compared for each day. There was no difference among treatments at day 20 (F3,44=0.13, p=0.944), but there was a difference at day 40 (F3,44=15.80, p<0.0001), and at day 60 (F3,44=20.26, p<0.0001) (Figure 10b). At day 40, plants grown out in 0 and 10 ppt grew more new stems than plants grown out in 20 and 30 ppt. At day 60, plants grown out at 30 ppt produced the fewest amount of new stems (Figure 10b). Comparison between the ecotypes with ANOVA reveals a difference at day 40 (F7,88=12.39 p<0.0001) and at day 60 (F7,88=27.13, p<0.0001 (Table 1; Figure 11). The Vermilion produced a greater number of new stems than the wild type when grown out at 0 and 10 ppt salinities (Figures 11a and 11b). 36
Change in Height (cm) 10 8 6 4 2 0 A AB B B B 0 10 20 30 Salinity (ppt) Figure 9. End of grow out mean (±SE) change in height for Vermilion smooth cordgrass over time. Means with a similar letter are not different. Values represent mean values over days 20, 40, and 60. 37
No. of New Stems 8 6 4 2 a 0 ppt 10 ppt 20 ppt 30 ppt A A A B 0 20 40 60 Time (days) A AB AB B No. of New Stems 8 6 4 2 b 0 ppt 10 ppt 20 ppt 30 ppt A A B B 0 20 40 60 Time (days) A A A B Figure 10. Mean (± SE) number of new stems for Vermilion (top) and common type (bottom) plants grown out at either 0, 10, 20, or 30 ppt over 20, 40, and 60 days. Treatments with similar letters are not different at that day. 38
Figure 11. Comparison of mean (± SE) number of new stems for Vermilion (black bars) and wild type (white bars) at day 40 (a) and day 60 (b) of the grow out. 39
Table 1. Mean (±SE) number of new stems for Vermilion and common type smooth cordgrass at grow out salinities of 0, 10, 20, and 30 ppt for 20, 40, and 60 days. Means marked with an asterisk indicate a difference between ecotypes at that grow out salinity. Ecotype Grow Out Salinity 0 10 20 30 Day 20 Wild 1.31 ± 0.17 0.65 ± 0.21 * 0.81 ± 0.21 0.83 ± 0.14 Vermilion 1.42 ± 0.18 1.45 ± 0.18 * 1.29 ± 0.32 1.3 ± 0.19 Day 40 Wild 2.1±0.21 * 2.11±0.32 * 1.19±0.36 * 0.89 ± 0.21 Vermilion 3.08±0.31 3.71±0.16 2.08±0.26 * 3.41 ± 0.29 Day 60 Wild 4.77 ± 0.32 * 4.74 ± 0.29 * 4.36 ± 0.49 3.41 ± 0.29 Vermilion 7.72 ± 0.48 6.89 ± 0.45 5.43 ± 0.28 3.68 ± 0.33 40
Greenhouse Grow Out Results-Belowground Biomass Based on ANOVA, there was no interaction between the acclimation salinity and the grow out salinity during the grow out period for the wild type (F15,32=0.76, p=0.649) or for Vermilion (F15,32=2.04, p=0.067), suggesting that the acclimation salinity does not affect the below ground biomass during the grow out (Figure 12). ANOVA was used to determine if acclimation salinity affected belowground biomass by the end of the grow out period. Based on ANOVA there was no difference in belowground biomass during the grow out compared among acclimation salinities for either Vermilion (F3,44=2.27, p=0.093) or wild type (F3,44=2.35, p=0.086) (Figure 12). Vermilion plants grown out in 10 ppt had a greater belowground biomass than the plants grown out in 30 ppt (F3,44=2.27,p=0.093; Figure 13a). Wild type plants had a similar belowground biomass among all grow out salinities (F3,44=1.24, p=0.307; Figure 13b). There was no difference in belowground biomass between the ecotypes across all grow out salinities (Figure 13c). Field Grow Out Period- Survival Survival was much lower for the field study than the greenhouse study. The overall mean survival when ecotypes were combined, was highest at day 20, then decreased at day 40, and again at day 63 (Figure 14). By the end of the grow out period for the field study, overall survival was 0.251 ± 0.04. Repeated measures ANOVA detected a time effect on survival for Vermilion (F2,8=49.97, p<.0001), but not by acclimation salinities (F2,8=0.51, p=0.617). Repeated measures ANOVA also revealed a time effect for the wild type (F2,8=42.96, p<.0001), but not by acclimation salinities (F2,8=0.15, p=0.8640), so survival was compared between ecotypes for each day (Figure 41
Figure 12. Mean (±SE) dry weight (g) of below ground biomass at the end of the greenhouse grow out for wild type (a) and Vermilion (b) acclimated to salinities of either 0, 5, 10, or 15 ppt. Comparison between ecotypes (c) shows no differences between the Vermilion (black bars) and wild type (white bars) across all acclimation salinities. 42
Dry Weight (g) 25 20 15 10 5 0 b AB A AB 0 10 20 Figure 13. Mean (±SE) dry weight (g) of below ground biomass at the end of the greenhouse grow out for wild type (a) and Vermilion (b) grown out at salinities of either 0, 10, 20, or 30 ppt. Means with similar letters are not different. Comparison between ecotypes (c) shows no differences between the Vermilion (black bars) and wild type (white bars) for all grow out salinities. 43
Mean Survival 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 V W 0 20 40 60 80 Day Figure 14. Mean (± SE) survival of smooth cordgrass acclimated to 0 or 15 ppt and grown out at Rockefeller Wildlife Refuge for 20, 40, and 63 days. V represents Vermilion and W represents wild type. 44
14). There were no differences between ecotypes as delineated by Tukeys post hoc analysis. Field Grow Out Period -Height For the Vermilion, repeated measures ANOVA did not detect a time effect on height (F2,6=2.21, p=0.191) or time by acclimation salinity effect (F2,6=2.50, p=0.162). For the wild type, repeated measures ANOVA detected a time effect (F2,4=14.47, p=0.015), but not by acclimation salinities (F2,6=0.81, p=0.508), so survival was compared between ecotypes for each day. For the overall mean height, there were no differences between ecotypes and height was greatest at day 63 (Figure 15). By the end of the grow out for the field study, mean height when ecotypes were combined was 26.16 ± 2.78 cm. 45
Figure 15. Mean (±SE) height for plants acclimated to 0 or 15 ppt and grown out at Rockefeller Wildlife Refuge for 20, 40, and 63 days. V represents Vermilion and W represents wild type. 46
Discussion Coastal wetland loss in Louisiana is happening at extensive rates, and will have severe impacts on the ecological, economical, and cultural services they provide. Current efforts to restore lost areas include vegetating newly created land to stabilize the sediment and prevent further loss. The ability to tolerate fluctuating salinity and water levels, and the vigorously spreading extensive root system of smooth cordgrass are characteristics that make it the ideal species to use in salt marsh vegetation projects (Partridge 1987).Vegetation projects that transplant smooth cordgrass to newly created land can accelerate the establishment of smooth cordgrass faster than if allowed to colonize naturally (Good 1989). The process of salt acclimation pre-exposes plants to an incremental increase of salinity so they are not exposed to a sudden increase in salinity, and can improve the plants adaptation to salt stressors (Guy 1990). Salt acclimation may increase a plants ability to survive transplanting to high salinity environments that might be lethal to plants not acclimated (Amzallag et al. 1990). Previous studies have found pre-exposure of plants to low levels of salt prior to salt stress improved salinity adaptation in soybean (Glycine max; Umezawa et al. 2000), cowpea (Vigna unguiculata; Silveira et al. 2001), rice (Orza sativa; Hassanein 2000; Djanaguiraman et al. 2006) and potato (Solanum tuberosum; Etehadnia et al. 2008). Rodriguez et al. (1997) also found salt acclimation of maize (Zea mays) seedlings prior to salt stress to improve survival and growth. The LDNR currently requires use of the Vermilion cultivar in all LDNR vegetation projects. Commercial growers typically culture Vermilion smooth cordgrass 47
in freshwater, but all smooth cordgrass transplants are to be salt acclimated prior to transplantation if the project is sponsored by the LDNR (DNR plant specifications- Section 02960) Acclimation may be beneficial to the transplants by reducing stress associated with an abrupt change in salinity, subsequently improving early growth and survival. I did not observe trends among the growth variables for either ecotype related to acclimation salinity by the end of the 35 d acclimation period. For example, height was similar at the end of the acclimation for the wild type among all acclimation salinities, but the Vermilion had the greatest height when acclimated at 5 ppt. The wild type had a greater number of new stems when grown in 5 ppt compared to 10 ppt, but the Vermilion plants had a similar number of new stems for all acclimation salinities. Because mortality was negligible and growth does not appear to be affected by the acclimation salinity, it does not appear that the acclimation process affects plant growth. Flooding and salinity stressors are predicted to rise in south Louisiana coastal marshes as sea level continues to rise, and it is important to understand the responses of coastal marshes. Past studies have found smooth cordgrass to tolerate salinities up to 60 ppt, with the optimal range between 10 and 20 ppt (Landin 1991) and contribute this tolerance to the ability of smooth cordgrass to use Na + for osmotic adjustment in the shoots through the increase of solutes in the plant, which consequently reduces Na + uptake (Vasquez et al. 2006). Vasquez et al. (2006) also demonstrated the ability of smooth cordgrass to produce new biomass up to 13.8 ppt. Based on the high survival rate and the repeated measures analysis of variance, it does not appear that the acclimation salinity influenced the growth performance of smooth cordgrass regardless of the salinity 48