2012 SOUTHEASTERN NATURALIST 11(4):747–754 Plant Source Influence on Spartina alterniflora Survival and Growth in Restored South Carolina Salt Marshes Jennifer Beck1,2 and Danny J. Gustafson3,* Abstract - Continued loss of coastal wetlands due to anthropogenic causes along the Atlantic and Gulf coasts has increased the need to restore eroded or hardened structures along the shoreline back to natural marsh systems. The source of plant material used in these restoration plantings may have unintended consequences for the reestablishment of marsh systems, especially if the plant material is not adapted to the local environment. In this two-year field study, we tested the hypothesis that locally collected Spartina alterniflora (Smooth Cordgrass) will have higher plant performance than non-local commercially available plant material from native plant nurseries. We found that locally collected S. alterniflora plants had higher survivorship, aboveground biomass, and cumulative stem length than plants from non-local sources. There was a significant association between plant performance and genetic similarity at the end of the first field season. Based on our findings, we recommend the use of locally collected S. alterniflora from adjacent salt marshes for small-scale salt marsh restoration projects; however, care should be taken to not degrade the donor marsh during the process. Introduction Salt marshes are a critical link between terrestrial and marine ecosystems along the Atlantic and Gulf Coasts (Mendelssohn and Morris 2000). They account for 41% of the total coastal wetlands in the conterminous United States, providing important ecosystem functions like creating habitat for estuarine species and nurseries for many marine species, reducing wave energy during storms, fostering important biogeochemical processes, and allowing for soil accretion in response to sea-level rise (Borja et al. 2010, Feagin et al. 2010, McKee et al. 2004). The loss of salt marshes, due to anthropogenic and biological causes, could result in the collapse of coastal ecosystems and associated species dependent on these habitats (NOAA 1995). Restoring the dominant perennial Spartina alterniflora Loisel. (Smooth Cordgrass) is essential for reestablishing salt marsh ecosystem functions (Mendelssohn and Morris 2000, Proffitt et al. 2003, Seliskar et al. 2002). Spartina alterniflora colonization of intertidal substrate occurs through dispersal of seeds and clonal growth (Elsey-Quirk et al. 2009, Proffitt et al. 2003). Variable seed production in wild populations coupled with low seed set and germination rates of the only available cultivar (Vermilion from Louisiana) can limit seed availability for large restoration and erosion control projects; however, polycross S. alterniflora seed shows promise (Utomo et al. 2010). Utomo et al. (2010) used 15 native Louisiana parental lines that demonstrated superior growth and seed production to establish 1College of Charleston, MES Program, Charleston, SC 29424. 2Current address - 33 Montana Avenue, Asheville, NC 28806. 3Department of Biology, The Citadel, Charleston, SC 29409. *Corresponding author - danny.gustafson@citadel.edu. 748 Southeastern Naturalist Vol. 11, No. 4 seed production plots that produced genetically diverse, high-viability seed which can be used in larger-scale marsh restoration. For smaller-scale salt marsh restoration projects, planting of vegetative material can be a preferred method because of the potential high survivorship rates and establishment of a marsh in as little as four growing seasons (Craft et al. 1999, Dai and Weigert 1996, Proffitt et al. 2003, Smart 1982). Selection of plant material may have unintended consequences for any restoration project if the planted material is poorly suited for environmental conditions of the restoration site. Spartina alterniflora ecotypes have been documented with genetic-based differences in morphology, physiology, and life history in comparison with other conspecific populations (Daehler et al. 1999; Proffitt et al. 2003, 2005; Travis et.al 2002). Therefore, matching plant material with environmental conditions at the target sites could increase the likelihood of a successful project and long-term stability of the salt marsh ecosystem. In this study, we approached small-scale salt marsh restoration from the perspective of a natural resources manager, local or regional government official, or any person interested in restoring S. alterniflora salt marsh. Because of the limited seed availability and the length of time it takes to establish a salt marsh from seed, most natural resource managers look for sources of vegetative material and prefer those sources able to ship the plants. The hypothesis tested in this study was that local S. alterniflora plants will have greater survivorship and growth than plants from non-local commercially available sources. In order to determine if differential plant performance was associated with genetic differences, we used inter-simple sequence repeats (ISSR) genetic markers to assess genetic relationships among sources. Methods Common gardens were established on Kiawah Island (32°37'27"N, 80°2'29"W) and Morgan Island (32°28'56"N, 80°29'10"W) in South Carolina. Both locations previously supported S. alterniflora growth, but had experienced complete loss of S. alterniflora (a.k.a. salt marsh dieback) in 1999 and 2003, respectively. The affected dieback areas selected to establish our common gardens were 1200 m2 on Kiawah Island and 2000 m2 on Morgan Island, with no surviving S. alterniflora ramets within plots or seedling recruitment during the course of this study. Morgan Island is part of the Ashepoo Combahee Edisto (ACE) Basin Reserve and is managed by the Natural Estuarine Research Reserve System (NERRS) and the South Carolina Department of Natural Resources. The common gardens were located approximately 28 miles apart, with similar climates, tidal inundation, and soils (Beck 2006). Plant material Seven sources of S. alterniflora were selected to represent populations from three distinct regions along the Gulf and Atlantic coasts. The geographic areas represented in this study area are (1) Mid-Atlantic (New Jersey, Maryland), (2) South Atlantic (North Carolina, South Carolina, Florida), and (3) Gulf (Louisiana) coasts. These regions differ in key environmental conditions such as annual temperature, winter conditions, and the duration of growing season along 2012 J. Beck and D.J. Gustafson 749 a north-to-south latitudinal gradient (Vogel et al. 2005). New England also has extensive S. alterniflora salt marshes; however, due to the dramatically shorter growing season and extensive ice scouring during winter (Bertness et.al 2002), the northeastern Atlantic region sources were excluded from this study. Florida was placed in the South Atlantic region, as opposed to the Gulf region, based on previous research (Seneca 1994) and the fact that the Florida nursery providing plant material was located on the Atlantic side. Plants from the Mid-Atlantic and South Atlantic regions were purchased from nurseries specializing in local wetland plant species. South Carolina plants were harvested from marshes within 400 m from the common garden. The Louisiana plant material, the only available registered S. alterniflora cultivar “Vermilion” still in production, was obtained from the Golden Meadows Plant Material Center in Louisiana. The “Bayshore” S. alterniflora cultivar, a registered cultivar developed and released in 1992 from the Cape May Plant Material Center, NJ, was from a Maryland source. “Bayshore” cultivar, however, was not kept in production due to the consumer demand for local ecotypes over cultivated varieties (Cape May Plant Material Center, Cape May, NJ, pers. comm.). Roots were washed thoroughly before the plants were transplanted into gallon containers with Pro-Mix™, and grown under greenhouse conditions from 2 to 4 weeks prior to field planting. Prior to placing the plants in the field, 1 gram of leaf material was collected from 18 individuals per plant source and stored at -20 °C until genomic DNA extractions were performed. Common garden In April 2005, S. alterniflora from each source was transplanted in a randomized block design, with plants spaced 0.5 m apart. The number of ramets, maximum plant height, and cumulative stem length was recorded in September 2005 and 2006. Less than 10% of the adjacent clones grew together over the 2 field seasons; however, definitive clone identification was possible by excavating rhizomes. Individual aboveground biomass was harvested in September 2006, washed free of debris, and dried to a constant mass. Genetic markers Plant genomic DNA was extracted from approximately 0.5-g leaf material using E.Z.N.A.® plant DNA miniprep kit (Omega Bio-Tek, Doraville, GA). Twenty-five inter simple sequence repeats (ISSR) primers were surveyed, with 4 primers selected for this study (sequence, number of bands: (GT)6-RG, 8 bands; (CA)8-RG, 7 bands; (GA)8-YC, 10 bands; (CT)9-G, 9 bands). ISSR polymerase chain reaction (PCR) protocol followed that of Wolfe et al. (1998): 94 °C for 1 min 50 sec, 40 cycles of 94 °C for 40 sec, 43 °C for 45 sec, and 72 °C for 1 min 50 sec, followed by a final extension at 72 °C for 5 min. PCR profiles were visualized in 1.5% agarose gels and stained with ethidium bormide. Images were captured using a digital camera (Olympus C-4000 Zoom, Melville, NY) and converted to a negative image, and fragment size was estimated based on a DNA marker (Benchtop pGEM, #G7521, Promega, Madison, WI). Fragment sizes were used to assign loci for each primer, and bands were scored as diallelic for each locus (1 = band present, 0 = band absent). 750 Southeastern Naturalist Vol. 11, No. 4 Data analyses Plant survivorship at the end of the first and second seasons was analyzed using c2 test of independence between plant source (local vs. non-local) and survivorship (alive or dead) (Gotelli and Ellison 2004). Two-way analysis of variance (ANOVA) was used to analyze end of the second growing season plant performance (log transformed) with site (common garden) and origin of plants (local vs. non-local; source) main effects, and all interactions (SAS Enterprise Guide, version 4.3, SAS Institute Inc., Cary, NC). Block effects were not significant and were removed from all analyses presented. Genetic relationships among sources were estimated using unweighted pair group means cluster analysis (UPGMA) cluster analysis of Jaccard’s distances based on band frequency data. Mantel’s test using the Monte Carlo approach with 1000 randomized runs was used to test the null hypothesis of no relationship between genetic similarity and plant performance matrices (PC-Ord, ver. 4.2, MjM Software Design, Gleneden Beach, OR). Results Local plants had significantly higher survivorship than non-local plants after the first season (c2 = 42.2, P < 0.0001) and across the first two seasons (c 2 = 16.3, P < 0.0001). Approximately twice as many local plants survived compared to the non-local plants (Fig. 1). When analyzing plant performance at the end of the second season, local plants produced significantly greater biomass (F[1,33] = 7.9, P < 0.01) and cumulative stem length (F[1,33] = 4.9, P = 0.04) than non-local plants (Fig. 2). There was a significant site effect for cumulative stem length (F[1,33] = 9.2, P < 0.01), with plants producing approximately twice the cumulative stem length at the Morgan Island common garden (Fig. 3). Morgan Island also had significantly higher ramet production (F[1,33] = 46.9, P < 0.0001) than plants grown at the Kiawah Island site (Fig. 3). There were no differences in maximum plant height at the end of the 2006 season. This means that differences in biomass and cumulative stem length between local and non-local sources was a function of ramet production and growth, despite no significant difference in ramet production (F[1,33] = 2.1, P = 0.16) between local and non-local sources. Figure 1. Local Spartina alterniflora plants had higher survivorship than non-local plants over both field seasons. 2012 J. Beck and D.J. Gustafson 751 Figure 2. Local Spartina alterniflora plants produced more total aboveground biomass (left) and cumulative stem length (right) than plants from non-local sources. Bars represent means ± 1 SE after 2 years of growth in South Carolina salt marshes. Figure 3. Spartina alterniflora plants growing on Morgan Island produced more ramets (left) and higher cumulative stem length (right) than plants growing on Kiawah Island. Bars represent means ± 1 SE after two years of growth in South Carolina salt marshes. Figure 4. Genetic relationships (above) and plant performance (below) of Spartina alterniflora from local and non-local sources grown in South Carolina salt marshes. Genetic relationships among source populations were based on UPGMA of Jaccard’s distances. Plant performance (average number of ramets, total plant height, and maximum height) relative to source populations were investigated using UPGMA cluster analysis of relative Euclidean distances. 752 Southeastern Naturalist Vol. 11, No. 4 Genetic relationships among source populations follow a roughly geographic pattern (Fig. 4). Groupings followed the Gulf coast (Florida and Louisiana), then South Carolina and Maryland, and the North Carolina and New Jersey sources. If the pattern of genetic relationships strictly followed source origins, then we would have predicted Maryland to group with New Jersey. There was significant (r = 0.69, Z = 0.02, P = 0.03) concordance between genetic relationships among sources and the end of the 2005 season plant performance (average number of ramets, cumulative stem length, and maximum height; Fig. 4). Inclusion of the 2005 survivorship data decoupled this relationship (r = -0.15, Z = 0.14, P = 0.32). Discussion Restoring S. alterniflora to southeastern intertidal salt marshes can have beneficial effects on native snail, fish, mammals and bird species (Galleher et al. 2009, Gawlik 2002, Hotaling et al. 2010, Pung et al. 2008, Stolen et al. 2009), as well as providing broader ecosystem functions (Leonard and Croft 2006, Seliskar et al. 2002). The primary objective of this research was to determine if locally collected S. alterniflora would outperform commercially available S. alterniflora in two southeastern salt marshes. From this 2-year field study, we found that locally collected S. alterniflora plants had higher survivorship, aboveground biomass and cumulative stem length in 2 South Carolina salt marshes. There was a general latitudinal pattern for genetic similarity and select plant performance measures (average number of ramets, cumulative stem length, and maximum height) at the end of the first field season; however, low survivorship rates of the purchased North Carolina and Florida plants during the second season decoupled this pattern. We did observe a consistent pattern of local plants outperforming non-local plants in this two-season, two-common-garden field study. In a study of early regeneration dynamics in restored and native salt marshes, Elsey-Quirk et al. (2009) found that S. alterniflora seedling recruitment was enhanced with open habitat at higher marsh elevations, while seedling recruitment was facilitated in the presence of established plants at lower marsh elevations. Unlike the strong diurnally tidal fluctuations of our common garden locations (Kiawah Island, ≈1.8 m; Morgan Island, ≈2.4 m), water levels in the Louisiana marshes are more influenced by wind direction than tidal flooding (Edwards and Proffitt 2003). Mechanical stresses like tides and wind have been shown to influence clonal plant growth (Puijalon and Bornette 2006, Puijalon et al. 2005), which could account for an increase in S. alterniflora ramet density and cumulative stem length differences between the 2 field sites. However, the potential effect of tidal flow velocity on S. alterniflora morphogenesis is beyond the scope of this current research. In conclusion, our research shows that S. alterniflora collected from adjacent salt marshes will outperform plants purchased from non-local nurseries. This pattern of local ecotypes outperforming non-local sources is well documented in many species, including S. alterniflora (Daehler et al. 1999; Gustafson et al. 2005, 2008; Proffitt et al. 2003 2005; Travis et al. 2002). We recommend using plant material collected from adjacent salt marshes for small-scale salt marsh restoration projects; however, care should be taken to not degrade the local donor marsh during the process. 2012 J. Beck and D.J. Gustafson 753 Acknowledgments We would like to thank Charlie Zemp, Norm Shea, Will Chapman, Shane Kersting, Ross Garner, and the 2005 Citadel Ecology class for help in the field. We acknowledge the thoughtful comments and suggestions by two reviewers that improved this manuscript. 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