1. Genetic structure of seagrasses from the western Gulf of Mexico Patrick D. Larkin, Tabitha Maloney, Sebastian Rubiano-Rincon, and Michael Barrett Department of Physical and Environmental Sciences Texas A&M University – Corpus Christi, 6300 Ocean Drive, Corpus Christi, TX 78412-5802 Introduction Seagrasses are marine angiosperms that perform a variety of critical ecological and environmental roles. Worldwide, many seagrass ecosystems are experiencing declines that have been closely linked to human activity. In the Gulf of Mexico, for instance, seagrasses have disappeared or declined from areas where they historically flourished, especially those near major population centers (Handley et al. 2007). Halodule wrightii [= beaudettei] (Cymodoceaceae) (Fig. 1) is one of the predominant seagrasses in the Gulf of Mexico. Seagrasses are especially important in the Laguna Madre of Texas where they, rather than phytoplankton, form the basis of the food chain (Tunnel & Judd, 2002). We undertook this study to assess genetic variation and structure among Halodule wrightii populations from the Texas coast (USA) in the western Gulf of Mexico. We expected populations from the Laguna Madre, with its favorable climate and environment, to be genetically more diverse compared to populations from the northern part of H. wightii’s Texas range. We expected genetic structure to align closely with the basins (Lower Laguna Madre, Upper Laguna Madre, Coastal Bend) from which the samples were collected. Materials and Methods Ten sites along the Texas coast were selected for sampling (Fig. 2). Locations were chosen from an array of monitoring sites designated in the Texas Seagrass Monitoring Program Strategic Plan (Pulich et al,. 2003). The sites chosen cover approximately 90% of H.wrightii’s range on the Texas coast. A 6x30m grid was set up at each site and ca. 10 cm of rhizome tissue was collected at 2 m intervals across the grid (48 samples/site). DNA was extracted from 20 mg of rhizome tissue using the Plant DNeasy® kit from Qiagen and DNA was quantitated using a Qubit-iT® fluorometer (Invitrogen). Each sample (452 total) was screened at 8 microsatellite loci using a Polymerase Chain Reaction (PCR)-based assay, as described previously (Larkin et al. 2012). PCR products were separated on a Beckman-Coulter CEQ 8000® Genetic Analyzer and alleles were scored using the Beckman-Coulter CEQ Fragment Analysis software, v 9.0. 10% of the samples from each population were run in duplicate to confirm reproducibility of results. Results Genetic Diversity Compared to other species, we found that H.w rightii populations from the Texas Gulf coast exhibit low clonal diversity (mean R = 0.40), moderate allelic diversity (mean A R = 4.5), and high heterozygosity (mean H e = 0.58). The inbreeding coefficient was uniformly low across sites (mean F IS = -0.03) Clonal diversity was lowest near the southern and northern borders of the Texas sampling range, though heterozygosity in these regions remained high. Clonal diversity was highest in the Laguan Madre, where historically large population sizes and favorable environmental conditions may be more conducive to sexual reproduction. Genetic Structure We found relatively weak genetic structure among the Texas sites. Population assignment analysis showed strongest support for two genetic clusters, neither of which correlated with a particular basin (Fig. 3). Analysis of molecular variance (AMOVA) showed that most of the variation could be attributed to differences among individuals, rather than between sites or basins (Table 2). One factor that did appear to be associated with genetic clustering was tidal range. Sites that experience tide-related changes in water movement showed more genetic admixture, while those in atidal areas had a relatively uniform genetic background Conclusions Gene flow among populations on the Texas Gulf coast appears to be relatively robust, despite geographical barriers that divide the coastal area into discrete basins. Compared to other species overall diversity appears to be moderate, with relatively high heterozygosity and low clonal diversity, though these values can differ substantially among sites. Underlying causes are yet to be defined, but historically high population sizes, conditions favorable to sexual reproduction, and a water-borne mechanism of dispersal seem likely candidates. References Goudet J (2001) FSTAT, a program to estimate and test gene diversities and fixation indices. Handley L, Altsman D, DeMay R (2007) Seagrass Status and Trends in the Northern Gulf of Mexico: 1940-2002. U.S. Geological Survey, Reston, VA Larkin P, Schonacher T, Barrett M, Paturzzio M (2012) Development and characterization of microsatellite markers for the seagrass Halodule wrightii. Conservation Genetics Resources 4 (2):511-513. Miermans, PG and Van Tienderen, PH (2004) GENOTYPE and GENODIVE: two programs for the analysis of genetic diversity of asexual organisms. Molecule Ecology Notes 4:792-794 Peakall R, Smouse PE (2006) GenAlEx6: Genetic Analysis in Excel - Population Genetic Software for teaching and research. Molecular Ecology Notes 6:288-295 Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155(2): 945-959 Pulich WM, Hardegree B, Kopecky AL, Schwelling S, Onuf C, Dunton KH (2003) Texas Seagrass Monitoring Program - 2000 Strategic Plan. Texas Parks & Wildlife, Texas Commission on Environmental Quality, Texas General Land Office, Austin, TX Rousset F (2008) Genepop'007: a complete reimplementation of the Genepop software for Windows and Linux. Molecular Ecology Resources 8:103-106 Tunnell J, Judd F (2002) The Laguna Madre of Texas and Tamaulipas. Texas A&M University Press, Acknowledgements Texas Sea Grant, Award No. S080033 Welch Foundation, Award No.BT-0041 Texas A&M University-Corpus Christi Research Enhancement Award Texas Parks and Wildlife Dept. Figure 1. Halodule wrightii (shoalgrass) Table 1. Genetic diversity estimates for 10 populations of the seagrass Halodule wrightii from the Texas Gulf of Mexico coastline. Figure 3. Population assignment plots of samples from Texas Gulf Coast using STRUCTURE 2.3.2. Box outlines repesent extent of tidal range. Table 2. Distirbution of genetic variation among Texas sites (AMOVA analysis). Allele scores were transferred to a Microsoft Excel® prior to analysis. Tests for linkage disequilibrium among loci and deviations from Hardy-Weinberg Equilibrium (HWE) were performed using Genepop on the Web® v. 4.0 (Rousset, 2008). Estimates of population genetic diversity, including mean number of alleles (A), allelic richness (A R ), proportion of rare alleles A rare, number of private alleles A p, clonal (genotypic) diversity (R), mean observed heterozygosity (H o ), mean expected heterozygosity (H e ), and an inbreeding coefficient (F IS ), were calculated using the GenAlEx® (v.6.4) and FSTAT® (v.2.9.3.2) software packages (Peakall & Smouse, 2006; Goudet, 2001). Analysis of genetic structure was performed using STRUCTURE 2.3.2 (Pritchard et al 2000). Distribution of genetic variation was calculated using an Analysis of Molecular Variance (AMOVA) procedure in Genodive 2.0b.27 (Miermans and Van Tienderen 2004) Figure 2. Sampling sites