NOAA RESTORE Science Program: Gulf-wide assessment of habitat use and habitat-specific production estimates of nekton in turtlegrass (Thalassia testudinum): Nekton and primary producer stable isotopes in turtlegrass-dominated seagrass beds in the northern Gulf of America, 2018-05-23 to 2018-10-03 (NCEI Accession 0304846)

Methods:
Sample Collection
Nekton and primary producer sources were collected from May23–October 3, 2018 at six sites spanning the range of turtlegrass distribution in the Northern GOM. Two sites were located in Texas (Lower Laguna Madre [LM] and the Texas Coastal Bend [CB]), one site in Louisiana (Chandeleur Islands [LA]), and three sites in Florida, (St. George Sound [AP], Cedar Key [CK], and Charlotte Harbor [CH]).
Sampling stations at each of the six sites were chosen using a stratified random sampling method of hexagonal tessellation in which a grid of hexagons (500 or 750 m edge) was overlaid on the mapped areal extent of known turtlegrass cover at each site, and 25 hexagons with > 50% turtlegrass cover were randomly selected to conduct surveys. In cases where no turtlegrass was found at a station, or stations were inaccessible, alternative hexagons were chosen and new stations were randomly generated. Mean distance between the farthest sampling stations at each site was 22.5 ± 2.4 km (Laguna Madre, TX = 20 km, Coastal Bend, TX = 20 km, Chandeleur Islands, LA = 24 km, Saint George Sound, FL = 21 km, Cedar Key, FL = 25 km, and Charlotte Harbor = 25 km).

All animal and vegetation samples were collected as part of a survey to assess nekton habitat use in turtlegrass environments. Larger nekton and drift macroalgae samples were collected at each site using a 4.8 m flat trawl towed from a boat for 2–3 minutes at a speed of 3.7–5.6 km min-1, and smaller nekton and additional macroalgae samples were collected using an 0.75 m wide epibenthic sled pulled by hand a distance of 13.3 m at a speed of ~0.3 m s-1. All individuals were measured for standard length (SL) and total length (TL) (carapace width, CW, for crabs) prior to processing to account for changes in δ15N values related to diet changes with size, and were identified to the lowest practical taxonomic level. Because of logistical difficulties in identifying different macroalgae species, macroalgae samples were identified based on functional traits and major taxa grouping (e.g., Rhodophyta, red branching macroalgae). At each site, trawl and epibenthic sled surveys were conducted on different days to prevent disturbance to the habitat. Approximately five individuals of each species collected at each site were retained for analysis. Vascular plant samples of all seagrass species and adjacent marsh species were collected by hand at a subset of five stations across each site. All epiphytes present on macrophyte leaves were removed by gently scraping with a razor blade and macroalgae samples were picked free of visible meiofauna and detritus to avoid isotopic signature contamination. Benthic microalgae (BMA) were collected at a subset of five stations at each site using a glass plate collector methodology (Dillon et al. 2015), in which paired collector plates were partially pushed in the sediment at each station, retrieved after one week, rinsed to remove sediment, separated, and scraped to collect microalgae. Suspended particulate organic matter (POM) samples were collected at five representative stations at each site using 60 mL plastic syringes with 2.5 cm glass fiber filters. All nekton and primary producer samples were transported on ice and frozen at the Gulf Coast Research Lab, The University of Southern Mississippi prior to isotopic analysis.
A subset of 30 common nekton taxa representing multiple tropic levels, feeding strategies, and ecological niches were collected and used for stable isotope analyses, including five seagrass species ( turtlegrass, shoal grass, manatee grass, widgeon grass, and star grass), two mangrove species (Avicennia germinans and Rhizophora mangle), two C3 marsh plant species (Phragmites sp. and Juncus roemerianus), two C4 marsh plant species (Sporobolus alterniflorus and Sporobolus pumilus), 20 macroalgae groups, POM samples, and BMA samples. Small invertebrates, primary producers, fish, and POM samples were processed whole, whereas subsamples of muscle tissue were taken from larger fish and invertebrates such as pinfish (Lagodon rhomboides), pigfish (Orthopristis chrysoptera), and inshore lizardfish (Synodus foetens). Smaller individuals for some taxa (e.g., Hippolytid shrimp) within a single collection at each station were combined to achieve enough mass for isotopic analysis.
Nekton and primary producer samples were rinsed with deionized (DI) water to remove sediment, dried to a constant weight at 60°C using a drying oven, then ground to a fine powder using either a mortar and pestle or a Wiley mill equipped with a #20 or #40 mesh delivery tube. Samples were stored in clean scintillation vials in desiccators prior to analysis and POM samples were acid fumed for 24 hrs using concentrated hydrochloric acid (HCl) to remove inorganic carbonates. All macroalgae taxa were rinsed once with 10% HCl and three times with DI water to remove inorganic carbonates. After each DI water rinse, samples were shaken vigorously using a Vortex mixer (GENIE SI-0235, Scientific Industries), centrifuged, and decanted. Following the final rinsing, acid washed samples were dried in a drying at 60°C for 24 hrs. Acid-washed portions of macroalgae samples were used for carbon isotope analysis and unwashed portions were used for nitrogen analysis because acid washing is known to bias δ15N values (Pinnegar & Polunin 1999). Samples were packed into tin capsules and analyzed for δ13C and δ15N signatures following standardized protocols (Levin & Currin 2012, Olsen et al. 2014) using continuous-flow stable isotope ratio mass spectrometry (CF-IRMS) with a Costech 93 Elemental Combustion System coupled to a Thermo-Fisher Scientific Delta V Advantage Isotope Ratio Mass Spectrometer at the Gulf Coast Research Lab Stable Isotope Facility.
Most (95%) nekton and primary producer samples were analyzed in duplicate, aside from some samples with limited material and POM samples, which were analyzed as single samples (5%). Carbon and nitrogen stable isotope values were calculated according to the formula
δX=[(𝑅𝑠𝑎𝑚𝑝𝑙𝑒/Rstandard )−1]×103
where X is 13C or 15N and R is the ratio of heavy to light isotopes (13C/12C or 15N/14N) for the samples or the standard (PeeDee belemnite [PDB] carbon or atmospheric dinitrogen [N2]).