To identify dietary sources consumed by Uca and Littoraria, samples for stable isotope analysis were collected by hand from five pre-determined random points along the length of the survey transect at each site. At each point, samples of Uca, Littoraria, and all likely end members (potential dietary contributors, see below) were collected. Multiple species of Uca are common in Gulf Coast salt marshes (most notably Uca rapax, Uca longisignalis, Uca panacea, and Uca spinicarpa in our sites), but the different species have similar diets in this region (Goeke and Armitage 2021), so we did not differentiate among species in our collection. Not all points had consumers present, but we were able to collect each consumer taxa from at least two points in eight of the nine sites. The exceptions were site AG2 for Littoraria and site SA2 for Uca, where we were only able to locate those consumers at a single point.
Sampled end members were particulate organic matter (POM), benthic organic material (BOM, which includes benthic microalgae and detritus), and any vascular plant species present. Macroalgae was not observed at any site so macroalgae was not included as an end member. At each of the five pre-determined points, 0.5 L of water and a scraping of the top 5 mm of sediment were collected to be processed in the lab for POM and BOM, respectively. Five live and five dead leaves were collected from each plant species present at each point to evaluate isotopic variation between live and senescent plant material. Leaves from plants of the same species in each site were combined to create a single live sample and a single dead sample per species in each site due to funding constraints. BOM and POM samples were not combined as we expected them to have higher spatial variation. A complete list of the sampled end members and sample sizes can be found in Table S1. Following collections, all samples were stored in a cooler with dry ice until they were transferred to a -20 °C freezer for storage prior to analysis.
Consumer muscle tissue was obtained from the legs and claws of Uca and from the muscular foot of Littoraria. Tissues from all individuals of a species collected at the same point in a site were combined, resulting in a maximum of five samples per species per site. Combined tissue samples were rinsed in distilled water, dried at 60°C for 48 hours, and then ground to a fine powder with a mortar and pestle. Plant leaves were rinsed thoroughly with distilled water, dried at 60 °C for 48 hours, then ground to a fine powder using a ball mill. Some samples with very small leaves (e.g., Batis maritima) were ground with a mortar and pestle to avoid sample loss that can occur with ball mills.
To isolate particulate organic matter, water samples were filtered through a 100 μm sieve onto pre-combusted glass fiber filters. Sieving removed any large detrital plant particles from the sample. Filters were dried in an oven at 60 °C for 48 hours, then stored in glass vials. Benthic organic material was separated from the sediment using density centrifugation (Levin and Currin 2012). Briefly, 15 mL of each sediment sample was rinsed twice with an equal amount of distilled water to remove salt. Twenty mL of Ludox (1.3 g/mL density) was added, and the sample was homogenized on a vortex mixer. Distilled water was carefully added without disturbing the surface of the Ludox to avoid mixing the Ludox and water layers, and the sample was centrifuged again. Following centrifugation, the organic material from the sediment, including decaying plant matter, microalgae, and benthic meiofauna, were caught at the interface of the water and Ludox layers due to the differential densities of the substances. This layer of organic material was pipetted onto a pre-combusted glass fiber filter through a 100 μm sieve to remove any larger masses of plant matter. Following filtration, the filter was dried at 60 °C for 48 hours, then stored in a glass vial.
Ground plant and animal tissues were weighed into tin capsules, and filters containing POM and BOM were placed in pre-combusted glass vials and cut into fine pieces using a pair of sterilized surgical scissors. The Vienna PeeDee Belemnite and atmospheric nitrogen were used as standards for carbon and nitrogen isotopes, respectively. The accuracy of isotopic measurements was calculated as 0.07‰ for δ13C and 0.03‰ for δ15N.
