100 isotope ratio mass spectrometer

Following behavioural observations and territory mapping, we collected turf algae and macroalgae if present (Halimeda spp.) from within the territory of each focal individual. All algal samples were dried at 60 °C for 24 h or until dry, ahead of nitrogen stable isotope analysis. Dried samples were washed in a 10% hydrochloric acid solution to remove any contaminants and calcareous matter, and then centrifuged at 1,008 × g (3,000 r.p.m.) for 6 min. Samples were washed thoroughly with distilled water between centrifuge cycles. In total, samples were centrifuged four times. Samples were then dried for a final time. Isotope samples were then analysed at Lancaster University (UK). Samples were combusted using an Elementar Vario MICRO cube elemental analyser before being analysed in an Isoprime 100 isotope ratio mass spectrometer. Samples were analysed with the two international standards IAEA 600 and USGS 41, and a random subset of samples were run in triplicates to ensure readings were accurate. From the analysis, we extracted values for the ratios of the nitrogen isotope N15:14 (δ¹⁵N) for both turf and macroalgae.

Bone collagen stable isotope analyses were performed in duplicate on 0.5 mg collagen samples for δ¹³C and δ¹⁵N analyses and, where collagen yield allowed, 6.0 mg samples for δ³⁴S. For scales, duplicate analyses were performed on collagen from two separate scales per individual. For δ¹³C and δ¹⁵N analyses, samples were combusted in tin capsules in an Elementar vario MICRO cube elemental analyzer coupled to an Isoprime isotope ratio mass spectrometer in continuous flow mode. Carbon and nitrogen isotopic compositions were calibrated relative to VPDB and AIR using USGS40 and USGS41. For δ³⁴S analyses, samples were combusted in tin capsules with 1 mg of V₂O₅ in an Elementar vario MICRO cube elemental analyzer coupled to an Isoprime 100 isotope ratio mass spectrometer in continuous flow mode. Sulphur isotopic compositions were calibrated relative to VCDT using IAEA-S-1 and NBS-127.

Stable isotope analyses of δ¹³C and δ¹⁵N, as well as measurements of %C and %N, were measured in plant and sediment samples using the Isoprime 100 isotope ratio mass spectrometer (IRMS) interfaced with a Micro Vario Elemental Analyzer (Elementar Americas, Mt. Laurel, NJ). Sediment trap, core, and eelgrass samples were weighed into 6 × 4 mm tin capsules (10–15 mg, 25–30 mg, and 4–6 mg, respectively). Due to low amounts of nitrogen in the core sediments, samples were double-tinned when necessary (tin acting as a catalyst for a greater combustion in the IRMS). Internal laboratory standards were used throughout runs, every 15–20 samples, to account for instrument offset (BCSS-1 for sediments and blue mussel homogenate for eelgrass). The %C in seagrass leaves was based on 3 composite samples.