Delta 5 mass spectrometer

After samples were dried, we manually separated the bone of the toe from skin and ligaments with an Xacto^© knife. Each skin sample was weighed to between 0.3 and 0.7 mg in tin capsules on a Mettler Toledo AG245 micro-scale. Samples were analysed at the University of New Mexico's Center for Stable Isotopes. The samples were combusted in a Costech 4010 elemental analyser (Costech, Valenicia, CA, USA) coupled to a Thermo Scientific Delta V mass spectrometer (Thermo Scientific, Bremen, Germany).
Stable isotope values were expressed with standard delta notation (δ) in parts per thousand (‰), where δX = (R_sample/R_standard − 1) × 1000, where X is δ¹³C or δ¹⁵N and R_sample and R_standard are the molar ratios of C¹³/C¹² and N¹⁵/N¹⁴ of the sample and the standard reference material, respectively. The reference material was Vienna-Pee Dee Belemnite for carbon and atmospheric N₂ for nitrogen.

Analyses were performed at the Godwin laboratory for Paleoclimate Research, Dept. of Earth Sciences, Cambridge University (UK). Tissue and symbiont samples (N = 4 corals per Site) were analysed for percentage carbon and nitrogen, ¹²C/¹³C and ¹⁴N/¹⁵N using a Costech Elemental Analyser attached to a Thermo DELTA V mass spectrometer in continuous flow mode. Reference standards from IAEA in Vienna were analysed along with the samples. The dried sample/standard was carefully weighed into a tin capsule, sealed and loaded into the auto-sampler. Reference standards were run at intervals throughout the sequence and these values were used to calibrate to the international standards for ¹⁴N/¹⁵N (δ¹⁵N air) and ¹²C/¹³C (δ¹³C VPDB). Precision of analyses is +/−0.05 % for C and N, better than 0.1 % for ¹²C/¹³C and better than 0.1 % for ¹⁴N/¹⁵N.

Sulphide in groundwater was fixed with a zinc acetate solution (final concentration 1%). The zinc sulphide precipitate was removed by centrifugation at 4,500 rcf for 10 min and the pellet was discarded. The supernatant was used for sulphur isotopic measurement of sulphate. The supernatant was acidified to ∼pH 3 with 1 M HCl and heated to 90°C in a water bath. Sulphate was precipitated as BaSO₄ by the addition of BaCl₂ (10% solution). Samples were left to cool overnight then centrifuged at 4,500 rcf for 10 min. The supernatant was discarded and the BaSO₄ pellet air-dried. The ³²S/³⁴S isotope ratio of sulphate (δ³⁴S_SO4 ‰ VCDT) was measured at the Stable Isotope Laboratory (Faculty of Geosciences and Environment, Université de Lausanne, Lausanne, Switzerland). The S isotope composition was measured with a He carrier gas and a Carlo Erba (CE 1100) elemental analyser linked to a Thermo Fisher Delta V mass spectrometer. Samples were reacted at 1,050°C in a stream of He-carrier gas spiked with oxygen gas. External reproducibility of standards was <0.15‰ and samples were calibrated against IAEA standards S1 and S3 (Ag₂S) and NBS-127 (BaSO₄) with accepted values of −0.3, −32.1, and 20.3‰, respectively.

Tissue samples from a subset of each sex and species were used in stable isotope analysis. Each sample was sliced into several thin pieces and dried at 50°C for at least 48 h, after which a 0.2–0.7 mg sample was weighed and processed. Samples were run on an isotope‐ratio mass spectrometer (Elemental Combustion System, Costech Instruments, Model 4010 linked to a ThermoScientific, Rockford, IL, USA, Delta V Mass Spectrometer) to test for isotopic nitrogen (∂¹⁵N) and carbon (∂¹³C) levels. Stable isotope analysis was run on 11 G. geographica females and 16 G. pseudogeographica females, and 24 G. geographica males and 35 G. pseudogeographica males. ∂¹⁵N was similarly analyzed for two plant specimens (Lemna sp.) from the study site to quantify trophic position for turtles using the formula: $\text{TP}=1+\left(\frac{{\mathit{\partial }}^{15}\mathrm{N}turtle-{\mathit{\partial }}^{15}\mathrm{N}Lemna}{\mathrm{\Delta }{\mathit{\partial }}^{15}\mathrm{N}}\right)$ with a discrimination factor (Δ∂¹⁵N) of 3.4 (Post 2002; Martinez del Rio et al. 2009). For each sex, pairwise t‐tests were run to test for differences in ∂¹⁵N, ∂¹³C and trophic position between G. geographica and G. pseudogeographica.