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  • A total of 18 skin samples (Figure 1) were analyzed for bulk stable isotope analysis. Skin tissue samples with enough material (3.5 mg) were selected for CSIA-AA. Data from 12 samples were obtained for individual AA δ15N values, and 9 samples for δ13C values. The Southwest Fisheries Science Center/Pacific Islands Fisheries Science Center Institutional Animal Care and Use Committee (IACUC) approved the original animal work that produced the samples. Sex was determined genetically using qPCR sexing assay by the PRD-Genetic Lab at NOAA [52]. These samples consisted of 5 females, 2 males and 2 unidentified individuals possibly corresponding to females or juvenile males. Large adult males were not included. Bulk isotope values were analyzed by continuous flow isotope ratio mass spectrometry (IRMS; Thermo Finnigan) and standardized relative to Vienna-Pee Belemnite (V-PDB) for carbon and atmospheric N2 for nitrogen. Results are expressed in part per thousand (‰) and standard notation: δHX = [(Rsample/Rstandard)−1]×1000, where H is the mass number of the heavy isotope, X is either C or N, and Rsample and Rstandard are the ratio of 13C/12C or 15N/14N in the sample and standard, respectively. We hydrolyzed and prepared approximately 3.5 mg of skin as well as a control (Cyanno; bacteria tissue) [53] to quantify δ15N values from source- and trophic-AAs and δ13C values from essential- and non-essential-AAs. All derivatives were injected with an AA control, N-leucine, to verify accuracy during each run, and analyzed via gas chromatography-IRMS to obtain δ15N and δ13C values from individual AAs. Each sample was run 3–4 times to maximize accuracy among chromatograms. The associated analytical error among replicates was <1.0 ‰. For all samples, δ15N values were obtained from a total of four source-AAs (phenylalanine, glycine, lysine, tyrosine), and five trophic-AAs (glutamic acid, alanine, isoleucine, leucine, proline) (Figure S1A). For δ13C values, the essential-AAs that we consistently determined were phenylalanine, valine and leucine, and the non-essential-AA were alanine, proline, aspatic acid, glutamic acid and tyrosine (Figure S1B). The relative pattern of AA δ15N and δ13C values was highly consistent with past work from other organisms and tissues [23], [25], [54]. We grouped data as source- or trophic-AAs for δ15N values, and essential- or non-essential-AAs for δ13C values to increase power in the analysis and evaluate temporal variation. We calculated average values for each AA group and they are reported in Table S1. Regression analyses were conducted to evaluate linear relationship between time and each isotopic tracer for both bulk and individual-AA δ15N and δ13C values (Table 1). There was a weak correlation between average source-AA and trophic-AA (r2 = 0.13; p = 0.67), indicating that trophic-AA δ15N values could not be predicted by the variability in source-AAs, and vice versa. However, the correlation between average essential-AA and non-essential-AA δ13C values was moderate (r2 = 0.63, p = 0.06). Since the controls on isotopic patterns for non-essential-AA δ13C values are complex and dependent on diet quality and quantity, including de novo synthesis and routing of AAs from diet-to-tissue, this group was not considered in the linear regression analysis.
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