Sampling

Mussels were collected in the winter (Dec – Feb) of 2009–2010. Sites were chosen to be approximately evenly distributed along the CA coastline, with ,80 km geographic separation between each

sampling site. Our main goal here was to sample mussels from a wide geographic range across the CCS, although for observing finer scale local or regional variations, a finer-scale sampling strategy would like be required. Typically 5 individual mussels were collected from each site, all between 30–40 mm maximum shell length, which were immediately placed on dry ice until further preparation. The adductor muscle of each individual was dissected for analysis. This tissue was selected because isotopic values in muscle tissue have shown relatively long turnover times; based on past growth data, mussels of this size would be expected to integrate approximately annual variability in suspended food source isotopic values for each location sampled.

The dissected adductor tissue was carefully separated from other tissue types, rinsed with deionized water, refrozen, and then freeze-dried for 48 hrs. Lipids were removed using petroleum ether in a Dionex Accelerated Solvent Extractor (Bannockburn, IL). Finally, in preparation for CSI-AA, composite samples were made from a subset of 13 collection sites. For each location chosen for CSI-AA (based on the bulk d15N record), 160.05 mg of lyophilized tissue was weighed and combined for each individual mussel (n = 5).

Elemental and Bulk Isotopic Analyses

Stable carbon (d13C) and nitrogen (d15N) isotope ratios were determined via elemental analyzer isotope ratio mass spectrometry (EA-IRMS) at the University of California, Santa Cruz, Stable Isotope Laboratory (UCSC-SIL; http://emerald.ucsc.edu/~silab/). Approximately 1 mg of each dry isolated DOM sample was weighed into tin capsules (Costec, 5 x 9 mm) for analysis. EA-IRMS analysis was conducted using a Carlo Erba CHNS-O EA1108-elemental analyzer interfaced via a ConFlo III device with a ThermoFinnigan Delta Plus XP isotope ratio mass spectrometer (Thermo Fisher Scientific). Standards, EA-IRMS protocols, and correction routines followed standard UCSC-SIL protocols. Analytical uncertainties of n=3 replicate measurements of isotopic standards ranged from ± 0.05 to 0.1‰ for both d13C and d15N. Carbon to nitrogen elemental ratios were similarly determined by elemental analysis. The presented ratios are atomic ratios (C/N)a normalized to the mass of C and N, but have been abbreviated as C/N throughout.

Compound-specific amino acid Isotopic 13C Analyses

Individual AA d13C values were measured as trifluoroacetyl isopropyl ester (TFA-IP) AA derivatives, after acid hydrolysis. Samples were hydrolyzed by adding 2.5 mg homogenized composite muscle tissue to 1 ml of 6 N HCl, and heating for 20 h at 110 deg C under nitrogen. After drying, AA isopropyl esters were prepared with a 1:5 mixture of AcCl:2-propanol (110 deg C, 60 min) and then acylated using a 1:3 mixture of di chloro - methane (DCM) and trifluoroacetic anhydride (TFAA) (100 deg C, 15 min). Derivatized AAs were dissolved in DCM to a final ratio of approximately 2.5 mg of original tissue to 250 ul DCM for injection on the gas chromatograph IRMS (GC-IRMS) system.

Isotopic analysis was conducted on a Thermo Trace GC Ultra with inline oxidation and reduction furnaces, coupled to a ThermoFinnigan Delta Plus XP IRMS, equipped with a CTC Analytics autosampler.Derivatives (1 ul) were injected (250 deg C constant temperature) onto an Agilent DB-5 column (50 m × 0.32 mm ID × 0.52 um film thickness), with a He carrier flow rate of 2 ml min−1 (constant flow). Separations were achieved with a 4-ramp oven program: 52 deg C, 2 min hold; ramp 1 = 15 deg C min−1 to 75 deg C, hold for 2 min; ramp 2 = 4 deg C min−1 to 185 deg C, hold for 2 min; ramp 3 = 4 deg C min−1 to 200 deg C; ramp 4 = 30 deg C min−1 to 240 deg C, hold for 5 min.

This method allowed for d13C determination of the following AAs in mussel tissue: non-essential AAs alanine (Ala), aspartic acid + asparagine (Asp), glutamic acid + glutamine (Glu), glycine (Gly), proline (Pro), serine (Ser), and tyrosine Tyr); and essential AAs leucine (Leu), isoleucine (Ile), valine (Val), phenylalanine (Phe), and threonine (Thr). Acid hydrolysis destroys tryptophan and cystine, so these were not detected, and it also deaminates\ asparagine to aspartic acid, and glutamine to glutamic acid. While the abbreviations Glx and Asx\ are sometimes used to denote the combined Gln+Glu and Asn+Asp peaks, in order to correspond better with extant CSI-AA literature, we have elected to simply use Asp and Glu abbreviations, as defined above.

All samples were analyzed on the GC-IRMS in triplicate, and measured AA d13C values were corrected for the C added during derivatization, following the approach of Silfer et al. (1991). Reproducibility for tissue samples was typically less than <0.3‰ (n = 3). The average mean deviation for all tissue sample replicates was 0.4‰.

Compound-specific amino acid Isotopic 15N Analyses

Amino acid d15N values were measured as Trifluoroacetyl isopropyl ester (TFA-IP) AA derivatives, following protocols described in detail elsewhere (e.g.,. Briefly, samples were hydrolyzed (6 N HCl, 20 hr at 110uC) under nitrogen, and TFA derivatives subsequently prepared from free AA using a modified version of the protocol described by Silfer (Silfer et al. 1991): isopropyl esters were made with a 1:5 mixture of Acetyl Chloride (AcCl):2-propanol (110uC, 60 minutes), and then acylated using a 1:3 mixture of Dichloromethane:Trifluroacetyl acetate (DCM:TFAA) (100uC, 15 minutes). Derivatized AAs were dissolved in DCM to a final ratio of approximately 4 mg of original tissue to 250 ml DCM. After derivatization, samples were analyzed by a Varian gaschromatograph coupled to a Finnegan Delta-Plus isotope ratiomass spectrometer (GC-IRMS). AAs were separated using a 50 m, 0.32 ID Hewlett Packard Ultra-1 column with 1 mm film thickness. Under our analytical conditions, d15N values could be reproducibly measured for alanine (Ala), aspartic acid + asparagine (Asp), glutamic acid + glutamine (Glu), leucine (Leu), isoleucine (Ile), proline (Pro), valine (Val), glycine (Gly), lysine (Lys), serine (Ser), phenylalanine (Phe), threonine (Thr), and tyrosine (Tyr) (Fig. S4). Most AAs were measured with a standard error of ,1.0% (based on n = 4 injections), and the average mean deviations for individual AA d15N measurements across all tissue sample replicates was 0.5%.

mussel_data.csv