DOI: 10.5061/dryad.zs7h44jbg
title: Washington harbor seal stable isotope data

description: Understanding the response of predators to ecological change at multiple
 temporal scales can elucidate critical predator-prey dynamics that would
 otherwise go unrecognized. We performed compound-specific nitrogen stable
 isotope analysis (CSIA) of amino acids on 153 harbor seal museum skull
 specimens to determine how trophic position of this marine predator has
 responded to ecosystem change over the past century. The relationships
 between harbor seal trophic position, ocean condition, and prey abundance,
 were analyzed using hierarchical modelling of a multi-amino acid framework
 and applying 1-, 2-, and 3- year temporal lags. We identified delayed
 responses of harbor seal trophic position to both physical ocean
 conditions (upwelling, sea surface temperature, freshwater discharge) and
 prey availability (Pacific hake, Pacific herring and Chinook salmon).
 However, the magnitude and direction of the trophic position response to
 ecological changes depended on the temporal delay. For example, harbor
 seal trophic position was negatively associated with summer upwelling, but
 had a 1- year delayed response to summer sea surface temperature,
 indicating some predator responses to ecosystem change are not immediately
 observable. These results highlight the importance of considering dynamic
 responses of predators to their environment as multiple ecological factors
 are often changing simultaneously and can take years to propagate up the
 food web.
description: Collagen samples were analyzed for using 5 mg of purified
 collagen from approximately 50 mg of bone. Preliminary analyses were
 conducted to determine the highest rate of collagen return from bone
 sampled from different parts of the skull to minimize destruction
 (mandible, internal occipital shelf, temporal process). All produced
 similar stable isotope measurements. Samples were primarily taken from the
 internal occipital shelf at the back of the skull to maintain external
 integrity. Bone was decalcified using 0.2 M HCl for 24-72 hours depending
 on bone thickness, followed by centrifugation and nanopure water rinse.
 Removal of humic acids was conducted using 0.125 M NaOH for 20 hours.
 Samples were washed to a neutral pH, then solubilized in 0.01N HCl. Once
 solubilized, samples were dried under a stream of N2 and freeze dried.
 Freeze dried collagen was analyzed for bulk isotopic composition of
 nitrogen by the UW IsoLab (isolab.ess.washington.edu) using a coupled
 elemental analyzer-isotope ratio mass spectrometer following the standard
 protocols of the laboratory. C:N ratios were available for most (n = 107)
 samples used as a measure of the quality for nitrogen analyses of bone
 collagen for stable isotope analysis (van Klinken 1999). No samples within
 this subset were outside the acceptable C:N range of 2.7-3.6 (by mass),
 indicating there was no substantial loss of glycine or addition of
 nitrogen due to microbial processing from mortality, decay, curation, and
 analysis. We therefore assumed samples without C:N data (n = 20) also were
 within the acceptable range as they were subject to the same storage and
 processing procedures at the same museum institution (University of Alaska
 Fairbanks, Museum of the North). δ15N of eleven amino
 acids (alanine, glycine, proline, aspartic acid, leucine, isoleucine,
 valine, threonine, serine, glutamic acid, phenylalanine, tyrosine) were
 measured in the UW Facility for Compound-Specific Isotope Analysis of
 Environmental Samples. The composition of amino acids in bone collagen is
 highly variable across amino acids (Gauza-Włodarczyk et al. 2017).
 Tyrosine, isoleucine, valine, and threonine are not abundant in bone
 collagen, whereas glycine is 20 times more abundant in bone collagen than
 most amino acids on a gram amino acid per 100 g of protein basis. Samples
 were prepared following the procedures developed by Chikaraishi et al.
 (2007) and protocols by Rachel Jeffrey’s lab at University of Liverpool UK
 which are modifications of that published by Metges et al. (1996) and Popp
 et al. (2007). Briefly, proteins were hydrolyzed in 6N HCl and purified
 using a cation exchange column. Norleucine was added as an internal
 standard. Amino acids were esterified using isopropanol acetyl chloride,
 and derivatized via acylation with 4:1 toluene: pivaloyl chloride. Samples
 were brought up in ethyl acetate and analyzed using a coupled gas
 chromatography-combustion-isotope ratio mass spectrometer system
 (GC-C-irMA; Thermo Scientific Trace GC + GC IsoLink coupled to a Delta V
 irMS) in continuous flow mode monitoring masses (m/z) 28 and 29. A 30 m x
 0.32 mm x 0.50 mm Agilent Technologies DB-35 capillary column with 35%
 Phenyl and 65% polysiloxane stationary phase and moderate polarity was
 used (Chikaraishi et al. 2010) with an inlet temperature of 260 C, column
 flow of 2 ml/min and oven ramp of 9 ˚C min-1.  Tyrosine and isoleucine for
 most samples were not discernable and thus were omitted from this
 analysis. Leucine and isoleucine also co-eluted for many samples and thus
 leucine stable isotope measurements were deemed unreliable and also
 omitted from this dataset.  For each run, a 12 amino acid external
 standard with known isotopic composition was injected four times to
 condition the column followed by sample injections. Samples were injected
 in triplicate, with the 12 amino acid standard mixture injected every two
 samples (or six injections). A two-hour column oxidation was performed
 after 6 samples (25 injections) followed by a 30-minute backflush. δ15N
 was measured as: S1.   δ15N ‰ vs.  air =
 [(15N/14N)Sample / (15N/14N)air - 1] *1000            
 For each machine run, a linear model was fit for each individual amino
 acid using the following equation: S2.  
 Stdaa=maat+baa Where m represents the slope of the
 precision drift, t represents the injection number since last column
 oxidation, and Std represents the δ15N of an individual amino acid aa for
 a standard observation. The data was then corrected using the following
 equations: S3.   Daa, t=Stdaa,t- Trueaa
 Where Daa,t is the difference between an observed standard δ15N
 (Stdaa,t) for a given amino acid (aa) at a given injection number (t) and
 the true δ15N for that standard. Then: S4.  
 Samplecorrected,  aa, t=Sampleobs,aa,t- Daa,t Wherethe
 drift value, Daa,t, is subtracted from the sample value for a given amino
 acid and a given injection to correct the observed sample values for
 precision drift since last column oxidation. Mean sample corrected values
 for the triplicate injections were used for all analyses and trophic
 position calculations and are reported in this dataset. Norleucine had
 lower precision in standards compared to phenyalanine, therefore no
 correction using the internal standard was applied. Mean precision for a
 given amino acid standard was calculated using the standard deviation of
 the external standard injections for a given run after drift correction
 and taking a mean of each run's standard deviation. Conditioning
 injections were omitted from this calculation.
description: For some sampled specimens, metadata was not available the museum
 institution. For these specimens, length, sex or latitude/longitude is
 reported as 'NA'. For some specimens there was not enough bone
 collagen to run both compound specific stable isotope analysis (CSIA) of
 amino acids and bulk stable isotope analysis. CSIA was prioritized and
 there is no bulk stable isotope data for carbon or nitrogen reported which
 is represented in the data as 'NA'. For some specimens, CSIA of
 an individual amino acid was deterimined to be poor quality due to peak
 area or co-elution with other peaks. Data that was not considered
 sufficient quality was omitted from both this dataset and
 analyses.