DOI: 10.5061/dryad.9kd51c5d3
title: Data from: Amino acid δ15N underestimation of cetacean trophic positions highlights poor understanding of isotopic fractionation in higher marine consumers

description: Compound specific stable isotope analysis (CSIA) of amino acids (AAs) has
 been rapidly incorporated in ecological studies to resolve consumer
 trophic position (TP). Differential 15N fractionation of ‘trophic’ AAs,
 which undergo 15N enrichment with each trophic step, and ‘source’ AAs,
 which undergo minimal trophic 15N enrichment and serve as a proxy for
 primary producer δ15N values, allows for internal calibration of TP.
 Recent studies, however, have shown the difference between source and
 trophic AA δ15N values in higher marine consumers is less than predicted
 from empirical studies of invertebrates and fish. To evaluate CSIA-AA for
 estimating TP of cetaceans, we compared source and trophic AA δ15N values
 of multiple tissues (skin, baleen, and dentine collagen) from five species
 representing a range of TPs: bowhead whales, beluga whales, short-beaked
 common dolphins, sperm whales, and fish-eating (FE) and marine
 mammal-eating (MME) killer whale ecotypes. TP estimates (TPCSIA) using
 several empirically-derived equations and trophic discrimination factors
 (TDFs) were 1 to 2.5 trophic steps lower than stomach content-derived
 estimates (TPSC) for all species. Although TPCSIA estimates using dual TDF
 equations were in better agreement with TPSC estimates for bowhead whales,
 belugas, and FE killer whales, our data do not support the application of
 a universal or currently available dual TDFs to estimate cetacean TPs.
 Discrepancies were not simply due to inaccurate TDFs, however, because the
 difference between consumer glutamic acid (Glu) and phenylalanine (Phe)
 δ15N values (δ15NGlu-Phe) did not follow expected TP order, indicating it
 is not a reliable index of relative TP in these species. In contrast with
 pioneering studies on invertebrates and fish, our data suggest trophic 15N
 enrichment of Phe is not negligible and should be examined among the
 potential mechanisms driving ‘compressed’ and variable δ15NGlu-Phe values
 at high TPs. We emphasize the need for controlled diet studies to clearly
 understand mechanisms driving AA-specific isotopic fractionation before
 widespread application of CSIA-AA in ecological studies of cetaceans and
 other marine consumers.
description: Most of the isotope data presented here have been compiled from
 previously published studies where detailed sample preparation and
 analysis procedures can be found (Matthews and Ferguson 2014, 2015;
 Ruiz-Cooley et al. 2014, 2017; Pomerleau et al. 2017; Zupcic-Moore et al.
 2017). Briefly, baleen samples were drilled from the proximal end of each
 plate where the most recent growth corresponds to foraging on the summer
 grounds (Matthews and Ferguson 2015), and no further sample preparation
 was carried out prior to isotope analysis. Bowhead whale skin samples were
 rinsed of DMSO using deionized water and were not lipid-extracted prior to
 analysis. Sperm whale skin samples were also rinsed of DMSO using
 deionized water, and then lipid-extracted using a 2:1 chloroform ethanol
 mixture (Lesage et al. 2010; Ruiz-Cooley et al. 2012). Dolphin skin was
 thawed and lipid-extracted with petroleum ether. Annual dentine growth
 layers of sperm whale teeth were sampled using a micromill and later
 combined, while a handheld rotary tool was used to collectively sample
 multiple dentine growth layers of beluga and killer whale teeth. All
 dentine was demineralized using repeated washes of 0.25 N HCl for 12-hr
 periods, and the remaining collagen was rinsed with distilled
 H<sub>2</sub>O. All samples except baleen were freeze-dried
 and finely homogenized. Compound specific stable
 isotope analysis All bowhead, dolphin, beluga, and
 killer whale tissues were analysed at the University of California-Davis
 Stable Isotope Facility, while sperm whale tissues were analysed at
 University of California-Santa Cruz Stable Isotope Laboratory. Briefly, at
 UC Davis, approximately 3 mg of each freeze-dried, homogenized tissue
 sample was acid hydrolysed using 6 M HCl at 150 <sup>o</sup>C
 under a N<sub>2</sub> headspace for 70 min, and derivatized
 using methoxycarbonylation esterification (Walsh et al. 2014, Yarnes and
 Herszage 2017). Methods at UC Santa Cruz differed primarily in the use of
 trifluoroacyl-isopropyl ester as the derivatization agent.
 δ<sup>15</sup>N values of individual derivatized AAs were
 measured at both labs by gas chromatography-combustion isotope ratio mass
 spectrometry (GC-IRMS). At UC Davis, two AA mixtures, previously
 calibrated to the international reference scale for
 δ<sup>15</sup>N (atmospheric N<sub>2</sub>), were
 used in calibration and scale-normalization procedures. Quality assurance
 of δ<sup>15</sup>N measurements followed Yarnes and Herzsage
 (2017). A third AA mixture served as the primary quality control reference
 material, while two well-known natural materials, baleen and fish muscle,
 were used as secondary quality control standards. At UC Santa Cruz,
 isotope values were calibrated to the isotopic composition of atmospheric
 N<sub>2</sub> through repeated measures of a co-injected
 reference compound of known isotopic composition (norleucine). Mean
 analytical precision assessed from duplicate or triplicate measures of
 samples and the reference compound was &lt; 1 ‰ at both
 labs.
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