Amino Acid Isotope Analysis: a New Frontier in Studies of Animal Migration and Foraging Ecology Kelton W

CHAPTER 7

Amino Acid Isotope Analysis: A New Frontier in Studies of Animal Migration and Foraging Ecology Kelton W. McMahon1 and Seth D. Newsome2 1University of Rhode Island, Narragansett, RI, United States, 2University of New Mexico, Albuquerque, NM, United States

7.1 INTRODUCTION Ecogeochemistry is the application of geochemical techniques to fundamental questions Whether it is diel, meter-scale migrations of in ecology (McMahon, Hamady, & Thorrold, zooplankton toward the ocean surface or the 2013a). This field offers a powerful suite of annual 70,000 km global migrations of Arctic tools to explore the intricate connections Terns (Sterna paradisaea), animal migration between movement and foraging dynamics. represents one of the most biologically signifi- Physical, chemical, and biological processes in cant redistributions of biomass on Earth nature create geospatial structure in the distri- (Dingle, 2014). Accordingly, there is a need to bution of stable isotopes; mapping these distri- develop powerful, precise, and integrative butions illustrates the invisible “isoscapes” in tools to describe and quantify these spectacu- which organisms operate (Bowen, 2010). As lar migratory movements (Hobson, 1999; animals interact with isoscapes, they incorpo- Hussey et al., 2015; Kays, Crofoot, Jetz, & rate the local isotopic signals of the environ- Wikelski, 2015; Chapter 1: Animal Migration: ment into their tissues (Graham, Koch, A Context for Using New Techniques and Newsome, McMahon, & Aurioles, 2010; Approaches). Given that the acquisition and Hobson, 1999). Herein lies one of the biggest allocation of dietary resources is a fundamen- challenges in interpreting isotope data in the tal requirement for all animals and an integral context of movement ecology: the isotope com- part of animal movement (Fryxell & Sinclair, position of a consumer reflects information 1988), movement ecology is intimately linked about both the baseline isoscape and the sub- with foraging ecology in space and time. sequent biochemical modifications of organic

Tracking Animal Migration with Stable Isotopes DOI: https://doi.org/10.1016/B978-0-12-814723-8.00007-6 173 © 2019 Elsevier Inc. All rights reserved. 174 7. AMINO ACID ISOTOPE ANALYSIS: A NEW FRONTIER IN STUDIES OF ANIMAL MIGRATION AND FORAGING ECOLOGY matter as it flows through food webs three major themes: (1) using CSIA-AA to (Chapter 4: Application of Isotopic Methods to refine isoscapes, (2) disentangling movement Tracking Animal Movements). and foraging ecology, and (3) examining The trophic modification of organic matter movement and resource utilization across dif- and its isotopic composition results in a series ferent ecosystems. We provide a brief over- of complex ecogeochemical “filters” between view of the analytical process needed to the baseline isoscape and the corresponding generate and interpret CSIA-AA data and an consumer tissue isotope δ-value. To use outlook toward future research needed to stable isotopes to track animal movement expand and refine the use of CSIA-AA for among isoscapes, we need to understand how studying animal movement and foraging these filters modify the isotope value of consu- ecology. mers relative to their baseline isoscapes. Conventional isotopic approaches to examining movement ecology have focused on the 7.2 PRIMER ON AMINO ACID isotope analysis of “bulk” tissues, i.e., the BIOCHEMISTRY AND ISOTOPE weighted average of all components within in DISCRIMINATION a specific tissue. While quite successful (Hobson & Wassenaar, 2008), it can be difficult To track animal movement through space to determine whether variation in bulk tissue and time with stable isotopes, we must be able isotope values is due to differences in (1) diet to link the isotope δ-value(s) of a consumer tis- (e.g., trophic level), (2) tissue types analyzed, sue to an underlying baseline isoscape. (3) physiology or metabolism, (4) isotopic com- However, the isotope values of the baseline iso- position at the base of the food web, or (5) scape (e.g., primary producers) are potentially some combination of these factors (Post, 2002). modified through consumer metabolism in a This can be particularly problematic when variety of ways depending on the number and studying mobile organisms in which resource isotopic effect of enzymatic reactions, as well as and habitat use change across space and time. the flux of elements through these metabolic Whereas bulk tissue isotope analysis pathways (reviewed in Hayes, 2001; McMahon averages all macromolecules in a sample, & McCarthy, 2016; Ohkouchi et al., 2017). The compound-specific isotope analysis of amino differential isotope fractionation of individual acids (CSIA-AA) takes a molecular approach AAs during metabolism provides a mechanism based on well-established biochemical path- to disentangle signals of the baseline isoscape ways. The power of the molecular approach from subsequent metabolic modifications of lies in the differential isotopic (e.g., δ2H, δ13C, consumer isotope values. Therefore in this sec- δ15N) discrimination of individual AAs during tion we present a brief primer on the basics of the transfer of organic matter between diet and how AA metabolism influences nitrogen, car- consumer (i.e., trophic transfer) or biochemical bon, and hydrogen isotopic discrimination. processing within the consumer. In this chapter, we explore the potential of CSIA-AA to study movement and foraging dynamics. We 7.2.1 Nitrogen Metabolism start with a brief primer on the roles of metabolism and physiology on AA stable isotopic Transamination (transferring an amine group) discrimination. We highlight the strengths and and deamination (removing an amine group) limitations of the CSIA-AA approach to move- are the two dominant enzymatic processes that ment ecology by describing case studies under control the flow of nitrogen, and thus nitrogen

TRACKING ANIMAL MIGRATION WITH STABLE ISOTOPES 7.2 PRIMER ON AMINO ACID BIOCHEMISTRY AND ISOTOPE DISCRIMINATION 175 isotope fractionation, in proteinaceous AAs thought to directly reflect the δ15N of the (Braun, Vikari, Windisch, & Auerswald, 2014). baseline isoscape without the confounding issue The diversity of transaminases and deami- of trophic isotope discrimination. However, nases, each with different N isotope effects, alternative metabolic pathways can impart N and the variable degree of transamination and isotope fractionation via transamination or deamination among AAs, result in a wide deamination for many source AAs. 15N-labeling range of nitrogen isotopic discrimination pat- experiments have shown that the central pool terns among individual AAs (McMahon & of dietary nitrogen can be incorporated into McCarthy, 2016). For δ15N analysis, protein Phe, albeit at low levels relative to most other AAs are commonly divided into two groups, AAs (Hoskin, Gavet, Milne, & Lobley, 2001). the minimally discriminating “source” AAs Identifying under what dietary and physiologi- and the heavily discriminating “trophic” AAs cal conditions source AA 15N discrimination (Popp et al., 2007), based on their nitrogen becomes significant is a current area of research metabolism and corresponding isotopic dis- in CSIA-AA. crimination. While this division is often con- Finally, it should be noted that several other fused with the more familiar essential versus AAs that were originally termed “source” nonessential AA groupings for carbon, these AAs, namely Glycine (Gly), Serine (Ser), and groupings not only represent different AAs, Threonine (Thr) (Popp et al., 2007), were done but are also based on fundamentally different so based largely on the empirical results biochemical mechanisms related to nitrogen reported in McClelland and Montoya (2002). cycling (source and trophic AAs) versus syn- Subsequently, across a much broader range of thesis of carbon sidechains of AAs (essential consumers, the variability in mean ( 6 SD) 15 and nonessential AAs). Below we explore the ∆ NC D values for these AAs has been shown À mechanisms of differential 15N discrimination to be extremely large: Gly (3.9 6 4.9m,), Ser among AAs, which allow ecologists to disen- (2.9 6 4.6m), and Thr (25.8 6 3.2m)(McMahon tangle the relative influence of baseline versus & McCarthy, 2016). As a result, we suggest trophic isotope variability on consumer δ15N caution be used when interpreting δ15N values values. of these particular AAs in the context of animal movement and foraging ecology without 7.2.1.1 Minimally Fractionating “Source” further mechanistic studies of their isotopic Amino Acids discrimination patterns. The source AAs (e.g., phenylalanine: Phe; methionine: Met; lysine: Lys) show relatively 7.2.1.2 Heavily Fractionating “Trophic” little N isotope discrimination between diet and Amino Acids consumer, likely because their dominant meta- The trophic AAs (glutamic acid: Glu; aspar- bolic pathways typically do not form or break tic acid: Asp; alanine: Ala; isoleucine: Ile; leu- C N bonds during metabolism (O’Connell, cine: Leu; proline: Pro; valine: Val) typically À 2017). A metaanalysis of published controlled undergo significant N isotopic discrimination feeding studies (70 species, 317 individuals, and during metabolism associated with extensive 88 distinct consumer diet combinations) found transamination and deamination (O’Connell, À minimal N isotopic discrimination of Phe 2017). The canonical trophic AA Glu often has 15 15 (∆ NC D 520.1 6 1.6m), Met (0.4 6 0.4m), and the highest mean ∆ NC D of all AAs À À Lys (0.8 6 1.5m) between consumer (C) and diet (6.4 6 2.5m; McMahon & McCarthy, 2016). The (D) (McMahon & McCarthy, 2016). The δ15N other trophic AAs typically exhibit discrimina- values of these “source” AAs are therefore tion patterns that closely resemble Glu because

TRACKING ANIMAL MIGRATION WITH STABLE ISOTOPES 176 7. AMINO ACID ISOTOPE ANALYSIS: A NEW FRONTIER IN STUDIES OF ANIMAL MIGRATION AND FORAGING ECOLOGY they exchange nitrogen with the central nitro- (Wu, 2009). Typically, δ13C data are reported gen pool in an organism via transamination for seven essential AAs (Ile, Leu, Lys, Met, chains linked to Glu (McCarthy, Lehman, & Phe, Thr, and Val), which contribute B25% À Kudela, 2013; O’Connell 2017). 40% of the AA budget of animal tissues com- Together, the δ15N values of a consumer’s monly analyzed by ecologists (Wolf, trophic and source AAs provide ecologists Newsome, Peters, & Fogel, 2015). Conversely, with a potential tool to calculate consumer tro- nonessential AAs are those that organisms can phic position (TPCSIA-AA) that is internally de novo synthesize from a common carbon indexed to the δ15N of the base of the food pool (Wu, 2009). δ13C data are routinely web. However, several controlled feeding reported for eight nonessential AAs (Gly, Ser, studies have noted that the degree of N iso- Ala, Glu, Asp, Pro, arginine: Arg, and tyrosine: tope discrimination of trophic AAs between Tyr), which contribute B60% 75% to the total À diet and consumer is not constant AAs budget of animal tissues (Wolf et al., (Chikaraishi, Steffan, Takano, & Ohkouchi, 2015). Some of these nonessential AAs are con- 2015; McMahon, Polito, Abel, McCarthy, & sidered conditionally essential (e.g., Pro, Arg, Thorrold, 2015; McMahon, Thorrold, Elsdon, & and Tyr), meaning their de novo synthesis can McCarthy, 2015; Yamaguchi et al., 2017). As be limited under certain physiological condi- with trophic patterns observed in bulk tissues tions. In contrast to the general classification (Vanderklift & Ponsard, 2003), diet quality and scheme used for δ15N (source vs trophic) that consumer mode of nitrogen excretion (e.g., is empirically derived, these essential and non- ammonia vs urea) are potentially key variables essential designations for δ13C are based on influencing the degree of trophic AA isotopic well-established biochemical pathways. discrimination (Germain, Koch, Harvey, & McCarthy, 2013; McMahon, Thorrold, et al., 7.2.2.1 Essential Amino Acids 2015; O’Connell, 2017). This variability in Animals have lost the enzymatic pathways nitrogen isotopic discrimination among trophic required to synthesize sufficient quantities of AAs can significantly impact the accuracy of essential AAs and hence must acquire the trophic position estimates (e.g., Dale, intact carbon skeletons of essential AAs Wallsgrove, Popp, & Holland, 2011; Lorrain directly from their dietary proteins (Howland et al., 2009). Therefore we need to increase the et al., 2003; McMahon, Fogel, Elsdon, & number of controlled feeding studies examin- Thorrold, 2010; O’Brien, Fogel, & Boggs, 2002) ing AA isotope fractionation to better under- or from their prokaryotic gut microbiome stand and account for the underlying (Ayayee et al. 2015; Newsome, Fogel, Kelly, & mechanisms controlling variability in trophic Martinez del Rio, 2011). As a result, essential AA 15N discrimination. AAs typically show minimal 13C isotopic discrimination between diet (or gut microbe essential AAs) and consumer tissue (Howland 7.2.2 Carbon Metabolism et al., 2003; McMahon et al., 2010). Consumer essential AA δ13C values therefore represent For carbon, AAs have conventionally been the isotopic signature of the producers of those classified into two categories, essential and AAs at the base of the food web, without the nonessential AAs, which relate to an organ- confounding variable of significant trophic ism’s ability for de novo synthesis of AA side discrimination. chains. Animals cannot synthesize essential On the other hand, plants, algae (protists), AAs at a rate adequate for normal growth bacteria, and fungi can synthesize essential

TRACKING ANIMAL MIGRATION WITH STABLE ISOTOPES 7.2 PRIMER ON AMINO ACID BIOCHEMISTRY AND ISOTOPE DISCRIMINATION 177

AAs de novo from a variety of inorganic and and (3) route them directly from nonessential organic carbon sources. These organisms use a AAs produced by the gut microbiome from variety of pathways and associated isotope dietary carbohydrate (Ayayee et al., 2015; effects to synthesize common essential AAs Newsome et al., 2011) and lipid precursors (Hayes, 2001). This metabolic diversity in syn- (Newsome et al. 2014). Nonessential AAs can thesis pathways imprints on the relative δ13C be further grouped into two general types: (1) values of essential AAs synthesized by differ- those synthesized from intermediaries in gly- ent groups of producers. Multivariate analyses colysis (Gly/Ser/Ala) and (2) those synthe- using carbon isotope data from a suite of sized from intermediaries in the tricarboxylic essential AAs results in unique essential AA acid (TCA) cycle (Glu/Asp/Pro/Arg), both of δ13C “fingerprints” among plants, algae, bacte- which have distinct sources of carbon and syn- ria, and fungi (Larsen, Taylor, Leigh, & thesis pathways. However, routing of intact O’Brien, 2009; Larsen et al., 2013; Scott et al. nonessential AAs directly from dietary protein 2006). Assuming the contribution of essential into consumer tissues (just like essential AAs) AAs synthesized de novo by gut microbiota is is energetically most favorable (Schwarcz, minimal (but see Ayayee, Jones, & Sabree, 1991), as the use of premanufactured carbon 2015 and Newsome et al. 2011 for counterargu- skeletons reduces the metabolic costs of syn- ments), then direct assimilation (routing) of thesis (Wu et al., 2014). Isotopic discrimination dietary essential AAs results in little C isotopic of C between nonessential AAs and bulk diet alteration of these molecules as they are is highly variable and is likely dependent on passed from diet to consumer (Howland et al., the degree of de novo synthesis, the nonpro- 2003; McMahon et al., 2010). By extension, the tein substrates utilized during synthesis (e.g., essential AA δ13C fingerprints of consumers at carbohydrates vs lipids), and the degree of any trophic level could be used to evaluate the direct routing of nonessential AAs sourced relative importance of different sources of pri- from dietary protein (Howland et al., 2003; mary production at the base of the food chain McMahon et al., 2010; McMahon, Polito, et al., (Arthur, Kelez, Larsen, Choy, & Popp, 2014; 2015; Newsome et al., 2011). Empirical studies McMahon, McCarthy, Sherwood, Larsen, & have demonstrated that the relative degree of Guilderson, 2015; McMahon, Thorrold, de novo synthesis versus direct routing of non- Houghton & Berumen, 2016). As discussed in essential AAs is primarily driven by dietary Section 7.4.2, when these distinct primary protein content (Newsome et al., 2011; producers occupy spatial gradients or discrete Newsome, Wolf, Peters, & Fogel, 2014; O’Brien habitats, the AA C isotope fingerprinting et al., 2002). Understanding the patterns in approach can be used to identify foraging direction and magnitude of nonessential AA across space and time. carbon isotope fractionation, as well as the underlying drivers, is an area of active 7.2.2.2 Nonessential Amino Acids research in ecogeochemistry. In contrast to essential AAs, animals can acquire nonessential AAs in several ways: (1) synthesize them de novo from a bulk carbon 7.2.3 Hydrogen Metabolism pool of protein and nonprotein dietary macromolecules (Newsome et al., 2011; O’Brien Only one study has reported δ2H values of et al., 2002; Wu et al., 2014), (2) route them individual AAs (Fogel, Griffin, & Newsome, directly or indirectly (via the gut microbiome) 2016). However, given the B200m δ-range in from dietary protein (McMahon et al., 2010), global δ2H isoscapes (Bowen, 2010; McMahon

TRACKING ANIMAL MIGRATION WITH STABLE ISOTOPES 178 7. AMINO ACID ISOTOPE ANALYSIS: A NEW FRONTIER IN STUDIES OF ANIMAL MIGRATION AND FORAGING ECOLOGY et al., 2013a), AA δ2H analysis, just like hydro- used to trace animal movement. But for her- gen isotope analysis of bulk tissues, may be bivorous or frugivorous species that con- the most promising system for studying terres- sume relatively low protein diets, AA δ2H trial animal movement and migration patterns. may be a more faithful recorder of local envi- While food is the only source of carbon and ronmental water, and thus may be a strong proxy nitrogen available for proteinaceous tissue syn- for tracking latitudinal or altitudinal movements. thesis in animals, hydrogen from both food and water is used to construct tissues (Hobson, 1999; Wolf, Newsome, Fogel, & 7.3 ACCOUNTING FOR Martinez del Rio, 2013). The experiment CONSUMER PHYSIOLOGY reported in Fogel et al. (2016) focused on bacteria (Escherichia coli)growninwaterofvary- Animals that migrate use a diverse array of ing δ2Hvaluesandonmediumthatdidor life history strategies to maximize fitness when did not contain protein. Interestingly, the engaging in long-distance movements and essential and nonessential classification extended residence in different ecosystems: scheme appears to work well for AA δ2H, aquatic versus terrestrial, freshwater versus likely because most of the hydrogen in AAs is marine, and tropics versus high latitudes bonded to carbon and does not exchange with (Johnson & Gaines, 1990). These migration body water. This experiment showed that strategies are associated with major shifts in most ( . 80%) of the hydrogen in E. coli cells organismal physiology (Gwinner, 2012). For was routed directly from a protein-rich sub- example, animal migration is often accompa- strate (tryptone). The only exception to this nied by major shifts in energy output associ- pattern was in the glycolytic nonessential AA ated with the physical act of migrating, alanine, which had B40% 50% of its hydro- alterations to metabolic rate associated with À gen derived from environmental water (i.e., environmental temperature shifts and/or cellular water) even when exogenous protein physical activity, and changes in body condi- was available for cellular synthesis. tion associated with fasting and/or shifts to By contrast, a relatively high proportion of endogenous energy reserves (e.g., body fat or hydrogen (B35% 75%) in both nonessential muscle). These changes are often concurrent À and essential AAs was sourced from environ- with additional changes in physiology associ- mental water when E. coli were grown on a ated with the drivers of migration, such as glucose medium containing no protein. Amino shifts in hormones and body condition associ- acid δ2H data from controlled feeding experi- ated with reproduction. ments on other organisms have yet to be There is a wealth of bulk tissue isotope data reported, however, the biochemical pathways on how major physiological stressors associ- (glycolysis and TCA cycle) used by E. coli to ated with migration, reproduction, and disease synthesize nonessential AAs are like those impact consumer C isotopic discrimination used by heterotrophic eukaryotes (Kanehisa, (e.g., Gannes, Martinez del Rio, & Koch, 1998), Furumichi, Tanabe, Sato, & Morishima, 2017). but comparatively little information for indi- Overall, these patterns suggest that the major- vidual AAs. For example, McMahon, Polito, 13 ity (B70% 80%) of hydrogen in the proteina- et al. (2015) found that the ∆ CC D of nones- À À ceous tissues of omnivorous and carnivorous sential AAs in captive penguins varied sig- animals is obtained from diet, which may nificantly depending on the duration of fasting obscure the relationships between tissue δ2H prior to feather molt. The strong 13C-depletion and that of precipitation that are commonly of AAs associated with the TCA cycle

TRACKING ANIMAL MIGRATION WITH STABLE ISOTOPES 7.3 ACCOUNTING FOR CONSUMER PHYSIOLOGY 179

(Glu, Asp, and Pro) appeared to be related to respectively (e.g., Newsome et al., 2010; increased reliance on endogenous lipid stores, Newsome, Wolf, Bradley, & Fogel, 2017). which are catabolized and readily converted to Conversion of one reservoir into another TCA precursors during nutritional stress from should be traceable at the molecular level, molting and migration. Similar patterns have especially for compounds (e.g., nonessential been observed in captive mice fed lipid-rich AAs) that consumers can synthesize de novo diets (Newsome et al., 2014). These variable while minimally fractionating essential AAs AA isotopic discrimination patterns associated provide concurrent information about the with changes in physiology, disease, and sources of primary production at the base of resource utilization make it challenging to the food web supporting those consumers accurately interpret migration and residence (e.g., McMahon, Polito, et al., 2015). Note that patterns of mobile consumers. There is a criti- isotopic effects of such changes in movement, cal need for more controlled feeding experi- foraging, and physiology will only be observ- ments in the lab and comparative field studies able in tissues with isotopic incorporation examining the impacts of these major changes rates that can record shifts on the same time in physiology on the isotopic discrimination of scales as the changes in resource allocation individual AAs, which can create additional and use. mismatches between consumer isotope values Another topic linked to exploring migra- and the underlying isoscape. tion and physiology with CSIA-AA that With proper calibration, these AA isotopic represents both a current complication and a discrimination patterns that currently pose potential opportunity is the role that the gut challenges to tracking animal migration could microbiome plays in consumer protein metab- instead provide powerful opportunities to olism. Gut microflora likely play an important study organismal physiology of migrating ani- role in the protein metabolism of many animals. Animals that roam over vast distances mals, particularly herbivorous and omnivo- are inherently difficult to capture and sample, rous hosts consuming diets deficient in often making it impossible to reliably capture dietary protein quantity or quality. In such the same individual multiple times to provide cases, the gut microbiome often supplements a longitudinal ecological or ecophysiological the essential AA budget of its host organism record. CSIA-AA could enable tracing of car- by converting nonprotein dietary macromole- bon as it is shuttled among tissue types, help- cules, such as carbohydrates or even lipids, ing to illuminate temporal variation in the into the carbon skeletons needed for de novo mobilization of different reservoirs of endoge- synthesis of essential AAs. Only a few studies nous versus exogenous resources. Empirical focused on a small suite of essential AAs (Lys data collected from a wide variety of organ- and Thr) have examined this topic in humans isms including bacteria, fungi, plants, and ani- (Metges, 2000), rats (Torrallardona, Harris, mals show that endogenous lipids and protein, Coates, & Fuller, 1996), fish (Newsome et al., which are the two largest endogenous reser- 2011), and insects (Ayayee et al., 2015)ina voirs that animals use during times of nutri- laboratory setting. This topic merits continued tional stress, have very different δ13C and δ2H study as the gut microbiome adds an impor- values (DeNiro & Epstein, 1977; Estep & tant filter between consumers and the Hoering, 1980; Hayes, 2001). Bulk adipose baseline isoscape, as well as a potential tissue, or lipids extracted from proteinaceous opportunity to explore patterns of resource tissues, have δ13C and δ2H values 6m 8m and utilization, metabolism, and physiology in À B100m 150m lower than tissue proteins, migratory species. À

TRACKING ANIMAL MIGRATION WITH STABLE ISOTOPES 180 7. AMINO ACID ISOTOPE ANALYSIS: A NEW FRONTIER IN STUDIES OF ANIMAL MIGRATION AND FORAGING ECOLOGY

7.4 CASE STUDIES IN MOVEMENT baseline isoscape signal as it moves up the AND FORAGING ECOLOGY USING food chain imparts additional isotopic discrim- CSIA-AA ination and uncertainty that can obscure the links between consumer isotope values and Perhaps the most striking advantage of the baseline isoscape. CSIA-AA has the poten- CSIA-AA is the ability to isolate information tial to expand and refine the use of isotopes about both the baseline isoscape and food web for studying animal movement by constraining structure from a single sample. In this section, (1) the development of baseline isoscapes (this we explore case studies under three important section) and (2) the offsets between baseline themes to the development and application of isoscapes and consumer stable isotope values CSIA-AA in movement and foraging ecology: (Section 7.4.2). (1) using CSIA-AA to refine isoscapes, (2) Generating isoscapes using minimally iso- disentangling movement and trophic dynamics tope fractionating AAs of primary or second- in space and time, and (3) examining move- ary consumers provides a temporal integration ment and resource utilization across different of the baseline signal without the confounding ecosystem types. issue of significant trophic discrimination. For example, Vokhshoori and McCarthy (2014) used δ15N values of source AAs from sessile, 7.4.1 Developing CSIA-Based Isoscapes filter-feeding mussel tissue (Mytilus california- nus) to generate CSIA-AA isoscapes across 10 Isoscapes form a valuable framework for latitude in the dynamic coastal zone of the tracking the movement of animals in space California Current System. They found strong and time (Graham et al., 2010; Hobson, 1999; latitudinal gradients in mussel Phe δ15N McMahon, Hamady, & Thorrold, 2013b; values, reflecting the mixing of northern 15N- Wunder & Norris, 2008). Primary producers depleted water brought south by surface flow integrate the physical, chemical, and biological of the California Current and southern 15N- cycling of elements in the environment and enriched water brought north via the form a link to upper trophic level consumers. California Undercurrent from the denitrifica- The tissues (e.g., leaves) of most primary pro- tion zone of the Eastern Tropical Pacific. In ducers in terrestrial environments integrate systems that are spatially and temporally con- environmental information over long time strained, the empirical generation of AA-based periods and thus may be suitable for the con- isoscapes may provide a robust geospatial struction of isoscapes at the primary producer framework for examining animal movement. level. In contrast, the typically short life spans The primary reason why CSIA-AA has not and fast isotopic incorporation rates of primary been routinely used to construct isoscapes is producers in marine environments (e.g., phyto- analytical. The requirement of significant spe- plankton) relative to upper trophic level consu- cialized isotope mass spectrometer instrumenta- mers can create a mismatch between the tion, time and expense of analyses, and analyst highly dynamic baseline isoscape and con- expertise required for proper interpretation of sumer isotope values. To circumvent this issue, CSIA-AA data mean it is not feasible to analyze marine isoscapes are often empirically con- hundreds or thousands of samples to generate structed using data from primary consumers large-scale isoscapes from AA isotope data. In with longer integration times that more closely all likelihood, the true value of CSIA-AA to the match those of the target organism. The bio- development of isoscapes will lie in the ability chemical and physiological processing of that to help constrain the underlying drivers of

TRACKING ANIMAL MIGRATION WITH STABLE ISOTOPES 7.4 CASE STUDIES IN MOVEMENT AND FORAGING ECOLOGY USING CSIA-AA 181 observed geospatial structure in isoscapes that often (1) use source AAs to examine local bio- are constructed using bulk tissue data. For geochemical cycling without the confounding example, MacKenzie, Longmore, Preece, Lucas, issue of consumer trophic discrimination or (2) and Trueman (2014) used lion’s mane jellyfish compare source and trophic AAs to calculate (Cyanea capillata) to construct a robust pelagic consumer trophic level while controlling for isoscape for the North Sea. However, jellyfish variation in baseline isotope values. are opportunistic feeders, preying on a wide The logical extension of these CSIA-AA range of pelagic organisms. To address this approaches is to use the isotope values of min- potential trophic variability issue, the authors imally isotope fractionating AAs in consumers compared their isoscapes to similar isoscapes as proxies for baseline isotope values to track generated from benthic, sessile scallops in the movement across known geochemical gradi- North Sea from Jennings and Warr (2003). They ents, while controlling for potential filters concluded that the remarkably similar gradients between baseline and consumer imposed by in isotope value between these two consumers, trophic discrimination. One of the first studies despite their differences in diet (trophic level) to use CSIA-AA to infer animal movement and habitat, suggested that the observed North was by Popp et al. (2007), who found strong Sea isoscape was a function of regional hydrog- latitudinal gradients in the source AA δ15N raphy and biogeochemical cycling rather than values of Pacific yellowfin tuna (Thunnus alba- trophic dynamics. A further test of this hypoth- cares) in the eastern tropical Pacific that mir- esis could apply CSIA-AA of select samples rored patterns in the local baseline isotope along the major isotopic gradients. Thus bulk values of particulate organic matter. They con- SIA facilitates the sample sizes necessary to cluded that these tuna were not undertaking generate robust isoscapes in space and time, significant ocean basin scale migrations, at while CSIA-AA provides a tool to further vali- least on the monthly time scale of tissue turn- date the structure of the observed isoscapes over (Bradley, Madigan, Block, & Popp, 2014). through insights into the underlying mechan- Dale et al. (2011) took this approach one step isms generating such geospatial patterns. further, using CSIA-AA to identify ontogenetic shifts in nursery habitat use of brown stingray (Dasyatis lata) across an isoscape in coastal 7.4.2 Disentangling Movement and Hawai’i. They were able to resolve shifts in Foraging Ecology both movement (migration from nursery habitats within the bay to adult habitats offshore) Movement and foraging ecology are funda- and foraging (diet shifts with concurrent mentally linked. Not only is foraging ecology a increases in trophic level) of this benthic pred- major driver of animal migrations (Fryxell & ator across a complex tropical isoscape. Sinclair, 1988), but food is often a primary vec- A number of other studies have employed tor for transferring the biogeochemical signals CSIA-AA to examine movement associated of baseline δ13C, δ15N, and δ2H isoscapes into with foraging (e.g., Lorrain et al., 2009; Ruiz- consumer tissues (Graham et al., 2010; Wunder Cooley, Koch, Fiedler, & McCarthy, 2014), & Norris, 2008). Perhaps the most common breeding (e.g., Seminoff et al., 2012; Vander applications of CSIA-AA in ecogeochemistry Zanden et al., 2013), and range expansion (e.g., have been to tease apart the relative influence Ruiz-Cooley, Ballance, & McCarthy, 2013) of baseline and trophic isotope variability on across a variety of spatial and temporal scales. consumer isotope values (see Fig. 1 of Several of these studies report substantial McMahon & McCarthy, 2016). These studies underestimates of top consumer trophic level

TRACKING ANIMAL MIGRATION WITH STABLE ISOTOPES 182 7. AMINO ACID ISOTOPE ANALYSIS: A NEW FRONTIER IN STUDIES OF ANIMAL MIGRATION AND FORAGING ECOLOGY based on CSIA-AA in both field and laboratory To date, most studies using CSIA-AA to settings (e.g., Dale et al., 2011; Germain et al., examine migration have focused on large-scale 2013; Lorrain et al., 2009; Ruiz-Cooley et al., (.100s km) movements of organisms relative 2013, 2014), which likely reflects challenges to known geochemical gradients. The use of with our current understanding of the under- isotopes to geolocate organisms to specific lying mechanisms of trophic AA isotopic dis- habitats distributed across smaller scales crimination (O’Connell, 2017). Characterizing (,10 km) is a potentially powerful but chal- the sources and magnitudes of these AA tro- lenging goal. McMahon, Berumen, and phic discrimination factors is an area of active Thorrold (2012) used an essential AA δ13C fin- research (e.g., Chikaraishi et al., 2015; Germain gerprinting approach to assess ontogenetic et al., 2013; McMahon, Polito, et al., 2015; migration patterns of Ehrenberg snapper McMahon, Thorrold, et al., 2015; Steffan et al., (Lutjanus ehrenbergii) among discrete juvenile 2013), as are new approaches to incorporating nursery habitats on the scale of 10s km. To do this variability into equations that estimate tro- so, they characterized spatially separated habi- phic level (e.g., McMahon, Polito, et al., 2015). tat signatures using resident fishes (reflecting As our understanding of AA isotopic discrimi- local food web structure; McMahon, Berumen, nation improves, so will our ability to calculate Mateo, Elsdon, & Thorrold, 2011) and then more accurate and precise trophic levels using compared those signatures to the core of adult CSIA-AA. fish otoliths (representing the period of time δ2H analysis of individual AAs could pro- when those fish were juveniles; McMahon, vide better spatial resolution than bulk tissue Fogel, Johnson, Houghton, & Thorrold, 2011). approaches to assigning origins in animals that In doing so, they were able to identify impor- undertake continental-scale migrations. tant nursery habitats and migration corridors Specifically, there are two potentially fruitful for an ecologically and economically important approaches that could be used to construct coral reef fish in the Red Sea. In situations like compound-specific hydrogen isoscapes. First, this with spatially and isotopically distinct nonessential AAs like Ala appear to be synthe- habitats, CSIA-AA can provide a powerful tool sized from a high proportion (40% 50%) of for identifying habitat use and residence pat- 2 À water-derived hydrogen. Thus δ H analysis of terns in mobile consumers. Ala may provide a more direct link between As with all indirect tools for tracking animal the hydrogen isotopic composition of local pre- movement, we need to validate the patterns of cipitation and that of tissues, of which only movement determined by CSIA-AA with 20% 30% is sourced from body water. A known locations. For instance, Seminoff et al. À 2 second approach would be to focus on the δ H (2012) used satellite telemetry and source AA of essential AAs, which are overwhelmingly δ15N analysis to show that patterns in bulk tis- derived from food ($90%), and thus may sue isotope values of Pacific leatherback sea more faithfully record regional or even land- turtles (Dermochelys coriacea) reflected diver- scape level variation in the δ2H of resources gent turtle migratory strategies to distinct for- than bulk tissues that are synthesized from a aging groups rather than spatial variation in combination of hydrogen from water and trophic dynamics. Similarly, Polito et al. (2017) food. However, AA δ2H compound-specific used archival geolocation tags to show that the approaches need to be tested with feeding strong segregation of essential AA δ13C values experiments in the laboratory followed by in Antarctic penguin tail feathers was indica- analysis of tissues from known origin indivi- tive of distinct migration strategies from the duals (e.g., Langin et al., 2007). Antarctic Peninsula east into the ice-covered

TRACKING ANIMAL MIGRATION WITH STABLE ISOTOPES 7.4 CASE STUDIES IN MOVEMENT AND FORAGING ECOLOGY USING CSIA-AA 183

Weddell Sea versus west into the ice-free Scotia trophic AA Glu. Assessing trophic dynamics Sea. There is an obvious need to expand upon of consumers that move and forage between these efforts across a variety of stable isotopes, these distinct ecosystem types (e.g., with dif- ecosystems, and spatiotemporal scales. While ferent primary producer β values) will require no isotopic approach will ever provide the geo- new trophic position models that take this var- location resolution of satellite telemetry, iso- iability into account. For instance, to examine topes have the advantage of recording dietary the trophic positions of prehistoric human inputs over a variety of timescales depending populations in Rapa Nui, South Pacific, on tissue type. Thus coupling isotopes with Jarman et al. (2017) used an amino acid δ13C telemetry could potentially discriminate fingerprinting approach to identify the relative between movement through and actual use of contribution of marine and terrestrial primary resources in particular habitats. production supporting humans, and then used an amino acid nitrogen isotope mass balance model with a βmixed value that multiplies the 7.4.3 Constraining Movement Across estimated fraction of marine and terrestrial Ecosystem foods with their corresponding known marine or terrestrial β values. This analytically inten- Most studies utilizing bulk and CSIA-AA sive, but innovative approach, likely reflects isotope methods to characterize animal move- the next generation in trophic dynamics mod- ment have focused on movement within a sin- els using CSIA-AA. But the success of this gle ecosystem type (e.g., an ocean basin or a approach necessitates a better understanding terrestrial grassland). Far fewer studies have of the parameters (e.g., B) that go into the used CSIA-AA to rigorously examine move- model. Furthermore, there have been very few ment across ecosystems types (e.g., between controlled feeding studies to examine trophic marine and terrestrial systems, or from C3 for- discrimination of individual AAs among terres- ests to C4 grasslands). Some early work in this trial consumers, particularly vertebrates and area assessed use of marine versus terrestrial upper trophic level consumers (but see resources by coastal consumers (including Nakashita et al., 2011; Steffan et al., 2015). humans) (e.g., Hare, Fogel, Stafford, Mitchell, Uric-acid producing insects, for instance, & Hoering, 1991; Honch, McCullagh, & appear to have very different isotopic discrimi- Hedges, 2012; Naito, Honch, Chikaraishi, nation patterns of Glu and Phe than many Ohkouchi, & Yoneda, 2010). However, inter- urea and uric-acid producing aquatic consu- pretation of source or essential AA isotope mers (see discussion in McMahon & data can be challenging when adjacent ecosys- McCarthy, 2016). In agreement with other tems have different primary producer sources recent comments (O’Connell & Collins, 2017), (Jarman et al., 2017). In comparison to marine we caution that more data are needed on pri- producers, very little is known about δ15N mary producer β values and AA isotopic dis- discrimination patterns among source AAs in crimination factors between diet and consumer terrestrial plants (e.g., Chikaraishi et al., 2009). from a wide range of natural marine and ter- Preliminary data suggest that C3 and C4 terres- restrial ecosystems before we can confidently trial plants have distinct β values (difference use AA-based equations to quantify trophic between trophic and source AAs in primary level, especially for consumers that forage producers), and these differences appear to be across multiple primary producer types (e.g., larger in magnitude than observed trophic dis- marine algae, terrestrial C3 plants, and terres- crimination of the most heavily fractionating trial C4 plants). Similar datasets are needed to

TRACKING ANIMAL MIGRATION WITH STABLE ISOTOPES 184 7. AMINO ACID ISOTOPE ANALYSIS: A NEW FRONTIER IN STUDIES OF ANIMAL MIGRATION AND FORAGING ECOLOGY further refine the capabilities and limitations carboxyl and amino terminus of sequential of using essential AA δ13C fingerprints to AAs in a polypeptide chain. AA purification address questions in both aquatic and terres- with a cation exchange resin (e.g., Dowex) is trial ecology. often necessary when dealing with heteroge- neous materials that contain a mixture of protein, lipids, and carbohydrates (e.g., blood 7.5 CSIA-AA METHODOLOGY plasma, plants, algae, and biofilms) or when working with biomineral tissues that have Successful application of CSIA-AA to ques- interfering compounds (e.g., otoliths) tions in movement and foraging ecology (Amelung & Zhang, 2001). However, signifi- requires significant analytical capital and cant isotopic fractionation can occur with some biochemical expertise to properly analyze, types of column resins or procedures (e.g., interpret, and evaluate molecular isotope data. Hare et al., 1991), so consistent analytical pro- Most notably, the isotope analysis of individ- cedures with careful evaluation of isotopic ual compounds requires significantly more fractionation should always be employed. time for sample preparation and analysis com- The vast majority of papers reporting AA pared to conventional analysis of bulk tissues isotope data use GC-C-IRMS, which requires via elemental analyzer-isotope ratio mass spec- the derivatization of individual AAs to reduce trometry (EA-IRMS). A telling proxy for the AA polarity and increase AA volatility prior to intense analytical nature of CSIA-AA is the separation and isotopic analysis (Silfer, Engel, average sample size in published datasets, Macko, & Jumeau, 1991). Derivatization neutra- often in the dozens rather than the hundreds lizes polar carboxyl ( COOH), amino ( NH ), À À 2 or thousands. While CSIA-AA is a potentially and hydroxyl ( OH) groups in AAs by repla- À powerful methodology in animal ecology and cing active hydrogen atoms with nonpolar moi- ecophysiology, it is currently best suited for a eties. The three most common derivatization well-defined question that can be answered reagents used in ecological and geochemical with a relatively small dataset. studies are: trifluoroacyl-isopropyl ester (TFA/ Contemporary analytical methods for deter- AA/iPr, e.g., Silfer et al., 1991), pivaloyl- mining δ13C, δ15N, or δ2H values of individual isopropyl ester (Pv/AA/iPr, e.g., Chikaraishi, AAs consists of two phases: a wet chemistry Kashiyama, Ogawa, Kitazato, & Ohkouchi, phase to purify AAs and an analytical phase to 2007), and methoxycarbonyl AA ester (MOC/ separate the AAs and measure their AA, e.g., Walsh, He, & Yarnes, 2014). As is the stable isotope values. In the wet chemistry case with all wet chemistry, great care should phase, samples are typically lipid extracted, be taken to choose the appropriate derivatiza- acid hydrolyzed, purified, and then deriva- tion protocol for the application at hand. Each tized (if analyzing via gas chromatograph- of these procedures has trade-offs, e.g., in the combustion (GC-C-)-IRMS, see below). Lipid amount of time required to derivatize, the sta- extraction with polar solvents (e.g., chloro- bility of the resulting derivatives, the safety form:methanol or petroleum ether) is neces- risks of exposure (both to humans and instru- sary when working with samples that contain ments), and the ability to maximize the number .2% lipids, as excess lipids can interfere with of AAs measured (Ohkouchi et al., 2017). A the derivatization process and degrade chro- smaller number of studies have successfully matography. Hydrolysis with strong (B6 N) used high-pressure liquid chromatography- hydrochloric acid liberates individual AAs by (HPLC)-C-IRMS, which does not require deriv- breaking the peptide bonds that bind the atization, to analyze the stable isotope values of

TRACKING ANIMAL MIGRATION WITH STABLE ISOTOPES 7.6 SUMMARY AND FUTURE WORK 185 individual AAs (Broek & McCarthy, 2014; isotope fractionation of the reaction (for sam- McCullagh, Juchelka, & Hedges, 2006). ple equations see Fogel et al., 2016; O’Brien However, GC-C-IRMS continues to be the pre- et al., 2002). Corrections accounting for the ferred analytical approach for CSIA-AA. proportion of extrinsic versus intrinsic carbon While the wet chemistry phase is time and hydrogen vary depending on individual intensive, the major bottleneck in sample AA structure. No extrinsic nitrogen is added throughput is instrument analytical time. during derivatization so post hoc corrections Methods vary, but separation of AAs via GC of δ15Ndataaresimilartothoseusedfor typically uses a 50 60 m nonpolar GC column correcting bulk tissue isotope data produced À and a temperature ramping procedure lasting by EA-IRMS. 40 75 minutes for an individual sample À injection. Samples are typically analyzed in triplicate and bracketed by an AA reference 7.6 SUMMARY AND FUTURE material that is derivatized along with the tar- WORK get samples. Reference materials are mixtures of commercially available AAs (e.g., Sigma CSIA-AA has great potential to enhance the Aldrich) with known isotope values indepen- study of animal migration, habitat use, and dently measured via EA-IRMS prior to deriva- foraging ecology. However, like all indirect tization. Under ideal analytical conditions tracer-based proxies, there are a number of when instrumentation is performing well, this assumptions and inherent limitations that run sequence will yield AA isotope data for must be considered when interpreting AA iso- B3 4 unique samples (run in triplicate with tope data. The success of any isotope-based À standards) in a B24-hour time period. But application to track resource utilization, be it note that GC-C-IRMSs are complex instru- diet or habitat use, is dictated by our under- ments that require constant attention and standing of two fundamental principles of expert knowledge to operate. Generating and how the isotopic composition of consumer diet maintaining good chromatography on any GC and tissues are related: (1) isotopic discrimina- system often transcends the boundary between tion factors that control the offset between con- science and art and takes time to learn. sumer and baseline isoscapes and (2) tissue High-quality peak separation in the chroma- incorporation rates that influence the time tography is critical for generating high-quality scale of integration. AA isotope data. Peak coelution prevents accu- First, we must consider the accuracy of the rate integration and isotopic measurement. AA discrimination factors used to link the iso- Ensuring that the trace returns as close as topic composition of the consumer to the possible to baseline between peaks requires underlying baseline isoscape. The analysis of the purification steps discussed earlier, proper source AAs (δ15N) and essential AAs (δ13C and selection of column type, modifications to the δ2H) offers a major advantage on this front. temperature ramps during analysis, and However, even these AAs with generally mini- adjustments to peak and baseline integration mal isotopic discrimination exhibit some on the chromatograph postanalysis. Once degree of change in isotope value during tro- optimal chromatography is achieved, deriva- phic transfer, which when propagated through tized AA isotope data must be corrected post several trophic levels, can impart a significant hoc for the extrinsic carbon or hydrogen shift in consumer AA isotope values relative to added to the carboxyl and amino terminus of the baseline isoscape. There is an obvious each AA as well as the associated kinetic need for more data on the mechanisms that

TRACKING ANIMAL MIGRATION WITH STABLE ISOTOPES 186 7. AMINO ACID ISOTOPE ANALYSIS: A NEW FRONTIER IN STUDIES OF ANIMAL MIGRATION AND FORAGING ECOLOGY influence carbon, nitrogen, and hydrogen iso- accurate consumer trophic positions using topic discrimination in individual AAs. This CSIA-AA will require choosing trophic and requires controlled laboratory experiments sources AAs with comparable turnover rates. cross-validated with natural experiments However, with proper calibration, variability across an increasingly wide range of consumer in individual AA incorporation rates may also taxa, consumer resource relationships (e.g., provide a framework to create an isotopic À diet composition and quality, and gut micro- clock, as has been done previously by compar- biota contribution), and consumer physiologi- ing bulk isotope values of multiple tissues cal conditions (e.g., fasting and disease). with different incorporation rates, to determine Recent developments in position-specific the length of time an individual has spent on a stable isotope analysis may open new doors to new resource (e.g., habitat or food) (Phillips & mechanistically study biochemical pathways Eldridge, 2006). along the synthesis and transformation of com- Finally, the accuracy of geolocation assign- pounds (Gauchotte-Lindsay & Turnbull, 2016). ments using CSIA-AA or any isotope approach Our improved understanding of isotopic dis- is only as good as the accuracy of the underly- crimination factors will also necessitate better ing isoscapes. Most current regional or conti- universal practices for accounting for uncer- nental scale isoscapes used to track large-scale tainty in AA isotopic measurements and migrations are hampered by the undersampling associated parameters (e.g., β and ∆ in trophic and uneven distribution of data across space level calculations) used in CSIA-AA applica- and time (e.g., Jennings & Warr, 2003; tions (e.g., Ohkouchi et al., 2017). McMahon et al., 2013b; Schell, Barnett, & Second, the accuracy of an isotope approach Vinette, 1998), as well as the paucity of calibra- to tracking animal movement and habitat use tion samples of known origin (e.g., Langin is dependent upon our understanding of the et al., 2007). With advances in our understand- integration time of the sampled tissue relative ing of isotope systematics (e.g., Hayes, 2001; to the organism’s residence time in a habitat. Macko, Estep, Engel, & Hare, 1986), biogeo- The rate of isotope incorporation into the AAs chemical and ecosystem models (e.g., Magozzi, of a consumer can vary significantly as a func- Yool, Vander Zanden, Wunder, & Trueman, tion of the degree of transamination/deamina- 2017), and statistical approaches (e.g., Wunder tion (nitrogen) or synthesis (carbon and & Norris, 2008), significant opportunities exist hydrogen), the kinetic isotope effects associ- to expand and refine the development of spa- ated with AA transport, and the biochemical tially and temporally robust isoscapes through reactions controlling metabolic breakdown the development of more mechanistic and com- (Chikaraishi et al., 2007; McCarthy et al., 2013). prehensive isoscape models (Trueman, While isotope turnover rates of individual MacKenzie, & Palmer, 2012). When coupled AAs is still relatively unknown, several studies with the improved understanding of individual have found that half-lives of individual AAs in AA isotopic discrimination and turnover rates, Pacific bluefin tuna (Thunnus orientalis) the CSIA-AA approach holds great promise for (Bradley et al., 2014) and Pacific white shrimp improving our understanding of animal move- (Litopenaeus vannamei)(Downs et al., 2014) var- ment, habitat use, and foraging ecology. ied from weeks (e.g., Pro) to .1 year (e.g., Ser). Understanding AA isotope turnover rates is critical to constraining the temporal and spa- Acknowledgment tial integrations of the local baseline isotope This chapter is dedicated to Dr. Marilyn L. Fogel. Through signals into consumer tissues. Calculation of her pioneering work in compound-specific stable isotope

TRACKING ANIMAL MIGRATION WITH STABLE ISOTOPES REFERENCES 187 analysis and her years of dedication to education and men- contents, bulk and amino acid stable isotopes. Marine toring, her fingerprints can be seen throughout this chapter Ecology Progress Series, 433, 221 236. À and the works cited within. DeNiro, M. J., & Epstein, S. (1977). Mechanism of carbon isotope fractionation associated with lipid synthesis. Science, 197(4300), 261 263. À References Dingle, H. (2014). Migration: The biology of life on the move. USA: Oxford University Press. Amelung, W., & Zhang, X. (2001). Determination of amino Downs, E. E., Popp, B. N., & Holl, C. M. (2014). Nitrogen acid enantiomers in soils. Soil Biology and Biochemistry, isotope fractionation and amino acid turnover rates in 33(4), 553 562. the Pacific white shrimp Litopenaeus vannamei. Marine À Arthur, K. E., Kelez, S., Larsen, T., Choy, C. A., & Popp, Ecology Progress Series, 516, 239 250. À B. N. (2014). Tracing the biosynthetic source of essential Estep, M. F., & Hoering, T. C. (1980). Biogeochemistry of amino acids in marine turtles using δ13C fingerprints. the stable hydrogen isotopes. Geochimica et Ecology, 95(5), 1285 1293. Cosmochimica Acta, 44(8), 1197 1206. À À Ayayee, P. A., Jones, S. C., & Sabree, Z. L. (2015). Can 13C Fogel, M. L., Griffin, P. L., & Newsome, S. D. (2016). stable isotope analysis uncover essential amino acid Hydrogen isotopes in individual amino acids reflect provisioning by termite-associated gut microbes? PeerJ, differentiated pools of hydrogen from food and water 3, e1218. in Escherichia coli. Proceedings of the National Academy of Bowen, G. J. (2010). Isoscapes: Spatial pattern in isotopic Sciences, 113(32), E4648 E4653. À biogeochemistry. Annual Review of Earth and Planetary Fryxell, J. M., & Sinclair, A. R. E. (1988). Causes and conse- Sciences, 38, 161 187. quences of migration by large herbivores. Trends in À Bradley, C. J., Madigan, D. J., Block, B. A., & Popp, B. N. Ecology & Evolution, 3(9), 237 241. À (2014). Amino acid isotope incorporation and enrich- Gannes, L. Z., Martinez del Rio, C., & Koch, P. (1998). ment factors in Pacific bluefin tuna, Thunnus orientalis. Natural abundance variations in stable isotopes and PLoS One, 9(1), e85818. their potential uses in animal physiological ecology. Braun, A., Vikari, A., Windisch, W., & Auerswald, K. Comparative Biochemistry and Physiology Part A: (2014). Transamination governs nitrogen isotope hetero- Molecular & Integrative Physiology, 119(3), 725 737. À geneity of amino acids in rats. Journal of Agricultural and Gauchotte-Lindsay, C., & Turnbull, S. M. (2016). On-line Food Chemistry, 62(32), 8008 8013. high-precision carbon position-specific stable isotope À Broek, T. A., & McCarthy, M. D. (2014). A new approach analysis: A review. TrAC Trends in Analytical Chemistry, to δ15N compound-specific amino acid trophic position 76, 115 125. À measurements: Preparative high pressure liquid chro- Germain, L. R., Koch, P. L., Harvey, J., & McCarthy, M. D. matography technique for purifying underivatized (2013). Nitrogen isotope fractionation in amino acids amino acids for stable isotope analysis. Limnology and from harbor seals: Implications for compound-specific Oceanography: Methods, 12(12), 840 852. trophic position calculations. Marine Ecology Progress À Chikaraishi, Y., Kashiyama, Y., Ogawa, N. O., Kitazato, H., Series, 482, 265 277. À & Ohkouchi, N. (2007). Metabolic control of nitrogen Graham, B. S., Koch, P. L., Newsome, S. D., McMahon, isotope composition of amino acids in macroalgae and K. W., & Aurioles, D. (2010). Using isoscapes to trace the gastropods: Implications for aquatic food web studies. movements and foraging behavior of top predators in oceanic Marine Ecology Progress Series, 342, 85 90. ecosystems. Isoscapes (pp. 299 318). Netherlands: Springer. À À Chikaraishi, Y., Ogawa, N. O., Kashiyama, Y., Takano, Y., Gwinner, E. (Ed.), (2012). Bird migration: Physiology and eco- Suga, H., Tomitani, A., ... Ohkouchi, N. (2009). physiology. Berlin: SpringerScience&BusinessMedia. Determination of aquatic food-web structure based on Hare, P. E., Fogel, M. L., Stafford, T. W., Mitchell, A. D., & compound-specific nitrogen isotopic composition of Hoering, T. C. (1991). The isotopic composition of car- amino acids. Limnology and Oceanography: Methods, 7 bon and nitrogen in individual amino acids isolated (11), 740 750. from modern and fossil proteins. Journal of À Chikaraishi, Y., Steffan, S. A., Takano, Y., & Ohkouchi, N. Archaeological Science, 18(3), 277 292. À (2015). Diet quality influences isotopic discrimination Hayes, J. M. (2001). Fractionation of carbon and hydrogen among amino acids in an aquatic vertebrate. Ecology isotopes in biosynthetic processes. Reviews in and Evolution, 5(10), 2048 2059. Mineralogy and Geochemistry, 43(1), 225 277. À À Dale, J. J., Wallsgrove, N. J., Popp, B. N., & Holland, K. N. Hobson, K. A. (1999). Tracing origins and migration of (2011). Nursery habitat use and foraging ecology of the wildlife using stable isotopes: A review. Oecologia, 120 brown stingray Dasyatis lata determined from stomach (3), 314 326. À

TRACKING ANIMAL MIGRATION WITH STABLE ISOTOPES 188 7. AMINO ACID ISOTOPE ANALYSIS: A NEW FRONTIER IN STUDIES OF ANIMAL MIGRATION AND FORAGING ECOLOGY

Hobson, K. A., & Wassenaar, L. I. (2008). Tracking animal using amino acid stable isotope fingerprinting. PLoS migration with stable isotopes (Vol. 2). Academic Press. One, 8(9), e73441. Honch, N. V., McCullagh, J. S., & Hedges, R. E. (2012). Lorrain, A., Graham, B., Me´nard, F., Popp, B., Bouillon, S., Variation of bone collagen amino acid δ13C values in Van Breugel, P., & Cherel, Y. (2009). Nitrogen and car- archaeological humans and fauna with different dietary bon isotope values of individual amino acids: A tool to regimes: Developing frameworks of dietary discrimina- study foraging ecology of penguins in the Southern tion. American Journal of Physical Anthropology, 148(4), Ocean. Marine Ecology Progress Series, 391, 293 306. À 495 511. MacKenzie, K. M., Longmore, C., Preece, C., Lucas, C. H., À Hoskin, S. O., Gavet, S., Milne, E., & Lobley, G. E. (2001). & Trueman, C. N. (2014). Testing the long-term stability Does glutamine act as a substrate for transamination of marine isoscapes in shelf seas using jellyfish tissues. reactions in the liver of fed and fasted sheep? British Biogeochemistry, 121(2), 441 454. À Journal of Nutrition, 85(5), 591 597. Macko, S. A., Estep, M. L. F., Engel, M. H., & Hare, P. E. À Howland, M. R., Corr, L. T., Young, S. M., Jones, V., Jim, (1986). Kinetic fractionation of stable nitrogen isotopes S., Van Der Merwe, N. J., ... Evershed, R. P. (2003). during amino acid transamination. Geochimica et Expression of the dietary isotope signal in the com- Cosmochimica Acta, 50(10), 2143 2146. À pound-specific δ13C values of pig bone lipids and Magozzi, S., Yool, A., Vander Zanden, H. B., Wunder, amino acids. International Journal of Osteoarchaeology, 13 M. B., & Trueman, C. N. (2017). Using ocean models to (1 2), 54 65. predict spatial and temporal variation in marine carbon À À Hussey, N. E., Kessel, S. T., Aarestrup, K., Cooke, S. J., isotopes. Ecosphere, 8(5), e01763. Cowley, P. D., Fisk, A. T., ... Flemming, J. E. M. (2015). McCarthy, M. D., Lehman, J., & Kudela, R. (2013). Aquatic animal telemetry: A panoramic window into Compound-specific amino acid δ15N patterns in marine the underwater world. Science, 348(6240), 1255642. algae: Tracer potential for cyanobacterial vs. eukaryotic Jarman, C. L., Larsen, T., Hunt, T., Lipo, C., Solsvik, R., organic nitrogen sources in the ocean. Geochimica et Wallsgrove, N., ... Popp, B. N. (2017). Diet of the prehis- Cosmochimica Acta, 103, 104 120. À toric population of Rapa Nui (Easter Island, Chile) shows McClelland, J. W., & Montoya, J. P. (2002). Trophic rela- environmental adaptation and resilience. American tionships and the nitrogen isotopic composition of Journal of Physical Anthropology, 164(2), 343 361. amino acids in plankton. Ecology, 83(8), 2173 2180. À À Jennings, S., & Warr, K. J. (2003). Environmental correlates McCullagh, J. S., Juchelka, D., & Hedges, R. E. (2006). of large-scale spatial variation in the δ15N of marine Analysis of amino acid 13C abundance from human and animals. Marine Biology, 142(6), 1131 1140. faunal bone collagen using liquid chromatography/iso- À Johnson, M. L., & Gaines, M. S. (1990). Evolution of dis- tope ratio mass spectrometry. Rapid Communications in persal: Theoretical models and empirical tests using Mass Spectrometry, 20(18), 2761 2768. À birds and mammals. Annual Review of Ecology and McMahon, K. W., Berumen, M. L., Mateo, I., Elsdon, T. S., & Systematics, 21(1), 449 480. Thorrold, S. R. (2011). Carbon isotopes in otolith amino À Kanehisa, M., Furumichi, M., Tanabe, M., Sato, Y., & acids identify residency of juvenile snapper (Family: Morishima, K. (2017). KEGG: New perspectives on gen- Lutjanidae) in coastal nurseries. Coral Reefs, 30,1135 1145. À omes, pathways, diseases and drugs. Nucleic Acids McMahon, K. W., Berumen, M. L., & Thorrold, S. R. (2012). Research, 45(D1), D353 D361. Linking habitat mosaics and connectivity in a coral reef À Kays, R., Crofoot, M. C., Jetz, W., & Wikelski, M. (2015). seascape. Proceedings of the National Academy of Sciences, Terrestrial animal tracking as an eye on life and planet. 109(38), 15372 15376. À Science, 348(6240), aaa2478. McMahon, K. W., Fogel, M. L., Elsdon, T. S., & Thorrold, Langin, K. M., Reudink, M. W., Marra, P. P., Norris, D. R., S. R. (2010). Carbon isotope fractionation of amino acids Kyser, T. K., & Ratcliffe, L. M. (2007). Hydrogen isoto- in fish muscle reflects biosynthesis and isotopic routing pic variation in migratory bird tissues of known origin: from dietary protein. Journal of Animal Ecology, 79(5), Implications for geographic assignment. Oecologia, 152 1132 1141. À (3), 449 457. McMahon, K. W., Fogel, M. L., Johnson, B. J., Houghton, À Larsen, T., Taylor, D. L., Leigh, M. B., & O’Brien, D. M. L. A., & Thorrold, S. R. (2011). A new method to recon- (2009). Stable isotope fingerprinting: A novel method struct fish diet and movement patterns from δ13C for identifying plant, fungal, or bacterial origins of values in otolith amino acids. Canadian Journal of amino acids. Ecology, 90(12), 3526 3535. Fisheries and Aquatic Sciences, 68, 1330 1340. À À Larsen, T., Ventura, M., Andersen, N., O’Brien, D. M., McMahon, K. W., Hamady, L. L., & Thorrold, S. R. (2013a). Piatkowski, U., & McCarthy, M. D. (2013). Tracing car- Ocean ecogeochemistry: A review. Oceanography and bon sources through aquatic and terrestrial food webs Marine Biology: An Annual Review, 51, 327 374. À

TRACKING ANIMAL MIGRATION WITH STABLE ISOTOPES REFERENCES 189

McMahon, K. W., Hamady, L. L., & Thorrold, S. R. (2013b). Newsome, S. D., Wolf, N., Peters, J., & Fogel, M. L. (2014). A review of ecogeochemistry approaches to estimating Amino acid δ13C analysis shows flexibility in the rout- movements of marine animals. Limnology and ing of dietary protein and lipids to the tissue of an Oceanography, 58(2), 697 714. omnivore. Integrative and Comparative Biology, 54(5), À McMahon, K. W., & McCarthy, M. D. (2016). Embracing 890 902. À variability in amino acid δ15N fractionation: O’Brien, D. M., Fogel, M. L., & Boggs, C. L. (2002). Mechanisms, implications, and applications for trophic Renewable and nonrenewable resources: Amino acid ecology. Ecosphere, 7, e01511. turnover and allocation to reproduction in Lepidoptera. McMahon, K. W., McCarthy, M. D., Sherwood, O. A., Proceedings of the National Academy of Sciences, 99(7), Larsen, T., & Guilderson, T. P. (2015). Millennial-scale 4413 4418. À plankton regime shifts in the subtropical North Pacific O’Connell, T. C. (2017). ‘Trophic’ and ‘source’ amino acids Ocean. Science, 350(6267), 1530 1533. in trophic estimation: A likely metabolic explanation. À McMahon, K. W., Polito, M. J., Abel, S., McCarthy, M. D., Oecologia, 184(2), 317 326. À & Thorrold, S. R. (2015). Carbon and nitrogen isotope O’Connell, T. C., & Collins, M. J. (2017). Comment on fractionation of amino acids in an avian marine preda- “Ecological niche of Neanderthals from Spy Cave tor, the gentoo penguin (Pygoscelis papua). Ecology and revealed by nitrogen isotopes of individual amino acids Evolution, 5(6), 1278 1290. in collagen”. Journal of Human Evolution. Available from À McMahon, K. W., Thorrold, S. R., Elsdon, T. S., & https://doi.org/10.1016/j.jhevol.2017.05.006. McCarthy, M. D. (2015). Trophic discrimination of Ohkouchi, N., Chikaraishi, Y., Close, H. G., Fry, B., Larsen, nitrogen stable isotopes in amino acids varies with diet T., Madigan, D. J., ... Ogawa, N. O. (2017). Advances quality in a marine fish. Limnology and Oceanography, 60 in the application of amino acid nitrogen isotopic anal- (3), 1076 1087. ysis in ecological and biogeochemical studies. Organic À McMahon, K. W., Thorrold, S. R., Houghton, L. A., & Geochemistry, 113, 150 174. À Berumen, M. L. (2016). Tracing carbon flow through Phillips, D. L., & Eldridge, P. M. (2006). Estimating the tim- coral reef food webs using a compound-specific ing of diet shifts using stable isotopes. Oecologia, 147(2), stable isotope approach. Oecologia, 180(3), 809 821. 195 203. À À Metges, C. C. (2000). Contribution of microbial amino acids Polito, M. J., Hinke, J. T., Hart, T., Santos, M., Houghton, to amino acid homeostasis of the host. The Journal of L. A., & Thorrold, S. R. (2017). Stable isotope analyses Nutrition, 130(7), S1857 S1864. of feather amino acids identify penguin migration strat- À Naito, Y. I., Honch, N. V., Chikaraishi, Y., Ohkouchi, N., & egies at ocean basin scales. Biology Letters, 13(8), Yoneda, M. (2010). Quantitative evaluation of marine 20170241. protein contribution in ancient diets based on nitrogen Popp, B. N., Graham, B. S., Olson, R. J., Hannides, C. C., isotope ratios of individual amino acids in bone colla- Lott, M. J., Lo´pez-Ibarra, G. A., ... Fry, B. (2007). gen: An investigation at the Kitakogane Jomon site. Insight into the trophic ecology of yellowfin tuna, American Journal of Physical Anthropology, 143(1), 31 40. Thunnus albacares, from compound-specific nitrogen iso- À Nakashita, R., Suzuki, Y., Akamatsu, F., Naito, Y. I., Sato- tope analysis of proteinaceous amino acids. Terrestrial Hashimoto, M., & Tsubota, T. (2011). Ecological Ecology, 1, 173 190. À application of compound-specific stable nitrogen isotope Post, D. M. (2002). Using stable isotopes to estimate trophic analysis of amino acids: A case study of captive and position: Models, methods, and assumptions. Ecology, wild bears. Researches in Organic Geochemistry, 27,73 79. 83(3), 703 718. À À Newsome, S. D., Bentall, G. B., Tinker, M. T., Oftedal, Ruiz-Cooley, R. I., Ballance, L. T., & McCarthy, M. D. O. T., Ralls, K., Estes, J. A., & Fogel, M. L. (2010). (2013). Range expansion of the jumbo squid in the NE Variation in δ13C and δ15N diet vibrissae trophic dis- Pacific: δ15N decrypts multiple origins, migration and À crimination factors in a wild population of California habitat use. PLoS One, 8(3), e59651. sea otters. Ecological Applications, 20(6), 1744 1752. Ruiz-Cooley, R. I., Koch, P. L., Fiedler, P. C., & McCarthy, À Newsome, S. D., Fogel, M. L., Kelly, L., & Martinez del M. D. (2014). Carbon and nitrogen isotopes from top Rio, C. (2011). Contributions of direct incorporation predator amino acids reveal rapidly shifting ocean bio- from diet and microbial amino acids to protein synthe- chemistry in the outer California current. PLoS One, 9 sis in Nile tilapia. Functional Ecology, 25(5), 1051 1062. (10), e110355. À Newsome, S. D., Wolf, N., Bradley, C. J., & Fogel, M. L. Schell, D. M., Barnett, B. A., & Vinette, K. A. (1998). (2017). Assimilation and isotopic discrimination of Carbon and nitrogen isotope ratios in zooplankton of hydrogen in tilapia: Implications for studying animal the Bering, Chukchi and Beaufort seas. Marine Ecology diet with δ2H. Ecosphere, 8(1), e01616. Progress Series, 162, 11 23. À

TRACKING ANIMAL MIGRATION WITH STABLE ISOTOPES 190 7. AMINO ACID ISOTOPE ANALYSIS: A NEW FRONTIER IN STUDIES OF ANIMAL MIGRATION AND FORAGING ECOLOGY

Schwarcz, H. P. (1991). Some theoretical aspects of isotope Vander Zanden, H. B., Arthur, K. E., Bolten, A. B., Popp, paleodiet studies. Journal of Archaeological Science, 18(3), B. N., Lagueux, C. J., Harrison, E., ... Bjorndal, K. A. 261 275. (2013). Trophic ecology of a green turtle breeding popu- À Scott, J. H., O’Brien, D. M., Emerson, D., Sun, H., lation. Marine Ecology Progress Series, 476, 237 249. À McDonald, G. D., Salgado, A., & Fogel, M. L. (2006). Vokhshoori, N. L., & McCarthy, M. D. (2014). Compound- An examination of the carbon isotope effects associated specific δ15N amino acid measurements in littoral mus- with amino acid biosynthesis. Astrobiology, 6(6), sels in the California upwelling ecosystem: A new 867 880. approach to generating baseline δ15N isoscapes for À Seminoff, J. A., Benson, S. R., Arthur, K. E., Eguchi, T., coastal ecosystems. PLoS One, 9(6), e98087. Dutton, P. H., Tapilatu, R. F., & Popp, B. N. (2012). Walsh, R. G., He, S., & Yarnes, C. T. (2014). Compound- Stable isotope tracking of endangered sea turtles: specific δ13C and δ15N analysis of amino acids: A rapid, Validation with satellite telemetry and δ15N analysis of chloroformate-based method for ecological studies. Rapid amino acids. PLoS One, 7(5), e37403. Communications in Mass Spectrometry, 28(1), 96 108. À Silfer, J. A., Engel, M. H., Macko, S. A., & Jumeau, E. J. Wolf, N., Newsome, S. D., Fogel, M. L., & Martinez del (1991). Stable carbon isotope analysis of amino acid Rio, C. (2013). The relationship between drinking water enantiomers by conventional isotope ratio mass spec- and the hydrogen and oxygen stable isotope values of trometry and combined gas chromatography/isotope tissues in Japanese Quail (Cortunix japonica). The Auk, ratio mass spectrometry. Analytical Chemistry, 63(4), 130(2), 323 330. À 370 374. Wolf, N., Newsome, S. D., Peters, J., & Fogel, M. L. (2015). À Steffan, S. A., Chikaraishi, Y., Currie, C. R., Horn, H., Variability in the routing of dietary proteins and lipids Gaines-Day, H. R., Pauli, J. N., ... Ohkouchi, N. to consumer tissues influences tissue-specific isotopic (2015). Microbes are trophic analogs of animals. discrimination. Rapid Communications in Mass Proceedings of the National Academy of Sciences, 112(49), Spectrometry, 29(15), 1448 1456. À 15119 15124. Wu, G. (2009). Amino acids: Metabolism, functions, and À Steffan, S. A., Chikaraishi, Y., Horton, D. R., Ohkouchi, N., nutrition. Amino Acids, 37(1), 1 17. À Singleton, M. E., Miliczky, E., ... Jones, V. P. (2013). Wu, G., Bazer, F. W., Dai, Z., Li, D., Wang, J., & Wu, Z. Trophic hierarchies illuminated via amino acid isotopic (2014). Amino acid nutrition in animals: Protein synthe- analysis. PLoS One, 8(9), e76152. sis and beyond. Annual Review of Animal Biosciences, 2 Torrallardona, D., Harris, C. I., Coates, M. E., & Fuller, (1), 387 417. À M. F. (1996). Microbial amino acid synthesis and utili- Wunder, M. B., & Norris, D. R. (2008). Analysis and design 15 15 zation in rats: Incorporation of N from NH4Cl into for isotope-based studies of migratory animals. lysine in the tissues of germ-free and conventional rats. Terrestrial Ecology, 2, 107 128. À British Journal of Nutrition, 76(5), 689 700. Yamaguchi, Y. T., Chikaraishi, Y., Takano, Y., Ogawa, À Trueman, C. N., MacKenzie, K. M., & Palmer, M. R. (2012). N. O., Imachi, H., Yokoyama, Y., & Ohkouchi, N. Identifying migrations in marine fishes through stable- (2017). Fractionation of nitrogen isotopes during amino isotope analysis. Journal of Fish Biology, 81(2), 826 847. acid metabolism in heterotrophic and chemolithoauto- À Vanderklift, M. A., & Ponsard, S. (2003). Sources of trophic microbes across Eukarya, Bacteria, and variation in consumer-diet δ15N enrichment: A meta- Archaea: Effects of nitrogen sources and metabolic analysis. Oecologia, 136(2), 169 182. pathways. Organic Geochemistry, 111, 101 112. À À

TRACKING ANIMAL MIGRATION WITH STABLE ISOTOPES