LAUSR

Compound-specific nitrogen isotope (δN) analysis of amino acids is a powerful tool for estimating the trophic positions (TPs) of animals. The TP of an animal can be represented as a linear function of the isotopic difference between glutamic acid (δNGlu) and phenylalanine (δ NPhe). However, the method using δ NGlu and δNPhe cannot be applied to animals in mixed food webs where basal resources are derived from both terrestrial and aquatic primary producers, because the mean value of δNPhe relative to δ NGlu differs greatly between terrestrial plants (+8.4‰) and aquatic algae (−3.4‰). To resolve this problem, the δN of methionine (δNMet) is useful. Because the C–N bond of methionine is not cleaved in its initial metabolic step, theoretically there should be little diversity in δNMet relative to δ NGlu among primary producers and a small trophic discrimination factor for methionine in animal metabolism. We developed a dual-column-coupled GC-C-IRMS method to determine δNMet. Data collected from controlled feeding experiments and wild samples demonstrated that the isotopic difference between methionine and phenylalanine in terrestrial food webs (ΔMet−Phe = −16.5 0.5‰) is clearly distinguishable from that in aquatic food webs (ΔMet−Phe = −5.0 0.5‰). This approach allowed us to determine ecologically reasonable TP values for carnivores in a stream food web, which were substantially underestimated with the conventional method. This method has potential utility in assessing TP for animals that rely on varying proportions of both terrestrialand aquatic-derived resources, with no requirement to characterize δN in their basal resources. The trophic positions (TPs) of organisms have been increasingly estimated using compound-specific isotope analysis (CSIA) of nitrogen (δN) in amino acids (AAs) (e.g., McClelland and Montoya 2002; Chikaraishi et al. 2007; McCarthy et al. 2007; Popp et al. 2007; Ogawa et al. 2013; Steffan et al. 2013, 2015). The first step in animal catabolism of “trophic” amino acids such as glutamic acid (Glu) is commonly deamination (a process of transamination), which leads to the enrichment of N at each trophic step. The extent of this isotopic fractionation is called the “trophic discrimination factor”, hereafter referred to as TDF (e.g., 8.0 1.2‰ for TDFGlu; Chikaraishi et al. 2009). In contrast, the dominant animal catabolism of “source” amino acids such as phenylalanine (Phe) does not start with deamination, resulting in an unchanged TDF (e.g., 0.4 0.5‰ for TDFPhe; Chikaraishi et al. 2009). Unlike traditional δN analyses, in which the bulk tissues of organisms are used (e.g., Minagawa and Wada 1984; Peterson and Fry 1987; Vander Zanden et al. 1999), the TP of an animal can be estimated with the following equation, without any characterization of δN in the basal resources of the food webs studied: TPGlu=Phe 1⁄4 δNGlu – δNPhe + βGlu=Phe TDFGlu –TDFPhe + 1 ð1Þ *Correspondence: [email protected] or ishikawan@jamstec. go.jp This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.