Leopard seal diets in a rapidly warming polar region vary by year, season, sex, and body size.

BACKGROUND: Resolving the preferred prey items and dietary proportions of leopard seals is central to understanding food-web dynamics in the rapidly-warming Antarctic Peninsula region. Previous studies have identified a wide range of prey items; however, due to anecdotal or otherwise limited information, leopard seal diets remain unresolved by seal sex, individual, body size, region, and season. Over the 2013, 2014, and 2017 field seasons we collected scat, tissue samples (red blood cells and plasma; n = 23) for stable isotope analyses, and previously-reported animal-borne video from 19 adult leopard seals foraging near mesopredator breeding colonies at Cape Shirreff, Livingston Island. We summarized a priori diet information from scat and video analysis and applied a three-isotope (δ13C, δ15N, δ34S), four-source (fish, fur seal, krill, penguin) Bayesian mixing model to examine temporal variability in both prey sources and leopard seal tissues. RESULTS: The austral spring diets of males and females focused on Antarctic krill (31.7-38.0%), notothen fish (31.6-36.5%), and penguin (24.4-26.9%) and were consistent across all 3 years. Several lines of evidence suggest the transition to summer foraging was distinct for males and females. Female diets transitioned rapidly to higher δ15N values (+2.1‰), indicating increased consumption of penguin (29.5-46.2%) and energy-dense Antarctic fur seal pup (21.3-37.6%). CONCLUSIONS: The seasonal increase in leopard seal δ15N values, and thus fur seal in their diet, was predictably related to larger body size; it may also be forcing reductions to the largest Antarctic fur seal colony in the Antarctic Peninsula. Our ensemble sampling approach reduces historical biases in monitoring marine apex predator diets. Further, our results are necessary to best inform regional fisheries management planning.

Effects of chemical preservation on bulk and amino acid isotope ratios of zooplankton, fish, and squid tissues.

RATIONALE: It is imperative to understand how chemical preservation alters tissue isotopic compositions before using historical samples in ecological studies. Specifically, although compound-specific isotope analysis of amino acids (CSIA-AA) is becoming a widely used tool, there is little information on how preservation techniques affect amino acid δ15 N values. METHODS: We evaluated the effects of chemical preservatives on bulk tissue δ13 C and δ15 N and amino acid δ15 N values, measured by gas chromatography/isotope ratio mass spectrometry (GC/IRMS), of (a) tuna (Thunnus albacares) and squid (Dosidicus gigas) muscle tissues that were fixed in formaldehyde and stored in ethanol for 2 years and (b) two copepod species, Calanus pacificus and Eucalanus californicus, which were preserved in formaldehyde for 24-25 years. RESULTS: Tissues in formaldehyde-ethanol had higher bulk δ15 N values (+1.4, D. gigas; +1.6‰, T. albacares), higher δ13 C values for D. gigas (+0.5‰), and lower δ13 C values for T. albacares (-0.8‰) than frozen samples. The bulk δ15 N values from copepods were not different those from frozen samples, although the δ13 C values from both species were lower (-1.0‰ for E. californicus and -2.2‰ for C. pacificus) than those from frozen samples. The mean amino acid δ15 N values from chemically preserved tissues were largely within 1‰ of those of frozen tissues, but the phenylalanine δ15 N values were altered to a larger extent (range: 0.5-4.5‰). CONCLUSIONS: The effects of preservation on bulk δ13 C values were variable, where the direction and magnitude of change varied among taxa. The changes in bulk δ15 N values associated with chemical preservation were mostly minimal, suggesting that storage in formaldehyde or ethanol will not affect the interpretation of δ15 N values used in ecological studies. The preservation effects on amino acid δ15 N values were also mostly minimal, mirroring bulk δ15 N trends, which is promising for future CSIA-AA studies of archived specimens. However, there were substantial differences in phenylalanine and valine δ15 N values, which we speculate resulted from interference in the chromatographic resolution of unknown compounds rather than alteration of tissue isotopic composition due to chemical preservation.

Marine subsidies likely cause gigantism of iguanas in the Bahamas.

We utilized natural experiment opportunities presented by differential conditions (presence/absence of seabirds and invasive species) on cays in the Bahamas to study whether interisland variations in food resources contributed to gigantism in Allen Cays Rock Iguanas (Cyclura cychlura inornata). We analyzed the stable carbon (δ13C) and nitrogen (δ15N) isotope values from iguana tissues and resources from each island food web to test the predictions that (1) food webs on islands with seabirds exhibit the influence of marine subsidies from seabird guano, whereas those from non-seabird islands do not, and (2) size differences in iguanas among cays were due to either (a) supplemental food availability from mice and/or seabird carcasses killed by barn owls (Tyto alba) and/or (b) access to more nutrient-rich vegetation fertilized by seabird guano. Food web components from the seabird island (Allen Cay) had 5-9‰ higher δ15N values than those on the other cays and Allen Cay plants contained nearly two times more nitrogen. Bayesian stable isotope mixing models indicated that C3 plants dominated iguana diets on all islands and showed no evidence for consumption of mice or shearwaters. The iguanas on Allen Cay were ~ 2 times longer (48.3 ± 11.6 cm) and ~ 6 times heavier (5499 ± 2847 g) than iguanas on other cays and this was likely from marine-derived subsidies from seabird guano which caused an increase in nitrogen concentration in the plants and a resultant increase in the δ15N values across the entire food web relative to non-seabird islands.

Long-term trends in the foraging ecology and habitat use of an endangered species: an isotopic perspective.

Evaluating long-term drivers of foraging ecology and population productivity is crucial for providing ecological baselines and forecasting species responses to future environmental conditions. Here, we examine the trophic ecology and habitat use of North Atlantic leatherback turtles (St. Croix nesting population) and investigate the effects of large-scale oceanographic conditions on leatherback foraging dynamics. We used bulk and compound-specific nitrogen isotope analysis of amino acids (CSIA-AA) to estimate leatherback trophic position (TP) over an 18-year period, compare these estimates with TP estimates from a Pacific leatherback population, and elucidate the pre-nesting habitat use patterns of leatherbacks. Our secondary objective was to use oceanographic indices and nesting information from St. Croix leatherbacks to evaluate relationships between trophic ecology, nesting parameters, and regional environmental conditions measured by the North Atlantic Oscillation (NAO) and Atlantic Multidecadal Oscillation. We found no change in leatherback TP over time and no difference in TP between Atlantic and Pacific leatherbacks, indicating that differences in trophic ecology between populations are an unlikely driver of the population dichotomy between Pacific and Atlantic leatherbacks. Isotope data suggested that St. Croix leatherbacks inhabit multiple oceanic regions prior to nesting, although, like their conspecifics in the Pacific, individuals exhibit fidelity to specific foraging regions. Leatherback nesting parameters were weakly related to the NAO, which may suggest that positive NAO phases benefit St. Croix leatherbacks, potentially through increases in resource availability in their foraging areas. Our data contribute to the understanding of leatherback turtle ecology and potential mechanistic drivers of the dichotomy between populations of this protected species.

Intrapopulation variability in the timing of ontogenetic habitat shifts in sea turtles revealed using δ15 N values from bone growth rings.

Determining location and timing of ontogenetic shifts in the habitat use of highly migratory species, along with possible intrapopulation variation in these shifts, is essential for understanding mechanisms driving alternate life histories and assessing overall population trends. Measuring variations in multi-year habitat-use patterns is especially difficult for remote oceanic species. To investigate the potential for differential habitat use among migratory marine vertebrates, we measured the naturally occurring stable nitrogen isotope (δ15 N) patterns that differentiate distinct ocean regions to create a 'regional isotope characterization', analysed the δ15 N values from annual bone growth layer rings from dead-stranded animals, and then combined the bone and regional isotope data to track individual animal movement patterns over multiple years. We used humeri from juvenile North Pacific loggerhead turtles (Caretta caretta), animals that undergo long migrations across the North Pacific Ocean (NPO), using multiple discrete regions as they develop to adulthood. Typical of many migratory marine species, ontogenetic changes in habitat use throughout their decades-long juvenile stage is poorly understood, but each potential habitat has unique foraging opportunities and spatially explicit natural and anthropogenic threats that could affect key life-history parameters. We found a bimodal size/age distribution in the timing that juveniles underwent an ontogenetic habitat shift from the oceanic central North Pacific (CNP) to the neritic east Pacific region near the Baja California Peninsula (BCP) (42·7 ± 7·2 vs. 68·3 ± 3·4 cm carapace length, 7·5 ± 2·7 vs. 15·6 ± 1·7 years). Important to the survival of this population, these disparate habitats differ considerably in their food availability, energy requirements and threats, and these differences can influence life-history parameters such as growth, survival and future fecundity. This is the first evidence of alternative ontogenetic shifts and habitat-use patterns for juveniles foraging in the eastern NPO. We combine two techniques, skeletochronology and stable isotope analysis, to reconstruct multi-year habitat-use patterns of a remote migratory species, linked to estimated ages and body sizes of individuals, to reveal variable ontogeny during the juvenile life stage that could drive alternate life histories and that has the potential to illuminate the migration patterns for other species with accretionary tissues.

Stable isotope discrimination factors and between-tissue isotope comparisons for bone and skin from captive and wild green sea turtles (Chelonia mydas).

RATIONALE: The ecological application of stable isotope analysis (SIA) relies on taxa- and tissue-specific stable carbon (Δ13 C) and nitrogen (Δ15 N) isotope discrimination factors, determined with captive animals reared on known diets for sufficient time to reflect dietary isotope ratios. However, captive studies often prohibit lethal sampling, are difficult with endangered species, and reflect conditions not experienced in the wild. METHODS: We overcame these constraints and determined the Δ13 C and Δ15 N values for skin and cortical bone from green sea turtles (Chelonia mydas) that died in captivity and evaluated the utility of a mathematical approach to predict discrimination factors. Using stable carbon (δ13 C values) and nitrogen (δ15 N values) isotope ratios from captive and wild turtles, we established relationships between bone stable isotope (SI) ratios and those from skin, a non-lethally sampled tissue, to facilitate comparisons of SI ratios among studies using multiple tissues. RESULTS: The mean (±SD) Δ13 C and Δ15 N values (‰) between skin and bone from captive turtles and their diet (non-lipid-extracted) were 2.3 ± 0.3 and 4.1 ± 0.4 and 2.1 ± 0.6 and 5.1 ± 1.1, respectively. The mathematically predicted Δ13 C and Δ15 N values were similar (to within 1‰) to the experimentally derived values. The mean δ15 N values from bone were higher than those from skin for captive (+1.0 ± 0.9‰) and wild (+0.8 ± 1.0‰) turtles; the mean δ13 C values from bone were lower than those from skin for wild turtles (-0.6 ± 0.9‰), but the same as for captive turtles. We used linear regression equations to describe bone vs skin relationships and create bone-to-skin isotope conversion equations. CONCLUSIONS: For sea turtles, we provide the first (a) bone-diet SI discrimination factors, (b) comparison of SI ratios from individual-specific bone and skin, and (c) evaluation of the application of a mathematical approach to predict stable isotope discrimination factors. Our approach opens the door for future studies comparing different tissues, and relating SI ratios of captive to wild animals.

Diet-tissue stable isotope (Δ(13)C and Δ(15)N) discrimination factors for multiple tissues from terrestrial reptiles.

RATIONALE: Stable isotope analysis is a powerful tool for reconstructing trophic interactions to better understand drivers of community ecology. Taxon-specific stable isotope discrimination factors contribute to the best use of this tool. We determined the first Δ(13)C and Δ(15)N values for Rock Iguanas (Cyclura spp.) to better understand isotopic fractionation and estimate wild reptile foraging ecology. METHODS: The Δ(13)C and Δ(15)N values between diet and skin, blood, and scat were determined from juvenile and adult iguanas held for 1 year on a known diet. We measured relationships between iguana discrimination factors and size/age and quantified effects of lipid extraction and acid treatment on stable isotope values from iguana tissues. Isotopic and elemental compositions were determined by Dumas combustion using an elemental analyzer coupled to an isotope ratio mass spectrometer using standards of known composition. RESULTS: The Δ(13)C and Δ(15)N values ranged from -2.5 to +6.5‰ and +2.2 to +7.5‰, respectively, with some differences among tissues and between juveniles and adults. The Δ(13)C values from blood and skin differed among species, but not the Δ(15)N values. The Δ(13)C values from blood and skin and Δ(15)N values from blood were positively correlated with size/age. The Δ(13)C values from scat were negatively correlated with size (not age). Treatment with HCl (scat) and lipid extraction (skin) did not affect the isotope values. CONCLUSIONS: These results should aid in the understanding of processes driving stable carbon and nitrogen isotope discrimination factors in reptiles. We provide estimates of Δ(13)C and Δ(15)N values and linear relationships between iguana size/age and discrimination factors for the best interpretation of wild reptile foraging ecology.

Terrestrial Scavenging of Marine Mammals: Cross-Ecosystem Contaminant Transfer and Potential Risks to Endangered California Condors (Gymnogyps californianus).

The critically endangered California condor (Gymnogyps californianus) has relied intermittently on dead-stranded marine mammals since the Pleistocene, and this food source is considered important for their current recovery. However, contemporary marine mammals contain persistent organic pollutants that could threaten condor health. We used stable carbon and nitrogen isotope, contaminant, and behavioral data in coastal versus noncoastal condors to quantify contaminant transfer from marine mammals and created simulation models to predict the risk of reproductive impairment for condors from exposure to DDE (p,p'-DDE), a major metabolite of the chlorinated pesticide DDT. Coastal condors had higher whole blood isotope values and mean concentrations of contaminants associated with marine mammals, including mercury (whole blood), sum chlorinated pesticides (comprised of ∼95% DDE) (plasma), sum polychlorinated biphenyls (PCBs) (plasma), and sum polybrominated diphenyl ethers (PBDEs) (plasma), 12-100-fold greater than those of noncoastal condors. The mean plasma DDE concentration for coastal condors was 500 ± 670 (standard deviation) (n = 22) versus 24 ± 24 (standard deviation) (n = 8) ng/g of wet weight for noncoastal condors, and simulations predicted ∼40% of breeding-age coastal condors have DDE levels associated with eggshell thinning in other avian species. Our analyses demonstrate potentially harmful levels of marine contaminant transfer to California condors, which could hinder the recovery of this terrestrial species.

High-Arctic butterflies become smaller with rising temperatures.

The response of body size to increasing temperature constitutes a universal response to climate change that could strongly affect terrestrial ectotherms, but the magnitude and direction of such responses remain unknown in most species. The metabolic cost of increased temperature could reduce body size but long growing seasons could also increase body size as was recently shown in an Arctic spider species. Here, we present the longest known time series on body size variation in two High-Arctic butterfly species: Boloria chariclea and Colias hecla. We measured wing length of nearly 4500 individuals collected annually between 1996 and 2013 from Zackenberg, Greenland and found that wing length significantly decreased at a similar rate in both species in response to warmer summers. Body size is strongly related to dispersal capacity and fecundity and our results suggest that these Arctic species could face severe challenges in response to ongoing rapid climate change.

Effects of demineralization on the stable isotope analysis of bone samples.

RATIONALE: The sampling of sequential, annually formed bone growth layers for stable carbon (δ(13)C values) and nitrogen (δ(15)N values) isotope analysis (SIA) can provide a time series of foraging ecology data. To date, no standard protocol exists for the pre-SIA treatment of cortical samples taken from fresh, modern, bones. METHODS: Based on the SIA of historical bone, it is assumed that fresh bone samples must be pre-treated with acid prior to SIA. Using an elemental analyzer coupled to an isotope ratio mass spectrometer to measure stable carbon and nitrogen ratios, we tested the need to acidify cortical bone powder with 0.25 M HCl prior to SIA to isolate bone collagen for the determination of δ(13)C and δ(15)N values. We also examined the need for lipid extraction to remove potential biases related to δ(13)C analysis, based on a C:N ratio threshold of 3.5. RESULTS: It was found that acidification of micromilled cortical bone samples from marine turtles does not affect their δ(15)N values, and the small effect acidification has on δ(13)C values can be mathematically corrected for, thus eliminating the need for pre-SIA acidification of cortical bone. The lipid content of the cortical bone samples was low, as measured by their C:N ratios, indicating that lipid extracting cortical bone samples from modern marine turtles is unnecessary. CONCLUSIONS: We present a standard protocol for testing fresh, modern cortical bone samples prior to SIA, facilitating direct comparison of future studies. Based on the results obtained from marine turtle bones, pre-acidification and lipid removal of cortical bone are not recommended. This is especially useful as there is frequently not enough bone material removed via micromilling of sequential growth layers to accommodate both acid treatment and SIA.

Age and residency duration of loggerhead turtles at a North Pacific bycatch hotspot using skeletochronology.

For migratory marine animals, like sea turtles, effective conservation can be challenging because key demographic information such as duration of life stages and exposure to spatially explicit threats in different habitats are often unknown. In the eastern Pacific near the Baja California Peninsula (BCP), Mexico, tens of thousands of endangered North Pacific loggerhead sea turtles (Caretta caretta) concentrate at a foraging area known to have high rates of fishery bycatch. Because stage survivorship of loggerheads in the BCP will vary significantly depending on the number of years spent in this region, we applied skeletochronology to empirically estimate residency duration in this loggerhead hotspot. The observed age distribution obtained from skeletochronology analysis of 146 dead-stranded loggerheads ranged from three to 24 years old, suggesting a BCP residency of >20 years. Given the maximum estimated age and a one-year migration to western Pacific nesting beaches, we infer age-at-maturation for BCP loggerheads at ~25 years old. We also examine survivorship at varying BCP residency durations by applying our findings to current annual mortality estimates. Predicted survivorship of loggerheads spending over 20 years in this BCP foraging habitat is less than 10%, and given that ~43,000 loggerhead turtles forage here, a significant number of turtles are at extreme risk in this region. This is the first empirical evidence supporting estimated age-at-maturation for BCP North Pacific loggerheads, and the first estimates of BCP stage survivorship. Our findings emphasize the urgent need for continued and effective international conservation efforts to minimize bycatch of this endangered species.

Introduced rats indirectly change marine rocky intertidal communities from algae- to invertebrate-dominated.

It is widely recognized that trophic interactions structure ecological communities, but their effects are usually only demonstrated on a small scale. As a result, landscape-level documentations of trophic cascades that alter entire communities are scarce. Islands invaded by animals provide natural experiment opportunities both to measure general trophic effects across large spatial scales and to determine the trophic roles of invasive species within native ecosystems. Studies addressing the trophic interactions of invasive species most often focus on their direct effects. To investigate both the presence of a landscape-level trophic cascade and the direct and indirect effects of an invasive species, we examined the impacts of Norway rats (Rattus norvegicus) introduced to the Aleutian Islands on marine bird densities and marine rocky intertidal community structures through surveys conducted on invaded and rat-free islands throughout the entire 1,900-km archipelago. Densities of birds that forage in the intertidal were higher on islands without rats. Marine intertidal invertebrates were more abundant on islands with rats, whereas fleshy algal cover was reduced. Our results demonstrate that invasive rats directly reduce bird densities through predation and significantly affect invertebrate and marine algal abundance in the rocky intertidal indirectly via a cross-community trophic cascade, unexpectedly changing the intertidal community structure from an algae- to an invertebrate-dominated system.

Indirect effects of invasive rat removal result in recovery of island rocky intertidal community structure.

Eleven years after invasive Norway rats (Rattus norvegicus) were eradicated from Hawadax Island, in the Aleutian Islands, Alaska, the predicted three-level trophic cascade in the rocky intertidal, with native shorebirds as the apex predator, returned, leading to a community resembling those on rat-free islands with significant decreases in invertebrate species abundances and increases in fleshy algal cover. Rats had indirectly structured the intertidal community via their role as the apex predator in a four-level trophic cascade. Our results are an excellent example of an achievable and relatively short-term community-level recovery following removal of invasive animals. These conservation successes are especially important for islands as their disproportionately high levels of native biodiversity are excessively threatened by invasive mammals.

Selecting the best stable isotope mixing model to estimate grizzly bear diets in the Greater Yellowstone Ecosystem.

Past research indicates that whitebark pine seeds are a critical food source for Threatened grizzly bears (Ursus arctos) in the Greater Yellowstone Ecosystem (GYE). In recent decades, whitebark pine forests have declined markedly due to pine beetle infestation, invasive blister rust, and landscape-level fires. To date, no study has reliably estimated the contribution of whitebark pine seeds to the diets of grizzlies through time. We used stable isotope ratios (expressed as δ13C, δ15N, and δ34S values) measured in grizzly bear hair and their major food sources to estimate the diets of grizzlies sampled in Cooke City Basin, Montana. We found that stable isotope mixing models that included different combinations of stable isotope values for bears and their foods generated similar proportional dietary contributions. Estimates generated by our top model suggest that whitebark pine seeds (35±10%) and other plant foods (56±10%) were more important than meat (9±8%) to grizzly bears sampled in the study area. Stable isotope values measured in bear hair collected elsewhere in the GYE and North America support our conclusions about plant-based foraging. We recommend that researchers consider model selection when estimating the diets of animals using stable isotope mixing models. We also urge researchers to use the new statistical framework described here to estimate the dietary responses of grizzlies to declines in whitebark pine seeds and other important food sources through time in the GYE (e.g., cutthroat trout), as such information could be useful in predicting how the population will adapt to future environmental change.

Stable isotope assessment of temporal and geographic differences in feeding ecology of northern fur seals (Callorhinus ursinus) and their prey.

We investigated the feeding ecology and foraging location of migrating and nursing northern fur seal (Callorhinus ursinus) adult females and migrating juvenile males from the Pribilof Islands, Alaska, using carbon (δ13C) and nitrogen (δ15N) isotope analysis of fur seal skin and whole potential prey. Post-parturient and lactating females had mean δ15N values significantly (0.8‰) higher than pregnant, migratory females, and δ13C values that were not significantly different. Two opportunistically collected, migrating, nulliparous females had mean δ13C values 1.1‰ lower than migrating, pregnant females, and δ15N values that were not different. Pregnant, migratory females had mean δ13C values significantly (~1.5‰) higher than migratory juvenile males, and mean δ15N values significantly (~0.6-1.6‰) higher than migratory juvenile males. The exception was one group of juvenile males from St. Paul Island with mean δ15N values that were not significantly different from migrating females. The mean δ15N values of pregnant females indicate they were feeding at a higher trophic level than juvenile males during migration. The higher mean δ13C values for pregnant females suggest they were feeding coastally during the spring migration, while juvenile males and nulliparous females were feeding offshore. The higher δ15N values for post-parturient, lactating females over migrating, pregnant females point to either a trophic shift in diet over time, or a more likely 15N-enrichment due to negative nitrogen balance caused by the nutritional stress of lactation and the feeding/fasting regime experienced by females. Similar mean δ13C values for migrating and breeding-season females indicate that both groups were feeding in coastal, on-shelf domains during their respective time periods. Similar mean δ15N values for nulliparous and pregnant females indicate they were feeding at similar trophic levels despite indications of feeding in separate ecosystems during migration. Using a δ15N shift of 2-3‰ per trophic level, we made general inferences about the trophic levels at which northern fur seals were feeding. The interpretation of our δ15N data indicates that migrating pregnant females, lactating females and the majority of migrating juvenile males consumed prey with mean δ15N values between 14.2‰ and 15.2‰, 15.1‰ and 16.1‰, and 13.6‰ and 14.6‰, respectively. Probable fur seal prey was analyzed as well. Walleye pollock showed progressive 15N and 13C-enrichments with age. Mean δ15N and δ13C values of 3- to 4-year-old fish were ~6.0‰ and 1.1‰ higher, respectively, than values for 0-age pollock. Atka mackerel also showed isotopic enrichment with age. The δ15N and δ13C values of large fish were 0.8‰ and 0.3‰ higher, respectively, than values for smaller fish.

Applications of stable isotope analysis in mammalian ecology.

In this editorial, we provide a brief introduction and summarize the 10 research articles included in this Special Issue on Applications of stable isotope analysis in mammalian ecology. The first three articles report correction and discrimination factors that can be used to more accurately estimate the diets of extinct and extant mammals using stable isotope analysis. The remaining seven applied research articles use stable isotope analysis to address a variety of wildlife conservation and management questions from the oceans to the mountains.

The effects of sex, tissue type, and dietary components on stable isotope discrimination factors (Δ13C and Δ15N) in mammalian omnivores.

We tested the effects of sex, tissue, and diet on stable isotope discrimination factors (Δ(13)C and Δ(15)N) for six tissues from rats fed four diets with varied C and N sources, but comparable protein quality and quantity. The Δ(13)C and Δ(15)N values ranged from 1.7-4.1‰ and 0.4-4.3‰, respectively. Females had higher Δ(15)N values than males because males grew larger, whereas Δ(13)C values did not differ between sexes. Differences in Δ(13)C values among tissue types increased with increasing variability in dietary carbon sources. The Δ(15)N values increased with increasing dietary δ(15)N values for all tissues except liver and serum, which have fast stable isotope turnover times, and differences in Δ(15)N values among tissue types decreased with increasing dietary animal protein. Our results demonstrate that variability in dietary sources can affect Δ(13)C values, protein source affects Δ(15)N values even when protein quality and quantity are controlled, and the isotope turnover rate of a tissue can influence the degree to which diet affects Δ(15)N values.