Compound‐specific 15N analysis of amino acids: A tool to estimate the trophic position of tropical seabirds in the South China Sea

Compound‐specific ¹⁵N analysis of amino acids (AAs) is a powerful tool to determine the trophic position (TP) of organisms. However, it has only been used in a few studies of avian ecology because the AA patterns in the consumer‐diet nitrogen trophic discrimination factor (TDF_Glu‐Phe = ?¹⁵N_Glu??¹⁵N_Phe) were unknown in birds until recently, and tropical seabirds have never been investigated with this methodology. Here, we explore the application of this method to tropical seabirds. In this study, we recovered the fossilized bones of tropical seabirds from ornithogenic sediments on two coral islands in the Xisha Islands, South China Sea, as well as the bones and muscle of their predominant food source, flying fish (Exocoetus volitans). Compound‐specific ¹⁵N and ¹³C analyses of AAs in both seabird and fish bone collagen were conducted. The TP of flying fish was calculated based on a widely used single TDF_Glu‐Phe approach. We then calculated the TP of tropical seabirds in three different ways: (a) according to the composition of their diet; (b) based on the single TDF_Glu‐Phe approach; and (c) using a multi‐TDF_Glu‐Phe approach. The results of the multi‐TDF_Glu‐Phe approach were much closer to the results based on the composition of the seabird diet than the results of the single TDF_Glu‐Phe approach, confirming its applicability for tropical seabirds. For seabird bone samples of different ages, TP determined from the multi‐TDF_Glu‐Phe approach was most similar to that of bulk δ¹⁵N of bird collagen, with seabirds occupying higher TPs during the Little Ice Age, as previously shown. In addition, the ¹³C Suess effect was reflected in the AAs δ¹³C in our samples. This study applied a compound‐specific ¹⁵N analysis of AAs to determine the TP of tropical seabirds that has potential to extend to all tropical seabirds many of which are widely distributed and play a key role in the evolution of coral island ecosystems.     

Carbon and nitrogen isotope fractionation of amino acids in an avian marine predator,the gentoo penguin (Pygoscelis papua)

Compound‐specific stable isotope analysis (CSIA) of amino acids (AA) has rapidly become a powerful tool in studies of food web architecture, resource use, and biogeochemical cycling. However, applications to avian ecology have been limited because no controlled studies have examined the patterns in AA isotope fractionation in birds. We conducted a controlled CSIA feeding experiment on an avian species, the gentoo penguin (Pygoscelis papua), to examine patterns in individual AA carbon and nitrogen stable isotope fractionation between diet (D) and consumer (C) (Δ¹³C_C‐D and Δ¹⁵N_C‐D, respectively). We found that essential AA δ¹³C values and source AA δ¹⁵N values in feathers showed minimal trophic fractionation between diet and consumer, providing independent but complimentary archival proxies for primary producers and nitrogen sources respectively, at the base of food webs supporting penguins. Variations in nonessential AA Δ¹³C_C‐D values reflected differences in macromolecule sources used for biosynthesis (e.g., protein vs. lipids) and provided a metric to assess resource utilization. The avian‐specific nitrogen trophic discrimination factor (TDF_Glu‐Phe = 3.5 ± 0.4‰) that we calculated from the difference in trophic fractionation (Δ¹⁵N_C‐D) of glutamic acid and phenylalanine was significantly lower than the conventional literature value of 7.6‰. Trophic positions of five species of wild penguins calculated using a multi‐TDF_Glu‐Phe equation with the avian‐specific TDF_Glu‐Phe value from our experiment provided estimates that were more ecologically realistic than estimates using a single TDF_Glu‐Phe of 7.6‰ from the previous literature. Our results provide a quantitative, mechanistic framework for the use of CSIA in nonlethal, archival feathers to study the movement and foraging ecology of avian consumers.     

Rescaling the trophic structure of marine food webs

Measures of trophic position (TP) are critical for understanding food web interactions and human‐mediated ecosystem disturbance. Nitrogen stable isotopes (δ¹⁵N) provide a powerful tool to estimate TP but are limited by a pragmatic assumption that isotope discrimination is constant (change in δ¹⁵N between predator and prey, Δ¹⁵N = 3.4‰), resulting in an additive framework that omits known Δ¹⁵N variation. Through meta‐analysis, we determine narrowing discrimination from an empirical linear relationship between experimental Δ¹⁵N and δ¹⁵N values of prey consumed. The resulting scaled Δ¹⁵N framework estimated reliable TPs of zooplanktivores to tertiary piscivores congruent with known feeding relationships that radically alters the conventional structure of marine food webs. Apex predator TP estimates were markedly higher than currently assumed by whole‐ecosystem models, indicating perceived food webs have been truncated and species‐interactions over simplified. The scaled Δ¹⁵N framework will greatly improve the accuracy of trophic estimates widely used in ecosystem‐based management.     

Are you what you eat? Effects of trophic discrimination factors on estimates of food assimilation and trophic position with a new estimation method

A key factor for estimates of assimilation of resources and trophic position based on stable isotope data is the trophic discrimination factor (TDF). TDFs are assumed based on literature reviews, but may vary depending on a variety of factors, including the type of diet. We analyzed effects of alternative TDFs on estimates of assimilated resources and trophic positions for an omnivorous fish, Jenynsia multidentata, that reveals dietary variation among locations across a salinity gradient of a coastal lagoon in southern Brazil. We also compared estimates of foods ingested vs. foods assimilated. Food assimilation was estimated using carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotope ratios of food sources and consumer muscle tissue and an isotopic mixing model (SIAR); consumer trophic position (TP) was estimated from consumer and production source δ¹⁵N values. Diet was estimated using an index of relative importance based on frequency of occurrence and volumetric and numeric proportions of food items from stomach contents. The effect of variation in TDF on food assimilation and TP was tested using three alternative TDFs reported in review papers. We then created a new method that used food source-specific TDFs (reported separately for herbivores and carnivores) weighted in proportion to estimated assimilation of resources according to mixing model estimates to estimate TP (hereafter TP_WAR). We found that plant material was not assimilated in a proportion similar to its importance in the diet of fish at a freshwater site, and the new method yielded best assimilation estimates. Animal material made greatest contributions to fish biomass irrespective of TDFs used in the mixing model. The new method produced TP estimates consistent with differences in estimated food assimilation along the salinity gradient. Our findings support the idea that food source-specific TDFs should be used in trophic studies of omnivores, since the method improved our ability to estimate trophic position and resource assimilation, two important ecological indicators.     

Fluid‐preserved fishes are one solution for assessing historical change in fish trophic level

1. There are few resources available for assessing historical change in fish trophic dynamics, but specimens held in natural history collections could serve as this resource. In contemporary trophic ecology studies, trophic and source information can be obtained from compound‐specific stable isotope analysis of amino acids of nitrogen (CSIA‐AA‐N).
2. We subjected whole Sebastes ruberrimus and Clupea pallasii to formalin fixation and 70% ethanol preservation. We extracted tissue samples from each fish pre‐fixation, after each chemical change, and then in doubling time for 32–64 days once placed in the final preservative. All samples were subjected to CSIA‐AA‐N, and their glutamic acid and phenylalanine profiles and associated trophic position were examined for differences over time by species.
3. Glutamic acid and phenylalanine values were inconsistent in direction and magnitude, particularly during formalin fixation, but stabilized similarly (in 70% ethanol) among conspecifics. In some cases, the amino acid values of our final samples were significantly different than our initial pre‐preservation samples. Nonetheless, significant differences in glutamic acid, phenylalanine, and estimated trophic position were not detected among samples that were in 70% ethanol for >24 hr.
4. Our results suggest that the relative trophic position of fluid‐preserved specimens can be estimated using CSIA‐AA‐N, and CSIA‐AA‐N estimates for fluid‐preserved specimens should only be reported as relative differences. Timelines of trophic position change can be developed by comparing specimens collected at different points in time, revealing trophic information of the past and cryptic ecosystem responses.

     

Diet quality influences isotopic discrimination among amino acids in an aquatic vertebrate

Stable nitrogen isotopic composition of amino acids (δ¹⁵N_AA) has recently been employed as a powerful tool in ecological food web studies, particularly for estimating the trophic position (TP) of animal species in food webs. However, the validity of these estimates depends on the consistency of the trophic discrimination factor (TDF; = Δδ¹⁵N_AA at each shift of trophic level) among a suite of amino acids within the tissues of consumer species. In this study, we determined the TDF values of amino acids in tadpoles (the Japanese toad, Bufo japonicus) reared exclusively on one of three diets that differed in nutritional quality. The diets were commercial fish‐food pellets (plant and animal biomass), bloodworms (animal biomass), and boiled white rice (plant carbohydrate), representing a balanced, protein‐rich, and protein‐poor diet, respectively. The TDF values of two “source amino acids” (Src‐AAs), methionine and phenylalanine, were close to zero (0.3–0.5‰) among the three diets, typifying the values reported in the literature (~0.5‰ and ~0.4‰, respectively). However, TDF values of “trophic amino acids” (Tr‐AAs) including alanine, valine, leucine, isoleucine, and glutamic acid varied by diet: for example, the glutamic acid TDF was similar to the standard value (~8.0‰) when tadpoles were fed either the commercial pellets (8.0‰) or bloodworms (7.9‰), but when they were fed boiled rice, the TDF was significantly reduced (0.6‰). These results suggest that a profound lack of dietary protein may alter the TDF values of glutamic acid (and other Tr‐AAs and glycine) within consumer species, but not the two Src‐AAs (i.e., methionine and phenylalanine). Knowledge of how a nutritionally poor diet can influence the TDF of Tr‐ and Src‐AAs will allow amino acid isotopic analyses to better estimate TP among free‐roaming animals.     

<Superscript>15</Superscript>N/<Superscript>14</Superscript>N ratios of amino acids as a tool for studying terrestrial food webs: a case study of terrestrial insects (bees,wasps, and hornets)

Compound-specific stable isotope analysis (CSIA) of amino acids is a new method that enables estimates of trophic position for consumers in food webs. We examined the nitrogen isotopic composition (δ¹⁵N) of amino acids of Japanese social insects (three bee, three wasp, and four hornet species) to evaluate the potential of CSIA of amino acids in studies of terrestrial food webs. For wasps, we also examined samples at different growth stages (ranging from egg to adult) to assess the effect of metamorphosis on CSIA estimates of trophic position. The δ¹⁵N values of bulk tissues for Japanese social insects are only weakly correlated with the biologically expected trophic positions. In contrast, the trophic positions estimated from the δ¹⁵N values of amino acids (yielding values of between 2.0 and 2.3 for bees, between 2.8 and 3.3 for wasps, and between 3.5 and 4.1 for hornets) are consistent with the biologically expected trophic positions for these insects (i.e., 2.0 for bees, 3.0 for wasps, and 3.0–4.0 for hornets). Although large variability is observed among the δ¹⁵N values of individual amino acids (e.g., ranging from 3.0 to 14.9‰ for phenylalanine), no significant change is observed in the trophic position during wasp metamorphosis. Thus, the CSIA of amino acids is a powerful tool for investigating not only aquatic food webs but also terrestrial food webs with predatory insects.     

Estimating tissue‐specific discrimination factors and turnover rates of stable isotopes of nitrogen and carbon in the smallnose fanskate Sympterygia bonapartii (Rajidae)

This study aimed to estimate trophic discrimination factors (TDFs) and metabolic turnover rates of nitrogen and carbon stable isotopes in blood and muscle of the smallnose fanskate Sympterygia bonapartii by feeding six adult individuals, maintained in captivity, with a constant diet for 365 days. TDFs were estimated as the difference between δ¹³C or δ¹⁵N values of the food and the tissues of S. bonapartii after they had reached equilibrium with their diet. The duration of the experiment was enough to reach the equilibrium condition in blood for both elements (estimated time to reach 95% of turnover: C t95%_blood = 150 days, N t95%_blood = 290 days), whilst turnover rates could not be estimated for muscle because of variation among samples. Estimates of Δ¹³C and Δ¹⁵N values in blood and muscle using all individuals were Δ¹³C_blood = 1·7‰, Δ¹³C_muscle = 1·3‰, Δ¹⁵N_blood = 2·5‰ and Δ¹⁵N_muscle = 1·5‰, but there was evidence of differences of c.0·4‰ in the Δ¹³C values between sexes. The present values for TDFs and turnover rates constitute the first evidence for dietary switching in batoids based on long‐term controlled feeding experiments. Overall, the results showed that S. bonapartii has relatively low turnover rates and isotopic measurements would not track seasonal movements adequately. The estimated Δ¹³C values in S. bonapartii blood and muscle were similar to previous estimations for elasmobranchs and to generally accepted values in bony fishes (Δ¹³C = 1·5‰). For Δ¹⁵N, the results were similar to published reports for blood but smaller than reports for muscle and notably smaller than the typical values used to estimate trophic position (Δ¹⁵N c. 3·4‰). Thus, trophic position estimations for elasmobranchs based on typical Δ¹⁵N values could lead to underestimates of actual trophic positions. Finally, the evidence of differences in TDFs between sexes reveals a need for more targeted research.     

Intra‐trophic isotopic discrimination of 15N/14N for amino acids in autotrophs: Implications for nitrogen dynamics in ecological studies

The differential discrimination of nitrogen isotopes (¹⁵N/¹⁴N) within amino acids in consumers and their diets has been routinely used to estimate organismal tropic position (TP). Analogous isotopic discrimination can occur within plants, particularly in organs lacking chloroplasts. Such discrimination likely arises from the catabolic deamination of amino acids, resulting in a numerical elevation of estimated TP, within newly synthesized biomass. To investigate this phenomenon, we examined the ¹⁵N/¹⁴N of amino acids (δ¹⁵N_AA) in spring leaves and flowers from eight deciduous and two annual plants. These plants were classified on the basis of their time of bloom, plants that bloomed when their leaves were absent (Type I) versus plants that bloomed while leaves were already present (Type II). Based on the δ¹⁵N_AA values from leaves, both plant types occupied comparable and ecologically realistic mean TPs (=1.0 ± 0.1, mean ± 1σ). However, the estimated TPs of flowers varied significantly (Type I: 2.2 ± 0.2; Type II: 1.0 ± 0.1). We hypothesize that these results can be interpreted by the following sequence of events: (1) Type I floral biomass is synthesized in absence of active photosynthesis; (2) the catabolic deamination of amino acids in particular, leaves behind ¹⁵N in the residual pool of amino acids; and (3) the incorporation of these ¹⁵N‐enriched amino acids within the biomass of Type I flowers results in the numerical elevation of the TPs. In contrast, the actively photosynthesizing Type II leaves energetically sustain the synthesis of Type II flower biomass, precluding any reliance on catabolic deamination of amino acids. Amino acids within Type II flowers are therefore isotopically comparable to the Type II leaves. These findings demonstrate the idiosyncratic nature of the δ¹⁵N_AA values within autotrophic organs and have implications for interpreting trophic hierarchies using primary producers and their consumers.     

Stable carbon and nitrogen isotope ratios in muscle and epidermis of arctic whales

Collection of minimally invasive biopsy samples has become an important method to establish normal stable isotopes reference ranges in various wildlife species. Baseline data enhance the understanding of feeding ecology, habitat use, and potential food limitation in apparently healthy, free‐ranging cetaceans. Epidermis and muscle were collected from subsistence‐hunted northern Alaskan bowhead (n= 133 epidermis/134 muscle) and beluga whales (n= 42/49) and subsistence‐hunted Russian gray whales (n= 25/17). Additional samples were obtained from gray whales stranded in California (n= 18/11) during mortality events (1999, 2000). Both δ¹⁵N and δ¹³C are trophic position and benthic/pelagic feeding indicators, respectively, in muscle and epidermis. Epidermis is generally enriched in ¹⁵N over muscle, while epidermal ¹³C is more depleted. Lipid extraction does not alter δ¹⁵N in either tissue, but affects epidermal δ¹³C. Nitrogen‐15 is enriched in muscle, but not epidermis of stranded compared to subsistence‐hunted gray whales, indicating probable protein catabolism and nutritional stress in stranded whales. Similarly, epidermal δ¹³C of harvested whales is lower than in stranded whales, suggesting depleted lipid stores and/or food limitation in stranded animals. Epidermal isotope signatures are similar in both present‐day bowheads and in an ancient sample from the Northern Bering Sea region. Although only one specimen, this suggests trophic level of the ancient whale compares to modern bowheads after a millennium.     

TDFCAM: A method for estimating stable isotope trophic discrimination in wild populations

1. Stable isotope mixing models (SIMMs) are widely used for characterizing wild animal diets. Such models rely upon using accurate trophic discrimination factors (TDFs) to account for the digestion, incorporation, and assimilation of food. Existing methods to calculate TDFs rely on controlled feeding trials that are time-consuming, often impractical for the study taxon, and may not reflect natural variability of TDFs present in wild populations.
2. We present TDF_CAM as an alternative approach to estimating TDFs in wild populations, by using high-precision diet estimates from a secondary methodological source—in this case nest cameras—in lieu of controlled feeding trials, and provide a framework for how and when it should be applied.
3. In this study, we evaluate the TDF_CAM approach in three datasets gathered on wild raptor nestlings (gyrfalcons Falco rusticolus; peregrine falcons Falco perigrinus; common buzzards Buteo buteo) comprising contemporaneous δ¹³C & δ¹⁵N stable isotope data and high-quality nest camera dietary data. We formulate Bayesian SIMMs (BSIMMs) incorporating TDFs from TDF_CAM and analyze their agreement with nest camera data, comparing model performance with those based on other relevant TDFs. Additionally, we perform sensitivity analyses to characterize TDF_CAM variability, and identify ecological and physiological factors contributing to that variability in wild populations.
4. Across species and tissue types, BSIMMs incorporating a TDF_CAM outperformed any other TDF tested, producing reliable population-level estimates of diet composition. We demonstrate that applying this approach even with a relatively low sample size (n < 10 individuals) produced more accurate estimates of trophic discrimination than a controlled feeding study conducted on the same species. Between-individual variability in TDF_CAM estimates for ∆¹³C & ∆¹⁵ N increased with analytical imprecision in the source dietary data (nest cameras) but was also explained by natural variables in the study population (e.g., nestling nutritional/growth status and dietary composition).
5. TDF_CAM is an effective method of estimating trophic discrimination in wild animal populations. Here, we use nest cameras as source dietary data, but this approach is applicable to any high-accuracy method of measuring diet, so long as diet can be monitored over an interval contemporaneous with a tissue's isotopic turnover rate.

     

Beneficial or not? Decoding carnivore roles in plant protection

Carnivores indirectly protect plants by reducing herbivory. This important ecosystem service can be undermined, however, as carnivores feed upon fellow carnivores. Such intraguild predation is exceedingly common, yet measurement of the degree to which this occurs has remained elusive due to difficulties in measuring the trophic tendencies of free-roaming animals. Conventional molecular methods, such as bulk-isotopic analyses, do not produce reliably accurate trophic position estimates, and often the inaccuracy is substantial. With the advent of compound-specific isotopic analysis (CSIA), it is now possible to accurately quantify the lifetime trophic tendencies of wild carnivore populations. Unfortunately, CSIA is extraordinarily expensive and time-consuming, limiting the number of samples that can be analyzed. The need for high-quality trophic information has to be balanced with the inaccessibility of CSIA. Here, we propose coupling CSIA-derived trophic position estimates with conventional bulk-¹⁵N analysis, effectively calibrating site-specific bulk-¹⁵N data and thereby allowing for trophic position estimation using bulk data alone. We also create a framework that uses trophic position as a basis to characterize carnivores as beneficial for crop protection. Within an agricultural field, we demonstrated the utility of this new approach by measuring the trophic positions of six common arthropod species. We then compare these trophic position estimates to those deriving from conventional bulk-¹⁵N analysis. Our hybrid approach produced more accurate trophic position estimates than the stand-alone bulk-¹⁵N method. Ultimately, we were able to examine enough specimens to determine which carnivore populations were likely beneficial for plant protection, and which were not.     

What does stable isotope analysis reveal about trophic relationships and the relative importance of allochthonous and autochthonous resources in tropical streams? A synthetic study from Hong Kong

1. Analysis of the stable isotope signatures of carbon (C) and nitrogen (N) of foods and consumers has led to some preliminary understanding of the relative importance of autochthonous and allochthonous resources in tropical streams. However, robust generalizations about the dynamics of food webs in these habitats, and their response to shading gradients or season, are still lacking. In addition, the feasibility of employing a baseline δ¹⁵N value for estimating trophic positions (TPs) of consumers in small tropical streams has yet to be explored. 2. We analysed data on stable isotope signatures of food sources and aquatic consumers obtained from 14 studies carried out in small streams in monsoonal Hong Kong (22°30′N, 114°10′E) between 1996 and 2006. Emphasis was placed on determining the relative importance of leaf litter and autochthonous foods in supporting consumer biomass, and the extent to which trophic base and TP vary among streams and seasons. 3. Although allochthonous leaf litter was generally ¹³C‐ and ¹⁵N‐depleted relative to autochthonous foods, there were marked isotopic shifts of food sources and consumers in response to season (dry versus wet) and stream shading. Consumer taxa were generally more ¹³C‐ and ¹⁵N‐enriched in the unshaded streams, but seasonal effects were more variable. Despite these changes, there was consistent evidence that stream food webs were based on periphytic algae and/or cyanobacteria with leaf litter serving as a minor food. 4. Heptageniidae (Ephemeroptera), Tipulidae (Diptera), Elmidae (Coleoptera) and shrimps (Atyidae) were used as a baseline for calculating the TPs of other consumer taxa. The maximum TPs in shaded streams remained fairly constant between seasons (dry = 3.93; wet = 3.97), while those in unshaded streams were higher and showed seasonal fluctuations (dry = 5.13; wet = 4.39). 5. Although variations in consumer isotope signatures in response to season and shading gradients did not confound our interpretation of the stream food base, changes in consumer δ¹⁵N did affect the calculation of consumer TPs. Misleading estimates of consumer TPs are likely if samples are collected from a narrow range of streams and/or during one season. Overestimation of the TPs of specialist herbivores (e.g. fish grazers) is also possible when autochthonous resources are substantially more ¹⁵N‐enriched than allochthonous foods.     

Substrate composition influences amino acid carbon isotope profiles of fungi: implications for tracing fungal contributions to food webs

Fungi link detrital resources and metazoan consumers through their role as decomposers. However, fungal contributions to metazoans may be misestimated in amino acid isotope studies because fungi are capable of both synthesizing amino acids (AAs) de novo and absorbing AAs from their environment. While fungi cultured in AA-free media have been used to represent fungi in studies of natural environments, fungi likely gain energetic benefits by taking up substrate AAs directly in situ. Consequently, fungi cultured on AA-free media may not be representative of the true variability of natural fungal δ¹³C_AA profiles. Therefore, the objective of this experiment was to determine the effect of substrate AA availability on yeast δ¹³C_AA profiles. We found that yeasts cultured in media of relatively higher AA content had different δ¹³C_AA profiles than yeasts grown in AA-free media, in part because yeasts utilized two essential AAs (Leu and Val) directly from media substrates when available in sufficient amounts. Furthermore, these differences among yeast δ¹³C_AA profiles remained after normalization of δ¹³C_AA values. We recommend further characterization of the variation in fungal δ¹³C_AA profiles and the incorporation of this potential variability into interpretations of basal resource use by metazoans.     

Variable littoral‐pelagic coupling as a food‐web response to seasonal changes in pelagic primary production

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How protein quality drives incorporation rates and trophic discrimination of carbon and nitrogen stable isotope ratios in a freshwater first-feeding fish

1. Using stable isotope ratios to explore the trophic ecology of freshwater animals requires knowledge about effects of food quality on isotopic incorporation dynamics. The aim of this experimental study was to: (1) estimate carbon and nitrogen isotopic incorporation rates and trophic discrimination factors (TDFs) of a freshwater first-feeding fish (i.e. salmonid fry) fed three diets that differed only in protein quality (animal or plant or a blend of both); (2) investigate effects of fasting and; (3) evaluate the proportion of each source assimilated when fry were fed a 50:50 animal:plant-based diet.
2. For each diet, incorporation rates of δ¹³C and δ¹⁵N values were estimated using a time or growth-dependent isotopic incorporation model. Effects of fasting on isotope ratio values were measured regularly until the death of fry. Bayesian stable-isotope mixing models were used to estimate the contribution of animal and plant material to fish fed a blend of both food types.
3. Our results show that incorporation rates were lower for fry fed a plant-based diet than for those fed an animal-based diet as growth rate decreased. Time- and growth-dependent models indicated that growth was solely responsible for isotopic incorporation in fry fed an animal-based diet, whereas catabolism increased in fry fed a plant-based diet. After lipid extraction, carbon TDFs were similar regardless of the diet, whereas nitrogen TDFs increased for fry fed a plant-based diet. Long-term fasting induced an increase of 0.63‰ in δ¹³C values of fry in 23 days, whereas δ¹⁵N values did not vary significantly. Proportions of food sources assimilated by fry fed an animal:plant-based diet were similar to those consumed when using a mixing model with the estimated TDFs, while proportions were unrealistic when using mean TDFs extrapolated from the literature.
4. The results of our study indicate that the quality of food must be considered to use an appropriate timescale to detect changes in fry diets in the field. Moreover, we recommend using different carbon and nitrogen TDFs, one for animal-derived sources and one for plant-derived sources, to increase the accuracy of mixing models.

     

Isotopic niche differs between seal and fish‐eating killer whales (Orcinus orca) in northern Norway

Ecological diversity has been reported for killer whales (Orcinus orca) throughout the North Atlantic but patterns of prey specialization have remained poorly understood. We quantify interindividual dietary variations in killer whales (n = 38) sampled throughout the year in 2017–2018 in northern Norway using stable isotopic nitrogen (δ¹⁵N: ¹⁵N/¹⁴N) and carbon (δ¹³C: ¹³C/¹²C) ratios. A Gaussian mixture model assigned sampled individuals to three differentiated clusters, characterized by disparate nonoverlapping isotopic niches, that were consistent with predatory field observations: seal‐eaters, herring‐eaters, and lumpfish‐eaters. Seal‐eaters showed higher δ¹⁵N values (mean ± SD: 12.6 ± 0.3‰, range = 12.3–13.2‰, n = 10) compared to herring‐eaters (mean ± SD: 11.7 ± 0.2‰, range = 11.4–11.9‰, n = 19) and lumpfish‐eaters (mean ± SD: 11.6 ± 0.2‰, range = 11.3–11.9, n = 9). Elevated δ¹⁵N values for seal‐eaters, regardless of sampling season, confirmed feeding at high trophic levels throughout the year. However, a wide isotopic niche and low measured δ¹⁵N values in the seal‐eaters, compared to that of whales that would eat solely seals (δ_N‐measured = 12.6 vs. δ_N‐expected = 15.5), indicated a diverse diet that includes both fish and mammal prey. A narrow niche for killer whales sampled at herring and lumpfish seasonal grounds supported seasonal prey specialization reflective of local peaks in prey abundance for the two fish‐eating groups. Our results, thus, show differences in prey specialization within this killer whale population in Norway and that the episodic observations of killer whales feeding on prey other than fish are a consistent behavior, as reflected in different isotopic niches between seal and fish‐eating individuals.     

Use of a δ13C–δ15N relationship to determine animal trophic positions in a tropical Australian estuarine wetland

Stable isotope composition of organisms from different trophic groups collected from a semi‐isolated wetland pool in the Ross River estuary, northern Australia, was analysed to determine if there was a consistent relationship between δ¹³C, δ¹⁵N and trophic level that could be used to assign trophic positions. A strong linear negative relationship between δ¹³C and δ¹⁵N was detected for the three trophic levels considered (primary producers, primary consumers and secondary consumers). This relationship was consistent among trophic levels, differing only in height, that is, on δ¹⁵N values, which indicate trophic positions. A difference of 3.6–3.8‰ between trophic levels was present, suggesting a δ¹⁵N fractionation of approximately 3.7‰, a value slightly higher than the commonly assumed δ¹⁵N fractionation of approximately 3.4‰. The relationship between δ¹³C and δ¹⁵N was similar for invertebrate and fish primary consumers, indicating similar δ¹⁵N trophic fractionation for both groups, meaning trophic positions and trophic length could be reliably calculated based on either invertebrates or fish.     

Ecotype and geographical variation in carbon and nitrogen stable isotope values in western North Pacific killer whales (Orcinus orca)

Killer whales are top predators in marine trophic chains, and therefore their feeding preferences can substantially affect the abundance of species on the lower trophic levels. Killer whales are known to feed on many different types of prey from small fish to large whales, but a given killer whale population usually focuses on a specific type of prey. Stable isotope analysis is widely used to study whale diets, because direct observations are often impossible. Killer whale feeding habits in the western North Pacific are poorly studied, and the large-scale stable isotope analysis provides a unique opportunity to gain insights into the trophic links of this top predator. In this study, we compare the δ¹³C and δ¹⁵N stable isotope values from killer whale skin samples obtained in different areas of the western North Pacific from fish-eating (R-type) and mammal-eating (T-type) killer whale ecotypes. The effect of ecotype was highly significant: both carbon and nitrogen stable isotope values were lower in R-type whales than in T-type whales. The geographical variation also affected killer whale stable isotope values due to both the differences in killer whale diet and the variation in baseline stable isotope values across the study areas.     

Evaluating δ15N–body size relationships across taxonomic levels using hierarchical models

Ecologists routinely set out to estimate the trophic position of individuals, populations, and species composing food webs, and nitrogen stable isotopes (δ¹⁵N) are a widely used proxy for trophic position. Although δ¹⁵N values are often sampled at the level of individuals, estimates and confidence intervals are frequently sought for aggregations of individuals. If individual δ¹⁵N values are correlated as an artifact of sampling design (e.g., clustering of samples in space or time) or due to intrinsic groupings (e.g., life history stages, social groups, taxonomy), such estimates may be biased and exhibit overly optimistic confidence intervals. However, these issues can be accommodated using hierarchical modeling methods. Here, we demonstrate how hierarchical models offer an additional quantitative tool for investigating δ¹⁵N variability and we explicitly evaluate how δ¹⁵N varies with body size at successively higher levels of taxonomic aggregation in a diverse fish assemblage. The models take advantage of all available data, better account for uncertainty in parameters estimates, may improve inferences on coefficients corresponding to groups with small to moderate sample sizes, and partition variation across model levels, which provides convenient summaries of the ‘importance’ of each level in terms of unexplained heterogeneity in the data. These methods can easily be applied to diet-based studies of trophic position. Although hierarchical models are well-understood and established tools, their benefits have yet to be fully reaped by stable isotope and food web ecologists. We suggest that hierarchical models can provide a robust framework for conceptualizing and statistically modeling trophic position at multiple levels of aggregation.