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Food webs are known to have myriad trophic links between resource and consumer species. While herbivores have well‐understood trophic tendencies, the difficulties associated with characterizing the trophic positions of higher‐order consumers have remained a major problem in food web ecology. To better understand trophic linkages in food webs, analysis of the stable nitrogen isotopic composition of amino acids has been introduced as a potential means of providing accurate trophic position estimates. In the present study, we employ this method to estimate the trophic positions of 200 free‐roaming organisms, representing 39 species in coastal marine (a stony shore) and 38 species in terrestrial (a fruit farm) environments. Based on the trophic positions from the isotopic composition of amino acids, we are able to resolve the trophic structure of these complex food webs. Our approach reveals a high degree of trophic omnivory (i.e., noninteger trophic positions) among carnivorous species such as marine fish and terrestrial hornets.This information not only clarifies the trophic tendencies of species within their respective communities, but also suggests that trophic omnivory may be common in these webs. Stable nitrogen isotope analysis of amino acids has been employed as a relatively new method with the high potential for accurate and precise estimates of the trophic position of organisms. In the present study, we applied this method for multiple species collected from coastal marine (a stony shore) and terrestrial (a fruit farm) environments. Results clearly demonstrate that based on the observed trophic position of multiple species, we are able to present a highly resolved image of the trophic structure of these food webs.

Carbon and chlorine isotope effects for biotransformation of chloroform by different microbes show significant variability. Reductive dehalogenases (RDase) enzymes contain different cobamides, affecting substrate preferences, growth yields, and dechlorination rates and extent. We investigate the role of cobamide type on carbon and chlorine isotopic signals observed during reductive dechlorination of chloroform by the RDase CfrA. Microcosm experiments with two subcultures of a Dehalobacter‐containing culture expressing CfrA—one with exogenous cobamide (Vitamin B12, B12+) and one without (to drive native cobamide production)—resulted in a markedly smaller carbon isotope enrichment factor (εC, bulk) for B12− (−22.1 ± 1.9‰) compared to B12+ (−26.8 ± 3.2‰). Both cultures exhibited significant chlorine isotope fractionation, and although a lower εCl, bulk was observed for B12− (−6.17 ± 0.72‰) compared to B12+ (−6.86 ± 0.77‰) cultures, these values are not statistically different. Importantly, dual‐isotope plots produced identical slopes of ΛCl/C (ΛCl/C, B12+ = 3.41 ± 0.15, ΛCl/C, B12− = 3.39 ± 0.15), suggesting the same reaction mechanism is involved in both experiments, independent of the lower cobamide bases. A nonisotopically fractionating masking effect may explain the smaller fractionations observed for the B12− containing culture. The presence of vitamin B12 has a significant effect on carbon isotope effects, consistent with an isotope masking effect. We interpret this in the context of predicted enzyme structures.

The widespread importance of variable types of primary production, or energy channels, to consumer communities has become increasingly apparent. However, the mechanisms underlying this “multichannel” feeding remain poorly understood, especially for aquatic ecosystems that pose unique logistical constraints given the diversity of potential energy channels. Here, we use bulk tissue isotopic analysis along with carbon isotope (δ13C) analysis of individual amino acids to characterize the relative contribution of pelagic and benthic energy sources to a kelp forest consumer community in northern Chile. We measured bulk tissue δ13C and δ15N for >120 samples; of these we analyzed δ13C values of six essential amino acids (EAA) from nine primary producer groups (n = 41) and 11 representative nearshore consumer taxa (n = 56). Using EAA δ13C data, we employed linear discriminant analysis (LDA) to assess how distinct EAA δ13C values were between local pelagic (phytoplankton/particulate organic matter), and benthic (kelps, red algae, and green algae) endmembers. With this model, we were able to correctly classify nearly 90% of producer samples to their original groupings, a significant improvement on traditional bulk isotopic analysis. With this EAA isotopic library, we then generated probability distributions for the most important sources of production for each individual consumer and species using a bootstrap-resampling LDA approach. We found evidence for multichannel feeding within the community at the species level. Invertebrates tended to focus on either pelagic or benthic energy, deriving 13–67% of their EAA from pelagic sources. In contrast, mobile (fish) taxa at higher trophic levels used more equal proportions of each channel, ranging from 19% to 47% pelagically derived energy. Within a taxon, multichannel feeding was a result of specialization among individuals in energy channel usage, with 37 of 56 individual consumers estimated to derive >80% of their EAA from a single channel. Our study reveals how a cutting-edge isotopic technique can characterize the dynamics of energy flow in coastal food webs, a topic that has historically been difficult to address. More broadly, our work provides a mechanism as to how multichannel feeding may occur in nearshore communities, and we suggest this pattern be investigated in additional ecosystems.

Food web ecology has revolutionized our understanding of ecological processes, but the drivers of food web properties like trophic position (TP) and food chain length are notoriously enigmatic. In terrestrial ecosystems, aboveand belowground systems were historically compartmentalized into “green” and “brown” food webs, but the coupling of these systems by animal consumers is increasingly recognized, with potential consequences for trophic structure. We used stable isotope analysis (δ13C, δ15N) of individual amino acids to trace the flow of essential biomolecules and jointly measure multichannel feeding, food web coupling, and TP in a guild of small mammals. We then tested the hypothesis that brown energy fluxes to aboveground consumers increase terrestrial food chain length via cryptic trophic transfers during microbial decomposition. We found that the average small mammal consumer acquired nearly 70% of their essential amino acids (69.0% ± 7.6%) from brown food webs, leading to significant increases in TP across species and functional groups. Fungi were the primary conduit of brown energy to aboveground consumers, providing nearly half the amino acid budget for small mammals on average (44.3% ± 12.0%). These findings illustrate the tightly coupled nature of green and brown food webs and show that microbially mediated energy flow ultimately regulates food web structure in aboveground consumers. Consequently, we propose that the integration of green and brown energy channels is a cryptic driver of food chain length in terrestrial ecosystems.

Mutualistic nutritional symbioses are widespread in marine ecosystems. They involve the association of a host organism (algae, protists, or marine invertebrates) with symbiotic microorganisms, such as bacteria, cyanobacteria, or dinoflagellates. Nutritional interactions between the partners are difficult to identify in symbioses because they only occur in intact associations. Stable isotope analysis (SIA) has proven to be a useful tool to highlight original nutrient sources and to trace nutrients acquired by and exchanged between the different partners of the association. However, although SIA has been extensively applied to study different marine symbiotic associations, there is no review taking into account of the different types of symbiotic associations, how they have been studied via SIA, methodological issues common among symbiotic associations, and solutions that can be transferred from one type of association with another. The present review aims to fill such gaps in the scientific literature by summarizing the current knowledge of how isotopes have been applied to key marine symbioses to unravel nutrient exchanges between partners, and by describing the difficulties in interpreting the isotopic signal. This review also focuses on the use of compound-specific stable isotope analysis and on statistical advances to analyze stable isotope data. It also highlights the knowledge gaps that would benefit from future research.

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) (Δ13CC‐D and Δ15NC‐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 Δ13CC‐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 (TDFGlu‐Phe = 3.5 ± 0.4‰) that we calculated from the difference in trophic fractionation (Δ15NC‐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‐TDFGlu‐Phe equation with the avian‐specific TDFGlu‐Phe value from our experiment provided estimates that were more ecologically realistic than estimates using a single TDFGlu‐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. Compound‐specific stable isotope analysis of amino acids has rapidly become a powerful tool in studies of food web architecture, resource use, and biogeochemical cycling. We conducted a controlled feeding experiment on gentoo penguins to examine patterns in individual amino acid carbon and nitrogen isotope fractionation in non‐lethal, archival feathers. Using the newly derived avian‐specific amino acid fractionation factors, we examined wild penguin foraging ecology and trophic dynamics in the Southern Ocean, including identifying the sources of primary production supporting wild penguins and calculating wild penguin trophic position.

1. Analysis of stable carbon isotopes is a valuable tool for studies of diet, habitat use and migration. However, significant variability in the degree of trophic fractionation (Δ¹³CC₋D) between consumer (C) and diet (D) has highlighted our lack of understanding of the biochemical and physiological underpinnings of stable isotope ratios in tissues. 2. An opportunity now exists to increase the specificity of dietary studies by analyzing the δ¹³C values of amino acids (AAs). Common mummichogs (Fundulus heteroclitus, Linnaeus 1766) were reared on four isotopically distinct diets to examine individual AA Δ¹³CC₋D variability in fish muscle. 3. Modest bulk tissue Δ¹³CC₋D values reflected relatively large trophic fractionation for many non-essential AAs and little to no fractionation for all essential AAs. 4. Essential AA δ¹³C values were not significantly different between diet and consumer (Δ¹³CC₋D = 0·0 ± 0·4[per thousand]), making them ideal tracers of carbon sources at the base of the food web. Stable isotope analysis of muscle essential AAs provides a promising tool for dietary reconstruction and identifying baseline δ¹³C values to track animal movement through isotopically distinct food webs. 5. Non-essential AA Δ¹³CC₋D values showed evidence of both de novo biosynthesis and direct isotopic routing from dietary protein. We attributed patterns in Δ¹³CC₋D to variability in protein content and AA composition of the diet as well as differential utilization of dietary constituents contributing to the bulk carbon pool. This variability illustrates the complicated nature of metabolism and suggests caution must be taken with the assumptions used to interpret bulk stable isotope data in dietary studies. 6. Our study is the first to investigate the expression of AA Δ¹³CC₋D values for a marine vertebrate and should provide for significant refinements in studies of diet, habitat use and migration using stable isotopes.

Knowledge of how zooplankton can utilize cyanobacteria to sustain their nitrogen (N) demand for essential compounds like amino acids (AAs) is a key to predicting responses of higher trophic levels in terms of production and food web structure to future enhanced water column stratification. We explored the natural abundances of bulk N and AA‐specific nitrogen isotopes (δ15N) in particulate organic matter and mesozooplankton size‐fraction samples from three vertically separated water bodies in the central Baltic Sea during two summertime cyanobacteria blooms. The combination of plankton community and isotope data together with environmental variables helped to identify a mechanism of diazotrophic AA supply (synthesized during N2 fixation) for mesozooplankton, that largely depended on the sea surface temperature which regulated the access to the diazotrophic N‐based food web in the surface water (SW). We found that in the warmer summer, thermophilic cladocerans (e.g., Bosmina spp.) benefited most from diazotrophic AAs in the SW (19.8°C), while only in the colder summer, temperate copepods (e.g., Temora longicornis) ascended from the subjacent winter water into the SW (16.2°C) and incorporated diazotrophic AAs. Trophic position estimates based on the phenylalanine and glutamic acid δ15N signatures revealed that the diazotrophic AA supply into mesozooplankton was mainly indirect via feeding on mixo‐ and heterotrophic diets. Significantly enriched δ15N signatures in phenylalanine in the deep mesozooplankton (mainly copepods of Pseudo‐ and Paracalanus spp.) from the bottom water (BW) that was a region of the suboxic zone point to a reliance on a local food web. Mesozooplankton in the BW was feeding on diets of heterotrophic origin and probably profited from the heterotrophic re‐synthesis of AAs originating from sinking organic matter, as well as from the indirect incorporation of de novo synthesized AAs that most likely originated from chemoautotrophic bacteria or archaea communities in the suboxic zone. Our findings suggest that indirect feeding on diazotrophs and chemoautotrophs will be principal ways of amino acid supply for zooplankton in future enhanced stratified aquatic systems. Only a relatively small increase in temperature may restrict temperate key species from diazotrophic N‐based food webs in the mixed layer.

Sympatric species in marine ecosystems often share habitats and food resources, leading to niche competition. We performed ecological niche width estimation based on bulk carbon (δ13C) and nitrogen (δ15N) isotopes and trophic position estimation via compound‐specific isotope analysis (CSIA) of amino acids to compare ecological interspecific competition between two sympatric fish species, Pennahia argentata and Larimichthys polyactis in Gwangyang Bay, South Korea. Moreover, ontogenetic niche changes were investigated by size classification for each species. The δ13C and δ15N values in P. argentata showed no significant size‐related differences (p > 0.1), while L. polyactis exhibited significant variation (p < 0.001), indicating different ontogenetic niche shifts between two sympatric species. Trophic positions (TPGlu/Phe) estimates based on nitrogen isotopic composition of glutamic acid (δ15NGlu) and that of phenylalanine (δ15NPhe) were consistent across groups (3.53–3.62), indicating similar diet consumption. However, we found lower δ15NPhe values in large L. polyactis suggesting assimilated prey from distant marine areas before migrating into the study site. These findings demonstrate the utility of combining bulk stable isotope analysis and amino acid‐specific CSIA to disentangle dietary patterns and habitat use in mobile fish species. This study provides fundamental information for more targeted and ecological management strategies under variable environmental conditions by contrasting ontogenetic niche shifts and habitat‐driven isotopic variation between two sympatric species. We investigated trophic interactions between two sympatric fish species, Pennahia argentata and Larimichthys polyactis, using bulk and compound‐specific stable isotope analyses. CSIA of amino acids revealed similar trophic positions but distinct δ15N baselines, indicating ontogenetic niche shifts and offshore migration patterns. Our findings demonstrate the utility of CSIA in resolving fine‐scale ecological dynamics in coastal fish communities.

Income and capital breeding represent opposing ends of a continuum of reproductive strategies. Quantifying nutrient allocation to reproduction is challenging, but recent advances in compound‐specific stable isotope analysis hold promise for tracing the source of individual compounds allocated to reproduction. Here, we describe a novel approach of using measured carbon (δ13C) and nitrogen (δ15N) isotope values of individual amino acids (AAs) in pectoral muscle of egg‐laying females and egg yolk as a useful tool to quantify the reliance on income versus capital breeding in migrating species. We used white‐fronted (Anser albifrons frontalis), lesser snow (A. caerulescens caerulescens) and black brant (Branta bernicla nigricans) geese breeding in tundra ecosystems of northern Alaska as model organisms. All three species relied on mixed capital–income breeding strategies, but models based on AA isotope data estimated higher proportional contributions of endogenous resources to yolk synthesis compared to results based on bulk tissue isotope analyses. Tracing income versus capital nutrient allocation in migratory species at the compound level is a major advance from the current ‘elemental’ perspective obtained from bulk tissue stable isotope analyses. Our framework is applicable to all taxonomic groups, as long as there is a sufficient spatial or temporal isotopic gradient between resources obtained during the breeding and non‐breeding periods.