How strongly does diet variation explain variation in isotope values of animal consumers?

Analysis of stable isotopes in consumers is used commonly to study their ecological and/or environmental niche. There is, however, considerable debate regarding how isotopic values relate to diet and how other sources of variation confound this link, which can undermine the utility. From the analysis of a simple, but general, model of isotopic incorporation in consumer organisms, we examine the relationship between isotopic variance among individuals, and diet variability within a consumer population. We show that variance in consumer isotope values is directly proportional to variation in diet (through Simpson indices), to the number of isotopically distinct food sources in the diet, and to the baseline variation within and among the isotope values of the food sources. Additionally, when considering temporal diet variation within a consumer we identify the interplay between diet turnover rates and tissue turnover rates that controls the sensitivity of stable isotopes to detect diet variation. Our work demonstrates that variation in the stable isotope values of consumers reflect variation in their diet. This relationship, however, can be confounded with other factors to the extent that they may mask the signal coming from diet. We show how simple quantitative corrections can recover a direct 1:1 correlation in some situations, and in others we can adjust our interpretation in light of the new understanding arising from our models. Our framework provides guidance for the design and analysis of empirical studies where the goal is to infer niche width from stable isotope data.

Non-indigenous species and their realized niche in tidepools along the South-East Pacific coast.

Non-indigenous species (NIS) have the potential to colonize and become established in a wide range of coastal habitats. Species with broad environmental tolerances can quickly adapt to local conditions and expand their niches along environmental gradients, and even colonize habitats with extreme abiotic conditions. Here we report and document the distribution of eight marine NIS (four seaweed and four invertebrate species) found in tidepools along a 3000 km latitudinal gradient along the Pacific coast of Chile (18.4°S to 41.9°S). The seaweed NIS Codium fragile, Capreolia implexa, Schottera nicaeensis and Mastocarpus latissimus were mostly distributed towards high latitudes (i.e., more southerly locations), where temperatures in tidepools were low. The invertebrate NIS Anemonia alicemartinae, Ciona robusta, Bugula neritina and Bugulina flabellata were more common towards low latitudes, where high temperatures were registered in the tidepools. Across the intertidal gradient, seaweed NIS were mostly found in pools in the mid and low intertidal zone, while invertebrate NIS occurred mostly in pools from the mid and upper intertidal zones. The realized niche spaces of NIS (based on the Outlying Mean Index, OMI) in the study area were mainly influenced by environmental conditions of temperature and salinity (along the latitudinal and intertidal gradients), while other tidepool characteristics (depth, surface area, exposition, and complexity) only had minor effects. Five of the eight NIS exhibited a realized niche space coinciding with the average tidepool environmental conditions, while marginal niches were occupied by species with affinities for specific temperatures and salinities along the latitudinal and intertidal gradients. Our results indicate that physiological tolerances to environmental factors play a fundamental role in the distribution of seaweed and invertebrate NIS in tidepools along the Chilean coast. This study confirms that tidepools offer suitable conditions for some seaweed and invertebrate NIS, potentially facilitating their invasion into new natural habitats.

Comparing trophic position estimates using bulk and compound specific stable isotope analyses: applying new approaches to mackerel icefish Champsocephalus gunnari.

Quantifying the tropic position (TP) of an animal species is key to understanding its ecosystem function. While both bulk and compound-specific analyses of stable isotopes are widely used for this purpose, few studies have assessed the consistency between and within such approaches. Champsocephalus gunnari is a specialist teleost that predates almost exclusively on Antarctic krill Euphausia superba. This well-known and nearly constant trophic relationship makes C. gunnari particularly suitable for assessing consistency between TP methods under field conditions. In the present work, we produced and compared TP estimates for C. gunnari and its main prey using a standard bulk and two amino acid-specific stable isotope approaches (CSI-AA). One based on the difference between glutamate and phenylalanine (TPGlx-Phe), and the other on the proline-phenylalanine difference (TPPro-Phe). To do that, samples from C. gunnari, E. superba and four other pelagic invertebrate and fish species, all potential prey for C.gunnari, were collected off the South Orkney Islands between January and March 2019, analyzed using standard isotopic ratio mass spectrometry methods and interpreted following a Bayesian approach. Median estimates (CI95%) for C. gunnari were similar between TPbulk (3.6; CI95%: 3.0-4.8) and TPGlx-Phe(3.4; CI95%:3.2-3.6), and lower for TPPro-Phe (3.1; CI95%:3.0-3.3). TP differences between C. gunnari and E. superba were 1.4, 1.1 and 1.2, all compatible with expectations from the monospecific diet of this predator (ΔTP=1). While these results suggest greater accuracy for Glx-Phe and Pro-Phe, differences observed between both CSI-AA approaches suggests these methods may require further validation before becoming a standard tool for trophic ecology.

Why aquatic scientists should use sulfur stable isotope ratios (ẟ34S) more often.

Over the last few decades, measurements of light stable isotope ratios have been increasingly used to answer questions across physiology, biology, ecology, and archaeology. The vast majority analyse carbon (δ13C) and nitrogen (δ15N) stable isotopes as the 'default' isotopes, omitting sulfur (δ34S) due to time, cost, or perceived lack of benefits and instrumentation capabilities. Using just carbon and nitrogen isotopic ratios can produce results that are inconclusive, uncertain, or in the worst cases, even misleading, especially for scientists that are new to the use and interpretation of stable isotope data. Using sulfur isotope values more regularly has the potential to mitigate these issues, especially given recent advancements that have lowered measurement barriers. Here we provide a review documenting case studies with real-world data, re-analysing different biological topics (i.e. niche, physiology, diet, movement and bioarchaeology) with and without sulfur isotopes to highlight the various strengths of this stable isotope for various applications. We also include a preliminary meta-analysis of the trophic discrimination factor (TDF) for sulfur isotopes, which suggest small (mean -0.4 ± 1.7 ‰ SD) but taxa-dependent mean trophic discrimination. Each case study demonstrates how the exclusion of sulfur comes at the detriment of the results, often leading to very different outputs, or missing valuable discoveries entirely. Given that studies relying on carbon and nitrogen stable isotopes currently underpin most of our understanding of various ecological processes, this has concerning implications. Collectively, these examples strongly suggest that researchers planning to use carbon and nitrogen stable isotopes for their research should incorporate sulfur where possible, and that the new 'default' isotope systems for aquatic science should now be carbon, nitrogen, and sulfur.

Ecological variation in invasive brown trout (Salmo trutta) within a remote coastal river catchment in northern Patagonia complicates estimates of invasion impact.

Salmonids were first introduced into the Chilean fresh waters in the 1880s, and c. 140 years later, they are ubiquitous across Chilean rivers, especially in the southern pristine fresh waters. This study examined the brown trout (Salmo trutta) and native taxa ecology in two adjacent but contrasting rivers of Chilean Patagonia. During spring 2016 and spring-fall 2017 we examined the variation in benthic macroinvertebrate and fish community composition and characterized fish size structure, stomach contents, and stable isotopes (δ13 C and δ15 N) to understand population structure, fish diet, and trophic interactions between S. trutta and native taxa. The native Galaxias maculatus (puye) dominated the fish community (74% of abundance). S. trutta was less abundant (16% of survey catch) but dominated the fish community (over 53%) in terms of biomass. S. trutta showed distinct diets (stomach content analysis) in the two rivers, and individuals from the larger river were notably more piscivorous, consuming native fish with a relatively small body size (<100-mm total length). Native fishes were isotopically distinct from S. trutta, which showed a wider isotopic niche in the smaller river, indicating that their trophic role was more variable than in the larger river (piscivorous). This study provides data from the unstudied pristine coastal rivers in Patagonia and reveals that interactions between native and introduced species can vary at very local spatial scales.

Multiple stable isotopes (C, N & S) provide evidence for fin whale (Balaenoptera physalus) trophic ecology and movements in the Humboldt Current System of northern Chile.

Reflecting the intense coastal upwelling and high primary productivity characteristic of the Humboldt Current System (HCS), the northern coast of Chile supports a diverse and productive community of marine consumers, including worldwide important pelagic fisheries resources. Although marine mammals are relatively understudied in the region, recent studies have demonstrated that fin whale (Balaenoptera physalus) is the most frequently encountered whale species, and forages in these waters year-round. However, a current lack of information limits our understanding of whether fin whales actively feed and/or remain resident in these waters or whether whales are observed feeding as they migrate along this part of the Pacific. Here, we use stable isotope ratios of carbon, nitrogen and sulphur of fin whale skin samples collected in early summer 2020 (n = 18) and in late winter 2021 (n = 22) to examine evidence of temporal isotopic shifts that could provide information on potential migratory movements and to estimate likely consumption patterns of putative prey (i.e. zooplankton, krill, pelagic fishes and Pleuroncodes sp.). We also analysed prey items in fin whale faecal plumes (n = 8) collected during the study period. Stable isotope data showed significant differences in the isotopic values of fin whales from summer and winter. On average, summer individuals were depleted in 15N and 34S relative to those sampled during winter. Whales sampled in summer showed greater isotopic variance than winter individuals, with several showing values that were atypical for consumers from the HCS. During winter, fin whales showed far less inter-individual variation in stable isotope values, and all individuals had values indicative of prey consumption in the region. Analysis of both stable isotopes and faeces indicated that fin whales sighted off the Mejillones Peninsula fed primarily on krill (SIA median contribution = 32%; IRI = 65%) and, to a lesser extent, zooplankton (SIA zooplankton = 29%; IRI copepod = 33%). These are the first isotopic-based data regarding the trophic ecology of fin whales in the north of Chile. They provide evidence that fin whales are seasonally resident in the area, including individuals with values that likely originated outside the study area. The information presented here serves as a baseline for future work. It highlights that many aspects of the ecology of fin whales in the Humboldt Current and wider SE Pacific still need to be clarified.

Shifts in maternal foraging strategies during pregnancy promote offspring health and survival in a marine top predator.

The success of maternal foraging strategies during the rearing period can greatly impact the physiology and survival of dependent offspring. Surprisingly though, little is known on the fitness consequences of foraging strategies during the foetal period. In this study, we characterized variation in maternal foraging strategy throughout pregnancy in a marine top predator (South American fur seal, Arctocephalus australis), and asked if these shifts predicted neonatal health and postnatal survival. We found that during early pregnancy all pregnant females belonged to a single, homogenized foraging niche without evident clusters. Intriguingly though, during late pregnancy, individual fur seal mothers diverged into two distinct foraging niches characterized by a benthic-nearshore and a pelagic-offshore strategy. Females that shifted towards the benthic-nearshore strategy gave birth to pups with greater body mass, higher plasmatic levels of glucose and lower levels of blood urea nitrogen. The pups born to these benthic females were eight times more likely to survive compared to females using the pelagic-offshore foraging strategy during late pregnancy. These survival effects were mediated primarily by the impact of foraging strategies on neonatal glucose independent of protein metabolic profile and body mass. Benthic-nearshore foraging strategies during late pregnancy potentially allow for the greater maternal transfer of glucose to the foetus, leading to higher chances of neonatal survival. These results call for a deeper understanding of the balance between resource acquisition and allocation provided by distinct foraging polymorphisms during critical life-history periods, and how this trade-off may be adaptive under certain environmental conditions.

No evidence for effects of mill-grinding on δ13 C, δ15 N and δ34 S values in different marine taxa.

RATIONALE: The analysis of natural variation in light stable isotopes such as carbon (δ13 C), nitrogen (δ15 N) and sulfur (δ34 S) plays an important role in deepening our understanding of ecosystems. To avoid misinterpretation, robust results are required, where pre-treatment steps such as sample homogenization are crucial to guarantee representative samples. The grinding of samples using stainless steel balls in polypropylene tubes (e.g., laboratory bead-beater) has been identified as a potential source of bias. METHODS: We tested possible effects of mill-grinding (e.g., contamination) of samples of coastal marine taxa including primary producers, primary consumers and higher trophic level fish. We compared potential impacts of homogenization by mill-grinding with hand-grinding over an extended time on δ13 C, δ15 N and δ34 S values. RESULTS: One-way Welch's analysis of variance (ANOVA) showed that there were no statistical differences between methods for all the studied taxa. Also, repeated measures ANOVA showed no evidence of effects of grinding for extended times (from 30 to 120 seconds) for δ13 C, δ15 N and δ34 S values. CONCLUSIONS: We found no evidence that grinding samples in polypropylene tubes in a bead-beater resulted in any marked alteration of the isotopic composition on the studied samples, e.g., through contamination by plastic. As such, we consider mill-grinding as an appropriate method for the homogenization of samples from a range of different marine taxa, which under controlled conditions did not affect δ13 C, δ15 N and δ34 S analysis.

Winter ecology of specialist and generalist morphs of European whitefish, Coregonus lavaretus, in subarctic northern Europe.

European whitefish is a model species for adaptive radiation of fishes in temperate and subarctic lakes. In northern Europe the most commonly observed morphotypes are a generalist (LSR) morph and a pelagic specialist (DR) morph. The evolution of a pelagic specialist morph is something of an enigma, however, as this region is characterized by long, dark winters with pelagic primary production limited to a brief window in late summer. We conducted the first winter-based study of polymorphic whitefish populations to determine the winter ecology of both morphs, and we combined seasonal diet and stable isotope analysis with several proxies of condition in three polymorphic whitefish populations. The generalist LSR morph fed on benthic and pelagic prey in summer but was solely reliant on benthic prey in winter. This was associated with a noticeable but moderate reduction in condition, lipid content and stomach fullness in winter relative to summer. In contrast, the DR whitefish occupied a strict pelagic niche in both seasons. A significant reduction in pelagic prey during winter resulted in severe decrease in condition, lipid content and stomach fullness in DR whitefish in winter relative to summer, with the pelagic morph apparently approaching starvation in winter. We suggest that this divergent approach to seasonal foraging is associated with the divergent life-history traits of both morphs.

Soil δ13C and δ15N baselines clarify biogeographic heterogeneity in isotopic discrimination of European badgers (Meles meles).

Isotopic techniques have been used to study phenomena in the geological, environmental, and ecological sciences. For example, isotopic values of multiple elements elucidate the pathways energy and nutrients take in the environment. Isoscapes interpolate isotopic values across a geographical surface and are used to study environmental processes in space and time. Thus, isoscapes can reveal ecological shifts at local scales, and show distribution thresholds in the wider environment at the macro-scale. This study demonstrates a further application of isoscapes, using soil isoscapes of 13C/12C and 15N/14N as an environmental baseline, to understand variation in trophic ecology across a population of Eurasian badgers (Meles meles) at a regional scale. The use of soil isoscapes reduced error, and elevated the statistical signal, where aggregated badger hairs were used, and where individuals were identified using genetic microarray analysis. Stable isotope values were affected by land-use type, elevation, and meteorology. Badgers in lowland habitats had diets richer in protein and were adversely affected by poor weather conditions in all land classes. It is concluded that soil isoscapes are an effective way of reducing confounding biases in macroscale, isotopic studies. The method elucidated variation in the trophic and spatial ecology of economically important taxa at a landscape level. These results have implications for the management of badgers and other carnivores with omnivorous tendencies in heterogeneous landscapes.

Salt to conserve: a review on the ecology and preservation of hypersaline ecosystems.

When it comes to the investigation of key ecosystems in the world, we often omit salt from the ecological recipe. In fact, despite occupying almost half of the volume of inland waters and providing crucial services to humanity and nature, inland saline ecosystems are often overlooked in discussions regarding the preservation of global aquatic resources of our planet. As a result, our knowledge of the biological and geochemical dynamics shaping these environments remains incomplete and we are hesitant in framing effective protective strategies against the increasing natural and anthropogenic threats faced by such habitats. Hypersaline lakes, water bodies where the concentration of salt exceeds 35 g/l, occur mainly in arid and semiarid areas resulting from hydrological imbalances triggering the accumulation of salts over time. Often considered the 'exotic siblings' within the family of inland waters, these ecosystems host some of the most extremophile communities worldwide and provide essential habitats for waterbirds and many other organisms in already water-stressed regions. These systems are often highlighted as natural laboratories, ideal for addressing central ecological questions due to their relatively low complexity and simple food web structures. However, recent studies on the biogeochemical mechanisms framing hypersaline communities have challenged this archetype, arguing that newly discovered highly diverse communities are characterised by specific trophic interactions shaped by high levels of specialisation. The main goal of this review is to explore our current understanding of the ecological dynamics of hypersaline ecosystems by addressing four main research questions: (i) why are hypersaline lakes unique from a biological and geochemical perspective; (ii) which biota inhabit these ecosystems and how have they adapted to the high salt conditions; (iii) how do we protect biodiversity from increasing natural and anthropogenic threats; and (iv) which scientific tools will help us preserve hypersaline ecosystems in the future? First, we focus on the ecological characterisation of hypersaline ecosystems, illustrate hydrogeochemical dynamics regulating such environments, and outline key ecoregions supporting hypersaline systems across the globe. Second, we depict the diversity and functional aspects of key taxa found in hypersaline lakes, from microorganisms to plants, invertebrates, waterbirds and upper trophic levels. Next, we describe ecosystem services and discuss possible conservation guidelines. Finally, we outline how cutting-edge technologies can provide new insights into the study of hypersaline ecology. Overall, this review sheds further light onto these understudied ecosystems, largely unrecognised as important sources of unique biological and functional diversity. We provide perspectives for key future research avenues, and advocate that the conservation of hypersaline lakes should not be taken with 'a grain of salt'.

'White gold' guano fertilizer drove agricultural intensification in the Atacama Desert from AD 1000.

The archaeological record shows that large pre-Inca agricultural systems supported settlements for centuries around the ravines and oases of northern Chile's hyperarid Atacama Desert. This raises questions about how such productivity was achieved and sustained, and its social implications. Using isotopic data of well-preserved ancient plant remains from Atacama sites, we show a dramatic increase in crop nitrogen isotope values (δ15N) from around AD 1000. Maize was most affected, with δ15N values as high as +30‰, and human bone collagen following a similar trend; moreover, their carbon isotope values (δ13C) indicate a considerable increase in the consumption of maize at the same time. We attribute the shift to extremely high δ15N values-the highest in the world for archaeological plants-to the use of seabird guano to fertilize crops. Guano-'white gold' as it came to be called-thus sustained agricultural intensification, supporting a substantial population in an otherwise extreme environment.

Functional changes in benthic macrofaunal communities along a natural gradient of hypoxia in an upwelling system.

Increasing global concern has been raised about the expansion of hypoxia in coastal waters and its potential to impact benthic ecosystems. Upwelling areas offer opportunities to study the effects of hypoxia on benthic communities under natural conditions. We used a biological trait-based approach and estimated functional diversity indices to assess macrobenthic community functioning along a depth gradient associated with naturally increasing hypoxia and concentrations of organic matter in the upwelling zone of northern Chile (South-East Pacific) over two years. Our results highlighted the increasing dominance of opportunistic biological traits associated with hypoxia and high organic matter content. Habitat filtering was the main process affecting the studied communities. Functional diversity patterns were persistent overtime despite the occurrence of a pulse of oxygenation. This study contributes to our understanding of how natural hypoxia impacts macrobenthic communities, providing useful information in the context of increasing eutrophication due to human influence on coastal areas.

Intraspecific variation and energy channel coupling within a Chilean kelp forest.

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 (δ13 C) 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 δ13 C and δ15 N for >120 samples; of these we analyzed δ13 C values of six essential amino acids (EAA) from nine primary producer groups (n = 41) and 11 representative nearshore consumer taxa (n = 56). Using EAA δ13 C data, we employed linear discriminant analysis (LDA) to assess how distinct EAA δ13 C 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.

Chilean Salmon Sushi: Genetics Reveals Product Mislabeling and a Lack of Reliable Information at the Point of Sale.

Species diagnosis is essential to assess the level of mislabeling or misnamed seafood products such as sushi. In Chile, sushi typically includes salmon as the main ingredient, but species used are rarely declared on the menu. In order to identify which species are included in the Chilean sushi market, we analyzed 84 individual sushi rolls sold as "salmon" from sushi outlets in ten cities across Chile. Using a polymerase chain reaction-restriction fragment length polymorphism protocol (PCR-RFLP), we identified mislabeled and misnamed products. Atlantic salmon was the most common salmonid fish used in sushi, followed by coho salmon, rainbow trout, and Chinook salmon. We found a total of 23% and 18% of the products were mislabeled and misnamed, respectively. In 64% of cases, the salesperson selling the product could not identify the species. We also identified the use of wild-captured Chinook salmon samples from a naturalized population. Our results provide a first indication regarding species composition in Chilean sushi, a quantification of mislabeling and the level of misinformation declared by sales people to consumers. Finally, considering that Chinook salmon likely originates from a non-licensed origin and that sushi is an uncooked product, proper identification in the food production chain may have important consequences for the health of consumers.

Southernmost distribution limit for endangered Peladillas (Aplochiton taeniatus) and non-native coho salmon (Oncorhynchus kisutch) coexisting within the Cape Horn biosphere reserve, Chile.

The Cape Horn Biosphere Reserve, one of the last wild areas of the planet, is not exempt from the pressures of global change, such as non-native species introductions. During 2018 and 2019 we studied the Róbalo river basin in order to update the diversity and distribution of fishes. Here, we report for the first time the native and endangered "Peladillas" Aplochiton taeniatus and the non-native coho salmon Oncorhynchus kisutch. The coexistence of native and non-native fishes poses a challenge for the management and conservation of aquatic biota from the Cape Horn Biosphere Reserve.

Sample acidification has a predictable effect on isotopic ratios of particulate organic matter along the Chilean coast.

RATIONALE: Stable isotopes of carbon and nitrogen have proved to be valuable tools for researchers working across the different subfields of ecology. However, the chemical pretreatment of samples prior to analytical determination of stable isotope ratios can influence the results, and therefore conclusions regarding the ecology of the taxon or system under study. Here, we determined the effect of vapor acidification with concentrated HCl on the δ13 C and δ15 N values of particulate organic matter (POM), which are commonly used as baselines for studies of trophic ecology, or to understand oceanographic patterns. METHODS: Samples of marine POM were obtained along a large-scale latitudinal gradient (ca 3000 km) along the Chilean coast, along with a range of oceanographic variables thought to potentially influence inorganic carbon at each sampling location. A random subset of 50 samples was divided into two parts: one acidified by HCl fumigation treatment, and the other acting as a control. We compared paired differences in δ13 C and δ15 N values measured by continuous flow isotope ratio mass spectrometry and used a model selection approach to examine which oceanographic factor best explained shifts in values following acid treatment. RESULTS: Acidification resulted in statistically significant reductions in both δ13 C and δ15 N values, but the effect was relatively small. The model that best explained the differences between acidified and non-acidified δ13 C values included depth, salinity and sea-surface temperature at the sampling point. A regression of acidified on non-acidified δ13 C values shows that the treatment effect was strongest on samples more depleted in 13 C. CONCLUSIONS: The differences between δ13 C and δ15 N values in acidified and non-acidified samples are linear and predictable. This implies that the nature of the POM and its possible alteration during the acid treatment are important factors that support the reliable determination of the values of δ13 C and δ15 N of POM.

Clarifying a trophic black box: stable isotope analysis reveals unexpected dietary variation in the Peruvian anchovy Engraulis ringens.

BACKGROUND: Small fishes play fundamental roles in pelagic ecosystems, channelling energy and nutrients from primary producers to higher trophic levels. They support globally important fisheries in eastern boundary current ecosystems like the Humboldt Current System (HCS) of the SE Pacific (Chile and Peru), where fish catches are the highest in the world (per unit area). This production is associated with coastal upwelling where fisheries target small pelagic fishes including the Peruvian anchovy (Engraulis ringens). The elevated biomass attained by small pelagics is thought to reflect their low trophic position in short/simple food chains. Despite their global importance, large gaps exist in our understanding of the basic ecology of these resources. For instance, there is an ongoing debate regarding the relative importance of phytoplankton versus animal prey in anchovy diet, and ecosystem models typically assign them a trophic position (TP) of ∼2, assuming they largely consume phytoplankton. Recent work based on both relative energetic content and stable isotope analysis (SIA) suggests that this value is too low, with δ 15N values indicating that anchovy TP is ca. 3.5 in the Peruvian HCS. METHODS: We characterised the trophic ecology of adult anchovies (n = 30), their putative prey and carnivorous jack mackerel (n = 20) captured from N Chile. SIA (δ 13C and δ 15N) was used to estimate the relative contribution of different putative prey resources. δ 15N was used to estimate population level trophic position. RESULTS: Anchovies showed little variability in δ 13C (-18.7 to -16.1‰) but varied greatly in δ 15N (13.8 to 22.8‰)-individuals formed two groups with low or high δ 15N values. When considered as a single group, mixing models indicated that anchovy diet was largely composed of zooplankton (median contribution: 95% credibility limits), with major contributions of crustacean larvae (0.61: 0.37-0.77) and anchovy (preflexion) larvae (0.15: 0.02-0.34), and the assimilation of phytoplankton was negligible (0.05: 0-0.22). The modal (95% credibility limits) estimate of TP for the pooled anchovy sample was 3.23 (2.93-3.58), overlapping with recent SIA-based estimates from Peru. When the two δ 15N groups were analysed separately, our results indicate that the lower δ 15N group largely assimilated materials from crustacean larvae (0.73: 0.42-0.88), with a TP of 2.91 (2.62-3.23). Mixing models suggested high δ 15N anchovies were cannibalistic, consuming anchovy preflexion larvae (0.55: 0.11-0.74). A carnivorous trophic niche was supported by high TP (3.79: 3.48-4.16), mirroring that of carnivorous juvenile jack mackerel (Trachurus murphyi; 3.80: 3.51-4.14). Our results support recent conclusions regarding high TP values of anchovy from Peru and reveal new insights into their trophic behaviour. These results also highlight the existence of cryptic trophic complexity and ecosystem function in pelagic food webs, classically considered as simple.