Stable Isotope Analysis Research Articles

Mammoth featured heavily in Western Clovis diet.

Ancient Native American ancestors (Clovis) have been interpreted as either specialized megafauna hunters or generalist foragers. Supporting data are typically indirect (toolkits, associated fauna) or speculative (models, actualistic experiments). Here, we present stable isotope analyses of the only known Clovis individual, the 18-month-old Anzick child, to directly infer maternal protein diet. Using comparative fauna from this region and period, we find that mammoth was the largest contributor to Clovis diet, followed by elk and bison/camel, while the contribution of small mammals was negligible, broadly consistent with the Clovis zooarchaeological record. When compared with second-order consumers, the Anzick-1 maternal diet is closest to that of scimitar cat, a mammoth specialist. Our findings are consistent with the Clovis megafaunal specialist model, using sophisticated technology and high residential mobility to subsist on the highest ranked prey, an adaptation allowing them to rapidly expand across the Americas south of the Pleistocene ice sheets.

A Simple Method for Triple Stable Isotope Analysis of Cellulose, Sugar, and Bulk Organic Matter-Advances and Limitations.

Determining several isotope ratios in one analysis multiplies the information that can be retrieved from a sample in a cost-efficient way. The stable isotope ratios of hydrogen (δ2H), carbon (δ13C), and oxygen (δ18O) in organic compounds are highly relevant due to their complimentary hydroclimatic and physiological signals. Different types of organic material reflect different processes and integration times, like short term in leaf sugars and long term in tree ring cellulose, but currently, no simple method exists for their triple isotope analysis. Here, we present a method that enables the isotopic analyses of the three elements H, C, and O in one run and is applicable to different types of carbohydrates and bulk organic matter. We discuss all steps required from water vapor equilibration necessary for obtaining reliable δ2H values of carbon-bound H to high-temperature conversion (HTC) of the sample to CO and H2 and to the mass-spectrometric isotope-ratio analysis. We show that reliable triple isotope analysis is possible for a large range of samples, although it results in some reduction of precision compared to individual isotope analysis. Important considerations are the equilibration procedure, the type of autosampler, selection of HTC reactor, the influence of nitrogen in the sample, the verification of δ13C values obtained by HTC versus combustion, and the selection of reference materials. By presenting a relatively simple triple-isotope method, we promote the use of multi-isotope studies in environmental sciences, which helps in addressing many important climate and ecological research challenges that we face today.

Inferring long‐distance movements of insects using combined hydrogen isotope and genetic analyses: A case study of the African edible bush‐cricket

Abstract The degree to which individuals within populations vary in whether, how far and what direction they disperse is central in order to understand many foundational questions in ecological entomology, including factors determining population and invasion dynamics, species' ability to respond to climate change or when designing conservation strategies for threatened species. This study combined stable hydrogen isotope analysis of nymphal and adult wing chitin with population genetic analysis of the cytochrome c oxidase subunit I (COI) gene to understand long‐distance movements of African edible bush cricket (Ruspolia differens) in East Africa. Results revealed that at most 77% of non‐swarming adults and 85% swarming adults originated locally, that is, within a range of a few hundred kilometres. In contrast, non‐local adults captured in Uganda likely originated from Ethiopia, Kenya or South Sudan based on the H isotopic evidence. Genetic analysis revealed 150 COI haplotypes with no significant differentiation among geographic source regions and no evidence of genetic isolation by movement distance. The observed frequent long‐distance movements, together with the historical records, which indicate that the swarm movements can be irregular in their direction, suggest that the movements represent nomadism rather than regular long‐distance migration. Our results thus provide a valuable case study of what characterizes nomadic movements in insects, which have been one of the outstanding open questions in movement ecology of animals.

Fishing or farming? Isotopic evidence of human subsistence strategies at the Dashuitian site during the middle Neolithic in the Three Gorges of the Yangtze River, China

The Three Gorges of the Yangtze River, with its deep canyons, abundant freshwater resources, and unique geographic location at the crossroads in the spread of rice-millet agriculture in the Middle Neolithic, is of great significance for understanding the interaction between fishing-hunting-gathering and farming in inland freshwater environments. However, few direct evidence for human subsistence strategies had been published in this region. This study presents results of carbon and nitrogen stable isotope analyses on human bones, animal and millet remains from the Dashuitian site (c. 6000–5500 BP) in the Three Gorges of the Yangtze River, China, to investigate the diet and subsistence and intrapopulation dietary variations in this region. The results indicate that the humans at the site consumed mostly freshwater fish foods and supplemented by terrestrial animals, with no discernable input from millets. They had consistently relied on a fishing, hunting, and gathering economy during the occupation of the site in the Middle Neolithic (c. 6000–5500 BP), differing from humans in other areas especially along the rivers and coasts of the lower Yangtze River valley for the roughly same period, further showing the extensive utilization of and adaptation to the available environmental resources. In addition, intrapopulation dietary variations based on burial style and sex provide evidence that differentiation and inequality, at least regarding to food consumption, existed at the Three Gorges region during the Middle Neolithic. Here, our findings provide isotopic evidence about the Middle Neolithic human subsistence strategies in the Three Gorges of the Yangtze River, providing a glimpse into the complexity related to inequality in food access among fisher-hunter-gatherers in inland areas, and new insights into understanding past human-environment interactions.

Lithification of pelletal grains by microbially mediated organomineralization

Fecal pellets are key components of the biological carbon pump and a source of nutrients. When cohesively bound and cemented, they become the most abundant non-skeletal grains in shallow-water carbonates and an important nucleus for ooid formation. Herein, we describe the mineralization processes contributing to pellet cementation using scanning electron microscopy in conjunction with stable isotope and mineralogical analyses of the bulk sediments on Great Bahama Bank, known for its abundance of peloids and fecal pellets. Results show biogenic signatures consistent with the initial stages of organomineralization. These include, curved-tubular filaments and bacilli bacteria associated with extracellular polymeric substances (EPS); fibrils of EPS binding aragonite needles; and the coexistence of decayed EPS with amorphous calcium carbonate that once it develops, coalesces and crystalizes into aragonite. While δ13Cinorg do not show large variations, slightly higher values are observed in areas where pellets are indurated, likely attributed to photosynthetic activity on the pellets. The δ13Corg values tend to be more elevated in indurated pellets, suggesting that heterotrophs are preferentially using enriched carbon sources. In contrast, friable pellets from nearshore areas show more negative δ13Corg values, suggesting microbial exposure to depleted C3 and CAM plant sources. Fingerprints of nitrogen fixation and denitrification are also documented and supported by δ15Norg values. Altogether, these findings suggest that EPS and a microbial consortium, including gut-microbial flora and free-living surface colonizers, are responsible for inducing early cements, which are later complemented by secondary cements. Microorganisms involved in pellet lithification may subsequently also aid in ooid cortex development.

Large-Volume Injection and Assessment of Reference Standards for n-Alkane δD and δ13C Analysis via Gas Chromatography Isotope Ratio Mass Spectrometry.

Compound-specific stable isotope analysis of hydrogen (δD) and carbon (δ13C) in organic compounds is a valuable tool in biogeochemical research. A key limitation of this method is the relatively large amount of sample required to achieve desirable precision. We developed a large-volume (20 μL) injection method that allows for high throughput analysis of less concentrated samples and tested it for δ13C and δD measurements of n-alkanes. We also conducted a comparison of reference standards and assessed several methods to normalize and correct n-alkane δD and δ13C measurements. The mean precision of the δD method based on 233 environmental n-alkane samples (two to three replications per sample) is 4.0‰ (1σ, estimated from the weighted mean of the pooled unbiased standard deviations) and 0.46‰ (1σ) for δ13C from 37 environmental samples (two to three replications per sample). The evaluation of reference standards shows that the use of n-alkane standards with large offsets in δD values in adjacent n-alkane chains can lead to biases in measurement correction. The large-volume injection method shows good reproducibility of δ13C and δD measurements of n-alkanes and reduces the required sample concentration by about 80%. We propose that for δD measurements, a reference standard set should be used in which each reference standard has a limited range of δD values and no adjacent n-alkane chains, to minimize memory effects.

A Deep Dive into the Trophic Ecology of Engraulis ringens: Assessing Diet Through Stomach Content and Stable Isotope Analysis

Our study investigates the trophic ecology of the anchoveta (Engraulis ringens). The anchoveta plays a key role in the Greater Humboldt Ecosystem and is extensively exploited by countries from the south-eastern Pacific Ocean. For a comprehensive study of trophic ecology, we employed a combined approach that included stomach content analysis, stable isotope analysis, and scaled mass index of body condition. Our results showed that the multivariate composition of the diet varies significantly between life stage and fishing zones in Chile. Copepods and euphausiids emerged as the dominant prey found in the stomachs across all fisheries zones. Stable isotope analysis revealed significant differences among different zones. The scaled mass index values were higher in the northern zone compared to the southern zones, for both juveniles and adults. This research carries significant implications for fisheries management and conservation efforts, such as the development of targeted management strategies that address variations in the trophic structure of anchoveta across different life stages and fishing zones.

Hydrochemical and Isotopic Characterization of Groundwater in the Nakivale Sub-Catchment of the Transboundary Lake Victoria Basin, Uganda

This study characterized groundwater resources for the Nakivale sub-catchment of the transboundary Victoria Basin in Uganda using classical hydrochemical and stable isotopic approaches. Groundwater in the study area is essential for domestic, agricultural, and industrial uses. As a sub-domain of the larger Victoria Basin, it also plays a crucial role in shaping the hydrological characteristics of this vital transboundary basin, both in terms of quality and quantity fronts. This makes its sustainable management and development vital. The predominant groundwater type is Ca-SO4, with other types including Ca-HCO3, Na-Cl, Na-HCO3, and Ca-Mg-SO4-Cl. Hydrochemical facies analysis highlights the importance of rock–water interactions in controlling groundwater chemistry, mainly through incongruent chemical weathering of Ca-rich plagioclase feldspars and the oxidation of sulfide minerals, such as pyrite, which are prevalent in the study area. Groundwater recharge is primarily influenced by the area’s topography, with recharge zones characterized by lineament networks, located in elevated areas. Stable isotope analyses indicate that groundwater mainly originates from local precipitation, while tritium data suggest the presence of both recent and older groundwater (likely over 20 years old). The study’s comprehensive approach and findings contribute significantly to the understanding of groundwater systems in the region, thus providing valuable insights for policymakers and stakeholders involved in water resource management and development strategies.

Interspecific relationships in a suboptimal habitat, an unexpected ally for the survival of the threatened Tenerife speckled lizard (Gallotia intermedia)

Anthropogenic-driven environmental changes are pushing species to the limits of their habitats. More often species are restricted to relic or suboptimal habitats that present the minimum requirements to sustain species populations. In this scenario of accelerated environmental change and biodiversity loss, is fundamental to understand why species can survive in such suboptimal conditions. In this study, we show how the Tenerife speckled lizard (Gallotia intermedia), a critically endangered endemic reptile from the Canary Islands, can maintain stable populations in relic habitats thanks to its interactions with the yellow-legged gull (Larus michahellis) colony. A stable isotope analysis revealed that G. intermedia relies on marine subsidies for its diet and that the foraging area of this reptile is likely to be restricted to the limits of L. michahellis breeding colony. Furthermore, an antipredator behaviour analysis showed that L. michahellis displayed a strong anti-predator or mobbing response against cats, one of the main threats for G. intermedia, thus potentially providing some protection to the reptiles inhabiting the seabird colony. Our results show how unusual and poorly studied biotic interactions can provide valuable resources and conditions for the conservation of a critically endangered species inhabiting a suboptimal or relict habitat.

Forage ecology of Neotropical fish in Brazilian biomes using stable isotopes

Neotropical regions are responsible for harboring most of the global diversity of freshwater fish, providing essential ecosystem services for society. Human activities (e.g., land use changes) jeopardize aquatic ecosystems as well as species, communities, etc., impairing ecosystem services. We investigate the impact of human disturbance on the foraging ecology of Neotropical freshwater fishes across five Brazilian biomes by stable isotope analysis. We analyzed carbon (δ13C) and nitrogen (δ15N) isotopic compositions of Neotropical fishes, sourced from the SIA-BRA dataset. Fishes were categorized into herbivores, omnivores, and carnivores. We tested the correlation between human disturbances, indicated by the human disturbance index (hdi), and changes in fish diets. We expected that the assimilation of C4‑carbon from exotic forage would increase with higher disturbance levels, while δ15N levels would similarly rise due to nitrogen input from anthropogenic sources. We found increases in fish δ13C with human disturbance increases, suggesting greater assimilation of C4 carbon in places where native vegetation was replaced by C4 sources, confirmed by isotopic mixing models. In contrast, δ15N values did not show a significant relationship with human disturbance, probably due to the complex interactions and multiple sources of nitrogen in disturbed environments. Our finds suggest that stable isotope analysis provides a powerful tool for monitoring the effects of landscape changes on aquatic food webs. Particularly, the δ13C values of detritivorous fish that feed on C4 plant detritus could serve as bioindicators of environmental degradation. However, a specific isotopic characterization of each site would be valuable for more accurate niche information.

Formation Mechanism of Muji Travertine in the Pamirs Plateau, China

The Muji spring travertines, located in the Muji Basin in the eastern Pamirs Plateau, represent a typical spring deposit found on plateaus that is characterized by arid and semi-arid climatic conditions. However, its formation mechanisms remain poorly understood. This study aims to explore the recharge processes of the spring, the sedimentary environment, and the genetics of Muji spring travertines through a comparative analysis of conventional hydrochemistry, H-O stable isotope analysis of both spring and river water, and petrographic observation, as well as in situ analysis of major and trace elements present in calcite within travertines. The basin is surrounded by mountains with a topography that facilitates groundwater convergence within it. Carbonate-bearing strata are extensively developed around the basin, which serves as a crucial material foundation for travertine development. It infiltrates underground through fractures and faults, interacting with carbonate rocks to produce significant amounts of HCO3−, Ca2+, and Mg2+. The observed range of isotopic compositions (δ2H, −102.27‰ to −96.43‰; δ18O, −14.90‰ to −14.36‰) in water samples suggests that their primary origin was from glacial and snowmelt sources. The concentration of HCO3− in spring water samples exhibits significant variability, with the highest value being 1646 mg·L−1, which deviates significantly from the typical composition of karst groundwater. During its migration, groundwater undergoes the dissolution of gaseous CO2 derived from deep metamorphic processes, leading to variable degrees of mixing with geothermal groundwater containing elevated concentrations of dissolved components that enhance the dissolution potential of carbonate rocks. Eventually, upwelling occurs along the Southwestern Boundary Fault of Muji, resulting in the formation of linear springs characterized by CO2 escape. The Muji laminated travertines exhibit distinct white and dark laminae, and radial coated grains consisting of micritic and sparry layers. Chemical composition analyses reveal significant differences in the trace and rare-earth element composition, as well as the Mg/Ca ratio, of the two types of travertines. Specifically, the micritic laminae of the pisoid (Mg/Ca = 0.019; Sr = 530 × 10−6; Ba = 64.6 × 10−6) and the dark laminae of the laminated travertine (Mg/Ca = 0.014; Sr = 523 × 10−6; Ba = 48.1 × 10−6) exhibit generally higher Mg/Ca ratios and Sr, Ba contents than the neighboring sparry laminae (Mg/Ca = 0.012; Sr = 517 × 10−6; Ba = 36.6 × 10−6) and white laminae (Mg/Ca = 0.006; Sr = 450 × 10−6; Ba = 35.6 × 10−6). The development of laminated travertines and radial coated grains here is attributed to periodic changes in groundwater recharge induced by seasonal temperature fluctuations, as evidenced by the structural characteristics of the two types of travertines and the trace element analysis of different layers. Algae play a role in forming the dark laminae of laminated travertines and the micritic laminae of pisoids.

A Stable Isotope Analysis to Quantify the Contribution of Basal Dietary Sources to Food Webs of Drinking Water Reservoirs

This study investigates the food web structure of the Xinlicheng Reservoir, a drinking water source of critical importance in Changchun, China, by employing stable isotope analysis (SIA) to quantify the contribution ratios of four basal dietary sources—phytoplankton, zooplankton, sediment organic matter, and particulate organic matter (POM)—to the diets of two key filter-feeding fish species, Hypophthalm ichthys molitrix and Aristichthys nobilis. The analysis reveals that phytoplankton is the dominant dietary source for both species, contributing 32.08% and 34.06%, respectively, whereas the POM contribution is discernably lower (13.25%). The average trophic level of the fish assemblage in Xinlicheng Reservoir is 3.03, while the trophic levels of the two filter-feeding species lie between 3.00 and 3.50. Furthermore, a random forest model was used to identify key environmental drivers of isotopic variations in these basal dietary sources, highlighting the significant role of pH, total nitrogen (TN), chloride (Cl−), calcium (Ca2+), phosphorus (TP), and silicate (SiO44−) in influencing carbon and nitrogen isotopic ratios. These findings provide critical insights to optimize biomanipulation strategies aimed at improving water quality in drinking water reservoirs by enhancing our understanding of the environmental factors that govern trophic interactions and broader food web dynamics.

New bass on the block: Trophic interactions among invasive largemouth bass Micropterus salmoides and local sportfish.

Novel introductions of largemouth bass, Micropterus salmoides, often cause negative impacts on endemic populations of prey fishes and interspecific competitors. Although many studies have investigated trophic interactions between M. salmoides and smallmouth bass, Micropterus dolomieu, few have included chain pickerel, Esox niger, as a competitor despite similarities in their habitat use. We used stable isotope analysis to investigate the trophic ecology of a recently introduced population of M. salmoides in the Wolastoq|Saint John River. Specifically, we measured carbon (δ13C) and nitrogen (δ15N) isotopes to describe isotopic niches and infer resource use of introduced M. salmoides, M. dolomieu, E. niger, and native yellow perch, Perca flavescens, at various life stages. Our results showed that isotopic niche overlap occurred between M. salmoides and other study species, indicative of resource competition. However, resource use plasticity, as demonstrated by a wide variety of resources (marine, terrestrial, littoral, and pelagic) used by all study species, could potentially reduce interspecific competition. Our findings indicate that competition is highest between adult M. salmoides and E. niger, but the presence of a seasonal marine resource can provide important contributions to diets and potentially reduce competition. Further research should focus on monitoring of long-term trends to identify the dynamics of all study species as M. salmoides populations become further established and dispersed.

Stable isotope analyses of lacustrine chitinous invertebrate remains: Analytical advances, challenges and potential

This review examines recent developments in the application of stable isotope analyses (δ18O, δ13C, δ15N, δD) to lacustrine invertebrate remains. These remains are ubiquitous in lacustrine sediments and thus provide an opportunity to measure changes in stable isotope ratios across a range of timescales and environments and allow interpretive power beyond taxonomic studies. To date they have been relatively understudied in comparison to carbonate fossils and offer both opportunities and challenges and we explore both themes in this review. This review will explore improvements to analytical instrumentation and the opportunities that this presents, it will look at a range of new studies of the modern lacustrine environment and how these studies allow a more nuanced palaeoenvironmental approach. We review recent studies that have used these advancements in understanding to help to reveal new knowledge of past climates, environments and ecology. In addition, we explore new studies that help to elucidate the role of methane-derived carbon to lacustrine food webs and the drivers behind this, including new data to estimate the contribution of methane derived carbon to an arctic lake. We conclude that major progress is currently being made in invertebrate-isotope analyses, and we expect this to continue apace.

Comparison of zooplankton assimilation of different carbon sources and fatty acids in a eutrophic lake and its restored basins

The ecological restoration of eutrophic lakes significantly influences the food composition of zooplankton. Zooplankton serve as the principal trophic link, transferring energy from phytoplankton to fish. Understanding the alterations in zooplankton carbon source compositions following ecological restoration and the seasonal variations in this relationship is crucial. This study employs stable carbon isotope (δ13C) and fatty acid (FA) analyses to investigate the seasonal changes in carbon source contributions to zooplankton between the restored and unrestored segments of Lake Xuanwu. Results from FA analysis reveal higher proportions of algal dietary markers in zooplankton FAs in both segments during spring and autumn. Summer exhibits a shift with zooplankton utilizing more bacterial FAs in the restored part and more algal FAs in the unrestored part. While approaching winter, zooplankton in the restored part consume more algal FAs but less in the unrestored part. Zooplankton FAs enriched in δ13C are associated with assimilation of more terrestrial carbon, contrasting with depleted δ13C when zooplankton relies more on phytoplankton. Isotope mixing models indicate a substantial contribution of terrestrial carbon to zooplankton carbon sources, especially in autumn (42.3% unrestored, 51.2% restored) and winter (41.4% unrestored, 36.8% restored), while phytoplankton has a higher contribution in summer (34.5% restored, 46.9% unrestored). These findings contribute to a comprehensive understanding of carbon cycling variations in food webs between eutrophic lakes and ecologically restored lakes.

Enantiomer-Specific Stable Carbon and Nitrogen Isotopic Analyses of Underivatized Individual l- and d-Amino Acids by HPLC + HPLC Separation and Nano-EA/IRMS.

We developed a new method for stable carbon and nitrogen isotopic (δ13C and δ15N) analysis of underivatized amino acid (AA) enantiomers simultaneously, based on high-performance liquid chromatography (HPLC) separation and off-line isotopic measurement. l- and d-Enantiomers of each AA were isolated using a ReproSil Chiral-AA column, purified by wet chemical procedure, and analyzed for δ13C and δ15N values with a nanomol-scale elemental analyzer/isotope-ratio mass spectrometry (nano-EA/IRMS) system. We successfully achieved the separation of l- and d-enantiomers of 15 proteinogenous AAs, with all l-enantiomers eluting before respective d-enantiomers. The δ13C and δ15N values of AA enantiomers were consistent before and after HPLC separation, demonstrating that this analytical method conserves isotopic information. By coupling this column with a multidimensional HPLC system for isolating individual AAs, we analyzed l- and d-AAs in a natural sample, peptidoglycan isolated from Gram-positive bacterium Bacillus subtilis. Results show a surprisingly large 15N-depletion, up to 20‰, in d-glutamic acid relative to its l-counterpart. The first example, to our knowledge, of δ13C and δ15N analyses of underivatized AA enantiomers is expected to contribute to various research areas in the future.

Plant organic nitrogen nutrition: costs, benefits, and carbon use efficiency.

Differences in soil mobility and assimilation costs between organic and inorganic nitrogen (N) compounds would hypothetically induce plant phenotypic plasticity to optimize acquisition of, and performance on, the different N forms. Here we evaluated this hypothesis experimentally and theoretically. We grew Arabidopsis in split-root setups combined with stable isotope labelling to study uptake and distribution of carbon (C) and N from l-glutamine (l-gln) and NO3 - and assessed the effect of the N source on biomass partitioning and carbon use efficiency (CUE). Analyses of stable isotopes showed that 40-48% of C acquired from l-gln resided in plants, contributing 7-8% to total C of both shoots and roots. Plants grown on l-gln exhibited increased root mass fraction and root hair length and a significantly lower N uptake rate per unit root biomass but displayed significantly enhanced CUE. Our data suggests that organic N nutrition is linked to a particular phenotype with extensive growth of roots and root hairs that optimizes for uptake of less mobile N forms. Increased CUE and lower N uptake per unit root growth may be key facets linked to the organic N phenotype.

Salps in the NW Atlantic Slope Water: metabarcoding and compound-specific stable isotope analysis of diet diversity and trophic interactions

Salps in the NW Atlantic Slope Water: metabarcoding and compound-specific stable isotope analysis of diet diversity and trophic interactions

Calibrations without raw data-A response to "Seasonal calibration of the end-cretaceous Chicxulub impact event".

A recent article by DePalma et al. reported that the season of the End-Cretaceous mass extinction was confined to spring/summer on the basis of stable isotope analyses and supplementary observations. An independent study that was concurrently under review reached a similar conclusion using osteohistology and stable isotope analyses. We identified anomalies surrounding the stable isotope analyses reported by DePalma et al. Primary data are not provided, the laboratory where the analyses were performed is not identified, and the methods are insufficiently specified to enable accurate replication. Furthermore, isotopic graphs for carbon and oxygen contain irregularities such as missing data points, duplicate data points, and identical-length error bars for both elements despite different scales, that appear inconsistent with laboratory instrument outputs. A close examination of such methodological omissions and data irregularities can help to raise the standards for future studies of seasonality and prevent inaccurate claims or confirmation bias.

Stable isotopes disentangle niche partitioning and co‐occurrence in a multi‐species marine mutualism

Ecologists have long sought general explanations for the co‐occurrence of ecologically similar taxa. Niche theory explains co‐occurrence via functional differences among taxa that reduce competition and promote resource partitioning. Alternatively, the unified neutral theory of biodiversity and biogeography suggests that co‐occurrence can be attributed to stochastic processes, and thus, presupposes that ecologically similar species that occur in sympatry are functionally analogous. We test these hypotheses using the most diverse crustacean‐sea anemone symbiosis from coral reefs in the Tropical Western Atlantic. δ13C and δ15N stable isotope analyses of six crustacean symbionts that co‐occur around the host anemone Bartholomea annulata exhibit highly differentiated isotopic niche space spanning several trophic levels. As multiple crustacean species within the symbiosis have been documented as cleaners that remove parasites from reef fishes, we extended our investigation into the broader cleaner community. Our stable isotope analyses of cleaners shows that Pederson's cleaner shrimp Ancylomenes pedersoni exhibits the highest δ15N isotopic values‐ significantly higher than all other putative cleaner species and consistent with expectations of a dedicated cleaning lifestyle. However, for other species previously described or observed to clean reef fishes, including Periclimenes yucatanicus, Stenopus hispidus and Stenorhynchus seticornis, δ15N isotopic values were substantially lower, raising questions about the degree to which these species rely on cleaning interactions to meet their nutritional requirements. Taken together, our data are consistent with the expectations of niche theory: co‐occurring symbiotic crustaceans have highly partitioned niche space with low levels of functional redundancy.