Evaluating the utility of stable isotope analyses of archived freshwater sample materials

We evaluated the potential utility of stable isotope analysis of tissues commonly archived by aquatic biologists. Previous studies with chemically preserved samples have shown contradictory results, which present an obstacle for the use of archived sample materials. We tested the effects of ethanol and formalin preservation on zooplankton and of ethanol on benthic macroinvertebrate δ¹³C and δ¹⁵N values. We found that neither formalin nor ethanol had a significant effect on δ¹³C and δ¹⁵N values of preserved zooplankton. Nor did ethanol significantly affect δ¹³C or δ¹⁵N values of macroinvertebrates. However, ethanol preservation slightly, but significantly decreased C:N ratios of both zooplankton and macroinvertebrates, probably reflecting some extraction of lipids. Overall, the effects of preservatives on δ¹³C and δ¹⁵N values that we observed were minor. We also compared δ¹³C and δ¹⁵N values analysed from roach scales and perch operculum bones with those analysed from muscle tissue. Decalcification of scales and operculum bones only slightly improved our comparison to muscle tissue δ¹³C and δ¹⁵N values. Decalcified scales had slightly higher δ¹³C and lower δ¹⁵N values. Similarly, decalcified operculum bones showed slightly increased δ¹³C and decreased δ¹⁵N values to those for fish muscle. Our results confirm that scales and operculum bones can provide a suitable proxy for fish muscle in isotope studies with minor correction. We conclude that various archived sample materials can indeed be used with confidence for historical reconstructions of freshwater food webs by stable isotope analysis. Handling editor: K. Martens     

The importance of quantifying inherent variability when interpreting stable isotope field data

Stable isotope data are often used to assess diet, trophic level, trophic niche width and the extent of omnivory. Notwithstanding ongoing discussions about the value of these approaches, variations in isotopic signatures among individuals depend on inherent variability as well as differences in feeding habitats. Remarkably, the relative contributions of diet variation and inherent variability to differences in δ¹⁵N and δ¹³C among individuals have not been quantified for the same species at the same life history stages, and inherent variability has been ignored or assumed. We quantified inherent variability in δ¹³C and δ¹⁵N among individuals of a marine fish (the European sea bass, Dicentrarchus labrax) reared in a controlled environment on a diet of constant isotopic composition and compared it with variability in δ¹³C and δ¹⁵N among individuals from wild bass populations. The analysis showed that inherent variability among reared individuals on a controlled diet was equivalent to a large proportion of the observed variability among wild individuals and, therefore, that inherent variability should be measured to establish baseline variability in wild populations before any assumptions are made about the influence of diet. Given that inherent variability is known to be dependent on species, life history stage and the environment, our results show that it should be quantified on a case-by-case basis if diet studies are intended to provide absolute assessments of dietary habits.     

Benthos as the basis for arctic lake food webs

Plankton have traditionally been viewed as the basis for limnetic food webs, with zooplankton acting as a gateway for energy passing between phytoplanktonic primary producers and fish. Often, benthic production has been considered to be important primarily in shallow systems or as a subsidy to planktonic food web pathways. Stable isotope food web analyses of two arctic lakes (NE14 and I minus) in the Toolik Lake region of Alaska indicate that benthos are the primary source of carbon for adults of all species of benthic and pelagic fish present. We found no effect of turbidity, which may suppress benthic algae by shading, on food web structure. Even though Secchi transparency varied from 10.2 m in NE14 to 0.55–2.6 m in I minus, food webs in both lakes were based upon benthos, had four trophic levels, and culminated with omnivorous lake trout. We suggest that the importance of benthos in the food webs of these lakes is due to their extreme oligotrophy, resulting in planktonic resources that are insufficient for the support of planktivorous consumers.     

Isotopic metrics as a tool for assessing the effects of mine pollution on stream food webs

Most tools used to assess pollution impacts are based on structural, or less frequently, functional aspects of biotic communities. However, the application of measures that take a food web approach to understand the effects of stress on stream ecosystems offers a new perspective and promising insights. We assessed quantitative isotopic metrics, which describe characteristics of food web structure, as indicators of acid mine drainage (AMD) in 12 streams along a stress gradient and compared these metrics with traditional structural and functional metrics. The gradient ranged from highly stressed (pH < 3) streams with elevated concentrations of dissolved metals (Fe and Al) to moderately acidic streams (pH 3.6–4.9) with substrata coated in metal hydroxide precipitates and circumneutral reference streams. Key differences in food web structure were detected by the isotopic metrics. Specifically, fewer trophic levels and reduced trophic diversity characterized food webs in all mining impacted streams but the differences were not significant along the gradient. In contrast, most structural and functional metrics were significant predictors of AMD as stress increased. Therefore, our results suggest that isotopic metrics offer little advantage over traditional metrics in terms of detecting impacts for biomonitoring purposes. However, they do provide additional insights into how whole food webs are disrupted, and are likely to be more useful for guiding stream management and rehabilitation strategies.     

Combining sources in stable isotope mixing models: alternative methods

Stable isotope mixing models are often used to quantify source contributions to a mixture. Examples include pollution source identification; trophic web studies; analysis of water sources for soils, plants; or water bodies, and many others. A common problem is having too many sources to allow a unique solution. We discuss two alternative procedures for addressing this problem. One option is a priori to combine sources with similar signatures so the number of sources is small enough to provide a unique solution. Aggregation should be considered only when isotopic signatures of clustered sources are not significantly different, and sources are related so the combined source group has some functional significance. For example, in a food web analysis, lumping several species within a trophic guild allows more interpretable results than lumping disparate food sources, even if they have similar isotopic signatures. One result of combining mixing model sources is increased uncertainty of the combined end-member isotopic signatures and consequently the source contribution estimates; this effect can be quantified using the IsoError model (). As an alternative to lumping sources before a mixing analysis, the IsoSource mixing model () can be used to find all feasible solutions of source contributions consistent with isotopic mass balance. While ranges of feasible contributions for each individual source can often be quite broad, contributions from functionally related groups of sources can be summed a posteriori, producing a range of solutions for the aggregate source that may be considerably narrower. A paleohuman dietary analysis example illustrates this method, which involves a terrestrial meat food source, a combination of three terrestrial plant foods, and a combination of three marine foods. In this case, a posteriori aggregation of sources allowed strong conclusions about temporal shifts in marine versus terrestrial diets that would not have otherwise been discerned.     

The role of alien fish (the centrarchid Micropterus salmoides) in lake food webs highlighted by stable isotope analysis

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Trophic structure of common marine species in the Bohai Strait,North China Sea,based on carbon and nitrogen stable isotope ratios

Trophic relationships among common coastal species in the Bohai Strait, North China Sea, were investigated in this study using stable isotope ratios of carbon (¹³C/¹²C) and nitrogen (¹⁵N/¹⁴N) as tracers. During the research cruises of this study, 16 species of invertebrates and 18 species of fishes were collected, as were various food sources (phytoplankton, macroalgae, and sediment). The carbon and nitrogen isotope ratios of all collected samples were measured and used for trophic level analyses. The results showed that the grazers Oregonia gracilis, Notoacmea schrenckii, Chlorostoma rustica, and Chelon affinis, preferentially consumed Zostera marina (p < 0.01), whereas Anthocidaris crassispina preferred Ulva conglobata (p < 0.01). Two trophic models based on nitrogen isotope ratios were built to identify the trophic level of each species. The model that combined food sources was more appropriate than the model that used a single primary producer to identify the relative trophic positions of primary consumers. Scombermorus niphonius, an important fishery resource, was at the top trophic level. The seastar Asterias amurensis was at the highest level among the invertebrates and directly threatened production of shellfish. Based on the trophic level and food source relationships identified in this study, we gave some advice for ecological restoration, such as optimizing the structure of food source distribution, limiting seastar numbers, and improving the applicability of habitat for fishery species.     

Dual isotope analyses indicate efficient processing of atmospheric nitrate by forested watersheds in the northeastern U.S.

Nitrogen from atmospheric deposition serves as the dominant source of new nitrogen to forested ecosystems in the northeastern U.S. By combining isotopic data obtained using the denitrifier method, with chemical and hydrologic measurements we determined the relative importance of sources and control mechanisms on nitrate (NO₃ ⁻) export from five forested watersheds in the Connecticut River watershed. Microbially produced NO₃ ⁻ was the dominant source (82–100%) of NO₃ ⁻ to the sampled streams as indicated by the δ¹⁵N and δ¹⁸O of NO₃ ⁻. Seasonal variations in the δ¹⁸O–NO₃ ⁻ in streamwater are controlled by shifting hydrologic and temperature affects on biotic processing, resulting in a relative increase in unprocessed NO₃ ⁻ export during winter months. Mass balance estimates find that the unprocessed atmospherically derived NO₃ ⁻ stream flux represents less than 3% of the atmospherically delivered wet NO₃ ⁻ flux to the region. This suggests that despite chronically elevated nitrogen deposition these forests are not nitrogen saturated and are retaining, removing, and reprocessing the vast majority of NO₃ ⁻ delivered to them throughout the year. These results confirm previous work within Northeastern U.S. forests and extend observations to watersheds not dominated by a snow-melt driven hydrology. In contrast to previous work, unprocessed atmospherically derived NO₃ ⁻ export is associated with the period of high recharge and low biotic activity as opposed to spring snowmelt and other large runoff events.     

The diet of great tit nestlings: Comparing observation records and stable isotope analyses

The diet of wild animals has been studied using many different strategies, approaches and methods in recent decades. In this regard, stable isotopes analysis (SIA) is becoming a widespread tool, but no study has yet, to our knowledge, compared diet estimations from SIA with direct observations of the diet of passerine nestlings. Accordingly, our aim was to test the predictive power of SIA for this purpose and identify potential confounding factors such as habitat effects. To do this, we compared isotopic signatures of δ¹³C and δ¹⁵N in the feathers of great tit (Parus major) nestlings, and the corresponding estimates of their diet based on stable isotope mixing models, with prey proportions delivered by their parents obtained through video-recordings. Between-nest differences in isotopic signatures of δ¹⁵N were larger than within-nest differences. We found that δ¹⁵N signatures of nestling feathers correlated positively with the proportion of spiders and negatively with the proportion of caterpillars in the nestlings’ diet, the most important prey types. On the other hand, between-nest and within-nest differences in δ¹³C ratios were of similar magnitude and δ¹³C ratios correlated mainly with the proportion of trees surrounding nest-boxes that were Quercus spp. Estimates of diet composition based on mixing models correlated with the observed nestling diet, yet effect sizes were quite low. Although mixing models are commonly used to ascertain diets, our data show that they can provide valuable information on the relative intake of prey types from different trophic levels; but when complex dietary patterns are recorded (e.g. due to the confounding effects of habitat and/or temporal variation) it can be difficult to draw firm conclusions about diet composition.     

Assessing trophic interactions in a guild of primary parasitoids and facultative hyperparasitoids: stable isotope analysis

Facultative hyperparasitism is likely to be the most common form of intraguild predation among parasitoids. However, difficulties associated with studying facultative hyperparasitoids in the field have hampered a thorough understanding of their trophic ecology. In this study, we used a combination of stable isotope analysis and published natural history information to infer trophic interactions in a guild of field-collected primary parasitoids and facultative hyperparasitoids that attack a gall-making midge on Baccharis pilularis. Our three a priori hypotheses were: (1) stable isotope values should increase incrementally from the host plant to higher trophic levels; (2) the two species of ectoparasitoids should exhibit higher stable isotope signatures than the two endoparasitoids, and; (3) the two facultative hyperparasitoids should exhibit stable isotope signatures that fall between zero and one trophic level steps above that observed for the primary parasitoids. Food webs inferred from stable isotope data generally agreed with previously published accounts of community structure. As expected, both δ¹³C and δ¹⁵N were progressively enriched in the heavy isotope from the host plant to the herbivorous midge to the parasitic wasps. Multivariate analysis of stable isotope data revealed that the two primary ectoparasitoids occupied a similar trophic niche, but were significantly different from the primary endoparasitoids. We attribute this result to “coincidental intraguild predation” by ectoparasitoids that develop on already-parasitized midge larvae. One of the facultative hyperparasitoids, Zatropis capitis, exhibited a stable isotope signature approximately one trophic step above the primary parasitoids. Unexpectedly, the second facultative hyperparasitoid, Mesopolobus sp., appeared to be developing as a primary parasitoid at all sites. Coupled with independent assessments of community structure, stable isotope analysis validated trophic links constructed by previous researchers and identified potential taxon-specific differences in trophic interactions for two facultative hyperparasitoids in the B. pilularis gall community.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Sources of organic matter and methylmercury in littoral macroinvertebrates: a stable isotope approach

The main objective of this study was to assess organic matter (OM) and methylmercury (MeHg) sources for freshwater littoral macroinvertebrate primary consumers. The carbon and nitrogen stable isotope ratios (δ¹³C, δ¹⁵N) of sources (epiphytes, macrophytes, suspended particulate matter _SPM) and of macroinvertebrate consumers were measured in a fluvial lake with extensive macrophyte beds (emergent and submerged). To determine the relative contribution of each OM source to macroinvertebrate diets we used the IsoSource model that examines all possible combinations of solutions for each source. Total and MeHg concentrations of consumers were also measured. Results show that epiphytes and macrophytes are dominant in the diet of macroinvertebrates, especially in early summer (July). In mid-summer (August), SPM constitutes a non-negligible OM source to the primary consumers. Hg concentrations were higher in epiphytes than in the other OM sources. The proportion of epiphytes in macroinvertebrate diet was positively correlated with the percentage of MeHg in their tissues. There was no relationship between SPM assimilation and Hg concentration in macroinvertebrate consumers. These results suggest that epiphytes and macrophytes constitute the main pathway of Hg bioaccumulation in littoral food webs.

A stable carbon and nitrogen isotope study of lake food webs in Canada's Boreal Plain

1.  Stable carbon and nitrogen isotope and fish stomach content analyses were used to investigate food webs in five relatively undisturbed lakes on the Boreal Plain of Canada. Stable isotope analysis was also used to determine the importance of external and internal carbon sources.
2.  Overlap in the carbon and nitrogen signatures of primary producers made it difficult to determine unambiguously the feeding habits of many invertebrates. However, isotope analysis suggested that external carbon inputs were detectable in the aquatic food chains of the one lake with a short water residence time («1 year). In the other four lakes, with water residence times ≥1 year, autochthonous carbon was the only detectable carbon source in the food webs.
3.  Food webs in these lakes spanned a range of four to five trophic levels. Both invertebrates and fish appeared to eat a variety of food, often feeding at more than one trophic level.
4.  With the exception of one lake (SPH20), top predators in these lakes, northern pike ( Esox lucius ) and fathead minnows ( Pimephales promelas ), occupied similar trophic positions despite large differences in body size and trophic morphology. In SPH20, where there were two additional fish species, pike occupied a higher trophic position. However, all the top predators in each lake appeared to be omnivores and generalists.
5.  The prevalence of omnivory and the apparent generalist feeding habits of fish in these lakes suggest that organisms are flexible in their feeding habits and that these food webs will be resilient to disturbance.     

Getting to the fat of the matter: models,methods and assumptions for dealing with lipids in stable isotope analyses

Within an organism, lipids are depleted in ¹³C relative to proteins and carbohydrates (more negative δ¹³C), and variation in lipid content among organisms or among tissue types has the potential to introduce considerable bias into stable isotope analyses that use δ¹³C. Despite the potential for introduced error, there is no consensus on the need to account for lipids in stable isotope analyses. Here we address two questions: (1) If and when is it important to account for the effects of variation in lipid content on δ¹³C? (2) If it is important, which method(s) are reliable and robust for dealing with lipid variation? We evaluated the reliability of direct chemical extraction, which physically removes lipids from samples, and mathematical normalization, which uses the carbon-to-nitrogen (C:N) ratio of a sample to normalize δ¹³C after analysis by measuring the lipid content, the C:N ratio, and the effect of lipid content on δ¹³C (Δδ¹³C) of plants and animals with a wide range of lipid contents. For animals, we found strong relationships between C:N and lipid content, between lipid content and Δδ¹³C, and between C:N and Δδ¹³C. For plants, C:N was not a good predictor of lipid content or Δδ¹³C, but we found a strong relationship between carbon content and lipid content, lipid content and Δδ¹³C, and between and carbon content and Δδ¹³C. Our results indicate that lipid extraction or normalization is most important when lipid content is variable among consumers of interest or between consumers and end members, and when differences in δ¹³C between end members is <10–12‰. The vast majority of studies using natural variation in δ¹³C fall within these criteria. Both direct lipid extraction and mathematical normalization reduce biases in δ¹³C, but mathematical normalization simplifies sample preparation and better preserves the integrity of samples for δ¹⁵N analysis.     

Analysis of primary food sources and trophic relationships of aquatic animals in a mangrove-fringed estuary,Khung Krabaen Bay (Thailand) using dual stable isotope techniques

Stable carbon (¹³C) and nitrogen (¹⁵N) isotopes were used to elucidate primary food sources and trophic relationships of organisms in Khung Krabaen Bay and adjacent offshore waters. The three separate sampling sites were mangroves, inner bay and offshore. The ¹³C values of mangrove leaves were –28.2 to –29.4, seagrass –10.5, macroalgae –14.9 to –18.2, plankton –20.0 to –21.8, benthic detritus –15.1 to –26.3, invertebrates –16.5 to –26.0, and fishes –13.4 to –26.3. The ¹⁵N values of mangrove leaves were 4.3 to 5.7, seagrass 4.3, macroalgae 2.2 to 4.4, plankton 5.7 to 6.4 , benthic detritus 5.1 to 5.3, invertebrates 7.2 to 12.2 , and fishes 6.3 to 15.9. The primary producers had distinct ¹³C values. The ¹³C values of animals collected from mangroves were more negative than those of animals collected far from shore. The primary carbon sources that support food webs clearly depended on location. The contribution of mangroves to food webs was confined only to mangroves, but a mixture of macroalgae and plankton was a major carbon source for organisms in the inner bay area. Offshore organisms clearly derived their carbon through the planktonic food web. The ¹⁵N values of consumers were enriched by 3–4 relative to their diets. The ¹⁵N data suggests that some of aquatic animals had capacity to change their feeding habits according to places and availability of foods and as a result, individuals of the same species could be assigned to different trophic levels at different places.     

Quantifying the trophic base for benthic secondary production in the Nakdong River estuary of Korea using stable C and N isotopes

The overall dependence of benthic secondary production on the main primary producers at three different habitats in the Nakdong River estuarine system, Korea, was estimated. Inventories of macrobenthic invertebrate biomass were combined with multiple-isotope-mixing models to evaluate the trophic base, comparing Scirpus triqueter-dominated and Phragmites australis-dominated marshes and bare intertidal flat. The feasible contributions of four main food sources, marsh macrophytes, the microphytobenthos, and riverine and marine suspended particulate organic matter (RPOM and MPOM), to the consumer biomasses were calculated using the isotopic mixing model. After weighting the feasible contributions of food sources to each taxon by the consumer biomass, the resultant values were summed for all the consumers at each habitat to quantify the trophic base of the benthic invertebrate community. Dual-isotope-mixing model calculations verified the varying dependence on those potential food sources among the functional feeding groups. In addition, the dependence on each source of the same functional group varied between bare intertidal and salt-marsh habitats, shifting from a dominance of benthic and pelagic microalgal sources on the former habitat to a mixed food source at the latter habitat. The biomasses of the species comprising each functional group differed among habitats and sampling dates, so that each functional group made a different contribution to the whole benthic community and its basal food source. Given the calculation of the overall dependence of macrozoobenthic community on each food source, our results indicate that the microphytobenthic source dominates (nearly half) the trophic base in all the intertidal habitats of different vegetational compositions. Marsh-macrophyte-derived organic matter and RPOM served as considerable subsidies only to salt-marsh food webs, reflecting the use of the mixed food source by salt-marsh-bed consumers. Conversely, the dominance of MPOM in the total food base was equal to that of the microphytobenthos in the bare intertidal ecosystem but increased during spring−summer in the salt-marsh systems. Our results also suggest that the river discharge concentrated during the summer monsoon does not lead to any shift in trophic base for estuarine secondary production.     

Climate change undermines the global functioning of marine food webs

Sea water temperature affects all biological and ecological processes that ultimately impact ecosystem functioning. In this study, we examine the influence of temperature on global biomass transfers from marine secondary production to fish stocks. By combining fisheries catches in all coastal ocean areas and life‐history traits of exploited marine species, we provide global estimates of two trophic transfer parameters which determine biomass flows in coastal marine food web: the trophic transfer efficiency (TTE) and the biomass residence time (BRT) in the food web. We find that biomass transfers in tropical ecosystems are less efficient and faster than in areas with cooler waters. In contrast, biomass transfers through the food web became faster and more efficient between 1950 and 2010. Using simulated changes in sea water temperature from three Earth system models, we project that the mean TTE in coastal waters would decrease from 7.7% to 7.2% between 2010 and 2100 under the ‘no effective mitigation’ representative concentration pathway (RCP8.5), while BRT between trophic levels 2 and 4 is projected to decrease from 2.7 to 2.3 years on average. Beyond the global trends, we show that the TTEs and BRTs may vary substantially among ecosystem types and that the polar ecosystems may be the most impacted ecosystems. The detected and projected changes in mean TTE and BRT will undermine food web functioning. Our study provides quantitative understanding of temperature effects on trophodynamic of marine ecosystems under climate change.     

Stable isotopes and trophic positions of littoral fishes from a Mediterranean marine protected area

Stable isotope analyses were employed to explore feeding and foraging habitats and trophic levels of littoral fishes in a western Mediterranean Marine Protected Area (Egadi Islands, Sicily, Italy). Carbon and nitrogen stable isotope ratios were measured in primary producers, invertebrates and fishes collected in December 2001 and January 2002. Fishes of the littoral region of the Egadi Islands had isotopic signatures that fell into a wider range for δ ¹³C (about 6‰) than for δ ¹⁵N (about 3‰). Carbon isotope ratios were consistent with a food web based on mixed sources and two trophic pathways leading to different fish species. Differences in the isotopic composition between islands were higher for benthivorous than for planktivorous fishes. The overall picture gained from this study is of a isotopic distinction between planktivorous and benthivorous fishes, resource partitioning facilitating the coexistence of similar species within the same ecosystem, and spatial variability in the isotopic signatures and trophic level of fishes. Asymmetrical analysis of variance showed that estimated trophic levels were lower in the area with the highest level of protection (Zone A) for only two out of the nine fishes analysed. As a consequence, overall spatial differences do not seem to be a consequence of protection, since in most cases trophic levels did not change significantly between zone A and zones C where professional fishing (trawling apart) is permitted, but of natural sources of variation (e.g. variability in food availability and site-specific food preferences of fishes). However, the results of this study suggest a different response at the species compared to the community level.     

Trophic relationships in a tropical stream food web assessed by stable isotope analysis

1. Stable isotope analysis, coupled with dietary data from the literature, was used to investigate trophic patterns of freshwater fauna in a tropical stream food web (Guadeloupe, French West Indies).
2. Primary producers (biofilm, algae and plant detritus of terrestrial origin) showed distinct δ¹³C signatures, which allowed for a powerful discrimination of carbon sources. Both autochthonous (¹³C-enriched signatures) and allochthonous (¹³C-depleted signatures) resources enter the food web. The migrating behaviour of fishes and shrimps between marine and freshwater during their life cycles can be followed by carbon isotopes. Here, shrimp δ¹³C signatures were shown to shift from −16‰ (for juveniles under marine influence) to −24.7‰ (for adults in freshwater habitats). For resident species, δ¹³C values partly reflected the species' habitat preferences along the river continuum : species living in river mouths were ¹³C-enriched in comparison with those collected upstream.
3. Nitrogen isotopic ratios were also discriminating and defined three main trophic guilds among consumers. The δ¹⁵N values of herbivores/detritivores were 5.0–8.4‰, omnivores 8.8–10.2‰ and carnivores 11–12.7‰.
4. Mixing model equations were employed to calculate the possible range of contribution made by respective food sources to the diet of each species. The results revealed the importance of omnivorous species and the dependence of riverine biota on terrestrial subsidies, such as leaf detritus and fruits. Finally, the abundance of shrimps and their feeding habits placed in relief their key role in tropical freshwater food webs. Isotopic analysis provides a useful tool for assessing animal feeding patterns.     

Quantifying the multiple facets of isotopic diversity: New metrics for stable isotope ecology

Stable isotope analyses have emerged as an insightful tool for ecologists, with quantitative methods being developed to analyse data at the population, community and food web levels. In parallel, functional ecologists have developed metrics to quantify the multiple facets of functional diversity in a n-dimensional space based on functional traits. Here, we transferred and adapted metrics developed by functional ecologists into a set of four isotopic diversity metrics (isotopic divergence, dispersion, evenness and uniqueness) complementary to the existing metrics. Specifically, these new metrics are mathematically independent of the number of organisms analysed and account for the abundance of organisms. They can also be calculated with more than two stable isotopes. In addition, we also provide a procedure for calculating the levels of isotopic overlap (similarity and turnover) between two groups of organisms. These metrics have been implemented into new functions in R made freely available to users and we illustrated their application using stable isotope values from a freshwater fish community. Transferring the framework developed initially for measuring functional diversity to stable isotope ecology will allow more efficient assessments of changes in the multiple facets of isotopic diversity following anthropogenic disturbances.