Stable isotopes differentiate bottlenose dolphins off west-central Florida

Distinguishing discrete population units among continuously distributed coastal small cetaceans is challenging and crucial to conservation. We evaluated the utility of stable isotopes in assessing group membership in bottlenose dolphins (Tursiops truncatus) off west-central Florida by analyzing carbon, nitrogen, and sulfur isotope values (δ¹³C, δ¹⁵N, and δ³⁴S) of tooth collagen from stranded dolphins. Individuals derived from three putative general population units: Sarasota Bay (SB), nearshore Gulf of Mexico (GULF), and offshore waters (OFF). Animals of known history (SB) served to ground truth the approach against animals of unknown history from the Gulf of Mexico (GULF, OFF). Dolphin groups differed significantly for each isotope. Average δ¹³C values from SB dolphins (−10.6‰) utilizing sea grass ecosystems differed from those of GULF (−11.9‰) and OFF (−11.9‰). Average δ¹⁵N values of GULF (12.7‰) and OFF (13.2‰) were higher than those of SB dolphins (11.9‰), consistent with differences in prey trophic levels. δ³⁴S values showed definitive differences among SB (7.1‰), GULF (11.3‰), and OFF (16.5‰) dolphins. This is the first application of isotopes to population assignment of bottlenose dolphins in the Gulf of Mexico and results suggest that isotopes may provide a powerful tool in the conservation of small cetaceans.     

Spatial,ontogenetic and interspecific variability in stable isotope ratios of nitrogen and carbon of Merluccius capensis and Merluccius paradoxus off South Africa

General linear models (GLMs) were used to determine the relative importance of interspecific, ontogenetic and spatial effects in explaining variability in stable isotope ratios of nitrogen (δ¹⁵N) and carbon (δ¹³C) of the co‐occurring Cape hakes Merluccius capensis and Merluccius paradoxus off South Africa. Significant GLMs were derived for both isotopes, explaining 74 and 56% of observed variance in Merluccius spp. δ¹⁵N and δ¹³C, respectively. Spatial effects (west or south coast) contributed most towards explaining variability in the δ¹⁵N model, with Merluccius spp. off the west coast having higher (by c. 1·4‰) δ¹⁵N levels than Merluccius spp. off the south coast. Fish size and species were also significant in explaining variability in δ¹⁵N, with both species showing significant linear increases in δ¹⁵N with size and M. capensis having higher (by c. 0·7‰) δ¹⁵N values than M. paradoxus. Species and coast explained most and similar amounts of variability in the δ¹³C model, with M. capensis having higher (by c. 0·8‰) δ¹³C values than M. paradoxus, and values being lower (by c. 0·7‰) for fishes off the west coast compared with the south coast. These results not only corroborate the knowledge of Merluccius spp. feeding ecology gained from dietary studies, in particular the ontogenetic change in trophic level corresponding to a changing diet, but also that M. capensis feeds at a slightly higher trophic level than M. paradoxus. The spatial difference in Merluccius spp. δ¹⁵N appears due to a difference in isotopic baseline, and not as a result of Merluccius spp. feeding higher in the food web off the west than the south coast, and provides new evidence that corroborates previous observations of biogeographic differences in isotopic baselines around the South African coast. This study also provides quantitative data on the relative trophic level and trophic width of Cape hakes over a large size range that can be used in ecosystem models of the southern Benguela.     

Stable isotope ratios in winter‐grown feathers of Great Reed Warblers Acrocephalus arundinaceus,Clamorous Reed Warblers A. stentoreus and their hybrids in a sympatric breeding population in Kazakhstan

Analyses of the stable isotope composition of feathers can provide significant insight into the spatial structure of bird migration. We collected feathers from Great Reed Warblers Acrocephalus arundinaceus, Clamorous Reed Warblers A. stentoreus and a small sample of their hybrids in a sympatric breeding population in Kazakhstan to assess natural variation in stable isotope signatures and delineate wintering sites. The Great Reed Warbler is a long‐distance migrant that overwinters in sub‐Saharan Africa, whereas the Clamorous Reed Warbler performs a short‐distance migration to the Indian sub‐continent. Carbon (δ¹³C), nitrogen (δ¹⁵N) and deuterium (δD) isotope signatures were obtained from winter‐grown feathers of adult birds. There were highly significant differences in δD and less significant differences in δ¹³C between Great and Clamorous Reed Warblers. Thus, our results show that the stable isotope technique, and in particular the deuterium (δD) signal, resolves continental variation in winter distribution between these closely related Acrocephalus species with sympatric natal origin. The isotope signatures of hybrid Great × Clamorous Reed Warblers clustered with those of the Great Reed Warblers. Hence, a parsimonious suggestion is that the hybrids undergo moult in Afrotropical wintering grounds, as do the Great Reed Warblers. The observed δD values fell within the range of expected values based on available precipitation data collected at precipitation stations across the wintering continents of each species. However, the power to predict the winter origin of birds in our study system using these data was weak as the expected values ranged widely at this broad continental scale.     

Biogenic methane contributes to the food web of a large,shallow lake

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DIFFERENCES IN FORAGING LOCATION OF MEXICAN AND CALIFORNIA ELEPHANT SEALS: EVIDENCE FROM STABLE ISOTOPES IN PUPS

Female northern elephant seals, Mirounga angustirostris, from Año Nuevo (AN) in central California feed offshore in mid‐latitude waters (40°–55°N). Migratory patterns and foraging locations of seals from Mexico are unknown. Rookeries on San Benitos (SB) islands in Baja California Sur, Mexico, are ～1,170 km south of AN. Although the colonies are similar in size, seals from SB begin breeding earlier and have an earlier breeding birthing peak than seals from AN. To determine if the foraging location of seals from Mexico was similar to that of seals from California, we measured δ¹³C and δ¹⁵N values in the hair of 48 suckling pups at SB and 37 from AN, assuming that their isotopic signatures reflected those of mothers' milk, their exclusive diet. The mean δ¹³C and δ¹⁵N values for SB pups (?16.1‰± 0.9‰ and 17.7‰± 0.9‰, respectively) were significantly higher than those for AN pups (?17.6‰± 0.4‰ and 15.6‰± 1.0‰, respectively). From data on environmental isotope gradients and known behavior of SB and AN populations, we hypothesize that the isotope differences are due to females in the SB colony foraging ～8° south of seals from AN. This hypothesis can be tested by deployment of satellite tags on adult females from the SB colony.     

Stable isotope differences of polar bears in the Southern Beaufort Sea and Chukchi Sea

The life-history, genetic, and habitat use differences between the 2 polar bear (Ursus maritimus) subpopulations in Alaska, USA, have been used to determine the geographic border separating them, but it has sparked a debate of the correct placement of the border for several years. Recently, the Southern Beaufort Sea (SBS) polar bear subpopulation has declined because of sea ice loss, while the Chukchi Sea (CS) subpopulation appears stable. To provide additional information about potential differences between the SBS and CS subpopulations, such as differences in prey sources, we used stable isotope analysis of carbon and nitrogen from bone collagen of polar bears in these 2 neighboring subpopulations. We analyzed polar bear bones from 112 individuals collected from 1954–2019. Our purpose was to determine if the SBS and CS subpopulations could be distinguished based on the stable isotope signatures of bone collagen. A difference >1‰ in stable carbon isotope (δ¹³C) values suggests a change in carbon sources, such as nearshore to offshore, while a 3‰ change in stable nitrogen isotope (δ¹⁵N) values equates to a change of about 1 trophic level. Our study indicated a difference in δ¹³C values (P ≤ 0.001) but not δ¹⁵N values (P = 0.654) between the CS (−13.0 ± 0.3‰ and 22.0 ± 0.9‰, respectively) and SBS bears (−14.7 ± 1.3‰ and 22.2 ± 1.0‰, respectively). Our findings indicate that the 2 subpopulations are consuming similar high trophic level prey, while feeding in ecosystems with different δ¹³C baselines. We performed a logistic regression analysis using δ¹³C and δ¹⁵N values of the polar bears to predict their placement into these 2 subpopulations. Using Icy Cape, Alaska as the geographical boundary, the analysis correctly placed polar bears in their respective subpopulations 82% of the time. Overall accuracy of placement changed to 84% when using the current geographical boundary at Utqiaġvik, Alaska. We predicted samples collected from the Wainwright, Alaska region as 58% CS and 42% SBS polar bears. This suggests that the area between Wainwright and Icy Cape is a polar bear mixing zone that includes bears from both subpopulations. Bone collagen has a long-term, potentially life-long, stable isotope turnover rate, and our results could be used to determine the association of harvested polar bears to Alaska subpopulations, thus aiding in transboundary harvest quota management.     

Stable-isotope analysis reveals sources of organic matter and ontogenic feeding shifts of a mangrove-dependent predator species,New Granada sea catfish,Ariopsis canteri

To gain a better understanding on the trophic ecology of New Granada sea catfish, Ariopsis canteri, and their linkage to mangroves, nitrogen and stable carbon isotopes (δ¹⁵N and δ¹³C), as well as Bayesian mixing models, were used to explore trophic dynamics and potential ontogenic feeding shifts across different size classes: class I (8–20 cm), class II (21–32 cm) and class III (>32 cm). The study area was the estuary of the Atrato River Delta, where information about fish ecology is scarce. The δ¹³C of size class I was lower (mean ± s.d . = −24.96 ± 0.69‰) than that of size classes II (−22.20 ± 0.90‰) and III (−22.00 ± 1.96‰). The δ¹⁵N of size class I was lower (mean ± s.d . = 8.50 ± 0.67‰) than that of size classes II (9.77 ± 0.60‰) and III (10.00 ± 0.66‰). Body size was positively and significantly correlated to δ¹⁵N and δ¹³C. Individuals with L_T > 32 cm presented the highest estimated trophic position (3.8). Five-source mixing models indicated that for class I, the mean estimated contribution of macroalgae was the highest (6%–57% c.i. ), and for classes II and III, the mean estimated contribution of macrophytes was the highest (3%–53% c.i. and 4%–53% c.i. , respectively). Ontogenetic feeding shifts of A. canteri were confirmed evidencing decreasing intraspecific competition between small and large individuals. Results suggest that mangroves are a nursery and feeding ground habitat for this species and that mangroves support A. canteri mainly due to the substrate/habitat that supports sources in the food webs. These results can be used in ecosystem-based fishery management focused on the protection of extensive mangrove areas in the southern Caribbean Sea.     

Investigation of diachronic dietary patterns on the islands of Ibiza and Formentera, Spain: Evidence from sulfur stable isotope ratio analysis

We present sulfur isotope ratio measurements of bone collagen from animals (n = 75) and humans (n = 120) from five sites dating to four chronological periods (Chalcolithic, Punic, Late Antiquity‐Early Byzantine, and Islamic) from the Balearic Islands of Ibiza and Formentera, Spain. This study is a follow up to previously published δ¹³C and δ¹⁵N values by [Fuller et al.: Am J Phys Anthropol 143 (2010) 512–522] and focuses on using δ³⁴S values to better understand the dietary patterns of these populations through time and to possibly identify immigrants to these islands. The range of δ³⁴S values (10.5–17.8‰) observed for the animals was relatively broad, which suggests that a significant sea spray effect has added marine sulfates to the soils of Formentera and Ibiza. The mean δ³⁴S values of the different human populations were found to be: Chalcolithic (16.5 ± 1.4‰), Punic rural (13.6 ± 1.7‰), Punic urban (12.9 ± 1.8‰), Late Antiquity‐Early Byzantine (12.3 ± 2.1‰), and Islamic (9.1 ± 2.7‰). These human δ³⁴S results are similar to the animal data, a finding that supports the notion that there was little marine protein consumption by these societies and that the diet was mainly based on terrestrial resources. During the Punic and Late Antiquity‐Early Byzantine periods the δ³⁴S values were used to identify individuals in the population who likely were not born or raised on the islands. In addition, 18 of the 20 individuals analyzed from the Islamic period have δ³⁴S values that indicate that they were immigrants to Ibiza who died before acquiring the new local sulfur isotopic signature. Am J Phys Anthropol 2012. © 2012 Wiley Periodicals, Inc.     

INVESTIGATING TROPHIC RELATIONSHIPS OF PINNIPEDS IN ALASKA AND WASHINGTON USING STABLE ISOTOPE RATIOS OF NITROGEN AND CARBON

We measured stable-nitrogen (δ¹⁵N) and stable-carbon (δ¹³C) isotope ratios in muscle and hair from 7 northern fur seals (Callorhinus ursinus) from the Pribilof Islands, Alaska, and 27 Steller sea lions (Eumetopias jubatus), and 14 harbor seals (Phoca vitulina) from the Gulf of Alaska and coast of Washington State, in order to contrast dietary information derived from isotopic vs. available conventional dietary studies. Stable-nitrogen-isotope analysis of muscle revealed that harbor seals were enriched over sea lions (mean δ¹⁵N = 18.6‰vs. 17.5‰) which were in turn enriched over northern fur seals (mean δ¹⁵N = 16.6‰). Trophic segregation among these species likely results primarily from differential reliance on herring (Clupea harengus), Atka mackerel (Pleurogrammus monopterygius), and large vs. small walleye pollock (Theregra chalcogramma). According to their δ¹⁵N values, adult male Steller sea lions showed a higher trophic position than adult females (mean δ¹⁵N: 18.0‰vs. 17.2‰), whereas adult female northern fur seals were trophically higher than juvenile male fur seals (mean δ¹⁵N: 16.5‰vs. 15.0‰). Each of these observed differences likely resulted from differential reliance on squid or differences in the size range of pollock consumed. Three northern fur seal pups showed higher δ¹⁵N enrichment over adults (mean 17.7‰vs. 15.8‰) due to their reliance on their mother's milk. Stable-carbon isotope measurements of hair revealed a cline toward more negative values with latitude. Segregation in hair δ¹³C between Steller sea lions and harbor seals off the coast of Washington (mean δ¹³C: ?13.6‰vs.?15.0‰) reflected the greater association of harbor seals with freshwater input from the Columbia River. Our study demonstrates the utility of the stable isotope approach to augment conventional dietary analyses of pinnipeds and other marine mammals.     

Tracking a century of change in trophic structure and dynamics in a floodplain wetland: integrating palaeoecological and palaeoisotopic evidence

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Relationships between growth traits and scale stable isotopes (δ13C, δ15N) of adult chum salmon Oncorhynchus keta in Hokkaido,Japan

Stable isotope analysis (SIA) in combination with growth analysis using scales collected from adult chum salmon Oncorhynchus keta migrating back to Hokkaido, Japan, was performed to describe the variation of isotopic composition of carbon (δ¹³C) and nitrogen (δ¹⁵N) in scales and to examine relationships with growth traits [age, fork length (FL), and relative growth ratio in the last growth period [(RGR^last)]. Scale stable isotope (SI) values in 3‐ to 6‐year‐old fish were highly variable, ranging from ?17.6‰ to ?14.3‰ for δ¹³C and 9.5‰ to 13.4‰ for δ¹⁵N. The δ¹⁵N was positively correlated with FL, and this tendency may indicate changes in trophic level with growth. Significant effect was not detected between δ¹⁵N and RGR^last, it can be inferred that factors potentially yielding high δ¹⁵N may not necessarily result in higher growth rates. No trend found between FL and δ¹³C may imply that there is no clear segregation in feeding locations between the 3‐ to 6‐year groups. This study provided basic information for scale SI values of chum salmon adults and indicated that SIA using scales could be a new approach to elucidating the trophic ecology of chum salmon.     

Carbon (δ13C) and nitrogen (δ15N) stable isotope composition in plant and soil in Southern Patagonia's native forests

Stable isotope natural abundance measurements integrate across several biogeochemical processes in ecosystem N and C dynamics. Here, we report trends in natural isotope abundance (δ¹³C and δ¹⁵N in plant and soil) along a climosequence of 33 Nothofagus forest stands located within Patagonia, Southern Argentina. We measured 28 different abiotic variables (both climatic variables and soil properties) to characterize environmental conditions at each of the 33 sites. Foliar δ¹³C values ranged from ?35.4‰ to ?27.7‰, and correlated positively with foliar δ¹⁵N values, ranging from ?3.7‰ to 5.2‰. Soil δ¹³C and δ¹⁵N values reflected the isotopic trends of the foliar tissues and ranged from ?29.8‰ to ?25.3‰, and ?4.8‰ to 6.4‰, respectively, with no significant differences between Nothofagus species (Nothofagus pumilio, Nothofagus antarctica, Nothofagus betuloides). Principal component analysis and multiple regressions suggested that mainly water availability variables (mean annual precipitation), but not soil properties, explained between 42% and 79% of the variations in foliar and soil δ¹³C and δ¹⁵N natural abundance, which declined with increased moisture supply. We conclude that a decline in water use efficiency at wetter sites promotes both the depletion of heavy C and N isotopes in soil and plant biomass. Soil δ¹³C values were higher than those of the plant tissues and this difference increased as annual precipitation increased. No such differences were apparent when δ¹⁵N values in soil and plant were compared, which indicates that climatic differences contributed more to the overall C balance than to the overall N balance in these forest ecosystems.     

Decrypting stable‐isotope (δ13C and δ15N) variability in aquatic plants

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Stable‐isotope comparisons between embryos and mothers of a placentatrophic shark species

Stable nitrogen (δ¹⁵N) and carbon (δ¹³C) isotopes of Atlantic sharpnose shark Rhizoprionodon terraenovae embryos and mothers were analysed. Embryos were generally enriched in ¹⁵N in all studied tissue relative to their mothers' tissue, with mean differences between mother and embryo δ¹⁵N (i.e. Δδ¹⁵N) being 1·4‰ for muscle, 1·7‰ for liver and 1·1‰ for cartilage. Embryo muscle and liver were enriched in ¹³C (both Δδ¹³C means = 1·5‰) and embryo cartilage was depleted (Δδ¹³C mean = ?1·01‰) relative to corresponding maternal tissues. While differences in δ¹⁵N and δ¹³C between mothers and their embryos were significant, muscle δ¹⁵N values indicated embryos to be within the range of values expected if they occupied a similar trophic position as their respective mothers. Positive linear relationships existed between embryo total length (L_T) and Δδ¹⁵N for muscle and liver and embryo L_T and Δδ¹³C for muscle, with those associations possibly resulting from physiological differences between smaller and larger embryos or differences associated with the known embryonic nutrition shift (yolk feeding to placental feeding) that occurs during the gestation of this placentatrophic species. Together these results suggest that at birth, the δ¹⁵N and δ¹³C values of R. terraenovae are likely higher than somewhat older neonates whose postpartum feeding habits have restructured their isotope profiles to reflect their postembryonic diet.     

Stable isotope records of sei whale baleens from Chilean Patagonia as archives for feeding and migration behavior

Carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotope variations in baleen plates of sei whales (Balaenoptera borealis) stranded after a mass mortality event in Chilean Patagonia were investigated to assess potential dietary and migratory patterns. Carbon and nitrogen isotope ratios of seven baleens from six individuals were analyzed. The δ¹³C values ranged from ? 19.1 to ? 15.9‰ and the δ¹⁵N values from 8.7 to 15.4‰. Variations of up to 2.9‰ for δ¹³C and 5.3‰ for δ¹⁵N were observed within one baleen. Carbon and nitrogen isotope records of each baleen were significantly correlated and showed recurring oscillations confirmed by wavelet analyses. Oscillations slightly differed in periodicity indicating variable baleen growth rates between 10.0 and 16.5 cm/year. Food sources of the whales are discussed in terms of available isotope data for potential prey taxa and potential migratory behavior on the basis of latitudinal isotope gradients of particulate organic matter. Cyclicity could be explained by regular migrations of the sei whales from subtropical calving areas to high‐latitude foraging grounds. δ¹⁵N records of baleens differed between individuals eventually pointing to diverse feeding and migratory preferences among sei whale individuals.     

Stable isotope evidence indicates the incorporation into Japanese catchments of marine‐derived nutrients transported by spawning Pacific Salmon

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Stable Isotopes (δD, δ13C, δ15N) Reveal Associations Among Geographic Location and Condition of Alaskan Northern Pintails

Abstract: Information on spring migration routes, geographic linkages among winter, spring, and breeding locations, and potential geographic effects on arrival body condition of northern pintails (Anas acuta) are currently unknown. Through a combination of stable-isotope measurements of tissues representing different periods of dietary integration and body composition analyses, we examined these linkages for pintails breeding in Alaska, USA. We collected 77 females at 4 locations upon spring arrival. We performed carbon (δ¹³C), nitrogen (δ¹⁵N), and hydrogen (δD) isotope measurements on flight feathers, breast feathers, and whole blood, and we conducted body composition analyses. Inference based on stable-isotope values in pintail tissues suggests that philopatry to Alaska was strong, as most of the collected females had stable-isotope values consistent with the boreal forest of Canada or western Alaska and most spring migrating females had whole-blood values indicating use of a food web in the boreal forest before collection. These patterns highlight the importance of the boreal forest for production and staging of pintails. Breast feather isotope values grown during prealternate molt were variable and covered the currently documented distribution of wintering pintails. Our results indicate associations among specific geographic areas, habitat use, and arrival condition of female pintails settling in Alaska. Females that wintered or staged in coastal habitat (as indicated by elevated δ¹³C values) arrived with less body fat compared to those that we inferred to have wintered or staged on inland freshwater habitat. Those females we inferred to use coastal areas appeared to rely more heavily on agricultural fields for nutrient acquisition (as indicated by elevated δ¹⁵N but low δ¹³C values). Our results provide the first link between low-condition females and inferred use of specific geographic areas before arrival. Conservation on wintering grounds should focus on restoration and protection of wetland complexes that provide adequate natural food resources in proximity to coastal systems that are heavily used by wintering pintails. Conservation efforts should also focus on the boreal forest, not only for pintail, but for other boreal-dependent species such as lesser scaup (Aythya affinis) (JOURNAL OF WILDLIFE MANAGEMENT 72(3):715–725; 2008)     

The origins of northern European Autographa gamma individuals evaluated using hydrogen stable isotopes

1. Many insect species are migratory. As the spring progresses, adults gradually depart from their over‐wintering habitats and arrive in northern zones where they reproduce during the summer. Understanding this transgenerational and highly adaptive migratory behaviour is crucial when interpreting life cycle dynamics of many insect pests. 2. Origins of migratory silver Y moths, Autographa gamma (Linnaeus, 1758), captured from Finland, were studied with stable hydrogen analysis of their wings (δ²H_w). 3. The difference between spring and autumn generation δ²H_w values indicate different geographical natal origins. The probability surface map shows that the spring generation probably emerged in central Europe (Benelux countries, Germany and parts of France). 4. A negative correlation between the δ²H_w values and the migrants' capture year suggests that a warming climate may have driven the transgenerational migratory stages northwards during the last century.     

Tracing dietary origins of aphids and the predatory beetle Propylea japonica in agricultural systems using stable isotope analyses

Tracing dietary origins of the predatory beetle Propylea japonica (Thunberg) (Coleoptera: Coccinellidae) aids understanding their roles in the food web and provides information to develop strategies for effective conservation in agroecosystems comprised of wheat [Triticum aestivum L. (Poaceae)], cotton [Hirsutum spp. (Malvaceae)], and maize [Zea mays L. (Poaceae)]. Intrinsic markers of carbon and nitrogen stable isotope ratios (δ¹³C and δ¹⁵N) in P. japonica need to be developed to ascertain the source(s) of diet. Experiments were carried out to examine the changes of δ¹³C and δ¹⁵N among the three crops, pests (wheat, cotton, and maize aphids; all Hemiptera: Aphididae), and P. japonica fed on aphids of each of the three crops. Results indicated that δ¹³C values in P. japonica fed on wheat, cotton, and maize aphids were ?27.2 to ?26.5‰, ?24.2 to ?23.9‰, and ?11.0 to ?10.7‰, respectively, whereas their δ¹⁵N values were 1.1 to 2.9‰, 6.0 to 7.4‰, and ?0.6 to 0.1‰, respectively. δ¹³C and δ¹⁵N plots clearly identify the three crops, the dietary origins of the aphids, and the host origins of the aphid prey consumed by the ladybird beetles, as each pathway displays a non‐overlapping pattern. Based on the values of δ¹³C and δ¹⁵N of the three food webs, dietary origins can be traced in the predatory beetle P. japonica derived from wheat, cotton, and maize crops.     

Discriminating geographic origins of migratory waders at stopover sites: insights from stable isotope analysis of toenails

In this study we test the potential of stable isotope analysis to reveal wintering origins of waders mixing at stopover sites, using the dunlin Calidris alpina as a case study. We determined stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope signatures of toenails of dunlins captured during winter at reference sites along the East‐Atlantic Flyway, from Mauritania to the United Kingdom. Afterwards, during spring migration, dunlins were sampled at the Tagus estuary, Portugal, and assigned to their wintering grounds according to their stable isotope signatures. Toenails from wintering dunlins at different sites had significantly different δ¹³C and δ¹⁵N signatures, despite some overlap in isotopic carbon ratios of birds from Morocco, Portugal and the UK. Among birds sampled during migration in Portugal, we found a clear bimodal pattern in δ¹³C values, corresponding to passage migrants from Mauritania (enriched δ¹³C values) and wintering birds from the Tagus estuary (depleted δ¹³C values). The first passage migrants from Mauritania appeared at the Tagus estuary by the end of March, with peak numbers during late April and early May. Our study provides evidence that isotopic signatures of toenails can play a determinant role in tracing the wintering origins of migrant dunlins at their stopover areas. Toenails, instead of feathers, can be the powerful and innovating tissue to sample in wader studies, allowing to bridge the gap in the field of migratory connectivity between sites used in different phases of the life cycle of waders.