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

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Intraskeletal isotopic compositions (δ13C, δ15N) of bone collagen: Nonpathological and pathological variation

Paleodiet research traditionally interprets differences in collagen isotopic compositions (δ¹³C, δ¹⁵N) as indicators of dietary distinction even though physiological processes likely play some role in creating variation. This research investigates the degree to which bone collagen δ¹³C and δ¹⁵N values normally vary within the skeleton and examines the influence of several diseases common to ancient populations on these isotopic compositions. The samples derive from two medieval German cemeteries and one Swiss reference collection and include examples of metabolic disease (rickets/osteomalacia), degenerative joint disease (osteoarthritis), trauma (fracture), infection (osteomyelitis), and inflammation (periostitis). A separate subset of visibly nonpathological skeletal elements from the German collections established normal intraindividual variation. For each disease type, tests compared bone lesion samples to those near and distant to the lesions sites. Results show that normal (nonpathological) skeletons exhibit limited intraskeletal variation in carbon‐ and nitrogen‐isotope ratios, suggesting that sampling of distinct elements is appropriate for paleodiet studies. In contrast, individuals with osteomyelitis, healed fractures, and osteoarthritis exhibit significant intraskeletal differences in isotope values, depending on whether one is comparing lesions to near or to distant sites. Skeletons with periostitis result in significant intraskeletal differences in nitrogen isotope values only, while those with rickets/osteomalacia do not exhibit significant intraskeletal differences. Based on these results, we suggest that paleodiet researchers avoid sampling collagen at or close to lesion sites because the isotope values may be reflecting both altered metabolic processes and differences in diet relative to others in the population. Am J Phys Anthropol 153:598–604, 2014. © 2013 Wiley Periodicals, Inc.     

Variations in stable isotope ratios of carbon (δ13C) and nitrogen (δ15N) in different body parts and eggs of adult stoneflies (Plecoptera)

Variations in δ¹³C and δ¹⁵N might arise from differences in nutrient allocation. Residence times of δ¹³C and δ¹⁵N vary among tissues depending on metabolic turnover rates. However, because of their small size, entire individual insects are generally used as single samples in isotope analyses. The present study aimed to determine the degree of isotope similarity among regions of the adult body and eggs in four species of Plecoptera (Amphinemura sp., Sweltsa sp., Kamimuria tibialis Pictet and Ostrovus sp.). Levels of δ¹³C and δ¹⁵N differ between the four species, being lowest in Amphinemura sp., and with δ¹⁵N being highest in Sweltsa sp. Egg masses contain consistently the lowest values of δ¹³C in the four species, with the δ¹⁵N value of eggs being highest in K. tibialis and Ostrovus sp., and lower in Amphinemura and Sweltsa spp. In Sweltsa sp., the δ¹⁵N levels of the dermal layers and cuticle are lowest, whereas the δ¹³C values of the dermal layers and cuticle are almost equal to those in other regions of the body, except egg masses. Oviposited individuals of Amphinemura and Sweltsa spp. have lower δ¹⁵N levels than individuals that have not oviposited. The rates of metabolism and incorporation of dietary metabolites will differ depending on the body regions and species. Differences in egg ecology such as egg developmental period and egg buoyancy among species are considered to impact on the values of δ¹³C and δ¹⁵N. These results will be useful for understanding the nutritional status of aquatic insects and their energy allocation.     

The diverse dietary profiles of MIS 3 cave bears from the Romanian Carpathians: insights from stable isotope (δ13C and δ15N) analysis

Late Pleistocene European cave bears (Ursus spelaeus ) have been considered to be largely vegetarian, although stable isotope data (δ¹³C and δ¹⁵N values) from the Romanian Carpathians has suggested considerable dietary variation. Here we evaluate previous and additional adult cave bear isotopic data from four Marine Isotope Stage 3 (MIS 3) sites in the Carpathians. Pe?tera Ur?ilor (N  =  35), Pe?tera Cioclovina (N  =  32), Pe?tera Muierilor (N  =  8), and Pe?tera cu Oase (N  =  72) provide both a dichotomy between samples suggesting vegetarian diets (from Cioclovina and Muierilor) and more omnivorous diets (from Ur?ilor and Oase), and considerable isotopic variation within samples from each site. While an inference of a strictly vegetarian diet may apply to groups that lived in ecosystems which restricted the available animal protein for these large ursids, the within and between sample isotopic variation among the Carpathian cave bears indicates considerable flexibility in their sources of protein and hence in their dietary regimes. In addition, developmental assessment of Cioclovina isotopic profiles (neonates, juveniles, sub‐adults and adults) provides patterns of transfer of stable isotope signatures throughout immature life for both δ¹³C and δ¹⁵N (increase and decrease, respectively), whereas those from Ur?ilor show little developmental shift.     

Nonlethal sampling for stable isotope analysis of juvenile Chinese sturgeon (Acipenser sinensis): Comparing δ13C and δ15N signatures in muscle and fin tissues

We compared δ¹³C and δ¹⁵N values of muscle with fin from juvenile Chinese sturgeon (Acipenser sinensis), to evaluate the feasibility of using nonlethal (fin) as an alternative to lethal (muscle) sampling. Size and lipid effect on the relationship between fin and muscle were also investigated. Dorsal muscle (DM) and fin clip (FC) were collected from A. sinensis with different body length (120–373 mm) in the Yangtze Estuary for isotope analysis. The result showed that (1) muscle isotope values could estimated by the values of fin, from either use the regression model (δ¹³C_DM = 0.939 × FC ? 2.577; δ¹⁵N_DM = 0.737 × FC + 4.638) or constants factors (δ¹³C_DM = δ¹³C_FC ? 1.27; δ¹⁵N_DM = δ¹⁵N_FC + 0.59); (2) no size‐based relationships with δ¹³C and δ¹⁵N from either fin or muscle; (3) lipid extraction significantly improving the fin and muscle regression model fit for both δ¹³C and δ¹⁵N values. Therefore, this study support the use of nonlethal fin tissues for isotope analysis of juvenile A. sinensis, and will allow trophic studies to avoid the effect of lipid accumulation from muscle.     

Changing gull diet in a changing world: A 150‐year stable isotope (δ13C, δ15N) record from feathers collected in the Pacific Northwest of North America

The world's oceans have undergone significant ecological changes following European colonial expansion and associated industrialization. Seabirds are useful indicators of marine food web structure and can be used to track multidecadal environmental change, potentially reflecting long‐term human impacts. We used stable isotope (δ¹³C, δ¹⁵N) analysis of feathers from glaucous‐winged gulls (Larus glaucescens) in a heavily disturbed region of the northeast Pacific to ask whether diets of this generalist forager changed in response to shifts in food availability over 150 years, and whether any detected change might explain long‐term trends in gull abundance. Sampled feathers came from birds collected between 1860 and 2009 at nesting colonies in the Salish Sea, a transboundary marine system adjacent to Washington, USA and British Columbia, Canada. To determine whether temporal trends in stable isotope ratios might simply reflect changes to baseline environmental values, we also analysed muscle tissue from forage fishes collected in the same region over a multidecadal timeframe. Values of δ¹³C and δ¹⁵N declined since 1860 in both subadult and adult gulls (δ¹³C, ~ 2–6‰; δ¹⁵N, ~4–5‰), indicating that their diet has become less marine over time, and that birds now feed at a lower trophic level than previously. Conversely, forage fish δ¹³C and δ¹⁵N values showed no trends, supporting our conclusion that gull feather values were indicative of declines in marine food availability rather than of baseline environmental change. Gradual declines in feather isotope values are consistent with trends predicted had gulls consumed less fish over time, but were equivocal with respect to whether gulls had switched to a more garbage‐based diet, or one comprising marine invertebrates. Nevertheless, our results suggest a long‐term decrease in diet quality linked to declining fish abundance or other anthropogenic influences, and may help to explain regional population declines in this species and other piscivores.     

Fatty acids and stable isotopes (δ13C and δ15N) reveal temporal changes in narwhal (Monodon monoceros) diet linked to migration patterns

Narwhals (Monodon monoceros) are sentinel species in the Arctic and to investigate marine food web changes from 1982–2011 we examined diet using fatty acids, δ¹⁵N, and δ¹³C, in narwhals from Baffin Bay (BB) and northern Hudson Bay (NHB). We predicted temporal changes would be greater in NHB due to a significant reduction in summer ice cover. In NHB, δ¹⁵N significantly increased, δ¹³C displayed a parabolic trend, and fatty acids gradually shifted, albeit not significantly, over time. δ¹⁵N was stable, δ¹³C decreased, and fatty acids significantly changed over time in BB. Stable isotope mixing models indicated a dietary reduction in capelin and increase in Greenland halibut from 1994–2000 to 2006–2011 in BB, while capelin was an important dietary component for narwhals in NHB in recent years (2006–2011). These dietary changes may be attributed to changes in sea ice and narwhal migration. Seasonal dietary changes, as evidenced by changes in blubber fatty acids and skin and muscle stable isotopes, were not as apparent in the NHB population, which may be indicative of a reduced migratory distance. Long‐term monitoring of narwhal diet and migratory patterns associated with reduced sea ice provides invaluable information about how the marine ecosystem will redistribute with global warming.     

Effects of ethanol storage and lipid and urea extraction on δ15N and δ13C isotope ratios in a benthic elasmobranch,the bluespotted maskray Neotrygon kuhlii

Ethanol storage and lipid and urea extraction had no effect on bluespotted maskray Neotrygon kuhlii muscle δ¹³C values whereas urea‐removal and ethanol storage increased δ¹⁵N values. Results presented here show a significant δ¹⁵N increase post‐urea removal and provide additional support for this approach in future elasmobranch stable‐isotope analysis (SIA) studies. Further experimental work on other elasmobranch species is needed to assess extraction and preservation effects on stable‐isotope (SI) values.     

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.     

Holocene boundary dynamics of a northern Australian monsoon rainforest patch inferred from isotopic analysis of carbon, (14C and δ13C) and nitrogen (δ15N) in soil organic matter

Abstract Soil organic matter (SOM) was sampled from lateritic soil profiles across an abrupt eucalypt savanna–monsoon rainforest boundary on the north coast of Croker Island, northern Australia. Accelerator mass spectrometry dating revealed that SOM that had accumulated at the base of these 1.5 m profiles had a radiocarbon age of about 5000 years. The mean carbon and nitrogen stable isotope composition of SOM from 10 cm deep layers from the surface, middle and base of three monsoon rainforest soil profiles was significantly different from the means for these layers in three adjacent savanna soil profiles, suggesting the isotopic ‘footprint’ of the vegetation boundary has been stable since the mid Holocene. Although there were no obvious environmental discontinuities associated with the boundary, the monsoon rainforest was found to occur on significantly more clay rich soils than the surrounding savanna. Tiny fragments of monsoon rainforest and abandoned ‘nests’ (large earthen mounds) of the orange‐footed scrubfowl, an obligate monsoon rainforest species, occurred in the savanna, signalling that the rainforest was once more extensive. Despite episodic disturbances, such as tropical storm damage and fires, the stability of the boundary is probably maintained because clay rich soils enable monsoon rainforest tree species to grow rapidly and achieve canopy closure, thereby excluding grass and reducing the risk of fire. Conversely, slower tree growth rates, grass competition and fire on the savanna soils would impede the expansion of the rainforest although high rainfall periods with shorter dry seasons may enable rainforest trees to grow sufficiently quickly to colonize the savanna successfully.     

The influence of lipid‐extraction and long‐term DMSO preservation on carbon (δ13C) and nitrogen (δ15N) isotope values in cetacean skin

Stable isotope analysis (SIA) has rapidly become a useful tool to study the ecology of wild animal populations, especially for elusive, wide‐ranging predators like marine mammals. The development of projectile biopsy techniques resulted in the collection of thousands of cetacean tissue samples that were archived in a dimethyl sulfoxide (DMSO) solution for long‐term, multidecadal preservation. Here we examine the influence of DMSO preservation on carbon (δ¹³C) and nitrogen (δ¹⁵N) values by comparing a set of paired delphinid skin samples stored frozen without preservative and in DMSO for up to 22 yr. Treatment of paired frozen and DMSO‐preserved skin in a 2:1 chloroform:methanol solution yielded similar δ¹³C and δ¹⁵N values, revealing that DMSO and lipid contamination have similar isotopic effects on skin, and that these effects can be removed using routine lipid‐extraction methods. Further, amino acid concentrations in DMSO‐preserved and frozen skin tissue were similar, providing independent evidence of minimal protein alteration due to preservation. Access to a rich archive of skin samples preserved in DMSO will expand our ability to examine temporal and spatial variability in the isotope values of cetaceans, which will aid our understanding of how their ecology has been influenced by historical changes in environmental conditions.     

A stable isotope (δ13C and δ15N) perspective on human diet on rapa nui (Easter Island) ca. AD 1400–1900

Ecological and environmental evidence suggests that Rapa Nui was among the most marginally habitable islands in Eastern Polynesia, with only a fraction of the biotic diversity found on archipelagos to the west, and capable of sustaining many fewer cultigens traditionally transported by Polynesian colonizers. However, archaeological evidence for human dietary adaptations under such restrictions is limited. Little is known about the particulars of the subsistence base and dietary changes on Rapa Nui that may be associated with a hypothesized late prehistoric decline in the quality and diversity of food sources. To better understand prehistoric Rapa Nui diet we examined stable carbon and nitrogen isotope compositions of human teeth along with archaeological faunal material thought to comprise the Rapa Nui food web. Our results indicate that contrary to previous zooarchaeological studies diet was predominantly terrestrial throughout the entire sequence of occupation, with reliance on rats, chickens and C3 plants. While a few individuals may have had access to higher trophic level marine resources, this is evident only later in time (generally post–AD 1600). A decline in δ¹⁵N through time was observed, and may be attributed to declines in available terrestrial proteins; however, presently we cannot rule out the effect of changing soil and plant baseline δ¹⁵N. Our results also suggest differential access to higher trophic level marine resources among contemporaneous populations, but more research is required to clarify this observation. Am J Phys Anthropol 152:173–185, 2013. © 2013 Wiley Periodicals, Inc.     

Early life histories of the London poor using δ13C and δ15N stable isotope incremental dentine sampling

High resolution incremental isotopic analysis of the dentine from early forming teeth, especially first molars (M1s), provides a means to assess the effects of poor childhood nutrition and healthcare on individuals in an assemblage where there are no infants to study. This approach is applied to an 18th and 19th century cemetery population associated with St Saviour's Almshouse burial ground in Southwark, London, to assess whether, or how, early dietary history, including weaning age, influenced health and nutritional status. The results show a general pattern in which non‐breast milk foods were introduced before or by 6 months of age, as indicated by elevated δ¹⁵N during this period. Almost all individuals for which we also have second molar (M2) records, showed lower δ¹⁵N values from a very young age (>1 year) until approximately 8–10 years, compared to adult values. The overall results show a significant difference in δ¹³C (p = 0 to 4sf, F = 17.327) and a weaker statistical difference in δ¹⁵N between males and females (p = 0.019, F = 5.581). One possible cause of this is a difference in the diet of males and females early in life, or alternatively, a greater susceptibility of males to nutritional deprivation compared to females. The latter argument is strengthened by a significant difference in the incidence of enamel hypoplasia between the males and females, with 7.7% of male teeth showing defects, compared to 3.9% of females. Am J Phys Anthropol 154:585–593, 2014. © 2014 Wiley Periodicals, Inc.     

Borrelia burgdorferi sensu lato and co‐infections with Anaplasma phagocytophilum and Rickettsia spp. in Ixodes ricinus in Hamburg,Germany

To obtain initial data on Borrelia burgdorferi sensu lato (Spirochaetales: Spirochaetaceae) in Ixodes ricinus (Ixodida: Ixodidae) ticks in Hamburg, Germany, 1400 questing ticks were collected by flagging at 10 different public recreation areas in 2011 and analysed using probe‐based quantitative real‐time polymerase chain reaction. The overall rate of infection with B. burgdorferi s.l. was 34.1%; 30.0% of adults were infected (36.7% of females and 26.0% of males), as were 34.5% of nymphs. Significant differences in tick infection rates were observed between the spring and summer/autumn months, as well as among sampling locations. Borrelia genospecies identification by reverse line blotting was successful in 43.6% of positive tick samples. The most frequent genospecies was Borrelia garinii/Borrelia bavariensis, followed by Borrelia afzelii, Borrelia valaisiana, B. burgdorferi sensu stricto, Borrelia spielmanii, Borrelia bissettii and Borrelia lusitaniae. Based on previously published data, co‐infection of Borrelia and Rickettsiales spp. was determined in 25.8% of ticks. Overall, 22.9% of ticks were co‐infected with Rickettsia spp. (Rickettsiales: Rickettsiaceae), 1.7% with Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), and 1.2% with both pathogens. Study results show a high prevalence of Borrelia‐positive ticks in recreation areas in the northern German city of Hamburg and the potential health risk to humans in these areas should not be underestimated.     

Dietary variation within and between populations of northeast Atlantic killer whales,Orcinus orca,inferred from δ13C and δ15N analyses

Epidermal skin samples from eastern North Atlantic killer whales, Orcinus orca, were analyzed for carbon and nitrogen stable isotope ratios. From those, comparisons within a data set of 17 samples collected from Tysfjord, Norway, in November suggested that diet is relatively specialized during this time period at this location. There were significant differences between a small set of samples from Iceland and those collected from Norway, which had all been assigned to the same population by a previous population genetics study. The results would be consistent with matrilines feeding on either the Norwegian or Icelandic stocks of Atlantic herring (Clupea harengus). There was no significant difference within Icelandic samples between those assigned to the population known to feed upon herring and those assigned to a population hypothesized to follow Atlantic mackerel (Scomber scombrus). The greatest differences were between the epidermal samples analyzed in this study and tooth and bone collagen samples from the North Sea that were analyzed previously, which also showed significantly more variation in isotopic ratios than found for skin samples. These differences could reflect differences in turnover rate, differences in diet‐tissue fractionation and discrimination due to the amino acid composition of the different tissues, and/or greater competition promoting dietary variation between groups in the North Sea.     

Effects of decomposition and storage conditions on the δ13C and δ15N isotope values of killer whale (Orcinus orca) skin and blubber tissues

Several different factors in the collection and preservation of whale skin and blubber samples were examined to determine their effect on the results obtained by stable nitrogen and carbon isotope (δ¹⁵N and δ¹³C) analysis. Samples of wet killer whale skin retained their original stable isotope values for up to 14 d at 4°C or lower. However, decomposition significantly changed the δ¹⁵N value within 3 d at 20°C. Storage at ?20°C was as effective as ?80°C for the preservation of skin and blubber samples for stable isotope analysis for at least a year. By contrast, once a skin sample had been freeze‐dried and lipid extracted, the stable isotope values did not change significantly when it was stored dry at room temperature for at least 12 mo. Preservation of whale skin samples for a month in DMSO‐salt solution, frozen or at room temperature, did not significantly change the δ¹⁵N and δ¹³C values of lipid extracted tissues, although the slight changes seen could influence results of a study if only small changes are expected.     

Investigating the biochar effects on C‐mineralization and sequestration of carbon in soil compared with conventional amendments using the stable isotope (δ13C) approach

Biomass‐derived black carbon (biochar) is considered to be an effective tool to mitigate global warming by long‐term C‐sequestration in soil and to influence C‐mineralization via priming effects. However, the underlying mechanism of biochar (BC) priming relative to conventional biowaste (BW) amendments remains uncertain. Here, we used a stable carbon isotope (δ¹³C) approach to estimate the possible biochar effects on native soil C‐mineralization compared with various BW additions and potential carbon sequestration. The results show that immediately after application, BC suppresses and then increases C‐mineralization, causing a loss of 0.14–7.17 mg‐CO₂–C g^?1‐C compared to the control (0.24–1.86 mg‐CO₂–C g^?1‐C) over 1–120 days. Negative priming was observed for BC compared to various BW amendments (?10.22 to ?23.56 mg‐CO₂–C g^?1‐soil‐C); however, it was trivially positive relative to that of the control (8.64 mg‐CO₂–C g^?1‐soil‐C). Furthermore, according to the residual carbon and δ¹³C signature of postexperimental soil carbon, BC‐C significantly increased (P < 0.05) the soil carbon stock by carbon sequestration in soil compared with various biowaste amendments. The results of cumulative CO₂–C emissions, relative priming effects, and carbon storage indicate that BC reduces C‐mineralization, resulting in greater C‐sequestration compared with other BW amendments, and the magnitude of this effect initially increases and then decreases and stabilizes over time, possibly due to the presence of recalcitrant‐C (4.92 mg‐C g^?1‐soil) in BC, the reduced microbial activity, and the sorption of labile organic carbon (OC) onto BC particles.     

The effects of Paraloid B‐72 and Butvar B‐98 treatment and organic solvent removal on δ13C, δ15N,and δ18O values of collagen and hydroxyapatite in a modern bone

Stable isotopes in bones are a powerful tool for diet, provenance, climate, and physiological reconstructions, but necessarily require well‐preserved specimens unaltered by postmortem diagenesis or conservation practices. This study examines the effects of Paraloid B‐72 and Butvar B‐98, two common consolidants used in field and museum conservation, on δ¹³C, δ¹⁵N, and δ¹⁸O values from bone collagen and hydroxyapatite. The effects of solvent removal (100% acetone, 100% ethanol, 9:1 acetone:xylenes, 9:1 ethanol:xylenes) and drying methods (ambient air, vacuum, oven drying at 80°C) were also examined to determine if bones treated with these consolidants can successfully be cleaned and used for stable isotope analyses. Results show that introduction of Paraloid B‐72 or Butvar B‐98 in 100% acetone or 100% ethanol, respectively, with subsequent removal by the same solvents and drying at 80°C facilitates the most successful removal of consolidants and solvents. The δ¹³C values in collagen, δ¹⁵N in collagen, δ¹⁸O in hydroxyapatite phosphate, and δ¹³C in hydroxyapatite structural carbonate were unaltered by treatments with Paraloid or Butvar and subsequent solvent removal. The δ¹⁸O in hydroxyapatite structural carbonate showed nonsystematic variability when bones were treated with Paraloid and Butvar, which is hypothesized to be a result of hydroxyl exchange when bones are exposed to consolidants in solution. It is therefore recommended that δ¹⁸O in hydroxyapatite structural carbonate should not be used in stable isotope studies if bones have been treated with Paraloid or Butvar. Am J Phys Anthropol 157:330–338, 2015. © 2015 Wiley Periodicals, Inc.     

Comparative response of δ13C, δ18O and δ15N in durum wheat exposed to salinity at the vegetative and reproductive stages

This study compared the performance of the stable isotope composition of carbon (δ¹³C), oxygen (δ¹⁸O) and nitrogen (δ¹⁵N) by tracking plant response and genotypic variability of durum wheat to different salinity conditions. To that end, δ¹³C, δ¹⁸O and δ¹⁵N were analysed in dry matter (dm) and the water‐soluble fraction (wsf) of leaves from plants exposed to salinity, either soon after plant emergence or at anthesis. The δ¹³C and δ¹⁸O of the wsf recorded the recent growing conditions, including changes in evaporative conditions. Regardless of the plant part (dm or wsf), δ¹³C and δ¹⁸O increased and δ¹⁵N decreased in response to stress. When the stress conditions were established just after emergence, δ¹⁵N and δ¹³C correlated positively with genotypic differences in biomass, whereas δ¹⁸O correlated negatively in the most severe treatment. When the stress conditions were imposed at anthesis, relationships between the three isotope signatures and biomass were only significant and positive within the most severe treatments. The results show that nitrogen metabolism, together with stomatal limitation, is involved in the genotypic response to salinity, with the relative importance of each factor depending on the severity and duration of the stress as well as the phenological stage that the stress occurs.