Isotopic fractionation with morphological change and sexual specificity in the lappet moth Euthrix potatoria

The δ¹⁵N values of adult holometabolous insects exceed those of larvae, but otherwise little information on terrestrial invertebrates has been obtained in food‐web analyses using stable isotope ratios (δ¹⁵N, δ¹³C). Changes in δ¹³C during metamorphosis and differences between males and females have not been examined. We collected the larvae and cocoons of Euthrix potatoria (L.) (Lepidoptera: Lasiocampidae) in the field and used them to assess the species’ isotopic fractionation. Each emerged moth was divided into five body parts. We conducted stable N and C isotope analyses for each body part, as well as for cocoons and exuviae, and also compared stable isotope ratios between sexes. We confirmed δ¹⁵N enrichment through metamorphosis and estimated that δ¹⁵N enrichment is accomplished by the relative concentration of ¹⁵N due to the excretion of copious meconium, which contains abundant ¹⁴N. We also observed changes in δ¹³C values through metamorphosis. Both isotope values tended to change more in males than in females. The proportion of the whole‐adult weight represented by meconium was higher in males than in females, suggesting that high meconium secretion in males contributes to the sexual difference in δ¹⁵N. These phenomena may be common in Holometabola, which require a pupal stage. For more accurate food‐web assessments, it is important to consider stable isotope changes during different life cycles, as well as sexual differences.     

Gas chromatography-mass spectrometry determination of [15N]ammonia enrichment in blood and urine

A rapid gas chromatography-mass spectrometry method for [¹⁵N]ammonia analysis is deseribed which is based on the formation of [¹⁵N]glutamic acid from ammonia and analysis of isotopic abundance in the N-trifluoroacetyl-n-butylester glutamate derivative. Mean recovery of [¹⁵N]ammonia added to either plasma or urine was greater than 99% with a relative standard deviation of less than 10%. The method can be applied to the determination of extremely low levels of ammonia through an isotope dilution technique. The [¹⁵N]ammonia abundance of blood and urine was determined in an adult following on oral dose (500 mg) of ¹⁵NH₄Cl. A peak isotopic abundance of 13 atoms% excess was reached by 30 min. Urinary excretion of [¹⁵N]ammonia during the first 4 h after administration of the isotope amounted to 4.1% of the isotope administered.     

A comparison of direct and indirect 15N isotope techniques for estimating crop N uptake from organic residues

Experiments were carried out to compare the direct approach for estimating crop N uptake from ¹⁵N labelled organic inputs, to two indirect approaches, ¹⁵N isotope dilution and A value. In the first experiment soils received 25, 50, 75, or 100 mg N kg soil⁻¹ in the form of Casuarina equisitifolia residues in addition to ammonium sulphate fertiliser, to give a total of 100 mg N kg soil⁻¹ added. This was a cross labelling design, thus two matching sets of treatments, were set up, identical in all but the position of the ¹⁵N label. Maize (Zea mays L.) plants were grown in the soils amended with residues for 11 weeks and N derived from residues (Ndfr) estimated using the A-value or the direct approach. The A-value approach appeared to significantly overestimate %Ndfr compared to the direct method. In the second experiment contrasting residues were added to soil, fababean (Vicia faba L. var. minor), alfalfa (Medicago sativa L.), soyabean fixing, (Glycine max (L.) Merrill), soyabean non-fixing, barley (Hordeum vulgare L.) and maize. This was also cross-labelling design, labelled and unlabelled residues were used. Maize plants were grown in these soils for 11 weeks and %Ndfr in the maize plants estimated using ¹⁵ N isotope dilution and the direct approach. The ¹⁵ N isotope dilution approach also overestimated %Ndfr compared to the direct method in this experiment. Pool substitution appeared to be responsible for the discrepancy between the direct and indirect techniques. It was concluded that ¹⁵N isotope dilution and A-value approaches as used in these experiments (i.e where residues and ¹⁵N label are added simultaneously) were not appropriate techniques for estimating N derived from organic residues in soils. This revised version was published online in June 2006 with corrections to the Cover Date.     

The use of natural abundance of nitrogen isotopes in plant physiology and ecology

Nitrogen stable isotope (¹⁵N, ¹⁴N) natural abundance has been much less used than carbon isotopes (¹³C, ¹²C) in plant physiology and ecology. Analytical problems, the lower fractional abundance of ¹⁵N than of ¹³C in the biosphere, the greater complexity of the N cycle relative to the C cycle, and smaller expressed discriminations in nature, are contributing factors. The major N pools, globally, have different isotope signatures: atmospheric N₂ is ¹⁵N-depleted relative to organic N (including sedimentary N), a situation resulting from a greater expressed discrimination in the organic N to N₂ (via denitrification) reaction than of diazotrophy during accumulation of the reduced N. Essentially all of the enzymes except nitrogenase which transform N compounds show discrimination against ¹⁵N, although for glutamine synthetase, and the amination of 2-oxoglutarate and pyruvate, this is only seen in terms of NH₄⁺ rather than the true substrate, NH₃. Discrimination is expressed in various N interconversions within plants, leading to substantial differences in δ¹⁵N (up to 12‰) among N compounds and macroscopic plant parts. N isotope fractionation during assimilation of exogenous combined N is often much lower than that expected from studies of isolated enzymes due to processes which show very little discrimination, such as limitation by transport through aqueous solution and membranes. Application of ¹⁵N/¹⁴N discrimination studies to plant ecology have concentrated largely on distinguishing diazotrophy from N supplied from combined N, based on the lower ¹⁵N/¹⁴N in diazotrophs due to the higher ¹⁵N/¹⁴N of combined N sources not being offset by fractionation during uptake. While potentially very useful, a number of pitfalls are discussed in its ecological use in both terrestrial and aquatic systems. N isotope discrimination is also useful in tracking N through food webs, and hence, back to combined N sources for plants.     

The 15N isotope effect in Escherichia coli: A neutron can make the difference

Several techniques based on stable isotope labeling are used for quantitative MS. These include stable isotope metabolic labeling methods for cells in culture as well as live organisms with the assumption that the stable isotope has no effect on the proteome. Here, we investigate the ¹⁵N isotope effect on Escherichia coli cultures that were grown in either unlabeled (¹⁴N) or ¹⁵N‐labeled media by LC‐ESI‐MS/MS‐based relative protein quantification. Consistent protein expression level differences and altered growth rates were observed between ¹⁴N and ¹⁵N‐labeled cultures. Furthermore, targeted metabolite analyses revealed altered metabolite levels between ¹⁴N and ¹⁵N‐labeled bacteria. Our data demonstrate for the first time that the introduction of the ¹⁵N isotope affects protein and metabolite levels in E. coli and underline the importance of implementing controls for unbiased protein quantification using stable isotope labeling techniques.     

Metabolic origin of δ15N values in nitrogenous compounds from Brassica napus L. leaves

Nitrogen isotope composition (δ¹⁵N) in plant organic matter is currently used as a natural tracer of nitrogen acquisition efficiency. However, the δ¹⁵N value of whole leaf material does not properly reflect the way in which N is assimilated because isotope fractionations along metabolic reactions may cause substantial differences among leaf compounds. In other words, any change in metabolic composition or allocation pattern may cause undesirable variability in leaf δ¹⁵N. Here, we investigated the δ¹⁵N in different leaf fractions and individual metabolites from rapeseed (Brassica napus) leaves. We show that there were substantial differences in δ¹⁵N between nitrogenous compounds (up to 30‰) and the content in (¹⁵N enriched) nitrate had a clear influence on leaf δ¹⁵N. Using a simple steady‐state model of day metabolism, we suggest that the δ¹⁵N value in major amino acids was mostly explained by isotope fractionation associated with isotope effects on enzyme‐catalysed reactions in primary nitrogen metabolism. δ¹⁵N values were further influenced by light versus dark conditions and the probable occurrence of alternative biosynthetic pathways. We conclude that both biochemical pathways (that fractionate between isotopes) and nitrogen sources (used for amino acid production) should be considered when interpreting the δ¹⁵N value of leaf nitrogenous compounds.     

Experimental evidence for diel δ15N‐patterns in different tissues,xylem and phloem saps of castor bean (Ricinus communis L.)

Nitrogen isotope signatures in plants might give insights in the metabolism and allocation of nitrogen. To obtain a deeper understanding of the modifications of the nitrogen isotope signatures, we determined δ¹⁵N in transport saps and in different fractions of leaves, axes and roots during a diel course along the plant axis. The most significant diel variations were observed in xylem and phloem saps where δ¹⁵N was significantly higher during the day compared with during the night. However in xylem saps, this was observed only in the canopy, but not at the hypocotyl positions. In the canopy, δ¹⁵N was correlated fairly well between phloem and xylem saps. These variations in δ¹⁵N in transport saps can be attributed to nitrate reduction in leaves during the photoperiod as well as to ¹⁵N‐enriched glutamine acting as transport form of N. δ¹⁵N of the water soluble fraction of roots and leaves partially affected δ¹⁵N of phloem and xylems saps. δ¹⁵N patterns are likely the result of a complex set of interactions and N‐fluxes between plant organs. Furthermore, the natural nitrogen isotope abundance in plant tissue is not constant during the diel course – a fact that needs to be taken into account when sampling for isotopic studies.     

Altitudinal gradients of grassland carbon and nitrogen isotope composition are recorded in the hair of grazers

Aim The hair of grazers provides an isotopic record of environmental and nutritional signals. Here, we assess the effect of altitude on the carbon and nitrogen isotope composition of the hair of ruminant grazers and its relation to grassland vegetation, to evaluate the use of hair isotope data for ecosystem reconstruction, animal nutritional ecology and biogeochemical studies in montane environments. Location European Alps. Methods We sampled grassland vegetation (pure C₃) and the hair of ruminants along an altitudinal gradient (400–2500 m), and analysed their isotope composition (δ¹³C and δ¹⁵N). Results were compared with published effects of altitude on ¹³C in C₃ plants at the species level and on ¹⁵N at the community level. The study was complemented with a comparison of diet and hair isotope composition in ruminants held in confinement. Results δ¹³C of hair increased (c. 1.1‰ km⁻¹) and δ¹⁵N decreased (c. 1.1‰ km⁻¹) with altitude. The same changes occurred in local grassland vegetation, and in regional to global grassland data sets. Offsets between hair and vegetation ¹³C or ¹⁵N (‘diet–hair shift’) were independent of altitude. Sheep (Ovis aries) and cattle (Bos taurus) exhibited a ¹³C shift near +3‰, but that of goats (Capra hircus) was larger (+4.2‰) in alpine environments and in confinement. The diet–hair shift for ¹⁵N was more variable (+2.1 to +3.6‰). Main conclusions Grazer hair provides a faithful spatially and temporally integrated record of grassland isotope composition, useful for ecosystem and environment reconstruction. The effect of altitude on hair ¹⁵N is important for studies of trophic relationships: an altitude shift of 2000 m produced the same effect in hair ¹⁵N as would a shift from an animal tissue‐based to a plant‐based diet. The similarity of altitude effects on δ¹³C of individual plant species, vegetation and hair indicates that the effect of altitude on species‐level ‘intrinsic water use efficiency’ scales up linearly to the community and landscape level.     

Isotope alteration caused by changes in biochemical composition of sedimentary organic matter

Stable carbon (C) and nitrogen (N) isotope ratios of sedimentary organic matter (OM) can reflect the biogeochemical history of aquatic ecosystems. However, diagenetic processes in sediments may alter isotope records of OM via microbial activity and preferential degradation of isotopically distinct organic components. This study investigated the isotope alteration caused by preferential degradation in surface sediments sampled from a eutrophic reservoir in Germany. Sediments were treated sequentially with hot water extraction, hydrochloric acid hydrolysis, hydrogen peroxide oxidation and di-sodium peroxodisulfate oxidation to chemically simulate preferential degradation pathways of sedimentary OM. Residue and extracts from each extraction step were analyzed using elemental analyzer-isotope ratio mass spectrometry and solid-state ¹³C nuclear magnetic resonance spectroscopy. Our results show that stable C and N isotope ratios reacted differently to changes in the biochemical composition of sedimentary OM. Preferential degradation of proteins and carbohydrates resulted in a 1.2‰ depletion of ¹³C, while the isotope composition of ¹⁵N remained nearly the same. Sedimentary δ¹⁵N values were notably altered when lignins and lipids were oxidized from residual sediments. Throughout the sequential fractionation procedure, δ¹³C was linearly correlated with the C:N of residual sediments. This finding demonstrates that changes in biochemical composition caused by preferential degradation altered δ¹³C values of sedimentary OM, while this trend was not observed for δ¹⁵N values. Our study identifies the influence of preferential degradation on stable C isotope ratios and provide additional insight into the isotope alteration caused by post-depositional processes.

     

Carbon and nitrogen stable isotope ratios of macroinvertebrates in the littoral zone of Lake Biwa as indicators of anthropogenic activities in the watershed

Carbon and nitrogen stable isotope ratios (δ¹³C and δ¹⁵N) of macroinvertebrates inhabiting littoral zones of lakes can serve as useful indicators of material loading from the watershed. We collected snails (Semisulcospira spp.) and bivalves (Unio douglasiae biwae Kobelt) from 29 littoral sites in Lake Biwa near the mouths of river tributaries with various human population density (HPD) and land-use patterns. The δ¹³C and δ¹⁵N signatures were determined for three potential food sources: particulate organic matter in the pelagic zone (PPOM), riverine particulate organic matter from tributaries (RPOM) and epilithic organic matter in the littoral zone (EOM). The stable isotope mixing model revealed that snails relied mainly on EOM, and bivalves on PPOM and RPOM. Multiple regression analysis showed that intersite variation in δ¹⁵N for snails was best explained by HPD, while variation in δ¹⁵N of EOM and nitrate was explained to a lesser extent by HPD. Comparison with isotope signatures of their food sources and riverine nutrients revealed that snails assimilated anthropogenic nitrogen from wastewater in the watershed. Our results also showed that the δ¹³C value of bivalves was marginally related to the fraction of paddy fields in the watersheds. In conclusion, the isotope signatures of macroinvertebrates inhabiting the littoral zone can be useful indicators of anthropogenic impacts from the watershed.     

Gross mineralization and plant N uptake from animal manures under non-N limiting conditions, measured using 15N isotope dilution techniques

To utilise wisely the manure resource, a better understanding of the processes that control the breakdown of organic N to inorganic N (mineralization) is required. ¹⁵N isotope dilution techniques should allow estimates of plant N uptake and gross mineralization from organic manures under non-N limiting conditions to be made. In natural systems the study of organic nitrogen breakdown to inorganic nitrogen, mineralization, is confounded by the processes of nitrification, nitrate leaching, gaseous N losses and plant N uptake. The ¹⁵N isotope dilution approach allows measurement of gross mineralization independently of these processes. Greenhouse experiments were conducted to determine plant N uptake from organic manures under non-N limiting conditions using the soil pre-labelling isotope dilution approach. The soil was pre-labelled with ¹⁵N and maize plants were then grown on the control treatments (no organic amendment) or on the manure treatments. The principle is thus that the control crop has a ¹⁵N abundance which reflects the ¹⁵N status of the soil and the treatment crop has a ¹⁵N enrichment diluted by the contribution of mineralized unlabelled manure N. Using this technique, it was estimated that maize plants derived 17 and 34% of their N from sewage sludge and turkey manure, respectively. The soil pre-labelling isotope dilution approach allowed yield-independent estimation of nitrogen derived from manures under non-N limiting conditions. Estimates of gross N mineralization were made to determine the breakdown of manure under field conditions. Results suggested that there was a rapid mineralization of turkey manure N in the initial weeks after application, in the order of 50 kg N ha^?1, which tailed off in the following weeks. The technique suggested that the soil used in the study had an extremely low basal mineralization rate, and a high nitrification rate.     

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.     

Nitrogen stable isotope ratios in surface sediments, epilithon and macrophytes from upland lakes with differing nutrient status

1. Thirty small upland lakes in Cumbria, Wales, Scotland and Northern Ireland were each visited once during June and July 2000. From each lake, samples of surface sediment epilithon, macrophytes and total dissolved nitrogen (TDN) were collected for nitrogen stable isotope analysis. As part of a wider programme, samples were also collected for chemical analysis and bioassays. 2. Considerable variation was found in δ¹⁵N values in all measured nitrogen compartments. Some regional variation was evident but was generally weak. Sediment and epilithon δ¹⁵N were positively correlated with δ¹⁵N of TDN, suggesting that baseline nitrogen isotope ratios influence those in some organic matter compartments in the lakes. 3. Sediment δ¹⁵N was higher when inorganic nitrogen concentration in the water was low, possibly reflecting reduced isotope fractionation under these conditions. However, this was not the case for epilithon or macrophytes. Sediment δ¹⁵N values were also negatively related to annual nitrogen deposition. 4. Sediment, epilithon and macrophyte δ¹⁵N values all showed significant relations to nutrient limitation in the lakes as determined by algal bioassays. We suggest that sediment δ¹⁵N might be developed as a simple integrating measure of the degree of nitrogen limitation in lakes.     

A non‐lethal approach to estimate whole‐body 13C and 15N stable isotope ratios of freshwater amphipods using walking legs

To analyze the stable isotope ratios of small‐bodied invertebrates, the entire animal is typically sacrificed and processed, which is problematic for threatened or endangered species. Appendages which are regenerated could be used to infer whole‐body isotope ratios, but differences in turnover rates and isotopic signatures among tissues may confound such an approach. We tested the hypothesis that the δ¹³C and δ¹⁵N of whole‐body tissue for freshwater amphipods could be predicted from the δ¹³C and δ¹⁵N of walking legs, with the goal of estimating body δ¹³C and δ¹⁵N of Gammarus acherondytes, a United States federally endangered species. To test this, we analyzed the δ¹³C and δ¹⁵N of walking legs and bodies of five species of amphipods from geographically distant areas (Idaho, Illinois, and Washington) in the United States. The general relationships of whole‐body isotope ratios of C and N as a function of leg isotope ratios were linear and had slopes of one. In the range of the data, leg δ¹³C was slightly lower than body δ¹³C, indicating some tissue‐specific fractionation, while δ¹⁵N was similar for legs and bodies. Our data suggest that legs can be used to predict body isotope ratios in freshwater amphipods. This approach provides an additional tool to help researchers understand the biology of small, endangered invertebrates without sacrificing individuals. This is especially useful in cave ecosystems where populations are naturally sparse.     

Measurements of nitrogen fixation in fababean at different N fertilizer rates using the 15N isotope dilution and ‘A-value’ methods

The ¹⁵N isotope dilution and A-value methods were used to measure biological nitrogen (N₂) fixation in field grown fababean (Vicia faba L.), over a 2-year period. Four N rates, 20, 100, 200 and 400 kg N ha^–1 were examined. The two isotope methods gave similar values of % N derived from the atmosphere (%Ndfa). With 20 kg N ha^–1, %Ndfa in fababean was about 85% in both years. Increasing the N rate to 100 kg N ha^–1 decreased N₂ fixation slightly to 75%. Further reductions in N₂ fixed to 60 and 43% occurred where 200 and 400 kg N ha^–1 were applied, respectively. Thus even higher rates of N than normally applied in farming practice could not completely suppress N₂ fixation in fababean.We also devised one equation for both the isotope dilution and A-value approaches, thereby (i) avoiding the need for different calculations for the ¹⁵N isotope methods, and (ii) showing once again that the isotope dilution and A-value methods are mathematically and conceptually identical.     

Variable δ15N Diet-Tissue Discrimination Factors among Sharks: Implications for Trophic Position,Diet and Food Web Models

The application of stable isotopes to characterize the complexities of a species foraging behavior and trophic relationships is dependent on assumptions of δ¹⁵N diet-tissue discrimination factors (∆¹⁵N). As ∆¹⁵N values have been experimentally shown to vary amongst consumers, tissues and diet composition, resolving appropriate species-specific ∆¹⁵N values can be complex. Given the logistical and ethical challenges of controlled feeding experiments for determining ∆¹⁵N values for large and/or endangered species, our objective was to conduct an assessment of a range of reported ∆¹⁵N values that can hypothetically serve as surrogates for describing the predator-prey relationships of four shark species that feed on prey from different trophic levels (i.e., different mean δ¹⁵N dietary values). Overall, the most suitable species-specific ∆¹⁵N values decreased with increasing dietary-δ¹⁵N values based on stable isotope Bayesian ellipse overlap estimates of shark and the principal prey functional groups contributing to the diet determined from stomach content analyses. Thus, a single ∆¹⁵N value was not supported for this speciose group of marine predatory fishes. For example, the ∆¹⁵N value of 3.7‰ provided the highest percent overlap between prey and predator isotope ellipses for the bonnethead shark (mean diet δ¹⁵N = 9‰) whereas a ∆¹⁵N value < 2.3‰ provided the highest percent overlap between prey and predator isotope ellipses for the white shark (mean diet δ¹⁵N = 15‰). These data corroborate the previously reported inverse ∆¹⁵N-dietary δ¹⁵N relationship when both isotope ellipses of principal prey functional groups and the broader identified diet of each species were considered supporting the adoption of different ∆¹⁵N values that reflect the predators’ δ¹⁵N-dietary value. These findings are critical for refining the application of stable isotope modeling approaches as inferences regarding a species’ ecological role in their community will be influenced with consequences for conservation and management actions.     

A skew in poo: Biases in primate fecal isotope analysis and recommendations for standardized sample preparation

Feces are a treasure trove in the study of animal behavior and ecology. Stable carbon and nitrogen isotope analysis allows to assess the dietary niches of elusive primate species and primate breastfeeding behavior. However, some fecal isotope data may unwillingly be biased toward the isotope ratios of undigested plant matter, requiring more consistent sample preparation protocols. We assess the impact of this potential data skew in 114 fecal samples of wild bonobos (Pan paniscus) by measuring the isotope differences (Δ¹³C, Δ¹⁵N) between bulk fecal samples containing larger particles (>1 mm) and filtered samples containing only small particles (<1 mm). We assess the influence of fecal carbon and nitrogen content (ΔC:N) and sample donor age (subadult, adult) on the resulting Δ¹³C, Δ¹⁵N values (n = 228). Additionally, we measure the isotope ratios in three systematically sieved fecal samples of chimpanzees (Pan troglodytes verus), with particle sizes ranging from 20 μm to 8 mm (n = 30). We found differences in fecal carbon and nitrogen content, with the smaller fecal fraction containing more nitrogen on average. While the Δ¹³C values were small and not affected by age or ΔC:N, the Δ¹⁵N values were significantly influenced by fecal ΔC:N, possibly resulting from the differing proportions of undigested plant macroparticles. Significant relationships between carbon stable isotope ratios (δ¹³C) values and %C in large fecal fractions of both age groups corroborated this assessment. Δ¹⁵N values were significantly larger in adults than subadults, which should be of concern in isotope studies comparing adult females with infants to assess breastfeeding. We found a random variation of up to 3.0‰ in δ¹³C and 2.0‰ in nitrogen stable isotope ratios within the chimpanzee fecal samples separated by particle sizes. We show that particle size influences isotope ratios and propose a simple, cost-effective filtration method for primate feces to exclude larger undigested food particles from the analysis, which can easily be adopted by labs worldwide.     

Variations in nitrogen-15 natural abundance of plant and soil systems in four remote tropical rainforests, southern China

The foliar stable N isotope ratio (δ¹⁵N) can provide integrated information on ecosystem N cycling. Here we present the δ¹⁵N of plant and soil in four remote typical tropical rainforests (one primary and three secondary) of southern China. We aimed to examine if (1) foliar δ¹⁵N in the study forests is negative, as observed in other tropical and subtropical sites in eastern Asia; (2) variation in δ¹⁵N among different species is smaller compared to that in many N-limited temperate and boreal ecosystems; and (3) the primary forest is more N rich than the younger secondary forests and therefore is more ¹⁵N enriched. Our results show that foliar δ¹⁵N ranged from ?5.1 to 1.3 ‰ for 39 collected plant species with different growth strategies and mycorrhizal types, and that for 35 species it was negative. Soil NO₃ ^? had low δ¹⁵N (?11.4 to ?3.2 ‰) and plant NO₃ ^? uptake could not explain the negative foliar δ¹⁵N values (NH₄ ⁺ was dominant in the soil inorganic-N fraction). We suggest that negative values might be caused by isotope fractionation during soil NH₄ ⁺ uptake and mycorrhizal N transfer, and by direct uptake of atmospheric NH₃/NH₄ ⁺. The variation in foliar δ¹⁵N among species (by about 6 ‰) was smaller than in many N-limited ecosystems, which is typically about or over 10 ‰. The primary forest had a larger N capital in plants than the secondary forests. Foliar δ¹⁵N and the enrichment factor (foliar δ¹⁵N minus soil δ¹⁵N) were higher in the primary forest than in the secondary forests, albeit differences were small, while there was no consistent pattern in soil δ¹⁵N between primary and secondary forests.     

THE MECHANISM OF ISOTOPE FRACTIONATION DURING ALGAL NITRATE ASSIMILATION AS ILLUMINATED BY THE 15N/14N OF INTRACELLULAR NITRATE1

The ¹⁵N/¹⁴N of nitrate in the external medium and intracellular pool of the cultured marine diatom Thalassiosira weissflogii (Grun.) Fryxell et Hasle was measured during nitrate assimilation under low light, a 12:12‐h light:dark cycle, low temperature, or low iron conditions. The ¹⁵N/¹⁴N of the nitrate in the medium and the particulate matter both followed the predicted Rayleigh fractionation model, and the intracellular nitrate always had a higher ¹⁵N/¹⁴N than did the medium nitrate. When the experiments were compared, the results showed a negative correlation between the isotope fractionation factor and the difference in the ¹⁵N/¹⁴N between the two pools of nitrate. These observations imply that the variations in the isotope effect result from variations in the degree to which the fractionation by nitrate reductase is expressed outside the cell, which is, in turn, controlled by the rate of nitrate efflux relative to nitrate reduction. The low iron and low temperature experiments showed relatively small isotope effects but a large intracellular‐medium difference in nitrate ¹⁵N/¹⁴N, consistent with a relative rate of efflux (compared with influx) that is small and similar to fast‐growing cells. In contrast, large isotope effects and small intracellular‐medium differences in nitrate ¹⁵N/¹⁴N were observed for low light and light:dark cycle grown cells and are explained by higher relative rates of nitrate efflux under these growth conditions.     

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.