Source Partitioning Using Stable Isotopes: Coping with Too Much Variation

Background

Stable isotope analysis is increasingly being utilised across broad areas of ecology and biology. Key to much of this work is the use of mixing models to estimate the proportion of sources contributing to a mixture such as in diet estimation.

Methodology

By accurately reflecting natural variation and uncertainty to generate robust probability estimates of source proportions, the application of Bayesian methods to stable isotope mixing models promises to enable researchers to address an array of new questions, and approach current questions with greater insight and honesty.

Conclusions

We outline a framework that builds on recently published Bayesian isotopic mixing models and present a new open source R package, SIAR. The formulation in R will allow for continued and rapid development of this core model into an all-encompassing single analysis suite for stable isotope research.     

Incorporating uncertainty and prior information into stable isotope mixing models

Stable isotopes are a powerful tool for ecologists, often used to assess contributions of different sources to a mixture (e.g. prey to a consumer). Mixing models use stable isotope data to estimate the contribution of sources to a mixture. Uncertainty associated with mixing models is often substantial, but has not yet been fully incorporated in models. We developed a Bayesian-mixing model that estimates probability distributions of source contributions to a mixture while explicitly accounting for uncertainty associated with multiple sources, fractionation and isotope signatures. This model also allows for optional incorporation of informative prior information in analyses. We demonstrate our model using a predator–prey case study. Accounting for uncertainty in mixing model inputs can change the variability, magnitude and rank order of estimates of prey (source) contributions to the predator (mixture). Isotope mixing models need to fully account for uncertainty in order to accurately estimate source contributions.     

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.     

Direct measurement of nitrogen fixation by Trifolium repens L. and Alnus glutinosa L. using 15N2

A closed-system flow-through enclosure apparatus was used tomeasure symbiotic nitrogen fixation directly. A legume-basedsystem comprising 6-week-old Trifolium repens L. (white clovercv. Blanca) growing with Lolium perenne L. (perennial ryegrasscv. Trani) in an agricultural soil was incubated for 19 d ina ¹⁵N-enriched atmosphere (mean value 3.663 atom%). An actinorhizal-basedsystem comprising 1 -year-old Alnus glutinosa L. (alder) saplingsgrowing with Festuca rubra L. (red fescue) in open-cast coalspoil was incubated for 21 d in a ¹⁵N-enriched atmosphere (meanvalue 3.265 atom%). Indirect estimates of N2 fixation were carriedout concurrently using N difference and ¹⁵N isotope dilutiontechniques. The theory underlying the three techniques and modificationswhich were adopted for comparative purposes are discussed. Thedirect measurements of N2 fixation were then compared with theindirect estimates using P_inc, the proportion of the N incrementduring the measurement period that was derived from fixation.The simple N difference method gave similar values for Pinc(0.94 and 0.97) as those derived from more complicated isotopemethodologies, both indirect (0.91) and direct (0.90). Valuesfor alder were far more variable, ranging from 0.16 to 0.92;this was due largely to variability within the trees and a verysmall N increment during the measurement period. Key words: N₂ fixation, ¹⁵N₂, white clover, alder, enclosure apparatus     

Phytoplankton photosynthesis parameters in central Canadian lakes

Values for two biological parameters — (i) P^B_m, the lightsaturated rate of photosynthesis per unit of chlorophyll and(ii) , the slope of the light limited part of the photosynthesisversus light curve per unit of chlorophyll — must be knownin order to be able to estimate rates of phytoplankton primaryproduction from chlorophyll data. These parameters were measuredfor periods of up to 9 years in central Canadian lakes locatedin temperate, subarctic and arctic climatic zones. Regardlessof their geographic locations, lakes where integral photosynthesiswas nutrient limited had lower values of these parameters thandid lakes where integral photosynthesis was light limited. Temperatureset an upper limit to the variability of P^B_m but was not a goodpredictor of its actual value. Year-to-year variability of photosynthesisparameters in the most intensively studied group of lakes waslarge: annual means varied by a factor of three over a 9-yearperiod. Until the sources and extent of this variability areknown, accurate production estimates can be obtained from chlorophylldata only if P^B_m and are measured in each water body and inevery year. Implications for estimating primary production fromremotely-sensed chlorophyll data are discussed.     

Source partitioning using stable isotopes: coping with too many sources

Stable isotopes are increasingly being used as tracers in environmental studies. One application is to use isotopic ratios to quantitatively determine the proportional contribution of several sources to a mixture, such as the proportion of various pollution sources in a waste stream. In general, the proportional contributions of n+1 different sources can be uniquely determined by the use of n different isotope system tracers (e.g., δ¹³C, δ¹⁵N, δ¹⁸O) with linear mixing models based on mass balance equations. Often, however, the number of potential sources exceeds n+1, which prevents finding a unique solution of source proportions. What can be done in these situations? While no definitive solution exists, we propose a method that is informative in determining bounds for the contributions of each source. In this method, all possible combinations of each source contribution (0–100%) are examined in small increments (e.g., 1%). Combinations that sum to the observed mixture isotopic signatures within a small tolerance (e.g., ±0.1‰) are considered to be feasible solutions, from which the frequency and range of potential source contributions can be determined. To avoid misrepresenting the results, users of this procedure should report the distribution of feasible solutions rather than focusing on a single value such as the mean. We applied this method to a variety of environmental studies in which stable isotope tracers were used to quantify the relative magnitude of multiple sources, including (1) plant water use, (2) geochemistry, (3) air pollution, and (4) dietary analysis. This method gives the range of isotopically determined source contributions; additional non-isotopic constraints specific to each study may be used to further restrict this range. The breadth of the isotopically determined ranges depends on the geometry of the mixing space and the similarity of source and mixture isotopic signatures. A sensitivity analysis indicated that the estimated ranges vary only modestly with different choices of source increment and mass balance tolerance parameter values. A computer program (IsoSource) to perform these calculations for user-specified data is available at http://www.epa.gov/wed/pages/models.htm.     

Ontogenetic Changes in the Chemical Composition of Ricinus communis Grown with NO-3 or NH+4 as N Source

Ricinus communis L. var. Gibsonii was grown in Long Ashton nutrientmedium with either 12mol m^–3 NO^–₃ or 8.0 mol m^–3NH⁺₄ as N source. Two plants from each N treatment were harvestedtwice a week and analysed for C, N, P, S, NO^–₃, SO^2–₄Cl^–K⁺Na⁺, Ca²⁺ Mg²⁺ and ash alkalinity. Statistical analysis of thedata showed that the effect of age and N source was differentfor the chemical variables analysed. Thus [Na⁺] was unaffectedby age or N source, and for both N sources [Mg²⁺] started atthe same level and decreased at the same rate as the plantsmatured. With NH⁺₄ as N source, [SO^2–₄] was higher thanwith NO^–₃, but did not alter with age. The concentrations,in mmol g^–1 dry wt, of C, organic N, K⁺ and Ca²⁺ weredifferent for the two N sources, but the levels of these variablesaltered with age in the same way for both N sources; i.e. therewas no age x N interaction. In the case of P, NO^–₃, Cl^– and COO^–, however,age-related variations were different for the two N sources.It is concluded, inter alia, that [Na⁺] is determined by external[Na⁺] alone, and that K⁺, Ca²⁺ and Cl^– are the inorganicions actively involved in charge balance during ion uptake bythe roots. Key words: Ontogeny, Chemical composition, Plant nutrition     

Light absorption by phytoplankton: development of a matching parameter for algal photosynthesis under different spectral regimes

A spectral matching parameter (absorption efficiency, A_e) wasdeveloped to quantify the relationship between the light absorptionspectra of phytoplankton communities and the spectral irradianceof their ambient light field. A_e was defined as the ratio betweenthe amount of radiation absorbed by the phytoplankton in situand the amount absorbed in a spectrally flat light regime. Thisapproach was applied to our measurements of spectral absorptionfor the phytoplankton communities in six lakes in High ArcticCanada that spanned a range of bio-optical conditions. A_e valueswere calculated for the light spectrum down through the watercolumn and for 11 types of artificial light source. Spectralmatching varied among lakes and with depth. There was a significantlinear relationship between the relative change in A_e with depthand the diffuse attenuation coefficient K_d (r² = 0.52, P = 0.012for K_d for the 400–700 nm waveband; r² = 0.78, P = 0.0003for K_d at 440 nm). The tabulated values for the matching parameterA_e allow the comparison of photosynthesis versus irradiance(P versus E) curves among studies using different light sources.A_e estimates also facilitate the evaluation of chromatic adaptationin natural waters, and the calculation of spectrally adjusted,in situ primary production down through a water column fromP versus E relationships under a single spectral regime.     

Staphylococcal enterotoxin genes are common in Staphylococcus aureus intestinal flora in Sudden Infant Death Syndrome (SIDS) and live comparison infants

Pathological and epidemiological findings in sudden infant death syndrome (SIDS) suggest an infectious aetiology with indications of involvement of staphylococcal enterotoxins (SEs). While SEA, SEB and SEC have been found in the sera and tissues of SIDS cases, little is known about the role of intestinal Staphylococcus aureus or the roles of later-described toxins SEE, SEG, SEH, SEI and SEJ in SIDS. We used a molecular-based approach to define whether the intestinal tract could be a source of SEs to support the staphylococcal toxic shock hypothesis for SIDS. Intestinal contents from 57 SIDS infants and faeces from 79 age- and gender-matched live comparison infants were cultured and tested for S. aureus and sea-b-c-e-g-h-j and TSST using PCR. High proportions of infants in both groups carried toxigenic and nontoxigenic S. aureus . Significantly greater proportions of SIDS compared with comparison babies were positive for S. aureus (68.4% vs. 40.5%) and for SE genes (43.8% vs. 21.5%), suggesting a possible role in SIDS. The results indicate that colonization by S. aureus with SE genes is common in infants; however, their detection is unlikely to be a strong predictive tool for SIDS. Other factors (including immune response) may reveal a specific susceptibility to SEs in SIDS infants.     

Primary production in lakes Huron and Michigan: in vitro and in situ comparisons

Oxygen- and carbon-14-based primary production estimates from9–16 h in vitro incubations were compared in lakes Huronand Michigan. For surface mixing layer compansons, gross O₂/¹⁴Cphotosynthetic quotients (gross PQ) averaged 2.2, and net O₂/¹⁴Cphotosynthetic quotients (net PQ) averaged 1.4. The mean grossPQ is consistent with a theoretical P0 based on the CO₂ andNO₃ assimilation ratio. However, within the deep chlorophylllayer, gross PQ and net PQ averaged 4.9 and 2.8 respectively.These higher values were likely due to excess NO₃ reductionat the expense of CO₂ uptake. Thus, during short experimentsunder low light conditions, oxygen evolution and CO₂ uptakemay not be tightly coupled. In vitro and in situ O₂ productionestimates were compared in four diurnal (dawn to dusk) experimentsin Lake Huron. In situ production estimates were determinedby measuring water-mass oxygen changes and oxygen transfer acrossthe air-water interface. In situ production estimates were approximatelytwice in vitro production estimates for both surface mixinglayer and deep chlorophyll layer comparisons. The differencebetween estimates was attributable to containment effects manifestin 13–16 h bottle incubations. Short-term (1–2 h)in vitro production was also compared to diurnal in vitro production.Rates of short-term production were {small tilde}1.6 times higherthan rates of diurnal production, suggesting that short-termin vitro production experiments may provide reasonable estimatesof in situ primary production.     

Incorporating concentration dependence in stable isotope mixing models

Stable isotopes are often used as natural labels to quantify the contributions of multiple sources to a mixture. For example, C and N isotopic signatures can be used to determine the fraction of three food sources in a consumer's diet. The standard dual isotope, three source linear mixing model assumes that the proportional contribution of a source to a mixture is the same for both elements (e.g., C, N). This may be a reasonable assumption if the concentrations are similar among all sources. However, one source is often particularly rich or poor in one element (e.g., N), which logically leads to a proportionate increase or decrease in the contribution of that source to the mixture for that element relative to the other element (e.g., C). We have developed a concentration-weighted linear mixing model, which assumes that for each element, a source's contribution is proportional to the contributed mass times the elemental concentration in that source. The model is outlined for two elements and three sources, but can be generalized to n elements and n+1 sources. Sensitivity analyses for C and N in three sources indicated that varying the N concentration of just one source had large and differing effects on the estimated source contributions of mass, C, and N. The same was true for a case study of bears feeding on salmon, moose, and N-poor plants. In this example, the estimated biomass contribution of salmon from the concentration-weighted model was markedly less than the standard model estimate. Application of the model to a captive feeding study of captive mink fed on salmon, lean beef, and C-rich, N-poor beef fat reproduced very closely the known dietary proportions, whereas the standard model failed to yield a set of positive source proportions. Use of this concentration-weighted model is recommended whenever the elemental concentrations vary substantially among the sources, which may occur in a variety of ecological and geochemical applications of stable isotope analysis. Possible examples besides dietary and food web studies include stable isotope analysis of water sources in soils, plants, or water bodies; geological sources for soils or marine systems; decomposition and soil organic matter dynamics, and tracing animal migration patterns. A spreadsheet for performing the calculations for this model is available at http://www.epa.gov/wed/pages/models.htm.     

Determination of the dietary habits of a Magdalenian woman from Saint-Germain-la-Rivière in southwestern France using stable isotopes

To obtain direct dietary information, carbon and nitrogen isotope ratios were measured from bone collagen acquired from the well-preserved skeleton of a Magdalenian woman from the site of Saint-Germain-la-Rivière in southwestern France. Comparison of delta13C and delta15N values of the human bone collagen to those of bone collagen from local herbivores and carnivores indicates that the woman's primary source of protein was the meat of large terrestrial herbivores. Application of a linear mixing model to the woman's isotopic signature indicates that (1) no significant marine-derived protein contributed to her average diet; (2) saiga antelope, which dominates the faunal remains at Saint-Germain-la-Rivière, was not the main source of terrestrial protein; and (3) her pattern of subsistence reflects a less opportunistic behavior than generally attributed to humans from this period. Dietary proportions of prey reflected by the number of identified specimens are revised using meat percentage estimates, which de-emphasize the importance of saiga antelope in human subsistence at Saint-Germain-la-Rivière during the middle Magdalenian.     

Development of the Xylem Ureide Assay for the Measurement of Nitrogen Fixation by Pigeonpea (Cajanus cajan (L.) Millsp.)

Quantification of N₂ fixation by pigeonpea (Cajanus cajan (L.)Millsp.) in the field has proved difficult using techniquessuch as ¹⁵N isotope dilution, acetylene reduction and N difference.We report experiments to develop the ureide assay of N₂ fixationbased on extraction and analysis of xylem exudate. Plants ofpigeonpea cv. Quantum, inoculated with effective Rhizobium spp.CB756, were grown in a temperature-controlled glasshouse inlarge pots filled with a sand: vermiculite mixture, in waterculture and in a slightly acidic, red-brown earth in replicatedfield plots. Xylem exudate was collected as bleeding sap fromboth nodulated and unnodulated roots, and from detached nodules.Exudate was extracted also from detached shoots and stems ofpigeonpea using a mild vacuum (60–70 kN m^–2). Largedifferences in the composition of N solutes exported from rootsof N₂-dependent and nitrate-dependent plants suggested thatshifts in plant dependence on N₂ fixation may be reflected byconcomitant changes in N solutes. Thus, nodulated plants weresupplied throughout growth with either N-free nutrients or nutrientssupplemented with 1, 2, 5, 5, 10, or 20 mol m^{–3 15}. Plants were harvested at regular intervals fordry matter and vacuum-extracted exudate. The relative abundanceof ureides ([ureide-N/ureide-N + nitrate-N + -amino-N] ? 100)in the exudate was highly correlated with the proportion ofplant N (calculated using a ¹⁵N isotope dilution technique)derived from N₂ fixation. Two distinct phases of plant growthwere recognized and standard curves were prepared for each.The relationship between proportional dependence of plants onN₂ and xylem relative ureides was unaffected by mineral-N source,i.e. nitrate or ammonium. This result is discussed in relationto interpretation of material from field-grown plants. The effectsof plant genotype, strain of rhizobia, section of stem extracted,removal of leaves, time delay between shoot detachment and extraction,and diurnal characteristics were examined in order to identifypotential sources of error and to optimize sampling procedures. Key words: Ureides, allantoin, allantoic acid, N₂ fixation, pigeonpea, Cajanus cajan     

Effects of nitrogen nutrition on salt-stressed Nicotiana tabacum var. Samsun in vitro plantlets

Tobacco shoots were grown in vitro for 35 d, in MS culture mediummodified to include various sources (nitrate-N, ammonium-N ora mixture) and levels (0–120 mM) of N, and in the presenceof 0–180 mM NaCI or iso-osmotic concentrations of mannitol.Growth of control plantlets was significantly inhibited whenNH₄⁺-N was the sole N source, and at high (120 mM) NO^–₃-N supply. Under conditions of salt stress (90 and 180 mM NaCI)growth was repressed, with roots being more severely affectedthan shoots. Salinity also inhibited root emergence in vitro.The only alleviation of the salt stress by nitrate nutritionobserved in this study was on shoot growth parameters of plantletsgrown on 60 mM NO^–₃-N and 90 mM NaCI. Although both weresignificantly inhibited by NaCI, nitrate reduc-tase activitywas more severely affected than nitrate uptake. When mannitolreplaced NaCI in the culture medium, similar Inhibition of growth,nutrient uptake and enzyme activity were recorded. These observations,together with the relatively low recorded values for Na⁺ andCI^– uptake, indicate that under in vitro salt stress conditionsthe negative effects of NaCI are primarily osmotic. Key words: Growth, nitrogen metabolism, osmotic stress, salinity     

Leaf δ<Superscript>13</Superscript>C variability with elevation,slope aspect,and precipitation in the southwest United States

Leaves from several desert and woodland species, including gymnosperms and angiosperms with both C₃ and C₄ physiology, were analyzed to detect trends in '¹³C_leaf with elevation and slope aspect along two transects in southeastern Utah and south-central New Mexico, USA. The main difference between the two transects is the steeper elevational gradient for mean annual and summer precipitation in the southern transect. For any given species, we found that isotopic differences between individual plants growing at the same site commonly equal differences measured for plants along the entire altitudinal gradient. In C₃ plants, '¹³C_leaf values become slightly enriched at the lowest elevations, the opposite of trends identified in more humid regions. Apparently, increasing water-use efficiency with drought stress offsets the influence of other biotic and abiotic factors that operate to decrease isotopic discrimination with elevation. For some species shared by the two transects (e.g., Pinus edulis and Cercocarpus montanus), '¹³C_leaf values are dramatically depleted at sites that receive more than 550 mm mean annual precipitation, roughly the boundary (pedalfer-pedocal) at which soils commonly fill to field capacity in summer and carbonates are leached. We hypothesize that, in summer-wet areas, this may represent the boundary at which drought stress overtakes other factors in determining the sign of '¹³C_leaf with elevation. The opposition of isotopic trends with elevation in arid versus humid regions cautions against standard correction for elevation in comparative studies of '¹³C_leaf.     

Are you what you eat? Effects of trophic discrimination factors on estimates of food assimilation and trophic position with a new estimation method

A key factor for estimates of assimilation of resources and trophic position based on stable isotope data is the trophic discrimination factor (TDF). TDFs are assumed based on literature reviews, but may vary depending on a variety of factors, including the type of diet. We analyzed effects of alternative TDFs on estimates of assimilated resources and trophic positions for an omnivorous fish, Jenynsia multidentata, that reveals dietary variation among locations across a salinity gradient of a coastal lagoon in southern Brazil. We also compared estimates of foods ingested vs. foods assimilated. Food assimilation was estimated using carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotope ratios of food sources and consumer muscle tissue and an isotopic mixing model (SIAR); consumer trophic position (TP) was estimated from consumer and production source δ¹⁵N values. Diet was estimated using an index of relative importance based on frequency of occurrence and volumetric and numeric proportions of food items from stomach contents. The effect of variation in TDF on food assimilation and TP was tested using three alternative TDFs reported in review papers. We then created a new method that used food source-specific TDFs (reported separately for herbivores and carnivores) weighted in proportion to estimated assimilation of resources according to mixing model estimates to estimate TP (hereafter TP_WAR). We found that plant material was not assimilated in a proportion similar to its importance in the diet of fish at a freshwater site, and the new method yielded best assimilation estimates. Animal material made greatest contributions to fish biomass irrespective of TDFs used in the mixing model. The new method produced TP estimates consistent with differences in estimated food assimilation along the salinity gradient. Our findings support the idea that food source-specific TDFs should be used in trophic studies of omnivores, since the method improved our ability to estimate trophic position and resource assimilation, two important ecological indicators.     

The Influence of Plant Water Deficit on Distribution of 14C-labelled Assimilates in Cacao Seedlings

The relationship between plant water status and distributionof ¹⁴C-labelled assimilates in cacao (Theobroma cacao L.) wasevaluated after ¹⁴CO₂ pulse labelling leaves of seedlings subjectedto varying levels of water deficiency. The proportion of ¹⁴Cexported by source leaves was strongly affected by seedlingwater status. An increasing proportion of labelled assimilatesremained in source leaves at both 24-h and 72-h harvests aswater stress intensity increased. Water stress reduced the distributionof exported label to leaves and to the expanding flush in particularbut increased the proportion of label in stems and roots. Theresults suggest that current photoassimilates may be temporarilystored in source leaves and stems of cacao seedlings duringperiods of plant water deficit. The stress-induced changes inpartitioning of labelled carbon were in concordance with changesin shoot to root biomass ratios, which was likely due to greaterreduction in growth of above-ground organs to that of roots. Theobroma cacao L, assimilate partitioning, cacao, ¹⁴C-photoassimilate, water stress, water potential     

Isotopic fractionation during nitrate uptake by phytoplankton grown in continuous culture

The isotopic fractionation associated with uptake of NO₃^–by six species of phytoplankton (two diatoms, one cryptophyte,one chlorophyte and two haptophytes) was measured at a varietyof steady-state growth rates in nitrogen-limited continuousculture. The magnitude of the isotopic fractionation factor(     

Modelling mixing parameters in concentric-tube airlift bioreactors

The mixing behaviour of the liquid phase in concentric-tube airlift bioreactors of different scale (RIMP: V_L=0.070 m³; RIS-1: V_L=2.50 m³; RIS-2: V_L=5.20 m³) in terms of mixing time was investigated. This mixing parameter was determined from the output curves to an initial Dirac pulse, using the classical tracer response technique, and analyzed in relation to process and geometrical parameters, such as: gas superficial velocity, x_SGR; top clearance, h_S; bottom clearance, h_B, and ratio of the resistances at downcomer entrance, A_d/A_R. A correlation between the mixing time and the specified operating and geometrical parameters was developed, which was particularized for two flow regimes: bubbly and transition (x_SGRА.08 m/s) and churn turbulent flow (x_SGR> 0.08 m/s) respectively. The correlation was applied in bioreactors of different scale with a maximum error of ᆲ%.     

The Influence of the Concentration of Gaseous Ammonia on its Uptake by the Leaves of Italian Ryegrass, with and without an Adequate Supply of Nitrogen to the Roots

Whitehead, D. C. and Lockyer, D. R. 1986. The influence of theconcentration of gaseous ammonia on its uptake by the leavesof Italian ryegrass, with and without an adequate supply ofnitrogen to the roots.—J. exp. Bot. 38: 818–827. Plants of Italian ryegrass (Lolium multiflorum Lam.) were grownin pots of soil with two rates of ¹⁵N-labclled nitrate, oneproviding adequate, and the other less than adequate, N formaximum growth. After 25 d in a controlled environment cabinet,the plants were transferred to chambers and exposed for 33 dto NH₃in the air at one of nine concentrations ranging from14 to 709 µg NH₃ m^–3. Increasing the concentrationof NH₃ in the air increased the dry weight of the shoots ofplants grown at the lower but not the higher rate of nitrate.The content of total N in the plant shoots (% dry weight) waslinearly related to NH₃ concentration; at 709 µg NH₃ andin both sets of plants it was more than double the content at14 µg NH₃ m^–3. Calculations, based on ¹⁵N enrichment,indicated that the amount of N taken up from the NH₃ per unitleaf area increased linearly with increasing concentration ofNH₃ in the air uptake (µg dm^–2 h^–1) = 0.1009xat the lower rate of nitrate and 0-0829x at the higher rateof nitrate, where x is the concentration of NH₃ in the air expressedas µg NH₃m^–3. The proportion of the total plant N that was derived from theNH₃ ranged from 4?0% at a concentration of 14 µg NH₃ m^–3with the higher rate of nitrate addition to 77?5% at a concentrationof 709 µg m^–3 with the lower rate of nitrate addition.The proportions of the total N in the water-insoluble proteinof the leaf tissue that were derived from nitrate and gaseousNH₃ were similar to the proportions in the whole leaf material. Key words: Ammonia, nitrogen, leaf sorption, Lolium multiflorum