Variability and directionality of temporal changes in δ<Superscript>13</Superscript>C and δ<Superscript>15</Superscript>N of aquatic invertebrate primary consumers

Seasonal oscillations in the carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope signatures of aquatic algae can cause seasonal enrichment–depletion cycles in the isotopic composition of planktonic invertebrates (e.g., copepods). Yet, there is growing evidence that seasonal enrichment–depletion cycles also occur in the isotope signatures of larger invertebrate consumers, taxa used to define reference points in isotope-based trophic models (e.g., trophic baselines). To evaluate the general assumption of temporal stability in non-zooplankton aquatic invertebrates, δ¹³C and δ¹⁵N time series data from the literature were analyzed for seasonality and the influence of biotic (feeding group) and abiotic (trophic state, climate regime) factors on isotope temporal patterns. The amplitude of δ¹³C and δ¹⁵N enrichment–depletion cycles was negatively related to body size, although all size-classes of invertebrates displayed a winter-to-summer enrichment in δ¹³C and depletion in δ¹⁵N. Among feeding groups, periphytic grazers were more variable and displayed larger temporal changes in δ¹³C than detritivores. For nitrogen, temporal variability and magnitude of directional change of δ¹⁵N was most strongly related to ecosystem trophic state (eutrophic > mesotrophic, oligotrophic). This study provides evidence of seasonality in the isotopic composition of aquatic invertebrates across very broad geographical and ecological gradients as well as identifying factors that are likely to modulate the strength and variability of seasonality. These results emphasize the need for researchers to recognize the likelihood of temporal changes in non-zooplankton aquatic invertebrate consumers at time scales relevant to seasonal studies and, if present, to account for temporal dynamics in isotope trophic models.     

Impact of two-bond <Superscript>15</Superscript>N–<Superscript>15</Superscript>N scalar couplings on <Superscript>15</Superscript>N transverse relaxation measurements for arginine side chains of proteins

NMR relaxation of arginine (Arg) ¹⁵N_ε nuclei is useful for studying side-chain dynamics of proteins. In this work, we studied the impact of two geminal ¹⁵N–¹⁵N scalar couplings on measurements of transverse relaxation rates (R ₂ ) for Arg side-chain ¹⁵N_ε nuclei. For 12 Arg side chains of the DNA-binding domain of the Antp protein, we measured the geminal ¹⁵N–¹⁵N couplings ( ² J _NN ) of the ¹⁵N_ε nuclei and found that the magnitudes of the ² J _NN coupling constants were virtually uniform with an average of 1.2 Hz. Our simulations, assuming ideal 180° rotations for all ¹⁵N nuclei, suggested that the two ² J _NN couplings of this magnitude could in principle cause significant modulation in signal intensities during the Carr–Purcell-Meiboom–Gill (CPMG) scheme for Arg ¹⁵N_ε R ₂ measurements. However, our experimental data show that the expected modulation via two ² J _NN couplings vanishes during the ¹⁵N CPMG scheme. This quenching of J modulation can be explained by the mechanism described in Dittmer and Bodenhausen (Chemphyschem 7:831–836, 2006). This effect allows for accurate measurements of R ₂ relaxation rates for Arg side-chain ¹⁵N_ε nuclei despite the presence of two ² J _NN couplings. Although the so-called recoupling conditions may cause overestimate of R ₂ rates for very mobile Arg side chains, such conditions can readily be avoided through appropriate experimental settings.     

Can tree-ring δ<Superscript>15</Superscript>N be used as a proxy for foliar δ<Superscript>15</Superscript>N in European beech and Norway spruce?

Key message

For long-term environmental investigations, tree-ring δ ¹⁵ N values are inappropriate proxies for foliar δ ¹⁵ N for both Fagus sylvatica and Picea abies under moderate N loads.

Abstract

Currently it is unclear whether stable nitrogen isotope signals of tree-rings are related to those in foliage, and whether they can be used to infer tree responses to environmental changes. We studied foliar and tree-ring nitrogen (δ¹⁵N) and carbon (δ¹³C) isotope ratios in European beech (Fagus sylvatica L.) and Norway spruce (Picea abies L.) from six long-term forest monitoring sites in Switzerland together with data on N deposition and soil N availability, as well as a drought response index over the last two decades. For both species, tree-ring δ¹⁵N and δ¹³C values were less negative compared to foliar δ¹⁵N and δ¹³C values, most likely due to recycling and reallocation of N within the tree and fractionation processes associated with the transport of sucrose and the formation of tree-rings, respectively. Temporal trends recorded in foliar δ¹⁵N were not reflected in tree-ring δ¹⁵N, with much higher variations in tree-rings compared to foliage. Soil N availability and N deposition were partially able to explain changes in foliar δ¹³C, while there were no significant correlations between environmental variables and either tree-ring or foliar δ¹⁵N. Our results suggest an uncoupling between the N isotopic composition of tree-rings and foliage. Consequently, tree-ring δ¹⁵N values are inappropriate proxies of foliar δ¹⁵N values under low-to-moderate N deposition loads. Furthermore, at such low levels of deposition, tree-ring δ¹⁵N values are not recommended as archives of tree responses to soil C/N or bulk N deposition.

     

The trophic history of Myall Lakes,New South Wales,Australia: interpretations using δ<Superscript>13</Superscript>C and δ<Superscript>15</Superscript>N of the sedimentary record

In an attempt to determine the trophic history of the Myall Lakes complex (New South Wales, Australia) δ¹³C_org, δ¹⁵N and C_org:N profiles were determined for bulk organic matter of two short sediment cores from Bombah Broadwater and Myall Lake. ²¹⁰Pb profiles and sediment types indicate significantly different trophic trajectories during the time periods examined. δ¹³C_org and C_org:N indicate Bombah Broadwater has been dominated by increasing inputs of terrestrial organic material over the last century, thought to be related to watershed disturbance including agricultural activity. Primary production appears to be dominated by phytoplankton. δ¹⁵N remained relatively stable at around 1‰ until the mid–1970s when there was a sharp increase to 4.7‰, interpreted as an influx of sewage-derived material. These observations offer an insight into the recent trophic changes at the site. Sedimentation rates are noticeably lower in Myall Lake and the most recent sediment is a flocculent organic rich deposit overlying mineral clay. δ¹³C_org and C_org:N values indicate a transition from plankton to macrophyte dominated primary production around 1800AD. δ¹⁵N values become increasingly negative from approximately 1900AD. This is interpreted as being due to increasing reliance by macrophytes on nitrogen recycled from decomposing sediments driven by natural infilling and eutrophication in this basin. The contrasting sedimentation rates, sediment types and geochemical profiles suggest the different basins of this water body are subject to substantially different internal and external influences which should be considered in management decisions.     

Ecosystem N Distribution and δ<Superscript>15</Superscript>N during a Century of Forest Regrowth after Agricultural Abandonment

Abstract Stable isotope ratios of terrestrial ecosystem nitrogen (N) pools reflect internal processes and input–output balances. Disturbance generally increases N cycling and loss, yet few studies have examined ecosystem δ¹⁵N over a disturbance-recovery sequence. We used a chronosequence approach to examine N distribution and δ¹⁵N during forest regrowth after agricultural abandonment. Site ages ranged from 10 to 115 years, with similar soils, climate, land-use history, and overstory vegetation (white pine Pinus strobus). Foliar N and δ¹⁵N decreased as stands aged, consistent with a progressive tightening of the N cycle during forest regrowth on agricultural lands. Over time, foliar δ¹⁵N became more negative, indicating increased fractionation along the mineralization–mycorrhizal–plant uptake pathway. Total ecosystem N was constant across the chronosequence, but substantial internal N redistribution occurred from the mineral soil to plants and litter over 115 years (>25% of ecosystem N or 1,610 kg ha⁻¹). Temporal trends in soil δ¹⁵N generally reflected a redistribution of depleted N from the mineral soil to the developing O horizon. Although plants and soil δ¹⁵N are coupled over millennial time scales of ecosystem development, our observed divergence between plants and soil suggests that they can be uncoupled during the disturbance-regrowth sequence. The approximate 2‰ decrease in ecosystem δ¹⁵N over the century scale suggests significant incorporation of atmospheric N, which was not detected by traditional ecosystem N accounting. Consideration of temporal trends and disturbance legacies can improve our understanding of the influence of broader factors such as climate or N deposition on ecosystem N balances and δ¹⁵N. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fish scale δ<Superscript>15</Superscript>N and δ<Superscript>13</Superscript>C values provide biogeochemical tags of fish comparable in performance to element concentrations in scales and otoliths

Natural variability in stable isotope ratios and element concentrations in calcified structures of fish (e.g. scales and otoliths) has provided biogeochemical ‘tags’ for studying origins and movements of marine species, but has been little used in freshwater studies. We examine whether variability in scale δ¹⁵N and δ¹³C values of Salmo trutta L., could provide a tag of fish over small spatial scales in a small river catchment (River Dee, U.K.) and compared their performance as tags with that of scale/otolith element concentrations. Whole scale δ¹⁵N and δ¹³C values differed among six study sites and fish could be classified to their site of origin with a high degree of accuracy. Classifying fish to their site of capture was marginally superior using scale δ¹⁵N and δ¹³C values compared to that achieved using Sr, Mn, Ba and Mg in scale hydroxyapatite or otolith aragonite. Scale δ¹⁵N and δ¹³C values could therefore provide non-lethally collectable biogeochemical tags superior in performance to element concentrations in otoliths and scales. A comprehensive study of δ¹⁵N and δ¹³C values within freshwater systems would develop our understanding of factors influencing geographical variability in baseline δ¹⁵N and δ¹³C signatures.     

Regional-scale patterns of δ<Superscript>13</Superscript>C and δ<Superscript>15</Superscript>N associated with multiple ecosystem functions along an aridity gradient in grassland ecosystems

Background and aims

Nitrogen (N) deficiency and drought are two key limiting factors for rice production worldwide, but the relationship of drought stress with N homeostasis in rice is rarely advanced. The aim of this study was to dissect the physiological effects of drought stress on rice growth that coupled unbalanced N metabolism.

Results

Water-deficient stress (WD) limited stomatal aperture function and activity of Rubisco carboxylase to photosynthesis. The rate of total electron transport (J_t) and the electron to carboxylation (J_c) were considerably decreased, whereas the proportion of e^? flow to photorespiration was stimulated by WD, especially at 1600 μmol m^?2 s^?1 PPFD. Concurrently, the expressions of glycolate oxidase genes (GOX1, GOX5) and glycine decarboxylase complex (GDCH, GDCP and GDCT) were significantly induced in leaves of WD treatment, which led to the accumulation of reactive oxygen species in leaves. With the photosynthetic change, nitrate uptake and reduction were suppressed. Moreover, the enhanced photorespiration generated excess NH₃ accumulation in leaves and stimulated the expressions of GS1;1, GS1;2 and GS2, which were tightly coupled with the expressions of PEPC1 and PEPC2 under WD stress.

Conclusions

Our results suggest that the inhibited nitrate reduction associated with diminished electron transport rate, and the photorespiration-associated accumulation of hydrogen peroxide and NH₃ were critical in the drought-induced rice growth inhibition.

     

Sources of variation in consumer-diet δ<Superscript>15</Superscript>N enrichment: a meta-analysis

Measurements of ¹⁵N of consumers are usually higher than those of their diet. This general pattern is widely used to make inferences about trophic relationships in ecological studies, although the underlying mechanisms causing the pattern are poorly understood. However, there can be substantial variation in consumer-diet ¹⁵N enrichment within this general pattern. We conducted an extensive literature review, which yielded 134 estimates from controlled studies of consumer-diet ¹⁵N enrichment, to test the significance of several potential sources of variation by means of meta-analyses. We found patterns related to processes of nitrogen assimilation and excretion. There was a significant effect of the main biochemical form of nitrogenous waste: ammonotelic organisms show lower ¹⁵N enrichment than ureotelic or uricotelic organisms. There were no significant differences between animals feeding on plant food, animal food, or manufactured mixtures, but detritivores yielded significantly lower estimates of enrichment. ¹⁵N enrichment was found to increase significantly with the C:N ratio of the diet, suggesting that a nitrogen-poor diet can have an effect similar to that already documented for fasting organisms. There were also differences among taxonomic classes: molluscs and crustaceans generally yielded lower ¹⁵N enrichment. The lower ¹⁵N enrichment might be related to the fact that molluscs and crustaceans excrete mainly ammonia, or to the fact that many were detritivores. Organisms inhabiting marine environments yielded significantly lower estimates of ¹⁵N enrichment than organisms inhabiting terrestrial or freshwater environments, a pattern that was influenced by the number of marine, ammonotelic, crustaceans and molluscs. Overall, our analyses point to several important sources of variation in ¹⁵N enrichment and suggest that the most important of them are the main biochemical form of nitrogen excretion and nutritional status. The variance of estimates of ¹⁵N enrichment, as well as the fact that enrichment may be different in certain groups of organisms should be taken into account in statistical approaches for studying diet and trophic relationships.     

Imprint of oaks on nitrogen availability and δ<Superscript>15</Superscript>N in California grassland-savanna: a case of enhanced N inputs?

Woody vegetation is distributed patchily in many arid and semi-arid ecosystems, where it is often associated with elevated nitrogen (N) pools and availability in islands of fertility. We measured N availability and δ¹⁵N in paired blue-oak versus annual grass dominated patches to characterize the causes and consequences of spatial variation in N dynamics of grassland-savanna in Sequoia-Kings Canyon National Park. We found significantly greater surface soil N pools (0–20 cm) in oak patches compared to adjacent grass areas across a 700 m elevation gradient from foothills to the savanna-forest boundary. N accumulation under oaks was associated with a 0.6‰ depletion in soil δ¹⁵N relative to grass patches. Results from a simple δ¹⁵N mass balance simulation model, constrained by surface soil N and δ¹⁵N measured in the field, suggest that the development of islands of N fertility under oaks can be traced primarily to enhanced N inputs. Net N mineralization and percent nitrification in laboratory incubations were consistently higher under oaks across a range of experimental soil moisture regimes, suggesting a scenario whereby greater N inputs to oak patches result in net N accumulation and enhanced N cycling, with a potential for greater nitrate loss as well. N concentrations of three common herbaceous annual plants were nearly 50% greater under oak than in adjacent grass patches, with community composition shifted towards more N-demanding species under oaks. We find that oaks imprint distinct N-rich islands of fertility that foster local feedback between soil N cycling, plant N uptake, and herbaceous community composition. Such patch-scale differences in N inputs and plant–soil interactions increase biogeochemical heterogeneity in grassland-savanna ecosystems and may shape watershed-level responses to chronic N deposition.     

The influence of weed control on foliar δ<Superscript>15</Superscript>N, δ<Superscript>13</Superscript>C and tree growth in an 8 year-old exotic pine plantation of subtropical Australia

Background and aims

The aim of weed control and fertilization in forest plantations was to increase tree growth by reducing competition for available nutrients and water. However, treatments that influence weed biomass can also have significant impacts on soil carbon (C) and nitrogen (N) cycling which can in turn lead to changes in the dynamics of stable C (δ¹³C) and N (δ¹⁵N) isotope compositions in soils and tree foliage.

Methods

We examined the key C and N cycling processes influenced by routine and luxury weed control and fertilization treatments as reflected by soil and foliar δ¹³C and δ¹⁵N and long-term tree growth in an 8-year old F₁ hybrid pine (Pinus elliottii x P. caribaea) plantation in southeast Queensland, Australia. Weed control treatments varied by treatment frequency and intensity while fertilization treatments varied by the application of N, phosphorus (P), potassium (K) and micronutrients. Different soil and canopy sampling positions were assessed to determine if sampling position enhanced the relationships among soil N transformations and tree N use, water use efficiency and carbon gain under the early establishment silviculture.

Results

Routine weed control was associated with increased weed biomass returned to the soil, compared with luxury weed control. Soil δ¹³C increased at the 0–5 cm soil sampling depth in both the inter-planting (IPR) and planting row (PR) as a result of the routine weed control treatments. In addition, soil δ¹³C was significantly higher as a result of fertilisation treatment in the 0–5 cm soil sampling depth in the PR. Soil δ¹³C was negatively correlated to soil δ¹⁵N at the 0–5 cm soil sampling depth in the IPR. Soil δ¹⁵N increased in the 0–5 and 5–10 cm soil sampling depths in the IPR, as a result of more frequent (luxury) weed control. Foliar δ¹⁵N and tree water use efficiency (WUE) (as indicated by foliar δ¹³C) were positively correlated with tree growth at age 8 years. While relationships between δ¹³C and δ¹⁵N in the soil and foliage varied depending on soil sampling depth and position, and with canopy sampling position where there were consistent relationships between soil δ¹³C (or δ¹⁵N) and foliar δ¹⁵N.

Conclusions

This study demonstrates how early establishment silviculture has important implications for soil C and N cycling and how soil δ¹³C and δ¹⁵N were consistent with changes in soil C cycling and N transformations as a result of weed control treatments, while foliar δ¹⁵N was linked to more rapid N cycling as reflected in the soil δ¹⁵N, which increased tree growth and tree WUE (as reflected by foliar δ¹³C).

     

Spatial and temporal variabilities of δ<Superscript>13</Superscript>C and δ<Superscript>15</Superscript>N within lower trophic levels of a large lake: implications for estimating trophic relationships of consumers

Stable isotopes of carbon (δ¹³C) and nitrogen (δ¹⁵N) often have unique values among lake habitats (e.g. benthic, littoral, pelagic), providing a widely used tool for measuring the structure and energy flow in aquatic food webs. However, there has been little recognition of the spatial and temporal variabilities of these isotopes within habitats of aquatic ecosystems. To address this, δ¹³C and δ¹⁵N were measured in seston, zebra mussels (Dreissena polymorpha) and young-of-year (YOY) yellow (Perca flavescens), and white perch (Morone americana) collected from four sites across the offshore habitat of the western basin of Lake Erie during June–September 2009. Values of δ¹³C and δ¹⁵N showed significant spatial and temporal variations, with month accounting for >50% of the variation, for both stable isotopes and all the species except seston. Such variation in isotope values has the potential to significantly influence or confound interpretation of stable isotopes in measures, such as trophic position (TP) which use lower trophic level organisms as their baseline. For example, TP was found to vary up to 0.7 for yellow and white perch (TP = δ¹⁵N_fish − δ¹⁵N_{zebra mussel}/diet-tissue fractionation factor) depending on the zebra mussel data used (e.g., from a different location or a different collection month). As the use of stable isotopes continues to move from qualitative to more quantitative measures of trophic structure, food web research must recognize the importance of stable isotopes' variability in lower trophic level organisms, especially in large lake systems.     

δ<Superscript>13</Superscript>C and δ<Superscript>15</Superscript>N in tissue of coral polyps and epilithic algae inhabiting damaged coral colonies under the influence of different light intensities

Studies were performed of the carbon and nitrogen stable isotope (δ¹³C and δ¹⁵N) composition (δ¹³C and δ¹⁵N) of the corals Porites cylindrica and P. lutea (5 years after damaging the colonies by the bleaching events) and of epilithic algae settled onto damaged areas of coral colonies. Coral polyps and three epilithic algal communities (‘red algal turf, green algal turf and red calcified crusts’) were sampled along the boundary between communities of coral polyps and algal colonizers from differently illuminated habitats from 2 to 90% of incident surface photosynthetically active radiation (PAR₀). It was found that communities with a predominance of red algae significantly differed from communities with a predominance of green algae in δ¹³C but not in δ¹⁵N values. An influence of habitat irradiance was found only for communities of coral polyps for δ¹³C and δ¹⁵N values: under bright light (70–90% PAR₀) polyp tissues of both coral species were significantly enriched in heavy carbon isotopes and insignificantly in nitrogen isotopes (δ¹³C values difference ~4‰) relative to tissues of corals under lower light 15–50% PAR₀. On the basis of these results we assumed that differences in light intensities in the habitat ranging from 15 to 90% PAR₀ do not influence on accessibility of the main carbon and nitrogen sources for corals and algae, and exchange by these elements between organisms. We also assumed that the relative enrichment in the heavy carbon isotopes of coral tissues in high light is a result of decreased isotope fractionation (or the absence of fractionation in photosynthesis of their zooxanthellae).     

<Superscript>1</Superscript>H–<Superscript>15</Superscript>N correlation spectroscopy of nanocrystalline proteins

The limits of resolution that can be obtained in ¹H–¹⁵N 2D NMR spectroscopy of isotopically enriched nanocrystalline proteins are explored. Combinations of frequency switched Lee–Goldburg (FSLG) decoupling, fast magic angle sample spinning (MAS), and isotopic dilution via deuteration are investigated as methods for narrowing the amide ¹H resonances. Heteronuclear decoupling of ¹⁵N from the ¹H resonances is also studied. Using human ubiquitin as a model system, the best resolution is most easily obtained with uniformly ²H and ¹⁵N enriched protein where the amides have been exchanged in normal water, MAS at 20 kHz, and WALTZ-16 decoupling of the ¹⁵N nuclei. The combination of these techniques results in average ¹H lines of only 0.26 ppm full width at half maximum. Techniques for optimizing instrument stability and ¹⁵N decoupling are described for achieving the best possible performance in these experiments.     

Nutrient cycling relative to δ<Superscript>15</Superscript>N and δ<Superscript>13</Superscript>C natural abundance in a coastal wetland with long-term nutrient additions

Because nitrogen and phosphorus are primary resources for plant, algal, and microbial production, increases in nutrient inputs can markedly alter aquatic ecosystems. Coastal wetland plots at Belle W. Baruch Marine Field Laboratory (South Carolina, USA) have been amended with nitrogen and phosphorus for ~20 years to determine the effects of nutrient loading on coastal wetlands. We conducted a survey of δ¹⁵N and δ¹³C natural abundance in coastal wetland organic pools (sediment, vegetation) with long-term nutrient amendments (control, no addition; nitrogen; phosphorus; and nitrogen + phosphorus additions). Additionally, we conducted laboratory assays to quantify pore water nutrient availability and nitrification rates. Marsh vegetation (Spartina alterniflora) had enriched δ¹³C values (mean −14‰) relative to bulk sediment samples (mean −18‰). Nitrogen-amended plots (alone and in combination with phosphorus) had enriched δ¹³C values in the surface sediment (0–5 cm; mean −16.1‰) relative to control (mean −16.5‰) and phosphorus-amended plots (mean −16.8‰). Nitrogen-amended plots also had depleted δ¹⁵N in S. alterniflora leaf tissues (−3.3‰) and surface sediment samples (mean 2.1‰) relative to leaf tissues (mean 2.1‰) or sediment samples (mean 5.8‰) from control or phosphorus-only amended plots. Nitrate availability (as increased pore water concentration) was higher in N-amended plots although ammonium availability did not differ. Phosphorus availability was higher only in phosphorus-only amended plots. Overall, we found that long-term nutrient amendments to coastal wetlands significantly altered nutrient availability and uptake rates as well as natural abundance of δ¹³C and δ¹⁵N in multiple organic matter sources.     

Estimation of denitrification potential in a karst aquifer using the <Superscript>15</Superscript>N and <Superscript>18</Superscript>O isotopes of NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack>

A confined aquifer in the Malm Karst of the Franconian Alb, South Germany was investigated in order to understand the role of the vadose zone in denitrifiaction processes. The concentrations of chemical tracers Sr²⁺ and Cl^– and concentrations of stable isotope ¹⁸O were measured in spring water and precipitation during storm events. Based on these measurements a conceptual model for runoff was constructed. The results indicate that pre-event water, already stored in the system at the beginning of the event, flows downslope on vertical and lateral preferential flow paths. Chemical tracers used in a mixing model for hydrograph separation have shown that the pre-event water contribution is up to 30%. Applying this information to a conceptual runoff generation model, the values of ¹⁵N and ¹⁸O in nitrate could be calculated. Field observations showed the occurence of significant microbial denitrification processes above the soil/bedrock interface before nitrate percolates through to the deeper horizon of the vadose zone. The source of nitrate could be determined and denitrification processes were calculated. Assuming that the nitrate reduction follows a Rayleigh process one could approximate a nitrate input concentration of about 170 mg/l and a residual nitrate concentration of only about 15%. The results of the chemical and isotopic tracers postulate fertilizers as nitrate source with some influence of atmospheric nitrate. The combined application of hydrograph separation and determination of isotope values in ¹⁵N and ¹⁸O of nitrate lead to an improved understanding of microbial processes (nitrification, denitrification) in dynamic systems.     

Intrapopulation variation in gray wolf isotope (δ<Superscript>15</Superscript>N and δ<Superscript>13</Superscript>C) profiles: implications for the ecology of individuals

In many organisms reproductive success is strongly dependent on several breeding site characteristics, which often vary in space and time. Although we have a good understanding of how ovipositing organisms respond to single factors, we still have little information about how they respond under more complex natural conditions. We examined the oviposition behavior of a tree-hole breeding frog, Phrynobatrachus guineensis, with respect to abiotic and biotic oviposition site characteristics, including desiccation risk and the presence of conspecific offspring using both observation and experiments. Based on daily monitoring data, compiled from 69 natural oviposition sites during a complete reproductive season, we developed oviposition site-selection models. A model based on water presence, sediment depth and maximal possible water depth showed the best predictive performance and was transferable to the subsequent season. Field observations and experiments revealed that frogs could estimate water-holding capacity of sites and timed oviposition with respect to future water presence. Despite the negative effects on larval growth and the availability of sites without conspecifics, data suggest that ovipositing individuals are attracted to conspecific offspring because they serve as a cue for low predation risk. Our results imply that a sites potential for being used at least once for oviposition was determined by abiotic factors, whereas the relative use of breeding sites was determined by a response to conspecifics. Our study demonstrates the importance of including multiple biotic and abiotic factors in the analysis of oviposition site-selection.     

Flow of Deposited Inorganic N in Two Gleysol-dominated Mountain Catchments Traced with <Superscript>15</Superscript>NO<Subscript>3</Subscript><Superscript>−</Superscript> and <Superscript>15</Superscript>NH<Subscript>4</Subscript><Superscript>+</Superscript>

In two mountain ecosystems at the Alptal research site in central Switzerland, pulses of ¹⁵NO₃ and ¹⁵NH₄ were separately applied to trace deposited inorganic N. One forested and one litter meadow catchment, each approximately 1600 m², were delimited by trenches in the Gleysols. K¹⁵NO₃ was applied weekly or fortnightly over one year with a backpack sprayer, thus labelling the atmospheric nitrate deposition. After the sampling and a one-year break, ¹⁵NH₄Cl was applied as a second one-year pulse, followed by a second sampling campaign. Trees (needles, branches and bole wood), ground vegetation, litter layer and soil (LF, A and B horizon) were sampled at the end of each labelling period. Extractable inorganic N, microbial N, and immobilised soil N were analysed in the LF and A horizons. During the whole labelling period, the runoff water was sampled as well. Most of the added tracer remained in both ecosystems. More NO₃⁻ than NH₄⁺ tracer was retained, especially in the forest. The highest recovery was in the soil, mainly in the organic horizon, and in the ground vegetation, especially in the mosses. Event-based runoff analyses showed an immediate response of ¹⁵NO₃⁻ in runoff, with sharp ¹⁵N peaks corresponding to discharge peaks. NO₃⁻ leaching showed a clear seasonal pattern, being highest in spring during snowmelt. The high capacity of N retention in these ecosystems leads to the assumption that deposited N accumulates in the soil organic matter, causing a progressive decline of its C:N ratio.     

Evaluating the need for acid treatment prior to δ<Superscript>13</Superscript>C and δ<Superscript>15</Superscript>N analysis of freshwater fish scales: effects of varying scale mineral content,lake productivity and CO<Subscript>2</Subscript> concentration

In order to evaluate the need for using scale acidification to remove carbonates prior to stable isotope analysis, we compared acidified and non-acidified scales of six freshwater fish species (perch, roach, rudd, pike, tench and bream) with contrasting mineral content in their scales. Fish samples were taken from six lakes with variable trophic conditions, ranging from oligotrophic to hypertrophic, and differing in CO₂ concentrations. The scale mineral content of the six species studied ranged between 31.8 and 61.3% dry weight (DW) in tench and perch, respectively. The elemental composition was characterised by high amounts of phosphorus, varying from 4.5 to 9.1% DW. The mineral fraction was dominated by apatite (range 24.4–49.2% DW), carbonates constituted a very small proportion of the total carbon content (average ± SD: 5.5 ± 1.7%). The average effect of acidification was very small for all species (average ± SD: 0.181 ± 0.122 and −0.208 ± 0.243 for carbon and nitrogen, respectively), albeit significant for five out of the six species (excepting tench that had the lowest mineral content). Linear regression slopes between acidified and untreated scales did not differ significantly from one for almost all the species and isotopes. The effects of acidification on the two isotopes were correlated with the relative carbonate content as well as with the CO₂ concentration for carbon and total phosphorus for nitrogen. We conclude that the need for scale acidification depends on the different species and on the system studied, although in most cases the acidification effect will be biologically irrelevant. However, dual analysis of acidified and untreated scales may provide useful information on differences in stable isotope composition of dissolved inorganic carbon and on phytoplankton carbon fractionation generated by varying levels of CO₂ availability.