15N Addition Research Articles

Warming, permafrost thaw and increased nitrogen availability as drivers for plant composition and growth across the Tibetan Plateau

Permafrost-affected ecosystems are subject to warming and thawing, which can increase the availability of subsurface nitrogen (N) with consequences in otherwise N-limited tundra and alpine vegetation. Here, we quantify the extent of warming and permafrost thawing and the corresponding effects on nitrogen availability and plant growth based on a 20-year survey across 14 sites on the Tibetan Plateau. The survey showed that most sites have been subject to warming and thawing and that the upper permafrost zone across all sites was rich in inorganic N, mainly as ammonium. We further explore the efficiency of plants to utilize 15N-labelled inorganic N over five years following 15N addition at the permafrost table far below the main root zone. The 15N experiment showed that deep-rooted plant species were able to utilize the labelled N. A SEM model suggests that changes in vegetation can be explained by both active layer warming and permafrost thawing and the associated changes in inorganic nitrogen availability. Our results highlight a feedback mechanism of climate warming, in which released plant-available N may favour deep-rooted plants. This can explain important changes in plant composition and growth across the sites on the Tibetan Plateau.

Tracer or toxicant: Does stable isotope labeling affect central processes in aquatic food webs?

Stable isotope analysis (SIA) is an elementary technique in food web ecology, but its insights become increasingly ambiguous in complex systems. One approach to elevate the utility of SIA in such systems is the use of heavy isotope tracers (i.e., labeling). However, the fundamental assumption that the addition of such tracers does not affect in situ conditions has been challenged. This study tests if labeling is suitable for autotrophy-based and detritus-based aquatic food webs. For the former, the survival and reproduction of Daphnia magna fed with phytoplankton cultured at different levels of 15N addition were assessed. For the latter, the microbial decomposition of leaf litter was assessed at the same tracer levels. While no significant differences were observed, effect patterns were comparable to a previous study, supporting the isotopic redundancy hypothesis that postulates discrete quantum mechanical states at which the reaction speeds of metabolic processes are altered. Although physiology (reproduction) and activity (microbial decomposition) might not be altered to an ecologically significant level, labeling with heavy stable isotopes could potentially affect isotopic fractionation in biochemical processes and bias conclusions drawn from resulting SI ratios.

Fertilizer N triggers native soil N-derived N2O emissions by priming gross N mineralization

Recent 15nitrogen (N) tracing studies have shown that unlabeled N2O emissions (UNEs) following 15N fertilizer application tend to be higher than those from zero-N controls, which indicates a potential risk for elevated N2O emissions from native soil. However, researchers do not clearly understand whether these increased UNEs are derived from a priming effect or how interactions between N in fertilizer and native soil affect various sources of N2O emissions. Here, we combined 15N tracing and 15N pool dilution in an incubation study to track gross N transformations, specific sources of N2O emissions, and N2O-production pathways following N fertilization. Four treatments, including two fertilizer N levels (0 and 120 mg N kg−1) in conjunction with two native soil N levels (0.56 and 1.25 g kg−1), were established in the study. Soil gross N mineralization (GM) increased by 33.0%–98.5% (4.59–14.97 mg N kg−1) after N fertilizer addition, which indicated a positive priming effect on soil N turnover and resulted in additional native soil N-derived N2O emissions. In addition to priming GM, the pool substitution effect after 15N addition, which was not triggered by stimulated soil N turnover, also generated substrates for the UNEs. Therefore, overlooking the pool substitution effect might overestimate native soil N-derived emissions by 104%–180%. Differentiation of the specific N2O emission sources showed that both the fertilizer N- and primed native soil N-derived N2O emissions were dominated by nitrifier-mediated processes, which were governed by ammonia-oxidizing bacteria. At a specified N fertilizer addition rate, soils with higher native N levels had greater levels of fertilizer N- and native soil N-derived N2O emissions due to the higher GM and gross nitrification rates. The study reveals that fertilizer N application triggers potential N2O emissions from native soil N by stimulating the GM and nitrifier-mediated N2O-production processes.

Assessing transport and retention of nitrate and other materials through the riparian zone and stream channel with simulated precipitation

Abstract Riparian zones, the interfaces between land and stream, perform vital ecosystem functions including transformation and retention of nutrients and sediment moving across the landscape. Although many studies assess transport through and transformation of materials in riparian zones, less is known about the direct influence of precipitation falling on these zones on material retention and transport. Additionally, few experiments can compare riparian retention to stream‐channel retention. We present a novel experimental approach to assess retention of nitrate entering as precipitation in riparian zones and compare riparian retention and movement of nitrate, other ions, sediments to and within the adjacent stream channel. We simulated an intense precipitation event with 15N‐labelled nitrate as a bioactive solute and bromide as an inert tracer. This method extends tracer release approaches applied to streams worldwide and links it to processes at the aquatic/ terrestrial interface. It further allows determination of movement of materials into streams from bankside precipitation. The riparian zone removed or retained a greater proportion of nitrate than the stream relative to bromide; over half the added bromide reached the stream through a few metres of riparian zone, compared to only 0.2% of the added nitrate. Of the 0.2% that reached the stream, 30% of that nitrate was removed or retained by instream processes after travelling 60 downstream. Roughly 10% of the total 15N addition ended up sequestered in the above‐ground portions of the riparian grasses by the end of the growing season, and very little of it was recovered from the soil. We saw little evidence of bulk transport of other ions or sediment from this riparian soil to the stream. Our data are consistent with the concept of high nitrate retention in vegetated riparian zones, even for nitrate falling directly upon them in the form of atmospheric deposition in precipitation.

Stable Isotope Tracer Addition Reveals the Trophic Status of Benthic Infauna at an Intertidal Area Adjacent to a Seagrass Bed

Stable isotope tracer addition can enhance the isotopic differences of primary producers for a wider food-web resolution than the use of natural abundance stable isotopes (NASIs) alone, particularly in ecosystems where primary producers have similar NASI values. To investigate the food sources and the trophic status of benthic infauna in an intertidal area near a Halophila minor seagrass bed within inner Tai Tam Bay, Hong Kong, China, a 15N addition experiment was conducted, and the results were compared with those from NASI data. Only benthic microalgae (BMA) were labeled by applying 15N-enriched NH4Cl to the sediment daily for the first 7 days during a 21-day study. In contrast to the NASI results, Bayesian mixing models based on the isotope tracer experiment suggested a larger dietary contribution of BMA for nematode Daptonema sp. and copepods, whereas a higher reliance on phytoplankton and seagrass detritus was noted for polychaete Neanthes sp. However, both NASI and isotope tracer addition demonstrated that seagrass detritus was a major food source for nematode Spilophorella sp. The present isotope tracer experiment also revealed a contrasting result of the relatively lower contribution of meiofauna in the diets of carnivores/omnivores as compared to the results of NASIs. This finding suggested that the isotope values in these consumers may have not reached an equilibrium with the added isotope in the study period. Thus, there is a need for applying NASI coupled with isotope tracer addition in the investigation of ecosystems in which primary producers have similar isotope values, especially in ecosystems with lower tissue turnover rates, in order for a more accurate determination of dietary contribution and trophic status of consumers in the food-web study.

Long‐Term 15N Balance After Single‐Dose Input of 15N‐Labeled NH4+ and NO3− in a Subtropical Forest Under Reducing N Deposition

AbstractNitrogen (N) deposition in Europe and North America decreased in the 1990s, whereas N deposition in China began to decline in the early 2010s. The response of temperate forests to decreasing N deposition implied a delay recovery, but it remains unknown whether recovery in subtropical forests follows a similar trend. Therefore, the effects of decreased N deposition on N leaching were simulated in an N‐saturated forest in southwest China following ten years of N application 4.00 g N m−2 yr−1. The addition of N (NH4+ or NO3−) was stopped in 2014. In 2017, a single in‐situ 15N addition as NH4+ or NO3− was performed to trace the fate of N under reducing N deposition. Combining the monitoring results of the N fluxes and 15N fates, both the actual N leaching and contribution of a “new” N input to N leaching were significantly reduced in response to decreasing the N input. The termination of N addition resulted in immediate decreases in the N mineralization and immobilization rates, which were even lower than those in the control plots with moderate (naturally occurring) deposition reduction. The ratio of N leaching from mineralization was also reduced, implying the critical role of N mineralization in the recovery from N saturation. Because the forest was still N‐saturated, decreasing N deposition slightly reduced the N leaching to the gross N input ratio (including both N deposition and N mineralization). The significant decrease in the ratio and thus leaching recovery may occur due to a significant N deposition reduction.

Hiding in plain sight: The secret contribution of the solitary ascidian Herdmania grandis to temperate reef nitrous oxide production

AbstractLarge solitary ascidians, like Herdmania grandis (Heller), can dominate the benthic substrates of subtropical and temperate reefs; however, their influence on nitrogen cycling, particularly nitrous oxide (N2O) production, is unknown. Here, we incubated individual H. grandis and compared fluxes of dissolved inorganic and gaseous nitrogen species to fluxes from reef sediments. Nitrous oxide production rates per individual ascidian (21 ± 8 nmol ind h−1) are the highest reported for any marine invertebrate. An individual ascidian produced more N2O than 1 m2 of inter‐reef sediment (1.7 ± 1.7 nmol m−2 h−1). Ascidian mediated N2O production was found to occur under nutrient depleted conditions. The addition of 15N labeled organic material showed that the microbiota associated with H. grandis is capable of both nitrification and denitrification, but the contribution of these pathways to N2O production could not be ascertained. As the ecology of temperate reefs change, any range expansion of H. grandis will increase coastal N2O production.

Quantifying nitrogen uptake and translocation for mature trees: an in situ whole-tree paired 15N labeling method.

Nitrogen (N) is one of the major nutrients limiting plant growth in terrestrial ecosystems. To avoid plant-microbe competition, previous studies on plant N uptake preference often used hydroponic experiments on fine roots of seedlings and demonstrated ammonium preference for conifer species; however, we lack information about N uptake and translocation in the field. In this paper, we described a method of in situ paired 15N labeling and reported the rates and time course of N uptake and translocation by mature trees in situ. We added 15N-enriched ammonium or nitrate, together with the nitrification inhibitor dicyandiamide, to paired Larix kaempferi (Lamb.) Carr (larch) trees from 30-, 40- and 50-year-old plantations. Fine roots, coarse roots, leaves and small branches were collected 2, 4, 7, 14 and 30days after labeling. Nitrate uptake and translocation averaged 1.59±0.16μg 15N g-1 day-1, which is slightly higher than ammonium (1.08±0.10μg 15N g-1 day-1), in all tree organs. Nitrate contributed 50-78% to N uptake and translocation, indicating efficient nitrate use by larch in situ. We observed no age effect. We suggest that sampling leaves after 4days of 15N labeling is sufficient to detect mature tree N uptake preference in situ. Whole-tree 15N-ammonium recovery equaled that of 15N-nitrate 30days after 15N addition, implying the importance of both ammonium and nitrate to mature larch N use in the long run. We conclude that our method is promising for studying mature tree N uptake preference in situ and can be applied to other conifer and broadleaf species. We suggest using highly enriched 15N tracer to overcome soil dilution and a nitrification inhibitor to minimize ammonium transformation to nitrate. Our study revealed mature tree N preference in situ and demonstrated the strong contribution of nitrate toward mature larch growth on soils rich in nitrate.

Effects of ryegrass amendments on immobilization and mineralization of nitrogen in a plastic shed soil: A 15N tracer study

In addition to chemical fertilizer, plant residues or manure with low C/N ratios are usually applied to intensively cultivated agricultural systems (e.g., vegetable cultivation in the plastic shed) to prevent nutrient deficiency and soil degradation and maintain high productivity. In this study, a batch incubation study was conducted for 120 days under controlled laboratory conditions to explore the dynamics of N conservation and supply in a plastic shed soil after the addition of 15N labeled fertilizer and/or ryegrass with a C/N ratio of lower than 15. We show that both C and N mineralization in the soil were related but not synchronous during the incubation period. Ryegrass addition provided a critical C source to enhance microbial use of fertilizer N and decreased the mineralization of native soil N (a negative “added N interaction”) at the early stage of the incubation. At the remaining incubation stage, the mineralization-dominated N turnover induced by insufficient labile C supply resulted in the continuous accumulation of inorganic N in the soil. Although the application of ryegrass decreased soil N availability during the short time, it might fail to conserve N supply capacity in the actual field condition due to the high and fast net-N-release pattern at a longer time scale. Therefore, considering the potential environmental risks from mineral N accumulation in plastic shed soils, organic material application with low C/N should be accompanied with other strategies to enhance soil N immobilization capacity.

Massive Nitrogen Loss Over the Western Indian Continental Shelf During Seasonal Anoxia: Evidence From Isotope Pairing Technique

The western Indian continental shelf houses the world's largest naturally-formed coastal low-oxygen zone that develops seasonally during the summer monsoon. We investigated multiple reductive nitrogen transformation pathways and quantified their rates in this system through anaerobic incubations with additions of 15N-labeled substrates during the anoxic period for three consecutive years (2008 to 2010). Addition of 15N labeled ammonium (15NH4+) resulted in low to moderate anaerobic ammonia oxidation (Anammox) rates in about half of our incubations from the oxygen depleted waters. In contrast, incubations with labeled nitrite (15NO2-) led to large production of 30N2 over 29N2 in all incubation experiments, indicating denitrification to be the dominant N-loss pathway. Rates of dissimilatory nitrate/nitrite reduction to ammonia (DNRA) were found to be highly variable and were lower by an order of magnitude than the denitrification rates. Extrapolation of average rates over the sampling periods and volume of anoxic waters showed large nitrogen removal (3.70 to 11.1 Tg yr-1) which is about three times as high as the previously reported estimate (1.3 to 3.8 Tg yr-1). Despite the small area it occupies, this shallow seasonal anoxic zone may account for as much as 20-60% the of the total annual fixed nitrogen loss in the perennial oxygen minimum zone of the Arabian Sea.

The retention dynamics of N input within the soil–microbe–plant system in a temperate grassland

In N-limited temperate regions, atmospheric N deposition remains high over the non-growing season. However, the retention dynamics of non-growing season N input within the ecosystem remain unclear. Using an isotopic approach, we investigated the initial retention and subsequent dynamics of 15N (1.5 g 15N m−2) in the soils, microbes, plants, and litter over three years in grazing-prohibited (PG) and heavily grazed treatments (HG) in northern China. For initial retention (21 days after 15N addition), most 15N was immobilized in soils and microbes, while less was taken up by plants. Soil and microbial 15N immobilization were significantly higher when grazing was prohibited, although plant 15N acquisition was not affected by grazing. After initial retention, rapid 15N loss was observed in microbes and soils, while 15N levels were sustained longer in plants and litter. The 15N residence times were longer when grazing was prohibited. The 15N acquisition capacity varies among plant taxa: perennial grasses and forbs accumulated 15N rapidly, while sagebrush and legumes acted slowly. Although the added 15N had significant contribution to early spring N demands of soil microbes and plants, it did not increase microbial or plant biomass N. Our results showed that non-growing season exogenous N was primarily retained by soil biota in temperate grasslands in the early stage, but N retention is finally sustained in soil and plants. The findings highlighted the importance of soil microbes in sustaining N upon N input, inferring the needs for considering the microbial role for better understanding N cycling in the temperate grasslands.

Species Differences in Nitrogen Acquisition in Humid Subtropical Forest Inferred From 15N Natural Abundance and Its Response to Tracer Addition

Differences in nitrogen (N) acquisition patterns between plant species are often reflected in the natural 15N isotope ratios (δ15N) of the plant tissues, however, such differences are poorly understood for co-occurring plants in tropical and subtropical forests. To evaluate species variation in N acquisition traits, we measured leaf N concentration (%N) and δ15N in tree and understory plant species under ambient N deposition (control) and after a decade of N addition at 50 kg N ha−1 yr−1 (N-plots) in an old-growth subtropical forest in southern China. We also measured changes in leaf δ15N after one-year of 15N addition in both the control and N-plots. The results show consistent significant species variation in leaf %N in both control and N-plots, but decadal N addition did not significantly affect leaf %N. Leaf δ15N values were also significantly different among the plant species both in tree and understory layers, and both in control and N-plots, suggesting differences in N acquisition strategies such as variation in N sources and dominant forms of N uptake and dependence on mycorrhizal associations among the co-occurring plant species. Significant differences between the plant species (in both control and N-plots) in changes in leaf δ15N after 15N addition were observed only in the understory plants, indicating difference in access (or use) of deposited N among the plants. Decadal N addition had species-dependent effects on leaf δ15N, suggesting the N acquisition patterns of these plant species are differently affected by N deposition. These results suggest that co-occurring plants in N-rich and subtropical forests vary in their N acquisition traits; these differences need to be accounted for when evaluating the impact of N deposition on N cycling in these ecosystems.

Trophic roles of Bufo bankorensis (Barbour 1908) tadpoles in an Asian subtropical stream

ABSTRACTTadpoles may be omnivorous with diverse trophic positions beyond herbivory, and may require multiple approaches for identification. Understanding the trophic and ecological roles of omnivorous tadpoles may require examination at a species level. The goals of this study were to determine the trophic positions of Bufo bankorensis tadpoles by using stable isotopes and food selection experiments, verify the tadpoles’ effects on benthic resources with enclosure–exclosure experiments, and examine their role on nutrient cycling. Stable isotope experiments showed that B. bankorensis tadpoles fed on algae and animal items, indicating a trophic level close to that of a damselfly. The food selection experiments showed that the tadpoles fed on dead tadpoles and mayflies but not on live animals, making them necrophagous, and further testing revealed that the tadpoles preferred dead tadpoles over algae. In the enclosure–exclosure experiment, the tadpoles reduced the amounts of algae and organic matter and changed the compositions of algal growth forms. The tadpoles released nitrogen (N) into the ambient water, which was then recycled by benthic algae, as revealed by an 15N addition experiment. We concluded that B. bankorensis tadpoles have trophic and ecological roles related to their trophic positions in a subtropical stream ecosystem.

The activity of nitrifying microorganisms in a high-altitude Andean wetland.

High-altitude wetland holds freshwater springs, evaporitic ponds and lagoon with variable salinity and nutrients, potentially influencing the ecology of nitrifying communities. In this study, nitrifying microorganisms in Salar de Huasco (Chile) were surveyed to determine bacterial and archaeal contribution to ammonium (AO), nitrite oxidation (NO), ammonium uptake (AU) during wet and dry seasons. The activity signals from these groups were assessed by specific amoA-qPCR transcription, 15N tracer studies and addition of group specific inhibitor experiments for nitrifying microorganisms (N1-guanyl-1, 7-diaminoheptane [GC7]-archaeal specific and allylthiourea [ATU]-bacterial specific). Nitrifying communities, i.e. Nitrosopumilus, Nitrosospira, Nitrosomonas, Kuenenia and Nitrospira, were more frequent (∼0.25% of 16S rRNA sequences) at low salinity sites. Bacterial amoA-qPCR transcripts also increased at low salinity and along in situ ammonium increase observed between wet/dry seasons. Nutrient changes through time and 15N tracer experiments results showed that AO and NO were detected and peaked mainly at low salinity-high ammonium sites (<37 000 μS cm-1 and >0.3 μM), whereas AU was predominant at evaporitic sites. Our results indicate that salinity and ammonium affect the nitrifying communities that are potentially more active at low-salinity sites but persistent at saltier evaporitic areas of the wetland when ammonium is available.

Uptake rates of ammonium and nitrate by phytoplankton communities in two eutrophic tropical reservoirs

While excess phosphorus typically results in the eutrophication of inland waters, there is growing evidence that excess nitrogen (N) and the availability of different N forms influence phytoplankton community composition, often favoring potentially toxic genera. In this study, the environmental dynamics, phytoplankton community structure, and N uptake rates were investigated in two tropical reservoirs. Phytoplankton ammonium () and nitrate () acquisition was assessed through 15N addition experiments over 2 years. We found that changes in ambient nutrient concentrations and temperature influenced different phytoplankton groups, which tended to have different N uptake strategies. The preferred N‐source by Cyanobacteria was while Dinophyceae and other groups seemed adapted to also take up , possibly due to competition. Potential uptake rates (maximum of 8.5 μM‐N hr−1 for and 1.3 μM‐N hr−1 for ) were high in comparison to previous reports from temperate freshwater or marine systems, likely due to elevated algal biomass and temperature. When normalized to biomass as chlorophyll‐a (Chl‐a), specific uptake rates varied between 0.01–3.4 μmol‐N μgChl‐a−1 day−1 for and &lt;0.01–0.8 μmol‐N μgChl‐a−1 day−1 for and were comparable to those reported for other eutrophic and hypereutrophic aquatic systems. In addition, higher temperatures favored Cyanobacteria (e.g., Cylindrospermopsis raciborskii and Microcystis aeruginosa), while a more diverse community was found during colder months. Results highlight how high loading of reduced forms of nitrogen and high temperatures can exacerbate harmful Cyanobacteria blooms in tropical reservoirs and be a concern for drinking water quality.

Symbiotic nitrogen fixation and interspecific transfer by Caragana microphylla in a temperate grassland with 15N dilution technique

Quantification of symbiotic nitrogen (N) fixation by legume and its N transfer to the neighboring plants is an important prerequisite to understand the N cycling in the N-limited grasslands. The objective of this study is to explore the symbiotic N2-fixation by a legume shrub (Caragana microphylla) and its N transfer to coexisting non-legumes with the 15N dilution technique. A paired experimental design (legume vs. non-legume quadrats) was used. A small amount (0.03gNm−2) of 15N-labeled KNO3 (99.5% 15N enrichment) was evenly distributed in these paired labeled quadrats, with only water additions as the control. One month after the 15N addition, aboveground parts of C. microphylla and four non-legumes of reference plants (i.e. Cleistogenes squarrosa, Artemisia frigida, Artemisia capillaris and Leymus chinensis) in both labeled and controlled quadrats were harvested. Symbiotic N2-fixation and transfer to non-legumes were estimated based on the aboveground biomass and its 15N in these plant species. The N derived from the atmosphere (%Ndfa) of C. microphylla was estimated to range from 84 to 95%. According to the aboveground biomass of C. microphylla, symbiotic N2-fixation was estimated to be 4.3gm−2yr−1 in the mixed C. microphylla community in the temperate grassland of northern China. The amount of N transfer from C. microphylla to their coexisting plants varied with plant species. About half of above N in C. squarrosa derived from C. microphylla, while L. chinensis showed almost no N derived from C. microphylla. Both A. frigida and A. capillaris showed about 36% and 37% of N derived from C. microphylla, respectively. Totally, about 17% of N fixed in the aboveground part of C. microphylla was transferred to its associated species. Considering the amount of N fixed and transferred by C. microphylla, we conclude that C. microphylla plays an important role in the N cycling of temperate grasslands in northern China.

Small‐scale heterogeneity of microbial N uptake in streams and its implications at the ecosystem level

Large-scale factors associated with the environmental context of streams can explain a notable amount of variability in patterns of stream N cycling at the reach scale. However, when environmental factors fail to accurately predict stream responses at the reach level, focusing on emergent properties from small-scale heterogeneity in N cycling rates may help understand observed patterns in stream N cycling. To address how small-scale heterogeneity may contribute to shape patterns in whole-reach N uptake, we examined the drivers and variation in microbial N uptake at small spatial scales in two stream reaches with different environmental constraints (i.e., riparian canopy). Our experimental design was based on two ¹⁵N additions combined with a hierarchical sampling design from reach to microhabitat scales. Regardless of the degree of canopy cover, small-scale heterogeneity of microbial N uptake ranged by three orders of magnitude, and was characterized by a low abundance of highly active microhabitats (i.e., hot spots). The presence of those hot spots of N uptake resulted in a nonlinear spatial distribution of microbial N uptake rates within the streambed, especially in the case of epilithon assemblages. Small-scale heterogeneity in N uptake and turnover rates at the microhabitat scale was primarily driven by power relationships between N cycling rates and stream water velocity. Overall, fine benthic organic matter (FBOM) assemblages responded clearly to changes in the degree of canopy cover, overwhelming small-scale heterogeneity in its N uptake rates, and suggesting that FBOM contribution to whole-reach N uptake was principally imposed by environmental constraints from larger scales. In contrast, N uptake rates by epilithon showed no significant response to different environmental influences, but identical local drivers and spatial variation in each study reach. Therefore, contribution of epilithon assemblages to whole-reach N uptake was mainly associated with emerging properties from small-scale heterogeneity at lower spatial scales.

Application of the &amp;lt;sup&amp;gt;15&amp;lt;/sup&amp;gt;N gas-flux method for measuring in situ N&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt; and N&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt;O fluxes due to denitrification in natural and semi-natural terrestrial ecosystems and comparison with the acetylene inhibition technique

Abstract. Soil denitrification is considered the most un-constrained process in the global N cycle due to uncertain in situ N2 flux measurements, particularly in natural and semi-natural terrestrial ecosystems. 15N tracer approaches can provide in situ measurements of both N2 and N2O simultaneously, but their use has been limited to fertilized agro-ecosystems due to the need for large 15N additions in order to detect 15N2 production against the high atmospheric N2. For 15N–N2 analyses, we have used an “in-house” laboratory designed and manufactured N2 preparation instrument which can be interfaced to any commercial continuous flow isotope ratio mass spectrometer (CF-IRMS). The N2 prep unit has gas purification steps and a copper-based reduction furnace, and allows the analysis of small gas injection volumes (4 µL) for 15N–N2 analysis. For the analysis of N2O, an automated Tracegas Preconcentrator (Isoprime Ltd) coupled to an IRMS was used to measure the 15N–N2O (4 mL gas injection volume). Consequently, the coefficient of variation for the determination of isotope ratios for N2 in air and in standard N2O (0.5 ppm) was better than 0.5 %. The 15N gas-flux method was adapted for application in natural and semi-natural land use types (peatlands, forests, and grasslands) by lowering the 15N tracer application rate to 0.04–0.5 kg 15N ha−1. The minimum detectable flux rates were 4 µg N m−2 h−1 and 0.2 ng N m−2 h−1 for the N2 and N2O fluxes respectively. Total denitrification rates measured by the acetylene inhibition technique in the same land use types correlated (r = 0.58) with the denitrification rates measured under the 15N gas-flux method, but were underestimated by a factor of 4, and this was partially attributed to the incomplete inhibition of N2O reduction to N2, under a relatively high soil moisture content, and/or the catalytic NO decomposition in the presence of acetylene. Even though relatively robust for in situ denitrification measurements, methodological uncertainties still exist in the estimation of N2 and N2O fluxes with the 15N gas-flux method due to issues related to non-homogenous distribution of the added tracer and subsoil gas diffusion using open-bottom chambers, particularly during longer incubation duration. Despite these uncertainties, the 15N gas-flux method constitutes a more reliable field technique for large-scale quantification of N2 and N2O fluxes in natural terrestrial ecosystems, thus significantly improving our ability to constrain ecosystem N budgets.

The Influence of Altered Rainfall Regimes on Early Season N Partitioning Among Early Phenology Annual Plants, a Late Phenology Shrub, and Microbes in a Semi-arid Ecosystem

In Mediterranean-type ecosystems, nitrogen (N) accumulates in soil during dry summer months and rapidly becomes available during early season rain events. The availability of early season N could depend on the size of rainfall events, soil microbial activity, and phenology of the plant community. However, it is poorly understood how precipitation patterns affect the fate of early season N. Microbes and plants with early phenology may compete strongly for early season N but theory suggests that microbial N storage can meet plant N demands later in the season. Using a 15N tracer and rainfall manipulation we investigated the fate of early season N. N allocation patterns differed substantially between microbes, early and late phenology plants. As expected early phenology annuals and microbes took up 15N, within 1 day, whereas a late-phenology shrub allocated 15N to leaves later in the season. We saw no evidence for microbial storage of early season N; the peak of 15N in shrub leaves did not coincide with detectable levels of 15N in the microbial biomass or labile soil pool. This suggests that shrubs were able to access early season N, store and allocate it for growth later in the season. Although we saw no evidence of microbial N storage, N retention in soil organic matter (SOM) was high and microbes may play an important role in sequestering N to SOM. Plant N uptake did not respond significantly to 1 year of rainfall manipulation, but microbes were sensitive to dry conditions. 1 year after 15N addition shrubs had resorbed up to half of the N from leaves whereas N in annuals remained as dead leaf litter. Differences in end-of-season N partitioning between dead and living biomass in the two vegetation types suggest that plant species composition could affect N availability in the following growing season, but it may take several years of altered precipitation patterns to produce rainfall-dependent changes.

Tree species partition N uptake by soil depth in boreal forests.

It is recognized that the coexistence of herbaceous species in N-depleted habitats can be facilitated by N partitioning; however, the existence of such a phenomenon for trees has not yet been demonstrated. Here, we show from both foliage and soil 15N natural abundance values and from a 12-year in situ 15N addition experiment, that black spruce (Picea mariana) and jack pine (Pinus banksiana), two widespread species of the Canadian boreal forest, take up N at different depths. While black spruce takes up N from the organic soil, jack pine acquires it deeper within the highly N-depleted mineral soil. Systematic difference in foliar 15N natural abundance between the two species across seven sites distributed throughout the eastern Canadian boreal forest shows that N spatial partitioning is a widespread phenomenon. Distinct relationships between delta15N and N concentration in leaves of both species further emphasize their difference in N acquisition strategies. This result suggests that such complementary mechanisms of N acquisition could facilitate tree species coexistence in such N-depleted habitats and could contribute to the positive biodiversity-productivity relationship recently revealed for the eastern Canadian boreal forest, where jack pine is present. It also has implications for forest management and provides new insights to interpret boreal forest regeneration following natural or anthropogenic perturbations.