Altered plant carbon partitioning enhanced forest ecosystem carbon storage after 25 years of nitrogen additions.

Decades of atmospheric nitrogen (N) deposition in the northeastern USA have enhanced this globally important forest carbon (C) sink by relieving N limitation. While many N fertilization experiments found increased forest C storage, the mechanisms driving this response at the ecosystem scale remain uncertain. Following the optimal allocation theory, augmented N availability may reduce belowground C investment by trees to roots and soil symbionts. To test this prediction and its implications on soil biogeochemistry, we constructed C and N budgets for a long-term, whole-watershed N fertilization study at the Fernow Experimental Forest, WV, USA. Nitrogen fertilization increased C storage by shifting C partitioning away from belowground components and towards aboveground woody biomass production. Fertilization also reduced the C cost of N acquisition, allowing for greater C sequestration in vegetation. Despite equal fine litter inputs, the C and N stocks and C : N ratio of the upper mineral soil were greater in the fertilized watershed, likely due to reduced decomposition of plant litter. By combining aboveground and belowground data at the watershed scale, this study demonstrates how plant C allocation responses to N additions may result in greater C storage in both vegetation and soil.

Response of soil fertility to 25 years of experimental acidification in a temperate hardwood forest.

The effects of enhanced acid deposition from the atmosphere, and associated elevated inputs of N, are widely evident, especially for forests where excess N has led to a variety of deleterious effects. These include declines in biodiversity, a response that will likely require considerable time for recovery. The purpose of this study was to determine responses of plant nutrient availability in surface mineral soil to 25 yr of experimental acidification and N addition in a central Appalachian hardwood forest ecosystem. We hypothesized that chronic additions of (NH4 )2 SO4 will increase mineral N, decrease soil pH, P, and base cations, increase micronutrients (Mn2+ and Fe2+ ), and increase levels of Al3+ . Results supported these predictions, although Mn2+ did not vary significantly. Earlier work on these plots found no response of any of the extractable nutrients to 3 yr of treatment, yet after 25 yr, our results suggest that impacts are apparent in the top 5 cm of the A horizon. We surmise that impacts in these soils may have lagged behind the onset of acidification treatments or that several years of treatment were required to overcome preexisting differences in soil ions. Generally, current findings confirm that (NH4 )2 SO4 treatments have lowered the pH, enhanced levels of exchangeable Al3+ , and increased stream-water exports of NO3 - and base cations-a process that further acidifies soil. The combination of these changes in surface soils, with their high proportion of fine roots, may contribute to the reduced growth and competitiveness of some hardwood species at the acidified site.

Assessing tree ring δ15N of four temperate deciduous species as an indicator of N availability using independent long-term records at the Fernow Experimental Forest, WV.

Nitrogen deposition in the northeastern US changed N availability in the latter part of the twentieth century, with potential legacy effects. However, long-term N cycle measurements are scarce. N isotopes in tree rings have been used as an indicator of N availability through time, but there is little verification of whether species differ in the strength of this signal. Using long-term records at the Fernow Experimental Forest in West Virginia, we examined the relationship between soil conditions, including net nitrification rates, and wood δ15N in 2014, and tested the strength of correlation between tree ring δ15N of four species and stream water NO3- loss from 1971 to 2000. Higher soil NO3- was weakly associated with higher wood δ15N across species, and higher soil net nitrification rates were associated with higher δ15N for Quercus rubra only. The δ15N of Liriodendron tulipifera and Q. rubra, but neither Fagus grandifolia nor Prunus serotina, was correlated with stream water NO3-. L. tulipifera tree ring δ15N had a stronger association with stream water NO3- than Q. rubra. Overall, we found only limited evidence of a relationship between soil N cycling and tree ring δ15N, with a strong correlation between the wood δ15N and NO3- leaching loss through time for one of four species. Tree species differ in their ability to preserve legacies of N cycling in tree ring δ15N, and given the weak relationships between contemporary wood δ15N and soil N cycle measurements, caution is warranted when using wood δ15N to infer changes in the N cycle.

Interactions among plants, bacteria, and fungi reduce extracellular enzyme activities under long-term N fertilization.

Atmospheric nitrogen (N) deposition has enhanced soil carbon (C) stocks in temperate forests. Most research has posited that these soil C gains are driven primarily by shifts in fungal community composition with elevated N leading to declines in lignin degrading Basidiomycetes. Recent research, however, suggests that plants and soil microbes are dynamically intertwined, whereby plants send C subsidies to rhizosphere microbes to enhance enzyme production and the mobilization of N. Thus, under elevated N, trees may reduce belowground C allocation leading to cascading impacts on the ability of microbes to degrade soil organic matter through a shift in microbial species and/or a change in plant-microbe interactions. The objective of this study was to determine the extent to which couplings among plant, fungal, and bacterial responses to N fertilization alter the activity of enzymes that are the primary agents of soil decomposition. We measured fungal and bacterial community composition, root-microbial interactions, and extracellular enzyme activity in the rhizosphere, bulk, and organic horizon of soils sampled from a long-term (>25 years), whole-watershed, N fertilization experiment at the Fernow Experimental Forest in West Virginia, USA. We observed significant declines in plant C investment to fine root biomass (24.7%), root morphology, and arbuscular mycorrhizal (AM) colonization (55.9%). Moreover, we found that declines in extracellular enzyme activity were significantly correlated with a shift in bacterial community composition, but not fungal community composition. This bacterial community shift was also correlated with reduced AM fungal colonization indicating that declines in plant investment belowground drive the response of bacterial community structure and function to N fertilization. Collectively, we find that enzyme activity responses to N fertilization are not solely driven by fungi, but instead reflect a whole ecosystem response, whereby declines in the strength of belowground C investment to gain N cascade through the soil environment.

Soluble soil aluminum alters the relative uptake of mineral nitrogen forms by six mature temperate broadleaf tree species: possible implications for watershed nitrate retention.

Increased availability of monomeric aluminum (Al3+) in forest soils is an important adverse effect of acidic deposition that reduces root growth and inhibits nutrient uptake. There is evidence that Al3+ exposure interferes with NO3- uptake. If true for overstory trees, the reduction in stand demand for NO3- could increase NO3- discharge in stream water. These effects may also differ between species that tolerate different levels of soil acidity. To examine these ideas, we measured changes in relative uptake of NO3- and NH4+ by six tree species in situ under increased soil Al3+ using a 15N-labeling technique, and measured soluble soil Al levels in a separate whole-watershed acidification experiment in the Fernow Experimental Forest (WV). When exposed to added Al3+, the proportion of inorganic N acquired as NO3- dropped 14% across species, but we did not detect a reduction in overall N uptake, nor did tree species differ in this response. In the long-term acidification experiment, we found that soluble soil Al was mostly in the free Al3+ form, and the concentration of Al3+ was ~65 µM higher (~250%) in the mineral soil of the acidified watershed vs. an untreated watershed. Thus, increased levels of soil Al3+ under acidic deposition cause a reduction in uptake of NO3- by mature trees. When our 15N uptake results were applied to the watershed acidification experiment, they suggest that increased Al3+ exposure could reduce tree uptake of NO3- by 7.73 kg N ha-1 year-1, and thus increase watershed NO3- discharge.

Non-random species loss in a forest herbaceous layer following nitrogen addition.

Nitrogen (N) additions have decreased species richness (S) in hardwood forest herbaceous layers, yet the functional mechanisms for these decreases have not been explicitly evaluated. We tested two hypothesized mechanisms, random species loss (RSL) and non-random species loss (NRSL), in the hardwood forest herbaceous layer of a long-term, plot-scale, fertilization experiment in the central Appalachian Mountains, USA. Using a random thinning algorithm, we simulated changes in species densities under RSL and compared the simulated densities to the observed densities among N-fertilized (+N), N-fertilized and limed (+N+L), and reference (REF) plots in regenerating forest stands. We found a lower S in the +N treatment across all survey years and determined that the reduction in S was a function of NRSL. Furthermore, non-random effects were observed in certain species, as they occurred at densities that were either higher or lower than expected due to RSL. Differential advantages were also observed among species between +N and +N+L treatments, suggesting that species responded to either the fertilization or acidification effects of N, though no consistent pattern emerged. Species nitrophily status was not a useful trait for predicting specific species losses, but was a significant factor when averaged across all treatments and sampling years. Our results provide strong evidence that declines in S in the forest herbaceous layer under N fertilization are due largely to NRSL and not simply a function of species rarity.

A reference-based approach for estimating leaf area and cover in the forest herbaceous layer.

Cover data are used to assess vegetative response to a variety of ecological factors. Estimating cover in the herbaceous layer of forests presents a problem because the communities are structurally complex and rich in species. The currently employed techniques for estimating cover are less than optimal for measuring such rich understories because they are inaccurate, slow, or impracticable. A reference-based approach to estimating cover is presented that compares the area of foliar surfaces to the area of an observer's hand. While this technique has been used to estimate cover in prior studies, its accuracy has not been tested. We tested this hand-area method at the individual plant, population, and community scales in a deciduous forest herbaceous layer, and in a separate farm experiment. The precision, accuracy, observer bias, and species bias of the method were tested by comparing the hand-estimated leaf area index values with actual leaf area index, measured using a leaf area meter. The hand-area method was very precise when regressed against actual leaf area index at the plant, population, and community scales (R(2) of 0.97, 0.93, and 0.87). Among the deciduous sites, the hand-area method overestimated leaf area index consistently by 39.1% at all scales. There was no observer bias detected at any scale, but plant overestimation bias was detected in one species at the population scale. The hand-area method is a rapid and reliable technique for estimating leaf area index or cover in the forest herbaceous layer and should be useful to field ecologists interested in answering questions at the plant, population, or community level.

Nutrient limitation in soils exhibiting differing nitrogen availabilities: what lies beyond nitrogen saturation?

The nature of nutrient limitation in large areas of temperate forest may be changing due to human activities. As N availability in these forests increases, other nutrients could increasingly constrain productivity and other ecosystem processes. To determine the nature of nutrient limitation (N, P, and Ca) in forest soils exhibiting differing N availability, we conducted three field studies in the Fernow Experimental Forest, West Virginia, USA. The first used a ubiquitous herbaceous species, Viola rotundifolia, to compare indices of N availability to the activity of root-associated phosphomonoesterase (PME) activity at two spatial scales. The second study used fertilized, root in-growth cores to assess the extent of N, P, and Ca limitation. Finally, we measured the root-associated PME activity of V. rotundifolia growing in experimental plots that have received various combinations of nutrient additions and harvest treatments. For entire watersheds, stream water nitrate concentrations were positively related to PME activities (R2 = 0.986). For small plots, PME activities were positively associated with soil nitrate availability (R2 = 0.425), and to a lesser extent with the leaf N concentrations (R2 = 0.291). Root growth into microsites fertilized with P was greater than growth into microsites fertilized with either N or Ca, especially in watersheds with high N availability. Experimental additions of N increased the root-associated PME activity of V. rotundifolia, supporting the causality of the relationship between N availability and PME activity. Collectively, our results indicate that, as N availability increases, P becomes increasingly limiting at the sites examined. Understanding how nutrient limitations change during N saturation should improve ecosystem models and better inform our attempts to mitigate any undesired effects.

N fertilization effects on denitrification and N cycling in an aggrading forest.

We investigated N cycling and denitrification rates following five years of N and dolomite amendments to whole-tree harvested forest plots at the long-term soil productivity experiment in the Fernow Experimental Forest in West Virginia, USA. We hypothesized that changes in soil chemistry and nutrient cycling induced by N fertilization would increase denitrification rates and the N2O:N2 ratio. Soils from the fertilized plots had a lower pH (2.96) than control plots (3.22) and plots that received fertilizer and dolomite (3.41). There were no significant differences in soil %C or %N between treatments. Chloroform-labile microbial biomass carbon was lower in fertilized plots compared to control plots, though this trend was not significant. Extractable soil NO3- was elevated in fertilized plots on each sample date. Soil-extractable NH4+, NO3-, pH, microbial biomass carbon, and %C varied significantly by sample date suggesting important seasonal patterns in soil chemistry and N cycling. In particular, the steep decline in extractable NH4+ during the growing season is consistent with the high N demands of a regenerating forest. Net N mineralization and nitrification also varied by date but were not affected by the fertilization and dolomite treatments. In a laboratory experiment, denitrification was stimulated by NO3- additions in soils collected from all field plots, but this effect was stronger in soils from the unfertilized control plots, suggesting that chronic N fertilization has partially alleviated a NO3- limitation on denitrification rates. Dextrose stimulated denitrification only in the whole-tree-harvest soils. Denitrification enzyme activity varied by sample date and was elevated in fertilized plots for soil collected in July 2000 and June 2001. There were no detectable treatment effects on N2O or N2 flux from soils under anaerobic conditions, though there was strong temporal variation. These results suggest that whole-tree harvesting has altered the N status of these soils so they are less prone to N saturation than more mature forests. It is likely that N losses associated with the initial harvest and high N demand by aggrading vegetation is minimizing, at least temporarily, the amount of inorganic N available for nitrification and denitrification, even in the fertilized plots in this experiment.

Soil warming and trace gas fluxes: experimental design and preliminary flux results.

We conducted several experiments to determine a procedure for uniformly warming soil 5° C above ambient using a buried heating cable. These experiments produced a successful design that could: 1) maintain a temperature difference of 5° C over a wide range of environmental conditions; 2) reduce inter-cable temperture variability to ca. 1.5° C; 3) maintain a temperature difference of 5° C near the edges of the plot; and 4) respond rapidly to changes in the environment. In addition, this design required electrical power only 42% of the time. Preliminary measurements indicate that heating increased CO2 emission by a factor of ca. 1.6 and decreased the C concentration in the O soil horizon by as much as 36%. In addition, warming the soil accelerated the emergence and early growth of the wild lily of the valley (Maianthemum canadense Desf.). The relationship between CO2 flux and soil temperature derived from our soil warming experiment was consistent with data from other hardwood forests around the world. Since the other hardwood forests were warmed naturally, it appears that for soil respiration, warming the soil with buried heating cables differs little from natural, aboveground warming. By warming soil beyond the range of natural variability, a multi-site, long-term soil warming experiment may be valuable in helping us understand how ecosystems will respond to global warming.