Edited by: Dietrich Hertel, Georg-August-Universität Göttingen, Germany
Reviewed by: Ute Hamer, Westfälische Wilhelms-Universität Münster, Germany; Felix Heitkamp, Georg-August-Universität Göttingen, Germany
*Correspondence: Andre Velescu
This article was submitted to Biogeoscience, a section of the journal Frontiers in Earth Science
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In the past two decades, the tropical montane rain forests in south Ecuador experienced increasing deposition of reactive nitrogen mainly originating from Amazonian forest fires, while Saharan dust inputs episodically increased deposition of base metals. Increasing air temperature and unevenly distributed rainfall have allowed for longer dry spells in a perhumid ecosystem. This might have favored mineralization of dissolved organic matter (DOM) by microorganisms and increased nutrient release from the organic layer. Environmental change is expected to impact the functioning of this ecosystem belonging to the biodiversity hotspots of the Earth. In 2007, we established a nutrient manipulation experiment (NUMEX) to understand the response of the ecosystem to moderately increased nutrient inputs. Since 2008, we have continuously applied 50 kg ha−1 a−1 of nitrogen (N), 10 kg ha−1 a−1 of phosphorus (P), 50 kg + 10 kg ha−1 a−1 of N, and P and 10 kg ha−1 a−1 of calcium (Ca) in a randomized block design at 2000 m a.s.l. in a natural forest on the Amazonia-exposed slopes of the south Ecuadorian Andes. Nitrogen concentrations in throughfall increased following N+P additions, while separate N amendments only increased nitrate concentrations. Total organic carbon (TOC) and dissolved organic nitrogen (DON) concentrations showed high seasonal variations in litter leachate and decreased significantly in the P and N+P treatments, but not in the N treatment. Thus, P availability plays a key role in the mineralization of DOM. TOC/DON ratios were narrower in throughfall than in litter leachate but their temporal course did not respond to nutrient amendments. Our results revealed an initially fast, positive response of the C and N cycling to nutrient additions which declined with time. TOC and DON cycling only change if N and P supply are improved concurrently, while NO3-N leaching increases only if N is separately added. This indicates co-limitation of the microorganisms by N and P. The current increasing reactive N deposition will increase N export from the root zone, while it will only accelerate TOC and DON turnover if P availability is simultaneously increased. The Saharan dust-related Ca deposition has no impact on TOC and DON turnover.
Tropical forests generate one third of the terrestrial gross primary production (Beer et al.,
The tropical Andean forest in south Ecuador has experienced improved nutrient supply in the past 15 years because of steadily increasing N and episodic calcium (Ca) and magnesium (Mg) deposition (Boy and Wilcke,
Dissolved organic matter (DOM) serves as a C and energy source for microorganisms and is an essential component of the global C and N cycles (Van Hees et al.,
As a result of further agricultural and industrial development in the Amazon Basin (Walker et al.,
In the fragile montane ecosystems, it is frequently assumed that nutrient constraints limit plant growth and primary production, which are known to be regulated by the supply of key elements, like N and P (Vitousek and Howarth,
Since nutrient limitation was thought to be widespread in tropical soils, P would usually limit productivity in lowland rain forests growing on heavily weathered soils, while N, in contrast, would be the limiting nutrient in montane rain forests growing on less weathered young soils (Tanner et al.,
Recently, single nutrient limitations have begun to be questioned. The study of Kaspari et al. (
However, constraints to primary production may be different from limitations of organic matter cycling. A large amount of nutrients is stored in the organic layer of tropical montane forest soils. Thickness of organic layers was shown to increase with elevation, because of decreasing temperatures and increasing soil humidity, which inhibit mineralization of organic matter and rates of nutrient turnover (Wilcke et al.,
Nutrient limitations play an important role in the control of C and N cycling in tropical montane forests. Improved nutrient availability favors microbial activity and an elevated N supply is known to increase the turnover of light, easily degradable organic matter, while it further stabilizes heavier, more recalcitrant compounds of the organic matter pool (Neff et al.,
Studies conducted during the last decade often stressed that N emissions to the atmosphere and the increasing use of fertilizers in the modern world strongly interfere with natural nutrient cycling (Galloway et al.,
In summary, investigations in tropical montane forests have shown that a considerable uncertainty still persists with regard to the potential response of the N and DOM cycling in these ecosystems to increasing supply of nutrients from atmospheric deposition and from climatically favored, enhanced mineralization of soil organic matter. Therefore, our objectives were to elucidate the response of (i) the N cycle in a tropical montane forest and (ii) the dissolved organic C and N concentrations and TOC/DON ratios to nutrient additions. We hypothesized that (i) N and Ca additions increase N concentrations while P additions (alone and in combination with N) reduce N concentrations in throughfall and soil solutions and (ii) that increasing nutrient availability decreases TOC and DON concentrations and widens the TOC/DON ratios. We tested these hypotheses in a nutrient manipulation experiment (NUMEX), in which N, P, N+P, and Ca were fertilized at moderate levels considered as reflecting realistic future changes in nutrient availability.
The study area is located in the province of Zamora-Chinchipe, on the Amazon-exposed slopes of the south Ecuadorian Andes (3.58° S, 79.08 W). The experimental plots are situated in the Reserva Biológica San Francisco at an altitude between 2010 and 2128 m a.s.l. (Figure
The vegetation cover consists of a near-natural, little disturbed lower montane forest (Bruijnzeel and Hamilton,
The soils of the experimental area are young and mostly shallow (< 60 cm), have a loamy texture and were classified according to IUSS Working Group WRB (
NUMEX was established in the year 2007 and is continued until present. It consists of three experimental sites along an altitudinal gradient at 1000, 2000, and 3000 m a.s. (Figure
The experiment site includes 20 plots (20 × 20 m) in a fourfold replicated, randomized block design (Figure
In 2007, all study plots were equipped with throughfall collectors, zero-tension litter lysimeters, and suction cups at the 0.15 and 0.3 m mineral soil depths.
Throughfall was collected with 20 rain gauges (Hellmann type) which were randomly distributed on each plot along two perpendicular transects, resulting thus in 400 collectors for the whole experiment area. To ensure sampling representativeness, number and spatial distribution of the throughfall collectors were determined taking into account the spatial and temporal variability of precipitation, which was analyzed in a separate experiment between 2004 and 2008 (Wullaert et al.,
Litter leachate (solution percolating through the organic layer) was collected using three zero-tension lysimeters per plot, which consisted of plastic boxes covered with a polyethylene net with a 0.5 mm mesh size, connected to sampling bottles, which were wrapped in aluminum foil. The lysimeters were installed underneath the organic layer at 7–51 cm below ground surface from the side of a small soil pit, at upper, middle and lower slope positions, taking care that the organic layer itself was only minimally disturbed during or after installation. Litter leachate was bulked plotwise after individual sampling of the collecting bottles, resulting thus in 20 samples per collecting date.
Throughfall and litter leachate have been collected fortnightly since August 2007. Incident rainfall was collected weekly at 2–4 gauging stations and five rain collectors at each station, with the same Hellmann-type collectors used for sampling throughfall. After collection, throughfall and litter leachate samples were transferred to the field laboratory of the Estación Científica San Francisco (ECSF), where they were immediately filtered through ashless filter paper, pore size 4–7 μm, Type 392 (Sartorius-Stedim GmbH, Göttingen, Germany) and then kept at −20°C until shipping in frozen state to Switzerland for chemical analysis.
For this study, we analyzed a dataset of 5 complete years including a fortnightly record of element concentrations in throughfall and litter leachate between 2007 and 2012, which we mainly measured in the laboratory of the Geographic Institute at the University of Bern, Switzerland. Electrical conductivity (TetraCon 325, WTW GmbH, Weilheim, Germany) and pH values (Sentix 81, WTW GmbH, Weilheim, Germany) were immediately measured in unfiltered aliquots of the collected water samples in the laboratory of the ECSF in Ecuador.
Concentrations of the dissolved N species ammonium-N (NH4-N), nitrate-N (NO3-N) and total nitrogen (TN) were measured by continuous flow analysis (CFA) using high resolution colorimetry and photometric detection (AutoAnalyzer 3 HR, Seal GmbH, Norderstedt, Germany). During the CFA measurements, samples were dialyzed and TN was oxidized to nitrate by UV digestion with potassium peroxydisulfate and sodium hydroxide in presence of a boric acid buffer. NH4-N was determined by the Berthelot reaction and NO3-N by cadmium reduction. The determination of NO3-N concentrations by cadmium reduction usually includes a small amount of nitrite-N (NO2-N), which was below the detection limits and hence considered negligible. We measured TOC (filtered through 4–7 μm pores) as non-purgeable organic carbon (NPOC) by elemental analysis through high temperature combustion and infrared (IR) detection (varioTOC cube, elementar Analysensysteme GmbH, Hanau, Germany), after acidifying 10 mL of the sample with 50 μL of a 10% HCl solution to remove inorganic C.
Average detection limits were 0.024 mg L−1 for NH4-N, 0.016 mg L−1 for NO3-N, 0.042 mg L−1 for TN and 0.16 mg L−1 for TOC. The quality of the CFA measurements was verified by running certified N standards (Merck, Darmstadt, Germany) with a concentration of 0.4 mg L−1 NH4-N and 0.5 mg L−1 NO3-N in each measured batch. Certified standards were combined with in-house control standards, with a concentration of 0.5 mg L−1 NH4-N and 0.25 mg L−1 NO3-N, which were run on average every 20 samples. The quality of TOC measurements was verified by use of in-house control standard only, which had a concentration of 5 mg L−1 and 10 mg L−1 TOC. Measurement batches were only accepted if the results for the control standards deviated by less than 10% from the target values. The relative SD of the measurement precision of the control standards was ± 4% for NH4-N, ± 2% for NO3-N, ± 5% for TN, and ± 3% for TOC.
Throughfall fluxes were measured fortnightly in the field, averaged per plot and summed up to monthly and annual data. Concentrations of dissolved organic nitrogen (DON) were calculated as difference between TN and the sum of NH4-N and NO3-N concentrations. Values below the detection limit were taken as zero for calculations, possibly resulting in an underestimation of the DON concentrations. Monthly mean concentrations were calculated by averaging fortnightly concentrations of each measured element. Annual mean element concentrations were calculated as volume-weighted means of monthly concentrations. The effects of nutrient addition on the investigated element concentrations were expressed as log response ratios (RRx), which were calculated as a natural logarithm of the ratios between the measured values in the nutrient addition treatments and in the control plots. A value of 0.2 indicates thus a relative difference in concentrations of 22% between treatment and control, while a value of 0.5 indicates a relative difference of 65%.
We performed statistical analyses with the software R, version 3.0.2 (R Core Team,
Time series of element concentrations in monthly data were analyzed using the seasonal Mann-Kendall test, as suggested by Hirsch et al. (
We did not detect significant differences between treatments and control plots either before the start of nutrient additions or during the 5 years of the experiment (Table
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2008 | 1225 | (± 17) | 1373 | (± 10) | 1280 | (± 13) | 1292 | (± 14) | 1182 | (± 18) |
2009 | 1176 | (± 19) | 1287 | (± 25) | 1277 | (± 22) | 1276 | (± 27) | 1189 | (± 22) |
2010 | 1060 | (± 12) | 1195 | (± 14) | 1124 | (± 17) | 1020 | (± 20) | 1082 | (± 23) |
2011 | 1162 | (± 22) | 1222 | (± 18) | 1157 | (± 12) | 1081 | (± 23) | 1110 | (± 20) |
2012 | 1354 | (± 18) | 1490 | (± 16) | 1436 | (± 15) | 1333 | (± 22) | 1375 | (± 16) |
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2008 | 0.11 | (±0.03) | 0.04 | (± 0.04) | 0.05 | (± 0.08) | –0.04 | (± 0.10) | ||
2009 | 0.09 | (±0.06) | 0.08 | (± 0.10) | 0.04 | (± 0.14) | 0.00 | (± 0.12) | ||
2010 | 0.12 | (±0.06) | 0.06 | (± 0.08) | –0.05 | (± 0.09) | 0.01 | (± 0.10) | ||
2011 | 0.06 | (±0.04) | 0.00 | (± 0.07) | –0.08 | (± 0.11) | –0.05 | (± 0.05) | ||
2012 | 0.10 | (±0.07) | 0.06 | (± 0.08) | –0.02 | (± 0.08) | 0.02 | (± 0.03) |
The analysis of element concentrations by linear mixed models (Figure
Nitrogen additions generally resulted in increased concentrations of inorganic N species. The NH4-N concentrations were 7 ± 3% higher than in the control in the plots with separate N additions (
The effects of separate P amendments and of simultaneous N+P additions on TOC and DON concentrations tended to be similar to those already observed in throughfall (Figure
Both N and P amendments to the forest soil showed strong and significant effects on inorganic N concentrations in litter leachate compared with the control treatment. Accordingly, NH4-N concentrations were 16 ± 4% higher (
The TOC concentrations in throughfall decreased by 16% in the unfertilized control plots between the first and the fifth year of NUMEX (τ = −0.248,
Concentrations of inorganic N in throughfall showed a high seasonality and doubled in 5 years (τ = 0.328,
In litter leachate (Figure
The concentration ratio of NH4-N to NO3-N is approximatively 10:1, which indicates that the system is dominated by
In throughfall of the plots with separate N additions, TOC (τ = 0.223,
The concentrations of NH4-N and NO3-N in throughfall behaved similarly as in the control plots and followed the positive concentration trends described in Figure
In litter leachate (Figure
There were no significant differences in pH values of litter leachate between the treatments and the control plots over the complete observation period, which confirms that the increased supply of N occurring as NH4-N did not acidify the organic layer during the observation period. The TOC/DON ratios in litter leachate did not significantly change with time after fertilizer additions, which was similarly observed in throughfall.
In the plots with separate N additions, NH4-N concentrations in litter leachate followed the same temporal patterns which we had already observed in the control plots, but decreased with time in the plots with separate P (τ = −0.398,
There was a fast initial positive response of N concentrations in litter leachate to P and N+P amendments. However, in the later course of the experiment, the differences to the control plots decreased (Figure
We observed no significant effects of inorganic N concentrations in litter leachate in response to Ca additions. Once more, TN concentrations in little leachate were driven by the NH4-N concentrations, while DON contributions to TN dropped by ca. 18%, as similarly observed in the control plots (Figure
Five years after the establishment of NUMEX, we showed that additions of N alone or simultaneously with P generally increased annual mean N concentrations in the above-ground part of the forest water cycle (Figure
Reactive N species
When N demands of microbes and plants are exceeded, N leaching from the ecosystem may be further enhanced by long-term availability of excessive N (Law,
We expected that Ca additions would generate a similar response of the N cycle as the addition of N, as observed by Perakis et al. (
Annual mean TOC and DON concentrations in throughfall responded weakly to nutrient amendments. The overall differences from the control treatments were not significant (Figure
In litter leachate, the overall response of TOC and DON concentrations was similar to their response in throughfall (Figure
Moreover, increasing air temperature and unevenly distributed rainfall have allowed for longer dry spells, leading to reduced air and soil humidity in our study area (Peters et al.,
Our study confirms the importance of the relationships between nutrient supply, leaching, and demands of organisms (Townsend et al.,
We expected that nutrient additions would result in wider TOC/DON ratios, because of the preferential mineralization of the more hydrophilic, polar fraction of DOM, but our results contradicted our hypothesis and indicated strong opposite effects for all treatments in throughfall (Figure
Our results demonstrate that N additions alone or in combination with P generally increased mean N concentrations in the above-ground part of the forest water cycle and led to an enrichment of the inorganic species
Separate N additions generated a steady increase of DON concentrations in throughfall. Interestingly, TOC exports from the canopy were not affected by nutrient additions. In the organic layer, separate additions of P and simultaneous additions of N and P stimulated mineralization of TOC and DON, while there was no significant response to separate additions of N. In absence of acidification of the litter leachate following N additions, we conclude that the availability of P plays a key role in the mineralization of TOC and DON by accelerating DOM turnover in the organic layer. Our findings contradicted the hypothesis that nutrient additions would lead to wider TOC/DON ratios over time. Rising availability of inorganic N species favored DON leaching to the mineral soil, leading thus to narrower TOC/DON ratios in litter leachate.
Increased supply of P—alone or together with N—will lead to a more efficient use of available N by microorganisms and plants and hence to reduced N concentrations in solutions originating from the organic layer in the long run. Thus, TOC and DON cycling will only change if the availability of N and P is concurrently improved, while NO3-N leaching from the organic layer will further rise if the N supply alone increases.
All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank particularly Jürgen Homeier and Hans Wullaert for the initial establishment of NUMEX, Arthur Broadbent and Hannes Thomasch for their valuable contribution during the field work in Ecuador and to the successful accomplishment of the chemical analyses in Bern and José Luis Peña Caivinagua for his support as a field technician. We thank the Deutsche Forschungsgemeinschaft (DFG) for funding the different phases of our projects within the research units FOR 402 and FOR 816. Furthermore, we thank Naturaleza y Cultura Internacional (NCI) in Loja for providing access to the study area and to the research station ECSF and the Ecuadorian Ministry of Environment for the research permits.