Soil Respiration and N-Mineralization Processes in the Patagonian Steppe Are More Responsive to Nutrient Than to Water Addition

Luisina Carbonell-Silletta (  luli_carbo13@hotmail.com ) UNPSJB: Universidad Nacional de la Patagonia San Juan Bosco https://orcid.org/0000-0003-33886015 Agustin Cavallaro Instituto de Biociencias de la Patagonia (INBIOP) Daniel A. Pereyra Instituto de Biociencias de la Patagonia (INBIOP) Javier O. Askenazi Centro de Investigaciones y Transferencia Golfo San Jorge Guillermo Goldstein Universidad de Buenos Aires (UBA CONICET) Fabian G. Scholz Instituto de Biociencias de la Patagonia (INBIOP) Sandra J. Bucci Instituto de Biociencias de la Patagonia (INBIOP) https://orcid.org/0000-0003-1079-9277

cycling. Soil respiration is one of the main processes resulting in C loss to the atmosphere from terrestrial ecosystems, particularly in low leaf area index (LAI) vegetation types such as arid shrublands. In addition, during mineralization carried out by soil microorganisms, organic N is transformed into inorganic through ammoni cation and nitri cation (Butterbach-Bahl and Gundersen 2011), at the same time that CO 2 release due to microbial activity occurs. In recent years it has been recognized that part of the organic matter is also degraded by abiotic factors such as solar radiation (Austin and Vivanco 2006), through which a fraction of CO 2 is released. Both CO 2  Moderate increases in soil moisture can enhance root growth and increase root exudations with enzymatic properties that can accelerate the organic matter degradation and microbial activity (Canarini et al. 2019). However, in some water addition experiments there was no response of mineralization rates (Fisher and Whitford 1995), and an increase in soil inorganic N has even been observed under dry conditions in arid ecosystems (Reynolds et al., 1999;Yahdjian et al., 2006).
It is also well known that soil nutrient availability drives many ecosystem processes, such as C mineralization and N transformations (mineralization and immobilization) which govern N availability (Fisk and (Li et al. 2016) and duration of experimental N enrichment. Long-term N addition leads to soil acidi cation, inhibiting soil microbial activity and structural diversity, thereby reducing soil N transformation. Other factors can further compromise N addition effects on C and N mineralization. For example, ecosystems may change towards P limitation as a consequence of improved N availability, limiting the N effect. In some semiarid ecosystems, P addition was required besides N addition to observe responses of soil respiration (Zhou et al. 2020).
The Patagonian steppe is a cold and arid-semiarid region where water (Bucci et al. 2009;Pereyra et al. 2017) and N and P availability are very scarce (Austin et al. 2004;Bertiller et al. 2006;Gherardi et al. 2013). In this ecosystem, the growing season occurs mainly in spring, when temperatures are increasing and soil water availability is decreasing, so that there is an asynchronism in the key environmental addition and their interaction on these soil processes have not been evaluated together. The main objective of this study was to quantify the effects of the experimental increase in water availability and soil N + P and its interaction on soil respiration and soil N-mineralization throughout the seasons. If an increase in soil moisture or nutrients stimulate the growth and metabolic activity of roots and microbial community, then increased soil respiration and soil N-mineralization are expected. In addition we expected that water and nutrient addition in combination generates synergic effects on these soil processes. A eld manipulative experiment was designed in which physico-chemical properties of the soil, root density, soil respiration, ammoni cation and nitri cation were evaluated in control, irrigated, fertilized and irrigated-fertilized plots. Our study can contribute to an improved understanding of the effects of global changes on Patagonian arid ecosystems.

Study site
The study was realized at the Rio Mayo Experimental Station of the Instituto Nacional de Tecnología Agropecuaria (INTA, 45º24′11″S; 70º17′37″W), located at the southwest of Chubut Province, Argentina. The site is characterized by a shrub-gramineous steppe from the southern of the Western District of the Patagonian Province (Oyarzabal et al. 2018). Soils are of coarse texture (sandy) with a cementedcalcareous layer at a depth of about 45 cm and have very low organic matter content (0.40%; Paruelo et al., 1988). Mean annual precipitation in the study site is close to 150 mm, and occurs as small events (88% are lower to 5 mm, Cavallaro et al. (2020)). Mean monthly temperature varies from 1°C in July to 15°C in January.
The Patagonian steppe is characterized by vegetation with low species diversity and low plant density, with a plant cover of between 30  from each other by 15 m, which were randomly assigned to one of the four treatments (control, fertilization (F), irrigation (I) and fertilization-irrigation (I + F)). The plots with the irrigation treatment were equipped with a semi-automatic sprinkler irrigation system. Irrigation water was extracted from a local well of 6 m depth and stored in 5 containers with a total capacity of 13000l. Due to the proximity to the Andean mountains, electric conductivity of groundwater, determined using a conductivity meter (HI 98311, Hanna instruments, Woonsocket, USA) was low (0.16 dS/m), similar to other wells in the research area. The annual precipitation in irrigation plots was increased by approximately 20-25%, and in each irrigation event ~ 5 mm of water were applied, being 6-8 irrigation events per year. The frequency of irrigation events depended on weather conditions (no wind and no natural rain events close to the date of irrigation treatments). Irrigation was carried out during spring and summer, and was canceled during fall and winter, which is the wet period and also to avoid damage to the irrigation system due to freezing of water near the rooting layers. Nutrient addition was done twice a year (once in autumn and once in early spring) applying urea and diammonium phosphate in an amount of 100 kg/ha/year of nitrogen and 75 kg/ha/year of phosphorus, since N and P are the two most limiting nutrients. Another reason for the addition of both nutrients is that nitrogen deposition could further reduce the availability of phosphorus in the soil (Zhang et al. 2013), but the addition of phosphorus could mitigate this limitation, as has been reported for other steppes (Huang et al. 2018).

Soil physico-chemical properties
In spring, 6 cores from the upper soil layer (5 cm diameter, 5 cm depth) were obtained randomly (3 cores close to vegetation and 3 in bare soil) and mixed well to produce one composite sample (n = 3, three of the ve plots of each treatment). All soil samples were analyzed for organic matter, total nitrogen, available phosphorus, pH and electrical conductivity. Soil organic matter, total nitrogen and available phosphorous were analyzed in the Soil Laboratory (INTA Chubut, Argentina). Soil organic matter was determined using the wet oxidation method (Walkley and Black 1934). Soil total nitrogen was determined with the Kjeldahl method (Bremner 1996). Available phosphorus was measured using the Olsen method

Soil root density
At the end of each soil respiration measurement, soil cores of 10 cm diameter and 10 cm depth inside each soil collar were collected and were transported to the laboratory. Root density was determined in the upper 10 cm of the soil because the highest root density is found in the upper soil layer (0-10 cm) at the study site and decreases exponentially with increasing depth (Pereyra et al. 2017). Fresh soil was carefully separated from the ne roots by sieving the soil through a mesh of 0.5 mm. Fine roots were washed and oven-dried at 70°C until constant weight. Root density was determined as root dry mass per unit volume of soil.

Soil nitrogen dynamics
Soil ammoni cation and nitri cation rates was estimated in situ during each incubation period as the difference between initial and nal content of ammonium and nitrate, respectively, in tubes that prevented plant uptake (Raison et al. 1987). PVC tubes of 5 cm diameter and 10 cm height were buried into the soil at 7.5 cm depth, in places close to the vegetation and on bare soil that were later integrated into a single data in three of the ve plots of each treatment. The tubes were placed inside the soil in three seasons (spring, summer and autumn) and they were collected after 1-2 months. At the same time as the placement of the tubes and close to each tube, a soil sample of 5 cm in diameter and 5 cm depth was extracted for the determination of the initial inorganic N content. The collected soil of each tube was transferred into a plastic bag and then transported to the laboratory. Fresh soil samples were sieved with a 0.5 mm mesh, and once frozen they were sent to the Soil Laboratory (INTA Balcarce, Argentina) to analyze the inorganic nitrogen (NH 4 -N and NO 3 -N) by a distillation method (Bremner and Keeney 1965).
The ammoni cation and nitri cation rates were determined from the difference between the initial and nal content of ammonium and nitrate in the soil, respectively, during each incubation period, divided by the number of days. Soil inorganic nitrogen and mineralization rates data were converted to aerial basis using bulk density measurements. The NH 4 -N/NO 3 -N ratio of the soil was calculated for each sample with the initial content values in each of the three seasons.

Statistical analyses
Statistical analyzes were performed using the software R version 4.0.1 (R Development Core Team, 2021). We used linear models (LMs) with F-test to evaluate the effect of treatment on soil physicochemical properties. We used linear mixed effects models (LMEs) with F-test to evaluate the effect of the interaction between season and treatment on soil water content, root density and soil respiration, with month as random effect. A multiple regression model was tested, using LME, to evaluate the effect of the interaction between soil temperature, soil water content and root density on soil respiration, with treatment as a covariate and month as a random effect. For this analysis, continuous variables were centered and scaled, and the quadratic factor of soil temperature and soil water content were included in the model. Linear models with F-test were used to evaluate the effect of the interaction between treatment and season on initial NH 4 -N and NO 3 -N content, NH 4 -N/NO 3 -N ratio and ammoni cation and nitri cation rates. Linear regressions were tested between the initial NH 4 -N and NO 3 -N content with the ammoni cation and nitri cation rates, respectively, using the previously estimated means per season and treatment. The LMEs were carried out using the function "lme" of the R package "nlme" version 3.1-148 (Pinheiro et al. 2020). The Cox and Snell`s R 2 was calculated with the function "nagelkerke" of the R package "rcompanion" version 2.3.25 (Mangia co 2020). When necessary, models were adjusted using variance models, and the selection of the best model was based on Akaike`s information criterion (AIC) (Burnham and Anderson 2002). The simpli cation of the xed effects of all models to reach adequate minimum model was carried out by hypothesis test (F-test). Tukey's post-hoc analysis was used for multiple comparisons in all models when the F-test was signi cant, using the function"glht" of the R package "multcomp" version 1.4-13 (Hothorn et al. 2008).

Results
Lower pH and higher electrical conductivity were observed in fertilized and irrigated-fertilized plots compared to control plots (p < 0.05 in all comparisons) ( Table 1). Organic matter and total nitrogen did not show signi cant differences among treatments (Table 1), but available phosphorus was higher in fertilized (p = 0.01) and irrigated-fertilized plots (p = 0.008) than in control plots (Table 1).
A multiple regression related soil respiration with soil temperature ("temp" in the model), water content ("water" in the model) and root density ("root" in the model). The nal simpli ed model was: respiration ~ temp*water*treatment + temp 2 + root (F ( Fig. 2). Root density had a linear and positive effect on soil respiration, without interaction with treatment, but with a higher intercept in the F and I + F treatments than in control (Fig. 2, right panel).
Soil mean ammonium-N content (NH 4 -N) at 0-5 cm depth varied between 0.3 and 10 g m − 2 , being higher in fertilized (p < 0.001) and irrigated-fertilized plots (p < 0.001) than in control in all seasons (treatment*season: F (6, 24) = 2.71, p = 0.037; Fig. 3a). Also, NH 4 -N was higher in summer and autumn than in spring in the I + F treatment. Ammoni cation rates were signi cantly lower and negative in F treatment (p < 0.001) than in control in all seasons (F (3,32) = 5.71, p = 0.003; Fig. 3b). In irrigated-fertilized plots, ammoni cation rates were lower than in control, but it was not signi cant. In control and irrigated plots, ammoni cation rates tended to be close to zero. Soil mean nitrate-N content (NO 3 -N) at 0-5 cm depth varied between 0.2 and 2 g m − 2 , with signi cant interaction between treatment and season (F (6, 24) = 3, p = 0.025). NO 3 -N was signi cant higher in F and I + F treatments compared to control plots in spring and summer (p < 0.01), and marginally higher in I + F treatment (p = 0.065) in autumn (Fig. 3C). Also, NO 3 -N was higher in spring than in autumn for F and I + F treatments, with intermediate values in summer, without signi cant differences between seasons for control and I treatment. Nitri cation rates were only signi cantly higher in irrigated plots (p < 0.001) compared to control in spring (treatment*season: F (6, 24) = 2.74, p = 0.036; Fig. 3d). Therefore, nitri cation was higher in spring than in summer (p < 0.001) and in autumn (p = 0.02) for I treatment.
NH 4 -N/ NO 3 -N ratio varied between treatments and seasons, with signi cant interaction between both factors (F (6, 24) = 2.63, p = 0.04). NH 4 -N/ NO 3 -N ratio was higher in fertilized and I + F treatments, but only was marginally signi cant in F (p = 0.05) and I + F (p = 0.05) in spring and in I + F (p = 0.09) in autumn (Fig. 4). Moreover, the ratio in the I treatment was marginally higher in spring than in summer (p = 0.05) and in the treatment I + F it was marginally higher in autumn than in spring (p = 0.06). A signi cant linear negative relationship was found between NH 4 -N and ammoni cation rate (R 2 = 0.98, p < 0.0001; Fig. 5a) and between NO 3 -N and nitri cation rate (R 2 = 0.495, p = 0.003; Fig. 5b).

Discussion
Responses of soil respiration to water and N + P addition . However, in our study, the addition of N + P alone or combinated with water had a positive effect on soil respiration at least during the growing season (spring), without a synergistic effect of these two treatments on soil respiration. The discrepance with other studies may be due to the fact that while N deposition may alleviate soil N limitation, this process is accompanied by a reduction in P availability due to an increase in its demand ( Consequently, the present result on non-signi cant changes in long-term soil respiration would suggest a rapid reversal of CO 2 e ux to background level after a water pulse. In this way, soil CO 2 e uxes to the atmosphere could be underestimated in both treatments (i.e., water adittion and water and N + P addition) since CO 2 pulses inmediatly after irrigation events were not considered.
The addition of water by applying small pulses of 5 mm each from the middle of the growing season to the end of the dry season (October-April) accumulated 20-25 mm more than the historical annual average precipitation at the site. Although during the experimental rain pulses the soil water content at 10 cm responded by increasing its content for a short period (Silletta et al. 2019), the added water was not translated into an increase in water availability throughout the year, as it can be seen from our current results. Borken and Matzner (2009) attributed the lack of response of soil respiration to the water addition to the stock of plant available water in soil, arguing that soil respiration would not be affected by the addition of water until the stock of water available to plants will change signi cantly.
The combined addition of N and P can stimulate soil respiration through its autotrophic or heterotrophic component (Wei et al. 2020b). Increase in underground biomass observed with nutrient addition in the Patagonian steppe would suggest that higher autotrophic respiration during spring could contribute to soil CO 2 emission. An increase in underground biomass with N and P addition has been previously reported in other ecosystems (Li et al. 2015;Huang et al. 2018). It should be noted that we only evaluated the effects of the treatments on root biomass in the rst 10 cm depth, but in this ecosystem more than 40% of roots are found in this soil layer and less than 20% at 50 cm depth (Pereyra et al. 2017). Moreover, the ne roots located in the upper part of the soil pro le have higher rates of respiration than larger roots or the few roots located deeper in the soil (Pregitzer et al. 1998). On the other hand, and although the experimental design does not allow us to conclude the reason for the observed increases in CO 2 e ux, it is probably that microbial respiration could also be stimulated with N + P. Shrub and grass leaves in these experimental plots with nutrient addition have a higher N content and a higher total aboveground productivity (Carbonell-Silletta, unpublished work). It may suggest higher litter input and quality which could accelerate decomposition rates and can lead to higher microbial respiration. Furthermore, the higher intercept value in the linear and positive relationship between soil respiration and root biomass in F and I + F plots compared to C and I plots would indicate a higher root metabolic activity per mass unit.
We also speculate that under nutrient addition, microbial activity could be stimulated by root exudation of enzimes that accelerate the degradation of organic matter and thus release carbon labile (Ataka et al. 2020). In addition, Song et al. (2011) found a positive relationship between N-immobilization and soil CO 2 e ux with N addition, which reinforces the idea of higher heterotrophic respiration with nutrient addition in our study, where we found a strong N-immobilization.
The lack of response of soil respiration to the addition of N + P from summer to winter can be a consequence of the reduction of the biological activity due to high or low temperatures, respectively, which also coincide with the periods with the lowest and highest soil water content respectively. In the present study it was possible to determine the combined effect of both factors (i.e., soil temperature and water content) on soil respiration. Unlike other studies in which a linear or exponential relationship was found between soil temperature and soil respiration (Hunt et  , our results support the idea that very low values of soil temperature and moisture restrict soil respiration and this behavior was independent of treatment. However, the pattern was different in the F and I + F treatments with respect to the control with medium to high soil temperature and water content values. In control and the I treatment, the highest values of soil respiration was achieved with intermediate values of soil temperature and moisture, which ocurrs in spring and concides with the highest biological activity of plants. However, the highest estimated values of soil respiration in the treatments with N + P addition were obtained with the highest values of soil temperature combined with the highest values of soil water content. In this steppe there is asynchrony of temperature and soil moisture favorable for the growth of plants, so that the combination of high temperatures with high soil moisture almost never occurs (Paruelo et al. 1998). But, according to our results, it is expected that with the soil nutrient amendament the soil respiration will increase under such conditions, probably due to a greater root density and metabolic activity of the roots and soil microorganims.

Soil nitrogen dynamics
Compared to other ecosystems, soils of the Patagonian steppe have higher percentage of their mineral nitrogen in form of nitrate (Austin and Sala 2002). The soil nitrogen addition increased N content in the form of ammonium and nitrate. Studies carried out in the same fertilized plots indicates that the two dominant functional groups in the Patagonian steppe (grasses and shrubs) uptakes more N from soil and produce leaves with higher N content than in control plots (Carbonell-Silletta, unpublished work). In this ecosystem, shrubs preferably absorb nitrate rather than ammonium,while ammonium uptake is similar for grasses and shrubs (Sala et al. 2012;Gherardi et al. 2013). The increase in inorganic N content was also accomplished by an substantial increase in the the NH4-N to NO3-N ratio with nutrient soil addition, suggesting higher consumption of nitrate by plants. Another plausible explanation for this change in the ammonium-nitrate ratio is that nitrate is relatively more mobile in the soil solution, being able to leach with water percolation (Yahdjian et al. 2006;Yahdjian and Sala 2010). Soil ammonium and nitrate usually vary seasonally in the Patagonian steppe as a consequence of the differential use by plants and soil microorganisms (Austin and Sala 2002;Yahdjian et al. 2006). However, we only found differences in the N availability between seasons in the treatments with addition of N + P. While the ammonium content was higher in summer and autumn in the I + F treatment, the nitrate content was higher in spring and summer in the F and I + F treatments. The seasonal nitrate behavior in fertilized plots could be explained by high plant demand during the growing season. Some studies in other ecosystems have found increases in soil NH 4 and NO 3 immediately after fertilization, but decrease at the beginning of the rainy season (Kozovits et al. 2007), or soil nitrate decrease after irrigation and fertilization by leaching (Choi et al. 2005).
Nitrogen mineralization in grasslands on a global scale is largely explained by microbial biomass, in addition to the temperature of the wettest quarter, clay content and bulk density (Risch et al. 2019). Contrary to our hypothesis, the negative relationship between inorganic N content and N-mineralization rates found in our study indicates that high N addition could inhibit N transformation, possibly by inhibiting microbial activity (Butterbach-Bahl and Gundersen 2011). Similar stimulation of NH4 + and NO3 − immobilization with increased substrate was observed by Booth et al. (2005) and Song et al. (2021) in a global synthesis. However, Austin and Vivanco (2006) found for the Patagonian steppe that alone N adition has no effect on potential N mineralization, but an increase is observed with the addition of labile C and N. Other studies have also indicated that the addition of nitrogen alone or with water has little or no effects on microbial community in arid ecosystems and it increases when combined with the addition of labile carbon (Austin and Vivanco 2006). The N addition in this study induced signi cant negative amoni cation and nitri cation rates (i.e.; nitrogen immobilization). In general terms, the balance between N mineralization and inmobilization depends on the relative C and N availability of the substrate and the metabolic need of the microbial biomass. Despite exogenous soil nutrient addition, this result implies that more N is needed for supply the microbial demand (Schimel 1986;Micks et al. 2004;Song et al. 2011). If fertilization supplied with more labile C throught leaf and root turnover for the microbial growth and activity, then consumption of labile C could result in increase of N microbial demand and inmobilization of available N. Net ammonium and nitrate release only occurs after N demand by microbial biomass, which has lower C:N ratios, was satis ed (Berg and  Ecosystems vary in their sensitivity to acidi cation and this is dependent on the initial pH, the decrease being greater the more basic the initial pH is (Tian and Niu 2015). The soils of the Patagonian steppe have a high calcium carbonate content (Paruelo et al. 1988; del Valle 1998) indicating that may be very sensitive to acidi cation by the N addition. The acidi cation observed in our study could explain, at least in part, the strong inhibition of soil mineralization due to its effect on microbial activity by affecting enzyme functioning (Li et al. 2018;Nannipieri et al. 2018).
The water addition did not signi cantly modify the content of mineral N and the N-mineralization rates, consistent with the results of studies carried out in another arid ecosystem (Reichmann et al., 2013, Hook andBurke 2000). But this nding contrasts with the results of Yahdjian and Sala (2010) for the Patagonian steppe. However, it should be noted that in this last study the mineralization rate was determined only 5 days after the addition of water, so the increase could represent only pulses of N availability, as previously reported (Epstein et al., 2006). Generally, C and N mineralization after rewetting are coupled (Fierer and Schimel 2002;Muhr et al. 2010), such that the response of N mineralization, similar to soil respiration, was not captured in our study. After microbial growth due to rewetting, an increase in metabolic microbial activity (Austin et al. 2004) and microbial death are observed simultaneously with the depletion of labile soil organic matter, leading to large pulses of N mineralization.
Our results indicate that the addition of relatively small water pulses does not lead to long-term responses.

Conclusions
The main control factors on the soil respiration in the Patagonian steppe were soil temperature, water content and root density. However, the experimental water supply did not produce a signi cant effect on the release of CO 2 and N mineralization over the long term. This emphasizes the need to evaluate these soil processes with higher frecuency after soil wetting to avoid underestimations of soil C loss in future studies. In this study the increase in mean precipitation was carried out with small precipitation events (~ 5mm), thus we cannot rule out that increasing the water pulse size may trigger long-term responses.
On the other hand, the soil respiration increased with the adittion of N and P during the growing season.
More roots in F and I + F treatments stimulated the loss of soil carbon but there were not sinergic effects of nutrient and water. Therefore, we suggest that soil N enrichment in arid ecosystems may strengthen the positive feedback between climatic change and C cycle. However, other studies are required to determine if only N addition without P result in higher CO 2 e uxes from the soil.
Nutrient adittion also resulted in changes in soil inorganic N, affecting soil N mineralization rates.
Although the soil inorganic N in the form of ammonium and nitrate increased with fertilization, these two forms of N compounds were immobilized by microorganisms. The low mineralization rates found even in the control plots would suggest that soil mineralization processes in the Patagonian steppe is restricted to short periods during the dry season in which rainfall pulses occur. However, how these changes modify the net C and N balance of the ecosystem were not explored, and further studies are necessary to assess the relative responses of various C and N cycles components to nutrients and water addition. While most studies in arid ecosystems have paid more attention to water availability, our results suggest that we must consider the effects of soil nutrients to better predit changes in C uxes and C net balance in this ecosystem.  Tables   Table 1 pH, electrical conductivity (dS m − 1 ), organic matter (%), total nitrogen (%) and available phosphorus (ppm). Each value represents the mean ± CI (n = 3). The statistical value F, the degrees of freedom (df) and the probability value p are shown. Statistically signi cant differences of a treatment with respect to control are labeled with asterisks (* p < 0.05)   (a) Soil ammoni cation rate (g N m-2 mo-1) related to N content in the form of ammonium (g NH4-N m-2), and (b) soil nitri cation rate (g N m-2 mo-1) related to N content in the form of nitrate (g NO3-N m-2). Each value represents the mean ±IC (N= 3) of each treatment: control (C), fertilization (F), irrigation (I) and irrigation-fertilization (I+F) and season (spring, summer and autumn). The lines are the linear regressions tted to the date and the gray bands are 95% con dence intervals