Article Short-Term N-Fertilization Differently Affects the Leaf and Leaf Litter Chemistry of the Dominant Species in a Mediterranean Forest under Drought Conditions Helena Vallicrosa 1,2, *, Jordi Sardans 1,2 , Romà Ogaya 1,2 , Pere Roc Fernández 1,2 and Josep Peñuelas 1,2 1 CSIC, Global Ecology Unit CREAF-CSIC-UAB, 08913 Bellaterra, Catalonia, Spain; j.sardans@creaf.uab.cat (J.S.); r.ogaya@creaf.uab.cat (R.O.); p.roc@creaf.uab.cat (P.R.F.); josep.penuelas@uab.cat (J.P.) 2 CREAF, 08913 Cerdanyola del Vallès, Catalonia, Spain * Correspondence: h.vallicrosa@creaf.uab.cat Abstract: Nitrogen (N) deposition is a key driver of global change with significant effects on carbon (C) cycling, species fitness, and diversity; however, its effects on Mediterranean ecosystems are unclear. Here, we simulated N deposition in an N-fertilization experiment with 15 N-labeled fertilizer in a montane evergreen Mediterranean holm oak forest, in central Catalonia, to quantify short- term impacts on leaf, leaf litter elemental composition, and resorption efficiency in three dominant species (Quercus ilex, Phillyrea latifolia, and Arbutus unedo). We found that even under drought conditions, 15 N isotope analysis of leaf and leaf litter showed a rapid uptake of the added N,  suggesting an N deficient ecosystem. Species responses to N fertilization varied, where A. unedo was  unaffected and the responses in P. latifolia and Q. ilex were similar, albeit with contrasting magnitude. Citation: Vallicrosa, H.; Sardans, J.; P. latifolia benefited the most from N fertilization under drought conditions of the experimental year. Ogaya, R.; Fernández, P.R.; These differences in species response could indicate impacts on species fitness, competition, and Peñuelas, J. Short-Term abundance under increased N loads in Mediterranean forest ecosystems. Further research is needed N-Fertilization Differently Affects the to disentangle interactions between long-term N deposition and the drought predicted under future Leaf and Leaf Litter Chemistry of the Dominant Species in a Mediterranean climate scenarios in Mediterranean ecosystems. Forest under Drought Conditions. Forests 2021, 12, 605. https://doi.org/ Keywords: elemental composition; isotopes; reabsorption; climate change; Arbutus unedo; Phillyrea 10.3390/f12050605 latifolia; Quercus ilex Academic Editor: Heinz Rennenberg Received: 6 April 2021 1. Introduction Accepted: 7 May 2021 Nitrogen (N), which is an essential element for plant nutrition, is involved in com- Published: 12 May 2021 plex cycling in terrestrial ecosystems that involves biotic and abiotic processes, such as N2 -fixation, mineralization, nitrification, ammonification, and denitrification [1–3]. An- Publisher’s Note: MDPI stays neutral thropogenic activities since the industrial and agricultural revolutions, particularly those with regard to jurisdictional claims in associated with fossil fuel combustion, cultivation of N2 -fixing crops, and production of N published maps and institutional affil- fertilizer using the Haber-Bosch process, increasingly influence the global N-cycle as they iations. are often key sources of N in terrestrial ecosystems, driving fluxes of similar magnitudes to natural fluxes [4–6]. Inorganic N deposition in European forests increased on average from about 2.8 kg ha−1 a−1 in 1900 to a peak of 10.3 Kg N ha−1 a−1 in 1990, followed by a decline to 6.6 Kg N ha−1 a−1 by 2018 [7,8]. However, 62% of Europe continues to remain at Copyright: © 2021 by the authors. risk of eutrophication [9] through rises in organic N deposition due to increasing levels of Licensee MDPI, Basel, Switzerland. N-fertilization of cropland [8]. This article is an open access article In general, N addition to ecosystems alters the first N cycle, increasing N miner- distributed under the terms and alization, nitrification, and nitrate leaching rates, and thereafter other ecosystem vari- conditions of the Creative Commons ables such as P-cycle [5], and finally is associated with a long-term plant community Attribution (CC BY) license (https:// diversity decrease [10]. Also, in some Mediterranean regions in California with elevated creativecommons.org/licenses/by/ 4.0/). N deposition, native plant species have been replaced by invasive species with higher Forests 2021, 12, 605. https://doi.org/10.3390/f12050605 https://www.mdpi.com/journal/forests Forests 2021, 12, 605 2 of 16 N performance [11–13]. Vegetation responses to increases in N supply are complex and include increased inter-plant competition, modified plant-soil feedbacks, and changes to plant-herbivore and plant-fungus relationships [14–16]. Typically, increases in N availabil- ity under N deposition are associated with greater plant growth [17,18], C-fixation [19], increased plant N concentrations [20,21], and reduced plant P concentrations [22,23] that contribute to increased plant N:P ratios [24–27]. While critical N loads have been described for a range of ecosystems and species, including 3–33 kg N ha−1 yr−1 for herbaceous species and shrublands and 4–39 kg N ha−1 yr−1 in forests in the US [10], there is a knowledge gap for Mediterranean Basin ecosystems. Along with water availability, nutrient supply is a frequent limiting factor for Mediter- ranean ecosystems [28,29] and is an important factor in the growth, structure, and distri- bution of plant communities [30–32]. Consequently, Mediterranean forest communities tend to be dominated by woody plant species characterized by conservative traits related to tolerance of low water and nutrient conditions, such as slow growth and high levels of sclerophylly, high levels of investment in root biomass, and greater metabolic flexibil- ity [28]. Responses of plant species to long-term N-fertilization under high levels of N deposition vary with life history strategy, where ruderal species are likely to be favored over stress-tolerators [33]. The resulting changes in competitive relationships within plant communities lead to long-term changes in species community composition [10,12,34], which could culminate in the extinction of endemic species and reduce overall levels of species diversity [16]. High levels of N deposition are known to constitute a threat for global plant diversity; as long as Mediterranean ecosystem is considered a diversity hotspot, an increase of N deposition could endanger its species diversity [35–37]. Deposition of N has been related to reduced levels of resilience in Mediterranean plant communities to disturbing climatic conditions [28]. However, few field studies have assessed the relationships between in- creases in N-availability and responses of dominant woody species and shifts in nutrient cycling in Mediterranean plant communities. In particular, even less field studies assessed the impacts of a sudden rise in N deposition on a semi-pristine Mediterranean ecosystem, in which current loads of N deposition are low and especially interacting with drought. Therefore, we aimed to study the short-term effects of N-fertilization to simulate rapid N deposition and availability on: (i) foliar and litter nutrient composition; (ii) foliar nutrient reabsorption; and (iii) foliar and litter carbon (C) and N isotope ratios in three dominant tree species of a semi-pristine, low-nutrient Mediterranean holm-oak forest to increase the understanding of species community composition and dynamics under increased N deposition in the increasingly dry Mediterranean environment. 2. Materials and Methods 2.1. Study Site The study was carried out on a south-facing slope (25% incline) of a semi-pristine (Quercus ilex L.) forest, where N deposition was 3.5 kg N ha−1 yr−1 between 1981 and 1994 [38], in the Prades Mountains, Catalonia, NE Spain (41◦ 210 N, 1◦ 020 E). The forest has not been disturbed for the last 70 years and the maximum height of the dominant species is about 6–10 m. Mean annual temperature and precipitation are 12 ◦ C and 658 mm, respectively, with the warmest months occurring between June and August and the rainiest period between September and November (Figure 1). These conditions correspond to a mesic-Mediterranean climate, with a pronounced summer drought period that usually lasts for 3 months. During the study period, mean annual temperature and annual precipitation in 2014 were 12.65 ◦ C and 661.38 mm respectively, and 13.25 ◦ C and 355.44 mm respectively in 2015 (Figure 1). Air temperature and rainfall were recorded by an automatic meteoro- logical station in the studied area. The soil is a stony Dystric Cambisol, on a bedrock of metamorphic sandstone, and ranges between 35 and 100 cm in depth. The dense forest vegetation is dominated by Q. Ilex, with abundant Phillyrea latifolia L., Arbutus unedo L. among other evergreen species that are well-adapted to drought conditions (Erica arborea Forests 2021, 12, x FOR PEER REVIEW 3 of 17 Forests 2021, 12, 605 3 of 16 forest vegetation is dominated by Q. Ilex, with abundant Phillyrea latifolia L., Arbutus unedo L. among other evergreen species that are well-adapted to drought conditions (Erica ar- borea L., L., Juniperus Juniperus oxycedrusoxycedrus L., andL., and Cistus Cistus albidusalbidus L.) andL.)theand the occasional occasional occurrence occurrence of de- of deciduous ciduous(Sorbus species speciestorminalis (Sorbus torminalis L. CrantzL.and Acerand Crantz monspessulanum Acer monspessulanum L.). The L.). The three three tree tree species (Q. ilex, (Q. species P. latifolia, and A. unedo) ilex, P. latifolia, and A.represent 97% of 97% unedo) represent the total aboveground of the biomass total aboveground of the biomass forest of the[39] and[39] forest theyandfrequently co-occur co-occur they frequently in Mediterranean maquis shrubland in Mediterranean and evergreen maquis shrubland and Q. ilex forests. Q. ilex is widely distributed across the subhumid areas evergreen Q. ilex forests. Q. ilex is widely distributed across the subhumid areas of theof the Mediterranean Basin, while P. latifolia Mediterranean Basin,occurs while in P. warmer latifolia and drier occurs in Mediterranean warmer and drier [40,41] and A. unedo areasMediterranean areas exploits [40,41] and forest gaps in A. unedo which forest exploits conditions gaps are limiting in which for Q. ilexare conditions [42]. limiting for Q. ilex [42]. Figure 1. Bagnouls-Gaussen ombrotermic Figure 1. ombrotermic diagram diagramofofmean meanclimatic climatictemperature temperature(in (inred) red)and andprecipitation precipitation(in(in blue) blue) byby month in month inthe thestudy studysite. site.(a) (a)Mean Meanmonthly monthlydata datafrom from1975 1975toto2020. 2020.(b) (b)Mean Meanmonthly monthlydatadatafor for2014. 2014.(c) (c)Mean Meanmonthly monthlydata data for for 2015. 2015. 2.2. Experimental 2.2. Experimental Design Design We established We established eight eight 1515 m m× × 10 10 m plots at m plots at the the same same altitude altitude (950 (950 m) m) [43]; [43]; four four plots plots receivedN-fertilizer received N-fertilizerand andthetheremainder remainderwerewere untreated untreated controls. controls. In 2015, In 2015, a total a total fertiliza- fertilization tion of 60ofkg60ofkg N of haN −1 ha a−1 a(at (at −1 −1 a rate a rate of kg of 15 15 kg N haN−ha −1 in each annual season) was applied as 1 in each annual season) was applied as a a solution solution of of ammonium ammonium nitrate nitrate (NH (NH 4NO3) with a sprayer. This fertilization was sprayed 4 NO3 ) with a sprayer. This fertilization was sprayed in 3 different days per season (5 kg N haha in 3 different days per season (5 kg N −1−1each eachapplication), application),withwithatatleast least11week weekbetween between different applications. This solution was sprayed over the canopy level after different applications. This solution was sprayed over the canopy level after climbing to the climbing to the of top topthe of uppermost the uppermost tree tree in in each each plot. plot. This This solution solution was enriched was enriched 15 with with N as N 15 as follows: follows: 90% 90% of of “normal” “normal” ammonium ammonium nitrate, nitrate, and 10%and of 10% of ammonium ammonium nitrate nitrate enriched with 15 enriched with 15N (the N (the 10% 10% of ofenriched this this enriched ammonium ammonium nitrate nitrate 15 N)15(Sigma-Aldrich, waswas N) (Sigma-Aldrich, Co.,Co., St. St. Louis, Louis, MO, MO, USA). USA). 2.3. Sampling We We sampled sampled leaves leaves of of Q. Q. ilex, ilex, P. P. latifolia, and A. latifolia, and A. unedo unedo and and foliar foliar litter litter in in mid-May mid-May (spring), (spring), endend of ofJuly July(summer), (summer),and and mid-November mid-November (autumn) (autumn) 2014, 2014, prior prior to treatment, to treatment, and and in 2015 in 2015 during during fertilization, fertilization, sampling sampling thethe sametree same treebefore beforeand andafter after treatment. treatment. On On each each sampling sampling occasion, occasion, we we randomly randomly selected selected five five trees trees of of each each species species per per plot plot and and collected collected mature mature and healthy leaves at a height of 2–3 m from plants with a diameter ofof and healthy leaves at a height of 2–3 m from plants with a diameter 2–12 2–12 cmcm at at 5 cm above ground level; these plants represent about the 70% 5 cm above ground level; these plants represent about the 70% of the community biomass of the community biomass [44,45]. To [44,45]. reduceToeffects reduce of effects of tissue tissue age age and orientation and orientation to sunlight,towesunlight, sampled weleaves sampled that leaves that were sun-lit and oriented southwards. Mean leaf were sun-lit and oriented southwards. Mean leaf life span is approximately 18 months life span is approxi- mately [45], so 18 mostmonths leaves[45], so most sampled leaves during 2015sampled during 2015were under fertilization under fertilization also present duringwere 2014 also present during 2014 prior to treatment. Litterfall at the soil surface prior to treatment. Litterfall at the soil surface was collected in 20 circular baskets (27 cm was collected in 20 circular baskets (27 cm in diameter, with 1.5-mm diameter mesh) in diameter, with 1.5-mm diameter mesh) that were randomly distributed in each plot. that were randomly distributed Even though inthe each plot. were baskets Even permanently though the basketsplaced were permanently over the ground, the placed denseover the canopy ground, the dense canopy cover almost totally avoided N fertilization reaching cover almost totally avoided N fertilization reaching the soil or the baskets. The collected the soil or the baskets. The collected litter was not in direct contact with the soil so its conservation litter was not in direct contact with the soil so its conservation state was good and the state was good and the decomposition process had hardly begun. Once collected, the leaf and litter samples were taken to the laboratory and stored at 4 ◦ C prior to analysis. Forests 2021, 12, 605 4 of 16 Foliar nutrient resorption was calculated as the difference in elemental compound concentration of leaves collected in 2014 and litter collected in 2015, using the equation 100 − (Xl × 100)/Xf , where Xl is litter element concentration in 2015 and Xf foliar element concentration in 2014. The result is an inference of proportional (%) element resorption efficiency prior to leaf fall. 2.4. Chemical Analyses The leaf and foliar litter samples were washed with distilled water, as described by Porter (1986) [46], dried in an oven at 60 ◦ C to a constant weight, and then ground using a mill (CYCLOTEC 1093, Foss Tecator, Höganäs, Sweden) for measurement of biomass. Leaf and litter concentrations of carbon (C) and N were quantified by combustion coupled to gas chromatography, by placing 1.4 mg of the milled samples in a tin microcapsule in an elemental analyzer (CHNS-O EuroVector, Milan, Italy), while concentrations of phosphorus (P), potassium (K), and micro-elements were determined by digesting samples of leaf and litter in acid in a microwave (MARSXpress, CEM, Matthews, NC, USA) at high pressure and temperature. Then 250 mg of each ground sample, 5 mL of nitric acid, and 2 mL of H2 O2 were placed into Teflon tubes; digestate was placed in 50-mL flasks and diluted with Milli-Q water to a volume of 50 mL. Concentrations of P, K, and micro-elements were determined using optic emission spectrometry with inductively coupled plasma and the accuracy of the digestions and analytical biomass procedures was assessed using a certified standard biomass (NIST 1573a, tomato leaf; NIST, Gaitherburg, MD, USA) as a reference. Isotopic analyses of δ13 C (13 C/12 C) and δ15 N (15 N/14 N) of the leaf and litter samples were conducted at the Stable Isotope Facility at the University of California, Davis using a PDZ Europa ANCA-GSL elemental analyzer connected to a PDZ Europa 20−20 isotope ratio mass spectrometer (Sercon Ltd., Cheshire, UK). 2.5. Statistical Analyses Between year treatment differences in plot, species, and season values were calculated based on proportional change in values between 2014 and 2015, and then linear mixed models were used to test for treatment differences in leaf and litter macro- (C, N, P, K) and micro-element concentrations (calcium, Ca; magnesium, Mg; chrome, Cr; iron, Fe; man- ganese, Mn; copper, Cu; zinc, Zn, strontium, Sr; nickel, Ni; and, lead, Pb) and reabsorption, and in foliar N and C isotopes by species, with plot and season as random factors. Models were defined using lme4 [47] and lmerTest [48] and r2 was calculated in MuMIn [49] R packages. Treatment differences in leaf and litter isotope content by species was tested using Student’s t-test. Overall treatment differences in leaf and litter macro- (N, P, K) and micro-element (Ca, Mg, Fe, Mn, Cu, Zn) composition and isotope content (δ13 C and δ15 N) by species were tested using general discriminant analysis (GDA) in Statistica 8.0 (StatSoft, Inc., Tulsa, OK, USA) that identifies variables most responsible for group differences, while controlling variance due to other categorical variables (here, season). In order to detect the effect of 2015’s exceptional arid conditions, we calculated a t-test between 2015 and 2014 for leaves and litter from only control plots (which have no treatment applied in 2015). 3. Results 3.1. Fertilization Effects on Foliar Element Concentrations There were between-year differences in C:P ratios in P. latifolia, where there were increases of 4.83 ± 3.36% and 3.05 ± 0.86% in untreated control and fertilized plots, respectively (difference between treatments: p < 0.05; ±standard error (SE) showed; Figure 2a, Table S1), and ratios of N:P differed for A. unedo, where there were 1.64 ± 1.65% decreases and 1.12 ± 1.38% increases in the untreated and treated plots from 2014 (pretreatment) to 2015 (post-treatment), respectively, (difference between treatments: p < 0.05; ±standard Forests 2021, 12, x FOR PEER REVIEW 5 of 17 Forests 2021, 12, 605 5 of 16 and 1.12 ± 1.38% increases in the untreated and treated plots from 2014 (pretreatment) to 2015 (post-treatment), respectively, (difference between treatments: p < 0.05; ±standard error (SE) error (SE) showed; showed;FigureFigure1b,1b,Table TableS1). There S1). were There no treatment were no treatment differences in leaf differences in con- leaf centration of N, P, or K or in C:N ratios of the three species. concentration of N, P, or K or in C:N ratios of the three species. Among the Among themicro-elements, micro-elements,there there were were differences differences in leaf in leaf concentrations concentrations of chro- of chromium mium (Cr) (Cr) in Q. in where ilex, Q. ilex,there wherewerethere were decreases decreases of 1.68and of 1.68 ± 1.83% ± 1.83% 3.56 ±and 3.56in±the 0.68% 0.68% in the untreated untreated and treated and treated plots, plots, respectively respectively (difference(difference between treatments: between treatments: p < 0.05; ±pstandard < 0.05; ±stand- error ard error (SE) showed; Figure 1c), while leaf concentration of nickel (Ni) in (SE) showed; Figure 1c), while leaf concentration of nickel (Ni) in A. unedo decreased by 0.76 A. unedo de- creased by 0.76 ± 0.41% and 1.49 ± 0.28%, respectively (difference between ± 0.41% and 1.49 ± 0.28%, respectively (difference between treatments: p < 0.05; ±standard treatments: p< error (SE) showed), and decreased in Q. ilex by 1.86 ± 1.79% and 4.67 ± 0.62% (difference± 0.05; ±standard error (SE) showed), and decreased in Q. ilex by 1.86 ± 1.79% and 4.67 0.62% (difference between treatments: between treatments: p < 0.05; ±standard p