Relevance of organic vs. inorganic nitrogen in intra- and interspecific competition of seven central European tree species

When competing for nitrogen with other tree species, organic N is more important than inorganic N for most species Competition for nitrogen (N) in the rhizosphere is a key process regulating a seedling´s chances for growth and survival. However, only few studies have investigated this at the individual species´ level when using a community approach. In this study, 15N incubation experiments were conducted to quantify inorganic (i.e. ammonium and nitrate) and organic (i.e. glutamine-N and arginine-N) net N-uptake capacity of seven temperate woody species co-occurring on calcareous substrate and to investigate the consequences of intra- vs. interspecific competition in a woody seedling community. The results showed that short-term net inorganic and organic N uptake capacity was unrelated to a seedling´s fast or slow growth. Furthermore, competition with other tree species did not change a seedlings´ overall capacity to take up inorganic and organic N sources. Organic N was preferred over inorganic N for most species and the preferences for specific N source shifted with competition regime and tree species. Overall, this study indicates the importance of organic N sources for N uptake in woody seedlings.


Introduction
Forest ecosystems play a crucial role for the future of human society, not only for mitigating climate change via the capture and long-term storage of carbon (C), but also via the provision of fundamental ecosystem services including resources (timber, food) and recreation (MEA 2005;FAO 2015;Sabatini et al. 2019;Simon and Adamczyk 2019). Thus, a sustainable management of forests is vital when facing today´s ecological challenges. Currently discussed strategies for a sustainable management of forest ecosystems in the future include the transition of forest monocultures to heterogenous mixed-species stands (Pretzsch 2020) because of their enhanced resilience to abiotic stressors (e.g. Fares et al. 2015;Mina et al. 2018). However, studies using multi-species approaches focus on the overall effect in plant communities, whereas the role of individual species in the game of competition and facilitation might be masked (e.g. Rewald and Leuschner 2009;Tobner et al. 2016;Fichtner et al. 2018;Trogisch et al. 2021). Furthermore, key processes in the rhizosphere are still not fully understood (Trinder et al. 2013;Weemstra et al. 2016;Pommerening and Sánchez Meador 2018), especially related to the acquisition of tree-growth limiting nitrogen (N) (Körner 2003;Millard et al. 2007;Millard and Grelet 2010).
Tree N acquisition from the soil is a key aspect when it comes to competition for N in the rhizosphere and is influenced by a variety of factors including the availability of different N forms (e.g. Näsholm et al. 2009;Stoelken et al. 2010;Hodge and Fitter 2013;Simon et al. 2017;Bueno et al. 2019) and/or a species´ functional traits-for example fast vs. slow growth (Trinder et al. 2013;Li et al. 2015;Simon et al. 2017;Freschet et al. 2021). Growth strategy and N acquisition might be linked. For example, when competing with a fast-growing pioneer species slow growing Fagus sylvatica seedlings showed a reduced uptake Communicated by Lee Kalcsits. of inorganic N, whereas inorganic N uptake increased in the pioneer species (Fotelli et al. 2002(Fotelli et al. , 2005. In addition, preferring certain N forms is a mechanism with the potential to avoid competition (Simon et al. 2014(Simon et al. , 2017. However, most competition studies only included inorganic N sources. Furthermore, only few studies have investigated the competition for N between trees at the species level (Simon et al. , 2014Li et al. 2015;Bueno et al. 2020;Reuter et al. 2021). Hence, the relevance of inorganic vs. organic N forms for the interactions between woody species still needs to be further elucidated (Tegeder and Perchlik 2018;Moreau et al. 2019). N acquisition is particularly crucial for woody seedlings when competing for limiting soil N (e.g. Körner 2003;Millard et al. 2007) due to their limited storage capacities for N (Millard and Grelet 2010).
Thus, the overall aim of this study was to investigate the consequences of competition in tree seedling communities including seven temperate woody species-co-occurring on calcareous soil-on inorganic and organic N acquisition. The specific hypotheses were: (1) Inorganic and organic N acquisition by the roots differs among tree species based on their physiological and morphological properties, such as growth rate and/or nutrient demand (e.g. Andersen et al. 2017;Simon et al. 2017;Liese et al. 2018;Bueno et al. 2019). For example, fast-growing species have a higher N demand and thus take up more N from the soil compared to slow-growing species, especially at the seedling stage (Millard and Grelet 2010). (2) Inorganic and organic N acquisition changes from intra-and interspecific competition depending on the competing species Simon et al. 2010;Li et al. 2015;Bueno et al. 2019). For example, inorganic and organic N acquisition in European beech was significantly reduced when competing for N with sycamore maple . (3) Within a tree species´, its preference for certain N sources changes when competing for N with other tree species (e.g. Ashton et al. 2008;Simon et al. 2010;Bueno et al. 2019) which could be a means to avoid competition with other species. For example, when grown in competition, seedlings of European beech prefer organic N, whereas sycamore maple favours inorganic N (Simon et al. , 2017.

Plant material
One-year-old mycorrhizal seedlings of seven temperate tree species (provenance southwestern Germany) were purchased from a commercial tree nursery (Müller Münchehof Pflanzen GmbH (Seesen/Münchehof, Germany). Species included  (Ellenberg and Leuschner 2014;Simon et al. 2021). Although beech can grow on different soils, it commonly occurs on soil derived from limestone which is highly susceptible to water deprivation; the other tree species were chosen because they co-occur in beech forest ecosystems on calcareous substrate (Ellenberg and Leuschner 2014). Furthermore, the species used here display different growth strategies and nutrient requirements, as well as tolerance to shade and drought (see Table 1), thus providing ideal target species to study the consequences of plant interactions on inorganic and organic N acquisition in woody seedlings. From here on, species used in this study will be referred to by their corresponding genus names, i.e. Fagus, Acer, Carpinus, Fraxinus, Prunus, Quercus, and Tilia.

Experimental design
In a multi-species community approach, individuals were grown in sand/vermiculite (1:1 mixture) in mesocosms (30

N uptake experiments and harvest
For quantification of inorganic (i.e. ammonium, nitrate) and organic (i.e. glutamine-N, arginine-N) net N uptake capacity of the target seedlings in the center of the mesocosms, the 15 N enrichment technique described by Gessler et al. (1998) and modified by Simon et al. (2010) was used. Seedlings were carefully removed from the mesocosms. The root system was thoroughly washed with tap water to remove any adhering substrate particles. Fine roots still attached to the seedlings were then incubated for 2 h (between 10 am and 2 pm to avoid diurnal variation in N uptake (Gessler et al. 2002) in 4 ml of the artificial low N soil solution (Dannenmann et al. 2009; see above). The artificial soil solution contained all four N sources, with only one labelled as either 15 NH 4 + , 15 NO 3 − , 13 C/ 15 N-glutamine, or 13 C/ 15 N-arginine. Natural abundance was accounted for by non-labelled controls. After incubating for 2 h, the submersed root tips and moistened upper parts (~ 8-10 cm) were cut off, washed twice with 0.5 µM CaCl 2 to remove excess 15 N on the root surface, dried with cellulose paper and oven-dried for 48 h at 60 °C. Fresh and dry weight was determined. The roots not incubating during the 15 N uptake experiments were wrapped in wet tissue to prevent desiccation. Following the 15 N uptake experiments, seedlings were separated into leaves, stems, and roots. Leaf area was measured for each seedling (LI-3100 C Area Meter, LI-COR, Lincoln, USA) to calculate specific leaf area (SLA). Fresh and dry weight (after 48 h at 60 °C) were determined for all plant tissues. Root:shoot ratio was calculated as the ratio between total belowground biomass (i.e. root biomass) and total aboveground biomass (i.e. leaves and stem biomass).

Quantification of 15 N, 13 C, and total N and C in the fine roots
For the quantification of 15 N, 13 C, and total N and C, the fine roots were dried (48 h, 60 °C) and ground into a fine homogenous powder using a ball mill. Aliquots of 1.2-2 mg were transferred into tin capsules (IVA Analysentechnik, Meerbusch, Germany). Samples were sent to Agroisolab GmbH (Jülich, Germany) where they were analysed using an elemental analyser (EA; Carlo Erba Instruments NA 1500 series 2, CE Instruments, Milan, Italy) coupled to an isotope ratio mass spectrometer (IRMS; Nu Horizon, Nu Instruments Ltd., Wrexham, UK). Working standards (L-leucine) calibrated against the primary standards IAEA-CH-6 (sucrose, delta 13 C PDB = −1 0.449), IAEA-CH-7 (polyethylene, delta 13 C PDB = − 32.151), IAEA-N-1 (ammonium sulfate, delta 15 N air = + 0.4), and IAEA-N-2 (ammonium sulfate, delta 15 N air = + 20.3) were analysed after every 12th sample to detect a potential instrument drift over time. Inorganic and organic net N uptake capacity (nmol / g fw / h) was calculated based on the incorporation of 15 N into the fine roots and the respective plant biomass according to Gessler et al. ( 1998):Net N uptake capacity = ( 15 N l − 15 N c × N tot × dw × 10 5 ) ∕ ∕ (MW × fw × t) −1 where 15 N l and 15 N c are the atom % of 15 N in labeled (N l ) and unlabeled control plants (N c , natural abundance), respectively, N tot is the total N percentage, MW is the molecular weight ( 15 N g mol −1 ), dw is the dry weight, fw is the fresh weight, and t is the incubation time. Amino acids were double-labelled with 13 C and 15 N to determine whether they were taken up as intact molecules (Simon et al. 2011). Net uptake capacity of glutamine and arginine was lower based on 13 C compared to that based on 15 N incorporation suggesting that amino acids degraded in the solution or on the surface of the roots, and/or the respiration of amino acid-derived C inside the roots (Simon et al. 2011).

Statistical analyses
Data were tested for normality and homogeneity of variance. Differences among species as well as preferences of N sources within a species were tested using ANOVA on Ranks followed by Dunn´s test. Differences between competition regimes within a given species were tested using Rank Sum test. Significant differences (p ≤ 0.050) were tested using Sigmaplot 14 (Systat Software GmbH, Erkrath, Germany) for all statistical analysis. In addition, principal component analysis (PCA) was conducted to expose potential differences in the combination of the four N sources taken up by the roots of the different species using Metabo-Analyst (Chong et al. 2018(Chong et al. , 2019Xia et al. 2009Xia et al. , 2011aXia et al. , b , 2012Xia et al. , 2016. Before PCA, data were pre-processed by log transformation.

Results and discussion
In a tree community, net inorganic and organic N uptake capacity is unrelated to a seedling´s fast or slow growth Inorganic and organic N acquisition strategies differ among tree species based on their physiological and morphological properties (e.g. Andersen et al. 2017;Simon et al. 2017;Liese et al. 2018;Bueno et al. 2019). For example, fast-growing species need more N because of their higher investment in new growth compared to slow-growing species (e.g. Reich et al. 1997;Miller and Hawkins 2007). Especially higher amounts of fine roots could explore more soil volume (Comas and Eissenstat 2004) and in turn lead to an increased N uptake (e.g. Ryser 1996;Craine et al. 2001;Reuter et al. 2021). In the present study, the tree species differed in their growth and biomass indices when grown in intraspecific competition (Supplemental Table 1). More specifically, Fraxinus had higher leaf, stem, and total biomass than Fagus, higher stem and total biomass than Carpinus, higher root biomass than that of Acer and Quercus (p < 0.001), and higher total biomass than Prunus (p ≤ 0.003). For Acer, root biomass and root:shoot ratio were higher compared to Carpinus (p < 0.001). Root:shoot ratio in Quercus and Fagus was higher compared to that of Carpinus when grown in intraspecific competition (p < 0.001). However, these differences in biomass allocation were only partly reflected in differences in organic but not inorganic net N uptake capacity ( Fig. 1; Table 2): Glutamine-N net uptake capacity was lower in Fraxinus compared to Carpinus and Tilia (p ≤ 0.002) and arginine-N net uptake capacity was lower in Fraxinus than in Carpinus and Quercus (p < 0.001) when seedlings were grown in intraspecific competition. Thus, there is no evidence that supports the hypothesis that a spe-cies´ growth rate or N demand is directly related to its shortterm N acquisition strategies. However, as seedlings used in this experiment were 1-year old and species grow at different rates, certain biomass effects cannot be excluded. Furthermore, seedlings of Fraxinus excelsior are classified slow-growing by the Professur für Waldbau und Professur für Forstschutz & Dendrologie der ETH Zürich (Zürich 2002) which was the basis in this study, whereas Schulz et al. (2011) considers them as fast-growing.
The lower uptake of organic N by Fraxinus been reported previously (Reuter et al. 2021) and could be explained by a higher density of nitrate transporters in the root membranes and chemical properties (Jacob and Leuschner 2015) as well as a positive interaction between arbuscular mycorrhiza and the uptake of nitrate (Liu et al. 2018;Reuter et al. 2021). Furthermore, N acquisition is related to species-specific root traits such as root hair length and density, root branching density, and/or specific root length (Freschet et al. 2021). These results are similar to those by Reuter et al. (2021) studying six of the seven species used here in the field who found that tree species and not mycorrhization type influenced net N uptake capacity overall. Similarly, with interspecific competition, tree species differed partly in their growth and biomass indices (Supplemental Table 1) but only in organic and not inorganic net N uptake capacity (Table 2): Glutamine-N net uptake capacity was lower in Fraxinus compared to Carpinus, Tilia, and Fagus grown in interspecific competition (p < 0.001). Arginine-N net uptake capacity was lower in Fraxinus compared to Carpinus, Tilia, and Quercus grown in interspecific competition and in Acer compared to Carpinus (p < 0.001). These results suggest that regardless of competition regime, differences among species are consistent with regard to the specific N sources they take up (e.g. Simon et al. 2017;Bueno et al. 2019;2020;Reuter et al. 2021). Differences among species were found only for organic but not inorganic N sources highlighting the relevance of organic N as sources of N for trees.
Considering the different N sources, principal component analysis (Figs. 1a and b ; Table 3) indicated that net uptake of ammonium, nitrate, glutamine-N, and arginine-N differed between species. When grown in intraspecific competition, 55.8% of the variation was explained by nitrate and 19.6% by arginine-N, whereas when grown in interspecific competition, 49.6% of the variation was explained by nitrate and 25.1% by ammonium. Inorganic N uptake differentiated species more than organic N uptake. Furthermore, comparing among species when grown in intra-vs. interspecific competition ( Fig. 1a and b ), the results indicate the relevance of specific N sources for tree N acquisition depending on their biotic environment.

In a community, a seedling's capacity to take up inorganic and organic N sources does not change in competition with other tree species
Competition for N in the rhizosphere leads to a shift in inorganic and organic N acquisition strategies in trees (e.g. Simon et al. 2010;Li et al. 2015;Bueno et al. 2019). For example, when grown in competition, Fagus sylvatica had a generally lower organic and inorganic N uptake, whereas inorganic, but not organic N acquisition of Acer pseudoplatanus increased ). In the present study, growth and biomass indices did not differ significantly Fig. 1 a. Two-dimensional score plot of principal component analysis computed with net N uptake capacity of ammonium, nitrate, glutamine-N, and arginine-N for tree seedlings grown in intra-specific competition. Species are shown in different colours: Acer-A. pseudoplatanus, Carpinus-C. betulus, Fagus-F. sylvatica, Fraxinus-F. excelsior, Prunus-P. avium, Quercus-Q. robur, Tilia-T. cordata. The explained variances (in percentage) are shown in x-and y-axes in the plot b. Twodimensional score plot of principal component analysis computed with net N uptake capacity of ammonium, nitrate, glutamine-N, and arginine-N for tree seedlings grown in inter-specific competition. Species are shown in different colours: Acer-A. pseudoplatanus, Carpinus-C. betulus, Fagus-F. sylvatica, Fraxinus-F. excelsior, Prunus-P. avium, Quercus-Q. robur, Tilia-T. cordata. The explained variances (in percentage) are shown in x-and y-axes in the plot between competition regimes for most species, except for a lower SLA in Fraxinus in inter-compared to intraspecific competition (p = 0.040). Similarly, inorganic and organic net N uptake capacity did not change from intra to interspecific competition for any of the study species which might be due to the high variation in net N uptake capacity displayed by the individuals in this study. Only trends were found for a higher glutamine-N (Quercus, p = 0.073) and nitrate (Fagus, p = 0.056) net uptake capacity when grown in intra-compared to interspecific competition. These results are in contrast with previous studies investigating the consequences of competition for N for inorganic and organic N acquisition using two-species approaches (e.g. Fotelli et al. 2004;Simon et al. 2010Simon et al. , 2014Li et al. 2015). Liese et al. (2018) used a community approach with four species grown together in a mesocosm; however, they only measured total N uptake and not that of the different tree species. Overall, the results of the present study suggest that the interactions between seedlings in a community and with regard to N uptake are more complex. In the multi-species approach used here, the consequences of potential competition and/or facilitation between certain individual species-such as a negative effect with one species, but a positive effect of another-might have canceled each other out and thus might have masked any individual species´ effects. However, the trends of reduced inorganic or organic N acquisition found in the present study for certain N sources and species suggests a negative effect of competition with certain species.

N source preferences shift with competition regime and tree species
Tree species prefer different N sources when competing for N (e.g. Ashton et al. 2008;Simon et al. 2010;Bueno et al. 2019;Reuter et al. 2021). This hypothesis was confirmed in the present study. When seedlings were grown in intraspecific competition, the preferred N sources in shortterm uptake experiments varied depending on the species (Table 4): Fagus showed no preferences, whereas Carpinus, Quercus, Prunus, and Tilia preferred organic N over ammonium (p ≤ 0.011). Acer preferred nitrate over ammonium and Fraxinus nitrate over ammonium and arginine-N (p < 0.001). Preferences for N also differed among species when grown in interspecific competition: Organic N was favoured over ammonium for Carpinus, Acer, and Tilia; glutamine-N was preferred over ammonium for Fagus, arginine-N over ammonium for Quercus and Prunus, and nitrate over ammonium for Acer (p ≤ 0.017). Fraxinus took up more nitrate than glutamine-N or ammonium (p < 0.001). These results highlight the significance of organic N for tree N acquisition for most of the studied species, except for Fraxinus.
Looking at how N source preferences of the study species shifted when seedlings were from intra-to interspecific competition treatments, five patterns were found (Table 4): (1) Acer included organic N as preferred N sources over ammonium; (2) Fagus preferred glutamine-N compared to no preferences in intraspecific competition; (3) Fraxinus switched between amino acids, but still preferred nitrate; (4) Prunus and Quercus no longer preferred glutamine-N over ammonium, and (5) Carpinus and Tilia showed no differences between competition regimes. Thus, the preference to take up specific N sources depends on the species and is regulated by underlying physiological traits, such as, the  density of specific transporters (Näsholm et al. 2009;Jacob and Leuschner 2015), as well as free amino acid synthesised when ammonium is assimilated (e.g. Imsande and Touraine 1994;Kreuzwieser et al. 1997;Collier et al. 2003;Reuter et al. 2021). Organic N uptake is an important N source for certain tree species, particularly when N is limiting. As the assimilation process can be bypassed, organic N acquisition requires less energy (Moreau et al. 2019).
In conclusion, short-term net inorganic and organic N uptake capacity are not related to a seedling´s inherent growth rate when grown in a tree community and do not shift in response to competing species, but the preferences for certain N sources can change.
Author contributions Statement JS conceived and designed the study, conducted the experiment, analysed the data, and wrote the manuscript.
Acknowledgement I gratefully thank Leonard Schink and Iljas Müller for their help with the 15 N incubation experiments. Furthermore, I thank Silvia Kuhn, Rosi Miller, and Regina Grimm for their help with the biomass sampling. I also thank Oliver Hartmann for his help with sample processing. Moreover, I thank the gardeners at the Botanical Garden of the University of Konstanz for their help with the experimental set up.
Funding Open Access funding enabled and organized by Projekt DEAL. This work was supported by the Young Scholar Fund at the University of Konstanz, Germany. JS is currently supported by a Heisenberg Fellowship (no. SI 1556/4-1) of the German Research Foundation (DFG).
Data Availability Data will be available from the Dryad Digital Repository upon acceptance of the manuscript.

Conflicts of interest The authors declare no conflicts of interest.
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