Seedlings of Prunus had a significantly lower SLA when competing with Fagus than when competing with Quercus (**Table 3** and **Supplementary Table 7**), while there were no differences between different competitor identities with respect to any of the other measured parameters (**Figure 2**, **Table 3** and **Supplementary Tables 7**, **8A**). Moreover, Robinia seedlings competing with Fagus had a significantly lower SLA (regardless of soil N availability), and higher total soluble amino acid-N content in the fine roots (only at low soil N availability) than seedlings competing with Quercus or Pinus (**Table 3** and **Supplementary Tables 7**, **8A,B**). Furthermore, seedlings of Robinia competing with Quercus had significantly lower glutamine-N net uptake capacity than those competing with either Fagus or Pinus (only at high soil N availability) (**Figure 2**). All other measured parameters did not differ significantly between different competitor identities for this species (**Figure 2**, **Table 3** and **Supplementary Tables 7**, **8A**).

### Effects of High Compared to Low Soil N Availability on Native and Invasive Tree Seedlings Growing in Competition

Native Tree Species – Effects of High Compared to Low Soil N Availability on Total Biomass, Growth Indices, N Acquisition, and N Pools in the Leaves and Fine Roots

For seedlings of Fagus a higher soil N availability resulted in significantly higher ammonium and arginine-N net uptake capacity (**Figure 1**), as well as a significantly lower SRL (**Table 2B** and **Supplementary Table 7**) and total soluble amino acid-N content in the leaves and fine roots (**Table 2B** and **Supplementary Table 8A**), regardless of competitor identity, while the rest of the measured parameters did not change significantly. For Quercus, ammonium and organic N net uptake capacity increased significantly (**Figure 1**), while RGR (**Table 2B** and **Supplementary Table 7**) and total soluble protein-N content in the fine roots (**Table 2B** and **Supplementary Table 8A**) decreased significantly at high compared to low soil N availability, regardless of competitor identity. Furthermore, higher soil N availability significantly reduced the total soluble amino acid-N content in the fine

fpls-10-00337 March 27, 2019 Time: 17:52 # 6

ammonium;

 NO

3 , nitrate; Gln-N, glutamine; Arg-N, arginine; total soluble amino acid-N (mg amino acid-N g

 dw); total soluble protein-N (mg protein-N g

 dw).

TABLE 2 | Effects of increased soil N availability on total biomass, growth indices, inorganic and organic N net uptake capacity, and N metabolite levels on (A) seedlings growing single and (B) seedlings growing in competition of Fagus sylvatica, Quercus robur, Pinus sylvestris, Prunus serotina, and Robinia pseudoacacia.

↑, significant increase with increased soil N availability; ↓, significant decrease with increased soil N availability; n.s., no significant differences between high and low soil N availability. Total biomass (g), root:shoot ratio, ratio of belowground biomass to aboveground biomass; RGR, relative growth rate (g dw g−1 dw d−1 ); SLA, specific leaf area (cm2 g −1 dw); SRL, specific root length (cm g−1 dw); N net uptake capacity (nmol N g−1 fw h−1 ); NH+ 4 , ammonium; NO− 3 , nitrate; Gln-N, glutamine; Arg-N, arginine; total soluble amino acid-N (mg amino acid-N g−1 dw); total soluble protein-N (mg protein-N g−1 dw). (a) Only in competition with Fagus and Pinus. (b) Only in competition with Prunus. (c) Only in competition with Fagus and Quercus. No letter: effect of soil N availability regardless of competitor identity.

roots for Quercus seedlings when grown in competition with Prunus but not in seedlings grown in competition with Robinia (**Table 2B** and **Supplementary Tables 8A,B**). All other measured parameters remained unchanged between soil N availability treatments. For seedlings of Pinus, a high soil N availability significantly increased ammonium and arginine-N net uptake capacity (**Figure 1**), whereas RGR (**Table 2B** and **Supplementary Table 7**) and total soluble amino acid-N content in the leaves decreased (**Table 2B** and **Supplementary Table 8A**) compared to low soil N availability regardless of competitor identity, while the other measured parameters did not differ significantly.

#### Invasive Tree Species – Effects of High Compared to Low Soil N Availability on Total Biomass, Growth Indices, N Acquisition, and N Pools in the Leaves and Fine Roots

Prunus seedlings at high compared to low soil N availability significantly increased their ammonium and organic N net uptake capacity (**Figure 2**), and decreased SRL (**Table 2B** and **Supplementary Table 7**), total soluble amino acid-N contents in the leaves and fine roots, as well as total soluble protein-N content in the leaves (**Table 2B** and **Supplementary Table 8A**) regardless of the competing species. There were no effects of soil N availability on any other measured parameter. For seedlings of Robinia, higher soil N availability resulted in significant increases in inorganic N and arginine-N net uptake capacity (**Figure 2**), as well as significantly decreased total soluble amino acid-N content in the leaves and total soluble protein-N content in the fine roots regardless of competition (**Table 2B** and **Supplementary Table 8A**). However, glutamine-N net uptake capacity increased significantly with higher soil N availability in seedlings competing with Fagus and Pinus, but not when competing with Quercus (**Figure 2**). Similarly, total soluble amino acid-N content in the fine roots decreased significantly with higher soil N availability in seedlings grown in competition with Fagus and Quercus, but not when grown in competition with Pinus (**Table 2B** and **Supplementary Tables 8A,B**). Soil N availability did not affect other parameters measured in Robinia.

### N Acquisition Preferences of Native and Invasive Species for Specific N Sources at Low and High Soil N Availability

Comparing inorganic and organic N acquisition among N forms for each combination of species and competitor identity as well as for the single grown seedlings, we found general preference patterns at low and high soil N availability. At low soil N availability, organic N was preferred over inorganic N across species and competitor identities (**Tables 4**, **5**), with a few exceptions that showed no preference for any N source: single seedlings of Quercus and Prunus, seedlings of Quercus competing with Robinia, and seedlings of Prunus and Robinia competing with Fagus (**Tables 4**, **5**). At high soil N availability, a general pattern of preference for ammonium was found followed by organic N sources over nitrate. However, no preferences were found in single seedlings of Prunus and Robinia, seedlings of Pinus competing with Robinia, seedlings of Robinia competing with

FIGURE 1 | Ammonium, nitrate, glutamine-N, and arginine-N net uptake capacity (nmol N g−1 fw h−1 ) by fine roots of Fagus sylvatica, Quercus robur, and Pinus sylvestris seedlings at low and high soil N availability under different competition regimes. vs. Prunus = competition with Prunus serotina; vs. Robinia = competition with Robinia pseudoacacia. Box plots show mean (dotted line) and median (continuous line). Different letters indicate significant differences between competition regimes within a specific soil N availability treatment, and asterisks indicate significant differences between soil N availability treatments detected using permutational analysis of variance (p < 0.05).

With regard to the specific preferences of each species across different competitor identities and single grown seedlings, native Fagus and Pinus had in general similar preferences for N sources regardless of competitor identity or whether they grow single or in competition (**Table 4**). However, native Quercus showed no preference for any N source either when grown single or in competition with Robinia, but preferred organic N over ammonium when grown in competition with Prunus (at low soil N availability). Moreover, at high soil N availability, the preferences of Quercus shifted from no preference when grown single to preferring ammonium and organic N over nitrate when grown in competition with Prunus or Robinia (**Table 4**). For the invasive species, Prunus had no preference of N sources when grown single but generally preferred arginine-N over glutamine-N and inorganic N when competing with Quercus or Pinus at low soil N availability, while preferences for N sources did not shift across competitor identities at high soil N availability (**Table 5**). Seedlings of Robinia preferred organic over inorganic N sources regardless of competitor identity as well as when grown single at low soil N availability, except for seedlings competing with Fagus which showed no preference. However, at high soil N availability, Robinia seedlings preferred ammonium and organic N over nitrate when competing with Fagus, while seedlings competing with other native species or grown single took up all N forms without preference (**Table 5**).

## DISCUSSION

#### Strategies of Native and Invasive Species Growing Without Competition Invasive Species Succeed With Fast Growth, but Natives Could Counter With Preference of Organic N Acquisition

Comparing single grown invasive and native species, invasive species showed traits characteristic for fast-growing species. The invasive species had higher total biomass, RGR, and SRL, as well as total soluble amino acid-N and protein-N levels in both leaves and fine roots compared to the natives. A fast RGR is related to a rapid increase in biomass as well as high leaf nutrient concentrations, whereas slow RGR is related to a higher investment of resources in defense (Reich et al., 1997; Aerts, 1999). A higher SRL allows to forage larger soil volumes and is linked to a higher competitive ability for belowground resources (Aerts, 1999). Our results suggest a potential of the studied invasive species to outcompete the native ones via rapid growth and exploitation of above- and below-ground resources (Vilà and Weiner, 2004; Gioria and Osborne, 2014), which is in line with previous studies that found higher biomass (e.g., Lee et al., 2004; Closset-Kopp et al., 2007; Ding et al., 2012; Kawaletz et al., 2013), and monthly height increments (e.g., Ding et al., 2012) of P. serotina and R. pseudoacacia compared to plant species native to Europe and Asia. Moreover, the higher content of N metabolites in the fine roots is also linked to fast growing species

fpls-10-00337 March 27, 2019 Time: 17:52 # 9

soil N availability.

 (b) Only at low soil N availability.

4 ,

 No letter: effect of competition

3 ,

 regardless of soil N availability.

Only significant differences are presented. n.s., no significant differences between net uptake capacity of different N forms.

TABLE 5 | Differences between ammonium (NH+ 4 ), nitrate (NO− 3 ), glutamine-N (Gln-N), and arginine-N (Arg-N) net uptake capacity of the fine roots of Prunus serotina and Robinia pseudoacacia seedlings grown single and in competition at low and high soil N availability.

Only significant differences are presented. n.s., no significant differences between net uptake capacity of different N forms.

compared to slow growers (e.g., A. pseudoplatanus compared to F. sylvatica in Li et al., 2015). For Robinia, a higher total N content in the leaves and fine roots compared to the other species is most likely associated with its N2-fixing ability (McKey, 1994; De Marco et al., 2013; Salpagarova et al., 2014). With regard to the N acquisition strategies, inorganic N acquisition was similar between invasive and native species. However, the lower organic N acquisition for the invasive compared to the native species suggests that native species could have a competitive advantage over the invasives via better exploitation of soil organic N. Although the invasives have faster growth, their species-specific N acquisition does not suggest a competitive advantage over natives.

#### With Higher Soil N Availability N Acquisition Strategies of Native and Invasive Seedlings Are Similar, Whereas N Allocation to N Pools in the Leaves and Fine Roots Is Species-Specific

In our study, the responses of single growing native and invasive species to high compared to low soil N availability were similar for all species for N acquisition, biomass and growth indices, whereas the allocation of N to N pools in the fine roots and leaves depended on the species. More specifically, inorganic and organic N acquisition increased with rising soil N levels, while biomass and growth indices showed no response, except for an increased root:shoot ratio for Robinia indicating a greater allocation to root biomass with increasing soil N supply. The allocation of N to N pools in the leaves and fine roots was species-specific with one of three responses with higher compared to lower soil N availability: (1) an increase in total soluble protein-N levels in the leaves (i.e., for native Fagus), (2) a decrease in total soluble amino acid-N levels in the leaves (i.e., for native Quercus), and (3) no change of total soluble amino acid-N or protein-N levels in neither leaves nor fine roots (i.e., for native Pinus and both invasives Prunus and Robinia).

The increase in total soluble protein-N levels in the leaves of Fagus with higher soil N availability in combination with no change in overall biomass suggests that N is stored as Rubisco for later mobilization (Millard, 1988; Masclaux-Daubresse et al., 2010). In contrast, Quercus had a reduced total soluble amino acid-N levels in the leaves which suggests that soluble amino acids are used to produce other compounds not quantified here, e.g., compounds related to defense.

Overall, for all studied species, the increased inorganic and organic N acquisition as well as the reduced levels of N metabolites in leaves and fine roots with rising soil N availability indicate plant physiological adjustments to meet the N demands with different soil N supply (BassiriRad, 2000). The lack of response with regard to biomass or growth indices suggests that the acquired N is either used for metabolism maintenance or assigned to storage (Millard and Grelet, 2010). Plant N acquisition is directly related to soil N supply, thus, higher organic and inorganic N acquisition with higher soil N availability reflects the active regulation of N uptake (Kulmatiski et al., 2017) which has already been reported for seedlings of F. sylvatica (e.g., Li et al., 2015) and P. sylvestris (e.g., Simon et al., 2013).

### Native Species Respond to Competition With Invasives With Distinct Species-Specific Strategies

The native species showed distinct species- and also parameterspecific strategies in their responses to competition with different invasive species that were partly mediated by soil N availability. When competing with Prunus compared to Robinia: (1) Fagus had lower RGR and N acquisition without changes in biomass or N metabolite levels, (2) Quercus had lower biomass and total soluble amino acid-N levels without changes in N acquisition, whereas (3) Pinus only had higher SRL, without changes in biomass, N acquisition or N metabolite levels. More specifically, the lower RGR combined with N acquisition in Fagus in competition with Prunus compared to Robinia suggests that the response strength to competition with an invasive species depends on the competitor. The slower growth combined with a reduced N acquisition from the soil indicates a remobilization of internal N in compensation for the impaired N acquisition from the soil assuming a similar N demand. This strategy of a reduced growth rate (Li et al., 2015) and N acquisition (Simon et al., 2010, 2014) was found also in response to competition with fast-growing A. pseudoplatanus, thus indicating a general response of F. sylvatica to fast-growing competitors. In contrast, Quercus had a decreased biomass as well as total soluble amino acid-N content in the fine roots (only at high soil N availability) in competition with Prunus compared to Robinia, with no change in N acquisition suggesting a shift in the allocation of N from growth to storage and/or the synthesis of defense compounds depending on the competing species (Millard and Grelet, 2010). The lower total soluble amino acid-N levels in the fine roots when competing with Prunus compared to Robinia but only a high soil N availability indicates a mediation of the competitive response by soil N supply. Lower amino acid-N levels in the fine roots have been related to slower growth (Simon et al., 2010), whereas an increase in total soluble amino acid levels in the fine roots as a result of competition (Li et al., 2015) was found in fast-growing sycamore maple (A. pseudoplatanus L.). However, Fagus showed a different response, despite also being a slow growing species which indicates that other species-specific factors regulate the response to competition in terms of total soluble amino acid-N content in the fine roots that were not considered in our study. For seedlings of Pinus the sole response to competition with Prunus compared to Robinia was a higher SRL which regulates the access to N (Andersen et al., 2017). Growing longer, thinner roots allows Pinus seedlings to maximize resource capture without changing N acquisition capacity per root, thereby being able to tolerate competition with no effects on productivity. Unlike both deciduous species Fagus and Quercus, coniferous Pinus did not show different responses to different invasive competitors regarding N acquisition and N pools in leaves and fine roots, likely because conifers maintain needles in which N is stored throughout the year (Millard and Grelet, 2010) and can be utilized when needed. By drawing on these resources, Pinus can buffer the different competitive effects of the invasive species. Noticeably, none of our native study species responded differently depending on the competitor with regard to their total soluble protein-N levels in the leaves or fine roots. Previous studies on temperate tree seedlings including F. sylvatica (Simon et al., 2010, 2014; Li et al., 2015) found changes in total soluble protein-N contents in the fine roots when grown in intraspecific or interspecific competition indicating de novo protein synthesis as an adaptive response to competition. In the present study, we compare native species responses to different invasive species rather than intraspecific vs. interspecific competition within native species. Thus, it is possible that protein-N levels changed for the single species when grown in competition, but the response was similar with different competitors.

Overall, the response of native seedlings in competition with invasive species is species-specific and reflects different coping mechanisms related to the species life history and growth strategies. For instance, coniferous species such as Pinus maintain their needles throughout the year resulting in higher N storage capacities compared to deciduous species like Fagus and Quercus (Millard and Grelet, 2010). As a consequence, conifers might be less responsive to the different competitive effects of the invasive species in terms of the competition for N. All three native species responded stronger when competing with Prunus than Robinia.

#### Why Is P. serotina a Stronger Competitor Than R. pseudoacacia for the Studied Native Species?

In our study, the native tree seedlings competing with Prunus responded with decreases in N acquisition, total biomass, RGR, and total soluble amino acid-N levels in the fine roots than when competing with Robinia which can be explained by its relatively high root biomass, and, in turn, a better exploitation of belowground resources of Prunus (Casper and Jackson, 1997; Kawaletz et al., 2014) compared to all other species in our study which is linked to increased success rates of invasive plants (e.g., Grotkopp and Rejmánek, 2007; Robakowski and Bielinis, 2011; Kawaletz et al., 2013; Gioria and Osborne, 2014). Moreover, Prunus produces cyanogenic compounds also in the roots, a strategy that might have had an allelopathic effect on the neighboring plants (Csiszár, 2009; Robakowski et al., 2016) and contributed to the general negative influence of Prunus on the native species in our study. Overall, the higher competitive ability of Prunus in our study is in accordance with earlier work by others; however, previous studies investigated the effects of Prunus and Robinia when competing with native species mainly

considering plant growth and biomass (e.g., Closset-Kopp et al., 2011; Robakowski and Bielinis, 2011; Kawaletz et al., 2013, 2014), whereas we found not only competition effects on growth and biomass but also on N acquisition and N allocation to different N pools depending on the species.

### Invasive R. pseudoacacia Reacts Stronger to Competition With Native Seedlings Than P. serotina

The invasive species used in our study responded to competition depending on the native competitor species and soil N availability: the response of Prunus to competition with native seedlings was an increase in SLA when grown in competition with Quercus compared to competition with Fagus, but not Pinus. Usually, plants with a high SLA have a lower competitive effect related to a shorter leaf lifespan resulting in a lower leaf mass fraction (Kuster et al., 2016); which is, however mostly related to limiting resource conditions (Knops and Reinhart, 2000). In contrast, having a higher SLA when belowground resources are sufficient becomes a competitive advantage as a strategy of fast resource acquisition (Liu et al., 2017). Therefore, given the higher root biomass of Prunus compared to our native study species (see section "Invasive Species Succeed With Fast Growth, But Natives Could Counter With Preference of Organic N Acquisition"), Prunus was a strong competitor for belowground resources, and enhanced its competitive ability and fast growth strategy by increasing light interception via a higher SLA (Liu et al., 2017).

In contrast, seedlings of Robinia responded to competition depending on the native species, with a decrease in organic N acquisition in competition with Quercus compared to Pinus and Fagus, as well as an increased SLA combined with a decrease in fine root total soluble amino acid-N content in competition with Quercus and Pinus compared to Fagus. Notably, the decrease in fine root total soluble amino acid-N content with competition was found only at low soil N availability, the decrease in organic N acquisition only at high soil N availability. This response dependency on soil N availability for Robinia but not Prunus indicates that Robinia shows a strong response to the combined effects of competition and soil N availability, whereas the response of Prunus is not mediated by soil N supply. The increase in SLA when competing with Quercus compared to Fagus was found regardless of soil N availability and suggests a potential reduced competitive ability of Robinia competing with Quercus, especially in combination with the reduced organic N acquisition and total soluble amino acid-N content in the fine roots indicating an impaired N nutrition. In general, leaves with a high SLA represent a disadvantage for plant growth under limiting nutrient conditions, because of the negative correlation with leaf lifespan, and therefore increased N losses through leaf senescence (Reich et al., 1997; Knops and Reinhart, 2000).

Overall, the investigated invasive species responded at different levels (i.e., morphological and physiological) depending on the identity of the native competitor suggesting that Robinia represents a lesser threat than Prunus at the investigated time scale. However, the impact of Robinia might become more important in the long run as a result of its capacity to fix atmospheric N2 and therefore potentially alter biogeochemical processes in invaded ecosystem (Strayer et al., 2006; Medina-Villar et al., 2016). By modifying soil N availability, for example, N2-fixing plants might influence community composition and diversity (i.e., by favoring the establishment of nitrophilous species) (Staska et al., 2014), and consequently impact on plant– plant interactions such as competition.

#### Higher Soil N Availability Results in Increased N Acquisition but No Change in Biomass or Growth Indices for All Species in Competition, While a Reduction in N Pool Levels Is Speciesand Organ-Specific

Seedlings of native and invasive species growing in competition in our study varied their responses with high soil N supply. Although, none of them increased in biomass with high soil N supply seedlings of Fagus and Prunus had lower SRL indicating that foraging for nutrients via an investment in longer, thinner roots is only required when soil nutrients are limiting (Zhu et al., 2016). In all species, organic and inorganic N uptake increased with high compared to low soil N supply which is a key driver of N acquisition in woody species (Simon et al., 2017) as reported for F. sylvatica (Stoelken et al., 2010; Li et al., 2015), P. sylvestris (Simon et al., 2013), and A. pseudoplatanus (Li et al., 2015). However, reduced total soluble amino acid-N and total soluble protein-N levels with higher soil N availability were species- as well as organ-specific in our study. In general, plants have to rely less on internal N storage to maintain growth and metabolic functions when N is readily available in the soil (Millard and Grelet, 2010). For Fagus, our results are in contrast with a previous study that found no changes in total soluble amino acid-N contents in the fine roots with increasing soil N availability neither in intraspecific competition nor in competition with A. pseudoplatanus (Li et al., 2015). However, in our study, the response of native species to competition with invasive species in terms of N strategies is evaluated for the first time and suggests a specific response of native species to combined soil N availability and competition with the invasive species Prunus and Robinia.

Species responded to soil N availability mostly regardless of competitor identity, however, for some species an interaction between soil N availability and competitor was detected. For example, the reduction in total soluble amino acid-N levels in the fine roots with at high compared to low soil N availability described above was found in some species only with specific competitors (i.e., for Quercus when competing with Prunus, and for Robinia when competing with Fagus or Quercus). Specifically, for Quercus this suggests that invasive species Prunus induces a stronger response than invasive Robinia, a result that is consistent with the general responses of Quercus to Prunus described in Section "Native Species Respond to Competition With Invasives With Distinct Species-Specific Strategies." We found no effects of competition on seedling total soluble amino acid-N or total soluble protein-N levels in the leaves which contrasts with studies reporting the invasion by Prunus to affect foliar N levels of

native mature tree species including the ones considered here (e.g., Aerts et al., 2017). This suggests an effect of plant age on the competition response, as well as the possibility that soil N availability in our study was a strong driver of foliar N levels, thereby overriding the potential effects of competition.

### Native and Invasive Species Share Common Preferences for N Sources Regardless of Soil N Availability, but There Are Species-Specific Patterns Between Competition Regimes and Competitor Identities

The common N preference patterns across species in our study depended on soil N availability and species strategies in response to competition. At low soil N availability, organic N was taken up preferentially over inorganic N by seedlings of both native and invasive suggesting that tree seedlings maintain their metabolism and growth by drawing upon a wider variety of N sources. However, at high soil N availability, ammonium-N was the preferred N form followed by organic N, while nitrate-N was the least preferred N form taken up by tree seedlings, as also found for F. sylvatica (Stoelken et al., 2010) and P. sylvestris (Simon et al., 2010). Nitrate acquisition from the soil is inhibited by high concentrations of amino acids (particularly glutamine) and ammonium (Näsholm et al., 2009; Stoelken et al., 2010). Both native and invasive species preferred specific N sources linked to other aspects considered in our study. For example, Quercus preferred organic N forms over ammonium only when grown in competition with invasive Prunus. Pinus did not shift N source preferences. Both invasive species also lacked a preference for certain N forms when competing with Fagus (at low soil N availability) or Pinus (at high soil N availability) which corresponds with their general stronger reaction to competition with Quercus (see section "Invasive R. pseudoacacia Reacts Stronger to Competition With Native Seedlings Than P. serotina"). Overall, N form preferences matched the general response strategies of native and invasive species to differing competitors that were found for growth, N acquisition and allocation of N to N pools.

### CONCLUSION

In our study, we found that invasive species display traits that grant them competitive advantage, such as fast growth rates, which however did not result in a generally higher N acquisition of invasive species, because native species had a higher organic N net uptake capacity that would allow them to better utilize soil N sources, and thus potentially avoid competition with neighboring invasive species. When growing in competition, native tree seedlings showed a stronger response to competition with Prunus than Robinia, although the response variable changed between native species indicating the use of species-specific strategies of native seedlings to cope with the competition with invasive plants. These are further mediated by soil N availability in some cases. These strategies reflected the differences between native species in terms of life history and growth traits. The stronger response to Prunus could be related to the higher total biomass of this species and its ability to produce allelopathic compounds. Furthermore, when comparing the responses of invasive species, Robinia responded to different native competitors at different morphological and physiological levels, further influenced by soil N availability, while Prunus only showed changes in SLA, without interaction with soil N availability. This suggests that Prunus might have a stronger competitive advantage over native species than Robinia at least at the time scale of our study. Moreover, considering that native Quercus responded more negative to competition with Prunus than Robinia, as well as the several responses of Robinia to competition with Quercus, our results suggest that specifically Quercus could have a competitive advantage over invasive Robinia, but not over invasive Prunus. However, it is possible that the impact of Robinia becomes more important in the longer run at the plant community level, as a result of its capacity to fix atmospheric N2 and therefore potentially alter biogeochemical processes in an invaded ecosystem. With regard to the effects of high compared to low soil N availability on competing seedlings, we found common patterns among species similar to those displayed by single grown seedlings regarding N acquisition (increased with soil N availability), but not regarding N allocation, i.e., in single seedlings N allocation was scarcely influenced by soil N availability, while in competing seedlings there was a general decrease in total soluble amino acid-N levels for most species, which suggests stronger responses to increased soil N availability when in combination with competition. Generally, our results highlight Prunus as a potential greater threat to seedlings of native species than Robinia in the time frame considered here. Overall, our results provide novel insights into the different species-specific effects of invasive species on native seedlings not only with regard to growth parameters, but also underlying physiological processes such as N acquisition and internal allocation. However, our study included only five species (three natives, two invasives) differing in their ecological background, with a focus on the competition for N rather than other resources, such as light and/or space, thus more detailed studies are still required in the future. Furthermore, the results should be validated in long term studies conducted in the field. In conclusion, the species-specific nature of competition between native and invasive tree seedlings should be considered in forest management strategies in the future.

## DATA AVAILABILITY

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

### AUTHOR CONTRIBUTIONS

AB and JS conceived the experimental design and manuscript writing. AB conducted the 15N uptake experiments and evaluated the data. KP contributed stable isotope analyses and ideas to data interpretation. All authors agreed on the final version of the manuscript.

#### FUNDING

Funding was provided by the Young Scholar Fund project no. 83979115 of the University of Konstanz.

#### ACKNOWLEDGMENTS

fpls-10-00337 March 27, 2019 Time: 17:52 # 15

We are grateful to Leonhard Schink and Leia Mijatovic for their help with 15N uptake experiments, harvest, and sample processing. Furthermore, we would like to thank Silvia Kuhn and

#### REFERENCES

Roswitha Miller for their help with sample analyses. We thank Franz Buegger for support with EA-IRMS analyses. We would also like to thank Iljas Müller, Gudrun Winter, Dietmar Funck, Marie-Luise Kocher, Anna Märkle, Dominic Stickel, Lea Held, Inna Koleber, Julia Maier, and Jasmin Thierschmidt for their assistance with parts of the harvest and sample processing. We thank the gardeners at the Botanical Garden of the University of Konstanz for their help with experimental set up.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2019.00337/ full#supplementary-material

and sycamore maple (Acer pseudoplatanus) seedlings. Plant Biol. 12, 453–458. doi: 10.1111/j.1438-8677.2009.00225.x

**Conflict of Interest Statement:** 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.

Copyright © 2019 Bueno, Pritsch and Simon. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Phosphorus Allocation to Leaves of Beech Saplings Reacts to Soil Phosphorus Availability

Sonia Meller1,2, Emmanuel Frossard2 and Jörg Luster1 \*

1 Forest Soils and Biogeochemistry, Swiss Federal Institute for Forest, Snow and Landscape Research WSL, Birmensdorf, Switzerland, 2 Institute of Agricultural Sciences, ETH Zürich, Zurich, Switzerland

Decreasing phosphorus (P) concentrations in leaves of beech (Fagus sylvatica L.) across Europe raise the question about the implications for forest health. Considering the distribution of beech forests on soils encompassing a broad range of nutrient availability, we hypothesized that this tree species exhibits high phenotypic plasticity allowing it to alter mass, and nutrient allocation in response to local nutrient availability. To test this, we grew two groups of 12–15 year old beech saplings originating from sites with high and low soil P availability for 2 years in mineral soil from their own site and in soil from the other site. After two growing seasons, P concentrations in leaves and stem, as well as mass allocation to leaves and fine roots were affected by both soil and plant origin. By contrast, relative P allocation to leaves and fine roots, as well as P concentrations in fine roots, were determined almost entirely by the experimental soil. Independent of the P nutritional status defined as average concentration of P in the whole plant, which still clearly reflected the soil conditions at the site of plant origin, relative P allocation to leaves was a particularly good indicator of P availability in the experimental soil. Furthermore, a high plasticity of this plant trait was indicated by a large difference between plants growing in the two experimental soils. This suggests a strong ability of beech to alter resource allocation in response to specific soil conditions.

Keywords: acclimation, beech, Fagus sylvatica, forest health, phenotypic plasticity, phosphorus allocation, phosphorus nutritional status, soil phosphorus availability

### INTRODUCTION

Forests dominated by beech (Fagus sylvatica L.) cover a large part of Europe (ca 14–15 Mha) from southern Norway to southern Italy and from northern Spain to northwest Turkey (Brunet et al., 2010; Durrant et al., 2016). Throughout this area, beech populations occur on a wide variety of soils realizing a broad ecological niche in terms of soil chemical properties including pH (3.2–7.3), base saturation (3–99%), and plant-available phosphorus (P) pools in the mineral topsoil (11–1287 mol P m−2 10 cm−1 ) (Leuschner et al., 2006; Batjes, 2011; Yang et al., 2013).

Analysis of data from forest monitoring plots (ICP forest level II) across Europe between 1991 and 2010 revealed a significant decline in P concentrations and an increase of N/P ratios in beech leaves on the majority of plots (Jonard et al., 2015; Talkner et al., 2015) confirming findings of earlier regional studies (Flückiger and Braun, 1998; Duquesnay et al., 2000; Jonard et al., 2009; Braun et al., 2010). These changes in leaf nutrient status have been attributed to continuing

#### Edited by:

Christian Ammer, University of Göttingen, Germany

#### Reviewed by:

Friderike Beyer, University of Freiburg, Germany Rodica Pena, University of Göttingen, Germany Werner Borken, University of Bayreuth, Germany

> \*Correspondence: Jörg Luster<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Functional Plant Ecology, a section of the journal Frontiers in Plant Science

Received: 17 February 2019 Accepted: 20 May 2019 Published: 06 June 2019

#### Citation:

Meller S, Frossard E and Luster J (2019) Phosphorus Allocation to Leaves of Beech Saplings Reacts to Soil Phosphorus Availability. Front. Plant Sci. 10:744. doi: 10.3389/fpls.2019.00744

**80**

high nitrogen (N) deposition and increasing atmospheric carbon dioxide concentrations. Both accelerated tree growth due to high N and carbon (C) input and negative effects of elevated N on fine root biomass, mycorrhization, and litter mineralization appear to have created a higher need for other nutrients which cannot be met by the supply from the soil (Kjøller et al., 2012; Peñuelas et al., 2013; Talkner et al., 2015). On 40% of the level II plots the average leaf P concentrations indicated P deficiency (Jonard et al., 2015) and average N/P ratios on 60% of the plots were higher than "good for harmonious nutrition" (Talkner et al., 2015). The currently widely accepted critical values for nutrient concentrations and ratios in leaves (Mellert and Göttlein, 2012) are based on a large number of studies comparing leaf values with deficiency symptoms, growth or reaction to fertilization. However, results from surveys and experiments on the relation between P concentrations in plant tissues and soil P availability are partially conflicting. While concentrations of P in leaves and fine roots of mature trees across different forest sites were not well related to measures of P availability in the soil (Talkner et al., 2015; Lang et al., 2017), this relation was stronger for saplings from two acid forest sites and considering different plant compartments including leaves, stem, fine and coarse roots (Yang et al., 2016; Zavišic and Polle, 2018 ´ ; Zavišic et al., 2018 ´ ). Furthermore, Zavišic et al. (2018) ´ observed a strong increase of P concentrations in all compartments upon fertilization. One reason for the conflicting results could be that often only total P concentrations in soil are available which not sufficiently reflect this elements bioavailability. Another explanation could be the strong seasonal and site dependent variability of P concentrations in plant tissues (Yang et al., 2016; Netzer et al., 2017; Zavišic and ´ Polle, 2018). This points to the importance of seasonal dynamics of nutrient uptake and plant internal nutrient allocation.

However, surprisingly little is known on how beech adapts its internal P allocation to different P availability in soil, and thus to what degree P concentrations in leaves directly reflect soil P availability or the ability of the tree to regulate its P content on the cellular and the whole-plant level (Marschner, 1996). On the cellular level, P homeostasis can be maintained by adjusting the flux of P into and out of the vacuole (Lee et al., 1990; Lee and Ratcliffe, 1992; Mimura et al., 1996). On the whole plant level, P redistribution is regulated via vascular tissues, and it follows seasonal patterns employing storage in and recycling from various organs including senescing leaves, roots, and stem (Raghothama, 1999; Lin et al., 2009, 2014; Brant and Chen, 2015; Netzer et al., 2017; Zavišic and Polle, 2018 ´ ; Zavišic et al., 2018 ´ ). The seasonally varying relations between soil P availability and internal P cycling have been shown for P concentrations in various plant compartments comprising leaves, fine roots, coarse roots and stem (Netzer et al., 2017; Zavišic and ´ Polle, 2018), for P concentrations in xylem and phloem (Yang et al., 2016; Netzer et al., 2017), and for P resorption from older and senescent leaves (Hofmann et al., 2016; Netzer et al., 2017). Furthermore, growth rates of adult beech not always reflected soil P availability (Netzer et al., 2017) highlighting the potential role of high-efficiency internal cycling in trees growing on soils with low P availability. Alteration of internal nutrient cycling can be part of the adaptive response (phenotypic plasticity) of plants to external factors. For many plants, phenotypic plasticity was shown to comprise rapid trait alterations during a lifetime of an organism (Sultan, 2004; Hodge, 2009), including morphological, anatomical, physiological, reproductive, and developmental traits. Considering the long time scales of migration and natural selection, phenotypic plasticity is the most important asset of tree populations to cope with the ongoing environmental changes (Matesanz et al., 2010; Nicotra et al., 2010; Vitasse et al., 2010). The phenotypic plasticity of beech in response to drought or increased temperature has been well studied, comprising plant traits such as phenology (e.g., Kramer, 1995; Vitasse et al., 2009), biomass and root morphology (Curt et al., 2005; Weemstra et al., 2016), leaf anatomy (Stojnic et al., 2015a,b ´ ), stem anatomy (Stojnic et al., 2013 ´ ; Diaconu et al., 2016), mass allocation, and growth rate (Rolo et al., 2015).

By contrast, little is known about the adaptive response of beech to changes in P availability. Our objective was therefore to identify the respective most responsive traits of this tree species. Considering the important role the plant nutritional status plays in governing nutrient acquisition (Marschner, 1996), we performed experiments with beech saplings adapted to grow in soil with either high or low soil P availability, and assessed their response to contrasting soil conditions. We hypothesized that the response of a beech sapling with a given P nutritional status, as defined by its site of origin, to higher or lower soil P availability in terms of biomass and P allocation to different plant compartments is mainly driven by the plant striving to increase a low foliar P concentration as fast as possible or to maintain a high foliar P concentration as long as possible.

#### MATERIALS AND METHODS

### Plant and Soil Materials

Plant and soil materials were collected on the core research sites of the priority program 1685 "Ecosystem nutrition" of the German and Swiss National Science Foundations1 in Unterlüss (LUE, Lower Saxony, Germany) with low P availability in the soil, and Bad Brückenau (BBR, northern Bavaria, Germany) with high P availability in the soil. The sites were chosen because they both sustain mature mono-specific beech stands but differ profoundly in soil P stocks and cycling. For details on the sites refer to Lang et al. (2017).

Saplings of beech (F. sylvatica) of similar size (approx. 45 cm in height and approx. 8 mm base diameter) were gently dug out at the sites during their dormancy period in December 2014, and stored at 4◦C with their roots embedded in soil from their own site until planting.

Soil materials were collected from the Bh horizon in LUE and the Bv horizon in BBR, air-dried at 15◦C, sieved to 4 mm, and homogenized. Plant residues were removed from the soils. Basic physical and chemical properties are listed in **Table 1** and were obtained as follows. Soil texture was determined using the pipette method (Gee and Bauder, 1986). Soil pH was measured in a 1:2 slurry in 0.01 M CaCl2 or water after 30 Min. equilibration.

1http://www.ecosystem-nutrition.uni-freiburg.de/

TABLE 1 | Physical and chemical properties of homogenized material from the Bv soil horizon at Bad Brückenau (BBR) and the Bh soil horizon at Unterlüss (LUE) used in the experiment; this includes grain size fractions, pH in two different extractants, exchangeable metal cations (Mex), organic C (Corg), total N (Ntot), and the following P fractions obtained by sequential extraction: resin exchangeable inorganic P (Presin), sum of inorganic P (extractable Pinorg) and organic P (extractable Porg) in various extracts (for details see text); all concentrations are given per mass dry soil; shown are means ± standard deviations of two technical replicates, except for sum parameters and element ratios.

Organic C (Corg) and total N (Ntot) contents of ground soil samples were measured using an elemental analyzer (NC 2500, CE Instruments Ltd, Hindley Green, Wigan, United Kingdom). Exchangeable cations (Alex, Caex, Feex, Kex, Mgex, Mnex, and Znex) were extracted with 1M NH4Cl for 1 h at 20◦C and a soil:extractant ratio of 1:10. The filtered extracts were analyzed for total elemental concentrations by inductively coupled plasma optical emission spectrometry (ICP-OES; Optima 7300 DV; Perkin Elmer, Waltham, MA, United States). Sequential P extraction was performed according to Hedley et al. (1982) as modified by Tiessen and Moir (2006). In **Table 1**, resin exchangeable inorganic P (Presin), the sum of inorganic P (Pinorg) in various extracts (0.5 M NaHCO3, 0.1 M NaOH before and after sonication, 1 M HCl, concentrated HCl) and the sum of organic P (Porg) in the NaHCO3 and NaOH extracts are shown. The soil from BBR exhibited much higher concentrations of both inorganic and organic extractable P than the LUE soil, but most importantly also resin exchangeable inorganic P, a measure of inorganic P in soil solution, and loosely sorbed to soil particles and thus of available P (Tamburini et al., 2012), was much higher in the BBR soil.

#### Experimental Setup

In April 2015, rhizoboxes were set up with beech saplings planted either in the soil from their site of origin or in the contrasting soil from the other site. In a completely randomized design, each treatment was replicated seven times. The rhizoboxes had inner dimensions of 60 cm × 25 cm × 1.5 cm. They consisted of PVC walls and a removable transparent lid made of polymethyl methacrylate. The soil was filled in at a bulk density of 1.2 kg/dm3 . After 1 week of soil conditioning under irrigation as described below, the saplings were planted. The roots of the saplings were washed with tap water to remove sticking soil, and approximately 2 cm of tap root were cut to stimulate new root formation. For each tree, the front plate of one rhizobox was opened, the roots pressed into the soil, and the front plate was closed again. At this time point, saplings possessed up to 10 cm long tap roots of 0.5–1.5 cm diameter but almost no fine roots, which presumably had died off during the storage. Rhizoboxes with trees were placed in a greenhouse with temperature control (22 ± 2 ◦C during the day/18 ± 2 ◦C at night), natural light and shading from the direct sun. Since shading with movable blinds was the only means for active cooling, at some days in summer temperatures higher than 22◦C occurred for short periods. The soil was kept dark, and to stimulate the formation of a quasi-planar root system along the transparent lid, the rhizoboxes were inclined at an angle of about 30◦ . Soil water potential in the rhizoboxes was kept at approximately -8 kPa by using irrigation tubes ("Rhizon irrigators," Rhizosphere research products, Wageningen, Netherlands) providing P-free artificial rain solution based on the composition of natural precipitation [2.1 µM K2SO4, 3.7 µM Na2SO4, 3.0 µM CaCl2, 4.4 µM CaSO4, 1.9 µM MgCl2, 26.4 µM NH4NO3, 2.0 µM Ca(NO3)2; Holzmann et al., 2016]. During summer, additional periodic irrigation from the top was needed to compensate for high evapotranspiration. At the end of the first growing season (end of September 2015), the rhizoboxes were placed outside of the greenhouse, but protected by a roof, to induce dormancy. In November 2015, they were moved to a dark cold room at 4◦C and periodically irrigated with artificial rain from the top. End of March 2016, after the last frost, the rhizoboxes were moved first to the protected area outside of the greenhouse, and in May, after appearance of the first leaves, back into the greenhouse with temperature control set to the same conditions as in the year before.

#### Plant Harvest and Analyses

During the first growing season in August 2015, when plants reached the phenological stage of fully developed leaves in both soils (Yang et al., 2016), five fully expanded leaves per plant were collected. Senescent leaves were collected at the end of the season after natural leaf abscission (December 2015) into nets spread around the plants. In August 2016 of the second growing season, the whole plants were harvested. At that time point, six saplings each from BBR growing in soil from BBR and LUE, 7 saplings from LUE growing in soil from BBR, and 3 saplings from LUE growing in soil from LUE had survived. The plants were divided into leaves, stem, coarse roots, and fine roots (diameter ≤ 2 mm).

The following analyses were performed on fresh tissue samples. The age of the saplings at final harvesting was determined by staining thin sections of the stem and subsequent tree-ring analysis (Gärtner and Schweingruber, 2013). According to this, the saplings from BBR were 11.7 ± 2.7 years old, and those from LUE 14.7 ± 1.6 years old. Subsamples of fully developed

leaves were used to measure the trichloroacetic acid (TCA) soluble P fraction (also called metabolic P; Wilcox et al., 2000). Approximately 200 mg of fresh leaves were frozen in liquid N2 in a 15 ml reagent tube and crushed to a fine powder using metal beads and vortexing. Powdered leaves were extracted for 1 h at 4◦C with 4 ml of 0.3 M TCA on a shaker. Extracts were filtered at 0.45 µm using glass fiber GF/F filters (Whatman International Ltd.). Inorganic P concentrations in TCA extracts were measured colorimetrically using malachite green (Van Veldhoven and Mannaerts, 1987). Bark and wood exudates were collected with the EDTA (Ethylene diamine tetra acetate) technique (Rennenberg et al., 1996; Gessler et al., 1998; Yang et al., 2016). Briefly, approximately 2 cm of the basal stem was collected fresh and separated into bark and wood. These parts were washed with deionized water and incubated in 2 ml of a solution 10 mM in Na2EDTA (pH 7) and 15 µM in chloramphenicol for 5 h at room temperature. Then, inorganic P in the incubation solution was measured colorimetrically as described above.

Plant parts not used for the analyses described above, were oven dried at 60◦C for 48 h (leaves and fine roots) or 72 h (stems and coarse roots), weighed, and ground to fine powder using a ball mill (Retsch MM400 Mixer Mill, Retsch GmbH, Retsch-Allee 1–5, 42781 Haan, Germany) with receptacle and balls made of agate. Total carbon and nitrogen contents of the ground material were measured by combustion using an elemental analyzer (NC 2500, CE Instruments Ltd, Hindley Green, Wigan, United Kingdom). The contents of total P were determined by ICP-OES (Optima 7300 DV; Perkin Elmer, Waltham, MA, United States) of digests obtained with a solution 8.3 M in HNO3 and 0.6M in HF using a microwave digestion unit (MW ultraCLAV, MLS, Milestone Inc., Shelton, CT, United States).

#### Mass and P Allocation Parameters

Biomass allocation to a specific plant compartment was calculated as mass fraction (g) of the compartment in percent of the total dry mass of the whole plant (g) (Poorter and Sack, 2012). Phosphorus allocation to a specific plant compartment was calculated as the mass fraction of P (g) in the plant compartment in percent of total plant P (g).

Resorption efficiency (RE) of nutrient elements X (X = P, N) from senescent leaves collected in December 15 was estimated by Eq. 1.

$$\text{RE(X)} = \left(1 - \left(\frac{(1 - 0.21) \times Xs}{Xf}\right)\right) \times 100 \tag{1}$$

Here Xs and Xf stand for nutrient concentrations in senescent and full season leaves, respectively. As no specific data on mass loss during senescence for beech was available, we used the average value of mass loss (21%) based on a multiple species analysis by Van Heerwaarden et al. (2003). A similar average mass loss of 21.6% was reported for "deciduous angiosperms" by Vergutz et al. (2012).

Considering the crucial role that xylem plays for P recycling in beech (Netzer et al., 2017), the P sink strengths of leaves (SLeaves), and fine roots (Sfine roots) were calculated as total P concentration in leaves and fine roots, respectively, divided by concentrations of inorganic P in wood exudates.

#### Plasticity Indices

We adapted the concept of plasticity indices (Valladares et al., 2006) to quantify the change of a given plant trait during the response of a beech sapling with a given P nutritional status to changes in soil P availability. For more on rationalizing the following equations see the respective subsection of "Discussion."

In a first step, we calculated the average potential span for a given trait (1Tr) as the arithmetic mean of pairwise differences between trait values of the m and n replicates, respectively, within the two treatments with beech saplings from BBR (TRBBRinBBR) and LUE (TRLUEinLUE) growing in soil from their own site using Eq. 2.

$$\Delta Tr = \frac{|\sum\_{i=1..n, j=1..m} (Tr\_{\text{BBR in BBR}}(i) - Tr\_{\text{LUE in LUE}}(j))|}{m\*n} \tag{2}$$

Only traits with significant differences between the mean trait values for the two treatments were taken into account.

In a second step, we calculated average plasticity indices for the response of saplings from LUE to soil with high P availability (PLLUEplant) and for the response of saplings from BBR to soil with low P availability (PLBBRplant). The plasiticity indices were calculated as the arithmetic mean of pairwise differences between trait values of the m and n replicates, respectively, within two treatments with beech saplings from a given site growing in soil from their own site and in soil from the other site, divided by the trait span (Eqs. 3 and 4).

$$PL\_{LUE\ plant}$$

$$= \left( \frac{|\sum\_{i=1\dots n, j=1\dots m} (Tr\_{\text{LUE in BBR}}(i) - Tr\_{\text{LUE in LUE}}(j))|}{m \ast n} \right) / \Delta Tr \tag{3}$$

PLBBR plant

$$= \left( \frac{|\sum\_{i=1\dots n, j=1\dots m} (Tr\_{\text{BBR in BBR}}(i) - Tr\_{\text{BBR in LUE}}(j))|}{m \ast n} \right) / \Delta Tr \tag{4}$$

#### Statistical Analysis

Using analysis of variance (ANOVA), we assessed to what extent current soil (the soil in which the beech saplings were growing during the experiment) on one hand, and plant origin (forest site where the beech saplings were collected) on the other hand, influenced the measured plant traits in the first and second growing season. All ANOVA analyses were performed in R, version 3.1.2 (R Core Team, 2014; RRID:SCR\_001905) with marginal type II test (Anova, package: "car") in order to account for unequal group sizes. Prior to ANOVA, data was subjected to Levene's test (leveneTest, package: "stats") for homogeneity of variance inside the groups and to the Shapiro-Wilk normality test (shapiro.test, package: "stats") for normality of residuals. Statistical significances indicated with

letters in figures and tables are the result of a one-factorial ANOVA (with treatment as explanatory variable) with a Tukey post hoc test (HSD.test, package: "agricolae"). Average plasticity indices for plants from BBR and LUE were compared using multiple t-tests. Each pair was analyzed individually, without assuming a consistent standard deviation, using GraphPad Prism 7.02 Software. The standard error of the mean for the plasticity indices was computed for the true sample size using Gaussian error propagation and assumption of no error in 1Tr.

#### RESULTS

### Foliar Element Concentrations and Nutrient Resorption in the First Growing Season

During the first growing season, both total and metabolic P concentrations in leaves were slightly higher for beech saplings originating from BBR, the site with high soil P availability, than for those from LUE, the site with low soil P availability (**Table 2**). According to ANOVA, both concentrations were significantly affected only by the factor "plant origin" (**Table 3**). The first measured reaction of the saplings to the soil they were growing in during the experiment was a higher P resorption from senescent leaves for all plants growing in LUE soil than for those growing in BBR soil, irrespective of plant origin (**Figure 1**). ANOVA revealed "current soil" as sole significant factor (**Table 3**). On the other hand, N resorption from senescent leaves did not differ among the treatments and was not affected neither by plant origin nor by current soil conditions (**Figure 1** and **Table 3**).

#### Phosphorus Concentrations in Different Plant Compartments in the Second Growing Season

In the second growing season, total P concentrations in stem and coarse roots, and average concentrations in the whole plant still reflected the soil P availability at the site of plant origin with higher values for the saplings from BBR than for those from LUE (**Table 4**). Plant origin dominated as factor in the ANOVA (**Table 5**). By contrast, the P concentrations in fine roots were significantly affected mainly by the factor current soil (**Table 5**) and were higher for saplings growing in the BBR soil (**Table 4**). Total and metabolic P concentrations in leaves as TABLE 3 | Analysis of variance for different traits of beech (Fagus sylvatica L.) saplings as determined in the first growing season of a rhizobox experiment; traits include concentrations of P (total P, Ptot; metabolic P, Pmetabolic) and total N (Ntot) in full season and senescent leaves as well resorption efficiency for P and N; saplings originated from the sites Bad Brückenau (BBR) with high soil P availability and Unterlüss (LUE) with low soil P availability (factor plant origin), and were grown in material from the Bv horizon at BBR or from the Bh horizon at LUE (factor current soil); shown are F values for the factors and their interactions; statistical significance is indicated as ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05, ns P > 0.05.

well as inorganic P concentrations in bark and wood exudates were similarly affected by both plant origin and current soil (**Table 5**), which led to higher values for the saplings from BBR growing in mineral soil from their own site than for all other treatments (**Table 4**).

The sink strengths of leaves and roots were still clearly dominated by the factor plant origin (**Table 5**) and were higher for the saplings from LUE (**Figure 2**).

#### Biomass and P Allocation to Leaves and Roots in the Second Growing Season

Total plant biomass and biomass of leaves and roots were mainly and significantly affected by the factor current soil (**Table 5**), with the smallest values for saplings from LUE growing in soil from their own site (**Table 4**). By contrast, stem biomass did not differ among the treatments.

Irrespective of the treatment, the largest percentage of biomass and P was allocated to coarse roots (**Figure 3D**). Of all measured plant traits, relative allocation of P to leaves most clearly reflected the factor current soil (**Table 5**) with significantly higher values for beech saplings growing in the BBR soil, irrespective of their site of origin (**Figure 3A**). Also, P allocation to fine roots exhibited a significant influence of current soil (**Table 5**), with

TABLE 2 | Concentrations of P (total P, Ptot; metabolic P, Pmetabolic) and total N (Ntot) in full season and senescent leaves of beech (Fagus sylvatica L.) saplings, measured in the first growing season of the experiment; saplings originated from the sites Bad Brückenau (BBR) with high soil P availability and Unterlüss (LUE) with low soil P availability, and were grown in material from the Bv horizon at BBR or from the Bh horizon at LUE; data represent mean concentrations per unit mass dry weight ± SE; different letters indicate significant differences between means according to the Tukey post hoc test.

values tending to be higher for saplings growing in BBR soil (**Figure 3C**). Noteworthy was the clearly highest value of all treatments for saplings from LUE growing in soil from BBR. On the other hand, allocation of biomass to the stem was still mainly determined by plant origin (**Table 5**) with a tendency to higher values for saplings from LUE (**Figure 3B**). A significant but similarly strong influence of both current soil and plant origin was detected for the allocation of biomass to leaves and fine roots (**Table 5**), with the highest and lowest values for the saplings from BBR and LUE, respectively, growing in their own soil (**Figures 3A,C**).

#### Plasticity Indices

**Figure 4** shows the plasticity indices for beech saplings from LUE and BBR considering all plant traits for which the precondition of a significant difference between the two treatments with the saplings growing in soil from their own site was fulfilled. Total and metabolic P concentrations in full season leaves, total P concentrations in senescent leaves, and P resorption were more plastic for beech saplings from BBR responding to soil with low P availability. On the other hand, P and mass allocation to stem were more reactive for beech saplings from LUE acclimating to soil with high P availability. For all other traits, plasticity was similar for beech saplings from both origins, and thus independent on the direction of acclimation. However, values differed strongly with particularly high indices for P allocation to leaves and P concentration in fine roots and particularly low indices for P concentrations in stem and coarse roots.

#### DISCUSSION

Our experimental model systems employed beech saplings from forest sites strongly differing in soil P availability in terms of both measures of available P such as Presin and total P stocks (Lang et al., 2017), BBR with high, and LUE with low P availability. At the end of the experiment, the saplings from the two sites still exhibited a significantly different P nutritional status in terms of average P concentration in the whole plant. Using material from mineral soil horizons from the two sites as model soils, while providing homogeneous material for the experimental replicates, possibly changed soil P availability compared to the natural situation, in particular in the case of the LUE site, where 50% of the P stock is stored in the organic surface layer (Lang et al., 2017). Nevertheless, in the second growing season total

TABLE 4 | Concentrations of P (total P, Ptot; metabolic P, Pmetabolic; inorganic P in bark exudates, PBark\_Exudates; inorganic P in wood exudates, Pwood\_exudates) in and biomass of different compartments of beech (Fagus sylvatica L.) saplings, measured in the second growing season of a rhizobox experiment; saplings originated from the sites Bad Brückenau (BBR) with high soil P availability and Unterlüss (LUE) with low soil P availability, and were grown in material from the Bv horizon at BBR or from the Bh horizon at LUE; data represent mean concentrations per unit mass dry weight (concentrations) or g dry weight (biomass) ± SE; different letters indicate significant differences between means according to the Tukey post hoc test.

TABLE 5 | Analysis of variance for different traits of beech (Fagus sylvatica L.) saplings as determined in the second growing season of a rhizobox experiment; traits include, for different plant compartments, concentrations of P (total P, Ptot; metabolic P, Pmetabolic; inorganic P in bark exudates, PBark\_Exudates; inorganic P in wood exudates, Pwood\_exudates), biomass, sink strength, and relative allocation of total P and biomass; saplings originated from the sites Bad Brückenau (BBR) with high soil P availability and Unterlüss (LUE) with low soil P availability (factor plant origin), and were grown in material from the Bv horizon at BBR or from the Bh horizon at LUE (factor current soil); shown are F values for the factors and their interactions; statistical significance is indicated as ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05, ns P > 0.05.

P concentrations in all plant compartments where similar to beech saplings collected at the same sites in the same year as our saplings, but were excavated together with an undisturbed soil core, and further grown this way for one season in the greenhouse (Zavišic et al., 2018 ´ ).

FIGURE 2 | Sink strength of leaves (black bars) and fine roots (gray bars) of beech (Fagus sylvatica L.) saplings in the second growing season of the experiment; saplings originated from the sites Bad Brückenau (BBR) with high soil P availability and Unterlüss (LUE) with low soil P availability, and were grown in material from the Bv horizon at BBR or from the Bh horizon at LUE; data represent mean values ± SE for the different treatments; different letters indicate significant differences between means according to the Tukey post hoc test (lowercase letters, leaves; uppercase letters, fine roots).

The results of our experiment thus allow to discuss the influence of P nutritional status on the plant internal allocation of P and biomass for beech saplings growing in soil with different P availability. We consider the treatments with saplings growing in the soil from their own site to represent the situation of plants adapted to a given soil situation. We furthermore assume that the results from the treatments with saplings growing in the soil from the other site provide clues on the plastic response of beech that allows it to acclimate to changes in soil nutrient conditions.

### Foliar P Concentrations in Beech Depend on Both Soil P Availability and P Nutritional Status of the Plant

As pointed out in the introduction, P concentrations in tissues of trees can strongly vary with the season (Eschrich et al., 1988; Netzer et al., 2017; Zavišic and Polle, 2018 ´ ) which indicates site dependent dynamics of nutrient uptake and plant internal nutrient allocation. Some studies even suggest that P utilization in beech can be decoupled from P uptake, i.e., the growth of young leaves may strongly depend on the transport of nutrients stored in the previous season in organs such as stem and coarse roots, but also from older leaves during a growing season (Schachtman et al., 1998; Güsewell, 2004; Zavišic et al., 2018 ´ ). This could well explain our observations, that during the first growing season, total and metabolic P in leaves reflected the P availability of the soil at the site of plant origin rather than the soil in which the beech saplings were growing during the experiment. The results of the second growing season point to combined effects of current soil and plant origin. On one hand, the low P concentrations in leaves and roots of the beech saplings growing in the LUE soil, irrespective of their site of origin and thus their

for the different treatments; different letters indicate significant differences between means according to the Tukey post hoc test (lowercase letters, mass allocation;

uppercase letters, P allocation).

FIGURE 4 | Average plasticity indices for the response of beech (Fagus sylvatica L.) saplings from the site Unterlüss (LUE) with low P nutritional status to mineral soil from Bad Brückenau (BBR) with high P availability (black bars) and for saplings from the site BBR with high P nutritional status to mineral soil from LUE with low P availability (gray bars); data represent mean values ± SE of all data pairs; statistical significance is indicated as ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05.

P nutritional status, were not surprising. On the other hand, the leaves of LUE saplings growing in BBR soil exhibited equally low P concentrations, despite high P uptake as indicated by relatively high P concentrations in fine roots. Taken together, and considering in addition the equally high mass allocation to leaves for LUE trees growing in BBR soil and for BBR trees

growing in LUE soil, these results point to the ability of beech to deal with different soil P availability optimally at a given P nutritional status of the plant by internal allocation of P and mass. It suggests a priority to alleviate the primary light limitation by producing as much photosynthetic organs as possible, while vacuolar P concentrations are kept low when soil P availability and/or the P nutritional status of the whole plant is low. The latter has been shown to be a successful mechanism in other plants (Lee et al., 1990; Lee and Ratcliffe, 1992; Mimura, 1995; Mimura et al., 1996). Comparing the leaf P concentrations in the second growing season with the threshold values for young beech trees published by Göttlein (2015) suggests the following. While a P concentration within the normal range (1.1–2.1 mg/g DW) indicates both a high P nutritional status of the plant and high soil P availability, a P concentration below the lower threshold of 1.1 mg/g DW may indicate that either the P nutritional status of the plant or the soil P availability or both are low. The same interpretations seem to apply to N/P ratios which, according to Mellert and Göttlein (2012) were in the normal range for BBR plants growing in BBR soil and above in all other cases.

#### P Resorption From Senescent Leaves Is the First Reaction to Current Soil Conditions

Phosphorus resorption from senescent leaves is a nutrient conservation mechanism, common for several plant species including beech, which reduces losses, and decreases the nutrient uptake demand for the next year (Aerts, 1996; Brant and Chen, 2015). As a consequence, there is less input of P with litter fall diminishing a recycling pool in the ecosystem (Lang et al., 2017). The higher P resorption efficiency of plants grown in the LUE soil than for those grown in the BBR soil, irrespective of their P nutritional status, are in general agreement with findings from recent chronosequence studies by Richardson et al. (2004) and Hayes et al. (2014). The sensitive reaction of this parameter to the current soil in the first growing season, when the concentrations in mature green leaves still reflected the nutrient situation at the site of plant origin, confirms the findings of Hofmann et al. (2016) who showed that fertilization of beech saplings growing in soil low in P led to a decrease in P resorption efficiency. By contrast, the similar values of N resorption efficiency, measured in our experiment for all treatments, indicate a similar degree of N availability. The observed level of about 50% falls within the average range compiled by Aerts (1996) for deciduous trees and shrubs.

#### P Allocation to Leaves Is a Sensitive Indicator of Soil P Availability

In this section we discuss in more detail the patterns of relative P and mass allocation to various plant compartments in the second growing season. While the link between nutrient allocation and a number of site factors such as precipitation, forest type, latitude, and plant age has been well studied (Sardans and Peñuelas, 2013), little is known about how soil P availability affects P allocation (Yang et al., 2016).

Let us first compare the saplings from LUE and BBR when growing in the soil from their site of origin. The lower allocation of mass and P to leaves and fine roots and higher allocation to the stem for the beech saplings from LUE are consistent with a conservative strategy reducing growth under nutrient limitation. Yang et al. (2016) also observed a slower growth of beech saplings from LUE than from BBR when growing in undisturbed soil cores from their own site of origin which represents a more natural situation than our experiment. A smaller root system in soil of low P availability is in line with the resource economics hypothesis (Grime, 1977; Craine, 2009) and the general characteristic that plant ecotypes from nutrient-limited environments grow slower than ecotypes from fertile soils. At the same time a tendency to a higher ratio between allocation of biomass to fine roots and leaves for the LUE saplings (mean ± SE: 2.0 ± 0.5) than for BBR saplings (mean ± SE: 1.6 ± 0.2) is in accordance with the notion that under nutrientlimited conditions plants should allocate proportionally more resources to roots (George et al., 2011). We do not know to what degree the difference in the size of the root system between these two treatments translates into volume of soil explored. In both treatments about 50% of the root tips were mycorrhized (mean ± SE: BBR in BBR, 49 ± 3%; LUE in LUE, 51 ± 10%), but extension of the hyphal system was not assessed.

When considering the two treatments with beech saplings growing in soil from the other site, the most striking result is that P allocation to leaves was the same as for the saplings adapted to the respective site, while mass allocation was intermediate in both cases. This indicates that P allocation to leaves might be a particularly good indicator of soil P availability, irrespective of P nutritional status of the plant. It further may be a key trait that the plant adjusts by balancing leaf biomass production against the transfer of nutrients from the soil or from/to internal storage organs such as stem or coarse roots.

Our results for the BBR plants growing in LUE soil indicate that the response of beech saplings with a high P nutritional status to low soil P availability is to decrease leaf P concentration, but to still produce as much leaf biomass as possible under the new low P flux into the roots. At this stage of plastic response, the substantial amount of P stored in coarse roots, and stem allows to partly compensate for the low P uptake. This compensation is also indicated by intermediate P concentrations in bark and wood exudates, maintaining the sink strength of leaves and roots at a low value similar to BBR plants growing in their own soil.

From the results for the LUE plants growing in BBR soil it appears that a beech sapling with low P nutritional status in response to high soil P availability increases leaf biomass production as much as possible under the new high P influx, while it keeps P concentrations in leaves low. Phosphorus concentrations in fine roots as high as for the saplings from BBR adapted to this soil, and the highest P allocation to fine roots of all treatments might be explained by inefficient P translocation to the aboveground plant compartments or by P recycling from stem and old leaves to support fine root growth (Marschner et al., 1996; Netzer et al., 2017). The higher sink strength of fine roots for LUE than BBR plants when growing in BBR soil argue rather in favor of inefficient translocation.

The significant difference in P allocation to leaves between beech saplings growing in BBR and LUE soil was not found for saplings growing in undisturbed soil cores from their own site (Zavišic and Polle, 2018 ´ ; Zavišic et al., 2018 ´ ). For a sampling time in July/August P allocation in saplings from both sites was similar to the one we measured for saplings growing in BBR soil, but also varied strongly during the growing season. This discrepancy may be explained by the presence of the organic surface layer in the undisturbed soil cores which could have increased the effective availability of soil P in the case of the LUE plants. The importance of the organic surface layer for the nutrition of young beech trees from soil low in P was clearly demonstrated by Hauenstein et al. (2018) who showed that the presence of the organic surface layer improved P nutrition and growth of beech seedlings in the LUE soil but had no effect in the BBR soil. The effect of the organic surface layer at LUE was attributed on one hand to a particularly high microbial activity promoted by a high root density, but also to a high water retention capacity minimizing loss of added or mobilized P to the mineral soil (Hauenstein et al., 2018). In particular, the high water holding capacity of the surface layer at LUE might have led to a similar effective P availability in the LUE soil as in the BBR soil during the well watered experiments with undisturbed soil cores. On the other hand, at on average drier conditions in the field one would expect a smaller effect, being consistent with the lower P nutritional status of beech saplings from LUE than from BBR.

Furthermore, we cannot exclude that stress induced by the transplantation process (accidental cutting of some roots during sampling, dying-off of fine roots during storage between sampling, and planting) may have affected P allocation even in the second growing season. In particular, "recovery" of a mycorrhized root system in the new soil could have led to a temporary elevated biomass and P allocation to the fine roots. A comparison with the more natural situation in the studies of Zavišic and Polle (2018) ´ and Zavišic et al. (2018) ´ revealed that biomass and P allocation were twice as high in our rhizoboxes than in the undisturbed soil columns of the mentioned studies for saplings from BBR but similar for saplings from LUE. Considering (i) the high variability among individual saplings, (ii) the differences in environmental conditions between the rhizobox and column studies, and (iii) that the larger differences occurred for the more fertile soil from BBR, this comparison does not indicate a large effect of transplantation in our rhizoboxes.

#### Phosphorus Allocation to Leaves and P Concentrations in Fine Roots Represent Best the Plastic Response of Beech to Changes in Soil P Availability

In this section we discuss the differences in various plant traits among the experimental treatments in terms of a plastic response of beech saplings to changes in soil P availability. For this, we assume that the trait values of saplings growing in the soil from their site of origin represent the extremes and thus define the potential trait span. We further assume, that the higher the degree to which the value of this trait changes relative to the trait span for a sapling exposed to soil from the other site, the higher is the plastic response of this trait. We did neither include P allocation to fine roots nor P and mass allocation to coarse roots, because there was no significant trait span according to the definition. We interpret the differences between the trait plasticity for BBR plants exposed to low P soil and for LUE plants exposed to high P soil as differences between the acclimation processes from high to low and from low to high soil P availability.

Considering all our plasticity indices, they indicate a rather high plasticity compared with other studies employing various plasticity quantification methods (Valladares et al., 2002; Stojnic´ et al., 2013, 2015a). The direction of acclimation affected the plasticity index for some traits but was unimportant for others. While the plasticity of P resorption and P concentration in leaves was higher for the beech saplings with a high P nutritional status acclimating to low P soil, P and mass allocation to the stem were more reactive for saplings with a low P nutritional status acclimating to soil with high P availability. The asymmetry in size of the reaction of the mentioned traits emphasizes the importance of taking into account the initial nutritional status of a plant when assessing plasticity. On the other hand, the strong responses of P allocation to leaves and P concentration in fine roots, which were similar for both directions of acclimation, suggest that these plant traits are most suitable to assess plasticity of beech in response to soil P availability.

### CONCLUSION

A two-year cross-growth experiment with beech saplings and mineral soil from two forest sites differing strongly in soil P availability, demonstrated a high plasticity of juvenile F. sylvatica to differences in soil P availability. Some influence of recovery from stress implicated by the transplantation on these results, can however, not be excluded. Relative P allocation to leaves appears to be a particularly good indicator of soil P availability, irrespective of the P nutritional status of the plant.

In contrast to P allocation to leaves, foliar P concentrations were not a clear indicator of soil P availability, which may partly explain the lack of relation between leaf P concentrations and soil P availability in studies comparing forest sites on the European or regional scale (Talkner et al., 2015; Lang et al., 2017). In particular, the ambiguity with respect to the interpretation of foliar P concentrations below threshold values for normal growth implies that the observation of such low values for beech on monitoring sites (Jonard et al., 2015; Talkner et al., 2015) not necessarily indicates P deficiency and respective growth reduction, but rather a tree with still good P nutritional status but reacting to a decrease in soil P availability.

Overall, the results clearly rebut our working hypothesis, that adaptation of beech saplings to new soil conditions is driven by the plant striving to achieve or maintain high foliar P concentrations. By contrast, they point to a sensitive signaling network that allows the plant to produce as much biomass as possible under given soil conditions by regulating mass and nutrient allocation accordingly.

#### DATA AVAILABILITY

fpls-10-00744 June 6, 2019 Time: 9:16 # 11

The datasets generated for this study are available on request to the corresponding author.

#### AUTHOR CONTRIBUTIONS

All authors designed the experiment and contributed significantly to the final version of the manuscript. SM and JL collected the plant and soil materials used in the study. SM set-up and carried out the experiment, performed most of the chemical analyses, analyzed the data, and wrote the first version of the manuscript.

#### FUNDING

This project was carried out in the framework of the Priority Program SPP 1685 "Ecosystem Nutrition" of the German

#### REFERENCES

and Swiss National Science Foundation (DFG and SNF, respectively). This particular project was funded by the SNF project no. 149138 and by internal funds of the Swiss Federal Research Institute WSL.

#### ACKNOWLEDGMENTS

The forest research stations "Bayerische Landesanstalt für Wald und Forstwirtschaft" and "Nordwestdeutsche Forstliche Versuchsanstalt" provided access to the field sites. The following groups at the Swiss Federal Research Institute WSL provided technical assistance: the technical support at Birmensdorf and the workshop at Davos helped with construction of the rhizoboxes, the experimental garden operated and maintained the greenhouse, the central analytical laboratories carried out part of the chemical analyses, and the soil physical and chemical laboratories provided various technical support. Andrea Polle (University of Göttingen) provided data from their experiments with undisturbed soil cores for comparison.

**Conflict of Interest Statement:** 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.

Copyright © 2019 Meller, Frossard and Luster. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Phosphorus Economy of Mediterranean Oak Saplings Under Global Change

#### Inga Dirks1† , Julia Köhler2 , Shimon Rachmilevitch1 and Ina C. Meier2 \*

1 French Associates Institute for Agriculture and Biotechnology of Drylands, Ben Gurion University of the Negev, Beersheba, Israel, 2 Plant Ecology, Albrecht-von-Haller Institute for Plant Sciences, University of Göttingen, Göttingen, Germany

#### Edited by:

Jian-Guo Huang, University of Chinese Academy of Sciences (UCAS), China

#### Reviewed by:

Raquel Lobo-do-Vale, Universidade de Lisboa, Portugal Xiali Guo, South China Botanical Garden, Chinese Academy of Sciences, China

> \*Correspondence: Ina C. Meier<EMAIL_ADDRESS>
> > †Present address:

Inga Dirks, Department of Crop Sciences, Division Agronomy/Crop Science, University of Göttingen, Göttingen, Germany

#### Specialty section:

This article was submitted to Functional Plant Ecology, a section of the journal Frontiers in Plant Science

Received: 29 October 2018 Accepted: 18 March 2019 Published: 05 April 2019

#### Citation:

Dirks I, Köhler J, Rachmilevitch S and Meier IC (2019) The Phosphorus Economy of Mediterranean Oak Saplings Under Global Change. Front. Plant Sci. 10:405. doi: 10.3389/fpls.2019.00405 While a severe decrease in phosphorus (P) availability is already taking place in a large number of ecosystems, drought and nitrogen (N) deposition will likely further decrease the availability of P under global change. Plants have developed physiological strategies to cope with decreasing P resources, but it is unclear how these strategies respond to elevated N deposition and summer droughts. We investigated the influence of N and P availability and soil drought on P uptake (H3 33PO4 feeding experiment) and use efficiencies in young Quercus calliprinos Webb. trees. We hypothesized that (H1) the expected increases in soil N:P ratios will increase the efficiencies of P uptake and use of oak saplings but will decrease the efficiencies of N uptake and use, whereas (H2) drought will affect P uptake efficiency more than N uptake efficiency. In confirmation of (H1) we found that a sharp increase of the soil N:P ratio from 4 to 42 g g−1 significantly increased the instantaneous 33P uptake efficiency (33PUptakeE) by five-fold and longterm P uptake efficiency (PUptakeE) by six-fold, while it decreased N uptake efficiency (NUptakeE) and N use efficiency (NUE). In contradiction to (H1), P use efficiency (PUE) did not respond to the simulated extended gradient of soil N:P ratios but remained relatively constant. (H2) was only partially confirmed as soil drought reduced PUptakeE by up to a fourth at high soil N:P ratios but had no significant effect on NUptakeE. As a consequence, increasing summer droughts may decrease the response of PUptakeE to increasing P limitation, which – in the absence of adjustments of the efficiency of P use – can aggravate growth reductions in this eastern Mediterranean tree species under global change.

Keywords: drought, eastern Mediterranean, global change, nitrogen deposition, phosphorus limitation, Quercus calliprinos, uptake efficiency

### INTRODUCTION

Increasing N deposition and summer droughts under global change will induce nutritional imbalances and large-scale undersupply of essential plant nutrients such as P in a large number of natural forest ecosystems (Talkner et al., 2015; Sardans et al., 2016), but especially in Eastern Mediterranean forests where extreme climate conditions are already common and the effect of global change will be disproportional (Lelieveld et al., 2012). The deterioration of P availability is already indicated by decreasing P concentrations and increasing N:P ratios in leaves and fine roots, as well as by reports on growth reductions due to P limitations for various regions of Central and

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Southern Europe and elsewhere (Elser et al., 2007; Peñuelas et al., 2012). This trend of increasing P deficiency of forest trees is even more pronounced in regions where P availability had already been low before (Jonard et al., 2015): Mediterranean ecosystems are commonly characterized by geological substrates poor in mineral P content and by accumulation of occluded P with longer soil development (Singer, 2007; Cramer and Hoffman, 2015) and, thus, may shift from latent to apparent P deficiency under global change.

Plants can respond to P deficiency by increasing their P uptake and use efficiencies. P uptake is generally constrained since most P occurs sorbed to or occluded in Fe and Al oxides, precipitated as secondary mineral, or integrated in humic substances but not in plant-available form (Schachtman et al., 1998; Ragothama, 1999). In addition, the low diffusion coefficient of plant-available soluble H2PO4 − causes a distinct depletion zone in the rhizosphere. Accordingly, several adaptation or acclimation strategies for the increase of the rate and efficiency of P uptake exist. Among them the most fundamental plant response is to increase the root:shoot ratio, adjust the root architecture toward higher branching intensity, and increase root proliferation into unexplored soil regions (Lambers et al., 2006). In addition, plants can enhance the exudation of organic acids or the secretion of phosphatases and phytases, which increases the solubility of labile and organic P (Neumann and Römheld, 2007; Hofmann et al., 2016). Plant P uptake efficiency (PUptakeE) can also be increased directly by an elevated expression of high-affinity Pi-transporters and other alterations of the membrane structure (Smith et al., 2000; Kavka and Polle, 2017) and by colonization with mycorrhizal fungi (Torres Aquino and Plassard, 2004; Cairney, 2011). In contrast to PUptakeE, increases in P use efficiency (PUE) are mostly achieved by internal reallocation of plant P to photosynthetic and growth functions (Lambers et al., 2012; Veneklaas et al., 2012).

Global change executes several constraints on the relation between P availability and PUptakeE: first, atmospheric N deposition from anthropogenic sources induces nutritional imbalances and accelerates the P demand of forest trees (Jonard et al., 2015). Second, the expected increases in the duration and intensity of summer droughts will likely further impair P availability due to decreased P mineralization (Schimel et al., 2007) and reduced mobility (Schachtman et al., 1998) and diffusion rates of P in drier soil (Kreuzwieser and Gessler, 2010). Secretion of phosphatases and expression of high affinity Pi transporters are additionally expected to be interfered by drought (Sardans and Peñuelas, 2007; Lang et al., 2016) as well as the mycorrhizal symbiosis (Kleczewski et al., 2010). These negative drought effects on P uptake seem to be even more pronounced than their effects on N uptake, leading to reductions in plant P storage (Sardans and Peñuelas, 2007) and further divergence of the N:P ratios with drought (He and Dijkstra, 2014).

In the dry eastern Mediterranean climate, Palestine oak (Quercus calliprinos Webb) is a common evergreen broadleaf tree species which is widely distributed from northern Algeria to southern Turkey and achieves predominance in the natural Mediterranean scrub woodland of Israel (Zohary, 1966; Schiller et al., 2003). Palestine oak grows on a relatively broad spectrum of soils and is used for reforestation, erosion control, and soil conservation. Yet despite its occurrence across a wide range of mean annual precipitation (from 460 to 1000 mm a−1 at higher elevation and altitude; Kutiel and Naveh, 1987; Klein et al., 2013), it is more drought sensitive than other co-occurring tree species and is limited by high temperatures (Helman et al., 2017) and low nutrient availability (Tognetti et al., 2000), which could reduce its growth and vitality under a future drier and warmer climate. The predicted climate change conditions in the eastern Mediterranean include faster warming than expected for the global mean, an increasing number of very hot days in summer, and a further decline in precipitation, especially in spring and summer (Lelieveld et al., 2012; Jacob et al., 2014). Concerns are rising that climate change in combination with N deposition will have disproportional effects on the P nutrition for the forest vegetation in this region compared to other areas – especially at sites that are currently already at the drought edge of distribution of Palestine oak.

To improve our understanding of the complex responses of P and N uptake and use efficiencies to the expected global change conditions, we exposed young Palestine oak trees to an increase in soil N:P ratios and a decrease in soil moisture in a two-factorial climate chamber experiment (3 N:P ratios × 2 soil moisture levels). We examined oak uptake and use efficiencies from a 33P feeding experiment, photosynthetic measurements, and elemental analyses of recent biomass. Subsequently, we analyzed the responses of P and N uptake and use efficiencies to decreasing P availability and decreasing soil moisture. We hypothesized that (H1) the increase in soil N:P ratios increases the efficiencies of P uptake and use of oak saplings but decreases the efficiencies of N uptake and use; and (H2) drought affects P uptake efficiency more than N uptake efficiency.

#### MATERIALS AND METHODS

### Plant Material and Experimental Set-Up

Our experiment was conducted with evergreen Palestine oak (Quercus calliprinos Webb.) saplings that were raised from seeds collected from a mature oak forest in the Lower Galilee in northern Israel (close to Turan; N 32◦ 460 , E 35◦ 220 ; plant nursery: Keren Kayemeth LeIsrael, Jerusalem, Israel). In February 2015, 48 bare-rooted 1.5-year-old oak trees of similar size were carefully transported to the botanical garden in Göttingen, Central Germany, where they were rinsed with bi-distilled water and planted in 6-L pots filled with sterilized mineral sand / very fine gravel with a particle size of ≥2 mm, a soil texture that facilitates root studies. The potted oak saplings were placed in a randomized array in a greenhouse chamber with climate control (air temperature 21/18◦C day/night, day length 12 h; in representation of the growing season conditions of Palestine oak between February and October) and were cultivated for 30 weeks until the start of the physiological and biochemical measurements in September 2015. The selected saplings had an average shoot height of 67 ± 2 cm (measured from the soil surface to the top of the sapling), a crown volume of 5130 ± 570 cm3 , and a mean maximum stem diameter of 0.6 ± 0.1 cm. Oak saplings were rerandomized bi-weekly. We simulated a gradient of decreasing

P availability in response to an increase in N deposition and decrease in soil moisture as expected under global change. We established two soil moisture conditions, a well-watered (M2; 10 % soil water content, v/v; close to field capacity) and a drought treatment (M1; 6.7% soil water content, v/v; close to the permanent wilting point) based on the texture of the sandy inorganic growth medium used in the experiment. Soil water contents were biweekly adjusted for plant water consumption by homogeneous irrigation with demineralized water to bring the soil back to its target soil moisture content.  Our N addition schedule is, thus, a compromise between N deposition rates that are two to six times higher than the expected deposition rates (if extrapolated to a whole year: 44 and 91 kg N ha−1 yr−1 ) and final soil N concentrations that were still 5–33 times lower than their average in the eastern Mediterranean (final soil N: 0.15 and 0.16 g N kg−1 ). N was added weekly as KNO3, NaNO3, and NH4NO3 by watering the soil surface with a dissolved fertilizer. We did not consider leaf N uptake in our experiment. Finally, we established two P availability levels, a low (P2; 1.79 mg kg−1 ) and an extremely low P supply treatment (P1; 0.36 mg kg−1 ) on the basis of the estimated plant-available P content in the sandy substrate used for the experiment (c. 3 mg Presin kg−1 ; cf. Köhler et al., 2018) and typical plant-available soil P concentrations in the eastern Mediterranean (4–11 mg Pbicarbonate kg−1 ; Henkin et al., 1994). P was added weekly as KH2PO4 by watering with a dissolved fertilizer. Our fertilization regime led to three N:P treatments: the relative N:P ratio increased from N1P2 (control; N/P = 4 g g−1 ) to N2P2 (elevated N addition; N/P = 8 g g−1 ) and N2P1 (elevated N addition and P limitation; N/P = 42 g g−1 ), with eight replicates per treatment. To avoid differences in salinity, we controlled for the electric conductivity of all stock solutions (σ ∼0.77 dS m−1 ). Micronutrients were added three times during the experiment with a diluted Hoagland solution.

#### Photosynthetic Carbon Assimilation

Leaf gas exchange measurements were conducted at the end of September 2015 on one canopy leaf per sapling with an infrared CO2 analyzer (LI-6400; LI-COR Biosciences, Lincoln, NE, United States) during the midday hours, i.e., between 10.30 a.m. and 1.30 p.m. We measured area-based midday leaf photosynthesis (A800; µmol CO2 m−2 s −1 ) at high photosynthetically active radiation (800 µmol PAR m−2 s −1 ). During the measurements, average leaf temperature was 21◦C, relative humidity 40%, vapor pressure deficit 12 hPa, and CO2 concentrations ambient (400 µmol CO2 mol−1 air). Mass-based leaf photosynthesis (in µmol CO2 g −1 s −1 ) was calculated from A800 and the ratio of leaf area per dry weight (specific leaf area, SLA; in cm2 g −1 ; for a description of the measurement see the description of the analysis of shoot biomass and morphology below). We calculated the instantaneous, latesummer photosynthetic P and N use efficiencies according to PPUE = A800 ∗ SLA/PLeaves and PNUE = A800 ∗ SLA/NLeaves, respectively (in µmol CO2 g −1 P or N s−1 ), where PLeaves is the foliar P concentration and NLeaves the foliar N concentration (cf. Li et al., 2012; Gan et al., 2016).

### 33P Labeling Experiment

To measure 33P uptake capacity at the end of the experiment, half of the experimental saplings representing all treatments were selected for a H3 33PO4 feeding experiment, which was conducted in the Laboratory for Radioisotope Research (LARI), University of Goettingen. Soils of the potted saplings were labeled with 100 mL H3 33PO4 tracer solution (total activity per sapling 1 MBq 33P), which was evenly injected to the soil at ten injection points and three soil depths (2, 4, and 8 cm soil depth, respectively). After an application time of 120 min, saplings were immediately excavated and separated into fine roots (≤2 mm), coarse roots (>2 mm), the stem base (base to 10 cm stem height), the older top stem which had originated before the experiment (above 10 cm stem height), the younger top stem which developed during the experiment, the older leaves which had originated before the experiment, and the younger leaves which developed during the experiment. All compartments were immediately frozen in liquid N2 and stored at −80◦C to avoid any further progressing of 33P. A subsample of mixed soil was frozen similarly and used for the determination of the specific 33P activity in soil (i.e., isotope dilution of 33P; determined from the amount of 33P per amount of soil inorganic Pi ; in Bq mg−1 Pi ; cf. Zavišic and Polle, 2018 ´ ). All plant samples were placed for 7 days in a freeze drier (BETA I, Christ, Osterode/Harz, Germany), weighed, and ground (Mixer Mill MM 400; Retsch, Haan, Germany). Subsequently, ground samples were digested with 65% HNO3 at 180◦C (cf. Heinrichs et al., 1986), filtered (filter paper grade MN 640 w; Macherey Nagel, Düren, Germany), and mixed with a scintillation cocktail (Rotiszint eco plus LSC cocktail; Roth, Karlsruhe, Germany). The 33P radioisotope signature was determined with a low activity liquid scintillation analyzer (Tri-Carb 3180 TR/SL; Perkin Elmar, Waltham, MA, United States). 33P uptake was determined separately for fine roots, coarse roots, and aboveground biomass. The mass-specific rate of 33P uptake (33PUptake) was calculated from the whole-plant amount of 33P after 2 h divided by the whole-plant biomass; and division of the quotient by the specific 33P activity (in g P g−1 DW d−1 ; cf. Zavišic and Polle, ´ 2018). The instantaneous 33P uptake efficiency (33PUptakeE; in Bq kBq−1 ) was calculated according to 33PUptakeE = wholeplant 33P/soil 33P, where whole-plant 33P is the wholeplant amount of 33P and soil 33P is the amount of 33P in soil.

#### Root and Shoot Biomass and Morphology

The remaining 24 plants were harvested during the following week and analyzed for biomass, morphology, and their N and P contents. Following the procedure of the 33P labeled saplings, plants were divided into their compartments, namely fine roots,

coarse roots, stem bases, older top stems, younger top stems, older leaves, and younger leaves. Leaves and fine roots were dried for 24 h at 65◦C until weight stability, while coarse roots and stems were dried for 72 h at 65◦C until weight stability. Relative growth rates were calculated according to RGR = (ln DW2 − ln DW1)/(t2–t1), where DW1 is the estimated dry weight at the beginning of the experiment in February 2015, DW2 is the dry weight at the end of the experiment in September 2015, and (t2–t1) is the length of the experiment (210 days). Biomass at the beginning of the experiment was estimated from the stem diameter measured at the beginning of the experiment and the relationship between dry weight and stem diameter D established at the end of the experiment from tree harvests (DW = 8.25 ∗ D – 11.46; R 2 = 0.52; in g). Twenty leaves per sapling were scanned to determine leaf area by using a flat-bed scanner and the computer program WinFOLIA (version 2014c; Régent Instruments Inc., Canada) and their dry mass was determined. Based on this information, SLA (in cm2 g −1 ) was calculated. Total leaf area was extrapolated based on the subsamples.

#### P and N Uptake and Use Efficiencies

Dry plant compartments were ground with a vibratory disk mill (Scheibenschwingmühle TS; Siebtechnik GmbH, Mülheim, Germany) and subjected to high-pressure chemical digestion with 65% HNO3 at 195◦C. The digested samples were analyzed for P by an inductively coupled plasma optical emission spectroscopy (ICP-OES, Optima 5300 DV; Perkin Elmer, Germany). Total N in the dried plant compartments were analyzed with an elemental analyzer (vario EL III; elementar, Langenselbold, Germany). We calculated the long-term P uptake efficiency (PUptakeE; in g P g−1 Padded) by dividing the content of P in recent biomass, i.e., in fine roots and leaves that developed during the experiment, by its amount added by fertilization during the 2015 growing season. P use efficiency (PUE; in g DW g−1 PDW) was calculated from the increment in total biomass (dry weight) for a given increase in P in biomass during a given time period (cf. Koide, 1991). Correspondingly, N uptake efficiency (NUptakeE) and N use efficiency (NUE) were calculated by considering N instead of P.

#### Data Processing and Analysis

Statistical analyses were performed with R, version 3.5.1 (R Development Core Team). Significance was determined at p ≤ 0.05. Means and standard errors were calculated from eight replicates per treatment for P, and four replicates for N. All datasets were log10 transformed to resemble normality and homogeneity of residuals. The probability of fit to normal distribution was tested by a Shapiro-Wilk test. We analyzed the effects of the soil N:P ratio and soil moisture on the analyzed parameters in two-way analyses of variance (ANOVA), where both effects were treated as fixed effect. Tukey's range post hoc tests (Tukey's honest significant difference test) were conducted to identify significant differences between individual factor levels. We calculated linear regressions to identify relationships between variables.

#### RESULTS

### Plant P Concentrations and N:P Ratios in Recent Biomass

Despite the extended soil N:P gradient and different soil moisture conditions, P concentrations in fine roots and young leaves remained relatively constant in different environmental conditions (**Figure 1**). Fine roots had on average a P concentration of 0.5 ± 0.02 mg P g−1 DW, while the average foliar P concentration in young leaves was slightly higher at 0.9 ± 0.02 mg P g−1 DW. The corresponding N:P ratios in fine roots responded to soil moisture conditions and were tendentiously higher in dry than in well-watered soil (increase from 13.5 g g−1 in moist soil to 15.3 g g−1 in dry soil; p = 0.051; **Figure 1C**), which resulted from a relative increase in fine root N concentrations in dry soil and not from a change in fine root P concentrations. In contrast to foliar P concentrations, N:P ratios in young leaves were significantly influenced by the soil N:P ratio (**Figure 1D** and **Supplementary Table 1**). The foliar N:P ratio increased from 11 ± 1 g g−1 at the lowest soil N:P ratio to 15–17 g g −1 at intermediate and high soil N:P ratios in well-watered soil,

since foliar N concentrations increased at higher soil N:P ratios in well-watered soil. In dry soil, the foliar N:P ratio increased from 16 ± 1.5 g g−1 to 20 ± 0.5 g g−1 from the lowest to the intermediate soil N:P ratio; but it decreased to 13 ± 0.6 g g−1 at the highest soil N:P ratio (p > 0.05).

### Plant Biomass and Photosynthetic Carbon Assimilation

Over the course of the experiment, oak biomass was not influenced by the soil N:P ratio but was significantly reduced by soil drought (on average −18%; **Supplementary Figure 1**). The reduction of plant biomass in dry soil was the most distinct at the highest soil N:P ratio, at which plant biomass was reduced by more than a third. This decrease of plant biomass occurred mostly for old leaves (because of premature litterfall), fine root biomass, and coarse root biomass, while the decrease of young leaf biomass was less pronounced.

By contrast to oak biomass, leaf morphology and photosynthesis (A800) responded to a change in the soil N:P ratio but were not influenced by soil drought (SLA, p = 0.34 for the soil moisture effect; A800, p = 0.89 for the soil moisture effect; **Figure 2** and **Supplementary Table 1**). Area-based carbon (C) assimilation of oak leaves increased from 4.7 to 7.4 µmol CO2 m−2 s −1 in well-watered soil (p < 0.05 for the N:P effect) and from 4.7 to 6.4 µmol CO2 m−2 s −1 in dry soil (p > 0.05 for the N:P effect), respectively, with an increase of the soil N:P ratio from 4 to 42 g g−1 (**Figure 2A**). Meanwhile, SLA significantly increased from 68 to 80 cm2 g −1 with an increase of the soil N:P ratio in dry soil (**Figure 2B**), which was also accompanied by an increase of mass-based photosynthesis at high N:P ratios in dry soil (**Supplementary Figure 2**).

#### P and N Uptake Efficiencies in Response to Soil N:P Ratios and Drought

Elevated soil N:P ratios significantly increased instantaneous 33PUptakeE and long-term PUptakeE and decreased NUptakeE (**Figure 3** and **Table 1**). More specific, instantaneous 33PUptakeE increased by 4.6 and by 2.8 times under well-watered and dry conditions, respectively, when the soil N:P ratio increased by 5.3 times from 8 to 42 g g−1 (**Figure 3A**). The majority of the 33P was retrieved from fine and coarse root biomass and only a fraction from aboveground biomass, independent of the respective treatment (**Supplementary Figure 3**). Long-term PUptakeE increased by six times under well-watered and dry conditions, respectively, when the soil N:P ratio was raised by five times, i.e., when relative P availability was reduced (**Figure 3B**). Despite the influence of the factors soil N:P ratio and soil moisture on instantaneous 33PUptakeE and long-term

TABLE 1 | Two-factorial analyses of variance (ANOVA) on the significance of the effects of the soil N:P ratio (NP), soil moisture (M), and their interaction on the variance of instantaneous 33PUptake efficiency (33PUptakeE), long-term P uptake efficiency (PUptakeE), P use efficiency (PUE), instantaneous late-summer photosynthetic P use efficiency (PPUE), N uptake efficiency (NUptakeE), N use efficiency (NUE), and instantaneous late-summer photosynthetic N use efficiency (PNUE) of young Palestine oak (Quercus calliprinos) trees.

Given are F values and the probabilities of error p. Response variables were logtransformed to resemble normality. Significant effects (p ≤ 0.05) are indicated by bold letters (n = 24 for 33PUptakeE, NUptakeE, NUE, and PNUE; n = 48 for PUptakeE, PUE, and PPUE).

PUptakeE, they had no cross-effect (p > 0.87; **Table 1**). We found no effect of the fine root:leaf biomass ratio on PUptakeE or NUptakeE in moist soil (PUptakeE: p = 0.39; NUptakeE: p = 0.81; not shown). Yet the absolute phosphorus uptake into recent biomass (young leaves and fine roots) related to the young leaf:fine root biomass ratio in dry soil (R 2 = 0.58, p < 0.001), where P uptake rates increased by 17 µg P d−1 with an increase of the young leaf:fine root biomass ratio by 1 g g−1 (not shown). PUptakeE of oaks increased with fine root biomass (R = 0.56, p = 0.01) and RGR (R = 0.57, p < 0.001) in well-watered soil, but decreased with increasing amount of old leaf biomass (R = −0.50, p = 0.02) in dry soil.

The reduction in NUptakeE with an increase in soil N:P ratio was far less pronounced than the concurrent increase in PUptakeE: NUptakeE was reduced by half in well-watered conditions when the soil N:P ratio increased from 4 to 8 g g−1 , i.e., when the relative N availability doubled, but it did not decrease much further at higher soil N:P ratio. It must be noted that both instantaneous 33PUptakeE and long-term PUptakeE were drought-sensitive, while NUptakeE was not influenced by soil moisture (p = 0.20; **Table 1**). According to our regression analyses, the uptake efficiencies for P and N did not relate (**Supplementary Figure 4A**).

#### P and N Use Efficiencies in Response to Soil N:P Ratios and Drought

Elevated soil N:P ratios significantly increased instantaneous, late-summer PPUE from 41 to 65 µmol CO2 g −1 P s−1 with an increase of the soil N:P ratio from 4 to 8 g g−1 , while soil moisture had no significant effect (**Figure 4A** and **Table 1**). PPUE significantly increased with foliar N concentration in young leaves (**Supplementary Figure 5A**) and with increasing

FIGURE 4 | (A) Instantaneous late-summer photosynthetic P use efficiency (PPUE), (B) instantaneous late-summer photosynthetic N use efficiency (PNUE), (C) P use efficiency (PUE), and (D) N use efficiency (NUE) of young Palestine oak (Quercus calliprinos) trees grown at increasing soil N:P ratio and decreasing soil moisture in a climate chamber. Shown are means and standard errors for replicate saplings (n = 48 for PPUE and PUE; n = 24 for PNUE and NUE). The results of two-way ANOVAs on the significance of the effect of the soil N:P ratio (NP) or soil moisture (M) are indicated by asterisks ( ∗∗ and ∗ for p ≤ 0.01 and 0.05; n.s., not significant).

instantaneous, late-summer PNUE (**Supplementary Figure 5B**) in both soil moisture treatments. PUE was on average 1328 ± 39 g DW g−1 PDW and was neither influenced by soil N:P ratio nor by soil moisture (**Figure 4C**). PUE significantly increased with NUE in well-watered soil (R 2 = 0.69; **Supplementary Figure 4B**) but was independent from PUptakeE (**Supplementary Figure 4C**).

N use efficiencies responded in antagonism to the P use efficiencies to environmental conditions. Late-summer PNUE was not influenced by the soil N:P ratio or soil moisture (p = 0.14 for the N:P effect; p = 0.54 for the soil moisture effect; **Figure 4B**), whereas NUE responded to both the soil N:P ratio and the moisture content (**Figure 4D**). Accordingly, NUE significantly decreased from 201 to 153 g DW g−1 NDW with a doubling of the soil N:P ratio in well-watered soil. Drought further reduced NUE to 142 ± 19 g DW g−1 NDW at high soil N:P ratios (significant decrease by −18%). NUE increased with NUptakeE in dry soil (R 2 = 0.54, p ≤ 0.05; **Supplementary Figure 4D**).

#### DISCUSSION

Elevated N deposition and summer droughts in the course of global change are assumed to deteriorate the availability of P for important forest trees, but it remains unclear to what

Dirks et al. P Economy of Mediterranean Oak

extent plants may adapt their uptake and use efficiencies for P and N under these conditions. Here we show that both short and long-term PUptakeE and late-summer PPUE increased with increasing soil N:P ratio, whereas PUE did not change. At the same time, both NUptakeE and NUE decreased with increasing soil N:P ratio, that is, with increasing N availability. Drought diminished the response of PUptakeE to increasing P limitation and decreased growth but had no significant effect on late-summer PPUE, PUE, and long-term NUptakeE. It seems that the efficiency of P use can be less responsive to the soil N:P and soil drought than the efficiency of P uptake in this Mediterranean oak species.

#### Acclimation of P Acquisition to Increasing Soil N:P Ratios

Our study demonstrates that Palestine oak can adjust its capacity for P acquisition when soil P is the limiting element to the extent that P-limitation of PUE and growth do not occur. At the same time, they down-regulate the uptake efficiency for non-limiting soil N, which is probably saving energy for the plant. In nutrient-limiting soil one of the most effective plant strategies to increase nutrient acquisition is increased resource allocation to the root system. According to optimal partitioning theory, plants allocate carbohydrates and nutrients preferentially to those organs that acquire the most growth-limiting resource, with the consequence of growth being equally limited by all resources (Bloom et al., 1985; Friedlingstein et al., 1999). Accordingly, optimal partitioning theory predicts that relative carbon allocation to fine roots and the fine root:leaf biomass ratio will increase with a limitation of soil P, but decrease with an increase in N supply. We found no effect of the fine root:leaf biomass ratio on either PUptakeE or NUptakeE, probably a consequence of the opposed influences by decreasing P supply at increasing N supply in our study. Other authors demonstrated that higher capacity for nutrient uptake and assimilation related positively to absolute root and net primary production (Norby and Iversen, 2006) and, thus, to RGR of the investigated deciduous tree species (Gan et al., 2016). Similarly, we also found a positive relationship of PUptakeE with fine root biomass and RGR in the investigated evergreen oaks. Yet this relationship is probably not the cause but rather the consequence of a more efficient P uptake, which may have been induced at the morphological (e.g., greater fine root surface area), physiological (e.g., enhanced exudation of exoenzymes involved in the decomposition of organic P), or even cellular level (e.g., increased mRNA levels of genes involved in P and N absorption and metabolism; Gan et al., 2016). While we can exclude root morphology as well as organic P decomposition as significant factors for PUptakeE of oak in our pot experiment, we can only speculate if changes at the cellular level, i.e., in the membrane structure and in the density and affinity of Pi-transporters, were the reason for increased PUptakeE of oaks under P limitation. This question of the effect of changes at the cellular level on PUptakeE of evergreen tree species deserves further study.

In contrast to our initial hypothesis, Palestine oak adjusted the efficiency of P uptake, but not the efficiency of P use under P limitation. Among different plant species, acclimation strategies to nutrient limitation differ profoundly: some species respond with changes in nutrient uptake efficiency, others with changes in nutrient use efficiency, and still others with changes in both (Nitschke et al., 2017). By contrast, PUE increased exponentially and at a higher rate than PUptakeE with increasing soil N:P ratio in temperate European beech (Köhler et al., 2018). Differences in PUE can be caused by either changes in plant P concentrations at constant biomass (Veneklaas et al., 2012; Gan et al., 2016) or changes in biomass production at constant plant P concentrations (Gan et al., 2016; Zavišic and Polle, ´ 2018; Zavišic et al., 2018 ´ ). The pronounced increase in PUE in deciduous European beech was mainly caused by a decrease in plant P concentration with increasing soil N:P, while the evergreen oak saplings of the current study had throughout a range of soil N:P ratios a comparably constant P concentration in plant tissues and constant biomass production. It seems that the young oaks have a P use economy which is independent from soil P availability. In times of low P availability, P from storage in vacuoles of coarse roots (Zavišic et al., 2018 ´ ) can make plants autonomous from the amount of soil P, but this will consequently increase PUE (Gan et al., 2016). Accordingly, prominent use of P from storage in P-limited Palestine oak seems unlikely. Whereas decreased NUE under P deprivation was probably due to less efficient, waste use of N and reduced N storage in bark and wood when P (and not N) was limiting growth. NUE of the investigated Palestine oaks (135–201 g DW g−1 NDW) was even much lower than NUE of other evergreen oak species (578– 1469 g DW g−1 NDW; Silla and Escudero, 2004). It appears that Palestine oaks can sufficiently adjust their PUptakeE to soil P availability to circumvent a shift in their PUE, while they are more opportunistic with respect to NUE.

When comparing European beech and Palestine oak it appears that PUE of the evergreen Mediterranean oak saplings (1249–1487 g DW g−1 PDW; current study) was much less variable and in the mid-range of the PUE of the deciduous temperate beech saplings (436–2260 g DW g−1 PDW; Köhler et al., 2018). While it has often been assumed that the dominance of evergreens in their low-nutrient habitats is due to higher nutrient use efficiencies than in deciduous species, comparisons at the whole plant level show that this is not the case (Aerts, 1995). Dominant plants on nutrient-poor and seasonally dry Mediterranean soils generally tend to have lower tissue N and P concentration in their evergreen leaves, which have longer lifespan and lower nutrient loss rates than deciduous leaves (Ribet and Drevon, 1996; Aerts and Chapin III, 2000), but they do not necessarily use foliar nutrients more efficiently for plant growth. This was also the case in the evergreen oaks, which had foliar P concentration at the lower range of the foliar P concentrations of deciduous beech from previous studies (oak: 0.8–0.9 mg P g −1 DW; beech: 0.5–2.8 mg P g−1 DW; Köhler et al., 2018; Zavišic et al., 2018 ´ ). Yet, foliar P concentration also decreases with increasing plant size (Elser et al., 2010), which may also have caused lower plant P concentrations in the three times taller Palestine oak saplings of the current study than in the

much smaller European beech saplings of the previous study (Köhler et al., 2018).

### Stoichiometric Homeostasis and Photosynthetic P Use Efficiency

Foliar and shoot P concentrations of Palestine oak were surprisingly constant at 0.8 mg P g−1 DW across the wide range of soil N:P ratios of our greenhouse study. Other field studies also showed that foliar P concentrations do not reflect soil P availability across extended P gradients (Bauer et al., 1997; Talkner et al., 2015; Lang et al., 2016), since leaves were preferred allocation sinks for P under P-poor conditions, which kept foliar P concentrations constant (Zavišic et al., 2018 ´ ). This phenomenon is referred to as homeostasis, i.e., the degree to which an organism maintains its nutrient concentration around a given species-specific and size-specific value, despite variation in the availability of soil resources (Elser and Hamilton, 2007; Wang et al., 2012). Foliar P homeostasis in Palestine oak was probably maintained by acclimation of P acquisition and plant growth, with the latter having a stronger effect on foliar P homeostasis than on foliar N homeostasis. It is known that foliar P homeostasis differs from N homeostasis, since foliar P concentrations decrease more rapidly with plant size than N concentrations, resulting in a 2 /3 slope for the N:P scaling relationship among different plant species (Reich et al., 2010). Accordingly, foliar N:P ratios increase systematically with decreasing leaf nutrient concentrations in larger plants, which represents a disproportionate investment in N-rich proteins for high photosynthetic rates and a rising return (sensu Niklas et al., 2007). This allometric scaling of N and P in plants is consequence of the dependence of both acquisition and use efficiency of foliar P on the acquisition and use of N and vice versa (biochemically dependent co-limitation hypothesis; Ågren et al., 2012). Yet stoichiometric homeostasis is not only dependent on plant size, but also on environmental conditions, which have been suggested to exert stronger control on foliar P concentrations than on foliar N concentrations (Elser et al., 2010). In contradiction, we observed foliar P concentrations in Palestine oak saplings that remained relatively constant across different environmental conditions (and probably mainly depended on sapling size), while foliar N concentrations varied as a function of soil N availability. This led to higher foliar N:P ratios and enhanced relative P limitation when soil N availability was increased. This increase in foliar N:P ratios in Palestine oak saplings observed in our study (11–17 g g−1 ) is comparable to other studies investigating trees in P-limited environments (8–24 g g−1 ; Dijkstra et al., 2016).

The constant foliar P concentrations across a range of soil N:P ratios probably maintain photosynthetic rates of Palestine oak. In general, foliar P concentrations directly correlate with photosynthesis across various plant species (Wright et al., 2004a; Hidaka and Kitayama, 2009), where decreased foliar P concentrations often decrease photosynthetic rates (Pieters et al., 2001). P limitation was shown to restrict photosynthesis and leaf dark respiration in different climatic regions (Kattge et al., 2009; Domingues et al., 2010). Accordingly, Palestine oak may circumvent P limitation of photosynthesis by homeostasis of foliar P concentration even at the most extreme soil N:P ratios. Even more, late-summer photosynthesis and PPUE of oak even increased with P limitation. Woody plants can increase PPUE at constant foliar P concentration by allocating more P to the metabolites that participate in photosynthesis and less P to compounds such as nucleic acids and phospholipids, which are not involved directly in C fixation (Lambers et al., 2012). In addition, increases in SLA at high soil N:P ratios as in our study relate to higher levels of mass-based photosynthesis (**Supplementary Figure 2**; Hidaka and Kitayama, 2009), even when foliar biomass is constant. Both a change in leaf morphology and an allocation shift to photosynthetic active metabolites can be reason for increases in late-summer PPUE and increases in photosynthetic C gain of Palestine oak under enhanced P limitation. Notwithstanding this increase in late-summer PPUE, it was still comparably low in this evergreen tree species in global comparisons (Palestine oak: 55.4 ± 3.3 µmol CO2 g −1 P s−1 ; global average: 103 µmol CO2 g −1 P s−1 ; Wright et al., 2004b; Proteaceae: 305 µmol CO2 g −1 P s−1 ; Lambers et al., 2012).

#### Dependence of the P Economy on Soil Drought