Christian Lang
email: christian.lang@materials.ox.ac.uk

The interdependency of morphology, strain and composition in buried GeSi/Si(001) quantum dots

Keywords: epitaxy, nanostructures, shape evolution quantum dots, shape transformation, S-K growth, mc.manuscriptcentral.com/pm-pml

Morphology, strain and composition of quantum dots

Introduction

The epitaxial formation of three-dimensional islands on a lattice mismatched substrate has attracted considerable scientific and technological interest for some time now.

The scientific interest arises from the complex nature of the process as much as the insights it gives into phenomena as diverse as the strain relaxation in semiconductors [START_REF] Stranski | Abhandlungen der Mathematisch-Naturwissenschaftlichen Klasse[END_REF]2] and the initial stages of oxidation of copper [3]. Its technological interest mainly stems from the potential applications of the islands as quantum dots in electronic or optoelectronic applications [4,5,6]. One of the most extensively studied systems is the formation of islands during the deposition of Ge on Si (e.g. [7,8]) both, because of the importance of silicon and germanium in the semiconductor industry and because this is a suitable model system to understand similar processes in more complicated compound semiconductor materials. This paper focuses on the morphological evolution of these islands. The aim is to clarify how the final island shape is determined by the interaction of the islands with the substrate, the intermixed planar layers (wetting layer) between the islands and any material that may be deposited subsequently onto the sample. To this end the paper draws on results of the existing and in parts contradictory literature to arrive at a comprehensive picture of the morphological development of the islands that takes into account both thermodynamic and kinetically driven processes.

While the basic mechanisms behind the transformation from planar growth to three dimensional island growth have been known for at least 50 years [2], the added complexities in the case of Ge islands on (001) oriented Si surfaces were first discussed in Refs. [7] and [8]. As in the most general case of the so called, Stranski-Krastanov growth, the growth in the Ge/Si system starts with a small number of planar Ge layers, which intermix with the underlying Si substrate. As more Ge is deposited the number of layers and their Ge content increases until a certain critical composition and layer thickness [9]. Then small pyramid shaped islands form which are bounded by {105} type facets (pyramid facets). As the island grows it changes shape from a pyramid to a more rounded dome-like shape and steeper facets are incorporated (dome facets) [7,8].

Shape transformation and reverse shape transformation

This shape change is a result of a competition between the surface area that is added by the island and the strain energy that is reduced by introducing steeper facets. Any formation of facets on a flat surface increases the surface area and therefore the surface energy in the system. However, in the case of Ge on the lattice mismatched Si it also decreases the strain energy. The critical pyramid size at which the steeper dome facets are introduced depends strongly on the island composition [10].The higher the average germanium content in the island, the earlier the dome facets appear. The average Ge content is not only a function of the composition of the deposited materials. Si from the substrate diffuses into the islands to relief the strain at the island -substrate interface and this can lead to significant non-uniformities in the composition throughout the islands. In fact, experimental studies [11] have shown that the content near the island-substrate interface can be below 35% when at the same time the top of the island is almost pure Ge.

Increasing strain between the island and the substrate leads to the nucleation of steeper facets and therefore to a shape transformation from a pyramid to a dome shaped island. Interestingly, a "reverse" transistion from dome to pyramid has been observed [12,13] when Si was deposited on an as-grown Ge island. This is also technologically important since, to exploit the quantum confinement properties, the islands have to be embedded in a matrix material. This is usually achieved by switching from the precursor gas which induces island growth (e.g. digermane) to a gas which deposits more of the substrate material (e.g. disilane). However, since this deposition is generally carried out under similar conditions as the island growth, material deposited on top of the island can interdiffuse with the material in the island in much the same way as the substrate material does. In the case of Si deposition onto a Ge island, the diffusion of Si into the island is driven not only by the reduction of the strain energy but also by the fact that the surface energy of Si on Ge is considerably higher than Ge on Si. Above a certain amount of Si diffusion into the island, the strain energy may be reduced to such a degree that the steep facets are no longer energetically stable. Statistical studies of Ge/Si islands before and after the deposition of a few monolayers of Si have led to the conclusion that the islands indeed undergo a reverse shape transformation from domes to pyramids [12]. Ref [13] showed that some islands shrink while they undergo a reverse shape transformation.

Other islands, particularly large ones, dissolved completely before they can be embedded in the matrix. This is counterintuitive because of the fact that material is deposited on top of the island and one would therefore expect an increase of the island size depending on the amount of material added.

In reality, the equilibrium shape of the island is determined not only by the energy in the island itself but also by the energy of the whole system made up of all islands and the wetting layer. That there is thermodynamically driven interaction between different islands has been shown before when it was found that larger islands grow at the expense of smaller ones (Ostwald ripening) [8]. To understand the behaviour of this system it is important not only to look at processes in the individual islands but also take into account the effect the Si deposition has on the wetting layer and consider the islands and the wetting layer as a closely coupled system. This is reasonable as they are closely coupled even during the island formation.Yu et al. [9] showed that in the initial stages the wetting layer forms by interdiffusion of the deposited Ge and Si. As more Ge is deposited, the Ge content in the top layers increases. Ge atoms do not generally penetrate deep into the surface of the substrate as shown by Migas et al. [14]. They tend to stay confined within the top four atomic layers. At a certain critical Ge composition the strain energy becomes high enough so that island formation becomes energetically favourable. At this stage, any atoms deposited onto the wetting layer either diffuse on the surface to attach onto an existing island or, by binding to other atoms diffusing on the surface, form a new island. The islands therefore not only grow by adsorbing atoms directly from the vapour phase but also by capturing atoms which diffuse on the surface. Even coordinated movements of whole islands are possible by atoms preferentially detaching from one side of the island and attaching to the other side [15]. As the area on the sample surface which is covered by islands is usually considerably smaller than the uncovered area of the wetting layer between islands, the island morphology and the morphological development is in large part determined by the interaction between the island and the wetting layer.

It is important to realise that this strong coupling between the wetting layer and the island morphology does not end when the growth conditions are changed so that instead of Ge, Si is deposited in order to cap the islands. As Si is deposited onto the wetting layer, it intermixes with the Ge top layer. This however disturbs the equilibrium between a wetting layer and the islands and the Ge content in the wetting layer falls below the critical composition. As a consequence, the wetting layer is ready to take up any Ge atoms that detach from the islands. This is thermodynamically favourable because the wetting layer relieves strain without increasing its surface area. As a result, island composition shifts towards being more Si rich. At the same time, the island loses material to the wetting layer. If atoms migrate from the island into the wetting layer faster than Si atoms are being deposited onto the island, the island volume shrinks. Because of the change in composition as well as in size, the steep facets are no longer stable. Thermodynamically this favours a reverse shape transformation from a dome to a pyramid shape. The kinetic pathway to the thermodynamically favourable morphology Essentially, while the "target shape" of the island is energetically determined, the pathway to this shape has to be kinetically feasible. The kinetics of the system depends strongly on the availability of both Ge and Si at the surface since surface diffusion is a much faster process than bulk diffusion. The shape transformations of the island depend therefore strongly on the composition profile within the island and not only on the overall energy of the system. Some areas, especially Si rich areas far away from the island surface are essentially immobile whereas areas close to the island surface that are Ge rich play a strong role in the shape change.

As outlined in [13], at sufficiently high temperatures and slow growth rates islands do indeed lose volume and revert to the pyramid shapes. For this to happen Ge atoms have to diffuse from the island surface into the wetting layer. While the island surface is expected to be Ge rich due to the lower surface energy of Ge, the supply of Ge from the bulk of the island is slow due to the much lower rate of bulk diffusion compared to surface diffusion. As discussed above, the apex of the island can be expected to be very Ge rich whereas a significant amount of Si would have diffused into the bottom of the island. It is therefore reasonable to expect that the top of the island is preferentially redistributed whereas the lower half remains intact for longer. As outlined in figure 1 pyramid facets are short compared to the dome facets, the material in the pyramid is not enough to transform the steep, dome facet into a pyramid facet. The Ge rich top of the island is consumed before the transformation is completed and a flat top is left behind, transforming the island into what can be described as a truncated pyramid. As the rest of the material in the island is only accessible through the surface, material is removed at a fairly large rate from the top facet. This can lead to dissolution of the island without the island ever achieving the equilibrium pyramid shape.

To illustrate this, figure 1 shows schematics of two possible island shapes, the composition distribution within them and their projected development throughout the Si deposition process. Figure 2 shows all the different diffusion processes that take place at different points in the transformation. The in-situ TEM images below the schematics (recorded using the equipment and conditions described in [13]) show a real life example of shape transformation similar to the one in the schematics.

However, it needs to be pointed out that the composition distribution in the islands cannot be determined from the TEM images. The two islands which at the start of the Si deposition have different lengths of dome and pyramid facets undergo very different transformations during the Si deposition even though they followed the same pathway of shape transformations during their formation. The island in figure 1 (a The larger island (figure 1b) is assumed to be considerably bigger and its composition to be Si rich and more uniform than in the smaller island (figure 1(a)). This assumption is justified because the diffusion of Si from the substrate into the island during the island formation is through the bulk and is therefore relatively slow. ¶ For Ge atoms in the centre of the island, the shortest path to the surface is to diffuse to the flat top facet. Material is therefore transported from there into the wetting layer.

Rather than transforming into a pyramid the island expands its top facet, becoming what looks like a truncated pyramid. Over the course of the Si deposition more material diffuses into the wetting layer until there is only a long flat mound left.

The island in figure 1(a) however does still have a high Ge content which is highest at the island apex. Only the layers close to the island-substrate interface are very Si rich. Ge atoms can therefore diffuse directly from the island surface to the wetting layer. This gives also an easy pathway for material to be redistributed from the island apex onto the short dome facets (figure 2), eventually flattening them and enabling the island to transform to its equilibrium pyramid shape. The mechanisms outlined above not only account for the in-situ observations of Ge/Si islands in [13] but also for ex-situ observations of capped islands in other materials systems which are predominantly found to have truncated pyramid shapes [e.g. 16] which are not usually observed in uncapped islands.

Conclusions

To summarise, the island size, shape and composition before the capping process is crucial for its morphological development during the capping. During the capping process, the islands become essentially a reservoir of Ge for the wetting layer, much like the wetting layer is a reservoir supplying Ge atoms to the islands during their formation process. As the redistribution of Ge atoms from the islands to the wetting layer largely depends on surface diffusion, the largest amount of material is removed from the Ge rich apex of the islands. As the composition distribution in the island is strongly linked to island shape which in turn is determined by its size, one can conclude that the evolution of island shapes during the capping process is a function of island sizes before the capping process. Therefore, while any one individual island may follow a different pathway of shape transformation during its capping process than during the growth process the former can nevertheless be viewed as the reverse process of the later when the whole system consisting of all islands and the wetting layer is taken into account.

Acknowledgements: C.L. would like to thank F.M. Ross and S. Kodambaka for their kind support in performing the in-situ TEM experiments and acquiring the TEM images shown in Figure 1.

Figure 1. shows the relationship between the relationship between the island shape before and after the Si deposition process. (a) illustrates the case of an island with short dome facets and long pyramid facets with a highly non-uniform composition distribution containing a large volume of Ge rich material at the top. (b) shows the case of an island with long dome facets and short pyramid facets with a more homogeneous composition distribution.

Figure 2. shows the interaction of different diffusion processes which lead to the reverse shape transformation or even a complete dissolution of the island. Black arrows symbolise Si deposition, grey arrows surface diffusion and the intertwined black and grey arrows symbolise the exchange of material between the surface and the bulk. Letter to referees: I would like to thank both referees for their constructive comments. I have incorporated the changes suggested by referee 1 in the text. I am grateful to referee 2 for pointing out the mismatch between the scope of the paper and the title. I have tried to address this concern by changing the title of the paper to "The interdependency of morphology, strain and composition in buried GeSi/Si(001) quantum dots". While some of the concepts presented in the paper are applicable to quantum dot formation in other materials systems, this change in the title reflects the focus of the paper on the case of islands formed by depositing Ge and Si on (001) oriented Si substrates.

  Deleted: mixture of the pre-cursor gases.Deleted: the reversal of this transformation,

Formatted

  , this can lead to two very different kinds of shapes, depending on the respective lengths of the steep (dome) facets and the shallow (pyramid) facets. If the pyramid facets are longer or about equal to the length of the dome facets, the transformation from the dome shape to the energetically more favourable pyramid shape can be achieved by rearranging only the surface atoms, mainly from the top half of the island which has a high Ge content. If however the Deleted: as Deleted: in the bottom of the island a significant amount of Si would have diffused in.

  ) is at an earlier stage in the transformation and therefore smaller than the island in figure 1 (b) with the dome facets being shorter than the pyramid facets (the images in figure 1 are scaled to show maximum detail and do not represent the correct size relationship ). The island in figure 1(b) has longer dome facets than pyramid facets. The composition distribution shown schematically (not visible in the TEM images) is chosen to be consistent with the arguments outlined above. When the Si deposition Deleted: same Deleted: as Deleted: are showing Deleted: island Deleted: resembles this Deleted:

  Ge rich volume towards the top of the island (shown in light grey) is redistributed first. That leaves the larger island with an essentially flat facet on top.

Figure 1 .Figure 2 .

 12 Figure 1. shows the relationship between the relationship between the island shape before and after the Si deposition process. (a) illustrates the case of an island with short dome facets and long pyramid facets with a highly non-uniform composition distribution containing a large volume of Ge rich material at the top. (b) shows the case of an island with long dome facets and short pyramid facets with a more homogeneous composition distribution. 254x190mm (96 x 96 DPI)

http://mc.manuscriptcentral.com/pm-pml Philosophical Magazine & Philosophical Magazine Letters


Rossella ; Guerrieri
email: rossella.guerrieri@ac.ed.uk

Maurizio Mencuccini

Lucy J Sheppard

Matthias Saurer

Mike Perks

Peter Levy

Mark A ; Sutton

Marco Borghetti

John Grace

THE LEGACY OF ENHANCED N AND S DEPOSITION AS REVEALED BY COMBINING δ13C, δ18O and δ15N IN TREE RINGS

Keywords: acid mist, CO2 assimilation, nitrogen, Sitka spruce, stable C-O-N isotopes, stomatal conductance, sulphur, WUEi annual tree rings, acid mist, CO 2 assimilation, nitrogen, Sitka spruce, stable C-O-24 N isotopes, stomatal conductance, sulphur, WUE i, 25

d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

This study aimed to evaluate the effects of repeated aerial nitrogen (N) and sulphur (S) misting over tree canopies of a Sitka spruce plantation in Scotland. We combined δ13C and δ18O in tree rings to evaluate the changes in CO2 assimilation (A) and stomatal conductance (gs) and to assess their contribution to variations in the intrinsic water-use efficiency (WUEi). Measurements of δ15N enabled shifts in the ecosystem N cycling following misting to be assessed. We found that: i) N applications, with or without S, increased the ratio between A and gs in favour of A, thus supporting a fertiliser effect of added N. ii) After the treatments have ceased, the trees quickly adjusted to the reductions of N deposition, but not to the reduction in S deposition, which had a negative effect on WUEi by reducing A. This indicates that the beneficial role of N deposition may be negated in forests that previously received a high load of acid rain. iii) δ15N in tree rings seems to reflect the N dynamics caused by canopy retention, with the fingerprint also present in the litter, after the experiment stopped. iv) Both our results (obtained using canopy N applications) and a collection of published data (obtained using soil applications) showed that generally WUEi increased in response to an increase of N applications, with the magnitude of the changes related to soil conditions and the availability of other nutrients. The opposite directions of shifts in δ15N in tree rings suggest that both the quantity of the applied N and its quality, mediated by processes occurring during canopy N retention, are important determinants of the interactions between N and C cycles, which occur in forests.

Stable isotopes are useful probes to understand these processes and to put in the results of short-term experiments into context. The extent to which forests mitigate climate change is affected by the increase in nitrogen 2 deposition (N dep ) from the atmosphere, due to anthropogenic activities. Together with 3 elevated atmospheric CO 2 , N dep has been recognized as one of the main drivers enhancing the 4 terrestrial carbon 'sink' [START_REF] Norby | Nitrogen deposition: a component of global change analyses[END_REF][START_REF] Gruber | An Earth-system perspective of the global nitrogen cycle[END_REF]. However, the carbon (C) 5 and nitrogen (N) cycles interact in very complex ways, with mechanisms that can be grouped 6 under two distinct scenarios. A beneficial role of N dep has been hypothesized for N-limited 7 systems (e.g., temperate forests), since N dep can act as a low-level but sustained input of 8 fertiliser, potentially increasing tree growth and C uptake [START_REF] Magnani | The human footprint in the carbon 1 cycle of temperate and boreal forests[END_REF]; Högberg 9 2007;Sutton et al. 2008;Thomas et al., 2009). However, high levels of N dep may result in soil 10 acidification (Vitousek et al. 1997;Galloway et al. 2002), increased tree mortality (Magill et 11 al. 2004) and declines in biodiversity [START_REF] Phoenix | Atmospheric nitrogen deposition in world 16 biodiversity hotspots: the need for a greater global perspective in assessing N deposition 17 impacts[END_REF]. 12 13 The extent of the enhancement in carbon uptake by N-limited forests that can be attributed to 14 N dep has been hotly debated, with variable estimates provided [START_REF] Magnani | Replying to: A[END_REF], which 15 suggests that a deeper investigation of the crucial underlying mechanisms may be useful. One 16 of the main issues relates to understanding whether the interactions between N dep and tree 17 canopies play an important role in linking the N and the C cycle. Most of our knowledge 18 regarding the effects of N dep on forests relies upon experiments carried out in managed 19 fertilized forests, where the additional N was supplied as fertilizer to the soil for 1-2 years 20 (Elhani et al. 2005;[START_REF] Brooks | Physiological responses to fertilization recorded in tree rings: 22 isotopic lessons from a long-term fertilization trial[END_REF] or for more than 10-years [START_REF] Magill | Ecosystem response to 15 years of chronic nitrogen additions at the 23 Harvard Forest LTER[END_REF]; 21 [START_REF] Nadelhoffer | Decadal-scale 11 fates of 15N tracers added to oak and pine stands under ambient and elevated N inputs at the 12 Harvard Forest (USA)[END_REF][START_REF] Högberg | Tree growth and soil 12 acidification in response to 30 years of experimental nitrogen loading on boreal forest[END_REF][START_REF] Betson | 19 Variations in the δ 13 C of foliage of Pinus sylvestris L. in relation to climate and addition of 20 nitrogen: analysis of 32-years chronology[END_REF]Feng et al. 2008). The 22 relevance of such investigations, which exclude the opportunity for canopy exchange and 23 processing of N on the leaf surface, is debateable as it is well-known that canopy uptake 24 occurs. The significance of the canopy uptake pathway has been shown in several studies: 25 Rennenberg & Gessler, 1999; Cape et al.2001;Gaige et al. 2007;[START_REF] Sievering | Canopy uptake of atmospheric N deposition at 23 a conifer forest: part I-canopy N budget, photosynthetic efficiency and net ecosystem 24 exchange[END_REF]. This 1 provides an important source of N readily available for plant metabolism (Sparks 2009), 2 whereas N applied to the soil may be rapidly incorporated into microbial biomass instead. 3 Recently, Dezi et al. (2009) showed that inclusion of canopy uptake in an ecosystem level 4 model significantly changed ecosystem C storage capacity. 5 6 Another relevant point is that deposition of N onto forest canopies occurs in combination with 7 other pollutants, e.g., sulphur (S), which may negate the potential beneficial effects of N dep 8 and accelerate tree decline. Even though S emissions have been significantly reduced in 9

Europe over recent years (ICP, 2009), Fowler et al. (2007) observed a non-linearity in the 10 relationship between emission and deposition changes, showing that S dep in remote areas has 11 not decreased at the same rate, as closer to S sources. 12 13 Only a few experiments have examined the contribution of atmospheric N, by spraying or 14 misting above tree canopies: the experiment at Deepsyke forest, in the UK (cf., Sheppard et 15 al. 2004Sheppard et 15 al. , 2007) ) and at Howland Forest, in the USA (cf., Gaige et al. 2007; [START_REF] Dail | 23 Distribution of nitrogen-15 tracers applied to the canopy of a mature spruce-hemlock stand, 24 Howland, Maine, USA[END_REF]. [START_REF] Barbour | Stable oxygen isotope composition of plant tissue: a review[END_REF] Between the two experimental sites, the Deepsyke experiment is unique in providing the 17 opportunity to evaluate the effects of frequent (> 50 applications during the growing season) 18 aerial N and S misting onto the canopy of a Sitka spruce forest for a relatively long period 19 (i.e., 5 to 8-years). At this experimental site, N treatments significantly increased tree stem 20 area increment, irrespective of whether N was added with or without S [START_REF] Sheppard | Effects of five 15 years of frequent N additions, with or without acidity, on the growth and below-ground 16 dynamics of a young Sitka spruce stand growing on an acid peat: implications for 17 sustainability[END_REF]. 21 22 This study aimed to investigate how experimental applications of N dep and S dep affected the 23 ratio between canopy CO 2 assimilation (A) and stomatal conductance (g s ), known as intrinsic 24 water-use efficiency (WUE i ), for the Sitka spruce stand at Deepsyke. Sitka spruce is one of 25

F o r R e v i e w O n l y
the most widely planted tree species in the UK, succeeding well on in nutrient-poor soils. This 1 species is very tolerant of low-N availability, and at the same time very responsive to small N 2 additions (Chandler & Dale, 1995), so that a beneficial effect of N dep on WUE i , achieved 3 through an increase in A, can be hypothesised. 4 5 Our approach combined the analysis of the stable isotopes of carbon (δ 13 C), oxygen (δ 18 O) 6 and nitrogen (δ 15 N) in annual tree rings. Tree rings are very useful archives of information 7 concerning changes in tree growth in response to environmental conditions. The combination 8 of all three isotopes δ 13 C, δ 18 O and δ 15 N in tree rings can provide more specific information 9 on the underlying ecophysiological processes. The effect of different treatments on WUE i , as 10 assessed through δ 13 C in tree rings, was investigated. Further, the combination of δ 13 C and 11 δ 18 O allowed us to suggest which physiological traits, i.e., A and g s respectively, contributed 12 most to the variations in WUE i . Finally, δ 15 N provided indications of changes in ecosystem N 13 cycling in response to the different treatments. 14 15 We addressed the following main questions: i) Do canopy N additions increase the balance 16 between A and g s (i.e., WUE i ) in favour of A, and does the addition of S counteract the effect 17 of N? ii) Does δ 15 N in tree rings carry a signal of changes in the N cycle within the ecosystem 18 in response to canopy N addition? iii) When the experimental applications are terminated, 19 how fast do trees adjust to a reduction in N and S deposition? iv) Are the effects on tree 20 physiological traits and N dynamics caused by canopy addition different from those predicted 21 by studies that have employed direct application of N to the soil? 22 The relative abundance (R) of the stable isotopes, i.e., 13 C/ 12 C, 18 O/ 16 O, 15 N/ 14 N, in plant 1 organic matter is expressed as the relative deviation from the international standards (V-PDB 2 for δ 13 C, V-SMOW for δ 18 O and atmospheric N 2 for δ 15 N), using delta (δ) notation, i.e., 3 13 C and its link to WUEi 6 Plant material is generally depleted in the heavier isotope ( 13 C) compared to atmospheric CO 2 , 7 because of isotope fractionations taking place during the biochemical and diffusional 8 processes leading to photosynthesis. Indeed, carbon isotope composition in tree rings (δ 13 C) 9 reflects variation of the ratio between CO 2 concentration in leaf intercellular spaces and the 10 atmosphere (i.e., c i /c a ratio) related either to changes of CO 2 assimilation (A) or stomatal 11 conductance (g s ). According to the simplified version of the Farquhar equation, δ 13

1000 1 δ standard sample sample •         - = R R . 4 5 δ
) ( C δ C δ c c a b a - - - =
[1] 14 where δ 13 C a is the isotopic signature of atmospheric CO 2 , a is the fractionation for 13

- + = t c [5] 5
where t is the calendar year. 6

Finally, eqn. [2] can be solved as: 7

8 a b b c a - + - = sample 13 a 13 i C δ C δ 6 . 1 WUE [6] 9
Hence, δ 13 C in tree rings allows changes in the A/g s ratio to be assessed taking into account 10 the long-term variation of atmospheric CO 2 concentration and its isotopic signature. Although 11 post-photosynthetic fractionations [START_REF] Cernusak | Why are non-photosynthetic tissues 7 generally 13 C enriched compared to leaves in C3 plants? Review and synthesis of current 8 hypotheses[END_REF]) may partially reduce the strength by 12 which the primary isotope signal imprinted in leaf organic matter is recorded in heterotrophic 13 tissues, δ 13 C in tree rings still contains long-term information on changes of leaf physiological 14 processes [START_REF] Saurer | Carbon isotope discrimination indicates 3 improving water-use efficiency of trees in northern Eurasia over the last 100 years[END_REF]Gessler et al. 2009). 15 16 Link between δ 18 O in tree rings and stomatal conductance 17 Variation in δ 13 C can be related to changes in either A or g s whereas δ 18 O in organic material 18 is specifically related to changes of δ 18 O in leaf water in response to transpiration. Hence, 19 when δ 18 O of source water does not vary among studied sites, δ 18 O in plant organic matter 20 records changes in g s . In fact, during transpiration molecules of water containing the lighter 21 isotopes, H 2 16 O, tend to diffuse faster from the sites of evaporation to the atmosphere than 22 H 2 18 O, so that water at the evaporative site becomes more enriched in the heavier isotopes 18 O 23 Variations in the oxygen isotope signature in leaf water (i.e., δ 18 O lw ) are linked to changes in 2 the ratio between water vapour in the atmosphere and in the intercellular spaces (e a /e i ≈ 3

Relative humidity, Rh), according to [START_REF] Craig | Deuterium and oxygen-18 variations in the ocean and the marine 20 atmosphere[END_REF]:

4 i a k s 18 v 18 k * s 18 lw 18 ) O δ O δ ( O δ O δ e e ε ε ε - - + + + = [7] 5
where δ 18 O s and δ 18 O v are the oxygen isotopic compositions of soil water and atmospheric 6 water vapour, respectively, ε k is the kinetic fractionation during evaporative water diffusion 7 through stomata and boundary layers, ε * is the fractionation associated with the lower vapour 8 pressure of H 2 18 O compared to that of H 2 16 O. However, at increasing levels of transpiration, 9 the primary 18 ) and dissolved organic N. Overall, 14 the δ 15 N in tree pools reflects the isotopic signature of the main N sources (soil and 15 atmosphere) and isotope fractionations occurring during N uptake by roots and foliage, and 16 assimilation within plant compartments [START_REF] Robinson | A theory for 15 N/ 14 N fractionation in 24 nitrate-grown vascular plants[END_REF][START_REF] Comstock | Steady-state isotopic fractionation in branched pathways using plant 18 uptake of NO 3 as an example[END_REF]). On the other 17 hand, δ 15 N in the soil reflects the relative contribution of different N compounds and is 18 strongly affected by fractionations during biogeochemical processes. Indeed, nitrification 19 produces 15 N-depleted NO 3 -, leaving in the soil 15 N-enriched NH 4 + . Ammonia volatilization 20 can also enhance the 15 N enrichment of the NH 4 + remaining in the soil and therefore, available 21 for plants, while atmospheric deposition of NH 3 may correspondingly supply 15 N depleted N 22 (Tozer et al. 2005). NO 3 -can also be lost via denitrification and leaching, leaving 15 N-23 enriched NO 3 -in the soil (Nadelhoffer & Frey, 1994;[START_REF] Högberg | 15 N natural abundance in soil-plant systems[END_REF]). Fractionations during 24 mineralization of the substantial pool of soil organic matter have been reported to reduce 1 [START_REF] Létolle | Nitrogen-15 in the natural environment[END_REF], albeit not significantly [START_REF] Högberg | 15 N natural abundance in soil-plant systems[END_REF]), the δ 15 N of the product NH 4 + . 2 3 Atmospheric N can be a significant source of N for trees. Before reaching the soil, 4 atmospheric N (e.g., NO x and NH x forms) interacts with tree canopies in such a way that a 5 consistent part can be retained and taken up by canopies, leading to a δ 15 N signal which 6 depends on the relative contribution of the exchanged N compounds (NH 4 + vs. NO 3 -) ( Heaton 7 et al. 1997). As a consequence, the amount of atmospheric N reaching the soil as wet and dry 8 deposition and the isotope signature are modified by the interaction with forest canopies. 9 10 Overall, it can be hypothesised that, at very low levels of N dep , when mineralization is the 11 chief source of available N, and provided canopies have also reached full closure, δ 15 N in tree 12 pools should be relatively stable and most closely related to soil δ 15 N with little long-term 13 variation. By contrast, when an increase in available N occurs, e.g., an increase of N dep from 14 the atmosphere or from fertilisation, variations of δ 15 N in tree pools should reflect: i) the 15 contribution of the added N, according to its δ 15 N and ii) the variations in soil δ 15 N because of 16 changes in the degree of closure of the N cycle (i.e., losses caused by NO 3 -leaching, 17 denitrification and NH 3 volatilization), the prevailing effect of which is to enrich the δ 15 N 18 signal of tree pools. 19 20

Experimental site 21

The study was carried out in a Picea sitchensis (Bong.) Carr. plantation at Deepsyke (290 m 22 asl, latitude 55°46'N, longitude 3°18'W) in the Scottish Borders (Scotland, UK), with a low 23 background of N dep (8-10 kg N ha -1 yr -1 ). A mixture of provenances (identities unknown) was 24

F o r R e v i e w O n l y
planted in 1986 at 2 m spacing on drained deep peat soil, on mounds formed from the 1 inversion of organic horizon removed to form the drainage ditches [START_REF] Sheppard | Early effects of 11 acid mist on Sitka spruce planted on acid peat[END_REF]). 2 3 Within the forest stand, an area of 1.5 ha was divided into four replicated blocks, each 4 containing six different spraying treatments: nitrogen (N), sulphur (S), NS (as a single dose), 5 2NS (as a double dose of both N and S), wet control (e.g., only water), no treatment (e.g., no 6 spraying). Treatments increased the amount of precipitation by 20% (for N, NS and wet 7 control) or 40% (for 2NS). In this study, we considered only N, NS, 2NS and wet control 8 (Table 1, Fig. 1a-b). Acid mist was applied approximately twice weekly during the growing 9 season (> 50 applications from April until November) from 1996 to 2000, with four replicates 10 for each treatment. In a second phase (2001)(2002)(2003), two plots (selected at random, irrespective 11 of the block) were maintained on the original treatment, while in the other two S or N addition 12 of the original treatment was removed in order to simulate the recovery from reduced S and N 13 (Table 1) (cf., [START_REF] Sheppard | Effects of five 15 years of frequent N additions, with or without acidity, on the growth and below-ground 16 dynamics of a young Sitka spruce stand growing on an acid peat: implications for 17 sustainability[END_REF][START_REF] Sheppard | Implications of acidified S inputs on 19 the fate and consequences of N deposition: results from a field manipulation of a Sitka spruce 20 canopy in southern Scotland[END_REF]. 14 15 Within each plot, five of the 10 trees were selected for sampling wood cores (two per tree) in 16 June 2009, using a 0.5 cm diameter increment borer. The five trees were selected according to 17 the diameter variation relative to the diameter distribution of the wet control. Therefore, trees 18 showing extreme growth rates (exceptionally low or high) were excluded. The undecomposed 19 litter and the organic soil (0-10 cm) were sampled from five locations in the furrows along the 20 planting lines. 21 22

Sample preparation and isotope analyses 23

Before isotopic measurements, wood cores were subjected to a chemical pre-treatment in 24 order to remove N mobile compounds, which may affect the detection of the δ 15 N signal. For 25

F o r R e v i e w O n l y
this purpose, we employed the rapid procedure proposed by Sheppard and Thomson (2000): 4 1 hours in a mixture of toluene: ethanol; 4 hours in ethanol; 4 hours in distilled water. After 2 drying, each wood core was dated and thereafter annual rings were identified and separated 3 from bark to pith under a stereoscope. Along a chronology of 15-years (1995-2009), annual 4 rings were split in to the following sections: I) the year 1995 (before the treatments); II) the 5 single annual rings from 1996 to 2003 (during the treatments); III) the group of annual rings 6 from 2004 to 2009 (after the treatments, i.e., all six rings together with no annual separation). 7

Within a plot, all the samples from the five trees were ground with a centrifugal mill and 8 pooled by year, in order to get an average isotopic signal. 9 10 Soil and litter samples were dried in the oven for 12 hours at 105°C. After removing traces of 11 fine root and litter, soil samples were ground with a ceramic mortar. For litter samples, a 12 centrifugal mill was used to produce a fine powder for the analysis. 13 14 For δ 13 C, δ 15 N and δ 18 O measurements, 0.6-0.8 mg, 15-20 mg and 0.5-0.7 mg of wood 15 powder, respectively were weighed in tin capsules (silver capsules for oxygen). An amount of 16 4-5 mg of soil and litter samples was weighed into tin capsules. Further, 4-5 mg of the 17 ammonium nitrate (NH 4 NO 3 ) used for the spraying and stored from 1996 was weighed into 18 tin capsules (n=6) for the δ 15 N analyses. We measured a δ 15 were used for determinations of δ 13 C and δ 15 N. An amount of 3-4 mg of needle samples was 7 weighed into tin capsules for combustion in the CE Instruments NA2500 Elemental Analyzer 8 and for determination of both δ 13 C and δ 15 N by VG Prism III Isotope ratio mass spectrometer. 9 10The isotope values were expressed in the δ -notation (in per mil; ‰) as relative deviation from 11 the international standard (atmospheric N 2 for δ 15 N, V-PDB for δ 13 C and V-SMOW for δ 18 O). 12 13

Meta-analysis 14

Among all the studies in the literature reporting on tree growth responses to increased N dep , 15 mainly simulated as ground fertilization, we selected those experiments that satisfied the 16 following main criteria: i) were carried out on tree species; ii) did not involve a 15 N-labelling 17 experiment; iii) were conducted in the field; iv) measured δ 15 N in tree rings and δ 13 C in either 18 needles or tree rings. These studies are summarised in Table 2. Most of the sites considered in 19 our meta-analysis were located in areas with low-level of N dep . Elhani et al. (2005) reported 20 values ranging between 9 and 13 kg ha -1 yr -1 , for the years after the fertilization experiment. 21

The δ 15 N signal of the mineral N-fertiliser supplied during the experiments had values close 22 to that of the atmospheric N 2 (i.e., 0‰) [START_REF] Nadelhoffer | Decadal-scale 11 fates of 15N tracers added to oak and pine stands under ambient and elevated N inputs at the 12 Harvard Forest (USA)[END_REF]Elhani et al. 2005). We 23 measured a δ 15 N value of the ammonium nitrate added during the spraying and found it to be 24

F o r R e v i e w O n l y
slightly enriched in 15 N, i.e., 1.6‰. Overall, we assume no differences in the δ 15 N of added N 1 either in the soil or to tree canopies. 2 3 From each study, data points and corresponding error bars of δ 13 C and δ 15 N values were 4 extracted using an image analysis programme (UTHSCSA Image Tool). We considered only 5 the years when the experimental treatments were undertaken. For given δ 13 C values, WUE i 's 6 were calculated, according to equation [START_REF] Cernusak | Why are non-photosynthetic tissues 7 generally 13 C enriched compared to leaves in C3 plants? Review and synthesis of current 8 hypotheses[END_REF]. Values are presented as differences between 7 treated and control treatments, for both WUE i and δ 15 N values, in order to highlight the effect 8 of the treatment and to account for differences in tree age and/or climate effects across 9 studies. 10 11

Statistical analyses 12

Pre-treatment (1995) and post-treatment (2004-2009) differences for δ 13 C, δ 18 O and δ 15 N 13 across plots were tested by ANOVA, considering the assigned treatments as fixed factors. For 14 the pre-treatment year (1995) we found no significant effect of 'block' as the random factor, 15 so block was excluded from the subsequent analyses. The effect of different treatments was 16 evaluated by repeated-measures ANOVA for the years 1996-2000, when replication of the 17 experiment was balanced (four replicates for each treatment). Soil moisture was included as a 18 covariate [START_REF] Sheppard | Effects of five 15 years of frequent N additions, with or without acidity, on the growth and below-ground 16 dynamics of a young Sitka spruce stand growing on an acid peat: implications for 17 sustainability[END_REF]) and with an interaction term with treatments. The year-by-19 year differences between the treatments and the wet control were investigated by ANOVA, 20 using the Least Significant Difference (LSD) test. Due to the experimental design not being 21 balanced during the recovery phase (2001)(2002)(2003), differences between recovery (n=2) and 22 treatment (n=2) for each treatment, were assessed using independent sample t-test. [START_REF] Robinson | A theory for 15 N/ 14 N fractionation in 24 nitrate-grown vascular plants[END_REF] 24

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The t-tests were employed for investigating the difference between needles collected in 1997 1 and the annual ring formed in the same year for δ 13 C and δ 15 N values, while differences 2 among treatments for either δ 13 C or δ 15 N in needles and annual rings were tested by ANOVA. 3 All statistical analyses were carried out with SPSS 16.0 statistical package (SPSS, Chicago, 4 IL). Where not differently specified, the level of significance for statistical tests was α = 0.05. 5 plots with different assigned treatments (Tables 3,4,5). However, over the five years of the 10 experiment, δ 13 C and δ 15 N were most affected by the addition of nitrogen (N) alone or in 11 combination with sulphur (S). While δ 13 C showed a decreasing trend over time in all the 12 treatments (Fig. 2, panels a, b, c), repeated-measures ANOVA from 1996 to 2000 also 13 revealed a significant effect of treatment, with 2NS and N having higher δ 13 C values than NS 14 and wet control (Table 3). The tests of between-subject effects showed a non-significant 15 effect of the covariate (i.e., the variability in soil moisture among plots) on changes in δ 13 C 16 (F= 1.2; P=0.3). 17 The effect of simulated N and S deposition to the tree canopy for δ 18 O was not as clear as for 19 δ 13 C (Fig. 2 panels d,e,f). Overall, there was a tendency for δ 18 O to decrease for the first few 20 years after the spraying started, with a trough in 1998, which was also the wettest year of the 21 experimental period. Repeated-measures ANOVA did not show any significant treatment 22 effect, even when variations in soil moisture were taken into account as a covariate (Table 4). 23

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Overall, δ 15 N values showed a slight tendency to increase over time (Fig. 2 g, h, i), with a 1 higher variability among years for the wet control compared to plots receiving the different 2 treatments. Changes in δ 15 N were not affected by treatment, when N was in combination with 3 S, with trees in NS and 2NS showing similar δ 15 N values to the wet control. By contrast, N 4 alone led to a more negative δ 15 N values compared to the wet control and the NS treatment 5 (Table 5). The covariate soil moisture was not significant for δ 15 N (F=0.06, P=0.8). 6 7 For the 1997 annual ring (i.e., after one year's treatment), δ 13 C and δ 15 N measured in wood 8 could be compared with the corresponding values measured in needles formed in the same 9 year (sampled in December 1997). When both values were plotted as differences from the wet 10 control (Fig. 3) a significant increase in δ 15 N in needles, but not in annual rings was observed 11 which seemed to be dose related (Fig. 3a). By contrast, the effect of treatment was not 12 obvious for δ 13 C measured in needles, while it was significant (P<0.1) in the case of the 13 corresponding annual ring, with the 2NS treatments showing an increase in δ 13 C within one 14 year of treatment (Fig. 3b). It is noteworthy that, the difference between the needle and annual 15 ring for C and N isotopes has a different direction, significant only for 2NS: for δ 15 N, more 16 positive values were observed in needles compared to the annual ring, while the opposite was 17 true for δ 13 C. 18 19 The removal of S or N addition from the original treatment (Table 1) did not significantly 20 affect stable isotope trends in the second phase of the experiment, with similar values 21 measured for the recovery and the treatment plots (Tables 3, 4, 5). There was a tendency for 22 δ 13 C in the wet control to increase, while δ 13 C tended to decrease in 2NS, NS and N (Fig. 2 a, 23 b, c). Variation of δ 18 O in treated plots followed the same time trend as in the wet control, 24 wood was 15 N-depleted compared to the soil samples, but 15 N-enriched compared to the litter 7 sampled in June 2009 (Fig. 4). 8 9

Changes in WUE i and its determinants 10

For each treatment, variations of WUE i were evaluated as differences from the wet control 11 (Fig. 5 a, b, c), without distinguishing between recovery and original treatments in the last 3-12 years of the experiment. The three treatments (i.e., 2NS, NS and N) showed different 13 temporal trends, with particular reference to the first phase of the experiment (i.e., 1996 -14 2000). The 2NS treatment led to a rapid but short-lived increase in WUE i , followed by a 15 decline from 1998. Furthermore, for the post-treatment phase (2004)(2005)(2006)(2007)(2008)(2009) this decline 16 strengthened producing a larger reduction in WUE i compared to the control (Fig. 5 a). In the 17 NS treatment, WUE i showed a pattern similar to that observed for 2NS, but with a lower 18 magnitude of variation and only a small non-significant decrease post treatment (Fig. 5 b). By 19 contrast, for the N treatment, WUE i kept increasing over the first phase of experiment and 20 also for the beginning of the second phase. However, during the post-treatment years, WUE i 21 in the N treatment also decreased but only by a small amount, falling back to that measured in 22 the wet control (Fig. 5 c). 23

F o r R e v i e w O n l y
The combination of δ 13 C and δ 18 O values, expressed in terms of their differences from the wet 1 control, is shown in the main panel of Fig. 6. Values relative to the 8-years of the experiment 2 (i.e., 1996-2003) were averaged, without distinction between recovery and treatment periods. 3

This allows the different effects of N alone and N in combination with S to be assessed for the 4 combined variations of δ 13 C and δ 18 O. N addition led to an increase in δ 13 C, with a marginally 5 significant effect on δ 18 O. By contrast, N combined with S (irrespective of the double or 6 single dose) led to an increase in δ 13 C combined with a decrease in δ 18 O. For the post-7 treatment phase (i.e., treatment vs. post-treatment phase), we observed a tendency to decrease 8 for both isotopes, with a higher magnitude of change for δ 13 C than δ 18 O. Different trends are 9 discussed later in the paper in terms of variations in CO 2 assimilation and stomatal 10 conductance, according to the conceptual model of [START_REF] Scheidegger | Linking stable oxygen and carbon 6 isotopes with stomatal conductance and photosynthetic capacity: a conceptual model[END_REF] (Fig. 6, side 11 panels). 12 13 3.3 Canopy vs. soil applications for WUEi and δ 15 N: a meta-analysis of data in the literature 14 We compared our results with those reported in previous studies, where N was added to the 15 soil as either ammonium nitrate or urea, sometimes also in combination with S or other 16 nutrients (e.g., K, P, Ca). 17

All values were considered as differences from the corresponding control, to standardise the 18 differences in climate and soil conditions across sites. Overall, both above-canopy and below-19 canopy fertilisation showed that addition of N leads to an increase in WUE i , with a magnitude 20 that was equal to or greater for canopy compared to soil applications (Fig. 7). In two cases, no 21 A different dynamic was observed for δ 15 N, when the two different experimental approaches 1 (canopy vs. soil N applications) were compared (Fig. 8). With increasing N dose applied to 2 the soil, the δ 15 N in tree rings became more 15 N-enriched compared to the control, whereas 3 canopy applications seemed to lower the 15 N enrichment, although the low number of points 4 prevents statistical analysis. 5 6 Interestingly, irrespective of the approach considered, the combination of N and S appears to 7 restrict the difference between treatment and control for δ 15 N. In fact, when N alone was 8 added either to the soil [START_REF] Nadelhoffer | Decadal-scale 11 fates of 15N tracers added to oak and pine stands under ambient and elevated N inputs at the 12 Harvard Forest (USA)[END_REF]) or to the canopy (i.e., Deepsyke forest), the 9 difference in δ 15 N from the control was always larger than in the case of combined N+S 10 applications, with a higher difference in the case of soil fertilization. Changes in the δ 15 N in 11 tree rings in response to N application seemed to be species-specific (conifer vs. deciduous 12 species) and compound-specific (ammonium nitrate vs. urea fertilizer). Conifers were more 13 sensitive than deciduous trees to N application. For the former group, the 15

Changes in WUEi and its determinants following N and S acid mist above tree canopies. 19

One year after the experiment started (i.e., 1997) a clear effect of the treatment on the C 20 isotope signal in the annual ring was already present, although no signal was detected in 1-21 year old needles produced in the same year (Fig. 3 b). 5 years exposure to acid mist affected 22 the isotopic signature in tree rings significantly, leading to higher δ 13 C values (Table 3), thus a 23 higher WUE i , for N and 2NS treatments. This result is consistent with the increase in stem 24 area increment reported by [START_REF] Sheppard | Effects of five 15 years of frequent N additions, with or without acidity, on the growth and below-ground 16 dynamics of a young Sitka spruce stand growing on an acid peat: implications for 17 sustainability[END_REF] treatments with the positive response for tree growth rates, δ 13 C enabled a treatment-specific 1 change in terms of WUE i to be identified. In fact, although both N and 2NS showed an 2 increase of WUE i , the different temporal trends (Fig. 5) demonstrate that the N application led 3 to a long lasting positive effect of N availability on the ratio between assimilation (A) and 4 stomatal conductance (g s ), whereas the 2NS application resulted in a short lived pulse only. 5

The short lived increase in WUE i with the 2NS treatment was followed post-treatment by a 6 significant decrease during 2004-2009 compared with the control, a tendency which was also 7 apparent (but not significant) for the NS treatment, but not for the N treatment. Combined 8 with the lower WUE i values for the NS compared with the N treatment during the experiment 9 (Fig. 5), this indicates that S deposition had a longer lasting detrimental effect on WUE i . We 10 are aware that by grouping the annual rings from 2004 to 2009 we might have lost a year-to-11 year trend of WUE i after N and S were removed, hiding some nutrient "payback" effect due 12 to the treatments. 13 14 The different patterns for WUE i suggest different physiological mechanisms behind the 15 responses observed for the three treatments. Combining δ 13 C and δ 18 O in a conceptual model 16 (Fig. 6), according to [START_REF] Scheidegger | Linking stable oxygen and carbon 6 isotopes with stomatal conductance and photosynthetic capacity: a conceptual model[END_REF], enabled us to better understand how N 17 treatments with and without S affects N use in the tree. An important prerequisite of the 18 conceptual model is similar δ 18 O of source water and vapour water for the investigated trees. 19 In our study, trees were growing in the same area with closed canopies giving rise to similar 20 Rh and treated with rainwater collected on site, so we assume that possible differences in g s , 21 as inferred by δ 18 O in tree rings, can be attributed to the treatments alone. 22 23 The increase in δ 13 C observed in all treatments during the experiment (i.e., 1996-2003) 24 indicates a reduction in the c i /c a ratio, explained by either an increase in A, at constant g s or by 25 a reduction of g s at constant A. δ 18 O, combined to δ 13 C, helps to clarify whether changes in A, 1 g s or both accounted for the variations of WUE i . 2 3 In the case of N addition, only small changes in δ 18 O were observed (compared to the pre-4 treatment time), therefore variation of WUE i were mostly related to an increase of A, in 5 response to a fertiliser effect of N (Fig. 6, side panels). In another experiment where spruce 6 branches were sprayed with NH 4 NO 3, N was shown to contribute significantly not only to 7 enhance CO 2 assimilation but also to reduce the thermally dissipated light, suggesting an 8 increase of photosynthetic efficiency (Tomaszewski & Sievering, 2007). 9 and to affect stomatal opening, with a mechanism related to changes in apoplastic pH and in 14 the electrochemical potential gradient across the guard cell plasmalemma (Eamus & Fowler 15 1990). 16 17 Based on our measurements, the beneficial effect of N on A tended to disappear the 18 experiment stopped (Fig. 6, sides panels), especially in the 2NS treatment. As suggested by 19 the larger reduction of δ 13 C compared to δ 18 O, g s did not change as much as A, which 20 decreased after the experiment stopped. This means that excess of S in the system affected 21 negatively tree physiological traits even six years after the spray treatment stopped, 22 suggesting a low resilience of the forest to recover from excess S dep to either tree canopies 23 and/or soil. 24 this was not observed in our study. In fact, variations in δ 13 C during the investigated period 4 were quite consistent, with a reduction of about 4‰ over a period of six years (Fig. 2a-c). 5

Since we considered a short chronology (i.e., 15-years) data were not corrected for the 13 C-6 depletion of atmospheric CO 2 , and thus may partially explain the decreasing trend for δ 13 C in 7 tree rings. Another explanation could be related to the age of the plantation. In fact, when the 8 experiment started in 1996, trees were about 10-year old, in a stage of pre-canopy closure 9 [START_REF] Sheppard | Effects of five 15 years of frequent N additions, with or without acidity, on the growth and below-ground 16 dynamics of a young Sitka spruce stand growing on an acid peat: implications for 17 sustainability[END_REF]), which could have played a significant role, by increasing shading, 10 which, in turn, reduced the δ 13 C, by increasing the c i /c a ratio, due to a low CO 2 assimilation 11 rate for a giving g s (Farquhar, 1980). precipitation and changes in summer relative humidity conditions (McCaroll & Loader, 17 2004). Because the treatment plots were small and randomly distributed, we can assume no 18 differences among treatments for the oxygen isotopic signature of soil water. Further, 19 although high variability in soil moisture was reported between plots (Sheppard et al. 2004), 20 this did not significantly affect the δ 18 O in the tree rings. We also assume that the water used 21 for the spraying had a similar δ 18 O in all treatments, since it was derived from the same 22 source, i.e., precipitation. Therefore, δ 18 O in tree ring records changes in g s due to variations 23 in atmospheric evaporative conditions. 24 25

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It is worth underlining that treatments (including the wet control) supplied an additional 20% 1 (e.g., NS, N and wet control) and 40% (e.g., 2NS) of water to the precipitation. Therefore, the 2 general decreasing trend observed (Fig. 2d-f) for δ 18 O in all treatments during the first years 3 of the experiment might be a spraying-related effect. In fact, frequent spraying (> 50 4 applications) over the growing season contributed to maintaining a low vapour pressure 5 deficit, enabling the needles to maintain optimal hydration states, with no limitation to 6 stomata opening. 7 8 Despite the fact that the additional water added during the spray treatments having possibly 9 reduced the detection of the climatic signal, δ 18 O proved to be a valuable archive for changes 10 in evaporative demand from the atmosphere. In fact, the trough of δ 18 O values in 1998, 11 corresponding to the wettest year of the experiment, may reflected "over watering" conditions 12 for trees, with particular reference to NS and 2NS. Moreover, the response recorded in 2003 13 for both δ 13 C and δ 18 O might be related to drought conditions all over Europe, with particular 14 reference to trees in the treatment plots where recovery phase started (i.e., the misting with S 15 had ceased), suggesting that S might have affected tree responsiveness to seasonal changes in 16 weather conditions. 17

Generalization of our findings: comparison with other studies 19

Only a few studies met the most relevant conditions for inclusion in the meta-analysis: 20 fertilizer response was assessed using δ 13 C and δ 15 N in tree rings. It was beyond our aim to 21 provide an estimation of the changes of absolute values of WUE i and δ 15 N for soil vs. canopy 22 N application, rather we were interested in the extent of variations in relation to the two 23 approaches, in order to account for differences in the underlying mechanisms. We are conscious that different parameters might account for variations of WUE i at each of 2 the sites considered in the meta-analysis, such as age, tree species, and different climatic and 3 environmental conditions. However, by considering the difference from the control, we 4 should be able to extract the influence of the treatment on WUE i , through an effect on A, at a 5 given g s , given that the contribution of ambient N dep is similar in all the sites considered. 6 7 The increase in N availability, through either soil or canopy application, enables trees to 8 enhance the efficiency by which they assimilate CO 2 , thus confirming the "almost axiomatic 9 and strongly positive relationship" (Warren and Adams, 2005) between A and N supply. 10

Overall, there seems to be a steep increase of WUE i up to 150 kg N ha -1 yr -1 (Fig. 7). At higher 11 doses, the ratio between A and g s tends to stabilize, though it must be noted that this 12 conclusion is contradicted by the results of [START_REF] Balster | Coupling tree-ring δ 13 C and δ 15 N to test the effect 8 of fertilization on mature Douglas-fir (Pseudotsuga menziesii var. glauca) stands across the 9 Interior northwest, USA[END_REF], where urea was used as a 13 fertiliser supply. 14 15 Although results for both above-canopy and below-canopy fertilisation followed a similar 16 trend for WUE i , the magnitude tended to be higher in the case of canopy applications. In fact, 17 for a similar dose of N applied (e.g., 48-50 kg N ha -1 yr -1 ), the difference from the control for 18

WUEi was higher for trees receiving N as mist application over tree canopies (Fig. 8), 19 compared to those receiving the same amount supplied to the soil [START_REF] Betson | 19 Variations in the δ 13 C of foliage of Pinus sylvestris L. in relation to climate and addition of 20 nitrogen: analysis of 32-years chronology[END_REF]). At 20 higher dose (e.g., 96-100 kg N ha -1 yr -1 ), the increase of WUE i compared with the control was 21 others reported in Fig. 7. In the latter investigation, the spraying with acid mist resulted in a 6 reduction of CO 2 assimilation (as assessed by the more negative δ 13 C in the treated trees 7 compared to the control), which was supported also by a decrease of radial growth increment 8 during the experiment. However, in the investigation of Heaton and Crossley, the Sitka spruce 9 at the Glencorse forest (Scotland), was potentially P limited, as a result of the acid treatment, 10 which mobilized Al [START_REF] Carreira | Reduced P soil availability in a Stika spruce 4 (Picea Sitchensis (Bong.) Carr) plantation induced by applied acid-mist: significance in forest 5 decline[END_REF]. This result has important implication for forest 11 management, indicating that in general, at low-level of N availability, an increase of N dep can 12 hardly match with a fertiliser effect on CO 2 assimilation and growth if other conditions are 13 not met (e.g., other nutrients or water availability). 14 The fertiliser applied to both soil and canopy had a similar δ 15 N values, thus we can assume 19 that the differences between the treatment and the control for δ 15 N measured in the tree rings 20 reflect changes in soil and canopy processes, rather than differences in isotope signature of the 21 N applied. 22 23 Results of our meta-analysis show an enrichment in the heavier isotope for δ 15 N in tree rings, 24 with cumulative added N in the soil (Fig. 8 a). Although data in our meta-analysis are scarce, 25

F o r R e v i e w O n l y
we observed a tendency for conifers to respond more strongly than deciduous trees to N 1 supply [START_REF] Nadelhoffer | Decadal-scale 11 fates of 15N tracers added to oak and pine stands under ambient and elevated N inputs at the 12 Harvard Forest (USA)[END_REF]), suggesting a difference in the NH 4 + vs. NO 3 -uptake 2 (Gebauer et al. 2000) and their specific N isotope signature. The differences observed for the 3 δ 15 N values measured in tree subjected to ammonium nitrate and urea applications could 4 reflect a different isotopic signature of these compounds as well as different N processing. 5 6 Soil N applications speed up many biogeochemical processes related to N, i.e., N microbial 7 immobilization and/or losses, which in turn not only modify the immobilized N in a form 8 more readily available to trees, but also its isotopic signature. In particular, losses of 15 N-9 depleted compounds, through denitrification, NO 3 -leaching or ammonia volatilization, which 10 increase when the N dose exceeds the requirements of the trees', are reflected in plant 11 material as an increase in δ 15 N (Johannison & Högberg 1994) (Fig. 8b). 12 13 14 If N losses had occurred also at our experimental site in response to the spraying, they would 15 have resulted in a significant enrichment of δ 15 N in tree rings compared to the control. 16 However, this was not the case. In fact, spraying of N over tree canopies tended to lower the 17 δ 15 N in tree rings compared to the wet control and the NS treatments (Fig. 2 g-i, Fig. 8 a). We 18 can exclude a difference in the isotopic signature of the ammonium nitrate salt used during 19 the experiment, which had a δ 15 N value of 1.6‰. Likewise, the δ 15 N value of the treatment 20 was unlikely to have been diluted by the δ 15 N of background N dep , given that the rate of 21 background N deposition (8 kg N ha -1 yr -1 ) was much smaller than the added rates (48 and 96 22 kg N ha -1 yr -1 ). Also, based on δ 15 N values of NH 4 + (-3.8±2.6 ‰) and NO 3 -(-3.8±2.4 ‰) in 23 rainfall, measured for six different sites across Scotland (Heaton, pers. comm.), only a small 24 dilution effect would be expected. Hence, assuming no changes in the δ 15 N of the atmospheric 25

F o r R e v i e w O n l y
N source, the long-term trend measured for δ 15 N in the different treatments reflect different 1 dynamics of canopy N exchange rather than changes of N in the soil. This suggests that soil 2 applications fail to capture all the dynamics and isotope fractionation that occur when 3 deposition takes place on the canopy. 4 5 Before reaching the soil, the atmospheric N can be modified by the interaction with tree 6 canopies, which can retain most of the N added by the spray treatments, leaving only a small 7 amount to reach the soil. [START_REF] Chiwa | Throughfall chemistry 12 and canopy interactions in a Sitka spruce plantation sprayed with six different simulated 13 polluted mist treatments[END_REF] reported significant levels of canopy N retention 8 at our experimental site, with 2NS and N showing higher N retention than NS (i.e., around 9 80%). Further, the dynamics of canopy N exchange varied according to whether the N 10 addition also contained S. In fact, the absolute amount of N retained as NH 4 + and NO 3 -was 11 similar in the 2NS treatment, whereas tree canopies in the N treatment retained more NH 4 -12

Nthan NO 3 -N [START_REF] Chiwa | Throughfall chemistry 12 and canopy interactions in a Sitka spruce plantation sprayed with six different simulated 13 polluted mist treatments[END_REF]). The δ 15 N measured in needles (for the year 1997) and tree 13 rings at each treatment would therefore reflect the proportion of NH 4 + and NO 3 -ions taken up 14 and their relative isotope signature, which can be very different [START_REF] Heaton | Potential canopy influences on the isotopic composition of nitrogen and 8 sulphur in atmospheric deposition[END_REF]. 15 16 The substantial level of canopy N retention would have restricted the amount of N washed 17 through to the soil, limiting any soil N accumulation or loss, thus explaining why the δ 15 N 18 shift goes in the opposite direction to that found when N fertilizer was added directly to the 19 soil. In fact, no clear indication of excess N was apparent after 5 years, except in the 2NS 20 treatment where needle fall was significantly increased (reducing canopy N uptake) and N 2 O 21 emissions were recorded [START_REF] Sheppard | Effects of five 15 years of frequent N additions, with or without acidity, on the growth and below-ground 16 dynamics of a young Sitka spruce stand growing on an acid peat: implications for 17 sustainability[END_REF]. 22 23 Six years after the experiment stopped, δ 15 N in the soil did not reveal any clear signal of the 24 treatments (Fig. 4). This further suggests that only a fraction of the N reached the soil, which 25

F o r R e v i e w O n l y
eventually was diluted in relation to the large soil N pool. Treatment effects, with particular 1 reference to the N addition, were not detectable in tree rings once treatment ceased (Fig. 2 g-i, 2 Fig. 4). However, it must be recognized that pooling the annual rings from 2004 to 2009 3 precluded our ability to track the rate of loss of the treatment signal. Interestingly, a carry-4 over effect of the treatment-specific contribution to canopy N dynamics was still present in 5 undecomposed litter sampled 5 years after the experiment stopped, i.e. more negative δ 15 N 6 values in the plot that received N treatment. 7 8

Conclusions 9

The combination of δ 13 C, δ 18 O and δ 15 N in tree rings enhanced our understanding of how 10 variations in atmospheric N and S deposition affect some key processes underpinning the C 11 and N cycle in a Sitka spruce. Specifically we have shown that: 12 13 i. N independent of the presence of S, increased the ratio between assimilation (A) and 14 stomatal conductance (g s ) in favour of A, thus supporting the fertiliser effect of N. 15

However, the combination of N and S, as a double dose, counteracted the duration and 16 the strength of the positive effect of N on WUE i , likely related to a negative effect of S 17 on stomatal control. 18

ii. Based on a mean value of WUE i , trees largely returned to pre-treatment values within 19 5 years (2004-2009) after the cessation of N dep . However, this was not the case for 20 S dep , where WUE i continued to fall. A carry-over effect of S excess could affect 21 negatively the balance between A and g s , by reducing A for trees subjected to 2NS . 22 This suggests that the beneficial role of N dep is counteracted by the negative effect of 23 S, which may still be operating in forests that have received high loads of acid rain. It 24 is possible that such a 'payback' effect after reduction in NS deposition was also 25 iii. Treatment-specific variations in the δ 15 N in tree rings carried the effect of different 3 canopy nitrogen dynamics, either in both the short (in needles) and the long-term (tree 4 rings), with an isotope signal imprinted also in the litter, even after treatment stopped. 5 iv. Soil and canopy N applications showed an increase of WUE i in response to increased 6 N dep . However, for the same N dose applied, spraying over tree canopies led to a larger 7 change in WUEi compared to soil applications. By contrast, the two application 8 methods produced different results with regard to the changes in the N dynamics. This 9 suggests, on one hand, that experiments considering soil application might 10 underestimate the contribution of N dep on the ecosystem N cycle, since they exclude 11 possible N exchanges at the canopy level. On the other hand, they might overestimate 12 the effect of N dep to the soil, since canopy N retention tends to reduce the quality and 13 quantity of N reaching the soil, thus contributing to slowing down soil bioactivity and 14 preventing N accumulation and losses. 15

F o r R e v i e w O n l y

  ENHANCED N AND S DEPOSITION AS REVEALED BY COMBINING δ13C, δ18O and δ15N IN TREE RINGS

23 2 .

 2 Materials and methods 242.1 Stable isotope theory 25

δ 2 (

 2 CO 2 as a 15 result of diffusion through air (4.4‰), b is the fractionation during carboxylation (27‰) by 16 the CO 2 -fixing enzyme Rubisco. Due to their reciprocal link to the c i /c a ratio, δ13 C can be 17 considered to infer changes in the intrinsic water-use efficiency (WUE i ), which is defined as 18 the ratio between A and stomatal conductance to water vapour ( eqn. [1] c i can be derived as follows: 2213 C a and c a for each year can be estimated according to the equations proposed by Feng

  in the transpiration stream i.e., coming from the soil (source of water). 1

20 For δ 13 C 3 4

 20133 N value of 1.6±0.42 ‰. 19 and δ 15 N analyses, each determined in a separate measurement, samples were 21 combusted under an excess of oxygen with an elemental analyzer (EA-1100, Carlo Erba, 22 Milano, Italy), connected to an isotope ratio mass spectrometer (Delta -S Finnigan MAT, 23 Bremen, Germany) through a variable open split interface (ConFlo II, Finnigan MAT, 24Bremen, Germany). For δ18 O, the weighed bulk organic material was decomposed to CO by 25 1080 °C in a different elemental analyzer (EA-1108, Carlo Erba, Milano, 1 Italy), which was connected to the continuous flow mass spectrometer (Delta -S Finnigan 2 MAT, Bremen, Germany).Ground bulk samples obtained from 1-year old needles collected in December 1997 and used 5 in previous investigation for measuring nutrient content in needles (e.g.,[START_REF] Sheppard | Effects of five 15 years of frequent N additions, with or without acidity, on the growth and below-ground 16 dynamics of a young Sitka spruce stand growing on an acid peat: implications for 17 sustainability[END_REF]) 6

  of treatments on δ 13 C, δ 18 O and δ 15 N in tree rings 8 Before the spray treatments began, δ 13 C, δ 18 O and δ 15 N in 1995 showed similar values for all 9

18

 18

2 3

 2 the exception of 2003, when an increase of δ 18 O, with particular reference to N and 2NS, 1 was observed.After the experiment had ceased, in all the treatments δ 13 C, δ18 O and δ 15 N values approached 4 those measured in the wet control (Tables3, 4, 5), bearing in mind that stable isotopes were 5 measured in the pooled annual ring from 2004 to 2009. With particular reference to δ 15 N, 6

  change[START_REF] Balster | Coupling tree-ring δ 13 C and δ 15 N to test the effect 8 of fertilization on mature Douglas-fir (Pseudotsuga menziesii var. glauca) stands across the 9 Interior northwest, USA[END_REF] or even a reduction (Heaton & Crossley 1995) of WUE i was 22 observed with applied N. This latter study interestingly used the same NS combination as

  N enrichment in 14 tree rings compared to control was lower when N was added in combination with other 15 nutrients (Elhani et al.

  at the same site. Despite the similarity in all 25

10 In

 10 the case of 2NS and NS treatments, the reduction of δ 18 O compared with the control 11 suggests that together with A, g s might have increased too. Acid mist has been shown to alter 12 cuticular wax structure (Fowler et al. 1980; Shepherd & Griffiths 2006 and references therein) 13

  effects on stable C and O isotope trends 1 Long dendro-isotope chronologies have often reported an increase of δ 13 C in tree rings during 2 the juvenile stage of tree growth (Duquesnay et al. 1998; McCaroll & Loader 2004), although 3

12 13δ

 12 18 O in tree rings reflects both δ 18 O of source water and the changes in δ 18 O in leaf water 14 during transpiration, modified by the Péclet effect[START_REF] Craig | Deuterium and oxygen-18 variations in the ocean and the marine 20 atmosphere[END_REF], Farquhar & Lloyd 15 1993,Barbour et al. 2000a), in response to changes in environmental factors, e.g.,[START_REF] Barbour | Stable oxygen isotope composition of plant tissue: a review[END_REF]

18

 18

  Changes in WUEi: same direction and magnitude? 1

3 4

 3 in the same range reported by Elhani et al.(2005), where however, N was added in 22 combination with other nutrients (e.g., K, Ca and P). Lower responses were observed by 23Elhani et al. (2005) when N was added alone (Fig.8).24 It must be acknowledged that differences in the N use efficiency and N-C allocation in young 1 (i.e., the present study) and mature(Betson et al., Elhani et al.) trees might account for the 2 differences in the WUE i following N additions.In the case of[START_REF] Balster | Coupling tree-ring δ 13 C and δ 15 N to test the effect 8 of fertilization on mature Douglas-fir (Pseudotsuga menziesii var. glauca) stands across the 9 Interior northwest, USA[END_REF] and[START_REF] Heaton | Carbon isotope variations in a plantation of Sitka spruce, and 6 the effect of acid mist[END_REF], values deviated from 5

15 16

 15 Footprint of N applications on δ 15 N in tree rings: what do they tell us about the N cycle in17 forests? 18

  the N treatment, but this was not clear from the bulk measurements for the 1 6-year period (2004-2009). 2

16 Acknowledgement.

 16 RG was supported during field and lab activities (May-August 2009) by 17 the European Science Foundation (ESF), within the programme Nitrogen in Europe, grant n 18 2221. RG is currently supported by a Newton International Fellowship funded by the Royal 19 Society, the British Academy and the Royal Academy of Engineering. Stable isotopes 20 analyses were funded by the project 'Greenhouse gas management in European land use 21 systems (GHG EUROPE) awarded to MAS, PL and LJS for δ 15 N and by Research funding 22 within the Newton International fellowship for δ 13 C and δ 18 O. MM acknowledges NERC for 23 their support via the grant n. NE/G00725X/1. We would like to thank C. McEvoy (Northern Hydrology) for their logistic support during the field work; M. Jarvis (University of Glasgow) 1 for providing facilities for wood cores chemical pre-treatment and C. Chilcott (University of 2 Edinburgh) for measuring stable isotopes in needles. R.G. acknowledges R. Siegwolf (Paul 3 Scherrer Institute) and T. Heaton (NERC Isotope Geosciences Laboratory) for stimulating Dezi S, Medlyn BE, Tonon G, Magnani F (2009) The effect of nitrogen deposition on forest 1 carbon sequestration: a model-based analysis. Global Change and Biology. 16, 1470-1486, 2 doi: 10.1111/j.1365-2486.2009.02102.x. 3 Duquesnay A, Breda N, Stievenard M, Dupouey JL (1998). Changes of tree-ring δ 13 C and 4 water-use efficiency of beech (Fagus sylvatica L.) in north-eastern France during the past 5 century. Plant Cell and Environment. 21, 565-572. 6 Eamus D, Fowler D (1990) Photosynthetic and stomatal conductance responses to acid mist 7 of red spruce seedlings. Plant Cell and Environment. 13, 349-357. 8 Ehleringer JR, Hall AE, Farquhar GD (1993) Water use in relation to productivity. In Stable 9 Isotopes and Plant Carbon-Water Relations (eds Ehleringer JR, Hall AE & Farquhar GD) pp. 10 3-8. Academic Press, New York. 11 Elhani S, Guehl JM, Nys C, Picard JF, Dupouey JL (2005) Impact of fertilization on tree-ring 12 δ 15 N and δ 13 C in beech stands: a retrospective analysis. Tree Physiology. 25, 1437-1446. 13 Farquhar GD (1980) Carbon isotope discrimination by plants: effects of carbon dioxide 14 concentrations and temperature via the ratio of intercellular and atmospheric CO 2 15 concentration. In: Carbon Dioxide and Climate: Australian Research (ed. Pearman GI) pp. 16 105-10. Australian. Academy of Science, Canberra. 17

Farquhar 20 Farquhar 25 F

 2025 Photosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology. 40, 503-19

Figure 1 .

 1 Figure 1. (a) Distribution of replicates for each treatment, modified from Chiwa et al. (2004). (b)

Figure 2 .

 2 Figure 2. Trend of δ 13 C (panels a, b, c), δ 18 O (panels d, e, f) and δ 15 N ( panels g h i) in tree rings from 1995

Figure 3 .

 3 Figure 3. Differences between treatments (2NS, NS and N) and control for δ 15 N (panel a) and δ 13 C

Figure 4 .

 4 Figure 4. δ 15 N values measured in soil and litter sample collected in June 2009 and in the group of

Figure 5 .

 5 Figure 5. Differences from the wet control (i.e., treatment-control) for WUEi values measured in

Figure 6 .

 6 Figure 6. Main panel. Combination of δ 13 C and δ 18 O values for 2NS, NS and N treatment,

Figure 7 .

 7 Figure 7. Difference between treatment and control for WUE i calculated from δ 13 C values

Figure 8 .

 8 Figure 8. (a) Difference between treatment and wet control for δ 15 N measured in tree rings,

negative relationship between δ 18 O in leaf organic matter and 16 g s , due to their reciprocal link to vapour pressure deficit, which, in turn depends on Rh 17

  O enrichment at the evaporative site is modified by the Péclet effect (Farquhar 10 & Lloyd 1993; Barbour et al. 2000a), which describes the mixing of enriched water at the 11 evaporative sites with unenriched water pools within leaves. The signal of δ 18 O lw is then 12

	1	Scheidegger et al. (2000) proposed a conceptual model, which helps to clarify whether
	2	changes in A, g s or both accounted for the variations of WUE i . The model inferred changes in
	3	c i and therefore A from δ 13 C, assuming a negative relationship between the two parameters,
	4	according to Eqn. [1]. Further, changes in Rh were derived from δ 18 O in tree organic matter,
	5	which, in turn, allows variations in g s to be predicted. As a further extension of the model,
	6	Grams et al. (2007) showed that g s can be inferred directly from δ 18 O in leaf cellulose, which
	7	was confirmed also by comparing stable isotopes with gas exchange measurements. In both
	8 9 10	conceptual approaches, an important prerequisite is similar δ 18 O of source water and vapour F water for the investigated trees. o r
	11	δ 15 N and ecosystem N cycle	R
	13 14 12 13	imprinted in the leaf organic molecules, due to the isotopic exchange between carbonyl oxygen and water in which they are formed (Sternberg et al. 1986). e The theoretical understanding of δ 15 N in tree pools is not as comprehensive as for δ 13 C and v δ 18 O. This is due to the complexity of the N cycle and the possibility for plants to use i e w different forms of N, i.e., ammonium (NH 4 + ), nitrate (NO 3 -
	15 18	O n l Several authors have reported a (Barbour et al. 2000b; Grams et al. 2007; Sullivan & Welker 2007; Ripullone et al. 2009). y
	19	The signal of changes in the δ 18 O of leaves may also be dampened in tree rings, due to isotope
	20	fractionation during translocation of sucrose and synthesis of cellulose in the tree stem
	21	(Sternberg et al. 1986; Saurer et al. 1997; Gessler et al. 2009). However, δ 18 O in tree rings
	22	still carries information related to changes in g s (McCaroll & Loader 2004; Barbour 2007).
	23
	24	Combining δ 13 C and δ 18 O in a conceptual model

Table 1 .

 1 Treatments of acid mist applied on a young Sitka spruce plantation at Deepsyke forest (Scotland, UK) from 1996 to 2003. In the first phase of the experiment(1996)(1997)(1998)(1999)(2000) all treatments had 4 replicates, while in the second phase(2001)(2002)(2003), two plots were maintened with the original treatments, while in the other two the recovery from acidity was started. Adapted fromSheppard et al. 2004.

	Number	Treatments	Treatments	Components	Doses	pH
	of plots	(1996-2000)	(2001-2003)		(kg ha -1 yr -1 )
		[I]	[II]		N	S
	2	NSacid	NSacid	NH 4 NO 3 +H 2 SO 4 [I-II]	48	50	2.5
	2	NSacid	NS-recovery (N)	NH 4 NO 3 +H 2 SO 4 [I]	48	50	2.5
				NH 4 NO 3 [II]	48		4.5
	2	2NSacid	2NSacid	NH 4 NO 3 +H 2 SO 4 [I-II]	96	100	2.5
	2	2NSacid	2NS-recovery (2N)	NH 4 NO 3 +H 2 SO 4 [I]	96	100	2.5
				NH 4 NO 3 [II]	96		4.5
	2	N	N	NH 4 NO 3 [I-II]	48		4.5
	2	N	N-recovery	NH 4 NO 3 [I]	48		4.5
			(wet control)	None [II]	-	-	4.5
	4	Wet control	No changes	None	-	-	4.5

Table 2

 2 Research papers included in the meta-analysis.

	Authors	Location	Ndep	Species	Age	Fertiliser	Application	Dose applied	Duration of the experiment	Isotope
			(kg ha -1 yr -1 )		(years)			(kg ha -1 yr -1 )
	Balster							178	Dose applied twice over a 6-	δ 13 C and δ 15 N
	et al. (2009) Interior NW, USA	N/A	Pseudotsuga menziesii	85	Urea	Soil	357	years interval	in tree rings
	Betson					Ammonium		30	32 years: dose applied from
	et al. (2007) Norrliden, Sweden	3	Pinus sylvestris	∼50	nitrate	Soil	60	1971 to 2002	δ 13 C in needles
								90
	Brooks &							157	1 year: dose applied only once,	δ 13 C, WUEi in
	Coulombe	Washington, USA	N/A	Pseudotsuga menziesii	85	Ammonium	Soil	314	in April 1964	tree rings
	(2009)					nitrate		471
	Choi								7 years: dose applied every
	et al. (2005) Scotland County, NC, USA	N/A	Pinus taeda	15	Urea	Soil	101	year from 1992 to 1998	δ 13 C in needles
	Elhani					Ammonium		100	2 years: dose applied once	δ 13 C, δ 15 N in
	et al. (2005) Brittany, France	9-13	Fagus sylvatica	80	nitrate	Soil	100with PKCa	(e.g., in May 1973 and 1974)	tree rings
	Heaton &								3 years: dose applied twice per
	Crossley	Scotland, UK	10	Picea sitchenisis	20	Ammonium	Canopy	48	week during the grwoing	δ 13 C in needles
	(1995)					nitrate			season from 1991 to 1993.
	Nadelhoffer					Ammonium		8
	et al. (2004) Massachusetts, USA	8	Deciduous species	∼80	nitrate	Soil	58	16 years: 6 montly additions	δ 15 N in tree
								158	during the growing season	rings
	Nadelhoffer					Ammonium		8	from 1988 to 2004
	et al. (2004) Massachusetts, USA	8	Pinus resinosa	∼80	nitrate	Soil	58
								158

Table . 3

 . . Results of the statistical analyses of the differences between treatments in δ 13 C for theperiod 1995, 1996-2000, 2001-2003 and 2004-2009. Differences for the first and last period were tested by univariate ANOVA, with four replicates for each treatment. The effect of different treatments during the second period was explored by repeated-measures ANOVA, with four replicates for each treatment. Finally, differences in the recovery period were assessed by independent sample t-tests, with two replicates for each treatment.

		F o r
	Treatments 2NS NS N	1995 Pre-treatment -25.91 a (±0.24) -26.05a (±0.15) -26.14a (±0.09)	2001-2003 Treatment II Recovery Treatment -29.43 (±0.13) n.s. -29.02 (±0.20) -29.47 (±0.19) n.s. -29.27 (±0.29) e 1996-2000 Treatment I R -27.85 a (±0.14) -28.15 a (±0.15) v -27.74 b (±0.12) -28.99 (±0.12) n.s. -29.29 (±0.14) i e	2004-2009 Post-treatment -29.56a (±0.11) -29.29a (±0.32) -29.12a (±0.02)
	Wet control Statistical	-26.14a (±0.11) ANOVA	-28.46 b (±0.12) Repeated-	Independent t-test w	-29.05a (±0.15) ANOVA
	tests	F=0.479; P=0.7	measures F=5.912 P<0.05	t=-1.73, P=0.11 t=-0.60, P=0.56 t=1.63, P=0.13	F=1.6 P=0.239 l O n
					y

Table 4 .

 4 Results of the statistical analyses of the differences between treatments in δ 18 O for theperiods 1995, 1996-2000, 2001-2003 and 2004-2009. Differences for the first and last period were analysed using univariate ANOVA, with four replicates for each treatment. The effect of different treatments during the second period was explored by repeated-measures ANOVA, with four replicates for each treatment. Finally, differences in the recovery phase were assessed by indipendent sample t-test, with two replicates for each treatment. For the treatment I (1996-2000), estimated marginal mean are provided (i.e., adjusted for the variability in the soil relative water content among plots).

	2001-2003
	Treatment II

Table 5 .

 5 Results of the statistical analyses of the differences between treatments in δ 15 N for the periods1995, 1996-2000, 2001-2003 and 2004-2009. Differences for the first and last period were analysed using univariate ANOVA, with four replicates for each treatment. The effect of different treatments during the second period was explored by repeated-measures ANOVA, with four replicates for each treatment. Finally, differences in the recovery phase were assessed by indipendent sample t-test, with two replicates for each treatment.

	2001-2003
	Treatment II

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