domain: sde sdv sdu

text:
Mary Gagen
email: m.h.gagen@swansea.ac.uk.

Walter Finsinger

Rike Wagner

Danny Mccarrol

Neil J Loader

Iain ; Robertson

Risto ; Jalkanen

Giles H F Young

Andreas Joachim Kirchhefer

Andreas ; Kirchhefer

Friederike Wagner-Cremer

Danny Mccarroll

Andreas Kirchhefer

Evidence of changing intrinsic water use efficiency under rising atmospheric CO2 concentrations in Boreal Fennoscandia from subfossil leaves and tree ring δ13C ratios

Keywords: intrinsic water use efficiency, physiological forcing, boreal forest, stomatal conductance, stable isotope dendroclimatology, stomatal density intrinsic water use efficiency, stomatal density, stomatal conductance, Fennoscandia, boreal forest, physiological forcing

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Investigating the many internal feedbacks within the climate system is a vital component of the effort to quantify the full effects of future anthropogenic climate change. The stomatal apertures of plants tend to close and decrease in number under elevated CO2 concentrations, increasing water use efficiency and reducing canopy evapotranspiration. Experimental and modelling studies reveal huge variations in these changes such that the warming associated with reduced evapotranspiration (known as physiological forcing) is neither well understood or constrained. Palaeo-observations of changes in stomatal response and plant water use efficiency under rising CO2 might be used to better understand the processes underlying the physiological forcing feedback and to link measured changes in plant water use efficiency to a specific physiological change in stomata. Here we use time series of tree ring δ 13 C (Pinus sylvestris) and subfossil leaf (Betula nana) measurements of stomatal density and geometry to derive records of changes in intrinsic water use efficiency and maximum stomatal conductance in the Boreal zone of northern Finland and Sweden. We investigate the rate of change in both proxies, over the recent past. The two independent lines of evidence from these two different Boreal species indicate increased intrinsic water use efficiency and reduced maximum stomatal conductance of similar magnitude from preindustrial times (ca. AD 1850) to around AD 1970. After this maximum stomatal conductance continues to decrease to AD 2000 in B. nana but intrinsic water use efficiency in i>P. sylvestris reaches a plateau. We suggest that northern boreal i>P. sylvestris might have reached a threshold in its ability to increase water use efficiency and we discuss the implication for modelling stomatal conductance in the land surface schemes of General Circulation Models. Investigating the many internal feedbacks within the climate system is a vital component of the effort to quantify the full effects of future anthropogenic climate change. The stomatal apertures of plants tend to close and decrease in number under elevated CO 2 concentrations, increasing water use efficiency and reducing canopy evapotranspiration. Experimental and modelling studies reveal huge variations in these changes such that the warming associated with reduced evapotranspiration (known as physiological forcing) is neither well understood or constrained. Palaeo-observations of changes in stomatal response and plant water use efficiency under rising CO 2 might be used to better understand the processes underlying the physiological forcing feedback and to link measured changes in plant water use efficiency to a specific physiological change in stomata.

Introduction

Plant gas exchange plays an important role in the positive climate feedback known as physiological forcing, because models indicate that the atmosphere dries out as evapotranspiration reduces under raised atmospheric CO 2 concentrations [START_REF] Cox | The impact of new land surface physics on the GCM simulation of climate and climate sensitivity[END_REF].

Evapotranspiration reduces because the stomatal apertures of plants tend to close, decrease in number and alter their geometry [START_REF] Woodward | Stomatal Numbers Are Sensitive to Increases in CO 2 from Preindustrial Levels[END_REF][START_REF] Woodward | The Influence of CO 2 Concentration on Stomatal Density[END_REF][START_REF] Kürschner | Predicting the response of leaf stomatal frequency to a future CO 2 -enriched atmosphere: constraints from historical observations[END_REF][START_REF] Gray | The HIC signalling pathway links CO 2 perception to stomatal development[END_REF][START_REF] Lake | Plant development -Signals from mature to new leaves[END_REF]) when exposed to elevated CO 2 , increasing water use efficiency (WUE unit carbon gain for each unit of water lost), and ultimately reducing evapotranspiration [START_REF] Cox | The impact of new land surface physics on the GCM simulation of climate and climate sensitivity[END_REF][START_REF] Bunce | Carbon dioxide effects on stomatal responses to the environment and water use by crops under field conditions[END_REF][START_REF] Young | CO 2 signaling in guard cells: Calcium sensitivity response modulation, a Ca 2+ -independent phase, and CO 2 insensitivity of the gca2 mutant[END_REF]. The climatic impact of the stomatally derived reduction in evapotranspiration is described by the feedback term physiological forcing. Climate model-based studies have shown that reductions in plant stomatal conductance can lead to enhanced regional and global warming via physiological forcing [START_REF] Sellers | Comparison of Radiative and Physiological Effects of Doubled Atmospheric CO 2 on Climate[END_REF][START_REF] Sellers | BOREAS in 1997: Experiment overview, scientific results, and future directions[END_REF][START_REF] Cox | The impact of new land surface physics on the GCM simulation of climate and climate sensitivity[END_REF][START_REF] Betts | Simulated responses of potential vegetation to doubled-CO 2 climate change and feedbacks on near-surface temperature[END_REF] whilst

F o r R e v i e w O n l y 3
experiments at the plant level reveal the incredible speeds with which plants can alter their stomatal numbers after exposure to raised CO 2 [START_REF] Lake | Plant development -Signals from mature to new leaves[END_REF][START_REF] Hetherington | The role of stomata in sensing and driving environmental change[END_REF]. However, physiological forcing is not well understood or constrained (National Research Council 2003;[START_REF] Doutriaux-Boucher | Carbon dioxide induced stomatal closure increases radiative forcing via a rapid reduction in low cloud[END_REF]. For example, warming might be offset by increases in leaf area index due to higher temperatures and precipitation changes that may accompany higher CO 2 levels [START_REF] Betts | Simulated responses of potential vegetation to doubled-CO 2 climate change and feedbacks on near-surface temperature[END_REF] and there is evidence of thresholds (so-called response limits) in plant capacity to raise water use efficiency [START_REF] Kürschner | Predicting the response of leaf stomatal frequency to a future CO 2 -enriched atmosphere: constraints from historical observations[END_REF].

Moreover the degree of spatial and species variability in the response of water use efficiency the atmosphere can be estimated from δ 13 C as iWUE, the potential rather than actual WUE [START_REF] Ehleringer | Atmospheric CO 2 and the Ratio of Intercellular to Ambient CO 2 Concentrations in Plants[END_REF][START_REF] Saurer | Carbon isotope discrimination indicates improving water-use efficiency of trees in northern Eurasia over the last 100 years[END_REF]; actual WUE being also influenced by changes in respiratory losses over time at the whole plant level and being sensitive to vapour pressure differences between atmosphere and leaf. Tree ring stable carbon isotope ratio derived time series of iWUE represent the maximum potential WUE as it changes through time. Stable carbon isotope (δ 13 C) ratios are known to provide a reliable method of monitoring long term changes in plant physiology [START_REF] Farquhar | On the Relationship between Carbon Isotope Discrimination and the Inter-Cellular Carbon-Dioxide Concentration in Leaves[END_REF].

and
Whilst rising iWUE over the recent past has been widely noted in tree ring based δ 13 C studies (e.g. [START_REF] Saurer | Carbon isotope discrimination indicates improving water-use efficiency of trees in northern Eurasia over the last 100 years[END_REF][START_REF] Waterhouse | Northern European trees show a progressively diminishing response to increasing atmospheric carbon dioxide concentrations[END_REF] The C3 plants have internalised their gas exchange surfaces in order to maintain control over water budget, a necessary step as any membrane which is permeable to large CO 2 molecules is also permeable to water molecules [START_REF] Mccarroll | Stable isotopes in tree rings[END_REF]). The gas exchange and water balance mechanisms in plants are therefore intrinsically linked; when plants take in CO 2 through stomata they transpire water at the same time. Long term water exchange in plants can be studied via δ 13 C ratios in annual tree ring cellulose, because δ 13 C essentially records CO 2 gas exchange [START_REF] Saurer | Carbon isotope discrimination indicates improving water-use efficiency of trees in northern Eurasia over the last 100 years[END_REF]). There is a slight offset between leaf and trunk δ 13 C and using δ 13 C cellulose to calculate iWUE, rather than initial photosynthate or even leaf δ 13 C, may introduce some bias into the calculated iWUE values. However, the temporal trends will remain unaffected [START_REF] Waterhouse | Northern European trees show a progressively diminishing response to increasing atmospheric carbon dioxide concentrations[END_REF]).

Increased iWUE under raised CO 2 has been reported in:Pinus [START_REF] Runion | Longleaf pine photosynthetic response to soil resource availability and elevated atmospheric carbon dioxide[END_REF], Quercus [START_REF] Anderson | Ontogeny affects response of northern red oak seedlings to elevated CO 2 and water stress carbon assimilation and biomass production[END_REF][START_REF] Lodge | Stomatal acclimation to increased CO 2 concentration in a Florida scrub oak species Quercus myrtifolia Willd[END_REF]), Fagus (Bucher-Wallin et al. 2000) and

Picea [START_REF] Roberntz | Effects of elevated CO 2 concentration and nutrition on net photosynthesis, stomatal conductance and needle respiration of field-grown Norway spruce trees[END_REF] with an average gain of around 19% in northern Eurasia [START_REF] Saurer | Carbon isotope discrimination indicates improving water-use efficiency of trees in northern Eurasia over the last 100 years[END_REF] over the industrial period, although over the wider northern hemisphere percentage changes are highly variable. [START_REF] Waterhouse | Northern European trees show a progressively diminishing response to increasing atmospheric carbon dioxide concentrations[END_REF] , between ~AD 1930 and1980. However, at some sites iWUE has remained static [START_REF] Marshall | Homeostatic gas-exchange parameters inferred from C-13/C-12 in tree rings of conifers[END_REF] or has actually shown decreases [START_REF] Saurer | Carbon isotope discrimination indicates improving water-use efficiency of trees in northern Eurasia over the last 100 years[END_REF]. The rates of change, even where iWUE is rising, also seem to alter over time [START_REF] Waterhouse | Northern European trees show a progressively diminishing response to increasing atmospheric carbon dioxide concentrations[END_REF]. It is not surprising that changes in iWUE are spatially variable because water usage is strongly dependant upon site conditions; the level of moisture stress experienced at a site, local pollution effects or other stress factors and the species sensitivity to moisture variability. With this in mind, CO 2 response limits may well need to be investigated on regional and species basis.

Materials and Methods

Stable carbon isotope measurements from annual tree rings as an estimate of intrinsic water use efficiency.

Stable carbon isotope dendroclimatology was used to produce a time series of iWUE averaged from eight individually measured Pinus sylvestris trees at a site (Laanila) in northern Finnish Lapland (Table 1). Measurements of δ 13 C in wood cellulose extracted from absolutely dated annual rings of trees record variability in the internal partial pressure of CO 2 (ci) [START_REF] Farquhar | On the Relationship between Carbon Isotope Discrimination and the Inter-Cellular Carbon-Dioxide Concentration in Leaves[END_REF]) thus ci and iWUE were calculated from measured tree ring δ 13 C values. from the cores and extracted to α-cellulose using a modified batch processing method [START_REF] Loader | An improved technique for the batch processing of small wholewood samples to alpha-cellulose[END_REF][START_REF] Rinne | On the purification of alpha-cellulose from resinous wood for stable isotope (H, C and O) analysis[END_REF]. The first 50 years of material from each tree was removed prior to analysis to avoid including 'juvenile effects' in δ 13 C [START_REF] Mccarroll | Stable isotopes in tree rings[END_REF].

δ 13 C time series were then corrected for changes in atmospheric δ 13 C of CO 2 [START_REF] Francey | A 1000-year high precision record of delta C-13 in atmospheric CO 2[END_REF][START_REF] Mccarroll | Stable isotopes in tree rings[END_REF]). Cellulose δ 13 C ratios were measured either offline by sealed-tube combustion followed by cryogenic distillation and analysis on a VG Micromass 602C dual inlet stable isotope ratio mass spectrometer (SIRMS) or online via an ANCA Elemental Analyser coupled to a PDZ Europa 20/20 SIRMS. Sample precision was 0.1‰ as measured via in-house and IAEA cellulose standards and reference gas.

Fractionation (∆) of carbon isotopes by C3 plants is described as follows,

∆ = a + (b-a) • ci/ca (1)
where a (28‰) and b (4.4‰) are constants representing fractionation due to diffusion and carboxylation respectively and ci and ca are intercellular and atmospheric CO 2 concentrations.

Fractionation (∆) acts upon the isotopic composition of the source gas (atmospheric CO 2 ) as follows: AD 1919AD , 1935AD , 1958AD , 1963AD , and 1965)), (ii) from a mire at Kevo (leaves growing AD 1976AD -1996)), and (iii) from modern plants growing at Kevo (69°45'N, 27°E, AD 199727°E, AD -200827°E, AD , 1998 missing) missing).

δ 13 C plant = δ 13 C atmosphere -∆ (2) F o r R e v i
Subfossil leaf fragments were picked from peat extracted from the mire, identified and separated under a binocular microscope by leaf and cuticle morphologies. ). Based on these measurements, maximum stomatal conductance to water vapour (g wmax [mol m -2 s -1 ]) was calculated using the equation proposed by [START_REF] Franks | Maximum leaf conductance driven by CO 2 effects on stomatal size and density over geologic time[END_REF]:

      + ⋅ ⋅ = π π max max max 2 a l a SD v d g w , (4)
where d (the diffusivity of water vapour in air [m 2 s -1 ]), v (the molar volume of air [m 3 mol - 1 ]), and l (the depth of the pore) were taken as constants, and a max is the maximal pore aperture that was approximated as π(PL/2) (5).

In addition, stomatal conductance g s was calculated from the reciprocal of maximum stomatal resistance (g s [m s -1 ] = 1/R s ) following [START_REF] Parlange | Stomatal Dimensions and Resistance to Diffusion[END_REF]:

( ) ( ) SD d PL PW PL PW PL l R S ⋅         ⋅ ⋅ + ⋅ = π π 4 ln , (6)
where the half pore width (PW) was calculated as being equal to half PL. The latter equation differs from equation (4) because it does not include an "end-correction term" (π/2√(a max /π))

accounting for diffusion shells at the outside end of stomatal pores [START_REF] Franks | The effect of exogenous abscisic acid on stomatal development, stomatal mechanics, and leaf gas exchange in Tradescantia virginiana[END_REF].

The significance of the differences between equations (4) and ( 6) is explored in the Supplementary Material.

Determination of the age of sediment samples from the Kevo and Kiruna monoliths is based on radiocarbon-dated samples consisting of individual B. nana leaves, Sphagnum leaves, stems and branches [START_REF] Hicks | A near annual record of recent tree line dynamics from Northern Finland[END_REF][START_REF] Goslar | Radiocarbon dating of modern peat profiles: Pre-and post-bomb C-14 variations in the construction of age-depth models[END_REF][START_REF] Barnekow | Strong correlation between summer temperature and pollen accumulation rates for Pinus sylvestris, Picea abies and Betula spp. in a high-resolution record from northern Sweden[END_REF]). In both monoliths, several samples analysed were deposited immediately prior to, or following, the peak in atmospheric 14 C values at ~ AD 1965 derived from the testing of nuclear weapons.

Therefore, it was possible to assign calendar ages to the radiocarbon dates with high levels of precision. Control points were subsequently used to construct age-depth chronologies following [START_REF] Goslar | Radiocarbon dating of modern peat profiles: Pre-and post-bomb C-14 variations in the construction of age-depth models[END_REF]. The resulting age range of samples analysed varies in the .

To obtain a combined dataset, values from B. nana leaves growing in the same year were averaged. Subsequently, in order to extract the main trend in the g wmax , g s , S and PL records, the combined core and herbarium observations were smoothed with a robust loess function (locally weighted regression) [START_REF] Cleveland | Locally Weighted Regression -an Approach to Regression-Analysis by Local Fitting[END_REF]) with a constant window width and fixed target points in time. The smoothed curve was constructed by determining fitted values at 10-year intervals with a 100-year window-width (here referred to the full span of the window). In this method the smoothing window does not change as a function of data density around the fitted values. The smoothed g wmax time series is shown in Fig. 1 and the g s , PL, S, and the unsmoothed g wmax time series are shown in the Supplementary Material (Fig. S1).

Results

Changes in iWUE since the start of industrialisation to present (AD 1820 to 2002) at Laanila reveal a significant rise over time (Fig. 1). There is a temporary decrease in iWUE centred on AD 1910 which is known to be climatically derived. The iWUE series is scaled from δ 13 C values which contain the effects of climate and the period around AD 1910 is known to be a prolonged cold spell in northern Fennoscandia, detected in many tree ring proxies from the region (see [START_REF] Gagen | Exorcising the 'segment length curse': Summer temperature reconstruction since AD 1640 using non-detrended stable carbon isotope ratios from pine trees in northern Finland[END_REF] for details). However, there is no long term change in the 2).

Maximum stomatal conductance to water vapour (g wmax ), as inferred from the B. nana leaf measurements of stomatal density and geometry, declines from AD 1800 (Fig. 1) with a slight increase in the rate of decline from AD 1850, when ca begins to rise more steeply. Between AD 1850 and AD 2000 a 15% decrease of g wmax is observed. It is notable that prior to ca AD 1850, changes in stomatal density were accompanied by changes in stomatal size such that The pre-to post-industrial percentage changes in tree ring iWUE and leaf stomatal conductance were calculated using the AD 1800-1850 (pre-ind) and AD 1950AD -1970 (post-ind) (post-ind) averages. Over this time period the stomata density and geometry-inferred g wmax record shows a 15% decrease and the δ 13 C inferred iWUE record from Laanila shows a 12% increase, very similar magnitudes of change in two different archives. It is reasonable to assume that, given the close locations of Kiruna and Laanila the B. nana and P. sylvestris sampled for the two records have experienced similar changes in climate as CO 2 has risen such that changes due to drying/wetting are controlled for. Similar magnitude shifts in g wmax and iWUE are thus of note as they suggest that changes in assimilation rate have not been experienced and g wmax and iWUE have changed in a ~1:1 relationship over the pre-ind and post-ind periods. However, there is a clear change in rate between the iWUE and g wmax records after ~ AD 1970 (Fig. 1) Coupled biosphere-atmosphere models have been used to study the physiological forcing feedback. Leaf-scale models of stomatal conductance (g s ) [START_REF] Collatz | Physiological and Environmental-Regulation of Stomatal Conductance, Photosynthesis and Transpiration -a Model That Includes a Laminar Boundary-Layer[END_REF]) describe a parameterization which results in a linear decrease in g s with rising CO 2 concentrations (e.g. [START_REF] Sellers | Comparison of Radiative and Physiological Effects of Doubled Atmospheric CO 2 on Climate[END_REF][START_REF] Cox | The impact of new land surface physics on the GCM simulation of climate and climate sensitivity[END_REF][START_REF] Betts | Simulated responses of potential vegetation to doubled-CO 2 climate change and feedbacks on near-surface temperature[END_REF]. [START_REF] Betts | Simulated responses of potential vegetation to doubled-CO 2 climate change and feedbacks on near-surface temperature[END_REF] used coupled models of global vegetation and climate to examine the response of g s to doubled CO 2 . Mean global values of g s lower by 20% with a change to doubled CO 2 if only stomatal changes are considered with no associated change in leaf area index, but lower by only 12% when the stomatal response and the response in vegetation are included. We compared the % changes in 14 our iWUE and g wmax records to the modelled g c (g c = canopy conductance; daytime mean canopy conductance is the net transpirational conductance of all stomata integrated over the canopy depth) changes from [START_REF] Betts | Simulated responses of potential vegetation to doubled-CO 2 climate change and feedbacks on near-surface temperature[END_REF] over the same time period (Table 2). The percentage changes were calculated between 1xCO 2 (323ppmv ~AD 1967) and the year AD 2000 (~370 ppmv). For the [START_REF] Betts | Simulated responses of potential vegetation to doubled-CO 2 climate change and feedbacks on near-surface temperature[END_REF] data, percentage change AD 1967-2000 was obtained by calculating the linear trend for modelled g c at 1 x CO 2 and at 2 x CO 2 for their 'RP' (no associated change in vegetation and leaf area index kept at 1 x CO 2 ) and 'RPS' (stomatal response and response in vegetation combined) experiments (see Table 1 -16% -3% Table 2. Comparison between conductance changes used by [START_REF] Betts | Simulated responses of potential vegetation to doubled-CO 2 climate change and feedbacks on near-surface temperature[END_REF] and proxy-inferred iWUE and g wmax changes. Canopy conductance (g c [(mm s -1 )]) and % change in g c were calculated via a linear trend for modelled g c at 1 x CO 2 and at 2 x CO 2 for 'RP' (no associated change in vegetation and leaf area index kept at 1 x CO 2 ) and 'RPS' (stomatal response and response in vegetation combined) modelling experiments (from Table 1 - [START_REF] Betts | Simulated responses of potential vegetation to doubled-CO 2 climate change and feedbacks on near-surface temperature[END_REF]. These changes are compared to g wmax and iWUE changes (lower panel). The percentage changes of g wmax and iWUE were calculated between 1xCO 2 (323ppmv ~AD 1967) and the year ~AD 2000 (~370 ppmv).

Modelled changes in stomatal conductance based on changes in stomatal apertures only [START_REF] Betts | Simulated responses of potential vegetation to doubled-CO 2 climate change and feedbacks on near-surface temperature[END_REF] could clearly not account for the larger changes in iWUE or g wmax seen in the palaeo record (Table 2 and Fig. 1). Were the [START_REF] Betts | Simulated responses of potential vegetation to doubled-CO 2 climate change and feedbacks on near-surface temperature[END_REF] data used to project the linear change in g c back to the pre-ind period (AD 1800-1850) the modelled changes would be much 15 lower than those indicated by the palaeo data. However, it should be remembered that the [START_REF] Betts | Simulated responses of potential vegetation to doubled-CO 2 climate change and feedbacks on near-surface temperature[END_REF] data are global averages. The range of changes in iWUE displayed in the tree ring series is considerable even within a relatively small geographical range, site conditions clearly playing an important part in the degree to which trees are able to make water savings. The [START_REF] Betts | Simulated responses of potential vegetation to doubled-CO 2 climate change and feedbacks on near-surface temperature[END_REF] comparison with the palaeo data reveals an interesting divergence between the g wmax and iWUE data after 1970 (Table 2). The 16% change in g wmax (AD 1967(AD -2000) ) is no longer reflected in a similar magnitude change in iWUE in P. sylvestris at Laanila, which has only shifted by 3% since 1970.

Discussion

A shift to static iWUE at Laanila? [START_REF] Waterhouse | Northern European trees show a progressively diminishing response to increasing atmospheric carbon dioxide concentrations[END_REF], in a study on Finnish and British oak, beech and pine trees first noted that, at higher levels of atmospheric CO 2 concentration, rising iWUE began to plateau as measured in tree ring δ 13 C, much as is seen at Laanila (Fig 2). Long term trends in iWUE as calculated from tree ring δ 13 C essentially record the relationship between ci and ca through time (equation 3). This relationship can be described as either passive or active depending on whether ci simply tracks ca, or whether the tree uses strong stomatal control to limit the change in ci as ca rises [START_REF] Mccarroll | Correction of tree ring stable carbon isotope chronologies for changes in the carbon dioxide content of the atmosphere[END_REF]. If neither stomatal conductance nor photosynthetic rate alters, then for each rise in ca, ci will simply drift upwards in a passive response and iWUE will not increase. The more common response seen in trees appears to be the active one; iWUE increasing in parallel with rising ca as stomatal conductance is reduced.

However, an examination of the entire Laanila iWUE time series reveals an apparent change in the response of the trees over the last few decades. Fig. 2 shows the measured iWUE time et al. 2009 and[START_REF] Gagen | Exorcising the 'segment length curse': Summer temperature reconstruction since AD 1640 using non-detrended stable carbon isotope ratios from pine trees in northern Finland[END_REF] for details). We conclude, on the basis of the divergence between the active and measured ci/ca response, that iWUE in P. sylvestris at this location is no longer rising.

The results from Laanila are unusual but not unique. [START_REF] Saurer | Carbon isotope discrimination indicates improving water-use efficiency of trees in northern Eurasia over the last 100 years[END_REF] find maintenance of ci/ca in δ 13 C measurements from 26 high latitude Eurasian sites between AD 1961-1990 (335 ppmv on average). However, a switch in iWUE, around AD 1970, has been seen elsewhere (e.g. [START_REF] Robertson | Tree response to environmental change[END_REF]) whilst [START_REF] Marshall | Homeostatic gas-exchange parameters inferred from C-13/C-12 in tree rings of conifers[END_REF] also note static iWUE in various pine and fir species from sites in the western United States between ~AD 1920-1990 . A picture is emerging of a complex plastic response in long term adaptation of trees to changes in CO 2 . The Laanila series supports the idea of a limit to the ability of the trees to maintain stomatal control over ci under higher CO 2 .

For much of the 150 year record length, before the divergence at ~ AD 1970, g wmax and δ 13 C do display a ~1:1 rate change as CO 2 has risen. It is of note that these two important Boreal species record changes of similar magnitudes over the 'pre-industrial' and 'post-industrial' periods. Two independent but closely related archives, in two different species, show changes of similar magnitude over the same period in response to rising CO 2 . It is notable that the measured changes in both archives in the past are also larger than those used in climate models for modelling canopy conductance changes.

In contrast to the δ 13 C record, the g wmax record from B. nana at Kiruna and Kevo seems to still be decreasing up to the present. The continuing decrease in stomatal conductance is observed commonly in Betula pubescens from various localities, suggesting that this birch species has not reached its response limit yet in this region. [START_REF] Kürschner | Predicting the response of leaf stomatal frequency to a future CO 2 -enriched atmosphere: constraints from historical observations[END_REF] The land surface schemes of GCMs require an accurate calculation of plant transpiration, and the actual value of change in stomatal conductance under CO 2 doubling is a vital parameter [START_REF] Cox | A canopy conductance and photosynthesis model for use in a GCM land surface scheme[END_REF]). At present GCM based studies of physiological forcing operate on a linear relationship between stomatal conductance and rising CO 2 to very high ca levels [START_REF] Sellers | Comparison of Radiative and Physiological Effects of Doubled Atmospheric CO 2 on Climate[END_REF][START_REF] Cox | The impact of new land surface physics on the GCM simulation of climate and climate sensitivity[END_REF]. The evidence presented here and elsewhere from inferred iWUE measurements (e.g. [START_REF] Waterhouse | Northern European trees show a progressively diminishing response to increasing atmospheric carbon dioxide concentrations[END_REF] suggest that, in fact, the linear relationship between stomatal conductance and rising CO 2 may not be valid at today's higher CO 2 levels for all species, in all regions.

Using the linear g s model, GCMs predict the drying effect of physiological forcing to cause an additional 1ºC warming in summer temperatures in the Boreal forest [START_REF] Pollard | Use of a land-surface-transfer scheme (LSX) in a global climate model: the response to doubling stomatal resistance[END_REF]. If this is not the case and there is a limit to the rise in iWUE derived from changes in stomatal conductance then physiological forcing will cause a smaller additional warming than expected. Whilst this may be good news, potentially limiting a positive feedback, it is important to get the estimate correct.

The two species represented in this study cover a huge area of the Boreal region. In order to account for the broad spatial variability in plants' response to changing CO 2 , more records of changing iWUE would be needed to model the canopy conductance changes through time over different regions. Unfortunately, for most stomatal frequency records, the geometry changes of stomata are unknown because they simply were not measured. In order to attribute the iWUE changes to the stomatal density and size regulation mechanism within the same species g wmax records would be needed for the same species monitored for iWUE (inferred from δ 13 C). We note a pressing need for measurements of stomatal morphology and stomatal frequency changes in more species, coupled to measurements of water use efficiency from the same region, for the same species.

  For microscopic analysis, all leaf fragments were bleached in a cold 2-4% sodium hypochlorite solution, rinsed in water, and mounted on microscopic slides. Standardized, computer-aided analysis of epidermal parameters was performed on a Leica Quantimet 500C/500+ image-analysis system. The number of analysed leaf fragments in each sample varies because of different cuticle preservation in the sediments. A maximum of ten stomata-bearing alveoles/leaf fragments were analysed for epidermal cell density (ED [n/mm²]) and stomatal density (SD [n/mm²]). On each leaf fragment, twenty stomatal pores were measured for maximum pore length(PL [µm]) and maximum stomatal length(SL [µm]

  ca. 1 year(AD 2003-1955) to 3 or 5 years (between AD 1954 and 1721), and in the Kevo monolith each sample is less than 1 year's peat accumulation (AD

  Fig 1. (a) δ δ δ δ 13 C inferred intrinsic water-use efficiency (iWUE) record from P. sylvestris tree rings at Laanila (red line) and 10-year smoothed stomatal density and geometry inferred maximum stomatal conductance (g wmax ) from subfossil and modern B. nana leaves at Kiruna/Kevo (dashed blue line). (b) Atmospheric CO 2 concentration (ca) (black). The two reference periods for comparing pre and post-industrial changes (preind and post-ind, respectively) in iWUE and g wmax are indicated. The change from preto post-ind are 12% for iWUE and 15% for g wmax . Grey band indicates the reference period between 1 x CO 2 (AD 1967) and CO 2 = 370 ppmv (AD 2000) (see Table2).

  almost constant. Instead, after ~AD 1850 the steeper decline of stomatal density was not counterbalanced by an equal increase in stomatal size (see Fig.S1and Supplementary Material). Hence, the change in g wmax we observe over the study period is mainly related to a change in stomatal density.

  and also the trend that would have occurred had the trees maintained an active response to rising CO 2 throughout. Active ci was calculated using equations (1) and (3) but maintaining the average starting δ 13 C values throughout the series length (such a curve displays the iWUE changes that would have been seen with the maintenance of constant ci/ca via strong active stomatal control).

Fig. 2

 2 Fig. 2 Active response and measured response iWUE. Time series for actual iWUE from δ δ δ δ 13 C are shown at Laanila (blue, annual values and 10-year centralised moving average) along with the trend which would have occurred had the trees maintained an active response to rising CO 2 (stable ci/ca) (black).

  approximately 400ppmv for B. pubescens. The indication of a nonlinear response, whether evident already or predicted to occur in the next decades, in the major evergreen and deciduous species of the Boreal forest clearly does not back up the continued use of a linear parameterisation for stomata conductance with rising CO 2 in coupled climatevegetation model studies.

  Here we use time series of tree ring δ 13 C (Pinus sylvestris) and subfossil leaf (Betula nana) measurements of stomatal density and geometry to derive records of changes in intrinsic water use efficiency and maximum stomatal conductance in the Boreal zone of northern Finland and Sweden. We investigate the rate of change in both proxies, over the recent past. The two independent lines of evidence from these two different Boreal species indicate increased intrinsic water use efficiency and reduced maximum stomatal conductance of similar magnitude from pre-industrial times (ca.

AD 1850) to around AD 1970. After this maximum stomatal conductance continues to decrease to AD 2000 in B. nana but intrinsic water use efficiency in P. sylvestris reaches a plateau. We suggest that northern boreal P. sylvestris might have reached a threshold in its ability to increase water use efficiency and we discuss the implication for modelling stomatal conductance in the land surface schemes of General Circulation Models.

  Diverging results for different plant species point to a genus or species-specific adaptation capacity, where in certain taxa stomatal frequency response limits are already reached at present day CO 2 levels[START_REF] Kürschner | Predicting the response of leaf stomatal frequency to a future CO 2 -enriched atmosphere: constraints from historical observations[END_REF][START_REF] Royer | Stomatal density and stomatal index as indicators of paleoatmospheric CO 2 concentration[END_REF].[START_REF] Waterhouse | Northern European trees show a progressively diminishing response to increasing atmospheric carbon dioxide concentrations[END_REF] also find that iWUE in trees across northern Europe is, in some cases, no longer rising. Clearly plant iWUE cannot rise indefinitely and stomatal conductance and frequency can never be zero. Hence future predictions of evapotranspiration flux within the Boreal forest zone might be more accurately gauged by taking account of palaeo evidence for threshold points in different species across different regions.

	between 350-400ppmv CO 2 .
	F o r
	R	its cause has not been attributed directly to a
	specific physiological source. Nor is it clear how iWUE will change with continued rising e v CO 2 in the future. We compare δ 13 C based records of iWUE from northern Boreal Pinus sylvestris and a record of maximum stomatal conductance to water vapour (g wmax ) derived i e
	from subfossil Betula nana leaves from the same geographical area (Table 1). The ideal data w
	set to attribute palaeo changes in iWUE to a specific physiological cause would be measured iWUE and stomatal density variations from the same palaeoclimate archive. Unfortunately, O n l
		y
		5

for most species the stomatal geometry and density records are unknown because they simply were not measured. With this in mind we investigate a different question -when comparing two independent lines of proxy evidence for changes in water use efficiency, what changes do two of the dominant Boreal species, from the same region, show? Using subfossil leaf assemblages

[START_REF] Kürschner | Predicting the response of leaf stomatal frequency to a future CO 2 -enriched atmosphere: constraints from historical observations[END_REF]

illustrated a non linear response in stomatal frequency changes of European birch and oak species and predicted that certain species may reach a 'response limit' and stop altering stomatal geometry and number at

  report increases of between

	24% to 48% in a variety of species
	F o r
	R
	e v
	i e
	w
	O n l
	y
	6

Table 1 -

 1 Site information.

	Site	Proxy	Location	Species	Source
	Laanila, Finland	δ 13 C-inferred	68ºN 27ºE	Pinus	Gagen et al. (2007) and this
		iWUE		sylvestris	study.
	Kiruna, Sweden	Stomata-	67ºN 20ºE	Betula nana	Finsinger and Wagner-Cremer
	Kevo, Finland	inferred g wmax	69°N 27°E		(2009) and this study

At Laanila, mature P. sylvestris trees of over 200 years in age were sampled for δ 13 C in 2001, the time series is fully described elsewhere

[START_REF] Gagen | Exorcising the 'segment length curse': Summer temperature reconstruction since AD 1640 using non-detrended stable carbon isotope ratios from pine trees in northern Finland[END_REF]

. Trees were sampled using a

  industrial time series of δ 13 C measurements contain the industrially derived change in atmospheric δ 13 C which is corrected against the actual atmospheric δ 13 C measurements (e.g.[START_REF] Francey | A 1000-year high precision record of delta C-13 in atmospheric CO 2[END_REF]. Corrected plant δ 13 C then reflects a pre AD 1850 B. nana leaves sampled from a peat mire at Kiruna in northern Swedish Lapland (Table1). In addition, B. nana leaves were collected from (i) herbarium specimens that were growing in

	such that post δ 13 C value of -6.4 ppmv. Fennoscandia >67°N (n years=5,
	Intrinsic water use efficiency is calculated as:
	iWUE = (ca-ci) / 1.6 F o r	(3)
	R
	e v
	e i e
	w w
	O n l O n l
	y y
		8 9

where ci is calculated by a re-arrangement of equation 1 after applying equation 2 (for full derivation see

[START_REF] Mccarroll | Correction of tree ring stable carbon isotope chronologies for changes in the carbon dioxide content of the atmosphere[END_REF]

.

Stable carbon isotope ratios in tree rings are commonly used as a climate proxy because ci is externally forced by a control that is often climatic, whereas ca remains stable or its changes can be accounted for

[START_REF] Mccarroll | Correction of tree ring stable carbon isotope chronologies for changes in the carbon dioxide content of the atmosphere[END_REF]

. Since the fractionation constants (a and b) do not vary in response to climate and the atmospheric concentration of CO 2 (ca) is known, δ 13 C essentially records changes in ci as regulated by two processes: stomatal conductance (g s ), and photosynthetic assimilation rate (A). If ca changes through time the response of the plant to these changes will also be recorded in tree ring δ 13 C.

A record of maximum stomatal conductance to water vapour (g wmax ) from subfossil Betula nana leaves

The long term g wmax time series

(AD 1750(AD -2008) )

was obtained from measured changes in stomatal density and stomatal geometry using standard paleoecological methods, in subfossil

  [START_REF] Betts | Simulated responses of potential vegetation to doubled-CO 2 climate change and feedbacks on near-surface temperature[END_REF] Table 2 below). Although the absolute values of inferred g wmax and g s are different, the % changes of those values over the period AD 1967 to 2000 are virtually the same (Table S1 and Fig. S2 in Supplementary Material).

	Model inferred gc changes	2 x CO 2 'RP'	2 x CO 2 'RPS'
	g c at 1x CO 2 (323 ppmv; AD 1967)	6.09	6.09
	g c at 2x CO 2 (646 ppmv)	4.89	5.35
	% change g c from 1x to 2x CO 2	-19.70%	-12.15%
	% change g c from 1x CO 2 to 370 ppmv (AD 2000)	-2.9%	-1.8%
	Proxy inferred iWUE and g wmax changes
	Site	Kiruna/Kevo	Laanila
	% change from 1x CO 2 to 370 ppmv (AD 2000)

Global Change Biology

Acknowledgments:

This research was funded by the European Union (Millennium 017008), and the British Natural Environment Research Council (NJL NE/B501504 GHFY). MHG is supported by an RCUK Fellowship. WF is thankful to the AXA Research Fund for financial support. This work was developed following the Millennium Project's Northern Group Workshop held at the Royal Swedish Academy of Sciences' Abisko Scientific Research Station. Ongoing thanks to all our friends in the Millennium consortium.