A functional hypothesis for the treeline position in north east Italian alps: some ecophysiological indication

UNIVERSIT DEGLI STUDI DI PADOVA

Sede Amministrativa: Universit degli Studi di Padova

Dipartimento Territorio e Sistemi Agro-Forestali

Dottorato di ricerca in Ecologia Forestale

CICLO: XVII

A functional hypothesis for the treeline position in north east Italian alps: some ecophysiological indication.

Coordinatore : Ch.mo Prof. Viola Franco Supervisore : Prof. Tommaso Anfodillo

Dottorando : Dott. Claudio Fior

ANNO ACCADEMICO 2004-2005

1 SummaryIn a changing world the treeline is a key element for biodiversity, the hydrologic cycle, and the relation with atmosphere gasses; in order to evaluate its dynamics, it is important to know which the main environmental variables are that control it. At a local level a lot of different hypothesis are suggested to give a functional explanation of the treeline position on mountains; moreover, at a global level only two of them attract the interest of the scientific community: the carbon limitation hypothesis suggesting that high altitude plants show a reduced carbon gain or higher carbon loss related to respiration, which compromises the carbon balance of plants; and the growth limitation hypothesis that low temperatures reduce plant meristematic activity in extreme environments also if there is no significant reduction of photosynthate availability in plant organs. In order to analyse the better functional explanation of the treeline position in the Italian Alps, we have made some gas exchange relives on adult plants at high altitudes, and some allometric relives on small plants along an altitudinal gradient. We observed that mature plants with an artificial increase of metabolic activity present higher photosynthetic activity; so the primary production does not limit plant growth. Moreover, on small plants the altitude affects only few allometric relations, many of them related to height growth and biomass allocation. This highlighted that near the soil, at high altitudes, there is a boundary layer with better environmental conditions for plant life and that plant modifications on such small plants are not so strictly related to a reduced primary plant production. Therefore our experimental data are better explainable by the growth limitation hypothesis than by the carbon hypothesis.

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RiassuntoIl limite superiore del bosco elemento fondamentale della biodiversit, del ciclo idrologico e nelle dinamiche dei gas in atmosfera; perci importante conoscere i fattori ambientali che ne determinano posizione e caratteristiche, specialmente in unepoca di cambiamento globale. Su scala locale molte sono le ipotesi proposte per una sua spiegazione funzionale, mentre su quella mondiale solo un paio sono ritenute determinanti da parte della comunit scientifica. Lipotesi del deficit nel bilancio del carbonio propone che le ridotte capacit di fissazione, o gli elevati costi metabolici sostenuti delle piante al limite superiore del bosco, determinino la difficolt nel mantenere il portamento arboreo al di sopra di una certa quota. A questa si contrappone lipotesi della limitazione dellattivit metabolica, la quale presuppone che le basse temperature riducano lattivit dei meristemi della pianta, pi che la capacit di fissare il carbonio. Per valutare quale delle due spiegazioni pi si adatti al contesto delle Alpi orientali italiane, sono stati misurati gli scambi gassosi in piante adulte al limite superiore del bosco, e sono state rilevate relazioni allometriche e strategie di partizione della biomassa su piante di piccole dimensioni lungo un gradiente altitudinale. Si osservato che piante adulte al limite superiore del bosco, sottoposte ad un innalzamento artificiale dellattivit metabolica, presentano una maggior attivit fotosintetica. Si pu concludere perci che non sia questultima a limitarne lo sviluppo. Si inoltre osservato che piante di piccole dimensioni subiscono, con il crescere dellaltitudine, modeste alterazioni nelle relazioni allometriche; per lo pi sono modificati gli incrementi in altezza e lallocazione della biomassa. Ci fa supporre che le piante sfruttino le condizioni ambientali sub ottimali allo sviluppo delle piante presenti a ridosso del suolo. I dati sperimentali inoltre non sono coerenti con lipotesi del deficit nel bilancio del carbonio. Quindi, nel contesto delle Alpi orientali italiane, la migliore spiegazione funzionale della posizione del limite superiore del bosco sembra essere quella della limitazione nellattivit metabolica.

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2 Index1 Summary............................................................................................................................................1 2 Index..................................................................................................................................................3 3 Introduction.......................................................................................................................................6 4 Gas exchange relives on treelines plants of Larix decidua Mill., Pinus cembra L. and Pinus leucodermis Ant..................................................................................................................................10 4.1 Material and methods...............................................................................................................10 4.1.1 Study area..........................................................................................................................10 4.1.2 Gas exchange measurements and environmental modifies...............................................11 4.1.3 Data analysis......................................................................................................................15 4.2 Results......................................................................................................................................17 4.2.1 Relations between gas exchange parameters and environmental factors in the different species.........................................................................................................................................17 4.2.1.1 Net photosynthesis (A)...............................................................................................17 4.2.1.2 Stomata conductance (gs)...........................................................................................19 4.2.1.3 Substomatal CO2 concentration (ci)..........................................................................21 4.2.1.4 Water use efficiency...................................................................................................24 4.2.2 Effect of heating in the woody parts of the plant on gas exchange parameters................24 4.2.2.1 Net photosynthesis (A)...............................................................................................24 4.2.2.2 Stomata conductance (gs)...........................................................................................28 4.2.2.3 Substomatal CO2 concentration (ci)..........................................................................31 4.3 Discussion.................................................................................................................................33 4.3.1 Gas exchange parameters in the different species.............................................................33 4.3.1.1 Net photosynthesis (A)...............................................................................................33 4.3.1.2 Stomata conductance (gs)...........................................................................................33 4.3.1.3 Substomatal CO2 concentration (ci)..........................................................................34 4.3.1.4 Water use efficiency (WUEg).....................................................................................35 4.3.2 Effect of heating in the woody parts of the plant on gas exchange parameters................35 4.4 Conclusions..............................................................................................................................37 5 Biomass partitioning and structural traits in Larix decidua Mill. and Pinus cembra L. at different elevation.............................................................................................................................................38 5.1 Material and methods...............................................................................................................38 5.1.1 Study site...........................................................................................................................38 3

5.1.2 Plant selection....................................................................................................................40 5.1.3 Manipulations and measurements.....................................................................................40 5.1.4 Statistical analysis.............................................................................................................41 5.1.5 Descrizione del modello WBE..........................................................................................43 5.2 Results......................................................................................................................................43 5.2.1 Environmental conditions..................................................................................................44 5.2.2 Height growth....................................................................................................................45 5.2.3 Diameter growth................................................................................................................46 5.2.4 Biomass growth and allocation.........................................................................................47 5.2.5 Branches characteristics....................................................................................................48 5.2.6 Root system characteristics...............................................................................................49 5.2.7 Allometric relations...........................................................................................................50 5.3 Discussion.................................................................................................................................53 5.3.1 Environmental conditions..................................................................................................53 5.3.2 Height growth....................................................................................................................54 5.3.3 Diameter growth................................................................................................................54 5.3.4 Biomass production...........................................................................................................55 5.3.5 Biomass allocation.............................................................................................................55 5.3.6 Branches characteristics....................................................................................................57 5.3.7 Root characteristics...........................................................................................................57 5.3.8 Allometric relations...........................................................................................................57 5.4 Conclusions..............................................................................................................................58 6 An integrated model for hydrodynamics, biomechanics and branching geometry applied on small plants of L. decidua Mill. and P. cembra L. at two different altitude.................................................59 6.1 Material and methods...............................................................................................................59 6.1.1 Architectural plant models.................................................................................................59 6.1.1.1 Stem sub model..........................................................................................................63 6.1.1.2 Hypothesis for a root system fractal sub model.........................................................67 6.1.2 Study site...........................................................................................................................68 6.1.3 Samples preparation and measurements............................................................................68 6.1.4 Statistical analysis.............................................................................................................69 6.2 Results......................................................................................................................................70 6.2.1 Stem sub model.................................................................................................................70 6.2.2 Root system sub model......................................................................................................74 4

6.2.3 Stem apex vessel diameter and plant development...........................................................78 6.2.4 Base stem vessel diameter and plant development............................................................78 6.3 Discussion.................................................................................................................................82 6.3.1 Stem sub model.................................................................................................................82 6.3.2 Root system sub model......................................................................................................83 6.3.3 Stem apex and plant development.....................................................................................84 6.3.4 Base stem vessel diameter and plant development............................................................84 6.4 Conclusions..............................................................................................................................84 6.5 Main symbols and abbreviation................................................................................................86 7 Conclusions.....................................................................................................................................87 8 Enclosed..........................................................................................................................................90 8.1 Spline resolution in gas exchange data analysis.......................................................................90 8.2 Different method of evaluation of crown and hydraulic architecture......................................94 8.3 Iconography............................................................................................................................100 9 Acknowledgments.........................................................................................................................107 10 References...................................................................................................................................108

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there (are) two kinds of original thinkers, those who upon viewing disorder try to create order, and those who upon encountering order try to protest it by creating disorder. The tension between the two is what drives learning forward. (Wilson, 1998)

3 IntroductionOne of the more intriguing and investigated traits of forest ecosystems is the existence of limits to their diffusion; the treeline is possibly the boundary best known and studied from a long time (Griggs, 1937), but still awaits a conclusive functional explanation. Mountain and boreal ecosystems that play a crucial role in the earth system for several reasons: they occupy a large proportion of terrestrial surface (10 % mountains and 22 % boreal forest); they are all inhabited to a greater or lesser extent (they provide direct life for close to 10% of the world population, and indirectly to over half); especially the mountains features high biodiversity , because of their great altitudinal range; still the mountains are key element in the hydrological cycle, being the source of many of the worlds major river systems; many of the possible structure and dysfunction changes (eg. greenhouse gas emission, modifies in albedo or fire regime in boreal zone) in these ecosystems have large effects on the atmosphere . There are clear indication from a number of high elevation and latitude climatic records (ice cores, sub-fossil wood samples (Tinner e Theurillat, 2003), fossil pollen and wood cores ) which suggests that the amplitude of temperature changes this century is greater than the observed global or hemispheric change; furthermore accelerated climatic change will be proportionally more perceptible at high elevations . Moreover modifies on vegetation related with a global change process, especially in boreal forest stands, could be observe with remote sensing instrument or aerial photographic documentation. The findings with these techniques are controversial (Moore, 2004), highlining sometime a significant expansion of the forest or sometimes only a slight treeline movement . In order to study treeline shift, and its relation to the clime it is necessary to at least hypothise a physical and functional explanation of treeline position at global level. Moreover it is necessary clearly describe this term: tree is an upright woody plant with a dominant above-ground stem that reaches a height of at least 3m; line is a subtle define because any natural boundary is in reality a transition zone or an ecotone, which is the result of a complex process that involves also a reduced availability of resources and abiotic conditions Therefore the treeline presents some conventional elements: tree species limit is the upper limit of a tree specie; treeline takes a middle position and roughly marks a line connecting the highest patches of forest; 6

timberline is the upper limit of the closed forest. On global scale, the elevation of the climatic treeline decreases with increasing latitude from more than 4.000 m in subtropical high mountains to sea level at the polar line (Fig. 1). At his scale the snowlimit, that provides purely physical driven references, presents an analogous trend shifted about 1.000m of altitude higher. So we could argue that, at least at global level, also the treeline position is related to a physical parameter as temperature; this fact is well know from a long time and the upper limit of the forest is located where the mean temperature during the growing season is between 5.5 C and 7 C (Jobbgy e Jackson, 2000; Krner, 1999).L i m it e d e ll e n e v i L im ite d e l b o sc o 6000 5000 A ltitu d in e (m ) 4000 3000 2000 1000 0 70 N o rd 60 50 40 30 20 10 0 10 20 30 40 50 Sud 60

L a titu d in e

Fig. 1 Treeline and snowlimit at global level (from Krner, 1998).

Temperature is a physical explanation of the treeline, but this lead to different functional explanations; the most important ones are: Human activity hypothesis, that plays an important role in highly anthropized as the Alps, but at global level presents a reduced importance (Didier, 2001); Stress hypothesis, repeated damage by frost, frost dissection or phototoxic effect after frost; actually these effects occur during the coldest part of the year, but usually in these moments tree frost tolerance exceed the environmental demand. Actually no clear relation between absolute minima of air temperature and treeline position was observed; so these kinds of effect act only at local level ; Disturbance hypothesis, the mechanical damage by wind (Alftine e Malanson, 2004), ice blasting, snow break, avalanches, herb ivory or fungal pathogens may remove similar or more biomass or meristems as can be replaced by growth and development. Experimental evidences give a key role of these factors only at local level, determining only in particular locations the treeline position (Baig e Tranquillini, 1976; Tranquillini, 1979); Reproduction hypothesis, pollination, pollen tube growth, seed development, seed dispersal, germination and seedling establishment may be limited and prevent tree recruitment at higher 7

altitudes . Usually seedling, dwarfed trees are quite abundant above the treeline in many parts of the world (Forbis, 2003), so this is not a useful treeline explanation at global level, albeit it could have an important rule at local scale ; Carbon balance hypothesis, either carbon uptake or the balance between uptake and loss are insufficient to support maintenance and minimum growth of trees . Actually some gas exchange relives has revealed no particular disadvantages compared to low altitude . Many studies have illustrated the relative insensitivity of photosynthesis in treeline trees to temperature over the range of predominant daytime field temperatures during the growing season. Recently it was demonstrated also that respiratory losses during the coldest mount of the winter con be recovered by a single days carbon gain in the growing season (Wieser, 1997). Moreover a greater sap flux concentration of no structural carbohydrates was observed at high altitudes, so there is no significant reduction of meristematic activity related to a low photosynthate availability ; Growth limitation hypothesis, synthetic process which lead from sugars and amino acids to the complex plant body may not match the minimum rates required for growth and tissue renewal, independently of the supply of row materials . Actually there are some evidences of carbon limitation in some gas exchange relive , moreover open field trails of CO 2 enrichment relived an increase in biomass growth . At global scale only a couple of the previous hypothesis attract the interest of the scientist: actually some of them have an important role only at local level (human activity and reproduction hypothesis), actually others could be joined in more explanative ones as the carbon balance hypothesis . It is possible to intend disturbance and stress hypothesis as an addictive cost in the plant carbon balance. Emphasizing the structural features of treeline leads to a new emphasis on the multiple levels of analysis necessary to understand treeline. The physiological tolerances of individual alone usually are punctual experiments and hardly could be applied at global level. Conversely studying just the dynamics of the treeline populations may not lead to ultimate causes if treeline is seen to drift, or if history and no contemporary processes sets distributional processes. In this study we tried to highline the most useful treeline explanations in northeast Italian Alps from the two most frequently proposed at global level, the carbon balance hypothesis and the growth limitation hypothesis. To do so we tackle the functional explanation of treeline from tree different point of view, at each of them is dedicate one of the following chapters. First of all we analyze the gas exchange in treeline plants of Pinus cembra, L. Larix decidua and Pinus leucodemis Ant. Actually we tried to increase the metabolic activity of a woody part of 8

the plant increasing its temperature; moreover if at the treeline the growth limitation is due to a carbon limitation we could not observe a significant increase net photosynthesis and stomata conductance. Otherwise if the growth limitation is due to lack in metabolic activity, increasing this last activity we would observe an higher net photosynthesis and stomata conductance. Than we analyzed the biomass production, the biomass allocation and production, and some allometric relation along an altitudinal gradient, in order to highline the effect of altitude. An higher proportion of heterotrophic mass in treeline plants is related with higher metabolic cost for the tree in this extreme environment, and so it is consistent with the carbon limitation hypothesis. At least we evaluated which allometric relation are affected by the high altitude, both in the crown and hydraulic system structure; actually if there is no significant boundary layer near the soil with best plant survivor condition we could observe only slight modifies in small plat at high altitude. At least the knowledge of the allometric relation firstly modified by altitude during plant development, we have some evidences of the plant physiological process involved.

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Trust in Lord with all your heart and do no lean on your own understanding. In all your ways acknowledge Him, and He will make your paths straight Reference book of LICOR 6400

4 Gas exchange relives on treelines plants of Larix decidua Mill., Pinus cembra L. and Pinus leucodermis Ant.In order to evaluate the ecosystem dynamics one of the most important trait is the plant carbon balance and the interaction of its components with the environmental factors. Actually the allometric relives on the trees that compose the ecosystem could give an answer about the annual net primary production of a plant but they hardly evidence the short-term effects of environmental factors on the previous parameter. Moreover with the gas exchange technique it is possible to organize laboratory or field experiments modifying one variable and observing the effects the shot term primary production and carbon balance of a plant. At least this technique it is possible to evaluate in a plant the short-term water use efficiency and how this parameter is modify by the environmental factors . We have made some gas exchange relive on treeline adult plants in order to asses the influence of the environmental factors, as air temperature, photosynthetic active radiation, vapor pressure deficit and soil water content, on carbon assimilation and water use efficiency in high elevation environments. Moreover we studied the effect on needle net photosynthesis after a metabolic activity increase of the woody parts of the tree obtained by raising their temperature of 10 K (Azcn-Bieto e Osmond, 1983).

4.1 Material and methods4.1.1 Study areaTreeline studies were carried out in 5 Torri (Cortina dAmpezzo, BL; Fig. 63) ) in the north east Italian Alps (2.080 m a.s.l.) on Larix decidua Mill. and Pinus cembra L. (Fig. 60; Fig. 56); other high altitude measures were made on Pinus leucodermis Ant. (Fig. 58) on Serra del Crispo (San Severino Lucano; PZ; Fig. 59) in the south Appennini (2.000 m a.s.l.). In the alpine site (N 46 31 E 12 04) the mean year precipitation is 1.109 mm at year mainly distributed during summer. Moreover the mean temperature in January is -7.5 C, and the mean one in July is 22.3C. The treeline is formed by a mix stand of young plants of L. decidua, P. cembra and P. abies on a shallow calcareous soil. 10

In the appennini site (N 39 55 E 16 13) the mean year precipitation are 1.100 mm at year mainly distributed during autumn and spring. Moreover the mean temperature in January is 3.2 C, and the mean one in June is 13,8C. In the area there is shallow calcareous soil and there is a sparse forest of old plants of P. leucodermis. Moreover gas exchange relive were made on adult tree in the south-east side of the crow with a uniform light availability. (Fig. 2;Fig. 75; Fig. 76). 5 T o rr i - C o r tin a d A m p e z z o ( B L )

S e rra d e l C ris p o - S a n S e v e rin o L u can o (P Z )

Fig. 2 Sites location

4.1.2 Gas exchange measurements and environmental modifiesThe gas exchange relives were made with an LCi (ADC BioScientific Ltd) photosynthesis system (Fig. 57); that also provide the data of photosynthetic active radiation (PAR, mol m-2 s-1). The instrument is a steady state system with a chamber insulated from the outer environment containing a photosynthesizing and transpiring leaf. A constant air stream passes continuously through the chamber, the air leaving the chamber will be depleted in CO 2 and enriched in water vapor. The instrument analyzes the inlet and outlet air of the camber determining modify in water and CO2 concentration. The water concentration was measured with two capacitance sensor in the inlet and outlet flux, they are a condenser permeable to the humidity which modify its capacitance. The difference in humidity between outlet and inlet air represent the amount of water lost from the leave by transpiration. The CO2 concentration was measured with an IRGA (INfrared Gas Analyzer) that calculate the air CO2 concentration measuring the transmitted of infrared radiation of a know source in a sample of air. There are a lot of different gasses which adsorb in this wavelength so the 11

instruments compare the absorption of the sample air with a reference one in which the CO2 was removed with soda lime powder. 2 NaOH + CO2 Na2CO3 + H2O The measures were made both in inlet air and outlet air but the reference sample was collected only for the inlet air. So because the vapor present a relevant absorption in infrared wavelength it is necessary a correction in the CO2 concentration of the outlet hair due to the higher humidity the reference one. The difference in CO2 concentration between inlet air and outlet air in the amount of this gas fixed with the photosynthesis by the leaf. The air flux in the chamber is exactly know with a mass flow meter and so from the variations of concentration of water and CO2 in air in the chamber it is possible to calculate the amount of these gasses fixed or lost from the leaf. Then we have to rapport these values to the leaf area in order to obtain the values of net photosynthesis (A, mol m-2 s-1) and transpiration (E, mmol m-2 s-1; Eq. 1; Eq. 2). A= u c a

Eq. 1 Formula for the net photosynthesis calculus. (A).

E=

u w a

Eq. 2 Formula for the transpiration calculus (E).

E=Transpiration (mmol m-2 s-1) A=Net photosynthesis (mol m-2 s-1) c=Difference of CO2 concentration between atmosphere and chamber (ppm) w=Difference of water vapor concentration between atmosphere and chamber (mol mol 1) a=Leaf area (cm2) u=molar flow (mol m-2 s1) Leaf resistance is the reciprocal of the conductance that is the permeability of the leaf to water vapor; a fraction of this permeability is related with the leaf boundary layer and another with the stomata opening and characteristics. The total conductance of the leaf it is ratio between transpiration and the difference of water vapor in the chamber and inside the leaf supposed saturated of water (Eq. 3). The leaf form could determine the resistance of the leaf boundary layer and the difference between total conductance and boundary layer conductance is the stomata conductance (gs, mol m-2 s-1) due to the stomata permeability (Eq. 4).

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gs w =

E w a

Eq. 3 Formula for the leaf conductance calculus. (gsw).

gs =

1 1 rb gs w

Eq. 4 Formula for the stomata conductance calculus (gs).

gsw = Total conductance (mol m-2 s-1) wa = Difference of water vapor concentration between chamber and the mesophil of the leaf supposed saturate of water (mol mol 1) rb = Resistance of boundary layer (m2 s mol1) gsc = Stomatal conductance (mol m-2 s-1) From the net photosynthesis and stomata conductance it is possible to evaluate the CO2 concentration in the leaf mesophil (Eq. 5). 1.6 ci = c ref c A1.37 * rb + gs c Eq. 5 Formula for the evaluation of the substomatal CO2 concentration (ci).

ci = Corrected sub stomata CO2 concentration (ppm) cref = Atmospheric CO2 concentration (ppm) One of the most important problem analyzing gas exchange data un conifer plants is that the leaf area analyzed is unknown before the end of the measures when it is possible to measure this parameter or evaluate this parameter from allometric equation which relate leaf mass and leaf area. So it is necessary to set a supposed leaf area in the instrument and afterward to correct the measured data using the effective leaf area of the sample. For net photosynthesis and transpiration the correction is a proportion with supposed area and effective area (Eq. 6; Eq. 7). For the conductance an analogous proportion could be applied only to the stomata resistance, that is the reciprocal of the total conductance. So once corrected the total resistance in this way we have to recalculate the stomata conductance (Eq. 8). The sub stomata CO2 concentration is a derived parameter based on the previous ones, so it is possible to calculate the correct value (Eq. 5), using the correct values of net photosynthesis and stomata conductance .

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Ec = Er

ar ac

Eq. 6 Formula for the correction of transpiration (E) for the effective sample leaf area.

Ac = Ar

ar ac

Eq. 7 Formula for the correction of net photosynthesis (A) for the effective sample leaf area.

gs c =

1 1 + rb a r gs r rb ac

Eq. 8 Formula for the correction of stomata conductance (gs) for the effective sample leaf area.

Where: Ec = Corrected transpiration (mmol m-2 s-1) Er = Measured transpiration (mmol m-2 s-1) Ac = Corrected net photosynthesis (mol m-2 s-1) Ar = Measured net photosynthesis (mol m-2 s-1) gsc = Corrected stomata conductance (mol m-2 s-1) gsr = Measured stomata conductance (mol m-2 s-1) ac = Measured leaf area (cm2) ar = Instrument leaf area (cm2) rb = Resistance of boundary layer (m2 s mol1) Another useful parameter for the ecological characterization of a specie is the water use efficiency (WUEg, mol CO2 molH2O-1) that is the ration between net photosynthesis and stomata conductance (Morgan e LeCain, 1991); it represents the number of moles of carbon gain for mole of leaf resistance to water. This definition of water use efficiency is not affected by differences in atmosphere vapor pressure deficit; another definition frequently used for this parameter is the ratio between net photosynthesis and transpiration. Actually a great part of variation in transpiration is due to modify in the water content in atmosphere than in the plant strategy to face the competition between carbon gain and water lost. Measures on L. decidua were made on the same shoot during all the trial with a conifer chamber, which could contain an small shoot.. Otherwise the data on P. leucodermis and P. cembra were measured with a broad leaves chamber using 4-6 needles. We used only one-year 14

needles in order to avoid a reduction in photosynthetic capacity due to the leaf longevity (Sonia e Alfonso, 2003). The analyzed parameters were net photosynthesis, stomata conductance, substomatal CO2 concentration and water use efficiency; a near meteorological station provides the environmental parameters as temperature (T, C), vapor pressure deficit (D, hPa) and soil water content (TDR, %). The measures were made in L. decidua from the 3 August to 16 September 2002; in. P. leucodermis form 6 to 17 June 2003, and in P. cembra from 18 to 28 August 2003. The heating trail was made on tree branch heated of 10 K than a reference one with 100 m of resistance wire (1.6 K) feed with 220 V; moreover the tips of the twigs were not altered. The reference branch was selected on the same plant with analogous conditions of exposition and size. Actually a datalogger CR-10X (Campbell Ltd. USA) for the data storage and a generator for electricity production were used. In detail the datalogger measured and stored the temperature of the reference branch and heated one every two seconds; so it fed the resistance wire if the temperature difference is lower than 10 K and stopped the fed if it exceeds 10 K. The data on three shoots of the heated branch and three on the reference one were measured. The LCi systems equilibrated to a steady state conditions in more or less 5 minutes, so in an hour we made 6 measures on the heated branch and 6 in the reference one. We evaluate the effects of heating on net photosynthesis, stomata conductance and substomatal CO2 concentration. In order to assess if the observed difference between heated branch and reference one is related to the modify in the woody parts temperature we compared the data of a heated breach and a reference one both on days with the heating system switched on or off. In each specie measures there were 2 4 days of measures with the heating system switched off before the heating trail; at the base of this experiment is the assumption of independent behavior of the branches in a plant .

4.1.3 Data analysisThe data of net photosynthesi and photosynthetic active radiation were fitted by a rectangular hyperbola (Eq. 9) as proposed by theorical models . This function is upper limited (Amax), otherwise a logarithmic crescent function is always crescent. A = Amax PAR + b PAR + c

Eq. 9 Rectangular hyperbola with three parameters.

The relations between stomata conductance and photosynthetic active radiation, net photosynthesis and substomatal CO2 concentration, substomatal CO2 concentration and 15

photosynthetic active radiation were presented with a spline function. Otherwise the relations between net photosynthesis and stomata conductance was fitted with a linear regression and the relation between stomata conductance and substomatal CO2 concentration is fitted with a linearized regression log transforming the dependent variable. The frequency of distribution of net photosynthesis and stomata conductance data was normal, or at least normalizable with a log transformation of the data. Actually the substomatal CO2 concentration presents an irregular frequency of distribution, so we used both Pearson correlation than Spearman correlation, obtaining analogous results also with the non parametric method (Zar, 1999). The analyses of the gas exchange parameters were made with multiple regression in order to assess the effect of temperature and other environmental variable. Actually we have log transformed the photosynthetic active radiation data in order to linearize the relation with the gas exchange parameters. The multiple regression technique evaluates which factors (eg. temperature) significantly modify the dependent variable calculating a determination coefficient (B) corrected for the interaction with other factors (eg. photosynthetic active radiation or soil water content) . The WUEg in a quite stable at changing of environmental parameters so the comparisons were made with Kruskal-Wallis test. Actually environmental factors cause a great part of the variation in the gas exchange parameters; in order to evaluate the effect of the warming and the related increasing metabolic activity of the woody parts we reduce this variability analyzing the residual expresses as the differences between the observed data on the heated branch and the one attended from the reference branch. The data attended from the reference branch was obtained fitting the daytime pattern of the observed data in this branch with a spline function (8.1 Spline resolution in gas exchange data analysis, pag. 90). The mean of the residues in the heated branch is significantly different from zero if different values of the gas exchange parameter were observed in the heated branch than in the reference one. This comparison was made with test U Mann-Whitney to evaluate if the residues are significantly different when the heating system is turned on or off. If the residues are higher when the heating system is turned on we could associate higher temperature in woody parts temperature with higher values of the gas exchange parameter. To assess when we observed the great part of the difference in the residues in the heating branch we can progressively sum the residuals of the heated branch from morning to midday, when the measures stopped. Afterward we could divide the values of the progressive sum for the sum of all the residue of the day, so we could associate to a certain hour of the day a percentage of the total residues observed. We can set the moment of the day when there the great part of difference from the two tests when we account the 50% of the total difference recorded during the day. This daytime 16

period is extended from the hour when we reach the 25% of the total difference recorded during the day and the hour when we reach the 75% of the same parameter.

4.2 Results4.2.1 Relations between gas exchange parameters and environmental factors in the different species4.2.1.1 Net photosynthesis (A) We evaluated the importance of the environmental factors determining the net photosynthesis in the tree different species with a multiple regression analysis. The dependent variables were air temperature, soil water content, vapor pressure deficit and photosynthetic active radiation (Tab. 1).R2 corrected N of samples log (Photosynthetic active radiation) Soil water content Vapor pressure deficit L. decidua 0.40 689 0.59 -0.20 -0.20 P. leucodermis 0.28 497 0.30 0.12 -0.45 P. cembra 0.18 431 0.43 -0.13 -0.05

Tab. 1 Multiple regression analysis of net phothosinthesys and main environmental variables. Bold values indicate environmental factors significantly related with the net photosynthesis for a certain specie.

A great part of net photosynthesis variability in L. decidua could be explained by environmental factors than in the two pines, in all the species a great part of variability is due to the photosynthetic active radiation. In L. decidua the soil water content explain a great part of net photosynthesis variability and in P. leucodermis a fewer one. Moreover in P. leucodermis an important environmental factor is the vapor pressure deficit that otherwise causes only a slight modify and with different sign on net photosynthesis in L. decidua. Net photosynthesis in P. cembra present analogous relations with the environmental factors than in L. decidua, albeit the explained variability is lower. We related the net photosynthesis to the photosynthetic active radiation, the most predictable environmental factor, in order to compare the first parameter in the different species (Fig. 3).

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10A = Amax PAR +b PAR +c

8

L. decidua P. leucodermis P. cembra

A ( mol m -2 s-1)

6

4

2

0

0

200

400

600

800

1000

1200 PAR (

1400 mol m-2

1600 s-1

1800 )

2000

2200

2400

2600

2800

Fig. 3 Relations between net photosynthesis (A) and photosynthetic active radiation (PAR) in the three species. The data were fitted with a rectangular hyperbola function and the significance of the regression is presented in Tab. 1.

Higher values of net photosynthesis were measured in L. decidua (Amax = 4.7 mol m-2s-1) than in P. cembra (Amax = 4.4 mol m-2s-1) and P leucodermis (Amax = 3.6 mol m-2s-1) at the same values of photosynthetic active radiation; it is possible to observe the higher variability in the dependent variable. Also the daytime pattern of net photosynthesis gives relevant information about the analyzed species (Fig. 4).5,0 L. d e c id u a P . le u c o d e rm is P . c e m b ra 10 T ra il o n L a rix d e c id u a P in u s le u c o d e rm is P in u s c e m b ra

4,5

9

4,0

8

3,5 A (mol m s )-1

7 VPD (hPa) 7 8 9 10 11 12 13 14

-2

3,0

6

2,5

5

2,0

4

1,5

3

1,0

2

7

8

9

10

11

12

13

14

D a y tim e (h )

D a y t im e ( h )

Fig. 4 Daytime patter of net photosynthesis (A) and vapor pressure deficit (VPD) in the three species.

18

The net photosynthesis daytime patter is quite similar in the three species with a decrease near midday or midmorning probably associated with higher values of vapor pressure deficit. P. leucodermis present a strong decrease of these value at midmorning and P. cembra present analogous values of assimilation at sunrise but a slight depression of this parameter is near midday. At least L. decida present higher values of net photosynthesis than the other two species with a slight decrease near midday. 4.2.1.2 Stomata conductance (gs) There was a good correlation between net photosynthesis and stomata conductance: P. leucodermis present higher values of net photosynthesis at analogous values of stomata conductance and L. decidua presents the lower ones. In order to linearize the relation it was necessary to log transform the stomata conductance data. This transformation was necessary to compensate the depression of net photosynthesis observed at higher stomata conductance could be ascribed to the lower values of photosynthetic active radiation associated or a limit in the photosynthetic capacity of the leaf. (Fig. 5; R2=0.22; t(1631)=21.87; p