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Leaf responses to irrigation of the endemic fog-oasis tree Myrcianthes ferreyrae: Can a fog-specialist benefit from regular watering?

David A. Ramrez Coaguila

4,5

1,2

*, Luis Balaguer 3, Rosa Mancilla 4, Virginia Gonzlez 1, Daniel

, Carmelo Talavera 4, Luis Villegas 4, Aldo Ortega 4, Percy Jimnez 4, Jos

M. Moreno1

Departamento de Ciencias Ambientales, Universidad de Castilla La Mancha, 45071 Toledo, Spain Departamento de Biologa, Universidad Nacional Agraria La Molina, Lima, Per Departamento de Biologa Vegetal I, Universidad Complutense de Madrid, E-28040 Madrid, Spain Laboratorio de Ecologa, Departamento Acadmico de Biologa, Universidad de San Agustn, Arequipa, Per

Departamento de Ecologa, Universidad de Alicante, 99-E-03080 Alicante, Spain

* Corresponding author: DavidAntonio.Ramirez@uclm.es, Phone: +34 925 268 800 ext.

5470, fax: +34 925 268 840

Running title: Responses to watering of a fog-oasis tree

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Abstract Myrcianthes ferreyrae is an endemic species with a scarce number of individuals located only in fog-oases (named lomas) along the Peruvian Desert in the South of Per, where fogs are the main source of water in this hyperarid region. After centuries of severe deforestation, reforestation this native species was carried out in the Atiquipa Lomas (Arequipa, Per). In 5 slopes, five 2-year-old seedlings were monthly irrigated with water trapped by raschel-mesh fog collectors that supplemented natural rainfall with 0, 20, 40, 60 and 80 mm month-1 from February to August 2008. Plant growth rate was estimated from the variation of shoot basal diameter and maximum height, and both were highly correlated with total biomass. We measured in 4 leaves of each seedling: leaf mass area (LMA), leaf carbon isotope composition (13C), nitrogen per leaf area, leaf total carbon and stomatal density. According with the plant growth response, only the LMA and 13C were higher in irrigated than control plants but with no further response to increasing water supply. This threshold response suggests an on-off strategy fitted to exploit pulses of fog water availability. The absence of a gradual response, however, is in consonance with the low phenotypic plasticity expected in plants from stressful environments

Key Words: Peruvian Desert, 13C, fog, leaf mass area, Lomas, Myrcianthes ferreyrae

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Introduction Lomas ecosystems are fog-oases into of the Peruvian and Atacama deserts which form an hyperarid belt close to the Pacific Ocean coast from North of Per to North of Chile (7-28S latitude) (Dillon et al., 2009). These deserts are considered as the driest of the planet (Rundel et al., 1991), where Cereceda et al. (2008) using 30 years of meteorological records calculated 0.8 mm of the average yearly precipitation in the Atacama Desert. Similar to other hyperarid areas (Henschel and Seely, 2008) the fog from the ocean is the most important water resource for the lomas (Cereceda et al., 1999). Thus, trees intercept the fog with their aerial architecture and throughfall and stemflow percolate into the soil and recharges the groundwater (Dawson, 1998; Edner et al., 2011). Fog collectors (Schemenauer et al., 1987) have been set up and tested in these deserts (Cereceda et al., 2008; Larrain et al., 2002; Schemenauer and Cereceda, 1993; Sotomayor and Jimnez, 2008; Westbeld et al., 2009). The amount of water trapped by these collectors in other hyperarid environments reaches maxima of 1.0, 5.7 and 7.0 mm day-1 reported for the Namib Desert (Henschel and Seely, 2008), the West Coast of South Africa (Olivier, 2002) and the Atacama Desert (Cereceda et al., 2008), respectively.

In the lomas ecosystems reforestation activities have been carried out with the aim to restore endemic and economic tree species which have been reduced by old cutting practices (Suasnabar et al., 2004). Unfortunately, there is little information about reforestations using fog water collected (Estrela et al., 2009), and there are no published studies assessing plant response to the irrigation in lomas ecosystems. Our study species was Myrcianthes ferreyrae (McVaugh) McVaugh an evergreen perennial tree. This fog-specialist species is endemic of lomas formation in the South coast of Per

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(Kawasaki and Holst, 2006), and presently it is only located in some patches of the Atiquipa and Taimara lomas, Arequipa-Per (Suasnabar et al., 2004). Myrcianthes ferreyrae is listed in the official classification of endangered species of wild flora protected by Peruvian legislation (D.S. N 043-2006-AG) into the critical endangered classification based on the red list criteria of the International Union for Conservation of Nature (IUCN, 1999). Leaf mass area (LMA), foliar isotope carbon composition (13C) and nitrogen concentration per leaf area (and other leaf traits) were measured in our study species with the aim to assess the response of planted seedlings to different irrigation treatments using fog water. These leaf traits are related to plant growth (e.g. Antunez et al., 2001; Poorter and Garnier, 2007; Poorter et al., 2009; Ruiz-Robleto and Villar, 2005), water use efficiency (Farquhar and Richards, 1984) and carboxylation capacity (Hanba et al., 1999; Wright et al., 2001) respectively. The measured traits have been used to assess plant response to water deficit (e.g. Gratani and Varone, 2006; Haase et al., 1999; Ramrez et al., 2009), altitudinal gradients or changes in temperature (e.g. Atkinson et al., 2011; Mendez-Alonzo et al., 2008; Suzuki, 1998; Zhang et al., 2005; Vitousek et al., 1990) and other stressing factors. In afforestation and previous reforestation studies, leaf nitrogen concentration, LMA, and 13C have been analysed to select the adequate species on a functional group basis (Delagrange et al., 2008), and to understand the effect of different practices on plant resource use efficiency (Querejeta et al., 2008). Our goals were: i) to assess the plasticity degree of the different leaf traits and the growth response to a regular water supplementation by irrigation with fog water, and ii) to infer from the leaf traits responses the functional process driven by regular watering in plant growth. Because a proportional water use according with fog availability have been reported in fog-specialist species in high water deficit situations (Dawson, 1998), we

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hypothesize a gradual improvement of the physiological status (indicated by leaf traits and growth) in response to increasing water inputs. Our second hypothesis is that the leaf traits could reflect the interaction of other external factors like light intensity with our irrigation treatments allowing us a better understanding of plant growth response to water inputs.

Materials and Methods Study area Our experiment was carried out in the restoration area of the Atiqupa lomas located in the Peruvian south coast (1542S; 7418W) belonging to the Peruvian Desert in the Caraveli province, Arequipa (Fig. 1). The yearly average rainfall is 54.0 mm which is distributed in 21 and 72% in the wet-cool (July-November, with 15.4C of average monthly temperature) and dry-hot (January-May, with 21.0C of average monthly temperature) seasons respectively. The fog from the Pacific Ocean, which occurs in the wet-cool season, can provide an additional water input of 57.6 mm year1

(Sotomayor and Jimnez, 2008). The soil shows sandy clay loam structure (51.2, 26.8

and 22% of sand, silt and clay respectively), 5.8% of organic matter, 15% of C/N, 5.3 pH and 0.62 mS/cm of electric conductivity (Coaguila, 2008). Vegetation is mainly dominated by annual species that growth in the wet-cool season such as Nicotiana paniculata, Alternanthera ferreyrae and Grindelia tarapacana. The perennial shrub and tree species play an important functional role in the water input of this ecosystem because serve as natural fog collectors. The prominent woody perennial species in the Atiquipa lomas are: Caesalpinia spinosa, Duranta armata, Citharexylum flexuosum and Vasconsellea candicans (Coaguila 2008). Only 34

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adults individuals of Myrcianthes ferreyrae have been reported into the Atiquipa lomas (Vizcarra, 2006).

Irrigation treatments with fog water and experimental design Twelve fog collectors were set-upped in different projects research activities carried out in the restoration zone of our study area

(http://www.irecaunsa.com/unsapadova.html). Each fog collector is a low-cost 48 m2 flat panel (12 m long x 4 m height) compounds by a polypropylene raschel mesh (Schemenauer and Cereceda, 1994) supported by two woody 5-m height beans. A gutter, located the low part of the flat panel, canalizes the collected water to a pipe that transport it to a four water reservoirs with 60, 300, 600 y 1000 m3 of capacity. Other pipes system transported the water from the reservoirs to 5 slopes with a plantation of 2-years old M. ferreyrae. Four irrigation treatments (20, 40, 60, 80 mm month-1) and one control (no watering) were established randomly in 5 seedlings per treatment in the 5 referred slopes. The watering was carried out manually at the beginning of the assessed months from February-August 2008.

Growth assessment The maximum height and basal stem diameter of each seedling was measured at the beginning and the end of the study period. The growth was assessed from the percentage of variation from the first measurement () as: X = [(XAugust- XFebruary)/XFebruary ] .100 (1) where X is the seedling maximum height or basal stem diameter. Twelve seedlings of contrasted size were removed, dried and weighted separating the above and below biomass with the aim to fit calibration functions among the seedling maximum height

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and stem basal diameter versus dry total biomass (aboveground + belowground biomass).

Leaf traits In August 2008 we collected 4 leaves of similar size and belonging to the same cohort located in the middle of the each seedling. In the lab the leaves were scanned and the leaf area (A) was measured using ImageJ software (1.41o version, National Institutes of Health, Maryland, USA). The leaves were re-hydrated for 2 days in deionized water at 10C and after that we estimated the leaf volume (V) using the same procedure of Mitchell et al. (2008) based on the Archimedes principle, which involves measuring the weight gain (in a balance with 0.01 mg of resolution) of a beaker with water of known mass after completely immerse the re-hydrated leaf. Following the proposal of Poorter et al. (2009) we calculated the leaf volume per area (LVA=V/A) as a surrogate of the leaf thickness. The epidermis of the leaf abaxial side was impressed in a dental resin (3M ESPE Dental Products, USA) with the aim to measure the stomatal density (SD) following the Geisler et al.s (2000) protocol. Thus, a layer of nail polish was spilt in the impressed leaf size and carefully removed when it was dried, after that it was located in a slide cover and analyzed in a inverted microscope (Leica DM IRB model, Leica Microsystems GmbH, Wetzlar, Germany) connected to a digital camera system (Leica DC 300F model, Leica Microsystems GmbH, Wetzlar, Germany) assessing the stomatal number in 4 images (located in the central part of the leaf) in 100x field (0.58 mm2) per leaf. To estimate foliar dry mass (DM) the leaves were dried per 2-days at 70C and weighted. The leaf mass area (LMA) and the leaf density (LD) were calculated as: LMA = DM/A (2)

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LD = DM/V

(3)

The dry leaves were ground using a mill ball and sent at Stable Isotope Facility of the Autonoma de Madrid University where the leaf carbon isotope composition (13C) was analyzed as: 13C () = [(RSample/RStandard) -1] . 1000 where R is
13

(4)

C/12C proportion of the leaf sample and the Pee Dee Belemnite (as

standard) and was determined by a continuous flow isotope ratio mass spectrometry (Europe Scientific Hydra 20/20, Cheshire, UK). Foliar N and C were determined with an automated 1500 Carbo Erba elemental analyzer. The nitrogen content per unit area (Narea) was calculated as total foliar N divided per A. 13C, foliar C and Narea were measured from 4 leaves collected per individual in a total of 75 seedlings (3 individuals x 5 treatments x 5 slopes).

Statistical analyses The measured variables were assessed using one-way randomized block ANOVAs testing irrigation treatments and slopes as blocks. Linear regressions analyses were carried out between LVA vs. LMA and DM vs. LMA. The association among LMA, 13C, SD, leaf carbon concentration, Narea was analysed using a principal component analysis (PCA). A correlation analyses (Spearman correlation index) was carried out to assess the relationship of the variables with the extracted principal components. We used SPSS 17.0 version software for Windows (SPSS, Chicago, IL, USA) to run the aforementioned analysis.

Results Growth response

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Stem basal diameter showed a stronger relationship with the aboveground + belowground biomass (y=7.5x-37.8; r2=0.95; p<0.01) than with the seedling maximum height (y=1.3x-24.7; r2=0.76; p<0.05). Significant differences among watering treatments were detected in the variation of the stem basal diameter ( Diam) and seedling maximum height ( Height) from the first assessment (Table 1). Plants in the control treatment showed significant lower values of Diam and Height than those submitted to the other watering treatments (Fig. 2 and Table 2). The block effect was not significant in Diam and Height (Table 1).

Leaf traits responses to irrigation treatments The leaf mass area (LMA, ranged between 105.7 and 352.3 g m-2) and leaf carbon isotope composition (13C, ranged between -27.0 and -23.7) were lower in the control than the other irrigation treatments (Table 1 and Fig. 2). The LMA was

explained by the leaf volume per area (r2=0.61), while no relationship was found between the former and leaf density (r2=0.08) (Fig. 3). The ANOVA analysis also detected significant differences among irrigation treatments in stomatal density (SD) and leaf carbon concentration (%C) (see Table 1 and 2). Thus, SD (ranged between 198.8 and 360.4 stomata mm-2) in the 80 mm month-1 treatment was lower than the other irrigation treatments, while the %C (ranged between 40.5 and 53.4%) was only different between the control and maximum irrigation treatment (Table 2). No significant differences were found in the nitrogen content per unit area (Narea, ranged between 0.20 and 0.50 g N m-2), nor in the irrigation factor (Table 1 and 2). Differences between blocks were not significant only in 13C and Narea which reflects the spatial variability at the restoration site (Table 1).

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Relationship among leaf traits The three component extracted by PCA explained 76.7% of the total variance (Table 3). The component 1 was highly correlated in negative way with LMA and 13C (r Spearman = -0.77 and -0.70 respectively), while the SD and Narea were negatively and positively correlated with component 2 (r Spearman = -0.71 and 0.67 respectively). The component 3 was highly positively correlated with %C (r Spearman = 0.80) (Table 3). A high proximity or/and association between LMA and 13C was found in the multivariate space established by the three extracted components (Fig. 4). The averages of the control (nowatering) in LMA vs. Narea and 13C vs. Narea plots were far away of the averages belonging to other the irrigation treatments (Fig. 5).

Discussion Seedlings of M. ferreyrae significantly increased their growth, LMA and 13C with the water input (Fig. 2). However, in disagreement with our first hypothesis, this response was not gradual, depending on amount of water, but rather was asymptotic. A lower phenotypic plasticity is expected in poor or less favourable environments in specialized plants (Lortie and Aarssen, 1996). Our results suggest that the water use in our fogspecialist species could be limited by a low phenotypic plasticity (in according with Lortie and Aarssen, 1996) in turn enhanced by a slow return of biomass investment into de leaf economics spectrum (Wright et al., 2004) typical of sclerophyllous species with high LMA.

Growth response to the irrigation The different irrigation treatments tested in this work tried to simulate water gains from fog intercepted by trees and that reach the understory and soil via stemflow and

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throughfall. For example, in our study area 96 mm day-1 has been collected as maximum bellow to Caesalpinia spinosa tree (Villegas et al., unpublished data), this value is close to the reported by Schemenauer and Cereceda (1994) below a Olea europaea tree (70 mm day-1). Larger trees with a high number of leaves and branches are able to intercept higher fog volumes and could allow a greater water inputs to the vegetation below them, thus for example in the redwood forest (Sequoia sempervirens) in California the fog water dripped from the vegetation was ranged from 22 to 46 mm year-1 which correspond to 13-45% of its annual transpiration (Dawson, 1998). Because the seedlings are not able to intercept important quantities of fog water, our irrigation treatments tried to simulate situations with different tree cover without shading effect. The lack of correspondence of growth according with the water increments tested by each watering treatments (Fig. 2a) could reflects a threshold (20 mm month-1) beyond which water increments does not causes a significant growth rise. We hypothesize two implications of the growth asymptotic response to water input in the restoration of this species: i) this threshold value (or close values) could be crucial in the regeneration phase of this specie, representing the minimum water quantity to improve the regeneration niche in this habitats, which is considered as the bottleneck for successful species establishment (Poorter, 2007); ii) this threshold could provide some reliability to drive the growth of individuals belonging to the seedling bank where the lowgrowth seedlings are waiting for the conditions improvement (Antos et al., 2005). However, M. ferreyrae shows a high LMA values (210 g m-2 of global average) which is coherent with the more sclerophyll Australian shrubs (66313 g m-2, (Niinemets et al., 2009). Because a high LMA is characteristic of slow-growth species (Poorter and Garnier, 2007; Wright et al., 2004), it is possible that we might have required a longer time of assessment to detect growth differences driven by our watering treatments. On

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the other hand, due to the fact that in dryland species the root biomass is often higher than shoot biomass (high values of root biomass/shoot biomass index, Larcher, 2003), we can not exclude that our different irrigation treatments may have had a differential effect in the belowground biomass. More studies are required to understand the root growth dynamic and its response to water inputs in these fog-oases ecosystems.

LMA, components and its response to the irrigation treatments LVA and LD are components of LMA whose contributions become more important under different environmental situations (Lambers et al., 2008). LVA has more weight in environments with higher irradiance (Poorter et al., 2009). In our study, LD explained 61% of the LMA variability (Fig. 3) which indicates that the leaves belonging to individuals submitted to the irrigation treatments could have been more exposed to the solar radiation. Thus also, Narea has been related to micro-environments with high irradiance to optimize the canopy photosynthesis (Hirose and Werger, 1987), in our study individuals submitted to the watering treatments trended to have higher values of this variable (without statistical significance) than control plants (Table 2, Fig. 5). Under full sun exposure, a larger parenchyma is developed (Onoda et al., 2008)

resulting in a thicker mesophyll that may, in turn, allow a more efficient use of incident light as well as a higher capacity to safely dissipate excess photon energy. Our measured leaf traits and their components support the hypothesis that light-processing efficiency is more important than structural components synthesis drove by water inputs. This last is in agreement with Lusk et al. (2008) work who suggested that in conditions of high light availability the symplast per area fraction is bigger than structural fraction in leaves of evergreen shade-tolerant species.

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Possible functional mechanisms driven by the irrigation with fog water A positive correlation between LMA and 13C has been found in inter-specific studies (Hoffmann et al., 2005; Lamont et al., 2002; Paula and Pausas, 2006) where the comparisons were carried out with contrasted leaf types. The high association of LMA and 13C (Fig. 4 and Table 3) and their correspondence with plant growth in our study species (Fig. 2) highlights the coherence in the leaf structure and photosynthetic (CO 2 diffusion and carboxilation) responses to water levels in fog-oases arid environments. Because in tick-leaved species (with high LMA like our study species) there is an important decoupling between 13C and water-use efficiency (WUE) (Marshall et al., 2007) we cant to state that the WUE is increased by our watering treatments (Fig. 2c). LMA affects gm and consequently chloroplastic carbon and 13C (Seibt et al., 2008). Small gm driven by a ticker mesophyll (Vitousek et al., 1990; Niinemets et al., 2009 but see Laureti et al., 1997; Hanba et al., 1999) could cause a rise of chloroplastic carbon demand and subsequently 13C increment (Lamont et al., 2002). Thus also, Narea has been related to higher Rubisco content (Hanba et al., 1999) and carboxilation (Wright et al., 2001). In our study we found that watered plants trend to show higher Narea and 13C than control plants (Fig. 5b), we hypothesize that in the former a high Rubisco activity could decrease the discrimination against the heavy carbon isotope and the 13C rise. SD trends to drop when the water conditions rise (Yang et al., 2004; Zhang et al., 2006) or under extreme drought (Xu and Zhou, 2008; Xu et al., 2009), because the SD was significantly lower in the greater irrigation treatment, we results confirm the first aforementioned pattern only in conditions of a high water inputs. Furthermore, SD had been positively related to WUE (Xu and Zhou, 2008; Yang et al., 2004). The lack of correlation of SD with 13C (a useful variable to explain the stomatal formation, Nobuhito and Katsuya, 2008) and the others assessed leaf traits prevent us to relate this

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variable with process involved in the gas exchange and carboxilation and consequently with WUE. We think that the leaf gas exchange assessments in future studies in our study species could be very useful for two main reasons: i) there is an uncoupling between 13C and WUE especially in sclerophyll species (Marshall et al., 2007; Seibt et al., 2008), ii) the relationship between SD and gas exchange is yet poorly known in plants (Xu and Zhou, 2008).

Conclusions Myrcianthes ferreyre, an endemic and specialist species of fog-oasis ecosystems, improves its growth and morfo-functional traits in response to water pulses from fog water, however this response is not gradual according with regular water inputs possibly caused by a low phenotypic plasticity typical of stressed environments. In practical terms we recommend a moderate water quantity (20 mm month-1) collected from fog to irrigate 2-year seedlings of Myrcianthes ferreyrae, this quantity will allow to save water or use this for the reclamations of more trees species. Due to the consistent response of LMA and 13C with plant growth to the irrigation with fog water, our work highlights the use of these variables in the assessment of the reclamation of endemic species (in our study Myrcianthes ferreyrae) in the Lomas ecosystems. We hypothesize that rise of the water input in the seedlings drive the arrangement of more irradiated leaves (possibly by more horizontal leaf angles) with a thicker mesophyll and higher content of nitrogen per area, all these could allow the processing of more quantity photons and higher Rubisco activity with a subsequent plant growth rise. However, all these aspects require specific experiments that help us to understand the functional process drove by the water input by fog, vital source for the maintaining of this fog-oasis into the Peruvian and Atacama Deserts.

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347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 Acknowledgements The financial support of this study was provided by the projects: Seguimiento adaptativo de la restauracin ecolgica del bosque de nieblas de las Lomas de Atiquipa (SEGARNIEBLA) funded by BBVA Foundation, and Elaboracin de una propuesta para el estudio de la demanda y uso de agua de plantones nativos reforestados, y suelo aledao en ecosistemas de desierto hmedo al sur de Per funded by General Foundation of the Castilla-La Mancha University (UCLM). We thank to M.D. Delgado to teach us the dental impression technique for the stomatal density assessment and to M.C. Fenol for allowing us to carry out the analysis in the facilities of the Plant Physiology Area - UCLM. L.B. was funded by the Madrid Regional Govt. (project REMEDINAL-2, S2009/AMB-1783). The authors are very grateful with the community of Atiquipa who help them to setup the experiment and were closely involved in the restoration activities.

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540 541 542 543 544 545 546 547 Irrigation Treatments Diam Height LMA 13C SD %C Narea 548 549 6.6 ** 3.2 * 5.0 ** 2.5 * 6.3 ** 2.6 ** 2.3 n.s. Blocks 1.4 n.s. 1.4 n.s. 3.5 ** 0.4 n.s. 6.4 ** 8.6 * 0.4 n.s. Table 1. F-values of one-way randomized block ANOVA testing irrigation treatments (control, 20, 40, 60, 80 mm month-1) in five blocks (slopes) in: variation of the stem basal diameter and seedling maximum height from the first assessment ( Diam and Height respectively ), leaf mass area (LMA), leaf carbon isotope composition (13C), stomatal density (SD), leaf carbon concentration (%C) and nitrogen content per unit area (Narea). ** p<0.01, * p<0.05, n.s. p>0.05

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550 551 552 553 554 555 556

Table 2.Mean ( standard error) value of the variation of the seedling height from the first assessment ( Height), stomatal density (SD), leaf carbon concentration (%C) and nitrogen content per unit area (Narea) grouped per each irrigation treatment. Different letters mean significant differences detected by post-hoc Tukey test of the one-way ANOVA (see Table 1) at p<0.05.

Height SD (stomata mm-2) %C Narea (g N m-2) 557 558 559 560 561 562 563 564 565 566 567 568 569

Control 5.2 2.3 a 260.6 8.3 a 45.8 0.8 a 2.9 0.2 a

Treatments (mm/month) 20 40 60 12.5 2.0 14.6 3.0 15.0 3.0 b b b 260.3 5.9 265.7 6.7 271.1 7.8 a a a 46.9 0.4 46.5 0.5 46.9 0.4 ab ab ab 3.8 0.3 3.2 0.2 3.3 0.2 a a a

80 19.7 3.0 b 238.0 4.4 b 47.5 0.5 b 3.7 0.2 a

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570 571 572 573 574 575 576

Table 3.- Spearman correlation index among the extracted components using multivariate principal components analysis (PCA) and foliar variables. ** and * mean significant values at p<0.01 and p<0.05 respectively. In grey the main correlations with /r
Spearman/>

0.6. LMA= leaf mass area, 13C= leaf carbon

isotope composition, SD = stomatal density, %C= leaf carbon concentration, Narea= nitrogen content per unit area.

Comp. 1 Comp. 2 Comp. 3 SD 0.26 -0.71** -0.30* 0.30* 0.67** -0.30 1.23 58.7
*

0.35** 0.30* 0.80** 0.12 0.16 0.90 76.7

13C -0.77** %C 0.41** Narea -0.51** LMA -0.70** Eigenvalue Cumulative Variance (%) 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 1.70 34.1

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598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626

Figure Captions

Figure 1. Study area map

Figure 2. Leaf traits and plant growth response to the irrigation treatments: (a) variation of the basal diameter from the first assessment ( Diam), (b) leaf mass area (LMA), (c) leaf carbon isotope composition (13C). * means significant differences (at p<0.05) detected by the post-hoc Tukey test of the ANOVA. Error bars represent SE.

Figure 3. Relationship among leaf mass area (LMA) and its components: leaf density (LD) and leaf volume per area (LVA ~ thickness).

Figure 4. Ordination of the leaf variables in a 3D space from the three principal components extracted in the PCA. For specification of PCA and abbreviation of the variables see Table 3.

Figure 5. Relationship among leaf mass area (LMA) and leaf carbon isotope composition (13C) versus nitrogen content per unit area (Narea) averaged for control plants and individuals subject to each of the irrigation treatments tested (circled values). Error bars represent SE.

25

627 628 629 630 631 632 633 634

Figure 1

75

73

71

14

Lima

Caraveli Lomas de Atiquipa 18

AREQUIPA

Arequipa

IF

IC

OC

EA

N
0 30 km

635 636 637

26

638 639 640

Figure 2

641 642 643 644 645 646 647 648 649 650 651

Figure 3

27

652

653 654 655 656 657 658

28

659 660 661 662

Figure 4

663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689

29

690 691 692 693 694

Figure 5

695 696 697 698 699 700 701 702 703 704 705 706 707

30