Stories from common gardens: Water shortage differentially affects Nothofagus pumilio from contrasting precipitation regimes

Forest Ecology and Management 458 (2020) 117796

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Stories from common gardens: Water shortage differentially affects Nothofagus pumilio from contrasting precipitation regimes Griselda Ignazia, Sandra J. Buccib, Andrea C. Premolia,

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Centro Regional Universitario Bariloche, Universidad Nacional del Comahue, INIBIOMA CONICET, Quintral 1250 8400 Bariloche, Argentina Universidad Nacional de la Patagonia, Instituto de Biociencias de la Patagonia, INBIOP CONICET, Facultad de Ciencias Naturales Ciudad Universitaria, Km 4, Chubut, Argentina

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A R T I C LE I N FO

A B S T R A C T

Keywords: Contrasting precipitations Climate change Ecophysiology Marginal populations Water deficit

Marginal populations are usually under stressful environmental conditions and may render novel phenotypes due to intense selection resulting from evolutionary and ecological changes. Thus the study of phenotypic variation under contrasting environments and their possible genetic basis is highly relevant particularly under changing climates. In Patagonia the study of populations located at the extremes of a pronounced west-to-east precipitation gradient due the rainshadow effect of the Andes has great importance to understand potential responses to drought that have caused already massive forest decay. Nothofagus pumilio is a winter deciduous tree that dominates high-elevation forests of southern Argentina and Chile. Populations at the driest extreme of the range are considered marginal in contrast to central ones at the humid end. We compare ecophysiological traits under common gardens and responses to a manipulative water deficit experiment of greenhouse-grown N. pumilio seedlings from contrasting precipitation regimes (humid and dry, hereafter central and marginal) to analyze genetically-based and / or plastic differences. During cultivation in common gardens central seedlings outgrew marginal ones in terms of height, basal diameter, and number of leaves. In contrast, plants from marginal populations endured water stress and had higher water use efficiency and relative growth rate than central ones which in turn showed greater susceptibility to desiccation. Given that the experiment was performed under homogeneous conditions, those differences are genetic. These results suggest that water-stress related traits have a genetic basis emphasizing their importance under predicted future altered water balances and intensity of droughts in northwestern Patagonia.

1. Introduction Species that occupy different habitats along environmental gradients may develop a wide range of physiological and ecological characteristics. At the extremes of such variable settings, populations are under conditions that limit their development and survival, compared to the ecological optima that generally exist towards the center of their distribution (Abeli et al., 2014; Lesica and Allendorf, 1995). Populations that inhabit the edges of species’ ranges are denominated marginal populations. Marginal populations are often less abundant and less dense than core populations (Cahill et al., 2014; Vucetich and Waite, 2003) and are usually under stressful environmental conditions. As a result, marginal populations may render novel phenotypes due to intense selection resulting from evolutionary and ecological changes (Hardie and Hutchings, 2010). Therefore, the study of phenotypic variations along environmental gradients and/or extreme conditions

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and their possible genetic basis is highly relevant. The persistence of a given species under environmental change, can be possible if populations consist of genetic variants able to respond to these environments and/or exhibit high phenotypic plasticity (Jump and Peñuelas, 2005). Therefore, a better understanding on how genetic adaptation and/or environmental factors influence populations throughout a species’ range is essential for evaluating the long-term persistence of species, particularly in the context of global change (Kapeller et al., 2016). When water availability is scarce, plants exhibit hydric stress, and their optimum functionality is limited. Facing this, plants have developed a wide spectrum of responses (Valladares et al., 2004), which may be genetically determined (i.e. adaptive) or environmentally driven (i.e. acclimatative). In order to deal with drier environments strategies include: elastic and osmotic adjustments (active and/ or passive, e.g., (Mitchell et al., 2008; Scholz et al., 2012), hydraulic changes (i.e.

Corresponding author. E-mail address: [email protected] (A.C. Premoli).

https://doi.org/10.1016/j.foreco.2019.117796 Received 3 September 2019; Received in revised form 17 November 2019; Accepted 20 November 2019 0378-1127/ © 2019 Elsevier B.V. All rights reserved.

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unknown. Previous studies found divergent drought resistance strategies of closely related species (Ambrose et al., 2015) and within species (Nguyen et al., 2017) differing in annual precipitation. Also a recent study under different climatic conditions suggested that six Nothofagus species should have adaptations to maintain relatively high water potentials at low water availability (Bucci et al., 2012). Studies of marginal populations are key for understanding how species will respond to changing environments (Kreyling et al., 2014). Particularly the study of populations located at the extremes of precipitation gradients has great importance to understand how the species could respond to increasing drought conditions predicted for northern Patagonia (CONAMA 2006). In particular, forest stands of N. pumilio located at the boundaries with the Patagonian steppe under reduced water availability presented a negative tendency to growth during the 20th century in response to an intensification of the water deficit (Srur et al., 2008). In addition, such stands have shown signals of partial crown mortality and thus are considered the most vulnerable to decline (Rodríguez-Catón et al., 2015). The aim of this study was to compare the ecophysiological traits of N. pumilio seedlings from contrasting precipitation regimes under common gardens and to analyze responses to water deficit to disentangle genetic from environmentally-based components. We tested the hypothesis that more xeric environmental conditions at the eastern (i.e. dry) extreme margin have resulted in the selection of individuals showing heritable variation related to water deficit. We predicted that seedlings from marginal populations will present ecophysiological characteristics that grant them greater resistance to drought compared to central (i.e. mesic) populations. We quantified ecophysiological traits that have previously shown genetic or environmentally induced differences along distinct environmental settings (e.g. elevation) inhabited by N. pumilio (Premoli et al., 2007, Premoli and Brewer, 2007) which were evaluated during the 3-year cultivation under common gardens. We also monitored mortality and partial crown mortality as an indicator of N. pumilio decline (RodríguezCatón et al., 2016). Plants from contrasting precipitation regimes were subjected to a manipulative water shortage experiment after which we evaluated growth rates.

decreasing the vulnerability to xylem cavitation, smaller vessel diameter, e.g., Bucci et al., 2013; Nardini et al., 2012; Blackman et al., 2014), stronger stomatal control to limit water loss and avoid hydraulic failure (Bucci et al., 2008; Martin-StPaul et al., 2017; Flexas et al., 2018), as well as increasing resource use efficiency and changes in the patterns of carbon allocation. Depending on the degree of stomatal regulation of water potential, plants can be classified as isohydric or anisohydric (Tardieu and Simonneau 1998), although the underlying mechanisms are not yet understood (Klein 2014). Isohydric plants exhibit a strong stomatal control such that leaf water potential is maintained relatively constant under different environment conditions. This behavior could avoid losses in hydraulic conductivity (Sperry et al., 2002) or cellular turgor (Cochard et al., 2002). But as a result of stomatal closure, carbon assimilation and accumulation of compounds from primary metabolism can be limited. In contrast, anisohydric plants have weak stomatal control, and continue transpiring even when soil water content decreases or under conditions of high vapor pressure deficit, but at the risk of dropping water potentials beyond the hydraulic safety thresholds and turgor loss point (Bucci et al., 2013). Facing elevated or prolonged stress levels, anisohydric behavior could not be maintained, reaching a limit from which the plant must adopt isohydric mechanisms (Schultz, 2003; Maseda and Fernández, 2006). Thus the timing of stomatal closure is critical for all plants under drought condition (Martin-StPaul et al., 2017; Bucci et al., 2019). The influence of the mountains in northern Patagonia creates a pronounced west to east precipitation gradient due to a rainshadow effect of the Andes (Veblen et al., 1996; Daniels and Veblen, 2004). Moving east from the Andes mountain range, the mean annual precipitation declines from 3000 mm to < 800 mm over a west-to-east distance of only 50 km (Veblen et al., 1996). An increase in average summer temperatures is predicted for this area which would produce changes in water balance and drought intensity (IPCC, 2014). Under this scenario it is a priority to analyze potential adjustments of key forest tree species. These changes could be more pronounced towards the extremes of species’ distribution where marginal populations exist (Premoli and Mathiasen, 2011). Nothofagus pumilio (Poepp. et Endl) Krasser (Nothofagaceae), commonly known as “lenga”, is a winter deciduous tree that dominates high-elevation environments of southern Argentina and Chile where it mostly occurs as monospecific forests. In Argentina N. pumilio is distributed from 36° 50'S on the Cordillera de los Andes to 56° S (Dimitri, 1972; Donoso 1995). It usually forms the upper altitudinal boundary of the forest and dominates the transition between forests and subalpine vegetation (Veblen et al., 1996). Contrary to mesic western-most populations, forest stands at the dry eastern-most end are under water stress conditions during summer, the period that coincides with the growing season. The latter are ecotonal forests immersed in a matrix of Patagonian steppe, and thus are considered ecologically and geographically marginal for the species’ range. The latitudinal and altitudinal variation for adaptive and neutral characters have already been studied for N. pumilio (Fajardo and Piper, 2011; Mathiasen and Premoli, 2010; Premoli et al. 2007; Premoli and Brewer, 2007; Premoli, 2003; Premoli, 1998; Barrera et al., 2000; Cuevas, 2000). Various studies suggest that intraspecific variation in functional traits of N. pumilio seedlings to environmental gradients appear to be modulated by the local climate (Barrera et al. 2000; Premoli and Brewer, 2007; Fajardo and Piper, 2011; Piper et al., 2013), some of which have a genetic basis (Premoli et al., 2007) and thus, are adaptive (Mathiasen and Premoli, 2016). A previous work showed a drought avoidance response to water stress of N. pumilio seedlings from two relatively humid locations (Peri et al., 2009). Also seedlings from contrasting elevations grown under homogenous irrigation showed plastical adjustments of water use, which may be favored under Mediterranean-type climates regimes with summer droughts (Premoli and Brewer, 2007). However, the variation and potential response of populations from contrasting precipitation regimes to water stress is

2. Methods 2.1. Collection and cultivation of plants in greenhouse The present study consisted of cultivation under common garden and drought manipulative experiments on plants from contrasting provenances, i.e. at the extreme of a precipitation gradient that occurs at 41° S in northern Patagonia. This area is under a Mediterranean-type climate characterized by summer drought and unpredictable rainfall during the growing season. We harvested seedlings bearing cotyledons that had germinated under natural conditions during the austral spring (October–December) in 2012 and 2013, from each of three marginal and three central populations (Table S1). All seedlings were collected at the lower limit of the elevation gradient, between 1000 and 1200 m.a.s.l. to control for adaptive differences with elevation previously reported (Premoli et al., 2007). We classified populations as marginal or central using a criterion based on mean annual precipitation estimated by WorldClim v.1, with a spatial resolution of 30 sec. (Hijmans et al., 2005; http://www. worldclim.org), i.e. those that receive less or more than 850 mm year−1, respectively. Mean annual precipitation of studied central and marginal populations is 1102 and 781.3 mm, respectively. To avoid transplantation stress, we collected seedlings carefully and brought them to the greenhouse retaining their original soil where we washed the roots and transplanted the seedlings into 0,5 L plastic pots (one seedling per pot) filled with a mixture of peat and native forest soil (Premoli and Brewer, 2007). We used the same type of soil for all the seedlings to minimize variability in soil nutrient content. Throughout three years, seedlings were grown in a randomized arrangement in a 2

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constant weight. The weight was stabilized at 96 h with a volumetric content of 30.5 ± 1.9%, which was considered the pot capacity. To maintain soil moisture levels and determine irrigation amount, we monitored volumetric water content (VWC) using a EC-5 soil moisture sensor (Decagon devices) daily, in at least 10 pots with plants from each provenance and treatment throughout the study period. We maintained the water deficit treatment near permanent wilting point within the range of 10–15% VWC and the control treatment near PC within 25–30% VWC throughout the experiment. At the beginning of the experiment, all the seedlings were irrigated to saturation and kept without irrigation until the relative humidity percentages determined for each treatment were reached. At the end of the experiment, we measured the mean soil water potential for the control and water deficit treatment, resulting in −0.4 and −1.1 MPa respectively. This was done taking as a reference the predawn leaf water potential (Ψpd, maximum leaf water potential) as it is considered the best estimator of soil water potential in plants that do not transpire at night (Bucci et al., 2004). The frequency and quantity of irrigations was variable in order to maintain the VWC within these ranges, and was done manually. At the beginning and end of the water-stress experiment we measured the height of each individual from the base of the principal trunk to the tip of the highest branch with a measuring tape ( ± 1 mm). We calculated the relative growth rate as RGR = ln (A2/A1), were A1 = height in t1 and A2 = height in t2 (Premoli et al., 2007).

greenhouse located at the Instituto de Investigaciones en Biodiversidad y Medioambiente (INIBIOMA), Bariloche, under conditions typical of understory of N. pumilio forest (PAR~700 µmolm−2 s−1 at solar noon). We watered and rotated the seedlings regularly to minimize confounding environmental effects. After two years in the greenhouse, we repotted the plants into 1 L containers. 2.2. Common garden measurements Each year we recorded the accumulated percentage of plant mortality as alive (1) or dead (0) and the accumulated percentage of partial death as plants presenting partial death of the main stem and/or lateral branches (1) or none (0). During two consecutive years (October 2015 and 2016) we measured plant height (cm), basal diameter (mm), number of branches and total number of leaves. The height of each individual was measured with a measuring tape from the base of the principal trunk to the tip of the highest branch at the beginning and end of the experiment. We recorded the leafing process from the onset of bud burst until leaf expansion following phenological stages described by Rusch (1993). The stage of the seedlings was monitored as the presence of the bud and/or leaf with the most advanced phenophase on each individual. The phenological stages were: resting buds, swollen buds, outbreaking leaves, and fully expanded leaves. We recorded phenological stages on a total of 91 central and 158 marginal seedlings on three dates between September and October 2014. Stomatal density and morphological leaf traits were measured on randomly collected leaves from different positions within each plant, to represent a sample of the entire individual. We estimated stomatal density from surface impressions made with clear nail varnish from the abaxial leaf surface (Brewer and Smith, 1997) of five randomly selected seedlings from both the marginal and central populations, by counting the number of stomata per field at 400 × magnification. Five leaves with five replicates per leaf for each individual were evaluated. We corrected the data by area, and reported values are the number of stomates per mm2. We also randomly selected five seedlings from both marginal and central populations and collected five leaves at random per individual in order to measure morphological leaf traits. These were leaf length, width, area, perimeter, shape factor, width to length and ratio on each leaf with a scanner and Digimizer software v4.6.1 (MedCalc Software, Belgium). We determined leaf dry mass and specific leaf area (calculated as one sided leaf area per unit of dry mass) of the leaves by drying at 60 °C to constant mass. Shape factor was defined as 4 π × area / perimeter2, it is a unitless measure of an object’s circularity, a perfect circle equals 1 whereas a line-shape object will approach zero.

2.4. Gas exchange measurements Instantaneous gas exchange measurements were performed in fully expanded leaves of a subset of 73 plants (13 and 29 plants under control, 11 and 20 plants under water deficit conditions from central and marginal provenances, respectively) , at the end of watering treatments, over two consecutive days, in a randomized order. We recorded net photosynthetic rate (Anet, µmol CO2 m−2 s−1), leaf conductance to water vapour rate (gs, mol H2O m−2 s−1), and leaf transpiration rate (E, mmol H2Om−2 s−1) using an LI-6400 portable photosynthesis measuring system (LI-COR, Lincoln, Nebraska, USA) with a 6400-02B LED source providing a photosynthetic photon flux density of 700 µmol m−2 s−1. This level of radiation was above saturation point for N. pumilio (Martínez Pastur et al., 2007). The atmosphere of the chamber was maintained at 20 °C, 40% relative humidity, with a CO2 concentration of 400 ppm. We calculated the instantaneous water use efficiency (WUE) as Anet/E (µmol CO2 fixed per mmol of transpired H2O). We cannot rule out the effect of vapour pression deficit to transpiration, nevertheless all seedlings were exposed to similar environmental conditions of temperature and humidity in the greenhouse. Therefore, we consider WUE determined as Anet/E may be valid for comparing between populations (Premoli and Brewer, 2007). Given that N. pumilio presents stomata only on the abaxial surface (Premoli and Brewer, 2007) Anet and gs were reported on a one-sided leaf area basis. After measurements, we collected the leaves and determined the leaf area with a scanner and Digimizer software v4.6.1 (MedCalc Software, Belgium).

2.3. Drought manipulative experiment In early November 2016 we selected a total of 103 plants of similar size from marginal and central areas, in two watering treatments (control and water deficit) and applied the treatments for 30 days. We performed the experiment in late spring to avoid excessively high mean and maximum greenhouse temperatures (Varela et al., 2010). A water retention curve of the soil (−0.03; −0.1; −0.15; −0.7 and −1.5 MPa) was developed in the Soil Laboratory of INTA EEA Bariloche. We converted gravimetric water content values of field capacity (FC) and permanent wilting point of the water retention curves (−0.03 and −1.5 MPa) to volumetric values in order to ensure the watering condition of the two treatments. The pots were well-watered until the drought treatment was initiated. Since the experiment was conducted in pots, pot capacity (PC) was also calculated, which is a measure analogous to FC, being the water content that delivered a uniform distribution of water inside the pot. The pot capacity is generally greater than the FC of the same soil measured in the laboratory by the gravimetric method. For its determination, three pots were watered to saturation, covered with plastic to prevent evaporation and allowed to drain until

2.5. Plant water relations We measured predawn (Ψpd) and midday leaf water potentials (Ψmd) with a Schölander pressure chamber (PMS 1000, PMS Instruments, Corvallis, Oregon, USA) for 5 seedlings per provenance/ treatment combination after the 30 days of watering treatments, using two twigs with fully expanded leaves per plant. The samples were taken between 05:00 and 07:00 h and between 12:00 and 14:00 h respectively. We used twigs instead of leaves because the petioles were very short. Pressure–volume (P–V) curves were constructed to estimate the osmotic potential at saturation (πsat, MPa), water potential at turgor loss full point (TLP, MPa), the relative water content at turgor loss 3

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3.2. Drought manipulative experiment

point, the maximum modulus of elasticity (Єmax, MPa) of cell walls, and the apoplastic relative water content. The P-V curves were developed by using the free transpiration technique (Tyree and Hammel, 1972). We used one twig with fully expanded leaves per seedling (3 to 6 per provenance/treatment combination) after the 30 days of watering treatments. We hydrated the samples in distilled water and kept them in the dark for at least 12 h. The portion of shoot that had been under water was removed prior to perform the P-V curves. An earlier study with this and other woody species from temperate Andean forests indicated no evidence of a rehydration-induced plateau and similar P-V parameters were obtained for non-rehydrated samples (Bucci unpublished results). We weighed the branches to the nearest 0.001 g to determine the initial fresh mass, and immediately placed them in the pressure chamber to obtain the initial water potential. We repeated the last two steps of this procedure at least 8 times per branch. After all the P-V measurements were completed, we determined dry mass of the samples by drying at 60 °C to constant mass.

The relative growth rate (RGR) of control seedlings from central provenances was similar to that of seedlings from marginal provenances under control and water deficit treatments (non-parametric KruskalWallis test comparisons of multiple groups, P greater than 0.05). However, a significant decrease of 60% of RGR was measured in central seedlings under water deficit compared to control (Fig. 2). Both provenances showed a significant reduction in net Anet and gs under water deficit conditions compared to the control (non-parametric Kruskal-Wallis test comparisons of multiple groups, P < 0.05) (Fig. 3). Seedlings from marginal provenances could maintain positive values of Anet under water deficit, although Anet decreased 65% with respect to control conditions. However, seedlings from central provenances had negative values of Anet under water deficit (Fig. 3A). Decline of gs was also lower in marginal populations than in central populations. While gs of seedlings from central provenance under water deficit decreased by 66% with respect to control, for seedlings from marginal provenance this reduction was 55% (Fig. 3B). Differences were also found in WUE between provenances under water deficit being greater for marginal seedlings (non-parametric Kruskal-Wallis test comparisons of multiple groups, P < 0.05) (Fig. 3C). Predawn leaf water potential of central seedlings was significantly lower in the water deficit treatment than in the control Fajardo and Piper, 2011; Mathiasen and Premoli, 2016; Tyree and Hammel, 1972; Wright et al., 2004. In these seedlings midday water potential decreased from −0.6 in control treatment to −1.3 MPa in the water deficit treatment (Fig. 4A, B respectively), Also, in marginal seedlings predawn and midday water potentials were lower under water deficit than under the control treatment (Fig. 4A, B respectively). Under water deficit treatment predawn leaf water potentials were significantly different to midday water potentials in plants of both provenances although the differences were lower in marginal seedlings (close to 0.2 MPa) than central seedlings (~0.4 MPa). No significant differences were found in leaf traits derived from P–V relationships for seedlings of different combinations of provenances /treatment (Table S3). Only marginally significant differences were found for the Єmax (one-way ANOVA, P = 0.05). Cell walls were more rigid under water deficit treatments in plants from all provenances. While seedlings from central provenance were able to maintain cell turgor (negatives values of TLP – Ψmd) under water deficit treatment, seedlings from marginal provenance lost turgor (Ψmd was 0.22 MPa more negative than osmotic potential at the turgor loss point) (Table S3).

2.6. Data analysis All data were checked for normal distribution using Shapiro-Wilk test and homogeneity of variance. When data did not fit a normal distribution, we used non-parametric tests. For the common garden experiment plant mortality and partial death were compared between provenances using a t-test. The variables plant height (cm), basal diameter (mm), number of branches and total number of leaves were repeatedly measured across different years and compared within each provenance using the Wilcoxon test and between provenances for each year using the Mann Whitney U test. We analyzed the effect of plant age on each of the architectural traits and total number of leaves by analysis of covariance (ANCOVA). Stomatal densities and morphological leaf traits of populations from different provenances were compared by oneway analysis of variance (ANOVA). We analyzed leafing phenology by χ2-tests. For the drought manipulative experiment we analyzed for statistically significant differences of the combined effects of provenances and treatments, for gas exchange measurements, leaf water potentials and RGR with a non-parametric Kruskal-Wallis test for multiple comparisons of groups. We analyzed parameters derived from the P–V curves by one-way ANOVA. We performed all the statistical analysis with STATISTICA v7.0 (Statsoft, Tulsa, OK). 3. Results 3.1. Common garden measurements

4. Discussion Both sets of plants similarly showed signals of increased partial death during the cultivation in common gardens reaching approximately 60% of them (Fig.S1A). Distinct provenances were also alike for the accumulated percentage of mortality, although plants form central populations yielded higher total mortality at the end of the experiment (t-test, P < 0.05) (Fig.S1B). The analysis of repeated measurements during the growing seasons of 2015 and 2016 indicated that the height, basal diameter, number of branches and number of leaves varied significantly for both provenances throughout the common garden experiment (Wilcoxon test, P < 0.05) (Fig. 1A to D respectively). Comparing provenances, central seedlings consistently grew taller, attained greater basal diameter and had more leaves than marginal ones (Mann Whitney test, P < 0.05) (Fig. 1). Anaysis of architectural and leaf traits yielded similar results when age was used as covariate. Leafing phenology was synchronized in central and marginal seedlings, suggesting similar duration of the growing period throughout the 2014 observation period (chi-square test) (Fig.S2). Leaf shape and size traits were highly conserved for both sets of plants and although stomatal density was 10% lower in marginal populations differences were not significant (Table S2).

The results of the common garden experiment indicate that N. pumilio seedlings from environments with contrasting precipitation regimes present highly conserved, i.e. genetically fixed, ecophysiological traits as well as adaptive responses to manipulative water stress. Under homogeneous growing conditions marginal seedlings, adapted to a more stressful environment, consistently presented reduced performance in plant architecture demonstrating a genetic basis for those differences. This could be similar to the genetic differences observed in N. pumilio plants facing the environmental stressors associated with high altitudes where lower temperatures impose a restrictive factor representing another form of marginal habitat for this species compared to those from lower elevations (Premoli et al., 2007, Mathiasen and Premoli, 2016). Marginal stressful habitats exert a selective pressure for plants to have long term responses as reduced height and basal diameter. Yet, the synchrony in leaf phenology measured here at contrasting precipitation regimes differs to the marked effect of environmental variation on phenological rhythms measured along altitudinal gradients of N. pumilio thermal (Rusch, 1993) which in turn were genetically controlled (Premoli et al., 2007) and of adaptive value 4

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Fig. 1. Repeated measurements for N. pumilio seedlings from central and marginal provenances under the common garden experiment. Plant height (A), basal diameter (B), number of leaves (C) and total number of branches (D). Comparisons within each provenance repeatedly measured across different years were analyzed using the Wilcoxon test and between provenances for each year using the Mann Whitney U test. Significant differences (P < 0.05) are indicated with different lowercase letters for Wilcoxon test. Capital letters show statistical differences for Mann Whitney U test. Results are presented as means ± standard error.

phenology are highly correlated in Nothofagus (G. Juri Universidad Nacional del Comahue, pers. comm.). Therefore, the lack of phenological differences may not preclude gene flow between provenances at the precipitation extremes. This would explain the reduced to moderate genetic divergence measured by neutral markers, still significantly different from zero, between central and marginal N. pumilio populations (Fst ~ 17%, G. Ignazi, unpublished). The lack of significant differences between provenances in plant mortality, partial death, morphological leaf traits and stomatal density under the common garden experiment, suggest that these characteristics are either highly conserved and/or show plastic adjustments. This is consistent with other studies that did not find variation in leaf mass per area deployed (g m−2) related to precipitation in deciduous species (Wright et al., 2004) and soil moisture in Nothofagus pumilio (Fajardo and Piper, 2011). Plants from the driest extreme of temperate Andean forest showed relatively similar physiological behavior and a more efficient resource acquisition under distinct soil water conditions (i.e. more isohydric) as well as higher RGR under water deficit than plants from mesic sites. Furthermore, marginal seedlings of N. pumilio endured experimental water stress better than central more mesic ones which could imply greater susceptibility of the latter to desiccation. Consistent with our results, Bucci et al. (2012) found that adult N. pumilio trees from mesic sites exhibit higher percentage of loss of stem and leaf hydraulic capacity compared with that observed in trees from dry populations during drowght. Given that the experiment presented here was performed in common gardens, those differences between N. pumilio from contrasting precipitation regimes are probably genetic. Both central and marginal seedlings presented a significant reduction in Anet and gs under water deficit, which could be a consequence of stomatal closure to avoid excessive loss of water. However, despite these reductions, we

Fig. 2. Relative growth rate for N. pumilio seedlings from central and marginal provenances under control and water deficit treatments. Relative growth rate (RGR), Control (C), Water deficit (WD). Comparisons between provenances, and treatments within provenances were analyzed with non-parametric Kruskal-Wallis test comparisons of multiple groups. Significant differences (P < 0.05) are indicated with different lowercase letters between treatments within provenances. Capital letters show statistical differences between provenances under the same treatment. Results are presented as means ± standard error.

(Mathiasen and Premoli, 2016). Given the sampling scheme of the current study, it seems that plants from similar elevations and latitudes, yet under contrasting precipitation regimes, set leaves simultaneously during the spring. Previous studies found that temperature was a critical variable on the initiation of the leafing process along altitudinal gradients (Barrera et al., 2000; Premoli et al., 2007; Mathiasen and Premoli, 2016), which could explain why we did not find phenological differences between contrasting precipitation regimes, as similar elevation strips experience similar regional temperatures. Leaf and flower 5

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Fig. 4. Leaf water potentials of N. pumilio seedlings from central and marginal provenances under control and water deficit treatments. Predawn (A, Ψpd) and midday (B, Ψmd) leaf water potential. Comparisons between provenances, and treatments within provenances were analyzed with non-parametric KruskalWallis test comparisons of multiple groups. Significant differences (P < 0.1) are indicated with different lowercase letters between treatments within provenances. Capital letters show statistical differences between provenances under the same treatment. Results are presented as means ± standard error. Comparisons between Ψpd and Ψmd for each different combination of provenance/treatment were analyzed with Wilcoxon matched pairs test and significant differences are indicated with asterisks. Fig. 3. Gas exchange measurements for N. pumilio seedlings from central and marginal provenances under control and water deficit treatments. Net photosynthetic rate (Anet), B stomatal conductance (gs), C water use efficiency (WUE). Comparisons between provenances, and treatments within provenances were analyzed with non-parametric Kruskal-Wallis test comparisons of multiple groups. Significant differences (p < 0.05) are indicated with different lowercase letters between treatments within provenances. Capital letters show statistical differences between provenances at the same treatment. Results are presented as means ± standard error.

compared to central ones under water deficit reported here, other studies found that most plants tend to show an increase in WUE under water deficit by restricting water loss more than inhibiting photosynthesis (Chaves et al., 2003; Park et al., 2016). This supports the hypothesis that plant evolution favored optimization of carbon uptake over water loss (Raven, 2002). Although higher WUE in seedlings under water deficit resulted in a trade-off, of reduced carbon assimilation and reduced water loss compared to control treatments, this trade-off was relatively smaller in seedlings from marginal provenances because the decrease in Anet was lower than in central populations. Carbon gain in seedlings from central populations was totally inhibited under water deficit measured at high light levels, which resulted in low RGR at the end of treatment. This could lead to carbon starvation as carbohydrates are depleted to maintain respiration under stronger or more extended exposure to water deficit. According to Bucci et al. (2019), isohydric plants from Andean forests have similar maximum photosynthetic rates Amax, earlier stomatal closure and lower embolisms formation than anisohydric plants. Taking into account these findings, in marginal seedlings, which show lower changes in water potential gradient under water deficit treatment, more photo-assimilates may be directed to growth. Whereas in central seedlings a fraction of photo-assimilates could be used to daily refilling embolisms and toproduce hydraulically safe tissues at the cost of plant growth. Plants can endure drought conditions by avoiding tissue dehydration, this involves minimizing water loss (e.g. closing stomata) and maximizing water uptake (Chaves et al., 2003). A maximization of water uptake could be related to the capacity of marginal seedlings to increase the driving force for water flow from soil to leaves. In seedlings from both provenances, the water potential gradient between soil and leaves increased under water deficit as result of a decrease in Ψpd as

observed that the marginal seedlings presented greater WUE compared to central seedlings, providing further evidence for the existence of genetic differences related to the control of WUE between provenances. The genetic basis found in ecophysiological traits related to the use of water differs with previous results on common garden-grown N. pumilio from contrasting elevations where they appear to be more responsive to environmental cues under optimal growing conditions (Premoli and Brewer, 2007). Yet, the water-stress manipulative experiment presented here allows the analysis of potentially adaptive ecophysiological differences, i.e. if they confer a selective advantage, under water shortage. The stomata are responsible for optimizing carbon fixation per unit of water loss and the maintenance of water status when the soil water supply is limited and /or evaporative demand by the atmosphere is high, in order to avoid hydraulic failure (Park et al., 2016). These roles operate in the same direction and both can be regulated (in principle) with the same set of control mechanisms (Raven, 2002). Therefore, the significantly greater WUE and tendency to greater growth under water stress of marginal seedlings could be due to a smaller reduction in Anet compared to central seedlings. Under drought plants with relatively high WUE should be able to accumulate more biomass than plants with low WUE (Touchette et al., 2007). In line with the significantly greater WUE of marginal seedlings 6

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well as in Ψmd, behaving as anisohydric plants, which was more notorious in central plants. However, for marginal seedlings this implied a loss of turgor as Ψmd dropped to values more negatives than osmotic potential at turgor loss point, although such relative losses compared to Ψpd potentials were not as marked as in central plants. Several studies have demonstrated that guard cell turgor loss is uncoupled from the turgor of epidermic cells (Franks and Farquhar, 2007; Scholz et al., 2012) and thus their effects on gas exchange could be relatively low (Bucci et al. 2019). In our study, seedlings from marginal provenances under water deficit had higher gs and carbon assimilation than seedlings from central provenances, suggesting that the former were relatively insensitive to turgor loss. Although no osmotic adjustments were measured under water deficit treatments (i.e πsat and TLP were similar to control treatment), a substantial change in cell wall properties, which resulted in an elastic adjustment, was evidenced in central and marginal seedlings (more rigid cell wall under water deficit). The interpretation of the elastic adjustment from an adaptive point of view is complex, given that in nature both decreases and increases of the Єmax can be observed as a response to drought (Fernández and Gyenge, 2010; Saito and Terashima, 2004; Scholz et al., 2012). Cells with rigid walls contribute to decreasing water potential with only moderate decrease in cell water content (Goldstein et al., 1989; Whitehead and Beadle, 2004). The lack of significant differences observed between treatments and /or provenances in the P–V relationships may reflect the short duration of the experiment, low response levels of these traits in Nothofagus pumilio, or both. A plausible explanation for the lack of response to water deficit in these traits in seedlings from central provenances may be that they operated above TLP, which was not necessary to allocate osmotically active solutes required for active osmotic adjustment. On the other hand, low sensitivity to loss turgor in seedlings from marginal provenances maintained the carbon assimilation at midday, without metabolic cost associated with the decrease in TLP. Furthermore, although most experimental stress studies apply only one stress cycle, other studies have shown that successive stress cycles can have a summative effect (Niinemets, 2010). It is possible then that redesigning the experiment with repeated exposures or drought cycles, the existence or otherwise of significant differences for these characteristics related with the provenance of the seedlings may be elucidated. These results provide important information, but other considerations should be kept in mind such us the different responses during the ontogeny of plants to transfer the knowledge from common garden grown-seedlings to the forest (Niinemets, 2010). Yet comparative gas exchange measurements using seedlings and adults of N. pumilio from contrasting elevations in northern Patagonia yielded similar patterns (Premoli and Brewer, 2007). Intraspecific functional trait variation across species’ ranges has not been sufficiently studied (Martinez-Vilalta et al., 2009; Violle et al., 2014) and could bring novel insights of range constraints by suggesting limits to physiological adjustment (Reichstein et al., 2014). These experiments suggest that central seedlings were more sensitive to experimental water deficit. It was recently shown that N. pumilio forests presently located in the relatively mesic sites will become more susceptible to extensive forest decline (Rodríguez-Catón et al., 2016) as massive tree mortality was recently documented in different parts of the world in association with global climate changes (e.g. Allen et al., 2010). On the contrary, marginal populations present adaptive characteristics related to gas exchange and water relations that give them greater tolerance to drought conditions. With ongoing climate change, it is important to protect and manage marginal population habitats, for example by reducing other extrinsic pressures (Moritz et al., 2012), with the objective to conserve the existing adaptive diversity (Kapeller et al., 2016). This knowledge can also be used for restoration purposes to minimize effects of climate change by the possible introduction of adapted individuals from marginal populations, which can help to adapt more quickly to ongoing changing climates (Thiel et al., 2014).

5. Conclusions Marginal seedlings of N. pumilio endured experimental water stress better than central more mesic ones presenting ecophysiological traits of genetic basis. These results emphasize the importance of marginal populations for the preservation of the evolutionary potential of species. Thus conservation of populations inhabiting species’ distribution margins is relevant in the face of increasing average summer temperatures, alteration in water balances and intensity of droughts as predicted for eastern populations of N. pumilio in northwestern Patagonia. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements Authors are most grateful to Patricia Suarez INIBIOMA CONICET for assistance during plant cultivation in common garden, Santiago Varela INTA Bariloche during gas exchange measurements. Ramiro Ripa assisted with pressure chamber. Financial support was provided by Agencia Nacional de Promoción Científica y Tecnológica of Argentina PICT2013-2404 CONICET PIP636, and Rufford Foundation Project 29211-1. Author Statement Authors declare that data and results of this manuscript have not been sent All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Furthermore, each author certifies that this material or similar material has not been and will not be submitted to or published in any other publication. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foreco.2019.117796. References Abeli, T., Gentili, R., Mondoni, A., Orsenigo, S., 2014. Effects of marginality on plant population performance, 239–249. doi:10.1111/jbi.12215. Allen, C.D., Macalady, A.K., Chenchouni, H., Bachelet, D., McDowell, N., Vennetier, M., Kitzberger, T., Rigling, A., Breshears, D.D., Hogg(Ted), E.H., Gonzalez, P., Fensham, R., Zhang, Z., Castro, J., Demidova, N., Lim, J.-H., Allard, G., Running, S.W., Semerci, A., Cobb, N., 2010. A global overview of drought and heat-induced tree mortality reveals emerging climate change risks for forests. For. Ecol. Manage. 259, 660–684. https://doi.org/10.1016/j.foreco.2009.09.001. Ambrose, A.R., Baxter, W.L., Wong, C.S., Næsborg, R.R., Williams, C.B., Dawson, T.E., 2015. Contrasting drought-response strategies in California redwoods. Tree Physiol. 35, 453–469. https://doi.org/10.1093/treephys/tpv016. Barrera, M.D., Frangi, J.L., Richter, L.L., Perdomo, M.H., Pinedo, L.B., 2000. Structural and functional changes in Nothofagus pumilio forests along an altitudinal gradient in Tierra del Fuego, Argentina. J. Veg. Sci. 11, 179–188. https://doi.org/10.2307/ 3236797. Blackman, C.J., Gleason, S.M., Chang, Y., Cook, A.M., Laws, C., Westoby, M., 2014. Leaf hydraulic vulnerability to drought is linked to site water availability across a broad range of species and climates. Ann. Bot. 114, 435–440. https://doi.org/10.1093/aob/ mcu131. Brewer, C.A., Smith, W.K., 1997. Patterns of leaf surface wetness for montane and subalpine plants. Plant Cell Environ. 20, 1–11. https://doi.org/10.1046/j.1365-3040. 1997.d01-15.x. Bucci, S.J., Goldstein, G., Meinzer, F.C., Scholz, F.G., Franco, A.C., Bustamante, M., 2004. Functional convergence in hydraulic architecture and water relations of tropical savanna trees: from leaf to whole plant. Tree Physiol. 24, 891–899. https://doi.org/10.

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