Subtle precipitation differences yield adaptive adjustments in the mesic Nothofagus dombeyi

Forest Ecology and Management 461 (2020) 117931

Contents lists available at ScienceDirect

Forest Ecology and Management journal homepage: www.elsevier.com/locate/foreco

Subtle precipitation differences yield adaptive adjustments in the mesic Nothofagus dombeyi Dayana G. Diaz, Paula Mathiasen, Andrea C. Premoli

T

⁎

Universidad Nacional del Comahue – Centro Regional Universitario Bariloche, INIBIOMA – CONICET, Quintral 1250, 8400 Bariloche, Argentina

A R T I C LE I N FO

A B S T R A C T

Keywords: Common garden Drought Genetic diversity Growth Plant architecture SNPs Water potential Water stress

Climate change is impacting on natural systems and forests worldwide have suffered from massive mortality due to extreme droughts. That is the case of mesic Nothofagus dombeyi forests of northern Patagonia, whereas no climate-driven mortality was recorded towards drier areas. The aim was to examine population differences in relation to a small precipitation gradient at morphological, physiological, and genetic scales. Adaptive differences between seedlings collected from dry and mesic sites of N. dombeyi on the eastern slopes of the Andes were studied combining common garden and water stress manipulative experiments with genomic analyses. We recorded mortality, plant architectural and leaf traits during cultivation in the greenhouse. A set of plants were subjected to water deficit experiments and water potential was measured. Genomic differences were analyzed by Single Nucleotide Polymorphisms (SNPs). Plants from the mesic site grew taller with more branches and number of larger leaves, and greater stomatal density under optimum conditions yet with slightly more negative midday water potentials due to induced desiccation. Adaptive SNPs yielded similar divergence than quantitative traits which were greater than that of neutral SNPs. Five adaptive loci had a significant association to whole plant and leaf traits. These results show that in spite of the small climatic differences between sites and continuous gene flow populations showed adaptive adjustments and differed significantly in growth, ecophysiology, and genetically. This information can be used in climate-oriented restoration efforts.

1. Introduction Climate change has impacted natural systems, and the evidence shows that it will become even stronger (IPCC, 2014). Predictions of climate change point to an increase in the duration and intensity of droughts, associated with a more irregular regime of precipitations and increasingly extreme and generally warmer temperatures (IPCC, 2013). Under these scenarios it is necessary to understand plant responses to changes in climate and thus to predict the future of forests particularly of dominant taxa. To avoid local extinction, the fate of natural populations under climate change could be one or a combination of non-exclusive potential responses such as to track their ecological optimum through migration or to rapidly adjust to novel conditions by phenotypic plasticity and/or adaptation (Nicotra et al., 2010; Hampe and Jump, 2011). In the case of forest trees, and particularly for species of reduced vagility a greater importance of some degree of in situ adequacy by adaptation through rapid genetic changes, and/or acclimation by phenotypic plasticity could be expected (Aitken et al., 2008). Potential responses of species depend, at least to some extent, on the existence of

⁎

genetic variation in populations (Jump et al., 2009) and its relevance has been considered by climate change reports. For example, the importance of protecting genetically diverse populations has been recognized for climate-change adaptation strategies in order to maintain ecosystem structure and function as well as that early response to warming can be detected by changes in the distribution and abundance at the local, genetically distinct, populations (IPCC, 2007). Hence, the quantification of the degree of adaptive genetic diversity within populations and the level of genetic divergence among populations, particularly those that inhabit different climates, are relevant under changing scenarios. One of the predictions of climate change is the increase of extreme droughts (Easterling et al., 2000); thus, knowledge on how plants respond to such events is essential. Extreme climate events have had rapid and large-scale effects on ecosystem structure and function that result from mortality of forest tress and woodlands due mainly to water limitations (McDowell et al., 2008). For example, altered timing of precipitation may have significant effects on plant growth and mortality rates, which in turn may differ in dry (water-limited) and comparatively mesic (water-abundant) sites (Zeppel et al., 2014; Grossiord

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

https://doi.org/10.1016/j.foreco.2020.117931 Received 29 October 2019; Received in revised form 22 January 2020; Accepted 23 January 2020 0378-1127/ © 2020 Elsevier B.V. All rights reserved.

Forest Ecology and Management 461 (2020) 117931

D.G. Diaz, et al.

factor regulating N. dombeyi tree growth is spring–summer water deficit induced by above-average temperature and reduced precipitation during the growing season, recent modifications to such climate-tree growth relationship highlight the vulnerability of N. dombeyi to climate changes (Suarez et al., 2015). Previous studies reported that N. dombeyi shows low stomatal regulation and that under severe water stress photosynthesis is largely suppressed thus seriously affecting foliar carbon balance (Sanhueza et al., 2015). Yet not much attention has been given to mechanisms of drought tolerance which show a strong correlation with water potential (Maréchaux et al., 2015) and the intraspecific variation in ecophysiological traits under distinct precipitation regimes. Experiments of ecological genetics such as cultivation in common gardens can directly estimate the genetic variation on characters that affect the fitness of plant populations. These consist of seedlings that are grown under homogeneous environmental conditions such that measured growth and ecophysiological differences among provenances can be considered genetic, or that the lack of them implies that differences in the field are caused by phenotypic plasticity. In addition, differential responses to manipulative experiments under controlled conditions, e.g. water stress, may suggest that they are adaptive. Genetic differences between populations that inhabit distinct environments can be also assessed by molecular markers. In recent years, advances in next generation technologies by the use of genotyping-by-sequencing (GBS) for a wide variety of non-model organisms (Ekblom and Galindo, 2011) allowed to discover large numbers of single nucleotide polymorphisms (SNPs). SNPs represent the smallest observable unit of DNA polymorphisms and are the most frequent type of mutations throughout the genome. These markers provide an opportunity to disentangle the effects of neutral, i.e. drift and gene flow, from adaptive, i.e. natural selection, forces and also to perform genome-wide association studies (GWAS) to identify relationships between candidate genes and phenotypes of interest. Drought responses of plants are modulated by different physiological mechanisms, which in general are controlled by multiple genes (Foolad, 2004). Thus SNPs constitute an efficient method for dissecting the genetic architecture of complex traits and have greatly accelerated the characterization of forest trees’ diversity and analyses in relation to adaptation to climate change (Plomion et al., 2016). For example, a genome scan along N. dombeyi distribution in Chile allowed the identification of signatures of divergent selection associated with both temperature and precipitation gradients, suggesting local adaptation (Hasbún et al., 2016). Widespread Nothofagus-dominated forests of northern Patagonia provide the ideal framework for analyzing potential adaptive and/or plastic responses under changing scenarios. This is mainly due to the fact that a significant variation in physical conditions exists in Patagonia such as the presence of steep precipitation gradients due to the rainshadow effect of the Andes upon the prevailing westerly winds. Although genetic studies showed that different Nothofagus species have persisted locally in response to historical changes in climate (Premoli et al., 2012) recent global change-driven forest decay suggest that some species may be vulnerable to current climatic trends that include the increase in frequency and severity of drought. Particularly mesic forests of N. dombeyi on the eastern, i.e. leeward, slope of the Andes has suffered from recent extreme droughts resulting in massive tree mortality within local populations (Suarez et al., 2004). On the contrary, towards the dry extreme of the precipitation gradient, populations seem to be relatively less susceptible to drought probably as the product of directional selection of genotypes adapted to water deficit. The aim of this study was to evaluate genetic differences and potential adaptive responses to water stress in mesic and dry provenances of Nothofagus dombeyi. We combined the analysis of plant growth and ecophysiological characteristics evaluated under cultivation in common garden and water stress manipulative experiments with neutral and adaptive molecular trait variation using genome-wide scans. We tested the hypothesis that adaptive differences have accumulated between

et al., 2017). Therefore, for species inhabiting variable precipitation regimes, water stress can favor local genotypes adapted to drought that can best respond to the present and future environmental conditions, making them vital for the survival of species (Gitlin et al., 2006). Given that in order to persist and thrive projected climate changes plants will need to undergo rapid adjustments in their physiology and morphology (Nicotra et al., 2010) common garden and environmental stress manipulation experiments provide a valuable tool for assessing adaptive and/or plastic responses of populations (Horváth and Mátyás, 2016). Genetic impact of responses to environmental stress has been widely acknowledged (McDowell et al., 2008) and genetically diverse longlived trees are expected to be the most resilient species to future droughts if genetic factors are important to survival (Hamrick, 2004). Yet intraspecific differences and the mechanisms under selection are not clear. Droughts can be moderate or extreme, chronic or acute, recurrent or sporadic and responses of species to such events will vary accordingly also in relation to the moment and the speed with which they occur (Valladares et al., 2004) and to the genetic background of the populations under stress. Plant species have developed drought stress response mechanisms at the morphological, anatomical and cellular levels (Chaves et al., 2003; Shinozaki and Yamaguchi-Shinozaki, 2007). Drought can produce an instantaneous shortage of water at the complete plant level so that supply falls below the normal range of variability altering leaf traits and whole plant architecture, which are translated into lower height, reduced leaf size, and smaller number of leaves (Ciríaco da Silva et al., 2013). Specific leaf area (SLA) as the ratio of leaf area to leaf dry mass, and thus refers to carbon gain relative to water loss, tend to decline towards lower rainfall (Fonseca et al., 2000). In addition, drought tolerance has been associated to reduced stomatal density under water stress (Bertolino et al., 2019). Water deficit reduces leaf cell turgor, restricting cell expansion and canopy area development, which can negatively affect biomass accumulation (Chaves et al., 2003). Chronic and prolonged water stress produces tree mortality by cavitation (i.e., desiccation due to an obstruction by the presence of air in the xylem- embolism) and carbon starvation (when the acquisition and mobilization of carbon stocks do not meet the needs of metabolic maintenance) that have been recognized as factors that promote this widespread phenomenon (McDowell et al., 2008). The water potential (state of water in the plant) is characterized by having elevated values at predawn and lower at midday, when the imbalance between perspiration and absorption is maximum. At night, when perspiration decreases considerably, diurnal water deficits are gradually eliminated, and the water potential of the plant reaches a balance with the water potential of the soil (SánchezDíaz and Aguirreolea, 2013). The extent of this trend increases with the degree of drought in the soil. Species can develop two opposite types of behavior to regulate water potential: isohydric control, with strict stomata closure mechanism (avoidance of drought-induced hydraulic failure), or anisohydric in which stomatal closure is less stringent (relatively drought-tolerant), or even intermediate situations. While isohydric responses may result in carbon starvation, anisohydric behavior predisposes plants to hydraulic failure (McDowell et al., 2008). World’s forested ecosystems are already responding to climate change, and concern is raised that forests may be increasingly vulnerable to massive mortality, in response to heating and drought, even in environments that are not normally considered water-limited (Allen et al., 2010, 2015). For example, in the period 1998–99, Patagonia was affected by one of the most severe droughts, coinciding with a strong La Niña climatic event, which caused massive mortality in populations of the evergreen tree Nothofagus dombeyi, the dominant species of low to mid-elevation mesic forests of Northern Patagonia (Suarez et al., 2004). The opening of clearings produced by the mortality of trees during the 1998–99 droughts favored the recruitment and survival of seedlings of coniferous Austrocedrus chilensis in opposition to N. dombeyi, altering the forest structure (Suarez and Kitzberger, 2008). While the critical 2

Forest Ecology and Management 461 (2020) 117931

D.G. Diaz, et al.

Fig. 1. (a) Distribution range of Nothofagus dombeyi in Chile and Argentina (in green). Study areas (red points) are located in mesic (west) and dry (east) environments in northern Patagonia, Argentina (b) Seedling and leaf samples for molecular analyses were collected at mesic sites A, Rio Melgarejo and B, Casa del Lago; and populations of dry environments at C and D, Estancia Chacabuco Zone 1, and Zone 2, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

distribution, in the area of the confluence of the rivers Traful and Limay, Neuquén Province, Argentina (40° 43′ 29″ S, 71° 03′ 09″ W and 40° 42′ 49″ S, 71° 05′ 54″ W) (Fig. 1b). The vegetation along this precipitation gradient changes from temperate mesic forest, with the canopy dominated by nearly pure and dense N. dombeyi with an understory of the bamboo Chusquea culeou, to open drylands, where scattered N. dombeyi are only found in riparian environments surrounded by open woodlands of the conifer Austrocedrus chilensis within a matrix of xerophytic shrubs and graminoid-dominated semi-arid steppe. Soils of the study area are mainly Andisols of volcanic origin although at the mesic site N. dombeyi grows in soils with greater accumulation of organic matter while towards the steppe is found in alluvial soils (Donoso et al., 2006). The climate of the study region is Mediterranean with winter rainfalls and summer droughts. The mean annual temperature for the mesic site is 7.7 °C with total annual rainfall of 1000 mm, and average spring precipitation of 190 mm; while in the dry site the annual mean temperature is 8.8° C with total annual rainfall of 800 mm, and average spring precipitation of 130 mm (Fig. S1; WorldClim v.2; Fick and Hijmans, 2017). However, precipitation was not similarly distributed throughout the year in both sites. The smallest differences occur during the fall (i.e. May) whereas the greatest ones are from August to October. Also, the mesic sites always received more precipitation except in June (Fig. S1).

provenances of N. dombeyi from the eastern Andes under subtle climatic and site differences as a result of diversifying selection in the face of gene flow. Under optimal and homogeneous growing conditions in common gardens N. dombeyi from distinct provenances will show genetic differences in quantitative whole plant and foliar characters, and molecular markers. Thus, N. dombeyi that inhabit mesic forests will respond better to optimal conditions during cultivation whereas those from dry environments will show ecophysiological traits associated to water shortage. 2. Materials and methods 2.1. Study species Nothofagus dombeyi (Mirb.) Oerst. common name “coihue” is an evergreen monoecious tree with small lanceolate leaves that reaches 40 m or more in height, with wind-borne pollen and seeds (Donoso et al., 2006). It has a wide latitudinal distribution between 34° 37′ and 47° 30′ south latitude in Chile and Argentina (Fig. 1a) (Donoso et al., 2004). It inhabits a great variety of climates although on the eastern Andes is most commonly found under mesic and relatively low-to midelevation forests. Nothofagus dombeyi endures low temperatures (Premoli, 1994) and possesses a wide range of protoplasmic resistance to dryness (Weinberger, 1973), mainly in comparison with other species of the Valdivian rainforest as N. nitida (Piper et al., 2007). Under Mediterranean climates of the eastern slopes of the Andes in Argentina or at the northern limit of the temperate forest in Chile the presence of N. dombeyi is scarce and limited to riparian environments, in which the water from the soil is not limiting at any period of the year, such as at the banks of watercourses, creeks, etc. (Donoso et al., 2004).

2.3. Sampling of natural populations and cultivation During February 2016, seedlings that were recently germinated under natural conditions were collected at the selected sites. Seedlings were harvested instead of seeds following Premoli et al. (2007) since germination rates of N. dombeyi under experimental conditions are very low (Donoso et al., 2006). Seedlings were selected based on the presence of cotyledons, and therefore they belonged to the same cohort. Measurements taken at the beginning of the common garden experiment showed that plants from distinct provenances were the same size (see below Fig. 3), thus maternal effects during early cultivation were probably nil. In order to minimize transplant shock and given the small size of seedlings (< 3 cm long) they were extracted carefully keeping the root ball intact without shaking out the natural soil. They were placed individually in 0.35 L pots, properly tagged and identified by site and were cultivated using a substrate consisting of a mixture of peat and their native forest soil. Initial survival of seedlings from mesic and

2.2. Study area Populations of N. dombeyi were selected based on contrasting site characteristics including distinct precipitation. Two study sites were selected at a mesic forest, on the west margin of Gutierrez Lake, 17 km south of Bariloche city, Rio Negro Province, Argentina (41° 24′ 12″ S, 71° 42′ 75″ W and 41° 22′ 12′' S, 71° 40′ 78″ W). At these mesic locations N. dombeyi suffered from massive mortality (Suarez et al., 2004) under the extreme drought recorded during the summer of 1999. The other two study sites were located at the driest extreme of the species’ 3

Forest Ecology and Management 461 (2020) 117931

D.G. Diaz, et al.

Fig. 3. Temporal variation of plant and leaf quantitative features of Nothofagus dombeyi seedlings from distinct provenances that were grown in common gardens: (a) plant height, (b) total number of leaves, and (c) total number of branches.

Nacional del Comahue), located at similar elevation than seedling source sites. Throughout the experiment seedlings were replicated to larger pots twice, reaching a final volume of 6 L.

Fig. 2. (a) Survival, (b) basal diameter, (c) stomatal density in the abaxial face of seedlings of Nothofagus dombeyi from contrasting precipitation regimes growing under common gardens. Different letters indicate significant differences P < 0.01, t test.

2.4. Measurements under common garden dry provenances was c. 100% respectively (Fig. S2) which discards the possibility for transplant shock and most mortality occurred during the first year under cultivation (see results below). Seedlings were grown with a randomized arrangement in a naturally lighted greenhouse of the Instituto de Investigaciones en Biodiversidad y Medio Ambiente in Bariloche (INIBIOMA- Universidad

Potentially genetic-based differences were monitored during the extent of the experiment in response to optimal growing conditions, i.e. common garden. Plant mortality was recorded as the number of alive (1) or dead (0) plants, from the total number of seedlings collected in the field from mesic (N = 189) and dry (N = 328) sites until the end of 4

Forest Ecology and Management 461 (2020) 117931

D.G. Diaz, et al.

Laboratory of the Argentinean Institute of Agronimical Technology (INTA) in Bariloche. Mean temperature (°C) and relative humidity (%) were calculated based on daily measurements taken in the common garden using data loggers (HOBO Onset). At the end of the water deficit treatment, rate of survival and the maximum water potential (at predawn) from 6:00 h to 8:00 h and minimum (at midday) from 12:00 h to 14:00 h was recorded in five randomly selected branches of each plant by treatment/origin using a pressure chamber (PMS 1000, PMS Instruments, Corvallis, Oregon, USA). Finally, the stomatal density was measured by counting the number of stomata on the abaxial surface of five randomly selected leaves from 10 plants of each site using transparent enamel following Premoli and Brewer (2007). Then, the stomatal density was measured based on the number of stomata observed using an increase of 400X (diameter = 0.05 mm) under an optical microscope (Olympus BH2) in an area of 0.019 mm2, equivalent to the diameter of the observation field. 2.6. Molecular analysis

Fig. 4. Relative growth rate (RGR) in relation to the initial height ln(Hi) of Nothofagus dombeyi seedlings from mesic (filled circles) and dry (empty circles) origins (each regression P < 0.02) and slope test for the comparison between regression lines P < 0.01.

Genetic differences among seedlings of different origins were analyzed at the molecular level using Single Nucleotide Polymorphisms (SNPs). The SNPs technique consists of genetic markers that show variations in DNA sequences that affects a single base of short DNA fragments throughout the entire genome. High quality and quantity (~1000 bp) DNA was extracted from foliar tissue of 10 randomly selected individuals per provenance that were subjected to the water stress experiment. We followed the DNA extraction protocol of Novaes et al. (2009) with the following modifications: ATMAB (Sigma M7635100 g) was used instead of CTAB, the pre-incubation wash was added to water soluble polymer polyvinylpyrrolidone PVP (Sigma P5288-500 g, for molecular biology) and a reducing agent Dithiothreitol DTT (BioRad), incubated 30 min at 65 °C and 30 min on ice, and after allowing to stand overnight at room temperature, 1 μL of RNase (Machere and Nagel Laboratories) was added and incubated for 30 min at 37 °C. The integrity of genomic DNA was evaluated by 1% agarose gel and quantified using a Qubit fluorometer (Invitrogen, USA). Library preparation and high-throughput sequencing were performed following Hasbún et al. (2016) in the Biotechnology Center of University of Wisconsin, Madison, USA (DNA Sequence Facility). High-throughput sequencing was conducted on an Illumina HiSeq 2000 (Illumina, USA) using 100 bp single-end sequencing runs. The samples were sequenced across one Illumina lane. Base calling was performed in Casava v1.8.2 (Illumina, USA).

the experiment. Plant architectural traits were measured on 16 randomly selected seedlings of each provenance that were monitored together with mortality at the starting date and thereafter at 14, 20 and 21 months of cultivation in the common garden. Variables measured were: stem length (or height), number of branches, total number of leaves, and the basal diameter with a digital electronic caliper 723Z-6/ 150MM (Starrett, USA). Leaf characteristics were measured at the end of the experiment on five randomly selected leaves from each of the same seedlings on which we measured architectural variables. Leaves were scanned to measure leaf area, perimeter, length, width and roundness using the program Digimizer v 4.6.1. (MedCalc Software, 2015). To quantify dry biomass leaves were dried in an oven for 48 h at 65 °C and weighted using a digital balance (Mettler AJ150) with precision of 0.0001 g.

2.5. Water stress experiment In November 2017, the same 32 seedlings (16 of each site) that were used to measure architectural and foliar traits were subjected to a water deficit experiment for a period of three weeks following Varela et al. (2010). The experiment consisted in the suspension of irrigation on eight seedlings chosen at random from each provenance (drought treatment), while the remaining ones were kept under irrigation, in conditions close to field capacity (FC) of the soil (control treatment). Soil moisture was determined as the volumetric water content (VWC = % vol. / vol.) and was recorded every two or three days in three locations within the pot, using a digital TDR (Time Domain Reflectrometry, ProCheck from Decagon Devices, Inc.). The experiment was conducted in 6 L pots which were periodically rotated to avoid distinct microclimates in the greenhouse. To determine FC and permanent wilting point (PWP) of the soil, a soil water retention curve (−0.03, −0.1, −0.15, −0.7 and −1.5 MPa) was developed in the Soil

2.7. Data analysis Growth performance measured at the plant level during cultivation in common garden (height, number of branches, total number of leaves, and basal diameter) was evaluated by repeated measures analysis of variance (ANOVA) which were performed on the same seedlings at four time stages (February 2016 and April, October, and November 2017). Percent survival, basal diameter, and foliar characteristics between provenances were evaluated at the end of the experiment, by t tests. To test for potential effects of the initial size at plant collection (Hi) on total shoot length of last measurement in November 2017 (Hf) we

Table 1 Mean ( ± SD) leaf characteristics of Nothofagus dombeyi seedlings from mesic and dry sites, maintained in common garden conditions. Source

Dry biomass (g)

Area (cm2)

SLA (cm2/g)

Perimeter (cm)

Length (cm)

Width (cm)

Roundness (cm)

Mesic

0,0219a ( ± 0,0048) 0,0157b ( ± 0,0056)

2,5371a ( ± 0,6421) 1,8403b ( ± 0,6903)

118,4899a ( ± 14,9330) 121,1668a ( ± 20,6347)

8,8621a ( ± 1,1783) 7,5957b ( ± 1,6547)

2,6456a ( ± 0,3250) 2,2292b ( ± 0,4663)

1,4329a ( ± 0,1936) 1,1965b ( ± 0,2375)

0,3908a ( ± 0,0337) 0,3732a ( ± 0,0496)

Dry

SLA: Specific leaf area. Different letters indicate significant differences P < 0.05 t test. 5

Forest Ecology and Management 461 (2020) 117931

D.G. Diaz, et al.

(Hi) (initial shoot length) (Zar, 1996). Between-site differences in leaf characteristics (biomass, SLA, leaf area, perimeter, length, width, roundness) were analyzed by t tests. The effect of the water stress experiment on water potential was assessed by generalized linear models (GLM), and paired t tests were used to evaluate differences in predawn and midday water potentials of each plant, provenance, and treatment. Also, a slope test was performed for the regression of the predawn water potential against midday values according to the site of origin under drought. Stomatal density differences among treatments were analyzed by t tests. All analyses were performed with STATISTICA v 7.0 StatSoft, Inc. (2004). The detection of SNPs loci and alleles were analyzed following the methodology used in N. dombeyi by Hasbún et al. (2016) with UNEAK (Universal Network Enabled Analysis Kit) which is part of the software package TASSEL 5.2.43 (Glaubitz et al., 2014). This was used since it does not require a reference sequence, as is the case of N. dombeyi. The network filter trimmed reads to 64 bp to reduce the effects of error sequencing and enabled efficient storage of data in bit format (Hasbún et al., 2016). The genotypes of SNPs were configured using the enzyme APeKI, with the error parameters of 3%, a minimum number of labels of two and the minimum frequency of alleles of zero. Then two functions of TASSEL 5.2.43 (filters per loci and individual) were applied that eliminated all the individuals and markers of SNPs with 90% or more of values where no alleles were distinguished. The identification of loci under selection was carried out through the program BayeScan (Foll and Gaggiotti, 2008). We used 5299 loci and the following detection parameters: 10 intervals with a sample size of 5000; 50,000 of 100,000 resulting iterations were ruled out. The loci considered adaptive (outliers) were those with FST > 0.05. We calculated different parameters of genetic variation: the observed and expected heterozygosis under the Hardy-Weinberg equilibrium condition which was compared by paired t tests. In addition, the number of private genotypes, i.e. those that are only present at one location, were quantified for each origin. The divergence among populations was analyzed by FST using Monte-Carlo tests and by principal component analysis (PCA) using hierFstat (Goudet, 2005) and adegenet (Jombart, 2008; Jombart and Ahmed, 2011) which are R packages for the estimation of hierarchical F-statistics and multivariate analysis of genetic markers in RStudio (RStudio Team, 2015). We searched for associations between different climatic and phenotypic variables with adaptive SNPs markers to explain population differences using a mixed linear models (MLM) with TASSEL 5.2.43. Finally, using the BLAST function we searched in the Genbank database (Benson et al., 2013) if adaptive SNPs had some homologous sequences with genes of known functions. Degree of between-site divergence in quantitative characters was measured by QST which is calculated as QST = VE / 2VD + VE; where VE represents the variance among populations, and VD the variance within populations according to Merilä and Crnokrak (2001). The comparison of QST with FST as the degree of genetic differentiation could be used to infer the degree of local adaptation: QST > FST would be favoring different phenotypes in different populations and thus provides evidence of diversifying selection and local adaptation; QST = FST, the degree of differentiation is due by genetic drift; and QST < FST is associated with stabilizing selection that favors the same phenotype in different populations (Merilä and Crnokrak, 2001; McKay and Latta, 2002; Lamy et al., 2012).

Fig. 5. Predawn (a) and midday (b) water potential (Ψ) in relation to VWC of Nothofagus dombeyi seedlings from contrasting precipitation regimes under control (CC = 21.5%) and drought (9.2–6.7%) treatments. Similar letters show non-statistical differences between the water potentials and treatments (using generalized linear models). (c) Regression of midday water potential (Ψmd) against predawn water potential (Ψpd) of each site under drought treatment. The slopes were statistically similar but the intercepts differed significantly between sites (P < 0.001 slope test).

3. Results 3.1. Measurements in common garden At the end of the common garden experiment, plants from the dry site had significantly higher mortality (c. 90%) than those of the mesic site (c. 80%) (t = 6.04; P < 0.001; Fig. 2a), which particularly occurred during the first year of cultivation and was similarly maintained thereof (Fig. S2). Seedlings from the mesic site attained greater final

calculated the relative growth rate as RGR = ln(Hf/Hi) and regressed this value against ln(Hi) for mesic and dry plants, respectively following Premoli et al. (2007). Differences in RGR between provenances were evaluated by slope tests by testing the relationship between RGR and ln 6

Forest Ecology and Management 461 (2020) 117931

D.G. Diaz, et al.

Fig. 6. Frequency of observed (Ho) and expected (He) heterozygosity for (a) 5197 loci of neutral SNPs and (b) 102 loci of adaptive SNPs separated by origin of the seedlings of Nothofagus dombeyi. Different letters indicate significant differences (P < 0.05) paired t test by locus.

plant that significantly varied over time (Fig. 3). Similarly, RGR of seedlings from the mesic site was significantly greater than that of plants from the dry site (F = 5.52; P = 0.004) and the difference in growth was also significant over time (test of slopes of RGR of last measurement in relation to the initial height of the seedlings F = 8.13; P = 0.002; Fig. 4). Plants of the mesic provenance also attained greater values for most leaf size traits (P < 0.05; Table 1) and stomatal density (t = 3.56; P = 0.00223; Fig. 2c); while the roundness and SLA variables did not present significant differences between origins (P > 0.05; Table 1). Plant height, basal diameter and number of leaves of seedlings from the mesic site attained higher values (P < 0.05) than those from the dry provenance which in turn yielded a smaller standard deviation for such morphological variables (Fig. S3).

3.2. Water stress experiment The average values ( ± SD) of the VWC during the drought experiment were 18.3 ± 4.0% for seedlings under control treatment (FC = 21.5%) and 12.8 ± 3.7% for seedlings under water stress (PWP = 13.8%). After 9 days, the VWC was lower than the PWP (Fig. S4). The average temperature of the common garden throughout the experiment was 16.5 ± 2.6 °C and the mean relative humidity 56 ± 12%. The survival rate was 100% for plants from different origins during the water stress experiment. The predawn water potential showed similar values independently of the VWC (drought and control) and the site of origin (Fig. 5a). At midday, a considerable decrease in water potential was observed in relation to predawn, but no statistically significant differences in drought and control treatments were detected (Fig. 5b). Nonetheless, under the drought treatment the slope test yielded between-site significant differences for the intercept (F = 37.88; P = 0.0008) (Fig. 5c). Also, seedlings of the mesic site had higher density of stomata per observation field area under an optical microscope than those of the dry site (t = 3.56; P = 0.002; Fig. 2c).

Fig. 7. Percentage of private genotypes in 102 loci of adaptive SNPs in seedlings of different provenances of Nothofagus dombeyi.

Table 2 Total inbreeding (FIT), within (FIS) and between (FST) the mesic and dry sites of Nothofagus dombeyi with significant neutral and adaptive SNPs (P = 0.01) for 99 aftershocks. Loci

FIT

FIS

FST

Neutral Adaptive

−0,017 0,573

−0,058 0,174

0,039 0,483

basal diameter (t = 3.14; P = 0.0037; Fig. 2b) and overall growth as indicated through repeated measures ANOVA at the individual level for total plant height (F = 4.34; P = 0.008), number of branches (F = 5.14; P = 0.006), and number of leaves (F = 4.82; P = 0.005) per

Table 3 List of SNPs outlier loci of Nothofagus dombeyi as indicators of adaptation in significant association with quantitative plant features and leaf characters. SNP

Adaptive loci

Characteristics Stomatal density

TP43864 TP14572 TP50194 TP14566 TP47454

“loc059” “loc021” “loc073” “loc020” “loc066”

Leaf area

# Leaves

# Branches

* *

* *

***

***

# Number. * p < 0.05 and *** p < 0.001 mixed linear model. 7

*** *

Height

*

Forest Ecology and Management 461 (2020) 117931

D.G. Diaz, et al.

N. dombeyi are expected to have more branches distributed on top of canopy in closed-mesic forests whereas a more random distribution of branches along the entire trunk is to be found in open dry forests. Due to the small size of seedlings we only recorded plant size traits (height and total number of leaves and branches). Yet, plants from the eastern range that have long withstood dry climates were either not responsive or inhibited in terms of growth and survival under optimal irrigation in the common garden. Best seedling performance of a relatively mesic population at its southernmost range of the deciduous Nothofagus pumilio was obtained under a low soil moisture level, 40–60% soil capacity (Martínez Pastur et al., 2011). Thus more studies are needed to disentangle the effects of variable irrigation levels on N. dombeyi. However, the genomic evidence that from the total adaptive SNPs 1/3 were unique to either the dry and mesic end of the distribution, reinforces the impact of genetic factors differentially affecting the fitness of N. dombeyi under distinct, but subtle, precipitation regimes. Tree species as Norway spruce showed high vulnerability to extreme heat and drought events and a quantitative and genomic analysis revealed high genetic variation among provenances some of them located at the species’ warmest end (Trujillo-Moya et al., 2018). Therefore, populations located at the margins of the species range will likely be more effectively responsive to the increase in frequency and severity of predicted water stress events, because these populations carry a greater spectrum of unique potentially drought-tolerant alleles and genotypes. Although in our case both locations showed similar genetic diversity levels, the dry site yielded lower observed heterozygosity than expected for adaptive loci which may reflect the effect of directional selection towards particular genotypes. Distinct cpDNA haplotypes found at each location indicate that the populations have diverged in the past, and that these differences have managed to persist over time despite the homogenizing effects of gene flow via pollen as shown by non-significantly different from zero FST values measured by biparental neutral markers as microsatellites and isozymes (Diaz, 2018). These results suggest that according to the historical context of the seedlings of N. dombeyi have accumulated molecular and genetically-based morphological differences that are maintained by selection. Individuals subjected to water stress survived under the imposed conditions. This could be due to the fact that the induced water stress of the experiment was low to moderate, since, in the natural environment during the summer (i.e. dry season), the lack of water could exceed the three week period (21 days of the water stress experiment) and the maximum potentials could reach more negative values. For example, between October 1998 and February 1999, strong La Niña conditions resulted in that only 17.7% of the average precipitation was recorded for the study area (Bran et al., 2001) that caused one of the most massive forest diebacks recorded at mesic locations (Suarez et al., 2004). All seedlings from the humid-most extreme of the distribution of N. dombeyi in Chile that were subjected to a gradual drought survived at water potentials of −2.7 MPa and all died at −3.1 MPa (Piper et al., 2007). Therefore N. dombeyi seedlings probably need a much more severe drought than the one applied here to show mortality. While the relevance of water stress treatments imposed on seedlings that were greenhouse-grown to natural droughts that occur in the field is under debate, yet similar ecophysiological patterns were obtained in common garden grown and adult Nothofagus plants from contrasting environmental conditions (Premoli and Brewer, 2007). The larger leaf area and biomass, and greater stomatal density of mesic populations could suggest greater water loss by transpiration and possibly higher photosynthesis by accumulating more biomass as previously documented in mesic N. dombeyi under drought (Sanhueza et al., 2015). As has already been demonstrated in Olea europaea, plants can also reduce water deficits to avoid loss of turgor through changes in leaf morphology and reducing total leaf area (Connor and Fereres, 2005), similar to the dry N. dombeyi population strategy. This would be important, given that the morphology of the leaves of the dry populations could mean an adaptive advantage under stress because, along

3.3. Molecular characteristics The results of SNPs, after filtering by sites and individuals, consisted of a total of 5299 markers data set, 102 of them resulted as potentially adaptive (outlier loci) leaving 5197 neutral loci. Out of those, 55% adaptive and 17.5% neutral presented significant deviations from the of Hardy-Weinberg equilibrium condition. The observed and expected heterozygosity for neutral loci showed no significant differences (P > 0.05; Fig. 6a), whereas significant differences were found in adaptive SNPs (t = 2.5064; P = 0.007), with heterozygous deficit in the dry site (Fig. 6b). We found similar proportion of private genotypes for the two provenances, being c. 30% of the total genotypes unique to each of them (Fig. 7). Inbreeding coefficients for adaptive SNPs were an order of magnitude greater than neutral ones particularly for the degree of betweensite divergence FST (Table 2) and these differences were significant (P = 0.01). The PCA for neutral markers showed low differentiation between origins, with greater overlap of individuals (Fig. S5a), while markers under selection showed two well differentiated groups corresponding to the mesic and dry sites, respectively (Fig. S5b). We found seven and 43 loci of adaptive SNPs that had a genetic load ≥ 0.01 and ≥ 0.005 in the first principal component (PC1) respectively, thus confirming their contribution to the population structure (Figs. S6 and S7). When performing the BLAST analysis, we found two adaptive SNP loci with high genetic load encoding for expansin (TP57866 = “loc 82″) and argonaute10 (TP46284 = ”loc 64″) which are proteins involved in the expansion of the cell wall and apical meristems of the root, respectively. The MLM analysis carried out with TASSEL did not detect significant associations between bioclimatic and genotypic characteristics, but it was significant for plant architectural features and quantitative leaf variables in five SNPs loci under selection (Table 3). Degree of between-site divergence in quantitative characters yielded a value of QST = 0.42, similar to the divergence measured by adaptive SNPs (FST = 0.483) which in turn was an order of magnitude greater than that calculated by neutral SNPs (FST = 0.039) that suggested no restrictions to gene flow. 4. Discussion Plants of N. dombeyi from mesic and dry sites differed in their phenotype and growth patterns during cultivation in common gardens and those differences are genetic. These features were selected and maintained in the long-term as a result of environmental differences despite continuous gene flow as indicated by reduced FST values of genetic divergence measured by neutral molecular markers. Betweensite differences for quantitative traits portray the action of disruptive selection (Fig. S3) which is reinforced by high FST values for adaptive molecular markers that were an order of magnitude greater than for neutral ones. The presence of unique genetic variants restricted to either mesic or dry areas show the action of diversifying selection such that distinct genotypes are locally adapted. Despite being geographically close, only 67 km away and without restriction for natural gene flow, their whole plant features and leaf morphological characteristics are different. This indicates the existence of two ecotypes of N. dombeyi and that climatically different conditions prompt local adaptations. Unexpectedly, plants from the dry extreme growing under optimum conditions in the common garden suffered increased mortality and decreased growth. While several dendrochronological studies analyzed impacted-by-drought N. dombeyi forests, eastern, i.e. driest-most, stands are studied here for the first time. Provenances considered mesic in our study, suffered standing‐dead tree mortality following drought/heat events where successful establishment was related to wet periods, thus showing the dependence of N. dombeyi on favorable conditions for growth (Suarez and Kitzberger, 2010). Under optimal settings mesic plants outgrew those from the dry provenance. Heliophillous species as 8

Forest Ecology and Management 461 (2020) 117931

D.G. Diaz, et al.

recently particularly in relation to which genotypes are most likely to be effective at particular locations. However, as environmental change accelerates, exceptions to ‘local is best’ may increase and may be better thought of as a testable hypothesis rather than as a general assumption (Jones, 2013). Therefore, seed provenances can be selected based on their expected improved performance over local sources under particular projected climates a strategy called ‘climate-adjusted’ provenancing (Prober et al., 2015).

with the growth form of these plants (slow increment in height, basal diameter, lower number of branches, leaves, and stomatal density), would provide a favorable habit for such environments. Although seedlings under water stress from both sites had a nearly similar behavior, those from the mesic site maintained more variable values of midday water potentials for distinct predawn water potentials (Fig. 5c) and thus they seem to be slightly more anisohydric in comparison to the isohydric-type of seedlings from the dry site. This is similar to that found in the deciduous Nothofagus pumilio inhabiting steep precipitation gradients in northern Patagonia where differences between predawn and midday water potentials were lower in seedlings from dry areas (Ignazi et al., 2020). Nonetheless, those differences in water potentials of N. dombeyi measured here are subtle as are the climatic envelopes of the study sites. The loci under selection reported here might be of great importance since they could correspond to regions of the genome that would facilitate the survival of N. dombeyi in different environments. For example, two SNPs with high genetic load that contribute to the separation of the provenances show a high resemblance to proteins involved in the expansion of the cell wall (TP57866) and regulation of apical meristems (TP46284). The first are closely linked to the cell wall and their activity is typically analyzed by stimulating the extension of the cell wall and stress relaxation (Sampedro and Cosgrove, 2005). In Zea mays, the flexibility of the cell wall and the maintenance of foliar growth in response to water stress are the results of the expression of the expansin gene in the leaves (Sabirzhanova et al., 2005). The second type of argonaute10 proteins are related to the growth of the apical meristems of the root (Zhu et al., 2011), which are very important increasing the surface of water uptake in the soil, especially in sites of low water availability. The absence of significant associations between genotypes and climatic variables using MLM may be due to (1) the architecture of polygenic traits, with many small loci but of additive effect and/or (2) by a strong population structure, in consonance with the association of drought survival to historically differentiated as distinct cpDNA groups of haplotypes (Exposito-Alonso et al., 2017). However, five adaptive SNPs (4.9%) yielded a significant relationship with architectural and foliar characteristics. Drought-related phenotypes were associated with SNPs in Phaseolus vulgaris (Villordo-Pineda et al., 2015). Natural selection was supposed to have sufficient time to maintain a functional relationship between the allele frequencies and architectural traits of seedlings (Plomion et al., 2016) such as relatively lower growth and leaf area that will confer an adaptive advantage in dry environments. Thus, constant water stress can favor certain genotypes able to respond better to the present and future environmental conditions, making them vital for the long term survival of the species. Nonetheless, this will occur at the expenses of mesic forests being further vulnerable under scenarios of more frequent drought events. The key for maintaining resilient plant populations that are dominant in certain ecosystems will be to preserve areas with genetically diverse individuals along environmental gradients, including sites that are currently under stress (Gitlin et al., 2006). Ideally, the greater the amplitude of the environmental variation is represented e.g. both populations located at the center and the periphery of the total species’ range, the greater the probability of populations will adapt to future environmental challenges (Araújo, 2002). In particular, populations in the distribution margins of a species may contain particular genetic characteristics due to isolation and novel environmental conditions, hence producing genetic divergence. Because geographic differentiation can lead to genetic adaptation for specific conditions, genes for example for stress tolerance can be selected in plant populations that grow in chronically stress conditions. This is in contrast to populations less exposed to limiting resources where genotypes associated to tolerate them are purged by stabilizing selection. As a result, those populations may be more vulnerable to changing scenarios. Seed provenance to be used in restoration efforts have been debated

5. Conclusions Our results show adaptive adjustments to subtle precipitation differences in the face of continuous gene flow. The morphology of individuals from drier populations would be providing a favorable growth habit, which could mean an adaptive advantage in places where the growing conditions are extreme. On the other hand, while under optimum conditions plants from the mesic site outperformed those from the dry extreme in terms of overall plant and leaf sizes, they potentially be in disadvantage under drought due to greater stomatal density and a slight tendency for an anisohydric behavior favoring continue carbon gain but maintaining open stomata at greater risk of cavitation that might trigger tree mortality. Thus, genetically distinct, i.e. dry and mesic, ecotypes of N. dombeyi are selected under different, yet subtle, precipitation regimes. In the face of predicted increase in the frequency and severity of droughts in Patagonia restoration efforts should be climate-oriented such as the use of experimental designs including “admixture” provenancing. Our study highlights the relevance to perform intraspecific trait variation studies under even slightly distinct climates. Author Statement Contributions to the paper were as follows: DGD, PM, ACP field work; DGD, PM laboratory and data analysis; ACP original ideas; DGD, PM, ACP contributed to writing. 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 We are grateful to M. Fasanella and R. Hasbún for help in the laboratory and/or with data analysis. The authors thank the University of Wisconsin Biotechnology Center DNA Sequencing Facility for providing sequencing and support services. We thank Administración Nacional de Parques Nacionales for extending permits to work within protected areas. This research was funded by Agencia de Promoción Científica y Tecnológica PICT-2015-1565 from Argentina and Rufford Foundation, United Kingdom project 29211-1. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foreco.2020.117931. References Aitken, S.N., Yeaman, S., Holliday, J.A., Wang, T., Curtis-McLane, S., 2008. Adaptation, migration or extirpation: climate change outcomes for tree populations. Evol. Appl. 1, 95–111. Allen, C.D., Macalady, A.K., Chenchouni, H., Bachelet, D., McDowell, N., Vennetier, M., Kitzberger, T., Rigling, A., Breshears, D.D., Hogg, E.H., Gonzalez, P., Fensham, R., Zhangm, Z., Castro, J., Demidova, N., Lim, J., Allar, G., Running, S.W., Semerci, A., Gonzalez, P., 2010. A global overview of drought and heat-induced tree mortality reveals emerging climate change risks for forests. For. Ecol. Manage. 259, 660–684. Allen, C.D., Breshears, D.D., McDowell, N.G., 2015. On underestimation of global vulnerability to tree mortality and forest dieoff from hotter drought in the Anthropocene. Ecosphere 6, 1–55.

9

Forest Ecology and Management 461 (2020) 117931

D.G. Diaz, et al.

(Eds.), Contribución del Grupo de trabajo II al Quinto Informe de Evaluación del Grupo Intergubernamental de Expertos sobre el Cambio Climático. Organización Meteorológica Mundial, Ginebra, Suiza. Jombart, T., 2008. adegenet: a R package for the multivariate analysis of genetic markers. Bioinformatics 24, 1403–1405. Jombart, T., Ahmed, I., 2011. adegenet 1.3-1: new tools for the analysis of genome-wide SNP data. Bioinformatics 27 (21), 3070–3071. Jones, T.A., 2013. When local isn’t best. Evol. Appl. 6, 1109–1118. Jump, A.S., Mátyás, C., Peñuelas, J., 2009. The altitude-for-latitude disparity in the range retractions of woody species. Trends Ecol. Evol. 24, 694–701. Lamy, J.B., Plomion, C., Kremer, A., Delzon, S., 2012. QST < FST as a signature of canalization. Mol. Ecol. 21, 5646–5655. Maréchaux, I., Bartlett, M.K., Sack, L., Baraloto, C., Engel, J., Joetzjer, E., Chave, J., 2015. Drought tolerance as predicted by leaf water potential at turgor loss point varies strongly across species within an Amazonian forest. Funct. Ecol. 29, 1268–1277. Martínez Pastur, G.J., Lencinas, M.V., Esteban, R.S., Ivancich, H., Peri, P.L., Moretto, A., Hernández, L., Lindstrom, I., 2011. Plasticidad ecofisiológica de plántulas de Nothofagus pumilio frente a combinaciones de niveles de luz y humedad en el suelo. Ecología Austral 21, 301–315. McDowell, N., Pockman, W.T., Allen, C.D., Breshears, D.D., Cobb, N., Kolb, T., Plaut, J., Sperry, J., West, A., Williams, D.G., Yepez, E.A., 2008. Mechanisms of plant survival and mortality during drought: why do some plants survive while others succumb to drought? New Phytol. 178, 719–739. McKay, J.K., Latta, R.G., 2002. Adaptive population divergence: markers, QTL and traits. Trends Ecol. Evol. 17, 285–291. MedCalc Software, 2015. Digimizer (imagen analysis sofware) version 4.6.1. www. digimizer.com. Merilä, J., Crnokrak, P., 2001. Comparison of genetic differentiation at marker loci and quantitative traits. J. Evol. Biol. 14, 892–903. Nicotra, A.B., Atkin, O.K., Bonser, S.P., Davidson, A.M., Finnegan, E.J., Mathesius, U., Poot, P., Purugganan, M.D., Richards, C.L., Valladares, F., van Kleunen, M., 2010. Plant phenotypic plasticity in a changing climate. Trends Plant Sci. 15, 684–692. Novaes, R.M.L., Rodrigues, J.G., Lovato, M.B., 2009. An efficient protocol for tissue sampling and DNA isolation from the stem bark of Leguminosae trees. Genet. Mol. Res. 8, 86–96. Piper, F.I., Corcuera, L.J., Alberdi, M., Lusk, C., 2007. Differential photosynthetic and survival responses to soil drought in two evergreen Nothofagus species. Ann. For. Sci. 64, 447–452. Plomion, C., Bartholomé, J., Bouffier, L., Brendel, O., Cochard, H., De Miguel, M., Delzon, S., Gion, J.M., González-Martínez, S.C., et al., 2016. Understanding the genetic bases of adaptation to soil water deficit in trees through the examination of water use efficiency and cavitation resistance: maritime pine as a case study. J. Plant Hydraul. 3, e008. Premoli, A.C., 1994. Genetic, morphological, and ecophysiological variation in geographically restricted and widespread species of Nothofagus from southern South America. PhD Thesis. University of Colorado, Boulder. Premoli, A.C., Brewer, C.A., 2007. Environmental v. genetically driven variation in ecophysiological traits of Nothofagus pumilio from contrasting elevations. Aust. J. Bot. 55, 585–591. Premoli, A.C., Raffaele, E., Mathiasen, P., 2007. Morphological and phenological differences in Nothofagus pumilio from contrasting elevations: evidence from a common garden. Austral Ecol. 32, 515–523. Premoli, A.C., Mathiasen, P., Acosta, M.C., Ramos, V.A., 2012. Phylogeographically concordant chloroplast DNA divergence in sympatric Nothofagus ss How deep can it be? New Phytol. 193, 261–275. Prober, S.M., Byrne, M., McLean, E.H., Steane, D.A., Potts, B.M., Vaillancourt, R.E., Stock, W.D., 2015. Climate-adjusted provenancing: a strategy for climate-resilient ecological restoration. Front. Ecol. Evol. 3, 65. RStudio Team, 2015. RStudio: Integrated Development for R. RStudio, Inc., Boston, MA. http://www.rstudio.com. Sabirzhanova, I.B., Sabirzhanov, B.E., Chemeris, A.V., Veselov, D.S., Kudoyarova, G.R., 2005. Fast changes in expression of expansin gene and leaf extensibility in osmotically stressed maize plants. Plant Physiol. Biochem. 43, 419–422. Sampedro, J., Cosgrove, D.J., 2005. The expansin superfamily. Gen. Biol. 6, 242. Sánchez-Díaz, M., Aguirreolea, J., 2013. Absorción de agua por la raíz y transporte por el xilema. Balance hídrico de la planta. In Azcón-Bieto, J., Talón, M. (Eds.), Fundamentos de Fisiología Vegetal. 2da edición. Mc Graw-Hill–Interamericana de España, S.L., pp. 67–76. Sanhueza, C., Bascunan-Godoy, L., Turnbull, M.H., Corcuera, L.J., 2015. Respiration in Nothofagus species. Plant Species Biol. 30, 163–175. Shinozaki, K., Yamaguchi-Shinozaki, K., 2007. Gene networks involved in drought stress response and tolerance. J. Exp. Bot. 58, 221–227. StatSoft, Inc., 2004. STATISTICA (data analysis software system), version 7. www. statsoft.com. Suarez, M.L., Ghermandi, L., Kitzberger, T., 2004. Factors predisposing episodic droughtinduced tree mortality in Nothofagus–site, climatic sensitivity and growth trends. J. Ecol. 92, 954–966. Suarez, M.L., Kitzberger, T., 2008. Recruitment patterns following a severe drought: longterm compositional shifts in Patagonian forests. Can. J. For. Res. 38, 3002–3010. Suarez, M.L., Kitzberger, T., 2010. Differential effects of climate variability on forest dynamics along a precipitation gradient in northern Patagonia. J. Ecol. 98, 1023–1034. Suarez, M.L., Villalba, R., Mundo, I.A., Schroeder, N., 2015. Sensitivity of Nothofagus dombeyi tree growth to climate changes along a precipitation gradient in northern Patagonia, Argentina. Trees 29, 1053–1067. Trujillo-Moya, C., George, J.P., Fluch, S., Geburek, T., Grabner, M., Karanitsch-Ackerl, S.,

Araújo, M.B., 2002. Biodiversity hotspots and zones of ecological transition. Conserv. Biol. 16, 1662–1663. Benson, D.A., Clark, K., Karsch-Mizrachi, I., Lipman, D.J., Ostell, J., Sayers, E.W., 2013. GenBank. Nucleic Acids Res. 42, D36–D42. Bertolino, L.T., Caine, R.S., Gray, J.E., 2019. Impact of stomatal density and morphology on water-use efficiency in a changing world. Front. Plant Sci. 10, 225. Bran, D., Pérez, A., Ghermandi, L., Barrios, D., 2001. Evaluación de poblaciones de coihue (Nothofagus dombeyi) del Parque Nacional Nahuel Huapi, afectadas por la sequía 98/99, a escala de paisaje (1: 250.000). Informe sobre mortalidad: distribución y grado de afectación. Bariloche, Argentina. Chaves, M.M., Maroco, J.P., Pereira, J.S., 2003. Understanding plant responses to drought-from genes to the whole plant. Funct. Plant Biol. 30, 239–264. Connor, D.J., Fereres, E., 2005. The physiology of adaptation and yield expression in olive. Horticulturae Rev. 31, 155229. Ciríaco da Silva, E., Bandeira de Albuquerque, M., Dias de Azevedo Neto, A., Dias da Silva J., C., 2013. Drought and its consequences to plants-from individual to ecosystem. In: Akıncı, S. (Ed.), Responses of Organisms to Water Stress, IntechOpen, DOI: 10.5772/ 53833. Available from: https://www.intechopen.com/books/responses-oforganisms-to-water-stress/drought-and-its-consequences-to-plants-from-individualto-ecosystem. Diaz, D.G., 2018. Potenciales respuestas diferenciales a la sequía y su base genética en Nothofagus dombeyi. Degree thesis, Universidad Nacional del Comahue, Bariloche, Argentina. Donoso, C., Premoli, A., Donoso, P., 2004. Variación en Nothofagus siempreverdes sudamericanos. In: Donoso, C., Premoli, A.C., Gallo, L.A., Iliniza, R. (Eds.), Variación intraespecífica en las especies arbóreas de los bosques templados de Chile y Argentina. Santiago de Chile, Chile. Editorial Universitaria, pp. 145–166. Donoso, P., Donoso, D., Navarro, C., Escobar, B., 2006. Nothofagus dombeyi. In: Donoso, C. (Ed.), Las especies arbóreas de los bosques templados de Chile y Argentina, Autoecología. Valdivia, Chile: Marisa Cuneo, pp. 423–432. Easterling, D.R., Meehl, G.A., Parmesan, C., Changnon, S.A., Karl, T.R., Mearns, L.O., 2000. Climate extremes: observations, modeling, and impacts. Science 289, 2068–2074. Ekblom, R., Galindo, J., 2011. Applications of next generation sequencing in molecular ecology of non-model organisms. Heredity 107, 1–15. Exposito-Alonso, M., Vasseur, F., Ding, W., Wang, G., Burbano, H.A., Weigel, D., 2017. Genomic basis and evolutionary potential for extreme drought adaptation in Arabidopsis thaliana. Nat. Ecol. Evol. 2, 352. Fick, S.E., Hijmans, R.J., 2017. WorldClim 2: new 1km spatial resolution climate surfaces for global land areas. Int. J. Climatol. 37, 4302–4315. Foll, M., Gaggiotti, O., 2008. A genome-scan method to identify selected loci appropriate for both dominant and codominant markers: a Bayesian perspective. Genetics 180, 977–993. Fonseca, C.R., Overton, J.M., Collins, B., Westoby, M., 2000. Shifts in trait combinations along rainfall and phosphorus gradients. J. Ecol. 88, 964–977. Foolad, M.R., 2004. Recent advances in genetics of salt tolerance in tomato. Plant Cell. Tissue Organ. Cult. 76, 101–119. Gitlin, A.R., Sthultz, C.M., Bowker, M.A., Stumpf, S., Paxton, K.L., Kennedy, K., Muñoz, A., Bailey, J.K., Whitham, T.G., 2006. Mortality gradients within and among dominant plant populations as barometers of ecosystem change during extreme drought. Conserv. Biol. 20, 1477–1486. Glaubitz, J.C., Casstevens, T.M., Lu, F., Harriman, J., Elshire, R.J., Sun, Q., Buckler, E.S., 2014. TASSEL-GBS: a high capacity genotyping by sequencing analysis pipeline. PLoS One 9, e90346. Goudet, J., 2005. Hierfstat, a package for R to compute and test hierarchical F-statistics. Mol. Ecol. Resour. 5, 184–186. Grossiord, C., Sevanto, S., Adams, H.D., Collins, A.D., Dickman, L.T., McBranch, N., Michaletz, S.T., Stockton, E.A., Vigil, M., McDowell, N.G., 2017. Precipitation, not air temperature, drives functional responses of trees in semiarid ecosystems. J. Ecol. 105, 163–175. Hampe, A., Jump, A.S., 2011. Climate relicts: past, present, future. Ann. Rev. Ecol. Evol. Syst. 42, 313–333. Hamrick, J.L., 2004. Response of forest trees to global environmental changes. For. Ecol. Manage. 197, 323–335. Hasbún, R., González, J., Iturra, C., Fuentes, G., Alarcón, D., Ruiz, E., 2016. Using genome-wide SNP discovery and genotyping to reveal the main source of population differentiation in Nothofagus dombeyi (Mirb.) Oerst. in Chile. Int. J. Genom. 3654093. Horváth, A., Mátyás, C., 2016. The decline of vitality caused by increasing drought in a Beech provenance trial predicted by juvenile growth. SEEFOR (South-East Eur. For.) 7, 21–28. Ignazi, G., Bucci, S.J., Premoli, A.C., 2020.. Stories from common gardens: water shortage differentially affects Nothofagus pumilio from contrasting precipitation regimes. For. Ecol. Manage. 117796. IPCC, 2007. Change 2007: impacts, adaptation and vulnerability. In: Parry, M.L., Canziani, O.F., Palutikof, J.P., van der Linden, P.J., Hanson, C.E. (Eds.), Contribution of Working Group II to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change, Cambridge University Press, Cambridge, UK. IPCC, 2013. Climate change 2013: the physical science basis. In: Stocker, T.F., Qin, D., Plattner, G-K., Tignor, M., Allen, S.K., Boschung, J., Nauels, A., Xia, Y., Bex, V., Midgley, P.M. (Eds.), Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change – Summary for policymakers. Cambridge University Press, Cambridge, New York. IPCC, 2014. Cambio climático 2014: Impactos, adaptación y vulnerabilidad - Resumen para responsables de políticas. In: Field, C.B., Barros, V.R., Dokken, D.J., Mach, K.J., Mastrandrea, M.D., Bilir, T.E., Chatterjee, M., Ebi, K.L., Estrada, Y.O., Genova, R.C., Girma, B., Kissel, E.S., Levy, A.N., MacCracken, S., Mastrandrea, P.R., White, L.L.

10

Forest Ecology and Management 461 (2020) 117931

D.G. Diaz, et al.

Caballero-Pérez, J., 2015. Identification of novel drought-tolerant-associated SNPs in common bean (Phaseolus vulgaris). Front. Plant Sci. 6, 546. Weinberger, P., 1973. Beziehungen zwischen mikroklimatischen Faktoren und natürlicher Verjüngung araukano-patagonischer Nothofagus-Arten. Flora 162, 157–179. Zar, J.H., 1996. Biostatistical Analysis. Prentice Hall, NewYork. Zeppel, M.J.B., Wilks, J., Lewis, J.D., 2014. Impacts of extreme precipitation and seasonal changes in precipitation on plants. Biogeosciences 11, 3083–3093. Zhu, H., Hu, F., Wang, R., Zhou, X., Sze, S.H., Liou, L.W., Barefoot, A., Dickman, M., Zhang, X., 2011. Arabidopsis Argonaute 10 specifically sequesters miR166/165 to regulate shoot apical meristem development. Cell 145, 242–256.

Konrad, H., Mayer, K., Sehr, E.M., Wischnitzki, E., Schueler, S., 2018. Drought sensitivity of Norway Spruce at the species’ warmest fringe: quantitative and molecular analysis reveals high genetic variation among and within provenances. G3: Gen. Genom. Genet. 8, 1225–1245. Valladares, F., Vilagrosa, A., Peñuelas, J., Ogaya, R., Camarero, J.J., Corcuera, L., Siso, S., Gil-Pelegrín, E., 2004. Estrés hídrico: ecofisiología y escalas de la sequía. Ecología del bosque mediterráneo en un mundo cambiante 2, 165–192. Varela, S.A., Gyenge, J.E., Fernández, M.E., Schlichter, T., 2010. Seedling drought stress susceptibility in two deciduous Nothofagus species of NW Patagonia. Trees 24, 443–453. Villordo-Pineda, E., González-Chavira, M.M., Giraldo-Carbajo, P., Acosta-Gallegos, J.A.,

11