Disentangling Mechanisms of Drought-Induced Dieback in Pinus nigra Arn. from Growth and Wood Isotope Patterns

: The increased frequency and intensity of warming-induced droughts have triggered dieback episodes a ﬀ ecting many forest types and tree species worldwide. Tree plantations are not exempt as they can be more vulnerable to drought than natural forests because of their lower structural and genetic diversity. Therefore, disentangling the physiological mechanisms leading to growth decline and tree mortality can provide tools to adapt forest management to climate change. In this study, we investigated a Pinus nigra Arn. plantation situated in northern Spain, in which some trees showed canopy dieback and radial-growth decline. We analyzed how radial growth and its responses to drought events di ﬀ ered between non-declining (ND) and declining (D) trees showing low and high canopy defoliation, respectively, in combination with carbon ( δ 13 C) and oxygen ( δ 18 O) isotope ratios in tree rings. The radial growth of P. nigra was constrained by water availability during the growing season and the previous autumn. The radial growth of D trees showed higher sensitivity to drought than ND trees. This fact is in accordance with the lower drought resilience and negative growth trends observed in D trees. Both tree classes di ﬀ ered in their growth from 2012 onwards, with D trees showing a reduced growth compared to ND trees. The positive δ 13 C- δ 18 O relationship together with the uncoupling between growth and intrinsic water-use e ﬃ ciency suggest that D trees have less tight stomatal regulation than ND trees, which could involve a high risk of xylem embolism in the former class. Our results suggest that di ﬀ erent water use strategies between coexisting ND and D trees were behind the di ﬀ erences in growth patterns and point to hydraulic failure as a possible mechanism triggering dieback and growth decline.


Introduction
Increasing rates of tree mortality and dieback events have been reported worldwide, covering all major forested biomes on Earth [1][2][3][4][5][6][7]. These trends have been linked to warming and drought, sometimes co-occurring with insect outbreaks [1]. Climatic models forecast an increase in extreme climatic events globally [8,9]; thus, drought-related growth decline and tree mortality are likely to intensify [10]. Widespread episodes of forest dieback and mortality have important consequences for biodiversity, ecosystem function and services, and feedbacks to climate change through biophysical effects and the loss of carbon sinks [11].
The impacts of climate change can be stronger in pine plantations that have been found to be more vulnerable to warming-related drought than naturally regenerated forests [12,13]. Pine plantations constitute ca. 7% of the total worldwide forest cover, and they amount to 10% in the Mediterranean Basin [14]. Over this region, massive pine reforestations were developed during the 20th century to increase forest productivity, to protect the soil against erosion and to restore watersheds on formerly deforested lands derived from traditional uses [15]. This resulted in very homogeneous forests of pioneer pine species that have been affected by striking episodes of drought-triggered canopy dieback, growth decline and mortality [16][17][18][19][20][21]. Therefore, a proper understanding of the mechanisms leading to mortality and shaping differences among individual trees can help to improve the management of tree plantations.
Hydraulic failure and carbon starvation have been proposed as the two main mechanisms inducing drought-related tree dieback [22][23][24]. Hydraulic failure refers to the cessation of symplastic biochemical functioning and the disruption of water transport through xylem embolism [25], whereas carbon starvation consists of a strong reduction in carbon pools that prevent the maintenance of cellular and defensive metabolisms [22]. Moreover, such mechanisms interact with biotic agents [26] and nutritional aspects [27]. The predisposition of trees to drought-induced mortality can be assessed using a dendrochronological retrospective approach. Tree ring width data offer an easily available long-term record of radial growth variability [28]. Indeed, dendrochronological studies comparing conspecific trees with different vitality (e.g., defoliation percentage) growing in the same stand have provided interesting insights into the short-and long-term growth patterns before tree death [2,6,29]. For instance, Camarero et al. [6] showed that legacy effects resulting from severe droughts can last for several years and they are usually greater in later dying trees. However, growth patterns per se may not be enough to disentangle the physiological mechanisms leading to tree mortality.
Stable isotope ratios in tree rings can help to overcome this issue because they contain retrospective information on the ecophysiological processes experienced by trees [30]. The processes of assimilation of carbon and oxygen are strongly influenced by climate. Therefore, the so-called dual-isotope composition approach is a useful tool to elucidate shifts in the relationship between photosynthetic rate (A) and stomatal conductance (g S ) rates, that is, intrinsic water-use efficiency (iWUE). On one hand, gas exchange modifications are captured in the composition of stable carbon isotopes (δ 13 C) of tree rings owing to the connection between carbon isotope fractionation during photosynthesis and the ratio of CO 2 concentration between the intercellular space and the atmosphere [31]. Conversely, the δ 18 O is related to g S through the influence of vapor pressure deficit [32] and transpirational dilution of evaporatively enriched leaf water [33]. Therefore, it can help to unravel the effects of drought on A and g S , after considering the variations in tree ring δ 18 O signature resulting from differences in δ 18 O water sources associated with changes in soil water uptake depth [34].
In this study, we aimed at analyzing the mechanisms underlying the growth decline and canopy dieback of some trees in a Pinus nigra Arn. plantation situated in north Spain. This species is widely distributed in mid-altitude Mediterranean forests and in temperate woodlands in central-eastern Europe, where it has been used extensively in reforestation and land rehabilitation due to its ecological flexibility [35]. For that purpose, we combined a retrospective reconstruction of long-term radial growth patterns and growth responses to severe drought events with carbon and oxygen isotope ratios. We hypothesized that successive drought events during the last decades affected symptomatic and asymptomatic trees differently, showing high and low defoliation levels, respectively, due to the less conservative use of water of symptomatic trees increasing their risk of hydraulic failure [30,36].

Study Site and Climate Data
The Sabaiza forest (42 • 37 57 N, 1 • 26 06 W, 865-900 m a.s.l.) is located in the Ezprogui municipality, Navarra province (northern Spain) (Figure 1a). The study stand is a Pinus nigra subsp. Nigra (Hoss) var. austriaca plantation carried out during the 1950s and 1960s with the objective of increasing forest productivity and minimizing soil loss as well as soil erosive processes. The soil type is xerochrepts, the aspect is E-SE and the slope 23 • . The stand accounts for a density of 720 trees ha −1 , a basal area of 24.9 m 2 ha −1 , an average diameter at 1.3 m (DBH) of 23.9 ± 1.2 cm and an average tree height of 11.5 ± 0.3 m. The main understory species are Buxus sempervirens L., Juniperus communis L., and Rubus ulmifolius Schott. No thinning has been carried out in the study stands for the past thirty years, but they have started showing dieback symptoms (leaf loss and shoot dieback, elevated mortality rate) since the mid-2010s. Some trees exhibit severe defoliation (>80%) due to outbreaks of the pine processionary moth (Thaumetopoea pityocampa Denis and Schiffermüller). Caterpillar nests were first observed in winter 2016-2017 and again in winter 2017-2018. Herbivory on old needles finishes in late winter, when caterpillars leave trees and bury in the soil.
and Rubus ulmifolius Schott. No thinning has been carried out in the study stands for the past thirty years, but they have started showing dieback symptoms (leaf loss and shoot dieback, elevated mortality rate) since the mid-2010s. Some trees exhibit severe defoliation (>80%) due to outbreaks of the pine processionary moth (Thaumetopoea pityocampa Denis and Schiffermüller). Caterpillar nests were first observed in winter 2016-2017 and again in winter 2017-2018. Herbivory on old needles finishes in late winter, when caterpillars leave trees and bury in the soil.
The climate in the study area is cold-wet Mediterranean, characterized by dry and warm summers and wet and mild springs and autumns. We retrieved the monthly maximum and minimum temperature and precipitation data from the nearest weather station (Lerga station, 42°33′54″ N, 1°30′04 W, 612 m a.s.l.) for the period 1975-2018. The mean annual temperature is 12.6 °C. July and August are the months with higher temperatures (average maximum temperature of 28.1 °C), while January is the coldest month (average minimum temperature of 1.4 °C). The mean annual precipitation is 736 mm, where the driest and wettest months are August (26 mm) and November (84 mm), respectively (Figure 1b).  The climate in the study area is cold-wet Mediterranean, characterized by dry and warm summers and wet and mild springs and autumns. We retrieved the monthly maximum and minimum temperature and precipitation data from the nearest weather station (Lerga station, 42 • 33 54 N, 1 • 30 04 W, 612 m a.s.l.) for the period 1975-2018. The mean annual temperature is 12.6 • C. July and August are the months with higher temperatures (average maximum temperature of 28.1 • C), while January is the coldest month (average minimum temperature of 1.4 • C). The mean annual precipitation is 736 mm, where the driest and wettest months are August (26 mm) and November (84 mm), respectively ( Figure 1b).
We estimated the monthly potential evapotranspiration (PET) using the modified form of the Hargreaves equation [38]. Then, we calculated the Standardized Precipitation Evaporation Index (SPEI), which is a multi-scalar drought index based on the accumulated water deficit (water balance), as the difference between monthly precipitation and monthly PET [39]. We calculated the SPEI at different timescales from 1 to 18 months.

Field Sampling and Dendrochronological Procedures
We sampled 10 couples of dominant non-declining (ND) and declining (D) trees (n = 20) in autumn 2018. We assessed defoliation by a visual assessment of crown transparency in 2018, and it was considered a proxy of tree vigor following previous studies on drought-stressed pines [6]. We selected the trees based on their defoliation levels by considering neighboring individuals showing high (>60%, D trees) and low (<40%, ND trees) canopy defoliation, respectively. This threshold was selected based on similar research which showed that a defoliation threshold of 60% allowed discriminating trees with different vigor [13]. Both ND and D trees produced shoots and needles in 2018, despite the fact that D trees were always more defoliated. We avoided sampling D trees affected by defoliation caused by the pine processionary moth, i.e., presenting caterpillar nests or needles eaten by this herbivore.
We took wood cross-sections at 1.3 m. The samples were air-dried and sanded until tree rings were clearly visible [28]. We visually cross-dated all samples and the tree ring width was measured to a precision of 0.01 mm along two opposite radii per sample (separated by 180 • ) using a LINTAB measuring device (Frank Rinn, Heidelberg, Germany). We further validated cross-dating using the COFECHA software, which calculates moving correlations among individual tree series [40]. We transformed tree ring width series to basal area increment (BAI) series using the following equation and assuming concentric rings: where R 2 t and R 2 t−1 are the radii corresponding to the years t and t−1, respectively. We used BAI because it is a two-dimensional measure of stem increment in area that is known to better reflect the growth of the whole tree than one-dimensional tree ring width [41].
We developed mean residual BAI chronologies for each tree vigor class to calculate climate-growth associations. Firstly, we removed non-climatic biological growth trends of individual tree ring width series by fitting a horizontal line using the mean of the series. Afterwards, an autoregressive model was applied to each detrended series to remove most of the first-order autocorrelation. Finally, we obtained mean chronologies of ND and D trees using a bi-weight robust mean.

Growth Responses to Drought
We identified drought events based on the SPEI series calculated for the month and timescale that best correlated with BAI chronologies. The years with 10% lower SPEI values were selected considering the reference period 1975-2018. If two events were separated by less than three years, the lowest value was chosen. We evaluated the short-term growth responses to drought by means of the three resilience indices proposed by Lloret et al. [42]. Resistance (Rt), calculated with the ratio BAI D /BAI preD , characterizes the ability of the tree to revoke the stress situation induced by the drought event. Recovery (Rc) reflects the extent of growth increase or decrease after the drought event. Rc is estimated as BAI postD /BAI D . Resilience (Rs) indicates the ability of a tree to reach a pre-drought growth level and it is calculated as BAI postD /BAI preD . BAI D is the BAI during the corresponding drought year, and BAI preD and BAI postD are the average BAI for the 3 years preceding and following the drought Forests 2020, 11, 1339 5 of 16 event, respectively. We chose consistent preD and postD periods of 3 years based on previous studies as this represents a good compromise between drought intensity and the short-term growth response [43]. We also quantified the long-term growth responses after selected drought events. For that purpose, we estimated trends in mean BAI series since the drought event to 2018.

Stable Isotope Analyses and Water-Use Efficiency
We performed the carbon and oxygen isotope analyses on five trees of each vigor class for the period 1975-2018. We selected the trees based on their highest BAI correlations with the mean BAI series of each vigor class. We separated the tree rings manually using a scalpel and pooled them at tree-level in groups of five contiguous rings (e.g., 1975-1979, 1980-1984, etc.; n = 9 samples per tree). We milled each group of five tree rings using a ball mill (Retsch ZM1, Haan, Germany). Then, we extracted α-cellulose with a double-step digestion [44].
The stable isotope composition was measured at the Stable Isotope Facility (University of California, Davis, CA, USA). The carbon isotope composition was analyzed using a PDZ Europa ANCA-GSL elemental analyzer interfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer (Sercon Ltd., Cheshire, UK). The oxygen isotope composition was analyzed using an elementar PyroCube (Elementar Analysensysteme GmbH, Hanau, Germany) interfaced to an Isoprime VisION (Isoprime Ltd., Stockport, UK, a unit of Elementar Analysensysteme GmbH, Hanau, Germany). The results were expressed as isotopic composition of carbon (δ 13 C) and oxygen (δ 18 O) relative to Vienna Pee Dee Belemnite and Vienna Standard Mean Ocean Water standards, respectively [45]. The accuracy of the analyses (SD of working standards) was 0.05-0.21‰.
We used δ 13 C to calculate the iWUE, that is, the ratio between the photosynthetic rate (A) and its stomatal conductance (g S ), using the following equation [31]: where Ca and Ci are CO 2 concentrations in the atmosphere and the intercellular space, respectively, and 0.625 is the relation among conductance of H 2 O and CO 2 . To determine Ci, we used the following equation: where δ 13 Ctree and δ 13 Catm are the tree and atmospheric C isotope compositions, respectively, a is the diffusion fractionation across the boundary layer and the stomata (+4.4‰) and b is the Rubisco enzymatic biologic fractionation (+27.0‰). We used δ 13 Catm estimated values for the period 1975-2003 from [46], and the measured values for the 2004-2018 period were retrieved from the Earth Research Laboratory website (http://www.esrl.noaa.gov/).

Statistical Analyses
We used the Kendall τ statistic to assess trends in climatic and growth series. We performed comparisons between ND and D trees regarding size (DBH and height), mean values of radial growth (BAI) and isotope data (δ 13 C, δ 18 O and iWUE) through time, as well as compared differences in short-term growth responses to drought, using Wilcoxon rank-sum tests. We analyzed the relationships between climatic variables and radial growth (residual BAI chronologies) for each tree vigor class using Pearson correlations, which were calculated from 1-to 18-month timescales and from January to December. We used linear mixed effects models [47] to test the relationship between stable isotope variables and growth, which was logarithmically transformed to normalize its distribution. Since these measurements represent repeated measures over the same individual trees, tree identity was included as a random factor.
We performed all statistical analyses in R (version 3.6.3, Vienna, Austria) [48]. The SPEI package [49] was employed for PET and SPEI calculation. The dplR package [50] was used to convert annual ring measurements into BAI and to detrend growth series and develop residual BAI chronologies. Kendall trends were calculated with the Kendall package [51]. The nlme package [52] was used to perform linear mixed effects models.

Tree Ring Width Statistics of Species Mean Chronologies
Significant positive trends were found for maximum spring and summer temperatures since 1975 at the study area (Figure 1c), whereas the mean winter minimum temperature has decreased during the last decades. No significant trends were found for precipitation (Figure 1d). There were no significant differences in DBH (W = 47.5, p = 0.561) or height (W = 30.0, p = 0.377) between the two tree vigor classes.
The radial growth of both ND and D trees followed similar patterns, with BAI reaching a maximum in the mid-1990s. Afterwards, both vigor classes showed a reduction in their growth rate. This tendency was more pronounced for D trees, and this vigor class displayed a significantly (p < 0.005) lower growth than ND trees after 2012 (9.93 ± 1.03 cm 2 of ND trees vs. 5.99 ± 0.87 cm 2 of D trees during the 2012-2018 period) (Figure 2a).  The highest correlations between the water balance and growth of both ND and D trees were found with the July SPEI calculated at a 12-month timescale, that is, including an accumulated water deficit of one year (Figure 3). Growth sensitivity to drought was higher in D trees than in ND trees (July SPEI 12-month correlations: ND trees r = 0.568, p < 0.001; D trees r = 0.637, p < 0.001). The July 12-month SPEI series were evaluated to identify extreme drought events for the study period . The lowest SPEI values corresponded to 1986, 2002 and 2012 (Figure 2b). Hence, they were identified as the more relevant drought events during the study period. Although the SPEI value of 2005 (SPEI = −1.430) was included within the 10% lower values, it was only three years apart from the 2002 drought (SPEI = −1.497) so it was not considered.   Short-term growth reductions after drought events were important in the two tree vigor classes (54.9% and 72.3% in ND and D trees, respectively). They resulted in Rt values < 1 (Table 1). Rt and Rc were significantly higher in ND trees than in D trees in 2002 and 2012, respectively. As a consequence, the Rs values of D trees were significantly lower than those of ND trees after the last two study droughts: 2002 and 2012. The long-term growth trends after droughts (considering the period since the drought event until 2018) were negative (p < 0.005) for D trees, but they were not significant for ND trees. Table 1. Short-and long-term term growth responses to drought. Short-term (three years long) responses were assessed by resistance (Rt), recovery (Rc) and resilience (Rs) indices values (mean ± standard error) between non-declining (ND) and declining (D) P. nigra trees. Different letters indicate significant (p < 0.005) statistical differences between tree vigor classes in Rt, Rc and Rs based on Wilcoxon rank-sum tests. Long-term trends are based on the Kendall τ statistic and the slope for linear rate of change was computed from the study drought year until 2018 (i.e., 33, 17 and 7 years long for the 1986, 2002 and 2012 drought events, respectively). Significant (p < 0.05) trends are indicated with asterisks (*).

Year
Vigor

Isotope Signals in Tree Rings
The carbon-related isotopic variables (δ 13 C and iWUE) did not show significant differences (p < 0.05) between D and ND trees during the period 1975-2000. However, when the growth between ND and D trees significantly differed, that is, when dieback intensified, D trees displayed lower δ 13 C values than ND trees (Figure 4). Likewise, D trees showed a lower iWUE during the 2000-2018 period than ND trees, with reductions of 6.6% in iWUE in D trees compared with ND trees since 2000. Considering δ 18 O, D trees showed lower values than ND trees throughout the last two decades, although significant differences were only found during the 2005-2009 and 2015-2018 periods. BAI was positively associated with iWUE in ND trees (ND trees: F = 4.072, p = 0.047; D trees: F = 1.969, p = 0.170) (Figure 5a). We also found a positive significant association between δ 13 C and δ 18 O only for D trees (ND trees: F = 0.390, p = 0.539; D trees: F = 3.537, p = 0.049) (Figure 5b).

Isotope Signals in Tree Rings
The carbon-related isotopic variables (δ 13 C and iWUE) did not show significant differences (p < 0.05) between D and ND trees during the period 1975-2000. However, when the growth between ND and D trees significantly differed, that is, when dieback intensified, D trees displayed lower δ 13 C values than ND trees (Figure 4). Likewise, D trees showed a lower iWUE during the 2000-2018 period than ND trees, with reductions of 6.6% in iWUE in D trees compared with ND trees since 2000.  isotope ratios in tree rings, as well as in intrinsic water-use efficiency (iWUE) (c) along the study period in non-declining (empty circles) and declining (black triangles) Pinus nigra trees. Symbols represent means ± standard error of each group of five consecutive rings. Significant differences (p < 0.05) between tree vigor classes are indicated by grey filled areas. isotope ratios in tree rings, as well as in intrinsic water-use efficiency (iWUE) (c) along the study period in non-declining (empty circles) and declining (black triangles) Pinus nigra trees. Symbols represent means ± standard error of each group of five consecutive rings. Significant differences (p < 0.05) between tree vigor classes are indicated by grey filled areas.
Forests 2020, 11, x FOR PEER REVIEW 9 of 15 Figure 5. Relationships between basal area increment (BAI) and intrinsic water-use efficiency (iWUE) (a) and between carbon (δ 13 C) and oxygen (δ 18 O) isotope ratios (b) for non-declining (empty circles) and declining (black triangles) Pinus nigra trees. Each symbol represents values of five consecutive rings for the entire study period . Big symbols in (b) show mean ± standard error values for both tree vigor classes, and the top left inset indicates changes in stomatal conductance (gS) and photosynthesis (A) according to [32]. Solid and dash lines represent significant associations for ND and D trees, respectively.

Discussion
The long-term radial growth patterns of the studied Pinus nigra trees were positively associated with water availability at long timescales encompassing the previous autumn to early summer, and the positive effect of water availability on growth was stronger in declining trees than in nondeclining trees. Consistently, declining trees exhibited greater impact of severe drought events at both short-and long-term scales, indicated by reduced resilience and negative growth trends, which led to the reduced growth of this vigor class from 2012 onwards. Differences between tree classes could be attributed to the lower stomatal control that declining trees displayed during the last two decades, reflected by lower δ 13 C and δ 18 O values, which resulted in decreased iWUE in declining trees compared to non-declining ones. Therefore, our results suggest hydraulic failure as the mechanism underlying growth decline and canopy dieback, which could be accentuated by leaf area reduction as a consequence of leaf desiccation.
The negative effect of water shortage on secondary growth, which results in narrow tree rings, is well documented in previous studies [28], and it has strong evidence for southern P. nigra populations [21,53,54]. This is also supported by our results (Figure 3). The strongest BAI-SPEI correlations were found at 12-month timescales covering from previous August to current July. Therefore, radial growth relies not only on water balance during spring and early summer, when the radial enlargement of tracheids peaks [16], but also on hydrological conditions recorded during the previous year. Favorable conditions during the previous autumn and winter would not directly influence tree growth, but they would increase soil water reserves for supporting growth during spring [55,56] and enhance carbohydrate accumulation and spring cambial dynamics [57,58] with a positive effect on the growth of earlywood next year [59]. Similar growth responses to long drought timescales (9-15 months) have been reported in studies assessing large water availability gradients in the Iberian Peninsula [55,60], and this pattern has been supported at a global scale [61]. Our study area is located in the transition between the Mediterranean and Atlantic regions, so prevailing conditions are not particularly xeric. However, growth and responses to drought are modulated by site conditions, such as soil type or interaction with neighboring trees [22,62,63]. In fact, strong competition for water resources in xeric sites resulting from high stand density has been proposed to drive higher drought vulnerability in P. nigra plantations compared to natural stands [53].
Long-term climate-growth relationships showed a stronger effect of water availability in D trees as compared to ND trees ( Figure 3). Consistently, the impact of severe drought events was stronger Figure 5. Relationships between basal area increment (BAI) and intrinsic water-use efficiency (iWUE) (a) and between carbon (δ 13 C) and oxygen (δ 18 O) isotope ratios (b) for non-declining (empty circles) and declining (black triangles) Pinus nigra trees. Each symbol represents values of five consecutive rings for the entire study period . Big symbols in (b) show mean ± standard error values for both tree vigor classes, and the top left inset indicates changes in stomatal conductance (g S ) and photosynthesis (A) according to [32]. Solid and dash lines represent significant associations for ND and D trees, respectively.

Discussion
The long-term radial growth patterns of the studied Pinus nigra trees were positively associated with water availability at long timescales encompassing the previous autumn to early summer, and the positive effect of water availability on growth was stronger in declining trees than in non-declining trees. Consistently, declining trees exhibited greater impact of severe drought events at both short-and long-term scales, indicated by reduced resilience and negative growth trends, which led to the reduced growth of this vigor class from 2012 onwards. Differences between tree classes could be attributed to the lower stomatal control that declining trees displayed during the last two decades, reflected by lower δ 13 C and δ 18 O values, which resulted in decreased iWUE in declining trees compared to non-declining ones. Therefore, our results suggest hydraulic failure as the mechanism underlying growth decline and canopy dieback, which could be accentuated by leaf area reduction as a consequence of leaf desiccation.
The negative effect of water shortage on secondary growth, which results in narrow tree rings, is well documented in previous studies [28], and it has strong evidence for southern P. nigra populations [21,53,54]. This is also supported by our results (Figure 3). The strongest BAI-SPEI correlations were found at 12-month timescales covering from previous August to current July. Therefore, radial growth relies not only on water balance during spring and early summer, when the radial enlargement of tracheids peaks [16], but also on hydrological conditions recorded during the previous year. Favorable conditions during the previous autumn and winter would not directly influence tree growth, but they would increase soil water reserves for supporting growth during spring [55,56] and enhance carbohydrate accumulation and spring cambial dynamics [57,58] with a positive effect on the growth of earlywood next year [59]. Similar growth responses to long drought timescales (9-15 months) have been reported in studies assessing large water availability gradients in the Iberian Peninsula [55,60], and this pattern has been supported at a global scale [61]. Our study area is located in the transition between the Mediterranean and Atlantic regions, so prevailing conditions are not particularly xeric. However, growth and responses to drought are modulated by site conditions, such as soil type or interaction with neighboring trees [22,62,63]. In fact, strong competition for water resources in xeric sites resulting from high stand density has been proposed to drive higher drought vulnerability in P. nigra plantations compared to natural stands [53].
Long-term climate-growth relationships showed a stronger effect of water availability in D trees as compared to ND trees ( Figure 3). Consistently, the impact of severe drought events was stronger on the short-and long-term growth responses of D trees than of ND trees (Table 1). Climate extremes such as droughts have been shown to have a stronger impact on ecological processes than average conditions [64,65]. Indeed, an increase in the global patterns of forest dieback and tree mortality episodes has been primary attributed to droughts and heat waves [1]. Likewise, we argue that the onset of growth decline and dieback symptoms of D trees in our study plantation was triggered by successive severe droughts that would progressively impair physiological processes, in line with previous studies reporting canopy dieback and tree mortality in other natural and planted P. nigra forests [6,16,21,53]. The fact that drought resilience was lower in D trees than in ND trees (significant in 2002 and 2012 extreme years), together with the negative growth trends found in D trees after all droughts considered, supports this argument (Table 1). Similarly to other research [66,67], we identified the severe drought in 2012 as a tipping point in the dieback process of D trees, but in this case the drought affected planted stands in a relatively mesic site.
The predisposition to growth decline and canopy dieback in D trees may arise from different water use strategies adopted by the two tree classes. The lower δ 13 C and δ 18 O in D trees in comparison with the values of ND trees (Figures 4 and 5b), together with the positive association between both isotope signals only found in D trees, indicate a poorer stomatal regulation in declining trees [32]. This water-spending behavior is consistent with the higher growth sensitivity to water supply ( Figure 3) and the uncoupling between the growth and iWUE of D trees (Figure 5a). High g S and associated increased hydraulic efficiency would maximize carbon assimilation and enhance growth under favorable environmental conditions. This prodigal strategy, as defined by Hentschel et al. [36], has previously been reported in P. nigra declining populations [21]. However, we did not find differences in growth between the tree vigor classes. Growth is not driven by source activity but by other environmental constraints such as water shortages [68], so seasonal summer drought can shorten the radial enlargement of tracheids, which largely determines final ring width, to a greater extent in D trees [16]. In contrast, lower stomatal control involves a higher risk of xylem cavitation during drought [30]. This is supported by the wider lumen and thinner cell walls of tracheids detected in declining trees of this species [21,69,70] and other gymnosperms [36,71], although the opposite pattern has also been reported for long-term dieback processes (see, for instance, [72]). Savi et al. [70] also found that hydraulic conductivity decreased in declining P. nigra trees after the severe drought in 2012, as a possible consequence of embolism accumulated during drought. Furthermore, the impairment of water transport linked to the progressive lower growth rates of D trees can be attributed to cavitation fatigue after successive severe droughts, and this is the phenomenon by which previous cavitation damage enlarges vulnerability to cavitation in following droughts [73].
We are aware that the interpretation of the dual isotope approach presents some caveats, since tree ring δ 18 O may also reflect variations in δ 18 O in the source water related to changes in soil water uptake depths [34,74]. However, the forest under study is an even-aged reforested stand with homogeneous trees in terms of size or age (no significant differences for DBH and height were found between ND and D trees), thus with comparable root systems. Therefore, we assume that both tree vigor classes took up water from soil sources with similar δ 18 O signatures.
Severe droughts can have long-lasting impacts on the leaf area on D trees either as an adaptation mechanism for reducing the transpiring area to compensate for delayed stomatal closure [75], or as a consequence of the reduced efficiency to transport water to foliage and the subsequent leaf desiccation [76,77]. Drought-induced defoliation, together with prolonged periods of near complete stomatal closure, has been related to reductions in non-structural carbohydrate (NSC) concentration in declining pine trees [70,78], thus reducing investment in defensive structures and compounds [26]. Indeed, tree individuals with reduced long-term vigor have been found to be more susceptible to insect outbreaks [29]. At stand level, scattered defoliation by the pine processionary moth detected in the study forest, which particularly affects P. nigra [79], could have accentuated the depletion in NSC. Nonetheless, the sampled trees showed neither caterpillar nests or signs of herbivory at the time of sampling, nor growth series exhibiting drops associated to insect attacks (Figure 1a). As a consequence of defoliation, the photosynthetic area suffered a greater reduction in D trees relative to ND trees, thus impairing carbon assimilation [78] and contributing to the decrease in iWUE and, probably, to a decline in carbon reserves. Although an impending carbon starvation should not be discarded since it has been found to be common among gymnosperms [80], our results only provide evidence for hydraulic failure underlying dieback, which, in fact, is ubiquitous across tree taxa [24].
Finally, we want to pay attention to the similar patterns of growth and carbon and oxygen isotope ratios followed by the two tree vigor classes (Figures 2 and 4). For instance, the δ 13 C and δ 18 O of ND and D trees were negatively associated before 2000, indicating strong stomatal regulation [32]. However, this relationship became positive after 2000, suggesting a loss in stomatal control in both tree vigor classes, although it was stronger in D trees (see Figure 4a,b). Although ND trees were asymptomatic (showed low defoliation) during the sampling dates, P. nigra has been shown to be sensitive to drought considering the thresholds of xylem cavitation [70]. Vulnerability to drought has been reported to be enhanced in forest plantations compared to naturally regenerated forests [13,53]. Furthermore, the subspecies of the study plantation (P. nigra subsp. nigra) is not native to the Iberian Peninsula, but it is originally from Austria and the Balkan Peninsula ( Figure 1a) and typically shows resistance to low temperatures but may be vulnerable to severe drought events. Nevertheless, the differences in physiological traits related to water-use and drought resistance have not been found among European subspecies of P. nigra [35].
Pine reforestations in Spain cover ca. 2.6 10 6 ha, of which 3.6 10 5 ha correspond to P. nigra plantations [81]. If the frequency and intensity of severe droughts and heat waves increase [8,82], loss of vitality and tree mortality could extent to ND trees of the study plantation and the same fate may follow other plantations of similar characteristics. These dieback and mortality episodes will involve important ecological and economic implications considering the productive and protective role of these planted forests. The systematic implementation of this experimental design (i.e., comparison of co-occurring ND and D trees) including several stands within the species distribution can help to elucidate processes leading to drought-induced dieback [6,75,83]. Studies dealing with drought-vulnerable pine plantations have provided insights into the patterns of growth decline and canopy dieback [13,17]. However, more information is needed for disentangling the underlying physiological mechanisms. The dual-isotope approach offers a unique opportunity in this regard [30,32], as this study proves.

Conclusions
Both dendrochronological and stable isotope approaches to tree response to climatic variability provide consistent outcomes and point towards a mechanistic explanation for the dieback of some trees in the studied P. nigra plantation. We postulate that reduced stomatal control (low δ 13 C and δ 18 O signatures) predisposed declining trees to xylem embolism during extreme drought events. The accumulated effect of xylem embolism after successive droughts likely reduced water transport efficiency. Therefore, hydraulic failure is proposed as the physiological mechanism leading to growth decline and canopy dieback. This agrees with the reduced resilience against drought observed in declining trees. In addition, drought-induced leaf area reduction suggests an impairment of photosynthesis, possibly contributing to dieback.