Physiological Mechanisms of Delayed Drought-Induced Forest Mortality

Forest mortality could become more severe and frequent due to global change in hydroclimatic patterns. Tree deaths will affect the global carbon cycle, ecosystem services, and levels of biodiversity. However, the mechanisms by which trees die-off over the course of the time are largely unknown. Anderegg et al. examine the physiological basis for the delayed, widespread die-off of trembling aspen (Populus tremuloides) across North America post-drought (2012). The authors studied experimental and observed data for both dying and healthy trees to assess physiological performance and accumulated hydraulic damage (hydraulic deterioration) demonstrated by xylem water conductivity. Cavitation fatigue caused hydraulic damage to persist or increase in dying trees, and was found to predict the probability of tree mortality over the course of the years studied. The authors also conclude that surviving regions of forests that have already been subjected to drought are the most vulnerable to future drought. Increased drought vulnerability affects ecosystem stability, biodiversity, and ecosystem carbon balance. Drought stress accumulation and repair affects tree mortality, which has subsequent implications for forest ecosystems.—Hilary Haskell
Anderegg, W., Plavcova, L., Anderegg, L., Hacke, U., Berry, J., Field, C. 2012. Drought’s legacy: multiyear hydraulic deterioration underlies widespread aspen forest die-off and portends increased future risk. Global Change Biology 19, 1188-1196.
Anderegg et al. studied the physiological basis of delayed widespread die-off of trembling aspen (Populus tremuloides) post-drought. The authors studied trees in Colorado’s San Juan national Forest, which experienced some of the worst sudden aspen decline (SAD) of any area in western Colorado. The forest studied has a mean annual temperature of f 3.2 °
C and mean annual precipitation 508 mm. In the forest, precipitation falls as either snow (November through May) or monsoon summer rain (July through September). Seasonal droughts typically occur in early summer, and represent peak water stress for the forest. The study’s specific site consisted of seven aspen clone groups with mortality rates ranging from healthy to SAD trees within 100 m of one another. The authors measured crown dieback, the percent of a tree’s branch tips within the crown of the tree (above ground parts: leaves, branches, trunk, etc.) that are dead as an indicator of tree health. Two categories of ramets, an individual specimen of a clone, were compared in this study: SAD affected (ramets with less than 50% crown dieback) and healthy (ramets with less than 20% crown dieback). Tree death in this study was designated as 100% crown dieback. The hydraulic properties of the trees studied are largely dependent on xylem, the woody vascular transport system that transports water from the soil throughout the plant.
The authors used observed and experimental evidence of trees’ accumulated physiological hydraulic changes to assess whether hydraulic properties can be used to predict inter-year stem mortality. Stem mortality is an indicator of subsequent tree death. These changes in tree’s hydraulic properties could thus explain delayed and multi-year aspen die-off after exposure to drought.  Anderegg et al. sought to explain whether different levels of water stress, accumulated growth of xylem, vulnerability of xylem vessels to cavitation or some combination of these factors caused the differences between the accumulated hydraulic properties of trees that survived and those that died.
The authors measured crown dieback and hydraulic conductance for thirty-six ramets, both SAD and healthy, between August 2010 and July 2011 in order to assess whether the hydraulic performance demonstrated by xylem water conductance affects inter-year tree mortality probability. Logistic regression techniques were used to estimate the probability of a ramet dying during this time period, compared to baseline native xylem water conductance in tree branches. During August 13–14, 2010, between 12:00 P.M and 2:00 P.M, the authors collected branch networks (branches, petioles, and leaves) from thirty-six clones that were farther than 10 cm from the mid canopy of the forest. After collecting the branches, they were kept moist by water in dark plastic bags. Then, the branches were re-cut under water. Using the vacuum method, the authors calculated hydraulic conductance. These hydraulic conductance measurements reveal instances of hydraulic failure where the xylem fails to transport water along the entire branch sample.
Anderegg et al, studied seasonal levels of plant water stress using xylem tension measures, between the dates of June 17–19, July 16–18, and August 17–19. These periods reflect the annual peak water stress period in June and July, and end of the peak period in August. Weather was generally clear and sunny with temperatures ranging from 16 and 19 °C when taking the samples. For each set of clones, two randomly selected branches per ramet, and three ramets per class (healthy versus dying) were evaluated. The same method was used to maintain moisture for sample branches as used in measuring inter-year tree mortality. Five minutes after collection, the authors removed twigs longer than 30 cm, two branch forks away from the branch’s break from the trunk. They used this twig to measure branch xylem tension, using a Schlander-type pressure chamber. The process was repeated three times per branch for consistency and accuracy. Branch xylem tension was taken at two points during the day: dawn (when the tension is lowest and plant water is equal with soil water, absent nightly transpiration of water out of the tree’s leaves), and at midday (when highest daily tensions occur, due to transportation).
Another parameter used to predict the probability of tree mortality over time was changes in growth, which also partially accounts for differences in conductivity. The authors measured branch growth and xylem vessel diameter for both the healthy and SAD ramets. Branch growth was measured using annual growth ring width data from the years 1998–2011. All branches studied were eighteen to thirty years old and fully developed before the drought hit from 2000–2003. Using a sliding microtome, 40<!–[if gte msEquation 12]>μ<![endif]–> branch cross-sections were cut and observed under a light microscope (DM3000; Leica) in order to measure ring width. Nine branches per treatment from a June 2011 sample were analyzed for both healthy and SAD treatments. The vessel diameters apparent in growth rings from the years 2003 through 2010 were observed. 350 vessels were measured per growth ring for healthy and SAD treatments. This finding suggests that lasting hydraulic changes are not apparent in vessel diameter. Furthermore, branch growth demonstrated resilience after three years of decreased growth post-drought. Both hydraulic changes and growth are affected by carbon balance within a tree. Growth rates parallel carbon uptake through a positive relationship. This study suggests that changes in hydraulic physiological processes lag behind droughts, but are reversible and play a minor role in hydraulic performance for this species.
Xylem’s vulnerability to cavitation over the course of multiple years limits the effectiveness of cavitation repair as well as restoration of hydraulic conductivity. Cavitation occurs within the xylem when water tension becomes so extreme that the water vaporizes, and the resulting air bubbles fill the water-conducting vessels, thus blocking hydraulic transport. To examine changes in xylem vulnerability, the authors used the standard vulnerability curve technique that measures the percent loss conductivity (PLC) of a branch sample as a function of artificial xylem tension. To determine the vulnerability curves, the authors used either centrifuge or air injection methods to generate simulated cavitation-inducing pressure differences in the tree branches. Embolisms (air blockages) were flushed out of the xylem via vacuum infiltration prior to measuring for the vulnerability curves. The authors used the maximum conductivities for both native and flushed specimens to ensure that flushing out embolisms did not bias results. The lab-tested xylem vulnerability curves were plotted against the baseline native hydraulic conductivity before and after vacuum infiltration, in order to validate the findings. Increased vulnerability to cavitation after exposure to drought stress is called “cavitation fatigue,” indicating a physiological threshold in a tree’s ability to cope with repeated periods of drought. In vulnerability curve analysis, cavitation fatigue is evident in conductivity changes. In the years after experimental drought, increased PLC occurred for ramets subjected to drought, despite similar xylem water tensions and water availability.
                  Finally, the authors monitored xylem water conductance for trees recovering from water stress induced by an experimental drought. To do so, the authors followed the hydraulic performance and health of mature individual ramets during and after the experimental drought. To simulate drought, two separate plots of mature aspen clones were excluded from rainfall from June to August, 2010. Therefore, the mature ramets for each treatment in the experimental group were subject to water stress over the course of the growing season. Anderegg et al. measured xylem tensions and hydraulic conductivity for the month of July in 2010, 2011, and 2012 under these simulated drought conditions. Their findings indicate that delays in tree mortality are associated with a shift toward increased vulnerability to cavitation in ramets exposed to drought. This cavitation fatigue is important in understanding why delayed hydraulic changes are related to forest die-off.
                  Anderegg et al.used ANOVA statistical analysis for time-series data and repeated measures of xylem tensions, loss of conductivity, and vulnerability. Direct group comparisons utilized Student’s t-stest, given normality from the Shapiro Wilkes test.
Twelve of the thirty-six ramets died between August 2010 and July 2011, including both healthy and dying trees. This finding suggests that differences in hydraulic conductivity can be linked to probability of mortality, even years after drought stress. With this finding, the authors were able to create a logistic regression based on August 2010 native branch xlyem water conductance versus the probability of ramet mortality between August 2010 and July 2011. Anderegg et al. concluded that native hydraulic conductance of branches could predict inter-year mortality between 2010 and 2011. The authors tried to determine what caused these differences in conductance. Initially, they considered water stress in dying trees, further amplified by root mortality. However, they found that SAD and healthy ramets did not have significantly different xylem tensions (P= 0.47), meaning that dying and healthy trees did not have different levels of water stress during the typical seasonal periods of early summer months.
To determine the influence of new xylem growth on the hydraulic disparities between dying and healthy trees, the authors tested differences in xylem diameter and growth to find that xylem vessel diameter was smaller overall in the year 2003 than in 2010 for both SAD and healthy branches. Branch growth decreased significantly in SAD ramets for three years after peak drought severity (P=0.01), but branch growth recovered by 2007, and remained constant through the year 2010. This finding suggests that growth in trees can be resilient post-drought, given adequate water supply and decreased hydraulic stress
                  Dying trees’ xylem did not grow slower or experience higher water stress. Still, the authors attempted to determine whether water stress was more damaging for dying than healthy trees. Xylem’s vulnerability to embolism was significantly higher for SAD dying branches than healthy branches (P=0.001). The authors also found this to be true for differences in embolism vulnerability apparent in hydraulic conductivity. Xylem pressure became fifty percent less conductive between pressure ranges of –1.0 MPA for SAD ramets and –2.3 MPA for healthy ramets. SAD branches had substantially lower water conductivity overall. After exposure to drought and thus higher xylem tensions, healthy aspen clones’ vulnerability was higher than that of health clones not exposed to drought with lower xylem tensions.
                  The experimentally-induced drought produced results similar to those seen in SAD areas. Tree mortality lagged post drought, and trees experienced many of the same physiological hydraulic changes. The time it took for crown dieback to occur lagged in the experimental study, as it did in SAD areas. In 2012, two years after water exclusion and drought ceased, moderate differences in dieback between control and drought-exposed ramets were significant (P = 0.1). After more than two years, xylem tensions for both control and drought-subjected ramets were almost identical, and only varied with seasonal rainfall. PLC during experimental drought conditions increased for drought ramets, and remained elevated after the experimental drought stress ended. In 2011 drought-stressed ramets had significantly higher PLC than control ramets (P = 0.01). Therefore, this study suggests that despite identical initial xylem tension conditions from the 2010 experiment, when ramets are subjected to drought, they demonstrate increased vulnerability to cavitation and subsequent mortality post-drought
Anderegg et al. found that delayed and accumulated physiological hydraulic changes affect wide-spread, drought-induced aspen die-off. The hydraulic performance of a tree, demonstrated by xylem water conductance, is an important predictor of tree mortality over time. Dying ramets do not experience different levels of water stress during seasonal drought, indicated by the lack of seasonal xylem tension changes. Furthermore, xylem tensions were similar for drought-exposed and control trees for two years following the experimental drought study. The authors suggest further study into the physiological mechanism behind dying trees’ similar xylem tensions, whether this phenomenon be due to stomatal regulation or concurrent root and leaf mortality. Root mortality does occur with SAD, which may be important for feedback effects from water stress. However, the balanced timing of root and crown mortality requires further research.
This study discusses the role of accumulated physiological hydraulic changes in xylem water conductivity (hydraulic deterioration) in tree die-off.  This deterioration is not reversible, even if normal hydroclimatic conditions are restored. Hydraulic deterioration is related to SAD tree mortality, due to trees’ vulnerability to seasonal water stress prior to severe drought. Trees can tolerate these seasonal droughts, but the accumulated damage from these annual droughts is too much to overcome in the face of severe drought. Logistic regression can be used to predict the probability of mortality across years, based on accumulated hydraulic deterioration.
The authors discuss other possible reasons for die-off, including branch scarring and elevated insect attack that exacerbate hydraulic deterioration. In addition, SAD areas had increased levels of fungal pathogens that could affect hydraulic deterioration. Still, multi-year drought feedback effects, repair of damage, and accumulated stress are crucial to understanding forest’s vulnerability to drought. The ability to repair drought-induced damage is just as important to a tree’s survival as the severity of the damage itself. These results from trembling aspen can be extrapolated to other delayed, post-drought tree death incidences in forest ranging from Canada to the tropical Amazon. The physiological mechanisms at work in trembling aspens are widely applicable across other species. 

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