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Ecophysiological Responses of Tsuga canadensis (Eastern Hemlock) to Projected Atmospheric CO2 and Warming
Lily Wilder and Jennifer N. Boyd

Southeastern Naturalist, Volume 15, Issue 4 (2016): 697–713

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Southeastern Naturalist 697 L. Wilder and J.N. Boyd 22001166 SOUTHEASTERN NATURALIST 1V5o(4l.) :1659,7 N–7o1. 34 Ecophysiological Responses of Tsuga canadensis (Eastern Hemlock) to Projected Atmospheric CO2 and Warming Lily Wilder1 and Jennifer N. Boyd1,* Abstract - Tsuga canadensis (Eastern Hemlock) is a keystone tree species currently experiencing high mortality in southeastern US forests due to Hemlock Woolly Adelgid (Adelges tsugae; HWA) invasion. Investigating the impacts of concurrent climate change on Eastern Hemlock is important given its potential direct effects on this species and possible interactions of climatic factors with the continued spread of HWA. We conducted a 2-factorial experiment in controlled-environment growth chambers to test for the main effects and interactions of projected atmospheric CO2 concentrations and transitional-season warming on gas-exchange traits of Eastern Hemlock saplings collected from a northern Georgia field site. We hypothesized that elevated CO2 would increase photosynthesis and that warming would not influence photosynthesis, given the demonstrated capacity for photosynthetic temperature-acclimation in this species. Saplings in elevated CO2 exhibited ~30% greater leaf-level photosynthesis (A) and ~35% greater maximum light-saturated photosynthesis (Amax) than saplings in ambient CO2. In contrast, warming did not influence A or Amax as a main effect. However, significant treatment interactions suggest that the response of Eastern Hemlock to rising CO2 could be impacted by associated warming. Specifically, when saplings were grown in elevated CO2, warming was associated with reductions in Amax, rate of respiration in the dark, and stomatal conductance, but these variables were not influenced by warming when combined with ambient CO2. An increase in the light-saturation estimate with elevated CO2 across temperature-treatment levels indicates that CO2 can be a limiting factor for Eastern Hemlock, but significant treatment interactions suggest that the capacity for this species to utilize increased CO2 may be impacted negatively by warming. Introduction Tsuga canadensis (L.) Carrière (Eastern Hemlock) is a shade-tolerant conifer typically associated with cool, moist locations throughout eastern North American forests from southern Canada to the southern Appalachians and west to the Great Lakes region (Godman and Lancaster 1990, McWilliams and Schmidt 2000). Eastern Hemlock is notable within these forests for its long life-span and key ecological role—creating distinct abiotic conditions that provide unique habitat for other organisms (Ellison et al. 2005). Dense evergreen foliage of hemlock casts deep shade in forests otherwise dominated by broadleaf deciduous trees and the species is associated with acidic leaf-litter and slow nutrient-cycling (Eschtruth et al. 2006, Orwig et al. 2002). The special abiotic environment associated with Eastern Hemlock has been linked to numerous positive impacts on associated biota, including plants, amphibians, reptiles, birds, and mammals (Benzinger 1994, 1University of Tennessee at Chattanooga, Department of Biology, Geology, and Environmental Science, 615 McCallie Avenue, Chattanooga, TN 37403. *Corresponding author - Jennifer-Boyd@utc.edu. Manuscript Editor: Howard Neufeld Southeastern Naturalist L. Wilder and J.N. Boyd 2016 Vol. 15, No. 4 698 DeGraaf and Rudis 1986, DeGraaf et al. 1992, Elowe 1984, Harrison et al 1989, Holmes and Robinson 1981, Howe and Mossman 1995, Martin 1960, Strickland and Douglas 1987). The decline of Eastern Hemlock due to the ongoing invasion of Adelges tsugae Annand (Hemlock Woolly Adelgid; HWA) is of growing ecological concern given the impacts that the loss of this foundational tree species could have on native ecosystems. In the nearly 70 years since its inadvertent introduction to Virginia from Asia in nursery stock in the 1950s (Cheah et al. 2004), the aphid-like HWA has spread throughout much of the range of Eastern Hemlock (Morin 2009). Once established on an individual tree or group of trees, HWA populations can grow rapidly and cause tree mortality within ~5 y (McClure 1990, 1991; Young et al. 1995). HWA attacks trees of all ages and sizes, and infested trees seldom recover (Orwig and Foster 1998). The spread of HWA into the northern range of Eastern Hemlock occurred earlier than its relatively recent spread into the southern range, which is likely due to differences in host abundance (Morin et al. 2009). However, in its northern range, Eastern Hemlock tends to experience slower rates of decline in response to HWA invasion than trees in the southern range; this difference is thought to be influenced by the intolerance of HWA to colder winter temperatures (Gouli et al. 2000, Skinner et al. 2003). In contrast, increased survival of HWA through winters in the southern range could allow it to spread more quickly (Paradis et al. 2008). Investigating the potential effects of concurrent projected change in climate on Eastern Hemlock is important given its likely direct impacts on this tree species and the possible interactions of climatic factors with HWA. Concentrations of CO2 in the atmosphere have increased about 40% since the Industrial Revolution, primarily due to the anthropogenic burning of fossil fuels (Stocker et al. 2013). Modeled projections suggest that future atmospheric CO2 concentrations could exceed 600 ppm and associated warming of up to 2.5 °C could occur by mid-century (Stocker et al. 2013). Positive responses of tree species to increased atmospheric CO2 via its “fertilization effect” on photosynthesis as a substrate have been well established (Ainsworth and Long 2005, Norby et al. 1999). However, these responses can be species-specific (Dawes et al. 2010, Körner et al. 2005) and can be influenced by resource limitations and other stresses (Karnosky 2003, Saxe et al. 1998). Conifers have been reported to exhibit more-positive growth responses to elevated CO2 in the absence of stress than deciduous trees (Saxe et al. 1998), and late-successional, shade-tolerant species including Eastern Hemlock have been shown to exhibit a larger positive-growth response to increased CO2 than less-shade–tolerant, early-successional species (e.g. Acer rubrum L. [Red Maple] and Pinus strobus L. [White Pine]; Bazzaz et al. 1990, Kinney and Lindroth 1997), although such responses can be influenced by other abiotic factors (Bazzaz and Miao 1993). The positive growth-response of Eastern Hemlock, in particular, to elevated CO2 has been supported by other studies (Eller et al 2011, Godbold et al. 1997), but no published studies to our knowledge have reported the responses of photosynthesis to elevated CO2 in this species as a physiological process underlying such growth responses. Southeastern Naturalist 699 L. Wilder and J.N. Boyd 2016 Vol. 15, No. 4 Photosynthesis also is generally and strongly affected by temperature (Berry and Björkman 1980), and the warming associated with increased atmospheric CO2 has been associated with positive photosynthetic responses (Ainsworth and Long 2005, Dawes et al. 2010, Körner et al. 2005). However, these responses too can be species-specific (Dawes et al. 2010, Hättenschwiler and Körner 2000, Körner et al. 2005, Tjoelker et al. 1998), often reflecting adaptations of photosynthetic enzymes to a range of temperatures that represent species’ native environments (Berry and Björkman 1980). For species adapted to seasonal environments, these adaptations can include the ability to acclimate to seasonal temperature shifts (Berry and Björkman 1980). Across typical seasons, light-saturated (to control for deciduous canopy cover) photosynthesis of Eastern Hemlock has been reported to be relatively consistent (Adams and Loucks 1971, Burkle and Logan 2003, Hadley 2000, Hadley and Schledbauer 2002), suggesting that this species has a high capacity for photosynthetic temperature acclimation. Previous research conducted in growth chambers also reported no detectable effect of temperature ranging from 15 °C to 30 °C on Eastern Hemlock photosynthesis (Hadley 2000). Such acclimation was further evidenced by measures of photosynthetic response to temperature of seedlings from southern Wisconsin that revealed a shift in their photosynthetic temperature optima from 13 °C to 17 °C from late spring to mid-summer. Yet, tree-ring chronologies have revealed that the growth of Eastern Hemlock is responsive to temperature and that such responses may be seasonally dependent, generally exhibiting negative correlations with summer temperatures and positive correlations with winter temperatures (see Cook and Cole 1991, D’Arrigo et al. 2001, Farahat et al. 2016, Hart et al. 2010). Such findings suggest that temperatures during especially warm summers may extend beyond optimum temperatures for photosynthetic-enzyme function, but tree-ring chronologies also have revealed the importance of moisture availability as influenced by relationships between temperature and precipitation on growth (see Abrams et al. 2000, Cook and Cole 1991, Cook and Jacoby 1977, D’Arrigo et al. 2001, Hart et al. 2010, Saladyga and Maxwell 2015). During the warmest parts of the year, water stress can can have a greater influence than temperature on plant photosynthetic performance (Berry and Björkman 1980); thus, it is possible that negative correlations of Eastern Hemlock to summer temperatures could be affected by the possible influence of temperature on moisture avai lability. Although tree-ring chronologies have described the responses of Eastern Hemlock to past climate, limited empirical research has examined the effects of CO2 or temperature on Eastern Hemlock growth (e.g., Adams and Louck 1971, Bazzaz et al. 1990, Eller et al. 2011, Hadley 2000) and/or underlying physiology (e.g., Adams and Loucks 1971, Burkle and Logan 2003, Hadley 2000, Hadley and Schledbauer 2002) within the context of contemporary climate, and no studies to date have examined future projections of these factors in combination. To explore the main effects and interactions of CO2 and temperature on saplings collected from a southern Eastern Hemlock population, we conducted a 2-way factorial experiment in controlled-environment growth chambers. We chose to focus on ecophysiological Southeastern Naturalist L. Wilder and J.N. Boyd 2016 Vol. 15, No. 4 700 traits linked to photosynthesis as performance measures due to the difficulty of resolving significant growth and reproductive responses of long-lived species to climate change. However, plant growth and reproduction fundamentally entail energetic expenses (Mooney 1972), and photosynthesis has been linked mechanistically to tree growth within the context of climate change (Reich et al. 2015). We focused on warming that would characterize transitional seasons (i.e., spring, fall), in particular, because Eastern Hemlock saplings in deciduous forests are proposed to achieve their greatest net photosynthetic rates during spring (Hadley 2000), and saplings were collected for our research with data collection planned for fall. We hypothesized that increased CO2 would be benefit the photosynthetic performance of saplings given the well-established “fertilization effect” associated with this factor (Ainsworth and Long 2005), while associated warming would not significantly influence photosynthetic performance given the demonstrated capacity of Eastern Hemlock to acclimate to a range of temperatures that would include warmer spring and fall temperatures in our region (Adams and Loucks 1971, Burkle and Logan 2003, Hadley 2000, Hadley and Schledbauer 2002). Methods Saplings We collected 80 Eastern Hemlock saplings ranging from 15 cm to 45 cm in height from a forest understory located on private property near Dahlonega, GA, in late August 2015. We loosely wrapped the plants’ root balls in wet newspaper and transported them to the lab on the same day. The following day, we potted the saplings separately in ~3.75-L (~1-gallon) nursery containers (Pro Cal I-300, Pro Cal, South Gate, CA) filled with 1 part field-collected soil to 3 parts standard commercially available potting soil (Potting Soil, Evergreen, San Jose, CA). Although there was no observable presence of HWA on saplings, we treated all individuals prophylactically with insecticidal soap, as well as with a broad-spectrum antifungal treatment (Neem, Natural Guard, Bonham, TX) to kill any fungal infections obtained in the field. We added the manufacturer’s suggested amount of slowrelease fertilizer (Osmocote, The Scotts Company, Marysville, OH) to each pot and watered to saturation; we watered the pots to saturation every other day for the first few weeks, and then every 2–4 days as needed. We divided the saplings into 4 even groups (20 individuals/group) such that each group included a broad representation of sapling sizes and observable health conditions. We assigned each group to a growth chamber (PGR15, Conviron, Winnipeg, MB, Canada) set to replicate CO2, temperature, photoperiod, light intensity, and relative humidity typical of transitional-season (i.e., spring and fall) conditions in northern Georgia for a 4-week acclimation period. We rotated pot locations within chambers twice a week to account for micro-environmental heterogeneity. Environmental treatments Following the 4-week acclimation period, we programmed the chambers to replicate all possible combinations of CO2 and temperature-treatment levels. Ambient Southeastern Naturalist 701 L. Wilder and J.N. Boyd 2016 Vol. 15, No. 4 and elevated CO2 were set at 400 ppm and 600 ppm, respectively. We set low and high temperature at 12/20 °C and 14/22 °C (night/day), respectively. Our elevated CO2 and high-temperature treatment levels were designed to simulate mid-century projections of these independent variables for the balanced A1B emissions scenario (Stocker et al. 2013). We chose to use near-future projections rather than late-century projections in our research due to the ongoing rapid decline of Eastern Hemlock as a result of HWA. Our experimental design included a different combination of CO2 and temperature in each of the 4 chambers: ambient CO2/low temperature, ambient CO2/high temperature, elevated CO2/low temperature, and elevated CO2/ high temperature. We programmed light regimes and relative humidity similarly in all chambers to provide conditions typical of the understory habitat in Eastern Hemlock forests; all saplings were watered every 2– 3 d with an equal amount of water such that plants in the ambient CO2/low temperature chamber were saturated. The saplings remained in their respective treatment combinations for an additional 6 weeks to acclimate prior to gas-exchange measurements. However, we rotated pots within chambers twice weekly to control for potential spatial differences in microclimate, and randomly reassigned the treatment levels assigned to each chamber each week and moved all saplings accordingly to help minimize any chamber effects and alleviate issues of pseudoreplication (Gibson 2014). Physiological performance Following the 6-week treatment-acclimation period, we measured leaf-level gas exchange during late mornings and early afternoons on a 3-cm length of the uppermost-branch tip of all saplings per treatment combination with a portable gasexchange analyzer (LI-COR 6400, Lincoln, NE) equipped with a CO2 module. The mortality of 6 saplings during the acclimation periods resulted in a slightly unbalanced sample design of 17–20 individuals per treatment combination. We generated a photosynthetic response to light level (an A/PAR curve) for each sapling by measuring the steady-state responses of photosynthesis to external photosynthetically active radiation (PAR) provided by blue–red light-emitting diodes mounted above the leaf cuvette at 12 setpoints from 0 μmol to 1600 μmol photons m-2 s-1. Eastern Hemlock is not a broadleaf species; thus, we estimated leaf area in accordance the protocol described for this species by Nelson et al. (2014). With the exception of PAR, conditions inside the leaf cuvette were set to replicate conditions inside the growth chamber in which each measured sapling was housed, including CO2, maximum midday temperature regulated with thermoelectric coolers, and relative humidity. We collected light-response measurements over the course of 1 month, typically with 3–5 saplings measured per day. We moved between chambers to select saplings for measurements each day to prevent inadvertent influence of time on measured variables. Data analyses We analyzed A/PAR curves to calculate net photosynthesis (A) and needle transpiration (E) at ambient PAR, the maximum rate of photosynthesis at saturating PAR (Amax), maximum apparent quantum yield (ɸ), the light-compensation point Southeastern Naturalist L. Wilder and J.N. Boyd 2016 Vol. 15, No. 4 702 (LCP), and the light-saturation estimate associated with 90% (LSE) of Amax. We conducted analyses of light-response variables by fitting measured A/PAR curves to the nonrectangular model described by Prioul and Chartier (1977) with the Excel Solver utility (Version 14.5.7., 2011, Microsoft, Redmond, WA). We determined the rate of respiration in dark (RD) as the intercept of the lower part of the A/PAR curves with the y-axis. We averaged all leaf-level gas-exchange variables across treatments. Means of select measured variables were used to model average A/PAR curves for each treatment-level combination with the Prioul and Chartier (1977) model. We employed a 2-way analysis of variance (ANOVA) model to assess the main effects and interaction of our CO2 and temperature treatments on all measured gas-exchange variables (SPSS for Windows, Rel. 7.5.1, 2016, SPSS, Inc., Chicago, IL). We considered mean differences significant if P ≤ 0.05. In the case of a significant interaction, we compared means of treatment combinations with leastsignificant difference (LSD) post-hoc analysis at the 0.05 probability level. Results Average A/PAR curves modeled using mean photosynthetic light-response characteristics illustrated differences among CO2 and temperature treatment groups in photosynthetic responses to external PAR (Fig. 1). Saplings growing in elevated CO2 and low temperature exhibited the greatest overall photosynthetic response to increasing PAR, while saplings growing in the ambient CO2 and low-temperature treatment exhibited the most-minimal response to elevated PAR. Elevated CO2 was associated with a ~30% increase in mean A (F1, 70 = 5.273, P = 0.025) and a ~35% increase in mean Amax (F1, 70 = 7.527, P = 0.008) of saplings (Table 1). However, measures of respiratory-carbon loss and the ratio of net photosynthesis to dark respiration (A/RD) were not significantly influenced by CO2 (Table 1). Specifically, there were no significant differences in RD (F1, 70 = 0.027, P = 0.870), or A/RD (F1, 70 = 0.277, P = 0.635) between our CO2 treatment levels (Table 1). Similarly, CO2 did not significantly influence E (F1, 70 = 1.093, P = 0.299) or stomatal conductance (gs) (F1, 70 = 0.548, P = 0.462; Table 1). In contrast to the influence of CO2 on photosynthesis, temperature did not significantly influence mean A (F1, 70 = 0.007, P = 0.935) or Amax (F1, 70 = 0.47, P = 0.830) as a main effect. There also was no significant effect of temperature on mean RD (F1, 70 = 0.001, P = 0.970). As a result, A/RD also was not influenced significantly by temperature (F1, 70 = 0.322, P = 0.572). Similarly, there was no significant influence of temperature on E (F1, 70 = 1.116, P = 0.294) or gs (F1 ,70 = 0.848, P = 0.360; Table 1). Although temperature did not influence significantly basic measures of photosynthesis and respiration as a main effect, the interaction of temperature and CO2 significantly influenced both Amax (F1, 70 = 5.303, P = 0.024) and RD (F1, 70 = 7.625, P = 0.007). Specifically, Amax was not influenced by CO2 when saplings were exposed to warming, (P = 0.762), but Amax increased with elevated CO2 in the absence of warming (P ≤ 0.001) (Fig. 2a). Similarly, RD was not influenced by CO2 when saplings were exposed to warming (P = 0.078); but in the absence of warming, RD Southeastern Naturalist 703 L. Wilder and J.N. Boyd 2016 Vol. 15, No. 4 increased with elevated CO2 (P = 0.037), although the increase was not statistically significant. In addition, although not significant, RD exhibited a trend in response to warming when CO2 was elevated (P = 0.052), but an increasing trend in response to warming in ambient CO2 (P = 0.057; Fig. 2b). Leaf gs also was significantly influenced by the interaction of temperature and CO2 (F1, 70 = 3.854, P = 0.050). Specifically, gs was not influenced by CO2 when saplings were exposed to warming (P = 0.403), but gs under conditions of elevated CO2 was higher in the absence of warming (P = 0.050). In elevated CO2, gs decreased in response to warming (P = 0.045), but was not affected by warming in ambient CO2 (P = 0.464) (Fig. 2c). In contrast, there were no significant interactions of CO2 and temperature on A (F1, 70 = 0.007, P = 0.562), A/RD (F1, 70 = 3.567, P = 0.063), or E (F1, 70 = 2.323, P = 0.132). Elevated CO2 was associated with a ~30% increase in LSE (F1, 70 = 4.126, P = 0.046; Table 1). However, other measures of photosynthetic light responses were not significantly influenced by CO2 (Table 1). Specifically, there were no significant differences in mean ɸ (F1, 70 = 1.141, P = 0.289) or LCP (F1, 70 = 0.243, P = 0.623) Figure 1. Curves depicting mean leaf-level photosynthetic response of Eastern Hemlock saplings to photosynthetically active radiation (PAR). Curves were generated using the light-response model developed by Prioul and Chartier (1977). Solid lines and dashed lines represent saplings grown in ambient CO2 (400 ppm) and elevated CO2 (600 ppm), respectively. Filled and open circles represent saplings grown in ambient (20 °C) and elevated temperature (22 °C), respectively. Error bars represent ± 1 SE of the mean. Southeastern Naturalist L. Wilder and J.N. Boyd 2016 Vol. 15, No. 4 704 between our CO2 treatment levels (Table 1). As with basic measures of photosynthesis and respiration, temperature had no significant influence as a main effect on any measured photosynthetic light responses, including LSE (F1, 70 = 0.475, P = 0.493), ɸ (F1, 70 = 0.405, P = 0.526), and LCP (F1, 70 = 0.363, P = 0.549). Although temperature did not significantly influence any of our measured photosynthetic light-response variables, the interaction of temperature and CO2 significantly influenced LSE (F1, 70 = 5.690, P = 0.020). Specifically, LSE was reduced by warming (P = 0.033) when saplings were grown in elevated CO2, but LSE was not influenced by warming when saplings were grown in ambient CO2 (P = 0.235; Fig. 2d). In contrast, there were no significant interactions of CO2 and temperature on ɸ (F1, 70 = 0.462, P = 0.499) or LCP (F1, 70= 0.097, P = 0.757). Discussion Potential impacts of climate change on Eastern Hemlock Overall, our findings support our hypothesis that increased CO2 enhances Eastern Hemlock photosynthesis but that warming as a main effect does not (Table 1; Table 1. The main effects of CO2 and temperature on leaf-level gas-exchange characteristics of Eastern Hemlock saplings, including net photosynthesis (A), maximum rate of photosynthesis at saturating light level (Amax), dark respiration (RD), A/RD, transpiration (E), stomatal conductance (gs), maximum apparent quantum yield (ɸ), light compensation point (LCP), and light saturation estimate (LSE). Saplings were grown in 2 CO2 treatment levels (ambient: 400 ppm, elevated, 600 ppm) crossed with 2 temperature levels (ambient: 20 °C, elevated 22 °C) in controlled-environment growth chambers. Values presented are means ± 1 SE; values followed by the same superscript letter are not statistically different at the P ≤ 0.05 level of significance. Ambient CO2, n = 35; elevated CO2, n = 35; ambient temperature n = 38; elevated temperature, n = 32. Independent variable Dependent variable Ambient Elevated CO2 A (μmol CO2 m-2 s-1) 2.9 ± 0.18A 3.7 ± 0.30B Amax (μmol CO2 m-2 s-1) 5.5 ± 0.34A 7.4 ± 0.59B RD (μmol CO2 m-2 s-1) 1.1 ± 0.15A 1.2 ± 0.16A A/RD 5.7 ± 1.14A 6.6 ± 1.64A E (mmol H2O m-2 s-1) 0.22 ± 0.026A 0.35 ± 0.117A gs (mmol H2O m-2 s-1) 12.7 ± 1.75A 13.8 ± 1.76A ɸ (mol CO2 mol-1 photons) 0.046 ± 0.0139A 0.030 ± 0.0046A LCP (μmol photons m-2 s-1) 8.9 ± 1.76A 10.4 ± 2.19A LSE90 (μmol photons m-2 s-1) 569.3 ± 45.17A 750.4 ± 73.91B Temperature A (μmol CO2 m-2 s-1) 3.3 ± 0.27A 3.3 ± 0.24A Amax (μmol CO2 m-2 s-1) 6.5 ± 0.57A 6.4 ± 0.41A RD (μmol CO2 m-2 s-1) 1.1 ± 0.17A 1.1 ± 0.15A A/RD 5.6 ± 1.0A 6.7 ± 1.76A E (mmol H2O m-2 s-1) 0.34 ± 0.114A 0.22 ± 0.028A gs (mmol H2O m-2 s-1) 14.1 ± 1.73A 11.6 ± 1.78A ɸ (mol CO2 mol-1 photons) 0.034 ± 0.0127A 0.043 ± 0.0070A LCP (μmol photons m-2 s-1) 8.8 ± 1.67A 10.6 ± 2.29A LSE90 (μmol photons m-2 s-1) 685.4 ± 67.82A 631.4 ± 56.94A Southeastern Naturalist 705 L. Wilder and J.N. Boyd 2016 Vol. 15, No. 4 Figure 2. (a) mean leaf-level maximum rate of photosynthesis at saturating light (Amax), (b) the rate of respiration in dark (RD), (c) rate of stomatal conductance (gs), and (d) the light-saturation estimate associated with 90% of the maximum rate of photosynthesis at saturating light (LSE) of fieldcollected Eastern Hemlock saplings collected grown in controlled-environment growth chambers under interacting ambient (400 ppm) and elevated (600 ppm) CO2 and ambient (20 °C) and elevated (22 °C) temperature treatment levels. Values shown below the same letter are not significantly different at the P ≤ 0.05 level of significance. Error bars represent ± 1 SE of the mean. Southeastern Naturalist L. Wilder and J.N. Boyd 2016 Vol. 15, No. 4 706 Figs. 1, 2). We also found that increased CO2 was associated with an increase in light-saturated Amax, suggesting that CO2 currently could be a limiting factor for this species in relatively high-light environments. In contrast, respiratory-carbon loss (RD) was not affected by CO2 or temperature. When CO2 and temperature are considered as single factors, these findings suggest that Eastern Hemlock may benefit from projected climate change. However, CO2 and temperature are projected to rise concurrently, and our results also reveal that CO2 and warming could counteract the effects of each other. In particular, Amax increased with elevated CO2 when samplings were grown in current temperatures, but warming negated the positive effect of elevated CO2 on Amax (Fig. 2a). This offset may be explained by concurrent reductions in stomatal conductance in response to warming for saplings grown in elevated CO2 (Fig. 2c), which could reduce carbon uptake. An associated decrease in RD (Fig. 2b) may have balanced reductions in carbon gained, resulting in overall similarities in A/RD. Reductions in leaf respiration have been reported as a common response to elevated CO2 (Wullschleger et al. 1994), and could result in greater carbon-use efficiency given a concurrent increase in photosynthesis as is typical under elevated CO2 (Ziska and Bunce 1998). Although decreased respiration rates are not a common response to increasing temperature, the increase in respiration typically associated with warming can be lost to acclimation resulting from enzymatic processes and biochemical changes (Amthor 1997, Curtis and Wang 1998, Drake et al. 1997, Gifford 1994, Gonzalez-Meler et al. 2004, Larigauderie and Körner 1995). Decreases in leaf respiration and stomatal conductance are common responses to water stress (Atkin et al. 2005, Chaves et al. 2002), which can be exacerbated by warming. Although not significant, the declining trend of RD of Eastern Hemlock with warming in elevated CO2 could be influenced by a possible combination of inhibition of respiration in response to elevated CO2 and potential water-stress associated with our warming treatment. While we did not measure soil-moisture availability, we concede that restrictions of rooting volume imposed by pots could have exacerbated water stress (see Poorter et al. 2012) in our experiment, especially for saplings grown with warming. Our ecophysiological findings could provide a mechanistic explanation for previous research that demonstrated a positive influence of increased CO2 on Eastern Hemlock growth when grown in temperatures representing warm, summer field conditions in controlled-environment growth chambers (Bazzaz et al. 1990). Although studies based on tree-ring chronologies have reported that Eastern Hemlock growth is negatively affected by warming (e.g., Cook and Cole 1991, D’Arrigo et al. 2001, Hart et al. 2010, Farahat et al. 2015), such responses could also be caused by reduced soil-moisture availability associated with high summer temperatures (see Abrams et al. 2000, Cook and Cole 1991, Cook and Jacoby 1977, D’Arrigo et al. 2001, Hart et al. 2010, Saladyga and Maxwell 2015). Analyses of Eastern Hemlock decline and delayed recovery during the mid-Holocene also have demonstrated its sensitivity to soil moisture (Marsicek et al. 2013, Oswald and Foster 2012). We suggest that the transitional-season warming treatment used in our experiment may not have exceeded the tolerable temperature range of this Southeastern Naturalist 707 L. Wilder and J.N. Boyd 2016 Vol. 15, No. 4 species and/or caused enough moisture stress to negatively affect its photosynthetic performance as a main effect. Further investigations of the responses of Hemlock to climate-change scenarios for other seasons could benefit our understanding of its responses to climate change, and such investigations also could consider further projections of future climate change. We chose to assess the effects of near-future (i.e., mid-century versus late century) projections of CO2 and temperature given the rapid mortality of Eastern Hemlock in our region due to the ongoing HWA invasion, but climate-change predictions for the year 2100 assert that CO2 and temperature will continue to rise (Stocker et al. 2013). If Eastern Hemlock survives to 2100 and beyond in the face of HWA invasion, we suggest that a continued fertilization effect of CO2 could further enhance the photosynthetic performance of this species; however, it is possible that associated warming could exceed its temperature range and/or increase water stress in the absence of increased precipitation. Although our experiment explored the climate-change responses of Eastern Hemlock saplings from its southern range, the wide geographic distribution of this species could make it important to consider potential local adaptations across its range in response to climate change. Particularly, because northern and southern populations of this species are genetically distinct (Potter et al. 2012), they may be characterized by strong local adaptations to climate. Studies have shown an increased sensitivity to warming in populations of tree species near their highesttemperature range limits relative to other locations (Reich et al. 2015), yet our ecophysiological findings suggest that the sensitivity of saplings collected from their southern limits to spring warming is minimal. In contrast, other plant species have been characterized by significantly higher sensitivity to warming in their northern versus southern ranges, suggesting that populations near warm range limits may be better adapted to warming (Peñuelas et al. 2004). In addition to the potential for local adaptations along a latitudinal temperature gradient to influence responses to climate change, previous research has suggested that temperature- and moisture-sensitive ecotypes of this species can exist among microsites distributed across regions (Kessell 1979). In the southeastern US, intermediate ecotypes also may exist (Kessell 1979), suggesting that climate-change responses could vary across populations even at relatively small spatial scales, a possibility that warrants further investigation. Implications for Eastern Hemlock within the context of HWA The impact of climate change on insect damage of trees has become apparent in many forest systems, with warming and associated drought often implicated as detrimental (Kurz et al. 2008, Logan et al. 2003). Such outcomes are influenced ultimately by a combination of the responses of trees to climate change, as well as the indirect impacts of climate change on insect interactions (Dukes et al. 2009). In part, such interactions between climate and insect activity are thought to have contributed to the decline of Eastern Hemlock during the mid-Holocene (Foster et al. 2006). Though insects are unlikely to be affected directly by increased CO2, insects can be affected indirectly by associated nutritional changes in their plant Southeastern Naturalist L. Wilder and J.N. Boyd 2016 Vol. 15, No. 4 708 hosts (Robinson et al. 2012). In Eastern Hemlock, increased CO2 has been associated with a greater foliar carbon-to-nitrogen ratio (Eller et al. 2011), a change in biochemistry that could represent diminished nutrient quality for the HWA and influence its feeding pressure and/or fitness (see Bezemer and Jones 1998, Stiling and Cornelissen 2007). Warming also could indirectly influence Eastern Hemlock through its impacts on HWA feeding activity and/or fitness becaus insect physiological and behavioral processes can be highly temperature dependent (see Irlich et al. 2009). Although our research increases understanding of the response of Eastern Hemlock to rising CO2 and temperature as climate change factors, the extent to which responses to climate change will impact this species within the context of ongoing HWA infestation remains unknown. We did not include an HWA treatment in our experiment, but our investigation of the combined effects of increased CO2 and warming establishes an important baseline of the direct responses of this species to concurrent climate change factors. Further research investigating the impacts of such factors on HWA fitness and resultant indirect impacts on Eastern Hemlock would improve the future prognosis of this species in the context of concurrent climate change and HWA invasion. Acknowledgments We are very grateful to Donna Shearer of the non-profit organization Save Georgia’s Hemlocks for providing the saplings used in this project. We also appreciate the support of the University of Tennessee at Chattanooga Departmental Honors Program and Drs. Hope Klug and Yukie Kajita for their advisement and review of an earlier draft of this manuscript. We thank 2 anonymous peer reviewers who provided comments on an earlier draft of this publication. 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