Linking leaf-level morphological and physiological plasticity to seedling survival and growth of introduced Canadian sugar maple to elevated precipitation under warming

Forest Ecology and Management 457 (2020) 117758

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Linking leaf-level morphological and physiological plasticity to seedling survival and growth of introduced Canadian sugar maple to elevated precipitation under warming ⁎

Yingying Zhua,b, Chen Chenb, Yuan Guoa, Songling Fua, , Han Y.H. Chenb,c,

T

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a

College of Forestry and Landscape Architecture, Anhui Agricultural University, Hefei, Anhui 230036, China Faculty of Natural Resources Management, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario P7B 5E1, Canada c Key Laboratory for Humid Subtropical Eco-Geographical Processes of the Ministry of Education, School of Geographical Sciences, Fujian Normal University, Fuzhou 350007, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Species introduction Leaf traits Elevated precipitation Seedling growth Sugar maple

Global climate change will lead to new combinations of temperature and precipitation patterns. Extreme precipitation events will become more common by the late 21st century due to anthropogenic warming, particularly in high latitudes; however, the specific responses of high latitude species are mostly unknown. We employed an introduction trial by transplanting temperate sugar maples (Acer saccharum Marsh) from three provenances of their native ranges within Canada, which had a mean annual temperature (MAT) ranging from 3.0 to 9.4 ℃, and mean annual precipitation (MAP) from 520 to 1190 mm. These maples were transplanted to four sites in subtropical China with a MAT of 15.8–16.2 ℃ and a MAP of 913 mm, 1001 mm, 1172 mm, 1490 mm, respectively. We measured the survival and growth of the first-year seedlings of all provenances, as well as the survival, growth, and leaf morphological and physiological traits of the seedlings of Ontario provenance after four years of acclimation. We found that the first-year survival and growth of all provenances increased with the MAP at the planting sites, peaked at 1172 mm, and then decreased at the 1490 mm MAP site, with the seedlings of Ontario provenance had the best overall survival and growth rates across all of the sites. At the end of the 4th growing season, the seedlings of the Quebec and Manitoba provenances died off, and the survival- and growth-MAP relationships of the Ontario provenance mirrored those observed in the first year. For the Ontario provenance at year 4, the major vein density, stomatal density and size, and non-structural carbohydrates (NSC) changed significantly with MAP at the introduced sites. Principal component analysis indicated that seedling survival was negatively associated with stomatal size, density and starch content and that growth was positively related to leaf major vein density and negatively to soluble sugar and NSC. Our results indicate that changes in the precipitation range determined the performance of introduced sugar maple at the introduced sites. A moderate increase in precipitation was observed to ameliorate heat stress and benefit plant growth; however, excessive water could result in a fatal effect. Moreover, our findings suggest that the plasticity of leaf morphological and physiological traits influenced the survival and growth of the introduced sugar maple under future precipitation and warming.

1. Introduction In the context of global warming, climatic change induces new combinations of temperature and precipitation patterns that can be critical in terms of species distribution and performance (Luo and Chen, 2015; Urban, 2015). Warming is consistently predicted at a global scale and projected to occur at twice the rate of the global average at high

latitudes by the late 21st century (IPCC, 2014). However, the natural pace of species expansion is insufficient to keep up with anticipated global warming rates (Walck et al., 2011). Changes in precipitation, however, are strongly region-dependent (Allan and Soden, 2008; Shongwe et al., 2009). Particularly, heavy precipitation events are expected to occur more frequently in warmer climates, leading to longer dry spells between events, higher risks of floods, and limited species

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Corresponding authors at: College of Forestry and Landscape Architecture, Anhui Agricultural University, Hefei, Anhui 230036, China (S. Fu). Faculty of Natural Resources Management, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario P7B 5E1, Canada (H.Y.H. Chen). E-mail addresses: [email protected] (Y. Zhu), [email protected] (C. Chen), [email protected] (Y. Guo), [email protected] (S. Fu), [email protected] (H.Y.H. Chen). https://doi.org/10.1016/j.foreco.2019.117758 Received 9 August 2019; Received in revised form 27 September 2019; Accepted 2 November 2019 Available online 12 December 2019 0378-1127/ © 2019 Elsevier B.V. All rights reserved.

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Table 1 Climatic characteristics of study sites and the three provenances of sugar maple. Site

Ontario

Quebec

Manitoba

Fuyang

Shucheng

Hefei

Jixi

Latitude Longitude MAT (°C)

43°40′ N 79°24′ W 9.4 8.8 25.3 26.0 −2.3 −4.0 831.1 721 216 203

46°48′ N 71°23′ W 4.2 4.5 23.6 23.7 −10.7 −9.9 1190 1273 337 339

49°55′ N 97°14′ W 3.0 3.4 24.8 25.8 −14.3 −14.0 521 446 247 217

32°93′ N 115°79′ E 15.5 15.8 31.2 30.9 3.4 4.3 913 1082 509 469

31°40′ N 117°08′ E 16.2 16.8 31.1 31.4 4.4 5.5 1001 1226 442 517

31°87′ N 117°25′ E 15.8 16.6 31.0 31.2 4.2 5.5 1172 1250 484 485

30°15′ N 118°53′ E 15.8 16.5 31.5 31.2 4.7 5.2 1490 1819 625 691

MSMT (°C) MWT (°C) MAP (mm) MSP (mm)

Mean annual temperature (MAT); mean summer maximum temperature (MSMT); mean winter temperature (MWT); mean annual precipitation (MAP); mean summer precipitation (MSP). The climatic variables shown correspond to the long-term mean average (above line, 1981–2010) and the experimental years (below line, 2014–2017).

temperatures in temperate-subtropical biomes (Zhu et al., 2011). Leaf stomata regulate the exchange of CO2 and water across all spatial and temporal scales, where their morphology and distribution tend to change with the environment, as the plant optimizes gas exchange and water-use efficiency (Hetherington and Woodward, 2003). Short-term controlled experiments have shown that stomatal density increases with water availability (Carins Murphy et al., 2014), but the responses of leaf stomatal traits to increasing temperatures are inconsistent (Zheng et al., 2013; Wu et al., 2018). Non-structural carbohydrates (NSC) include soluble sugar and starch (Dickman et al., 2015). The NSC reserves allow woody species to overcome periods of stress and disturbance (O'Brien et al., 2017). Mechanistically, NSC assists with maintaining hydraulic functionality under drought conditions by optimizing the balance between carbon supply (i.e., photosynthesis) and carbon demand (i.e., growth) (O'Brien et al., 2017). However, the linkages between NSC allocation and increased precipitation have not been well investigated. To determine the effects of increased precipitation on tree species under extreme warming, we introduced sugar maple from Southern (Toronto, Ontario), Central (Quebec City, Quebec), and Northern (Winnipeg, Manitoba) Canada to subtropical China. We transplanted sugar maple seedlings at four nursery sites with an increased MAP and a ~6 ℃ higher mean annual temperature (MAT) than their origins. We measured the whole-plant performance (survival and growth) as well as the morphological, anatomical, and physiological leaf traits of the transplanted seedlings over the course of four years. Our central hypotheses included that: 1) a higher MAP would increase the survival and growth of the introduced sugar maple seedlings of all provenances, as sufficient water availability could relieve heat stress; 2) the morphological and physiological leaf traits would be mechanistically linked with survival and growth across the MAP gradient.

regeneration (Benito-Garzon et al., 2013; Bucharova et al., 2016; Putnam and Reich, 2017). However, there is a limited understanding of the responses of plant/tree species to the combined effects of warming and changing precipitation in extreme cases. The anthropogenic introduction of species may mitigate spatial and temporal barriers (Chapman et al., 2016). In recent years, assisted migration has advocated filling the gap between the natural migration capacities of species and the increasingly rapid pace of climate change (Gray et al., 2011; Bucharova, 2017). Given the rate of anticipated climate change, the range and effect of assisted migration require further exploration. Motivated by Chinese introduction programs of “948” at the beginning of the 21st century, North American tree species have been widely introduced into China (Liu, 2010). The introduction of sugar maple (Acer saccharum Marsh) may offer new economic opportunities for China, due to the value of maple syrup and wood quality. We have previously shown that summer heat stress was the limiting factor for introduced sugar maple seedlings at one site in subtropical China (Zhu et al., 2019). It is unclear whether the acclimation of the species has occurred over time. The combined effects of temperature and precipitation are vital drivers of species performance. Warming alone may not damage carbon assimilation (Way et al., 2013). Rather, water availability limits the capacity of plants to withstand warming (Hoeppner and Dukes, 2012; Canham and Murphy, 2016). Moreover, warming accompanied by increased precipitation typically stimulates plant growth according to a global meta-analysis of 85 studies (Wu et al., 2011). Previous studies of boreal and temperate species, including sugar maple, show robust photosynthesis and respiration acclimation to moderately warmer temperatures (Sendall et al., 2015; Reich et al., 2016), with increased survival and growth under longer growing seasons when water is not a limiting factor (Keenan et al., 2014). In this study, we hypothesize that increased mean annual precipitation (MAP) will help mediate the adverse effects of heat stress on our introduced sugar maple. Leaf traits are useful predictors of whole-plant performance, and the leaf morphological and physiological trade-offs underlie the survival and growth of species that are introduced into new environments (Poorter and Bongers, 2006; Nicotra et al., 2010; Onoda et al., 2017). On a global scale, leaf traits are strongly influenced by precipitation and temperature (Ordoñez et al., 2009). Leaf area tends to decrease with decreasing MAP as smaller leaves are better at shedding expelling heat under low precipitation (Peppe et al., 2011). Further, changes in leaf area lead to the modification of its shape (McDonald et al., 2003). The major veins in leaves conduct water throughout the lamina and affect leaf life span, stomatal conductance, photosystem yields, and hydraulic conductance (Sack et al., 2003; Brodribb et al., 2010). The minor vein network, which provides “conductive overload” capacities to the major veins, experiences significantly increased activity with higher

2. Materials and methods 2.1. Study sites To examine the effects of precipitation on the transplanted sugar maple seedlings, we conducted our experiments at four research nursery sites in Central China (Fuyang, Shucheng, Hefei, and Jixi), which featured an increasing MAP gradient with little variation in mean annual temperature (MAT) (Table 1). These four sites are home to a typical subtropical monsoon climate, with a hot and rainy summer (June to August). Approximately 75% of the MAP is distributed during the growing season (April to October). The mean MAT of the four sites was 15.8 ℃, whereas the maximum and minimum monthly temperatures were 32.3 ℃ in July and 2.7 ℃ in January. All sites were frost-free for 218–240 days, and the soils belonged to yellow–brown Alisols based on the IUSS Working Group WRB (2014). 2

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Fig. 1. Survival, height, and collar diameter of the sugar maple seedlings of three provenances at four precipitation levels during the first growing season. Values are bootstrapped means ± 95% confidence intervals (CIs). Differences are statistically significant at α = 0.05 when CI does not overlap the other mean. Table 2 Effects of precipitation (P), provenance (R), and sample date (D) on the survival and growth of first-year sugar maple seedlings. Predictors

Survival df

P R D P×R P×D R×D P×R×D

3, 24 2, 24 2, 48 6, 24 6, 48 4, 48 12, 48

Collar diameter P

df −16

< 2.2 × 10 < 2.2 × 10−16 < 2.2 × 10−16 1.4 × 10−14 1.7 × 10−12 2.1 × 10−9 6.2 × 10−8

Height P

3, 24 2, 25 2, 912 6, 25 6, 912 4, 912 12, 912

−16

< 2.2 × 10 7.0 × 10−8 < 2.2 × 10−16 3.8 × 10−9 < 2.2 × 10−16 < 2.2 × 10−16 < 2.2 × 10−16

df

P

3, 24 2, 26 2, 912 6, 26 6, 912 4, 912 12, 912

< 2.2 < 2.2 < 2.2 < 2.2 < 2.2 < 2.2 < 2.2

× × × × × × ×

10−16 10−16 10−16 10−16 10−16 10−16 10−16

Linear mixed-effects model fit tests used Kenward–Roger’s method for denominator degrees of freedom (df).

Quebec City, Quebec (QC), and Winnipeg, Manitoba (MB), respectively. To mimic the chilling requirement (overwinter to break seed dormancy), we stratified the seeds into moist sands at 4 °C for three months from December to February. We sowed the seeds as described by Zhu et al. (2019), at a density of 250 seeds m−2 in nursery benches in the spring of 2014. The first-year seedlings were thinned in November 2014, and the plantation density was 1 × 1 m. During the growing season, weed competition was manually controlled.

For each precipitation level, we selected a local nursery with site conditions that were representative of the study area, i.e., zonal site (BC Ministry of Forests and Range and BC Ministry of Environment, 2010). We obtained long-term climatic data from the China Meteorological Data Sharing Service System and the Canada Natural Resources Database. The differences in temperature and precipitation among the four experimental sites were consistent between the averages of 30 years (1981–2010) and the average of our study period (2014–2017) (Table 1).

2.3. Survival and growth measurement 2.2. Plant materials and growth conditions In 2014, three 1 × 1 m plots with each provenance of each site were randomly selected for survival and growth measurements of the firstyear seedlings on April 30th, August 8th, and November 6th, 2014.

Sugar maple seeds were obtained from the Ontario Tree Seed Facility located in Canada and originated from Toronto, Ontario (ON), 3

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2.4. Measurement of leaf morphological traits In each plot, leaf samples were collected from three randomly selected seedlings with Ontario provenance during the peak of the growing season, when the leaves were fully developed (July 10th, 2017). For each seedling, the leaves of three branches in the middle part of the crown were randomly selected. The sampled leaves were placed in an ice bag for transportation and stored at −20 °C in the laboratory prior to measurements. Fresh leaves without petiole were freshly weighed and scanned using a flatbed scanner (Epson perfection 640U, Epson, USA) for leaf length, width, and area. ImageJ software (NIH Image, Bethesda, MD, USA) was employed for all image analyses. LWR describes the leaf length to width ratio. Primary, secondary, and tertiary veins protrude as ridges, which are visible on the abaxial face of the lamina. For sugar maple seedlings, each leaf has seven primary veins (1° vein), which are directly connected to the petiole, with secondary veins (2° veins) branching from the primary veins. Tertiary veins (3° veins) are smaller in diameter than the secondary veins and branch from (and sometimes link) the primary and secondary veins (Sack et al., 2003). Quarternary veins (minor veins) are fully embedded in the mesophyll and consist of several diameter classes, which are all smaller than the tertiary veins. In the current research, 1° and 2° vein densities were considered to comprise the major vein density (Dmaj), which represented the lower order veins (Roth-Nebelsick, 2001). The 3° and higher-level veins cannot be distinguished in sugar maple leaves; thus, the vein density of 3° and higher-level veins were collectively measured as the minor vein density (Dmin). To determine the Dmaj, all scanned images were analyzed. The 1° veins were measured throughout the entire leaf, and the 2° veins were measured on half of the leaf and multiplied by two. To determine the Dmin, one leaf per tree and three leaves per plot were randomly selected. Three 5 × 5 cm sections at the right side of the midrib were excised from the central section of each sample leaf (Zhu et al., 2011). For each section, one image was obtained at 100× magnification using a microscope (Leica DM 2500) linked to a computer with imaging software (Leica Application Suite). The view field area was 3.84 mm2, and a total of 27 images per site were obtained. Three 5 × 5 areas were randomly selected within the view field images, and all vein lengths were measured using ImageJ. The area data were averaged via the images to facilitate the generation of statistics. The minor vein density (mm mm−2) was expressed as the sum of the length of all its segments (mm) per unit area (mm2). We sampled three leaves in each plot from one of the three randomly sampled trees for stomatal characteristics. During the measurements, we found that the stomata of the seedlings were located primarily on the abaxial leaf surface; thus, we focused on measuring the stomatal density of the abaxial leaf surface in this study. We used the low-temperature scanning electron microscopy (LTSEM, Hitachi S-4800 SEM, Hitachi High-Technologies Corporation, Tokyo, Japan), with digital image analysis to observe the leaf surfaces as described by Pearce et al. (2006). We cleaned the leaf surface carefully and cut three 5 × 5 cm pieces from the center portion of each leaf. Images at 500× magnification were obtained from the lower surface with three views, and ImageJ software was used for image analysis. The stomatal density was calculated as the stomata number per unit leaf area (Rasband, 1997).

Fig. 2. Survival, height, and collar diameter of fourth-year sugar maple seedlings of Ontario provenance at the four precipitation levels. Values are bootstrapped means ± 95% confidence intervals (CIs).

Following four years of acclimation, the Quebec and Manitoba seedlings finally failed at all sites. Therefore, we measured only the provenance from Ontario. On July 10th, 2017 three 10 × 10 m plots with fourth-year seedlings of Ontario provenance from each site were randomly selected for survival and growth measurements. The distance between plots was at least 100 m to minimize spatial autocorrelation. Nine seedlings from each plot were randomly selected for growth measurements. The height was measured as the length from the root collar to the highest leaf sheath using a ruler with 1 mm precision, and the root collar diameter was measured using a digital caliper with 0.01 mm precision. We measured the root collar diameter for the fourth-year seedlings as some of them did not reach breast height (1.3 m).

2.5. Measurement of leaf physiological traits Similar to the stomatal measurements, three scanned fresh leaves from each plot were randomly selected and weighed. They were rehydrated in water for 12 h, re-weighed for turgid weight, subsequently dried at 70 °C for 48 h, and weighed for dry weight. The dry leaf mass per area (LMA) was calculated as the dry leaf mass divided by the leaf 4

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Fig. 3. Morphological and physiological leaf traits of fourth-year sugar maple seedlings with Ontario provenance in relation to different precipitation levels. The hollow circles show individual data and solid circles are bootstrapped means ± 95% confidence intervals (CIs). Abbreviations are LMA (Leaf dry matter per area) and NSC (Non-structural carbohydrates).

via the anthrone method and colorimetrically determined at 620 nm using a UV-2450 spectrophotometer (Shimadzu, Japan). The remaining pellet was saved and hydrolyzed to glucose to determine the starch content. The pellet was mixed with 3 ml of distilled water and a boiling water bath for 15 min. After cooling, 2 ml of 9.2 mol l−1 HClO4 was added to each sample and hydrolyzed for 10 min. Following centrifugation for 10 min and collecting the supernatant, the remainder was re-extracted with 2 ml of 4.6 mol l−1 HClO4 for 15 min. The supernatant that was collected and combined from the two extractions, was adjusted to 50 ml. The concentration was tested via the anthrone method and colorimetrically determined at 620 nm (Xie et al., 2018). The soluble sugar and starch were calculated by comparing them with glucose standards, expressed as mg glucose g−1 DW.

area. The leaf relative water content (RWC) was calculated as:

RWC(%) = [(W − Dw ) / (Tw − Dw )] × 100

(1)

where W is the fresh weight; Tw is the turgid weight following rehydration; Dw is the dry weight. Three leaves from each of the three randomly selected seedlings in each plot were sampled to quantify the presence of non-structural carbohydrates (NSC), i.e., the sum of soluble sugars and starch. Leaves from each plot were pooled, ground, and sieved using a metal-free plastic mill to pass through a 0.2 mm mesh screen for carbohydrate analysis. Soluble sugar was extracted from 50 mg ground powdered materials in 10 ml of 80% (v/v) ethanol. The extraction was performed in a shaking water bath at 80 °C for 30 min (Chow and Landhausser, 2004). Subsequent to centrifugation for 10 min, the supernatant was tested 5

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Yijklm = Pi + Rj + Pi × Rj + Dk + Pi × Dk + Rj × Dk + Pi × Rj × Dk + πl + εm (lijk )

(2)

where Yijklm is the seedling survival rate in each plot; Pi (i = 1, 2, 3, 4) is the precipitation; Rj (j = 1, 2, 3) is the provenance; Dk is the measurement date (k = Apr 2014, Aug 2014, Nov 2014); πl (l = 1, 2,…36) is the random plot effect, accounting for temporal autocorrelation between the repeated measurements; and εm(ijkl) (m = 1, 2, 3) is the survival sampling error. To determine the effects of precipitation, provenance, and measurement date on the growth of the first-year seedlings, we used the following linear mixed effect model:

Yijklmn = Pi + Rj + Pi × Rj + Dk + Pi × Dk + Rj × Dk + Pi × Rj × Dk + πl + θm + εn (ijklm )

(3)

where Yijklmn is the collar diameter or height; Pi, Rj, Dk, and πl are the same as in Eqn. (2); θm (m = 1, 2…12) is the random seedling effect to account for temporal autocorrelation between the repeated measurements; εn(ijklm) (n = 1, 2…9) is the growth sampling error. The linear mixed-effect models were implemented with the ‘lme4′ package using restricted maximum likelihood estimation (Bates et al., 2017). ShapiroWilk’s tests on model residuals indicated that the assumption of normality was met at α = 0.05. We employed one-way analysis of variance (ANOVA) to examine the effects of precipitation on the survival of four-year-old sugar maple seedlings of Ontario provenance, followed by post hoc Tukey’s HSD test, implemented with the ‘multcomp’ package (Bretz and Westfall, 2016). We used a linear mixed-effect model to examine the effects of precipitation on the growth, as well as morphological and physiological traits of the leaves of four-year-old sugar maple seedlings of Ontario provenance with the plot as a random effect (Bates et al., 2017). To examine the multivariate associations between the leaf traits and seedling performance of the different precipitation sites, we used principal component analysis (PCA) and permutational multivariate analysis of variance (perMANOVA) with the ‘vegan’ package (Jari Oksanen et al., 2018). All statistical analyses were performed using R Statistical Software, version 3.5.2 (R Development Core Team, 2018), and graphics were prepared with the ‘ggplot2′ package (Wickham and Chang, 2018). 3. Results The survival and growth of the sugar maple seedlings differed significantly with provenance and MAP, and changed temporally over the first growing season (Fig. 1, Table 2). The Ontario provenance had the highest survival, followed by the Quebec and Manitoba provenances across all sites and measurement dates. The seedling growth of all provenances showed no significant difference at the four sites in April (Fig. 1d,g), and then became distinguished between the different sites and provenances. The seedling growth in August and November showed similar patterns with survival, as growth peaked at the MAP of 1172 mm. The Ontario provenance showed the highest growth among the three provenances (Fig. 1e,f,h,i). Meanwhile, the survival rates of all provenances increased with the MAP until it attained 1172 mm, and then decreased at the MAP of 1490 mm (Fig. 1a–c). All of the Quebec and Manitoba provenance seedlings died off at the end of the 4th year. The survival and growth of the Ontario provenance seedlings responded to MAP similarly to those observed after one year (Fig. 2). The survival did not decline significantly following the first year (Fig. 1c,2a). The seedling height and collar diameter were the lowest at the site with the highest MAP (Fig. 2b,c). The morphological and physiological traits of leaves had different

Fig. 4. Results of principal component analysis (PCA) showing (a) associations between the leaf traits and survival and growth of the fourth-year Ontario provenance seedlings; (b) distributions of the plots with different precipitations in the first two axes. Abbreviations were LA (Leaf area), LWR (Length to width ratio), Dmaj (Major vein density), Dmin (Minor vein density), LSD (Leaf stomatal density), LSS (Leaf stomatal size), LMA (Leaf dry matter per area), RWC (Relative water content), SS (Soluble sugar), and NSC (Non-structural carbohydrates). Large dots are the average scores of precipitation sites. Units associated with variables are in Figs. 2 and 3.

2.6. Statistical analysis To determine the effects of precipitation, provenance, and measurement date on the survival rate of the first-year seedlings, we employed the following linear mixed effect model:

6

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water assisted with offsetting warming-induced respiration (Jarvi and Burton, 2013). Sugar maples could benefit greatly from increased precipitation due to their shallow root system, as a result of adaptation to the soil, with a limited capacity for water storage and fast water depletion in the top layer (Burton et al., 1998). Furthermore, the increased precipitation of the summer monsoon might have also contributed to relieving heat-induced drought stress (Barker et al., 2006). Unexpectedly, we found that a further increase in MAP, from 1172 to 1490 mm, led to decreased survival and growth, and the mechanisms associated with such declines were unclear. The highest MAP site had annual precipitation of over 2000 mm in two of the four experimental years, and also had slightly higher summer temperatures than the other sites. It may have been possible that the heavy precipitation with reduced sunlight coupled with extreme temperatures during the growing season exerted excessive stresses on the transplanted seedlings, as evidenced by their morphological and physiological traits (Fig. 3). Following the four years of acclimation, the Quebec and Manitoba provenances died due to summer heat stress. The survival of the Ontario provenance seedlings with MAP was consistent with those measured at the end of the first year; however, the growth rate was relatively stationary at the highest MAP of 1490 mm. This may have indicated that the seedlings tended to conserve energy for survival, rather than growth under extreme weather conditions. As reported by Beaudet and Messier (1998), sugar maples have a conservative growth pattern and enhance their survival by minimizing the costs associated with the maintenance of wooden structures, while allowing for the increased allocation of attributes that favor long-term survival under adverse environmental conditions. The fourth-year survival rate of Ontario provenance did not change much over several months, which supported the findings in their home range (Putnam and Reich, 2017). The morphological and physiological leaf traits of the sugar maples responded to elevated MAP with different trends. This corroborated with the idea that individual leaf traits are inadequate to reflect plant acclimation to precipitation gradients (Warren et al., 2005; McLean et al., 2014). Our data did show a positive trend of leaf area with MAP, which was consistent with the results by Hilaire and Graves (1999) from their origins near 43°N (670–1150 mm of MAP). Leaf width to length ratio was not significantly altered under elevated precipitation, although leaf shape is known to respond plastically to precipitation, albeit with exceptions (McDonald et al., 2003; Meier and Leuschner, 2008). A potential explanation is that our experiment considered only a limited MAP range over a short period. We found a lower major vein density of the seedlings at the highest MAP site, but no different in minor vein density along the MAP gradient, likely because minor vein density is a conservative trait for many plants (Zhu et al., 2012; Carins Murphy et al., 2014). In contrast to our hypothesis, we found decreases in stomatal density and size with increasing precipitation. It might be possible that the transplanted seedlings were under higher heat stress in sites with lower MAP due to reduced water cooling effects, and had to increase stomatal density and size to increase evapotranspiration to maintain leaf temperatures (Wu et al., 2018). The LMA and relative water content did not show a significant relationship with the MAP, which was consistent with the global scale findings of deciduous species (Wright et al., 2005). The maintenance of LMA and the relative water content of the sugar maples were explained as a survival strategy for drought-intolerant species, or a phenotypic response to stress (Ellsworth and Reich, 1992). The soluble sugar and NSC content of leaves peaked when the starch troughed at the 1490 mm MAP site, as previous studies revealed that water availability increased the NSC primarily through increasing soluble sugar, even if the starch declined (Adams et al., 2013). This carbohydrate allocation pattern was consistent with the results of sugar maple under climate stress (Wong et al., 2003). Overall, our results showed that the transplanted sugar seedlings acclimated to higher MAP by increasing the soluble sugar and NSC, while decreasing the stomatal size and density and starch. This provided insights into the evolution of

levels of plasticity with the MAP following four years of acclimation (Fig. 3). Neither the leaf area nor length to width ratio changed significantly with the MAP (Fig. 3a,b). The minor vein density did not differ with the MAP; however, the major vein density did differ marginally (P = 0.05). The stomatal density and size were significantly altered, with the leaves at the site of the highest MAP having the lowest major vein density, stomatal density, and size (Fig. 3c,e,f). The leaf area per mass (LMA) and relative water content did not change significantly with the MAP (Fig. 3g,h). The soluble sugar and total non-structural carbohydrates were the highest at the highest MAP site, while the starch content decreased with MAP (Fig. 3i–k). The perMANOVA results revealed that both leaf traits (F = 9.28, P = 0.003) and plant performance (including survival and growth) (F = 202, P = 0.001) differed significantly between the four MAP levels. The first two PCA axes explained 38.5% and 23.3% of the variation of all the leaf traits, respectively (Fig. 4). Seedling survival was negatively related to leaf stomatal density, length to width ratio, and relative water content, while the collar diameter and height were positively related to major vein density and minor vein density, and negatively related to NSC, soluble sugar content, and leaf area (Fig. 4a). The higher MAP sites were associated with higher soluble sugar and NSC, while the lower MAP sites were associated with higher starch, leaf stomatal density, and size (Fig. 4b). 4. Discussion During the four years of acclimation to an introduced subtropical climate in Central China, we found that the temperature and precipitation had a pronounced effect on the survival and growth of sugar maple seedlings over time. The Ontario provenance exhibited the best survival and growth across all sites, which was likely due to the minimal MAT differences between the original and introduced sites. The survival and growth of the first-year seedlings of all provenances and the fourth-year seedlings of the Ontario provenance increased with the MAP, peaked at a MAP of 1172 mm, and then decreased at a MAP of 1490 mm. The leaf shape did not change; however, the vein and stomatal traits and NSC responded plastically to changes in the MAP. The plasticity of the morphological and physiological leaf traits was mechanistically linked to the overall plant performance (survival and growth) of the introduced sugar maples. The Ontario provenance had the best survival and growth rates across all sites in Central China over time, which extended our previous study to a larger climate range and longer experimental duration (Zhu et al., 2019). The improved survival of Ontario provenance was attributable to the smallest MAT gap between its origins and the introduced sites. A limited change in temperature range may facilitate the successful introduction of seedlings of a species from a cold to warm climate. Thus, extreme temperature differentials would become the limiting factor (Putnam and Reich, 2017). Adequate winter chilling temperatures in their native range is well known as an important variable for predicting whether buds will mature in the following spring (Kriebel and Wang, 1962). In our study, the winter temperature increase was not detrimental as all of the transplanted sites could supply the winter chilling (< 7 ℃) requirement period (Kriebel and Wang, 1962). However, summer heat stress might have been critical for the performance of the introduced sugar maples. The warmer Ontario provenance was more adapted to temperature and moisture extremes in warmer regions (McCarragher et al., 2013; Solarik et al., 2016), and had higher heat stress tolerance, which resulted in the best whole-plant performance across all sites (Zhu et al., 2019). As we hypothesized, increased precipitation ameliorated the heat stress for introduced sugar maples, as evidenced by the increased firstyear survival and growth from the lowest level of MAP to the 1172 mm. However, a further increase in the MAP was observed to reduce survival and growth. Our result was consistent with a previous experimentally warmed soil study (+4 to +5 ℃) of sugar maples, where additional 7

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leaf traits of sugar maples under climate change. In summary, four years following the introduction of temperate sugar maple to subtropical China, we demonstrated that the elevated MAT and MAP had significant effects on both leaf-level and whole-plant acclimation. The Ontario provenance sugar maple with the smallest MAT gap between the original and introduced sites had the best survival and growth, which indicated that temperature stress was a detrimental factor. The survival and growth of introduced sugar maple increased with MAP to a certain threshold but then declined, which might have resulted from extreme precipitation events. We observed that MAP-dependent survival and growth were strongly associated with the plasticity of veins, stomatal traits, and NSC allocation. Our analysis highlighted that functional trait plasticity offers mechanistic insights into plant acclimation in response to significant variations in temperature and precipitation.

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