Adaptive plasticity of morphological and anatomical traits of Brant’s oak (Quercus brantii Lindl.) leaves under different climates and elevation gradients

Abstract The morphological and anatomical characteristics of leaves are sensitive and adaptable to environmental changes. Determining eco-physiological patterns of leaf characteristics along elevational gradients allows for a better understanding and prediction of how plants might respond to climate change. In this work, the ecological adaptation mechanisms related to morphological and anatomical characteristics of Brant’s oak (Quercus brantii Lindl.) leaves were studied at three elevation classes (low, middle, and high) in two different Mediterranean and subhumid climates in Zagros forests in western Iran. There were no significant changes in leaf length, although the leaf-specific area was higher at low and middle elevations in subhumid climates. In addition, stomata length, width, density, and stomatal pore index were higher in the upper elevations of subhumid climate than in the Mediterranean climate. At low and middle elevations, dry matter content was higher at sites from the Mediterranean climate. The results of plasticity indices showed that individuals of Q. brantii from middle-elevation sites exhibited greater plasticity than those from low and high-elevation sites. Overall, Q. brantii, the dominant oak species in Zagros forests, appears to respond to elevational and environmental changes, suggesting that leaves can adapt to these changes through morphological and anatomical traits. These results provide new insights into the environmental adaptation strategies of plants at the morphological and anatomical levels against climate change.


Introduction
Climate change is altering the range of resources and habitats for plant function (Parmesan and Yohe 2003). In a rapidly changing climate, the ability of a plant species to adapt through plasticity or through genetic change will play a critical role in the persistence of that species (Franks et al. 2014;Nicotra et al. 2015). Plasticity is recognized for its key role in short-term adaptive responses to prompt environmental changes, as it can allow the plant to maximize its adaptation under optimal conditions and tolerate stressful environments under unfavorable conditions (Hendry 2016). Many factors, such as climate and topography, influence plant growth. These factors determine the light intensity, temperature, and precipitation to which plants are exposed, which in turn affects plant functional traits Shen 2016, 2017;Niu et al. 2020). Plant responses to changing global climatic conditions can be predicted by analyzing changes in leaf morphological traits and anatomical structures along climatic gradients (Guo et al. 2017;Peng et al. 2020).
Changes in elevation, a parameter with a comprehensive influence on immediate climatic conditions, affect factors such as temperature, precipitation, and illumination. It can have a far-reaching effect over a relatively small vertical range (Zhang et al. 2022). Plant adaptations at different elevations are valuable parameters for understanding and predicting the effects of climate change on plant species in the future (Sundqvist et al. 2013;Mat ıas et al. 2017).
Consideration of the mechanisms of carbon acquisition, water use, and gas exchange associated with leaf traits at the tissue (e.g. anatomical traits), organ (morphological traits), and cell (e.g. stomatal traits) levels may be particularly informative in relation to climate (Liu et al. 2019;Baillie and Fleming 2020). Stomata are responsible for controlling the flow of gases between plants and the outside atmosphere and thus influence water loss and CO 2 uptake (Hetherington and Woodward 2003). To study the processes of photosynthesis and evapotranspiration, stomatal traits may prove more useful than leaf morphological and chemical traits (Wang et al. 2015). Stomatal density and stomatal size are two critical parameters that affect the stomatal conductance of CO 2 and water (Hetherington and Woodward 2003;. A number of studies have examined differences in leaf anatomy and morphology across longitude, latitude, and altitude gradients; however, these studies have mainly focused on variations in general leaf anatomy among different species (Tian et al. 2016;Guo et al. 2017). Liu et al. (2020) showed that leaf length, width, and stomatal density decreased as elevation increased in the three species studied, while leaf thickness increased as elevation increased. According to Lin et al. (2021), stomatal density and stomatal pore index increase with increasing elevation. Leaf area and leaf dry matter content also increased and decreased, respectively, with increasing altitude. Kouwenberg et al. (2007) and Hu et al. (2019) found that stomatal characteristics could differ along elevational gradients due to changes in CO 2 pressure.
Climate change is particularly important in arid and semiarid areas such as the Zagros forests of in western Iran, where Brant oak is a dominant tree species suffering declines that most researchers link to climate change (Hosseini et al. 2017;Moreno-Fern andez et al. 2019;Karami et al. 2022). Studying the characteristics of a plant species responding to current climate gradients is fundamental to understanding and predicting how plants can survive in their habitat despite a changing climate (Nicotra et al. 2010;Anderson and Gezon 2015). We hypothesized that the evolution of tree species at higher elevations is subject to strong abiotic selection pressures and may be distinctly separate from evolution at lower elevations. With the work described here, we aimed to test three hypotheses: (1) How do leaf characteristics of Brant oaks change along elevational gradients? (2) Do climatic variations lead to corresponding differences in leaf property relationships among elevations? (3) to compare the plasticity of oaks from low and high elevations.

Study site
Sampling was conducted in two forest stands in Ilam province in Zagros forest, western Iran. According to the de Marton dryness index (De Martonne 1926) (Equation (1)), there are two climatic zones in this region (Table 1; Figures 1 and 2). Average precipitation and temperatures were calculated for the period 1987-2021 (Ilam Meteorological Bureau 2021). Trees grew as natural regeneration and were sampled from areas with uniform habitat and topography conditions, all belonging to the same biosocial classes. The dominant trees in these two forest stands were Quercus brantii, Crataegus aronia, Acer monspessulanum, and the shrub Daphne sp. (1) where DI: De Martonne dryness index; MAP: mean annual precipitation; MAT: mean annual temperature.

Sampling method
Samples were collected from July to August 2021. First, we established three plots (50 Â 50 m) in each climate with three successive elevation classes (low, middle, and high elevation) ( Table 1). Twelve seedorigin oak trees (Q. brantii L.) with a diameter at breast height (DBH) ranging from 30 to 40 cm were selected (two individuals from each elevation). Geographic information (latitude, longitude, and elevation), plant species composition and ecosystem structure were recorded for each plot. Briefly, in two climatic zones, two healthy trees with no signs of disease, fungi, and decline were selected in each height elevation class. Leaves were sampled from the middle of the canopy using long-handled shears or by climbing. In the subhumid climate, five healthy mature leaves were taken from each tree species in each elevation. Some leaf samples were lost during the study. In the Mediterranean climate, seven healthy mature leaves were collected from each individual, representing one replicate. Leaf samples were placed in sealable plastic bags and immediately stored on ice in a cold box. To better preserve the leaves, samples were fixed in FA (50%; alcohol:formalin:glacial acetic acid:glycerin ¼ 90:5:5:5 v/v) (Liu et al. 2019).

Morphological traits
After sampling, leaf length (LL; mm) was measured using Vernier calipers to an accuracy of 0.02 mm. Leaf area was measured using a leaf area meter (model CI-202, Inc, CID, USA; mm 2 ). Leaf fresh weight (LFW, mg per individual) was measured on an A&C 320.3 electronic balance (Accu LAB, Germany) with an accuracy of 0.0001 g; leaves were then dried in an oven at 60 C to a constant weight (Wang et al. 2016) to measure leaf dry weight (LDW; mg per individual). Dry matter content (DMC; g kg À1 ) and specific leaf area (SLA; mm 2 mg À1 ) were calculated according to the following equations (Liu et al. 2019):

Anatomical traits
To measure the anatomical features of Brant's oak leaves and to better visualize the trichomes and stomata on the epidermis, the density of trichomes was reduced using a twin-blade razor and tape. Chlorophylls were removed by immersion in a mixture (1.5:100 ml) of acetic acid (99%) and hydrogen peroxide (30%) at 100 C for 1 h in a bain-marie water bath (Camargo and Marenco 2011). After washing twice in distilled water, samples were stained in aqueous safranin O and dehydrated in an ethanol series (60%, 85%, 95%, and absolute for 15 min each step) before mounting on glass slides in Canada balsam. Finally, 10 stomata were randomly selected for each tree to measure the length (SL, mm) and width (SW, mm) of the stomata at 40Â magnification using an optical image analyzer connected to a computer (True chrome metrics, Fuzhou, China). The number of stomata (SD, pores mm À2 ) in a microscopic field of 0.5 mm 2 was calculated using Equation (4) (Liu et al. 2018). The stomatal pore index (SPI) was calculated using Equation (5) (Sack et al. 2003) as follows: Where 0.5 is the area of leaf surface examines (mm 2 ).

Plasticity index
Plasticity index (PI) was calculated according to the following formula of Gratani et al. (2006).
where x denotes the lowest average value of all the populations and X denotes the highest average value of all the populations of the corresponding species.

Data analysis
Raw data were tested for normality using the Kolmogorov-Smirnov test, and homogeneity of variances was examined using Levene's test. Means and standard errors of morphological and anatomical traits were calculated for leaf traits in different climates and elevations. Analysis of variance (ANOVA) was used to evaluate the main and interaction effects of leaf traits and climates at a 5% level of probability. The relationships between leaf traits and elevation were examined using linear regression analysis. The SPSS 21 statistical software package was used for all statistical analyses. Principal component analyses (PCA), based on the correlation matrix, using PC-Ord version 5.0 were used to investigate multivariate correlations (i.e. relationships between leaf morphological and anatomical traits across different climatic conditions and at three elevation classes).

Changes in leaf morphological traits
The effects of elevation, climate, and their interactions on DMC were significant (p < 0.01) for leaf traits ( Table 2). The LL showed no changes with elevation. At low and middle elevations in sub-humid climates, SLA indicated a significant increase compared with the Mediterranean climate (p < 0.01). However, no significant differences were found between the two climates at high elevations (p > 0.01; Figure 3(a)). DMC at low and middle elevations was significantly higher in the Mediterranean climate than in the sub-humid climate (p < 0.01; Figure 3(b)).

Changes in leaf anatomical traits
SL in leaf traits were significantly affected by elevation, climate, and their interaction (p < 0.01); in leaf characteristics, differences in SW and SPI in two climates and interactions between elevation and climate were significant (p < 0.01) ( Table 2). The interaction effects of elevation and climate and the effect of elevation individually showed a significant effect on SD (p < 0.01; Table 2). SL, SW, and SPI were significantly higher in the Mediterranean climate and at low and middle elevations than in the sub-humid middle elevations. In contrast, they were significantly higher at high elevations in the sub-humid climate than in the Mediterranean climate (p < 0.01; Figure 4(c,d,f)). At low elevations in the Mediterranean climate, SD was significantly higher than in the sub-humid climate.
However, SD was significantly higher in the subhumid climate and at upper elevation than in the Mediterranean climate (p > 0.01; Figure 4(e)).

Changes in leaf morphological and anatomical traits along elevational gradients
SPI and SD decreased linearly and significantly with increasing elevation (R 2 ¼ 0.66 and 0.58; p < 0.01 and p < 0.05, respectively) in the Mediterranean climate ( Figure 5(a,b)). While other factors in the same climate type did not show significant trends with altitude. In contrast, DMC, SW, SPI, and SL increased linearly and significantly (R 2 ¼ 0.50, 0.56, 0.57, and 0.69; p < 0.05 and p < 0.01, respectively) with increasing elevation in the sub-humid climate ( Figure 5(c-f)). SLA and SD showed no significant changes in elevation in the sub-humid climate.
Principal components analysis (PCA) to ordinate oak trees based on leaf functional traits in different climates and elevation The first and second axes of the PCs explained 36.63% and 20.41% of the variance in morphological and anatomical characteristics under different climatic conditions and elevations. Climatic regions and elevation  were distinguished based on morphological and anatomical characteristics along axes 1-3. Climatically, the low-and middle-elevation in the sub-humid climate were clearly separated from the Mediterranean climate with a higher SLA. For morphological and anatomical traits, low and middle-elevation trees in the sub-humid climate had more similarity and a higher SLA. In contrast, the characteristics of oak leaves collected at middle and high elevations in the Mediterranean climate clustered together and along the positive direction of the second axis. These leaves had the lowest SD values. Furthermore, trees at low elevations in the Mediterranean climate and at high elevations in the sub-humid climate had the greatest similarity in terms of morphological and anatomical characteristics, including SL, SW, SD, SPI, and DMC, which were grouped together in the PCA diagram ( Figure 6; Table 3).

Discussion
A better understanding and prediction of how plant species respond to climate change can be achieved through the eco-physiological patterns of leaf traits along elevational gradients (Blonder et al. 2017). Due to the high plasticity, the changes in leaf functional traits may make plant species migrate to other elevations where growing conditions may not be favorable (Guo et al. 2018). Furthermore, characteristic traits of plants are able to adapt and co-evolve with local habitats. Apart from reflecting the ability of plants to acquire, use, and maintain resources, tree traits also provide information on the interaction between the environment, individual plants, and biological processes, structures, and functions in the ecosystem (Funk et al. 2017;Bruelheide et al. 2018;Eller et al. 2020). SLA is likely to be smaller at high altitudes under high wind speed influences (Figures 3(a) and 6); when SLAs are high in low-elevation areas with relatively abundant resources, a large area of leaf tissue is available for light capture and plants, therefore they have higher production capacity (Zhang et al. 2022). In addition, SLA is an integrative boundary between leaf area and leaf dry weight, and increasing SLA at low and middle elevations may be a strategy by which species adapt to changing conditions by maximizing their photosynthetic rate (Guo et al. 2018).
At low and middle elevations, DMC was significantly higher in the Mediterranean climate than in the sub-humid climate (Figures 3(b) and 6). DMC is considered an indicator of leaf water content (Liu et al. 2019). Higher DMC likely results in higher moisture diffusion resistance in leaves in low-elevation areas. Lin et al. (2021) showed that DMC decreased with increasing elevation in evergreen oak species. Abiotic environmental stresses such as solar radiation, low temperatures, and nutrient losses will increase with increasing elevation. In contrast, photosynthetic activities and carbon assimilation were decreased, leading to a significant reduction in dry leaf weight (Guo et al. 2018).
The morphological characteristics of stomata determine the balance between CO 2 uptake for plant photosynthesis and water deficit through transpiration (Hetherington and Woodward 2003). In addition to short-term dynamics caused by changes in stomatal width, plants can respond to environmental changes by adapting their stomatal morphology through longterm evolution and development (Wang et al. 2015). The SL and SW at low and middle elevations were significantly higher in the Mediterranean climate ( Figures  4(c,d)  reported differential responses to increasing altitude in stomata and photosynthetic activity in an evergreen tree (Quercus spinosa), a deciduous shrub (Salix atopantha), and an annual perennial (Rumex dentatus). While SL and SW increased with elevation in the sub-humid climate (Figures 4(c,d),5(d,e), and 6). This could be due to the fact that plants in lowlight environments form large stomata to produce higher photosynthetic capacity (Hetherington and Woodward 2003).
SD at low elevations was significantly higher in the Mediterranean climate. Although it increased with increasing elevation in the sub-humid climate. (Figures 4(e), 5(b), and 6). The effects of co-varying environmental factors (e.g. temperature, irradiance, vapor pressure deficit, wind speed) and internal plant factors (growth habit, leaf economic strategy) could lead to the interactive responses of stomata with increasing altitude associated with a decrease in CO 2 sequestration (Shi et al. 2015). The high density of stomata under favorable environmental conditions contributes to increased photosynthesis by allowing higher conductance (Pato and Obeso 2012) SPI is an integrative parameter that measures stomatal conductance by integrating stomatal density and stomatal length: higher SPI leads to higher stomatal conductance and photosynthetic capacity of leaves (Sack et al. 2003). In the Mediterranean climate, SPI was significantly higher at low and middle elevations. While SPI increased with increasing elevation in the sub-humid climate (Figures 4(f), 5(a,f), and 6). Therefore, increasing SPI may enable plants to maximize photosynthetic rates at higher altitudes and increase their ability to grow over a relatively short period of time. This could be considered an adaptation strategy of leaf stomatal characteristics to the changing environment (Tian et al. 2016).
Leaves are more sensitive and plastic/variable to environmental change than other organs (Shi and Cai 2009;Klancnik and Gaberscik 2015). A reliable reason for their reactions comes from plasticity, which is the ability of special genotypes to react to natural conditions (Gianoli and Valladares 2012). In the Mediterranean climate LL, DMC, SL, SD, and SPI traits had the most plasticity at the middle elevation, and SLA and SW had the least plasticity at the high elevation (Table 4). In sub-humid climates LL, DMC, SL, SW, and SPI traits showed the highest plasticity at the middle elevation. SLA and SD traits had the least plasticity at high and low elevations, respectively (Table 2). Overall, our results showed that Q. brantii trees exhibited lesser plasticity at low and high elevations than at middle elevations. Escobar-Sandoval et al. (2021) and Vitasse et al. (2013) stated that the leaves show less plasticity at high elevations and that this is constrained more by environmental features than the genetic separation among populations.
Tree species adapted to different environmental conditions must change leaf characteristics to provide optimal performance (Renninger et al. 2020). Highaltitude plants tend to invest more in twig and stem growth in the current year, and thicker stems can support more reproduction and photosynthetic activity, as well as withstanding higher wind speeds ). Plants at low altitudes, in contrast, have longer twigs, because they can better capture light energy, increase flower pollination efficiency, and ultimately, seed dispersal (Zhu et al. 2019). The adaptation abilities of plants to habitat variation associated with elevation are therefore highly dependent on flexible stomatal traits and related ecological and physiological traits.

Conclusion
In this research, we aimed to identify variations in leaf traits under different climates and elevations to understand the adaptation mechanisms of Q. brantii to various conditions. There was no significant change in leaf length, at low and middle elevations, while the leafspecific area was higher in the sub-humid climate than Mediterranean climate. At low and middle elevations, dry matter content was higher in forest sites from the Mediterranean climate. Stomata length, width, density, and stomatal pore index in sub-humid climate and higher elevations were higher than Mediterranean Table 3. Leaf functional traits used in the principal component analysis analyses (PCA) between three elevation classes (low, middle, high) in two climate types. climate. Quercus brantii trees at the middle elevation exhibited more plasticity than at other elevations. Traits associated with Q. brantii might comprise important mechanisms for coping with changing environments, which is worthy of further studies.