Altitude-dependent variations in some morphological and anatomical features of Anatolian chestnut

Abstract

Morphological measurements of Anatolian chestnut (Castanea sativa Mill.) leaves were done within the borders of Abana district of Kastamonu province. The study was conducted using mixed (oak, beech, hornbeam, black pine, and yellow pine) medium (41% to 70%) and fully closed (71% to 100%) stands. Some leaf parameters, such as leaf blade width, petiole length, leaf blade length, leaf length, distance between lateral veins, teeth width, teeth length, the angle between the leaf base and the petiole, and the angle between the midrib and lateral veins, were measured. Moreover, stomata of the leaves picked up from precise altitudes were observed under a scanning electron microscope. The differences between fibre elevation, fibre wall thickness, elasticity coefficient, rigidity coefficient, Muhlstep rate, and Runkel ratio were found in the wood samples taken from different altitude zones. It was found that altitude did not affect leaf blade width, fibre length, fibre width, felting ratio, and lumen width. However, it was determined that altitude affected other studied characteristics.

Full Article

Altitude-dependent Variations in Some Morphological and Anatomical Features of Anatolian Chestnut

Gizem Özdikmenli,^a Nurcan Yiğit,^a Halil Barış Özel,^b and Hakan Şevik ^c

Morphological measurements of Anatolian chestnut (Castanea sativa Mill.) leaves were done within the borders of Abana district of Kastamonu province. The study was conducted using mixed (oak, beech, hornbeam, black pine, and yellow pine) medium (41% to 70%) and fully closed (71% to 100%) stands. Some leaf parameters, such as leaf blade width, petiole length, leaf blade length, leaf length, distance between lateral veins, teeth width, teeth length, the angle between the leaf base and the petiole, and the angle between the midrib and lateral veins, were measured. Moreover, stomata of the leaves picked up from precise altitudes were observed under a scanning electron microscope. The differences between fibre elevation, fibre wall thickness, elasticity coefficient, rigidity coefficient, Muhlstep rate, and Runkel ratio were found in the wood samples taken from different altitude zones. It was found that altitude did not affect leaf blade width, fibre length, fibre width, felting ratio, and lumen width. However, it was determined that altitude affected other studied characteristics.

DOI: 10.15376/biores.19.3.4635-4651

Keywords: Anatolian chestnut; Morphology; Altitude; Variation

Contact information: a: Department of Forest Engineering, Faculty of Forestry, Kastamonu University, Türkiye; b: Department of Forest Engineering, Faculty of Forestry, Bartın University, Türkiye; c: Department of Environmental Engineering, Faculty of Engineering and Architecture, Kastamonu University, Türkiye;

* Corresponding author: nyigit@kastamonu.edu.tr

INTRODUCTION

Anatolian chestnut (Castanea sativa Mill.) belongs to the Fagaceae family. They are long-lasting trees that can reach up to 30 to 35 m in height and can grow up to 1.5 to 2 m in diameter. They have a wide crown and a bark, which is smooth in juvenile trees and cracked in more mature trees (Yaltirik 1993; Subasi 2004).

Anatolian chestnut widely spreads in the highly precipitated and elevated places of the temperate zones in the northern hemisphere. Other locations where it spreads are Northern Africa, Southern Europe, East Asia, and South-West and North America. In terms of altitude, it can grow at 700 to 800 m, and can reach an altitude of 1700 to 1800 m in the Caucasus and Rize province. In addition, it locally spreads in the Aegean and the Mediterranean regions of Türkiye. The only chestnut species naturally grown in Türkiye is Castanea sativa species (Erdem 1951; Kayacik 1981; Yaltirik 1993; Yilmaz 2014).

Castanea sativa generally grows in places with good drainage located in the humid and temperate broad-leaved forests in the Black Sea region of Türkiye. They are rarely found in pure stands. Rather, they are often in mixed form with hornbeam (Carpinus), beech (Fagus), linden (Tilia), alder (Alnus), and ash (Fraxinus) trees. Among the other parts of Türkiye, they grow especially in the main Aegean part of the Aegean region, on the north-facing slopes of Boz Mountains, and in the river valleys of Aydin region. In these areas, they are found in the mixed form with black pine, Calabrian pine, and scrub communities (Özdikmenli 2019).

Plants that are grown in different environments undergo some anatomical and morphological changes adaptable to that environment. Environmental conditions affect all phenotypic characteristics of living organisms (Key et al. 2022). The most important environmental factors are temperature, precipitation, and humidity (Cetin et al. 2023; Dogan et al. 2023). Although other factors remain constant in the same region, climatic parameters change depending on altitude (Tekin et al. 2022; Zeren Cetin et al. 2023). For this, studies on the relationship between altitude and plant morphology can be beneficial for ecological researchers, and morphological knowledge is still very important in many fields of plant sciences, including: population variability (Zebec et al. 2015; Poljak et al. 2018), taxon delimitation (Sękiewicz et al. 2016), morphological and physiological seed characterization (Güney et al. 2015; Daneshvar et al. 2016; Atar et al. 2020), morphological involucre variation (Xue et al. 2020; Atar 2022), cultivar characterization (Ertan 2007; Poljak et al. 2016) and selection (Solar et al. 2005), and variation of macro- and micro-morphological leaf (Poljak et al. 2015; Güney et al. 2016; Bayraktar et al. 2018) and fruit traits (Atar and Turna 2018; Eminagaoglu and Ozcan 2018). Overall, morphological variability and plasticity can be used to predict population dynamics and the evolutionary adaptations of plants to a novel environment (Nicotra et al. 2010). For this reason, many changes are observed in their stomata. Most of the plants’ water loss (about 85% to 90%) occurs through the stomata. Therefore, it is important to know the structure and number of stomata of each plant species (Dickison 2000; Sevik et al. 2017; Yiğit et al. 2023).

This study was carried out to determine the altitude-dependent variations of some morphological and micro-morphological characteristics of the leaves and some anatomical characteristics of the woods of Anatolian chestnut (Castanea sativa Mill.) species as well as the anatomical characteristics of its branches.

EXPERIMENTAL

In this study, the changes in morphological, micro-morphological, and anatomical characteristics of Castanea sativa depending on the altitude were investigated. Samples were taken from the branches and green leaves of the species. The study area is composed of the zones where Anatolia chestnut naturally spreads, within the borders of Abana Forest Sub-District Directorate of Bozkurt Forestry Operation Directorate.

In the studies, Kastamonu University Faculty of Forestry Bilgehan Bilgili Herbarium, Forest Industry Engineering Wood Chemistry Laboratory, and Kastamonu University Central Research Laboratory were utilized.

The study was carried out between 2013 and 2015, by the end of August beginning of September, when Castanea sativa completely stopped growing. Within the scope of the study, 3 different altitude zones were identified (0 to 200 m, 200 to 400 m, and 400 to 600 m), and samples were collected from 3 different points in each altitude zone. From these mentioned points, 5 leaves were collected from 20 trees. Mature leaves were collected from the central parts of the trees that were exposed to the sun from different directions. Their coordinate values were noted; they were kept separately after being numbered and then brought to the laboratory environment. These leaf samples were dried using standard pressing processes. The different altitude zones of each sample and the points from which they were taken were recorded. During this drying phase, the newspapers were periodically changed to avoid decay, fungus, etc. in the plants. The dried leaves were photographed by placing a ruler next to them to create a scale, and files with “.jpeg” extension were obtained.

Nine different morphometric parameters of the leaves were measured using “imageJ” computer measurement program over the scaled leaf photographs. These parameters were specified as;

• LW (blade width (cm)),
• PL (petiole length (cm)),
• LBL (leaf blade length (cm)),
• LL (leaf length (cm)),
• DBLV (distance between lateral veins (cm)),
• TW (teeth width (cm)),
• TL (teeth length (cm)),
• ABP (the angle between the leaf base and the petiole (°)), and the
• ABV (angle between the midrib and lateral veins (°))

Additionally, the stomata on the leaf samples collected from 3 different points in each of 3 different altitude zones (0 to 200 m, 200 to 400 m, and 400 to 600 m) were examined. Scanning electron microscopy (SEM) was used for the examination and the “ImageJ” computer measurement program was used on the obtained figures; the following characters were measured:

• SL (Stoma Length (µm): obtained by measuring the length of 10 stoma figures at each altitude zone),
• SW (Stoma Width (µm): obtained by measuring the width of 10 stoma figures at each altitude zone),
• SPL (Stoma Pore Aperture Length (µm): obtained by measuring the length of 10 stoma pore aperture figures at each altitude zone),
• SPW (Stoma Pore Aperture Width (µm): obtained by measuring the width of 10 stoma pore aperture figures at each altitude zone),
• SW/SL: (Stoma width/Stoma length): the value found by dividing stoma width to stoma length,
• SD (Stomatal density): (obtained by counting the stomata in mag*1000 per unit area)

The Spearin-Isenberg (sodium chlorite and acetic acid) method was used for the fibre-releasing process called maceration (Alkan et al. 2003). The leaves of the samples used for maceration were separated from their woody branches. The pieces of wood, which were brought to the size of the matchstick, were extracted from the last two-year ring of the trunk.

First, the sample pieces, then 0.5 mL of sodium chloride, pure water exceeding the sample size, and then approximately 2 mL of acetic acid with the help of dropper were added in each glass test tube. Some water was added in an empty beaker, and the glass test tubes were placed in this beaker and allowed to boil in the laboratory furnace. For about 2.0 h, NaClO₂ and CH₃COOH were added in half an hour intervals until the lignin in the test tubes was softened.

The samples in the test tubes were washed with water and filtered to remove the solution. Afterwards, the samples were placed into an empty beaker and some water was added onto them. Their disintegration into fibres was observed in the laboratory mixer for 5 to 10 min, and when it was ready, a few drops of alcohol (C₂H₆O) were added onto the jars to prevent deterioration. The disintegrated fibres were observed on a micrometre calibrated SOIF brand binocular laboratory microscope to identify their properties and to perform measurements.

The samples were transferred to a computer by way of an MshOT microscope image transfer camera, and then measured via the program. Fibre length, fibre width, and lumen diameter width of the samples were measured when using a 4x objective glass. A total of 100 measurements were performed for the average fibre length (L), and 50 for fibre width (D) and lumen width (d). At least 150 measurements were carried out from each altitude zone. The fibre wall thickness (W) was calculated using the (D-d)/2 equation.

The cellulose content of the fibres, fibre sizes, and the ratios calculated based on these sizes are important in the determination of the plant’s suitability to be turned into paper. The following equations were used in fibre sizes and the relationships between these sizes (Goksel 1986; Kirci 2006).

• Felting Ratio (FR) = Fibre Length (L) / Fibre Width (D) × 1000
• Elasticity Coefficient (EC) = Lumen Width (d) × 100 / Fibre Width (D)
• Rigidity Coefficient (RC) = Fibre Wall Thickness (W) × 100 / Fibre Width (D)
• Muhlstep Ratio (MR) = Fibre Wall Area (D₂ –d₂) × 100 / Fibre Cross Section Area (D₂)
• Runkel Ratio (RR) = 2 × Fibre Wall Thickness (W) / Lumen Width (d)
• “F” Factor (FF) = Fibre Length (L) × 100 / Fibre Wall Thickness (W)

The data obtained for all characters were evaluated using SPSS 20.0 package program, variance analysis was applied to the data, and the homogenous groups were formed by applying Duncan test to the data with statistically significant differences (p < 0.05). In addition, correlation analysis was conducted to determine the level of relationship between the characters in the study.

RESULTS AND DISCUSSION

In this study, the changes in morphological, micro-morphological, and anatomical characteristics of the chestnut species, which naturally spread in three different altitude zones, were evaluated based on these different altitude zones. Variance analysis was applied to the values measured from the samples taken from different altitude zones, and the analysis results are given in Table 1.

As a result of the variance analysis conducted according to different altitude zones, it was found that the altitude did not affect the lamina width (LW), one of the morphological characteristics. However, altitude zones were observed to affect (at the 99.9% confidence level) some other studied characters of PL, LBL, LL, TW, TL, ABP, and ABV. In terms of the distance between lateral veins (DBLV) character, a difference was determined at the 95% confidence level. The Duncan test was applied to the characteristics to determine this effect rate (Tables 1 and 2).

Table 1. Effects of 3 Altitude Zones on the Leaf Morphological Parameters

According to the results of the Duncan test conducted in accordance with the variance analysis applied to the characteristics with at least 95% confidence of difference between them, it was found that a single class was established in terms of LW characteristics, and that the LW characteristics in the 1^st, 2^nd, and 3^rd altitude zones were in the same class.

As a result of variance analysis, the morphological characteristics related to the altitude zones were not found to be statistically significant (p = 0.117). For this reason, the Duncan test was not applied to the LW character.

Table 2. Duncan Test Results Regarding the Effect of 3 Altitude Zones on the Leaf Morphological Parameters

According to the Duncan test results given in Table 2, it was found that two different classes were formed in terms of PL characteristics and that the PL characteristics in the 2^nd (1.207 cm) and 3^rd (1.308 cm) altitude zones were in the same groups. In addition, it was found that two different classes were formed in terms of LBL characteristics, that the LBL characteristics in the 1^st and 3^rd altitude zones were in the same class, but the value measured in the individuals in the second altitude zone was 18.1 cm. It was found that two different classes were formed in terms of LL characteristics, and that the LL characteristics in 1^st and 3^rd altitude zones were in the same class. It was also seen that there were two different classes formed in terms of DBLV characteristics, and that the DBLV characteristics in the 3^rd (400 to 600 m) and 1^st (0 to 200 m) altitude zones were in totally different classes in terms of measured values. In terms of TW characteristics, it was found that 3 different classes were formed and that the TW characteristics in the 1^st, 2^nd, and 3^rd altitude zones were in separate classes. It was also found that the highest TW value was measured at 400 to 600 m, which was the 3^rd altitude zone. When the TL characteristic was examined, two different classes were formed and the TL characteristic in the 1^st and 3^rd altitude zones were in the same class (Table 2).

When examined in terms of ABP and ABV characteristics, it is apparent that both characteristics formed the first and a different class with the lowest value compared to the characteristics measured at 200 to 400 m altitude zones. The ABP and TW characteristics were in three different classes in terms of measured values.

As a result of the variance analysis performed in accordance with different altitude zones, it was found that the altitude zones affected (with at least 99.9% confidence) 9 characteristics, and the Duncan test was applied to the characters to determine the effect (Table 3).

When the stomata in the leaf samples collected from three different points in three different altitude zones (0 to 200 m, 200 to 400 m, and 400 to 600 m) were examined, it was found that the stomata were hypostomatic type because of the fact that the stomata were present only on the abaxial surface of the leaves.

Table 3. Mean Values of Micro-morphological Characters According to Altitudes

**significant at 0.01 level; ***significant at 0.001 level; The letters a, b, c, etc. means, according to Duncan test results, show that the group is located. It is statistically different from the values contained in different groups, starting with the letter a numerical value grows

As a result of the measurements made, it was found that SL, SW, SPL, and SPW characteristics had the lowest values in chestnut individuals located in an altitude zone of 200 to 400 m. In terms of micro-morphological characteristics, it was determined that the highest values were found in chestnut individuals at an altitude of 400 to 600 m; whereas the minimum stoma density value was found at an altitude zone of zero to 200 m.

The stoma images obtained from the leaf samples collected from the trees at different altitude zones are shown in Figs. 1, 2, and 3.

Fig. 1. Stoma figure in the zero to 200 m altitude zone

Fig. 2. Stoma figure in the 200 to 400 m altitude zone

Fig. 3. Stoma figure in the 400 to 600 m altitude zone

The abaxial surface stoma images obtained from the leaf samples collected from the chestnut trees at different altitude zones are shown in Figs. 4, 5, and 6.

Fig. 4. Abaxial surface stoma in the 0-200 m altitude zone

Variance analysis was applied to the characters measured to determine the altitude-related differences demonstrated by the anatomical characteristics. The results of variance analysis are shown in Table 4.

Table 4. Results of Variance Analysis Applied to the Anatomical Characteristics

Fig. 5. Abaxial surface stoma in the 200 to 400 m altitude zone

Fig. 6. Abaxial surface stoma in the 400 to 600 m altitude zone

When examining Table 4, it can be seen that there were statistically significant differences between L, W, Elasticity Coefficient (EC), Rigidity Coefficient (RC), Muhlstep ratio (MR), and Runkel ratio (RR), and that difference was significant at 99.9% confidence level for all characteristics. The Duncan test was applied to the characteristics that had significant differences as a result of the analysis. Duncan test results are given in Table 5.

Table 5. Duncan Test Results for Anatomical Characters